Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. possible existence of neurons containing serotonin and GABA

It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decar☐ylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80–100 nm), the second displays only clear round vesicles (40–60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.     

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and d-[H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benizidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in te lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surounding the perihypoglosal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminals, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of d-[³H]aspartate (d[³H]Asp) and autoradiography consistently revealed retrogra labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of d-[³H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an exitatory amino acids as transmitter. Significant numbers of d-[³H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large d-[³H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to sorrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents ith relatively lower affinity for d-[³H]Asp. Several afferents to NRD failed to label with d-[³H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.     

GABAergic input to the synaptic terminals of mb1 bipolar cells in the goldfish retina

An EM-autoradiographical/immunocytochemical technique was used to study amacrine cell synapses onto mb1 bipolar cell terminals in goldfish retina. Tissue was double labeled for [3H]GABA uptake and glutamate decarboxylase (GAD) immunolocalization. Nearly 90% of the amacrine cell synaptic processes onto both proximal and distal halves of mb1 terminals were labeled with either [3H]GABA or GAD-immunoreactivity (IR). Proximal half: 73% of the amacrine synapses were labeled with [3H]GABA uptake and 82% with GAD-IR; 88% of [3H]GABA labeled contacts were double labeled. Distal half: 17% of the amacrine synapses were labeled with [3H]GABA uptake and 67% with GAD-IR; 63% of [3H]GABA labeled contacts were double labeled. After consideration of the possible sources of [3H]GABA labeled synapses onto mb1 terminals, we concluded that the synaptic terminals of pyriform Ab amacrine cells double label for [3H]GABA and GAD-IR despite our previous report that Ab cell bodies do not stain for anti-catfish brain GAD antiserum. We suggest that Ab cells contain isoenzymes of GAD which differ in subcellular distribution, thereby accounting for the differential staining of the cell bodies and dendrites obtained with the GAD antiserum we used.     

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and D-[3H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benzidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in the lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surrounding the perihypoglossal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminalis, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of D-[3H]aspartate (D[3H]Asp) and autoradiography consistently revealed retrograde labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of D-[3H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an excitatory amino acid as transmitter. Significant numbers of D-[3H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large D-[3H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to surrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents with relatively lower affinity for D-[3H]Asp. Several afferents to NRD failed to label with D-[3H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.     

The distribution of GABA-containing perikarya,fibers, and terminals in the forebrain and midbrain of pigeons,with particular reference to the basal ganglia and its projection targets

Immunohistochemical techniques were used to study the distributions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid (GABA) in pigeon forebrain and midbrain to determine the organization of GABAergic systems in these brain areas in birds. In the basal ganglia, numerous medium-sized neurons throughout the striatum were labeled for GABA, while pallidal neurons, as well as a small population of large, aspiny striatal neurons, labeled for GAD and GABA. GAD+ and GABA+ fibers and terminals were abundant throughout the basal ganglia, and GABAergic fibers were found in all extratelencephalic targets of the basal ganglia. Most of these targets also contained numerous GABAergic neurons. In pallial regions, approximately 10-12% of the neurons were GABAergic. The outer rind of the pallium was more intensely labeled for GABAergic fibers than the core. The olfactory tubercle region, the ventral pallidum, and the hypothalamus were extremely densely labeled for GABAergic fibers, while GABAergic neurons were unevenly distributed in the hypothalamus. GABAergic neurons and fibers were abundant in the dorsalmost part of thalamus and the dorsal geniculate region, while GABAergic neurons and fibers were sparse (or lightly labeled) in the thalamic nuclei rotundus, triangularis, and ovoidalis. Further, GABAergic neurons were abundant in the superficial tectal layers, the magnocellular isthmic nucleus, the inferior colliculus, the intercollicular region, the central gray, and the reticular formation. GABAergic fibers were particularly abundant in the superficial tectal layers, the parvocellular isthmic nucleus, the inferior colliculus, the intercol-licular region, the central gray, and the interpeduncular nucleus. These results suggest that GABA plays a role as a neurotransmitter in nearly all fore- and midbrain regions of birds, and in many instances the observed distributions of GABAergic neurons and fibers closely resemble the patterns seen in mammals, as well as in other vertebrates. © 1994 Wiley-Liss, Inc.     

