# Behavioral Consequences of Delta-Opioid Receptor Activation in the Periaqueductal Gray of Morphine Tolerant Rats

Authors: Michael M. Morgan; Michelle D. Ashley; Susan L. Ingram; MacDonald J. Christie
Journal: Neural Plasticity
 (2009)

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## Abstract

Chronic morphine administration shifts delta-opioid receptors (DORs) from the cytoplasm to the plasma membrane. Given that microinjection of morphine into the PAG produces antinociception, it is hypothesized that the movement of DORs to the membrane will allow antinociception to the DOR agonist deltorphin II as a way to compensate for morphine tolerance. Tolerance was induced by twice daily injections of morphine (5, 10, or 20 mg/kg, subcutaneous) for 3.5 days. Microinjection of deltorphin into the vPAG 6 hours after the last morphine injection produced a mild antinociception that did not vary in a consistent manner across morphine pretreatment doses or nociceptive tests. In contrast, deltorphin caused a decrease in activity in morphine tolerant rats that was associated with lying in the cage. The decrease in activity and change in behavior indicate that chronic morphine administration alters DORs in the vPAG. However, activation of these receptors does not appear to compensate for the decrease in antinociception caused by morphine tolerance.


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## Body

## 1. Introduction

Opioid receptors in the periaqueductal gray
(PAG) contribute to a wide range of behaviors. These include nociceptive
modulation, cardiovascular regulation, thermoregulation, and locomotor activity
[1–5]. Although mu-opioid receptors (MOR) are known to contribute to
PAG mediated antinociception [6, 7], less is known about the contribution of delta-opioid receptors
(DORs). Although antinociception has been produced by the administration of DOR
agonists into the PAG, these effects are mild compared to the antinociception
produced by MOR agonists [8–10].DORs are particularly interesting because the expression
of these receptors is surprisingly dynamic. Chronic treatment with morphine
causes the spinal density of DORs to shift from the cytoplasm to the plasma
membrane [11, 12]. A similar shift in DORs from the cytoplasm [13] to the plasma membrane appears to occur in the PAG. Swim stress causes an
increase in DOR density in the plasma membrane of PAG neurons [14]. In vitro recordings show that DOR agonists do not alter
GABAergic synaptic transmission in PAG neurons from drug-naive animals [15–17], but inhibit GABAergic IPSCs in mice treated chronically with
morphine [18].The behavioral significance of enhanced DOR
expression in the PAG has not been characterized. The increased expression of
DOR in the PAG of morphine tolerant rats could be a compensatory mechanism for
the loss of antinociception at the mu-opioid receptor. Increased expression of
DORs in the spinal cord has been shown to enhance the antinociceptive effect of
intrathecal administration of the DOR agonist deltorphin II [19].The objective of the present study was to
determine the behavioral consequences of activating DORs in the PAG following
induction of morphine tolerance. Given the widespread effects mediated by the
PAG, mobilization of DORs to the plasma membrane could contribute to a wide
range of behaviors. The enhanced antinociceptive effects of DOR agonists at the
spinal level [19] suggest that the administration of DOR agonists into the vPAG of
morphine tolerant rats will produce antinociception. This hypothesis will be
tested by examining the antinociceptive and locomotor effects of microinjecting
the DOR agonist deltorphin into the vPAG of rats made tolerant to morphine.


## 2. Methods

Male Sprague-Dawley rats (240–360 g) were
anesthetized with pentobarbital and implanted with a guide cannula aimed at the
ventrolateral PAG using stereotaxic techniques (from lambda: AP  =  +1.2 mm, ML  =  0.6 mm, and DV  =  −4.6 mm). The guide cannula was 9 mm long and affixed to two
screws in the skull with dental cement. Rats were handled daily for one week
following surgery. All injections and testing were conducted during
the dark phase of a 12-hour light/dark cycle in a dimly illuminated room.
Experiments were conducted in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory animals. Efforts were made to minimize
the number and potential suffering of the experimental subjects.


### 2.1. Materials

Nociception
was assessed using the hot plate, tail withdrawal, and formalin tests. The hot-plate
test (IITC, Woodland Hills, Calif, USA) consisted of measuring the latency for
a rat to lick a hind paw when placed on a 52°C plate. Tail withdrawal measured the latency to
move the tail when placed in 52°C water. The formalin test consisted of rating
pain behavior on a 0–3 scale following
injection of formalin (2% in 50 ul) into the plantar surface of the hind paw [20]. The values on this
scale are 0  =  normal behavior; 1  =  paw touches the ground without bearing
weight; 2  =  paw does not touch the ground; 3  =  paw is above the ground and
licked.


