Treatment of sections of chick retina with acetylcholinesterase increases the enkephalin and substance P immunoreactivity.

Frozen sections 10 microns thick were cut from the retina of chicks which had been kept either in total darkness or in a well lit room. The sections were incubated with acetylcholinesterase before antibodies to [Leu] enkephalin, substance P or somatostatin were applied. Sections of bovine adrenals were treated similarly but they were developed only with antibodies to [Leu]enkephalin. There were low numbers of immunoreactive amacrine cells and processes when any of the three antibodies were used on sections of dark-adapted retinae. When the sections were treated with acetylcholinesterase, however, the enkephalin-like and substance P-like immunoreactivity was enhanced while there was no effect on somatostatin. Counts of immunofluorescent cells indicated that the numbers had increased to levels like those found in light-adapted retinae. The adrenal also showed an enhanced enkephalin-like immunoreaction after treatment with the enzyme. Incubation with buffer alone or with enzyme together with 10 mM acetylcholine abolished the reaction. Acetylcholinesterase treatment of sections from light-adapted retinae had no discernible effect on the already high immunoreaction found using any of the three antisera. It is concluded that the peptidase activity of acetylcholinesterase has the capacity to hydrolyze proteins of which some may be the precursor molecules for the enkephalins and substance P. Since the amacrine cells that contain the enkephalin-like and the substance P-like immunoreactivity were found to contain acetylcholinesterase, it is possible that the action found here in vitro represents a physiological function of the enzyme. The immunoreactivity on which there was no effect, somatostatin, does not co-exist with acetylcholinesterase. A second conclusion that may be drawn from these data is that the dark-adapted retinae lose immunoreactive peptide because of the rate of processing; the results suggest that there is adequate precursor molecule available to maintain "control" levels.

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.

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 in neural tube defects: a model using chick embryo amniotic fluid.

Acetylcholinesterase was measured in amniotic fluid from normal chick embryos and embryos with neural tube defects. Neural tube defects were induced in the chick embryos by three procedures, removal of albumen, mechanical disruption of the closed neural tube or injection of tetanus toxin. The concentration of acetylcholinesterase in amniotic fluid from untreated normal embryos changed throughout the period examined (5-14 days incubation) but was stable at 0.5 U1(-1) over the time period 6-11 days. Amniotic fluid taken from treated embryos with neural tube defects at 8 days always contained a higher concentration of acetylcholinesterase than fluid from sham operated but otherwise normal embryos, mean 40.9 U1(-1), S.E.M. = 10. U1(-1), versus 1.0 U1(-1), S.E.M. = 0.2 U1(-1). The range of values (6.1-393 U1(-1)) was clearly separated from the normal values, range 0.0-5.5 U1(-1). In 13 cases with developmental abnormalities other than neural tube defects, the concentration of acetylcholinesterase was elevated in only one. Two different forms of acetylcholinesterase, as shown by gel electrophoresis, were present in fluid form both normal and defective embryos. These forms were also present in blood plasma, cerebrospinal fluid and in the high speed supernatant from brain extracts, the latter tissue contained an additional form of greater electrophoretic mobility. After irreversible inhibition, enzyme activity in amniotic fluid recovered slowly; only half the control value was reached by 140 h compared with complete recovery in the tissues of the embryo within 19 h. Histochemical staining for acetylcholinesterase showed that the spinal cord in the region of the lesion contained high concentrations of the enzyme. The possible sources of acetylcholinesterase in amniotic fluid are discussed. This chicken model of neural tube defects provides support for the use of acetylcholinesterase tests in the detection of neural tube defects clinically, and provides a model for experimentation with this system.

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.

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.

The enkephalins are amongst the peptides hydrolyzed by purified acetylcholinesterase.