Immunocytochemical and autoradiographic localization of GABAergic neurons in the goldfish retina

The putative neurotransmitter gamma-aminobutyric acid (GABA) was localized in goldfish retina by using an antiserum directed against GABA itself. The same types of cells were stained with this antibody as were labelled with an antiserum directed against the synthesizing enzyme for GABA, glutamic acid decarboxylase. Stained neurites of these cells were located throughout the inner plexiform layer (IPL) but staining was more intense in the proximal IPL. The GABA-immunoreactive staining could be reduced or completely abolished by preabsorbing the primary antibody with GABA. Uptake of [3H]-GABA or the GABA agonist [3H]-muscimol was localized in GABA-stained retinas using light microscope autoradiography. These experiments demonstrated that all types of GABA-immunoreactive amacrine cells had high-affinity uptake mechanisms for both [3H]-GABA and -muscimol. Thirty percent of proximal inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL) were labelled by all three GABAergic markers. Most GABA-immunoreactive amacrine cells were lightly labelled due to [3H]-GABA uptake but a few amacrines (Ab) were heavily labelled. These findings demonstrate that the autoradiographic localization of [3H]-GABA or [3H]-muscimol uptake and the immunocytochemical localization of GAD or GABA are appropriate methods for localizing GABAergic neurons in the retina. Few GABA-immunoreactive amacrine cells accumulated the putative amino acid transmitter [3H]-glycine, verifying that the goldfish retina contains distinct subpopulations of glycinergic and GABAergic amacrine cells.     

Distribution of GABAergic neurons and axon terminals in the macaque striate cortex

Antisera to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) have been used to characterize the morphology and distribution of presumed GABAergic neurons and axon terminals within the macaque striate cortex. Despite some differences in the relative sensitivity of these antisera for detecting cell bodies and terminals, the overall patterns of labeling appear quite similar. GABAergic axon terminals are particularly prominent in zones known to receive the bulk of the projections from the lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C beta and in lamina 5 suggest that the prominence of GAD and GABA axon terminal labeling in the geniculate recipient zones is due, at least in part, to the presence of larger GABAergic axon terminals in these regions. GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in all layers of striate cortex. The density of GAD and GABA immunoreactive neurons is greatest in laminae 2-3A, 4A, and 4C beta. The distribution of GABAergic neurons within lamina 3 does not appear to be correlated with the patchy distribution of cytochrome oxidase in this region; i.e., there is no significant difference in the density of GAD and GABA immunoreactive neurons in cytochrome-rich and cytochrome-poor regions of lamina 3. Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex. Layer 1 is distinct from the other cortical layers by virtue of its high percentage (77-81%) of GABAergic neurons. Among the other layers, the proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A to 12% in laminae 5 and 6. Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina 4C alpha and lamina 6 are distinguished from the other layers by the presence of populations of large GABAergic neurons, some of which have horizontally spreading dendritic processes. GABAergic neurons within the superficial layers are significantly smaller and the majority appear to have vertically oriented dendritic processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased glutamate decarboxylase activity in the red nucleus of the adult cat after cerebellar lesions

Glutamate decarboxylase (GAD)activity, a marker for GABAergic structures, was studied in the cat red nucleus. GAD is more concentrated in the rostral than in the caudal third of the structure.GAD levels were measured after chronic unilateral lesions of the cerebellum. Destruction of the dentate area and of the nucleus interpositus induced increases of GAD in the contralateral but not in the ipsilateral red nucleus. Similar changes also occurred in the denervated nucleus ventralis lateralis (VL) and nucleus ventralis anterior (VA) of the thalamus.Results show that loss of the excitatory cerebellar input could lead to changes in inhibitory GABAergic nerve terminals. This increase may be induced transsynaptically within existing neurons or, more likely, additional GAD-containing nerve terminals may be formed by axonal sprouting.     

