Benefits and risks of intranigral transplantation of GABA-producing cells subsequent to the establishment of kindling-induced seizures

Neural transplantation has been investigated experimentally and clinically for the purpose of developing new treatment options for intractable epilepsy. In the present study we assessed the anticonvulsant efficacy and safety of bilateral allotransplantation of genetically engineered striatal GABAergic rat cell lines into the substantia nigra pars reticulata (SNr). Rats with previously-established seizures, induced by amygdala kindling, were used as a model of temporal lobe epilepsy. Three cell lines were transplanted: (1) immortalized GABAergic cells (M213-2O) derived from embryonic rat striatum; (2) M213-2O cells (CL4) transfected with human GAD67 cDNA to obtain higher GABA synthesis than the parent cell line; and (3) control cells (121-1I), also derived from embryonic rat striatum, but which did not show GAD expression. A second control group received injections of medium alone. Transplantation of M213-2O cells into the SNr of kindled rats resulted in significant but transient anticonvulsant effects. Neither control cells nor medium induced anticonvulsant effects. Strong tissue reactions were, however, induced in the host brain of kindled but not of non-kindled rats, and only in animals that received grafts of genetically modified CL4 cells. These tissue reactions included graft rejection, massive infiltration of inflammatory immune cells, and gliosis. The anticonvulsant effect of M213-2O cells emphasizes the feasibility of local manipulations of seizures by intranigral transplantation of GABA-producing cells. On the other hand, the present data suggest that kindling-induced activation of microglia in the SNr can enhance immune reactions to transplanted cells. In this case, under conditions of further immunological stimulation by CL4 cells, transfected with a human cDNA, substantial immune reactions occurred. Thus, it appears that the condition of the host brain and the production of foreign proteins by transplanted cells have to be considered in estimating the risks of rejection of transplants into the brain.     

Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat

Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 1998;31:73-84]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg C, Tornatore C, Abi-Saab W, Monaco MC, Dillon-Carter O, Vawter M, et al. Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content. Exp Neurol 2000;161:453-61], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures.     

Intranigral transplants of a GABAergic cell line produce long-term alleviation of established motor seizures

We have previously shown that intranigral transplants of immortalized GABAergic cells decrease the number of kainic acid-induced seizures [Castillo CG, Mendoza S, Freed WJ, Giordano M. Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav Brain Res 2006;171:109-15] in an animal model. In the present study, recurrent spontaneous behavioral seizures were established by repeated systemic injections of this excitotoxin into male Sprague-Dawley rats. After the seizures had been established, cells were transplanted into the substantia nigra. Animals with transplants of control cells (without hGAD67 expression) or with sham transplants showed a death rate of more than 40% over the 12 weeks of observation, whereas in animals with M213-2O CL-4 transplants, the death rate was reduced to less than 20%. The M213-2O CL-4 transplants significantly reduced the percentage of animals showing behavioral seizures; animals with these transplants also showed a lower occurrence of stage V seizures than animals in the other groups. In vivo and in vitro analyses provided evidence that the GABAergic cells show sustained expression of both GAD67 and hGAD67 cDNA, as well as increased gamma-aminobutyric acid (GABA) levels in the ventral mesencephalon of transplanted animals. Therefore, transplantation of GABA-producing cells can produce long-term alleviation of behavioral seizures in an animal model.     

Immortalized GABAergic Cell Lines Derived from Rat Striatum Using a Temperature-Sensitive Allele of the SV40 Large T Antigen

Embryonic striatal cells were immortalized using the A58 temperature-sensitive allele of the SV40 large T antigen. Two cell lines, M213-2O and M26-1F, with γ-aminobutyric acid (GABA)ergic properties were selected from 85 clones thus developed. M213-20 is a multipolar, polygonal cell line which expresses SV40 large T antigen and glutamate decarboxylase (GAD) at the permissive temperature (33°C) and GAD and MAP-2 immunoreactivity at the nonpermissive temperature (39.3°C). M26-1F has a fibroblast-like morphology and expresses SV40 large T antigen and GAD both at the permissive and nonpermissive temperatures and MAP-2 immunoreactivity at the nonpermissive temperature. Both lines contain GABA as measured by reversed-phase HPLC and M213-2O expresses nipecotic-sensitive [¹⁴C]GABA uptake.     

Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.     

Role of TNF-alpha receptors in mice intoxicated with the parkinsonian toxin MPTP

Local application of GABA-potentiating agents can prevent or reduce the development and maintenance of behavioral seizures induced by limbic kindling in rats. Microinjection and lesion studies suggest that the transition zone between anterior and posterior piriform cortex (PC), termed here central PC, is a potential target for transplantation of GABA-producing cells. In the present study, we transplanted conditionally immortalized mouse cortical neurons, engineered with the GABA-synthesizing enzyme GAD(65), to the central PC of rats. Suspensions of 1.5 x 10(5) cells in 1 microl were transplanted bilaterally. Control animals received transplantation of beta-galactosidase (beta-gal)-expressing cells. All rats were subsequently kindled through a chronically implanted electrode placed in the basolateral amygdala. The pre- and postkindling threshold currents for eliciting behavioral seizures were determined before and after kindling. We found the prekindling partial seizure threshold to be significantly increased by about 200% in the rats that received the GABA-producing cells compared to rats receiving beta-gal-producing transplants. After kindling, the seizure threshold tended to be higher by 100% in rats that received GABA-producing cells, although the difference from controls was not statistically significant. GABA-producing transplants had no significant effect on the rate of amygdala kindling, but the latency to the first generalized seizure during kindling was significantly increased in animals receiving GABA-producing cells. The transplanted cells showed long-term GAD(65) expression as verified immunohistologically after termination of the experiments. The findings substantiate and extend previous findings that the central PC is part of the anatomical substrate that facilitates propagation from partial to generalized seizures. The data demonstrate that genetically engineered cells have the potential to raise seizure thresholds when transplanted to the central PC.     

Transduction of Human GAD67 cDNA into Immortalized Striatal Cell Lines Using an Epstein–Barr Virus-Based Plasmid Vector Increases GABA Content

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol.124:395–400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD₆₇ cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD₆₇ expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12–15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 ± 0.4 μmol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.     

能合成和分泌GABA的永生化星形胶质细胞的构建

目的 构建能合成和分泌GABA的永生化星形胶质细胞。方法 在原代培养的大鼠星形胶质细胞内转入猴肾病毒40大T抗原基因（si mian virus40large T antigen gene，SV40Tag）使其永生化（这一部分由同济医院麻醉科田玉科教授实验室完成），并在这些细胞内转入含谷氨酸脱羧酶65亚型（glutamate decaxrboxylase65，GAD65）目的基因质粒，对照组转染含β-gal（β-半乳糖苷酶，对照质粒）质粒；应用免疫组化及Western-blot方法检测转染后细胞内GAD65的表达水平；应用毛细管电泳法检测这些细胞内外的氨基丁酸（GABA）含量；用全细胞膜片钳记录构建细胞的GABA电流。结果 与转染β-gal的对照组比较，转染GAD65的实验组细胞在具备胶质细胞特性之外，能稳定表达GAD65，细胞内的GAD65含量明显增加；同时细胞内的GABA含量明显高于对照组；实验组细胞外液GABA的浓度明显高于对照组；实验组和对照组均能记录到GABA电流，说明构建细胞功能完善。结论 永生化的星形胶质细胞中转入GAD65质粒后具备胶质细胞特性，构建细胞能稳定表达GAD65，并且能合成和分泌GABA。     

Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures

Transplantation of genetically engineered cells can provide sustained focal delivery of naturally occurring molecules, including neurotransmitters and growth factors. We have engineered immortalized mouse cortical neurons and glia to deliver GABA by driving GAD(65) expression. Engineered cell lines showed GAD(65) mRNA expression, enzymatic activity, and GABA release. In vitro, basal flux of GABA was approximately 20% of total cellular GABA. We transplanted these GABA-producing cells bilaterally into either the anterior or the posterior substantia nigra of 43 rats. The rats were subsequently kindled through an electrode placed in the entorhinal cortex. GABA-producing cells, but not beta-galactosidase-producing cells, affected kindling rates. The number of stimulations needed to reach the first stage-5 seizure and to achieve full kindling differed significantly between the anterior and posterior transplantation sites when GAD(65)-producing cells were transplanted but not when beta-galactosidase-producing cells were transplanted. Our data show that transplanted engineered cells can make and release GABA at physiologically meaningful concentrations.     

Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two “fast-acting” amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects. © 1996 Wiley-Liss, Inc.     

Recombinant adeno-associated virus (AAV) drives constitutive production of glutamate decarboxylase in neural cell lines.

Many neurological disorders result directly or indirectly from the loss of inhibitory function. Engineering the production of GABA, an inhibitory neurotransmitter, may therefore be able at least partly to restore the lost inhibition seen in epilepsy, Parkinson's disease, or Huntington's disease. In this article, we describe a set of recombinant adeno-associated viruses (AAVs) that can deliver cDNAs encoding the GABA-producing enzyme, glutamate decarboxylase (GAD), directly into neural cells. We have characterized these recombinant AAVs in several cell lines derived from the CNS. These recombinant AAVs effectively transduced all neural cell lines, although with different efficiencies. Transduction occurred in both proliferating and nonproliferating cells, but actively proliferating cell lines had approximately six times greater transduction efficiency than nonproliferating cells. Furthermore, these AAVs maintained long-term expression of GAD in an astrocytic cell line for at least seven passages. These recombinant AAVs are promising vehicles for investigating the potential therapeutic effects of GABA in animal models of epilepsy and neurodegenerative diseases.     

Enhancement of neuronal protection from oxidative stress by glutamic acid decarboxylase delivery with a defective herpes simplex virus vector

We have developed defective herpes simplex virus 1 (HSV-1) vectors, based on amplicon plasmids with a replication-deficient mutant, as helper for the transfer of the glutamic acid decarboxylase (GAD67) or beta-galactosidase (beta-gal) gene as control directed by HCMV promoter into neuronal-like cells (PC12) and primary neurons. GAD67 protein was detected immunochemically, while GAD67 activity in virus-producing and nonproducing cell lines was detected enzymatically or by GABA release. Infection with GAD67-expressing amplicon vectors enhanced the resistance of PC12 cells to H(2)O(2). This protection was related to increased energy metabolism, as shown by MTT reduction and ATP level, and involved the GABA shunt, as shown by the reduction in ATP level seen in the presence of gamma-vinyl GABA (GVG), a specific GABA transaminase inhibitor. Level of glutathione (GSH), which requires ATP for its synthesis, was increased by the GAD67 transgene. The activity of glucose-6-phosphate dehydrogenase involved in the maintenance of the NADPH that can be used for the regeneration of the GSH pool, was increased by infection with amplicon vectors. Thus, replication-deficient HSV-1 and the GAD67 transgene have complementary neuroprotective effects and infection with GAD67-expressing amplicon vectors was able to protect nondifferentiated cortical neurons from glutamate toxicity mediated by oxidative stress. Such defective GAD67-expressing HSV-1, as neurotropic vector, should be helpful in neurodegenerative diseases implicating alterations of energy metabolism and oxidative stress in neuronal cells expressing GABA transaminase.     

Evidence for changing positions of GABA neurons in the developing rat dentate gyrus

In recent studies, we demonstrated a distinct change in the distribution of glutamate decarboxylase 67 (GAD67) mRNA-containing neurons within the rat dentate gyrus from embryonic day 20 (E20) to postnatal day 15 (PN15) (Dupuy and Houser, J Comp Neurol 1997;389:402-418). We also observed a similar changing pattern for cells with birthdates of many of the mature GAD-containing neurons in the dentate gyrus (Dupuy and Houser, J Comp Neurol 1997;389:402-418). These observations suggested that some early-appearing GABA neurons within the developing molecular layer of the dentate gyrus may gradually alter their positions to become the mature GABAergic cells along the inner border of the granule cell layer. The goal of the present study was to provide additional evidence for our hypothesis by demonstrating the spatial relationships between GAD-containing neurons and granule cells at progressively older ages during development. In this study, immunohistochemical or in situ hybridization methods for the localization of GAD67 or its mRNA were combined with bromodeoxyuridine birthdating techniques that labeled early-generated granule cells with birthdates on E17. At E20, GAD67-containing neurons were located above the granule cell layer that contained E17 birthdated granule cells. During the first two postnatal weeks, both GAD67 mRNA-containing neurons and early-born granule cells were primarily concentrated within the granule cell layer. Double-labeled neurons were rarely observed, and this suggests that these two groups are separate populations. By PN15-PN30, most GAD67 mRNA-containing neurons were distributed along the base of the granule cell layer, significantly below the E17 birthdated granule cells. These findings support our new hypothesis that mature GABA neurons along the inner border of the granule cell layer reach their positions by migrating or translocating through the developing granule cell layer.     

