GAD Gene Expression Research Articles

Age-related changes in glutamic acid decarboxylase 1 gene expression in the medial prefrontal cortex and ventral hippocampus of fear-potentiated rats subjected to isolation stress

Gamma-aminobutyric acid (GABA) plays a crucial role as a neurotransmitter in anxiety circuits, prominently in the hippocampus, amygdala, and prefrontal cortex. The synthesis of GABA in the central nervous system is primarily governed by glutamic acid decarboxylase 67 (GAD67). Aging is associated with emotional alterations, and isolation stress has been linked to increased anxiety. This study aimed to investigate the impact of aging on the gene expression of GAD67 (Gad1) in the medial prefrontal cortex (m PC) and ventral hippocampus (v Hip) of fear-potentiated rats subjected to isolation stress. To conduct the study, Wistar rats of different age groups 21-day-old (immature), 42-day-old (peri-adolescent), and 365-day-old (mature adult) were utilized. Each age level was categorized into four groups: 1) Control group - no pre-stressor, no maze, no drug, 2) Innate fear group (M) - no pre-stressor, maze, no drug, 3) Fear-potentiated group (IM) - isolation pre-stressor for 120 min, maze, no drug, and 4) Diazepam-treated group (IMD) - isolation pre-stressor for 120 min, maze, and diazepam administration. Following the tests, the (m PC) and (v Hip) regions were dissected, and Gad1 gene expression changes were assessed using Real-time PCR technique. The results revealed that, across all age groups, Gad1 expression in both the (m PC) and (v Hip) was significantly higher in the fear-potentiated groups (IM) compared to the control and innate fear (M) groups. Notably, in aged 365-day-old rats from the innate fear group (M), the expression of Gad1 in (v Hip) was also higher than that in the control group. Additionally, aged fear-potentiated rats exhibited elevated Gad1 gene expression in both structures compared to other age groups. These findings suggest that isolation stress before exposure to the elevated plus maze (EPM) can elevate Gad1 gene expression in both the (v Hip) and (m PC), and age may play a role in modulating its expression.

Lactobacillus delbrueckii reduces anxiety-like behavior in zebrafish through a gut microbiome – brain crosstalk

Certain bacteria possess the ability to reduce anxiety- and stress-related behaviors through the gut microbiome-brain axis. Such bacteria are called psychobiotics, and can be used to improve mood and cognition. However, only a few bacteria have been characterized as psychobiotics, and their exact mechanism of action remains unclear. Hence, in this study we analyzed three different species under the Lactobacillacea family, namely, Lactobacillus delbrueckii, Lacticaseibacillus casei, and Lacticaseibacillus paracasei for their potential psychobiotic activities. L. delbrueckii treatment reduced anxiety-like behavior and increased brain and gut glutamic acid decarboxylase (gad) gene expression in zebrafish. It also altered zebrafish gut microbial community as determined by PCR-DGGE and 16S rRNA-based metagenomics analysis. Overall, this paper showed that L. delbrueckii but not L. paracasei and L. casei, induced a consistent improvement in anxiety-like behavior in zebrafish, implicating its potential role as a psychobiotic to reduce anxiety.This article is part of the Special Issue on 'Microbiome & the Brain: Mechanisms & Maladies'.

Predator odor (TMT) exposure potentiates interoceptive sensitivity to alcohol and increases GABAergic gene expression in the anterior insular cortex and nucleus accumbens in male rats.