High affinity GABA receptors — Autoradiographic localization

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I-IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distribution of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.     

Changes in [3H]muscimol binding in substantia nigra, entopeduncular nucleus, globus pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autoradiography

A quantitative autoradiographic technique for measuring the binding of [3H]muscimol to central nervous system GABA receptors is described using tritium-sensitive film. [3H]Muscimol binding was studied in primary and secondary striatal projection areas of rat brain following kainic acid lesions of the striatum. Seven days after the lesion, binding affinities in the striatum and its projection areas were not altered significantly. There was a loss of [3H]muscimol receptors in the striatum. Receptors increased in numbers in the ipsilateral globus pallidus (19%), entopeduncular nucleus (22%), and substantia nigra pars reticulata (38%). [3H]Muscimol binding was decreased in the ipsilateral anteroventrolateral and ventromedial (8%) thalamic nuclei. [3H]Muscimol binding in other brain areas (layer IV of the cerebral cortex, central gray, superior colliculus, and stratum moleculare of hippocampus) was not affected. The findings suggest that a loss of striatal innervation resulted in increased numbers of GABA receptors in striatal projection sites. It is further suggested that loss of inhibitory striatal inputs to neurons in the entopeduncular nucleus and substantia nigra pars reticulata may activate GABAergic projections to thalamus and thus result in decreased numbers of thalamic GABA receptors.     

Zinc-enriched GABAergic terminals in mouse spinal cord

Electrophysiological experiments have shown that zinc ions modulate glutamate and GABA receptors in brain slices. All the zinc-enriched neuronal pathways in the brain analyzed up until now have been found to be glutaminergic. Many years ago, zinc-enriched terminals with flat vesicles and symmetric synapses were found to be present in rat spinal cord by Henrik Daa Schr?der, and recently these findings have been supported by immunohistochemical and electron microscopical data in lamprey, mouse and rat. In the present study we expanded these observations by revealing a colocalization of zinc ions, zinc transporter-3 (ZnT3) and glutamic acid decarboxylase (GAD) in synaptic vesicles of zinc-enriched terminals throughout the mouse spinal cord. Confocal analysis of ZnT3 and GAD immunofluorescence was used at light microscopical levels, and a combination of zinc selenium autometallography and GAD immunocytochemistry at electron microscopic levels. Zinc-enriched/GABAergic terminals were observed in all laminae of the spinal gray matter, but most densely populated were laminae I and III in the dorsal horn. In the lateral and ventral funiculi of the white matter, rows of inhibitory zinc-enriched boutons were seen radiating from the gray matter. Ultrastructurally, colocalization of zinc ions and GAD immunoreactivity was seen in a pool of presynaptic terminals in the above locations. Some zinc-enriched terminals were not GAD-positive and some GAD-positive terminals were void of zinc ions. The majority of the zinc-enriched, not GABAergic terminals could be classified as excitatory based on their morphology, i.e. round clear vesicles and symmetric synapses. We conclude that a majority of the spinal cord zinc-enriched terminals are GABAergic. The zinc-enriched terminals with excitatory morphology are most likely glutaminergic, a few have an inhibitory morphology but are not GABAergic. These are most likely glycinergic.     

Transmitter histochemistry of the rat olfactory bulb III. Autoradiographic localization of [3H]GABA.

