Role Of Gamma-aminobutyric Acid Research Articles

Effects of acute tiagabine administration on aggressive responses of adult male parolees

Experimental and clinical studies have supported a relationship between gamma-aminobutyric acid (GABA) and aggressive behavior in non-humans and humans. Tiagabine is a GABA uptake inhibitor that has been shown to produce acute behavioral effects in animals. In addition, tiagabine has been shown to decrease aggression in agitated patients when administered chronically. The present study was designed to investigate the effects of acute administration of tiagabine on aggressive responding on a laboratory task in adult humans. Ten adult males participated in experimental sessions on the Point Subtraction Aggression Paradigm (PSAP), which provided subjects with aggressive, escape, and monetary-reinforced response options. All subjects received four acute oral doses of Tiagabine (4, 8, 12 and 16 mg) separated by placebo sessions. Tiagabine decreased aggression at doses that either did not affect, or affected to a lesser extent, monetary-reinforced responding. The results are consistent with some prior research using the PSAP showing a possible unique role for GABA in the regulation of human aggression. A possible behavioral mechanism for the rate-decreasing effects on aggressive responding produced in the present study is that tiagabine may modify aggressive responding by suppressing reactions to aversive stimuli.

Does gamma-aminobutyric acid (GABA) influence the development of chronic inflammation in rheumatoid arthritis?

BackgroundRecent studies have demonstrated a role for spinal p38 MAP kinase (MAPK) in the development of chronic inflammation and peripheral arthritis and a role for GABA in the inhibition of p38 MAPK mediated effects. Integrating these data suggests that GABA may play a role in downregulating mechanisms that lead to the production of proinflammatory agents such as interleukin-1, interleukin-6, and matrix metalloproteinase 3 – agents implicated in the pathogenesis of rheumatoid arthritis (RA). Genetic studies have also associated RA with members of the p38 MAPK pathway.HypothesisWe propose a hypothesis for an inefficient GABA signaling system that results in unchecked proinflammatory cytokine production via the p38 MAPK pathway. This model also supports the need for increasing research in the integration of immunology and neuroscience.

Supraspinal modulation of pain by cannabinoids: the role of GABA and glutamate

Recent physiological, pharmacological and anatomical studies provide evidence that one of the main roles of the endocannabinoid system in the brain is the regulation of gamma-aminobutyric acid (GABA) and glutamate release. This article aims to review this evidence in the context of its implications for pain. We first provide a brief overview of supraspinal regulation of nociception, followed by a review of the evidence that the brain's endocannabinoid system modulates nociception. We look in detail at regulation of supraspinal GABAergic and glutamatergic neurons by the endocannabinoid system and by exogenously administered cannabinoids. Finally, we review the evidence that cannabinoid-mediated modulation of pain involves modulation of GABAergic and glutamatergic neurotransmission in key brain regions.

Stiff-person syndrome with acute recurrent peripheral vertigo: possible evidence of gamma aminobutyric acid as a neurotransmitter in the vestibular periphery

We report a case of a 58-year-old man suffering from stiff-person syndrome and recurrent peripheral vertigo. A case report and a review of the recent literature on stiff-person syndrome are presented. The patient presented with recurrent episodes of vertigo with a pure peripheral pattern and with concomitant episodes of burning muscle pain, muscle twitching, weight gain and fatigue, worsening with tension or stress that also occurred in periods without vertigo. Cochlear examinations only showed presbyacusis-like hearing loss. The diagnosis of stiff-person syndrome was made with electromyographic examination and from findings in the blood and cerebrospinal fluid of high titres of anti-glutamic acid decarboxylase (GAD67) autoantibodies. In a two-year follow-up period, therapy for stiff-person syndrome abolished episodes of both stiffness and vertigo. As far as we know, no other clinical case of acute vestibular damage with a possible correlation with anti-glutamic acid decarboxylase antibodies has been described. Peripheral vertigo possibly related to a lack of gamma aminobutyric acid underlines a possible role of gamma aminobutyric acid as a neurotransmitter in the peripheral vestibular system.

