GAD67 mRNA Research Articles

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Induction of hypothalamic GABA synthetic enzymes mRNA (Gad 1 and Gad 2) expression by negative energy balance in broiler and layer chicks

Broiler and layer chicks have been selected for higher and lower food intake and body weight gain, respectively. It has recently been reported that glutamate decarboxylase (Gad1) mRNA, a gamma-aminobutyric acid (GABA) synthetic enzyme gene, is a reliable proxy for GABA release. Previous studies have revealed that GABAergic system has a stimulatory role on food intake in both mammals and birds. Over the recent years, evidence has identified the presence of GABAergic neurons as either the first- or second-order neurons within the various feeding nuclei of hypothalamus of laboratory rodents. They respond to the negative energy balance representing a critical role for GABA in the regulation of food intake. In the current study, the mRNA abundance of Gad 1 and Gad 2 was measured within the hypothalamus of both broiler and layer free fed, 12 h-fasted and 12 h-fasted / 3 h refed chicks. Furthermore, the effect of intracerebroventricular (ICV) injection of GABA was studied on food intake of chicks. The results indicated an increase in both Gad 1 and 2 expressions during fasting which tended to return to the baseline after refeeding. However, this increase was greater in broilers than in layers. The results also showed that ICV injection of GABA had no effect on food intake with the exception of an increase in free fed broilers. This study suggests a role for hypothalamic GABAergic system in birds that respond to negative energy balance, which seems to be more considerable in broilers than in layers.

The molecular mechanism underlying GABAergic dysfunction in nucleus accumbens of depression-like behaviours in mice.

Depression is the most frequent psychiatric disorder in the world. Recent evidence has shown that stress‐induced GABAergic dysfunction in the nucleus accumbens (NAc) contributed to the pathophysiology of depression. However, the molecular mechanisms underlying these pathological changes remain unclear. In this study, mice were constantly treated with the chronic unpredictable mild stress (CUMS) till showing depression‐like behaviours expression. GABA synthesis, release and uptake in the NAc tissue were assessed by analysing the expression level of genes and proteins of Gad‐1, VGAT and GAT‐3 by qRT‐PCR and Western blotting. The miRNA/mRNA network regulating GABA was constructed based on the bioinformatics prediction software and further validated by dual‐luciferase reporter assay in vitro and qRT‐PCR in vivo, respectively. Our results showed that the expression level of GAT‐3, Gad‐1 and VGAT mRNA and protein significantly decreased in the NAc tissue from CUMS‐induced depression‐like mice than that of control mice. However, miRNA‐144‐3p, miRNA‐879‐5p, miR‐15b‐5p and miRNA‐582‐5p that directly down‐regulated the expression of Gad‐1, VGAT and GAT‐3 were increased. In the mRNA/miRNA regulatory GABA network, Gad‐1 and VGAT were directly regulated by binding seed sequence of miR‐144‐3p, and miR‐15b‐5p, miR‐879‐5p could be served negative post‐regulators by binding to the different sites of VGAT 3′‐UTR. Chronic stress causes the impaired GABA synthesis, release and uptake by up‐regulating miRNAs and down‐regulating mRNAs and proteins, which may reveal the molecular mechanisms for the decreased GABA concentrations in the NAc tissue of CUMS‐induced depression.

