GABA-transaminase Inhibitor Research Articles

Quantitative structure-activity relationship and molecular docking studies of a series of quinazolinonyl analogues as inhibitors of gamma amino butyric acid aminotransferase

Quantitative structure-activity relationship and molecular docking studies were carried out on a series of quinazolinonyl analogues as anticonvulsant inhibitors. Density Functional Theory (DFT) quantum chemical calculation method was used to find the optimized geometry of the anticonvulsants inhibitors. Four types of molecular descriptors were used to derive a quantitative relation between anticonvulsant activity and structural properties. The relevant molecular descriptors were selected by Genetic Function Algorithm (GFA). The best model was validated and found to be statistically significant with squared correlation coefficient (R2) of 0.934, adjusted squared correlation coefficient (R2adj) value of 0.912, Leave one out (LOO) cross validation coefficient (Q2) value of 0.8695 and the external validation (R2pred) of 0.72. Docking analysis revealed that the best compound with the docking scores of −9.5kcal/mol formed hydrophobic interaction and H-bonding with amino acid residues of gamma aminobutyric acid aminotransferase (GABAAT). This research has shown that the binding affinity generated was found to be better than the commercially sold anti-epilepsy drug, vigabatrin. Also, it was found to be better than the one reported by other researcher. Our QSAR model and molecular docking results corroborate with each other and propose the directions for the design of new inhibitors with better activity against GABAAT. The present study will help in rational drug design and synthesis of new selective GABAAT inhibitors with predetermined affinity and activity and provides valuable information for the understanding of interactions between GABAAT and the anticonvulsants inhibitors.

MTOR Inhibition Mitigates Molecular and Biochemical Alterations of Vigabatrin-Induced Visual Field Toxicity in Mice

BackgroundGamma-vinyl–γ-aminobutyric acid (GABA) (vigabatrin) is an antiepileptic drug and irreversible GABA transaminase inhibitor associated with visual field impairment, which limits its clinical utility. We sought to relate altered visual evoked potentials associated with vigabatrin intake to transcriptional changes in the mechanistic target of rapamycin (mTOR) pathway and GABA receptors to expose further mechanisms of vigabatrin-induced visual field loss. MethodsVigabatrin was administered to mice via an osmotic pump for two weeks to increase GABA levels. Visual evoked potentials were examined, eye samples were collected, and gene expression was measured by quantitative reverse transcription-polymerase chain reaction. Similarly, human retinal pigment epithelial cells (ARPE19) were exposed to vigabatrin and treated with mTOR inhibitors for mTOR pathway analysis and to assess alterations in organelle accumulation by microscopy. ResultsDysregulated expression of transcripts in the mTOR pathway, GABAA/B receptors, metabotropic glutamate (Glu) receptors 1/6, and GABA/glutamate transporters in the eye were found in association with visual evoked potential changes during vigabatrin administration. Rrag genes were upregulated in both mouse eye and ARPE19 cells. Immunoblot of whole eye revealed greater than three fold upregulation of a 200 kDa band when immunoblotted for ras-related guanosine triphosphate binding D. Microscopy of ARPE19 cells revealed selective reversal of vigabatrin-induced organelle accumulation by autophagy-inducing drugs, notably Torin 2. Changes in the mTOR pathway gene expression, including Rrag genes, were corrected by Torin 2 in ARPE19 cells. ConclusionsOur studies, indicating GABA-associated augmentation of RRAG and mTOR signaling, support further preclinical evaluation of mTOR inhibitors as a therapeutic strategy to potentially mitigate vigabatrin-induced ocular toxicity.

Genotoxic and mutagenic effects of vigabatrin, a γ-aminobutyric acid transaminase inhibitor, in Wistar rats submitted to rotarod task.