Role of GABA in anxiety and depression

This review assesses the parallel data on the role of gamma-aminobutyric acid (GABA) in depression and anxiety. We review historical and new data from both animal and human experimentation which have helped define the key role for this transmitter in both these mental pathologies. By exploring the overlap in these conditions in terms of GABAergic neurochemistry, neurogenetics, brain circuitry, and pharmacology, we develop a theory that the two conditions are intrinsically interrelated. The role of GABAergic agents in demonstrating this interrelationship and in pointing the way to future research is discussed.

What shapes amino acid and sugar composition in Mediterranean floral nectars?

We studied the amino acid (AA) composition of the floral nectars of 73 plant species occurring in a phryganic (East Mediterranean garrigue) community and investigated whether AA and sugar composition is shaped by evolutionary (plant phylogeny), ecological (flowering time as a direct effect of summer drought) and coevolutionary (pollinator partnership) constraints. Our study utilised an extensive plant–pollinator matrix compiled in the same area where the plants had been sampled.Using HPLC we detected 22 AA compounds/groups of compounds, out of which 15 were commonly present in almost all nectars. Among all AAs, phenylalanine was the most abundant, especially in keystone (“cornucopian”) plant species visited by many insect species, such as the majority of the Lamiaceae. Amino acid quantities were transformed into percentages (% of each AA over the total AA content of a flower). Sugar composition was similarly expressed as % of each of the three sugars (glucose, fructose, sucrose) over the total content of these sugars; a number of other sugars, occurring in only a few plant species and in very low quantities were disregarded. The number of insect species of a particular family or guild was taken as a measure of the attraction of a nectar compound for such a family (guild).We found that taxonomical plant group had a weakly significant effect on nectar composition while neither life form nor flowering season had a discernable effect. Pollinators’ preference had the most important effect, with phenylalanine being the most consequent discriminatory compound for the response of the nectar consumers in phrygana, predominantly for long tongued bees, especially for Megachilidae. Gamma‐aminobutyric acid (GABA) had a similar, even stronger influence on bees (long tongued bees, Anthophoridae, Andrenidae) and flies (Syrphidae and other Diptera), whereas asparagine behaved as a general repellent together with tryptophane (rather as repellent). Considering total sugar and AA contents, as well as the volume of nectar, we found that total AA content was positively related to the number of species of long tongued bees and included families visiting the phryganic species; nectar volume was negatively related to flies (both hover flies and remaining Diptera), whereas total sugar content was not significant for any guild. We argue that due to the highly concentrated nectars in the dry Mediterranean communities that are characterised by outstanding melittophily, sugars play a less important role as phagostimulants compared to AAs in floral nectars. This is why phenylalanine, a phagostimulant tested earlier on honeybees, appears to be of high importance in phrygana, especially with long tongued bees and Megachilids as the main selective agents for phenylalanine‐rich nectars. The role of GABA, a strongly NaCl‐dependent AA, may be similar, probably because of the associated presence of NaCl.

The role of GABAAβ2 subunit‐containing receptors in mediating the anticonvulsant and sedative effects of loreclezole

The majority of inhibitory neurotransmission in the brain is mediated by the gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor. The anticonvulsant loreclezole largely acts by potentiating GABA(A) receptors containing beta2 and beta3 subunits. We used a genetically modified mouse containing a loreclezole-insensitive beta2 subunit (beta2N265S) to determine the role of this subunit in mediating the sedative and anticonvulsive effects of loreclezole. Sedation was assessed by measuring spontaneous locomotor activity and beam walking performance, and anticonvulsant efficacy was determined by pentylenetetrazole (PTZ) and amygdala kindling-induced seizures. The beta2N265S mice did not exhibit loreclezole-mediated sedation as shown by normal locomotor activity and beam walking performance. However, loreclezole also failed to provide significant protection against PTZ-induced seizures in the beta2N265S mice. Reduced efficacy against amygdala-kindled seizures, both acutely and over a 13-day chronic dosing study, was also observed in beta2N265S mice. These results suggest that the majority of the sedative effects and a significant proportion of the anticonvulsant efficacy of loreclezole are mediated via beta2-containing GABA(A) receptors.

