GABA-immunoreactive Cell Bodies Research Articles

The timing of neuronal loss across adolescence in the medial prefrontal cortex of male and female rats

Adolescence is a critical period of brain maturation characterized by the reorganization of interacting neural networks. In particular the prefrontal cortex (PFC), a region involved in executive function, undergoes synaptic and neuronal pruning during this time in both humans and rats. Our laboratory has previously shown that rats lose neurons in the medial prefrontal cortex (mPFC) and there is an increase in white matter under the frontal cortex between adolescence and adulthood. Female rats lose more neurons during this period, and ovarian hormones may play a role as ovariectomy before adolescence prevents neuronal loss. However, little is known regarding the timing of neuroanatomical changes that occur between early adolescence and adulthood. In the present study, we quantified the number of neurons and glia in the male and female mPFC at multiple time points from preadolescence through adulthood (postnatal days 25, 35, 45, 60 and 90). Females, but not males, lost a significant number of neurons in the mPFC between days 35 and 45, coinciding with the onset of puberty. Counts of GABA immunoreactive cell bodies indicated that the neurons lost were not primarily GABAergic. These results suggest that in females, pubertal hormones may exert temporally specific changes in PFC anatomy. As expected, both males and females gained white matter under the PFC throughout adolescence, though these gains in females were diminished after day 35, but not in males. The differences in cell loss in males and females may lead to differential vulnerability to external influences and dysfunctions of the PFC that manifest in adolescence.

Tectorotundal connections in turtles: An electron microscopic tracing and GABA-immunocytochemical study

The nucleus rotundus of the turtles Emys orbicularis and Testudo horsfieldi was analysed by axonal tracing methods and post-embedding GABA immunocytochemistry. After injections of horseradish peroxidase or biotinylated dextran amine into the optic tectum, electron microscopic observations showed that the vast majority of ipsilateral tectorotundal axon terminals were small in size, had smooth contours and contained small, round, densely packed synaptic vesicles. These terminals were GABA-immunonegative, often gathered in clusters, and established asymmetrical synaptic contacts with either small- or medium-sized GABA-negative dendritic profiles and with GABA-immunoreactive (GABA-ir) dendrites, which did not contain synaptic vesicles. Occasional GABA-ir-labelled axon terminals were observed; these may arise from the rare GABAergic neurons in the central tectal layer, or from neurons in the ventral pretectal nucleus, which projects both to the optic tectum and nucleus rotundus. In addition to tracer-labelled axon terminals, we observed both GABA-negative and GABA-ir cell bodies and dendrites also labelled by the tracer. No GABA-ir presynaptic dendritic profiles containing synaptic vesicles were observed. The existence in reptiles of reciprocal connections between the nucleus rotundus and the optic tectum as a phylogenetically ancient feedback system is discussed.

GABAergic structures in the ventral part of the oral pontine reticular nucleus: An ultrastructural immunogold analysis

GABA mediates inhibitory effects in neurons of the ventral part of the oral pontine reticular nucleus (vRPO). Evidence increasingly suggests that GABA plays an important role in the modulation of rapid eye movement (REM) sleep generation in the cat vRPO. Here, we investigate the anatomical substrate of this modulation using GABA immunocytochemistry. Immunoperoxidase labeling revealed a few small GABA-immunoreactive cell bodies scattered throughout the vRPO. The numerical densities of all vRPO synapses and the GABA-immunoreactive synapses were estimated, at the electron microscopical level, by using a combination of the physical disector and the post-embedding immunogold techniques. We estimated that 30% of all vRPO synaptic terminals were immunoreactive to GABA. Our findings support the hypothesis that vRPO neuron activity is significantly controlled by inhibitory GABAergic terminals that directly target somata and the different parts of the dendritic tree, including distal regions. GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep–wakefulness cycle such as wakefulness or non-REM sleep.

Thalamic nucleus submedius receives GABAergic projection from thalamic reticular nucleus in the rat

