Glutamic acid decarboxylase immunoreactivity is present in perikarya of neurons in nucleus tractus solitarius of rat

Immunohistochemical procedures reveal perikarya containing glutamic acid decarboxylase immunoreactivity in the nucleus tractus solitarius of the rat. After colchicine pretreatment, neurons were observed in all subdivisions of the nucleus with a particular concentration in the ventrolateral and intermediate subdivisions.     

Distribution and development of glutamic acid decarboxylase immunoreactivity in the spinal cord of the dogfish Scyliorhinus canicula (elasmobranchs)

The adult distribution and development of gamma-aminobutyric acid (GABA)-synthesizing cells and fibers in the spinal cord of the lesser spotted dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against glutamic acid decarboxylase (GAD). Complementary immunostaining with antibodies against GABA, tyrosine hydroxylase (TH), and HuC/HuD (members of the Hu/Elav family of RNA-associated proteins) and staining with a reduced silver procedure ("en bloc" Bielschowski method), Nissl, and hematoxylin were also used. In adults, GAD-immunoreactive (GAD-ir) cells were observed in the ventral horns, in the spinal nucleus of the dorsal horn, at the base of the dorsal horns, and around the central canal, where some GAD-ir cells were cerebrospinal fluid-contacting (CSF-c). In addition, a few GAD-ir cells were observed in the lateral funiculus between the ventral horn and the marginal nucleus. The adult spinal cord was richly innervated by GAD-ir fibers. Large numbers of GAD-ir fibers and boutons were observed in the dorsal and ventral horns and also interstitially in the dorsal, lateral, and ventral funiculi. In addition, a rich GAD-ir innervation was observed in the marginal nucleus of the spinal cord. In the embryonic spinal cord, GAD-ir cells develop very early: The earliest cells were observed in the very thin mantle/marginal layer of stage 22 embryos in a short length of the spinal cord. At stages 25 and 26, several types of GAD-ir cells (commissural and noncommissural) were distinguished, and two of these cells were of CSF-c type. At stages 28, 30, and 31, the GAD-ir populations exhibited a marked longitudinal columnar organization. Double-immunolabeling experiments in embryos showed the presence of two different GAD-ir CSF-c cell populations, one ventral that is simultaneously TH-ir and other more dorsal that is TH-negative. By stage 33 (prehatching), GAD-expressing cells are present in virtually all loci, as in adults, especially in the ventral horn and base of the dorsal horn. The present results for the lesser spotted dogfish suggest an important role for gamma-aminobutyric acid in sensory and motor circuits of the spinal cord.     

Reductions in N-acetylaspartylglutamate and the 67 kDa form of glutamic acid decarboxylase immunoreactivities in the visual system of albino and pigmented rats after optic nerve transections

This study compares the immunohistochemical distributions of N-acetylaspartylglutamate (NAAG) and the large isoform of the gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD(67)) in the visual system of albino and pigmented rats. Most retinal ganglion cells and their axons were strongly immunoreactive for NAAG, whereas GAD(67) immunoreactivity was very sparse in these cells and projections. In retinorecipient zones, NAAG and GAD(67) immunoreactivities occurred in distinct populations of neurons and in dense networks of strongly immunoreactive fibers and synapses. Dual-labeling immunohistochemistry indicated that principal neurons were stained for NAAG, whereas local interneurons were stained for GAD(67). In contrast to the distribution observed in retinorecipient zones, most or all neurons were doubly stained for NAAG and GAD(67) in the thalamic reticular nucleus. Ten days after unilateral optic nerve transection, NAAG-immunoreactive fibers and synapses were substantially reduced in all contralateral retinal terminal zones. The posttransection pattern of NAAG-immunoreactive synaptic loss demarcated the contralateral and ipsilateral divisions of the retinal projections. In addition, an apparent transynaptic reduction in GAD(67) immunoreactivity was observed in some deafferented areas, such as the lateral geniculate. These findings suggest a complicated picture in which NAAG and GABA are segregated in distinct neuronal populations in primary visual targets, yet they are colocalized in neurons of the thalamic reticular nucleus. This is consistent with NAAG acting as a neurotransmitter release modulator that is coreleased with a variety of classical transmitters in specific neural pathways.     

Dopaminergic regulation of glutamic acid decarboxylase mRNA expression and GABA release in the striatum: A review

Lindefors Nils: Dopaminergic Regulation of Glutamic Acid Decarboxylase mRNA Expression and GABA Release In the Striatum: A Minireview. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1993, 17(6): 887–903.

