bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of melatonin secretion in a photoreceptive pineal organ: an in vitro study in the pike
Authors: Falcon J , Marmillon JB , Claustrat B , Collin JP
Published: March 2018

The pineal organ (or pineal) of a teleost fish, the pike, contains typical (cone-like) and modified photoreceptor cells. Both are involved in indole metabolism, including melatonin production. How photoperiod controls melatonin biosynthesis in organs containing mainly photoreceptor cells, remains unclarified. To tackle this question we have used cultured pike pineal organs to investigate the variations in (1) the activity of the arylalkylamine-N-acetyltransferase (NAT), which is involved in the biosynthesis of melatonin, in static culture and (2) melatonin release in a perifusion system. Serum melatonin was also quantified in pike kept under a 24 hr light/dark (LD) cycle. Under LD conditions, NAT activity, melatonin release, and serum melatonin levels were high during the scotophase and low during the photophase. High- amplitude rhythms in NAT activity and melatonin release were maintained during three 24 hr cycles in constant darkness and a low-amplitude rhythm of NAT persisted in constant illumination. Midnight illumination induced a dramatic decrease of NAT activity and melatonin release. Darkness at midday did not induce a rise of the melatonin release, which occurred only at the onset of the subjective scotophase. From the present data it is strongly suggested that the pineal of the pike contains a circadian oscillator--synchronized by the photoperiod--which generates the rhythms of NAT activity and of melatonin release. Melatonin release, which reflects the rhythmic activity of NAT, might largely contribute to the melatonin circulating levels. The circadian oscillations observed under constant conditions suggest that the oscillator might behave differently in the pike, compared with intrapineal oscillators of 2 other species (lizard and chicken) under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of DIDS on the chick retinal pigment epithelium. I. Membrane potentials, apparent resistances, and mechanisms
Authors: Gallemore RP , Steinberg RH
Published: March 2018

While little is known about the transport properties of the retinal pigment epithelium (RPE) basal membrane, mechanisms for anion movement across the basal membrane appear to be present (Miller and Steinberg, 1977; Hughes et al., 1984; Miller and Farber, 1984). This work examines the electrophysiological effects of the anion conductance blocker, 4,4′- diisothiocyanostilbene-2,2′-disulfonate (DIDS) on the basal membrane of an in vitro preparation of chick retina-RPE-choroid. DIDS (10–125 microM), added to the choroidal bath, decreased the transtissue potential by decreasing the potential across the RPE. Intracellular RPE recordings showed that DIDS affected the membrane potential in 2 phases, initially hyperpolarizing the basal membrane and then, after prolonged exposure, depolarizing the apical membrane. Resistance assessment by transtissue current pulses and intracellular c-wave recordings suggested that DIDS increased basal membrane resistance (Rba) during the first phase and increased apical membrane resistance (Rap) during the second phase. Measurements of intracellular Cl- activity (aiCl) showed that Cl- was actively accumulated by the chick RPE since it was distributed above equilibrium across both the apical and basal membranes. Perfusion of the basal membrane with 50 microM DIDS significantly increased aiCl-. The DIDS-induced basal membrane hyperpolarization, apparent increase in Rba, and increase in aiCl- are all consistent with Cl- -conductance blockade. During the second phase, apical membrane responsiveness to the light-evoked decrease in subretinal [K+]o (Oakley, 1977) was reduced an average of 58%. This finding, given the second-phase apical membrane depolarization and apparent increase in Rap, is consistent with a decrease in apical membrane K+ conductance.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrophysiological correlates of presynaptic alpha 2-receptor- mediated inhibition of norepinephrine release at locus coeruleus synapses in dentate gyrus
Authors: Washburn M , Moises HC
Published: March 2018

Despite an abundance of evidence that presynaptic alpha 2-adrenergic receptors mediate inhibitory control of the release of norepinephrine (NE) from the terminals of locus coeruleus (LC) neurons, few studies have demonstrated the physiological significance of this “autoreceptor”- mediated inhibition on NE-mediated synaptic activity within the mammalian brain. This question was addressed by examining the effects of systemic administration of alpha 2-adrenergic agonists and antagonists on the ability of LC stimulation to augment the population spike recorded in the dentate gyrus in response to activation of the perforant path (PP). Extracellular field potentials were recorded in the cell body and dendritic layer of dentate gyrus following single shocks of the entorhinal cortex in halothane-anesthetized rats. Stimulation of the ipsilateral LC 35 msec prior to PP activation produced a short-term enhancement of the population spike amplitude recorded in the cell layer but did not significantly alter dendritic potentials. The effects of LC stimulation were blocked by administration of the beta-adrenergic antagonist propranolol but not the alpha 2-antagonist idazoxan and were abolished by pretreatment of animals with the catecholamine neurotoxin 6-hydroxydopamine. Administration of clonidine reversibly abolished the enhancement produced by LC conditioning. The effect of clonidine was dose dependent and was blocked by administration of idazoxan, which restored the LC potentiative effect. Conditioning stimulation of LC noradrenergic axons in the dorsal bundle also potentiated the PP-evoked population spike, and this effect was equally sensitive to the depressant action of clonidine. In comparison, clonidine, in the range of dosages tested, did not significantly affect the potentials evoked in the dendritic or cell layer by presentations of unconditioned PP test stimuli. We interpret these data to provide evidence for a functional impairment of LC-mediated physiological action on postsynaptic target cells as a result of presynaptic alpha 2-autoreceptor-mediated feedback inhibition of NE release.
Physiological and anatomical studies of the interactions between Purkinje cells and basket cells in the cat's cerebellar cortex: evidence for a unitary relationship
Authors: O'Donoghue DL , King JS , Bishop GA
Published: March 2018

