Glycinergic control of [Leu5]enkephalin levels in chicken retina.

Retinal levels of [Leu5]enkephalin-like immunoreactivity (LE-LI) increase during the light and decrease during darkness, in vivo15. Intravitreal injection of the GABA antagonist picrotoxin had no effect on the accumulation of LE-LI during the light, suggesting the absence of significant GABAergic control over LE-LI cells. However, injection of the glycine antagonist strychnine, prevented the light-induced increase of retinal levels of LE-LI during 6 h exposure to light, indicating the presence of glycinergic control over the LE-LI neurons. When applied during the dark, strychnine increased the depletion of LE-LI by 34% compared to vehicle-injected eyes, suggesting that the LE-LI neurons receive some glycinergic input during the dark as well. The release of LE-LI from retinas superfused in vitro is depressed by exposing the preparation to light. Superfusing isolated retinas with physiological buffer containing picrotoxin (100 microM), GABA (50 mM), or the GABA agonists muscimol (100 microM), (+)-baclofen (200 microM), or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (100 mM), had no effect on the efflux of LE-LI. Strychnine (100 mM) however increased the efflux of LE-LI by 64%, compared to the spontaneous efflux during the light. Glycine (15 and 50 mM) decreased the spontaneous efflux of LE-LI from retinas superfused in darkness by 44-48% and by 31% at 5 mM. These data are consistent with the results from pharmacological manipulations in vivo. We conclude that the LE-LI amacrine cells are under inhibitory control from glycinergic but not from GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

How peptidergic neurons cope with variation in physiological stimulation.

A general scheme for neuropeptide metabolism is outlined and the potential sites of regulation are discussed. Two major sites of regulation are distinguished: transcription which ultimately limits the rate of translation to form the prepropeptide, and post-translational processing steps. The consequences of up-regulation of these steps in response to increased metabolic demand are discussed. An alternative strategy for peptidergic neurons, reliance on a large pool of neuropeptide, is proposed. Data on the response of enkephalin-containing cells to increased levels of stimulation are reviewed. It is concluded that there is good evidence for genomic up-regulation, perhaps in association with regulation of processing. Evidence based on studies on enkephalin-containing amacrine cells in the chicken retina is also reviewed. It is suggested that these cells rely on a large pool of neuropeptide to cope with changes in demand.

Synapses, axonal and dendritic patterns of GABA-immunoreactive neurons in human cerebral cortex.

Gamma-aminobutyric acid (GABA) containing neurons were characterized in human association cortex by a combination of Golgi impregnation and immunohistochemistry. Neurons were Golgi impregnated, gold toned, drawn and then classified on the basis of their dendritic and axonal arborization in layers I-VI. An antiserum to GABA was used to determine which of the impregnated neurons were immunopositive. Twenty-four GABA-positive cells were Golgi impregnated: 7 were bitufted with their dendrites predominantly radially oriented, and 17 were multipolar stellate cells. Three of the multipolar cells with large somata in the deep layers showed dendritic patterns similar to previously described basket cells. Nine of the multipolar stellate cells in layers III-VI showed characteristics of 'neurogliaform' neurons (Ramón y Cajal, 1899). The somata and the dendritic field of these cells were spherical, with diameters of about 10-15 microns and 200 microns, respectively. Their dendrites were smooth and slightly beaded. The axon collaterals were densely distributed in and around the dendritic field, in a spherical area with a diameter of at least 300 microns. The thin axon collaterals had only occasional 'en passant' swellings. Contacts between the axons of neurogliaform cells and the distal dendrites of Golgi-impregnated pyramidal cells were observed. Electron microscopic immunocytochemistry revealed that GABA immunopositive nerve terminals formed symmetric synaptic contacts with somata, with GABA immunonegative and immunopositive dendritic shafts and with dendritic spines. The results show that GABAergic neurons are heterogeneous with respect to their dendritic and axonal patterns. In addition to the chandelier and basket cells, which have been shown in animal studies to contain GABA, other cell types, most prominently the neurogliaform cells, terminating on the distal parts of neurons, also contain GABA and may have a inhibitory function. Many of the GABAergic terminals make synapses on dendritic spines and shafts in the human cerebral cortex.

