Opiate Receptor

Subtypes and

Brain Function

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Opiate Receptor Subtypes

and Brain Function

Editors:

Roger M. Brown, Ph.D.

Doris H. Clouet, Ph.D.

David P. Friedman, Ph.D.

Neuroscience Branch

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 71 1986

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

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NIDA Research Monographs are prepared by the research divisions of the

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NB Associates, Health Sciences

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Alcohol and Drug Problems Association

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Cornell University Medical College

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The Johns Hopkins University School of

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Washington University School of

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Los Angeles, California

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National Federaiton of Parents for

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Langley Porter Neuropsychiatric Institute

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College of Physicians and Surgeons of

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Opiate Receptor Subtypes

and Brain Function
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from a technical

review on opiate receptor subtypes and brain function which took

place on September 26 and 27, 1985, at Bethesda, Maryland. The

review meeting was sponsored by the Office of Science and the

Division of Preclinical Research, National Institute on Drug Abuse.

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The U. S. Government does not endorse or favor any specific

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the context of the studies reported herein.

DHHS publication number (ADM)86-1462

Printed 1986

NIDA Research Monographs are indexed in the Index Medicus. They are

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Biosciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Contents

Page

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Properties of Receptors Mediating Opioid Effects:

Discrimination of Receptor Types

Brian M. Cox and Charles Chavkin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Dynorphin: Specific Binding to Opiate Receptors

Nancy M. Lee and Herbert Lundahl . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Selective Radioligands for Characterization and

Neuroanatomical Distribution Studies of Brain Opioid

Receptors

R. Suzanne Zukin, Ann Tempel, and Mahboubeh Eghbali........28

Anatomical and Biochemical Perspectives on Opioid Peptides

Robert P. Elde ............................................. 48

Opiate-Inhibited Adenylate Cyclase in Mammalian

Brain Membranes

Steven R. Childers, Peter Nijssen, Pautine Nadeau,

Page Buckhanna, Phi-Van Le, and Jeffrey Harris........65

Membrane Conductances and Opioid Receptor Subtypes

R. Alan North .............................................. 81

Modulation of Opioid Peptide Metabolism by Seizures:

Differentiation of Opioid Subclasses

Jacqueline F. McGinty, Tomoyuki Kanamatsu, Johnny Obie,

and Jau-Shyong Hong ........................................ 89

Kappa Isoreceptors: Neuroendocrine and Neurochemical

Evidence

Paul L. Wood and Smriti Iyengar . . . . . . . . . . . . . . . . . . . . . . . . . . .102

v
Page

Multiple Receptor Types in Opioid Discrimination

Stephen G. Holtzman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Neurobiological Substrates of Drug Self-Administration

Jamee E. Smith and Steven I. Dworkin . . . . . . . . . . . . . . . . . . . . . . 127

Neurochemical Substrates for Opiate Reinforcement

George P. Koob, Franco J. Vaccarino, Marianne Amalric,

and Floyd E. Bloom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Opiate Receptor Subtypes Associated With the Brain

Mechanisms of Feeding and Reward

Roy A. Wise, Francois Jenck, Alain Gratton,

and Remi Quirion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

In Vivo Interactions Among Opiate Receptor Agonists

and Antagonists

John W. Holaday, Frank C. Tortella,, Rhoda Maneckjee,

and Joseph B. Long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

List of NIDA Research Monographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

vi
Preface

OPIOID RECEPTOR SUBTYPES AND BRAIN FUNCTIDN

A technical review was held on September 26 and 27, 1985, at the

National Institute on Drug Abuse to examine the current state of

knowledge of opiate receptor subtypes and specific opioid peptides

that act on these receptor subtypes, and to consider how brain

function might be related to the diversity of receptor/ligand

combinations. This monograph consists of papers delivered at this

review. The conclusion of each presentation, suggested or expressed, was that there are indeed a variety of opioid receptors

differing in ligand specificity and location in the nervous

system, and that these receptors do mediate different functions.

The current descriptions of the complex processes underlying the

functioning of opioid neuronal pathways have been derived from

investigations at all levels from the molecular to the behavioral. It now appears that by combining new neuroscience technology with well-described behavioral paradigms, questions about the

effects of the various peptide-ligands and receptors on behavior

itself can be answered. As a group, the papers reveal the widespread distribution and influence of opiopeptides within the nervous system and highlight some of the exquisitely sensitive control

mechanisms that allow these systems to react to and counteract the

varieties of influences that make up our daily lives as well as

those of drug abusers.

Multiple Receptor Subtypes Defined

The uses of the various methods that serve to define opioid receptors and pitfalls of these methods were described by BRIAN COX.

The original definition of subtypes termed the mu, kappa, and

sigma receptors was based on physiological responses to opiates in

the chronic spinal dog. More recently, two new subtypes, delta

and epsilon receptors, have been recognized as well. Newer methodologies for characterizing receptors have utilized biochemical

measures of binding affinities (KD), total numbers of receptors

(Bmax), potencies of various agonists (A50 and IC50), agonist

affinities as measured from agonist effects? (KA), and estimates of

vii
antagonist affinity (Ke). Although a few of the endogenous opioid

ligands may have exquisite specificity for some subtypes of the

opiate receptor, the synthetic narcotic agonists and antagonists

and the opiopeptide-related synthetic compounds generally react

with more than one subtype of opiate receptor. Although the

specificity of synthetic ligands continues to improve, crossreactivity makes the determination of the physical characteristics

of the receptors difficult.

Many neuropeptide hormones exist as large molecules, sometimes

active, that can be hydrolyzed by peptidase action into smaller

peptides that are active harmonally. Proopiomelanocortin is such

a peptide. It is processed into several active subunits including

beta-endorphin. In turn, beta-endorphin is hydrolyzed into

smaller subunits including methionine-enkephalin. Dynorphin is

another opiopeptide that can be hydrolized into smaller active

peptides. NANCY LEE described this process, showing that short

peptides tended to react with mu and delta receptors, while longer

segments reacted at kappa receptors. Using refined methodology,

she showed that none of the dynorphin subunits seem to have a

clear specificity for any one receptor subtype.

SUZANNE ZUKIN summarized our knowledge of opioid receptor subtypes

(table 1), opioid receptor subtype distribution in brain, and ligand specificities. In addition to biochemically defined receptor

properties, the dynamic nature of the receptors is emphasized;

they can change in number or affinity depending on the physiological state or previous drug history. An example of receptor

dynamics is "up-regulation" in response to opiate antagonist

administration. Treatment with an opiate antagonist such as

naloxone results in (1) a supersensitivity to morphine, (2) an

increase in mu receptor density observed in layers I and I II of

frontal cortex, nucleus accumbens, and amygdala, and (3) increased

enkephalin levels. This up-regulation is not permanent but requires about 6 days to return to predrug status.

Opioid receptor subtypes were further defined and classified by

PAUL WOOD from the perspective of Martin's 1976 definition of mu,

kappa, and sigma receptors, the receptor bindinq properties of mu

and delta ligands, and the in vitro immunological characterization

of mu, delta, and epsilon subtypes. He further categorized opiate

subtypes into mu1 and mu2 as well as kappa1 and kappa2 receptors.

Since kappa agonists act as antagonists at the mu2 receptor, it

may be possible to either develop an analgesic that has less unwanted side effects, or to combine kappa and mu agonists to obtain

analgesic effects while blocking out the unwanted side effects.

The Neuron Model

Anatomical and biochemical perspectives of opioid peptide and

opiate receptor subtypes were presented by ROBERT ELDE, who discussed our current understandins of opioid function in terms

of the neuron model. The classic criteria for identifying transmitters are: (1) distribution, (2) production within the neuron,

viii
TABLE 1. Receptor subtypes and ligand specificity

(3) release and inactivation, (4) identity of action, and

(5) antagonism. It is now clear that opioids and their receptors

have a selective, though widespread distribution. The peptides

are manufactured from large propeptides, which may contain a variety of products. Activation, as well as inactivation, occurs by

proteolytic cleavage. The difficulty in establishing a good

neuron model for the Dpioid system is the same problem that has

caused difficulties with other neurotransmitters; that is, the

diffuse nature of peptide distribution and the difficulty of finding a brain region with a chemically homogeneous neuron population

to examine closely. Another problem lies in the specificity of

drugs available to study these systems. For example, although

antagonists to most receptor subtypes have been found, the usual

difficulty ensues: the antagonists are not specific for the subtype. The so-called "mismatch" of opioid receptors with peptidergic nerve terminals was also discussed. In some brain regions,

there are high concentrations of opioid peptides but few receptors, whereas in other regions there are many receptors but few

peptides. This mismatch may be more apparent than real for several reasons. The release of a ligand in amounts considerably less

than the amounts of receptor would be a very sensitive way of

producing short-term regulation. On the other hand, low receptor

ix
population with high ligand levels might create a long-term regulation of neurotransmitter release at the synapse. In addition,

it is also possible that, since histochemical identification of

opiopeptides usually involves antibody interaction, the same antibody may not recognize peptide fragments that are not completely

processed, thus underestimating the amount of ligand.

Transmitter Release Utilization, and Receptor Activation

One approach to relate opioid receptor function to ligands that

bind to the opioid receptors is to examine the role of second

messenger mechanisms in opioid action. STEVEN CHILDERS described

the pharmacology of the well-known ability of opiates to cause

physiological inhibition of adenylate cyclase. Activation of

adenyl cyclase requires the binding of the activating transmitter

to a receptor that is linked to an N(+) protein which, in turn, is

linked to the cyclase itself. Inhibition of adenylate cyclase

occurs through a similar mechanism , except that the cyclase link

is a N(-) protein. However, when a striatal tissue preparation

was exposed to low pH that uncouples the N(-) protein, various

opioid ligands still inhibited adenylate cyclase activity, suggesting that an as yet undiscovered mechanism is responsible for

the inhibition.

R. ALAN NORTH described changes in voltages and currents induced

by altered flow across nerve cell membranes that result from

activation of specific subtypes of opiate receptors. For these

studies, a variety of nervous tissues were used including the submucous plexus of the guinea pig ileum, which has a pure population

of delta receptors, and the locus coeruleus, which contains only

mu receptors. In all cases studied so far, activation of opioid

receptors leads to inhibition of the neuronal firing. The ionic

events which take place during inhibition include an efflux of K+

that hyperpolarizes the cell and reduces Ca++ influx. Reduced

Ca influx reduces the amount of transmitter released even if the

cell continues to fire. An inhibition of firing in the ileum is

produced by delta receptor activation, whereas mu agonists have

the same effect in the locus coeruleus. Both receptors seem to be

linked to the same kind of K+ channel. Selective delta and mu

receptor activation decrease Ca++ transport as a result of K+

conductance increases, whereas selective kappa receptor activation

in the neurons of the myenteric plexus appears to decrease directly the entry of Ca++ into these cells. Thus, opioids may inhibit

neural activity and transmitter release by a variety of mechanisms, some of which are also shared by nonopioid transmitters

such as norepinephrine.

JACQUELINE MCGINTY has developed models to study opioid metabolism. release. and utilization. She has studied alterations in

these parameters in the hippocampus following electroconvulsive

seizures. The excitatory state of neurons induced by the convulsions is correlated with an initial loss of enkephalin, then a

subsequent rebound in the content of this peptide within the

neurons. In addition, there is an increase in mRNA for enkephalin

x
biosynthesis which is concomitant with enkephalin release, increased levels of intermediary enkephalin products, and eventually, increased levels of enkephalin. Whereas immunostaining shows

a quick increase in met-enkephalin levels after seizures, it shows

only a slow return to baseline for dynorphin levels, which are

also lowered during seizures. This suggests that the loss of dynorphin does not lead to a stimulation of prohormone biosynthesis

or processing in this paradigm.

Behavioral Expression of Opioid Receptor Activation

Behavioral models which distinguish opioid receptor subtypes

were described by four neuropsychopharmacologists. STEVEN

HOLTZMAN described a "psychophysics" approach to determining

selectivity of opioid receptor activation using the paradigm of

stimulus discrimination. An animal is trained to press one of two

levers for food or water under the influence of the "training"

drug (kappa agonist, for example) and to press the other lever

following an injection of vehicle. When the discrimination

("drug" vs. "no drug") is established, the ability of other drugs

to generalize to the training drug is tested. Using this approach, the ability of an opiate to exert a behavioral effect on a

particular receptor subtype is evaluated using one or all of the

following criteria: (1) similarity of effect, (2) stereospecificity, (3) specificity of antagonist actions, and (4) order of

potency. When morphine (a mu agonist) was used as the training

drug, etonitazine was found to strongly mimic morphine action,

while codeine or propoxyphene did not generalize to morphine until

they were given in high doses. Presumably, mu agonism is weak in

the latter two drugs, and high doses are needed before a threshold

mu effect is obtained. Other receptor agonists can be used as the

basis for discrimination. PCP was found to generalize to sigma

agonists (SKF-10,047, cyclazocine, ketamine, or dextrorphan).

That the locus of the discrimination is central is shown by the

demonstration that the behavior can be established following

intraventricular as well as systemic administration.

An approach to studying the neurobiological basis of drug-seeking

behavior was presented by JAMES E. SMITH who has found, using a

yoked-control procedure, that the biochemical consequences of

active drug self-administration are different from those resulting

from passive drug infusion. Further, a role for the ventral tegmental dopamine system (the A10 cell group and its projections)

has been established in mediating the reinforcing action of a

variety of drug classes, including opiates. The nucleus accumbens, a projection area of the A10 cell group, contains primarily

delta opiate receptors, whereas the ventral tegmental region

itself contains mostly mu receptors. The behavioral relevancy of

this receptor distribution is that a low to moderate dose of morphine is self-administered into the ventral tegmental area (VTA)

but not into the nucleus accumbens, but a high dose is administered into both regions. Presumably it takes high doses of morphine, which is primarily a mu agonist, to activate delta

receptors. Consistent with this differential activation of opioid

xi
receptor subtypes in the brain is the finding that met-enkephalin,

a delta agonist, is self-administered into the nucleus accumbens

at low doses. Thus, at least two opiate receptor subtypes and the

neural systems with which they are associated play an important

role in the reinforcing action of drugs.

The use of a place-preference paradigm to evaluate the reinforcing

qualities of kappa, mu, and other opiate receptor agonists was

discussed by GEORGE KOOB. In this model, an animal-is placed in

one side of a two-compartment chamber and injected with a test

drug. The tendency to return to the location where drug was previously received is correlated with the drug's reinforcing

qualities. In general, kappa agonists were not found to be particularly reinforcing in this system. On the other hand, betaendorphin was found to be reinforcing at low doses. A variety of

experiments have implicated the delta receptor, in addition to the

mu and epsilon receptors, in the rewarding action of this peptide.

The lack of place preference at high doses was due to the induction of a state of catatonia which is incompatible with placepreference behavior.

A fourth approach to testing the functional role of ooioid receptor activation was discussed by ROY WISE, who has discovered that

a neural network involved in feeding behavior can be activated in

a frequency-dependent manner by stimulation of either the ventral

tegmental area (VTA) or the periaqueductal gray (PAG) region. In

VTA neurons, the activation threshold is lowered by D-Pen-D-Pen

enkephalin (or DPDPE, a delta agonist), morphine (a mu agonist),

or U-50,488H (a kappa agonist), whereas in the PAG the delta and

kappa agonists had no effect, but morphine still facilitated the

behavior. This is a new behavioral paradigm that has already

shown its utility in evaluating the action of opioid receptor subtypes, and this approach might be used to examine the hypothesis

that several reinforcers may all activate a common pathway.

Clinical Aspects

The search for a nonaddicting narcotic analgesic has led us to

discover opioid receptors and endogenous opiatelike substances in

the brain. The descriptions of several subtypes of the opiate

receptor and of drugs which can selectively stimulate these sites

have brought the search for selective analgesia closer to its goal

than ever before. In the process, anatomical and/or biochemical

dissociation of the substrates underlying analgesia, physical

dependence, and euphoria has been demonstrated. In addition, the

functions of a number of opiate receptors have come to light and a

number of research programs are actively exploring ways to use

this information clinically. Some of the avenues being explored

in relation to drugs with specific action on receptor subtypes

were described by JOHN HOLADAY, who discussed the roles of opiate

receptor subtypes in a of clinical conditions:

(1) Kappa receptors and dynorphins appear to play a role in regulating the severity of both spinal injury due to trauma and

xii
any central nervous system (CNS) damage due to ischemia. The

evidence is that the spinal cord contains large numbers of

kappa receptors, dynorphin levels increase in spinal injury,

and dynorphin can induce a paralysis that is reversed by

kappa antagonists. Thus, kappa antagonists might be useful

in treating ischemia and stroke.

(2) Opiate receptors are also involved in some forms of shock.

Naloxone reverses endotoxin-induced hypotension, and this may

be due to an involvement of delta receptors. The hypotension

occurring in shock may be mediated by delta receptors, because delta agonists produce hypotension and delta antagonists can block the shock-induced hypotension.

(3) Mu and delta receptors may modify seizures. The flurothylinduced seizure model has been studied using a variety of

selective ligands. Etorphine potently elevated seizure

threshold, an effect that was totally blocked by mu antagonists but only partially blocked by delta antagonists. Delta

antagonists entirely blocked the ability of DADL to elevate

seizure thresholds, whereas mu antagonists had no effect on

DADL. This model has also produced interesting data suggesting that some kappa agonists may act also as longlasting mu

antagonists and as antagonists to delta antagonists. These

findings suggest interesting interactions among the subtypes

of opiate receptors and may be interpreted as indicating

close anatomical linkage between at least the mu and delta

receptors, a suggestion that finds support from other findings as well.

Summary

During the past decade, we have made impressive theoretical and

practical progress in defining opioid systems and relating specific opioid peptides and specific subtypes of opiate receptors to

specific behaviors. Table 1 presents a summary of current knowledge of the relationship of receptor subtypes to behavior. During

the next decade, we will see not only the full development of the

neuron model, but our understanding of the role of opioids in

brain function will increase dramatically. We also anticipate a

host of new opioid-based pharmaceuticals and new modes of treatment not only for drug-abuse-related conditions, but, as suggested

by Dr. Holaday's presentation, for conditions that now have no

conceivable relation to drug abuse.

THE EDITORS

xiii
Properties of Receptors

Mediating Opioid Effects:

Discrimination of Receptor Types

Brian M. Cox and Charles Chavkin

INTRODUCTION

The initial proposal by Beckett and Casy (1954) that opiate drugs

might act through specific receptors to produce analgesic effects

was based on a comparison of the analgesic potencies of enantiomeric pairs of opiates and the relative potencies of chemically

similar synthetic opiatelike drugs. An extensive study of a

complex series of phenylpiperidines led Portoghese (1965) to note

that not all the activities observed with these compounds could be

explained by a common mechanism of interaction with a single

receptor type. He therefore raised the possibility that opiate

drugs might be able to interact with more than one type of

receptor. Martin and coworkers (Martin 1967; Martin et al. 1976)

also found it necessary to postulate the existence of more than

one type of receptor in order to account for the differences in

the profile of actions exerted by several chemically dissimilar

opiates in the chronic spinal dog preparation. Martin et al.

(1976) named these receptor types after the prototypic opiate

compounds which induced each pattern of actions; morphine acted on

μ receptors; ketocyclazocines, on receptors; and SKF-10,047 (Nallylnormetazocine), on μ receptors. Despite the different

pattern of effects for each of these drugs, Martin regarded the

effects of each compound as opiatelike, because all the actions

appeared to be antagonized by naloxone or naltrexone (Gilbert and

Martin 1976; Martin et al. 1976). These in vivo results were

supported by results obtained in in vitro bioassay preparations.

Lord et al. (1977) noted the different relative potencies of

selected alkaloid and peptide opioids in inhibiting the electrically stimulated guinea pig ileum (GPI) and mouse vas deferens

(MVD) preparations, and differences in their sensitivity to

antagonism by naloxone in the same tissues. They were also able

to show that the apparent receptor heterogeneity could be confirmed by comparing the relative potencies of several opioids in

displacing radiolabelled alkaloid and peptide opioids from binding

sites in brain neural membranes. Sites at which the alkaloid

opiates interacted in these tissues showed properties similar to

those predicted for Martin's μ receptors. Ethylketocyclazocine

1
appeared to act at a separate site comparable to Martin's receptor, while the enkephalins apparently acted at yet another site,

clearly distinct from Martin's site, which Lord et al. (1977)

characterized as

Evidence from numerous in vivo and in vitro studies has documented

the existence of type opioid receptors. The existence of additional opioid receptor types has also been proposed,

and the properties of receptors are under study in

several laboratories. receptors were first identified by Schulz

et al. (1979) and Lemaire et al. (1978) as receptor sites in the

rat vas deferens, having high affinity and selectivity for

endorphin and fragments longer than ß-endorphin (1-19). In this

smooth muscle bioassay, the effects of ß-endorphin can be blocked

by naloxone with an apparent affinity between 15-27 nM (Gillan et

al. 1981; Lemaire et al. 1978). Surprisingly, the benzomorphan

opioids, ethylketazocine and bremazocine (potent receptor

agonists) are pure antagonists in the rat vas deferens (Gillan et

al. 1981). receptor binding sites in rat brain membranes have

been biochemically detected by Chang and coworkers (1984). However, the existence of receptors in the rat vas deferens was

recently challenged (Smith and Rance 1983).

Very high affinity (Kd<1 nM) binding sites able to bind

opioids without discrimination were first identified as a subset

of μ receptor designated μ1 by Wolozin and Pasternak (1981). The

noncompetitive antagonists naloxazone and naloxonazine, synthesized by Pasternak, are claimed to be selective, irreversible

inhibitors of μ1 binding. μ1 sites are postulated to be important

for mediating opioid analgesia but not respiratory depression

(Nishimura et al. 1984; Ling et al. 1985).

The receptor first described by Martin (Martin et al. 1976) has

also been extensively studied. Whether receptors are opioid

receptors is a matter of definition and is currently the subject

of ongoing debate. The original description of the behavioral

syndrome included the observation that these effects could be

blocked by naltrexone (Martin et al. 1976). In addition, the

prototypic ligand N-allylnormetazocine (SKF-10,047) is a derivative of the opioid structure. Yet others have found that SKF10,047 produces a syndrome in rats and monkeys that is not sensitive to naloxone antagonism (Harris 1980; Brady et al. 1982).

Additional studies have shown that the 3H-SKF-10,047 has two different binding sites: one shared by the nonopioid, phencyclidine

(Zukin and Zukin 1981; Mendelsohn et al. 1985), and a separate

site that binds haloperidol (Su 1982; Tam 1985). Neither site

binds naloxone with high affinity. Thus, if we accept the definition that the opioid receptor types must all share the characteristic of sensitivity to the Dpioid antagonists naloxone and

naltrexone, then the existence of opioid receptors has only been

shown in the chronic spinal dog preparation.

The methods described in this review have proven to be quite

powerful and will undoubtedly be important for the further

2
characterization of these additional opioid receptor types. As

will be apparent from the following discussion, strategies for the

identification of opioid receptor types require converging methodologies, since no single method is unambiguous.

The in vivo results of Martin et al. (1976) indicating the

presence of more than one type of opioid receptor are now well

supported by in vitro studies. Methods for the characterization

of each receptor type have become available.

The different types of opioid receptors have been shown to differ

primarily in their relative affinities for different opioids,

although in some cases there is accumulating evidence for differences in the signal transduction mechanisms employed by opioid

receptor types (North, this volume). In examining the properties

of a receptor mediating a specific effect, it is therefore necessary to examine the affinities and potencies of opioid ligands

interacting with that receptor. Opioid receptors, like others,

may be characterized by measurement of ligand binding affinities,

measurement of agonist potencies in inducing an effect, estimates

of agonist affinity constants obtained from measurements of the

pharmacological effect, and estimates of antagonist apparent

affinity for the receptor by measurements of the potencies of

antagonists in inhibiting the actions of opioid agonists. These

analyses each provide insight into a different component of agonist action and, when compared for different opioids in different

tissues, may allow a profile of the properties of each receptor

type to be assembled.

OPIOID LIGAND BINDING: LIGAND DISSOCIATION CONSTANTS (Kd)

The initial interaction of a ligand with its receptor is obviously

essential for agonism or antagonism to be observed, and the affinity of a ligand for its receptor is an important determinant of

its potency, although pharmacokinetic and other factors may also

significantly affect potency. The estimation of Kd therefore provides critical information regarding a ligand's interaction with a

receptor. Many studies of radiolabelled opioid ligand interactions with binding sites in membrane preparations from brain and

other tissues have now been conducted, following the pioneering

studies of Terenius (1973), Pert and Snyder (1973), and Simon et

al. (1973). It is now clear that a few opioid ligands bind with

high affinity in a manner predicted by a simple Langmuir adsorption isotherm and are thus presumed (in the absence of evidence to

the contrary) to bind to a single type of binding site. An

example of such a ligand is [

3

H-D-Ala2-MePhe4-Gly-ol5]enkephalin

(DAGO), which appears to have high selective affinity for μ-type

opioid receptors (Gillan and Kosterlitz 1982). However, most

opioids show a more complex interaction with neural membrane

preparations. By suitable curve fitting procedures, the plot of

amount of specifically bound ligand vs. ligand concentration can

usually be resolved into a summation of two or more Langmuir

adsorption isotherms (Munson and Rodbard 1980). The use of such

procedures, especially when comparisons are made between the

3
binding affinities of both selective and nonselective opioids in

displacing a series of selective radiolabelled opioids. enables

the apparent affinities of many ligands at several types of

binding sites to be determined (Werling et al. 1985).

A question arises as to the significance of binding affinities

measured in this way. Most studies of this kind have employed

buffer media lacking ions critical for tissue function, at temperatures lower than that at which the tissue usually functions.

Such abnormal conditions tend to increase the ratio of specific to

nonspecific binding of opioid radioligands, thus making quantitation of binding more reliable and possibly increasing the stability of the ligands used. Opioid agonist binding is generally

reduced in the presence of sodium and is also sensitive to changes

in incubation temperatures (Simantov et al. 1976). It is uncertain to what extent variations in environmental conditions

affecting agonist affinity also affect receptor selectivity.

However, it appears that the discrimination of opioid receptor

types observed under optimum binding conditions can still be

observed when incubations are conducted in a modified Krebs medium

at 37 °C (Werling et al. 1985; Zarr et al. 1986). A comparison of

ligand selectivities measured in Tris buffer and modified Krebs is

presented in table 1. The selectivity of the agonists is generally not impaired, although [D-Ser2

-Leu5]enkephalyl-Thr (OSLET)

TABLE 1. Comparison of opioid agonist receptor selectivities

determined by displacement of radiolabelled ligands in

Tris buffer or modified Krebs buffer

Receptor Types

Ligand Conditions (°C) μ

DAG0

DSLET

U50488H

Tris, 25o*

Krebs, 37°**

Tris, 25°

Krebs, 37°

Tris, 25°

Krebs, 37°

[8.36] 130 170

[8.34] 330 >200

20 [8.74] >1,OOO

9 [8.05] >11O

1,300 12,000 [9.14]

>280 >280 [8.46]

*Data from James and Goldstein (1984) using guinea pig brain membranes.

**Data computed from Werling et al. (1985) using guinea pig cortex membranes.

NOTE: Binding selectivity profiles are tabulated according to the convention proposed by James and Goldstein (1984). Figures In square brackets are the

negative logarithms of the K1 values (in molar units) for each displacing

ligand at the site at which thls ligand has highest affinity (the preferred

site). Other values are ratios of the Kl at the designated site to the K1

at the preferred site. Values preceded by the > symbol are minimal values;

the K1 at the nonpreferred site exceeds 1 μM and is not accurately

determined.

4
(Gacel et al. 1980) appeared to show less discrimination between μ

and μ binding sites in the modified Krebs medium. On the basis of

such studies, ligands with good selectivity for each type of opioid receptor can be established, and the ligand binding properties

of each type of receptor can be defined in terms of their relative

affinities for a few select ligands; μ receptors bind DAGO with

much higher affinity than DSLET, [D-Pen2

-D-Pen5]enkephalin (DPDPE)

(Mosberg et al. 1983). or U50488H (Lahti et al. 1982); receptors

bind DPDPE or DSLET with much higher affinity than DAGO or

U50488H; and receptors bind U50488H with much higher affinity

than DAGO, DSLET, or DPDPE. This type of relative affinity comparison has become very useful in the functional characterization

of opioid receptor types (Gillan and Kosterlitz 1982; James and

Goldstein 1984; Werling et al. 1985).

AGONIST POTENCY ESTIMATES (A50 AND Ka)

Agonist-dependent and tissue-dependent factors that determine the

magnitude of a tissue's response to an agonist are summarized in

table 2. Differences in the ability of drugs to gain access to

receptors and differences in their stability in tissues are often

neglected as possible contributors to relative potency differences

TABLE 2. Components of agonist action

Agonist dependent

1. Access to receptor; ligand stability

2. Affinity (Kd)

3. Intrinsic efficacy

Tissue dependent

4. Receptor concentration ([Ro]); spare receptors

5. Receptor heterogeneity (Ro1, Ro2, etc.)

6. Stimulus-effect relationship (E/S)

Model

NOTE: In the model, A represents an agonist; R1, R2 represent different receptors

interacting with A; AR1* and AR2 are activated forms of each agonist

receptor complex (e.g., following an agonist-induced conformational change

and/or an interaction with another macromolecule); and E1, E2 are effects

produced as a result of receptor activation by agonist A.

between tissues, but clearly these differences require consideration. The demonstration that peptidase inhibitors significantly

increase the apparent potencies of some but not all opioid

5
peptides in bioassay preparations (Corbett et al. 1984; James and

Goldstein 1984) emphasizes the importance of demonstrating agonist

stability in quantitative estimates of potencies, if artifactually

low estimates of potency are to be avoided.

If completely specific agonists or antagonists for each type of

opioid receptor were available, then determination of the type of

opioid receptor mediating a particular effect could be made simply

by observing in a qualitative fashion whether or not a response

was induced (or antagonized) by each type of specific agent.

Since absolute specificity is at present not attained in opioid

receptor ligands, some consideration must be given to determining

the degree of selectivity that is required for unambiguous receptor identification by qualitative demonstration of activity

induced by a partially selective ligand. Unfortunately, the required level of selectivity for agonists is very high; substantial

differences in agonist efficacies and tissue receptor concentrations will greatly affect the potency of an agonist and thus

reduce the reliability of potency estimates in qualitative discrimination of receptor types. The situation is less critical for

antagonists, where the specificity is determined exclusively by

its affinity for each receptor type. Here, information from

radioligand binding studies is relevant; it is readily apparent

from radioligand displacement studies that very clear discrimination of receptor types can be achieved with ligands with more than

about 200-fold selectivity for a particular receptor type.

Competitive antagonists for specific opioid receptor types with

this degree of selectivity are not yet available, although the

selective peptide antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH

(ICI 174,864) approaches this level of selectivity (Corbett et al.

1984). It is therefore necessary to make quantitative estimates

of agonist potencies and from these to compute functional agonist

affinities.

A convenient measure of agonist potency is the concentration of

agonist required to produce 50 percent of maximum effect in a

particular preparation (A50). This value can be obtained by

interpolation in concentration-response curves or by computation

from a theoretical curve fitted to the experimental data (Black

and Leff 1983). However, A estimates alone are not reliable

indicators of agonist affinity for the receptors mediating the

observed effect. In addition to binding affinity, observed

agonist potency is often controlled by the efficacy of the

agonist, receptor reserve in the tissue, and the ability of the

intact drug to penetrate to the site of action. These are

characteristics that greatly differ among the opioids available

and limit the value of simple potency estimates in characterizing

receptors.

Furchgott (Furchgott and Bursztyn 1967; Furchgott 1978) has proposed a method by which functional estimates of agonist affinity

can be obtained from comparisons of agonist dose-response curves

constructed before and after irreversible inactivation of a fraction of the receptors. This is essentially a null-point method in

6
which equal effects are assumed to result from an equal stimulus

to the effector system; in the model outlined in table 2, for

example, an equal number of activated agonist-receptor complexes.

The second assumption is that the noncompetitive antagonist used

does not alter the binding affinity of the residual receptor sites

or the efficacy of the agonist. The development of the

irreversible opioid antagonists ß-chlornaltrexamine (ß-CNA) and

ß-funaltrexamine (ß-FNA) (Portoghese et al. 1979; Portoghese et

al. 1980) has enabled this method to be applied to the computation

of opioid agonist affinities. Figure 1 shows dynorphin A(1-13)

dose-response curves in guinea pig ileum and mouse vas deferens

preparations, before and after ß-CNA treatment. It is apparent

that the receptor reserve for the receptors through which dynorphin exerts its inhibitory effect is greater in guinea pig ileum

than in mouse deferens, since the same ß-CNA treatment produced a

greater reduction in maximum effect in vas than in ileum. However, when reciprocals of doses producing equal effects before and

after ß-CNA treatment are plotted and the Ka values computed from

the slopes and intercepts, it appears that dynorphin had a very

similar affinity for the receptors mediating its effects in each

tissue. Other evidence also points to this conclusion (Cox and

Chavkin 1983), suggesting that despite considerable differences in

potency, dynorphin acts in both mouse vas deferens and guinea pig

ileum through type opioid receptors. Opioid agonist affinities

have been estimated in other systems by the same technique; some

representative values from the literature are listed in table 3.

In each case, the affinity estimates are higher than the A50 values obtained in the same preparation. The ratio of Kato A50

gives an approximate indication of the magnitude of the receptor

reserve in each tissue.

Some indication of the magnitude of the receptor reserve in a bioassay preparation can also be obtained by examination of the effect of treatment with an irreversible antagonist on the agonist

log. concentration-response curve. An example of such an experiment is shown in figure 2. Normorphine, dynorphin A(1-13), and

[Leu5]enkephalin act through opioid receptors, respectively, to inhibit contractions of mouse vas deferens preparations. After ß-CNA treatment, the maximum response obtainable

with normorphine and dynorphin A(1-13) was considerably reduced.

However, the log. concentration-response curve for [Leu5]enkephalin was shifted to the right, with no reduction in maximum response after the same ß-CNA treatment. A higher concentration of

ß-CNA produced a further parallel shift to the right in the

[Leu]enkephalin curve. The simplest explanation of these results

is that after destruction of a fraction of the receptors by covalent occupation by ß-CNA, insufficient receptors were left for agonists acting through these receptors to

produce full inhibition. However, sufficient receptors remained

for a full inhibitory response to be obtained. Thus, there was a

greater reserve of receptors than receptors in this

tissue.

7
FIGURE 1. Estimation of dynorphin affinity at receptors in

guinea pig ileum and mouse vas deferens

NOTE: (A) Log. concentration-response curves for dynorphln A(1-13) on the inhibition of stimulated responses In isolated guinea pig ileum (GPI) and mouse

vas deferens (MVD), before and after exposure to ß-CNA (3 nM, 20 minutes,

followed by 90 minutes of repeated washing). Each data point represents a

mean value from estimates obtained in four preparations (for methods, see

Chavkln and Cox (1983)). (B) Furchgott plots, showing reciprocals of equieffective concentrations of dynorphln A(1-13) before and after ß-CNA treatment, are shown. The Ka values for dynorphin A(1-13) In each tissue were

computed from the equation Ka [(slope-1)/intercept] (Furchgott 1978). Data

from Chavkin and Cox (unpublished).

For all three types of opioid receptors considered here, there

appears to be a substantial receptor reserve in the tissues listed

in table 3. However, it should be noted that the estimates of

8
TABLE 3. Estimates of agonist affinity

Ligand

Ka A 5 0

Ratio

(μM) (μM) (Ka/A50)

μ Receptors

Normorphinea

GPI 1.5 0.25 6

NormorphinebRat LC slice 12 1.2 10

MorphineC

Rat hot plate 1.7 0.2 8

[Met5]enkephalinbRat LC slice 16 0.6 25

OADLEbRat LC slice 2 0.15 13

Receptors

[Leu5]enkephalind

Receptors

Dynorphin A

d

Dynorphin A

d

MVO

MVO

GPI

2

0.02

0.02

0.02

0.0014 16

0.0003 88

100

KEY: GPI, guinea pig ileum; CC, locus coeruleus; MVD, mouse vas deferens.

aPorreca and Burks (1983).

bWilIiams and North (1984).

c

Tallarida and Cowan (1982).

dCox and Chavkin (unpublished).

functional agonist affinity are most readily made in tissues where

agonist potency is initially very high; that is, where there is

very probably a substantial receptor reserve. No conclusions

about the generality of the occurrence of spare receptors in opioid receptor populations can therefore be made from this very

limited sampling of tissues. Recent studies have demonstrated

that opioid tolerance is associated with a reduction in opioid

receptor reserve, at least in guinea pig ileum myenteric plexus

(Chavkin and Goldstein 1984).

As further studies are completed, it seems probable that it will

be possible to characterize the various types of opioid receptors

by examination of the agonist affinity constants for a few relatively selective agonists. It should be noted that the Furchgott

method depends on a double-reciprocal plot. Alternative methods

in which nonlinear curve-fitting procedures are used to fit the

experimental data have been proposed (Parker and Waud 1971; Black

et al. 1985). The errors inherent in the estimates of opioid

9
FIGURE 2. Effects of ß-CNA treatments on log. concentrationresponse curves for normorphine (NM; upper panel),

dynorphin A(1-13) (D13; middle panel), and

[Leu5]enkephalin (LE; lower panel) in isolated mouse

vas deferens

KEY: = prior to treatment; = after ß-CNA. 1 nM for 20 minutes; = after ßCNA, 3 nM for 20 minutes.

NOTE: After pretreatment, the tissues were washed repeatedly for 90 minutes to

remove noncovalently bound ß-CNA before the log. concentration curves were

redetermined. Results are the mean values from tour preparations; vertical

bars are estimates of the standard errors of the mean values. The ß-CNA

1-nM treatment clearly reduced the maximum response to normorphine and

dynorphin A(1-13), while producing a parallel shift to the right In the

[Leu5]enkephalin log. concentration-response curve. (For details of methods, see Cox and Chavkin (1983)).

10
agonist Ka have generally not been calculated, but may be significant in relation to affinity estimates which differ by less than

one or two orders of magnitude. For this reason, receptor discrimination will be most reliably achieved if receptor affinities

are estimated with agonists with considerable selectivity for each

receptor type.

ESTIMATES OF ANTAGONIST AFFINITY CONSTANTS (Ke)

Classically, estimation of antagonist affinity constants has been

the method of choice in receptor discrimination (Schild 1947).

Kosterlitz and coworkers (Kosterlitz and Watt 1968; Lord et al.

1977) have shown the utility of estimates of naloxone affinity in

characterization of opioid receptors. However, naloxone has the

disadvantage that its affinities for type opioid receptors are very similar, and only about tenfold lower than its

affinity for μ receptors. Thus, estimates of naloxone Ke have

been valuable in discriminating receptors mediating opioid effects

in tissues where the functional contribution to the actions of an

agonist of either receptors can be shown to be negligible,

on the basis of independent evidence (e.g., in guinea pig ileum

(Lord et al. 1977) or in NG 108-15 cells (Sharma et al. 1975)).

However, naloxone cannot reliably discriminate between

receptor-mediated effects in mouse vas deferens preparations (Cox

and Chavkin 1983). There is generally good agreement between

estimates of naloxone affinity for opioid receptors based on displacement of the specific binding of radiolabelled agonists and on

antagonism of their pharmacological effects (table 4) .

There are few other pure antagonists of opioid actions. Of the

currently available compounds, diprenorphine, a partial agonist

with weak agonist effects in some opioid systems, comes closest to

being a universal antagonist at all types of opioid receptors.

This attribute makes it useful as a radiolabelled ligand in displacement studies examining selective agonists but limits its

value in the direct discrimination of receptor types. A recently

developed enkephalin analogue (ICI 174,864) has very good selectivity for receptors (Cotton et al. 1985) but relatively low

potency (table 4). Further studies are needed to determine how

useful this compound will be in pharmacological discrimination of

receptor types. A selective antagonist for receptors is not yet

available. The benzomorphan compound Mr 2266 has comparable

antagonist potencies at receptors (Lord et al. 1977)

but also has weak agonist actions in some systems.