Intracellular recordings have been obtained from neurons in lobule V of the cat's vermis, which were identified as basket cells following intracellular injections of HRP. Stimulation of the inferior cerebellar peduncle or peripheral nerves elicited an initial depolarizing and subsequent hyperpolarizing response. Neither potential could be graded with changes in stimulus intensity; both displayed all-or-none properties at threshold levels of stimulation. The depolarization and hyperpolarization were confirmed as an excitatory postsynaptic potential and an inhibitory postsynaptic potential (IPSP), respectively, on the basis of their response to intracellular injections of hyperpolarizing and depolarizing currents into the cell body. A possible source of the unitary IPSP is the Purkinje cell, via its recurrent axonal collaterals. To test this hypothesis, an electron microscopic analysis was carried out to define the synaptic relationships between the recurrent collaterals of an HRP-filled Purkinje cell and 3 basket cells. Serial section analysis reveals that collaterals from a single Purkinje cell contact several basket cells, but each basket cell received somatic input from only one Purkinje cell. These data provide an anatomical substrate for the unitary IPSP observed during intracellular recording from basket cells. The unitary nature of the Purkinje cell-basket cell interaction indicates that a very limited population of cortical neurons may be involved in local circuits that integrate afferent information in the cerebellar cortex.
Development of rat cerebellar Purkinje cells: electrophysiological properties following acute isolation and in long-term culture
Authors: Hockberger PE , Tseng HY , Connor JA
Published: March 2018

The objectives of this study were 2-fold: (1) to characterize the electrical properties of Purkinje cells (PCs) acutely isolated from rat cerebella at different stages of development, and (2) to compare these properties with those recorded from PCs grown in long-term culture. PCs under both conditions were identified with the aid of cell-specific immunocytochemical staining, and the electrical properties were analyzed using whole-cell-recording techniques. PCs acutely isolated during late embryonic and early postnatal periods displayed a progressive change in electrical properties. Between embryonic days 20 and 22 (stage 1), PCs were inexcitable, did not respond to glutamate, and displayed only small outward currents under voltage clamp. During postnatal days 1–4 (stage 2), current stimulation elicited nonovershooting action potentials, and small inward and outward currents were evoked under voltage clamp. Glutamate application depolarized the cells resulting in an increase in intracellular free calcium measured with fura-2. Stage 3 and 4 cells spanned postnatal days 5–9 and 10–14, respectively, and the cells showed progressively larger voltage-dependent conductances and greater sensitivity to glutamate. We found no evidence for either spontaneous or complex spikes in PCs isolated at any of these stages. In agreement with previous studies, we found that PCs dissociated from postnatal rats did not survive well in culture. On the other hand, PCs from embryonic rats cultured for 2–3 weeks in high-potassium, serum-supplemented medium developed extensive dendritic processes and excitability. Current stimulation or glutamate application elicited depolarizing waveforms reminiscent of climbing fiber-evoked responses in vivo. The results suggest that dendritic processes are important in the generation of complex spikes and that PC excitability can develop in the absence of the highly structured architecture of the intact cerebellum.
Fura-2 measurements of cultured rat Purkinje neurons show dendritic localization of Ca2+ influx
Authors: Hockberger PE , Tseng HY , Connor JA
Published: March 2018

The specific objectives of this study were the following: (1) to characterize the types of calcium currents in cultured PCs using whole- cell voltage-clamp techniques; (2) using fura-2 imaging techniques, to monitor intracellular Ca2+ levels during application of high potassium, glutamate, or glutamate analogs; and (3) to evaluate the types of calcium channels contributing to the calcium fluxes using pharmacological blocking agents. Voltage-clamp analysis of calcium currents proved to be difficult due to space-clamping problems. The latter was presumably due to the unfavorable geometry of cultured PCs. Nevertheless, we found no evidence for inward currents in cells bathed in TTX-TEA-BaCl2 saline. On the other hand, fura-2 measurements demonstrated that free Ca2+ levels were elevated in PCs following local application of either high-potassium saline or glutamate. When individual cells were injected with fura-2 and analyzed in TTX- containing saline, the Ca2+ elevation was usually greater in the dendrites. Since Ca2+ levels were not elevated in all dendrites of the same cell, the smaller responses in the soma wre not simply due to volumetric differences. Together with the voltage-clamp results, the fura-2 data indicate that calcium channels were localized to certain dendrites. Using selective calcium channel blockers, we found evidence for 2 types of calcium conductances in the dendrites of cultured PCs. The Ca conductance induced by high potassium was reduced in a dose- dependent manner by nifedipine (ED50 = 5 X 10(-7) M), indicating that a high-threshold voltage-dependent calcium channel was present. The Ca response to glutamate (or NMDA) was reduced by 2-amino-5- phosphonovaleric acid (ED50 = 10(-4) M), as well as by nifedipine or 10(-4) M LaCl3, indicating that both voltage-dependent and glutamate- coupled channels were opened by glutamate application.
Non-steady-state measurement of in vivo receptor binding with positron emission tomography: "dose-response" analysis
Authors: Perlmutter JS , Kilbourn MR , Welch MJ , Raichle ME
Published: March 2018

