GABA immunoreactivity in the human cerebellar cortex: a light and electron microscopical study

The distribution of gamma-aminobutyric acid (GABA) in surgical samples of human cerebellar cortex was studied by light and electron microscope immunocytochemistry using a polyclonal antibody generated in rabbit against GABA coupled to bovine serum albumin with glutaraldehyde. Observations by light microscopy revealed immunostained neuronal bodies and processes as well as axon terminals in all layers of the cerebellar cortex. Perikarya of stellate, basket and Golgi neurons showed evident GABA immunoreactivity. In contrast, perikarya of Purkinje neurons appeared to be negative or weakly positive. Immunoreactive tracts of longitudinally- or obliquely-sectioned neuronal processes and punctate elements, corresponding to axon terminals or cross-sectioned neuronal processes, showed a layer-specific pattern of distribution and were seen on the surface of neuronal bodies, in the neuropil and at microvessel walls. Electron microscope observations mainly focussed on the analysis of GABA-labelled axon terminals and of their relationships with neurons and microvessels. GABA-labelled terminals contained gold particles associated with pleomorphic vesicles and mitochondria and established symmetric synapses with neuronal bodies and dendrites in all cortex layers. GABA-labelled terminals associated with capillaries were seen to contact the perivascular glial processes, basal lamina and endothelial cells and to establish synapses with subendothelial unlabelled axons.     

Glutamic acid decarboxylase-positive neuronal cell bodies and terminals in the human cerebellar cortex

The distribution of -aminobutyric acid (GABA) in the human cerebellar cortex was studied using immunohistochemistry for glutamic acid decarboxylase (GAD), the enzyme that catalyses GABA synthesis. Observations by light microscopy revealed, in all layers of the cerebellar cortex, strong, punctate positivity for GAD, related to putative GABAergic nerve terminals, as well as a diffuse cytoplasmic immunoreactivity within neuronal cell bodies. GAD-positive nerve terminals were found in close relationship with the walls of the cerebellar cortex microvessels. Observations by electron microscopy revealed positive nerve terminals in contact with the astrocyte perivascular sheath of capillaries. GAD immunoreactivity was also detected within astroglial perivascular endfeet and endothelial cells. The findings provide further insights into the GABAergic synapses of the circuitry of the human cerebellar cortex. The detection of vascular GAD immunoreactivities suggests that GABAergic mechanisms may regulate cerebellar microvessel function.     

A light and electron microscopic study of GAT-1-positive cells in the cerebral cortex of man and monkey

Summary Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the GABA transporter GAT-1. Cortical material from macaque monkeys was prepared similarly. Large numbers of GAT-1-positive non-pyramidal neurons were observed in layers I, II, V, and VI of the cortex. Electron microscopy also showed that the GAT-1-positive axon terminals formed symmetrical and not asymmetrical synapses, suggesting that they were the terminals of non-pyramidal neurons. Processes of cells in the walls of blood vessels were also labelled. We conclude that GAT-1 is present in cell bodies and axon terminals of non-pyramidal neurons, and a population of mural cells in blood vessels, in the primate cerebral cortex.     

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

Immunocytochemical localization of GABAergic neurones at the electron microscopical level

Summary Antibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, -aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternberger's peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H₂O₂. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord.An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.     

The projection of spinocerebellar neurons from the sacrococcygeal region of the spinal cord in the cat. An experimental study using anterograde transport of WGA-HRP and degeneration.

The projection from the sacro-coccygeal region of the spinal cord to the cerebellum was studied by two different techniques in the cat. In five cats wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) was injected caudal to a preceding unilateral cordotomy at the sacral level, aimed at interrupting the spinocerebellar tracts on one side completely, and the distribution of WGA-HRP labeled mossy fibers and mossy fiber terminals was studied in the cerebellum. In three additional cats, degenerating fibers were examined in Fink-Heimer stained sections following unilateral transection of the lateral and ventral funiculi at L7 or S3 level. In the WGA-HRP experiments the labeled mossy fiber terminals were located bilaterally in lobules I-V. Most of them were found in the anterior part of lobule II. In addition, labeled terminals were observed in sublobule VIIIB and in pars copularis of the paramedian lobule, contralateral to the cordotomy. The terminals in the anterior lobe were concentrated in longitudinal zones parallel to the mid sagittal plane. In lobule II, the terminals were most abundant in the superficial, apical parts of the folia. Some presumed terminals were also seen in the cerebellar nuclei. Labeled fibers were found contralateral, but not ipsilateral to the cordotomy in the superior and inferior cerebellar peduncles, as well as in the spinal cord rostral to the cordotomy. The results of the degeneration experiments were the same as those of the WGA-HRP experiments with regard to the detailed projections in the cerebellar cortex. This is strong support against the possibility that WGA-HRP labeled cerebellar mossy fiber terminals, following WGA-HRP injections in the spinal cord, would represent terminals of collaterals of retrogradely labeled neurons. It also lends strong support in favour of WGA-HRP as a reliable anterograde tracer for studying cerebellar cortical projections of spinocerebellar neurons in the cat.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Glutamatergic components of the retrosplenial granular cortex in the rat

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 μm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V–VI; some were also present in layers I–III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.     

GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarboxylase 67

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67 kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.     

Synaptic input to the axon hillock and initial segment of inhibitory interneurons in the cerebellar cortex of the rat

Summary The axon hillock (AH) and initial segment (IS) of 10 Golgi neurons and 6 basket cells in the cerebellar cortex of the rat were investigated by electron microscopy using serial sections. An average of 10.4 and 11.3 synaptic terminals were observed to establish synaptic contact with the axon hillock region of Golgi and basket cells, respectively. Most of these terminals were identified as the varicosities of the ascending parallel fibers. It is suggested that the focal innervation of AH regions represents an excitatory input pattern which is basically different from the randomly distributed, huge, parallel-fiber input onto the dendritic trees of Golgi and basket cells. In contrast to Golgi and basket neurons, no accumulation of parallel-fiber synapses was observed around the AH of stellate cells. The IS proper of the three neuronal types were devoid of true axo-axonal synapses.     

Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum

The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met5)-enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum.     

The glutamine commute: Lost in the tube?

The "glutamate-glutamine" cycle appears to have an important, albeit not exclusive role, in the recycling of glutamate (Glu) between neurons and astrocytes. Recent studies show that the efflux of glutamine (Gln) from astrocytes is mediated by SNAT3 (formerly SN1), a system N amino acid transporter localized to perisynaptic astrocytes, whereas its influx into neurons is thought to be mediated by transporters of the system A family, specifically SNAT1 and SNAT2. However, the results of our confocal and electron microscopy immunocytochemical studies of the localization of these transporters in the cerebral cortex show that SNAT1 and SNAT2 are robustly expressed in the somatodendritic domain of cortical neurons, but rarely to axon terminals. To rule out a possible influence of fixation and procedural variables on detection of SNAT1 and SNAT2 immunoreactivity in axon terminals, we used non-conventional immunocytochemical methods, which, in certain cases, improve antigen detection. Though evidencing a slightly increased percentage of axon terminals expressing the two transporters, these techniques demonstrated that SNAT1 and SNAT2 are indeed rarely localized to axon terminals. Our data thus suggest that neither SNAT1 nor SNAT2 meet the criteria for their postulated role in the "glutamate-glutamine" cycle, and indicate that other Gln transporters (either orphan or yet to be identified) must be expressed at axon terminals and sustain the Glu (and gamma-aminobutyric acid) neurotransmitter pool (s).     

Localisation of Formyl-Peptide Receptor 2 in the Rat Central Nervous System and Its Role in Axonal and Dendritic Outgrowth

Arachidonic acid and docosahexaenoic acid (DHA) released by the action of phospholipases A₂ (PLA₂) on membrane phospholipids may be metabolized by lipoxygenases to the anti-inflammatory mediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), and these can bind to a common receptor, formyl-peptide receptor 2 (FPR2). The contribution of this receptor to axonal or dendritic outgrowth is unknown. The present study was carried out to elucidate the distribution of FPR2 in the rat CNS and its role in outgrowth of neuronal processes. FPR2 mRNA expression was greatest in the brainstem, followed by the spinal cord, thalamus/hypothalamus, cerebral neocortex, hippocampus, cerebellum and striatum. The brainstem and spinal cord also contained high levels of FPR2 protein. The cerebral neocortex was moderately immunolabelled for FPR2, with staining mostly present as puncta in the neuropil. Dentate granule neurons and their axons (mossy fibres) in the hippocampus were very densely labelled. The cerebellar cortex was lightly stained, but the deep cerebellar nuclei, inferior olivary nucleus, vestibular nuclei, spinal trigeminal nucleus and dorsal horn of the spinal cord were densely labelled. Electron microscopy of the prefrontal cortex showed FPR2 immunolabel mostly in immature axon terminals or ‘pre-terminals’, that did not form synapses with dendrites. Treatment of primary hippocampal neurons with the FPR2 inhibitors, PBP10 or WRW4, resulted in reduced lengths of axons and dendrites. The CNS distribution of FPR2 suggests important functions in learning and memory, balance and nociception. This might be due to an effect of FPR2 in mediating arachidonic acid/LXA4 or DHA/RvD1-induced axonal or dendritic outgrowth.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

Two Forms of the γ-Aminobutyric Acid Synthetic Enzyme Glutamate Decarboxylase Have Distinct Intraneuronal Distributions and Cofactor Interactions

Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.     

A light and electron microscopic study of GAT-1 in the monkey basal ganglia

The distribution of the GABA transporter GAT-1 was studied by immunocytochemistry and electron microscopy in the monkey basal ganglia. Dense staining was observed in the globus pallidus externa and interna, intermediate in the subthalamic nucleus, and substantia nigra, and light staining in the caudate nucleus and putamen. Staining was observed in axon terminals, but not cell bodies. Electron microscopy showed that the GAT-1 positive axon terminals formed symmetrical synapses, suggesting that they were the terminals of GABAergic neurons. Comparison of areas high in GAT-1 protein with that of GABA showed a good correlation between the density in neuropil staining for GAT-1, and that of GABA.