Post-traumatic stress disorder (PTSD) confers enhanced vulnerability to developing comorbid alcohol use disorder (AUD). Exposure to the scent of a predator, such as the fox odor TMT, has been used to model a traumatic stressor with relevance to PTSD symptomatology. Alcohol produces distinct interoceptive (subjective) effects that may influence vulnerability to problem drinking and AUD. As such, understanding the lasting impact of stressors on sensitivity to the interoceptive effects of alcohol is clinically relevant. The present study used a 2-lever, operant drug discrimination procedure to train male Long-Evans rats to discriminate the interoceptive effects of alcohol (2g/kg, i.g. [intragastrically]) from water. Upon stable performance, rats underwent a 15-min exposure to TMT. Two weeks later, an alcohol dose-response curve was conducted to evaluate the lasting effects of the TMT stressor on the interoceptive effects of alcohol. The TMT group showed a leftward shift in the effective dose (ED50) of the dose-response curve compared to controls, reflecting potentiated interoceptive sensitivity to alcohol. TMT exposure did not affect response rate. GABAergic signaling in both the anterior insular cortex (aIC) and the nucleus accumbens (Acb) is involved in the interoceptive effects of alcohol and stressor-induced adaptations. As such, follow-up experiments in alcohol-naïve rats examined neuronal activation (as measured by c-Fos immunoreactivity) following TMT and showed that TMT exposure increased c-Fos expression in the aIC and the nucleus accumbens core (AcbC). Two weeks after TMT exposure, Gad-1 gene expression was elevated in the aIC and Gat-1 was increased in the Acb, compared to controls. Lastly, the alcohol discrimination and alcohol-naïve groups displayed dramatic differences in stress reactive behaviors during the TMT exposure, suggesting that alcohol exposure may alter the behavioral response to predator odor. Together, these data suggest that predator odor stressor results in potentiated sensitivity to alcohol, possibly through GABAergic adaptations in the aIC and Acb, which may be relevant to understanding PTSD-AUD comorbidity.

Production of GABA-enriched tomato juice by Lactiplantibacillus plantarum KB1253

To produce tomato juice with health-promoting functions, lactic acid bacteria (LAB) capable of converting l-glutamic acid in tomatoes into γ-aminobutyric acid (GABA) was screened from LAB stocks isolated from Japanese pickles. Lactiplantibacillus plantarum KB1253 was selected as the highest GABA producer among 74 strains of LAB stocks. gad gene expression and glutamic acid decarboxylation activity increased at low pH (3.0-3.5), whereas the growth decreased. Under optimal reaction conditions using resting cells as catalysts, this strain produced 245.8 ± 3.4mM GABA. Furthermore, this strain produced 41.0 ± 1.1mM GABA from l-glutamic acid in tomato juice under optimal fermentation conditions (pH 4.0, 20°Bx). This study may provide the basis for developing health-promoting functional foods rich in GABA from tomatoes and other agricultural products.

The anxiolytic effect of salicylic acid is mediated via the GABAergic system in the fear potentiated plus maze behavior in rats.

Salicylic acid(SA) is a natural phenolic compound in plants with many beneficial effects for humans. The anxiolytic effect of this compound has been reported in animal models, but its mechanism of action remains unclear. In this study, by using the fear potentiated plus maze test, we evaluated the effect of salicylic acid on the gene expression of the main form of GABA (gamma aminobutyric acid) synthesizing enzyme i.e., the enzyme glutamic acid decarboxylase 67 (GAD67) which is called GAD1, in the ventral subiculum of the hippocampus, one of the main brain structures, in anxiety circuits. Also, the hypnotic effect of Salicylic acid was evaluated. Animals were divided into the solvent, (SA) and diazepam treated groups (n = 6). For evaluating the anxiolytic effect of Salicylic acid, animals were subjected to 2h of isolation, before placing them in the elevated plus maze (EPM). Afterward, the ventral part of the hippocampus was removed for evaluating the change in GAD1 gene expression by the reverse transcription-quantitative polymerase chain reaction (RTqPCR) technique. The hypnotic effect of Salicylic acidwas evaluated in the ketamine induced sleeping test. Salicylic acid at 10 and 30 (mg/kg) increased time spent and entries to the open arms in the (EPM) (p < 0.05). (RTqPCR) revealed that 30mg/kg of Salicylic acidincreased GAD1 gene expression (p < 0.001). Salicylic acid(30 and 300mg/kg) also increased the duration of sleep, in ketamine induced sleeping test (p < 0.05). Our results showed that Salicylic acidhas anxiolytic and hypnotic effects and it exerts its anxiolytic effect partly, via up the regulation of GAD1 in the ventral part of the hippocampus.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae.