We previously developed a non-steady-state technique using positron emission tomography (PET) and the radioligand 18F-spiperone (18F-SP) for the measurement of in vivo radioligand-receptor binding in brain. The purpose of this investigation is to determine the sensitivity of this method to alterations in the apparent number of available specific binding sites. Nine studies were performed on the same baboon. The animal was pretreated with varying doses of unlabeled SP (15–600 micrograms) to compete for specific binding sites. The experimental procedure included measurement of regional cerebral blood flow, cerebral blood volume, and the protein binding of 18F-SP in arterial blood. At least 3.5 hr after pretreatment, no-carrier-added 18F-SP (containing less than 3 micrograms SP) was administered intravenously. Sequential PET scans and measurements of arterial-blood radioactivity due to radioligand and its labeled metabolites continued for 3 hr. A 3- compartment model representing the in vivo behavior of radioligand was used to analyze the data. As expected, we found that an index of binding called the combined forward rate constant (which equals the product of the apparent maximum number of available specific binding sites and the association rate constant of radioligand for receptor) declined with increasing dose of unlabeled SP. Other estimated variables including the dissociation rate constant did not change. This demonstrates that our non-steady-state method for estimating radioligand-receptor binding kinetics can detect a decrease in the apparent number of available specific binding sites. This is an important step in the validation of this in vivo receptor binding assay and its subsequent application.
Evidence for transsynaptic regulation of calmodulin-dependent cyclic nucleotide phosphodiesterase in cerebellar Purkinje cells
Authors: Balaban CD , Billingsley ML , Kincaid RL
Published: March 2018

Calmodulin-dependent phosphodiesterase (CaM-PDE) is selectively expressed in specific neuronal populations in adult rat brain. In cerebellar cortex, it is expressed at high levels in Purkinje cells (soma and dendrites). Climbing fiber ablation by intraperitoneal injections of 3-acetylpyridine resulted in a selective depression of cerebellar CaM-PDE expression using Western immunoblot procedures; neither calcineurin (calmodulin-dependent protein phosphatase) nor other calmodulin binding proteins, detected by biotinylated calmodulin overlays, were affected. Immunocytochemical staining of cerebellum revealed a loss of detectable CaM-PDE immunoreactivity in Purkinje cells, with no appreciable change in calcineurin immunoreactivity. Cerebral cortex was examined as a control for a direct effect of 3- acetylpyridine on CaM-PDE expression, independent of climbing fiber deafferentation. There were no detectable changes in CaM-PDE or calcineurin immunoreactivity in cortical pyramidal cells, and no changes were detected, either in Western blot analyses for CaM-PDE or calcineurin or in biotinylated calmodulin overlays. These data suggest that CaM-PDE expression in Purkinje cells is regulated transsynaptically by climbing fiber inputs.
The location and function of NMDA receptors in cat and kitten visual cortex
Authors: Fox K , Sato H , Daw N
Published: March 2018

The role of N-methyl-D-aspartate (NMDA) receptors in cat visual cortex was studied as a function of both layer and age by iontophoresis of the NMDA antagonist (D)-2-amino-5-phosphonovaleric acid (APV). Effects on both visual responses and spontaneous activity were observed. In superficial layers (II and III), D-APV reduced visual responses substantially at all ages. Iontophoresis of D-APV with 10 nA of ejecting current for 2–3 min was sufficient to reduce the response to approximately one third of control levels. The magnitude of the reduction did not vary with age. In granular and deep layers (IV, V, and VI), D-APV affected the visual response in young animals but only spontaneous activity in older animals. On average, visual responses were reduced to about half at 20–23 days of age and to about 75% at 4 weeks of age but in most cases were not significantly affected in adults. The rapid change in the functional effect of NMDA receptors over the third and fourth week in granular and deep layers suggests a role in development. There was a reasonable age correlation between the change in effect and the period of geniculocortical afferent segregation. Further experiments will be necessary to determine whether NMDA receptors are necessary for segregation to occur. The presence of an NMDA component to the visual response in the adult in layers II and III argues either that these layers retain some form of plasticity in the adult or that NMDA receptors play a role in the transmission of normal visual input to these layers.
Disuse-induced enhancement of Ia synaptic transmission in spinal motoneurons of the rat
Authors: Manabe T , Kaneko S , Kuno M
Published: March 2018

Monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in rat spinal motoneurons by stimulation of a muscle nerve are enhanced during the first few days after section of the muscle nerve before subsiding to subnormal levels. We have examined whether this biphasic alteration results from changes of different factors involved in the regulation of central synaptic function. We chronically blocked the conduction of the intact peripheral nerve with tetrodotoxin (TTX). Under this disuse condition, enhancement of monosynaptic EPSPs was fully expressed within 2 d, and synaptic function remained elevated for at least 3 d, even after resumption of the impulse activity following discontinuance of the TTX. The magnitude of synaptic enhancement and the minimal period of nerve block required for synaptic enhancement were both comparable to those observed following nerve section. Thus, the early synaptic enhancement produced by section of the peripheral nerve could be accounted for entirely by the deprivation of sensory impulse activity. However, a prolonged nerve block up to 6 d still maintained synaptic enhancement. This was in contrast with synaptic depression observed after the first 3–4 d following section of the peripheral nerve. The late synaptic depression can thus be attributed to changes in some factors other than sensory impulse activity. Therefore, sensory impulse activity and the peripheral axonal continuity of sensory fibers appear to be 2 distinct factors for the regulation of central synaptic function.
The monoclonal antibody B30 recognizes a specific neuronal cell surface antigen in the developing mesencephalic trigeminal nucleus of the mouse
Authors: Stainier DY , Gilbert W
Published: March 2018

A monoclonal antibody, B30, obtained with whole cells from embryonic brain as an immunogen, recognizes a neuronal cell surface antigen that appears only in 2 distinct systems in the developing mouse brain: the trigeminal system and the cerebellum. In the trigeminal system, B30 labels the surface of neurons, including their axons and their transient dendrites, in 2 groups of cells: the centrally located mesencephalic trigeminal nucleus and the peripheral trigeminal ganglion. Immunoreactivity is detectable during axon outgrowth, peaks around the seventh postnatal day, and disappears around 2 weeks after birth. In the cerebellum, B30 labels 2 layers of cells during development. Perinatally, and for about a week after birth, the layer of premigratory granule cells stains. After their maturation, Purkinje cells start to stain and by 12 d postnatally all the Purkinje cell bodies, their axons, and their dendritic trees show strong immunoreactivity. Subsequently, and in the adult, this staining is lost from some cells to reveal bands of antigen positive and negative Purkinje cells. Initial biochemical characterization of the epitope shows that it is carried on 2 minor gangliosides.
Contact-mediated loss of the nonsynaptic response to transmitter during reinnervation of an identified leech neuron in culture
Authors: Drapeau P , Melinyshyn E , Sanchez-Armass S
Published: March 2018

We have examined the modification of responses to transmitter during the reformation of a specific synapse by identified leech neurons in culture. Single pressure-sensitive mechanosensory (P) neurons in culture and the soma of this cell in vivo have 2 conductances that are activated by application of 5-HT: a Cl conductance and a monovalent cation conductance (gCations); synaptic release of 5-HT by a serotoninergic Retzius cell in vivo and in culture activates only the Cl conductance and not gCations in the P cell. We have characterized the loss of gCations in P cells by manipulating the culture conditions. When 5-HT was applied from a pipette, innervated P cells and P cells paired with Retzius cells that had not formed synapses had a gCations that was markedly reduced compared with gCations in the single P cell. When 5-HT was included in the culture medium in which single P cells were grown, gCations was not reduced. When Retzius-P cell pairs were treated with reserpine, which was shown to deplete the presynaptic neuron of 5-HT, gCations in the P cell was as low as in untreated pairs. Pairing aldehyde-fixed Retzius cells with untreated P cells also resulted in the loss of gCations. The Retzius cell had both types of receptors, but synapse formation did not affect gCations in the presynaptic neuron. The results demonstrate that the loss of the nonsynaptic response of the P cell to transmitter is due to contact between the neurons rather than to the release of transmitter or trophic factors.(ABSTRACT TRUNCATED AT 250 WORDS)
Beta 1- and beta 2-adrenergic 125I-pindolol binding sites in the interpeduncular nucleus of the rat: normal distribution and the effects of deafferentation
Authors: Battisti WP , Artymyshyn RP , Murray M
Published: March 2018

The plasticity of the beta 1- and beta 2-adrenergic receptor subtypes was examined in the interpeduncular nucleus (IPN) of the adult rat. The beta-adrenergic receptor antagonist 125I-pindolol (125I-PIN) was used in conjunction with the selective subtype antagonists ICI 118,551 and ICI 89,406 to determine the subnuclear distribution of beta 1- and beta 2-adrenergic receptors in this nucleus and to correlate the receptor distribution with the distribution of both noradrenergic afferents from the locus coeruleus (LC) and non-noradrenergic afferents from the fasiculus retroflexus (FR). The density of these binding sites was examined following lesions that decreased (LC lesions) or increased (FR lesions) the density of the noradrenergic projection in the IPN. Quantitative radioautography indicated that beta 1-labeled binding sites account for the larger percentage of binding sites in the IPN. The beta 1-binding sites are densest in the those subnuclei that receive a noradrenergic projection from the LC: the central, rostral, and intermediate subnuclei. beta 1-binding sites are algo homogeneously distributed throughout the lateral subnuclei, where there is no detectable noradrenergic innervation. beta 2-binding sites have a more restricted distribution. They are concentrated in the ventral half of the lateral subnuclei, where they account for 70% of total 125I-PIN binding sites. beta 2-binding sites are also present along the ventral border of the IPN. Some of this labeling extends into the central and intermediate subnuclei. Bilateral lesions of the LC, which selectively remove noradrenergic innervation to the IPN, result in an increase in the beta 1-binding sites. Bilateral lesions of the FR, which remove the major cholinergic and peptidergic input from the IPN, elicit an increase in noradrenergic projections and a decrease in beta 1-binding sites. beta 1-binding sites thus exhibit both up-regulation and down- regulation which is correlated with the density of the noradrenergic projection. Our results suggest, therefore, that the density of beta 1- binding sites is regulated by noradrenergic input. beta 2-binding sites increase in density in response to both the LC and FR lesions, suggesting that they are postsynaptic to both of these afferents. The distribution suggests that some of these binding sites may reflect binding to glial cells. The beta 2-binding sites may therefore be regulated by both noradrenergic and non-noradrenergic mechanisms.
Three conductance classes of nicotinic acetylcholine receptors are expressed in developing amphibian skeletal muscle
Authors: Owens JL , Kullberg R
Published: March 2018