The use of partially selective antagonists may give ambiguous results in receptor classification if nonselective agonists are used

in an experimental system where the same effect can be induced by

more than one receptor type. An example is provided by studies of

the actions of opioid peptides derived from prodynorphin on isolated guinea pig ileum preparations (Chavkin and Goldstein 1981;

James et al. 1984). In these studies, naloxone gave Ke values of

3 to 4 nM against the normorphine standard, a fairly selective μ

agonist. Ke values of about 30 to 40 nM were observed against

11
TABLE 4. Estimates of opioid antagonist affinity constants

Affinity

Receptor Type/ Constant,

Antagonists Procedures nM

μ Receptors

Naloxone

Receptors

Naloxone

Receptors

Naloxone

ICI 174,864

Displacement of 3H-DAG0, brain 2

e

Guinea pig ileum, antagonism of DADLE 2

e

Guinea pig ileum, antagonism of normorphine 1

a,e

Mouse vas, antagonism of normorphine 4

c,d

Rat locus coeruleus, antagonism of 3

f

normorphine

Rat locus coeruleus, antagonism of DADLE 3

f

Displacement of 3H-EKC, brain (μ block)

Guinea pig ileum, antagonism of EKC

Guinea pig ileum, antagonism of

17e

23a

91a

dynorphin A

Mouse vas, antagonism of dynorphin A 18d

Displacement of 3H-DADLE, brain (μ block)

Displacement of 3H-DPDPE, brain

Mouse vas, antagonism of [Leu5]enkephalin

Displacement of 3H-DADLE, brain (μ block)

Displacement of 3H-DPDPE, brain

30b

23b

24c

170b

190b

a

Chavkin et al. (1982).

b

Cotton at al. (1985).

c

Cox and Chavkin (1983).

d

Gillan and Kosterlitz (1982).

e

Magnan at al. (1982).

f

Williams and North (1984).

12
dynorphin A and dynorphin B, which are thought to act preferentially with receptors. However, peptides of intermediate length

such as dynorphin A(1-8), a-neo-endorphin, and ß-neo-endorphin gave

intermediate naloxone Ke values ranging from 8 to 22 nM. The

simplest interpretation of these intermediate values is that these

agonists exert effects on the release of acetylcholine in guinea

pig ileum by interactions with both μ and receptors. The intermediate Ke values may arise as a weighted average Ke determined by

the relative contributions of each receptor type to the agonist

effect of the peptide. Thus, the antagonist Ke alone is not a

useful guide to the receptors mediating a response unless it is

determined against a fairly selective agonist. In estimates of

naloxone Ke, it is desirable to construct agonist concentrationresponse curves at several concentrations of antagonist and to

determine the slope of the Schild plot (log (DR-1) v.log. antagonist concentration (Schild 1947)). The Schild equation assumes

that only a single receptor binding site is involved; therefore,

slope values significantly different from 1.0 will invalidate the

estimate of agonist affinity.

Quantitation of antagonist potencies after in vivo administration

can also be used in the characterization of receptors. Competitive

opioid antagonists produce a parallel shift to the right of in vivo

log. administered dose-response curves that is analogous to theshift of in vitro log. concentration-response curves (Cox and

Weinstock 1964; Smits and Takemori 1970). Pseudo-Schild plots can

be constructed and in vivo apparent pA2 values computed. However,

it should be noted that these values are not quantitatively identical to pA2 estimates obtained from in vitro concentration-effect

curves. This estimate is related to the true antagonist affinity

constant by proportionality factors (usually unknown) which reflect

the relationship between administered dose and the free drug concentrations of both agonist and antagonist in equilibrium with the

receptor (Tallarida et al. 1979). Since proportionality between

dose and tissue concentration may not hold over a large range of

agonist or antagonist concentrations , and the time of peak tissue

concentration of drug (which is most likely to be directly proportional to administered dose) may differ for agonist and antagonist,

receptor discrimination by estimates of in vivo pA2 is potentially

subject to additional sources of error which limit its usefulness.

Irreversible antagonists which preferentially interact with one

class of receptors can also be used to identify receptor types

mediating a pharmacological effect. The naltrexone analogue ß-FNA

appears to act irreversibly with μ receptors (Portoghese et al.

1980). Thus, actions mediated exclusively through μ receptors can

be antagonized by ß-FNA. It is important in the use of irreversible antagonists to confirm that the observed antagonism is

irreversible. This is necessary, because such agents may also

display reversible components of action which do not demonstrate

the receptor selectivity of the irreversible component. ß-FNA

shows receptor agonist activity which may dominate the profile of

actions observed shortly after ß-FNA administration (Ward et al.

1982). However, this action is reversible; at longer time periods,

13
a selective antagonism of opioid action at μ receptors is observed.

The irreversible nature of this antagonistic effect can be demonstrated by showing inability to overcome the antagonism by the

addition of higher doses of μ agonist (Ward et al. 1982; Gmerek and

Woods 1985). Another irreversible antagonist with selectivity for

μ receptors is naloxonazine (Hahn et al. 1982). Unlike ß-FNA, however, this agent appears to have selective affinity for a subset of

μ receptors, designated by Wolozin and Pasternak (1981) as μ1.

Irreversible antagonists which can act selectively with

receptors are not currently available; their development would

considerably aid the characterization of opioid receptor types.

When used in vivo, irreversible receptor ligands have a duration of

effect which considerably exceeds the duration of action of naloxone or naltrexone. However, their duration of action is not

indefinite; antagonized responses usually return 3 or 4 days after

treatment (Portoghese et al. 1979). This is generally assumed to

result from the synthesis and integration into neural membranes of

newly synthesized receptors which are not occupied by the irreversible antagonist.

Quantitative characterization of opioid receptor types is now feasible by several methods which examine different aspects of ligand

interaction with receptor and the induction of agonist effect.

Application of these approaches to opioid receptor classification,

using receptor-type selective agonists and antagonists, should lead

to the development of profiles of the properties of each type of

receptor which will be useful in defining the receptors mediating

opioid action in other systems.

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16
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Takemori, A.E. Synthesis and pharmacological characterization of

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3

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17
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ACKNOWLEDGMENTS

Some of the work reported here was supported by grants from the

National Institute on Drug Abuse. The opinions and assertions

contained herein are those of the authors and are not to be

construed as official; nor do they reflect the views of the

Department of Defense or the Uniformed Services University of the

Health Sciences. The experiments reported herein were conducted

according to the principles set forth in the Guide for Care and Use

of Laboratory Animals, Institute of Laboratory Animal Resources,

National Research Council (DHEW Pub. No. NIH 78-23, 1978).

AUTHORS

Brian M. Cox. Ph.D.

Department of Pharmacology

Uniformed Services University of the

Health Sciences

4301 Jones Bridge Road

Bethesda, MD 20814-4799

Charles Chavkin, Ph.D.

Department of Pharmacology SJ-30

School of Medicine

University of Washington

Seattle, WA 98195

18
Dynorphin: Specific Binding to

Opiate Receptors

Nancy M. Lee and Herbert Landahl

INTRODUCTION

The fact that multiple receptors and multiple endogenous ligands

exist in the opioid system is now well established. The receptors

include μ, selective for morphine and related compounds; selective for enkephalins; selective for ethylketocyclazocine, and

probably others as well (Martin et al. 1976; Lord et al. 1977).

The endogenous opioid peptides can be grouped into three major

classes with members of each sharing a common precursor: ß-endorphin and related peptides, the enkephalins, and dynorphin and

neo-endorphin (Rossier 1982).

Since one assumes that every endogenous ligand must interact with

a specific receptor, it is tempting to hypothesize that each of

the known receptor types interacts with a different class of

endogenous opioids. In the case of the receptor and the enkephalins, the correlation is clear, but in the case of the

receptors, the picture is not so simple. ß-endorphin is known to

interact with the μ receptor, but it binds equally well to receptors (Lord et al. 1977). Furthermore, it has not been established whether still another so-called receptor is responsible

for some of its physiological effects (Wood 1982).

With regard to the receptor, it has been suggested that dynorphin is the endogenous ligand, because it behaves like the drug

ethylketocyclazocine in the guinea pig ileum (Huidobro-Toro et al.

1981), and because it binds to sites in mammalian brain (Chavkin

et al. 1982). However, this peptide has many in vivo effects that

are quite Unlike those of typical agonists. Although not analgesic itself, it antagonizes morphine- or ß-endorphin-induced

analgesia in naive mice and, even more remarkably, potentiates the

analgesic effects of these opioids in tolerant animals (Tulunay et

al. 1981); this potentiation is also seen in the chronically

treated guinea pig ileum (Rezvani and Way 1984). Dynorphin also

potentiates morphine-induced respiratory depression, again while

having no effect by itself (Woo et al. 1983).

19
These findings suggest that while dynorphin may interact with

receptors in the brain, it probably interacts with other types of

opioid receptors as well. In order to explore this question, we

have examined the binding of dynorphin to brain homogenates and

its effect on the binding of other opioid ligands.

DYNORPHIN COMPETITION WITH OTHER LIGANDS

The ability of dynorphin to interact directly or indirectly with

different types of opiate receptors was first tested by determining the effect of this peptide on the in vitro binding of various

tritiated opioid ligands to a brain membrane fraction. As shown

in table 1, dynorphin is able to compete with a number of different opiates, including dihydromorphine (DHM), a prototypical μ

ligand; D-Ala2-D-Leu5 enkephalin (DADLE), a drug, and ethylketocyclazocine (EKC), a agonist. In further studies, a range of

concentrations of the unlabelled drug was used and dynorphin's

effect on the resulting Scatchard plots determined. The binding

was analyzed on the assumption that there were two populations of

binding sites for each ligand; in every case, dynorphin's main

effect was to reduce BMax of the high-affinity site in a doserelated manner. In the case of EKC, dynorphin also increased the

high-affinity Kd (Garzon et al. 1984). These results establish

that dynorphin interacts with not only receptors for ligands.

but for the others as well.

TABLE 1. The potency of dynorphin 1-13 to inhibit binding of

various opiate ligand binding to mouse brain P2

fraction

Tracer Drugs Apparent ICso (nM)

Dihydromorphine

Etorp ine

D-Ala2

-D-Leu5-enkephalin 

D-Ala2-Met5-enkephalinamide

Ethylketocyclazocine

Naloxone

10

160

50

14

17

52

NOTE: The concentration of radiolabelled ligand used was 0.5 nM. The ligand and

dynorphin1-13 were added simultaneously.

DYNORPHIN INTERACTION WITH SPECIFIC OPIOID SITES

While the studies just discussed suggest possible heterogeneity

in dynorphin binding sites, it is difficult to conclude anything

about the specific sites involved, because each of the displaced

ligands may interact with several sites. Thus DADLE has been

reported to interact with μ as well as sites (Chang and

Cuatrecasas 1979), while EKC binds to in addition to

20
sites (Pfeiffer and Herz 1981). A further problem with Scatchard

plots is that it is difficult'to cover a wide range of drug concentrations, because data at very low and high concentrations

(i.e., binding to highest and lowest affinity sites) are subject

to large experimental error. Error also is increased because both

ordinates depend on the free drug concentration, which may be

difficult to determine with precision.

In order to gain insight into dynorphin's interaction with

specific sites, we used a competition technique, coupled with

computer analysis. To define the sites involved, we initial1y

studied the effects of tritiated dihydromorphine (DHM), D-Ala2-DLeu5

-enkephalin (DADLE), and ethylketocyclazocine (EKC) on the

binding of the corresponding unlabelled ligands. The resulting

data generated nine competition curves (figure 1). These could

then be compared with theoretical curves generated by a computer

program which assumed various numbers of binding sites and various

values for different parameters associated with-these sites.

FIGURE 1. Competition curves

NOTE: Binding of tritiated EKC, DADLE, and DHM was assayed in the presence of

various concentrations of each of these unlabelled ligands (substituting

morphine for DHM). Theoretical curves are Indicated by solid lines.

21
To analyze the binding data, we used the following mathematical

model. For each site i, of total number Roi, the number of tracer

molecules of concentration (T) bound to the receptor will be

(TRi) = (T) (Ri)/KiT,

receptors and KiT

where Ri is the total number of occupied

is the dissociation constant of receptor i for

tracer T; likewise, the number of drug molecules of concentration

D bound to the receptor will be:

The total number of Roi

and (DRi) from which:

is just the sum of the free Ri plus (TRi)

so that

(1)

(2)

The measured value YTD is the ratio of the sum of the amounts of

tracer bound to each receptor in the presence of tracer and drug

(in amount D) divided by the total amount bound when the tracer

has a concentration T, but D equals 0. That is, the specific

binding for each of the nine curves in figure 1 is given by:

or substituting (2) into (3).

(3)

(4)

If T and D refer to the same drug, T and D are concentrations of

labelled and unlabelled drug, respectively, and KiT equals KiD.

In all experiments, T equals 0.5 nM. The quantity T is retained

in equation (4), since a number of K values were found which were

substantially less than T.

The best fit under these conditions for the data shown in figure 1

proved to be a model assuming four independent sites. In this

model, site 1 was most selective for DADLE, sites 3 and 4 were

most selective for DHM, and site 2 bound only EKC, although the

latter drug also interacted with sites 1 and 3.

We next applied this technique to dynorphin 1-17 and some related

peptides. The latter included dynorphin 1-5 through 1-13, and

neo-endorphin, and dynorphin B. An inspection of the competition curves using these ligands with 3

H-DHM showed that most of

these compounds competed to some extent with DHM at site 3. These

affinities are shown in table 2. A similar approach was applied

to the isplacement of 3H-DADLE, to obtain K values for site 1,

and to 3H-EKC, for K values for site 2 (table 2).

22
TABLE 2. Dissociation constants for dynorphin fragments (mM)

Competing

Drugs #1

Sites

#2 #3

Dyn 1-5

1-6

l-7

1-8

1-9

1-10

1-11

1-12

1-13

1-17

neo-end

Dyn B

DHM

DADLE

EKC

4.1

113.0

126.0

106.0

20.0

24.0

8.1

43.0

14.0

10.0

46.0

36.0

252.0 19,860.O 4.70

4.0 56,700.O 50.80

1.7 2.3 3.50

1,500.0

2.0

4.0

10.0

13.0

6.0

2.0

10.0

13.0

25.0

0.4

100.0

63.00

342.00

355.00

1,735.00

1.60

5.50

0.24

0.89

1.20

0.75

30.00

1.40

In conclusion, our binding data indicate that dynorphin-related

peptides bind to all three major sites in our analysis. Since

enkephalins have the greatest affinity for site 1, and morphine

has the greatest affinity for site 3, these sites correspond to

the classes of conventional terminology. Furthermore, as

the peptide becomes longer, the affinity for site 3 generally

becomes higher relative to that of site 1.

KINETICS OF DYNORPHIN INHIBITION OF OPIATE BINDING

The time course of the inhibitory effect of dynorphin 1-13 and

1-17 on tritiated naloxone binding at 37 °C is presented in tables

3 and 4. Peptides were added in a concentration of 100 nM 15

minutes after naloxone (the antagonist at this temperature requires less than 4 minutes to reach equilibrium), and specific

binding of 3H-naloxone was monitored. The maximum inhibition was

obtained during the first 5 minutes and was practically identical

for both peptides. After this period, the inhibition decreased

very slowly, the half-time or recovery of naloxone binding being

approximately 40 minutes for dynorphin 1-13 and 90 minutes for

dynorphin 1-17. In a parallel study, the remaining free form of

the peptide was evaluated over these time intervals using the

mouse vas deferens (MVD) bioassay.

In further studies, we unexpectedly found that dynorphin inhibits

in vitro binding even if the peptide is administered in vivo and

23
TABLE 3. Time course of the inhibitory effect of dynorphin 1-13

on 

3H-naloxone (1 nM) specific binding

Time After 3

H-Naloxone

Specific Detectable

Dynorphin 1-13 Peptide

(100 nM) Binding* (MVD)**

(Minutes) (Percent) (Percent)

5

10

15

20

30

45

60

75

90

17

23

27

28

44

60

90

92

108

18

16

5

*Tritiated naloxone (1 nM) was equilibrated (15 minutes at 37 °C), then peptide

was added to a final concentration of 100 nM. Binding was determined and specific binding expressed as percentage of the controls which received buffer

Instead of the peptlde.

**Samples received peptlde without 3H-naloxone. The amount of peptide present was

calculated from a dose response curve and then expressed as percent of the

initial concentration added.

TABLE 4. Time course of the inhibitory effect of dynorphin 1-17

on 

3H-naloxone (1 nM) specific binding

Time After 3

H-Naloxone

Specific Detectable

Dynorphin 1-17 Peptide

(100 nM) Binding (MVD)

(Minutes) (Percent) (Percent)

5

10

15

20

30

45

60

75

90

105

120

150

180

10 70

17 50

22 30

18 20

20 10

35 5

40

47

50

70

82

90

98

NOTE: The experimental procedure Is identical to that described In table 3,

except that dynorphin 1-17 instead of 1-13 was studied.

24
washed away during preparation of the brain membranes. In these

experiments, mice were injected intracerebroventricularly (ICV)

with various doses of dynorphin and sacrificed after various periods of time for tissue preparation. Significant inhibition of in

vitro binding of both 3H-DHM and 3H-DADLE was observed following

an ICV dose of dynorphin as low as 1 μg (Garzon et al. 1984).

This effect was observed with animals sacrificed up to 6 hours

after this dose, while binding returned to normal after 12 hours.

This inhibition was not affected by subjecting the brain membranes

to extensive washing, suggesting that it was not due to the presence of free dynorphin in the tissue.

Further studies of this novel finding showed that shorter fragments of dynorphin did not have this effect (Garzon et al. 1984),

indicating that the inhibition was probably not due to a dynorphin

metabolite. However, inhibition could be reversed by incubation

of the membranes from dynorphin-treated mice with either morphine

or with Na+ ion (Garzon et al. 1984). Our interpretation of this

is that the inhibition may be due to a longlasting association of

dynorphin with its receptor(s), with dynorphin being displaced by

morphine or Na+. This conclusion is consistent with the observation that dynorphin dissociates very slowly from its receptors and

suggests that it may be the endogenous ligand reported by others

to interfere with in vitro opiate binding assays (Simantov et al.

1976).

CONCLUSIONS

Our initial studies of the in vivo action of dynorphin indicated

that it was unique among opiates. Although having no analgesic

properties of its own, dynorphin was able to modulate the analgesia induced by morphine or ß-endorphin. Furthermore, the direction of this modulation--enhancement or inhibition--depended on

the state of the animal.

The studies discussed here further support the notion of the

uniqueness of dynorphin and provide some clues to the mechanisms

underlying its modulatory action. Thus, although we have found

that dynorphin interacts with type opioid sites, as others have

reported, we also have evidence that it interacts with μ and

sites as well. This interaction with multiple binding sites in

itself is not unusual, as other endogenous opioids, such as ßendorphin and the enkephalins, also bind to more than one site.

However, the broad range of its interactions, together with the

apparently complex manner in which it interacts with these various

sites, suggests that dynorphin may have special modulatory properties. It is also possible that some of the effects of dynorphin

are exerted through as yet unidentified receptors, that is, ones

that are not labelled by even such a nonspecific opioid agonist as

EKC.

25
REFERENCES

Chang, K.J., and Cuatrecasas, P. Multiple opiate receptors:

Enkephalin and morphine bind to receptors of different

specificities. J Biol Chem 254:2610-2618, 1979.

Chavkin, C.; James, I.F.; and Goldstein, A. Dynorphin is a specific endogenous ligand of the opioid receptor. Science

215:413-415, 1982.

Garzon, J.; Sanchez-Blazquez, P.; Gerhart, J.; Loh, H.H.; and Lee,

N.M. Dynorphin A: Inhibitory effect on other opiate ligand

bindings in the mouse brain. Biochem Pharmacol 33:2609-2614,

1984.

Huidobro-Toro, J.P.; Yoshimura, K.; Lee, N.M.; Loh, H.H.; and Way,

E.L. Dynorphin interaction at the opiate site. Eur J

Pharmacol 72:265-266, 1981.

Lord, J.A.H.; Waterfield, A.A.; Hughes, J.; and Kosterlitz. H.W.

Endogenous opioid peptides: Multiple agonists and receptors.

Nature 267:495-499, 1977.

Martin, W.R.; Eades, C.G.; Thompson, J.A.; Huppler, R.E.; and

Gilbert, P.E. The effects of morphine- and nalorphine-like

drugs in the non-dependent chronic spinal dog. J Pharmacol Exp

Ther 197:517-532, 1976.

Pfeiffer, A., and Herz, A. Demonstration and distribution of an

opiate binding site in rat brain with high affinity for ethylketocyclazocine and SKF 10047. Biochem Biophys Res Comm 101:38-

44, 1981.

Rezvani, A., and Way, E.L. Dynorphin-(1-13) restores the potency

of morphine on the tolerant guinea pig ileum. Eur J Pharmacol

102:475-479, 1984.

Rossier, J. Opioid peptides have found their roots. Nature

298:221-222, 1982.

Simantov, R.; Snowman, A.M.; and Snyder, S.H. Temperature and

ionic influences on opiate receptor binding. Mol Pharmacol

12:977-986, 1976.

Tulunay, F.C.; Jen, M.F.; Chang, J.K.; Loh, H.H.; and Lee, N.M.

Possible regulatory role of dynorphin on morphine and ß-endorphin induced analgesia. J Pharmacol Exp Ther 219:296-298, 1981.

Woo, S.K.; Tulunay, F.C.; Loh, H.H.; and Lee, N.M. Effect of

dynorphin-1-13 and related peptides on respiratory rate and

morphine-induced respiratory rate depression. Eur J Pharmacol

96:117-122, 1983.

Wood, P.L. Multiple opiate receptors: Support for unique

and sites. Neuropharmacol 21:487-497, 1982.

ACKNOWLEDGMENTS

This research was supported by grant DA02643 (NML) and Research

Scientist Career Award 5-K02-DA00020 (NML).

26
AUTHORS

Nancy M. Lee, Ph.D.

Adjunct Associate Professor

Department of Pharmacology

and Langley Porter Psychiatric Institute

School of Medicine

University of California, San Francisco

San Francisco, CA 94143

Herbert Landahl, Ph.D.

Professor

Department of Biochemistry and Biophysics

University of California, San Francisco

San Francisco, CA 94143

27
Selective Radioligands for

Characterization and

Neuroanatomical Distribution

Studies of Brain Opioid Receptors

R. Suzanne Zukin, Ann Tempel, and Mahboubeh Eghbali

INTRODUCTION

Opiates and opioid peptides produce actions on the nervous system

by interaction with opioid receptors and related receptors. These receptors exhibit differing ligand selectivity

patterns, diverse neuroanatomical distribution patterns, differing

physiological and behavioral profiles, and differing sensitivities

of opioid agonists at each site to naloxone antagonism (Snyder

1984; Zukin and Zukin 1984). Most of the classical opiate drugs

and endogenous opioid peptides interact with more than one of

these receptor sites. Thus, the complex neuropharmacological

actions of a given opioid would appear to reflect interaction with

varying potencies at a combination of these sites.

Opioid receptors are membrane associated, and their integrity

appears dependent upon protein (Pasternak and Snyder 1974),

carbohydrate (Howells et al. 1982), and phospholipid (Lin and

Simon 1978) components. Studies in neuronal cell lines demonstrating opioid-induced inhibition of adenylcyclase (Klee and

Nirenberg 1976; Sharma et al. 1975), as well as studies in brain

and cell lines showing guanyl nucleotide modulation of agonist

binding (Blume 1978; Childers and Snyder 1978; Zukin et al. 1980),

suggest that μ and receptors are coupled to cyclase through an

inhibitory guanyl nucleotide binding protein (Ni). The molecular

dimensions of the receptors have been estimated in prelabelling

experiments and, more directly, in partially purified preparations. Experiments in which opioid receptors were prelabelled

with the potent agonist [3H]etorphine (Simon et al. 1975) or with

u-selective radiolabelled enkephalin derivatives (Childers and

Snyder 1978) prior to solubilization afforded a molecular weight

estimate of 300 to 400,000 daltons. More recently, solubilization

studies of active receptor species from rat brain (Howells et al.

1982; Bidlack et al. 1981; Chow and Zukin 1983), toad brain (Ruegg

et al. 1981), and cell cultures (Simonds et al. 1980) have provided similar molecular weight estimates and suggest that the

receptors are aggregates of multiple protein components.

28
The receptors can be distinguished by their ligand

selectivity patterns (table 1). The μ receptor exhibits highest

affinity for morphine and related morphinelike alkaloids (normorphine, dihydromorphine, and levorphanol), as well as for certain

synthetic enkephalins which have bee modified to confer μ-like

character. The latter include D-Ala2, N-Phe4-Met(O)ol5-enkephalin

(FK 33824) (Romer et al. 1977), which exhibits somewhat higher

affinity for μ than

D-Ala , N-Me-Phe ,

for receptors (Kream and Zukin 1979), and

Gly-ol5-enkephalin (DAGO) (Handa et al. 1981),

which exhibits a 22O:1 preference for receptors. The receptor exhibits preferential affinity for the naturally occurring

Met-5 and Leu-5 enkephalins and certain of their congeners. A

relatively high selectivity for the receptor is exhibited by

D-Ala , D-Leu5-enkephalin (DADLE) and D-Ser2, L-Leu5-enkephalylThr (DSLET) (Garcel et al. 1980). Highest selectivity for the

receptor is exhibited by the conformationally restricted, cyclic,

disulfide-containing an logs

mine]enkephalin ([D-Pen2, L-Pen5]enkephalin), and [2-D-penicilla-

[2-D-penicillamine, 5-L-penicillamine, 5-D-penicillamine]enkephalin ([D-Pen , D-Pen5

]enkephalin)

(Mosberg et al., 1983). In binding assays, [D-Pen2, L-Pen5] and

D-Pen2,D-Pen5]enkephalin exhibited receptor selectivities of

371 and 175, respectively; compared with the other analogs,

these bis-Pen-containing analogs provide an order of magnitude

increase in receptor selectivity. Thus, although most

ligands cross-react with μ and receptors, highly selective μ and

ligands have been designed. Neither μ nor δ opioids exhibit

significant affinity for the κ or σ receptors. The prototypic κ

ligands ketocyclazocine and ethylketocyclazocine (EKC) do exhibit

high affinities for both μ and κ receptors (Eghbali et al., in

press; Kosterlitz et al. 1981). More recently, U-50,488 has been

TABLE 1. Binding parameters for opioid receptor sites in rat

striatum

Receptor Type

Kd1 BMax 1 Kd

2

B Max

2

(nM) (fmol/mg) (nM) (fmol/mg)

μ Receptors

δ Receptors

Total [

3

H]EKC

Sites*

1.7 108

7.1 184

0.7 94 8.7 252

κ Receptors** 8.2 74

*Total specific [3H]EKC binding was carried out In the absence of μ and δ

blockers.

∗∗κ receptor binding was carried out using [3HlEKC In the presence of DAGO and

DADLE, which block binding of radioligand to μ and δ receptors, respectively.

29
reported to be a highly selective κ ligand essentially devoid of μ

or δ activity (VonVoigtlander et al. 1983). Prototypic μ agonists

((-)SKF-10,047 and (-)cyclazocine) exhibit potent affinities at μ

and, in the case of cyclazocine, κ receptors (Goodman and Snyder

1982; Zukin and Zukin 1981). Phencyclidine and (+)SKF-10,047 are

specific ligands for the σ receptor (Zukin and Zukin 1981; Zukin

and Zukin 1984) and exhibit no detectable affinity for μ, δ, or κ

receptors.

A variety of studies indicate that μ and δ receptors have somewhat

different distributions throughout the central and peripheral

nervous systems. Thus, the thalamus and hypothalamus of rat brain

(Chang et al. 1979) and guinea pig ileum (Lord et al. 1977) appear

to be highly enriched in μ receptors, whereas the rat frontal

cortex, rat corpus striatum (Chang et al. 1979), and mouse vas

deferens (Lord et al. 1977) show relatively higher densities of δ

receptors. Of particular importance has been the finding of

tissues homogeneous in δ receptors, such as the neuroblastoma

cell line N4GT1 (Chang et al. 1978). The distributions of μ and

δ receptors have been confirmed in the central nervous system

(CNS) by light microscopy autoradiography (Goodman et al. 1980)

and in the peripheral nervous system (PNS) by the twitch and other

bioassays on isolated tissue strips (Chang et al. 1978; Robson and

Kosterlitz 1979). These studies, however, did not use specific

radioligands to label

used [3H]EKC and [3H]bremazocine (another κ drug) in the presence

μ or δ receptors. Goodman and Snyder (1982)

of selective μ and δ blockers in an autoradiography study to

elucidate a unique κ receptor distribution in guinea pig brain.

The highest selective localization of putative κ receptors was

found to occur in layers V and VI of the cerebral cortex. These

researchers did not, however, study the κ receptor distribution in

rat brain.

The present chapter reviews in vitro receptor binding studies and

quantitative receptor autoradiography studies using highly specific radioligands to label μ, δ, and κ receptors. The relative densities and affinities of the three receptor classes in rat brain,

as well as their neuroanatomical distribution patterns, are discussed. These studies reveal strikingly different patterns for

the three opioid receptors.

EQUILIBRIUM BINDING STUDIES OF μ, d, AND k RECEPTORS IN RAT BRAIN

MEMBRANES

Binding parameters for μ, δ, and κ opioid receptors can be determined by incubating brain membranes with radioligands highly specific for the various receptor classes.Striatal tissue in our 

studies was incubated with [3H]D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin (DAGO) (for μ receptor assays), [3

H][D-Pen2, D-Pen2]enkephalin

(for δ receptor assays), or [3H]ethylketocyclazicine (EKC) (for κ

receptor assays) in 50 mM Tris-HCl buffer, pH 7.4, at 4 °C for 60

minutes in the absence or presence of the same nonradioactive ligand (10 μM). Radioligand concentration was varied over a concentration range of 0.1 to 25 nM. In the case of κ receptor assays,

30
binding was carried out in the presence of DAG0 (100 nM) and DADLE

(100 nM) in order to block the binding of [3H]EKC to μ and δ receptors and ensure its Specific binding to κ receptors (Eghbali et

al., in press). This concentration was determined to be more than

100 times greater than the IC50 for either ligand in displacement

of [3H]EKC, in confirmation of Kosterlitz et al. (1981).

Figure 1 shows aturation and Scatchard analyses of the equilibrium binding of [3

H]DAGO (μ-specific opioid, 0.1 to 25 nM) to rat

striatal membranes.

(figure 1).

Scatchard analysis revealed a linear plot

Computer-assisted linear regression analysis (Munson

and Rodbard 1980) afforded a best fit for a single binding component; the calculated parameters were Kd=1.72 nM, BMax=I08 fmol/mg

protein. Figure 2 depicts representative saturation and Scatchard

isotherms for [3H][D-Pen2, D-Pen5]enkephalin (δ-specific ligand,

0.1 to 25 nM) binding to striatal membranes. Scatchard analysis

of δ receptor binding again yielded a straight line, the calculated parameters Of which were Kd=10.5 nM and BMax=142 fmol/mg

protein (figure 2).

FIGURE 1. Saturation (A) and Scatchard (B) analyses of specific

[

3H]DAGO binding to rat striatal tissie

NOTE: Aliquots of homogenate (250 μl, 0.9 to 1 mg/ml protein) In 50 ml Tris-HCl

buffer. pH 7.4, were Incubated In triplicate at 4 oC for 60 minutes with

[

3

H]DAGO (0.1 to 20 nM). In the absence or presence of 10 μM nonradiolabelled DAGO. Free [3

H]DAGO was separated from bound ligand by the rapid

filtration method. Specific binding, defined as total binding minus binding in the presence of nonradioactive DAGO, is reported. Data as shown

were fit by straight lines using computer-assisted linear regression

analysts. This experiment was replicated two times.

31
FIGURE 2. Saturation (A) and Scatchard (B) analyses of specific

[

3H][D-Pen2

, D-Pen5]enkephalin binding to rat

striatal tissue

NOTE: Aliquots of homogenate (250 μ1. 0.9 to 1 mg/ml protein) in 50 mM Tris-HCl

buffer, pH2 7.4, were incubated In triplicate at 4 oC for 3 hours with

[

3

H], [D-Pen2 , D-Pen5]enkephalin (0.1 to 20 nM) in the absence or presence of

10 μM nonradiolabel led ligand. Separation of free ligand from bound ligand

and data analysis were carried out as described In the legend to figure 1.

Th Is experiment was replicated two times.

κ receptors can be labelled specifically in preparations of rat

brain membranes using the κ opioid [3H]EKC in the presence of nonlabelled DAGO and DADLE. These peptides serve to block binding of

the radioligand to μ and δ receptors, respectively, and direct its

binding to κ receptors (Kosterlitz et al. 1981; Goodman and Snyder

1982; Eghbali et al., in press). Scatchard analysis of specific

[

3

H]EKC binding (unblocked) to rat striatal membranes revealed a

biphasic curve suggesting binding to at least two classes of binding sites (figure 3). Computer-assisted nonlinear regression

analysis (Munson and Rodbard 1980) afforded a best fit for a curve

calculated for two binding components. The first was characterized by κ =0.94±0.14 nM and BMax =94 fmol/mg protein; the second,

by Kd=8.6±2.4 nM and BMax=±1 fmol/mg protein.

Experiments in which [3H]EKC binding was determined in the

presence of DAGO (100 nM) and DADLE (100 nM) yielded a linear

Scatchard plot for rat brain membranes (Kd=11.4±1.9 nM and

BMax=119±19 fmol/mg protein) (figure 4). That these sites are

likely to be pharmacologically relevant κ receptors was established in guinea pig brain tissue by Kosterlitz and coworkers

(Kosterlitz et al. 1981). They demonstrated that the rank order

of potencies of a large series of opioids in inhibiting binding of

32
FIGURE 3. Scatchard analysis of specific [

3H]ethylketocyclazocine

(EKC) biding to rat brain membranes

NOTE: Aliquots of homogenate (1 ml, 0.9 mg protein) in 50 nM Tris-HCl buffer,

pH 7.4, were incubated in triplicate at 4 oC for 60 minutes with various

concentrations of [3HlEKC in the absence or presence of 10 μM nonradioactive EKC. Free [3

H]EKC was separated from bound ligand by the rapid filtration method as described In the legend to figure 1. Specific binding,

defined as total binding minus binding In the presence of nonradioactive

EKC, is reported. Data as shown were fit by straight lines using computerassisted linear regression analysis. The biphasic plot was analyzed using

computer-assisted nonlinear least squares regression analysis (Munson and

Rodbard 1980). This experiment was replicated two times.

33
FIGURE 4. Saturation (A) and Scatchard (B) analyses of specific

[

3H]EKC binding to rat brain homogenate in the

presence of DAGO (μ-selective opioid) and DADLE (δselective opioid)

NOTE: Rat brain homogenates were Incubated in triplicate for 60 minutes under the

same experimental conditions as described In the legend to figure 3, except

that μ and δ receptor binding was suppressed by the Inclusion of DAGO (100

nM) and DADLE (100 nM), respectively. Data as shown were fit by straight

lines using computer-assisted linear regression analysis. This experiment

was replicated two times.

[

3

H]EKC to these sites paralleled that found for behavioral and

pharmacological assays of κ activity. Inclusion of the cerebellum

in the brain homogenate did not result in any significant change

in either Kd or BMax values. [3H]EKC binding to rat brain in the

presence of U-50,488H, a highly specific κ ligand, revealed a

monophasic Scatchard plot with a Kd similar to the high-affinity

component. Table 1 summarizes μ, δ and κ receptor affinities and

receptor densities obtained in rat brain tissue.

AUTORADIODRAPHIC STUDIES OF μ, δ, AND κ DPIOID RECEPTORS

In order to visualize the neuroanatomical distribution patterns of

the μ, δ, and κ opioid receptors, autoradiography at the level of

the light microscope was carried out on thaw-mounted sections of

frozen rat brain. Autoradiograms of selected coronal sections are

shown in figures 5 through 7. μ receptor labelling at the level

of the anterior commissure revealed strikingly dense patches of

34
FIGURE 5. Photomicrographs of [

3H]D-Ala2

, N-Me-Phe4, Gly-ol5-

enkephalin (DAGO) autoradiograms of selected coronal

sections of rat brain

KEY: CP=caudate; NC=neocortex; CC=corpus callosum; HI-hippocampus; FrPa=frontal

parietal cortex; BL=basolateral amygdaloid nucleus; VP=ventroposterior

thalamic nucleus; CM=central medial thalamic nucleus; SuG=gray layer of

superior colliculus; MC=medial geniculate nucleus; CG=central gray;

SN=substantia nigra; AHI=amygdalohippocampal area; cb-cerebellum; LC=locus

coeruleus.

NOTE: Brain sections were labelled in vitro In a solution of 4 nM [3

H]DAGO (60

Cl/mmol). To assess nonspecific binding, adjacent sections were Incubated

under the same conditions in a solution containing radiolabelled ligand in

the presence of a 1,000-fold excess of levorphanol. Sections corresponding

to both “total binding” and “nonspecific binding” from each brain region

were exposed to the same sheet of LKB film together with tritium standards

(Amersham). After appropriate exposure time (8 to 12 weeks). the film was

developed using standard Kodak developer (D-19) and fixative (5 minutes D19, 20 seconds H20 rinse, 10 minutes fixative). The optical density of

each structure was determined by a Photovolt densitometer with an aperture

diameter of 0.1 mm (Photovolt Corp., New York, NY). For each brain structure, densitometric readings were taken from a minimum of three consecutive

sections per rat after correction for the contributions due to nonspecific

binding and background film density. Corrected densitometric readings (In

optical density units) were then averaged and converted to receptor density

values (fmol/mg) by reference to a standard curve for brain tissue computed

using tritium standards (Amersham). Receptor density values reported are

means ± S.D. of femtomoles of radlolabelled ligands, specifically bound per

milligram of tissue, of averaged values from the corresponding frozen sections of a minimum of these rats. Figure 5A depicts μ receptor labelling

at the level of the anterior commissure. Dense patches overlying the

striosomes of the caudate putamen can be seen surrounded by diffusely organized receptors. Figure 58 depicts μ receptor binding at the level of the

diencephalon. In the thalamic nuclei, μ receptor labelling can be seen

specifically in the medial and midline nuclear groups. In the midbrain

area of rat brain (figure 5C), a hlgh density of μ receptors was observed

In the substantia nigra reticulata, the interpeduncular nucleus, and the

dorsal lateral aspects of the central gray. [3H]DAGO blndlng to the rat

brainstem (figure 5D) revealed dense labelling In the locus coeruleus of

the pons.

35
FIGURE 6. Photomicrographs of selected coronal sections demonstrating δ receptor labelling in rat brain

NOTE: δ receptors were labelled using 6 nM [

3

H]D-Pen2, D-Pen5]enkephalin (41.5

Cl/mmol). Procedures for receptor labelling and for autoradiography were

as described in the legend to figure 5. Figure 5A depicts δ receptor

labelling et the level of the telencephalon. δ receptors exhibit few

patches In the caudate putamen and a dense, diffuse distribution throughout

the striatum. [

3

H]DPDPE labelling at the level of the neocortex (figure

58) revealed heaviest labelling In layers I, II, V, and VI. Note the lack

of δ receptors in nuclei of the thalamus and hypothalamus (B), midbrain

(C), and hindbrain (D). For the key to the abbreviations, see the legend

to figure 5.

receptors overlying the striosomes (figure 5A). These patches

were surrounded by areas of diffusely organized receptors. This

topographical organization is consonant with the previously reported pattern of μ receptor labelling in the striatum (Tempel et

al. 1984; Herkenham and Pert 1982; Pert et al. 1976; Atweh and

Kuhar 1977). μ receptor labelling in the neocortex of rat brain

(figure 5A) revealed a highly specific pattern corresponding to

known cytoarchitectural boundaries. Labelling was of highest

36
FIGURE 7. Photomicrographs of [

3H]EKC autoradiograms of selected

coronal sections of rat brain

NOTE: Thaw-mounted sections of frozen rat brain were prepared as described In the

legend to figure 5. Sections were Incubated in 50 nM Tris-HCI (pH 7.4,

4 oC) containing 10 nM [3H]EKC In the presence of 100 nM DAGO and 100 nM

DADLE to block binding of the radloligand to μ and δ receptors and direct

its binding to κ receptors. To assess nonspecific binding, adjacent sections were Incubated under the same conditions in a solution containing

radlolabelled ligand in the presence of a 1,OOO-fold excess of EKC (10 μM).

κ receptor labelling can be seen In (A) the striatal area and the bed

nucleus of the stria terminalis and (B) the hippocampal formatIon, especially overlylng the pyramidal cell field and the granular cell layer.

Note the dense labelling in several of the thalamic nuclei, including the

medial and midline nuclear groups. Also seen In thls autoradiogram is the

neocortex, which is uniformly labelled by [3

H]EKC. κ receptor labelling

can be seen in (C) the midbrain area, particularly In the substantia nigra

reticulata. the interpeduncular nucleus, and the central gray. Dense κ receptor labelling can also be seen in (D) the locus coeruleus, the cerebellum, and the spinal tract of the trigeminal nerve of the brainstem. For

abbreviations, see the legend to figure 5.