### 2.2. Microinjection Procedure

Four
days after surgery, an injection cannula was inserted through the guide
cannula, but no drug was injected. This process habituates rats to the
injection procedure and diminishes behavioral effects produced by cell damage
on the test day. Testing began one week following surgery, deltorphin II (1μg/0.5 μl)
or saline was microinjected into the vPAG. An 11 mm injection cannula was
inserted into the guide cannula while the rat was gently restrained by hand.
The injection cannula extended 2 mm beyond the end of the guide cannula. Drugs
were injected at a rate of 0.1 ul/10 s. The injection cannula remained in place
an additional 20 seconds to minimize drug flow up the cannula track. The stylet
was reinserted into the guide cannula and the rat was returned to its home
cage.Experiment 1. Repeated Morphine Injections
The objective of this experiment was to
determine the behavioral effects of microinjecting deltorphin into the vPAG in
rats made tolerant to repeated subcutaneous injections of morphine. Morphine
(5, 10, or 20 mg/kg) or saline (1 ml/kg)
was administered twice a day (at 9:30 and 15:00) for 3.5 days. Nociception was assessed with
the hot plate and tail flick tests 30 minutes after the injection on trials 1
and 7, but not after injections on trials 2–6. This procedure
limits changes in nociception from repeated testing [21, 22]. Six hours after the last subcutaneous injection, all rats were
injected with deltorphin (1 μg/0.5 μl) into the vPAG. Nociception was assessed
using the hot-plate test 20 minutes later. A subset of these rats was tested
again on the hot plate 50 minutes after deltorphin administration (N  =  9, 5,
10, and 8 for groups tested with saline, 5, 10, and 20 mg/kg of morphine, resp.).
Nociception was assessed using the formalin test in the other rats (N  =  8, 6, and
9 for groups tested with saline, 10, and 20 mg/kg of morphine, resp.).
Locomotor activity was assessed for 30 minutes
beginning immediately after the 20 minutes hot-plate test. Activity
was assessed by placing the rat into a chamber (25.1  ×  47 cm) with 7 photobeams
spaced 5.1 cm apart (San Diego Instruments, San Diego, Calif, USA). The average
number of photobeams disrupted each minute was measured and averaged over 10
minute intervals for 30 minutes. The behavior of the rat was examined every 5 minutes during the
locomotor test in an attempt to determine the reason for the changes in
locomotion (e.g., grooming, sleeping, and freezing). Normal behavior was
defined as walking, sniffing, and grooming.Experiment 2. Continuous Morphine Administration
Rats were surgically implanted with a guide
cannula aimed at the vPAG as described in Experiment 1. One week later,
tolerance was induced by implanting two 75 mg morphine pellets under the skin
of the upper back while rats were briefly anesthetized with halothane. Control
rats were implanted with two placebo pellets. Nociception was assessed using
the hot-plate test 2 hours following pellet implantation.
Rats were returned to the test room 3 days after
pellet implantation and allowed to habituate for 30 minutes. Nociception was
assessed at the end of this period using the hot-plate test to determine
whether tolerance had developed. Following this baseline test, both morphine
and placebo-treated rats were injected with deltorphin (1 ug/0.5 ul) into the
vPAG. Rats were returned to their cage immediately following the injection.
Nociception was assessed using the hot-plate test 30 and 60 minutes after the
deltorphin microinjection.


### 2.3. Histology

Following testing, rats
were given an overdose of halothane (Sigma, St. Louis, Mo, USA). The
microinjection site was marked by injecting cresyl violet (0.2μl) into the PAG. The brain was removed, placed
in formalin (10%), sectioned coronally (50 μm), and viewed under a microscope to localize
the injection site [23]. Only rats with
injection sites in or immediately adjacent to the vPAG were included in data
analysis.


### 2.6. Data Analysis

The effects of morphine pretreatment were compared to
saline or placebo-treated controls using at-test or analysis of
variance. The Bonferroni and Tukey tests were used for post hoc comparisons. Statistical significance was defined as a probability of less than .05.