Two previously described classes of nicotinic AChRs in vertebrate skeletal muscle have conductances of 40 and 60 pS. In addition, a third conductance class of AChR channels is present in developing Xenopus muscle. This class appears to represent an independent channel type, rather than a subconductance state of the larger conductance channels. The channel has a slope conductance of 25 pS and a reversal potential of about 0 mV membrane potential. Its kinetic properties resemble those of the 40 pS channels present in early embryonic myotomal muscle. The channel has a mean open time of about 6 msec (at 40 mV applied potential). The open time is dependent on membrane potential and increases e-fold for every 60 mV of hyperpolarization. Consecutive openings were often separated by brief closures of about 0.4 msec in duration. The identity of the channel as a nicotinic AChR was established by blocking the channel openings with alpha-BTX and by demonstrating bursting and desensitization in the presence of high agonist concentrations. In some muscles (e.g., extraocular), this channel may be a predominant form at early developmental stages and could therefore be important to the function of developing synapses in those muscles.
Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography
Authors: Weaver DR , Rivkees SA , Reppert SM
Published: March 2018

Little is known of the neural sites of action for the pineal hormone, melatonin. Thus, we developed an in vitro autoradiographic method using 125I-labeled melatonin (I-MEL) to study putative melatonin receptors in rodent brain. We first determined optimal in vitro labeling conditions for autoradiographic detection of I-MEL binding sites in rat median eminence, the most intensely labeled area in the rat brain. We then assessed the pharmacologic and kinetic properties of I-MEL binding sites in rat median eminence by quantitative autoradiography. These sites have high affinity for I-MEL (equilibrium dissociation constant = 43 pM). I-MEL binding was inhibited by nanomolar concentrations of melatonin or 6-chloromelatonin, but was not inhibited by serotonin, dopamine, or norepinephrine (100 microM). These results suggest that I- MEL binding sites identified by in vitro autoradiography represent specific, high-affinity melatonin receptors. Studies of the distribution of I-MEL binding in rat, Syrian hamster, and Djungarian hamster brain confirm that the median eminence and suprachiasmatic nucleus are major sites of I-MEL binding in rodent brain; other brain areas labeled in one or more of these species were the thalamus (paraventricular, anteroventral, and reuniens nuclei, nucleus of the stria medullaris, and medial part of the lateral habenular nucleus), hypothalamus (dorsomedial nucleus), subiculum, and area postrema. The presence of putative melatonin receptors in the suprachiasmatic nuclei and median eminence of these rodent species suggests that these brain regions are important loci for melatonin effects on circadian rhythms and reproduction.
Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localizations with 125I-peptide YY and 125I- neuropeptide Y imply receptor heterogeneity
Authors: Lynch DR , Walker MW , Miller RJ , Snyder SH
Published: March 2018

Neuropeptide Y (NPY) receptor binding sites have been localized in the rat brain by in vitro autoradiography using picomolar concentrations of both 125I-NPY and 125I-peptide YY (PYY) and new evidence provided for differentially localized receptor subtypes. Equilibrium binding studies using membranes indicate that rat brain contains a small population of high-affinity binding sites and a large population of moderate-affinity binding sites. 125I-PYY (10 pM) is selective for high-affinity binding sites (KD = 23 pM), whereas 10 pM 125I-NPY labels both high- and moderate-affinity sites (KD = 54 pM and 920 pM). The peptide specificity and affinity of these ligands in autoradiographic experiments match those seen in homogenates. Binding sites for 125I-PYY are most concentrated in the lateral septum, stratum oriens, and radiatum of the hippocampus, amygdala, piriform cortex, entorhinal cortex, several thalamic nuclei, including the reuniens and lateral posterior nuclei, and substantia nigra, pars compacta, and pars lateralis. In the brain stem, 125I-PYY sites are densest in a variety of nuclei on the floor of the fourth ventricle, including the pontine central grey, the supragenual nucleus, and the area postrema. 125I-NPY binding sites are found in similar areas, but relative levels of NPY binding and PYY binding differ regionally, suggesting differences in sites labeled by the two ligands. These receptor localizations resemble the distribution of endogenous NPY in some areas, but others, such as the hypothalamus, contain NPY immunoreactivity but few binding sites.
A nerve terminal protein with a selective distribution in spinal cord and brain
Authors: Ritchie TC , Thomas MA , Coulter JD
Published: March 2018

