<Emphasis Type="Italic">N</Emphasis>-Methyl-<Emphasis Type="SmallCaps">D</Emphasis>-Aspartate Receptor Type 1 Immunoreactivity and Protein Level in the Gerbil Main Olfactory Bulb After Transient Forebrain Ischemia

It has been reported that the over-stimulation of N-methyl-d-aspartate receptor (NR) modulates glutamate postsynaptic neurotransmission by generating long lasting Ca²⁺ channel openings. In the present study, we investigated ischemia-induced change in NR1 immunoreactivity and level in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. NR1 immunoreactivity in the sham-operated group was shown mainly in tufted cells of the external plexiform and in mitral cells of the mitral cell layer. NR1 immunoreactivity in these neurons was increased with time and was very strong 15 days after ischemia/reperfusion. At that time, NR1 protein level in the MOB was also highest. Thereafter, NR1 immunoreactivity and protein level in the MOB were decreased with time after ischemia/reperfusion. Thus, NR1 in tufted and mitral cells in the gerbil MOB is changed after transient forebrain ischemia. This suggests that mitral and tufted cells may be the principal neurons in the MOB affected in receiving inputs and sending projections to the olfactory area after transient ischemia. Y. Her and K.-Y. Yoo contributed equally to this article.     

Immunocytochemical identification of GABAergic neurons in the main olfactory bulb of the rat

With the aid of a sheep antiserum against rat brain glutamate decarboxylase (GAD), the endogenous marker for GABAergic neurons, we have labeled immunocytochemically various types of nerve cells in the main olfactory bulb of rats, with and without topic injections of colchicine. The peroxidase-antiperoxidase procedure was applied to floating Vibratome and frozen sections. A large part of the periglomerular cell population and practically all granule cells in the deep layers contain GAD-like immunoreactivity in untreated rats, while tufted and mitral cells (the projection neurons) are unstained. This observation confirms a previous study with a rabbit antiserum against mouse brain GAD, which suggested that GABAergic neurons with presynaptic dendrites contain high somatal concentrations of GAD. We show, however, that immunostaining of granule cell bodies decreases progressively from the internal plexiform layer to the deep portion of the granule cell layer. Many cell processes in the glomeruli are densely stained. They presumably represent synaptic gemmules of the numerous GAD-positive periglomerular cells, which thus could provide initial, inhibitory modulation of the afferent input. In the external plexiform layer immunostaining of the neuropil is substantially denser in the superficial half than in the deep half. This may reflect a corresponding gradient of inhibition related to unequal frequency of occurrence of synaptic gemmules of granule cell dendrites. Alternatively such a graded immunostaining of cell processes could be related to the corresponding gradient in the density of immunostaining of granule cell bodies in the deep layers, in accordance with recent data indicating that superficial and deep granule cells project their ascending dendrites respectively to superficial and deep portions of the external plexiform layer. Furthermore, we have demonstrated the presence of additional classes of GAD-positive neurons, microneurons in the external plexiform layer, small neurons in the periglomerular region, the external plexiform layer, the mitral cell layer, the internal plexiform layer, and medium-size neurons in the granule layer and the white matter. The small- and medium-size GAD-positive neurons appear weakly immunoreactive in untreated rats, but become densely stained after topic colchicine injection. Such cells presumably lack presynaptic dendrites and may correspond to different types of short axon cells demonstrated by the Golgi method.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient Cerebral Ischemia Induces Active Astrocytosis Without Distinct Neuronal Death in the Gerbil Main Olfactory Bulb: A Long-Term Analysis

In the present study, we examined ischemia-induced neuronal and glial changes in the gerbil MOB at various time points during 60 days after 5 min of transient cerebral ischemia. The number of neuronal neuclei-immunoreactive neurons was not changed after ischemia/reperfusion (I/R). Myelin basic protein immunoreaction was well preserved after I/R. Five days after I/R, reactive form of GFAP-immunoreactive astrocytes began to increase in the external plexiform layer and granule cell layer: These reactive astrocytes peaked 10 days after I/R, thereafter, they decreased with time after I/R. Iba-1-immunoreactive microglia were ubiquitously distributed in all layers of the MOB. After I/R, significant changes in their morphology and immunoreactivity were not detected. The results of western blot analyses for GFAP, Iba-1 and MBP were similar to the immunohistochemical data. In addition, 8-hydroxy-2′-deoxyguanosine (a marker for DNA damage) immunoreactivity and SOD1, an antioxidant, protein levels were not changed in the ischemic MOB. These results indicate that neurons in the MOB are resistant to ischemic insult, showing that astrocytes are activated late in the ischemic MOB.     

Calbindin D-28k-positive neurons in the rat olfactory bulb

Summary We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.     

Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.     

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.     

Relation Among Neuronal Death,Cell Proliferation and Neuronal Differentiation in the Gerbil Main Olfactory Bulb after Transient Cerebral Ischemia

Neurogenesis occurs during the embryonic stage and throughout life. Brain injuries such as ischemic insults enhance cell proliferation in some areas of the brain. We examined proliferation of newly generated cells in each layer of the gerbil main olfactory bulb (MOB) after 5 min of transient cerebral ischemia using bromodeoxyuridine (BrdU) immunohistochemistry. Ischemia-related neuronal death in the MOB was not detected using Fluoro-Jade B histofluorescence and TUNEL staining. Many BrdU-positive (⁺) cells were found in the rostral migratory stream in control and ischemic MOBs. Significant increase of BrdU⁺ cells was observed in the granule cell layer (GCL) and glomerular layer (GL) from 15 days post-ischemia, and BrdU⁺ cells were very much higher than those of the control group 30 days post-ischemia. At this time point after ischemia/reperfusion, a few BrdU⁺ cells in the GL and GCL were co-localized with calretinin⁺ cells, and many BrdU⁺ cells expressed doublecortin, a marker of immature neurons. These results indicate that cell proliferation is increased in the GCL and GL without apparent neuronal loss from 15 days after transient cerebral ischemia in gerbils.     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