Vigabatrin (VGB) is an antiepileptic drug thatincreases brain γ-aminobutyric acid (GABA) levels through irreversible inhibition of GABA transaminase. The aim of this study was to evaluate neurotoxicological effects of VGB measuring motor activity and genotoxic and mutagenic effects after a single and repeated administration. Male Wistar rats received saline, VGB 50, 100, or 250 mg/kg by gavage for acute and subchronic (14 days) treatments and evaluated in the rotarod task. Genotoxicity was evaluated using the alkaline version of the comet assay in samples of blood, liver, hippocampus, and brain cortex after both treatments. Mutagenicity was evaluated using the micronucleus test in bone marrow of the same animals that received subchronic treatment. The groups treated with VGB showed similar performance in rotarod compared with the saline group. Regarding the acute treatment, it was observed that only higher VGB doses induced DNA damage in blood and hippocampus. After the subchronic treatment, VGB did not show genotoxic or mutagenic effects. In brief, VGB did not impair motor activities in rats after acute and subchronic treatments. It showed a repairable genotoxic potential in the central nervous system since genotoxicity was observed in the acute treatment group.

Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by β-alanine transamination.

Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney. Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation. The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl-l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake.

Central GABAA Receptors Provoke Inflammatory and Cardiovascular Consequences of Endotoxemia in Conscious Rats

Gamma‐aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, plays a key role in inflammatory and neurodegenerative disease. In this study, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The BP lowering effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist, 3 μg/rat) or saclofen (selective GABAB receptor antagonist, 50 μg/rat). By contrast, the concomitant LPS‐evoked tachycardia and decreases in time‐ and frequency domain indices of HRV, measures of cardiac autonomic control, were abolished upon treatment with bicuculline but not saclofen. The increases in serum tumor necrosis factor‐α and interleukin‐6 caused by LPS disappeared in rats treated concurrently with bicuculline or saclofen, whereas the LPS‐evoked increases in serum nitric oxide metabolites was reversed by bicuculline only. We also report that none of the effects of endotoxemia was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor, 100 μg/rat) or vigabatrin (GABA transaminase inhibitor, 200 μg//rat). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895).

Continuous bilateral infusion of vigabatrin into the subthalamic nucleus: Effects on seizure threshold and GABA metabolism in two rat models

The subthalamic nucleus (STN) plays a crucial role as a regulator of basal ganglia outflow but also influences the activity of cortical and limbic structures, so that it is widely used as a therapeutic target in different brain diseases, including epilepsy. In addition to electrical stimulation of the STN, targeted delivery of anti-seizure drugs to the STN may constitute an alternative treatment approach in patients with pharmacoresistant epilepsy. In the present experimental study, we investigated the anti-seizure and adverse effects of chronic infusion of vigabatrin into the STN of rats. Vigabatrin is a clinically approved anti-seizure drug, which acts by increasing brain GABA levels by irreversibly inhibiting GABA-aminotransferase (GABA-T). Based on functional and neurochemical effects of acute STN microinjection, doses for continuous infusion were calculated and administered, using an innovative drug infusion technology. Bilateral infusion of only 10μg/day vigabatrin over 3weeks into the STN resulted in an almost complete inhibition of GABA-T and 4-fold increase in GABA in the target region, which was associated with a significant increase in seizure threshold, determined once weekly by i.v. infusion of pentylenetetrazole (PTZ). Lower doses or unilateral infusion were less effective, both on PTZ seizures and on kindled seizures. Bilateral infusion into substantia nigra pars reticulata was less effective and more toxic than STN infusion. In part of the rats, tolerance to the anti-seizure effect developed. The data demonstrate that chronic administration of very low, nontoxic doses of vigabatrin into STN is an effective means of increasing local GABA concentrations and seizure threshold.