## 2.1. Materials

Nociception
was assessed using the hot plate, tail withdrawal, and formalin tests. The hot-plate
test (IITC, Woodland Hills, Calif, USA) consisted of measuring the latency for
a rat to lick a hind paw when placed on a 52°C plate. Tail withdrawal measured the latency to
move the tail when placed in 52°C water. The formalin test consisted of rating
pain behavior on a 0–3 scale following
injection of formalin (2% in 50 ul) into the plantar surface of the hind paw [20]. The values on this
scale are 0  =  normal behavior; 1  =  paw touches the ground without bearing
weight; 2  =  paw does not touch the ground; 3  =  paw is above the ground and
licked.


## 2.2. Microinjection Procedure

Four
days after surgery, an injection cannula was inserted through the guide
cannula, but no drug was injected. This process habituates rats to the
injection procedure and diminishes behavioral effects produced by cell damage
on the test day. Testing began one week following surgery, deltorphin II (1μg/0.5 μl)
or saline was microinjected into the vPAG. An 11 mm injection cannula was
inserted into the guide cannula while the rat was gently restrained by hand.
The injection cannula extended 2 mm beyond the end of the guide cannula. Drugs
were injected at a rate of 0.1 ul/10 s. The injection cannula remained in place
an additional 20 seconds to minimize drug flow up the cannula track. The stylet
was reinserted into the guide cannula and the rat was returned to its home
cage.Experiment 1. Repeated Morphine Injections
The objective of this experiment was to
determine the behavioral effects of microinjecting deltorphin into the vPAG in
rats made tolerant to repeated subcutaneous injections of morphine. Morphine
(5, 10, or 20 mg/kg) or saline (1 ml/kg)
was administered twice a day (at 9:30 and 15:00) for 3.5 days. Nociception was assessed with
the hot plate and tail flick tests 30 minutes after the injection on trials 1
and 7, but not after injections on trials 2–6. This procedure
limits changes in nociception from repeated testing [21, 22]. Six hours after the last subcutaneous injection, all rats were
injected with deltorphin (1 μg/0.5 μl) into the vPAG. Nociception was assessed
using the hot-plate test 20 minutes later. A subset of these rats was tested
again on the hot plate 50 minutes after deltorphin administration (N  =  9, 5,
10, and 8 for groups tested with saline, 5, 10, and 20 mg/kg of morphine, resp.).
Nociception was assessed using the formalin test in the other rats (N  =  8, 6, and
9 for groups tested with saline, 10, and 20 mg/kg of morphine, resp.).
Locomotor activity was assessed for 30 minutes
beginning immediately after the 20 minutes hot-plate test. Activity
was assessed by placing the rat into a chamber (25.1  ×  47 cm) with 7 photobeams
spaced 5.1 cm apart (San Diego Instruments, San Diego, Calif, USA). The average
number of photobeams disrupted each minute was measured and averaged over 10
minute intervals for 30 minutes. The behavior of the rat was examined every 5 minutes during the
locomotor test in an attempt to determine the reason for the changes in
locomotion (e.g., grooming, sleeping, and freezing). Normal behavior was
defined as walking, sniffing, and grooming.Experiment 2. Continuous Morphine Administration
Rats were surgically implanted with a guide
cannula aimed at the vPAG as described in Experiment 1. One week later,
tolerance was induced by implanting two 75 mg morphine pellets under the skin
of the upper back while rats were briefly anesthetized with halothane. Control
rats were implanted with two placebo pellets. Nociception was assessed using
the hot-plate test 2 hours following pellet implantation.
Rats were returned to the test room 3 days after
pellet implantation and allowed to habituate for 30 minutes. Nociception was
assessed at the end of this period using the hot-plate test to determine
whether tolerance had developed. Following this baseline test, both morphine
and placebo-treated rats were injected with deltorphin (1 ug/0.5 ul) into the
vPAG. Rats were returned to their cage immediately following the injection.
Nociception was assessed using the hot-plate test 30 and 60 minutes after the
deltorphin microinjection.


## 2.3. Histology

Following testing, rats
were given an overdose of halothane (Sigma, St. Louis, Mo, USA). The
microinjection site was marked by injecting cresyl violet (0.2μl) into the PAG. The brain was removed, placed
in formalin (10%), sectioned coronally (50 μm), and viewed under a microscope to localize
the injection site [23]. Only rats with
injection sites in or immediately adjacent to the vPAG were included in data
analysis.


## 2.6. Data Analysis

The effects of morphine pretreatment were compared to
saline or placebo-treated controls using at-test or analysis of
variance. The Bonferroni and Tukey tests were used for post hoc comparisons. Statistical significance was defined as a probability of less than .05.