GABAergic projection from thalamic reticular nucleus to thalamic nucleus submedius in the medial thalamus of the rat was studied by using immunohistochemistry for GABA, retrograde labeling with Fluoro-Gold combined with immunohistochemistry for GABA, and anterograde labeling with biotinylated dextranamine. Immunohistochemistry displayed that only GABA immunoreactive terminals were observed in the thalamic nucleus submedius, while GABA immunoreactive neuronal cell bodies were located in the thalamic reticular nucleus and lateral geniculate nucleus. Injection of Fluoro-Gold into the thalamic nucleus submedius resulted in massive retrogradely labeled neuronal cell bodies in the rostroventral portion of the ipsilateral thalamic reticular nucleus and a few in the contralateral thalamic reticular nucleus, and most of these cell bodies showed GABA immunopositive staining. Many biotinylated dextranamine anterogradely labeled fibers and terminals in the thalamic nucleus submedius were observed after injection of biotinylated dextranamine into the thalamic reticular nucleus. The present results provide a morphological evidence for a hypothesis that a disinhibitory effect on output neurons elicited by opioid or 5-hydroxytryptamine inhibiting a GABAergic terminal in the thalamic nucleus submedius may lead to activation of the descending inhibitory system and depression of the nociceptive inputs at the spinal cord level.

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.

Perineuronal nets in the rat medial nucleus of the trapezoid body surround neurons immunoreactive for various amino acids, calcium-binding proteins and the potassium channel subunit Kv3.1b

Perineuronal nets (PNs) are known as chondroitin sulfate-rich, lattice-like coatings of the extracellular matrix ensheathing mainly GABAergic, parvalbumin-containing neurons especially in the cerebral cortex. PNs have also been detected around GABA-immunonegative cells which were shown to be not aminergic, cholinergic, nitrinergic or peptidergic in various brain regions of some mammalian species. To find out whether glycine and aspartate may occur in net-bearing neurons the present study was focused on the rat medial nucleus of the trapezoid body (MNTB) which contains a large portion of cells immunoreactive for these amino acids, but appears to be devoid of GABA-immunoreactive cell bodies. PNs were detected around many glycine- and aspartate-immunopositive neurons in the MNTB by carbocyanine double labeling and confocal laser scanning microscopy. An additional finding was that the lectin-cytochemically stained extracellular matrix surrounds the calretinin-immunoreactive calyces of Held known as giant glutamatergic endbulbs which cover glycinergic principal cells in the MNTB. As elucidated by triple fluorescence labeling, the vast majority of somata co-expressed the calcium-binding proteins parvalbumin and calbindin, but not calretinin. The observed co-localization of PNs and immunoreactivity for the voltage-dependent potassium channel Kv3.1b — as an established marker of fast-firing parvalbumin-containing neurons — supports the assumed function of PNs as a cation exchanger ensuring rapid ion transport as required by highly active nerve cells.

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for gamma-aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar-King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA-immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA-positive fibers, and some of them were followed to the pineal gland. GABA-positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA-immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal.

γ-Aminobutyric acid-immunoreactive neurons in the amygdala of the rat – sex differences and effect of early postnatal castration

The distribution of GABA-immunoreactive neurons in the rat amygdala was determined by immunohistochemical methods. Sex differences in the number of GABA-immunoreactive cell bodies were observed in the medial, central, cortical and basolateral amygdaloid nuclei. Females had more numerous GABA-expressing neurons than males ( P<0.01). Castration of neonatal males had controversial effects on this sex difference in the different parts of the amygdaloid complex. In the cortical and basolateral amygdala castrated males showed a female pattern of GABA-expression. In the central amygdala no effect of castration on the number of GABA-immunoreactive cells was found. In the medial amygdaloid nucleus numbers of GABAergic neurons in castrated males differed both from intact males and females. The results of the present study suggest that the expression of GABA in the neurons of the amygdala is under complicated gonadal hormone regulation.

Colocalization of NADPH-diaphorase and GABA-immunoreactivity in the olfactory and visual system of the locust

Nitric oxide synthesizing neurons of the locust CNS have been identified by NADPH-diaphorase staining. However, the conventional transmitters of these neurons are unknown. Here we use double labelling for NADPH-diaphorase and GABA-immunofluorescence on sections of the brain to investigate a potential coexpression of both markers. The antennal lobe is innervated by a cluster of about 45–50 NADPH-diaphorase positive local interneurons which express GABA-immunofluorescence. The mushroom bodies are a higher order olfactory center which receive an extrinsic innervation from GABA-immunoreactive and NADPH-diaphorase positive fiber systems. Each optic lobe contains about 4500 GABA-immunoreactive cell bodies. In the visual system, identifyable GABA-immunoreactive neurons arborize in the external plexiform layer of the lamina, in several strata of the medulla, and in the lobula complex. A survey of all NADPH-diaphorase positive cell groups detected a colocalization of GABA-immunoreactivity in a small subpopulation of somata along the anterior rim of the medulla. These cytochemical findings suggest that nitric oxide may be a characteristic cotransmitter of GABAergic circuits of the antennal lobe, while in mushroom bodies and the visual system the majority of nitric oxide and GABA releasing neurons are distinct populations.