Ligand based Drug Design of New Heterocyclic Imines of GABA Analogues: A Molecular Docking Approach for the Discovery of New GABA-AT Inhibitors

Degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is mainly catalysed by GABA aminotransferase (GABA-AT), excessive activity of which leads to convulsions. Inhibition of GABA-AT increases the concentration of GABA and can terminate the convulsions. Several studies have revealed that GABA analogues could be the outstanding scaffolds for the design of potent inhibitors of GABA-AT. The poor ability of GABA analogues to cross the blood-brain barrier (BBB), always produces low therapeutic index. However, Vigabatrin, a mechanism-based inhibitor of GABA-AT, is currently approved treatment of epilepsy, but it has harmful side effects, leaving a need for improved GABA-AT inactivators. In our present in silico investigation, AutoDock 4.2,-based on Lamarckian genetic algorithm was employed for virtual screen of a compound library with 35 entries (Schiff's bases of GABA) in search for novel and selective inhibitors of GABA-AT. By means of flexible type of molecular docking, we proposed that these designed molecules could successfully bind into the active pocket of GABA-AT with good predicted affinities in comparison to standard vigabatrin. Among the designed analogues, HIG18, HIG28 and HIG30 showed significant binding free energy of -10.25, -9.88 and -9.31 kcal/mol with predicted inhibitory constant values of 0.03, 0.05 and 0.15 µM respectively. Using ligand-based drug design, we proposed that electron withdrawing phenyl substituted heterocyclic imines of GABA could be considered as promising structures for synthesis and testing of new GABA-AT inhibitors from this class. We hypothesize that novel GABA analogues with an azomethine linkage incorporated with heterocyclic system can have increased affinity and more lipophilic character that would provide a probability of having less toxic effect in the therapy of convulsions.

Central GABAA receptors are involved in inflammatory and cardiovascular consequences of endotoxemia in conscious rats.

γ-Aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, modulates inflammatory and neurodegenerative disease. Here, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The hypotensive effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist) or saclofen (GABAB receptor antagonist). By contrast, the concomitant LPS-evoked tachycardia and decreases in time domain and frequency domain indices of HRV (measures of cardiac autonomic control) were abolished upon treatment with bicuculline but not saclofen. Increases in serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) caused by LPS disappeared in the presence of bicuculline or saclofen, whereas LPS-evoked increases in serum nitric oxide metabolites (NOx) were counteracted by bicuculline only. None of the endotoxemia effects was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor) or vigabatrin (GABA transaminase inhibitor). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.

A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension

Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH.

GABA in Paraventricular Nucleus Regulates Adipose Afferent Reflex in Rats.

BackgroundChemical stimulation of white adipose tissue (WAT) induces adipose afferent reflex (AAR), and thereby causes a general sympathetic activation. Paraventricular nucleus (PVN) is important in control of sympathetic outflow. This study was designed to investigate the role of γ-aminobutyric acid (GABA) in PVN in regulating the AAR.Methodology/Principal FindingsExperiments were carried out in anesthetized rats. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. AAR was evaluated by the RSNA and MAP responses to electrical stimulation of the right epididymal WAT (eWAT) afferent nerve. Electrical stimulation of eWAT afferent nerve increase RSNA. Bilateral microinjection of the GABAA receptor agonist isoguvacine or the GABAB receptor agonist baclofen attenuated the AAR. The effect of isoguvacine on the AAR was greater than that of baclofen. The GABAA receptor antagonist gabazine enhanced the AAR, while the GABAB receptor antagonist CGP-35348 had no significant effect on the AAR. Bilateral PVN microinjection of vigabatrin, a selective GABA-transaminase inhibitor, to increase endogenous GABA levels in the PVN abolished the AAR. The inhibitory effect of vigabatrin on the AAR was attenuated by the pretreatment with gabazine or CGP-35348. Pretreatment with combined gabazine and CGP-35348 abolished the effects of vigabatrin.ConclusionsActivation of GABAA or GABAB receptors in the PVN inhibits the AAR. Blockade of GABAA receptors in the PVN enhances the AAR. Endogenous GABA in the PVN plays an important role in regulating the AAR.

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD(+)/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.

Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase.

Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from Psammomys obesus. In vitro studies demonstrated inactivation of OAT by gabaculine (1), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidene)cyclopentane-1-carboxylic acid (2) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. In vitro administration of 2 significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; in vivo, 2 significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator.

Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors.

GABAergic neurons in cerebellar interposed nucleus modulate cellular and humoral immunity via hypothalamic and sympathetic pathways

Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways.