1. 1. The majority of neurons in the striatum (caudate-putamen, dorsal striatum; nucleus accumbens, ventral striatum) and in striatal projection regions (the pallidum, the entopeduncular nucleus and substantia nigra reticulata) use γ-aminobuturic acid (GABA) as transmitter and express glutamic acid decarboxylase (GAD; rate limiting enzyme) in the synthesis of GABA. GABA is the major inhibitory transmitter in the mammlian brain.

2. 2. GAD in brain is present as two isoenzymes, GAD₆₅ and GAD₆₇. GAD₆₅ is largely present as an inactive apoenzyme, which can be induced by nerve activity, while most GAD₆₇ is present as a pyridoxal phosphate-bound permanently active holoenzyme. Thus GAD₆₅ and GAD₆₇ seem to provide a dual system for the control of neuronal GABA synthesis.

3. 3. GAD mRNA expression can be visualised and quantified using in situ hybridisation, and GABA release can be quantified using in vivo microdialysis.

4. 4. Different populations of GABA neurons can be distinguished in both dorsal and ventral striatum as well as in other parts of the basal ganglia.

5. 5. Inhibition of dopaminergic transmission in the striatum by lesion of dopamine neurons or by neuroleptic treatment is followed by an increased release of GABA and increased expression of GAD₆₇ mRNA in a subpopulation of striatal medium-sized neurons which project to the globus pallidus, and increased striatal GAD enzyme activity.

6. 6. Increased dopaminergic transmission by repeated but not single doses of amphetamine is followed by decreased striatal GABA release and decreased GAD₆₇ mRNA expression in a subpopulation of medium-sized neurons in the striatum.

7. 7. Two populations of medium-sized GABA neurons in the striatum seem to be under tonic dopaminergic influence. The majority of these GABA neurons are under inhibitory influence, whereas a small number seem to be stimulated by dopamine.

8. 8. Specific changes in activity in subpopulations of striatal GABA neurons probably mediate the dopamine-dependent hypokinetic syndrome seen in Parkinson's disease and following neuroleptic treatment.

Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex

The birth dates of neurons showing glutamic acid decarboxylase (GAD) immunoreactivity have been determined in mouse somatosensory cortex. Pregnant C57Bl mice received pulse injections of (3H)thymidine from E10 through E17 (E0 being the day of mating). The distributions of thymidine-labeled, GAD-positive and nonimmunoreactive (non-GAD) cells as a function of depth under the pial surface were recorded in adult animals. The maximum rate of generation of GAD-positive neurons occurred at E14, whereas the generation of non-GAD neurons reached its maximum rate at E13. Except for those in layer I, GAD-positive neurons followed an inside-out sequence of positioning. GAD-positive neurons born at E12 and E13 were located in layers VI-IV. GAD-positive neurons born at E14 were found throughout the cortical thickness, with a maximum in layer IV. The GAD-positive neurons labeled after pulses at E15 or E16 or E17 were limited to the superficial strata, forming a band that became narrower as it moved toward the pial surface with increase in age of pulse labeling. GAD-positive neurons in layer I were generated at a constant rate during the whole embryonic period analyzed. Non-GAD neurons also followed an inside-out spatiotemporal gradient. Two partially overlapping phases were distinguished in non-GAD neurogenesis. During the first phase (from E12 to E14) neurons populating adult layers VI and V originated, while neurons located in layers IV through I were generated during the second phase (from E13 to E17). Since GAD-immunoreactive neurons form a heterogeneous population, we envisage further studies in order to test whether differences exist in birth dates among the classes.     

Limbic encephalitis with antibodies to glutamic acid decarboxylase presenting with brainstem symptoms

Limbic encephalitis (LE) is a neurological syndrome that may present in association with cancer, infection, or as an isolate clinical condition often accompanying autoimmune disorders. LE associated with glutamic acid decarboxylase antibodies (anti-GAD) is rare in children. Here, we characterized the clinical and laboratory features of a patient presenting with brainstem involvement with non-paraneoplastic LE associated with anti-GAD antibodies. In our patient, after plasma exchange, we determined a dramatic improvement of the neurological deficits.     