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

The influence of laser beam and high light intensity on lentil (Lens culinaris) and wheat (Triticum aestivum) seedlings growth and metabolism

Light is considered as an environmental signal that controls plants growth and development. Sufficient duration, quality and wavelength are mainly responsible for switching on/off many physiological processes. The aim of this study was to examine the role of gamma aminobutyric acid (GABA) shunt pathway specific response in wheat (Triticum aestivum) and lentil (Lens culinaris) seedlings to blue LED laser (intensity= 1.48 W/cm2) and high intensity invisible ultraviolet light (intensity= 3.99 W/cm2) treatments separately, with respect to: seed germination, seedling growth, oxidative damage in term of reactive oxygen substances accumulation, GABA accumulation level, total proteins and total carbohydrates level and glutamate decarboxylase gene (GAD) expression. Data showed a remarkable changes in proteins content, carbohydrates content, chlorophyll level, dry and fresh weight, GABA accumulation, MDA level, and GAD m-RNA level under blue LED laser and high intensity invisible ultraviolet light treatments; separately in the both wheat and lentil cultivars. There were no significant differences of root emergence in all cultivars under all treatments. The concentrations of MDA increased in all cultivars under blue LED laser and high intensity invisible ultraviolet light treatments with increasing exposure durations. GABA levels increased and correlated significantly in all cultivars as the exposure duration of blue LED laser and high light intensity increased. Significant decrease in chlorophyll a and b contents in Um Qayes, Jordan1 and Jordan2 cultivars under all laser and high intensity invisible ultraviolet light treatments. A significant difference was found in proteins content in all cultivars in response to laser and high intensity invisible ultraviolet light treatments. Carbohydrates content in all cultivars used in this study were significantly decreased under laser treatments. The transcription level of GAD increased significantly under blue LED laser and high intensity invisible ultraviolet light in all cultivars used in this study. GAD expression was high in Hurani75 cultivar in agreement with the low level of GABA accumulation under high intensity invisible ultraviolet light at 60 min which strongly suggested that GABA was catabolized to enter another pathways (C:N balance) that was confirmed by increase in higher level of carbohydrates and proteins beside reduction in MDA content at the same exposure duration. On other hand, GAD expression in Jordan 2 was lower in response to laser treatment under the same exposure duration. The high increase of GAD gene expression explains the high accumulation of GABA level under both treatments. Results indicated that GABA shunt is a key metabolic pathway that allows wheat and lentil to adapt blue LED laser and high intensity invisible ultraviolet light stressful treatments.

Physiological and proteomics responses of nitrogen assimilation and glutamine/glutamine family of amino acids metabolism in mulberry (Morus alba L.) leaves to NaCl and NaHCO3 stress

ABSTRACT In order to find out the response mechanism of nitrogen assimilation and glutamine/glutamine family of amino acids metabolism in mulberry (Morus alba L.) leaves under NaCl and NaHCO3 stress, and to reveal its role in salt alkali adaptation. The effects of the nitrogen metabolism of mulberry leaves were studied under 100 mmol L−1 NaCl and NaHCO3 stress.The results showed that the activity of NR and the content of TN and SP did not change significantly, the expression of NiR, Fd-NiR, Fd-NiR gene and theactivity of NiR increased significantly under NaCl stress, but nitrogen assimilation was inhibited under NaHCO3 stress. NaCl stress had no significant effect on the expression and activity of GS and GOGAT in mulberry leaves. Under NaHCO3 stress, the expression of Fd-GOGAT, Fd-GOGAT2, Fd-GOGAT gene, and the activity of GS and GOGAT were significantly decreased. NaCl stress can promote the accumulation of Pro, Put and Spd in mulberry leaves. The accumulation of Pro under NaHCO3 stress is greater than that under NaCl stress. NaCl stress also induced the up-regulation of GAD, GAD1 and GAD1 gene expression, so promoting the synthesis of GABA may be an adaptive mechanism for mulberry to cope with NaCl stress, but the expression of GAD did not change significantly and GAD gene expression lower than CK under NaHCO3 stress. Although both NaCl and NaHCO3 stress could promote the synthesis of GSH by up-regulation of GCLM expression, GSH under NaHCO3 stress was significantly higher than that under NaCl stress, the content of H2O2 was still significantly higher than that of NaCl stress, that means GSH may not play a key role in alleviating the oxidative damage in mulberry leaves caused by salt and alkali.

γ-aminobutyric acid production by Kluyveromyces marxianus strains.