37
density in layers I and III of the cortex. Dense μ receptor

labelling was also notable in the cingulate cortex and adjacent

medial frontal cortex. A particularly high density of receptors

was observed in the ventral striatal and pallidal areas, endopiriform nucleus, and diagonal band of Broca. By contrast, μ receptor

labelling was noticeably lacking in the anterior commissure and

corpus callosum. Table 2 summarizes μ receptor densities in rat

brain structures, as determined by quantitative, in vitro

autoradiography.

TABLE 2. Regional distribution of μ opioid receptor densities in

sections of frozen rat brain as determined by

quantitative autoradiography

Region

μ Receptor Densities

[

3

H]DAGO

(fmol/mg protein)

(n=3)

Medial frontal (anticingulate) cortex

Neocortex

Layer I

Layer II

Layer III

Layer IV

Layer V

Layer VI

Striatum

Patches

Surrounds

Fundus striati

Medial aspect of the nucleus accumbens

Bed nucleus of the stria terminalis

Endopiriform nucleus

Amygdala

Posteromedial cortical

Basolateral nucleus

Lateral septum

Hippocampus

Molecular layer

Pyramidal cell layer

CA2

CA3

Thalamic nuclei (in total)

Centromedial

Reunions

Parvocellular area of ventroposterior

nucleus

Lateral mediodorsal

Lateral posterior

Anterior laterodorsal

Anterior ventral

127.0±1.O

75.7±1.0

28.7±0.2

100.9±3.2

28.7±0.2

28.7±O.2

28.7±0.2

226.2±7.1

114.8±3.4

Not detected

182.7±1.0

Not detected

Not detected

Not detected

67.9±O.3

100.9±1.1

127.O±2.2

52.2±2.1

22.6±1.9

55.7±1.1

28.7±0.1

95.7±2.2

127.0±1.3

Not detected

Not detected

75.7±2.1

52.2±3.1

34.8±2.8

38
TABLE 2 (Continued)

Region

μ Receptor Densities

[

3

H]DAGO

(fmol/mg protein)

(n=3)

Posteromedial cortical

Posterior nucleus

Centrolateral

Intermediodorsal

Rhomboid

Medial geniculate

Dorsal lateral geniculate

Hypothalamic nuclei

Dorsal

Ventromedial

Medial habenulae

Substantia nigra reticulata

Interpeduncular nucleus

Superficial gray layer of the

superior colliculus

Inferior colliculus, medial portion

Central gray

Locus coeruleus

Cerebellum

Spinal tract of the trigeminal nerve

Corpus callosum

Not detected

74.0±0.1

132.2±11.2

Not detected

132.2±7.7

Not detected

63.3±0.1

Not detected

Not detected

Not detected

121.8±1.3

63.5±0.2

55.7±0.1

83.5±O.2

104.4±O.2

38.3±O.l

92.2±7.0

Not detected

Not detected

9.1±0.1

NOTE: μ opioid receptors were labelled as described in the legend to figure 5.

For each brain structure, densitometric readings were taken from a minimum

of three consecutive sections per rat after correction for contributions

due to nonspecific binding and background film density. Corrected densitometric readings (in optical density units) were then averaged and converted

to receptor density values (fmol/mg) by reference to a standard curve for

brain tissue, computed using tritium standards (Amersham). Receptor density values reported are means ± S.E.M. of femtomoles of [3

H]DAGO specifically bound per milligram of tissue, of averaged values from the corresponding

frozen sections of a minimum of three rats.

Figure 5B depicts μ receptor binding to a section of frozen rat

brain at the level of the diencephalon. μ receptors were highly

concentrated in the pyramidal cell field, especially in the CA2

region of the hippocampal formation.

In the thalamic area, dense μ receptor labelling was localized to

specific nuclei (figure 5C) as follows: the lateral mediodorsal,

lateral posterior, anterior laterodorsal, and posterior nuclei,

and the medial and midline nuclear groups, including the centromedial nucleus, centrolateral nucleus, reunions nucleus, and

rhomboid nucleus.

39
At the level of the midbrain, regions exhibiting a high density of

μ receptors were the medial aspect of the inferior colliculus, the

superficial gray layer of the superior colliculus, and the substantia nigra reticulata. The interpeduncular nucleus and the

dorsal lateral aspects of the central gray exhibited a somewhat

lower level of μ receptor labelling. In the brainstem region

(figure 50) the locus coeruleus of the pons revealed a particularly dense clustering of this receptor type. All other areas

were devoid of μ receptors.

Figure 6A depicts the pattern of δ receptors labelled by [3H][2-DPen, 5-D-Pen]enkephalin in the telencephalon at the level of the

striatum (caudate putamen). Characteristically, δ receptors

exhibited few patches and a diffuse distribution throughout the

striatum with more dense labelling in the ventrolateral regions

and fundus striati. δ receptor labelling was absent from the

anterior commissure and corpus callosum. [

3

H]DPDPE labelling at

the level of the neocortex revealed a pattern distinctly different

from that of μ receptors. Highest labelling was observed in layers I, II, V, and VI. No δ receptors were detected in layers III

or IV of the cortex. Nuclei of the thalamus, hy thalamus, midbrain, and brainstem were noticeably lacking in [3

H]DPDPE-labelled

receptors. Table 3 summarizes δ receptor densities in these rat

brain structures as determined by quantitative, in vitro autoradiography.

TABLE 3. Regional distribution of δ opioid receptor densities in

sections of frozen rat brain, as determined by

quantitative autoradiography

Region

δ Receptor Densities

 [3H]DPDPE

(fmol/mg protein)

n=3

Medial frontal (anticingulate) cortex

Neocortex

Layer I

Layer II

Layer III

Layer IV

Layer V

Layer VI

Striatum

Patches

Surrounds

Fundus striati

Medial aspect of the nucleus accumbens

Bed nucleus of the stria terminalis

Endopiriform nucleus

Amygdala

79.5±5.0

79.5±5.0

Not detected

Not detected

132.6±9.1

136.6±9.1

Not detected

169.9±2.1

192.8±11.2

125.3±1.3

Not detected

Not detected

Not detected

40
TABLE 3 (Continued)

Region

δ Receptor Densities

[

3

H]DPDPE

(fmol/mg protein)

(n=3)

Posteromedial cortical Not detected

Basolateral nucleus Not detected

Lateral septum Not detected

Hippocampus

Molecular layer

Pyramidal cell layer

CA2

CA3

Thalamic nuclei (in total)

Centromedial

Reunions

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Posterior nucleus

Centrolateral

Intermediodorsal

Rhomboid

Medial geniculate

Dorsal lateral geniculate

Hypothalamic nuclei

Dorsal

Ventromedial

Medial habenulae

Substantia nigra reticulata

Interpeduncular nucleus

Parvocellular area of the ventroposterior

nucleus

Lateral mediodorsal

Lateral posterior

Anterior laterodorsal

Anterior ventral

Posteromedial cortical

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

Superficial gray layer of the

superior colliculus

Inferior colliculus, medial portion

Central gray

Locus coeruleus

Cerebellum

Spinal tract of the trigeminal nerve

Corpus callosum

Not detected

Not detected

Not detected

Not detected

Not detected

Not detected

3.3±0.4

NOTE: δ opioid receptors were labelled as described In the legend to figure 6.

Densitometric readings and quantification of autoradiograms were performed

as described In the legend to table 2.

41
In order to visualize the neuroanatomical pattern of κ opioid

receptors, light microscopy autoradiography was carried out on

thaw-mounted sections of frozen rat brain. Figure 7 shows LKB

film autoradiograms of [3H]EKC binding after suppression of μ and

δ binding in selected coronal sections. Figure 7A shows an autoradiogram at the level of the telencephalon; a very low density of

κ sites was found to overlie the striosomes or patches of the

striatum. By contrast, dense κ receptor labelling was identified

with the bed nucleus of the stria terminalis. In the neocortex

(figures 7A and 7B), κ receptor labelling was nearly uniform

throughout the molecular layers; the pattern of receptor labelling

failed to correspond to known cytoarchitectural boundaries. By

contrast, κ receptor labelling in guinea pig neocortex (Goodman

and Snyder 1982; Eghbali et al., in press) exhibits a highly

specific pattern of intense labelling in layers V and VI of the

neocortex.

Figure 7B depicts κ binding to a section of frozen rat brain at

the level of the diencephalon. A high density of κ receptors was

found to overlie the pyramidal cell layers adjacent to the molecular layers of the hippocampal formation, CA1, CA2, and CA3 areas,

and the granular layer of the dentate gyrus. By contrast, κ

receptors in guinea pig brain sections are of high density only in

the molecular layer of the dentate gyrus and the pyramidal cell

layer of the hippocampal formation (Eghbali et al., in press).

In the thalamic and hypothalamic nuclei, striking differences were

seen between the rat and the guinea pig. In the thalamus of the

rat brain, only selected nuclei exhibited a high density of κ

receptors (figure 7B); these were the laterodorsal nucleus, the

posterior nucleus, and the medial and midline nuclear groups (for

example, the centrolateral nucleus, intermediodorsal nucleus,

rhomboid nucleus, and the gelatinosus nucleus of the thalamus).

Moderate levels of κ receptor labelling were seen in the hypothalamic area, for example, the dorsal hypothalamic area and ventromedial nucleus of the hypothalamus. By contrast, the guinea pig

brain displays only a low level of κ receptor labelling uniformly

distributed throughout the thalamic and hypothalamic nuclei. A

notable exception is the medial geniculate nucleus of the thalamus, which exhibits a dense concentration of κ receptors (Eghbali

et al., in press).

In the midbrain area of the rat brain (figure 7C), examples of

regions exhibiting high densities of κ receptors were the periaqueductal gray (primarily the dorsal area), the substantia nigra

reticulata, and the interpeduncular nucleus. Moreover, the

superior colliculus exhibi ed dense κ receptor labelling in the

superficial gray layer. [3H]EKC binding to the rat brainstem at 

the level of the locus coeruleus (figure 7D) revealed dense labelling in the locus coeruleus, the central gray area of the pons,

and the cerebellum. Moderate levels of κ receptors were also seen

in the spinal tract of the trigeminal nerve (fifth nerve). Table

4 summarizes κ receptor densities in rat brain structures, as

determined by quantitative, in vitro autoradiography.

42
TABLE 4. Regional distribution of κ opioid receptor densities in

sections of frozen rat brain, as determined by

quantitative autoradiography

Region

κ Receptor Densities

in Rat Brain

(fmol/mg)

(n=3)

Neocortex

Layers I-IV

Layers V-VI

Striatum

Patches

Surrounds

Nucleus accumbens

Bed nucleus of the stria terminalis

Hippocampus

Molecular layer

Pyramidal cell layer

Granular cell layer

Thalamic nuclei (in total)

Laterodorsal nucleus

Posterior nucleus

Centrolateral nucleus

I ntermediodorsal nucleus

Rhomboid nucleus

Medial geniculate nucleus

Hypothalamic nuclei

Dorsal hypothalamic area

Ventromedial nucleus

Substantia nigra reticulata

Interpeduncular nucleus

Superficial gray layer of the

superior colliculus

Central gray

Locus coeruleus

Cerebellum

Spinal tract of the trigeminal nerve

Corpus callosum

41.9±2.1

41.9±2.1

71.0±5.0

45.5±2.3

58.2±3.5

81.9±7.4

Not detected

41.9±1.7

51.0±2.6

56.4±0.2

72.8±2.9

83.7±3.4

98.3±4.9

92.8±9.3

Not detected

51.0±1.0

56.4±3.4

51.0±1.3

72.8±2.2

78.3±3.1

56.4±1.1

78.3±8.6

32.8±1.0

43.7±1.8

7.3±1.0

NOTE: κ opioid receptors were labelled as described In the legend to figure 7.

Densitometric readings and quantification of autoradiograms were performed

as described in the legend to table 3.

Autoradiographic studies using highly specific radiolabelled

opioids clearly demonstrate strikingly different neuroanatomical

distribution patterns for the μ and δ receptors in rat brain (Pert

et al. 1976). μ receptors appear to be most densely localized in

the patches of the striatum, layers I and III of the cortex, the

pyramidal cell layer of the hippocampal formation, specific nuclei

43
of the thalamus, the substantia nigra, the interpeduncular nucleus, and the locus coeruleus of the pons. δ receptors labelled by

the specific δ ligand [3H]DPDPE exhibit selective localization in

layers I, II, V, and VI of the cortex and a dense, diffuse pattern

in the striatum with no visible patches. δ receptors are notably

absent in most other brain structures.

The present study further demonstrates, on the basis of both in

vitro binding assays and quantitative receptor autoradiography at

the level of the light microscope, the presence of κ binding sites

in rat brain. Although κ receptors in rat and guinea pig brain

exhibit similar distributions in the midbrain and hindbrain, they

exhibit strikingly different patterns in several forebrain structures. First, in sections of rat brain, κ receptor labelling

exhibits dense patches in the corpus striatum and a dense concentration in the nucleus accumbens, whereas in guinea pig brain it

exhibits a low, uniform labelling in the striatum and a low level

of density in the nucleus accumbens. Secondly, κ receptors exhibit selective localization in the deep layers (V and VI) of the

guinea pig neocortex, whereas in the rat neocortex, κ receptors

exhibit no visible boundaries corresponding to cytoarchitectural

boundaries. Thirdly, in the rat hippocampal formation, κ receptors show highest selective localization in the pyramidal and

granular cell layers, whereas in the guinea pig (Eghbali et al.,

in press), κ receptor labelling is most dense in the pyramidal

cell layer and the molecular layer of the dentate gyrus. Finally,

the most striking differences between the species are seen in the

thalamic and hypothalamic areas. In rat brain, dense κ receptor

labelling is seen in the central and midline nuclei of the rat

thalamus, and moderate labelling, in certain hypothalamic nuclei.

By contrast, in guinea pig brain, a significant density of κ receptors is observed only in the medial geniculate nuclei of the

thalamus (Eghbali et al., in press). It is interesting to note

that the oldest parts of the brain (the hindbrain and midbrain

areas) share commonalities of κ receptor distribution in the two

species. The more recently developed areas of the forebrain show

species-specific distribution patterns of κ receptors, which may

correlate with differences in the ontogeny of the various opioid

receptor types.

κ receptor labelling was strikingly similar to μ receptor labelling. Dense labelling was seen in the patches of the striatum,

the nucleus accumbens, the pyramidal cell layer of the hippocampal

formation, the midline nuclei of the thalamus, and the locus

coeruleus and cerebellum of the brainstem. It is interesting that

the distribution patterns of the μ and κ receptors share many

commonalities in the rat brain. The time course of their appearance in the developing brain could account for many of these

similarities. The δ receptors appear to be localized to the more

recently developed areas of the forebrain, such as the neocortex

and striatum, which may indicate a more advanced need for specialized receptors either as modulators or for specialized functions.

44
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opiate receptors in rat brain. I. Spinal cord and lower

medulla. Brain Res 134:393, 1977.

Bidlack, J.M.; Abood, L.G.; Osei-Gyimah, P.; and Archer, S.

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46
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AUTHORS

R. Suzanne Zukin, Ph.D.

Departments of Neuroscience

and Biochemistry

Ann Tempel, Ph.D.

Department of Neuroscience

Mahboubeh Eghbali, Ph.D.

Department of Neuroscience

Albert Einstein College

of Medicine

Bronx, NY 10461

47
Anatomical and Biochemical

Perspectives on Opioid Peptides

Robert P. Elde

INTRODUCTION

A decade has now passed since the initial report describing the

isolation, structural characterization, and synthesis of the opioid peptides leucine and methionine enkephalin (Hughes et al.

1975). The subsequent discovery of other opioid peptides (i.e.,

ß-endorphin (Li and Chung 1976; Ling et al. 1976) and the dynorphins (Goldstein et al. 1979; Goldstein et al. 1981)) led to

numerous imnunocytochemical studies describing the distribution of

these peptides. This information has established the notion that

the physiologically relevant occupants of opioid receptors are

produced by discrete neuronal circuits that use opioid peptides as

intercellular messengers. The purpose of this chapter is to review some of the features of the chemical neuroanatomy of opioidergic neurons. In addition, an attempt will be made to identify

some of the fundamental problems that remain concerning the

neurobiology of opioid neurons.

STRUCTURAL FEATURES OF OPIOID PEPTIDES

The discoveries of extended forms of met-enkephalin (i.e., aendorphin and ß-endorphin (Guillemin et al. 1976; Li and Chung

1976; Ling et al. 1976) and BAM-22P (Mizuno et al. 1980)) as well

as extensions of leu-enkephalin (i.e., dynorphin, neo-endorphin,

and rimorphin (Goldstein et al. 1979; Goldstein et al. 1981;

Kilpatrick et al. 1982)) initially led to confusion among investigators, because it was not clear which might be the most important

opioid peptide. This confusion did not last long, however, because immunohistochemical and radioimmunoassay studies quickly

suggested a differential localization of many of these peptides,

implying an independent existence of the enkephalins from ß-endorphin (Watson et al. 1978) and from the several forms of dynorphin

(Watson et al. 1982; Watson et al. 1983; Weber et al. 1982a; Weber

et al. 1982b; Weber et al. 1982c). Proof of the existence of

discrete families of opioid peptides came from the cloning and

sequencing of DNA complementary to messenger RNA for preproenkephalin (figure 1) (Gubler et al. 1981; Noda et al. 1982a; Noda et

48
al. 1982b); preproopiomelanocortin (figure 2) (Nakanishi et al.

1979; Nakanishi et al. 1981; Roberts et al. 1979); and preprodynorphin (figure 3) (Kakidani et al. 1982).

FIGURE 1. Schematic of the proposed structure of bovine

preproenkephalin deduced from complementary DNA

sequencing

SOURCE: Gubler et al. (1981); Noda et al. (1982a); Noda et al. (1982b).

The principles of posttranslational cleavage of precursor proteins

(Loh and Gainer 1983) (see below) suggest that the opioid peptides

isolated and sequenced from various tissues arise from one or more

of these precursors. Nevertheless, it remains possible that other

independent opioid peptides, as well as precursor proteins, will

be identified in future studies. In addition, the possible existence and synthesis of endogenous alkaloid opioids in brain has

recently received experimental support (Goldstein et al. 1985).

The intraneuronal mechanisms for the synthesis, packaging, processing, and transport of neuropeptides (including the opioids) are

distinct from those involved in the production of "classical"

neurotransmitters (Hokfelt et al. 1980; Loh and Gainer 1983). In

particular, the synthesis of neuropeptides is accanplished by

translation of messenger RNA by ribosomally directed precursor

protein synthesis. The nascent protein chain is inserted via the

"signal" peptide sequence into the cisternae of the rough endoplasmic reticulum with which the ribosomes are associated. Since

rough endoplasmic reticulum is restricted to the neuronal cell

body and its proximal dendrites (Peters et al. 1976), structures

such as axons and their terminals are incapable of supporting the

49
FIGURE 2. Schematic of the proposed structure of bovine preproopiomelanocortin deduced from complimentary DNA

sequencing

SOURCE: Nakanishi et al. (1979); Nakanishi et al. (1981); Roberts et al. (1979).

synthesis of neuropeptides. Thus, the presumed major site of release of neuropeptides--the axon terminal--obtains its stores of

peptides only through mechanisms of axonal transport. These processes contrast sharply with the means by which neurons synthesize

and store the low molecular weight transmitters such as the monoamines (Cooper et al. 1978). In general, low molecular weight

transmitters are synthesized by enzymatic conversion of a precursor molecule into the transmitter. This process may occur

anywhere within the neuron in which the enzyme, substrate, and

cofactors are present in adequate concentrations and, in contrast

to the synthesis of neuropeptides, is thought to occur predominantly in axons and their terminals.

POSTTRANSLATIONAL PROCESSING OF OPIOIDS

The biological activity of opioid peptides is manifest only after

cleavage of the appropriate peptide bonds within the precursor

molecule liberates the active opioid peptide sequence from neighboring, cryptic peptide sequences. Characterization of the enzymes responsible for these cleavages is currently under intensive

investigation (Fricker and Snyder 1983; Goodman et al. 1983). It

has been suggested that all the proteolytic enzymes responsible

for particular types of cleavages are not uniformly present in

cells that produce a given opioid precursor (Weber et al. 1982a;

Zakarian and Smyth 1982; Akil et al. 1984; Zamir et al. 1984a;

Zamir et al. 1984b; Liebisch et al. 1986). Thus, it is possible

that a given precursor can give rise to different opioid products,

50
FIGURE 3. Schematic of the proposed structure of porcine

prepmdynorphin deduced from complimentary DNA

sequencing

SOURCE: Kakidani et al. (1983).

if cells di'fferentially produce enzymes responsible for posttranslational processing. In fact, data suggest differences in posttranslational processing of preproenkephalin between central

neurons and adrenal chromaffin cells (Mizuno et al. 1980; Liebisch

et al. 1986); of preproopiomelanocortin between anterior and

intermediate lobe cells in the pituitary (Akil et al. 1984); and

of preprodynorphin between certain brain regions, leading to a

relative abundance of dynorphin A 1-8 in some areas and to longer

forms of dynorphin in others (Weber et al. 1982a; Zamir et al.

1984b).

Posttranslational modification of opioids is not limited to cleavage of the precursor proteins but also includes several types of

covalent modification of the cleaved peptides. These covalent

modifications of opioids include the a-acetylation of the amino

terminal tyrosine of ß-endorphin (Akil et al. 1981; Akil et al.

1984) and the amidation of the carboxy terminus of metorphamide

(Weber et al. 1983). It is important to note that posttranslational modifications of opioids may serve to enhance the biological activity of the molecule (as in cleavage of the biologically

active opioids from precursor molecules), alter its receptor

selectivity, or inactivate it (Akil et al. 1981; Akil et al.

1984).

51
LOCALIZATION STUDIES OF OPIOID PEPTIDES IN NEURONS

The ability to produce antisera selective for the various opioid

peptides has permitted both quantitative radioimmunoassay surveys

of extracts of macroscopic regions of the nervous system (Weber et

al. 1982a) and cellular identification of opioid neurons (Elde et

al. 1976; Hokfelt et al. 1977; Sar et al. 1978; Watson et al.

1978; Watson et al. 1981; Watson et al. 1982; Uhl et al. 1979;

Khachaturian et al. 1982; Khachaturian et al. 1983a; Khachaturian

et al. 1983b; Vincent et al. 1982; Weber et al. 1982b; Furness et

al. 1983; recently reviewed by Khachaturian et al. 1985). The

opioid peptides derived from preproenkephalin and preprodynorphin

are widely distributed among neurons of both the peripheral

(sympathetic, parasympathetic, and enteric nervous systems) and

central nervous systems. In contrast, neurons producing opioids

derived from preproopiomelanocortin are not found in the peripheral nervous system at all and are restricted to only two areas of

the mammalian central nervous system--the arcuate nucleus of the

hypothalamus and the nucleus of the solitary tract within the

medulla oblongata. For each family of opioid peptides, the areas

containing nerve fibers and terminals that release opioid peptides

outnumber the areas containing neuronal cell bodies, thus suggesting that opioids are found within projection neurons as well as

within local circuit neurons.

In recent years, details of the connections of some opioid neurons

have been established by combining lesion or tract tracing studies

with immunohistochemical methods. In one of the first of these

studies, it was found that enkephalin-immunoreactive cell bodies

in the amygdala project to distant targets through the stria terminalis (Uhl et al. 1978). Prior to this work, many investigators

felt that enkephalinergic neurons were restricted to participation

in local circuits rather than projection pathways. Enkephalinand dynorphin-immunoreactive neurons in the pontine and medullary

raphe and reticular nuclei project to the spinal cord, and some of

these fibers terminate in the vicinity of sympathoadrenal preganglionic neurons (Holets and Elde 1982; Appel et al., unpublished).

Some of the opioidergic neurons in the striatum project to the

substantia nigra (Zamir et al. 1984a). Opioid peptides are found

in neurons of the superficial lamina of the dorsal horn of the

spinal cord that are thought to be importantly involved in ascending, nociceptive pathways. Recently, a significant proportion of

these opioidergic "marginal zone" neurons has been shown to project to the parabrachial nucleus of the upper pons (Standaert et

al. 1986). Another ascending, putatively nociceptive pathway that

produces opioid peptides arises from the vicinity of the central

canal of the spinal cord (Nahin and Micevych, in press). These

findings of opioids in neurons believed to respond to noxious

stimuli may prompt development of significant new approaches for

investigating basic aspects of pain and analgesia. Finally,

enteric neurons that produce enkephalin immunoreactivity have been

found to reside within the myenteric ganglia of the gut wall.

These neuronal cell bodies send enkephalin-immunoreactive fibers

orally to provide terminals in other myenteric ganglia, as well as

52
anally to supply the circular and muscular plexuses of the gut

wall (Furness et al. 1983). Thus, the endogenous opioid neurons

that regulate gut motility have been positively identified.

Apart from the numerous specific findings, some general principles

have emerged. For the most part, if an individual neuron produces

opioid peptides, all the opioid peptides produced appear to derive

from a single biosynthetic precursor. Thus, neurons that produce

ß-endorphin fail to produce either proenkephalin-derived peptides

or prodynorphin-derived peptides (Watson et al. 1978). Similarly,

the vast majority of neurons that produce either proenkephalin- or

prodynorphin-derived peptides fail to produce peptides derived

from other precursors of opioid peptides (Watson et al. 1982;

Weber et al. 1982c; Khachaturian et al. 1985). However, two

"exceptions" to this "rule" have recently been described in central neurons related to autonomic control. Guthrie and Basbaum

(1984) described the coexistence of proenkephalin- and prodynorphin-derived peptides in some neurons within the nucleus of the

solitary tract. We have described a similar coexistence in

neurons of the dorsal gray commissure at the level of the sacral

parasympathetic nucleus (Sasek and Elde, in press). The presence

of two families of opioid peptides in individual neurons suggests

that opioid production must be especially crucial for the signalling functions of such neurons.

Another general principle is that opioidergic neurons often produce additional, nonopioid transmitter substances, including

acetyl choline (Altschuler et al. 1984), monoamines (Schultzberg et

al. 1978; Glazer et al. 1981; Armstrong et al. 1984; Leger et al.

1986), GABA (Zahm et al. 1985), or other peptides (Hokfelt et al.

1980; Coulter et al. 1981; Erichsen et al. 1982; Weber et al.

1982c; Martin et al. 1983; Beauvillain et al. 1984; Morris et al.

1985). Most studies of the coexistence of neurotransmitters have

focused on the neuronal cell body for the analysis. Recently, we

have refined and characterized a two-fluorochrome, light microscopic method that allows studies of coexistence at the level of

individual varicosities and nerve terminals (Wessendorf and Elde

1985). Preliminary studies in spinal cord suggest the existence

of diverse combinations of putative transmitters in peptidergic

nerve fibers and terminals (Appel et al. 1986; Appel et al., in

preparation). Although the regional compartmentalization and combinations of chemical markers that coexist within opioid neurons

are only beginning to be studied, we suspect that the unravelling

of this chemical complexity will provide the basis for discerning

functional divisions among opioidergic neurons. Such a vision is

already realized, in part, for opioid neurons in the enteric nervous system. Furness and colleagues (1983) previously described

the organization and connections of enkephalin-immunoreactive neurons in this preparation. In addition, they have characterized

the electrophysiological properties of identified enteric enkephalinergic neurons (Bornstein et al. 1984)--a task that has been

extremely difficult in mammalian central nervous system preparations. Their studies have now been expanded to include dynorphin

and numerous other putative transmitter substances (Costa et al.

53
1985; Furness and Costa 1986). The expanded survey has demonstrated up to six discrete transmitter-related markers in individual neurons. Despite this myriad of neurochemical combinations,

adding information about the connections and electrophysiological

properties of individual neurons gives a unique, functionally

meaningful accounting of those neurons. Given the power of this

new level of knowledge about chemically coded neurons, it can be

expected that significant advances in understanding the neurobiology of opioidergic neurons will arise from studies of this

sort.

Species variations in the distribution of opioids have been noted

(Elde et al. 1980), and it now appears that an enkephalinergic

circuit in one species may be substituted by dynorphinergic neurons in another species. This is illustrated by the occurrence of

prodynorphin-derived peptides in hypothalamic magnocellular neurons of the rat (Weber et al. 1982c) and proenkephalin-related

peptides in the homologous neurons in the cat (Micevych and Elde

1980). Similarly, prodynorphin-derived peptides are abundant in

the substantia nigra of the rat (Khachaturian et al. 1982;

Khachaturian et al. 1985), whereas proenkephalin-derived peptides

are more abundant in the homologous neurons of the nonhuman primate (Haber and Elde 1982). The production of one or another of

the opioid precursor proteins can now be precisely determined by

the differential localization of their respective messenger RNA by

in situ hybridization. However, the identification of posttranslationally processed, releasable peptides continues to be an

important task for immunocytochemical studies of nerve fibers and

terminals.

LINGERING QUESTIONS CONCERNING THE NEUROBIOLOGY OF OPIOIDERGIC

NEURONS

Although the regional and cellular distribution of opioidergic

neurons is now fairly well understood, we know very little about

the numerous neuronal circuits in which opioids appear to participate. In addition, several questions have emerged concerning the

cellular mechanisms important in processing opioids. For many

neurotransmitters, including the opioids, the sites of release

remain to be clarified. Although it is expected that opioids and

other transmitters are released at the terminal boutons of axons,

it is unknown whether or not preterminal varicosities are also

release sites. The axons of opioid neurons are generally unmyelinated and exhibit many varicosities along their course (figure 4).

Unmyelinated, varicose axons with a similar morphology arise from

postganglionic sympathetic neurons. Release of catecholamines and

neuropeptides is suspected at the varicosities along the route of

these axons. In contrast, axons of hypothalamic magnocellular

neurons that terminate in the neural lobe of the pituitary exhibit

striking varicosities (termed Herring bodies) which are not

thought to be release sites for vasopressin and oxytocin. It is

unknown whether or not other portions of the opioidergic neurons

are capable of selective release of opioid peptides. For example,

54
Release Sites of Transmitters

FIGURE 4. Schematic illustration of a neuron, its dendrites, and

an axon

NOTE: The axon is illustrated to have varicosities along Its course. Similar

varicosities represent sites for the release of neurotransmitters In the

peripheral autonomic nervous system. It is not known whether or not varicosities along the course of opioidergic axons in the central nervous

system represent potential sites of release.

it is suspected that dendrites of neurons in several brain regions

may release transmitters to act "postsynaptically" on receptors on

other dendrites (Shepherd 1979). Thus, the sites at which opioid

peptides might be released from opioidergic neurons must be established in order to understand the sites where opioids might exert

their action.

If release of opioid peptides occurs at varicosities along the

course of an axon, it then becomes possible that the molecular

structure of the opioid peptides may differ between proximal and

distal release sites (figure 5). This possibility exists because

axonal transport and posttranslational processing are both timedependent phenomena. It has been found, for example, that the

proximal axons of hypothalamic magnocellular neurosecretory

neurons contain relatively unprocessed versions of preprovasopressin and preprooxytocin. More distally (i.e., with additional

time), these axons contain increasing concentrations of the

cleaved, biologically active neurohormones (Gainer et al. 1977;

Brownstein et al. 1980). Thus, if opioid peptides are released

from proximal axon collaterals or varicosities, one might expect

that the profile of these peptides will be different from that of

the peptides released at more distal terminals.

55
Releasable Forms of Peptides

FIGURE 5. Schematic illustration similar to figure 4

NOTE: If preterminal varicosities might be sites for the release of neurotransmitters, It then becomes possible that the molecular forms of opioids and

other peptides stored and available for release at proximal varicosities

may be less processed than at the more distal terminals.

The relationship between release sites of opioid peptides and

their sites of action also remains controversial. The somewhat

parallel uncovering of multiple opioid peptides with different

spectra of activity and multiple classes of opioid receptors has

tempted many to propose a neurotransmitter-receptor linkage for a

given opioid peptide type and opioid receptor class. Indeed, the

semiselective binding properties of enkephalins (Chang et al.

1979), metorphamide (Weber et al. 1983), dynorphins (Chavkin et

al. 1982; Corbett et al. 1982), and ß-endorphin (Akil et al. 1981;

Akil et al. 1984) might be used to predict a parsimonious relationship between these endogenous ligands and particular types of

binding sites. Unfortunately, the anatomical relationship between

opioid peptides and opioid binding sites is not in any way

straightforward (Lewis et al. 1984). Even the initial immunohistochemical studies of opioid peptides (Elde et al. 1976; Watson

et al. 1978; Watson et al. 1981; Vincent et al. 1982; Weber et al.

1982b) failed to provide regional, rank order density ratings of

these peptides that correlated well with regional, rank order density ratings of different classes of opioid binding sites (Atweh

and Kuhar 1977; Chang et al. 1979; Goodman et al, 1980). We have

also seen this discrepancy at the level of identified neurons

within the intermediolateral cell column of the spinal cord.

Although these neurons exist within a highly enriched plexus of

enkephalin- (Holets and Elde 1982) and dynorphin-immunoreactive

nerve fibers and terminals (Appel and Elde, unpublished), there is

not a corresponding enrichment in dihydromorphine binding sites

56
(Seybold and Elde 1984). Current studies are seeking to determine

whether or not other classes of opioid binding sites are enriched

over these identified neurons. However, such mismatches between

the density of binding sites and the abundance of opioidergic

nerve fibers and terminals is a widely recognized phenomenon

(Kuhar 1985; Herkenham and McLean 1986). Thus, an enigma is

apparent in that potential release sites for opioids are, in many

cases, separated from binding sites by significant biological distances. Among the more plausible explanations may be that autoradiographic binding studies typically reveal only high-affinity

binding sites. It may be that low-affinity sites are more closely

related to release sites for opioids, and that the high-affinity

sites are occupied only during times of massive release of opioids

that would lead to diffusion to these more distant sites (figure

6). This enigma is difficult to resolve with present techniques

and may require the development of antibodies to discrete opioid

receptor proteins which can then be employed for the immunohistochemical localization of all classes and affinities of opioid receptors. The resolution of this problem is extremely important

for understanding the biology of opioid peptides and their

receptors.

Relation of Release sites to

Binding Sites

FIGURE 6. Schematic illustration, similar to figures 4 and 5, to

which neurotransmitter binding sites have been added

NOTE: The details of the spatial ralationships between sites of release of opioid

peptides and their binding sites are not known.

57
CONCLUSIONS

The complexity of neuronal relationships with respect to the production of the various opioid peptides and opioid receptors suggests that a great deal of effort will be required to solve the

existing riddles. For the time being, it would seem prudent that

efforts be directed at relatively simple opioidergic circuits and

functions. One example of an exploitable model of opioidergic

circuitry and many of its complexities is found in the enteric

nervous system. Significant progress in clarifying the neurobiology of mammalian, central opioids will be made as techniques

allow for the isolation and examination of the anatomy, chemistry,

and physiology of individual neurons and their functional interactions.

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63
ACKNOWLEDGMENTS

Studies in the author's laboratory were supported by USPHS

grant DA 02148 from the National Institute on Drug Abuse.

Drs. N. Appel, V. Seybold, and M. Wessendorf of the University

of Minnesota provided helpful ideas and advice.

AUTHOR

Robert P. Elde, Ph.D.

Professor

Department of Cell Biology and Neuroanatomy

University of Minnesota Medical School

321 Church Street, SE

Minneapolis, MN 55455

64
Opiate-Inhibited Adenylate

Cyclase in Mammalian Brain

Membranes

Steven R. Childers, Peter Nijssen, Pauline Nadeau,

Page Buckhanna, Phi-Van Le, and Jeffrey Harris

INTRODUCTION

The identification of opiate receptor binding sites in brain membranes (Pert and Snyder 1973; Terenius 1973; Simon et al. 1973)

demonstrated the existence of the first step in the molecular

actions of opiates and endogenous opioid peptides. However, the

second messenger of opioid systems has been more difficult to

study. Although several potential opiate-coupled intracellular

responses have been reported, the principal opiate-coupled second

messenger system characterized in isolated membranes has been the

inhibition of adenylate cyclase by opioid agonists. The first experiments showing opiate inhibition of cyclic AMP synthesis were

conducted by Collier and Roy (1974), who showed that morphine inhibited adenylate cyclase in membranes from rat striatum.

Although other studies of opiate-inhibited adenylate cyclase in

brain membranes appeared at this time, they were limited, not only

by the relatively small degree of inhibition by opiate agonists,

but also by the extreme heterogeneities of brain membranes that

produced small responses of adenylate cyclase to any known neurotransmitter (Drummond 1981). An easier system for the study of

this reaction was the neuroblastoma-glioma hybrid cell line NG108-

15, which contained opiate receptor binding sites (Klee and

Nirenberg 1974) and represented a much less heterogeneous neural

tissue than brain membranes. Several groups (Sharma et al. 1975;

Traber et al. 1975) showed that morphine and enkephalin analogs

inhibited adenylate cyclase in NG108-15 cells. Moreover, chronic

exposure of these cells to morphine altered the adenylate cyclase

response: basal adenylate cyclase activity was gradually increased during morphine exposure, while addition of naloxone

caused a rebound increase in adenylate cyclase activity (Sharma et

al. 1975). These results suggested that the development of opiate

tolerance could be at least partially due to uncoupling of opiate

receptors from adenylate cyclase. Since experiments had already

shown that tolerant animals showed no changes in opiate receptor

binding sites themselves (Pert and Snyder 1976), the findings with

adenylate cyclase represented one of the first potential molecular

mechanisms of tolerance.

65
At the same time, research in other areas determined the biochemical characteristics of hormone receptor-coupled adenylate

cyclase (Rodbell 1980; Gilman 1984; Schramm and Selinger 1984).

Receptors are coupled to the catalytic unit of adenylate cyclase

through specific guanine nucleotide-binding (N) proteins. One,

Ns, mediates stimulation of adenylate cyclase, while another, Ni,

mediates inhibition (Gilman 1984). Each N protein is composed of

three subunits, alpha, beta, and gamma. While the beta subunit is

identical in both Ns and Ni, the alpha subunits are different, and

function to bind and hydrolyze GTP. Thus, coupling of receptors

with adenylate cyclase requires GTP. N proteins also perform a

second function by interacting directly with receptor binding

sites and allowing the formation of high-affinity agonist complexes; in the presence of GTP, the receptor/N protein complex

dissociates and agonist affinity is decreased. The opiate receptor system operates in an analogous fashion. Opiate agonist binding sites are regulated by guanine nucleotides in both brain

membranes (Blume 1978a; Childers and Snyder 1978; Childers and

Snyder 1980) and membranes from NG108-15 cells (Blume 1978b).

Moreover, opiate-inhibited adenylate cyclase in NG108-15 cells

requires GTP (Blume et al. 1979).

It was important to extend the studies of opiate-inhibited adenylate cyclase in transformed cell lines to brain membranes, since

the brain contains multiple binding sites not present in neuroblastoma cells (Chang and Cuatrecasas 1979). More recent studies

on opiate-inhibited adenylate cyclase in brain membranes have become more consistent as the biochemical requirements of receptormediated adenylate cyclase become more clear. Thus, several

studies (Cooper et al. 1982; Law et al. 1981) demonstrated the

guanine nucleotide requirement for opiate-inhibited adenylate

cyclase and determined some of the pharmacological properties of

opiate-inhibited adenylate cyclase in brain membranes. Unfortunately, brain membranes remained a very heterogeneous system, and

adenylate cyclase responsiveness was still not as good as that in

the neuroblastoma-glioma cells.

To characterize the molecular properties of opiate receptor

coupling to adenylate cyclase, our studies focused on techniques

that modified the interactions of N proteins with receptors and

adenylate cyclase. One such technique involved low pH pretreatment of brain membranes to alter coupling of opiate receptors with

adenylate cyclase. This report summarizes our recent findings in

brain membranes altered by low pH pretreatment. This treatment

allows the quantification of opiate-inhibited adenylate cyclase

and the determination of receptor subtypes involved in coupling

with adenylate cyclase.

METHODS

Opiate-Inhibited Adenylate Cyclase

For determination of low pH effects on adenylate cyclase, membranes were resuspended in 0.5 ml of either adenylate cyclase

66
buffer, pH 7.4, or low pH buffer (50 mM sodium acetate, 5 mM

MgCl2, 1 mM dithiothreitol, pH 4.5), and incubated at 0 oC for 20

minutes. The low pH reaction was terminated by addition of 4 ml

cyclase buffer; the membranes were centrifuged at 30,000 x gravity

for 10 minutes and resuspended in cyclase buffer. For assay of

adenylate cyclase, membranes (10-50 μg protein per tube) were

added to tubes containing 20 mM creatine phosphate, 10 units creatine phosphokinase, 10 mM theophylline. 700 μg bovine serum albumin, and 100 μM (1 μCI) 3

H-ATP in a total of 100 μl. For assay of

opiate-inhibited activity, the tubes contained 100 mM NaCl and 50

mM GTP in addition to the appropriate drugs. The adenylate cyclase reaction was initiated by addition of ATP, the tubes were

incubated for 10 minutes at 30 oC, and the reaction was terminated

by boiling for 2 minutes. Separation of the labelled cyclic AMP

was performed on a reverse-phase high performance liquid chromatography (HPLC) column. This technique was modified (Childers

1986) from the method of Schulz and Mailman (1984), using 3H-ATP

as substrate and adenosine deaminase to reduce levels of adenosine, which interfere in the HPLC peak of cyclic AMP.