Alterations in cerebellar glutamic acid decarboxylase (GAD) activity in a genetic model of torsion dystonia (rat)

Glutamic acid decarboxylase (GAD) activity was studied in specific brain regions of a newly identified genetic (rat) model of human torsion dystonia. GAD activity was found to be significantly increased in the deep cerebellar nuclei of dystonic rats at 16, 20, and 24 days of age. GAD activity in the other regions examined (vermis, cerebellar hemispheres, caudate nucleus, and globus pallidus) did not differ from that of age-matched normal littermate controls. Diazepam treatment significantly reduced the frequency of dystonic movements in the mutant.     

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for γ-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo. J. Comp. Neurol. 396:253–266, 1998. © 1998 Wiley-Liss, Inc.     

Decreased glutamic acid decarboxylase mRNA expression in prefrontal cortex in Parkinson's disease

Parkinson's disease (PD) patients typically suffer from motor disorders but mild to severe cognitive deficits can also be present. Neuropathology of PD primarily involves loss of dopaminergic neurons in the substantia nigra, pars compacta, although more widespread pathology from the brainstem to the cerebral cortex occurs at different stages of the disease. Cognitive deficits in PD are thought to involve the cerebral cortex, and imaging studies have identified the dorsolateral prefrontal cortex (DLPFC) as a possible site for some of the symptoms. GABAergic neurons in the cerebral cortex play a key role in the modulation of pyramidal neurons and alterations in muscimol binding to GABA_A receptors have been reported in Brodmann area 9 (BA9) of the prefrontal cortex in PD patients (Nishino et al., 1988). In order to further assess the likelihood that GABAergic activity is altered in the prefrontal cortex in PD, gene expression of the 67 kilodalton isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD67 encoded by the GAD1 gene), was examined in BA9 of post-mortem brains from 19 patients and 20 controls using isotopic in situ hybridization histochemistry. GAD67 mRNA labeling was examined and quantified on X-ray films and emulsion radioautographs. We show that GAD67 mRNA labeling is significantly lower in PD compared to control cases. Analysis of emulsion radioautographs indicates that GAD67 mRNA labeling is decreased in individual neurons and is not paralleled by a decrease in the number of GAD67 mRNA-labeled neurons. Analysis of expression data from a microarray study performed in 29 control and 33 PD samples from BA9 confirms that GAD67 expression is decreased in PD. Another finding from the microarray study is a negative relationship between GAD67 mRNA expression and age at death. Altogether, the results support the possibility that GABAergic neurotransmission is impaired in the DLPFC in PD, an effect that may be involved in some of the behavioral deficits associated with the disease.     

Effects of early monocular deprivation on choline acetyltransferase and glutamic acid decarboxylase in pigeon visual Wulst

Choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities were measured in various visual structures of the pigeon brain after long-term monocular deprivation followed by short-term binocular presence or absence of light stimulation. The short-term phase (45 min) was coupled with a 2-deoxyglucose experiment in order to select the adequate brain samples. After monocular deprivation during the first 6–11 months, ChAT activity was higher by 40–60% in the dorsolateral visual Wulst contralateral to the deprived eye, as compared to the other side. In the same structure, animals, either monocularly deprived or undeprived and exposed binocularly to environmental light for 45 min, had higher ChAT activities on both sides than those maintained in the dark. Monocular deprivation performed in adult animals did not affect the ChAT activity in visual Wulst. GAD activity was bilaterally decreased in the visual Wulst after early monocular deprivation. These results suggest that early monocular deprivation has an effect on biochemical systems involved in synaptic transmission at selected relays of the visual pathways.     

重组大鼠谷氨酸脱羧酶载体的构建和功能分析(英文)

目的谷氨酸脱羧酶2 (glutamic acid decarboxylase 2,GAD65) 是γ-氨基丁酸(gamma-aminobutyric acid, GABA)的合成酶。本研究拟构建重组大鼠GAD65基因的慢病毒载体(recombinant lentivirus-rGAD65,rLV-rGAD65),并在体内外分析其功能。方法用RT-PCR法克隆大鼠GAD65基因的cDNA 并亚克隆至慢病毒载体上,形成重组慢病毒质粒(rLV-GFP-rGAD65)。在包装质粒的帮助下,获得重组慢病毒颗粒(rLV-rGAD65)并检测其滴度。用rLV-rGAD65感染原代培养的大鼠肺成纤维细胞,并用免疫细胞化学和蛋白印迹法检测rGAD65在成纤维细胞中的表达,用高效液相法(high-performance liquid chromatograph, HPLC)检测培养上清中GABA的含量。在体内, rLV-rGAD65 定点注射到Sprague-Dawley大鼠的丘脑底核(subthalamic nucleus,STN)。用免疫组织化学和蛋白印迹法检测GAD65基因在STN中的表达水平,HPLC检测黑质网状部(su...     

Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. II: Area dentata

The temporal patterns of neurogenesis of cells showing glutamic acid decarboxylase (GAD) immunoreactivity were determined in the area dentata of the mouse. Pregnant C57Bl mice received pulse injections of (3H)thymidine from E11 through E17 (E0 being the day of mating). The distribution of (3H)thymidine-labeled, GAD-positive neurons in the hilus and in the different strata of the fascia dentata (stratum infragranulosum, stratum granulosum, stratum moleculare) were recorded in adult animals. A radial gradient of neurogenesis of GAD-positive cells in the area dentata was not apparent. In the transverse axis, neurogenesis of GAD-positive cells seemed to follow a faint suprapyramidal to infrapyramidal gradient, which was due to differential timing of neurogenesis of GAD-positive cells destined for the stratum infragranulosum of the suprapyramidal and infrapyramidal blades of the fascia dentata. GABAergic neurons in the fascia dentata comprise a limited number of well-defined cell types. All of the different morphologic types of GAD-positive neurons present in the area dentata were generated prenatally. These diverse forms did not have specific times of neurogenesis. These results support the concept that the adult morphology of GAD-positive cells in the area dentata of the mouse do not bear any relationship to their times of origin.     

Elevated immunoreactivity for glutamic acid decarboxylase in the rat cerebral cortex following transient middle cerebral artery occlusion

We investigated the expression of glutamic acid decarboxylase (molecular weight 67,000; GAD67) immunohistochemically in the rat cerebral cortex following transient middle cereral artery occlusion (MCAO) capable of producing slowly progressive neuronal damage. An increase in GAD67 immunoreactivity was observed in the cerebral cortex ipsilateral to the ischemic insult, most prominent in lamina IV, 3 to 14 days after MCAO. At this stage, light microscopy showed GAD67-positive puncta to be larger and more strongly immunoreactive in the ipsilateral cortex than those in the contralateral side. The elevated expression of GAD67 in the insulted cortex may reflect part of the adaptive functional changes in GABA transmission with slowly progressive cortical ischemic damage.Supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Developmental changes of glutamate acid decarboxylase 67 in mouse brain after hypoxia ischemia

Objective To study the developmental changes of glutamic acid decarboxylase-67 (GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6±7.0)% TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.     

Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation

The mRNAs for two forms of glutamic acid decarboxylase (GAD65 and GAD67) were localized in the rat hippocampal formation by nonradioactive in situ hybridization methods with digoxigeninlabeled cRNA probes. Some neurons in all layers of the hippocampus and dentate gyrus were readily labeled for each GAD mRNA, and the patterns of labeling for GAD65 and GAD67 mRNAs were very similar. All major groups of previously described GAD-and GABA-containing neurons appeared to be labeled for each GAD mRNA. Such findings suggest that most GABA neurons in the hippocampal formation contain both GAD mRNAs. When the labeling of neurons in the hippocampal formation and cerebral cortex was compared in the same sections, the intensity of neuronal labeling for GAD67 mRNA was generally similar in the two regions. However, the intensity of labeling for GAD65 mRNA was generally stronger for many neurons in the hippocampal formation than for most neurons in the cerebral cortex. Neurons in the hilus of the dentate gyrus were particularly well labeled for GAD65. The nonradioactive labeling for the GAD mRNAs was confined to the cytoplasm of neuronal cell bodies, and this allowed a clear visualization of the relative number and location of labeled neurons. Several distinct patterns of GAD mRNA-containing neurons were observed among different regions of the hippocampal formation. In the hilus of the dentate gyrus, GAD mRNA-containing neurons were numerous in the regions deep to the granule cell layer as well as in more central parts of the hilus. Within CA3, the densities (quantities) of labeled neurons varied among the regions. In the inner or hilar segment of CA3, the density of labeled neurons was often lower than that in the outer part of CA3 where numerous labeled neurons were distributed throughout all layers. In CA1, GAD mRNA-labeled neurons were distributed in a relatively laminar pattern with the highest density in stratum pyramidale and moderate densities in stratum oriens and at the interface between strata radiatum and lacunosum-moleculare. Lower densities were found within the latter two layers. The prominent localization of the two GAD mRNAs in the hippocampal formation suggests that dual system for GABA synthesis is necessary for normal GABAergic function in this brain region. Most putative GABA neurons contain relatively high levels of GAD67 mRNA as might be expected if this GAD form is responsible for the synthesis of GABA for metabolic and baseline synaptic function. The relatively high levels of GAD65 mRNA in many hippocampal neurons, particularly those of the dentate hilus, may indicate that these neurons have a well-developed reserve system for GAD and GABA synthesis. © 1994 Wiley-Liss, Inc.     