GABA-mediated echo duration selectivity of inferior collicular neurons of Eptesicus fuscus, determined with single pulses and pulse–echo pairs

When insectivorous bats such as Eptesicus fuscus emit ultrasonic signals and analyze the returning echoes to hunt insects, duration selectivity of auditory neurons plays an important role in echo recognition. The success of prey capture indicates that they can effectively encode progressively shortened echo duration throughout the hunting process. The present study examines the echo duration selectivity of neurons in the central nucleus of the bat inferior colliculus (IC) under stimulation conditions of single pulses and pulse-echo (P-E) pairs. This study also examines the role of gamma-aminobutyric acid (GABA)ergic inhibition in shaping echo duration selectivity of IC neurons. The data obtained show that the echo duration selectivity of IC neurons is sharper when determined with P-E pairs than with single pulses. Echo duration selectivity also sharpens with shortening of pulse duration and P-E gap. Bicuculline application decreases and GABA application increases echo duration selectivity of IC neurons. The degree of change in echo duration selectivity progressively increases with shortening of pulse duration and P-E gap during bicuculline application while the opposite is observed during the GABA application. These data indicate that the GABAergic inhibition contributes to sharpening of echo duration selectivity of IC neurons and facilitates echo recognition by bats throughout different phases of hunting.

Electrically stimulated relaxation is not mediated by GABA in cat lower esophageal sphincter muscle

This study examined the effect of Gamma-Amino butyric acid (GABA) and selective GABA receptor related drugs on the electrically stimulated relaxation in the lower esophageal sphincter muscle (LES) of a cat. Tetrodotoxin (10(-6) M) suppressed the electrically stimulated (0.5-5 Hz) relaxation of the LES. However, guanethidine (10(-6) M) and atropine (10(-6) M) had no effect indicating that the relaxations were neurally mediated via the nonadrenergic and noncholinergic (NANC) pathways. NG-nitro-L-arginine methyl ester (10(-4) M, L-NAME) also inhibited the relaxant response but did not completely abolish the electrically stimulated relaxation with 60 % inhibition, which suggests the involvement of nitric oxide as an inhibitory transmitter. This study examined the role of GABA, an inhibitory neurotransmitter, on neurally mediated LES relaxation. GABA (10(-3)-10(-5) M, non selective receptor agonist), muscimol (10(-3)-10(-5) M, GABA-A agonist), and baclofen (10(-3)-10(-5) M, GABA-B agonist) had no significant effect on the electrically stimulated relaxation. Moreover, bicuculline (10(-5) M, GABA-A antagonist) and phaclofen (10(-5) M, GABA-B antagonist) had no inhibitory effect on the electrically stimulated relaxation. This suggests that GABA and the GABA receptor are not involved in the electrically stimulated NANC relaxation in the cat LES.

Gamma-aminobutyric acid up- and downregulates insulin secretion from beta cells in concert with changes in glucose concentration

The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA(A)Rs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA(A)Rs in the pancreatic islets. However, the reported effects of GABA(A)R activation on insulin secretion from islet beta cells have been controversial. This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 micromol/l, induced a transmembrane current (I(GABA)) which was sensitive to the GABA(A)R antagonist bicuculline. The current-voltage relationship revealed that I(GABA) reversed at -42+/-2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V (M)), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V (M) of INS-1 cells was below the I(GABA) reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V (M) of INS-1 cells was above the I(GABA) reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). These findings indicate that activation of GABA(A)Rs in beta cells regulates insulin secretion in concert with changes in glucose levels.

Recovery of fear memories in rats: Role of gamma-amino butyric acid (GABA) in infantile amnesia.

Infantile amnesia is a ubiquitous phenomenon, but its neural bases remain largely unknown. The authors identify a role for GABAergic transmission in suppressing retrieval of memories acquired in infancy. Eighteen-day-old rats received pairings of white noise and shock; considerable forgetting of this experience (assessed by freezing) occurred after 10 days. The memory was recovered by pretest administration of the GABAA inverse agonist FG7142 10 days, but not 2 months, after training. This effect of FG7142 generalized when a passive avoidance procedure was used. Also, FG7142 decreased fear of a latently inhibited conditioned stimulus, showing that the observed memory recovery effect was not due to a state-dependent process. It appears that GABA may be involved in infantile amnesia regardless of the emotional content of the memory.