Chronic Drug Treatments

Rats were treated with naltrexone according to the method of Zukin

et al. (1982). Naltrexone (775 mg/ml, dissolved in saline) was

placed in Alzet osmotic mini-pumps (Model 2001, 1 μl/hr), which

were implanted subcutaneously (SC) in male Sprague-Dawley rats

(200 to 250 gm). Rats were treated for 7 days, then sacrificed.

Forebrains were assayed for 3

H-D-Ala enkephalinamide (D-Ala enk)

binding, and striatal membranes were assayed for opiate-inhibited

adenylate cyclase after low pH pretreatment as described above.

For chronic morphine studies, rats were implanted SC with one 75-

mg morphine (free base) pellet, then 2 days later with two more

pellets, then sacrificed 3 days after the second implantation.

Some morphine-tolerant rats were injected intraperitoneally (IP)

with 1 mg/kg naloxone and sacrificed 1 hour later. For acute

morphine studies, 10 mg/kg morphine was injected IP, then rats

were sacrificed 20 to 60 minutes later. Striatal membranes were

assayed for opiate-inhibited adenylate cyclase as described above.

Miscellaneous Assays

Opiate receptor binding assays were conducted as previously

described (Childers and Snyder 1978), using H-D-Ala enk (40.6

Ci/mmole) or H-naloxone (56.4 Ci/mmole) as labelled ligands. For

experiments using p-nitro-phenyl-oxymorphone (PNPO), membranes

were incubated with 10 μM PNPO at 25 oC for 30 minutes, then

washed twice by centrifugatfon before assay of receptor binding

and adenylate cyclase. To ensure removal of the reversible

component of PNPO binding, control membranes were incubated in

parallel with 10 μM oxymorphone; after washing was completed,

these membranes exhibited binding identical to untreated control

membranes (Childers 1984). In experiments with phospholipase-A,

membranes were incubated with 0.5 μg of the enzyme with 1 mM CaC12

67
for 20 minutes at 25 oC. The reaction was terminated by addition

of 5 mM EGTA, and membranes were assayed for receptor binding and

adenylate cyclase. Protein values were determined by the method

of Lowry et al. (1951).

RESULTS AND DISCUSSION

Effects of Lou pH Pretreatment on Brain Adenylate Cyclase

When brain membranes were preincubated in sodium acetate buf er at

pH 4.5 prior to assay of opiate receptor binding at pH 7.7, 3Hopiate agonist binding was unaffected, while the maximum inhibition of agonist binding by guanine nucleotides was increased

approximately twofold (Childers and Jackson 1984; Lambert and

Childers 1984). Since these results suggested that low pH pretreatment altered interactions of N proteins with receptors in

brain membranes, we tested whether the same pretreatment would

alter N protein interactions with adenylate cyclase. Results

(Childers and LaRiviere 1984) showed that while low pH pretreatment did not change basal adenylate cyclase activity, it decreased

stimulation of adenylate cyclase by agents that act through Ns;

these agents included fluoride, stable guanine nucleotides such as

Gpp(NH)p, and dopamine, which stimulates adenylate cyclase through

D1 receptors.

Measurement of opiate-inhibited adenylate cyclase has always been

difficult to quantitate in brain membranes because of the small

effect of opiate agonists in this tissue. Recent literature has

reported maximum inhibition of 15 to 25 percent, depending on the

study (Cooper et al. 1982; Law et al. 1981). In our experiments,

we were unable to demonstrate reproducible opiate inhibition of

adenylate cyclase of more than 10 to 15 percent. However, when

brain membranes were preincubated at pH 4.5 before assay of adenylate cyclase at pH 7.4, the decrease in Ns-stimulated adenylate

cyclase resulted in an increase in opiate-inhibited adenylate

cyclase to a maximum of 30 to 40 percent inhibition. The inhibition of adenylate cyclase by D-Ala enk was mediated by opiate receptors, since the dose response curve of the agonist was shifted

to the right by naloxone (figure 1).

This experiment also showed that D-Ala enk inhibited activity at

μM concentrations. While such concentrations may seem high compared to affinities of D-Ala enk at receptor binding sites in Tris

buffer, they are reasonable concentrations when used in physiological buffers containing sodium and guanine nucleotides. In fact,

when binding studies were conducted in adenylate cyclase buffer,

under conditions identical to those required to demonstrate

opiate-inhibited adenylate cyclase, D-Ala enk displaced 3H-naloxone binding at μM concentrations (not shown). Even conducting

binding assays in adenylate cyclase buffer, with GTP but without

sodium, decreased both agonist and antagonist binding (table 1).

Under these conditions, the binding of the mu compounds 3H-DAG0

and 3H-naloxone was most affected, with greater than 70 percent

inhibition in adenylate cyclase buffer compared to Tris buffer

68
FIGURE 1. Inhibition of adenylate cyclase in low pH pretreated

striatal membranes by D-Ala enk in the absence and

presence of 0.2 μM naloxone

NOTE: Maximum inhibition in nontreated membranes was approximately 10 percent.

containing 50 μM GTP. The mixed specificity ligands D-Ala enk and

EKC were inhibited by approximately 60 percent, while diprenorphine and DSLET were less affected (20 to 30 percent inhibition).

This decreased binding of both agonists and antagonists in adenylate cyclase buffer is caused by the addition of theophylline to

prevent cyclic AMP hydrolysis in the adenylate cyclase assay, and

demonstrates that high-affinity (nM) binding sites should not be

expected under conditions that maximize receptor coupling to

adenylate cyclase.

TABLE 1. Opiate receptor binding in Tris buffer and adenylate

cyclase buffer

3

H-Ligand

Specific cpm Bound

Tris Buffer Cyclase Buffer % Bound

3

H-D-Ala enk

3

H-DAGO

3

H-DSLET

3

H-EKC

3

H-naloxone

3

H-diprenorphine

6,744 41

5,181

2,744

1,195 23

5,480 3,894 71

4,010 1,594 40

4,895 1,224 25

3,089 2,528 82

69
Mechanism of the Low pH Effect

To explore the mechanism of low pH-induced changes in receptor/

N protein/adenylate cyclase interactions in brain membranes, several different possibilities were explored. One of these possibilities was that low pH pretreatment changed membrane structure

to alter the interactions of membrane proteins. Changes in

membrane fluidity alter beta-adrenergic receptor interactions with

adenylate cyclase (Hanski et al. 1979), so we explored this possibility by adding membrane fluidizing agents to low pH-pretreated

brain membranes (Childers and LaRiviere 1984). Results (not

shown) demonstrated that addition of lipids like phosphatidyl choline or cis-vaccenic acid had no effect on adenylate cyclase in

untreated membranes, but reversed the effects of low pH pretreatment. Thus, fluoride-stimulated adenylate cyclase was restored,

while opiate-inhibited adenylate cyclase was reduced. In fact, a

significant inverse correlation was discovered between Ns-stimulated and opiate-inhibited adenylate cyclase: as Ns-stimulated

activity was reduced, opiate-inhibited activity was increased.

Interestingly, this reversal effect of lipids was not observed for

opiate receptor binding sites: in low pH-pretreated membranes,

the guanine nucleotide regulation of agonist binding was increased, whether lipids were added or not (Childers and LaRiviere

1984).

These results suggest that a low pH-induced change in lipid structure of brain membranes may alter N protein interactions with

receptors and adenylate cyclase. The difference between reversals

of low pH effects on receptors and adenylate cyclase might suggest

that these two functions of GTP are mediated through different N

proteins, or, alternatively, that interaction of an N protein with

either receptors or adenylate cyclase may be regulated by different factors in membranes. Therefore, predictions cannot be made

concerning efficiency of receptor/adenylate cyclase coupling from

the degree of regulation of agonist binding by guanine nucleotides.

Properties of Opiate-Inhibited Adenylate Cyclase in Brain

Membranes

The effect of low pH pretreatment to increase opiate-inhibited

adenylate cyclase in brain membranes to a maximum of 30 to 40

percent inhibition allowed the quantitative examination of this

activity in brain.

Lineweaver-Burke plot of enzyme kinetics experiments (figure 2)

showed that inhibition by 10 μM D-Ala enk was noncompetitive, decreasing the Vmax but not the Km of adenylate cyclase for ATP.

This class of inhibition is typical for other neurotransmitterinhibited activities (Drummond 1981).

Opiate-inhibited adenylate cyclase required at least 1 μM GTP, and

50 μM GTP was required for full inhibition (not shown). Gpp(NH)p,

which stimulated basal activity by 50 percent, and forskolin,

70
FIGURE 2. Lineweaver-Burke kinetics of basal (circles) and D-Ala

enk-inhibited (squares) adenylate cyclase in low pH

pretreated striatal membranes

which stimulated activity threefold, did not support any significant inhibition by D-Ala enk (not shown).

Table 2 shows the agonist specificity of opiate-inhibited adenylate cyclase in low pH-pretreated striatal membranes. Most opioid

peptides exhibited μM affinities, with maximal inhibitions between

30 and 35 percent. Enkephalin, enkephalin analogs, beta-endorphin, and dynorphin were all effective agonists in this system.

N-acetyl-beta-endorphin, which has very little affinity for opiate

receptors, was ineffective in inhibiting adenylate cyclase. Moreover, morphine and the mu opioid peptides were weaker and less

effective than the other opioid peptides in inhibiting activity.

Therefore, mu receptors are probably not involved in opiate

receptor-inhibited adenylate cyclase.

The actions of antagonists in blocking D-Ala enk-inhibited adenylate cyclase in striatal membranes are summarized in tables 3 and

4. To determine the antagonist specificity of D-Ala enk-inhibited

adenylate cyclase in low pH-pretreated membranes, a series of opioid and nonopioid antagonists were added at 1-μM concentrations to

block 10 μM D-Ala enk-inhibited adenylate cyclase (table 3). All

nonopioid antagonists were completely ineffective in blocking DAla enk-inhibited activity. Opioid antagonists such as naloxone,

71
naltrexone, and levallorphan totally blocked inhibition at 1-μM

concentrations. Interestingly, the delta antagonist ICI-174864

did not block D-Ala enk inhibition at 1-μM concentrations;

increasing the concentration of ICI-174864 to 10 μM was only

slightly more effective in blocking D-Ala enk inhibition (not

shown).

TABLE 2. Specificity of opiate-inhibited adenylate cyclase in rat

striatal membranes

Maximal Inhibition

Opioid IC50, μM % Inhibition Concentration, μM

Met-enk 0.8 33 10

Leu-enk 0.6 30 10

D-Ala enk-NH2 0.8 32 10

DADL enk 0.7 32 10

DSLET 0.3 32 10

Beta-end

N-AC beta end

Dyn (1-13)

Dyn A (1-17)

D-Ala dyn A

Morphine

DAGO

Morphiceptin

0.8 40 10

11 50

0.7 3 0 10

0.6 31 10

0.6 40 10

6 18 100

10 24 50

10 15 50

TABLE 3. Effect of antagonists on striatal opiate-inhibited

adenylate cyclase

Neurotransmitter Antagonist % Inhibition

None None 35±4

Muscarinic ACh Atropine 30±4

Dopamine Chlorpromazine 38±6

Beta-adrenergic Propranolol 32±7

5-HT, dopamine Spiroperidol 39±3

5-HT, histamine Mianserin 39±2

Alpha2-adrenergic Yohimbine 32±6

Opioid Naloxone 6±5

Opioid Naltrexone 2±6

Opioid Levallorphan 8±4

Dpioid ICI-174864 36±6

72
Ke values for naloxone were determined using 100 to 500 nM naloxone to shift dose response curves of several agonists (table 4).

Naloxone was effective in blocking the actions of all the agonists

tested, and the Ke values of naloxone against the delta peptide

DADL-enkephalin, the mu peptide FK-33824, and beta-endorphin were

approximately equal, ranging from 50 to 80 nM. Again, although

these Ke values of naloxone appear high compared to receptor

binding data, the values of 50 to 80 nM are not very different

from Ke values of naloxone in blocking delta receptors in mouse

vas deferens (Kosterlitz and Paterson 1980). On the other hand,

naloxone was considerably weaker against dynorphin, with a Ke of

400 nM.

TABLE 4. Striatal opiate-inhibited adenylate cyclase:

Ke values of naloxone

Agonist Naloxone Ke, nM

D-Ala, D-leu enkephalin 86±25

FK-33824 77±26

Beta-endorphin 68±31

Dynorphin-A 441±129

The distribution of opiate-inhibited adenylate cyclase in several

regions of rat brain is seen in table 5. Of all the regions tested, only striatum and amygdala provided good (at least 30 percent)

inhibition of adenylate cyclase. Thalamus exhibited moderate

inhibition, and hypothalamus contained moderate to low inhibition,

but all other regions had no real, significant opiate-inhibited

adenylate cyclase. This distribution does not follow the distribution of any known opiate receptor binding sites in rat brain.

Effects of Chronic Drug Treatments on Opiate-Inhibited Adenylate

Cyclase

Zukin et al. (1982) demonstrated that chronic treatment of rats

with naltrexone increased numbers of opiate receptors in brain

membranes. Moreover, these increased sites were more sensitive to

regulation by guanine nucleotides, suggesting that receptor/

N protein coupling might be affected by chronic antagonist treatment. To test this latter hypothesis, we treated rats for 1 week

with naltrexone and assayed opiate receptor binding in forebrain

membranes and opiate-inhibited adenylate cyclase in low pH-pretreated striatal membranes. Results (table 6) showed that chronic

treatment with naltrexone did significantly increase receptor

binding by an average of 60 percent, similar to results reported

by Zukin et al (1982). However, we did not observe any change in

regulation of 'H-D-Ala enk binding by GTP. Moreover, in the

treated animals, there was no change in opiate-inhibited adenylate

73
cyclase in either the affinity of D-Ala enk or the maximal inhibition. Therefore, the increase in receptor binding sites is not

accompanied by a change in receptorladenylate cyclase coupling, as

predicted from changes in GTP regulation of binding (Zukin et al.

1982). This confirms, as mentioned above, the danger in predicting the degree of receptor/adenylate cyclase coupling based on

data of guanine nucleotide regulation of binding.

TABLE 5. Regional distribution of opiate-inhibited adenylate

cyclase in rat brain membranes

Region

Basal Activity,

pmoles/min/mg/

Maximal % Inhibition*

+DSLET +DAGO

Striatum 63.4 32 29

Amygdala 32.1 37 31

Thalamus 37.4 22 20

Sensomotor cortex 13.8 15 11

Hypothalamus 53.4 10 11

Hippocampus 12.6 9 12

Frontal cortex 10.7 5 0

Brain stem 11.3 5 1

Cerebellum 34.4 4 1

*Assayed in the presence of 1O μM OSLET and 50 μM DAGO, together with 100 mM NaCl,

50 μM GTP, and crude rat brain membranes.

TABLE 6. Effect of chronic naltrexone treatment on opiate

receptor binding and adenylate cyclase

% Specific Binding Adenylate Cyclase

Treatment -GTP +GTP D-Ala enk IC50 Max. % Inhib.

Control 100±12 39.9±2.1 5.522±3 μM 29.1±7.9

Naltrexone 159±16 4O.O±2.3 6.8±3.7 μM 25.5±6.1

NOTE: Rats were treated with 75 μg/hr naltrexone for 7 days, then striatal

membranes were assayed for H-D-Ala enk binding ± 50 μM GTP and for

inhibition of adenylate cyclase by 1 to 50 μM D-Ala enk.

To determine the effects of chronic morphine treatment on opiateinhibited adenylate cyclase, rats were implanted SC with morphine

pellets for 1 week and assayed for opiate-inhibited adenylate

cyclase in low pH-pretreated striatal membranes. Results (table

7) showed that neither acute injection of morphine (10 mg/kg

74
injected 20 minutes before sacrifice), chronic morphine treatment,

nor naloxone withdrawal following chronic morphine treatment

significantly altered inhibition of adenylate cyclase by D-Ala

enk.

TABLE 7. Effect of chronic morphine treatment on D-Ala enkinhibited adenylate cyctase

Adenylate Cyclase Activity

Treatment % Basal IC50, μM Max. % Inhibition

Control

Acute morphine

Chronic morphine

Naloxone withdrawal

1OO±12 10.1±1.3 20.8±1.7

112±17 10.2±2.0 14.O±1.8

113±10 16.1±6.5 31.4±3.4

98±9 9.0±1.3 18.7±3.8

These results suggest that the development of morphine tolerance

does not involve a change in the coupling between receptors and

adenylate cyclase; however, morphine is not a good agonist in the

opiate-inhibited adenylate cyclase system, and therefore is not

the agonist of choice in chronic drug treatments. We are currently exploring this possibility by chronic treatment of rats with a

better agonist in this system, D-Ala enk.

Receptor Subtypes Coupled to Adenylate Cyclase in Brain

The pharmacological properties of opiate-inhibited adenylate cyclase in brain membranes demonstrated, as discussed above, that mu

receptors were not coupled to adenylate cyclase in brain, thus

leaving delta receptors as prime candidates for the receptor subtype coupled to adenylate cyclase. However, although delta opioid

peptides were relatively potent in inhibiting adenylate cyclase,

and although Ke values for naloxone were similar to those at delta

receptors, several lines of evidence made this conclusion untenable. First, other nondelta opioid peptides were as potent as

delta opioid peptides, including beta-endorphin and dynorphin.

Second, the regional distribution of opiate-inhibited adenylate

cyclase does not follow that of delta binding sites. Finally,

although classical mu antagonists such as naloxone and naltrexone

were effective in blocking opiate-inhibited adenylate cyclase, the

delta-specific antagonist ICI-174864 was ineffective up to 10-μM

concentrations.

To explore the relationship between opiate binding sites and the

receptor-coupled adenylate cyclase, we utilized agents that block

high-affinity opiate receptor binding. Naloxone azine, an irreversible antagonist at mu(l) sites (Hahn et al. 1982), was an

effective antagonist in blocking opiate-inhibited adenylate

75
cyclase, but this blo kade was totally washed out by centrifugation steps that kept 3

H-naloxone binding inhibited by 75 percent

(data not shown). An even more effective irreversible compound is

p-nitro-phenyl-oxymorphone (PNPO), an agonist that blocks opiate

receptor binding sites at nM concentrations (Pasternak, submitted

for publication). When PNPO was added to low pH-pretreated striatal membranes, it inhibited adenylate cyclase in a manner similar

to that of morphine, with an affinity of 10 μM and maximal inhibition of 25 percent (not shown). Irreversibility of PNPO at both

receptor binding sites and with adenylate cyclase is demonstrated

in table 8. In these experiments, 10 μM PNPO was incubated with

striatal membranes, then the reversible component was washed out

by centrifugation. PNPO could not be washed away from receptor

binding sites, as seen by persistent 80 to 99 percent inhibition

of 3H-DSLET, 3H-DAGO, and 3H-EKC binding. Surprisingly, even

after this significant inhibition of binding, D-Ala enk continued

to inhibit adenylate cyclase, with maximal inhibition identical to

that in nontreated membranes.

TABLE 8. Effect of p-nitro-phayl-oxymorphole on opiate receptor

binding and opiate-inhibited denylate cyclase in

striatal membranes

Ligand

A. Receptor Binding

Control Treated

Specific cpm % Control Specific cpm % Control

3

H-DAG0

3

H-DSLET

3,166 100 27 1

3

H-EKC

3,169 100 619 20

1,572 100 235 15

B. Adenylate Cyclase

% Basal Activity

Additions Control Treated

None 100 101

D-Ala enk-NH2 61 65

Naloxone + D-Ala enk-NH2 94 96

Even more dramatic separation of binding and opiate inhibition of

adenylate cyclase was demonstrated with phospholipase-A, an enzyme

known to inhibit opiate receptors at low concentrations (Pasternak

and Snyder 1975), but which does not inhibit basal adenylate cyclase (Anderson et al. 1978; Partington and Daly 1979; Lad et al.

1979). When low pH-pretreated striatal membranes were incubated

76
with 0.5 μg/ml phospholipase-A, receptor binding was eliminated,

whether assayed with 3H-DSLET, 3H-DAGO, or 3H-EKC (table 9).

However, opiate-inhibited adenylate cyclase was not affected by

this treatment: although basal adenylate cyclase activity was

slightly increased, activity was still inhibited by more than 30

percent by D-Ala enk. Thus, several different techniques reveal

that opiate-inhibited adenylate cyclase persists in membranes in

which mu, delta, and kappa agonist binding has been eliminated.

TABLE 9. Effect of phospholipase A on opiate receptor binding

and opiate-inhibited adenylate cyclase in striatal

membranes

A. Receptor Binding

Control Treated

Ligand Specific cpm % Control Specific cpm % Control

3

H-DAG0 3,166 3

3

H-DSLET 1 2

3

H-EKC

3,169

100 91

100 3 0

1,572 100 109 7

B. Adenylate Cyclase

% Basal Activity

Additions Control Treated

None

D-Ala enk-NH2

Naloxone + D-Ala enk-NH2

100 118(100)

71 88 (74)

9 7 117 (99)

These results strongly suggest that the receptor coupled to adenylate cyclase in brain is neither mu, delta, nor kappa sites as

measured by classical binding methods. This conclusion should not

be too surprising, since the conditions used to maximize receptor/

adenylate cyclase coupling are totally different from those used

to maximize high-affinity receptor binding. In fact, as stated

earlier, high-affinity (nM) agonist receptor binding is not

detectable in buffers containing sodium and GTP. Moreover, there

is precedence in the study of cell systems to show that classical

opioid binding sites are not coupled to adenylate cyclase. For

example, when 95 percent of delta binding sites were blocked by

chlornaltrexamine in NG108-15 cells, opiate-inhibited adenylate

cyclase was unaffected (Fantozzi et al. 1981). In addition, detailed pharmacological experiments questioned the delta identity

of receptor-coupled adenylate cyclase in these same cells (Costa

et al. 1985). Finally, in C-6 glioma cells, where opiate receptors and opioid inhibition of cyclic AMP levels were induced by

77
treatment of cells with desmethylimipramine (Tocque et al. 1984),

the receptor binding sites were not delta sites, suggesting that

opiate-inhibited adenylate cyclase in this system was not mediated

by delta receptors. This evidence, coupled with the results reported here, strongly suggests that receptors coupled to adenylate

cyclase represent a novel class of receptor sites not described so

far in binding studies. This receptor has μM affinity for opioid

peptides, is blocked by 50 to 100 nM concentrations of naloxone

and naltrexone, and is present primarily in striatum and amygdala.

Despite the fact that this new receptor class is not one of the

classical binding sites, it is nevertheless a genuine opiate

receptor, since it exhibits agonist specificity (for example, Nacetyl beta-endorphin is much less effective than beta-endorphin

itself) as well as antagonist specificity (only opiate antagonists

block the opioid peptide inhibition). Whether this class of

receptor is the same as that seen in cells such as C-6 glioma or

NG108-15 is not yet clear.

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ACKNOWLEDGMENTS

This research was supported in part by PHS grant DA-02904 from the

National Institute on Drug Abuse. Dr. J. Simpkins provided

morphine pellets and Dr. G. Pasternak provided naloxone azine and

p-nitro-phenyl-oxymorphone for use in these studies.

AUTHORS

Steven R. Childers

Peter Nijssen

Pauline Nadeau

Page Buckhannan

Phi-Van Le

Jeffrey Harris

Department of Pharmacology

University of Florida College

of Medicine

Gainesville, FL 32610

80
Membrane Conductances and

Opioid Receptor Subtypes

R. Alan North

INTRODUCTION

The null methods provide

receptor and effect. The second technique was more recently

developed. Membrane currents can be measured from individual neurones in either the central or peripheral nervous system, maintained in vitro. It is important to study single cells, because

not all neurones, even in a given brain nucleus, respond equally

to opioids. Voltage-clamp methods are needed, because the

membrane-bound ion channels that are affected by opioids may also

be affected by membrane potential; potential must therefore be

controlled. In vitro study of the cells is essential so that

concentrations of opioids and ions can be carefully controlled.

This is readily done in a tissue bath.

There is a great deal of information available regarding the

actions of opioids on individual nerve cells (Duggan and North

1984). The principal direct action is a reduction in the rate of

firing of action potentials; this has been observed in numerous

brain regions. A second action that also occurs at many sites is

a depression of the amount of transmitter released at nerve

terminals (presynaptic inhibition). In the last few years, it has

become possible to study the primary effects of opioids that

underlie these two actions at the level of the cell membrane.

This has been made possible by a combination of two techniques.

The first is an old one. Pharmacological null methods (Gaddum

1937; Schild 1947) have been applied to the study of single nerve

cells, and these have allowed the measurement of agonist and

antagonist dissociation equilibrium constants (Kes). This is

important because opioids can interact with more than one kind of

receptor subtype, and the least ambiguous way to determine the

subtype is to measure the antagonist Ke.

a measure of Ke which is independent of the coupling between

EFFECTS OF μ RECEPTOR ACTIVATION

There is now abundant evidence for the presence of opioid receptor

subtypes. These are generally classified on the basis of antagonist Kes, either in ligand binding studies or in pharmacological

81
experiments on isolated tissues. The μ receptor is defined for

present purposes as one that has a naloxone Ke of 0.5 to 3 nM, is

activated by selective agonists such as morphiceptin (Tyr-Pro-PhePro-NH2) in the low nanomolar concentration range, and is

irreversibly blocked by ß-funaltrexamine (ß-FNA) (Goldstein and

James 1984). In rat locus coeruleus cells (Williams and North

1984) (antagonist affinities, ß-FNA), and on mouse dorsal root

(MDR) ganglion cells (Werz and Macdonald 1983a) (ß-FNA, selective

agonists), μ receptor activation increases a potassium conductance. In the locus coeruleus, this hyperpolarizes the cell and

inhibits action potential discharge. A similar action has been

observed in other neurones including rat substantia gelatinosa

(Yoshimura and North 1983), myenteric neurones (Morita and North

1982), and hippocampal interneurones (Nicoll and Madison 1984).

The underlying receptor has not been carefully defined in these

experiments, but the effectiveness of morphine as an agonist

implicates the μ receptor (see below). In the hippocampus, extracellular recording of field synaptic potentials indicates that

they are enhanced by opioids acting on μ receptors (Bostock et al.

1984). Since the increase of the field potential results from an

inhibition of interneurones (Nicoll and Madison 1984), it is likely that the interneurones themselves have μ receptors.

The potassium conductance increased by μ receptor activation in

the rat locus coeruleus has been described in some detail (North

and Williams 1985). The maximal conductance increase caused by

opioids is about 5 nS, and the value is independent of membrane

voltage between -120 and -50 mV. The conductance is also not

sensitive to 4-aminopyridine (1 mM) (which reduces the A-current

in the same cells by about 70 percent); nor is it sensitive to

tetraethylammonium (10 mM) or apamin (up to 1 μM). The conductance is significantly reduced by barium (100 to 300 μM) and by

quinine (100 μM) and is completely blocked when barium is used to

replace extracellular calcium (usually 2.5 mM). These results

allow one to "fingerprint" the particular conductance or set of

channels affected by opioids and to distinguish it from other

potassium conductances in the same or other cells.

Although the conductance increase by opioids becomes smaller at

membrane potentials less negative than about -50 mV, it still

contributes to action potential repolarization. Thus, opioids

acting on μ receptors shorten the action potential in locus coeruleus neurones by increasing the potassium conductance; their

effect on the action potential is lost when the action potential

is recorded in conditions of substantial potassium channel blockade (intracellular caesium or extracellular barium) (North and

Williams 1983). This action of opioids serves to modulate calcium

entry indirectly (Kandel 1981); if such an action occurred at

transmitter release sites, it would cause presynaptic inhibition

of release. Such indirect modulation of calcium entry (that is,

by an increase in potassium conductance), as a means of reducing

transmitter release, contrasts with direct modulation; in direct

modulation, the activated receptor reduces the calcium conductance

82
without any change in potassium conductance (see discussion of κ

receptors below).

In dorsal root ganglion cells, opioids act on μ receptors to

increase a potassium conductance that contributes to the action

potential repolarization and also to the action potential after

hyperpolarization (Werz and Macdonald 1983a; Werz and Macdonald

1983b). This conductance has been studied by measuring the duration of the action potential prolonged by tetraethylammonium. As

in the locus coeruleus, opioids that are selective for μ receptors

do not shorten the action potential which has been prolonged by

intracellular caesium. It is concluded that the potassium conductance which is increased by opioids in the dorsal root ganglion

cells has slightly different properties from that affected in the

locus coeruleus. It is not known whether this might be accounted

for by the difference in species or the immaturity of the dorsal

root ganglion cells.

EFFECTS OF d RECEPTOR ACTIVATION

The existence of the δ receptor as a separate entity on single

neurones has also been shown by experiments of the kind described

above. δ receptors are coupled to a potassium conductance in

cells of the guinea pig submucous plexus (Mihara and North, in

press). The properties of this potassium conductance are very

similar to that increased by μ opioids on locus coeruleus or other

cells (see above). The naloxone Ke for antagonism of [Met]enkephalin hyperpolarizations is different from that found in locus

coeruleus neurones, and ICI174864 (N-bisally-Tyr-Aib-Aib-Phe-Leu)

(Aib = aminoisobutyrate), a δ receptor antagonist (Cotton et al.

1984), acts as a competitive antagonist in submucous neurones with

a Ke of about 30 nM. This is in sharp contrast to the results in

the locus coeruleus, where ICI174864 also acts as an antagonist of

[Met]enkephalin, but the Ke is more than 10 μM (Williams and North

1984). The agonist potencies on submucous plexus neurones are

also strongly indicative of the δ receptor type. Morphine,

FK33824 (Tyr-D-Ala-Gly-MePhe-Met(O)-ol), and DAGO (Tyr-D-Ala-GlyMePhe-Gly-ol), which are typical μ receptor agonists, and dynorphin A and tifluadom, which are κ receptor agonists, do not affect

potassium conductance at concentrations up to 1 μM. The selectivity displayed by these agonists for opioid receptors on locus

coeruleus (μ) and submucous plexus (6) cells is actually considerably greater than that seen in ligand binding studies using

prototypic ligands (DAGO and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE)),

suggesting perhaps that the preparation of membranes for ligand

binding experiments may result in a loss of agonist selectivity.

On the other hand, there is good quantitative agreement between

antagonist Kes measured on single neurones and in binding studies

on brain homogenates.

Mouse DRG cells also have δ receptors, because selective δ agonists such as Tyr-D-Pen-Gly-Phe-Pen (DPLPE) cause spike shortening. Morphiceptin is inactive in many of the neurones affected by

DPLPE, suggesting the existence of only δ and not μ receptors in

83
some cells (see below). The δ agonists have no effect after

blockade of potassium conductance, indicating that, like μ agonists. they indirectly modulate the entry of-calcium ions byshortening the duration of the action potential (Werz and

Macdonald 1983a; Werz and Macdonald 1983b).

EFFECTS OF k RECEPTOR ACTIVATION

The κ agonists dynorphin, tifluadom, and U50,488H have no effect

on neurones of the rat locus coeruleus (μ receptors) (Williams and

North 1984) or guinea pig submucous plexus (δ receptors) (Mihara

and North 1986) in concentrations up to 1 μM. However, some

guinea pig myenteric neurones are affected by κ agonists. The

effect observed is a shortening of the action potential, but this

differs from the findings with μ and δ receptor agonists, because

it still occurs after the action potential has been prolonged by

intracellular caesium. This indicates that the κ agonists

directly modulate calcium entry into the neurone by an action that

does not involve potassium channels.

Direct suppression of calcium currents by κ agonists was first

suggested by Werz and Macdonald (Werz and Macdonald 1984a; Werz

and Macdonald 1984b; Werz and Macdonald 1985). They recorded membrane currents from voltage-clamped mouse dorsal root ganglion

cells and found that these were inhibited by κ opioids but not by

μ or δ selective agonists. There are other reports that opioids

directly depress calcium conductance (Mudge et al. 1979; Bixby and

Spitzer 1983), but in those experiments the receptor subtype was

not known, Because relatively high concentrations of agonists

were used, it is possible that the effects resulted from activation Of a κ receptor.

The significance of this action of opioids on transmitter release

can be assessed by measuring the amplitude of the nicotinic fast

excitatory postsynaptic potential (epsp) in the myenteric

plexus. This can be taken as a measure of the amount of acetylcholine released by a single action potential in a few presynaptic

fibers. Superfusion of either μ or κ agonists depresses the epsp

by acting on the terminals of presynaptic fibers. These fibers

have their cell bodies within the myenteric plexus, and it is

therefore reasonable to suppose that the depression by the agonists (see above) of the calcium action potential observed in the

cell bodies might also be occurring near the terminals; this could

be the reason for the presynaptic inhibition. In keeping with

this notion is the finding that barium, which prevents the increase in potassium conductance, blocks the presynaptic inhibitory

action of morphine but not that of dynorphin (Cherubini and North

1985).

DO DIFFERENT OPIOID RECEPTORS EXIST ON THE SAME CELL?

Some neurones of the myenteric plexus show effects of both morphine (hyperpolarization) and dynorphin (reduction in calcium

spike). Similarly, many of the epsps in the myenteric plexus are

84
depressed by both morphine and dynorphin (Cherubini and North

1984). Although the receptors involved have not been characterized by Ke determinations, the agonists were used at concentrations at which they were likely to be selective. This indicates

that μ and κ receptors probably coexist on the same myenteric

neurones.

Mouse DRG neurones seem to fall into four groups (Werz and

Macdonald 1985; Werz and Macdonald, personal commnunication). The

first group (about 50 percent of cells) shows no effects of opioids, which may be because they do not express any of the receptor

types. The second group (about 25 percent) shows effects of

κ agonists only--that is, the action potential is shortened even

when recording with a caesium-filled electrode. The third group

exhibits effects of both κ and μ agonists, and the fourth group, kK

and δ. In view of the finding that the effects of activating μ

and δ receptor types seem to be identical, it is of some interest

that individual neurones seem not to express both μ and δ

receptors. One would question the evolutionary value of conserving two opioid receptors which both open the same kind of

potassium channel in the membrane of a single cell if they respond

only to one naturally occurring ligand; it is more likely that

they would represent minor variations of a single gene product.

Conversely, if neurones are found which possess both μ and δ

receptors, one might hypothesize that there exists a more highly

selective natural ligand for the μ receptors than any of those

presently known.

OPIOIDS AND NONOPIOIDS HAVE SIMILAR ACTIONS

Noradrenaline has the same effect as opioids in neurones of the

rat locus coeruleus (Williams et al. 1985), the guinea pig myenteric plexus (Surprenant and North 1985), and the guinea pig

submucous plexus (North and Surprenant 1985). In the rat locus

coeruleus and the guinea pig submucous plexus, the receptors that

underlie this action of noradrenaline have been characterized by

measurement of antagonist Kes. They belong to the α2 class.

There is no difference between the properties of the potassium

conductance increased by opioids and that increased by α2 -adrenoceptor agonists on the same cell, with respect to sensitivity to

barium, quinine, or rubidium, or insensitivity to 4-aminopyridine

and tetraethylammonium. Furthermore, the maximum conductance

increase that can be evoked by an agonist acting on the opioid

receptor (μ in the case of the locus coeruleus. and δ in the case

of the submucous plexus) is the same as that which can be evoked

by an agonist acting at the α2 receptor on that cell. When the

current responses to agonists of each type are added, it is found

that the same population of potassium channels is opened by both

(Williams and North 1984; Mihara and North 1986). In the guinea

pig submucous plexus, somatostatin also increases the same potassium conductance by acting on a receptor that is neither δ nor α2

(not blocked by naloxone or idazoxan).

85
It is less clear whether precisely the same calcium conductance is

depressed by κ agonists as that affected by other substances.

However, depression of calcium conductance is an effect of several

different neurotransmitters (for example, γ-aminobutyric acid,

adenosine, and catecholamines). In some cases, κ opioids inhibit

calcium entry in the same neurone as one or more of these substances, and since there are more discrete receptors than types of

calcium channels, it seems likely that the different receptor

mechanisms will also converge prior to the effect on the channel.

CONCLUSIONS

The principal actions of opioids on the properties of mammalian

neurones can be ascribed to an inhibition of the rate of discharge

of action potentials and to a reduction in the amount of transmitter released by each action potential passing along the axon.

Opioids of the μ and δ types achieve both these actions by opening

membrane ion channels that allow outward movement of potassium

ions and thereby hyperpolarize the membrane; the outward potassium

current also serves slightly to reduce the duration of the action

potential, and this seems to be responsible for the presynaptic

inhibition of transmitter release. These opioid actions are

shared by α2-adrenoceptor agonists (and somatostatin) acting on

their own receptors; all the cells examined so far appear to express both an opioid (μ or δ) and an α2 receptor. Opioid receptors of the κ class mediate inhibition by a quite different

cellular mechanism; they directly depress calcium conductance.

This contributes to presynaptic inhibition of transmitter release,

and it may also inhibit the firing of cells that are normally

spontaneously active as a result of inward calcium currents at

normal membrane potentials.

The elucidation of the ionic conductances which are coupled to the

three opioid receptor subtypes allows comparisons to be made with

receptor subtypes for other transmitters. Individual transmitters

such as γ-aminobutyric acid, acetylcholine, and noradrenaline each

act on a variety of receptor subtypes; in each case, one subtype

inhibits a calcium conductance and another increases a potassium

conductance. In one sense, therefore, the opioids are conventional. In another sense, they are very different in that the

naturally occurring ligands for at least two of the receptor

subtypes (μ and κ) are encoded on different genes; dynorphin and

[Met]enkephalin might be considered to have little in common

except the same N terminal amino acids.

REFERENCES

Bixby, F.C., and Spitzer, N.C. Enkephalin reduces calcium action

potentials in Rohon-Beard neurons. J Neurosci 3:1014-1018,

1983.

Bostock, E.; Dingledine, R.; and Chang, K.-J. Mu opioid receptors

participate in the excitatory effect of opiates in the hippocampal slice. J Pharmacol Exp Ther 231:512-517, 1984.

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Cherubini, E., and North, R.A. μ and κ opioids inhibit transmitter release by different mechanisms. Proc Natl Acad Sci USA

82:1860-1863, 1985.

Cotton, R.; Giles, M.G.; Miller, L.; Shaw, J.S.; and Timms, D.

ICI 174864: A highly selective antagonist for the opioid δ−

receptor. Eur J Pharmacol 97:331-332, 1984.

Duggan, A.W., and North, R.A. Electrophysiology of opioids.

Pharmacol Rev 35:219-281, 1984.

Gaddum, J.H. The quantitative effects of antagonistic drugs.

J Physiol 89:7P-9P, 1937.

Goldstein, A., and James, I. Multiple opioid receptors: Criteria

for identification and classification. Trends Pharmac Sci

5:503-505, 1984.

Kandel, E.R: Calcium and the control of synaptic strength by

learning. Nature 293:697-700, 1981.

Mihara, S., and North, R.A. Opioids increase potassium conductance in guinea-pig submucous plexus neurones by activating δ

receptors. Brit J Pharmacol 88:315-322, 1986.

Morita, K., and North, R.A. Opiate activation of potassium

conductance in myenteric neurons: Inhibition by calcium ions.

Brain Res 242:145-150, 1982.

Mudge; Leeman, S.E.; and Fischbach, G.D. Enkephalin inhibits release of substance P from sensory neurons in culture and

decreases action potential duration. Proc Natl Acad Sci USA

79:526-530, 1979.

Nicoll, R.A., and Madison, D.V. The action of enkephalin on

interneurons in the hippocampus. Soc Neurosci Abstr 10:660,

1984.

North, R.A., and Surprenant, A. Inhibitory synaptic potentials

resulting from α2-adrenoceptor activation in guinea-pig submucous plexus neurones.

North, R.A.,

J Physiol 358:17-32, 1985.

and Williams, J.T. Opiate activation of potassium

conductance inhibits calcium action potentials in rat locus

coeruleus neurones. Brit J Pharmacol 80:225-228, 1983.

North, R.A., and Williams, J.T. On the potassium conductance

increased by opioids in rat locus coeruleus neurones. J Physiol

364:265-280, 1985.

Schild, H.O. pA, a new scale for measuring drug antagonism. Brit

J Pharmacol 2:189-206, 1947.