## 3. Results

Experiment 1. Repeated Morphine Injections
Systemic administration of high doses of morphine (5,
10, and 20 mg/kg) produced maximal antinociception on trial 1 (see Figure1). A
significant decrease in antinociception was evident with repeated
administration from trial 1 to 7 (F(1,56) = 53.446, P  <  .05). The
magnitude of the decrease in antinociception was dose dependent (F(3,56) = 5.536, P  <  .05). That is, the lowest dose (5 mg/kg) produced the
least antinociception and the highest dose (20 mg/kg) produced the greatest
antinociception in morphine tolerant rats.
Microinjection of deltorphin into the vPAG of
morphine naive rats
produced a slight increase in hot-plate latency. Rats pretreated with saline
showed a significant increase in hot-plate latency following microinjection of
deltorphin from 10.8 (see trial 7 in Figure1) to 14.5 seconds (20-minute test in Figure 2)
(one-tailed t(18)  =  1.925, P  <  .05).
The effect of microinjecting deltorphin into the
vPAG of morphine tolerant rats varied with the pretreatment dose (see Figure2). A 2  ×  2 ANOVA revealed a significant difference in hot-plate latency
between the pretreatment groups (F(3,84)  =  3.203, P  <  .05), but no
difference in hot-plate latency between the 20- and 50-minute tests (F(1,84)  =  0.866; P  >  .05).
The difference between groups was caused by a slight increase in the hot-plate
latency of rats pretreated with 20 mg/kg of morphine and a slight decrease in
latency in rats pretreated with 10 mg/kg (see Figure 2). Although the
difference between these groups was statistically significant (Bonferroni, t  =  2.420, P  <  .05), neither the 10 mg/kg (t  =  0.96 and 1.15 for 20 and 50
minutes, resp.) nor the 20 mg/kg (t  =  1.49 and 0.87 for 20 and 50 minutes, resp.)
groups differed from the saline-pretreated group. Moreover, these changes in
nociception were quite small compared to the antinociception produced by
systemic administration of morphine on trial 7 (26.2±2.7 and 31.3±2.7 seconds
following 10 and 20 mg/kg doses of morphine).
A similar difference between pretreatment groups was evident
when nociception was assessed with the tail flick test 50 minutes after
deltorphin administration (F(2,52)  =  8.699,P  <  .05). Microinjection
of deltorphin into the PAG of rats pretreated with 20 mg/kg of morphine caused
a small, but significant, increase in tail flick latency (4.4±0.2 seconds)
compared to rats pretreated with saline (3.5±0.2 seconds) or 10 mg/kg of
morphine (3.4±0.2 seconds) (Tukey test, P  <  .05 for both
comparisons).
Nociception was assessed using the formalin test
in the rats not tested on the hot plate at 50 minutes. Formalin was injected 5
minutes following the 20 minute hot-plate test so the first and second phases [20] could be assessed 25–29 and 40–44 minutes after
microinjection of deltorphin. In contrast to the hot-plate test, pain ratings
on the formalin test did not differ between the pretreatment groups on either
the first (mean  ratings  =  1.65, 1.60, and 1.73) or the second (mean ratings  =  0.40, 0.63, and 0.67) phases (F(2,20)  =  0.272, P  =  .76).
Locomotor activity was assessed during the 30
minutes following the 20 minute hot-plate test in the subset of rats not
injected with formalin. Microinjection of deltorphin into the vPAG caused a
significant decrease in activity in the morphine compared to the saline-pretreated
rats (F(2,29)  =  6.84,P  <  .05). This decrease was similar in rats
pretreated with 10 and 20 mg/kg of morphine (see Figure 3). The greatest
decrease in activity was evident at the latest time period (41–50 minutes) for
both pretreatment groups.
The decrease in activity was accompanied by a
change in behavior in which rats tended to crouch or lie down in the activity
chamber. An increase in the number of rats crouching or lying increased in rats
pretreated with 10 and 20 mg/kg of morphine beginning approximately 25 minutes after the deltorphin
microinjection (see Table1). This shift in the number of rats displaying
normal exploratory behavior to crouching and lying increased further during the
last 15 minutes of the test (40–54 minutes after
the deltorphin microinjection).Table 1
Percentage of