Role of γ-aminobutyric acid B (GABAB) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA(B)) receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA(B) receptors antagonist, 20 mug) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca(2+)/calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA(B) receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Immunohistochemical localization of GAD67‐expressing neurons and processes in the rat brainstem: Subregional distribution in the nucleus tractus solitarius

The role of gamma-aminobutyric acid (GABA) in homeostatic control in the brainstem, in particular, in the nucleus tractus solitarius (NTS), is well established. However, to date, there is no detailed description of the distribution of GABAergic neurons within the NTS. The goal of the current study was to reexamine the efficacy of immunohistochemical localization of glutamic acid decarboxylase (GAD) protein, specifically the 67-kDa isoform (GAD67), as a marker for GABAergic neurons in the medulla and to provide a detailed map of GAD67-immunoreactive (-ir) cells within rat NTS by using a recently developed mouse monoclonal antibody. We describe a distribution of GAD67-ir cells in the medulla similar to that reported previously from in situ hybridization study. GAD67-ir cells were localized in regions known to contain high GABA content, including the ventrolateral medulla, raphe nuclei, and area postrema, but were absent from all motor nuclei, although dense terminal labeling was discerned in these regions. In the NTS, GAD67-ir was localized in all subregions. Semiquantitative analysis of the GAD67-ir distribution in the NTS revealed greater numbers of GAD67-ir cells medial to the solitary tract. Finally, dense GAD67 terminal labeling was found in the medial, central, intermediate, commissural, and subpostremal subregions, whereas sparse labeling was observed in the ventral subregion. Our findings support the use of immunohistochemistry for GAD67 as a marker for the localization of GABAergic cells and terminal processes in the rat brainstem. Furthermore, the reported heterogeneous distribution of GAD67-ir in the NTS suggests differential inhibitory modulation of sensory processing.

Analysis of gamma-aminobutyric acid-mediated responses in the pulmonary vascular bed of the cat.

In this investigation, we sought to identify the role of gamma-aminobutyric acid (GABA)(A) and GABA(B) receptors in the feline pulmonary vascular bed. Using adult mongrel cats and in separate experiments, we investigated the effects of l-N(5)-(1-iminoethyl) ornithine hydrochloride (l-NIO) (a nitric oxide synthase inhibitor), glibenclamide (an adenosine triphosphate (ATP)-sensitive K(+) channel blocker), meclofenamate (a nonselective cyclooxygenase inhibitor), bicuculline (a GABA(A) receptor antagonist), and saclofen (a GABA(B) receptor antagonist) on pulmonary arterial responses to pinacidil (an ATP-sensitive K(+) channel activator), bradykinin (a nitric oxide synthase inducer), muscimol (a GABA(A) receptor agonist), and 3-aminopropyl(methyl)phosphinic acid, hydrochloride (SKF-97541; a GABA(B) receptor agonist). Under increased tone conditions in the isolated left lower lobe vascular bed of the cat, muscimol induced a dose-dependent vasodepressor response that was not significantly altered after the administration of l-NIO, glibenclamide, meclofenamate, and saclofen. SKF-97541-induced vasodepression was not significantly attenuated after the administration of l-NIO, meclofenamate, and bicuculline. Responses to SKF-97541 were significantly attenuated after the administration of glibenclamide and saclofen. Responses to muscimol were significantly reduced after the administration of bicuculline. The results suggest that muscimol and SKF-97541 have potent vasodepressor activity in the feline pulmonary vascular bed and that these responses are modulated by, respectively, GABA(A) and GABA(B) receptor-sensitive pathways. Further, SKF-97541-induced vasodepression is mediated or modulated by an ATP-sensitive K(+) channel.

Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid

Leptin, a hormone secreted by adipocytes, produces a number of central and neuroendocrine effects, the mechanisms behind which are not completely understood. Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin. Therefore, we hypothesized that leptin could affect hypothalamic NE activity to bring about its central and neuroendocrine effects. Because gamma aminobutyric acid (GABA) is known to affect the release of NE, we also tested the possibility that leptin-induced changes in NE could be mediated through GABA. The mediobasal hypothalami from adult male rats were incubated in an in vitro incubation system for four consecutive incubation periods of 60 min each at 37 °C in Krebs Ringers Henseleit (KRH) solution in an atmosphere of 95% O 2 and 5% CO 2. After determining the basal release, the hypothalami were challenged with 0, 0.1, 1 or 10 nm of leptin, bicuculline (a GABA-A receptor antagonist; 10 μM) and bicuculline (10 μM) +10 nM of leptin during the second incubation period. Residual effects of leptin were measured in the third incubation where tissues were incubated with KRH alone, and the viability of tissues was determined in the fourth incubation when tissues were exposed to high K + KRH. NE levels in the incubation medium were measured using high-performance liquid chromatography with electrochemical detection (HPLC-EC). Leptin inhibited NE efflux from the hypothalamus in a dose-dependent manner. Moreover, incubation of hypothalami with 10 nM of leptin and bicuculline, a completely blocked the leptin-induced decrease in NE efflux. These results demonstrate for the first time that leptin could act directly on the hypothalamus to inhibit NE efflux through GABA. It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus.

GABAergic mechanisms constraining the activity of the hypothalamo-pituitary-adrenocortical axis.

There are two major inhibitory mechanisms that constrain the activity of the hypothalamo-pituitary-adrenocortical axis: the hormonal negative feedback and the neural inhibition including that posed by the GABAergic neurons. This chapter summarizes our recent morphologic and functional findings on the role of gamma-aminobutyric acid (GABA) in the transcriptional regulation of hypophyseotropic neuropeptide genes in the parvocellular neurosecretory cells of the hypothalamic paraventricular nucleus (PVH). We used organotypic hypothalamic slice cultures and in vivo microinjection protocols in combination with in situ histologic and ultrastructural procedures to address the role of local interneurons in the regulation of hypothalamic effector neurons. Under basal conditions, an intrinsic GABAergic mechanism in the PVH microenvironment was revealed that by itself, without limbic contribution, impinged a tonic inhibitory influence on the parvocellular corticotropin-releasing hormone (CRH) neurons in vitro. In vivo, remote inputs were superimposed on the local circuit, allowing differential transcriptional regulation of CRH and arginine vasopressin (AVP) genes in the hypophyseotropic neurons. During stress, GABAergic cells that are known to project to the PVH become activated and are involved in restraining the cellular, transcriptional, and hormonal responses to stress.

Molecular and Physiological Evidence for Functional γ-Aminobutyric Acid (GABA)-C Receptors in Growth Hormone-secreting Cells

The neurotransmitter gamma-aminobutyric acid (GABA), released by hypothalamic neurons as well as by growth hormone- (GH) and adrenocorticotropin-producing cells, is a regulator of pituitary endocrine functions. Different classes of GABA receptors may be involved. In this study, we report that GH cells, isolated by laser microdissection from rat pituitary slices, possess the GABA-C receptor subunit rho2. We also demonstrate that in the GH adenoma cell line, GH3, GABA-C receptor subunits are not only expressed but also form functional channels. GABA-induced Cl- currents were recorded using the whole cell patch clamp technique; these currents were insensitive to bicuculline (a GABA-A antagonist) but could be induced by the GABA-C agonist cis-4-aminocrotonic acid. In contrast to typical GABA-C mediated currents in neurons, they quickly desensitized. Ca2+i recordings were also performed on GH3 cells. The application of either GABA or cis-4-aminocrotonic acid led to Ca2+ transients of similar amplitude, indicating that the activation of GABA-C receptors in GH3 cells may cause membrane depolarization, opening of voltage-gated Ca2+ channels, and a subsequent Ca2+ influx. Our results point at a role for GABA in pituitary GH cells and disclose an additional pathway to the one known via GABA-B receptors.