Surprenant, A., and North, R.A. μ and α2 receptors coexist on

myenteric but not on submucous plexus neurones. Neuroscience

16:425-430, 1985.

Werz, M.A., and Macdonald, R.L. Opioid peptides selective for mu

and delta receptors reduce calcium dependent action potentials

by increasing potassium conductance. Neurosci Lett 42:173-178,

1983a.

Werz, M.A., and Macdonald, R.L. Opioid peptides with differential

affinity for mu and delta receptors decrease sensory neuron

calcium-dependent action potentials. J Pharmacol Exp Ther

227:394-402, 1983b.

Werz, M.A., and Macdonald, R.L. Dynorphin reduces calciumdependent action potential duration by decreasing voltagedependent calcium conductance. Neurosci Lett 46:185-190, 1984a.

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Werz, M.A., and Macdonald, R.L. Dynorphin reduces voltage dependent calcium conductance of mouse dorsal root ganglion neurons.

Neuropeptides 5:253-256, 1984b.

Werz, M.A., and Macdonald, R.L. Dynorphin and neo-endorphin peptides decrease dorsal root ganglion neuron calcium-dependent

action potential duration. J Pharmacol Exp Ther 234:49-56,

1985.

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single locus coeruleus neurons. Molec Pharmacol 26:489-497,

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in vitro hyperpolarized by enkephalin. Nature 529-530, 1983.

AUTHOR

R. Alan North, B.Sc., M.B., Ch.B., Ph.D.

Massachusetts Institute of Technology

Cambridge, MA 02139

88
Modulation of Opioid Peptide

Metabolism by Seizures:

Differentiation of Opioid Subclasses

Jacqueline F. McGinty, Tomoyuki Kanamatsu, Johnny Obie,

and Jau-Shyong Hong

INTRODUCTION

Evidence is accumulating that the proenkephalin and prodynorphin

families of opioid peptides play fundamentally different roles in

the regulation of brain excitability, particularly in the seizuresensitive hippocampus.

cated that leu5- and met5-enkephalin have a net excitatory action

Electrophysiological studies have indiwhen iontophoretically applied to pyramidal cells in the hippocampus, whereas in most other brain areas these peptides manifest

inhibitory actions (Nicoll et al. 1977). Intracellular recordings

from pyramidal cells in the CA1 (or superior) region of the hippocampus have indicated that enkephalin-induced excitation, which is

naloxone-reversible, may be due to inhibition of interneurons

(Zieglgansberger et al. 1979); however, this interpretation is

controversial (Corrigall 1983). In contrast, iontophoretically

applied dynorphin peptides produce both naloxone-reversible excitatory effects and non-naloxone-reversible inhibitory effects in

the hippocampus (Henriksen et al. 1982; Walker et al. 1982; Gruol

et al. 1983; Moises and Walker 1985), indicating that more than

one receptor subtype in the hippocampus may be affected by dynorphin peptides. Furthermore, intracerebroventricular (ICV) application of enkephalins and ß-endorphin have naloxone-reversible

seizurogenic properties (Urca et al. 1977; Henriksen et al. 1978),

whereas ICY dynorphin (Henriksen et al. 1982) and several synthetic opioid agonists directed against both mu and delta receptors (Tortella et al. 1984; Tortella et al. 1985) do not. Thus,

opioid agonists may have proconvulsant or anticonvulsant effects

depending on the dose and class of opioid receptors that they

activate (Frenk 1983).

The demonstration of convulsant and anticonvulsant properties of

opioid agonists has suggested that one endogenous opioid class may

mediate seizure activity, while another may mediate the electrographic refractory state and behavioral depression that follow a

seizure (postictal state). An involvement of opioids in the postictal state is supported by evidence that naloxone pretreatment

diminishes the behavioral postictal depression that follows

89
seizures elicited by amygdaloid kindling (Frenk et al. 1979) or by

electroconvulsive shock (Holaday and Belenky 1980; Tortella and

Cowan 1982b; Tortella et al. 1985; Urca et al. 1981). If different opioid peptides are involved in seizure occurrence and postictal depression, then one would expect that recurrent seizures

would have a different effect on enkephalin and dynorphin systems

in seizure-sensitive brain areas. This hypothesis is strengthened

by demonstrations that electroconvulsive shock treatments (Hong et

al. 1979; Hong et al. 1985b; Kanamatsu et al., in press), kainic

acid administration (Hong et al. 1980; McGinty et al. 1983; Obie

et al. 1985; Kanamatsu et al.,

(McGinty et al.,

in press), and amygdaloid kindling

in press) differentially alter enkephalin and

dynorphin peptide levels in the hippocampus, reflecting changes in

opioid metabolism in response to seizures. The data, reviewed

herein, suggest that experimentally induced epileptiform activity

may be a useful model in the study of the control of opioid

metabolism in general.

LOCALIZATION OF HIPPOCAMPAL OPIOID PEPTIDES

The initial approach was to identify the pathways in the hippocampal formation that contain opioid peptides. Research has shown

that two of the three major excitatory circuits of the hippocampal

formation, the lateral perforant/temperoammonic (PTA) pathway and

the dentate granule cell-mossy fiber (MF) pathway, contain opioid

peptides. Proenkephalin is present in the PTA pathway (Gall et

al. 1981; McGinty et al. 1982; McGinty et al. 1983; McGinty et al.

1984), which is the first link in a trisynaptic excitatory chain

from the entorhinal cortex to the dentate granule cells and pyramidal cells of the hippocampus (figure 1, A and B). Prodynorphin

and proenkephalin are contained in the granule cell-mossy fiber

path (McGinty et al. 1983), which is the second link in the trisynaptic chain. Within the mossy fiber system of normal rats,

however, the concentration of prodynorphin is much greater than

the concentration of proenkephalin (figure 1, C and 0). Recently,

studies have shown that different populations of granule cells and

hippocampal interneurons contain dynorphin B and bovine adrenal

medullary peptide (BAM 22P), a proenkephalin-derived peptide

(McGinty et al. 1984; McGinty et al. 1985; figure 1, E). Thus, in

addition to scattered opioid interneurons, the hippocampus contains one pathway (PTA) that contains only proenkephalin and one

pathway (MF) that contains proenkephalin and prodynorphin peptides

in different neurons.

The distribution of opioid peptides partially overlaps the distribution of opioid receptors in the hippocampal formation. In vitro

autoradiography demonstrates that ligands that predominantly bind

mu, kappa, and lambda receptors are most densely distributed in

the CA3-4 pyramidal cell layer and the dentate molecular layer

(Meibach and Maayani 1980; Duka et al. 1981; McLean et al. 1985;

Perry and Sadee 1985; Chang et al., in press). Delta-selective

binding overlaps the distribution of mu and kappa sites, but it is

90
FIGURE 1. Localization of hippocampal opioid peptides

NOTE: A. Nissi-stalned coronal section of the dorsal hippocampal formation showing the dentate granule cell layer (GL) and pyramidal cell layers of CA3

and CA1 (x6.8). B. Schematic illustration of the major dynorphin-ir and

enkephalin-ir hippocampal pathways (McGinty et al. 1983). (1) Dynorphin/

enkephalin-ir mossy fibers originating from granule cells In both the

dorsal and ventral dentate gyrus. (2) Enkephalin-ir lateral perforant/

temperoammonic path originating In the lateral entorhinal cortex and innervating the outer molecular layer of the dentate gyrus and hippocampal

fields CA1-CA4. Enkephalin-ir is densest in the caudal-ventral divisions

of the entorhinal pathway. C. Dynorphin-ir in the mossy fibers of the dorsal hippocampus In a normal Sprague-Dawley rat (x4.3). D. Leu5

-enkephalinir In mossy fibers of a normal Sprague-Dawley rat (x4.8). E. Dentate

granule cells In the ventral hippocampal formation stained with a two-color

immunoperoxidase method (McGinty 1985). Right cell (arrow) contains

dynorphin B-ir, and left cell (open arrowhead) contains BAM 22P-ir (x533).

91
lighter in the pyramidal and granule cell layers and more extensive in other layers of the hippocampal formation (McLean et al.

1985; Chang et al., in press).

DIFFERENTIAL RESPONSES OF OPIOID PEPTIDES TO SEIZURES

Our evidence from radioimmunoassay (RIA) and immunocytochemical

(ICC) measurements of opioid peptides subsequent to seizures is

consistent with the concept of different actions of enkephalin and

dynorphin in the hippocampal formation. We have demonstrated that

enkephalin-ir is increased transiently in the lateral PTA pathway,

whereas dynorphin-ir is depleted transiently in the mossy fiber

system of the hippocampus as a result of seizures induced by three

techniques: (1) kainic acid injections (Hong et al. 1980; McGinty

et al. 1982; McGinty et al. 1983), (2) maximal transauricular

electroconvulsive shock (Hong et al. 1985b; Kanamatsu et al., in

press), and (3) amygdaloid kindling (McGinty et al., in press).

Kainic Acid-Induced Seizures

Kainic acid is an excitatory amino acid that causes hippocampal

epileptiform activity and motor seizures (Nadler et al. 1978). It

is toxic to hippocampal pyramidal cells in fields CA3 and CA4

(Nadler et al. 1978), which are innervated by enkephalin- and

dynorphin-containing mossy fibers. At regular time points beginning 2 days after a single ICV or intrastriatal dose of kainic

acid, the level of met5-enkephalin-ir increased significantly in

the hippocampus and remained elevated for 2 weeks, as demonstrated

by RIA (Hong et al. 1980). Subsequently, we have found that leu5-

enkephalin-ir detected both by RIA and ICC was increased in the

mossy fibers and perforant path fibers, whereas dynorphin-ir was

unchanged in mossy fibers 4 days after kainic acid administration

(McGinty et al. 1983). These alterations were the first indication that the metabolism of dynorphin and enkephalin could be differentially regulated, not only in the same brain structure but in

the same pathway.

Most recently, we have assayed hippocampal opioid peptides by RIA

and ICC at early time points after a single intrastriatal injection of kainic acid (Obie et al. 1985; Kanamatsu et al., in press)

(table 1). Recurrent seizures were still apparent 3 to 6 hours

after kainic acid administration; dynorphin (1-8)-ir in mossy

fibers was depleted 70 percent, and met-enkephalin-ir in the mossy

fibers and perforant path was depleted by 30 to 40 percent. Within

24 to 48 hours, the levels of both peptides had risen above normal, with dynorphin-ir peaking at 48 hours at 171 percent of normal and enkephalin-ir, at 270 percent of normal. However, by 72

hours, enkephalin had continued to rise to 300 percent of normal,

especially in the mossy fibers, while dynorphin-ir in the mossy

fibers had returned to normal levels. The reduction in dynorphinir and enkephalin-ir within 3 to 6 hours of kainic acid administration suggests an increase in the release of these peptides

during the seizure period. The rebound in peptide levels in later

phases may be due to a transient overproduction of dynorphin and a

92
TABLE 1. Hippocampal opioid peptide metabolism in different

seizure models

Seizure

Model Dynorphin A

Met5-

Enkephalin

Kainic Acid

6 hr

24 hr

48 hr

72 hr

2 wk

ECS

1 day after: 1 ECS

3 ECS

6-10 ECS

2 wks after: 6-10 ECS

Kindling

1 day after:

stage 3 (subconvulsive)

stage 5 (convulsive)

28 days after stage 5

Seizure-Sensitive Gerbil

Postseizure interval:

72 hr

5 min

-70%

+35%

+65%

+0%

+l0%

-30%

-70%

+0%

+0%

-70%

+0%

+34%

+34%

-30%

+95%

+170%

+200%

+60%*

+0%

+0%

+40%

+0%

+0%

-50%

+0%

+24%

+100%

*Ipsilateral to intrahippocampal injection.

larger, more sustained overproduction of enkephalin. This

possibility is supported by the demonstration of a sharp increase

in proenkephalin messenger RNA in the hippocampus, as measured

with a cDNA clone for proenkephalin (Hong et al. 1985b; Kanamatsu

et al., in press) which peaked at 6 to 12 hours and returned to

normal levels by 48 hours after kainic acid administration. These

dramatic biphasic changes, the details of which are distinct for

dynorphin-ir and enkephalin-ir, demonstrate the need to take

neurochemical measurements at frequent intervals after seizure

induction in order to detect a pattern of rapid changes and even

reversals in peptide levels and mRNA. Only by constructing a

93
continuous time course of changes during and after seizure

activity were we able to detect how dynamic the regulation of

opioid peptide biosynthesis is.

Electroconvulsive Shock

Electroconvulsive shock (ECS) is the second seizure model that we

have begun to explore in relation to the metabolic plasticity of

opioid peptides (table 1). Several lines of evidence suggest that

opioid peptides may be involved in ECS-elicited behavioral alterations such as analgesia, retrograde amnesia, changes in seizure

threshold, and postictal depression (Urca et al. 1981; Urca et al.

1983; Holaday and Belenky 1980; Carrasco et al. 1982; Tortella and

Cowan 1982b; Berman and Adler 1984). Twenty-four hours after 6 to

10 daily maximal ECS treatments (150 V at 60 Hz for 1-second duration), the levels of dynorphin (1-8)-ir and met5

-enkephalin-ir in

several limbic-basal ganglia brain regions and the level of mRNA

coding for preproenkephalin in the hypothalamus were significantly

increased. In the hippocampal formation, however, met5-enkephalin-ir increased by 40 percent in the perforant pathway, whereas

dynorphin-ir decreased by 70 percent in the mossy fiber system in

the absence of any cellular damage (Hong et al. 1985a; Kanamatsu

et al., in press). These alterations in opioid peptide levels

returned to normal by 2 weeks posttreatment. Repeated ECS treatments also downregulated mu and delta opioid receptor binding in

the hippocampus, hypothalamus, and caudate nucleus, but not in the

frontal cortex or brain stem (Nakata et al. 1985). Scatchard

analysis demonstrated that the number, not the affinity, of opioid

sites was reduced for up to 2 weeks after the last of 10 daily ECS

treatments. The fact that both enkephalin and dynorphin systems

are affected in many brain regions after ECS is consistent with a

general involvement of these peptides in ECS-elicited behavioral

alterations. However, the opposite changes in hippocampal enkephalin and dynorphin levels after ECS suggest that these two peptides must play fundamentally different roles, possibly by

interacting with different receptor subtypes, in mediating hippocampal excitability.

Amygdaloid Kindling

In order to see if postseizure changes in opioid peptides were a

common response to seizure induction in the hippocampal formation,

we employed yet another model of epilepsy. Amygdaloid kindling is

an experimental model of temporal lobe epilepsy in which initially

subthreshold intra-amygdaloid stimulations result in progressively

more intense seizure activity (stages 1 to 4) until a generalized

(stage 5) seizure occurs (Goddard 1967; Racine 1972). More work

has been focused on discovering the neurochemical correlates of

kindling than on any other seizure model; despite this effort,

there is no prevailing consensus about whether defects in specific

neurotransmitters or their receptors are responsible for the

kindling phenomenon (McNamara 1984).

94
Several lines of evidence have suggested a relationship between

endogenous opioid peptides and kindled seizures. Naloxone, a

stereoselective opioid antagonist, has been reported to facilitate

amygdaloid kindling in rats (Hardy et al. 1980). In addition, the

duration of postictal depression can be altered by opioid agonists

and antagonists (Frenk et al. 1979). Enkephalin-ir has been reported to be increased in whole rat brain (Vindrola et al. 1981),

and dynorphin-ir has been observed to increase in rabbit hippocampus 1 day after reaching stage 5 seizures (Przewlocki et al.

1983). In order to construct a time course of opioid peptide

changes during and after amygdaloid kindling, we examined rats 1

day after reaching subconvulsive stage 3 seizures and 1 day and 4

weeks after two consecutive stage 5 seizures (table 1). One day

after stage 3 seizures, no significant change in opioid peptide

levels was observed by RIA or ICC, indicating that opioid peptides

are not involved in the process of kindling before the stage of

generalized seizures. In contrast, 1 day after two cons cutive

stage 5 seizures, there was a 50 percent increase in met5-

enkephalin-ir in the temperoamnonic pathway and a 70 percent decrease in dynorphin (1-8)-ir in the mossy fibers of the hippocampus (McGinty et al., in press). The only other change was an

increase in the immunostaining of dynorphin in the substantia

nigra, pars reticulata, a structure recently implicated in the

regulation of kindled seizure thresholds (McNamara et al. 1984).

Normal levels of both peptides in the hippocampus and substantia

nigra returned by 4 weeks after completion of kindling in the absence of any apparent seizure activity. Since all of the above

neurochemical changes after kindling appear to be transient in

nature, the permanence of the kindling phenomenon cannot be

explained by these alterations.

SEIZURE-SENSITIVE GERBILS

In contrast to models of epilepsy that depend on seizure induction

by an exogenous trigger, there are strains of rodents that have

spontaneous seizures. Immediately after a gerbil has a spontaneous seizure, we have demonstrated recently that both enkephalin

and dynorphin levels of the hippocampus are significantly elevated

compared to those levels in seizure-resistant gerbils (Lee et al.,

in press) (table 1). However, the elevation in enkephalin occurs

only after a seizure, whereas the elevation in dynorphin levels is

present in seizure-sensitive gerbils regardless of when a seizure

occurs. It has also been reported that seizure-sensitive gerbils

have an increased number of GABA-immunostained basket cells in the

dentate gyrus of the hippocampus (Peterson et al. 1985). This increase in GABA-containing structures is in contrast to a reduced

number of GABA-immunostained terminals in cortical epileptic foci

(Ribak et al. 1979). Electron microscopic evidence has demonstrated, however, that the GABA-immunostained basket cells in the

dentate gyrus of the seizure-sensitive gerbil innervate (i.e.,

inhibit) each other instead of inhibiting the granule cells as

they would in normal gerbils (Peterson and Ribak 1985). Therefore, one should expect fundamental differences between the patterns of neurochemical changes in seizure induction models and

95
genetic models of epilepsy, perhaps reflecting a similar diversity

in the human epilepsies.

SEPTOHIPPOCAMPAL DIFFERENTIATION

Brain cholinergic, monoaminergic, and GABA systems are the most

widely implicated in seizure transmission and inhibition (Woodbury

1984). All of these neurotransmitter systems are present in neurons intrinsic to or afferent to the hippocampal formation and

have been implicated in the regulation of hippocampal excitability. Of these, we have preliminary evidence that destruction of

the primarily cholinergic septohippocampal pathway leads to alterations in endogenous opioid metabolism in the absence of seizures.

Four days after fornix transections (Hong et al. 1981) or lesions

of the medial septal nuclei (McGinty et al. 1985), enkephalin-ir

was increased in the temperoammonic pathway, but no change was

observed in dynorphin-ir in the mossy fibers. These preliminary

data indicate that dynorphin and enkephalin metabolism in these

two hippocampal pathways are differentially regulated. The data

also indicate that perturbations of hippocampal pathways that do

not induce seizures, but may alter seizure sensitivity to other

stimuli such as kindling (Gellman and McNamara 1984), can alter

opioid peptide metabolism in the hippocampal formation.

SUMMARY AND CONCLUSIONS

Until now, we have measured dynorphin-ir and enkephalin-ir at only

a few time points after a single seizure or after multiple seizures in most of the models we have employed. Except for the

genetically seizure-prone gerbil, our data consistently show a

transient and robust decrease in dynorphin-ir and a sustained increase in enkephalin-ir in the hippocampal formation subsequent to

kainic acid-, ECS- , or amygdaloid-kindled convulsive seizures. At

this point, kainic acid appears to have the most dramatic effects

on hippocampal enkephalin and dynorphin levels, causing an initial

decrease followed by a rebound increase beyond control levels,

which, for met5-enkephalin, is maintained for at least 2 weeks.

Recurrent seizures leading to neurotoxic effects on CA3 pyramidal

cells, which are not present after ECS or kindling, may underlie

the sustained alteration in enkephalin metabolism after kainic

acid. Further investigation into the time course of seizureinduced enkephalin and dynorphin metabolic changes using RIA, ICC,

and measurements of opioid mRNA levels may reveal a common pattern

of depletion due to immediate release, rebound synthesis according

to the severity of demand, and stabilization at a new equilibrium

over several days or even weeks in each seizure model.

Our preliminary time points suggest striking differences in the

rate of metabolism of hippocampal dynorphin and enkephalin in

response to seizures. We would like to find out if other perturbations of the hippocampus, primarily the elimination of the

influence of its known neurochemical afferents by lesion (as performed on the septohippocampal system described above) or pharmacological blockade, can alter the metabolism of hippocampal opioid

96
peptides and influence subsequent seizure transmission. Distinguishing the physiological conditions that induce metabolic

changes in discrete opioid peptidergic pathways may help us to

understand how endogenous opioids are involved in the regulation

of neuronal excitability in specific brain regions, as well as to

understand more about the differential regulation of opioid peptide metabolism in different brain pathways.

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ACKNOWLEDGMENTS

Jacquelyn Loesche of the East Carolina University School of

Medicine performed the immunocytochemistry and prepared the

illustrations; Jeff Simpson of the East Carolina University School

of Medicine assisted with photography; Robert Dyer of the

Environmental Protection Agency, Research Triangle Park, NC,

prepared the kindled rats; Randall Lee of the University of

California at Los Angeles supplied the gerbils; and Chris

Frederickson of the University of Texas supplied the septallesioned rats. Supported by USPHS grants NS 20451 and DA 03982

(JFM).

AUTHORS

Jacqueline F. McGinty, Ph.D.

Department of Anatomy

East Carolina University

School of Medicine

Greenville, NC 27834

100
Tomoyuki Kanamatsu, Ph.D.

Department of Physiology

Toho University Medical School

Tokyo, Japan

Johnny Obie

Laboratory of Behavioral and

Neurological Toxicology

National Institute of Environmental

Health Sciences

National Institutes of Health

Research Triangle Park, NC 27709

Jau-Shyong Hong, Ph.D.

Director

Laboratory of Behavioral and

Neurological Toxicology

National Institute of Environmental

Health Sciences

National Institutes of Health

Research Triangle Park, NC 27709

101
Kappa Isoreceptors:

Neuroendocrine and

Neurochemical Evidence

Paul L. Wood and Smriti lyengar

INTRODUCTION

The concept of a major opioid receptor triad consisting of mu,

delta, and kappa receptors (Wood 1982) is now well established.

The wide diversity of behavioral and pharmacological effects of

opiate analgesics and opioid peptides has been a key argument for

the existence of multiple opioid receptor systems. The establishment of reliable binding assays has further aided in the pharmacological and biochemical delineation of these receptor differences

(Wood 1983a). In the case of the mu receptor population, there

has been a further subclassification into mu-1 and mu-2 isoreceptors (Wood 1984; Pasternak 1982).

MU-1 Systems

Analgesia induced by drug injections directly into the periaqueductal gray (PAG) is effective with mu and delta agonists

but not with kappa agonists (Wood et al. 1983).

Cortical and hippocampal acetylcholine (ACh) turnover is suppressed by mu and delta agonists but not by kappa agonists

(Wood et al. 1984b; Wood 1984).

Catalepsy is induced in the rat by mu, delta, and epsilon

agonists but not by kappa agonists (Pasternak 1982).

MU-2 Systems

Striatal dopamine metabolism is accelerated by mu and delta

agonists but not by kappa agonists (Wood et al. 1980; Wood

1983b; Wood 1984; Wood et al. 1984b).

Respiratory depression is induced by mu and delta agonists but

not by kappa agonists (Wood et al. 1982; Ling et al. 1983).

102
In studies of physical dependence, it has become evident that

many of the signs of precipitated withdrawal in rats are mu-2

and delta mediated (Ling et al. 1983). Withdrawal signs with

kappa agonists are different from those with a mu or delta

agonist and are much less severe (Woods et al. 1982).

In the above systems, the isoreceptor populations were defined via

inhibition or lack of inhibition by the selective mu-l antagonist

naloxonazine (Hahn et al. 1982; Wood and Pasternak 1983), or by

the mu-2 antagonist actions of presently available kappa agonists

(Wood 1984).

KAPPA ISORECEPTORS IN THE CENTRAL NERVOUS SYSTEM

The examples given do not represent all the available evidence for

mu isoreceptor populations, but were listed to demonstrate a key

point of interest in early studies with kappa agonists. Specifically, a large number of systems appeared that were lacking kappa

receptor regulation; it was not until more physiological assays

specific for kappa agonists appeared that the concept of kappa

isoreceptors could be evaluated.

The rabbit vas deferens is a kappa-selective peripheral tissue

(Oka et al. 1981).

The guinea pig esophagus appears to be most sensitive to kappa

agonists (Kamikawa and Shimo 1983).

In the dog mesenteric artery, kappa agonists appear to selectively depress norepinephrine release and elicit dilatation

(Sun and Zhang 1985).

Spinal analgesia and inhibition of substance P-induced scratching in the rat were potently elicited by kappa agonists (Wood

et al. 1981; Rackham et al. 1981), presumably via kappa receptors located in the dorsal horn of the spinal cord (Gouarderes

et al. 1985).

The endogenous kappa agonist dynorphin1-8 is contained within

vasopressin neurosecretory vesicles in the rat neurointermediate lobe (Whitnall et al. 1983; Zamir et al. 1984) along

with specific kappa receptors on pituicytes (Bunn et al.

1985). This system is presumably involved in the diuretic

actions of kappa agonists (Leander 1983).

Binding Studies

Initial binding studies with membrane homogenates also were able

to demonstrate a unique kappa binding site, which was characterized by low affinities for mu, delta, and epsilon agonists but

high affinity for kappa agonists (Wood 1982); greater resistance

to proteolytic agents (Pasternak 1980); greater resistance to

alkylation (Wood and Charleson 1982); and greater resistance to

heat inactivation (Wood and Pilapil 1983). These studies led to

103
the ultimate evidence for receptor heterogeneity: solubilization

of the kappa receptor (Itzhak et al. 1984).

Receptor heterogeneity among the published kappa binding sites

became evident when multiple cold displacers were coevaluated

within one laboratory. This was the case with analysis of 3H-SKF10,047 binding in rat brain (Pilapil and Wood 1983), 3

H-etorphine

binding in guinea pig spinal cord (Attali et al. 1982), 3H-etorphine binding in guinea pig striatum in the presence of mu and

delta receptor blockers (Audigier et al. 1982), and 3H-EKC binding

in guinea pig brain in the presence of mu and delta receptor

blockers (Su 1985). These studies all consistently described two

kappa receptor sites; both sites possessed high affinities for the

synthetic kappa agonists, but one site possessed preferential

affinities for dynorphin1-17, and the other site, in contrast to

previously described kappa sites, possessed high affinities for

met-enkephalin Arg6Phe7, beta-endorphin, DADLE, and morphine.

Both sites were unaffected by PCP, suggesting kappa isoreceptor

populations and no PCP receptor component; however, a sigma

receptor involvement has not been excluded.

These binding studies were the first to suggest the potential of

kappa isoreceptors in the central nervous system (CNS). Subsequent in vivo validation of this concept has been slower in

momentum, with the first data coming from detailed studies of

kappa regulation of the hypothalamic-pituitary-adrenocortical

(HPA) axis in the rat (Iyengar et al. 1985).

Corticosterone Release

as follows:

stereospecific increases in serum corticosterone;

stereospecific reversal by opiate antagonists;

dose dependency with a rank order of drug potency similar to

that observed in the rabbit vas deferens (i.e., a kappaspecific tissue);

Studies of the actions of a series of kappa and agonist/antagonist

analgesics on corticosterone release in the rat have clearly

demonstrated a kappa regulation of this neuroendocrine system.

The key characteristics of these actions (Iyengar et al. 1985) are

no cross tolerance with mu agonists;

tolerance in subchronic studies;

inactivity in hypophysectomized rats; and

no sigma receptor role involved in the actions of kappa

agonists.

104
These data argue for a potent kappa regulation of the HPA axis and

suggest an action at the level of the hypothalamus or pituitary

with regard to corticosterone release. In further studies of the

antagonism of these actions of kappa agonists by the narcotic

antagonists naloxone and WIN44,441, we observed that while the

actions of tifluadom and EKC are blocked by both of these antagonists, the actions of MR-2,034 and U50,488H were only reversed by

naloxone (table 1). WIN44,441 was inactive up to 5 mg/kg, a dose

that is efficient in blocking the actions of mu agonists in this

system. Similarly, WIN44,441 was active in blocking morphinedependent analgesia and increases in striatal dopamine metabolism

(Wood et al. 1984a).

TABLE 1. Summary of actions of kappa agonists a plasma

corticosterone, plasma TSH, DOPAC levels in the

olfactory tubercle, and cerebral arteries in vitro

Parameter MR-2,034 U50,488H Tifluadom EKC

Corticosterone

% control (mg/kg, IP)

Naloxone reversal

WIN44,441 reversal

TSH

% Control (mg/kg, IP)

Naloxone reversal

WIN44,441 reversal

DOPAC (Olf. Tub.)

Naloxone Reversa!

WIN44,441 Reversal

Cerebral Arteries*

800(4)

Y

N

25(16)

Y

N

149(4) 150(16) IA(16)

Y Y

N N

Cont

600(8) 600(2)

Y Y

N Y

26(8) 50(10)

Y Y

N Y

Cont NT

700(8)

Y

Y

20(16)

Y

NT

IA(16)

Relax

NOTES: Y = yes; N = no, up to 5 mg/kg IP; NT = not tested; IA = inactive;

Cont = contraction; Relax = relaxation.

*Altura et al. (1984).

TSH Release

Preliminary studies of kappa regulation of TSH release in the rat

(Iyengar and Wood 1985) have also delineated a stereospecific

kappa-dependent decrease in plasma TSH. As with corticosterone,

these actions express no cross-tolerance with morphine and a differential antagonism by narcotic antagonists (table 1).

105
DOPAC in Olfactory Tubercle

Previous studies have documented the regulation of the nigrostriatal and ventral tegmental-nucleus accumbens dopamine projections by mu and delta agonists, but not by kappa agonists (Wood

1983a). In fact, the mu-2 and delta antagonist actions of presently available kappa agonists have been demonstrated with regard

to these pathways (Wood 1983a; Wood 1984). In contrast, it

appears that the dopamine input to the olfactory tubercle of the

rat is regulated by mu, delta, and kappa opioid systems. The

actions of MR-2,034 and U50,488H were naloxone--but not WIN44,441-

-reversible, while tifluadom and EKC were inactive on this

dopaminergic pathway up to 16 mg/kg (table 1).

SUMMARY

It has become clear, in the area of opiate research, that there

are multiple opioid receptors and endogenous ligands. The functional roles of these opioid systems are being established as

these concepts evolve and as our analytical capabilities become

more sophisticated and thorough. When complete dose-response

studies are performed and a number of antagonists are examined, a

clearer definition of isoreceptor populations may also occur.

Such appears to be the case with kappa agonists, which may in fact

comprise two groups of compounds (table 1). This separation into

two groups has also been observed in the dog cerebral artery

(table 1) (Altura et al. 1984). Taken together, these data are

sufficient to propose the testable hypothesis that there are kappa

isoreceptor populations in the brain that probably subserve a vast

array of discrete and different regulatory functions.

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opiate agonists and antagonists: The 14- hydroxydihydromorphinoneazines. J Neurosci 2:572-576, 1982.

Itzhak, Y.; Hiller, J.M.; and Simon, E.J. Solubillzation and

characterization of mu, delta and kappa opioid binding sites

from guinea pig brain: Physical separation of kappa

receptors. Proc Natl Acad Sci USA 81:4217-4221, 1984.

Iyengar, S.; Kim, H.; and Wood, P.L. In vivo evidence for kappa

isoreceptors in rat: Neurochemical and neuroendocrine indices.

J Neurochem 44:S89, 1985.

Kamikawa, Y., and Shimo, Y. Pharmacological characterization of

the opioid receptor in the submucous plexus of the guinea-pig

oesophagus. Br J Pharmacol 78:693-699, 1983.

Leander, J.D. Further studies of kappa opioids on increased

urination. J Pharmacol Exp Ther 227:35;41, 1983.

Ling, G.S.F.; Spiegel, K.; Nishimura, S.L.; and Pasternak, G.W.

Dissociation of morphine's analgesic and respiratory depressant

actions. Eur J Pharmacol 86:487-488, 1983.

Oka, T.; Negishi, K.; Suda, M.; Matsumiya, T.; Inazu, T.; and

Ueki, M. Rabbit vas deferens: A specific bioassay for opioid

kappa receptor. Eur J Pharmacol 73:235-236, 1981.

Pasternak, G.W. Multiple opiate receptors: [

3

H]Ethylketocyclazotine receptor binding and ketocyclazocine analgesia. Proc Natl

Acad Sci USA 77:3691-3694, 1980.

Pasternak, G.W. High and low affinity opioid binding sites:

Relationship to mu and delta sites. Life Sci 31:1303-1306,

1982.

Sun, F.Y., and Zhang, A.Z. Dynorphin receptor in the blood

Dynorphin-A-(1-8) is contained-within vasopressin neurosecretory

vesicles in rat pituitary. Science 222:1137-1139, 1983.

Wood, P.L. Multiple opiate receptors: Support for unique mu,

delta, and kappa sites. Neuropharmacology 21:487-497, 1982.

Wood, P.L. Opioid receptor affinities of kappa agonists,

agonist/antagonists and antagonists in vitro and in vivo. Prog

Neuropsychopharmacol Biol Psychiatry 7:657-662, 1983a.

Wood, P.L. Opioid regulation of CNS dopaminergic pathways: A review of methodology, receptor types, regional variations and

species differences. Peptides (Fayetteville) 4:595-601, 1983b.

Wood, P.L. Kappa agonist analgesics: Evidence for mu-2 and delta

opioid receptor antagonism. Drug Dev Res 4:429-435, 1984.

Wood, P.L., and Charleson, S. High affinity [3

H]ethylketazocine

bindina: Evidence for specific kappa receptors.

Pilapil, C., and Wood, P.L. [3

H]SKF 10047 binding to rat brain

membranes: Evidence for kappa isoreceptors. Life Sci 33:263-

265, 1983.

Rackham, A.; Therriault, M.; and Wood, P.L. Substance P:

Evidence for spinal mediation of some behavioral effects.

Neuropharmacolbgy 20:753-755, 1981.

Su, T.P. Further demonstration of kappa opioid binding sites in

the brain: Evidence for heterogeneity. J Pharmacol Exp Ther

232:144-148, 1985.

vessel. Neuropeptides 5:595-598, 1985.

Whitnall, M.H.; Gasiner, H.; Cox, B.M.; and Molineaux, C.J.

Neuropharmacology 21:215-219, 1982.

107
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regulation of nigrostriatal neurons: In vivo evidence with

naloxonazine. Neurosci Lett 37:291-293, 1983.

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heat stability in vitro. Eur J Pharmacol 88:281-282, 1983.

Wood, P.L.; Stotland, M.; Richard, J.W.; and Rackham, A. Actions

of mu, kappa, sigma, delta and agonist/antagonist opiates on

striatal dopaminergic function. J Pharmacol Exp Ther 215:697-

703, 1980.

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Comparison of the mu agonist morphine and the kappa agonist

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kappa and agonist/antagonist analgesics. In: Boigegrain, R.,

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Groupe Sanofi, 1982. pp. 319-329.

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analgesics: In vivo assessment. J Pharmacol Exp Ther 226:545-

550, 1983.

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44,441: A stereospecific and long-acting narcotic antagonist.

Pharm Res 1:46-48, 1984a.

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and sigma narcotic receptors. In: Hanin, I., ed. Dynamics of

Neurotransmitter Function. New York: Raven Press, 1984b.

pp. 99-107.

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1982.

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AUTHORS

Paul L. Wood, Ph.D.

Smriti Iyengar, Ph.D.

Neuroscience Research

Pharmaceuticals Division

Ciba Geigy

Summit, NJ 07901

108
Multiple Receptor Types in

Opioid Discrimination

Stephen G. Holtzman

INTRODUCTION

Drugs can serve as interoceptive stimuli to govern behavioral responses in much the same way as exteroceptive environmental stimuli, such as auditory or visual cues, can govern behavior. In

most discrimination paradigms designed to study the interoceptive

stimulus properties of drugs, an animal is trained to emit one response after being injected with a reference drug and to emit a

different response after being injected with the drug vehicle.

Responses have included turning to the left or right in a T-maze

escape procedure or, more commonly, activating one of two manipulanda in order to receive appetitive reinforcement or to avoid or

escape from presentation of an aversive stimulus. A well-trained

subject will reliably emit the response appropriate for the drug

condition not only when injected with the reference drug used for

training but also when injected with a novel drug that has pharmacologic properties similar to those of the training drug. Thus,

stimulus control of behavior is generalized from the training drug

to the novel drug, presumably because the novel drug and the

training drug give rise to similar interoceptive stimuli. On the

other hand, subjects usually emit the response appropriate for the

vehicle condition when tested with a novel compound that is dissimilar pharmacologically from the training drug.

The exquisite pharmacologic specificity of this behavioral paradigm lends itself well to systematic investigation of mechanisms

of action of drugs from a variety of classes. There is perhaps no

better example of this than the application of drug discrimination

methodology to the study of opioid drugs. That opioid agonists

and antagonists interact with several distinct but stereochemically similar receptors was suggested by the differences in the

profiles of action of opioid drugs in a variety of in vivo and in

vitro assay systems (Martin et al. 1976; Gilbert and Martin 1976;

Lord et al. 1977). Results of drug discrimination studies have

been consistent with hypothesized multiple populations of opioid

receptors and have provided new insights into the actions of

109
opioid agonists and antagonists at the neuronal level (Herling and

Woods 1981; Holtzman 1982a; Holtzman 1983).

STIMULUS PROPERTIES OF OPIOIDS THAT INTERACT WITH THE MU RECEPTOR

The discriminative stimulus effects of several types of opioid

drugs appear to be mediated in whole or in part by the mu receptor. Included here are the traditional morphinelike agonists,

partial morphine agonists, mixed agonist-antagonists, and at least

some opioid peptides.

Morphinelike Agonists

The discriminative stimulus properties of morphine and related

drugs have been characterized the most extensively of any group of

opioids. Like many of their other effects, the discriminative

stimulus effects of these drugs are robust and reproducible under

a variety of experimental conditions. With minor exceptions,

relationships among drugs are consistent and independent of the

particular opioid used for training, species of experimental subject, or parameters of the discrimination paradigm (Holtzman 1983;

Holtzman 1985a). There is now overwhelming evidence that the discriminative stimulus effects of these drugs are a receptormediated phenomenon, mediated by the same type of receptor that

subserves other well-documented effects of the drugs, such as

analgesia, depression of respiration, and induction of physical

dependence (Holtzman 1982a; Holtzman 1983):

all drugs usually classified as morphinelike agonists on the

basis of their overall pharmacologic profile produce similar

discriminative stimulus effects;

they do so with an order of potency similar to that obtained in

other bioassays involving interactions with the mu receptor

(figure 1);

steric requirements for morphinelike discriminative stimulus

effects are the same as those for other mu receptor mediated

effects, with strong selectivity for the levorotatory optical

isomer of enantiomeric pairs;

discriminative stimulus effects are blocked completely and in a

surmountable manner by low doses of antagonists, such as naloxone and naltrexone, consistent with a competitive interaction

at the mu receptor. (In the rat, apparent pA2

T

values for

interactions between naltrexone and morphine Holtzman 1983)

and between naloxone and morphine (Shannon et al. 1984) are

7.69±0.07 and 7.85±O.36, respectively, which accord well with

the value of 7.95±0.19 for naloxone-morphine interactions in

the tail compression test for analgesia (Tallarida et al.

1979)).

110
FIGURE 1. Drugs classified as morphinelike agonists produced

discriminative stimulus effects comparative to those

of morphine, with the predicted order of potency

NOTE: Drugs were administered SC 30 minutes before the start of a session to rats

trained to discriminate between saline and 3.0 mg/kg of morphine In a twochoice discrete-trial avoidance paradigm. Each point is the mean number of

trials completed on the morphine-appropriate lever in a 20-trial session;

the remaining trials of the session were completed on the lever appropriate

for the saline condition. Four or more rats were tested at each dose. The

upper and lower broken horizontal lines indicate the minimum levels at

which the discrimination performance of the animals was maintained during

training sessions with 3.0 mg/kg of morphine and saline, respectively.

Data are adapted from Shannon and Holtzman (1976; 1977b) and reproduced

from Holtzman (1983). Copyright 1963, Academic Press, London.

Morphinelike discriminative stimulus effects are produced at central sites of action. The potency of morphine as a discriminative

stimulus is fully two orders of magnitude greater when the drug is

administered intracerebroventricularly (ICV) than when it is

administered by systemic routes (Shannon and Holtzman 1977a).