Kluyveromyces marxianus dairy strains were tested for γ-aminobutyric acid (GABA) production. The genes involved in GABA catabolism (UGA1 and UGA2) and anabolism (GAD1) were found in K. marxianus genome. Their relative expression was evaluated with primer designed ad hoc. Strains were grouped on the basis of GAD1 gene sequence. Representative strains for each group were tested for GABA production by high-performance liquid chromatography. All strains produced it at low levels. qRT-PCR showed the absence of a relation between GABA production and GAD1 gene expression. UGA1 and UGA2 genes were not upregulated and low amounts of succinic acid were detected. All strains released a low amount of GABA suggesting that probably it was stored within cells. The different behaviour of strains in terms of GABA and succinic acid production as well as gene expression highlighted the genetic and phenotypic biodiversity of this species. GABA production and genes involved in its catabolism and anabolism were described in a population of dairy K. marxianus for the first time. The variability observed in terms of genetic and phenotypic biodiversity is important especially to exploit this non-conventional yeast as microbial platform.

Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.

Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

Alterations of GABAergic Neuron-Associated Extracellular Matrix and Synaptic Responses in Gad1-Heterozygous Mice Subjected to Prenatal Stress.

Exposure to prenatal stress (PS) and mutations in Gad1, which encodes GABA synthesizing enzyme glutamate decarboxylase (GAD) 67, are the primary risk factors for psychiatric disorders associated with abnormalities in parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC). Decreased expression of extracellular matrix (ECM) glycoproteins has also been reported in patients with these disorders, raising the possibility that ECM abnormalities may play a role in their pathogenesis. To elucidate pathophysiological changes in ECM induced by the gene–environment interaction, we examined heterozygous GAD67-GFP (Knock-In KI; GAD67+/GFP) mice subjected to PS from embryonic day 15.0 to 17.5. Consistent with our previous study, we confirmed a decrease in the density of PV neurons in the mPFC of postnatal GAD67+/GFP mice with PS, which was concurrent with a decrease in density of PV neurons surrounded by perineuronal nets (PNNs), a specialized ECM important for the maturation, synaptic stabilization and plasticity of PV neurons. Glycosylation of α-dystroglycan (α-DG) and its putative mediator fukutin (Fktn) in the ECM around inhibitory synapses has also been suggested to contribute to disease development. We found that both glycosylated α-DG and the mRNA level of Fktn were reduced in GAD67+/GFP mice with PS. None of these changes were detected in GAD67+/GFP naive mice or wild type (GAD67+/+) mice with PS, suggesting that both PS and reduced Gad1 gene expression are prerequisites for these changes. When assessing the function of interneurons in the mPFC of GAD67+/GFP mice with PS through evoked inhibitory post-synaptic currents (eIPSCs) in layer V pyramidal neurons, we found that the threshold stimulus intensity for eIPSC events was reduced and that the eIPSC amplitude was increased without changes in the paired-pulse ratio (PPR). Moreover, the decay rate of eIPSCs was also slowed. In line with eIPSC, spontaneous IPSC (sIPSC) amplitude, frequency and decay tau were altered. Thus, our study suggests that alterations in the ECM mediated by gene-environment interactions might be linked to the enhanced and prolonged GABA action that compensates for the decreased density of PV neurons. This might be one of the causes of the excitatory/inhibitory imbalance in the mPFC of psychiatric patients.

The effect of sodium thiopental as a GABA mimetic drug in neonatal period on expression of GAD65 and GAD67 genes in hippocampus of newborn and adult male rats.

Objective(s):Development of the nervous system in human and most animals is continued after the birth. Critical role of this period in generation and specialization of the neuronal circuits is confirmed in numerous studies. Any pharmacological intervention in this period may result in structural, functional or behavioral abnormalities. In this study, sodium thiopental a GABA mimetic drug was administrated to newborn rats and their GAD65 and GAD67 expression in hippocampus was evaluated before and after puberty.Materials and Methods:Newborn male Wistar rats were received sodium thiopental (35 mg/kg) daily for 11 days (from 4 to 14 days after birth). Expression of GAD65 and GAD67 in their hippocampus was compared with control groups in 15 and 45 days after birth with RT-qPCR method.Results:Significant down regulation of GAD65 and GAD67 gene expression was observed in treated rats compared with control group in 45 days after birth animals. But no significant difference was shown between experimental and control groups 15 days after birth animals.Conclusion:The effect of sodium thiopental on GAD65 and GAD67 expression only at adult rats showed a latent period of influence which can be attributed to dosage or intension of sodium thiopental neurotoxicity. Significant down regulation of GAD65 and GAD67 showed unwanted effect of sodium thiopental as GABA mimetic drug in critical period of development.