Interactions between GABAergic and aminoacidergic pathways in the control of gonadotropin and GH secretion in pre-pubertal female rats

Present experiments were carried out in 23-day-old female rats to analyze the interaction between excitatory amino acids (EAAs) and gamma-aminobutyric acid (GABA) in the control of gonadotropin and GH secretion. For this purpose, serum concentrations of LH, FSH and GH were measured after injection of different agonists of EAA receptor subtypes [N-methyl-D-aspartate (NMDA); kainic acid (KA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)], antagonists of GABA receptors (bicuculline, phaclofen) or the combined administration of both types of drugs. The results obtained indicated that: 1) GABA has a minor physiological role in the control of LH and GH secretion, since neither LH nor GH serum concentrations changed after administration of bicuculline (antagonist of GABA(A) receptors) or phaclofen (antagonist of GABA(B) receptors); 2) GABA has a sex-specific physiological role in the control of FSH secretion in female rats, in which FSH secretion increases after phaclofen administration; 3) GH secretion was enhanced after administration of NMDA, KA and AMPA, while LH increased only after activation of NMDA receptors; 4) the stimulatory effect of NMDA on LH secretion was counteracted by administration of phaclofen; and 5) bicuculline and phaclofen reduced the ability of NMDA and AMPA to stimulate GH secretion. In conclusion, present experiments evidenced a physiological role of GABA, mediated by GABA(B) receptors, in the control of FSH secretion and a cross-talk between excitatory and inhibitory amino acids in the control of anterior pituitary secretion.

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate.

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.

The vesicular GABA transporter vGAT, is present in sperm and testis.

Objective: The amino acid gamma aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the CNS. GABA is transported across the plasma membrane into cells by transporter proteins. Four such proteins have been cloned and sequenced (GAT 1-4). A protein different from GAT 1-4, the vesicular GABA transporter (VGAT) has been identified, mediating GABA transport from cytoplasm into storage vesicles called small synaptic vesicles in neurons. Recently, a role for GABA has been proposed in reproductive medicine. Specific GABAergic proteins have been identified in human sperm and oocytes and a possible role for GABA in the initiation of the acrosome reaction has been proposed. We have previously reported the presence of specific GABA transport proteins in human and rat spermatozoa and testis. In this study we have identified VGAT in rat testis by immunoblotting immunohistochemistry and RT-PCR. Design: Experimental design in laboratory setting Materials/Methods: Immunohistochemistry: Male Sprague-Dawley rats were sacrificed and testis were removed and fixed in formalin/picric acid. Fixed testis were rinsed and finally cut for indirect fluorescence. Sections of rat testis and spermatozoa were incubated in rabbit antiserum to VGAT for 18-22 hours. The tissues were then washed and incubated with fluorescin isothiocyanate conjugated donkey anti rabbit secondary antibodies. Sections were examined in a laser scanning confocal imaging system equipped with a krypton/argon mixed gas laser. SDS-PAGE and Immunoblotting: Homogenized samples were denatured in SDS-PAGE sample buffer. Equal amounts of protein were loaded and subjected to analysis on a 7.5% SDS-PAGE gel. Proteins were transferred to nitrocellulose and following blocking, the blots were probed overnight with rabbit anti VGAT. Immunoreactive protein bands were visualized following incubation with peroxidase-conjugated goat-anti-rabbit IgG. Reverse transcriptase PCR: Messenger RNA was prepared from rat testis and the mRNA was transcribed into cDNA using a commercially available kit. Primers used for PCR were designed on basis of sequences for cloned vesicular GABA transport. The validity of the PCR procedure was tested by gel electrophoreses and DNA sequencing. Results: Immunohistochemistry: Confocal images of rat testis sections demonstrated strong immunofluorescence in the cytoplasm of germ cells. Peritubular cells did not exhibit staining. Confocal images of rat sperm cells incubated with antibodies to VGAT demonstrated immunofluorescence in the head region. SDS-PAGE: The testis VGAT protein reacted with the rabbit anti VGAT antibody. The immunoreactivity was observed as a band in the 60 kDa region RT-PCR: With the oligonucleotide primer pair based on the cDNA sequence from the rat VGAT, a PCR product matched the size predicted from the location of the primers. This PCR product was sequenced and found identical to rat VGAT. Conclusions: The results from both immunohistochemistry and immunoblots in this study strongly indicate the presence of the vesicular GABA transporter in rat testis. The identification of mRNA expression of VGAT in rat testis confirms this finding. Several GABAergic proteins have now been identified in testis and spermatozoa, this is suggestive of a role for GABA in male reproductive organs and/or spermatozoa. Supported By: This project was supported by grants from the Swedish Medical Council 14X-07164 and the Karolinska Institutes Research Funds.