The distribution of [3H]gamma-aminobutyric acid (GABA) labeled elements in rat olfactory bulb was studied by light and electron microscopic autoradiography. [3H]GABA was strongly taken up into glial cells and pericytes in all layers of the bulb. The neuronal uptake of [3H]GABA was mainly seen in certain types of nerve terminals. About one-third of the granule dendritic terminals, some nerve endings of short axon cells, and certain nerve endings of extrabulbar origin showed a strong labeling. Labeling was seen in a small population of the periglomerular, short axon and granule cell bodies. Most cell bodies of these 3 types as well as the mitral cells did not, however, accumulate any appreciable amo9nt of [3H]GABA. The labeling pattern seen after injection of [3H]glycine and [3H]leucine was clearly different from the pattern seen after [3H]GABA injection. The labeling was more uniformly distributed over the components of the neuropil with a considerably higher activity over certain cell somata such as the mitral cells. The present results demonstrate that neuronal uptake and accumulation of [3H]GABA occur into populations of olfactory bulb cells and processes, which from neurophysiological and/or immunohistochemical studies are supposed to use GABA as a neurotransmitter.     

Studies of suspected neurotransmitters in the vestibuloocular pathways.

Isolated fresh cat trochlear and oculomotor nuclei, which contain the axon terminals of inhibitory neurons whose cell bodies are in the superior vestibular nucleus (SVN), actively synthesize and store [3H]GABA, [14C]acetylcholine, [3H]dopamine and [3H]tyramine from labeled precursors of these compounds. Twelve to 14 days following lesions of the ipsilateral superior vestibular nucleus or its efferent pathway to the oculomotor and trochlear nuclei, at a time when there is extensive degeneration of superior vestibular nucleus axon terminals in these nuclei, the synthesis and storage of GABA in the ipsilateral trochlear nucleus is markedly reduced compared to that in the contralateral trochlear nucleus; the synthesis of acetylcholine, dopamine and tyramine is not measurably affected. The oculomotor nuclei, which unlike the trochlear nuclei receive a heavy bilateral projection from the SVN, show no asymmetric decrease after SVN lesions in their ability to synthesize any of the compounds tested. The data support the identity of GABA as an inhibitory transmitter in the superior vestibular nucleus-trochlear nucleus pathway.     

In vivo release of newly synthesized [H]GABA in the substantia nigra of the rat: relative contribution of GABA striato-pallido-nigral afferents and nigral GABA neurons

The release of [3H]gamma-aminobutyric acid ([3H]GABA) continuously formed from [3H]glutamine has been measured with a push-pull cannula implanted in the substantia nigra of the rat anesthetized with ketamine. Consistent with the high density of GABA terminals coming from both the striato-pallido-nigral afferents, and from GABA nigrofugal neurons, our results showed that a large amount of [3H]GABA was spontaneously released in the reticulata, about 4 times higher than in the compacta. In the absence of calcium the spontaneous [3H]GABA release was reduced (-30%), as well as the K(+)-induced release of [3H]GABA (-66%). Bicuculline (10(-4) M) did not affect the K(+)-evoked release of [3H]GABA, suggesting that autoreceptors on GABA afferent fibers are distinct from the GABAA subtype. Partial lesions of striato- and pallido-nigral GABA neurons with kainic acid (1.2 micrograms) decrease by 40% the glutamic acid decarboxylase (GAD) activity in the ipsilateral SN without decreasing the spontaneous release of [3H]GABA; even following extensive lesions with kainic acid (2.5 micrograms), GAD activity (-72%) and spontaneous [3H]GABA release (-83%) were not completely abolished. These results suggest that a non-negligible contribution of GABA nigral neurons accounts for the spontaneous GABA release measured in the substantia nigra. This is further supported by the decrease (-20%), and the increase (+40%) of [3H]GABA release produced by the local application of glycine (10(-6) M), and bicuculline (10(-4) M), which respectively, inhibits and activates the nigral neuron activity. The contribution of nigral GABA neurons to the amount of [3H]GABA release from the substantia nigra, is likely linked to their high spontaneous firing rate.     

Transient increase in [3H]Ro 15-4513 specific binding in the superficial gray layer of the rat superior colliculus induced by visual deafferentation.