Abstract 4379: Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism

Abstract Although an upregulation of aerobic glycolysis (the Warburg effect) is a canonical hallmark of cancer, it is now recognized that most cancerous cells continue to rely on the TCA cycle as a source of both biosynthetic precursors (DeBarardinis & Chandel, 2016) and the majority of ATP production (Zu & Guppy, 2004). Drug-resistant cancers have been associated with metabolic reprogramming (Rahman & Hasan, 2015) involving an upregulation in oxidative phosphorylation (Wolf, 2014). Two advanced, treatment-resistant variants of prostate cancer are castration-resistant adenocarcinoma (CRPC-adeno) and neuroendocrine prostate cancer (NEPC). Emerging evidence suggests that the γ-aminobutyric acid (GABA) shunt—an evolutionarily conserved pathway for energy production, biosynthetic precursors, and ROS management—may be used by both CRPC-adeno and NEPC. The GABA shunt is a bypass for two steps of the TCA cycle, wherein GABA is synthesized from glutamate by a GAD1-encoded decarboxylase, transaminated into succinic semialdehyde, and metabolized into the TCA cycle intermediate succinate. Analysis of publicly available transcript datasets (via cBioPortal) indicates that GAD1 mRNA expression increases with increasing Gleason score (p < 0.00005, prostate adenocarcinoma TCGA provisional dataset), with higher expression in both CRPC-adeno compared to primary adenocarcinoma (Ippolito and Piwnica-Worms, 2014), and NEPC compared to CRPC-adeno (p < 0.005, Beltran, et al., 2016). Expression of GAD1 transcripts appears to be contingent on low levels of AR transcripts (Trento/Cornell/Broad data set), consistent with the AR independence of the NEPC phenotype. Similarly, GABA levels increase as AR expression is lost during transdifferentiation to NEPC (Solorzano, et al., 2018). GAD67 protein levels in prostate tumors are a more accurate predictor of patient risk for metastasis than Gleason score (Azuma, et al., 2003), and flux through the GABA shunt is critical for the growth of brain tumors in mouse xenograft models (Schnepp, et al., 2017; Neman, et al., 2014). Our experiments show that treatment of an early-stage prostate cancer cell line model (LNCaP) with GABA (100 µM, 24h) induces a shift from aerobic glycolysis to oxidative metabolism. This metabolic reprogramming toward increased oxidative phosphorylation requires at least 6 hours to emerge, and is maximal by 18 hours. During this time period, the presence of GABA in the mitochondrial matrix steadily increases, as shown by our newly-developed GABA-sensing fluorescent receptor, mitoGABASnFR. The observed increases in oxygen consumption are accompanied by GABA-induced decreases in extracellular acidification and lactate levels. In light of these findings, we propose that the GABA shunt represents an understudied pathway for inducing a metabolic shift towards oxidative phosphorylation in advanced prostate cancer, and therefore may be a target for future anticancer therapies. Citation Format: Erika L. Knott, Sumitra Miriyala, Manikandan Panchatcharam, Nancy Leidenheimer. Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4379.

Morphological and neurochemical changes in GABAergic neurons of the aging human inferior colliculus

It is well known that quality of hearing decreases with increasing age due to changes in the peripheral or central auditory pathway. Along with the decrease in the number of neurons the neurotransmitter profile is also affected in the various parts of the auditory system. Particularly, changes in the inhibitory neurons in the inferior colliculus (IC) are known to affect quality of hearing with aging. To date, there is no information about the status of the inhibitory neurotransmitter GABA in the human IC during aging. We have collected and processed inferior colliculi of persons aged 11–97 years at the time of death for morphometry and immunohistochemical expression of glutamic acid decarboxylase (GAD67) and parvalbumin. We used unbiased stereology to estimate the number of cresyl-violet and immunostained neurons. Quantitative real-time PCR was used to measure the relative expression of the GAD67 mRNA. We found that the number of total, GABAergic and PV-positive neurons significantly decreased with increasing age (p < 0.05). The proportion of GAD67-ir neurons to total number of neurons was also negatively associated with increasing age (p = 0.004), but there was no change observed in the proportion of PV-ir neurons relative to GABAergic neurons (p = 0.25). Further, the fold change in the levels of GAD67 mRNA was negatively correlated to age (p = 0.024). We conclude that the poorer quality of hearing with increasing age may be due to decreased expression of inhibitory neurotransmitters and the decline in the number of inhibitory neurons in the IC.

GAD mRNA in Orbital Prefrontal Cortex and Anterior Cingulate Cortex in Alcoholics Compared with Nonpsychiatric Controls: A Negative Postmortem Study.

