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The development of intrinsic gamma-aminobutyric acid (GABA)-ergic neurons was studied in the first month of postnatal life in the rat periaqueductal gray matter (PAG) by light and electron microscopy using an anti-GABA serum. At birth (postnatal day 0: P0) GABA-immunopositive (GABA(IP)) neurons were detected only on the outer edge of dorsolateral PAG (PAG-DL) and were rare in the other PAG subdivisions. Their distribution did not change from P0 to P5, while they increased progressively from P5 to P10 in PAG-DL and began to be detected in ventrolateral PAG (PAG-VL). At the end of the second postnatal week the immunostaining pattern was nearly adult-like, and between P20 and P30 the adult pattern of GABA immunoreactivity was established. Quantitative light microscopic examination indicated that in the first postnatal month the cross-sectional area of GABA(IP) neurons gradually increased from 67.63 and 78.69 microm(2) at P0 to 122.15 and 119.16 microm(2) at P30 in PAG-DL and PAG-VL, respectively. Electron microscopic observations disclosed GABA labeling from P0 in cell bodies, dendrites, growth cones, and axon terminals. GABA(IP) terminals were few in neonatal rats and became more numerous and morphologically mature around the second week. Synapse development and maturation were examined by quantitative ultrastructural analysis. Synaptic vesicle number and size of GABA(IP) axon terminals progressively grew in the first postnatal month. In conclusion, the number and size of GABA(IP) cells progressively increase in postnatal PAG, with two populations of intrinsic neurons expressing their GABAergic nature in two different periods., (Copyright 2010 Wiley-Liss, Inc.)

To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in approximately 90% of VGLUT1+, approximately 30% of VGLUT2+ and 30-50% of VGAT+ puncta; SYPI was expressed in approximately 95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in approximately 7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in approximately 94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in approximately 3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that approximately 30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats.

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against gamma -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA(IP)) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35-36% of all terminals were GABA(IP); they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7-10% of the sample). Moreover, 49.15% of GABA(IP) axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA(IP) dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA(IP) and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.

The gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) mediate GABA uptake into presynaptic axon terminals and glial processes, thus contributing to the regulation of the magnitude and duration of the action of GABA at the synaptic cleft. The aim of the present study was to investigate the expression of three high-affinity GABA transporters (GAT-1, GAT-2, and GAT-3) in the periaqueductal gray matter (PAG) of adult cats by using immunocytochemistry with affinity-purified antibodies. Light microscopic observations revealed GAT-1 immunoreactivity in punctate structures, particularly dense in the lateral portion of the dorsolateral PAG column. Weak GAT-2-immunopositive puncta were homogeneously distributed in the PAG. GAT-3 immunoreactivity was detected in each column of the PAG but was more intense in the dorsolateral PAG column and around the aqueduct. Electron microscopic studies showed GAT-1 immunoreactivity in distal astroglial processes, in unmyelinated and small myelinated axons, and in axon terminals making symmetric synapses on both PAG neurons and dendrites. GAT-2 immunoreactivity was present mostly in the form of patches of different sizes in the cytoplasm of neuronal elements like the perikarya and dendrites of PAG neurons, in myelinated and unmyelinated axons, and in the axon terminals forming both symmetric and asymmetric synapses. Labeling was also observed in nonneuronal elements. Astrocytic cell bodies and their distal processes as well as the ependymal cells lining the wall of the aqueduct showed patches of GAT-2 immunoreactivity. Electron microscopic observation revealed GAT-3 immunoreactivity exclusively in distal astrocytic processes adjacent to the somata of PAG neurons and in axon terminals making both symmetric and asymmetric synapses. The present results suggest that three types of termination systems of GABAergic transmission are present in the cat periaqueductal gray matter., (Copyright 2000 Wiley-Liss, Inc.)

Neuronal and glial high-affinity Na+/Cl(-)-dependent plasma membrane gamma-aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT-2 by immunocytochemistry with affinity-purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser-scanning light microscopic studies revealed intense GAT-2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT-2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double-labeled with GAT-2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT-2-positive profiles also were GFAP positive. Ultrastructural studies showed GAT-2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT-2-positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT-2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT-2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain.

Using light- and electron-microscopic immunocytochemistry, we investigated the regional distribution and the ultrastructural localization of GAT-1, a prominent GABA transporter, in the cat PAG. Light microscopic observations indicate that GAT-1-immunoreactive elements are particularly dense in PAG-DL and form a pair of longitudinal columns extending in the intermediate region of this structure. At electron-microscopic level, GAT-1 immunoreactivity was present in axon terminals forming symmetric synapses and in the distal processes of astroglial cells. These data further confirm the existence of longitudinal columns within PAG. They also indicate that GAT-1 could influence the action of GABA on its receptors, probably regulating the magnitude and duration of GABA's synaptic action on PAG neurons, and suggest that astrocytes may play an important role in this process.

Neurons containing glutamatic acid decarboxylase (GAD) are known to exist in the spinal dorsal horn, dorsal column nuclei (DCN), n. ventralis posterior (VP), and somatosensory cortex of cats. Recent work suggested that species differences exist concerning the presence and/or density of GAD-positive neurons in VP. The present experiments demonstrate that, in contrast with carnivores and primates, the rat's VP contains virtually no GAD-positive neurons and that virtually all neurons in it project to the cortex. This conclusion is supported by the failure to find, in Golgi-impregnated material, neurons with characteristics commonly attributed to Golgi type II neurons in VP of cats. The lack of GAD-positive neurons in VP of rats contrasts also with the presence of such neurons in the DCN in the same species. As in cats, about one third of the neurons in the cuneate n. are GAD-positive; these have mostly small perikarya and they are present throughout the nucleus. It is likely that these are intrinsic neurons, i.e. non-projecting beyond the limits of the DCN since a comparable percentage of neurons are unlabeled by simultaneous injections of horseradish peroxidase in multiple targets of the DCN. Like GAD-positive neurons, neurons unlabeled by the retrograde transport of HRP have, for the most part, small perikarya. It is possible that inhibitory mechanisms necessary for basic transfer functions in VP of rats are sustained through projections to this nucleus from the n. reticularis thalami. Extrinsic source of GABAergic input to the DCN seem to be absent or very weak. From this and previous evidence it may be proposed that intrinsic inhibitory interneurons have gradually developed in VP of rabbits, carnivores, and primates in parallel with more elaborate levels of thalamic integration of somatosensation.