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

Forward acoustic masking enhances the auditory brainstem response in a diotic, but not dichotic, paradigm in salicylate-induced tinnitus

We recently reported that forward acoustic masking can enhance the auditory brainstem response (ABR) in rats treated with a high dose of sodium salicylate (NaSal), a tinnitus inducer, when tested in open acoustic field (Liu and Chen, 2012, Brain Research 1485, 88–94). In the present study, we first replicated this experiment in closed acoustic field under two conditions: (1) the forward masker and the probe were presented to both ears (diotic paradigm); (2) the forward masker was presented to one ear and the probe to the other ear (dichotic paradigm). We found that only when the stimuli were presented by using the diotic, rather than the dichotic, paradigm could forward acoustic masking enhance the ABR in the rat treated with NaSal (300 mg/kg). The enhancement was obvious for ABR waves II and IV, but not for wave I, indicating a central origin. The enhancement occurred at the high frequencies (16, 24, 32 kHz) at which the animals demonstrated a tinnitus-like behavior as revealed by using the gap prepulse inhibition of acoustic startle paradigm. We then administered vigabatrin, a GABA transaminase inhibitor, in the animals to suppress NaSal-induced tinnitus. The vigabatrin treatment successfully prevented forward acoustic masking from enhancing the ABR. These findings demonstrate that the observed enhancement of ABRs by forward acoustic masking originates in the central auditory pathway ipsilateral to the stimulated ear. We propose that the enhancement is closely associated with NaSal-induced tinnitus.

Long-lasting glutamatergic modulation induced by neonatal GABA enhancement in mice

A subgroup of anticonvulsant and neuroleptic drugs acts through the potentiation of GABA pathways. The regulatory role of GABA in neuronal circuit formation is related to its depolarizing action that supports activity-dependent synaptogenesis. We hypothesized that elevated levels of GABA in the immature brain modify synaptogenesis in excitatory synapses and consequently affect mice behavior. In support of this theory, we showed previously that neonatal exposure to a GABA-transaminase inhibitor (Vigabatrin, GVG) modifies the expression of presynaptic proteins and suppresses excitatory synaptic potentials. To further characterize this phenomenon, we examined the effect of GVG applied during postnatal days 4–14, during the switch in GABA function from a depolarizing to a hyperpolarizing substance, on the development of excitatory synapses and mice sociability. Early exposure to GVG induced differential effects on synaptic proteins in the hippocampus and the cerebral cortex, including the downregulation of GluR1/GluR2 and NR2A/NR2B ratios in the hippocampus cytoplasm, a minute effect on the regulatory proteins CAMKII and PKA in the cerebral cortex, and increases in pGluR1, CAMKII, PKA and Reelin levels. Early GVG exposure was also associated with region specific regulation of monoamines, reduction in hippocampal DA, and enhancement of cortical NE levels. Age-dependent modified sociability and lack of preference for social interactions were observed in mice treated with GVG. Overall, early life exposure to GVG is expected to alter cortico-hippocampal axis connectivity and balance due to the different effects GVG has on key synaptic proteins in the associated brain regions, thus potentially causing behavioral impairment.

Valproate improves prepulse inhibition deficits induced by corticotropin-releasing factor independent of GABAA and GABAB receptor activation

Corticotropin-releasing factor (CRF) is implicated in the pathogenesis of bipolar disorder, an illness associated with deficits in prepulse inhibition (PPI) of the acoustic startle response. Valproate is used in the treatment of bipolar disorder and may alter CRF activity via a GABAA-ergic mechanism. This study determined the effect of valproate on CRF-disrupted PPI and examined the role of the hypothalamic–pituitary–adrenal axis and GABA-ergic signaling in the effect of valproate. Valproate (60–240 mg/kg) dose-dependently reversed PPI deficits displayed by transgenic mice overexpressing CRF (CRFtg), and normalized PPI deficits induced by CRF i.c.v. infusion in 129Sv mice. Valproate enhanced corticosterone secretion more effectively in CRFtg than in wild-type mice. The effect of valproate on PPI was not blocked by the GABAA receptor antagonist bicuculline, the GABAB receptor antagonists phaclofen and SCH 50911 or combined administration of a GABAA and GABAB receptor antagonist. The beneficial effect of valproate on PPI was not mimicked by the GABAA receptor agonist muscimol, the GABA transaminase inhibitor vigabatrin, the histone deacetylase (HDAC) inhibitor sodium butyrate or by the mood stabilizers lithium, carbamazepine, lamotrigine or topiramate.Thus, we showed that valproate improves CRF-induced PPI deficits, albeit via a so far unknown mechanism. These marked beneficial effects of valproate on CRF-induced sensorimotor gating deficits suggest that valproate may be of particular value in specific subgroups of bipolar patients that are characterized by alterations in the CRF system.