Alcohol increases inhibitory neurotransmission, an effect mediated through GABA receptors. With chronic alcohol exposure, the inhibitory effects diminish. Glutamic acid decarboxylase (GAD) catalyzes glutamate in the synthesis of GABA. We sought to determine the amount of GAD65/67 mRNA in anterior cingulate cortex (BA24) and orbital prefrontal cortex (BA45) of medication-free alcoholics and nonpsychiatric controls postmortem.Studies were performed in 16 pairs of nonpsychiatric controls and alcoholics, matched for age, sex and PMI. DSM-IV diagnosis of alcohol use disorder (AUD) was made by the SCID I in a psychological autopsy. Frozen blocks of BA24 or BA45 were sectioned (10 µm) for in situ hybridization of 35S-labelled riboprobe for GAD65/67 mRNA and autoradiograms were analyzed by quantitative densitometry. Three isodensity bands of labeling were evident, with different relative amounts of GAD65 and GAD67 (outer and inner, predominantly GAD65, intermediate predominantly GAD67), and the isodensity bands were analyzed separately.GAD65/67 mRNA levels were not different between alcoholics and controls in the gray matter of BA24 (p = 0.53) or BA45 (p = 0.84) or in any of the three isodensity bands in which the GAD65/67 mRNA was distributed. GAD65/67 mRNA in white matter underlying either region was also not different in alcoholics (p > 0.05). GAD65/67 mRNA levels did not correlate with age, sex or duration of alcoholism in either BA24 or BA45.Effects on inhibitory neurotransmission in alcoholics do not appear to be associated with change in the levels of GAD65 or GAD67 mRNA.

Capacitation of mouse sperm is modulated by gamma-aminobutyric acid (GABA) concentration.

In mammals, ejaculated sperm acquire their fertilizing ability during migration through the female reproductive tract, which secretes several factors that contribute to sperm capacitation. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter in the central nervous system, but additionally enhances the sperm acrosome reaction in the rat and cow. However, the detailed effects of GABA concentration on sperm function remain unclear. In this study, we detected the presence of the GABA type A receptor (GABA A) in mouse epididymal sperm by western blot analysis and in the sperm acrosome by immunocytochemistry. We also investigated the effects of GABA on sperm fertilizing ability. We found that GABA facilitated the tyrosine phosphorylation of sperm proteins, which is an index of sperm capacitation. GABA also promoted the acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. We then found that the effective GABA concentration for the acrosome reaction corresponds to sperm concentration, but we did not detect any marked effect of GABA on sperm motility using a computer-assisted sperm analysis system. Using immunohistochemistry, we also detected GABA expression in the epithelia of the mouse uterus and oviduct. Furthermore, we found that the mRNA levels of glutamate decarboxylase (Gad), which generates GABA from L-glutamate, were higher in the oviduct than in the uterus, and that Gad mRNA levels were higher at estrus than at the diestrus stage. These results indicate that the GABA concentration can act as a modulator of the acrosome reaction and sperm capacitation in the female reproductive tract.

Altered mRNA expressions for N-methyl-D-aspartate receptor-related genes in WBC of patients with major depressive disorder

ObjectiveMajor depressive disorder (MDD) is a complex mental disorder. The lack of well-established biomarkers hinders its diagnosis, treatment, and new-drug development. N-methyl-D-aspartate receptor (NMDAR) dysfunction has been implicated in the pathogenesis of MDD. This study examined whether expressions of the NMDAR-related genes are characteristic of MDD. MethodsExpressions of NMDAR-related genes including SRR, SHMT2, PSAT1, GCAT, GAD1, SLC1A4, NRG1 and COMT in peripheral WBCs of 110 patients with MDD (25 drug-naïve, 21 drug-free, and 64 medicated patients) and 125 healthy individuals were measured using quantitative PCR. ResultsThe mRNA expression levels of SRR, PSAT1, GCAT, GAD1, NRG1 and COMT were significantly different among the four groups (all p < 0.05). For drug-naïve patients, the ΔΔCT values of SRR, PSAT1, GCAT, GAD1, and NRG1 mRNA expressions were significantly different from those in healthy individuals (all p < 0.05). The ROC analysis of the ΔΔCT values of the target genes for differentiating drug-naïve patients from healthy controls showed an excellent sensitivity (0.960) and modest specificity (0.640) (AUC = 0.889). Drug-free and medicated patients obtained less favorable AUC values while compared to healthy controls. The results for the age- and sex-matched cohort were similar to those of the unmatched cohort. ConclusionsThis is the first study demonstrating that the peripheral mRNA expression levels of NMDAR-related genes may be altered in patients with MDD, especially drug-naïve individuals. The finding supports the NMDAR hypothesis of depression. Whether mRNA expresssion of NMDAR-related genes could serve as a potential biomarker of MDD deserves further investigations.

