GAD67 Expression Research Articles

Fluoxetine exerts subregion/layer specific effects on parvalbumin/GAD67 protein expression in the dorsal hippocampus of male rats showing social isolation-induced depressive-like behaviour

The molecular background of depression is intensively studied in terms of alterations of inhibitory circuits, mediated by gamma aminobutyric acid (GABA) signalization. We investigated the effects of chronic social isolation (CSIS) and chronic fluoxetine (Flx) treatment (15 mg/kg/day) (3 weeks), on Parvalbumin (PV) and GAD67 expression in a layer-specific manner in rat dorsal hippocampal subregions. CSIS-induced depressive- and anxiety-like behaviours were confirmed with decrease in sucrose preference and increase in marble burying during behavioural testing, while Flx antagonized these effects. CSIS altered PV expression in stratum pyramidale (SP) of dorsal cornu ammonis 1 (dCA1) and stratum radiatum (SR) of dCA3. Flx antagonized this effect, and boosted PV expression in SP of the entire dCA and the dorsal dentate gyrus (dDG), as well as in the SR of dCA1/CA3. CSIS showed no significant effects on GAD67 expression, while Flx boosted its expression within the SR of the entire CA and SO of the dCA3. A correlation between SP of dCA1 and SR of dCA3 with regard to PV changes, implicates their possible role in the inhibitory circuit alterations. Flx-induced increase in GAD67 expression, specifically in SR of the entire dHIPP, may impose its involvement in the cell metabolic processes. Strong negative correlation between GAD67 and sucrose preference following Flx-treatment of CSIS rats was revealed. PV + cells of the SP layer of dCA1 and CA2 could be a potential target for the antidepressant action of Flx, while strong effect of Flx on GAD67 expression in the SR should be more extensively studied.

Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain.

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of ‘patches’ of higher expression, interspersed within a less immunoreactive ‘matrix’, which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

GAD1 expression and its methylation as indicators of malignant behavior in thymic epithelial tumors.

Thymic epithelial tumors (TETs) comprise thymomas and thymic carcinoma (TC). TC has more aggressive features and a poorer prognosis than thymomas. Genetic and epigenetic alterations in thymomas and TC have been investigated in an attempt to identify novel target molecules for TC. In the present study, genome-wide screening was performed on aberrantly methylated CpG islands in thymomas and TC, and the glutamate decarboxylase 1 gene (GAD1) was identified as the 4th significantly hypermethylated CpG island in TC compared with thymomas. GAD1 catalyzes the production of γ-aminobutyric acid from L-glutamic acid. GAD1 expression is abundant in the brain but rare in other tissues, including the thymus. A total of 73 thymomas and 17 TC tissues were obtained from 90 patients who underwent surgery or biopsy at Tokushima University Hospital between 1990 and 2017. DNA methylation was examined by bisulfite pyrosequencing, and the mRNA and protein expression levels of GAD1 were analyzed using reverse transcription-quantitative PCR and immunohistochemistry, respectively. The DNA methylation levels of GAD1 were significantly higher in TC tissues than in the normal thymus and thymoma tissues, and GAD1 methylation exhibited high sensitivity and specificity for discriminating between TC and thymoma. The mRNA and protein expression levels of GAD1 were significantly higher in TC tissues than in thymomas. Patients with TET with high GAD1 DNA hypermethylation and high mRNA and protein expression levels had significantly shorter relapse-free survival rates than those with low levels. In conclusion, significantly more epigenetic alterations were observed in TC tissues compared with in thymomas, which may contribute to the clinical features and prognosis of patients.

Elevated expression of complement C4 in the mouse prefrontal cortex causes schizophrenia-associated phenotypes.

Accumulating evidence supports immune involvement in the pathogenesis of schizophrenia, a severe psychiatric disorder. In particular, high expression variants of C4, a gene of the innate immune complement system, were shown to confer susceptibility to schizophrenia. However, how elevated C4 expression may impact brain circuits remains largely unknown. We used in utero electroporation to overexpress C4 in the mouse prefrontal cortex. We found reduced glutamatergic input to pyramidal cells of juvenile and adult, but not of newborn C4-overexpressing (C4-OE) mice, together with decreased spine density, which mirrors spine loss observed in the schizophrenic cortex. Using time-lapse two-photon imaging in vivo, we observed that these deficits were associated with decreased dendritic spine gain and elimination in juvenile C4-OE mice, which may reflect poor formation and/or stabilization of immature spines. In juvenile and adult C4-OE mice, we found evidence for NMDA receptor hypofunction, another schizophrenia-associated phenotype, and synaptic accumulation of calcium-permeable AMPA receptors. Alterations in cortical GABAergic networks have been repeatedly associated with schizophrenia. We found that functional GABAergic transmission was reduced in C4-OE mice, in line with diminished GABA release probability from parvalbumin interneurons, lower GAD67 expression, and decreased intrinsic excitability in parvalbumin interneurons. These cellular abnormalities were associated with working memory impairment. Our results substantiate the causal relationship between an immunogenetic risk factor and several distinct cortical endophenotypes of schizophrenia and shed light on the underlying cellular mechanisms.