Light inhibits the release of both [Met5]enkephalin and [Met5]enkephalin-containing peptides in chicken retina, but not their syntheses.

The levels of native and cryptic [Met5]enkephalin in the chicken retina were found to vary during a 12:12 h light-dark cycle, both rising in the light and falling during the dark. Such variations could conceivably arise from (a) changes in the rate of release and subsequent degradation of native and/or cryptic [Met5]enkephalin, (b) changes in the rate of proenkephalin A synthesis, or (c) changes in the rate of proenkephalin A processing. Measurement of the rate of release of native and cryptic [Met5]enkephalin in vitro indicated that the increased rate of release of both of these forms of [Met5]enkephalin during the dark quantitatively accounted for the fall in their retinal levels during the dark. This indicated that the biosynthesis of proenkephalin A was not activated during the light-dark cycle. Molecular weight fractionation of retinal extracts also supported this idea, since the pool of high molecular weight precursors did not vary in size, suggesting that processing was not modulated during the light-dark cycle. Instead, the fall in both cryptic and native [Met5]enkephalin during the dark was due to their increased rate of release together with a rate-limiting conversion of high molecular weight [Met5]enkephalin-containing peptides to low molecular weight [Met5]enkephalin-containing peptides. The enkephalinergic cells of the retina seem to cope with physiological variations in demand by accumulating a large pool of peptide during periods of low stimulation (light), so that when stimulation and release is high (dark), the decrease in pool levels does not compromise the function of the cells and their postsynaptic targets.

The release of Leu5-enkephalin-like immunoreactivity from chicken retina is reduced by light in vitro.

A superfusion system was established to examine the efflux of endogenous Leu5-enkephalin-like immunoreactivity (LE-LI) from isolated chicken retinas. Superfusion with buffer containing high concentration of K+ (60 mM KCl) increased the efflux of LE-LI by 96%. This effect was not observed when Co2+ (4 mM CoCl2) was present. Exposing the retinas to light decreased the efflux of LE-LI by 59% compared to that observed during superfusion in the dark. No effect of ambient light could be detected in the presence of Co2+. Upon reverse-phase high-performance liquid chromatography the material released by the retina comigrated with synthetic Leu5-enkephalin. These results demonstrate that the release of LE-LI from retinal neurons is increased during the dark, and it is concluded that the lighting conditions exert their effects by modifying the state of polarization of the LE-LI amacrine cells and hence the release of LE-LI from these neurons.

Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with [3H]diisopropyl fluorophosphate.

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.

The ultrastructural localization of acetylcholinesterase-like immunoreactivity in the chicken retina.

The localization of acetylcholinesterase (AChE) in the chicken retina was studied using histochemical and immunohistochemical techniques. Using histochemistry, reaction end product was found in amacrine cells, ganglion cells, horizontal cells and in 4 bands in the inner plexiform layer. Ultrastructurally, the reaction end product was located between membranes of the endoplasmic reticulum, between the membranes of the nuclear envelope, surrounding neurites in the inner plexiform layer and filling synaptic clefts. Immunohistochemical techniques using a monoclonal antibody against AChE showed a similar staining pattern to that obtained with histochemistry. Ultrastructurally, AChE-like immunoreactivity was located on, not between, the membranes of the endoplasmic reticulum and nuclear envelope of amacrine cells, ganglion cells and horizontal cells. In the inner plexiform layer, immunoreactivity was on both pre- and postsynaptic membranes, and there was no immunoreactivity in non-terminal regions of the dendritic membranes and none within the synaptic clefts.

Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study.