Impaired GAD1 expression in schizophrenia-related WISKET rat model with sex-dependent aggressive behavior and motivational deficit.

After peri-adolescence isolation rearing (IS) and subchronic ketamine (KET) treatment, adult, selectively bred Wistar rats (named WISKET) mimic abnormal behaviors reminiscent of human schizophrenia, including reduced prepulse-inhibition of startle reflex, disturbances in cognition, locomotor activity and thermoregulation, decreased pain sensitivity and electrophysiological alterations. To further validate our WISKET rat line, regarding its translational utility in schizophrenia research, we examined their social behavior and introduced a short and simple holeboard (HB)-like test to investigate their motivational deficit that predicts the cognitive disturbance. Sex-dependent alterations in schizophrenia may yield important insights into its etiology; thus, male and female WISKET rats were also investigated and compared with their naive Wistar counterparts. Considering the contribution of the hippocampal and cortical GABAergic inhibitory circuitry in these behavioral alterations, molecular-biology studies were also performed regarding the GAD1 gene products. Impaired social activity with increased aggression, stress-related behavior, active social withdrawal, motivation deficit and decreased exploration were observed, especially in male WISKET rats, compared with Wistar ones and their corresponding females. These alterations were accompanied by sex-dependent alterations regarding GAD67 mRNA and protein expression in the prefrontal cortex and hippocampus. In conclusion, the WISKET animals are valuable tools for animal-based preclinical drug discovery studies for predictive screening of novel compounds improving negative symptoms with potential antipsychotic efficacy.

Abstract 5486: The role of the GABA shunt in prostate cancer metabolic reprogramming and aggressive phenotype

Abstract The GABA (γ-aminobutyric acid) shunt, a bypass mechanism for a portion of the tricarboxylic acid (TCA) cycle, is responsible in the mammalian central nervous system for the synthesis and degradation of the inhibitory neurotransmitter GABA. Glutamic acid decarboxylase (GAD67 and GAD65; gene names GAD1 and GAD2, respectively) synthesizes GABA from glutamate, while GABA-transaminase (GABA-T; gene name ABAT) metabolizes GABA into succinic semialdehyde, which is dehydrogenated into succinate. Although poorly studied in mammals, the GABA shunt plays a protective role against pH, thermo-, and oxidative stresses in plants and bacteria. We hypothesize that both cell non-autonomous GABA from the tumor microenvironment (TME), and cell-autonomous production of GABA contribute to cancer progression. Cell types within the TME such as endothelial and T-cells, are known to secrete GABA. To investigate the utilization of cell non-autonomous GABA by prostate cancer cells, we determined via LC-MS/MS that although the prostate adenocarcinoma LNCaP cell line does not contain significant amounts of GABA, it is capable of taking up exogenous GABA. The effect of exogenous GABA treatment on the metabolic capacity of LNCaP cells was examined using the SEAhorse XF24 Bioanalyzer. GABA treatment increased oxygen consumption rate while decreasing the rate of glycolysis and glycolytic reserve. This effect was blocked by co-treatment with GABA-T inhibitors, suggesting the ability of GABA to increase mitochondrial respiration occurs through the GABA shunt. GABA treatment did not affect the metabolic phenotype of a normal epithelial cell line control. Regarding cell-autonomous production of GABA, recent studies show dysregulation of genes encoding GABA shunt enzymes in various cancers; specifically relevant to this study, GAD1 is upregulated in castration-resistant prostate cancer cell lines (Ippolito 2014). Consistent with the involvement of GAD1 in aggressive prostate cancers, our analysis of the TCGA Provisional prostate cancer dataset reveals that GAD1 mRNA expression level increases with increasing Gleason Score (Pearson r = 0.19; p &amp;lt; 0.00005). Additionally, analysis of the Beltran (2016) data set reveals that GAD1 transcripts are upregulated in treatment-induced neuroendocrine prostate cancer compared to prostate adenocarcinoma (unpaired t-test, p &amp;lt; 0.0005). We hypothesize that both cell non-autonomous and autonomous GABA play a role in cancer progression. Uptake of GABA from the TME may support metabolic reprogramming of primary tumor cells, while upregulation of GAD1 and subsequent increase in cell-autonomous GABA production may provide a malignant advantage to late-stage cancer. The GABA shunt may offer a novel therapeutic target for prostate cancer treatment by exploiting a poorly understood metabolic pathway linked to the TCA cycle. We thank the LSUHSC-S Feist-Weiller Cancer Center for financial support. Citation Format: Erika L. Knott, Sumitra Miriyala, Hyung Nam, Manikandan Panchatcharam, Nancy Leidenheimer. The role of the GABA shunt in prostate cancer metabolic reprogramming and aggressive phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5486.