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia.

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.

Dopamnine‐Immunoreactive axon varicosities form nonrandom contacts with GABA‐immunoreactive neurons of rat medial prefrontal cortex

Recent postmortem studies have suggested that reduced gamma-aminobutyric acid (GABA)ergic activity in limbic cortex may be one component to the pathophysiology of schizophrenia. This hypothesis has underscored the importance of knowing whether midbrain dopamine afferents interact extensively enough with inhibitory interneurons to suggest a direct functional relationship. Toward this end, a double immunofluorescence approach combined with confocal laser scanning microscopy has been used to localize dopamine and GABA simultaneously in rat medial prefrontal cortex. The results confirm studies from other laboratories showing a rich network of dopamine-immunoreactive fibers forming a gradient across the cortical laminae, with deeper layers having the highest density. When viewed with oil immersion optics, dopamine-immunoreactive fibers were frequently found to be in close apposition with GABA-immunoreactive cell bodies. The percentage of GABA-containing neurons showing such contacts was highest in layer VI (65%) and progressively decreased toward layer I (9%). Varicose regions of the dopamine fibers were typically present at the point of contact with a GABA-immunoreactive cell body. Using an immunoperoxidase technique to localize dopamine fibers and cresyl violet staining to visualize neurons simultaneously, two separate statistical analyses were performed to assess whether the frequency of contacts between dopamine fibers and cell bodies in general may be due to random effects. In layer VI, a high percentage of both pyramidal and nonpyramidal neurons were found to be in contact with dopamine varicosities (71% and 76%, respectively), but these were not significantly different from that observed for GABA-containing cells (65%) in double-immunofluorescence specimens. A Chi-square statistical test was used to compare the observed and predicted number of varicosities forming cell body contacts. This analysis indicated that the percentage of dopamine varicosities (30%) that form appositions with cell bodies is much greater than would be expected if these appositions were due to random effects (15%). Moreover, using an estimate of intensity for a stationary Poisson process, it was again found that random effects can not account for these interactions (P = 0.01). Taken together with earlier electron microscopic studies from other laboratories, the present findings support the idea that GABAergic interneurons have extensive interactions with dopamine varicosities. While these interactions are not unique to GABAergic cell bodies, they suggest that inhibitory interneurons can play a direct role in mediating the effects of midbrain dopamine afferents in rat medial prefrontal cortex.

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.

Presumptive GABAergic feedback input to the frog olfactory bulb: a double labelling study with retrograde axonal tracing and GABA immunohistochemistry

The localization of γ-aminobutyric acid (GABA)-immunoreactive neurons that project to the main olfactory bulb in the frog was studied by combining GABA immunohistochemistry with retrograde axonal tracing. Double-labelled neurons were observed in the dorsal, medial and lateral pallial areas involved in reciprocal connections with the main olfactory bulb. GABA-immunoreactive cell bodies constituted only a part of the population of bulbopetal neurons in each pallial region. This suggests that bulbopetal neurons in the frog pallium belong to neuronal populations which differ in their neurotransmitter specificities. The bulbopetal GABA-immunoreactive pallial neurons may be the source of GABAergic feedback input to the frog main olfactory bulb.

Gamma-Aminobutyric acid immunoreactivity in multiple cell types of the developing rabbit retina.

We have previously demonstrated that the neonatal rabbit retina contains a larger complement of cells that accumulate [3H]-GABA than does the adult. In order for these neurons to be classified as GABAergic, they must also contain endogenous GABA. We now report that these same neonatal cell populations are also immunoreactive to GABA antisera. In frozen sections from rabbit retina, treated with GABA antisera, immunoreactive processes in both synaptic layers were observed at postnatal day 1. The appearance of immunofluorescent fibers precedes that of photoreceptor and bipolar cell terminals in the outer plexiform layer and is diminished by postnatal day 5. Also noted, was a 50% decrease in the density of GABA-immunoreactive cell bodies in the inner nuclear and ganglion cell layers, accompanied by an increase in cell volume and a shift toward a more spherical cell shape of the remaining cells. At postnatal day 1 and 3, we also observed immunoreactive cells having the characteristic morphology of interplexiform cells. This cell type sends branches to both the outer and inner plexiform layers, thus a morphological basis for communication between the two developing plexiform layers is present as early as postnatal day 1. Thus, retinas from neonatal rabbits have a larger complement of cells that stain for endogenous GABA than does the adult. These results coupled with our previous studies suggest that GABAergic properties are expressed by a larger number of cell types in the neonate than in the adult and are consistent with the general hypothesis that GABA functions as a trophic agent during development.

