Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.     

Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

Summary The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.This work was supported by the grants from the Ministry of Education, Science, and Culture, Japan     

Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.     

GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABA(A)Rs). We and others recently reported that islet β-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 μM) suppressed the GABA-induced current (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 μM, p<0.01) and insulin (1 μM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABA(A)Rs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

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.     

Immunohistochemical localization of glutamate decarboxylase in the rat oviduct and ovary: Further evidence for non-neural GABA systems

Summary The distribution of L-glutamate decarboxylase (GAD), a major biosynthetic enzyme for gamma-aminobutyric acid (GABA), was examined in the oviduct and ovary of the rat by means of an immunohistochemical technique. The polyclonal antiserum raised against brain GAD showed specific immunoreaction in some non-neuronal elements of the sex organs. In the oviduct, the inner layer of the mucosa was predominantly labelled. The selective distribution of GAD immunoreactivity in epithelial cells of the oviduct is consistent with former findings for GABA-like immunoreactivity in the same organ, indicating that the GAD-catalyzed reaction may be a major biosynthetic pathway for GABA even in these cells. In the ovary, vacuole-like formations within the follicular fluid and oocytes showed intense, specific staining. The occurrence of GAD immunoreactivity inside developing ovarian follicles including the oocyte may suggest a role for GABA related to follicular development and certain functions concerning the ovum.     

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina

Summary The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina.The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina.The GABA immunoreactive cells in rabbit retinas can be maintained in culture. Cultures of retinal cells derived from 2-day-old animals can be maintained for up to 20 days and show the presence of GABA-positive cells at all stages. In one-day-old cultures the GABA immunoreactive cells lacked processes but within three days had clearly defined processes. After maintenance for 10 days a meshwork of GABA-positive fibres could also be seen in the cultures.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.     

Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.     

GABA-immunoreactive structures in rat kidney.

We examined the distribution of gamma-aminobutyric acid-like immunoreactivity (GABA-LI) in the rat kidney by light and electron microscopy. In vibratome sections, GABA-LI was present in both the renal medulla and cortex. The inner stripe of the outer medulla was most heavily and almost homogeneously labeled, whereas GABA-LI in the cortex was mainly confined only to some tubules. GABA-positive structures involved the epithelial cells of the thin and the thick ascending limbs of the loop of Henle, the connecting tubules, and the collecting ducts. In GABA-positive connecting tubules and collecting ducts the immunoreactivity was present in the cytoplasm of about half of the epithelial cells. As revealed by electron microscopy, the labeled cells in the collecting tubules were the light (principal) cells. No GABA-LI occurred in neuronal structures. These findings are consistent with the presence of a non-neuronal GABA system in the rat kidney. Furthermore, the specific distribution of GABA in the tubular epithelium suggests a functional significance of this amino acid in tubular transport processes.     

GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats

Gamma amino butyric acid (GABA) and its related enzymes have been demonstrated in pancreatic beta cells of normal rat. Antibodies against GABA-synthesizing enzymes have been implicated in the pathogenesis of Type I diabetes. In spite of the importance of GABA in the aetiology of diabetes mellitus, detailed morphological data on the pattern of distribution of GABA in the pancreas of normal and diabetic rats are lacking. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (60 mg kg body weight(-1)). Four weeks after the induction of DM, normal (n = 6) and diabetic (n = 6) rats were anesthetized with chloral hydrate and their pancreata were removed and processed for the localization and effect of GABA on insulin secretion using immunohistochemistry and radioimmunoassay techniques. The number of GABA-like immunoreactive (GABA-LIR) cells in the pancreatic islets of STZ-diabetic rats decreased significantly (P<0.0001) when compared to non-diabetic control rats. The pattern and percentage distribution of GABA in the islet of Langerhans of normal and diabetic rat was similar to that of insulin. GABA induced a significant (P<0.0007) increase in insulin secretion from the pancreas of normal rats. In diabetic pancreas, GABA evoked a higher but not significant (P<0.1) increase in insulin secretion. These findings showed that the number of GABA-LIR cells is reduced significantly in diabetes. Moreover, GABA is a strong secretagogue of insulin from the pancreas of normal rat.     

饥饿状态大鼠胰腺高血糖素和胰岛素变化的定量分析

用免疫组织化学方法结合图象分析技术对饥饿状态大鼠胰岛Ａ、Ｂ细胞中胰因糖素和胰岛素的免疫反应强度进行定量分析。结果表明：与正常对照相比,饥饿大鼠胰岛细胞中的Ｇｌｕ含量明显下降,Ｂ细胞中Ｉｎｓ含量明显升高。提示饥饿可导致Ｇｌｕ释放增加,Ｉｎｓ和减少。与饥饿５天大鼠线要比较,饥饿５天后静脉注射葡萄糖组９０ｍｉｎ后胰岛内Ｇｌｕ含量明显升高,Ｉｎｓ含量无显著变化。提示：静脉注射葡萄糖要快速作用下胰岛Ａ细胞,     

Comparison of cellular and medium insulin and GABA content as markers for living beta-cells