Neurochemical changes underlying schizophrenia-related behavior in a modified forced swim test in mice

The modified forced swim test (MFST) has excellent predictive validity for investigating the antipsychotic activity of drugs, with particular emphasis on their activity toward negative symptoms of schizophrenia. However, its face and construct validity are less understood. Therefore, in the present study, some biochemical changes within GABAergic and serotonergic neurotransmission that could be related to observed MK-801-induced disturbances and the activity of compounds active at those neurotransmitters were investigated.In biochemical experiments, mice were treated acutely or chronically with MK-801 (13 days, 0.4 mg/kg). Their brains were dissected and frontal cortices and hippocampi were taken for further analysis. The levels of neurotransmitters were investigated with HPLC, and the expression of surrogate markers of schizophrenia (5-HT1A receptors, GAD65, and GAD67, at both protein and mRNA levels) was measured via western blotting and qRT-PCR. The modified forced swim test and locomotor activity were used to assess the activity of GABAB and 5-HT1A-related compounds.Repeated MK-801 treatment (13 days, 0.4 mg/kg dose) led to decreases in the DOPAC/DA, 3MT/DA and HVA/DA metabolic ratios. Increased 5-HT1A protein expression and decreased GAD65 and GAD67 protein expression was observed in both the cortex and hippocampus. mRNA levels for all proteins were decreased. The increased immobility in the forced swim test was reversed both by a GABAB agonist (SKF97541, 0.025 or 0.05 mg/kg), a positive allosteric modulator of GABAB receptor (racBHFF, 5 or 10 mg/kg) and by a 5-HT1A agonist ((R)-(+)-8-OH-DPAT 0.01 or 0.025 mg/kg).Our research supports the hypothesis that changes in the levels of GABA and/or 5-HT1A receptors may contribute to the schizophrenia-like phenotype, and GABAergic and serotonergic agents may be good candidates for treating negative symptoms of schizophrenia.

