Jang-Yen Wu

Localization of immunoreactive enkephalins in GABA synthesizing neurons of the rat neostriatum

The localization of immunoreactive glutamic acid decarboxylase (GAD) and enkephalin-like immunoreactivity was examined in serial, 4-micron frozen sections of the caudate nucleus from rats pretreated with colchicine. Colocalization was found in numerous caudate neurons of medium size. Cell counts of corresponding labeled neurons in paired adjacent sections showed that GAD and enkephalin-like immunoreactivity coexist in about one half of the caudate cell populations containing each of these substances.

Immunocytochemical localization of l-glutamate decar☐ylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decar☐ylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD), GABA degrading enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotransferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartergic/glutamergic cells and their endings in the central nervous system.

Two forms of rat brain glutamic acid decarboxylase differ in their dependence on free pyridoxal phosphate

Abstract: There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal‐5′‐phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45°C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonie, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl‐trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of γ‐aminobutyric acid (GABA) level is also discussed.

GABAergic synaptic boutons in the granule cell layer of rat dentate gyrus

GABAergic synapses in the granule cell layer of the rat dentate gyrus were examined light and electron microscopically with glutamate decarboxylase (GAD) immunocytochemistry. GAD-immunoreactive synaptic boutons formed synapses with axon initial segments and somatic spines as well as somata and dendritic shafts of the granule cell. Most of these synapses were symmetrical, while a few were asymmetrical.

Association of l-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles

Recently we have reported that the membrane-associated form of the γ-aminobutyric acid-synthesizing enzyme, l-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366–24371). In this report, several lines of evidence are presented to indicate that l-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD65 as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD65-specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD65 and GAD67 are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in γ-aminobutyric acid neurotransmission.

Production and characterization of polyclonal and monoclonal antibodies to rat brain L-glutamate decarboxylase

Abstract Specific monoclonal and polyclonal antibodies to rat brain glutamate decar☐ylase (GAD) were produced and characterized. Polyclonal antibodies against GAD were raised in rabbits by injecting a total of 70–210 μg of purified GAD i.m. The specificity of anti-GAD serum was established from a variety of tests including Ouchterlony immunodiffusion, immunoelectrophoresis, immunoprecipitation, dot immunoassay, ELISA tests and Western immunoblottings. In immunodiffusion and immunoelectrophoresis tests using partially purified GAD preparations and anti-GAD serum a single, sharp precipitin line corresponding to GAD activity was obtained. Quantitative immunoprecipitation of GAD activity was achieved using anti-GAD IgG and Staphylococcus aureus . Specificity of the antiserum was further indicated from a dot immunoassay and ELISA tests in which the intensity of the reaction product was proportional to the amount of GAD protein present. In the Western immunoblotting experiments using partially purified GAD preparations only two protein bands corresponding to the position of the two subunits of GAD were stained by anti-GAD IgG, further supporting the specificity of polyclonal antibodies against GAD. In addition to polyclonal antibodies, several specific GAD-antibodies-producing clones were also obtained by the hybridoma technique. The specificity of monoclonal antibodies against GAD were established from the following criteria: positive on ELISA test using homogenous GAD as antigen; formation of GAD-anti-GAD IgG complex as indicated from gel filtration chromatography and sodium dodecyl sulfate polyacrylamid gel electrophoresis; and specific recognition of GAD subunit in a partially purified GAD preparation in Western immunoblotting test. Monoclonal antibodies were further characterized by immunohistochemical localization of known GABAergic neurons and their processes in the cerebellum and retina.

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

The localization of γ-aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [3H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [3H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.

A RAPID METHOD FOR ASSAYING ENZYMES WHOSE SUBSTRATES AND PRODUCTS DIFFER BY CHARGE. APPLICATION TO BRAIN l-GLUTAMATE DECARBOXYLASE

Abstract— A simple, rapid, sensitive and economical assay technique has been developed for any enzyme whose substrate and product differ by charge. It is based on a combination of rapid vacuum filtration with an ion exchange resin. The technique has been successfully applied to brain glutamate decarboxylase.

Purification of L-glutamic acid decarboxylase from catfish brain.

—L‐Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS‐polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD‐anti‐GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti‐GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD‐anti‐GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.

Immunocytochemical localization of GABA neurons in the rabbit and frog retina

Abstract The visualization of γ-aminobutyric acid (GABA) neurons in rabbit and frog retinas has been carried out, using an immunocytochemical technique for the localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In the rabbit, immunoreactivity was restricted to a small group of amacrine cell bodies and their laminated processes in the inner plexiform layer. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In the frog, dense GAD immunoreactivity was observed in the inner plexiform layer, both as punctate deposits and as filled processes of stratified and diffuse amacrine cells; in the inner nuclear layer, where many cell bodies were labeled, including those of some horizontal cells; and diffusely in the outer plexiform layer.