Regulation of γ-Aminobutyric Acid Synthesis in the Brain

Abstract: γ-Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD₆₅ and GAD₆₇, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′-phosphate (pyridoxal-P), and level of expression among brain regions. GAD₆₅ appears to be localized in nerve terminals to a greater degree than GAD₆₇, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal-P is a major factor in the short-term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD₆₅, but GAD₆₇ also contributes to the pool of apoGAD. The apparent localization of GAD₆₅ in nerve terminals and the large reserve of apo-GAD₆₅ suggest that GAD₆₅ is specialized to respond to short-term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo- to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short-term regulation by second messengers. The syntheses of GAD₆₅ and GAD₆₇ proteins are regulated separately. GAD₆₇ regulation is complex; it not only is present as apoGAD₆₇, but the expression of GAD₆₇ protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.     

GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarboxylase 67

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67 kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.     

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.     

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification,cloning, expression,assay development,identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD₆₅ and GAD₆₇ genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD₆₅ and GAD₆₇ resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.     

Induction of the GABA cell phenotype: an in vitro model for studying neurodevelopmental disorders

Recent studies of the hippocampus have suggested that a network of genes is associated with the regulation of the GAD₆₇ (GAD1) expression and may play a role in γ-amino butyric acid (GABA) dysfunction in schizophrenia (SZ) and bipolar disorder (BD). To obtain a more detailed understanding of how GAD₆₇ regulation may result in GABAergic dysfunction, we have developed an in vitro model in which GABA cells are differentiated from the hippocampal precursor cell line, HiB5. Growth factors, such as PDGF, and BDNF, regulate the GABA phenotype by inducing the expression of GAD₆₇ and stimulating the growth of cellular processes, many with growth cones that form appositions with the cell bodies and processes of other GAD₆₇-positive cells. These changes are associated with increased expression of acetylated tubulin, microtubule-associated protein 2 (MAP2) and the post-synaptic density protein 95 (PSD95). The addition of BDNF, together with PDGF, increases the levels of mRNA and protein for GAD₆₇, as well as the high affinity GABA uptake protein, GAT1. These changes are associated with increased concentrations of GABA in the cytoplasm of “differentiated” HiB5 neurons. In the presence of Ca²⁺ and K⁺, newly synthesized GABA is released extracellularly. When the HiB5 cells appear to be fully differentiated, they also express GAD₆₅, parvalbumin and calbindin, and GluR subtypes as well as HDAC1, DAXX, PAX5, Runx2, associated with GAD₆₇ regulation. Overall, these results suggest that the HiB5 cells can differentiate into functionally mature GABA neurons in the presence of gene products that are associated with GAD₆₇ regulation in the adult hippocampus.     

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD₆₅ and GAD₆₇, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD₆₅ and GAD₆₇ exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the K_m of GAD₆₇ and increasing the V_max of both isoforms. Of the 15 cysteines in GAD₆₅, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD₆₇, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD₆₅ and GAD₆₇ are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD₆₅ suggests that alteration of normal redox conditions could affect GAD targeting.     

Schizophrenia-like GABAergic gene expression deficits in cerebellar Golgi cells from rats chronically exposed to low-dose phencyclidine

One of the most consistent findings in schizophrenia is the decreased expression of the GABA synthesizing enzymes GAD₆₇ and GAD₆₅ in specific interneuron populations. This dysfunction is observed in distributed brain regions including the prefrontal cortex, hippocampus, and cerebellum. In an effort to understand the mechanisms for this GABA deficit, we investigated the effect of the N-methyl-d-aspartate receptor (NMDAR) antagonist phencyclidine (PCP), which elicits schizophrenia-like symptoms in both humans and animal models, in a chronic, low-dose exposure paradigm. Adult rats were given PCP at a dose of 2.58 mg/kg/day i.p. for a month, after which levels of various GABAergic cell mRNAs and other neuromodulators were examined in the cerebellum by qRT-PCR. Administration of PCP decreased the expression of GAD₆₇, GAD₆₅, and the presynaptic GABA transporter GAT-1, and increased GABA_A receptor subunits similar to those seen in patients with schizophrenia. Additionally, we found that the mRNA levels of two Golgi cell selective NMDAR subunits, NR2B and NR2D, were decreased in PCP-treated rats. Furthermore, we localized the deficits in GAD₆₇ expression solely to these interneurons. Slice electrophysiological studies showed that spontaneous firing of Golgi cells was reduced by acute exposure to low-dose PCP, suggesting that these neurons are particularly vulnerable to NMDA receptor antagonism. In conclusion, our results demonstrate that chronic exposure to low levels of PCP in rats mimics the GABAergic alterations reported in the cerebellum of patients with schizophrenia (Bullock et al., 2008. Am. J. Psychiatry 165, 1594–1603), further supporting the validity of this animal model.     