Cholinergic amacrine cells of the chicken retina were detected by immunohistochemistry using an antiserum against affinity-purified chicken choline acetyltransferase. Three populations of cells were detected: type I cholinergic amacrine cells had cell bodies on the border of the inner nuclear and inner plexiform layers and formed a prominent laminar band in sublamina 2 of the inner plexiform layer, while type II cholinergic amacrine cells had cell bodies in the ganglion cell layer, and formed a prominent laminar band in sublamina 4 of the inner plexiform layer. Type III cholinergic amacrine cell bodies were located towards the middle of the inner nuclear layer, and their processes were more diffusely distributed in sublaminas 1 and 3-5 of the inner plexiform layer. Type I and type II cells were present at densities of over 7000 cells/mm2 in central areas declining to less than 2000 cells/mm2 in the temporal retinal periphery. The cells were organized locally in a non-random mosaic, with regularity indices ranging from 3 peripherally to over 5 centrally. Neither at the light nor electron microscopic levels was a lattice of cholinergic dendrites of the kind reported by Tauchi and Masland [J. Neurosci. 5, 2494-2501 (1985)] detectable. Within the two prominent dendritic plexuses, a major feature of the synaptic interactions of the type I and type II cholinergic cells was extensive synaptic interaction between cholinergic processes. Apart from this, there was little, if any, input to cholinergic processes from non-cholinergic amacrine cells, but there was input from bipolar cells. Output from the cholinergic amacrine cell processes was directed towards non-cholinergic amacrine cells as well as other cholinergic amacrine cells, and ganglion cells.

Acetylcholinesterase exhibits trypsin-like and metalloexopeptidase-like activity in cleaving a model peptide.

Acetylcholinesterase (EC 3.1.1.7) has been shown to possess an intrinsic peptidase activity. [Chubb et al. (1983), Neuroscience 10, 1369-1383]. To examine this activity further, the breakdown of a model hexapeptide (leu-trp-met-arg-phe-ala) LWMRFA was studied. Affinity-purified eel acetylcholinesterase rapidly cleaved the hexapeptide in a trypsin-like manner to produce two peptides (LWMR and FA). Acetylcholinesterase more slowly cleaved the C-terminal alanine residue from the peptide to yield LWMRF. Although the enzyme showed preference for cleaving the hexapeptide at its C-terminal, it was also able to cleave the N-terminal leucine residue form the tryptic product LWMR. Hydrolysis of the peptide at the tryptic site (arg4-phe5) was strongly inhibited by the trypsin inhibitor diisopropylfluorophosphate. Cleavage of the C-terminal alanine was only poorly inhibited by diisopropylfluorophosphate, but more strongly inhibited by metal-ion chelating agents, and it was increased in the presence of Zn2+ and Co2+. The pH optimum for cleavage at the tryptic site was 6, while that for the carboxypeptidase site was 8-9. These results show that acetylcholinesterase can hydrolyse peptides like a trypsin-like endopeptidase and a Zn2+- or Co2+-dependent exopeptidase, and they suggest that these two peptidase activities are associated with two separate active sites on the acetylcholinesterase molecule. As both peptidase activities eluted with acetylcholinesterase from a TSK 4000SW column when it was chromatographed by high-performance liquid chromatography, it is unlikely that the presence of either peptidase activity could be attributable to a contaminant in the acetylcholinesterase preparation. We suggest that acetylcholinesterase may be involved in the breakdown of bioactive peptides or their precursors in neuroendocrine cells.

Chromogranin A, B and C: immunohistochemical localization in ovine pituitary and the relationship with hormone-containing cells.

Chromogranins A, B and C, three distinct groups of proteins found in bovine chromaffin granules, were also found to be present in the pituitary using immunoblotting techniques. Their distribution was therefore studied in the normal ram pituitary using an immunoperoxidase technique applied to semithin serial sections and compared with that of some of the hormones of the anterior pituitary. Chromogranin-immunoreactivity was found in gonadotrophs (all three), thyrotrophs (A with some positive for C) and corticotrophs (a fraction with A and fewer with B and C). The mammotrophs and somatotrophs were negative. Chromogranin C was the only one of the three to be located in the pars nervosa, whilst chromogranin B was rarely found in the pars intermedia. The results suggest that chromogranins A, B and C are not always stored together, some hormone-containing cells do not contain immunohistologically detectable levels of the chromogranins.

Acetylcholinesterase hydrolyses chromogranin A to yield low molecular weight peptides.

