Kihachi Saito

Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport.

Abstract The enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), has been immunocytochemically localized in the somata and dendrites of certain neurons in rat cerebellum and Ammoms horn following colchicine injections into these two brain regions. In the cerebellum. GAD-positive reaction product was observed in the somata and proximal dendrites of Purkinje, Golgi II, basket and stellate neurons. Occasional staining of the proximal portions of axons was also observed in these colchicine-injected preparations. None of the somata or dendrites of these same cell types exhibited reaction product in preparations that were not pretreated with colchicine, although the axon terminals of these neurons were GAD-positive. In Ammons horn, the somata of a few cells that are classified as probable basket and other short-axon neurons contained detectable concentrations of GAD in preparations that were not pretreated with colchicine. However, following colchicine injections into the Ammons horn, there was approximately a five-fold increase in the number of GAD-positive somata of basket and other short-axon neurons. There was also a substantial increase in the extent of dendritic staining exhibited by these neurons. Control injections of saline and lumicolchicine produced the same results as those observed in preparations which were not pretreated with colchicine. Thus, the results from the control injections indicate that the increases in somal and dendritic staining are due to a colchicine-mediated inhibition of the somatofugal transport of GAD rather than to a non-specific effect of the drug and/or the injection procedure. The results of the present study permit the direct identification of the neuronal somata in the cerebellum and Ammons horn whose synaptic terminals probably use GABA as their neurotransmitter. On the basis of the present findings, a reasonable explanation for the failure of earlier immunocytological studies to detect somal GAD in certain GABAergic neurons is that the axonal transport of GAD appears to occur at a sufficiently rapid rate to limit the somal concentration of GAD to low, undetectable levels.

GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord

Multiple, dorsal rhizotomies were performed unilaterally at lumbar levels L1–L4 in adult rats. Following 24–48 h degeneration periods and fixation by intracardiac perfusions, spinal cord were removed and transversely cut into 150 μm thick sections. These sections were incubated in immunocytochemical reagents for the peroxidase-labeling of glutamic acid decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA). The sections were then prepared for electron microscopic examination, while other sections were processed for light microscopic, GAD immunocytochemistry and for Fink-Heimer staining of degenerating axons and axon terminals. Thirty-six hours following dorsal rhizotomies, the sections that were prepared for the light microscopic study of terminal degeneration showed large numbers of degenerating profiles in the ipsilateral substantia gelatinosa while degenerating profiles were virtually absent contralaterally. In electron microscopic preparations, degenerating primary afferent terminals were commonly observed at the centers of rosettes where they formed synaptic contacts with other axon terminals and with surrounding dendrites. Several types of synaptic relationships were observed in the rosettes which involved both GAD-positive and degenerating primary afferent terminals. Such synaptic relationships included those in which: (a) a single GAD-positive terminal was presynaptic to the central, primary afferent terminal, (b) two different GAD-positive terminals formed synapses with opposite sides of the same central, primary afferent terminal and were also closely apposed to the surrounding dendrites of the rosette, and (c) a GAD-positive terminal was presynaptic to a primary afferent terminal and both types of terminals were presynaptic to the same dendrite of the rosette. The synaptic relationships described in this study are discussed with respect to their possible functional roles in such GABA-mediated phenomena as: (a) primary afferent depolarization, (b) the dorsal root reflex and (c) primary afferent hyperpolarization. Our observations support the concept that GABAergic axon terminals are involved in the synaptic circuits which produce presynaptic inhibition and presynaptic facilitation of the primary afferent input to the dorsal spinal cord. Collectively the observed synaptic relationships could provide a morphological substrate that is compatible with an inhibitory surround system in the substantia gelatinosa.

The fine structural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum

Abstract Glutamic acid decar☐ylase (GAD), the enzyme that synthesizes the putative neurotransmitter γ-aminobutyric acid (GABA), has been localized by peroxidase labeling antibody techniques at the light and electron microscopic levels in rodent cerebellum. Specific anti-GAD peroxidase product was highly localized in certain synaptic terminals in close association with the membranes of synaptic vesicles and mitochondria but not within these organelles. GAD-positive terminals were seen on the somata and proximal dendrites of neurons in the deep cerebellar nuclei. Other positive terminals were presumed Golgi type II endings of synaptic glomeruli in the granular layer. Positive terminals were also seen in the molecular layer, including presumed basket cell endings which contained product on smooth membrane cisternae in the preterminal axon as well as around synaptic vesicles and mitochondria. These observations correlate well with a large body of evidence that certain synaptic connections in the cerebellum are inhibitory and that many, if not all, of the presynaptic components of these connections may use GABA as their neurotransmitter.

Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra

L-Glutamate decarboxylase (GAD, EC 4.1.1.15), the enzyme which catalyzes the alpha-decarboxylation of L-glutamate to form gamma-aminobutyric acid (GABA), was localized both light and electron microscopically in rat substantia nigra by an immunoperoxidase method. Large amounts of GAD-positive reaction produce were seen throughout the substantia nigra in light microscopic preparations, and it appeared to be localized in punctate structures that were apposed to dendrites and somata. Electron microscopic studies revealed that most of the axon terminals in the substantia nigra were filled with GAD-positive reaction product and formed both axodendritic and axosomatic synapses. Many dendrites were extensively surrounded by GAD-positive terminals which most commonly formed symmetric synaptic junctions, although some formed asymmetric synpatic junctions. The results of this investigation are consistent with biochemical, pharmacological and physiological data which have indicated that neurons of the neostriatum and globus pallidus exert a GABA-mediated, postsynaptic inhibition upon the neurons of the substantia nigra. These findings provide another example in the vertebrate central nervous system where Golgi I projection neurons are inhibitory and use GABA as their neurotransmitter.

Glutamate decarboxylase localization in neurons of the olfactory bulb.

Glutamate decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA), has been localized in the rat olfactory bulb by immunocytochemical methods with both light and electron microscopy. The light microscopic results demonstrated GAD-positive puncta concentrated in the external plexiform layer and in the glomeruli of the glomerular layer. In addition, GAD-positive reaction product stained the dentrites and somata of granule and periglomerular cells. The electron microscopic observations confirmed the presence of GAD-positive reaction product within granule and periglomerular somata and dendrites. In electron micrographs of the external plexiform layer, the gemmules which arise from the distal dentrites of granule cells were also observed to be filled with reaction product, and these structures corresponded in size and location to the puncta observed in light microscopic preparations. The gemmules were observed to form reciprocal dendrodentritic synaptic junctions with mitral cell dentrites which lacked reaction product. In the glomeruli, GAD-positive reaction product was observed in the dentritic shafts and gemmules of periglomerular cells which also formed reciprocal dendrodentritic synaptic contacts with mitral/tufted cell dentrites. The localization of GAD in known inhibitory neurons of the olfactory bulb supports the case that these local circuit neurons use GABA as their neurotransmitter. The present study demonstrates that GAD molecules located within certain neuronal somata and dentrites can be visualized with antisera prepared against GAD that was purified from synaptosomal fractions of mouse brains. This finding suggests that the lack of GAD staining within somata and dentrites of GABA-ergic neurons noted in previous studies of the cerebellum and spinal cord was probably due to low GAD concentrations, rather than to antigenic differences among GAD molecules located in different portions of the neuron. A striking differences among GAD molecules located in different portions of the neuron. A striking difference between the granule and periglomerular neurons of the olfactory bulb and the neurons of the cerebellum and spinal cord is that the former have presynaptic dentrites while the latter do not. Since GAD-positive reaction product can be detected in the somata and dentrites of GABA-ergic neurons which have presynaptic dentrites, it is suggested that these neurons may differ from other GABA-ergic neurons with respect to either transport or metabolism of GAD.

