GABAergic neuronal ontogeny in the central nervous system of the viviparous teleost Poecilia sphenops.

Recent neurochemical investigations of the central vestibular pathways have demonstrated that several neurotransmitters are involved in various operations required for stabilizing posture and gaze. Neurons of the vestibular nuclei (VN) receive GABAergic inhibitory afferents, and GABAergic neurons distributed throughout the vestibular complex are implicated in inhibitory vestibulo-ocular and vestibulo-spinal pathways. The aim of this study was to analyse the modifications of GABA immunoreactivity (GABA-ir) in the cat VN after unilateral vestibular neurectomy (UVN). Indeed, compensation of vestibular deficits is a good model for studying adult central nervous system (CNS) plasticity and the GABAergic system is involved in CNS plasticity. We studied GABA-ir by using a purified polyclonal antibody raised against GABA. Light microscopic preparations of thin (20 microm) sections of cat VN were used to quantify GABA-ir by an image analysing system measuring GABA-positive punctate structures and the number of GABA-positive neurons. Both the lesioned and intact sides were analysed in three populations of UVN cats killed at different times after injury (1 week, 3 weeks and 1 year). These data were compared to those collected in normal unlesioned and sham-operated cats. Results showed a spatial distribution of GABA-ir in the control cats that confirmed previous studies. GABA-ir neurons, fibres and nerve terminals were scattered in all parts of the VN. A higher concentration of GABA-positive neurons (small cells) was detected in the medial and inferior VN (MVN and IVN) and in the dorsal part of the lateral VN (LVNd). A higher level of GABA-positive punctate structures was observed in the MVN and in the prepositus hypoglossi (PH) nucleus. Lesion-induced changes were found at each survival time. One week after injury the number of GABA-positive neurons was significantly increased in the MVN, the IVN and the dorsal part of the LVN on the lesioned side and in the ventral part of the LVN on the intact side. One year later a bilateral increase in GABA-positive neurons was detected in the MVN whilst a bilateral decrease was observed in both the SVN and the ventral part of the LVN. Changes in the GABA-staining varicosities did not strictly coincide with the distribution of GABA-ir cells, suggesting that GABA-ir fibres and nerve terminals were also modified. One week and later after injury, higher GABA-staining varicosities were seen unilaterally in the ipsilateral MVN. In contrast, bilateral increases (in PH) and bilateral decreases (in SVN and the ventral part of the LVN) were recorded in the nearly (3 weeks) or fully (1 year) compensated cats. At this stage GABA-staining varicosities were significantly increased in the lesioned side of the MVN. These findings demonstrate the reorganization of the GABAergic system in the VN and its possible role in recovery process after UVN in the cat. The changes seen during the acute stage could be causally related to the VN neuron deafferentation, contributing to the static vestibular deficits. Those found in the compensated cats would be more functionally implicated in the dynamic aspects of vestibular compensation.

Gaba in the Retina and Central Visual System

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.

The localization of gamma-aminobutyric acid (GABA)- and L-glutamate 1 carboxy-lyase (GAD)-immunoreactive neurons was compared in the skate, frog, pigeon, chicken, rabbit, and man. Horizontal cells show both GABA and GAD immunoreactivity in the skate, frog, and bird. Certain amacrine cells show GABA and GAD immunoreactivity in all species. The distribution of GABA- and GAD-immunoreactive cell bodies and cell processes was very similar, if not identical, in the skate and man. In the other species, cell populations with GAD immunoreactivity also showed GABA immunoreactivity. However, in the bird, frog, and rabbit, the GABA-immunoreactive amacrine cells were at least twice as numerous as the GAD-immunoreactive cells. In birds, the distributions of the GAD and GABA immunoreactivities were different in the sublayers of the inner plexiform layer. The reason for the difference is currently unknown. GABA-immunoreactive bipolar-like cells were seen in the frog.

GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons

GABA-immunoreactive neurons in the Central Nervous System of the viviparous teleost Poecilia sphenops

The afferent and efferent cortical projections of the dorsal lateral geniculate nucleus (GLD) of adult specimens of the turtle Emys orbicularis were investigated after intraocular or intracortical injections of horseradish peroxidase (HRP), and the distribution of gamma aminobutyric acid (GABA) immunoreactivity in the nucleus was carried out by immunocytochemical techniques, both techniques being combined with light and electron microscopy. In addition, some specimens were prepared for double-labeling of HRP and GABA immunoreactivity, and additional samples impregnated by a rapid Golgi technique. On purely morphological grounds, four types of neurons can be distinguished by light microscopy: two types of large cells in the cell plate which project to the cortex, and two types of smaller cells in the neuropil and optic tract which do not. The small cells are consistently GABA-immunoreactive, while the former are, with extremely rare exceptions, immunonegative for GABA. The supposition that the small neurons of the neuropil are interneurons is supported by electron microscopic observations; these strongly GABA-immunoreactive cells have large plicated nuclei surrounded by a thin layer of cytoplasm poorly endowed with organelles. The dendrites of these cells may contain pleomorphic synaptic vesicles (DCSVs) and appear to be presynaptic to other dendritic profiles. These DCSVs are occasionally contacted by GABA-immunoreactive axon terminals, and more frequently by retinal terminals consistently immunonegative for GABA. The latter, frequently organized in glomeruli, also make synaptic contacts with immunonegative dendrites arising from corticopetal neurons of the cell plate. Two major categories of GABA-immunoreactive axon terminals can be distinguished, and we are led to the conclusion that one of these represents an intrinsic GABAergic innervation of the GLD, while the second is tentatively interpreted as an extrinsic source of GABA to the nucleus, possibly from ventral thalamic structures. The fine structure of the dorsal lateral geniculate nucleus of Emys orbicularis thus shows many similarities with that of mammals.

In the cerebellum and basal ganglia, projection neurons are GABAergic; but in the cerebral cortex, there has been a historically strong dichotomy between glutamatergic projection neurons and GABAergic local circuit neurons. While this dichotomy is overwhelmingly valid, it is now clear that a small population of long-distance projecting GABAergic neurons occurs in primates, as well as in cats and rodents. Beyond their well-documented existence, however, the functional significance, ontogeny, and connectivity of this intriguing subpopulation remain obscure. This volume brings together nine articles that are intended to provide a summary of some of the current thinking regarding cortical GABAergic neurons with long-distance connections. These cover issues of nomenclature, subtype heterogeneity, species differences, and functional importance, in the adult brain, and in development. A final article (Clancy et al.) is organized as a series of extracts and commentaries on the individual articles, each section of which is followed by general comments and discussion. The order of articles in the present volume parallels the order in this final article. The topics in the other eight articles are briefly described below. The first article (Clancy et al.) presents a database of neural developmental events in three commonly used experimental species: rat, mouse, and macaque, using data from the online database www.translatingtime.net, as well as GABAergic and subplate developmental data from the empirical literature. Striking similarities between the two rodent populations permit the identification of developmental dates for GABAergic and subplate neural events in rats that were previously identified only in mice, and proposed timing of development events in mouse, previously identified in rats. The authors also exploit their model to produce estimates of dates for GABAergic and subplate neural events that have not yet been, or cannot be determined empirically in humans. The next two articles focus on projections and neurochemical profiles of long-distance projecting GABAergic neurons in several cortical areas (Higo et al.) and in the hippocampal system (Jinno). The authors use a variety of techniques to visualize and characterize in detail these scattered, hard to localize subpopulations: Golgi-like filling by NADPH-d (Higo et al.), GFP-labeling of GABAergic neurons in GAD67-GFP Cre-reporter mice (Higo et al.), and combined molecular, electrophysiological, and retrograde tracer approaches (Jinno). A cluster of three articles address the identity of white matter neurons, their role in development, and variations across species (Kanold; Luhmann et al.; Petanjek et al.). Subplate neurons are a largely transient subpopulation, morphologically, and neurochemically heterogeneous, that resides in the neonatal cortical white matter. They play a key role in the segregation and functional maturation of thalamocortical and intracortical circuits and, especially as discussed in Luhmann et al., are postulated not only to serve as a transient relay station for afferent inputs, but also as an active element amplifying the afferent and intracortical activity. Local and long-distance projections arise from subpopulations of glutamatergic and GABAergic subplate neurons. Projections to more distant neocortical regions are thought to form a corticocortical synaptic network. Clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits, pathological activity, and long-term neurological deficits. Espinoza et al. propose a novel classification of non-subplate pioneer neurons, with distinctive neurochemical phenotypes, projections, and localization within the preplate before its partition. They report the two populations as recognizable in rat and mouse, but with species-specific neurochemical features. Finally, Zhang et al. report a subpopulation of immature, interneuron-like cells in the association cortex and amygdala of non-human primates, and infer that these are involved in a life-long role, at least partly associated with plasticity. In summary, these articles present a range of data and issues that are under active investigation. Even since the short time since 2009 (the original publication of these articles), technical advances have occurred that promise significant new approaches to unraveling the structure and function of this subpopulation, not only as relates to basic cortical circuitry and function but also in relation to developmental and psychiatric disorders.