Selective Brain Distribution and Distinctive Synaptic Architecture of Dual Glutamatergic-GABAergic Neurons

For decades, it has been thought that glutamate and GABA are released by distinct neurons. However, some mouse neurons innervating the lateral habenula (LHb) co-release glutamate and GABA. Here, we mapped the distribution of neurons throughout the rat brain that co-express vesicular transporters for the accumulation of glutamate (VGluT2) or GABA (VGaT) and for GABA synthesis (GAD). We found concentrated groups of neurons that co-express VGluT2, VGaT, and GAD mRNAs within subdivisions of the ventral tegmental area (VTA), entopeduncular (EPN), and supramammillary (SUM) nuclei. Single axon terminals established by VTA, EPN, or SUM neurons form a common synaptic architecture involving asymmetric (putative excitatory) and symmetric (putative inhibitory) synapses. Within the LHb, which receives co-transmitted glutamate and GABA from VTA and EPN, VGluT2 and VGaT are distributed on separate synaptic vesicles. We conclude that single axon terminals from VGluT2 and VGaT co-expressing neurons co-transmit glutamate and GABA from distinct synaptic vesicles at independent synapses.

Stereological Estimates of Glutamatergic, GABAergic, and Cholinergic Neurons in the Pedunculopontine and Laterodorsal Tegmental Nuclei in the Rat.

The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.

GABAergic inhibition in dual-transmission cholinergic and GABAergic striatal interneurons is abolished in Parkinson disease

We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl−]i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl−]i levels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson’s disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl−]i levels and GABAergic inhibition.

Peripheral GABAA receptor-mediated signaling facilitates persistent inflammatory hypersensitivity

Unlike in the central nervous system (CNS), in the adult peripheral nervous system (PNS), activation of GABAA receptors (GABAAR) is excitatory because of the relatively high concentration of intracellular chloride in these neurons. Indeed, exogenous GABA and muscimol, a GABAAR agonist, exacerbate acute inflammatory hypersensitivity in rodents. However, it remains unclear whether peripheral GABAAR and the endogenous GABA play an important role in persistent inflammatory hypersensitivity. In this study, we thus investigated how peripheral GABAAR affects pain hypersensitivity by using the complete Freund's adjuvant (CFA)-induced persistent inflammatory pain mouse model. We found that intraplantar (i.pl.) administration of GABAAR antagonists, picrotoxin, and 1(S),9(R)-(−)-bicuculline methiodide significantly inhibited both spontaneous nociceptive (paw licking and flinching) behavior and mechanical hypersensitivity in CFA-injected mice at day 3 (D3), but not in naïve mice. Interestingly, CFA-induced mechanical hypersensitivity was significantly reversed by anti-GABA antibody (anti-GABA, i.pl.). In addition, RT-qPCR revealed that glutamate decarboxylase Gad1 (GAD 67) and Gad2 (GAD 65) mRNA expression was also upregulated in the ipsilateral hind paw of CFA-injected mice at D3. Finally, 5α-pregnan-3α-ol-20-one (3α,5α-THP), a selective positive allosteric modulator of GABAAR, produced mechanical hypersensitivity in naïve mice in a dose-dependent manner. Taken together, our results indicate that peripheral GABAAR and endogenous GABA, possibly produced by the inflamed tissue, potentiate CFA-induced persistent inflammatory hypersensitivity, suggesting that they can be used as a therapeutic target for alleviating inflammatory pain.

F196. DIFFERENTIAL EFFECTS OF MGLU5 RECEPTOR BLOCKADE ON BEHAVIOR, SCHIZOPHRENIA-RELEVANT GENE EXPRESSION AND NEURONAL ACTIVATION PATTERNS FROM DEVELOPMENT TO AGING MICE