Distribution of GABA immunoreactive systems in the forebrain and midbrain of the chameleon

An immunocytochemical method, using glutaraldehyde fixation and an antiserum developed against a GABA-glutaraldehyde protein conjugate, permitted direct visualization of GABAergic structures in the brain of a reptile (chameleon). GABA immunoreactive cell bodies and nerve terminals were observed to be evenly distributed throughout the forebrain and midbrain. In the forebrain, GABA-positive perikarya were shown in all cortical areas, the septal area, the striatum, the dorsal ventricular ridge, and in the nucleus accumbens. In the midbrain, the optic tectum contained a dense and laminar distribution of GABA neurons. These neurons were also observed in the lateral geniculate nucleus, nucleus profundus mesencephali, nucleus opticus tegmenti and substantia nigra. Immunoreactive nerve fibers and terminals were observed in the same structures and, additionally, in the tractus septo-hypothalamicus, habenula complex, median eminence, intermediate lobe of the pituitary, basal part of the subcommissural organ, torus semicircularis and nucleus reticularis isthmi. These results provide a framework for a further electron microscopic analysis of the GABAergic innervation of some encephalic areas involved in physiological regulations particular to this species especially the visual system.

Evidence for the presence, synthesis, immunoreactivity, and uptake of GABA in the nervous system of the snail Helisoma trivolvis

In the present study several techniques were employed to test the hypothesis that gamma-aminobutyric acid (GABA) is a neurotransmitter in the central nervous system (CNS) of the pond snail Helisoma trivolvis (Mollusca, Pulmonata). First, by using chromatographic techniques, the presence of GABA and its differential distribution among the ganglia constituting the CNS was demonstrated. Second, de novo synthesis of 3H-GABA from 3H-glutamate was shown by the CNS. Levels of both endogenous and newly synthesized GABA were greatest in the buccal, cerebral, and pedal ganglia. Third, indirect immunohistochemistry of wholemounts revealed a central network of GABA-like immunoreactive neurons. With the possible exceptions of two pairs of fibers in nerve trunks, all projections from GABA-immunoreactive neurons were confined to the CNS, suggesting a predominantly central role for GABA. Stained neurons were found on the dorsal surface of the buccal ganglia and throughout the cerebral and pedal ganglia. No GABA-immunoreactive cell bodies were observed in the parietal, pleural, or visceral ganglia. Finally, uptake of 3H-GABA was examined autoradiographically in sectioned ganglia. A pattern of radiolabelled cells was observed that closely resembled the distribution of GABA-immunoreactive neurons. The data described above fulfill several criteria necessary to establish GABA as a transmitter in the nervous system of Helisoma. Taken together with previously obtained pharmacological evidence demonstrating that GABA acts on Helisoma central neurons, GABA is considered to be a strong candidate for a neurotransmitter in Helisoma.

Morphological organization of rat hippocampal slice cultures

Using various histological methods, we investigated the cellular and morphological organization of rat hippocampal slice cultures. Many of the typical features of the hippocampus were retained in vitro over a long period of time. The principal cell types of the hippocampus and dentate gyrus, the pyramidal cells and granule cells, were well preserved and matured in vitro. Nonpyramidal cells and gamma-aminobutyric-acid (GABA) cells were also present in slice cultures and exhibited a strikingly similar dendritic appearance at the light microscopic level. Moreover, GABA-immunoreactive cell bodies and presynaptic terminals could be identified at the electron microscopic level; they expressed typical symmetric synaptic contacts with cell bodies and dendrites. The course of the intrinsic hippocampal fiber pathways--the mossy fibers, Schaffer collaterals, and alveus--was generally retained in vitro. Additional aberrant fiber projections could be identified. Finally, three types of nonneuronal cells could be distinguished on the basis of immunocytochemical methods.