Schizophrenia-like GABAergic gene expression deficits in cerebellar Golgi cells from rats chronically exposed to low-dose phencyclidine

One of the most consistent findings in schizophrenia is the decreased expression of the GABA synthesizing enzymes GAD₆₇ and GAD₆₅ in specific interneuron populations. This dysfunction is observed in distributed brain regions including the prefrontal cortex, hippocampus, and cerebellum. In an effort to understand the mechanisms for this GABA deficit, we investigated the effect of the N-methyl-d-aspartate receptor (NMDAR) antagonist phencyclidine (PCP), which elicits schizophrenia-like symptoms in both humans and animal models, in a chronic, low-dose exposure paradigm. Adult rats were given PCP at a dose of 2.58 mg/kg/day i.p. for a month, after which levels of various GABAergic cell mRNAs and other neuromodulators were examined in the cerebellum by qRT-PCR. Administration of PCP decreased the expression of GAD₆₇, GAD₆₅, and the presynaptic GABA transporter GAT-1, and increased GABA_A receptor subunits similar to those seen in patients with schizophrenia. Additionally, we found that the mRNA levels of two Golgi cell selective NMDAR subunits, NR2B and NR2D, were decreased in PCP-treated rats. Furthermore, we localized the deficits in GAD₆₇ expression solely to these interneurons. Slice electrophysiological studies showed that spontaneous firing of Golgi cells was reduced by acute exposure to low-dose PCP, suggesting that these neurons are particularly vulnerable to NMDA receptor antagonism. In conclusion, our results demonstrate that chronic exposure to low levels of PCP in rats mimics the GABAergic alterations reported in the cerebellum of patients with schizophrenia (Bullock et al., 2008. Am. J. Psychiatry 165, 1594–1603), further supporting the validity of this animal model.     

Sex difference and response to testosterone in gabaergic cells of the medial preoptic nucleus and ventral bed nuclei of the stria terminalis in gerbils

The medial preoptic nucleus (MPN) and ventral bed nuclei of the stria terminalis (BST) are needed to maintain mating in sexually experienced male gerbils and rats. The gerbil ventral BST is also activated with mating, as assessed by Fos expression, as is the medial MPN (MPNm) of both species. In gerbils, many of those mating-activated cells contain glutamic acid decarboxylase (GAD), the enzyme that synthesizes γ-aminobutyric acid (GABA). Some of those cells are projection neurons, but others may release GABA locally. Through actions in the medial preoptic area, GABA inhibits and testosterone (T) promotes male sex behavior. Thus, T may promote mating, in part, by decreasing GAD in MPNm or ventral BST cells. In rats, T increases GAD mRNA in the central MPN (MPNc), where MPN GABAergic cells are densest, but mating behavior does not change in sexually experienced males when the MPNc is ablated. Therefore, this study focused on the MPNm and ventral BST to ask whether their GABAergic cells respond to T or are sexually dimorphic. This was done by visualizing cells immunoreactive (IR) for GAD₆₇, an isoform found primarily in cell bodies, in male and female gerbils and in castrated males with and without T. At both sites, males had more GAD₆₇-IR cells than females, and T decreased GAD₆₇-IR cell numbers in males. Thus, the MPNm and ventral BST have GABAergic cells that are sexually dimorphic and in which T decreases GAD, consistent with local effects of T and GABA on mating.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.     