Furthermore, naltrexone methobromide, a quaternized analog of naltrexone that does not readily penetrate the blood-brain barrier,

blocks the discriminative stimulus effects of morphine when it is

administered ICV but not when it is administered systemically

(Valentino et al. 1981; Locke and Holtzman 1985).

Partial Morphine Agonists

The few compounds available that are classified as partial morphine agonists have discriminative stimulus effects that. for all

intents and purposes, are indistinguishable from those of the full

agonists considered in the preceding section. Buprenorphine

111
probably has been the most widely studied drug of this class.

Ranging from 10 to 100 times more potent than morphine, depending

upon species, buprenorphine is reliably generalized to morphine in

the rat (Shannon et al. 1984) and pigeon (France et al. 1984) and

to etorphine in the rhesus monkey (Young et al. 1984). It occasions responding appropriate for the vehicle condition in animals

trained to discriminate kappa or PCP/sigma agonists. The apparent

pA2 for the interaction between naloxone and buprenorphine in

morphine-trained rats is 7.48±O.16, similar to values obtained for

antagonist interactions with morphine. Thus, the discriminative

stimulus effects of buprenorphine, like most of the other actions

of the drug, are essentially exclusively morphinelike.

Mixed Agonist-Antagonists

In contrast to the full and partial morphine agonists, mixed

agonist-antagonists have been viewed as having affinity for, but

no intrinsic activity at, the mu receptor. These drugs have both

affinity for and intrinsic activity at other receptor types. As a

consequence, they should function as antagonists of drug effects

mediated at the mu receptor while manifesting agonist activity

that is mediated by one or more of the other postulated types of

opioid receptors (Martin 1983). For example, pentazocine has been

classified as a full agonist with low affinity for the kappa and

PCP/sigma receptors, cyclazocine as a full agonist with high

affinity for the kappa and PCP/sigma receptors, and nalorphine as

a partial agonist at each of these two receptor populations

(Martin et al. 1976). However, just as these drugs have many

actions in common with morphine in human subjects (Martin 1967),

they also share stimulus effects with morphine in animals.

Morphine-trained rats generalize completely to pentazocine

(Shannon and Holtzman 1976), and rats trained with pentazocine

generalize completely to morphine (Kuhn et al. 1976). a symmetry

in responding which provides compelling evidence of the commonality in the discriminative stimulus effects of the two drugs. In

rats trained with morphine, the apparent pA2 for the naltrexonepentazocine interaction is 7.52±0.12 (Holtzman 1983). This

finding suggests that the morphinelike discriminative stimulus

effects of pentazocine are mediated by the same type of receptor

with which morphine interacts: the mu opioid receptor.

Other mixed agonist-antagonists are generalized partially to

morphine in the rat. That is, they occasion responding on the

drug-appropriate lever that is significantly greater than that

produced by vehicle but significantly less than that produced by

the training drug. Included here are nalorphine, cyclazocine, and

levallorphan (Shannon and Holtzman 1976; Shannon and Holtzman

1977b; Overton and Batta 1979), to name but a few. Systematic

interaction experiments between morphine and nalorphine or

cyclazocine yield good in vivo approximations of theoretical logconcentration-response curves for the combination of a partial

agonist and a full agonist acting at the same receptor (figure 2).

Thus, in the rat, many mixed agonist-antagonists appear, in fact,

to be partial agonists at the mu receptor, not simply antagonists

112
at this site. Other lines of evidence derived from experiments in

rats trained with fentanyl are consistent with the foregoing

interpretation (Colpaert and Janssen 1984).

FIGURE 2. Nalorphine and cyclazocine appear to be partial

morphine agonists in the mt, whereas pentazocine

acts as a full agonist

NOTE: Stimulus generalization curves are shown for each agonist-antagonist tested

alone (morphine dose: 0 mg/kg) or in combination with 0.3, 1.0, or 3.0

mg/kg of morphine In rats (n=5) trained to discriminate between saline and

3.0 mg/kg of morphine. The unconnected points above M indicate the mean

number of trials completed on the morphine-appropriate choice lever In

sessions that followed the administration of 0 (saline), 0.3, 1.0, or 3.0

mg/kg of morphine alone. Other details are the same as in figure 1. The

family of curves for the combination of nalorphine and morphine and for

cyclazocine and morphine converge upon the corresponding curve for nalorphine or cyclazocine alone, consistent with a partial agonist and a full

agonist acting on the same receptor population (Arlens et al. 1964).

Reproduced from Holtzman (1983). Copyrlght 1983, Academic Press, London.

In contrast to the robustness and uniformity of the discriminative

stimulus effects of morphinelike agonists and partial agonists,

stimulus effects of mixed agonist-antagonists are critically

dependent upon factors such as the particular drug and dosage used

for training and upon species of the experimental subject

(Holtzman 1982a; Holtzman 1982b). Pentazocine, for example, is

generalized only partially to morphine in the pigeon (Herling et

al. 1980) and squirrel monkey (Schaefer and Holtzman 1981).

Butorphanol and nalmexone, agonist-antagonists which have many

pharmacologic characteristics in common with pentazocine in

animals and humans (Griffiths and Balster 1979), exhibit similar

species-dependent patterns of discriminative stimulus effects

which are illustrated in figure 3. Such interspecies differences

113
FIGURE 3. The morphinelike discriminative stimulus effects of

mixed agonist-antagonists are often species dependent

NOTE: Butorphanol, nalmexone, and pentazocine were generalized to morphine completely by rats (n=4 or 5) but only partially by squirrel monkeys (n=3 or

4). Each species was trained to discriminate between saline (S) and 3.0

mg/kg of morphine (M) In a 20-trial (rats) or 25-trial (monkeys) session.

Data are reproduced from Holtzmen (1985b). Copyright 1985, Elsevier

Scientific Publishers, Ireland Ltd.

may well reflect differences in the densities and ratios of the

several types of opioid receptors with which the agonist-antagonists interact. The rat brain is especially rich in mu receptors

relative to other types of opioid receptors (Maurer 1982). The

presence of "spare" mu receptors in the rat could permit some

drugs that are partial agonists at the mu receptor, e.g., pentazocine, to appear as full agonists in this species.

Opioid Peptides

Data on discriminative stimulus effects of opioid peptides are

sparse in comparison to the existing body of literature on the

opioid alkaloids. This situation is due, in large part, to the

technical difficulties inherent in maintaining healthy and behaviorally stable subjects during long-term experiments that require

repeated injections of compounds into the brain. Some of these

problems can be overcome by using opioid peptides that are metabolically stable so that they retain behavioral activity when

administered systemically. To date, all such peptides have been

found to have discriminative stimulus effects which are morphinelike, regardless of their relative selectivity for mu vs. delta

receptors in binding assays or in bioassays on isolated tissue

preparations. The metabolically stable analogs of methionine enkephalin, FK 33,824, which is selective for the mu receptor, and

metkephamid, which shows selectivity for the delta receptor, were

114
generalized to morphinelike agonists when administered systemically to rhesus monkeys (Young et al. 1983). The putative delta

receptor agonist D-ala , D-leu5

-enkephalin (DADL) was tested using

the ICV route of administration in separate groups of rats trained

to discriminate vehicle from either the mu receptor agonist fentanyl or the kappa receptor agonist ethylketocyclazocine. DADL was

generalized to fentanyl at a dose of approximately 1.0 μg, but

occasioned only vehicle-appropriate responding in the rats trained

with ethylketocyclazocine (Shearman and Herz 1982a).

Locke and Holtzman (unpublished observations) examined a series of

opioid peptides having relative selectivity either for the mu or

delta opioid receptor. The peptides were tested ICV in rats

trained to discriminate 3.0 mg/kg of morphine (SC) from saline.

The mu-selective peptides (DAGO, FK 33,824, and morphiceptin), the

delta-selective peptides (DADL and metkephamid), and ß-endorphin,

which interacts equally well with both receptor types, all produced dose-dependent morphinelike stimulus control of behavior

(table 1). These generalization data and the ease with which the

morphinelike stimulus effects of the peptides could be blocked by

naltrexone suggest that mu opioid receptors in the brain are

critical for the genesis of morphinelike discriminative stimulus

effects by these particular opioid peptides. It would be premature to dismiss entirely the possibility of a role for delta

TABLE 1. Opioid peptides which produce morphinelike discriminative stimulus effects when administered intracerebroventricularty (ICV) to rate trained to discriminate

between SC injections of 3.0 mg/kg of morphine and

salinea

a

Three or more doses of each peptide were tested In each of four to seven rats

(Locke and Holtzman, unpublished observations).

b

Morphine was tasted over a range of doses in each of 27 rats (Locke and Holtzman

1985).

115
receptors in the discriminative stimulus effects of opioid drugs.

Any such role, however, will remain obscure in the absence of

studies on subjects trained to discriminate delta-selective

agonists.

Clearly, studies of the discriminative stimulus effects of opioid

peptides have only scratched the surface. Many peptides remain to

be evaluated. The consistency of experimental outcomes across

animal species must also be determined.

STIMULUS PROPERTIES OF OPIOIDS THAT INTERACT WITH THE KAPPA

RECEPTOR

Opioids usually classified as kappa receptor agonists exhibit

discriminative stimulus properties that are clearly different from

those of drugs that interact primarily with the mu receptor. For

example, rats trained to discriminate between saline and ethylketocyclazocine respond almost exclusively on the salineappropriate lever when tested with graded doses of morphine or

fentanyl; the same outcome is obtained when rats trained with

fentanyl are tested with ethylketocyclazocine, bremazocine, or

other prototypic kappa drugs (Shearman and Herz 1982b). The

discriminative stimulus effects of kappa agonists are receptor

mediated in the rat (Herling and Shannon 1982) and rhesus monkey

(Hein et al. 1981; Young and Stephens 1984). satisfying the criteria of stereoselectivity, competitive blockade by antagonists,

and order of potency among a series of related compounds (figure

4). The apparent pA2 value for the interaction between naloxone

and ethylketocyclazocine in ethylketocyclazocine-trained rats is

6.77 (Herling and Shannon 1982), significantly below the corresponding values for antagonist interactions with morphine (vide

supra). Thus, the affinity of naloxone for the receptor mediating

the stimulus effects of ethylketocyclazocine is approximately 10

times lower than its affinity for the receptor mediating the stimulus effects of morphine. This difference accords well with results from assays of radioligand binding to putative mu and kappa

sites (Kosterlitz et al. 1981).

Full characterization of the stimulus control of behavior mediated

by kappa receptors has been hampered by the lack of selective

kappa antagonists as well as by the sometimes extreme differences

in stimulus properties of drugs from species to species. Ethylketocyclazocine provides a good example of the problem. In the

pigeon, the discriminative stimulus effects of this prototypic

kappa agonist are entirely morphinelike (Herling et al. 1980;

Herling and Woods 1981). The discriminative stimulus effects of

nalorphine, too, vary markedly across species. Nalorphine is a

partial mu agonist in the rat (figure 2) and also exhibits weak

kappa activity in this species (Shearman and Herz 1982c), consistent with its earlier classification by Martin and colleagues

(Martin et al. 1976; Gilbert and Martin 1976). In primates, however, nalorphine has no discriminative stimulus effects in common

with morphine (Schaefer and Holtzman 1981). Indeed, it has a

profile of stimulus properties entirely characteristic of a

116
FIGURE 4. Discriminative stimulus effects of the kappa receptor

agonist ethytketocyclazocine (EKC) are stereoselective for the levorotatory isomer and are

mimicked by cyctazocine (CYC) and SKF-10,047 (SKF),

with the predicted order of potency

NOTE: Rats (n=5) were trained to discriminate between saline and 0.3 mg/kg of

racemic EKC in a discrete-trial avoidance paradigm. Drugs were injected SC

15 minutes before the start of a 2O-trial session. The ordinate is average

percent of trials per session completed on the choice lever appropriate for

EKC. Data are reproduced from Herling and Shannon (1982).

kappa receptor agonist. Nalorphine and ethylketocyclazocine

generalize completely to one another in the rhesus monkey (Hein et

al. 1981; Tang and Code 1983). The nalorphine cue is also generalized completely to other kappa agonists such as bremazocine,

tifluadom, and U-50,488, with the predicted order of potency.

Finally, stimulus control of behavior is selective for the levorotatory optical isomer of cyclazocine, SKF-10,047, and U-50,488,

the member of the enantiomeric pair in which kappa activity

resides (Tang and Code 1983).

Findings of the type described for nalorphine and the greater

relative potency of kappa agonists in the squirrel and rhesus

monkeys as compared to the rat--often two orders of magnitude--

suggest that primates have a higher ratio of kappa to mu receptors

than does the rat. Kappa sites represent only a small percentage

of total opioid binding sites in rat brain (Maurer 1982). Perhaps

this explains why dynorphin, postulated to be the endogenous ligand of the kappa receptor (Chavkin et al. 1982), and several of

its biologically active fragments occasioned responding appropriate for the vehicle condition when tested ICV in rats trained

117
to discriminate between ethylketocyclazocine and saline (Shearman

et al. 1985). It will be of interest to determine the discriminative stimulus properties of kappa-selective opioid peptides in a

primate species.

STIMULUS PROPERTIES OF OPIOIDS THAT INTERACT WITH THE PCP/SIGHA

RECEPTOR

A sigma receptor was proposed as one of the components of a threereceptor model of opiate activity derived from a series of experiments in the chronic spinal dog bioassay. Martin et al. (1976)

suggested that the sigma receptor mediated the canine delirium

induced by certain opioids such as SKF-10,047 (N-allylnormetazocine, NANM) and cyclazocine. The fact that both of these drugs

are psychotomimetic in humans did not go unnoticed. Interest in

the sigma site was stimulated further by the finding that saturable binding of the dissociative anesthetic phencyclidine (PCP) to

brain preparations is displaced by nanomolar concentrations of

NANM and cyclazocine (Zukin and Zukin 1979; Zukin and Zukin 1981;

Quirion et al. 1981). Kappa receptor agonists are less potent in

this regard; morphine and related mu agonists are essentially

inactive, as are naloxone and naltrexone.

The discriminative stimulus effects of sigma opioids, like those

of mu receptor agonists, are relatively robust, uniform across

animal species, and independent of the drug used for training.

The results of numerous studies are consistent with the notion

that the stimulus effects of this group of drugs are mediated at a

neuronal site that has not traditionally been associated with

opiate activity, a site of action in common with PCP (table 2).

Rats trained with either cyclazocine (Teal and Holtzman 1980) or

NANM (Shannon 1983a) generalize completely to PCP and ketamine,

another dissociative anesthetic and an analog of PCP. Conversely,

rats (Holtzman 1980; Shannon 1981), squirrel monkeys (Holtzman

1982c), and pigeons (McMillan 1982) trained with PCP generalize

completely to cyclazocine and NANM but emit responses appropriate

for the vehicle condition when tested with a variety of mu or

kappa agonists (figure 5). Stimulus control of behavior by these

PCP/sigma agonists is essentially insensitive to blockade by even

high doses of naloxone or naltrexone. In rats trained in a threechoice paradigm to discriminate concurrently between PCP, saline,

and cyclazocine, the administration of a fixed dose of naltrexone

in combination with high, graded doses of cyclazocine resulted in

a dose-dependent increase in responses on the choice lever appropriate for PCP (White and Holtzman 1983). This outcome is compelling evidence that an opioid-antagonist-insensitive component of

action of cyclazocine is responsible for the PCP/sigma-like

discriminative stimulus effects of the drug.

In contrast to steric requirements for discriminative stimulus

effects mediated by mu or kappa receptors (vide supra), the

PCP/sigma site frequently favors the dextrorotatory conformation,

although not consistently so. (+)-NANM is slightly more potent

118
TABLE 2. Potency order of drugs for producing PCP- and d,l,-NANM

(SKF-10,047)-like discriminative effects in the rat

and for displacing binding of 3H-PCP from rat brain

membranes

Drug

Discriminative Effects

Training Drug: PCPa NANMb

(2.0-3.0 mg/kg) (3.0-5.0 mg/kg) BindingC

l-Cyclaroclne 1

PCP 2

Dexoxadrol 3

d-NANM 4

d,l-Metazocine 5

l-NANM 6

Dextrorphan 7

d-Cyclazocine 8

Ketamine 9

Levallorphan 10

1 3

2 2

3 1

4 4

8

_d

5 5

9 6

7 7

10 8

6

_d

a

From Holtzman (1980); Shannon (1982; 1983b).

b

From Shearman and Herz (1982d); Shannon (1983a).

c

From Mendelsohn et al. (1984).

d

Not tested.

NOTE: Spearman rank correlation coefficient:

r =0.81 for column I VS. II (p<O.O05)

r

s

=O.90 for column I vs. III (p<O.005)

r

s

s=0.68 for column II vs. III (p<O.005)

than its levorotatory counterpart in producing PCP-like discriminative stimulus effects in a number of species (Brady et al.

1982a). On the other hand, (-)-cyclazocine is several times more

potent than (+)-cyclazocine (Shannon 1982; McMillan 1982). The

order of potency of the enantiomers of NANM and cyclazocine for

producing PCP- and NANM-like discriminative effects in the rat

parallels their order of potency in displacing H-PCP binding from

membranes of rat brain cortex (table 2).

Some members of two other chemical families also have been found

to produce PCP/sigma-like discriminative stimulus effects: opiate

morphinans and nonopiate dioxolanes. Dextrorphan, the nonanalgesic enantiomer of the classical morphinelike agonist levorphanol

has a profile of discriminative stimulus properties entirely characteristic of a PCP/sigma agonist (table 2 and Herling et al.

1983). Among the dioxolanes, etoxadrol, an intravenous anesthetic

which produces psychotomimetic side effects in human subjects, and

dexoxadrol, a structural analog, share discriminative stimulus

effects with PCP, NANM, and dextrorphan in rats (Shannon 1982;

Shannon 1983a), pigeons (Herling et al. 1983), and monkeys (Brady

119
injections of satins and 2.0 mg/kg of PCP generalize

completely to ketamine and the opioids cyclazocine,

SKF-10,047, and dextrorphan, but respond almost

exclusively on the lever appropriate for the saline

condition when tested with a variety of other

opioids, some of which are illustrated here

FIGURE 5. Rats (n=5 or mope) trained to discriminate between IP

NOTE: Data are reproduced from Holtzman (1980). Copyright 1980, American Society

for Pharmcology and Experimental Therapeutics.

et al. 1982b). Levoxadrol, the levorotatory isomer of dexoxadrol,

is apparently devoid of PCP/sigma activity.

Cone et al. (1984) have proposed an elegant model of a receptor

complex composed of several subsites with differing steric

requirements. Separate subsites can bind PCP and its analogs,

psychotomimetic opioids, and dioxolane derivatives. An important

implication of this model is that the opioid structure is probably

coincidental to the PCP-like stimulus properties of these drugs.

Critical conformational features shared with PCP are most likely

the principal determinants of the PCP/sigma activity of the

opioids.

CONCLUSIONS

The discriminative stimulus properties of opioid drugs are

diverse, consistent with concepts that multiple populations of

receptors subserve the actions of these drugs. With the exception

120
of peptides, for which sufficient data are lacking, it is possible

to partition opioids into three broad categories on the basis of

their patterns of stimulus generalization to the various prototypic reference drugs used for training. Such categorization predicts sensitivity of discriminative stimulus effects to antagonism

by naloxone or naltrexone and appears to reflect the sites of

action of the drugs at the neuronal level (table 3). It should

also be apparent from table 2 that the discriminative stimulus

effects of opioids have significant parallels with other important

actions of the drugs, for example, reinforcement efficacy in primate models of drug self-administration (Woolverton and Schuster

1983). Opioids with morphinelike discriminative stimulus effects

are almost invariably reinforcing and are reliably self-administered. Opioids with discriminative stimulus effects in common

with ethylketocyclazocine and nalorphine are rarely self-administered (Woods et al. 1982; Young et al. 1984). Opioids that

engender PCP/sigma-like discriminative stimulus effects seem to

fall between those two extremes (Slifer and Balster 1983).

TABLE 3. Characteristics of opioids classified on the basis of

their discriminative stimulus propertiesa

Complete Generalization to:

Morphine Ethylketocyclazocineb SKF-10,047 (NANM)

Fentanyl Nalorphinec

PCP

Proposed site of

action (receptor)

mu kappa PCP/sigma

Sensitivity to naloxone hlgh moderete low or none

or naltrexone (<0.1 mg/kg) (>O.1 mg/kg)

Stereoselectivity yes (1) yes (1) inconsistent

or (d)

Consistency across high low moderate-high

animal species

Reinforcement efficacy

(monkey)

high low moderate

Subjective effects

(human)

“euphoria" "dysphoria" (?) psychotomimetic

Human abuse potential high none (?) low-moderate (?)

Other drug examples “classical” ketocyclazocine cyclazocine

morphinelike bremazocine dextrorphan

opiates U-50,488

a

Modified from Holtzman (1985a; 1985b).

b

In rats or primates.

c

In primates.

Early suggestions of a correspondence between the discriminative

stimulus effects of morphinelike agonists in animals and the

121
subjective effects of these drugs in humans also appear applicable

to the discriminative stimulus and subjective effect of PCP/sigma

agonists (Holtzman 1982d). The nature of the subjective effects

produced by kappa agonists has not been fully determined. However, evidence is emerging that kappa receptors may subserve

antagonist-sensitive dysphoric and perhaps even psychotomimetic

symptomology in humans (Shannon 1982; Shannon 1983a; Tang and Code

1983). If the relationships between discriminative stimulus, reinforcing, and subjective effects suggested in table 3 are valid,

the discriminative stimulus properties of opioids should afford

important insights into components of drug action at the neuronal

level that are directly relevant to abuse potential.

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ACKNOWLEDGMENTS

Research in the author's laboratory was supported in part by

USPHS grants DA00541 and DA02208 and by Research Scientist Award

K05 DA00008.

AUTHOR

Stephen G. Holtzman, Ph.D.

Professor

Department of Pharmacology

Emory University School of Medicine

Atlanta, GA 30322

126
Neurobiological Substrates of

Drug Self-Administration

James E. Smith and Steven I. Dworkin

INTRODUCTION

Behavioral pharmacologists have been involved in investigations of

the behavioral parameters influencing drug action for over three

decades, with the basic assumption that these effects result from

actions on brain neuronal systems. Today, it is generally accepted that the behavioral effects of many drugs result from alterations in neuronal activity through direct or indirect interaction

of these substances with specific receptors. Neuroscientists are

currently attempting to identify the loci of these actions and to

characterize the neuronal systems that mediate these processes.

This chapter will focus on the status of knowledge of the neuronal

substrates involved in drug self-administration.

DRUG SELF-ADMINISTRATION: A COMPLEX BEHAVIORAL PHENOMENON

Drug self-administration is a complex behavioral phenomenon that

likely encompasses many of the behavioral actions of the drug,

including, but not limited to, the reinforcing effects. It is

generally assumed that biological correlates of self-administration or experimental manipulations affecting drug intake primarily

represent the reinforcing properties of the substance. However,

the delivery of a drug, whether self-administered or not, results

in a number of pharmacological actions. For example, opiates have

multiple effects on the central nervous system that result in

alterations in sensitivity to environmental stimuli, including decreased pain perception, footshock reactivity, separation distress

and social bonding, and increased food and water consumption (Reid

and Siviy 1983). Opiates also have stimulus- and/or responsemodulating properties. The presence of the drug results in the

initiation of neuronal events that are distinguished from those

produced by drugs of different classes and sometimes even from

others of the same class (Overton 1983). The presence of the drug

may also initiate neuronal events that alter response rates, even

drug-maintained responding (Goldberg and Kelleher 1977). Opiate

self-administration most likely includes some of these CNS

127
effects. That the neuronal substrates reflect primarily the reinforcing properties is a limited assumption.

NEUROBIOLOGICAL MECHANISMS INVOLVED

Neurobiological substrates are defined here as the neuronal processes responsible for a specific action or function. The neurobiological substrates of opiate self-administration include all

the neuronal events resulting in and from opiate self-administration. A complete understanding of these events requires knowledge

of the neuronal circuits and pathways , activation or inhibition of

which are necessary to these processes (figure 1). The location

of the cell bodies and projections of each component neuron in

such circuits is necessary, as is knowledge of the nature and

localization of the necessary receptors. Knowledge of the influence of other inputs to each neuron participating in these circuits is also necessary for a complete understanding of the

neuronal events mediating these processes. Needless to say,

FIGURE 1. Neurobiological substrates involve: (a) The identification of each neuron that participates in circuits

mediating a behavioral effect; (b) identification of

the nature and location of the receptors involved;

(c) elucidation of the effects of other inputs to

each participating neuron

128
the neurobiological substrates of self-administration, or any

other behaviors, are yet to be elucidated.

IDENTIFICATION OF NEUROBIOLOGICAL SUBSTRATES

As mentioned above, the neuronal systems affected by drug selfadministration are likely to include many of the systems that

control the behavioral actions of the drug. Neuronal circuits

involved in the reinforcing effects, stimulus properties, response

modulating effects, and other behavioral effects would most likely

also be activated. This complicates the identification of neurobiological substrates exclusive to the reinforcing properties.

Additional complications stem from the likelihood that there is

some utilization of common neurons in the circuits that mediate

any one behavioral effect (figure 2). For example, the circuits

involved in the reinforcing properties of an opiate may include

some neurons that also participate in circuits mediating the

stimulus properties, response-rate altering, or consummatory

behavior-modulating properties. Some participating neurons could

also function in additional neuronal circuits involved in other

behavioral effects. Moreover, unique subsets of neurons may mediate the same behavioral effect independent of the drug class.

This behavior-specific function probably operates for reinforcing

properties, since opiates and stimulants have both been shown to

alter dopamine transmission. Thus, the neuronal circuits mediating the reinforcing effects of opiates may include some of the

neurons that mediate the reinforcing properties of stimulants

and/or benzodiazepines (figure 3). But some unique systems must

also participate. Stereospecific binding sites for subclasses of

FIGURE 2. The neuronal circuits involved in a particular behavioral drug effect may share some common neurons with

circuits mediating other behavioral effects of the

drug

129
FIGURE 3. The neuronal circuits involved in a behavioral effect

of one drug class may share common neurons with

circuits involved in the same behavioral effect of

other drug classes

opiates (Weber et al. 1982; Khachaturian et al. 1983), cocaine

(Reith et al. 1980), amphetamine (Hauger et al. 1984), benzodiazepines (Braestraup and Squires 1977), and other CNS active

drugs have been identified that are heterogenously distributed

throughout the brain. If a common group of circuits mediates a

specific behavioral drug effect, then the sites where a particular

drug class initiates these activities may be separate and distinct

(figure 4).

Identification of neuronal circuits involved in a specific behavioral drug effect is a difficult challenge. The degree to which a

manipulation can similarly influence a behavioral effect exerted

by several different drug classes points to commonalities in

neurobiological mechanisms. Furthermore, these effects must be

separated from manipulations that alter an animal's ability to

respond (i.e., nonspecific motor effects). Appropriate control

groups and experimental procedures are necessary to isolate and

characterize the neuronal systems initiating such effects.

INTRAVENOUS SELF-AOMINISTRATION

Initial studies of the neuronal systems mediating intravenous

drug self-administration utilized neurotransmitter receptorblocking agents. The effects of such treatments on drug intake

served as a rough estimate of the potential participation of that

130
FIGURE 4. Unique receptor-mediated sites for the initiation of a

behavioral effect may exist for a drug that activates

common circuits mediating the some behavioral effect

for other drugs

population of neurons in the processes responsible for self-administration. Pharmacological blockade experiments demonstrated

cholinergic, dopaminergic, and noradrenergic neurons to be important to intravenous morphine-maintained responding, since atropine

(Davis and Smith 1975; Glick and Cox 1975), haloperidol (Glick and

Cox 1975; Smith and Davis 1973)) and dopamine-ß-hydroxylase inhibitors (Davis et al. 1975) all decreased intake. In contrast,

haloperidol (DeWit and Wise 1977) and atropine (Wilson and

Schuster 1973) decreased intravenous cocaine self-administration,

while propranolol (Goldberg and Gonzalez 1976) increased it.

Opiate-receptor antagonists also attenuated intravenous opiate

self-administration, but did not affect cocaine intake, while

doses of alpha-flupenthixol (a dopaminergic-receptor antagonist)

that decreased cocaine intake did not affect heroin self-administration (Ettenberg et al. 1982). These kinds of experiments have

generally suggested the involvement of cholinergic, dopaminergic,

and noradrenergic neuronal systems in the neuronal processes maintaining intravenous opiate and stimulant self-administration.

However, such systemic blockade experiments are extremely limited

in localizing central mechanisms. A homogeneity of function of

heterogeneous neuronal systems is assumed. Furthermore, the total

effect is often concluded to be a specific result of alterations

in reinforcing efficacy. These weak assumptions are intrinsic to

this relatively nonspecific pharmacological manipulation.

Intracranial Lesions--Electrolytic

Lesion methodologies have also been used to identify brain regions

or pathways that participate in a behavioral process. Electrolytic lesions of the brains of rats intravenously self-adminis131
tering morphine have been found to modulate drug intake. Lesions

of the anterior cingulate cortex (Trafton and Marques 1971), frontal cortex, hippocampus, and medial raphe nucleus (Glick and Cox

1977) shifted the dose intake curve to the right, so that higher

doses were necessary to maintain prelesion rates of intake;

lesions of the caudate nucleus (Glick et al. 1975) and substantia

nigra (Glick and Cox 1977) shifted the dose intake curve to the

left, so that lower doses maintained prelesion self-administration

rates. Lesions of the dorsal raphe nucleus, locus coeruleus

(Glick and Cox 1977), amygdala (Glick and Ross 1983), medial

thalamus, nucleus accumbens, posterior cortex, and olfactory

tubercle (Glick and Cox 1978) had no effect on intake. Although

electrolytic lesions are site-specific, they do not permit conclusions concerning the nature of the neuronal systems involved.

Such lesions are also nonspecific and can destroy both excitatory

and inhibitory systems in the same region, with no net change in

drug intake occurring. This may result in the erroneous conclusion that a specific brain region is not involved in the processes

responsible for drug-maintained responding.

Intracranial Lesions--Neurotoxins

Lesion techniques that involve the intracranial administration of

neurotoxins into discrete brain regions can produce both site- and

neuronal system-specific information. The intracranial administration of 6-hydroxydopamine (6-OHDA), after pretreatment with a

noradrenergic reuptake inhibitor (i.e., desmethylimipramine), has

been used to selectively destroy dopaminergic innervations of discrete brain regions. Such lesions of the nucleus accumbens decreased the intake of intravenous amphetamine (Lyness et al. 1979)

and cocaine (Roberts et al. 1977; Roberts et al. 1980), but did

not change heroin (Pettit et al. 1984), and produced only moderate

increases in morphine intake (Smith et al., in press). Lesions

(produced by 6-OHDA) of the ventral tegmental system, where the

cell bodies of these mesolimbic-mesocortical dopaminergic neurons

are localized, decreased intravenous cocaine intake (Roberts and

Koob 1982).

These data collectively suggest a role for dopamine neurons in

drug self-administration. However, not all mesolimbic or mesocortical dopamine projections are involved, since 6-OHDA lesions of

the medial prefrontal cortex did not affect intravenous cocaine

intake (Szostak et al. 1984). Other, nondopaminergic, neuronal

systems may also be important. Kainic acid lesions of the nucleus

accumbens, which primarily destroy interneurons and projections

from the site of injection, also decreased intravenous cocaine

self-administration (Zito et al. 1983), suggesting the involvement

of elements postsynaptic to dopamine innervations. It was found

that 5,7-dihydroxytryptamine lesions of serotonergic innervations

of the nucleus accumbens did not affect intravenous amphetamine

intake (Lyness et al. 1980), but had a profound effect on intravenous morphine self-administration (figure 5). Cholinergic

innervations or projections from the nucleus accumbens may also be

involved, since lesions with the neurotoxin AF64A shifted the

132
FIGURE 5. The effects of bilateral 5,7-dihydroxytryptamine (5,7-

DHT) lesions of the nucleus accumbens on intravenous

morphine self-administration

NOTE: The points are means and the error measures standard deviations for double

determinations of 24-hour exposure probes of vehicle, 2.5, 5.0, or 40.0

mg/kg doses. Baseline drug exposure was 24-hour access to 1O mg/kg

morphine. The 10 mg/kg points represent the mean of the baseline Intake on

the days before dose probe manipulations. The lesion of serotonergic

Innervations with 5,7-DHT (6 μg In 0.5 μl in each side after desmethylimipramine pretreatment) resulted in significant increases in morphine intake that was indistinguishable from vehicle intake. The point and dotted

error line at saline represents intake with 7 continuous days of saline

exposure postlesion and postdose intake analysis.

dose-intake curve for morphine to the right (figure 6). These

data implicate multiple neuronal systems in the maintenance of

drug self-administration and suggest some potential specificity of

function with respect to drug class, since some lesions can affect

the intake of one drug class but not that of another.

Intracranial Administration of Receptor Antagonists

Intracranial administration of receptor-blocking agents can potentially provide similar information on brain site and the nature of

133
FIGURE 6. The effects of bilateral AF64A lesions of the nucleus

accumbens (2.3 μg each side in 0.5 μl) on intravenous

morphine self-administration

NOTE: Each point represents the mean and standard deviation of morphine intake

for double determinations of 24-hour dose probes of vehicle, 2.5, 5.0, and

40.0 mg/kg/infusion, before and after such lesion In one animal. The

lesion resulted in a shift to the right in the dose Intake curve, so that

higher doses were necessary postlesion to maintain prelesion rates of selfadministration.

participating neuronal systems. Infusion of the hydrophobic

opiate-receptor antagonist, diallyl-nor-morphinium bromide, into

the ventral tegmental area resulted in a dose-dependent increase

in intravenous heroin self-administration, while similar infusions

into the nucleus accumbens had no effect (Britt and Wise 1983).

However, infusions of another slowly diffusing opiate receptor

antagonist (methyl naloxonium chloride) into the nucleus accumbens

resulted in dose-dependent increases in intravenous heroin intake,

while infusions into the ventral tegmental area had no effect

(Vaccarino et al. 1984). Although these data are contradictory,

they do suggest a major role for opiate receptors in a particular

brain area and the noninvolvement of receptors in another. The

discrepancy in the findings from the two studies may be related to

the different opiate receptor subtypes found in these two areas.

This dissimilarity accounted for the disparity observed in opiate

intracranial self-administration in these two brain regions (see

below).

134
Neurochemical Measureaent

Neurochemical approaches have also been used to identify neuronal

systems involved in drug self-administration. Concurrent measurement of the turnover rates of seven neurotransmitters (dopamine,

norepinephrine, serotonin, aspartate, glutamate, glycine, and

gamma-aminobutyric acid) in small brain regions of rats intravenously self-administering morphine, and in yoked morphine- and

yoked vehicle-infused littermates has suggested the involvement of

two neuronal circuits in the processes maintaining this behavior

(Smith et al. 1982) (figure 7). Turnover rates of acetylcholine

and muscarinic cholinergic binding were assessed in brain regions

of separate groups of similarly treated littermates, demonstrating

the involvement of specific cholinergic innervations (Smith et al.

1984a; Smith et al. 1984b). Integrating these neurochemical data,

pharmacological blockade data, and data from lesion studies with

current knowledge of neurotransmitter-specific pathways suggested

two neuronal circuits to be involved in opiate self-administration. A frontal cortex/striatum/frontal cortex circuit (figure 8)

and a nucleus accumbens/amygdala/entorhinal cortex/hippocampus/

nucleus accumbens circuit (figure 9) were identified. Cholinergic

innervations of the frontal cortex from the globus pallidus, from

FIGURE 7. Circuits proposed to mediate intravenous morphine

self-administration

NOTE: The circuits include a hippocampal formation/nucleus accumbens/amygdalold

complex/entorhinal cortex/hippocampal formation circuit and a frontal

cortex/striatum/frontaI cortex circuit. Activity in these circuits may be

modulated by the substantia nigra, ventral tegmental area, locus coeruleus,

and raphe nuclei. Feedback pathways to these brainstem nuclei may In turn

modulate activity in these centers.

SOURCE: Smith et al. 1984a, Copyright 1984, Ankho International, Inc.

135
FIGURE 8. Frontal cortex circuit proposed to partially mediate

opiate reinforcement

SOURCE: Smith et al. 1984a, Copyright 1984. Ankho International, Inc.

FIGURE 9. Hippocampal circuit proposed to partially mediate

opiate reinforcement

SOURCE: Smith et al. 1984a, Copyright 1984, Ankho International, Inc.

136
the preoptic region to the amygdala, from the amygdala to the

entorhinal cortex, and from the septum to the hippocampus were

implicated, as well as glutamatergic innervations of the striatum

from the frontal cortex, to the nucleus accumbens from the hippocampus, and to the preoptic region from the nucleus accumbens.

Aspartergic interneurons or innervations of the hippocampus,

nucleus accumbens, and brainstem also appear to be involved, as do

intrinsic GABA neurons in the frontal cortex, hippocampus, nucleus

accumbens, and striatum. Apparently, GABA feedback pathways to

the brainstem from the striatum and nucleus accumbens are also

involved. Dopamine innervations of the striatum, frontal cortex,

nucleus accumbens, and septum also appear to participate, as do

serotonergic innervations of the nucleus accumbens and noradrenergic innervations of the nucleus accumbens and septum.

These circuits may be generalized reinforcement pathways, since

autoradiographic analysis of the accumulation of radioactive 2-

deoxyglucose in animals intracranially electrically self-stimulating one of two different brain regions (ventral tegmental area or

substantia nigra) have identified the involvement of many of the

same areas as these neurotransmitter turnover rate studies

(Esposito et al. 1984; Porrino et al. 1984). If such general

reinforcement systems exist, then areas and receptors that initiate activity in these systems may also exist. Intracranial

self-administration methodologies can be used to identify and

characterize such initiation systems.

INTRACRANIAL SELF-ADMINISTRATION

If a substance is delivered into a brain region where its receptors are localized and this delivery results in the initiation of

reinforcing neuronal activity, this substance would be expected to

be self-administered into this region. However, the injection of

a substance directly into the brain could modify neuronal activity

nonspecifically. Chemicals can initiate neuronal activity in a

nonselective manner similar to that produced by electrical stimulation. Thus, self-administration of a drug could actually be

chemically elicited stimulation of little behavioral relevance.

Such misinterpretations can be avoided by using physiological

concentrations and demonstrating that the self-administration is

receptor mediated. Selective receptor antagonists should be used

to attenuate intake as has been done with intravenous selfadministration (Woolverton and Goldberg 1984). Furthermore, an

intracranially infused substance could elicit a motor reflex

nonspecifically or increase motor activity (Costall et al. 1982);

either could result in elicitation of additional responses that

result in more infusions, thus continuing the process. These

drug-elicited responses are not emitted operant responses whose

frequency has increased because of reinforcing consequences. Such

behavior, therefore, should not be interpreted as self-administration. Schedules of drug presentation can be used to prevent such

elicited behavior from resulting in repeated drug infusion.

137
Some brain regions support intracranial self-administration of

opioids and stimulants while others do not. Morphine is selfadministered into the ventral tegmental area at 150 pmole doses,

but is not self-administered into the nucleus accumbens, caudate

nucleus-putamen, periventricular gray substance, or lateral hypothalamus (Bozarth and Wise 1981). These data resulted in the

hypothesis that the site of opiate reinforcement after systemic

injection is the cell bodies of the mesolimbic mesocortical dopamine system in the ventral tegmental area. But, self-administration of similar doses also occurs in the septum, preoptic region,

anterior hypothalamus, and lateral hypothalamus (Stein and Olds

1977), and much information exists concerning the heterogeneous

distribution of opiate receptors and the relative affinity of

various agonists for them. The nucleus accumbens contains primarily delta opiate receptors, and the ventral tegmental area,

primarily mu opiate receptors (Goodman et al. 1980). It is not

surprising that a dose of a mu agonist (morphine) is self-administered into a brain region where these receptors predominate and

not into another brain region where delta receptors predominate.

Morphine has a lower affinity for the delta receptor. Accordingly, higher doses are necessary to produce an equivalent degree of

delta receptor binding. Higher doses of morphine have been found

to be self-administered into the nucleus accumbens (Olds 1982), as

are higher doses of the delta opiate agonist, met-enkephalin

(Goeders et al. 1984). Thus, opioids will initiate reinforcing

neuronal activity in brain regions other than the ventral tegmental area. Intracranial self-administration should be expected to

be dose related, as is intravenous and oral self-administration.