Refractory status epilepticus and glutamic acid decarboxylase antibodies in adults: presentation,treatment and outcomes

Aim. Glutamic acid decarboxylase antibodies (GAD‐Abs) have been implicated in refractory epilepsy. The association with refractory status epilepticus in adults has been rarely described. We discuss our experience in managing three adult patients who presented with refractory status epilepticus associated with GAD‐Abs. Methods. Case series with retrospective chart and literature review. Results. Three patients without pre‐existing epilepsy who presented to our institution with generalized seizures between 2013 and 2014 were identified. Seizures proved refractory to first and second‐line therapies and persisted beyond 24 hours. Patient 1 was a 22‐year‐old female who had elevated serum GAD‐Ab titres at 0.49 mmol/l (normal: <0.02) and was treated with multiple immuno‐ and chemotherapies, with eventual partial seizure control. Patient 2 was a 61‐year‐old black female whose serum GAD‐Ab titre was 0.08 mmol/l. EEG showed persistent generalized periodic discharges despite maximized therapy with anticonvulsants but no immunotherapy, resulting in withdrawal of care and discharge to nursing home. Patient 3 was a 50‐year‐old black female whose serum GAD‐Ab titre was 0.08 mmol/l, and was discovered to have pulmonary sarcoidosis. Treatment with steroids and intravenous immunoglobulin resulted in seizure resolution. Conclusion. Due to the responsiveness to immunotherapy, there may be an association between GAD‐Abs and refractory seizures, including refractory status epilepticus. Causation cannot be established since GAD‐Abs may be elevated secondary to concurrent autoimmune diseases or formed de novo in response to GAD antigen exposure by neuronal injury. Based on this report and available literature, there may be a role for immuno‐ and chemotherapy in the management of refractory status epilepticus associated with GAD‐Abs.     

抗谷氨酸脱羧酶65抗体脑炎临床特点分析

目的 探讨抗谷氨酸脱羧酶65(GAD65)抗体脑炎患者的临床特点。方法 回顾性总结分析2016年4月至2022年3月就诊于中南大学湘雅医院神经内科的9例抗GAD65抗体脑炎患者的临床资料。结果 9例患者发病年龄为14～76(31.2±20.5)岁;男女比例为4∶5。1例有前驱感染史。首发症状：癫痫发作3例、头痛2例、头晕1例、听力下降与幻嗅1例、不自主运动1例、面部感觉异常1例。临床表现：边缘性脑炎5例、单纯癫痫发作1例、边缘性脑炎伴小脑性共济失调1例、僵人综合征伴脑干脑炎1例、癫痫发作伴僵人综合征及小脑性共济失调1例。9例患者中,合并胰岛素依赖型糖尿病2例、合并甲状腺功能亢进1例、血抗甲状腺过氧化物酶抗体阳性4例。9例均进行了头部磁共振(MRI)检查,海马或颞叶异常信号3例、小脑萎缩1例。有7例患者在急性期进行了脑电图检查,4例合并癫痫样放电,均累及额颞区,其中有3例还累及双侧。9例患者均接受免疫治疗,单用一线治疗3例、一线联合二线治疗6例。6例接受了抗癫痫药治疗,其中4例需联合应用2种及2种以上的抗癫痫药。对9例患者进行了随访,失访1例;6例预后良好,其中2例症状完全缓解,4例部分...     

Fine distribution of gamma-aminobutyric acid, glutamic acid decarboxylase, and glutamic acid in the rabbit cerebellum

The fine distribution of GABA, glutamic acid decarboxylase, and glutamic acid within each layer of the rabbit cerebellar cortex was determined with microanalytical methods. The greatest glutamic acid decarboxylase activity and the highest GABA concentration were found in the Purkinje cell layer. In the distribution of GABA and glutamic acid decarboxylase the peak of glutamic acid decarboxylase activity was more pronounced than that of GABA; the concentration of glutamic acid did not show much variation between each layer.