Experimental and therapeutic use of islet cell preparations could benefit from assays that measure variations in the mass of living beta-cells. Because processes of cell death can be followed by depletion and/or discharge of cell-specific substances, we examined whether in vitro conditions of beta-cell death resulted in changes in tissue and medium content of insulin and of gamma-aminobutyric acid (GABA), two beta-cell-specific compounds with different cellular localization and turnover. Exposure of rat purified beta-cells to streptozotocin (5 mM, 120 min) or to the nitric oxide donor GEA-3162 (GEA; 50 microM, 120 min) caused 80% necrosis within 24 h; at the end of this period, cellular insulin content was not significantly decreased, but cellular GABA content was reduced by 70%; when cultured at basal glucose (6 mM), the toxin-exposed cells did not discharge less insulin but released 80% less GABA in the period 8-24 h. As in rat beta-cell purification, GABA comigrated with insulin during human islet cell isolation. Twenty-four hours after GEA (500 microM, 120 min), human islet cell preparations exhibited 90% dead cells and a 45 and 90% reduction, respectively, in tissue insulin and GABA content; in the period 9-24 h, insulin discharge in the medium was not reduced, but GABA release was decreased by 90%. When rat beta-cells were cultured for 24 h with nontoxic interleukin (IL)-1beta concentrations that suppressed glucose-induced insulin release, cellular GABA content was not decreased and GABA release increased by 90% in the period 8-24 h. These data indicate that a reduction in cellular and medium GABA levels is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after streptozotocin injection and remained unaffected by 12 h of hyperglycemia. At further variance with insulin, GABA release from living beta-cells depends little on its cellular content but increases with IL-1beta-induced alterations in beta-cell phenotype.     

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 mum in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

饥饿状态大鼠胰腺胰高血糖素和胰岛素变化的定量分析

用免疫组织化学方法结合图象分析技术对饥饿状态大鼠胰岛Ａ、Ｂ细胞中胰高血糖素（Ｇｌｕｃａｇｏｎ,Ｇｌｕ）和胰岛素（Ｉｎｓｕｌｉｎ,Ｉｎｓ）的免疫反应强度进行定量分析。结果表明：与正常对照相比,饥饿大鼠胰岛Ａ细胞中的Ｇｌｕ含量明显下降,Ｂ细胞中Ｉｎｓ含量明显升高。提示饥饿可导致Ｇｌｕ释放增加,Ｉｎｓ释放减少。与饥饿５天大鼠组相比较,饥饿５天后静脉注射葡萄糖组９０ｍｉｎ后胰岛内Ｇｌｕ含量明显升高,Ｉｎｓ含量无显著变化。提示：静脉注射葡萄糖可快速作用于胰岛Ａ细胞,减少Ｇｌｕ释放,但其对Ｂ细胞作用缓慢。从而为进一步阐明葡萄糖对胰岛Ａ、Ｂ细胞的不同作用机制提供形态学依据。     

Morphology of calretinin-immunoreactive neurons in the superficial layers of hamster superior colliculus after enucleation: lack of co-localization with GABA

We recently reported on the distribution and effects of eye enucleation on the immunoreactivity of calretinin in the superficial layers of the hamster superior colliculus (SC). In the present study, we describe the types of labeled cells and compare this labeling to that of GABA, the major inhibitory neurotransmitter in the central nervous system. An almost complete depletion of calretinin-immunoreactive (IR) fibers in the superficial layers of the contralateral SC was found following unilateral enucleation. On the contralateral SC, many calretinin-IR cells were newly appeared. The majority of the newly-appeared cells had small- to medium-sized round, oval, or vertical fusiform cell bodies. Two-color immunofluorescence revealed that none of these newly-appeared cells were labeled with an antibody to GABA. The present results show that the calretinin-IR cells are unique in the superficial hamster SC when compared to most of the other brain areas, where many calretinin-IR cells are GABAergic interneurons.     

Depolarization elicits,while hyperpolarization blocks uptake of endogenous glutamate by retinal horizontal cells of the turtle

We have employed an immunoreaction against glutamate to qualitatively demonstrate varying levels of glutamate in retinal horizontal cells of the turtle. Glutamate-like immunoreactivity (GLI) in horizontal cells could be demonstrated after glutamate decarboxylase was inhibited by aminooxy acetic acid (AOAA) and its degradation to GABA was blocked. Depolarization of horizontal cells by kainic acid (KA) induces strong glutamate immunoreactivity in these cells, whereas hyperpolarization by 2,3-cis piperidine dicarboxylate (PDA) abolishes glutamate-like immunoreactivity in horizontal cells. When glutamate release from cones and bipolar cells is blocked in the absence of calcium, or when glutamate uptake is blocked by DL-threo -hydroxy aspartate, KA/AOAA treatment of the retina does not induce GLI in horizontal cells. Our data show that horizontal cells are capable of taking up glutamate from the endogenous retinal pool in an activity dependent way. Our interpretation of these findings is that retinal horizontal cells are capable of regulating glutamate levels in the extracellular space of the cone pedicle complex by an activity-dependent uptake system. We suggest that inhibition of glutamate uptake upon hyperpolarization rather than inhibition of GABA release may evoke the antagonistic surround response of retinal bipolar cells.