rats showing normal and abnormal behavior following deltorphin microinjection
into the vPAG in morphine-pretreated rats.
Crouching/lyingPretreatment25–39 minutes40–54 minutesSaline0% (0/6)0% (0/6)Morphine 10 mg20% (1/5)67% (6/9)Morphine 20 mg36% (4/11)40% (6/15)Note: sample sizes are shown in parentheses.Figure 1
Tolerance to
antinociception develops with repeated injections of morphine. The hot-plate
latency following morphine administration on trial 7 was significantly less
than on trial 1 for the morphine-treated groups (F(1, 39)  =  31.38,P  <  .01). Antinociception was still evident with the administration of large doses
of morphine (10 and 20 mg/kg), but not as pronounced as on trial 1. Large
sample sizes were used for most groups (N  =  19, 5, 19, and 17 for rats
pretreated with saline and morphine at 5, 10, and 20 mg/kg, resp.) because
subsets of these rats were subsequently tested using either the formalin test
or repeated hot-plate tests.Figure 2
Changes in
nociception following microinjection of deltorphin into the vPAG. Nociception
was assessed 20 and 50 minutes after deltorphin microinjection into the vPAG of
rats made tolerant to morphine. Pretreatment with morphine produced a slight decrease
in hot-plate latency in rats made tolerant to 10 mg/kg of morphine compared to
the slight increase in rats made tolerant to 20 mg/kg of morphine, although
neither of these changes differed significantly from the effects of deltorphin
administration in saline-pretreated controls. All rats were tested on the hot
plate 20 minutes after deltorphin microinjection (see sample sizes in the
caption of Figure1), but only a subset was tested at 50 minutes for the saline (N  =  9), 5
(N  =  5), 10 (N  =  10), and 20 (N  =  8) mg/kg morphine groups.Figure 3
Decrease in
locomotion following microinjection of deltorphin into the vPAG. Microinjection
of deltorphin (1μg/0.5 μl) into the vPAG caused a decrease in locomotion in
rats pretreated with morphine (N  =  13 and 9 for rats pretreated with 10 and 20 mg/kg) compared to rats pretreated with saline (N  =  11). This decrease in
activity is caused by a change in normal behavior to lying and crouching in the
cage following deltorphin administration (see Table 1).Experiment 2. Continuous Morphine Administration
Implantation of morphine pellets produced an
increase in hot-plate latency compared to placebo-treated rats when assessed at
1 hour (36.1±3.9 versus 20.5±2.4 seconds; t(8)  =  3.865, P  <  .05).
Tolerance to the antinociceptive effect of morphine was evident by day 4 as
indicated by a decrease in hot-plate latency to 18.8±1.8 seconds.
Microinjection of deltorphin into the PAG caused a modest, but significant,
increase in hot-plate latency compared to baseline latency (F(2,16)  =  5.806, P  <  .05). Although the baseline hot-plate latency for rats treated with
morphine pellets was greater than in placebo-treated rats (see Figure 4), this
difference did not reach statistical significance using the Bonferroni test (t  =  1.482 nanoseconds). However, microinjection of deltorphin into the vPAG of
placebo-treated rats caused a significant increase in hot-plate latency at the
60-minute time point compared to the baseline (Bonferroni,
t  =  3.044. P  <  .05). There was no difference in mean hot-plate latency following deltorphin
administration between morphine and placebo-treated rats (F(1,8)  =  0.791, P  >  .05), suggesting
that deltorphin administration did not increase antinociception beyond what was
already present in rats with morphine pellets.Figure 4
No change in
nociception following deltorphin microinjection into the vPAG in rats treated
with continuous morphine. Rats implanted with morphine pellets 3 days prior to
this test had higher baseline hot-plate latency than rats implanted with
placebo pellets indicating that tolerance to the antinociceptive effects was not complete.
Microinjection of deltorphin into the vPAG caused an increase in hot-plate
latency in rats with placebo pellets (N  =  5), but did not increase the hot-plate
latency for rats with morphine pellets (N  =  5) above the baseline latency. That
is, there was no additional antinociception by injecting deltorphin into the
vPAG of rats receiving morphine.