Blockade of A2A receptors plus l-DOPA after nigrostriatal lesion results in GAD67 mRNA changes different from l-DOPA alone in the rat globus pallidus and substantia nigra reticulata

Studies in animal models of Parkinson's disease (PD) suggest the potential utility of adenosine A(2A) antagonists in the treatment of this disease. In the present study, unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats received chronic intermittent treatment with the adenosine A(2A) antagonist SCH58261 (5 mg/kg) plus l-DOPA (3 mg/kg) or l-DOPA (6 mg/kg) alone, at doses producing the same intensity of contralateral turning on first administration. Three days after discontinuation of treatments, GABA synthesizing enzyme glutamic acid decarboxylase (GAD67) mRNA was evaluated at cellular level in the globus pallidus (GP) and substantia nigra pars reticulata (SNr) by in situ hybridization. 6-OHDA lesion significantly increased GAD67 mRNA levels in both the GP and SNr ipsilateral to the lesion. Chronic l-DOPA (6 mg/kg), in contrast to SCH58261 plus l-DOPA (3 mg/kg), produced a sensitized contralateral turning indicative of dyskinetic potential and further increased GAD67 mRNA in the GP. In the SNr, a significant decrease in GAD67 mRNA was observed after either treatments. However, while l-DOPA (6 mg/kg) decreased SNr GAD67 mRNA below the intact side, SCH58261 plus l-DOPA (3 mg/kg) brought GAD67 mRNA to the same level of the intact SNr. l-DOPA (3 mg/kg) or SCH58261 (5 mg/kg) alone failed to modify GAD67 mRNA. Results suggest that an increase in GAD67 mRNA in GP and a decrease in SNr might underlie dyskinetic movements induced by chronic l-DOPA. In contrast, the lack of GAD67 mRNA changes in the GP and a less marked inhibition of SNr might correlate with the absence of dyskinetic potential observed after SCH58261 plus l-DOPA.     

Glutamate-modulated production of GABA in immortalized astrocytes transduced by a glutamic acid decarboxylase-expressing retrovirus

Replication-defective Moloney murine leukemia virus expressing the GAD67 gene under the control of the GFAP promoter was produced using selected clones of a fibroblast-packaging cell line. A spontaneously immortalized astrocyte cell line was infected with this virus and cellular clones expressing GAD67 selected. Astrocyte and fibroblast clones expressed functional GAD (detected by glutamic acid decarboxylation), but only fibroblasts were able to also produce GABA in the extracellular medium. When exposed to 200 μM glutamate, despite an observed difference in the rates of glutamate accumulation in control and GAD67-expressing astrocytes, similar proportions of glutamate taken up were detected. In GAD67-expressing astrocytes, the glutamate was mainly converted into GABA, suggesting GAD transgene activity to be dominant over other glutamate metabolic pathways, such as glutamine synthetase and glutamate dehydrogenase. Moreover, rapid GABA release into the cell medium was also observed, suggesting the involvement of reverse GABA transporters. The use of the GFAP promoter might be able to take advantage of its activation in response to factors inducing reactive gliosis observed in pathological insults. GAD67-expressing astrocytes might therefore be used for future grafting in pathological situations in which an excess of glutamate results in neuronal dysfunction or cell death. GLIA 22:86–93, 1998. © 1998 Wiley-Liss, Inc.     