A monoclonal antibody, designated S-7B8, recognizes a protein antigen localized to highly selected populations of nerve terminals in spinal cord and brain. The antibody produces dense immunocytochemical staining of primary afferent endings that synapse in superficial laminae of the spinal cord dorsal horn. Electron microscopy shows staining to be localized in nerve terminals where reaction product is associated primarily with spherical vesicles. In brain, S-7B8 immunoreactivity occurs in nerve terminals in sensory relay nuclei, most thalamic nuclei, and other selected areas, including the cerebellar molecular layer, the substantia nigra, the globus pallidus, and certain synaptic layers of the hippocampus and dentate gyrus. Endocrine glands and other tissues do not exhibit S-7B8 immunoreactivity. Although the antibody localizes to certain populations of nerve terminals that may use excitatory amino acid neurotransmitters, the distribution of S-7B8 immunoreactivity in the CNS does not correspond to that of any previously identified nerve terminal protein. Experiments to characterize the S-7B8 antigen indicate it may be an integral membrane component since extraction of synaptosomes with alkaline pH or high ionic strength does not release the antigen from the membranes. To identify the molecular weight of the S-7B8 antigen, synaptosomal membranes were solubilized in CHAPS and sequentially chromatographed on hydroxylapatite and then on DEAE anion-exchange resin to produce enriched fractions. When enriched fractions were separated on SDS-PAGE and Western blotted, the S-7B8 antibody specifically stained a protein migrating at 75,000 Da. This protein has been designated NT75. Preliminary studies of developing pathways show that the appearance of S-7B8 immunoreactivity in growing nerve endings corresponds closely to the time when synaptic connections are formed. Thus, the NT75 protein recognized by the S-7B8 antibody may have a role in the development and maintenance of specific synaptic endings.
Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity
Authors: Ikonomidou C , Mosinger JL , Salles KS , Labruyere J , Olney JW
Published: March 2018

The endogenous excitotoxin, glutamate (Glu), acting at the N-methyl- aspartate (NMA) subtype of Glu receptor, is thought to play a major role in hypoxic/ischemic neuronal degeneration. In the present study, the sensitivities of the developing rat CNS to hypoxic/ischemic neuronal degeneration and to the neurotoxic action of NMA were compared at various postnatal ages. In the hypoxic/ischemic experiments, ischemia was produced by unilateral common carotid artery ligation and hypoxia by subjecting the pups to a partial vacuum. Keeping the duration of the hypobaric episode constant at 75 min for all age groups, we observed that the vulnerability of the immature brain to hypobaric/ischemic damage increased during the early neonatal period (days 2–4), reached a peak at day 6 and then diminished progressively with increasing age. In the second part of the study, NMA was microinjected unilaterally into the head of the caudate nucleus at various postnatal ages (2–80 d). In the early neonatal period (days 2– 6), injections of relatively small doses of NMA (6–15 nmol) produced a dose-dependent widespread excitotoxic reaction throughout the forebrain with peak sensitivity being observed on day 6. The cytotoxic reaction to NMA was identical in appearance and time course to that induced by hypobaric/ischemic methods. With increasing age, the excitotoxic response to a given dose of NMA decreased progressively and the lesions became more strictly confined to the injection site. Cell populations most sensitive to NMA toxicity in the 2–10 d period closely correlated with those most vulnerable to hypoxia/ischemia, and sensitivity to both types of injury reached a peak at 6 d. These findings reinforce other evidence linking an excitotoxic mechanism and the NMA subtype of Glu receptor to hypoxic/ischemic brain damage and suggest that there may be a period during development when NMA receptors are hypersensitive to excitotoxic stimulation, thus rendering the neurons possessing such receptors hypervulnerable to hypoxic/ischemic damage.
Transneuronal regulation of protein synthesis in the brain-stem auditory system of the chick requires synaptic activation
Authors: Hyson RL , Rubel EW
Published: March 2018

The cellular mechanisms by which afferents influence their target neurons were investigated using a slice preparation of the chick brain- stem auditory system. Each brain slice contained portions of the auditory nerve and the second-order auditory nucleus, nucleus magnocellularis (NM), bilaterally. NM neurons on one side of the slice were stimulated either orthodromically, via activation of the ipsilateral auditory nerve, or antidromically, via electrical stimulation of their axons. NM neurons on the other side of the slice were not stimulated and served as a within-animal control population. Evoked activity was monitored extracellularly in all preparations. Orthodromic activation of NM neurons for either 1.5 or 3.5 hr resulted in enhanced protein synthesis by these neurons. This result is similar to those of previous in vivo experiments (Steward and Rubel, 1985; Born and Rubel, 1988). When slices were maintained in a medium having low Ca2+ and high Mg2+ concentrations, both synaptic transmission from the auditory nerve to NM and also the difference in protein synthesis between the stimulated and unstimulated sides of the brain were blocked. Antidromic activation of NM neurons did not enhance protein synthesis, but rather resulted in reliably less synthesis by the stimulated cells. Together, these results suggest that activity- dependent release of some “trophic” substance from the auditory nerve is necessary for this form of transneuronal regulation. Electrical activity of the postsynaptic neuron per se is not sufficient for increasing protein synthesis in these cells.
Induction of rhythmic jaw movements by stimulation of the mesencephalic reticular formation in the guinea pig
Authors: Hashimoto N , Katayama T , Ishiwata Y , Nakamura Y
Published: March 2018