GABAergic Interneuron Differentiation in the Basal Forebrain Is Mediated through Direct Regulation of Glutamic Acid Decarboxylase Isoforms by Dlx Homeobox Transcription Factors.

GABA is the key inhibitory neurotransmitter in the cortex but regulation of its synthesis during forebrain development is poorly understood. In the telencephalon, members of the distal-less (Dlx) homeobox gene family are expressed in, and regulate the development of, the basal ganglia primodia from which many GABAergic neurons originate and migrate to other forebrain regions. The Dlx1/Dlx2 double knock-out mice die at birth with abnormal cortical development, including loss of tangential migration of GABAergic inhibitory interneurons to the neocortex (Anderson et al., 1997a). We have discovered that specific promoter regulatory elements of glutamic acid decarboxylase isoforms (Gad1 and Gad2), which regulate GABA synthesis from the excitatory neurotransmitter glutamate, are direct transcriptional targets of both DLX1 and DLX2 homeoproteins in vivo Further gain- and loss-of-function studies in vitro and in vivo demonstrated that both DLX1 and DLX2 are necessary and sufficient for Gad gene expression. DLX1 and/or DLX2 activated the transcription of both Gad genes, and defects in Dlx function disrupted the differentiation of GABAergic interneurons with global reduction in GABA levels in the forebrains of the Dlx1/Dlx2 double knock-out mouse in vivo Identification of Gad genes as direct Dlx transcriptional targets is significant; it extends our understanding of Dlx gene function in the developing forebrain beyond the regulation of tangential interneuron migration to the differentiation of GABAergic interneurons arising from the basal telencephalon, and may help to unravel the pathogenesis of several developmental brain disorders.SIGNIFICANCE STATEMENT GABA is the major inhibitory neurotransmitter in the brain. We show that Dlx1/Dlx2 homeobox genes regulate GABA synthesis during forebrain development through direct activation of glutamic acid decarboxylase enzyme isoforms that convert glutamate to GABA. This discovery helps explain how Dlx mutations result in abnormal forebrain development, due to defective differentiation, in addition to the loss of tangential migration of GABAergic inhibitory interneurons to the neocortex. Reduced numbers or function of cortical GABAergic neurons may lead to hyperactivity states such as seizures (Cobos et al., 2005) or contribute to the pathogenesis of some autism spectrum disorders. GABAergic dysfunction in the basal ganglia could disrupt the learning and development of complex motor and cognitive behaviors (Rubenstein and Merzenich, 2003).

GAD1 Gene Expression in Blood of Patients with First-Episode Psychosis.

γ-Aminobutyric acid (GABA), the primary inhibitory neurotransmitter, has often been studied in relation to its role in the pathophysiology of schizophrenia. GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD), derived from two genes, GAD1 and GAD2. GAD1 is expressed as both GAD67 and GAD25 mRNA transcripts with the former reported to have a lower expression level in schizophrenia compared to healthy controls and latter was reported to be predominantly expressed fetally, suggesting a role in developmental process. In this study, GAD67 and GAD25 mRNA levels were measured by quantitative PCR (qPCR) in peripheral blood of subjects with first-episode psychosis (FEP) and from healthy controls. We observed low GAD25 and GAD67 gene expression levels in human peripheral blood. There was no difference in GAD25 and GAD67 gene expression level, and GAD25/GAD67 ratio between patients with FEP and healthy controls. PANSS negative symptoms were associated with levels of GAD25 mRNA transcripts in patients with FEP. While the current study provides information on GAD25 and GAD67 mRNA transcript levels in whole blood of FEP patients, further correlation and validation work between brain regions, cerebrospinal fluid and peripheral blood expression profiling are required to provide a better understanding of GAD25 and GAD67.