If dose-intake curves were routinely generated, then such erroneous conclusions could often be avoided. Self-administration of

methionine enkephalin into the nucleus accumbens is dose related,

with maximal intake at 500 pmole (figure 10). This self-administration can also be placed on intermittent schedules, with the

animals increasing response output to maintain consistent interinfusion intervals (figure 11). The self-administration is

probably emitted reinforced behavior, not elicited responding,

since the animals will respond on an active lever but not on an

inactive lever, and will change their responding appropriately

when the contingency is switched between the levers (figures 11

and 12). This self-administration is also receptor mediated,

since naloxone will attenuate intake to vehicle levels (figure

13). In summary, opioids appear to initiate neuronal activity in

specific brain regions where appropriate opioid receptors are

localized and such neuronal activation involves general reinforcement circuits.

Stimulants are also intracranially self-administered into discrete brain regions. Amphetamine is self-administered into the

nucleus accumbens at 5 nmole doses (Hoebel et al. 1983) and cocaine into the medial prefrontal cortex at 50 to 100 pmole doses

(Goeders and Smith 1983). Neither the nucleus accumbens nor the

ventral tegmental area support cocaine self-administration at a

range of doses (25 pmole to 5 nmole) (Goeders and Smith 1983),

138
FIGURE 10. Dose intake relationship for the intracranial selfadministration of vehicle and methionine enkephalin

into the nucleus accumbens on a fixed ratio 1

schedule of reinforcement

NOTE: Points are means and the error measures standard deviations for double

determinations In seven rats.

SOURCE: Goeders et al. 1984, Copyright 1984, Ankho International, Inc.

supporting the existence of specific sites in the brain for initiation of reinforcing neuronal activity. The self-administration

of cocaine into the medial prefrontal cortex appears to be mediated by a direct action of cocaine on presynaptic dopamine nerve

endings, since 6-OHDA lesions of this region will attenuate drug

intake (Goeders and Smith, in press). Postsynaptic D2 dopaminergic receptors are also involved since sulpiride will attenuate

intake in intact animals (Goeders and Smith 1983), and dopamine,

when substituted for cocaine, will initiate self-administration

after 6-OHDA lesions (Goeders and Smith, in press).

If neuronal circuits mediate reinforcement processes, then it

should be possible to initiate this activity with exogenous administration of endogenous substances that may be released by neurons

participating in these processes. Rats have been demonstrated to

self-administer neurohumors directly into brain regions where they

139
FIGURE 11. Patterns of self-administration of 500 pmole doses of

methionine enkephalin into the nucleus accumbens for

one animal exposed to four experimental conditions

NOTE: The conditions were (A) a fixed ratio 1 schedule; (B) a fixed ratio 5

schedule; (C) and (D) active and inactive lever presses during a two-lever

discrimination experiment; (C) infusions from responding on the active

lever on a fixed ratio 1 schedule; (D) nonreinforced responses on the

inactive lever; and (E) infusions of vehicle (artificial CSF) on a fixed

ratio 1 schedule.

SOURCE: Goeders et a I. 1984, Copyright 1984, Ankho International, Inc.

FIGURE 12. Percent responding maintained by intracranial infusions of 500 pmole of methionine enkephalin into the

nucleus accwnbens in a two-lever choice procedure on

a fixed ratio 1 schedule

SOURCE: Goeders et al. 1984, Copyright 1984, Ankho International, Inc.

140
FIGURE 13. Effects of naloxone on methionine enkephalin selfadministration into the nucleus accumbens presented

as the percent responding maintained by 500 pmole of

methionine enkephalin (100 percent)

NOTE: Including equimolar or twice equimolar concentrations of naloxone resulted

in a transient increase in intake, while tripling the molar ratio of naloxone to methionine enkephalin resulted In extinction. Points are means and

error measures standard deviations for double determinations in two

animals.

SOURCE: Goeders et al. 1984, Copyright 1984, Ankho InternatIonal, Inc.

are released endogenously. Methionine enkephalin is self-administered into the nucleus accumbens (Goeders et al. 1984) as are

dopamine (Dworkin et al., in press) and cholecystokinin (Hoebel

and Aulisi 1984), while neurotensin is self-administered into the

ventral tegmental area (Glimcher et al. 1983). These substances

are released by neurons in these regions, suggesting that endogenous release may occur when reinforcement circuits are activated.

CONCLUSIONS

Specific neuronal pathways and circuits likely mediate reinforcement processes, as demonstrated by lesion studies and neurotransmitter turnover rate and receptor binding studies in intravenous

drug self-administering rats. Multiple neurohumors are likely involved in these circuits and in the mediation of activity therein.

It is likely that specific receptors in discrete brain regions are

141
the sites of initiation of reinforcing neuronal activity, as demonstrated by the intracranial self-administration studies. Cocaine will initiate such reinforcing neuronal activity at specific

brain regions but not at others. Activation of these initiation

sites likely results in neuronal activity in reinforcement circuits that may be involved in general reinforcement processes, as

demonstrated by the autoradiographic distribution of 2-deoxyglucose in animals intracranially self-stimulating. Investigations in progress to identify neurohumors and brain regions that

initiate reinforcing neuronal activity should lead to a better

understanding of the neurobiological substrates of drug selfadministration. Furthermore, similar techniques can be employed

to investigate the neurobiological mechanisms involved in other

behavioral drug effects.

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ACKNOWLEDGMENTS

This research was supported in part by USPHS grants DA-03681(SID),

DA-01999, DA-03628, and DA-03832 (JES).

AUTHORS

James E. Smith, Ph.D.

Professor

Department of Psychiatry

Psychiatry Research Unit

University of Louisiana State

School of Medicine in Shreveport

P.O. Box 33932

Shreveport, LA 71130

Steven I. Dworkin, Ph.D.

Instructor

Department of Psychiatry

Psychiatry Research Unit

University of Louisiana State

School of Medicine in Shreveport

P.O. Box 33932

Shreveport, LA 71130

145
Neurochemical Substrates for

Opiate Reinforcement

George F. Koob, Franco J. Vaccarino, Marianne Amalric,

and Floyd E. Bloom

INTRODUCTION

Drugs such as the psychomotor stimulants and opiates act like

other natural reinforcers that increase the probability of a

response: they strengthen and maintain operant behavior, e.g.,

drug self-administration (Pickens and Harris 1968; Woods and

Schuster 1968; Deneau et al. 1969; Thompson and Pickens 1970;

Yokel and Pickens 1973; Schuster and Thompson 1969). The repeated

use of opiate drugs in man is thought to be based both on the

pleasurable effects of the drugs and on their effects in terminating the pattern of opiate withdrawal. Although there is some

argument over the relative importance of these two factors, tolerance and physical dependence have long been held to be major motivators for continued administration of opiates (Lindesmith 1968).

Others have argued that an essential component of opiate abuse is

the direct reinforcing effect of the drug, or its "euphorigenic"

effect (Kornetsky and Wheeling 1982). Indeed, humans will repetitively use opiates in the absence of physical dependence (Zinberg

et al. 1978; Harding and Zinberg 1983). Rats will also readily

self-administer heroin without prior physical dependence and can

maintain indefinitely this self-administration with limited daily

access to heroin, without the development of obvious physical

dependence (Koob et al. 1984). This "euphorigenic" effect of opiates has been linked to a direct action on brain reinforcement

systems (Marcus and Kornetsky 1974; Kornetsky et al. 1979; Wise

1980). Indeed, the neuropharmacological advances that allow for

relatively specific inactivation of specific neurochemical systems

make it feasible to examine the neurobiological mechanisms for

these reinforcing effects. Such studies not only provide information on the basic mechanisms of action for the behavioral effects

of these drugs, but also may provide insight into the neurobiological organization of reinforcement processes themselves.

In stimulant and opiate self-administration, animals maintain a

relatively stable level of drug intake over time, with very

regular interinjection intervals, particularly with short daily

sessions (approximately 3 hours) (Ettenberg et al. 1982; Koob et

146
al. 1984). In response to changes in injection dose, animals

typically show an inverse relationship between dose and number of

injections per session; i.e., low doses produce a higher number of

self-injections than do higher doses (figure 1). More important

for the present context is the nature of the change in behavior

following treatment with a pharmacological antagonist. Treatment

with the opiate receptor antagonist produces an increase in the

number of self-injections of morphine (Goldberg et al. 1971; Weeks

and Collins 1976; Ettenberg et al. 1982). This increase is generally considered to reflect a competitive functional interaction;

the rat presumably increases its drug self-administration to compensate for the decreased effectiveness, in the presence of partial receptor occupancy by the antagonist, of the opiate as a

reinforcer. Consequently, an increase in self-administration resulting from administration of an opiate antagonist is qualitatively similar to the effects of decreasing the dose of drug per

injection. A hypothetical description of this relationship is

shown in figure 2.

FIGURE 1. Dose-response relationship for heroin selfadministration in rats

NOTE: Rats were allowed dally 3-hour access to heroin (0.06 mg/kg/injection) on a

continuous reinforcement schedule. After stable baseline responding

(± 20 percent mean for 3 days) was established, rats were subjected to a

doubling of the dose. followed by a return to baseline. followed by a

halving of the dose on 3 successive test days. Results are expressed as

mean ± S.E.H., n=4.

147
FIGURE 2. Hypothetical model of the effects of a pharmacological

antagonist on the dose-response function with selfadministration

NOTE: Note the shift of the dose-response function to the right. With this

model, a decrease in reinforcement value Is reflected in an increase in the

number of self-administered drug injections.

NEUROCHEMICAL SUBSTRATES OF HEROIN REINFORCEMENT

The Role of Peripheral Opiate Receptors

In humans, opiate injection is accompanied by "a warm flushing of

the skin and sensations in the lower abdomen described by addicts

as similar in intensity and quality to sexual orgasm" (Jaffe 1980;

Wikler 1952). Known as the "rush," this sensation lasts for

approximately 45 seconds and is generally thought to be one of the

motivating factors involved in opiate use (Jaffe 1980). However,

it is not clear that this rush is mediated by direct drug action

on the central nervous system (CNS), since it is well established

that interoceptive autonomic stimuli have an important role in the

maintenance of heroin consumption in humans (Meyer and Mirin

1979). More operationally, it is not known whether the reinforcing properties of opiates that result from the sensations associated with this rush arise directly from activation of opiate

receptors in the CNS or from opiate receptors localized in the

periphery.

In light of the above, we compared the potency and efficacy of

systemic administration of naloxone and naltrexone with their

quaternary derivatives in antagonizing the reinforcing properties

148
of heroin. The quaternary derivatives of naloxone and naltrexone

were chosen because of their potentially selective antagonist

action, which excludes them from penetrating through the blood

brain barrier. As a result, they can antagonize opiate effects on

peripheral opiate receptors, as inferred from their ability to

antagonize morphine effects on gastrointestinal transit, but do

not antagonize central opiate actions on pain (Tavani et al.

1979). Recent studies have confirmed, conditionally, the peripheral selectivity of these compounds (Bianchi et al. 1982).

Furthermore, others have reported that quaternary naltrexone was

ineffective as an antagonist of the morphine discriminative

stimulus or in precipitating abstinence in morphine-dependent

rhesus monkeys (Valentino et al. 1981).

In rats self-administering heroin (0.06 mg/kg/injection in daily

3-hour sessions), low doses (0.05 to 0.2 mg/kg) of naloxone and

naltrexone produced dose-dependent increases in self-administration; at higher doses (10 to 30 mg/kg), these drugs produced

transient decreases in heroin self-administration, followed by

recovery (Ettenberg et al. 1982; Koob et al. 1984) (figure 3).

FIGURE 3. Effects of naloxone, methylnal oxonium chloride, naltrexone, and naltrexone methobromide on responding

over the total 3-hour test session for rats selfadministering heroin

NOTE: Data are expressed as mean ± S.E.M. of the total Infusions for 3 hours.

Asterisks Indicate that the-treatment doses were reliably different from

the saline (SAL)-pretreatment condition, p<.05 (paired t-test). Note that

the SAL group represents mean t S.E.M. of al I the SAL observations (which

Is equal to the number of different rats used in each drug group) for that

drua: i.e., for naloxone. n=14: for methylnaloxonium chloride. n=15: for

naltrexone; n=9; and for naltrexone methobromide, n=11. Each dose consists

of five separate observations. Koob et al. 1984. Copyright 1984,

Williams & Wilkins Company.

149
The quaternary derivatives were ineffective as antagonists of

heroin self-administration in doses 200 times greater than the

effective antagonist dose of naloxone or naltrexone (Koob et al.

1984). These results support the hypothesis that the acute reinforcing properties of intravenous (IV) opiates associated with

the sensation of the rush involve opiate receptors located within

the central nervous system (CNS) and do not involve peripheral

opiate receptors (Koob et al. 1984).

In support of a selective peripheral action for these quaternary

compounds, others have shown that the quaternary derivatives of

opiate antagonists do not induce withdrawal signs in morphinedependent dogs or monkeys at doses that blocked morphine-induced

intestinal spike potentials (Russell et al. 1982). Also, the

present results are unlikely to be explained simply on the basis

of differential binding potency of the antagonists to opiate

receptors. For example, methylnaloxonium (MN) appears to be onetenth as potent as naloxone in displacing tritiated etorphine

binding in rat brain homogenates (de Graaf et al., unpublished),

and data from our laboratory indicate that MN is one-eighth as

potent as morphine in displacing tritiated DAGO binding in rat

brain homogenates and one-eleventh as potent as naloxone in displacing tritiated DAGO binding (Koob et al., unpublished). These

results suggest that MN does block opiate receptors in a manner

similar to that of naloxone itself. In addition, others have

shown in vivo that the dose (1 mg/kg) of naloxone methobromide

required for complete antagonism of the effect of morphine (5

mg/kg) on gastrointestinal transport in rats was only two to four

times higher than the dose of naloxone hydrochloride required to

produce the same effect (Bianchi et al. 1982). Although not

directly evaluated in our experiments, these potency ratios

suggest that the quaternary derivatives are effective opiate

antagonists in the periphery.

The Role of Brain Dopamine Receptors

Previous studies have shown that centrally acting opiate receptor

antagonists increase heroin self-administration, and centrally

acting dopamine receptor antagonists increase cocaine selfadministration. Unknown was whether or not these effects were

pharmacologically independent of each other. To examine whether

dopamine receptor antagonists alter the self-administration of

opiates, rats trained to self-administer heroin (0.06 mg/kg/injection) intravenously for daily 3-hour sessions were subjected to a

series of doses of the dopamine receptor antagonist alpha flupenthixol. Alpha flupenthixol, in doses that dose-dependently increased cocaine self-administration, failed to increase heroin

self-administration. Indeed, the only significant effect on

heroin self-administration was a decrease in self-administration

at 0.4 mg/kg of alpha flupenthixol, a cataleptic dose. Naltrexone

produced a dose-dependent increase in self-administration but had

no effect on cocaine self-administration (Ettenberg et al. 1982).

These results confirmed those observed by others using other

150
antagonists of opiates and dopamine (Goldberg et al. 1971; Yokel

and Wise 1976; Weeks and Collins 1976; De Wit and Wise 1977).

The increased responding observed with the respective antagonist

for each drug is thought to occur because the antagonist drugs

presumably compete with the same synaptic sites as those influenced by the self-administered drug. Heroin, for example, is

assumed to be converted to morphine in the brain, and the morphine

is assumed to bind to central opiate receptors (Way and Adler

1960). Cocaine is thought to enhance dopaminergic transmission by

blocking the reuptake of presynaptic dopamine (Patrick et al.

1975). Therefore, the net reinforcement produced by combining the

self-administered drug and its specific antagonist is equivalent

to the net effect on neurotransmission; animals respond to increasing doses of the antagonist by increasing the amount of drug

self-administered. Of particular significance was the observation

that low doses of alpha flupenthixol did not increase responding

for heroin, nor did naltrexone increase cocaine-reinforced responding. The specificity with which these antagonists exerted

their behavioral effects strongly suggests that separate neural

substrates are responsible for the reinforcing actions of heroin

and cocaine (Ettenberg et al. 1982).

Location Within the CNS of the Opiate Receptors Critical for

Heroin Reinforcement

The mesolimbic dopamine system has not only been accorded an

important role in psychomotor stimulant reward; it has also been

hypothesized that this system is critical for the reinforcing

properties of opiates (Bozarth and Wise 1981a). For example, rats

will self-administer morphine into the ventral tegmental area

(VTA) (Bozarth and Wise 1981b), and more recent studies have shown

that rats will directly self-administer D-ala2-methionine enkephalin into the nucleus accumbens (Goeders et al. 1984). Indeed,

Britt and Wise (1983) have shown that administration of quaternary

nalorphine (diallyl nor-morphinium bromide) into the VTA but not

into the nucleus accumbens (N.Acc.) attenuated the self-administration of heroin. Our next series of studies was designed to

extend these observations by examining the effects of methylnaloxonium chloride, a naloxone derivative known not to cross the blood

brain barrier on IV heroin self-administration after microinjection into the cerebral ventricles, the VTA, or the N.Acc.

Rats were implanted with intracerebral injection cannulas aimed

above the lateral ventricle, the VTA, or the N.Acc. They were

trained to self-administer heroin (0.06 mg/kg/injection) intravenously. Following stable responding, each rat received bilateral microinjection of methylnaloxonium chloride (MN) into the

lateral ventricle, the N.Acc., or the VTA 10 minutes prior to

self-administration tests.

The results showed that lateral ventricular injections of MN

produced a dose-dependent increase in heroin self-administration

similar to that observed for systemic injections of naloxone

151
(figure 4). Effective doses ranged from 1.0 to 4.0 μg (Vaccarino

et al. 1985a). Similar results were obtained following injections

of MN into the VTA with no effect until MN reached a dose of 1.0

μg (figure 5). However, MN injected into the N.Acc. was approximately eightfold as potent at increasing self-administration of

heroin. Significant increases were observed in doses from as low

as 0.125 to 0.25 μg, with peak effects at 0.5 μg (figure 6)

(Vaccarino et al. 1985b). MN had no effect on cocaine selfadministration.

FIGURE 4. The effects of intracerebroventricular (ICV) MN

treatment on responding for heroin over the first

hour (A) and over the total 3-hour self-administration session (B)

NOTE: Response rates were expressed as the percentage of baseline responding.

Asterisks indicate that the treatment doses were significantly different

from the saline treatment, p<.05 (Newman-Keuls test). SiX rats were tested

across all drug treatments. The day prior to ICV Injections was used as

the baseline day. Vaccarino et al. 1985a. Copyright 1985, Ankho

International Inc.

The net results suggest that the N.Acc. is an important and possibly critical substrate for the reinforcing actions of opiates.

Effective doses of MN in the N.Acc. were approximately one-eighth

those observed for lateral ventricular injections. Injections of

MN into the VTA were no more effective than lateral ventricular

injections. These results suggest that the receptors important

for opiate reward may be localized on neurons in the region of the

N.Acc.,

(1981a).

as well as in the VTA, as hypothesized by Bozarth and Wise

To address the possibility that these receptors were

localized on dopamine neurons terminating in the N.Acc., subsequent work in our laboratory examined whether destruction of

presynaptic dopamine terminals in the N.Acc. would alter heroin

self-administration.

152
FIGURE 5. Percent baseline (predrug day) responding for IV heroin

during the first hour (top graph) and for the total 3

hours (bottom graph) of the heroin self-administration

session following MN injections into the ventral

tegmentat area

NOTE: Asterisks indicate a significant difference (p<.05) from both saline

vehicle (0.0 dose) and 0.5 MN (Duncan Multiple Range test). Vaccarino

et al. 1985b, Copyright 1985, Springer-Verlag.

The Role of Presynaptic Dopamine Terminals in the Nucleus

Accumbens in Opiate Reinforcement

Catecholamines have been strongly implicated in the reinforcing

properties of psychomotor stimulants (Pickens et al. 1978). More

specifically, the reinforcing properties of psychomotor stimulants

have been linked to the activation of central dopamine (DA) neurons and their postsynaptic receptors. When the synthesis of

catecholamines is inhibited by administering alpha-methyl-paratyrosine, an attenuation of the reinforcing effects of psychomotor

stimulants occurs (Pickens et al. 1978; Jonsson et al. 1971).

Furthermore, low doses of DA antagonists will increase the response rates for intravenous injections of d-amphetamine (Davis

and Smith 1975; Yokel and Wise 1975; Yokel and Wise 1976). These

authors hypothesized that a partial blockade of DA receptors produced a partial blockade of the reinforcing effects of d-amphetamine. Thus, animals are thought to compensate for decreases in

the magnitude of the reinforcer by increasing their self-administration behavior, as discussed above and shown in figure 2. The

role of DA in the reinforcing properties of cocaine was extended

by the observation that 6-hydroxydopamine (6-OHDA) lesions of the

N.Acc. produced extinction-like responding and a significant and

longlasting reduction in self-administration of cocaine over days

(Roberts et al. 1977; Roberts et al. 1980; Lyness et al. 1979).

153
FIGURE 6. Percent baseline (predrug day) responding for IV heroin

during the first hour (top graph) and for the total 3

hours (bottom graph) of the heroin setf-administration

session following MN injections into the nucleus

accumbens

NOTE: Asterisks indicate a significant difference (p<.05) from saline vehicle

(0.0 dose) (Duncan Multiple Range test). Vaccarino et al. 1985b. Copyright

1985, Springer-Verlag.

These results demonstrated that postsynaptic blockade of DA receptors or destruction of presynaptic DA terminals in the region of

the N.Acc. significantly decreased the reinforcing value of

psychomotor stimulants.

Thus, the mesolimbic DA system appears to be critical for psychomotor stimulant reinforcement (Roberts et al. 1977; Roberts et

al. 1980; Lyness et al. 1979), and any hypotheses regarding a role

for DA in opioid reinforcement would likely focus on this same

mesolimbic system. This is particularly relevant, since rats will

maintain self-administration, of morphine applied directly into the

brain region containing the mesolimbic DA cell bodies, the VTA

(Phillips and LePiane 1980; Bozarth and Wise 1981b), as well as

the N.Acc. itself (Olds 1982). To clarify further the role of

mesolimbic DA neurons in opiate reinforcement, we examined the

effects of DA denervation of the N.Acc. on both heroin and cocaine

self-administration.

Rats were trained to self-administer intravenously heroin (0.06

mg/kg/injection) and cocaine (0.75 mg/kg/injection) for 3 hours on

154
alternate days. This alternating drug self-administration procedure was continued until stable intake and titration on both drugs

had occurred. For each rat, each drug was delivered via a given

lever (left or right), and a colored light (red or yellow) was

used as a constant discriminative stimulus, which was turned on at

the onset of the infusion and remained on for 20 seconds. Animals

that showed stable baselines over 3 days with each drug were given

an intracerebral injection of either 6-hydroxydopamine (6-OHDA) or

vehicle into the N.Acc.,

1981).

as described previously (Joyce and Koob

These lesions produced a 94 percent depletion of DA in the

N.Acc. but no significant decrease in the anterior striatum

(Pettit et al. 1984). Four days following the lesion, the rats

were allowed to resume the alternating schedule of self-administration as described above. The 6-OHDA lesions initially produced

an attenuation in both cocaine and heroin self-administration on

the first self-administration trial postlesion. Subsequently,

heroin responding recovered with time, gradually increasing to 76

percent of prelesion baseline levels, on average. In contrast,

cocaine responding continued to decrease over trials after 6-OHDA.

By the fifth trial postlesion, cocaine self-administration rates

were reduced to 30 percent of prelesion baseline levels; this

percent change was significantly different from that for heroin

rates (Pettit et al. 1984). No significant differences in

responding to cocaine or heroin postlesion were seen in shamoperated rats (Pettit et al. 1984). In three animals, heroin responses were distinctly higher than cocaine responses postlesion,

demonstrating an absolute as well as a relative difference in

responding (Pettit et al. 1984). These results demonstrate that

selective lesions of the presynaptic DA input to the N.Acc. can

significantly attenuate cocaine self-administration, without

influencing heroin responding.

An important factor in this study was that the effects of each

lesion could be measured on the two independent drug variables

almost simultaneously. Thus, differential effects could not be

attributed to different DA depletion levels, since for each subject the lesion had specific effects on the self-administration of

cocaine and heroin, and these effects were compared within individuals to prelesion rates. Thus, these results have direct

implications as to the neural substrates responsible for the reinforcing properties of both psychomotor stimulants and opioids;

the results suggest that the reinforcing properties of heroin are

at some point independent of the dopaminergic neural systems mediating the reinforcing properties of cocaine.

OPIATE RECEPTOR SUBTYPES AND OPIATE REINFORCEMENT

The hypothesis that specific subtypes of opiate receptors may be

involved in the reinforcing properties of opiates reflects a

question of significant interest to pharmacologists (Zukin and

Zukin 1984)--a question difficult to address because of technical

limitations. A variety of opiate drugs are self-administered by

animals, including, in rats, morphine, codeine, etonitazene,

meperidine, propoxyphene, as well as the mixed agonist/antagonists

155
butorphanol, nalbuphine, nalorphine, and pentazocine (Collins et

al. 1984). The opiate antagonists cyclazocine and naloxone were

inactive; ethylketazocine had clear reinforcing effects in rats

(Collins et al. 1984) but not in monkeys (Woods et al. 1979).

However, because of different pharmacokinetic characteristics of

these drugs, any correlation of potency for self-administration

with binding affinity to opiate subtype would be meaningless. An

alternative approach has been to examine the reinforcing efficacy

of opiate agonists more or less specific to a given opiate receptor, and from these data to generate hypotheses regarding a possible subtype of opiate receptor particularly involved in opiate

reinforcement.

Evidence From Conditioned Place-Preference Studies

A number of opiate drugs, when paired as unconditioned stimuli to

a particular environment, show the capability of imparting to that

environment a positive reinforcing property (Mucha et al. 1982;

Spyraki et al. 1983; Stolerman et al. 1978; Amalric et al., in

press; Mucha and Iversen 1984). The measure of reinforcement in

this paradigm is the return of an animal in a choice situation to

the environment previously paired with the drug, i.e., a preference for that environment over an unpaired environment. Analogous

to the classical taste preference procedures used by Wikler and

associates in dependent rats (Wikler 1965), nonphysically dependent animals will show readily such a conditioned place preference

with as few as one prior pairing with a drug such as heroin (Mucha

et al. 1982).

In a recent study (Mucha and Herz 1985), the reinforcing efficacy

of various opioid agonists acting preferentially on the kappa and

mu opioid receptors was assessed using the place-preference conditioning paradigm. Kappa receptor agonists such as U50-488 and

(-)bremazocine produced place aversions, whereas mu agonists such

as morphine, fentanyl, and sufentanil produced place preferences

(Mucha and Herz 1985).

Similarly, in our laboratory, experiments were directed at examining the reinforcing properties of beta-endorphin (B-END) using a

discrete-trials conditioned place-preference test. B-END binds

preferentially to mu and delta opiate receptor subtypes. The

paradigm paired an intracerebroventricular injection of an opioid

peptide with one distinct environment and saline with another

distinctly different environment on alternate days for 6 days

(training). Peptide injections were paired with the least preferred environment, based on the preference of the rats in a pretreatment session; however, there were no differences in group

preference for either environment. B-END (1.5, 2.5, 5.0, or 10.0

μg/rat) was injected intracerebroventricularly (ICV 2 μl volume)

immediately before the rat was placed in the training box for 30

minutes. Control rats were injected with saline, morphine (10 μg

ICV), or heroin (0.5 mg/kg subcutaneously (SC)). After training,

each rat was tested drug-free in a double-environment box where

each end was identical to the training environments, with a

156
smaller, gray, neutral area in the center. Time spent in each end

of the test box was recorded over a lo-minute period.

In a separate experiment, rats were similarly treated except that

they received only one pairing of ICV B-END (2.5 μg) to the least

preferred environment, plus SC injection of 0.04, 0.20, or 1.0

mg/kg of naloxone.

Heroin (0.5 mg/kg SC) produced strong preference for the heroinpaired environment. Rats also showed dose-dependent place preference for the environment paired with B-END (figure 7). Rats

injected with the higher doses showed no preference for the paired

environment, but did show catalepsy and immobility with B-END

(Amalric et al., in press). Naloxone effectively blocked this

place preference at a dose as low as 0.04 mg/kg (figure 8). This

dose had no effect on its own, but higher doses of naloxone alone

produced a place aversion (Amalric et al., in press).

FIGURE 7. The effects of intracerebroventricular (ICV) injections

of beta-endorphin on place preference in the rat

NOTE: Data represent mean ± S.E.M. of the difference In time between pretest and

posttest on the conditioned side during a 1O-minute test after 6 days of

conditioning (B-END for 3 days on the paired side alternating with saline

for 3 days on the unpaired side). There was no systematic preference for

the white or black side prior to training. Heroin was injected SC.

Asterisks indicate significant difference from saline (Student’s t-test).

These results demonstrate positive reinforcing properties for BEND. Given that B-END interacts mainly with mu and delta receptors, these results, combined with the results of studies with

other selective opiate agonists (Mucha and Herz 1985), suggest

that mu and delta receptors are the opiate receptor subtypes

important for opiate reinforcement.

157
FIGURE 8. The effects of natoxone injected systemically SC on

place preference produced by ICV injection of B-END

NOTE: Data represent mean ± S.E.M. of the dlfference in time between pretest and

oosttest on the conditioned side during a 1O-minute test after 2 days of

conditioning. Asterisks indicate significant difference from B-END

(Student's t-test).

Evidence From Self-Administration Studies

Further support for this hypothesis has been obtained using the

self-administration procedure. As discussed above, many opiate

agonists and mixed agonist-antagonists are self-administered by

rats and monkeys. However, in rats, at least one study has found

the putative kappa agonist ethylketazocine to have reinforcing

properties (Collins et al. 1984). even though it does not produce

place preference or aversion.

To examine this question further, rats prepared with intravenous

catheters and trained to self-administer heroin were subjected to

a series of acute treatments with mu and kappa agonists during

self-administration. Following establishment of a dose-dependent

pause in opiate self-administration, verification of the ability

of a given compound to substitute for heroin was obtained by

replacing the heroin with the drug in question.

Six rats were allowed to self-administer heroin for 3 hours per

day on an FR 5 schedule of reinforcement. During response pause

testing, the rats were allowed a l-hour period of access to

heroin; they were then removed from the apparatus and injected

with 0.01, 0.02, or 0.04 mg/kg of fentanyl (mu agonist) or 0.25,

0.50, or 1.0 mg/kg of U50-488 (kappa agonist). Following each

series, the respective agonists were substituted as the drug to be

self-administered, at a dose deemed equivalent to that used for

heroin.

As can be seen in table 1, fentanyl produced a dose-dependent

pause in self-administration when injected SC during a heroin

158
TABLE 1. Effects of fentanyl and U50-488 on heroin selfadministration in the rat

Response Pause Self-Administration

Compound (Secs)* Compound (Number Infusions)*

Fentanyl

0

0.01 mg/kg

0.02 mg/kg

0.04 mg/kg

U50-488

0

0.25 mg/kg

0.50 mg/kg

1.00 mg/kg

Heroin

0

0.1 mg/kg

0.2 mg/kg

0.4 mg/kg

10.0±1.3‡

33.7±5.9

51.5±7.8

74.0±7.6

10.0±1.3*

17.2±2.9

36.8±15.0

13.O±2.5

10.0±1.3*

21.3±6.8

43.0±11.9

65.2±10.9

Fentanyl

0.006 mg/kg/inj

U50-488

0.15 mg/kg/inj

Heroin

0.06 mg/kg/inj

Saline

Total 3 hrs 15.2±2.4

1st hr 6.7±O.7

2nd hr 4.5±1.0

3rd hr 4.0±0.9

Total 3 hrs 5.5±1.7

1st hr 4.5±1.4

2nd hr 0.5±0.5

3rd hr 0.5±0.3

Total 3 hrs 16.0±1.9

1st hr 7.2±O.9

2nd hr 5.0±0.7

3rd hr 3.8±O.7

Total 3 hrs 6.0±1.9

1st hr 4.5±2.1

2nd hr O.O±O.O

3rd hr 0.3±O.2

*Values represent mean ± S.E.M. for Six rats.

‡

Represents the same observations for a single systemic saline injection 1 hour

into a 3-hour heroin self-administration session.

self-administration test. Fentanyl also readily substituted for

heroin on a subsequent test day at a dose of 0.006 mg/kg/injection. U50-488 failed to produce any systematic response pauses at

doses as high as 1.0 mg/kg and failed to substitute for heroin at

a dose of 0.15 mg/kg/injection on a subsequent trial. These results suggest that the reinforcing properties of heroin are related to an activation of the mu opiate receptor subtype.

159
SUMMARY

The studies reported herein summarize our work to date aimed at

determining the neurochemical substrates for the reinforcing

properties of opiates. Rats were trained to self-administer

heroin intravenously in daily 3-hour sessions, and pharmacological

blockade and neurotoxin-induced lesions were used to define the

neurochemical substrates for this reinforcing action. Low-dose DA

receptor blockade failed to alter heroin self-administration but

significantly increased cocaine self-administration, presumably

reflecting a decrease in the reinforcing effectiveness of cocaine. Destruction of presynaptic DA terminals within the N.Acc.

produced extinction of cocaine, but not heroin, self-administration. Opiate receptor blockade with systemic naloxone increased

heroin, but not cocaine, self-administration. Methylnaloxonium

injections into the N.Acc. were effective in increasing heroin

self-administration at doses one-eighth those observed for

intracerebroventricular injections.

Reinforcement has been explored using a place-preference procedure

and a self-administration drug-substitution paradigm. Mu/delta

agonists such as B-END readily produce a naloxone-reversible place

preference. Fentanyl derivatives also produce place preference

and substitute for heroin during self-administration. The kappa

agonist U50-488 produces place aversion, not place preference, and

does not readily substitute for heroin. Altogether, these results

suggest that mu/delta receptor subtypes in the region of the

N.Acc. may be an important neurochemical substrate for opiate

reinforcement.

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ACKNOWLEDGMENTS

Preparation of this manuscript was supported in part by National

Institute on Drug Abuse grant DA 03665.

163
AUTHORS

George F. Koob, Ph.D.

Marianne Amalric, Ph.D.

Floyd E. Bloom, M.D.

Division of Preclinical Neuroscience

and Endocrinology

Scripps Clinic and Research Foundation

10666 North Torrey Pines Road

La Jolla, CA 92037

Franco J. Vaccarino, Ph.D.

Department of Psychology

University of Toronto

Toronto, Ontario, Canada

164
Opiate Receptor Subtypes

Associated With the Brain

Mechanisms of Feeding and

Reward

Roy A. Wise, Francois Jenck, Alain Gratton,

and Remi Quirion

INTRODUCTION

Determination of the opiate receptor subtypes involved in a given

behavior depends, at the present time, on the ability to determine

the relative potencies of various receptor agonists for the behavior in question. The critical measure of potency is the concentration needed, at the receptor, to produce the behavioral action

in question. Since the defining feature of drug abuse is compulsive drug self-administration, the most relevant drug action for

determining the receptor types associated with abuse would be the

rewarding or reinforcing action of opioids. Unfortunately, it is

very difficult to determine the necessary opfoid concentration, at

the relevant receptor, for a just-detectable rewarding effect.

This difficulty arises from several facts. First, the paradigms

in which reinforcing effects of drugs are identified are paradigms

in which the animal, rather than the experimenter, controls the

number and timing of the injections and, thus, the dose administered. Second, the concentration at the relevant receptor is not

simply proportional to the dose administered. Even if all animals

self-administered the same amount of a drug at the same intervals,

dose at the relevant receptor would be difficult to determine for

different opioids. If the drugs were given intravenously, differences in ability to penetrate the brain, as well as differences in

rate of metabolism, would have to be considered for each compound. If the drugs were given centrally, rate of metabolism and

unpredictable dosing would still be major factors, and concentration gradients between the injection site and the site of action

would have to be determined for each compound. This would be a

problem, as it is not yet clear how many sites of rewarding opiate

action there are in the brain, and for neither of the two current

candidate sites are the boundaries of the population of target

neurons known. Another problem is that in order to have good

control over concentration of the drug at the receptor, it would

be preferred that long-acting agents be used so that concentration

did not vary significantly over time; this would conflict with the

needs of the behavioral paradigm, where short-acting agents are

165
preferred because they increase the number of responses and, thus,

increase the precision of response-rate estimates.

The best paradigm for determining opioid receptor involvement in behavior would involve experimenter, rather than animal,

controlled dosing and long-acting, rather than short-acting,

drugs. These conditions are compatible with studies of

independent behaviors, such as feeding, but not with drug selfadministration itself. There is evidence, however, to suggest

that the same brain mechanisms may be involved in feeding and

opiate self-administration. To the degree that this evidence is

credible, certain feeding paradigms may thus serve as models that

predict the relative rewarding potency of various opioid

compounds. This chapter summarizes our recent explorations of

this possibility.

The evidence linking the mechanisms of reward and feeding is, for

the most part, evidence from brain stimulation studies.

Stimulation of the lateral hypothalamic medial forebrain bundle

can be rewarding, and stimulation at the same brain loci can

induce feeding and other biologically primitive, species-typical

behaviors (Glickman and Schiff 1967). In general, drugs and

treatments that influence stimulation-induced feeding similarly

influence intracranial self-stimulation (Hoebel 1969). Moreover,

the refractory periods for the populations of neurons underlying

each of the two behaviors have been characterized and found to be

similar (Hawkins et al. 1983). We have redone the early estimates

of refractory period distribution using a finer-grained analysis,

and we have found that at least two subpopulations of medial

forebrain fibers are implicated in brain stimulation reward: a

very fast, atropine-sensitive subpopulation, and a slower,

atropine-insensitive subpopulation (Gratton and Wise 1985a). We

have extended the fine-grained analysis to stimulation-induced

feeding, and we found evidence that the same subpopulations

participate in this behavior as well (Gratton and Wise 1985b).

Moreover, when the dorsal-ventral extent of the system is mapped

using a moveable electrode, each electrode movement that causes an

increase in the reward threshold also causes an increase in the

feeding threshold (figure 1).

The circuitry underlying brain stimulation reward and stimulationinduced feeding is not understood in detail, but the current view

is that lateral hypothalamic stimulation activates descending

fibers (Shizgal et al. 1980), which transsynaptically activate

ventral tegmental area (VTA) dopamine cells (Wise and Bozarth

1984). Morphine is known to interact with this circuitry at the

level of the VTA dopaminergic cell bodies (Matthews and German

1984; Ostrowski et al. 1982), where it produces direct rewarding

effects (Bozarth and Wise 1981; Phillips and LePiane 1980) and it

facilitates both brain stimulation reward (Broekkamp et al. 1976)

and stimulation-induced feeding (Wise et al. 1985). Naloxone does

not block but attenuates both behaviors (Carr and Simon 1983;

Stapleton et al. 1979). VTA morphine injections also facilitate

free feeding; naloxone blocks this effect and inhibits feeding in

166
morphine-free animals (Wise et al. 1985). Morphine injections

into the periaqueductal gray (PAG) substance, on the other hand,

inhibit self-stimulation (Broekkamp et al. 1976), stimulationinduced feeding (Wise et al. 1985), and free feeding (Wise and

Raptis, unpublished observations).

FIGURE 1. Variations in brain stimulation reward (BSR) and

stimulation-induced feeding (SIF) threshold as a

function of electrode placement fop rat AG19

NOTE: The absolute values for the two behaviors differ because of task variables (the BSR thresholds would be lower than the SIF thresholds if the

animals could use nose-poke, rather than lever-press, stimulation). The

ratio of the SIF threshold to the BSR threshold was constant, however, as

shown in the Inset.

Thus, it appears that the mesolimbic dopaminergic fibers play

a role in both reward and feeding, and that opiates can activate

these fibers at the level of the VTA cell bodies. These facts

suggest that the effects of various opioids on stimulation-induced

feeding--of considerable interest in its own right--may provide a

useful model from which to predict the relative potency of the

opioids in reward function. We have studied the relative potency

of VTA and PAG injections of three opioids in the stimulationinduced feeding paradigm.

METHODS

Rats with lateral hypothalamic electrodes were tested for

stimulation-induced feeding in a 25x25 cm box with 45 mg Noyes

food pellets covering the floor. Cathodal pulses of 0.1 ms

duration were administered at a frequency of 50 Hz, and, in

animals that ate under stimulation, stimulation intensity was

adjusted to produce the eating of just three pellets in 10 seconds

167
time. Time to eat three pellets was then determined over the

range of effective stimulation frequencies. For these tests,

stimulation was administered in 20-s trains with 20-s intertrain

intervals; no eating was ever seen in the intertrain intervals, as

the animals were allowed to satiate on the test food just prior to

testing. Four blocks of stimulation tests were given each day:

stimulation frequency was increased, from ineffective levels to

maximally effective levels, in the first and third blocks and was

decreased, from maximally effective levels to ineffective levels,

in the second and fourth blocks. The animals were tested daily in

this fashion until day-to-day fluctuations in their frequencylatency functions were minimal; then drug testing began.