GABA Attenuates L-DOPA-Induced Striatal and Nigral ERK1/2 Signaling in a Rat Model of Parkinson’s Disease

L-DOPA is the primary drug used to treat Parkinson’s disease (PD) symptoms, but motor side effects limit its long term use. Previous experimental studies show that L-DOPA acts on supersensitive D1 receptors in the basal ganglia to induce extracellular signal-regulated kinases 1 and 2 (ERK1/2), a pair of MAP-kinase proteins that may be involved in induction of motor side effects. Since GABA is known to be intimately involved in basal ganglia function, we investigated whether elevating GABA levels via a GABA-transaminase (GABA-T) inhibitor affects the L-DOPA-induced ERK1/2 phosphorylation in the striatum and substantia nigra (SN) using a rat model of PD. Unilateral dopaminergic lesions of median forebrain bundle neurons were done using the neurotoxin 6-hydroxydopamine. Rats were prescreened for the extent of the lesion by apomorphine-induced rotation test. Lesioned rats were treated with aminooxyacetic acid (AOAA, a GABA-T inhibitor), L-DOPA, or in combination. Immunohistochemistry of tyrosine hydroxylase (TH, a direct indicator of dopaminergic lesion), substance P (SP, an indirect marker that decreases after lesion), and phospho-ERK1/2 was done using slices at the level of striatum and SN. Unilateral dopaminergic lesioned rats, as expected, exhibited >90% TH loss and a modest SP loss in the striatum and SN. L-DOPA alone induced a 343% and 330% increase in phospho-ERK1/2 in the striatum and SN, respectively. We report here a novel finding that pretreatment with AOAA attenuated the L-DOPA induced increase in phospho-ERK1/2 by 62% and 68% in the striatum and SN, respectively, suggesting a DA-GABA-ERK1/2 link in the therapeutic and/or side effects of L-DOPA.

Rat Submandibular Gland Perfusion Method for Clarifying Inhibitory Regulation of GABAA Receptor

GABA is an inhibitory transmitter found in rat salivary gland. However, the inhibitory potential of GABA on salivary secretion is unclear. Using an in vivo cannulation method, intraperitoneal administration of GABA was ineffective in the absence of gabaculine, a GABA transaminase inhibitor, on pilocarpine-induced salivary secretion, suggesting that GABA was rendered metabolically inactive before reaching the salivary gland. We hypothesized that the action of a drug on the salivary glands could be measured directly using a submandibular gland perfusion system. The submandibular gland artery, veins, and duct were cannulated in situ so that physiological functions such as innervation would not be compromised. Hank's balanced salt solution (pH 7.4) was perfused at a rate of 0.5 ml/min together with 1 μM carbachol (CCh) over a 5-min period every 30 min. Amount of secreted saliva showed no change to the recurrent addition of CCh to the perfusate. GABA or muscimol dose-dependently inhibited CCh-induced salivary secretion. This effect was blocked by bicuculline, a GABA(A)-receptor (GABA(A)-R) antagonist, and enhanced by clonazepam, a central-type benzodiazepine-receptor agonist. These results suggest that salivary secretion is suppressed by GABA(A)-R in rat salivary gland and that the perfusion method used was effective in clarifying inhibitory regulation of GABA(A)-R.