We have shown that purified acetylcholinesterase has the ability to hydrolyze a number of peptides including the physiologically occurring enkephalins. The enkephalins lost both the amino- and carboxyl-terminal amino acids, but several other peptides were not degraded. The enzyme was purified using an affinity chromatographic matrix that recognised one component of the active centre that is specific to cholinesterases, the anionic-binding site. The acetylcholinesterase was extracted from four tissues of diverse origin to minimise the risk of co-purifying a peptidase. The enzyme was essentially homogeneous on polyacrylamide gels, and there was only one protein that bound diisopropylfluorophosphate in the samples. The peptidase activity was not affected by the aminopeptidase inhibitor puromycin, but it was inhibited by acetylcholine at concentrations that also reduced the esterase activity. It was concluded that acetylcholinesterase also has the capacity for a novel type of hydrolysis of peptide bonds. The ability of acetylcholinesterase to hydrolyse naturally occurring compounds of different chemical nature, like esters and peptides, may help explain the long-standing puzzle of why the enzyme is more widely distributed than acetylcholine, once thought to be its sole natural substrate. The localization of the enzyme probably more accurately reflects the distribution of all its substrates, although their identity remains to be determined.

The concentration of enkephalin-like material in the chick retina is light dependent.

The enkephalin-like immunoreactivity in the retina of chicks has been studied using immunohistochemical and radioimmunoassay techniques. The histochemical experiments showed that the immunoreactivity was confined to a subpopulation of amacrine cells in the inner nuclear layer which projected processes into sublaminae 1 and 3-5 of the inner plexiform layer. The distribution of the immunoreactivity was markedly influenced by the ambient lighting conditions: it was reduced in the dark and restored by a period in the light. The reactivity was lost from both cell soma in the inner nuclear layer and from the processes. Radioimmunoassays showed that the quantity of enkephalin-like material was reduced by more than 60% after 12 h in the dark. Attempts to entrain a rhythm by keeping chicks on 12/12 h light/dark cycles for up to 4 days were largely unsuccessful. A rhythm may have been partially entrainable, but the major factor involved was light. These results highlight the lability of the neuropeptide in the retina and the need for controlled lighting conditions in studies of this kind. They also indicate that this system may be a fruitful model to explore two important issues: (i) it could allow studies of neuropeptide metabolism in a physiologically intact system; (ii) the role of particular amacrine cells in visual processing could be determined by depleting them of their neurotransmitter/neuromodulator.

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.

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.

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.

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.

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.

Substance P in the chick retina: effects of light and dark.

Monoclonal antibodies against substance P were used to study the distribution of the peptide in the retinae of chicks that had been kept in either total darkness or brightly lit conditions. In retinae from light-adapted chicks, substance P was found to be in normal sized amacrine cells and giant cells in the inner nuclear layer, in fibres in sublaminae 1,3-5 in the inner plexiform layer, in cells in the ganglion cell layer which may have been mostly displaced amacrine cells and in a few fibres in the optic nerve fibre layer. There was no apparent concentration of immunoreactive cell bodies in any part of the retina. After dark-adaption of the birds, the immunoreactivity in the retinae was much reduced; there were fewer visible cells in all parts of the retina and there was no reaction in the inner plexiform layer. When birds were taken from the dark and kept in the light for different periods, there was a gradual increase in the number of cells and in the overall immunoreaction. Substance P is thus like several other putative retinal neurotransmitters in that its concentration in the retina is regulated by light/dark. These experiments do not indicate how the changes occur, but they underline the need for a strict control of lighting conditions in experiments with these relatively slowly metabolized neurotransmitters.

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.

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.

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)

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.

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)

Discrete distributions of putative cholinergic and somatostatinergic amacrine cell dendrites in chicken retina.

The distributions of putative cholinergic and somatostatinergic amacrine cells of the chicken retina were compared. Acetylcholinesterase-positive amacrine cell bodies were concentrated at the border between the inner nuclear and plexiform layers. Similar amacrine cell bodies were detected in a displaced position in the ganglion cell layer. Both populations had dendrites joining the 4 bands of acetylcholinesterase activity in the inner plexiform layer. The cell bodies of somatostatin-immunoreactive amacrine cells were distinct from the intensely acetylcholinesterase-positive cell bodies. The immunoreactive terminal bands did not overlap the acetylcholinesterase-positive bands, except in the inner parts of the inner plexiform layer.