Septal GABAergic neurons innervate inhibitory interneurons in the hippocampus of the macaque monkey

The septohippocampal projection was visualized in three Macaca mulatta monkeys by anterograde transport of Phaseolus vulgaris leucoagglutinin. Following injections of the lectin into the medial septal nucleus, P. vulgaris leucoagglutinin-labelled fibres were found in the hippocampal complex, mainly in stratum oriens of the CA1 subfield, throughout the CA3 subfield, and in the hilus and stratum moleculare of the dentate gyrus. The majority of labelled axons were varicose, and formed multiple contacts with cell bodies and dendrites of calbindin D 28k- and parvalbumin-immunoreactive non-pyramidal cells. GABA immunoreactivity of P. vulgaris leucoagglutinin-labelled axons and their postsynaptic targets was investigated by sectioning varicose axon segments for correlated light and electron microscopy, and processing alternate ultrathin sections for postembedding immunogold staining for GABA. All P. vulgaris leucoagglutinin-labelled boutons examined were GABA-immunoreactive and the majority of them formed symmetrical synapses with GABA-immunoreactive cell bodies and dendrites. The results demonstrate that a GABAergic septohippocampal pathway exists in the monkey, and, similar to the rat, terminates on different types of GABAergic neurons, including the parvalbumin- and calbindin D 28k-containing non-pyramidal cells.

Light microscopic and ultrastructural analysis of GABA‐immunoreactive profiles in the monkey spinal cord

It is hypothesized that terminals containing gamma-aminobutyric acid (GABA) participate in presynaptic inhibition of primary afferents. To date, few convincing GABA-immunoreactive (GABA-IR) axo-axonic synapses have been demonstrated in support of this theory. The goal of this study is to document the relationship between GABA-IR profiles and central terminals in glomerular complexes in lumbar cord of the monkey (Macaca fascicularis). In addition, the relationship between GABA-IR profiles and other neural elements are analyzed in order to better understand the processing of sensory input in the spinal cord. GABA-IR cell bodies were present in Lissauer's tract (LT) and in all laminae in the spinal gray matter except lamina IX. GABA-IR fibers and terminals were heavily concentrated in LT; laminae I, II, and III; and present in moderate concentration in the deeper laminae of the dorsal horn, ventral horn (especially in association with presumed motor neurons), and lamina X. Electron microscopic analysis confined to LT and laminae I, II, and III demonstrated GABA-IR cell bodies, dendrites, and myelinated and unmyelinated fibers. GABA-IR cell bodies received sparse synaptic input, some of which was immunoreactive for GABA. The majority of the synaptic input to GABA-IR neurons occurred at the dendritic level. Furthermore, the presence of numerous vesicle-containing GABA-IR dendrites making synaptic interactions indicated that GABA-IR dendrites also provided a major site of output. Two consistent arrangements were observed in laminae I-III concerning vesicle-containing GABA-IR dendrites: 1) they were often postsynaptic to central terminals and 2) they participated in reciprocal synapses. The majority of GABA-IR axon terminals observed contained round clear vesicles and varying numbers of dense core vesicles. Only on rare occasions were GABA-IR terminals with flattened vesicles observed. GABA-IR terminals were not observed as presynaptic elements in axo-axonic synapses; however, on some occasions, GABA-IR profiles presumed to be axon terminals were observed postsynaptic to large glomerular type terminals. Our findings suggest that a frequent synaptic arrangement exists in which primary afferent terminals relay sensory information into a GABAergic system for further processing. Furthermore, GABA-IR dendrites appear to be the major source of input and output for this inhibitory system. The implications of this GABAergic neurocircuitry are discussed in relation to the processing of sensory input in the superficial dorsal horn and in terms of mechanisms of primary afferent depolarization (PAD).

Immunocytochemical detection of GABAergic nerve cells in the human striatum and cerebellum using a gamma-aminobutyric acid antiserum

We have adapted the immunocytochemical technique which allows direct detection of gamma-aminobutyric acid (GABA) which has enabled us to visualize GABA-immunoreactive nerve cell bodies, fibers and terminals in post-mortem human striatum and cerebellum. A high density of GABA-immunoreactive nerve cell bodies is detected in the striatum. They are mostly medium-sized with a mean cross-sectional area of 236 +/- 80 ?m(2) but some larger nerve cell bodies with a mean cross-sectional area of 547 +/- 104 ?m(2) are also present. These neurons are most probably essentially spiny neurons type I and type II identified in the human striatum by the Golgi method. In the cerebellum, Purkinje, stellate, Golgi and basket cells are GABA-immunoreactive. In the dentatus nucleus of the cerebellum a high density of presumptive synaptic GABA-immunoreactive terminals surround the GABA-negative neurons. As predicted by animal studies the present results demonstrate that GABAergic neurons are widely distributed in the human striatum and cerebellum. They demonstrate also that GABA-immunocytochemistry can be successfully performed on post-mortem human tissue fixed by immersion after short post-mortem delay and that application of this technique may lead to a better understanding of pathological conditions involving the GABAergic transmission.