The major soluble protein of bovine chromaffin granules chromogranin A was purified by reverse-phase high performance liquid chromatography. Brief incubations with either acetylcholinesterase or trypsin cleaved chromogranin A to yield two chromogranin-immunoreactive polypeptides which were similar in molecular weight to two of the major endogenous chromogranin polypeptides. A number of peptidase inhibitors which strongly inhibited tryptic digestion of chromogranin A also inhibited the acetylcholinesterase digestion, although they were less potent. More prolonged digestion of chromogranin A with acetylcholinesterase produced a large number of peptides which were similar to some of the endogenous chromogranin peptides in their elution profile by high performance liquid chromatography. In contrast, complete tryptic digestion of chromogranin A yielded peptides with a totally different elution profile. The experiments indicate that acetylcholinesterase possesses a peptidase activity which is similar, but not identical to trypsin, and suggest that a second non-tryptic activity is also present. They also suggest that acetylcholinesterase, an enzyme found in chromaffin cells, may process chromogranin A to yield lower molecular weight chromogranins in bovine chromaffin cells.

Acetylcholinesterase generates enkephalin-like immunoreactivity when it degrades the soluble proteins (chromogranins) from adrenal chromaffin granules.

Acetylcholinesterase was purified by passage through 3 affinity columns. The enzyme so purified was found to be homogeneous by electrophoresis and the peptidase and AChE activities co-eluted from a high pressure liquid chromatography column. The purified AChE degraded the chromogranins, the soluble proteins from the adrenal chromaffin granules, at a rate of nearly 8 micrograms/microgram AChE/h. The rate was fastest with the largest chromogranins, but proteins across the whole molecular weight spectrum were hydrolyzed. Immunoassay of extracts after incubation with AChE showed that enkephalin-like material had been produced. Incubations were also done with chromogranins that had been fractionated by size exclusion chromatography. The AChE degraded protein in all fractions and generated enkephalin-like immunoreactive material in fractions where it was produced by sequential treatment with trypsin and carboxypeptidase B. It seems likely, therefore, that AChE can hydrolyze some of the enkephalin precursors that are sensitive to trypsin and carboxypeptidase B, but the one-step nature of its action suggests a mode of action with fewer restrictions. It is concluded that AChE can hydrolyze proteins of widely differing sizes and the data add to the evidence that AChE is able to hydrolyze enkephalin precursors resulting in the generation of immunoreactive peptide.

Somatostatin-immunoreactive amacrine cells of chicken retina: retinal mosaic, ultrastructural features, and light-driven variations in peptide metabolism.

Somatostatin-like immunoreactive amacrine cells of the chicken retina have been characterized by immunohistochemistry at the light and electron microscope levels. The cell bodies were set back from the junction of the inner nuclear and inner plexiform layers, and prominent fibre plexuses were found in sublaminas 1 and 3-5 of the inner plexiform layer. The cells were distributed across the retinal surface with a centroperipheral gradient of cell density. Locally, the cells were organized in a non-random mosaic. Ultrastructurally, immunohistochemical reaction product was found throughout the cytoplasm of the cell bodies, particularly associated with membranous structures, including the cytoplasmic surfaces of the Golgi apparatus, and within large dense-core vesicles. In dendritic varicosities in the inner plexiform layer, reaction product was associated with the external surfaces of small, clear synaptic vesicles. The synaptic relationships of the somatostatin-immunoreactive terminals in sublamina 1 were distinct from those in sublaminas 3-5. Those in sublamina 1 received input predominantly, possibly exclusively, from bipolar cells. Feedback synapses onto bipolar terminals or to the other amacrine cell process at a synaptic dyad were observed. In sublaminas 3-5, input came predominantly, possibly exclusively, from other, non-immunoreactive amacrine cells, and output was primarily onto other amacrine cells. No synaptic contacts with ganglion cells or with other somatostatin-immunoreactive amacrine cells were identified. Changes in levels of somatostatin-like immunoreactivity in retinas of chicks kept on 12:12 light:dark cycles were detected by radioimmunoassay, and by light and electron microscopic immunohistochemistry. Levels of retinal somatostatin-like immunoreactivity increased in the light and decreased in the dark. The changes appear to be light-driven rather than circadian, since with prolonged exposure to light or dark, the levels of somatostatin-like immunoreactivity continued to increase or decrease until plateaus were reached. The light-driven change in levels of somatostatin-like immunoreactivity may be related to the predominance of bipolar input to the immunoreactive processes in sublamina 1 of the inner plexiform layer. The reduction in peptide levels in the dark may indicate greater release of somatostatin-like immunoreactivity from the amacrine cells in the dark, resulting in an inability of peptide synthesis to keep pace with breakdown. In the light, release of somatostatin-like immunoreactivity may be lower, leading to a net synthesis of peptide.