Genetic markers for glutamic acid decarboxylase do not predict insulin-dependent diabetes mellitus in pairs of affected siblings

Reliable genetic and immunological markers are important in the prediction of insulin-dependent diabetes mellitus (IDDM). Since glutamic acid decarboxylase (GAD) is a candidate primary autoantigen, we examined the possible linkage between IDDM and the genes encoding GAD₆₅ (GAD2, 10p11–12) and GAD₆₇ (GAD1, 2q31) in 58 Danish IDDM affected sib pairs. The allelic inheritance of 10 polymorphic dinucleotide repeat sequences spanning the chromosomal regions of the two GAD genes, were examined by affected sib pair analysis (ASP). In addition a restriction fragment length polymorphism (RFLP) was identified in the gene encoding GAD₆₅ using the restriction enzyme PvuII. The GAD gene markers were analyzed in relation to the presence of specific HLA types and GAD autoantibodies. No evidence of linkage was found between IDDM and either of the genes encoding GAD. This was also the case when subgroups carrying specific HLA susceptibility alleles were analyzed. Nor did we observe any association between these GAD genetic markers and the presence of GAD autoantibodies. Considering the high prevalence of GAD autoantibodies in IDDM, a putative genetic association between GAD and IDDM would be expected to affect most diabetic individuals. Therefore, our data indicate that the association between GAD and IDDM is not genetically determined, and that microsatellites used in this study do not contribute to the prediction of IDDM. Received: 1 July 1996 / Revised: 21 August 1996     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.     

Divergent Evolution of the Activity and Regulation of the Glutamate Decarboxylase Systems in Listeria monocytogenes EGD-e and 10403S: Roles in Virulence and Acid Tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GAD_i), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GAD_i system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GAD_i/GAD_e). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GAD_i but not GAD_e the results indicate that the GAD_i system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.     

Characterization of GABAergic Neurons in the Mouse Lateral Septum: A Double Fluorescence In Situ Hybridization and Immunohistochemical Study Using Tyramide Signal Amplification

Gamma-aminobutyric acid (GABA) neurotransmission in the lateral septum (LS) is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD), the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2) and GAD67 (GAD1), and used fluorescence in Situ hybridization (FISH) with tyramide signal amplification (TSA) to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg) and medial preoptic area (MPOA) in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%), while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%). Using the neuronal marker NeuN, almost every neuron in LS (> 90%) was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB) or calretinin (CR), but not parvalbumin (PV); almost all CB- or CR-immunoreactive neurons (98-100%) were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI) (approximately 58% for CB and 35% for CR). These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of the LS.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

Glutamate Decarboxylases in Nonneural Cells of Rat Testis and Oviduct: Differential Expression of GAD65 and GAD67

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.     

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Down-Regulated GABAergic Expression in the Olfactory Bulb Layers of the Mouse Deficient in Monoamine Oxidase B and Administered With Amphetamine

This study explores primarily the role of the activity of monoamine oxidase B (MAOB) in the regulation of glutamic acid decarboxylase₆₇ (GAD₆₇) expression in distinct layers of main olfactory bulb (OlfB), which links the limbic system. Moreover, the response of GAD₆₇ was investigated to amphetamine perturbation in the absence of MAOB activity. Immunocytochemical analysis was performed on OlfB sections prepared from the adult wild type (WT) and the MAOB gene-knocked-out (KO) mice after receiving repeated intraperitoneal injections (two doses per day, total seven doses) of saline or amphetamine, 5 mg/kg. The levels of the GAD₆₇ immunoreactivity were approximate 25 and 38% lower in respective glomerular (GloL) and mitral cell layers (ML) of saline-treated KO mice than that of WT, whereas similar in the external plexiform or granule cell layers (GraL) of the KO and WT. In the GloL, the level of tyrosine hydroxylase was 39% lower in the KO mice than WT, implicating different dopamine content in the KO from WT. The amphetamine exposure down-regulated the levels of GAD₆₇ in the WT layers by 46 to 52%, and in KO layers 65 to 71%, except ML. The GraL GAD₆₇ level may be regulated by the activation of CREB, as the phosphorylated (p) CREB coexisted with GAD₆₇, and the percentage of GAD₆₇-expressing pCREB neurons was decreased by the amphetamine exposure. The data indicate that the activity of MAOB could modulate the regular and amphetamine-perturbed expression of GAD₆₇ and pCREB. Thus, interactions are suggested among the MAOB activity, GABA content of OlfB, and olfaction.