This study was designed to investigate whether stimulation of the mesencephalic reticular formation (MRF) induces rhythmic jaw movements (RJMs) and, if it does so, to determine the RJM-inducing region in the MRF in ketamine-anesthetized guinea pigs. The results were as follows: (1) Repetitive electrical stimulation of the MRF at the level of the red nucleus induced rhythmic EMG bursts in the anterior digastric muscle (DIG). (2) The duration and cycle time of the rhythmic DIG EMG burst induced from the medial MRF were longer than those induced from the lateral MRF. (3) Repetitive MRF stimulation after paralysis still induced rhythmic multiple-unit activities in the anterior digastric motoneuron pool. (4) Neither precollicular decerebration nor cerebellectomy affected the MRF induction of RJMs. (5) Transverse hemisection at the rostral border of the pons abolished the RJMs induced from the contralateral, but not ipsilateral, MRF. Midline section of the midbrain abolished RJMs induced from the MRF on either side. (6) A lesion in the pontine pyramidal tract abolished the RJMs induced by stimulation of the ipsilateral cortical masticatory area (CMA), but not those induced from the contralateral MRF. (7) A unilateral lesion of the oral portion of the gigantocellular reticular nucleus, where the rhythm generator for the CMA-induced RJMs is located, abolished RJMs induced from not only the CMA, but also MRF on the contralateral side. (8) Microinjection of L-glutamate into the lateral, but not medial, MRF induced RJMs similar to those elicited by repetitive electrical stimulation of the same site.(ABSTRACT TRUNCATED AT 250 WORDS)
Neurotransmission regulates stability of acetylcholine receptors at the neuromuscular junction
Authors: Avila OL , Drachman DB , Pestronk A
Published: March 2018

The majority of acetylcholine receptors (AChRs) at normally innervated neuromuscular junctions are stable, with a half-life averaging about 12 d in most rodent muscles. Following denervation, the AChRs turn over much more rapidly after a lag period. The mechanism by which motor nerves normally maintain stabilization of junctional AChRs is not yet known. In order to determine whether synaptic transmission plays a role in this process, we have compared the effects of pre-and postsynaptic chloinergic blockade with those of surgical denervation. 125l-alpha- bungarotoxin was used to label junctional AChRs and follow their loss over time. Presynaptic blockade of quantal ACh transmission was produced in the soleus (SOL) and flexor digitorum brevis muscles of mice by repeated injections of type A botulinum toxin. Postsynaptic blockade of quantal and nonquantal ACh transmission was produced by continuous infusion of alpha-bungarotoxin in the SOL. Our findings show that treatment with botulinum toxin resulted in an accelerated loss of junctional AChRs that was similar to the effects of surgical denervation, though briefly delayed in its onset. Treatment with alpha- bungarotoxin produced an effect that was quantitatively equivalent to the accelerated loss of junctional AChRs following surgical denervation, with an identical time course. These results support the concept that cholinergic transmission is a mediator of the neural control of stability of junctional AChRs. The possibility that receptor stabilization may represent a mechanism of long-term postsynaptic “memory” dependent on neural transmission is discussed.
Quantitative lineage analysis of the origin of frog primary motor and sensory neurons from cleavage stage blastomeres
Authors: Moody SA
Published: March 2018

The average number of primary motoneurons and Rohon-Beard neurons that descend from each “identified” blastomere of the 16- and 32-cell stages of the frog Xenopus laevis was determined. The dorsal, animal blastomeres are the major motoneuron progenitors, and the ventral, animal blastomeres are the major Rohon-Beard progenitors. Cells along the midline primarily give rise to only one of these phenotypes, whereas cells along the frontal plane, which separates dorsal from ventral, give rise to both phenotypes. Each blastomere produces a characteristic number of each type of neuron, with only small variations between embryos. The mean values were used to construct quantitative retrospective lineage diagrams for the first 5 cell cycles after fertilization. These diagrams illustrate that the fate to become a major neuronal progenitor is segregated as early as the 4-cell stage. The lineage patterns of which sister cell makes the majority of primary neurons at each cleavage after the 4-cell stage are quite similar for both neurons in the D lineage but only moderately similar for both neurons in the V lineage. The pattern of predominant Rohon-Beard neuron fate is very similar in the D and V lineages. Analysis of the axial distribution of the primary motoneurons and Rohon-Beard neurons that descend from each blastomere indicates that the major progenitors contribute neuronal descendants periodically, to nearly every segmental bin, but the minor progenitors distribute neuronal descendants randomly along the axis. These data demonstrate that primary neuronal phenotype, cell number, predominant lineal pattern, and in some cases segmental distribution are highly regular across a large population of embryos. This population consistency suggests that several features of neuronal fate may be influenced either by cell position or lineage.
Asymmetry in voltage-dependent movements of isolated outer hair cells from the organ of Corti
Authors: Santos-Sacchi J
Published: March 2018