## 4. Discussion

The
present data demonstrate that repeated morphine administration alters the response
of vPAG neurons to the DOR agonist deltorphin. Although PAG neurons contribute
to a wide range of behaviors [24], the change in response
to deltorphin microinjection was specific to locomotor activity. Microinjection
of deltorphin into the vPAG produced a consistent decrease in activity in rats
pretreated with morphine. This decrease in activity was caused by a drastic
change in behavior from exploring and grooming to crouching and lying along the
edge of the cage. In contrast, microinjection of deltorphin produced a mild
antinociception that was not altered in a consistent manner by prior morphine
administration.MOR
agonists such as morphine are the most effective treatment for pain. The
descending modulatory system that runs from the PAG to rostral ventromedial
medulla (RVM) to spinal dorsal horn plays an important role in the
antinociceptive effects of both MOR and DOR agonists [9, 25–28]. The antinociception
produced by microinjection of DOR agonists into the PAG is weak compared to
morphine administration [9, 10].
However, DORs are located in the PAG [13] and these receptors
appear to move from the cytoplasm to the plasma membrane following stress [14]. The density of DORs on
the membrane has also been
shown to increase in spinal neurons following chronic exposure to morphine [11, 12].The present data show
that microinjection of deltorphin into the vPAG had modest effects on
nociception. Rats pretreated with 10 mg/kg of morphine showed a slight
hyperalgesia compared to saline-pretreated rats injected with deltorphin into
the vPAG. This effect seems to be dose and test dependent. Rats pretreated with
20 mg/kg of morphine showed a slight increase in hot-plate latency following
deltorphin microinjection. No changes in nociception were evident on the
formalin test. The lack of effect of deltorphin in modulating nociception is
surprising given that spinal administration of deltorphin following chronic
morphine administration produces antinociception [11].The
lack of a consistent change in nociception following deltorphin administration
could be caused by an inability of the morphine administration procedure to
mobilize DORs to the plasma membrane. Although possible, this explanation seems
unlikely given that a decrease in activity was produced by microinjection of
deltorphin into the PAG. Moreover, we used two different procedures to induce
tolerance (repeated injections and continuous administration) that closely
match previous studies reporting changes in DORs [11, 12, 18]. Finally,
mobilization of DORs to the plasma membrane is associated with morphine
tolerance and the rats in the present study showed clear signs of tolerance to
the antinociceptive effects of morphine.In vitro electrophysiological
recordings reveal DOR-mediated inhibition of GABAergic IPSCs in tissue from
mice pretreated with morphine [18], but no effect of DOR
agonists in PAG slices from animals that have not been exposed to morphine [15–17]. This inhibition of GABA input
is similar to the effect produced by administration of MOR agonists into the
PAG [29]. These data suggest
that microinjection of DOR agonists into the PAG of morphine tolerant rats
should produce antinociception. However, the present data show no consistent
antinociceptive effect following deltorphin microinjection into the PAG of
morphine tolerant rats.In
contrast, microinjection of deltorphin into the vPAG of morphine pretreated-rats
caused a clear and consistent decrease in locomotor activity. Acute
administration of morphine into the vPAG also produces a decrease in activity [4]. Thus, it appears that
DORs compensate for the locomotor, but not the antinociceptive effects
associated with morphine tolerance in the vPAG. The immobility produced by
morphine microinjection into the vPAG appears to be part of a defensive
freezing response [30–33]. However, the decrease
in activity produced by microinjection of deltorphin into the vPAG reported
here does not appear to be caused by fear-induced freezing. Microinjection of
deltorphin caused rats to crouch and lay along the edge of the cage as if the
rats were ill or dysphoric. This effect appears to be consistent with previous
research showing that deep tissue pain sufficient to induce recuperative
behavior activates vPAG neurons [34]. Given that stress
increases the density of DORs on the plasma membrane [14], activation of these
receptors may contribute to recuperative behavior.One hypothesis is that
the recuperative behavior mediated by the vPAG is part of a coordinated response triggered by
severe hemorrhage that includes hypotension. Severe blood loss has been shown
to activate neurons in the vPAG [35], and inactivation of
the vPAG [2, 36]
or microinjection of the DOR antagonist naltrindole into the PAG [37] blocks the hypotension
produced by hemorrhage. Future studies are needed to determine whether
activation of DORs in the PAG alters blood pressure.


## 5. Conclusion

The decrease in
locomotor activity caused by microinjection of deltorphin into the vPAG of
morphine tolerant rats is consistent with previous data showing that DOR
density on the plasma membrane increases following chronic morphine
administration. PAG DORs could contribute to morphine tolerance [38–40], behavioral changes
related to stress [14], or hypovolemic shock [35, 37], but
do not appear to contribute to antinociception. That is microinjection of
deltorphin into the PAG did not produce antinociception regardless of how
tolerance was induced (repeated injections or continuous administration), test
used to assess nociception (hot plate, tail flick, and formalin tests), or test
times (20 and 50 minutes).



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*Source: 516328-2009-02-25.xml*