The imidazodiazepine compound [3H]Ro 15-4513, a partial inverse agonist of benzodiazepine receptors of the central type, binds with high affinity (order of 10(-8) M) to a single population of benzodiazepine binding sites in the mammalian central nervous system. A quantitative autoradiographic study was carried out to determine the effects of one eye removal on [3H]Ro 15-4513 specific binding to rat brain sections in the superficial gray layer or stratum griseum superficiale (SGS) of the superior colliculus. Retinal afferent degeneration due to right eye removal, performed 3 and 7 days before sacrifice, led to a significant and symmetrical increase in the [3H]Ro 15-4513 specific binding in both right and left SGS by enhancing the binding affinity of the radioligand. This transient phenomenon disappeared when a longer survival period of 45 days was allowed to elapse. Conversely, unilateral lesion of the primary visual areas had no apparent effects on the specific binding of the radioligand. The absence of any loss of binding sites after either type of lesion suggests that the benzodiazepine receptors are probably not situated on the optic nerve axon terminals, nor on the cortical axon terminals originating from primary visual areas. In the SGS, as in other rat brain structures, benzodiazepine receptors of the central type are functionally coupled with GABAA receptors and form 'GABAA receptors/benzodiazepine receptors/chloride channel' complexes. The involvement of the local GABAergic system in the postlesion plasticity of benzodiazepine receptors was studied by testing the effects of exogenously applied GABA on [3H]Ro 15-4513 specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA(A) receptor beta2/beta3 subunit and GAD67 immunoreactivity in the trigeminal motor nucleus during early postnatal development

GABA neurotransmission plays a role in brainstem circuitry responsible for jaw movements. We investigated the developmental relationship between terminals expressing GAD67 and GABA(A) receptor beta(2)/beta(3) subunit expression within the trigeminal motor nucleus. GAD67 immunoreactivity was intense throughout development. Neuropilar beta(2)/beta(3) immunoreactivity emerged during the 2nd postnatal week. Our data provide anatomical evidence for a GABAergic innervation of neonatal trigeminal motoneurons and suggest that beta(2)/beta(3) subunit expression is developmentally regulated in trigeminal motoneurons.     

GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry

Previous light microscopic immunoperoxidase studies of glutamic acid decarboxylase (GAD)-immunoreactive neural elements in the rat basilar pontine nuclei revealed immunocytochemical reaction product in neuronal somata and axon terminals. In the present study, pre-embedding immunoperoxidase labeling of GAD or gamma-aminobutyric acid (GABA) and postembedding immunogold labeling of GABA allowed the ultrastructural visualization of these neural elements in the basilar pontine nuclei of colchicine-treated animals. At the electron microscopic level, immunolabeled neuronal somata exhibited smoothly contoured nuclei, whereas some dendrites also contained reaction product after immunocytochemical treatment and were postsynaptic to both immunoreactive and nonimmunoreactive axon terminals. Synaptic boutons immunoreactive for GAD or GABA exhibited cross-sectional areas that ranged from 0.1 to 3.8 microns 2 and generally appeared round or elongated in most sections. The majority (95%) of immunolabeled boutons contained pleomorphic synaptic vesicles and formed symmetric synapses at their postsynaptic loci; however, boutons exhibiting round vesicles and boutons forming asymmetric synapses (5%) were also immunopositive. Small (less than 1.5 microns 2) GAD- or GABA-labeled axon terminals formed synaptic contact mainly with small dendritic profiles, dendritic spines, and neuronal somata, whereas large labeled boutons (greater than 1.5 microns 2) formed synapses with all sizes of dendritic profiles. Occasionally, a single immunolabeled bouton formed synaptic contact with two separate postsynaptic dendrites. It is suggested that the immunolabeled neuronal somata and dendrites observed in the rat basilar pontine nuclei represent a population of pontine local circuit neurons; however, it is known that GABAergic cell groups extrinsic to the pontine gray provide afferent projections to the basilar pons, and therefore at least some immunoreactive axon terminals present in the pontine nuclei are derived from these extrinsic sources. The ultrastructural observation of GABAergic neural elements in the rat basilar pontine nuclei confirms previous light microscopic findings and provides an anatomical substrate through which GABAergic neurons, whether arising from an intrinsic or extrinsic source, might exert an inhibitory influence on target cells within the pontine nuclei.     