The electrically induced movements of outer hair cells (OHC) were studied using the whole-cell voltage-clamp technique and video analysis. Cell shortening occurs during depolarization and elongation occurs during hyperpolarization from holding potentials near -70 mV. However, a marked asymmetry in response magnitude exists such that depolarization produces larger cell length changes than do comparable levels of hyperpolarization. The response is such that at normal resting potentials in vivo, displacements are about 2 nm/mV, but increase to about 15 nm/mV as the cell is depolarized. This mechanical rectification in the depolarizing direction manifests itself during symmetrical sinusoidal voltage stimulation as a “DC” reduction in cell length superimposed upon “AC” length changes. The observed OHC mechanical rectification may be involved in the reported production of “DC” basilar membrane displacements during suprathreshold acoustic stimulation (LePage, 1987). Estimates of the magnitude of OHC movements at acoustic threshold levels induced by receptor potentials in the high- frequency region of the cochlea indicate a disparity between basilar membrane and OHC movements on the order of 21 dB. Thus, it appears questionable whether OHC mechanical movements solely underlie the “active process” thought to be responsible for the high degree of neural tuning at sound pressures near 0 dB.
Anti-tubulin monoclonal antibodies that bind to specific neurons in Caenorhabditis elegans
Authors: Siddiqui SS , Aamodt E , Rastinejad F , Culotti J
Published: March 2018

We have identified 2 anti-tubulin monoclonal antibodies that bind to 2 different subpopulations of identified neurons in the simple nervous system of the nematode Caenorhabditis elegans. The antibodies also recognize specific tubulin isotypes from C. elegans that were separated by isoelectric focusing. Antibody 6–1 1B-1 intensely stained the mechano-sensory neurons ALML, ALMR, PLML, PLMR, PVM, and AVM, plus neuron PVR by indirect immunofluorescence, and it bound to 1 of 2 major alpha-tubulin isotypes separated by iso-electric focusing. The epitope for antibody (Ab) 6–1 1B-1 is acetylated alpha-tubulin (Piperno and Fuller, 1985). Antibody 2–28–33 stained a set of neurons that contain the neurotransmitter GABA. It bound to the 2 major and 1 of the minor beta-tubulin isotypes of C. elegans. These results suggest that specific alpha- and beta-tubulin isotypes are greatly enriched in the subsets of neurons recognized by these antibodies. Most (possibly all) of the neurons in each subset are functionally related. The antibodies should allow us to examine whether specific tubulin isotypes have specific functions in these neurons. They may also be useful for examining neuron morphologies in mutants of C. elegans which affect nerve development.
GABA-mediated inhibition of locus coeruleus from the dorsomedial rostral medulla
Authors: Ennis M , Aston-Jones G
Published: March 2018

Recent anatomic studies in our laboratory (Aston-Jones et al., 1986) identified the nucleus prepositus hypoglossi (PrH) in the dorsomedial medulla as a major afferent of the locus coeruleus (LC). In the present studies, the influence of projections from PrH to LC was assessed in anesthetized rats. Focal electrical stimulation of PrH inhibited the spontaneous discharge of 42 of 47 LC neurons; the latency to onset of such inhibition was 19.8 +/- 2.5 msec and its duration was 172.4 +/- 10.4 msec. PrH-evoked inhibition of LC neurons was unaffected by administration of the opiate receptor antagonist naloxone or the alpha 2-receptor antagonist idazoxan but was substantially reduced by systemic picrotoxin, an antagonist of GABA. The GABAA receptor antagonist bicuculline methiodide blocked the inhibition from PrH, whether applied by local microinfusion or iontophoresis into the LC. These results lead us to propose that PrH provides a direct inhibitory synaptic input to LC, for which GABA is the likely transmitter.
Numerical matching between granule and Purkinje cells in lurcher chimeric mice: a hypothesis for the trophic rescue of granule cells from target-related cell death
Authors: Vogel MW , Sunter K , Herrup K
Published: March 2018

Previous studies of wild-type mice or mutant-wild-type mouse chimeras using the neurological mutant staggerer have supported a numerical matching hypothesis for target-related cell death. However, analyses of chimeras of a second neurological mutant, lurcher, have suggested that there may be significant flexibility in the relationship between the numbers of pre- and postsynaptic neurons. Whereas in staggerer chimeras there is a strict proportionality between the number of cerebellar granule cells and their postsynaptic target, the Purkinje cells, in lurcher chimeras, Wetts and Herrup (1983) report a relative increase in granule cell survival. We have reexamined the numerical matching between Purkinje and granule cells in an additional 5 lurcher----wild- type and 4 wild-type----wild-type chimerase. Our findings confirm and extend the results of the previous study to show that there is a close correlation between the number of granule and Purkinje cells in +/Lc chimeras, but for any given number of Purkinje cells in the +/Lc chimeras, more granule cells survive than in staggerer chimeras or inbred mouse strains. Whereas the ratio of granule to Purkinje cells in staggerer chimeras or inbred mouse strains is constant across all target sizes, in +/Lc chimeras the ratio of granule cells to Purkinje cells increases as the number of target neurons decreases. It seems likely that the increased granule cell survival is somehow related to the delayed degeneration of the +/Lc fraction of target cells in the +/Lc chimeras. Among the possible explanations for the observed results, we favor the hypothesis that a trophic factor is produced in +/Lc chimeras in response to the deafferentation of Purkinje cells that is capable of rescuing granule cells from target-related cell death. Our preference is based, in part, on observations of the state of the dendritic tree of the wild-type Purkinje cells that survive in +/Lc chimeras (Caddy et al., 1986).