Behavioral Consequences of a Combination of Gad1 Haplodeficiency and Adolescent Exposure to an NMDA Receptor Antagonist in Long-Evans Rats.

Glutamate decarboxylase 67-kDa isoform (GAD67), which is encoded by the GAD1 gene, is one of the key enzymes that produce GABA. The reduced expression of GAD67 has been linked to the pathophysiology of schizophrenia. Additionally, the excitatory glutamatergic system plays an important role in the development of this disorder. Animal model studies have revealed that chronic blockade of NMDA-type glutamate receptors can cause GABAergic dysfunction and long-lasting behavioral abnormalities. Based on these findings, we speculated that Gad1 haplodeficiency combined with chronic NMDA receptor blockade would lead to larger behavioral consequences relevant to schizophrenia in a rat model. In this study, we administered an NMDAR antagonist, MK-801 (0.2 mg/kg), to CRISPR/Cas9-generated Gad1 +/− rats during adolescence to test this hypothesis. The MK-801 treated Gad1 +/− rats showed a shorter duration in each rearing episode in the open field test than the saline-treated Gad1 +/+ rats. In contrast, immobility in the forced swim test was increased and fear extinction was impaired in Gad1 +/− rats irrespective of MK-801 treatment. Interestingly, the time spent in the center region of the elevated plus-maze was significantly affected only in the saline-treated Gad1 +/− rats. Additionally, the MK-801-induced impairment of the social novelty preference was not observed in Gad1 +/− rats. These results suggest that the synergistic and additive effects of Gad1 haplodeficiency and NMDA receptor blockade during adolescence on the pathogenesis of schizophrenia may be more limited than expected. Findings from this study also imply that these two factors mainly affect negative or affective symptoms, rather than positive symptoms.

Interaction of maternal immune activation and genetic interneuronal inhibition

Genes and environment interact during intrauterine life, and potentially alter the developmental trajectory of the brain. This can result in life-long consequences on brain function. We have previously developed two transgenic mouse lines that suppress Gad1 expression in parvalbumin (PVALB) and neuropeptide Y (NPY) expressing interneuron populations using a bacterial artificial chromosome (BAC)-driven miRNA-based silencing technology. We were interested to assess if maternal immune activation (MIA), genetic interneuronal inhibition, and the combination of these two factors disrupt and result in long-term changes in neuroinflammatory gene expression, sterol biosynthesis, and acylcarnitine levels in the brain of maternally exposed offspring. Pregnant female WT mice were given a single intraperitoneal injection of saline or polyinosinic–polycytidilic acid [poly(I:C)] at E12.5. Brains of offspring were analyzed at postnatal day 90. We identified complex and persistent neuroinflammatory gene expression changes in the hippocampi of MIA-exposed offspring, as well in the hippocampi of Npy/Gad1 and Pvalb/Gad1 mice. In addition, both MIA and genetic inhibition altered the post-lanosterol sterol biosynthesis in the neocortex and disrupted the typical acylcarnitine profile. In conclusion, our findings suggest that both MIA and inhibition of interneuronal function have long-term consequences on critical homeostatic mechanisms of the brain, including immune function, sterol levels, and energy metabolism.

Mouse Mandibular Retromolar Taste Buds Associated With a Mucus Salivary Gland.

We have characterized a recently rediscovered chemosensory structure at the rear of the mandibular mucosa in the mouse oral cavity originally reported in the 1980s. This consists of unorganized taste buds, not contained within troughs, associated with the ducts of an underlying minor salivary gland. Using whole-mount preparations of transgenic mice expressing green fluorescent protein under the promoter of taste-signaling-specific genes, we determined that the structure contains taste bud clusters and salivary gland orifices at the rear of each mandible, distal to the last molar and anterior to the ascending ramus. Immunohistochemical analysis shows in the retromolar taste buds expression of the taste receptors Tas2R131 and T1R3 and taste cascade molecules TrpM5, PLCβ2, and GNAT3, consistent with type II taste cells, and expression of GAD1, consistent with type III taste cells. Furthermore, the neuronal marker, calcitonin gene-related peptide, in retromolar mucosa tissue wrapping around TrpM5+ taste buds was observed. RT-PCR showed that retromolar taste buds express all 3 mouse tas1r genes, 28 of the 35 tas2r genes, and taste transduction signaling genes gnat3, plcb2, and trpm5, making the retromolar taste buds similar to other lingual and palate taste buds. Finally, histochemistry demonstrated that the mandibular retromolar secretory gland is a minor salivary gland of mucous type. The mandibular retromolar taste structure may thus play a role in taste sensation and represent a potential novel pharmacological target for taste disorders.