Specific cell types in cat retina express different forms of glutamic acid decarboxylase

We studied the expression of glutamate decarboxylase (GAD), GAD65 and GAD67, in cat retina by immunocytochemistry. About 10% of GABAergic amacrime cells express only GAD65 and 30% express only GAD67. Rougly 60% contain both forms of the enzyme, but GAD67 is present only at low levels in the majority of these double-labeled amacrine cells. The staining pattern in the inner plexiform layer (IPL) for the two GAD forms was also different. GAD65 was restricted to strata 1–4, and GAD67 was apparent throughout the IPL but was strongest in strata 1 and 5. This indicates that soams, as well as their processes, are differentially stained for the two forms of GAD. Cell types expressing only GAD65 include interplexiform cells, one type of cone bipolar cell, and at least one type of serotonin-accumulating amacrine cell. Cell types expressing only GAD67 include amacrine cells synthesizing dopamine, amacrine cells synthesizing nitric oxide (NO), and amacrine cells accumulating serotonin. Cholinergic amacrine cells express a low level of both GAD forms. Our findings in the retina are consistent with previous observations in the brain that GAD65 expression is greater in terminals than in somas. In addition, in retina most neurons expressing GAD67 also contain a second neurotransmitter as well as GABA, and they tend to be larger than neurons expressing GAD65. We propose that large cells have a greater demand for GABA than small cells, and thus require the constant, relatively unmodulated level of GABA that is provided by GAD67. © 1995 Willy-Liss, Inc.     

Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study.

Two major forms of glutamic acid decarboxylase (GAD) are present in the mammalian brain, a 65-kDa isoform (GAD(65)) and a 67-kDa isoform (GAD(67)), and it is usually assumed that all GABAergic neurons contain both. The two forms have not yet been colocalized to the same neurons, because the GAD(65) protein is found almost exclusively in axon terminals, while GAD(67) is found predominantly in the cell body. Using double in situ hybridization (DISH) with both radioactive [35S] and non-radioactive (digoxigenin, DIG) probes, the distributions of GAD(65) and GAD(67) mRNA have been simultaneously examined in the rat hippocampus. The results suggest that [35S] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labels is necessary in DISH studies to determine whether a neuronal subtype which expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD(65) and GAD(67) mRNA. In sectors CA1 and CA3 approximately 5-10% of the cells positive for GAD(67) showed little or no detectable GAD(65) mRNA. In the hilus, however, GAD(65) levels were higher, and all cells seem to express both GAD(65) and GAD(67) mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD(65) and GAD(67). However, it appears that some interneurons in the CA subfields differ from "classic" GABAergic interneurons by preferentially expressing the 67-kDa isoform of GAD under baseline conditions, with GAD(65) mRNA levels very low or absent.     

Baculovirus p35 gene greatly enhances PC12 cell's resistance against oxidative stress

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.     

Dark-rearing-induced reduction of GABA and GAD and prevention of the effect by BDNF in the mouse retina

Gamma‐aminobutyric acid (GABA) is an important retinal neurotransmitter. We studied the expression of GABA, glutamate decarboxylase 65 (GAD65) and GAD67 by immunocytochemistry and Western blot, in the retinas of control and dark‐reared C57BL/6J black mice. This study asked three questions. First, is visual input necessary for the normal expression of GABA, GAD65 and GAD67? Second, can the retina recover from the effects of dark‐rearing if returned to a normal light–dark cycle? Third, does BDNF prevent the influence of dark‐rearing on the expression of GABA and GAD? At postnatal day 10 (P10), before eye opening, GABA immunoreactivity was present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL) and throughout the inner plexiform layer (IPL) of control and dark‐reared retinas. In P30 control retinas, GABA immunoreactivity showed similar patterns to those at P10. However, in P30 dark‐reared retinas, the density of GABA‐immunoreactive cells was lower in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis showed corresponding differences in the levels of GAD65 but not of GAD67 expression between control and dark‐rearing conditions. In our study, dark‐rearing effects were reversed when the mice were put in normal cyclic light–dark conditions for 2 weeks. Moreover, dark‐reared retinas treated with BDNF showed normal expression of both GABA and GAD65. Our data indicate that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggest that BDNF controls this dependence.