Morphine was chosen as a prototypic mu agonist, U-50,488H as a

specific kappa agonist (Vonvoigtlander et al. 1983), and D-Pen2,

D-Pen5 enkephalin as a specific delta opioid agonist (Mosberg et

al. 1983). These drugs were microinjected into the VTA or the PAG

by way of chronically implanted guide cannulae (o.d.=0.4 mm).

Microinjections were carried out by inserting into the guide

cannula an injection cannula (o.d.=0.28 mm) connected by polyethylene tubing to a 1 μl Hamilton microsyringe. The injection

cannula extended beyond the tip of the guide by 1 mm; the injector

was left in place for 2 minutes after the injection was complete. Injection volume was 0.25 μl and doses of 8 and 16 nmoles

were infused in 30 seconds into VTA or the PAG on separate days.

We have previously shown that such doses of morphine into VTA

facilitate stimulation-induced eating, while such injections into

PAG inhibit it (Wise et al. 1985). Each animal was tested

alternately with VTA then PAG microinjections such that a minimum

of 1 week elapsed between two consecutive microinjections into the

same brain site. Testing began 20 minutes after each

microinjection and was otherwise similar to the tests made under

no-drug conditions.

RESULTS

VTA microinjections facilitated stimulation-induced eating,

whether morphine, U-50,488H or D-Pen2,D-Pen5 enkephalin was

infused. Time to complete eating of three food pellets decreased,

as did the minimum stimulation frequency required to induce eating

at a given latency. The three agents were similar in potency

(figure 2), although D-Pen2,D-Pen5 enkephalin appeared more effective in inducing locomotor activity. Morphine and U-50,488H were

as effective in the third and fourth test blocks as in the first

and second; D-Pen2,D-Pen5 enkephalin appeared to lose effectiveness by the third and fourth blocks of tests.

PAG injections of morphine inhibited stimulation-induced eating

(figure 3). The other two agonists, however, produced no effects

in the PAG. The threshold dose for inhibition of stimulationinduced eating by morphine in the PAG was about twice the threshold dose for facilitation of stimulation-induced eating by

morphine in the VTA.

168
FIGURE 2. Effects of ventral tegmental injections of selective mu

(morphine), kappa (U-50,488H), and delta (bis-penenk) opioid receptor agonists on stimulation-induced

feeding

NOTE: The same injection dose of 8 nmoles was used for each agent; injectIon

volume was 0.25 μl. Mean data from four animals are shown.

DISCUSSION

These data suggest opioid interactions with the VTA mechanisms

involved in feeding, or in the facilitation of feeding, and with

PAG mechanisms that inhibit or conflict with feeding. While the

drugs used are relatively selective for mu, kappa, and delta

opioid receptors, their equal potency in the VTA tests does not

mean that all three receptors are involved in the mechanism of

this behavior. Since equal injection doses do not guarantee equal

concentrations at the relevant receptors, it is possible that one

of the agents was more potent--at the critical receptors--than

were the other two. Thus, while mu, delta, and kappa receptors

might all be linked to the VTA mechanism of opioid-induced facilitation of stimulation-induced feeding, it is also possible that

only one or two of these receptors is involved and that one or

more of the agonists had its action at a nonpreferred receptor.

Clearly, not all the agonists are acting on the same class of

receptor. The fact that neither the delta agonist nor the kappa

agonist had morphine-like effects in the PAG suggests that neither

the kappa nor the delta agonist was acting on the same class of mu

169
FIGURE 3. Effects of periaqueductal grey injections of selective

mu (morphine), kappa (U-50,488H), and delta (bis-penenk) opioid receptor agonists on stimulation-induced

feeding

NOTE: The ssme injection dose of 16 moles was used for each agent; injection

volume was 0.25 μl. Mean data from four animals are shown.

receptor as was involved in the PAG effects. Further tests are

needed to narrow the possibilities in this regard. Nonetheless,

the fact that U-50,488H and D-Pen2,D-Pen5 enkephalin had different

effects, relative to morphine, at different sites indicates at

least some degree of behavioral selectivity of the agonists and

some degree of specificity of the receptors in the facilitation

and inhibition of feeding.

The opposite effects of PAG and VTA morphine on stimulationinduced feeding parallels the effects of these injections on brain

stimulation reward (Broekkamp et al. 1976) and strengthens the

evidence for a common mechanism for stimulation-induced feeding

and brain stimulation reward. The effects of the kappa and delta

agonists on brain stimulation reward have not yet been tested.

The hypothesis that stimulation-induced feeding predicts the rewarding effects of various drug manipulations would indicate that

VTA injections of U-50,488H and D-Pen2,D-Pen5 enkephalin should

both facilitate brain stimulation reward and produce rewarding effects in their own right. Tests of this hypothesis are under way.

170
REFERENCES

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of morphine into the ventral tegmental area in rats. Life Sci

28:551-555, 1981.

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R.H.; Cools, A.R.; and Van Rossum, J.M. Separation of

inhibiting and stimulating effects of morphine on selfstimulation behavior by intracerebral microinjections. Eur J

Pharmacol 36:443-446, 1976.

Carr, K.D., and Simon, E.J. Effects of naloxone and its

quarternary analoaue on stimulation-induced feeding.

Neuropharmacology 22:127-130, 1983.

Glickman, S.E., and Schiff. B.B. A biological theorv of

reinforcement.

Gratton, A.,

Psychol Rev 74:81-109, 1967.

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Science 227:545-548, 1985a.

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of the substrate for brain stimulation reward and stimulationinduced feeding. Soc Neurosci Abstr 11:1176, 1985b.

Hawkins, R.D.; Roll, P.L.; Puerto, A,; and Yeomans, J.S.

Refractory periods of neurons mediating stimulation-induced

eating and brain stimulation reward: Interval scale measurement

and tests of a model of neural integration. Behav Neurosci

97:416-432, 1983.

Hoebel, B.G. Feeding and self-stimulation. Ann NY Acad Sci

157:758-778, 1969.

Matthews, R.T., and German D.C. Electrophysiological evidence for

excitation of rat ventral tegmental area dopaminergic neurons by

morphine. Neuroscience 11:617-626, 1984.

Mosberg, M.I.; Hurst, R.; Hruby, V.J.; Gee, K.; Yamamura, H.I.;

Galligan, J.J.; and Burks, T.F. Bis-penicillamine enkephalins

possess highly improved specificity toward delta opioid

receptors. Proc Natl Acad Sci (USA) 80:5871-5874, 1983.

Ostrowski, N.L. Hatfield, C.B.; and Caggiula, A.R. The effects

of low doses of morphine on the activity of dopamine containing

cells and on behavior. Life Sci 31:2347-2350, 1987.

Phillips, A.G., and LePiane, F.G. Reinforcing effects of morphine

microinjection into the ventral tegmental area. Pharmacol

Biochem Behav 12:965-968, 1980.

Shizgal, P.; Bielajew, C.; and Kiss, I. Anodal hyperpolarization

block technique provides evidence for rostro-caudal conduction

of reward related signals in the medial forebrain bundle. Soc

Neurosci Abstr 6:422, 1980.

Stapleton, J.M.; Merriman, V.J.; Coogle, C.L.; Gelbard, S.D.; and

Reid, L.D. Naloxone reduces pressing for intracranial

stimulation of sites in the periaqueductal gray area, accumbens

nucleus, substantia nigra and lateral hypothalamus. Physiol

Psychol 7:427-436, 1979.

Vonvoigtlander, P.F.; Lahti, R.A.; and Ludens, J.H. U-50,488: A

selective and structurally novel non-mu (kappa) opioid

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Wise, R.A., and Bozarth, M.A. Brain reward circuitry: Four

circuit elements "wired" in apparent series. Brain Res Bull

12:203-208, 1984.

Wise, R.A.; Jenck, F.; and Raptis, L. Morphine potentiates

feeding via the opiate reinforcement mechanism. In: Harris,

L.S., ed. Problems of Drug Dependence 1985. National Institute

on Drug Abuse Research Monograph, in press.

ACKNOWLEDGMENTS

This work was supported by National Institute on Drug Abuse

Grant DA01720 and by Natural Sciences and Engineering Research

Council of Canada Grant A0362.

AUTHORS

Roy A. Wise, Ph.D.

Francois Jenck. Ph.D.

Alain Gratton, Ph.D.

Center for Studies in Behavioral Neurobiology

Department of Psychology

Concordia University

Montreal, Quebec, Canada H3G 1M8

Remi Quirion, Ph.D.

Douglas Hospital Research Centre

Verdun, Quebec, Canada H4H 1R3

172
In Vivo Interactions Among

Opiate Receptor Agonists and

Antagonists

John W. Holaday, Frank C. Tortella, Rhoda Maneckjee,

and Joseph B. Long

INTRODUCTION

It is a never-ending quest by medicinal chemists and pharmacologists to synthesize ligands that have selective actions at known

receptor types and subtypes. Often, newly discovered agonist and

antagonist ligands are initially heralded as being "pure" and

"selective." Over time, as the pharmacological profiles of these

compounds become more thoroughly characterized, these descriptions

become meaningless as biochemical and pharmacological data establish that the ligands "cross-react" with different receptors, or

function as mixed agonist and/or antagonist molecules. Based upon

evidence with biologically derived substances, this may not be

surprising. For example, although epinephrine has a preponderant

effect upon ß adrenergic receptors and norepinephrine acts predominantly upon α adrenergic receptors, it is well known that

given in adequate concentrations, these ligands will cross-react

with both receptor systems (Goldstein et al. 1974). Furthermore,

adrenergic "agonist" drugs with actions at α2 receptors (e.g.,

clonidine) may serve to antagonize endogenous α agonist actions,

presumably by activating presynaptic receptors which decrease

norepinephrine release (Goodman and Gilman 1975).

The cross-reactivities and differences in intrinsic activities

among agonist and antagonist ligands are fundamental concerns of

pharmacologists. Several in vivo and in vitro methods have been

used to identify the pharmacologic profiles of various agonist and

antagonist ligands. Prominent among these are various receptor

binding assays, which have been used extensively to identify ligand selectivity. However, receptor binding studies alone provide

little or no evidence of "intrinsic activity," a phenomenon that

requires the measurement of biological responses following ligandreceptor interactions. It is generally assumed that agonists have

intrinsic activity, while antagonists lack intrinsic activity

(Goldstein et al. 1974). One plausible explanation for this

difference between agonist and antagonist ligands is that they

bind in a different manner to receptors.

173
Over the past few years, we have become interested in studying

apparent "cross-reactivities" among opioid responses using various

physiological, pharmacological , and neurochemical measurements.

These in vivo experiments have used specific combinations of opioid agonist and antagonist ligands to reveal consistent patterns

of responses. Using three experimental paradigms, including the

rat endotoxic shock model, the rat flurothyl seizure model, and in

vivo changes in striatal cyclic adenosine monophosphate (CAMP), we

provide evidence indicating that μ antagonists (some of which

share κ agonist properties) also block the effects of δ antagonists. Our working hypothesis is that these repeating patterns of

opioid cross-reactivities reinforce other evidence suggesting that

there apparently are interactions among the various subtypes of

opiate receptors (Lee and Smith 1980; Bowen et al. 1981; Rothman

and Westfall 1982; Vaught et al. 1982). We and other researchers

postulate that these interactions may occur via allosteric

mechanisms or may be mediated by interconversions of a common

receptor macromolecule.

MULTIPLE OPIATE RECEPTORS

Several subtypes of opiate receptors have been characterized over

the last decade. Thus, many laboratories have presented evidence

to indicate that these receptors exist as separate macromolecular

species with anatomically distinct distributions and unique pharmacological profiles (Holaday and Tortella 1984; Paterson et al.

1983). Moreover, sequential administration of opioid agonist

ligands has provided evidence for a lack of cross-tolerance among

the μ, δ, and κ receptor subtypes (Schulz et al. 1980; Schulz et

al. 1981; Vonvoigtlander et al. 1983). Indeed, these observations

may be considered as strong evidence for the individuality of

multiple opiate receptors. Additionally, cross-protection studies

have provided further evidence to distinguish these receptors as

separate entities (Goldstein and James 1984; Robson and Kosterlitz

1979).

Despite the strong case that can be made for separating the various subtypes of opioid receptors , contrasting evidence has also

accumulated to support the concept that opioid receptor subtypes

may not be physically or functionally independent. Several investigators have-performed in vivo and in vitro experiments to indicate that μ and δ receptor sites may functionally interconvert

(Bowen et al. 1981) or interact through noncompetitive molecular

mechanisms (Lee and Smith 1980; Rothman and Westfall 1982; Vaught

et al. 1982). Consistent with these observations, evidence from

our laboratories also indicates that interactions among μ and δ

binding sites may occur in vivo (D'Amato and Holaday 1984; Holaday

and D'Amato 1983; Holaday et al. 1983; Tortella and Holaday 1984;

Tortella et al. 1985a; Tortella et al. 1985b).

As reviewed above, it is probable that agonist and antagonist ligands bind in a different manner to their respective binding sites

on receptors. Thus, one way to demonstrate the presence or absence of interactions among opiate binding sites is to evaluate

174
responses following administration of combinations of agonist and

antagonist ligands. Using arterial pressure responses in endotoxic shock, flurothyl seizure thresholds, and in vivo changes in

striatal CAMP as three different experimental endpoints, we have

observed that certain μ antagonists (which may also demonstrate κ

agonist effects) prevent δ antagonists from blocking or reversing

δ agonist effects. This concept of "antagonists of antagonists,"

first mentioned by Villarreal and colleagues (Villarreal et al.

1982; Herrera et al. 1982) for opiate systems, has also been

described for ligand interactions at the benzodiazepine receptor

(Schweri et al. 1982; Ninan et al. 1982). Specifically, ß-carbolines, the "active" or "inverse" agonists at this receptor, administered at doses which do not show intrinsic activity, block the

antagonistic action of the antagonist Ro 15-1788 against benzodiazepines. Similarly, in our studies with opioid ligands summarized below, we observe a consistent pharmacological profile

indicating that certain μ antagonists also block the antagonistic

actions of δ antagonists. Based upon these concepts of agonistantagonist interactions, a new perspective on the separability of

opioid receptor subtypes may be gleaned.

Endotoxic Shock

Over the past several years, we have demonstrated that the activation of endogenous opioid systems by circulatory shock or CNS

ischemia contributes to the pathophysiology of these ischemic disorders (Holaday 1983; Holaday 1985). Specifically, treatment with

the opiate antagonist naloxone results in a significant improvement in cardiovascular performance, metabolic status, neurologic

function, and survival in many basic and clinical studies of shock

and CNS ischemia (Holaday 1985). Because of the duality of action

of endogenous opioids upon cardiovascular and analgesic systems,

administration of naloxone for the treatment of shock or CNS injury may also enhance pain by antagonizing endogenous or exogenous

opioid analgesia, However, if ischemia and analgesia are mediated

by different types of opiate receptors, the use of "selective"

opiate receptor antagonists should provide for a dissociation of

these effects. Naloxone, which is generally accepted as a "pure"

opiate antagonist, lacks adequate selectivity for the different

opiate receptor subtypes. Thus, our initial investigations into

the type(s) of opiate receptors responsible for ischemia and analgesia used more "selective" lfgands and resulted in the unique

pharmacological observations described below.

In order to define the opioid receptor subtype responsible for the

hypotensive effects of endotoxic shock, we initially used the μ

antagonist naloxazone. Naloxazone (now known to be active following biochemical conversion to naloxonazine) is reported to be a

longlasting alkylator of μ-antagonist sites (Holaday et al. 1983).

In later experiments, we used ß-funaltrexamine (ß-FNA), a different μ antagonist alkylator defined by Portoghese, Takemori, and

175
colleagues (Takemori et al. 1981; Ward et al. 1982). To achieve

an antagonism of δ sites, we used the δ-selective antagonist

ICI 154,129 or high doses of naloxone (D'Amato and Holaday 1984;

Holaday and D'Amato 1983; Holaday et al. 1983).

We demonstrated that the 6 antagonist ICI 154,129 or high doses

of naloxone successfully reversed endotoxic shock hypotension,

whereas the μ antagonists naloxazone or ß-FNA failed to do so

(Holaday et al. 1983; D'Amato and Holaday 1984). Conversely, the

δ antagonist ICI 154,129 failed to alter morphine (u-induced)

antinociception, whereas naloxazone and ß-FNA blocked morphine

antinociception (D'Amato and Holaday 1984). From these results,

it appears that endogenous opioids released in endotoxic shock act

upon δ (not μ) receptors to produce their pathophysiological

effects. However, these studies also revealed an unexpected

finding; if naloxazone or ß-FNA were administered prior to the

ICI 154,129 or naloxone in rats subjected to endotoxemfa, the

latter substances were no longer effective in reversing endotoxic

shock hypotension (D'Amato and Holaday 1984; Holaday and O'Amato

1983; Holaday et al. 1983). This concept is schematically

represented in figure 1.

ß-FNA exhibits κ agonist properties in addition to its irreversible antagonism of the μ site (Ward et al. 1982). It thus has a

pharmacological profile like that of the peptide dynorphin 1-13,

an opioid compound known for its κ agonist actions, but which also

has persistent μ antagonist properties (Garzon et al. 1982). Not

surprisingly, dynorphin 1-13 produced results similar to our

earlier findings with naloxazone and ß-FNA. Pretreatment with

dynorphin 1-13 had no effect by itself in altering endotoxic

hypotension, yet this pretreatment significantly antagonized the

usual therapeutic effects of naloxone in this model (Holaday et

al. 1984).

More recently, we have used the new s-selective antagonist, ICI

174,864 (Cotton et al. 1984) alone and in combination with dynorphin pretreatment (Long et al. 1984). As expected, ICI 174,864

reversed endotoxic shock hypotension. Moreover, as observed with

dynorphin-naloxone studies (Holaday et al. 1984), pretreatment

with 1.0 mg/kg dynorphin 1-13 intravenously (IV) 2 hours prior to

endotoxin injection also prevented the therapeutic effects of ICI

174,864 (Long et al. 1984) (table 1).

From these studies of endotoxic shock in rats, we concluded:

(1) endogenous opioids contribute to endotoxic hypotension by

acting on δ opiate receptors, (2) dynorphin peptides may exert

actions well beyond those predicted from metabolic half-lives

obtained in tissues, and (3) compounds such as naloxazone, ß-FNA,

and dynorphin act as μ antagonists which also antagonize the

actions of δ antagonists (figure 1).

176
FIGURE 1. Schematic representation of possible allosteric

interactions among μ and δ binding sites on the

opioid receptor complex

NOTE: TOP: Morphine analgesia Is represented by μ in the square, whereas circulatory shock, presumably resulting from endogenous δ ligand activation, is

represented by the δ in the triangle. TOP MIDDLE: The putative μ antagonists ß-FNA and naloxone (represented by 'L’ shape pointing left) blocked

analgesic responses to the μ agonist morphine while having no effect upon

shock. BOTTOM MIDDLE: By contrast, ICI 154,129, representing a δ antagonist ('L’ shape pointing right), was without effect upon morphine (μ) analgesia while reversing shock hypotension. BOTTOM: However. prior treatment

with the irreversible μ antagonists (bottom of schematic) prevented the

actions of the δ antagonist in reversing shock, indicating possible allosteric interactions between these opioid binding sites.

Flurothyl Seizures

To confirm the pattern of opioid interactions initially observed

in the endotoxic shock model, we investigated the same interactions using a model of flurothyl-induced seizures in rats

(Tortella and Holaday 1984). Flurothyl is a volatile convulsant,

and rats exposed to flurothyl experience clonic convulsions within

approximately 360 seconds (Cowan et al. 1979; Tortella et al.

1981). Unlike the endotoxic shock model where endogenous opioids

are physiologically released to act upon δ receptors, in the

flurothyl seizure studies, opioid agonists and antagonists were

pharmacologically administered. Using this approach, we and

others have shown that several classes of opioid agonist ligands

177
TABLE 1. Effects of dynorphin pretreatment on reversal of endotoxic hypotension by the δ antagonist ICI 174,864

Postshock Increase in MAP

Pretreatment Treatment 15 Min After

Population (1st Injection) (2nd Injection) 2nd Injection

15 saline saline 3.0±3.8

13 saline ICI 174,864217.1±4.2*

7 dynorphin 1-133saline 2.3±5.7

11 dynorphin 1-13 ICI 174,864 5.6±3.2**

1

Following a fall in mean arterial pressure (MAP) of 20 mm Hg produced by endotoxin (22.5 mg/kg IV).

2

ICI 174,864 (3.0 mg/kg IV) was dissolved in 0.1 M Na Bicarb and injected IV.

3

Dynorphin 1-13 (1.0 mg/kg) was dissolved in saline and injected IV.

*p<0.05 compared to saline alone.

*p<0.05 compared to saline pretreatment.

SOURCE: From Long et al. (1984).

may act upon separate receptor types to elevate seizure thresholds

in this model (Cowan et al. 1979; Tortella et al. 1983). For

example, the anticonvulsant effects of etorphine were completely

blocked by the μ antagonist ß-FNA, whereas ß-FNA only partially

antagonized the effects of the δ agonist, D-Ala2-D-Leu5-enkephalin

(DADL) (figures 2 and 3) (Tortella et al. 1985a). Likewise, the δ

antagonist ICI 154,129 completely blocked the anticonvulsant effects of DADL but was without effect on the μ actions of etorphine

(Tortella and Holaday 1984; Tortella et al. 1983). Once again,

consistent with the pattern of interactions observed in the endotoxic shock model, ß-FNA pretreatment prevented ICI 154,129 from

further blocking the anticonvulsant effect of DADL (figure 4)

(Tortella and Holaday 1984; Tortella et al. 1985a).

Additional studies provide support for these unique interactions

and further demonstrate that dynorphin 1-13 exerts longlasting μ

antagonism. After the initial nonopioid anticonvulsant effects of

dynorphin 1-13 pretreatment have ended (2 hours) (Tortella et al.

1985b), the μ agonist DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol) failed

to demonstrate its usual potent anticonvulsant effects (unpublished observation). Thus, as others have shown using receptor

binding assays and pharmacological responses (Garzon et al. 1982),

dynorphin 1-13 is a longlasting μ antagonist. Furthermore,

following pretreatment with dynorphin 1-13, the anticonvulsant

178
FIGURE 2. The effect of ICI 154,129 (horizontally hatched bar),

naloxone (vertically hatched bar), or ß-FNA (diagonally hatched bar) to attenuate the anticonvulsant

action of etorphine (solid bar) in the rat

*Significantly different from saline control group, p<O.O1.

+

Significantly different from the 122-nmol/kg etorphine group, p<O.05 (Student’s

test).

NOTE: Subscripts represent the dose given intracerebroventricularly (ICV).

SOURCE: From Tortella et al. (1985a).

effects of subsequent DADL injections persist and can no longer be

blocked with the δ antagonist ICI 154,129 or high doses of naloxone (table 2). These observations with dynorphin 1-13 confirm

evidence reviewed above with endotoxic shock and flurothyl seizure

models indicating that molecules with μ antagonist properties

antagonize the actions of δ antagonists.

179
FIGURE 3. Dose-related antagonism of the anticonvulsant effect of

DADL (solid bar) in the rat

*Significantly different from saline control group, p<0.05.

+

Significantly different from the 35-nmol DADL group, p<0.01 (Dunnett’s test for

multiple comparisons).

NOTE: LEFT: Antagonism by ICI 154,129. MIDDLE: Antagonism by naloxone.

RIGHT: Antagonism by ß-FNA.

SOURCE: From Tortella et al. (1985a).

Striatal cAMP

Opioid peptides inhibit striatal adenylate cyclase, resulting in a

decrease in striatal CAMP concentrations (Childers, this volume).

Evidence indicates that opioids accomplish this action by activation of the coupling Ni GTPase, resulting in an inhibition of the

adenylate cyclase enzyme (Abood et al. 1985). Changes in striatal

CAMP serve as a postreceptor endpoint which may serve to localize

the mechanism of ligand interactions to events which precede second messenger systems. In order to confirm and refine the pattern

of ligand interactions found in the in vivo experiments described

above, but using a neurochemical correlate, we investigated the

effects of sequential in vivo opioid antagonist and/or agonist

injections on striatal cAMP levels.

Using the pharmacological strategy outlined above for the flurothy1 experiments, we attempted to block the inhibitory effects of

the δ agonist DADL on CAMP levels using ICI 154,129. Additionally, DADL challenge (following ß-FNA Pretreatment with or without

ICI 154,129 injections) was used to determine whether or not the δ

antagonist effects of ICI 154,129 would be blocked by the μ antagonist ß-FNA. As before, ß-FNA (or saline) were admin stered 18

180
FIGURE 4. The effect of prior treatment with ß-FNA (diagonal

bars) to diminish the abilities of either ICI 154,129

(horizontal hatching) or naloxone (vertical hatching)

to antagonize the anticonvulsant action of DADL

(solid bar)

*Signoficantly different from saline control, p<O.05.

+

Significantly different from the 35-nmol OADL group, p<O.05 (Dunnett's test).

Significantly different from ICI5o

 +DADL and NLX290 +DADL groups, respectively,

p<0.05 (Student's test).

NOTE: Subscripts represent the dose given ICV.

SOURCE: From Tortella et al. (1985a).

hours prior to ICV injections of ICI 154,129 (or saline), and DADL

was injected ICV 10 minutes later. Following microwave fixation

of rat brains at 20 minutes after DADL or saline challenge, brains

minus cerebella were frozen for measurement of striatal CAMP

levels by radioimmunoassay.

As previously reported (Childers, this volume), the δ agonist OADL

reduced striatal CAMP by about 50 percent. This effect was

blocked by pretreatment with ICI 154,129 or high doses of naloxone

(table 3). Consistent with both the endotoxic shock and flurothyl

seizure threshold studies, preliminary evidence indicates that

pretreatment with the μu antagonist ß-FNA blocked the ability of

the δ antagonist ICI 154,129 to antagonize the actions of the

δ agonist DADL (table 3). In other words, in rats receiving

181
TABLE 2. The effects of dynorphin on the delta antagonist actions

of ICI 154,129 and naloxone

Population

Treatment (μq ICV)1

Antagonist Agonist

Change in

Seizure Threshold

(% Control ± SEM)

1 0

8

8

8

8

8

8

saline saline 100

saline DADL2 (20)

ICI3 (25)

157±13

DADL (20) 120±8*

naloxone (100) DADL (2*) 106±6"

dyn4 (25) DADL (20) 151±9

dyn (25) + ICI (50) DADL (20) 166±9**

dyn (25) + naloxone (100) DADL (20) 132±6**

1

ICI and naloxone were given 10 minutes prior to DADL; dyn was given 2 hours prior

to OADL.

2

DADL is D-Ala3-D-Leu5-enkephalin.

3

ICI is the δ antagonist ICI 154,129.

4

Dyn Is dynorphin 1-13.

*p<O.O5 compared to saline + DADL.

**p<O.O5 compared to ICI t DADL and naloxone + DADL groups, respectively.

SOURCE: From Tortella et al. (1985b).

TABLE 3. Preliminary evidence for changes in striatal CAMP levels

in rats receiving combinations of opioid agonists

and/or antagonists

Population

Treatment (μg ICV)1

Antagonist Agonist

pmoles cAMP/mg

Wet Weight % Control

23 saline saline 0.48 100%

12 saline DADL (20) 0.23 52%

7 ICI (50) saline 0.50 104%

9 ICI (50) DADL (20) 0.50 104%

9 ß-FNA (9.8) saline 0.51 lO6%

8 ß-FNA (9.8) DADL (20) 0.52 106%

7 ß-FNA (9.8) + ICI (50) DADL (20) 0.27 47%

1

ß-FNA was given 18 hours earlier , and ICI 154,129 was given 10 minutes before

challenge with agonists or saline.

NOTE: These data represent preliminary results; more thorough dose response

studies using more "selective” δ ligands are under way.

combined pretreatment with the opiate antagonists ß-FNA plus ICI

154,129 (each of which blocked the decrease in CAMP produced by

DADL when given alone), the decrease in CAMP produced by DADL was

no longer observed. Interestingly, in this model and unlike

182
earlier studies, ß-FNA pretreatment also blocked the ability of

DADL to decrease striatal CAMP, indicating that DADL may be acting

in part through μ receptors in the striatum.

Although still limited by attempts to define potential receptor

interactions using in vivo approaches, data obtained from striatal

CAMP studies provide an important neurochemical confirmation of

earlier pharmacological data and indicate that patterns of interactions occur via mechanisms preceding second messenger systems.

SUMMARY AND CDNCLUSIDNS

The pattern of ligand interactions described here is consistent

across three separate models of opioid actions. In each case, it

was shown that three different μ antagonists (naloxazone, ß-FNA,

and dynorphin 1-13) each blocked the actions of δ antagonists (ICI

154,129 or ICI 174,864).

In addition to our demonstrations that μ antagonists are also able

to block the actions of δ antagonists, it is also known that many

κ agonists (nalorphine, nalbuphine, bremazocine, and others) also

act as μ antagonists (Gillan et al. 1981; Robson and Kosterlitz

1979; Vonvoigtlander et al. 1983). As reviewed above, our flurothy1 seizure data confirm that certain opioids (ß-FNA and dynorphin 1-13), classified as κ agonists in other systems, also

demonstrate μ antagonist effects (Garzon et al. 1982; Ward et al.

1982). It is tempting to speculate that the frequent occurrence

of ligands with both κ agonist and μ antagonist properties indicates that interactions among opioids are not restricted to μ and

δ ligands, but may also involve κ ligands. Recently, DemoliouMason and Barnard (1986), using receptor binding studies, have

provided evidence for allosteric interactions between κ and δ

receptors. At present, we are evaluating the possibility that

more selective κ agonists (e.g., U-50,488) may also act as μ

antagonists and/or antagonists of δ antagonists.

To clarify the reasons underlying these sometimes confusing and

interdependent phenomena of "cross reactivity," several working

hypotheses may be proposed: (1) one ligand may display several

moieties that account for cross-reactivity among receptor types;

(2) the complex effects of ligands apparent from in vivo studies

or in vitro tissue preparations must be interpreted in view of the

potential involvement of networks of neurons with different opioid

receptor subtypes; (3) combinations of ligands may alter the uptake or metabolism of other ligands; (4) combinations of ligands

may result in alterations of receptor coupling to second messengers, and (5) cooperativity or allosteric coupling may exist among

multiple binding sites of a macromolecular receptor complex.

Although each emphasizes a different perspective, these multiple

working hypotheses are not all mutually exclusive.

The mechanism(s) that account for the pattern of responses we

observed can be considered according to the five hypotheses posed

above. (1) It appears unlikely that molecules as different as the

183
nonpeptides naloxazone and ß-FNA would all share similar moieties

with the peptide dynorphin 1-13 to explain their actions as both μv

antagonists and antagonists of δ antagonists. (2) Although neuronal networks and altered neuronal uptake may still exist within

the striatum, evidence obtained in our preliminary studies of

striatal CAMP are consistent with the hypothesis that these

pharmacological profiles are mediated by events that precede

second messenger systems, possibly at the level of receptors.

However, these data do not exclude the potential involvement of

neuronal networks with different opioid receptor subtypes, since

drugs were administered in vivo. (3) Alterations of metabolism

are difficult to invoke due to the similar pharmacological profiles, yet entirely different chemical structures of the nonpeptide and peptide ligands were used. Furthermore, these actions

of naloxazone, ß-FNA, and dynorphin occur at a time when these

ligands do not express an intrinsic activity of their own. (4) If

receptor coupling to second messengers is involved, the striatal

CAMP data indicate that these interactions precede second messenger events. (5) Although by no means proven from the evidence

presented in this monograph, we believe that a single agonist or

antagonist ligand, acting through its binding site, may affect the

binding of other agonist or antagonist ligands at other opioid

binding sites by allosteric interactions (De Lean et al. 1979).

Regardless of the mechanisms involved, this consistent pattern

must have biological meaning.

Another noteworthy finding from these studies is that all three μ

antagonists had very persistent effects. Naloxazone and ß-FNA are

known to alkylate the receptor, a finding that has been reported

previously. However, dynorphin 1-13, which is not described as an

alkylating agent, has effects which ersist far beyond its limited

plasma half-life (Garzon et al. 1982.) Furthermore, attempts to

overcome the antagonism of δ antagonists produced by μ antagonists

using extremely high concentrations of naloxone or the ICI compounds have been unsuccessful in the endotoxic shock studies

(unpublished data). Thus, the pattern of these interactions

suggest that all three substances share a noncompetitive, persistent biological effect.

Many additional studies will be required to illuminate the mechanisms of action that account for the data reviewed in this monograph. We are attempting to extend these studies with direct

receptor binding experiments on rat brains following the in vivo

administration of these ligands. Additionally, we are investigating the responses using more recently available κ agonists

e.g., U-50,488) and δ agonists (e.g., the penicillamine

derivatives).

What is the functional significance of these findings? We suggest

that the classically accepted independence of multiple opioid receptor subtypes must be reconsidered. Although evidence has been

presented to suggest that at least μ and δ receptors are differentially distributed within the brain (Goodman et al. 1980), others

184
have shown that μ and δ binding sites may represent interconvertible components of low-affinity binding (Bowen et al. 1981). Additionally, it has been demonstrated, using whole tissue target size

analysis (radiation inactivation), that κ, μ, and δ "receptors"

all have about the same molecular weight (approximately 90,000

Daltons) (Ott et al. 1983). Of course, biochemical analysis of

extracted and partially purified receptors in various laboratories

reveals differences in molecular weights (Gioannini et al. 1985).

However, in the biological milieu, these opiate "receptors" may

exist as collaborative complexes, much as those described for the

subunits of the nicotinic-cholinergic receptor.

Thus, anatomical, biochemical, behavioral, and physiological

evidence is available to indicate that multiple opioid "receptors"

may instead be multiple opioid "binding sites" which interact as

part of the same macromolecular cluster or complex.

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ACKNOWLEDGMENTS

Ms. Jean Paige assisted in the preparation of this manuscript.

187
AUTHORS

John W. Holaday, Ph.D.

Frank C. Tortella, Ph.D.

Rhoda Maneckjee, Ph.D.

Joseph B. Long, Ph.D.

Neuropharmacology Branch

Department of Medical Neurosciences

Division of Neuropsychiatry

Walter Reed Army Institute of Research

Washington, DC 20307-5100

188
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11 DRUGS AND DRIVING. Robert Willette, Ph.D., ed.

NCDAI out of stock

GPO Stock #017-024-00576-2 $5.50 NTIS PB #269 602/AS $16.95

12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine, M.D., and

Demetrios A. Julius, M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 084/AS $16.95

13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman, M.D., eds. NCDAI out of stock

GPO Stock #017-024-00592-4 $6 NTIS PB #269 175/AS $22.95

14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. NCDAI out of stock

GPO out of stock NTIS PB #271 279/AS $22.95

15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm. Sharp,

Ph.D., and Mary Lee Brehm, Ph.D., eds.

GPO out of stock NTIS PB #275 798/AS $28.95

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 357/AS $22.95

17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D., et

al., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 353/AS $28.95

190
18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 337/AS $16

19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #293 807/AS $28.95

20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #288 471/AS $22.95

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman, M.D., eds.

GPO Stock #017-024-00785-4 $7 NTIS PB #288 472/AS $28.95

22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

ANALGESICS, NARCOTIC ANTAGONISTS, AND HALLUCINOGENS. Gene Barnett,

Ph.D.; Milan Trsic, Ph.D.; and Robert Willette, Ph.D.; eds.

GPO out of stock NCOAI out of stock

NTIS PB #292 265/AS $35.50

23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A. Krasnegor,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00895-8 $6 NTIS PB #297 721/AS $22.95

24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP PAPERS

AND DISCUSSION. Jos. Steinberg. ed. NCDAI out of stock

GPO out of stock NTIS PB #299 009/AS $22.95

25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman A.

Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #80-112428 $22.95

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. (Reprint from 1979 Surgeon General's Report on Smoking and

Health.)

GPO out of stock NTIS PB #80-118755 $16.95

27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. L.S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00981-4 $9 NTIS PB #80-175482 $40.95

28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Willette, Ph.D., and

Gene Barnett, Ph.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #81-238875 $22.95

29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. NCDAI out of stock

GPO out of stock NTIS PB #80-178882 $16.95

191
30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

J. Lettieri, Ph.D.; Mollie Sayers; and Helen W. Pearson, eds.

NCDAI out of stock

GPO Stock #017-024-00997-1 $10 Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

ed.

GPO out of stock NTIS PB #80-215171 $22.95

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

Ph.D .; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz.

GPO out of stock NTIS PB #81-133746 $22.95

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen I

Szara, M.D., D.Sc., and Jacqueline P. Ludford, M.S., eds.

GPO Stock #017-024-01108-8 $5 NTIS PB #82-139106 $16.95

34 PROBLEMS 0F DRUG DEPENDENCE, 1980: PROCEEDINGS OF THE 42ND

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01061-8 $8 NTIS PB #81-194847 $34.95

35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed.

GPO out of stock NTIS PB #82-103417 $16.95

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF

MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y.

Ng, M.D., ed.

GPO out of stock NTIS PB #81-240913 $22.95

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

NCDAI out of stock

GPO Stock #017-024-01109-6 $7 NTIS PB #82-136961 $28.95

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri, Ph.D.,

and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01107-O $4.50 NTIS PB #82-148198 $16.95

39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D.

GPO Stock #017-024-01097-9 $5.50 NTIS PB #82-147372 $22.95

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin. M.D., Ann Pollinger. Ph.D., Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #82-133117 $16.95

41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

Not available from GPO NTIS PB #82-190760 $40.95

192
42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $16.95

43 PROBLEMS OF DRUG DEPENDENCE, 1982: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #B3-252-692/AS $40.95

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude. Ph.D., and Jacqueline P. Ludford, M.S., eds. A

RAUS Review Report.

GPO Stock #017-024-01202-5 $4 NTIS PB #85-150563/AS $16.95

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01191-6 $2.75 NTIS PB #84-184670/AS $11.95

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D., and Jack E.

Henningfield, Ph.D., eds.

GPO Stock #017-024-01192-4 $4.25 NTIS PB #84-184688/AS $16.95

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01180-1 $5.50 NTIS PB #85-159663/AS $22.95

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

John Grabowski, Ph.D., and Catherine S. Bell, M.S., eds.

GPO Stock #017-024-01181-9 $4.50 NTIS PB 84-145-184 $16.95

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $34.95

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed.

GPO Stock #017-020-01214-9 $4.50 NTIS PB 85-150381/AS $16.95

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $16.95

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #017-024-0204-1 $4.25 NTIS PB 85-150373/AS $16.95

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

Ph.D., and Sharon M. Hall, Ph.D., eds.

GPO Stock #O17-024-01266-1 $3.50

193
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed. NCDAI out of stock

GPO Stock #O17-024-01213-1 $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #017-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S.. and Louise G. Richards, Ph.D., eds.

GPO Stock #017-024-01246-7 $4.25

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W., ed.

GPO Stock #017-024-01247-5 $4.25

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert. M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang,

Ph.D., and Charles C. Lee, Ph.D., eds.

GPO Stock #017-024-01257-2 $3.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL PERSPECTIVES.

Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S., eds.

GPO Stock #017-024-01258-1 $5

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M. Brown,

Ph.D.. and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01260-2 $3.50

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

AOOLESCENTS. Catherine S. Bell, M.S., and Robert Battjes,

O.S.W.. eds.

GPO Stock #017-024-01263-1 $5.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed

GPO Stock #017-024-01281-5 $6.50

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A. Ray, Ph.D.,

and Monique C. Braude, Ph.D., eds.

GPO Stock #017-024-01283-1 $3.25

194
66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND ALCOHOLISM.

Monique C. Braude. Ph.D., and Helen M. Chao, Ph.D. eds.

GPO Stock #017-024-01291-2 $3.50

67 PROBLEMS OF DRUG DEPENDENCE, 1985. PROCEEDINGS OF THE 47TH ANNUAL

SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG DEPENDENCE,

INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01292-1 $18.00

68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS OF ABUSE.

Monique C. Braude, Ph.D., and Harold M. Ginzburg, M.D.,J.D.,M.P.H.,

eds.

GPO Stock #017-024-01296-3 $6.50

IN PRESS OR PREPARATION

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

Gene Barnett, Ph.D.; and Richard L. Hawks, Ph.D., eds.

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY, BIOSYNTHESIS, AND

ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M. Brown,

Ph.D.; Doris H. Clouet, Ph.D.; and David P. Friedman, Ph.D., eds.

195

GPO : 1986 0 - 163-314 QL 3
DHHS Publication No. (ADM) 86-1462

Printed 1986