The organization of GABAergic elements in the histaminergic tuberomammillary nucleus has been examined by using antibodies against gamma-aminobutyric acid (GABA) and light and electron microscopy. Most neuronal perikarya of the ventral subgroup of the tuberomammillary nucleus were GABA immunoreactive (GABA-i). The morphology of the GABA-i perikarya was similar to the morphology of histaminergic perikarya described by Hayashi et al. ('84: J. Comp. Neurol. 229: 223-241) and Wouterlood et al. ('86: J. Comp. Neurol. 252:227-243). The GABA-i perikarya were contacted by relatively few terminals. The mean bouton covering ratio of GABA-i perikarya was 6.1%, whereas the mean bouton covering ratio for GABA-i dendrites in the tuberomammillary nucleus was 31%. Some of the presynaptic terminals were GABA-i. In addition, GABA-i perikarya and dendrites formed close contacts that never presented synaptic specializations. These results suggest that neurons of the histaminergic tuberomammillary nucleus contain the neurotransmitter GABA. Furthermore, GABA may act as a modulator of cellular processes within the tuberomammillary nucleus.

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

In the cerebellum and basal ganglia, projection neurons are GABAergic; but in the cerebral cortex, there has been a historically strong dichotomy between glutamatergic projection neurons and GABAergic local circuit neurons. While this dichotomy is overwhelmingly valid, it is now clear that a small population of long-distance projecting GABAergic neurons occurs in primates, as well as in cats and rodents. Beyond their well-documented existence, however, the functional significance, ontogeny, and connectivity of this intriguing subpopulation remain obscure. This volume brings together nine articles that are intended to provide a summary of some of the current thinking regarding cortical GABAergic neurons with long-distance connections. These cover issues of nomenclature, subtype heterogeneity, species differences, and functional importance, in the adult brain, and in development. A final article (Clancy et al.) is organized as a series of extracts and commentaries on the individual articles, each section of which is followed by general comments and discussion. The order of articles in the present volume parallels the order in this final article. The topics in the other eight articles are briefly described below. The first article (Clancy et al.) presents a database of neural developmental events in three commonly used experimental species: rat, mouse, and macaque, using data from the online database www.translatingtime.net, as well as GABAergic and subplate developmental data from the empirical literature. Striking similarities between the two rodent populations permit the identification of developmental dates for GABAergic and subplate neural events in rats that were previously identified only in mice, and proposed timing of development events in mouse, previously identified in rats. The authors also exploit their model to produce estimates of dates for GABAergic and subplate neural events that have not yet been, or cannot be determined empirically in humans. The next two articles focus on projections and neurochemical profiles of long-distance projecting GABAergic neurons in several cortical areas (Higo et al.) and in the hippocampal system (Jinno). The authors use a variety of techniques to visualize and characterize in detail these scattered, hard to localize subpopulations: Golgi-like filling by NADPH-d (Higo et al.), GFP-labeling of GABAergic neurons in GAD67-GFP Cre-reporter mice (Higo et al.), and combined molecular, electrophysiological, and retrograde tracer approaches (Jinno). A cluster of three articles address the identity of white matter neurons, their role in development, and variations across species (Kanold; Luhmann et al.; Petanjek et al.). Subplate neurons are a largely transient subpopulation, morphologically, and neurochemically heterogeneous, that resides in the neonatal cortical white matter. They play a key role in the segregation and functional maturation of thalamocortical and intracortical circuits and, especially as discussed in Luhmann et al., are postulated not only to serve as a transient relay station for afferent inputs, but also as an active element amplifying the afferent and intracortical activity. Local and long-distance projections arise from subpopulations of glutamatergic and GABAergic subplate neurons. Projections to more distant neocortical regions are thought to form a corticocortical synaptic network. Clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits, pathological activity, and long-term neurological deficits. Espinoza et al. propose a novel classification of non-subplate pioneer neurons, with distinctive neurochemical phenotypes, projections, and localization within the preplate before its partition. They report the two populations as recognizable in rat and mouse, but with species-specific neurochemical features. Finally, Zhang et al. report a subpopulation of immature, interneuron-like cells in the association cortex and amygdala of non-human primates, and infer that these are involved in a life-long role, at least partly associated with plasticity. In summary, these articles present a range of data and issues that are under active investigation. Even since the short time since 2009 (the original publication of these articles), technical advances have occurred that promise significant new approaches to unraveling the structure and function of this subpopulation, not only as relates to basic cortical circuitry and function but also in relation to developmental and psychiatric disorders.