Cholinergic and acetylcholinesterase-containing neurons of the chicken retina.

In the chicken retina, choline acetyltransferase-like immunoreactivity (ChAT-LI) defines three populations of cholinergic amacrine cells and two terminal bands in the inner plexiform layer (IPL). Acetylcholinesterase (AChE) histochemistry defines two prominent bands within the IPL which corresponded to those containing ChAT. Other AChE-positive bands in the IPL are not associated with cholinergic transmission sites. Cholinergic cell bodies contain AChE, but the most intensely AChE-positive cells do not appear to be cholinergic. AChE histochemistry may be used to define the major cholinergic synaptic sites in the IPL, and may be a useful marker of IPL lamination.

Identified axo-axonic cells are immunoreactive for GABA in the hippocampus and visual cortex of the cat.

Chandelier or axo-axonic cells (AACs) are specialized interneurons terminating on the axon initial segments of pyramidal neurons. Two AACs have been localized by Golgi impregnation, one in the CA1 region of the hippocampus and one in the visual cortex of cat, for structural analysis and for the identification of their transmitter. They had 323 and 268 terminal bouton rows, respectively, probably making synapses with an equal number of initial segments. The distribution of the dendrites of the hippocampal cell was strikingly similar to that of pyramidal cells suggesting a similar input. Using an antiserum to GABA and postembedding GABA-immunocytochemistry, developed for Golgi-impregnated neurons, both cells were found to be GABA-immunoreactive. The strategic location of their synapses and the presence of GABA in AACs suggest that in normal cortical tissue they play a major role in GABA-mediated inhibition.

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system.

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.

Chromogranin immunoreactivity in the central nervous system. Immunochemical characterisation, distribution and relationship to catecholamine and enkephalin pathways.

Chromogranin A, the major soluble protein of the chromaffin granules, was isolated from bovine adrenals and used for immunization of rabbits. Chromogranin (CHR) immunoreactivity was studied by immunochemical and immunohistochemical methods in the adrenal, pituitary, brain and spinal cord of cattle, sheep, rats and guinea pigs using two antisera neither of which cross-reacted with dopamine beta-hydroxylase. Detailed studies were done using tissues from sheep only because very weak immunoreaction was obtained in tissues from the latter two species. Immunoblots of soluble proteins separated by two-dimensional polyacrylamide gel electrophoresis showed that the sera recognized a family of polypeptides in the adrenal which differed in size, but had almost identical isoelectric points. The patterns of immunoreactive proteins in extracts from the adrenal and pituitary were similar. Only two bands corresponding to the major high molecular weight bands in adrenal could be detected in the hippocampus which appeared to have a lower concentration of antigen. Other brain areas also showed two major immunoreactive proteins, one with a molecular weight similar to chromogranin A, and one smaller. Adrenal chromaffin cells, peripheral noradrenergic nerve axons and terminals in the pineal gland, a proportion of the anterior pituitary cells and the neurosecretory terminals of the posterior pituitary were strongly immunoreactive. In addition, CHR-immunoreactivity was widely distributed in the brain and spinal cord. The reactivity was readily visible in some nerve cell bodies and in well-defined pathways and terminal fibre networks. There were neurons whose perikarya were intensely stained but whose terminal projections appeared to be negative, while in other cases, the terminals appeared rich in CHR, while the perikarya were barely stained. All chromogranin immunoreactivity was abolished by absorption of the sera with a lysate from the chromaffin granules, but was not affected by absorption with Met- or Leu-enkephalin, dynorphin1-17, Met-enkephalin-Arg6-Phe7 or BAM-22P. Electron microscopic experiments revealed that the CHR-reaction in cell bodies was almost exclusively confined to the Golgi apparatus, while in synaptic boutons it was found in large dense-cored vesicles common to many types of terminals. In the hippocampal mossy fibre terminals, the immunoreactive granulated vesicles sometimes appeared to have fused with the plasma membrane of the boutons suggesting that the CHR was being secreted by exocytosis. The CHR-immunoreactivity was found to overlap partially with the distribution of many other neuroactive substances.(ABSTRACT TRUNCATED AT 400 WORDS)