BackgroundThe glutamate system is implicated both in schizophrenia and mood disorders. Mice lacking metabotropic mGlu5 receptors (mGluR5 KO) display schizophrenia-like abnormalities. Additionally, mGluR5 antagonists represent promising alternative anxiolytics/antidepressants. However, the underlying age-specific molecular/cellular mechanisms are only partially understood. We aimed at identifying molecular alterations associated with a genetically induced mGluR5 deletion, which results in a schizophrenia-like phenotype. Additionally, we investigated age-specific effects of mGluR5 antagonists on emotional behaviour and c-fos activation.MethodsFor analysis of mRNA and protein levels we performed Real-time RT-PCR and Western blot investigations in the hippocampus and prefrontal/frontal cortex (PFC/FC) of mice with a genetic deletion of the metabotropic glutamate receptor 5 (mGlu5), addressing key components of the GABAergic and glutamatergic systems. Additionally, we used classical behavioral tests for determining anxiety- and depression-like changes triggered by the mGluR5 antagonist 2-Methyl-6-(phenylethynyl)pyridine (MPEP). Finally, we used profiling of c-Fos expression, as marker of neuronal activity, induced by MPEP from postnatal day 16 (P16) to adulthood (P90).ResultsmGlu5 knockout (KO) mice showed a significant reduction of reelin, GAD65, GAD67 and parvalbumin mRNA levels, which is specific for the PFC/FC, and that is paralleled by a significant reduction of protein levels in male KO mice. We also analysed the main NMDA and AMPA receptor subunits, namely GluN1, GluN2A, GluN2B and GluA1, and observed that mGlu5 deletion determined a significant reduction of their mRNA levels, also within the hippocampus, with differences between the two genders. We measured age-specific alterations in emotional behaviour of mGluR5 KO mice, with marked increase of anxiety during aging. There was a remarkably conserved activation of the paraventricular nucleus of the hypothalamus, implicated in stress regulation, by MPEP at all investigated ages, whereas the extended amygdala was specifically activated in adulthood only.DiscussionOur data suggest that neurochemical abnormalities impinging the glutamatergic and GABAergic systems may be responsible for the behavioral phenotype associated with mGlu5 KO animals and point to the close interaction of these molecular players for the development of neuropsychiatric disorders such as schizophrenia. These data could contribute to a better understanding of the involvement of mGlu5 alterations in the molecular imbalance between excitation and inhibition underlying the emergence of a schizophrenic-like phenotype and to understand the potential of mGlu5 modulators in reversing the deficits characterizing the schizophrenic pathology.

F206. A TRANSLATIONAL STUDY OF BEHAVIOR, BRAIN STRUCTURE AND GENE PATHWAY IN ERBB4 KNOCKOUT MICE AND FIRST-EPISODE TREATMENT-NAïVE PATIENTS WITH SCHIZOPHRENIA

BackgroundThe current study was to explore how disruption of specific molecular circuits in the cerebral cortex may cause large-scale brain structure deficits and behavior changes via a translational study in conditional Erbb4 mutant mice and patients with schizophrenia.MethodsWe conducted prepulse inhibition (PPI) and brain structural and diffusion magnetic resonance imaging (MRI) scans in 27 mice with ErbB4 knockout in parvalbumin (PV) interneurons and 23 age, sex-matched controls. Real-time quantitative polymerase chain reaction was used to assess the levels of five GABA-related transcripts in brain regions. We also measured structural and diffusion MRI and cumulative contribution of risk alleles in the GABA pathway genes using polygenic risk scores (PRS) in first-episode treatment-naïve schizophrenic patients (N=117) and age, sex-matched healthy controls (N=86).ResultsErbB4 knockout mice displayed behavioral deficit of PPI, as well as gray and white matter impairment in right sensorimotor cortical-striatal networks. We found significant correlations between gray matter volumes (GMVs) of the somatosensory cortex and PPI as well as GAD1 mRNA expression in controls but not in knockout mice. These findings were confirmed in a human sample where we observed significantly decreased gray and white matter impairment in sensorimotor cortical-striatal networks in schizophrenics. The PRS of GABA-pathway genes also displayed a negative correlation with the GMVs of the somatosensory cortex in patients.DiscussionOur study identified ErbB4 ablation induced prepulse inhibition deficits and GABAergic dysregulation in sensorimotor cortical-lateral striatal networks. We propose that ErbB4 signaling participates in sensorimotor gating dysfunction in schizophrenia by getting involved in somatosensory cortex deficits and GABAergic dysfunction.

Key molecules in the GABA signalling pathway are present in mouse and human cervical tissue.