Long-range GABAergic projection in a circuit essential for vocal learning

The anterior forebrain pathway (AFP) in the passerine song system is essential for song learning but not for song production. Several lines of evidence suggest that area X, a major nucleus in the AFP, forms part of the avian striatum. A key feature of striatal projection neurons is that they use the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Some area X neurons express GABA-like immunoreactivity, but the neurotransmitter phenotype of the projection neurons is largely unknown. To determine whether area X projection neurons are GABAergic, we used immunocytochemistry and confocal microscopy to examine whether these neurons in adult male zebra finches express the GABA synthetic enzyme glutamic acid decarboxylase (GAD). We observed numerous large and small GAD+ somata in area X, and dense GAD+ terminals, but no GAD+ somata in the target of area X, the medial nucleus of the dorsolateral thalamus (DLM). The density of GAD+ terminals in DLM was strongly reduced by ibotenic acid lesions of area X. After tracer injection into the DLM, all of the retrogradely labeled neurons in area X were GAD+. After tracer injection into area X, the vast majority of anterogradely labeled terminals in DLM were GAD+. We conclude that area X neurons projecting to DLM express GAD and are thus likely GABAergic. If this projection is indeed inhibitory, information processing in the AFP is substantially more complicated than previously realized. Moreover, because a GABAergic projection to a thalamic target is reminiscent of pallidal rather than of striatal circuitry, area X may contain both striatal and pallidal components.     

GABAergic cell types in the superficial layers of the mouse superior colliculus

To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa isoform of glutamic acid decarboxylase (GAD67-GFP), or Cre-recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2-cre). We used Cre-dependent virus injections in GAD2-Cre mice and tracer injections in GAD67-GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67-GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67-containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67-GFP interneurons are a source of these presynaptic dendrites.     

Characterization of GABAergic neurons in cerebellar primary cultures and selective neurotoxic effects of a serum fraction

The morphological and functional differentiation of GABAergic interneurons present in cerebellar primary cultures has been examined by means of [3H]gamma-aminobutyric acid (GABA) autoradiography and [3H]GABA depolarization-evoked release. At 2 days in vitro these neurons showed scarce accumulation of radioactivity and no Ca2+-dependent K+-evoked or veratridine-induced release of [3H]GABA. At 5 days in vitro GABAergic interneurons appeared more intensely labeled and had grown out long and often branched neuritic processes; a large Ca2+-dependent release of [3H] GABA could be evoked by high K+. At later stages the progressive increase in labeling and branching of the neuritic processes was paralleled by a further increase in the amount and Ca2+ dependence of [3H]GABA release; a tetrodotoxin-sensitive, veratridine-stimulated release was also demonstrated. The [3H]GABA-accumulating stellate astrocytes present in the culture were not responsible for the observed release of the amino acid. GABAergic neurons were also identified by indirect immunofluorescence, using antibodies to the specific marker glutamic acid decarboxylase. Total renewal of the culture medium at 7 days in vitro caused a drastic (90%) reduction in the number of GABAergic neurons and a concomitant decrease in the amount of [3H]GABA uptake and release in the cultures. The disappearance of GABAergic neurons was caused by a low molecular weight (Mr less than 1000) fraction of the serum used to supplement the basal culture medium. This serum component did not significantly influence the survival of the major neuronal population of the culture (the granule cells) and appeared to be selectively toxic for GABAergic neurons only after they had reached a quite advanced degree of morphological and functional differentiation in vitro. The toxic activity was no longer present in neuronal or glial conditioned media.