The purpose of the present study was to examine the effects of retinal impulse blockade on gamma-aminobutyric acid (GABA)-immunoreactive (GABA-IR) neurons in cytochrome oxidase (CO)-rich puffs of the adult monkey striate cortex. Specifically, we wished to know if changes occurred in their CO activity, GABA immunoreactivity, and synaptic organization. A double-labeling technique, which combined CO histochemistry and postembedding GABA immunocytochemistry on the same ultrathin sections, was used to reveal simultaneously the distribution of the two markers. We quantitatively compared changes in GABA-IR neurons of deprived puffs (DPs) with respect to non-deprived puffs (NPs) 2 weeks after monocular tetrodotoxin treatment. We found that the proportion of darkly CO reactive mitochondria in GABA-IR neurons of DPs drastically decreased to about half of those in NPs. There was a greater reduction of CO levels in GABA-IR axon terminals than in their cell bodies and dendrites. In contrast, most non-GABA-IR neurons displayed no significant change in their CO levels. Morphologically, GABA-IR neurons and axon terminals in DPs showed a significant shrinkage in their mean size. GABA immunoreactivity, as indicated by the density of immunogold particles in GABA-IR neurons, declined in DPs, and a greater decrease was also found in axon terminals than in cell bodies or dendrites. Moreover, the numerical density of GABA-IR axon terminals and synapses in DPs was significantly reduced without changes in that of asymmetric and symmetric synapses. Thus, the present results support the following conclusions: 1) Oxidative metabolism and neurotransmitter expression in GABA-IR neurons are tightly regulated by neuronal activity in adult monkey striate cortex; 2) GABA-IR neurons are much more vulnerable to functional deprivation than non-GABA-IR ones, suggesting that these inhibitory neurons have stringent requirement for sustained excitatory input to maintain their heightened oxidative capacity; and 3) intracortical inhibition mediated by GABA transmission following afferent deprivation may be decreased in deprived puffs, because the oxidative capacity and transmitter level in GABAergic neurons, especially in their axon terminals, are dramatically reduced.

The development of gamma-aminobutyric acid (GABA)-immunoreactive neurons was investigated in the embryonic and posthatch chick lumbosacral spinal cord by using pre- and postembedding immunostaining with an anti-GABA antiserum. The first GABA-immunoreactive cells were detected in the ventral one-half of the spinal cord dorsal to the lateral motor column at E4. GABAergic neurons in this location sharply increased in number and, with the exception of the lateral motor column, appeared throughout the entire extent of the ventral one-half of the spinal gray matter by E6. Thereafter, GABA-immunoreactive neurons extended from ventral to dorsal regions. Stained perikarya first appeared at E8 and then progressively accumulated in the dorsal horn, while immunoreactive neurons gradually declined in the ventral horn. The general pattern of GABA immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterwards. In the dorsal horn, most of the stained neurons were observed in laminae I-III, both at the upper (LS 1-3) and at the lower (LS 5-7) segments of the lumbosacral spinal cord. In the ventral horn, the upper and lower lumbosacral segments showed marked differences in the distribution of stained perikarya. GABAergic neurons were scattered in a relatively large region dorsomedial to the lateral motor column at the level of the upper lumbosacral segments, whereas they were confined to the dorsalmost region of lamina VII at the lower segments. The early expression of GABA immunoreactivity may indicate a trophic and synaptogenetic role for GABA in early phases of spinal cord development.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function.