Cervical mucus modulates fertility by cyclical changes of its biophysical and functional properties. Based on an analogy with bronchial goblet cells we set out to investigate the possible role of the gamma-aminobutyric acid (GABA) signalling pathway in the mediation of oestrogen-induced mucus secretion from endocervical secretory cells. The aim of the study was to examine the existence of GABAA receptor (GABAAR), glutamic acid decarboxylase 65/67 (GAD65/67) and vesicular GABA transporter (VGAT) in human and mouse cervical tissue. The mouse cervical tissue expressed GabaAR mRNA transcripts throughout the oestrous cycle. GABAAR-positive immunolabelling was present in the superficial layer of the mouse cervico-vaginal epithelium in pro-oestrus. Human cervical tissue showed the presence of GABAAR, GAD67 and VGAT mRNA transcripts and clear immunofluorescent signals of all three molecules were detected in the endocervical secretory epithelium. The results of this study confirmed that elements of the GABA signalling pathway are present in the secretory epithelium of mouse and human cervical tissue and that GABA signalling pathway could be considered a possible mediator in oestrogen regulation of mucus secretion in the endocervical glands.

Adolescent environmental enrichment prevents the emergence of schizophrenia-like abnormalities in a neurodevelopmental model of schizophrenia

In the present study, we investigated whether exposure to an enriched environment (EE) during adolescence might affect the behavioural dysfunction (sensorimotor gating deficit, memory and social interaction impairments) and neurochemical changes (GAD67 expression, histone methylation) induced by methylazoxymethanol (MAM) in the MAM-E17 rat model of schizophrenia. EE was introduced for 7 days in early adolescence (days 23–29), and behavioural and biochemical studies were performed on adult rats at postnatal day 70. The results showed that exposure to EE prevented the development of adult behavioural deficits induced by prenatal MAM administration. EE also prevented the decrease in GAD67 mRNA and protein levels induced by MAM in the medial prefrontal cortex (mPFC). Moreover, EE inhibited the reductions in the amount of Gad1 bound to H3K4me3 and in the total H3K4me3 protein level induced by prenatal MAM administration in the adult mPFC. However, there was no effect of EE on behaviour or levels of the various neurochemical markers in adult rats prenatally treated with vehicle. Thus, these results indicate that EE exposure during early adolescence may inhibit the development of schizophrenia related symptoms through epigenetic mechanisms that regulate the expression of genes (e.g., Gad1) that are impaired in schizophrenia.

Age-dependent decrease of GAD65/67 mRNAs but normal densities of GABAergic interneurons in the brain regions of Shank3-overexpressing manic mouse model

Dysfunction of inhibitory GABAergic interneurons is considered a major pathophysiological feature of various neurodevelopmental and neuropsychiatric disorders. The variants of SHANK3 gene, encoding a core scaffold protein of the excitatory postsynapse, have been associated with numerous brain disorders. It has been suggested that abnormalities of GABAergic interneurons could contribute to the SHANK3-related disorders, but the limitation of these studies is that they used mainly Shank3 knock-out mice. Notably, Shank3-overexpressing transgenic mice, modeling human hyperkinetic disorders, also show reduced inhibitory synaptic transmission, abnormal electroencephalography, and spontaneous seizures. However, it has not been investigated whether these phenotypes of Shank3 transgenic mice are associated with GABAergic interneuron dysfunction, or solely due to the cell-autonomous postsynaptic changes of principal neurons. To address this issue, we investigated the densities of parvalbumin- and somatostatin-positive interneurons, and the mRNA and protein levels of GAD65/67 GABA-synthesizing enzymes in the medial prefrontal cortex, striatum, and hippocampus of adult Shank3 transgenic mice. We found no significant difference in the measurements performed on wild-type versus Shank3 transgenic mice, except for the decreased GAD65 or GAD67 mRNAs in these brain regions. Interestingly, only GAD65 mRNA was decreased in the hippocampus, but not mPFC and striatum, of juvenile Shank3 transgenic mice which, unlike the adult mice, did not show behavioral hyperactivity. Together, our results suggest age-dependent decrease of GAD65/67 mRNAs but normal densities of certain GABAergic interneurons in the Shank3 transgenic mice.