The fine structural localization of glutamate decarboxylase in developing axonal processes and presynaptic terminals of rodent cerebellum

The immunocytochemical localization of L-glutamate decarboxylase (GAD), the enzyme which which forms gamma-aminobutyric acid (GABA), has been studied in developing rodent cerebellum. During the first 3-4 postnatal days, GAD is distributed along non-terminal portions of axonal processes in close association with small vesicles. Some of the axonal processes emanate from profiles which resemble growth cone varicosities, and are presumed to be foliopodia which extend distally from axonal growth regions. At the end of the first postnatal week the GAD-containing axonal processes are seen to form protosynaptic contacts, and GAD is localized around synaptic vesicles and at presynaptic junctional membranes. During the second and third postnatal weeks GAD gradually becomes localized to mature synaptic terminals in association with synaptic vesicle, mitochondrial, and presynaptic junctional membranes. The results suggest that GAD is present in growing neurites in close association with small vesicles prior to the time the neurites make protosynaptic contacts, and that differentiation of these contacts results in a sequestering of GAD into synaptic terminals.

Immunochemical comparisons of vertebrate glutamic acid decar☐ylase

Abstract Antisera to l -glutamate decar☐ylase (GAD) purified from mouse brain were produced in rabbits and the species specificities of GAD were examined. Antibodies to mouse enzyme were found to cross-react with the decar☐ylases from brain of rat, human, calf, rabbit, guinea pig, frog, pigeon and quail in double diffusion tests. Trout enzyme did not cross-react. The decar☐ylases from mouse and rat brain were inhibited to the extent of 50% and 35%, respectively, by anti-GAD IgG. The glutamate decar☐ylases from other species were inhibited only slightly. Microcomplement fixation tests showed that the mouse, rat and human enzymes were quite similar. The results indicated that the rat enzyme is most closely related to the mouse enzyme, followed in order by human, rabbit, calf, guinea pig, pigeon, quail, frog and trout.

DISTRIBUTION AND TISSUE SPECIFICITY OF GLUTAMATE DECARBOXYLASE (EC4.1.1.15)

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO2 method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO2 assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO2 method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO2 method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.

Properties of L-glutamate decarboxylase from brains of adult and newborn mice.

Abstract— l‐Glutamate 1‐carboxy‐lyase (EC 4.1.1.15) (GAD) and 4‐aminobutyrate‐2‐oxo‐glutarate aminotransferase (EC 2.6.1.19) (GABA‐T) have been purified from mouse brain (Wuet al. 1973; Schousboeet al., 1973) and their properties have been extensively studied (Wu & Roberts, 1974; Schousboeet al., 1974). The above enzymes were prepared from a water lysate of crude mitochondrial fraction, which accounted for only 25–30% of total GAD or GABA‐T activities in brain. A procedure has been developed which liberates approx 85% of total GAD and GABA‐T activities into supernatant. Two distinct, well‐separated peaks with GAD activity and a single peak with GABA‐T activity were observed when a concentrated extract from brain of adult or newborn mice was chromatographed on Sephadex G‐200 or Bio‐Gel A–1.5 m. The first peak appeared in the void volume and is. therefore. an entity of high molecular weight. The second peak gave elution characteristics which were identical to those of the enzyme that had been purified previously (mol wt = 85,000). These two GAD peaks were also clearly separated on polyacrylamide gel electrophoresis. The GAD activities in the two peaks showed similar pH profiles (optimum, 6.5). Km values (1–2 mM), immunodiffusion patterns and inhibitions by anti‐GAD IgG prepared against GAD purified from synaptosome‐containing crude mitochondrial fraction (60–80%). The physiological implications of high molecular weight and low molecular weight forms of GAD are discussed.

Immunochemical studies of brain glutamate decar☐ylase and GABA-transaminase of six inbred strains of mice

Six different inbred strains of mice (C57BL/6J, CBA/CaJ, CE/J, DBA/2J, LP/J and RF/J) were compared in terms of specific activities and immunochemical properties of brain L-glutamate decarboxylase (GAD) and gamma-aminobutyrate transaminase (GABA-T), the enzymes responsible for the synthesis and degradation of GABA, respectively. GAD from the brains of the different strains was indistinguishable on the basis of specific activities, double diffusion tests, immunoelectrophoresis and inhibition by antibody. However, microcomplement fixation tests showed GAD from DBA and C57BL mice to be most distinctly different from GAD extracted from the Swiss mouse, from which the original antigen was prepared and that the enzyme from the CE, LP and RF also differed. Similar fixation curves were obtained for the GAD from CBA and Swiss mice. GABA-T from the different strains was indistinguishable on the basis of all the tests employed.