The influence of chlorsulfuron herbicide on GABA metabolism and oxidative damage in lentil (Lens culinaris Medik) and wheat (Triticum aestivum L.) seedlings

Abiotic stresses like salinity, drought, cold, heat, nutrient deficiency, and nutrient toxicity act as a major threats for crops quality and productivity. Herbicides are large category of chemical classes that affect various metabolic pathways and energy cascade in plants. γ-aminobutyric acid (GABA) is proposed to be critically involved in nitrogen metabolism, cytosolic pH regulation, and protection against oxidative imbalance in response to a wide range of abiotic stresses in plants. The objectives of this study were to determine the influence of chlorsulfuron herbicide on GABA metabolism and oxidative damage in two wheat (Triticumaestivum L.) cultivars (Hurani 75 and Um Qayes) and two lentil cultivars (Jordan 1 and Jordan 2) with respect to seed germination, GABA and malondialdehyde (MDA) levels accumulation, transcription of glutamate decarboxylase gene (GAD) mRNA level, total proteins, and carbohydrates contents. A significant increase in GABA, MDA and GAD mRNA level, proteins, and carbohydrates contents under chlorsulfuron treatments in the wheat and lentil cultivars was found in this study. The significant increase in GAD gene expression indicates the increase in GABA level under chlorsulfuron treatments. Hurani 75 and Jordan 2 cultivars showed significant decrease in protein level at high concentration of chlorsulfuron, whereas Um Qayes and Jordan 1 cultivars showed more tolerance under the same treatments. This study indicates that GABA metabolism might be one of multiple integrated signaling and metabolic pathways of herbicide stress adaptation and tolerance in wheat and lentil seedlings. This suggests that GABA molecule may act as a protective and metabolic signaling molecule in carbohydrates and proteins metabolism in plants under herbicidal treatments.

Molecular and morphological changes in zebrafish following transient ethanol exposure during defined developmental stages

Alcohol is a teratogen that has diverse effects on brain and craniofacial development, leading to a constellation of developmental disorders referred to as fetal alcohol spectrum disorder (FASD). The molecular basis of ethanol insult remains poorly understood, as does the relationship between molecular and behavioral changes as a consequence of prenatal ethanol exposure. Zebrafish embryos were exposed to a range of ethanol concentrations (0.5–5.0%) during defined developmental stages, and examined for morphological phenotypes characteristic of FASD. Embryos were also analyzed by in situ hybridization for changes in expression of defined cell markers for neural cell types that are sonic hedgehog-dependent. We show that transient binge-like ethanol exposures during defined developmental stages, such as early gastrulation and early neurulation, result in a range of phenotypes and changes in expression of Shh-dependent genes. The severity of fetal alcohol syndrome (FAS) morphological phenotypes, such as microphthalmia, depends on the embryonic stage and concentration of alcohol exposure, as does diminution of retinal Pax6a or forebrain and hindbrain GAD1 gene expression. We also show that changes in eye and brain morphology correlate with changes in Pax6a and GAD1 gene expression. Our results therefore show that transient binge-like ethanol exposures in zebrafish embryos produce the stereotypical morphological phenotypes of FAS, with the severity of phenotypes depending on the developmental stage and alcohol concentration of exposure.

Influence of Light on the Free Amino Acid Content and γ-Aminobutyric Acid Synthesis in Brassica juncea Seedlings

Glutamate decarboxylase (GAD; EC 4.1.1.15) is an important enzyme in γ-aminobutyric acid (GABA) biosynthesis. Here we report the influence of light on amino acid accumulation and investigate the molecular mechanism by which light influences GABA biosynthesis at the seedling stage of two mustard (Brassica juncea) cultivars (green-leaf and purple-leaf). Gene expression profiles of four GAD-encoding genes (GAD1, GAD2, GAD4a, and GAD4b) and their impact on GABA biosynthesis were analyzed. Light exerted an obvious influence on amino acid accumulation in mustard seedlings. GAD gene expression was also significantly regulated by light/dark or dark treatment, which differentially regulated GABA biosynthesis in B. juncea seedlings. High-performance liquid chromatography (HPLC) revealed that the seeds of purple cultivars contain a higher amount of free amino acids and GABA than do the seeds of green cultivars. After seed germination, however, the accumulation of free amino acids peaked in dark-treated seedlings on day 9 in both cultivars, whereas GABA synthesis peaked at 9 days under light conditions. This study may provide a foundation for understanding the effect of light on amino acids, particularly GABA biosynthesis in Brassica plants.