GABA concentration and GAD activity levels in normal and degenerated retinas from mice

Freeze-dried sections (14 m thick) were prepared from mice with normal (C57BL strain) and degenerated (C3H strain) retinas. GABA concentration and GAD activity were determined in the microsamples (1.8–20 ng dry weight) of retinal layers and sublayers, using an enzymatic amplication reaction, NADP cycling. 1) GABA was distributed over all layers of normal retina with a broad concentration peak covering both inner nuclear and plexiform layers. In contrast, GAD activity was mostly localized in the inner plexiform layer. 2) GABA concentration was similar in one-fourth of the sublayers of each inner nuclear or plexiform layer. GAD activity was highest in the innermost sublayer of the inner nuclear layer. An increasing gradient of GAD activity was present in the inward direction in the inner plexiform layer. 3) In the degenerated retina, lacking in photoreceptors, the inner nuclear and plexiform layers remained, and GABA and GAD levels in these layers were similar to those in normal retina.Special Issue dedicated to Dr. O. H. Lowry.     

Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons

Gamma‐aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate‐limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin‐releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1–7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock‐out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 249–270, 2015     

Glutamic acid decarboxylase (GAD)-like immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary A substance immunologically related to vertebrate glutamic acid decarboxylase (GAD) has been visualized in the pedal ganglion of Mytilus with the pre-embedding peroxidase-antiperoxidase method, by use of an antiserum raised in sheep against rat brain GAD. The results show that GAD-like immunoreactivity is present both in neuronal perikarya and in nerve fibers. Positive neurons are located mainly among the fibers of the ganglion neuropil at the commissural level, and more rarely close to unreactive cortical cell bodies. Immunoreactive nerve fibers are observed throughout the neuropil and also in cerebropedal and pedal nerves.Supported by Ministero Pubblica Istruzione (40%)     

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.     

Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils.

Although galanin (GAL) protects hippocampal neurons from ischemic damage, no study has examined ischemia-related changes in endogenous GAL in the hippocampal dentate gyrus. We investigated the chronological changes of GAL, well-known as the potassium channel opener, expression in the dentate gyrus at various times after 5 min of transient forebrain ischemia in gerbils. A few GAL-immunoreactive (IR) neurons were found in the polymorphic layer of the sham-operated group. Three hours after ischemia-reperfusion, the pattern of GAL immunoreactivity was similar to that of the sham-operated group and the number of GAL-IR neurons and immunoreactivity were highest 12 h after ischemic insult. At this time, GAL-IR neurons in the polymorphic layer showed strong GAL immunoreactivity. Thereafter, GAL-IR neurons and immunoreactivity significantly decreased in the dentate hilar region. Four days after ischemic insult, GAL-IR neurons were not detectable. In addition, the results of a Western blot study showed a pattern of GAL expression similar to the immunohistochemical changes. GAL protein content also was highest 12 h after ischemia. In conclusion, the increased expression of endogenous GAL in the dentate gyrus after ischemia is related to response to the ischemic damage.     

Compartmentalization of GABA Synthesis by GAD67 Differs between Pancreatic Beta Cells and Neurons

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.     

Immunoreactivity of Glutamic Acid Decarboxylase (GAD) Isoforms in the Central Nervous System of the Barn Spider,Araneus cavaticus

The γ‐aminobutyric acid (GABA) has long been considered as an inhibitory neurotransmitter in the central nervous system (CNS) of both vertebrates and arthropods. Since the glutamic acid decarboxylase (GAD) has a restricted tissue distribution and catalyzes the conversion of L‐glutamate to GABA, immunoreactivity of GAD isoforms can reveal distribution of GABAergic neurons in the CNS. In the CNS of the spider Araneus cavaticus, immunoreactivity of GAD isoforms can be detected in the optic lobes including neurons and neuropiles of the supraesophageal ganglia. Strong GAD‐like immunoreactive cell bodies are concentrated in two bilaterally symmetric cell clusters of the protocerebrum. Some intrinsic cell bodies near the central body also show strong immunoreactivity. However, the intrinsic nerve masses and some of the longitudinal and transverse tracts within the supraesophageal ganglion are only lightly labelled, and the fibers transverse the hemisphere and the central fibrous masses are not labelled. Among the three basic types of cell bodies surrounding the central body, several clusters of the Type‐C cells show strong GAD‐like immunoreactivity, however both of the Type‐A and Type‐B cells are not labelled at all.     

Long-term persistence of GAD activity in injured crayfish CNS tissue

Crayfish CNS fibers were isolated in vivo from their cell bodies, from cellular connections in the CNS, and from peripheral sensory and effector cells. The glutamic acid decarboxylase (GAD) activity of the experimental tissues was about half of that of the sham-operated and unoperated control tissues by two weeks after surgery and remained at about that level during the ensuing six weeks. During that time, there was no significant behavioral, electrophysiological, or histological evidence of regeneration of nerve fibers across the lesion sites. The crush-isolated connectives possessed many intact axon profiles and nonneuronal cell nuclei. The long-term persistence of GAD activity in the injured CNS tissue may reflect the involvement of glial cells in maintaining neurotransmitter levels.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).