Expression of Glutamate Decarboxylase (GAD) mRNA in the Brain of Bile Duct Ligated Rats Serving as a Model of Hepatic Encephalopathy

Hepatic encephalopathy (HE) is a neurologic disorder that involves different pathophysiological mechanisms, including disturbances in the GABAergic neurotransmitter system. Albeit an overall increase in the level of neurotransmitter GABA has not been found in HE, alterations in GABA receptors and metabolism have been described. Moreover, it has been reported that bile duct ligated (BDL) rats, an animal model for the study of HE, exhibited an altered GABA biosynthesis involving preferentially the tricarboxylic (TCA) cycle. In this context it should be noted that the GABA synthesizing enzyme glutamate decarboxylase (GAD) is expressed in the brain in two isoforms GAD67 and GAD65, GAD65 being related to the synthesis of GABA that occurs via the TCA cycle and coupled to the vesicular pool of the neurotransmitter. The aim of the present study was to investigate whether changes in mRNA expression of GAD67 and GAD65 were related to the altered GABA biosynthesis previously observed. To study this, cerebral cortices and hippocampi were dissected from control and BDL rats, total mRNA was isolated and cDNA was synthesized by reverse transcription reaction. Subsequently samples were analyzed for gene expression of GAD67 and GAD65 by qPCR multiplex assay, using GAPDH as endogenous control. No changes in GAD67 and GAD65 mRNA expression between control and BDL rats either in cerebral cortex or in hippocampus were observed indicating that the HE condition did not lead to changes in GAD mRNA expression. However, other regulatory mechanism might be affecting GAD activity and to clarify this additional studies need to be conducted.

Characterization of γ-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress

The molecular response of plants to abiotic stresses has been considered a process mainly involved in the modulation of transcriptional activity of stress-related genes. Nevertheless, recent findings have suggested new layers of regulation and complexity. Upstream molecular mechanisms are involved in the plant response to abiotic stress. Plants gain resistance to abiotic stress by reprogramming metabolism and gene expression. GABA is proposed to be a signaling molecule involved in nitrogen metabolism, regulating the cytosolic pH, and protection against oxidative damage in response to various abiotic stresses. The aim of our study was to examine the role of the GABA shunt pathway-specific response in five wheat (Triticum aestivum L.) cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes and Nodsieh) to salt and osmotic stress in terms of seed germination, seedling growth, oxidative damage (malondialdehyde (MDA) accumulation), and characterization of the glutamate decarboxylse gene (GAD) m-RNA level were determined using RT-PCR techniques. Our data showed a marked increase in GABA, MDA and GAD m-RNA levels under salt and osmotic stress in the five wheat cultivars. Um Qayes cultivar showed the highest germination percentage, GABA accumulation, and MDA level under salt and osmotic stresses. The marked increase in GAD gene expression explains the high accumulation of the GABA level under both stresses. Our results indicated that the GABA shunt is a key signaling and metabolic pathway that allows wheat to adapt to salt and osmotic stress. Based on our data, the Um Qayes wheat cultivar is the cultivar most recommended to be grown in soil with high salt and osmotic contents.

Nicotine Restores Wt-Like Levels of Reelin and GAD67 Gene Expression in Brain of Heterozygous Reeler Mice

Important reduction of reelin, a neural development- and plasticity-associated protein, and glutamic acid decarboxylase (GAD67) are reported in brains of schizophrenic patients. These individuals are consistently engaged in tobacco smoking and nicotine is thought to alleviate negative behavioral symptoms or cognitive alterations. In mouse brain, nicotine has been shown to reduce GAD67 promoter methylation and increase its transcription. We assessed the effects of administration of nicotine (1mg/kgs.c.) for 6days, in male mice heterozygous for reelin (HRM), a putative model for symptoms related to schizophrenia. Expression of reelin, GAD67 and brain-derived neurotrophic factor (BDNF) was measured in different brain areas. RNA expression analysis evidenced genotype-related changes, with a marked reduction in reelin and GAD67 gene expression in prefrontal cortex, hippocampus, cerebellum, and striatum from HRM. Nicotine treatment selectively reversed the HRM-related phenotype in most brain areas and increased BDNF gene expression in cortex and hippocampus of both genotypes. Locomotor performance in their home cage revealed that HRM subjects were characterized by general hyperactivity; with nicotine administration restoring WT-like levels of locomotion. These findings are interpreted within the hypothesis of pre-existing vulnerability (based on haploinsufficiency of reelin) to brain and behavioral disorders and regulative effects associated with nicotine exposure.