GAD65Cre Drives Reporter Expression in Multiple Taste Cell Types.

In taste buds, Type I cells represent the majority of cells (50-60%) and primarily have a glial-like function in taste buds. However, recent studies suggest that they have additional sensory and signaling functions including amiloride-sensitive salt transduction, oxytocin modulation of taste, and substance P mediated GABA release. Nonetheless, the overall function of Type I cells in transduction and signaling remains unclear, primarily because of the lack of a reliable reporter for this cell type. GAD65 expression is specific to Type I taste cells and GAD65 has been used as a Cre driver to study Type I cells in salt taste transduction. To test the specificity of transgene-driven expression, we crossed GAD65Cre mice with floxed tdTomato and Channelrhodopsin (ChR2) lines and examined the progeny with immunochemistry, chorda tympani recording, and calcium imaging. We report that while many tdTomato+ taste cells express NTPDase2, a specific marker of Type I cells, we see some expression of tdTomato in both Gustducin and SNAP25-positive taste cells. We also see ChR2 in cells just outside the fungiform taste buds. Chorda tympani recordings in the GAD65Cre/ChR2 mice show large responses to blue light. Furthermore, several isolated tdTomato-positive taste cells responded to KCl depolarization with increases in intracellular calcium, indicating the presence of voltage-gated calcium channels. Taken together, these data suggest that GAD65Cre mice drive expression in multiple taste cell types and thus cannot be considered a reliable reporter of Type I cell function.

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.

Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

Early life stress compromises brain development and can contribute to the development of mental illnesses. A common animal model used to study different facets of psychiatric disorders is social isolation from early life on. In rats, this isolation can induce long-lasting alterations in molecular expression and in behavior. Since social isolation models severe psychiatric symptoms, it is to be expected that it affects the overall wellbeing of the animals. As also promoted by the 3Rs principle, though, it is pivotal to decrease the burden of laboratory animals by limiting the number of subjects (reduce, replace) and by improving the animals’ wellbeing (refine). The aim of this study was therefore to test possible refinement strategies such as resocialization and mere adult social isolation. We examined whether the alternatives still triggered the necessary phenotype while minimizing the stress load on the animals. Interestingly, we did not find reduced wellbeing-associated burrowing performance in isolated rats. The hyperactive phenotype seen in socially isolated animals was observed for rats undergoing the adult-only isolation, but resocializing ameliorated the locomotor abnormality. Isolation strongly affected markers of neuroplasticity in the prefrontal cortex independent of timing: mRNA levels of Arc, Bdnf and the pool of Bdnf transcripts with the 3’ long UTR were reduced in all groups. Bdnf splice variant IV expression was reduced in lifelong-isolated animals. Some of these deficits normalized after resocialization; likewise, exon VI Bdnf mRNA levels were reduced only in animals persistently isolated. Conversely, social deprivation did not affect the expression of Gad67 and Pvb, two GABAergic markers, whereas changes occurred in the expression of dopamine d1 and d2 receptors. As adult isolation was sufficient to trigger the hyperactive phenotype and impaired neuroplasticity in the prefrontal cortex, it could be a candidate for a refinement strategy for certain research questions. To fully grade the severity of post-weaning social isolation and the alternatives, adult isolation and resocialization, a more profound and multimodal assessment approach is necessary.

α6GABAA Receptor Positive Modulators Alleviate Migraine-like Grimaces in Mice via Compensating GABAergic Deficits in Trigeminal Ganglia.

Migraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) play an important role. This hyperactivity might originate from an underfunctional GABAergic system in TG. To investigate this possibility, we adapted a mouse model of migraine by inducing migraine-like grimaces in male mice via repeated injections of nitroglycerin (NTG, 10mg/kg, i.p.), once every 2days, for up to 5 sessions. Migraine-like facial pain scores were measured using the mouse grimace scale. Repeated NTG treatments in mice caused significant increases in migraine-like grimaces that were aborted and prevented by two anti-migraine agents sumatriptan and topiramate, respectively. After 5 sessions of NTG injections, the GABA-synthesizing enzyme, 65-kDa glutamate decarboxylase (GAD65), but not the GABA transporter 1 (GAT1) or the α6 subunit-containing GABAA receptors (α6GABAARs), was downregulated in mouse TG tissues. Taking advantage of the unaffected TG α6GABAAR expression in NTG-treated mice, we demonstrated that an α6GABAAR-selective positive allosteric modulator (PAM), DK-I-56-1, exhibited both abortive and prophylactic effects, comparable to those of sumatriptan and topiramate, respectively, in this migraine-mimicking mouse model. The brain-impermeable furosemide significantly prevented the effects of DK-I-56-1, suggesting its peripheral site of action, likely via preventing α6GABAAR modulation in TG. Results suggest that a decreased GABA synthesis caused by the reduced GAD65 expression in TG contributes to the trigeminovascular activation in this repeated NTG-induced migraine-mimicking model and that the unaltered α6GABAARs in TG are potential targets for migraine treatment. Thus, α6GABAAR-selective PAMs are potential anti-migraine agents for both abortive and preventive therapies.

The Wnt/Ca2+ signaling pathway is essential for the regeneration of GABAergic neurons in planarian Dugesia japonica.

The growth and differentiation of neurons are critical events in the establishment of proper neuron connectivity and function. Planarians have a remarkable ability to completely regenerate a functional nervous system from a pluripotent stem cell population. Thus, planarians provide a powerful model to identify genes required for neuronal differentiation in vivo. The Wnt/Ca2+ signaling pathway is crucial for cancer development, arousing inflammatory responses, and neurodegeneration. We analyzed the expression patterns and RNAi phenotypes for members of the Wnt/Ca2+ signaling pathway in the planarian, Dugesia japonica. The expression of DjWnt5a, DjPLC-β, DjCamKII, and DjCaln during regeneration was surprisingly similar and revealing in the regenerated brain. RNAi knockdown of DjWnt5a, DjPLC-β, DjCamKII, and DjCaln led to defects in regenerated brains including brain partial deletions, incompact phenotypes at the posterior of the new brain, and lateral branches, which could not regenerate. Furthermore, the expressions of GAD and the number of GABAergic neurons decreased. Together, these results suggest that the Wnt/Ca2+ signaling pathway is required for GABAergic neuron regeneration.

α7 nicotinic receptor agonist and positive allosteric modulators differently improved schizophrenia-like cognitive deficits in male rats

The majority of schizophrenia patients have cognitive deficits as a separate symptom cluster independent of positive or negative symptoms. Current medicines, unfortunately, cannot provide clear benefits for cognitive symptoms in patients. Recent findings showed decreased α7 nicotinic acetylcholine receptor (nAChR) expressions in subjects with schizophrenia. α7 nAChR full/partial agonists and positive allosteric modulators (PAMs) may be valuable drug candidates to treat cognitive deficits of disease. This study comparatively investigated the effect of α7 nAChR agonist (A-582941), type I PAM (CCMI), type II PAM (PNU-120596), and the antipsychotic drug (clozapine) on behavioral, molecular, and immunohistochemical parameters in a subchronic MK-801 model of schizophrenia in male rats. Novel object recognition (NOR) and Morris water maze (MWM) tests were performed to evaluate recognition and spatial memories, respectively. Gene and protein expressions of parvalbumin, glutamic acid decarboxylase-67 (GAD67), and α7 nAChR were examined in the rats' hippocampal tissue. The subchronic MK-801 administration produced cognitive deficits in the NOR and MWM tests. It also decreased the protein and gene expressions of parvalbumin, GAD67, and α7 nAChR in the hippocampus. Clozapine, A-582941, and PNU-120596 but not CCMI increased the parvalbumin and α7 nAChR expressions and provided benefits in recognition memory. Interestingly, clozapine and CCMI restored the MK-801 induced deficits on GAD1 expression and spatial memory while A-582941 and PNU-120596 were ineffective. These results indicated that α7 nAChR agonist, type I and type II PAMs may provide benefits in different types of cognitive deficits rather than a complete treatment in schizophrenia.

Prenatal sevoflurane exposure causes neuronal excitatory/inhibitory imbalance in the prefrontal cortex and neurofunctional abnormality in rats

The balance of excitatory and inhibitory neurons in the central nervous system is critical for maintaining brain function and sevoflurane, a general anesthetic and an GABA receptor modulator, may change the balance of excitatory and inhibitory neurons in the cortex during early brain development. Herein, we investigated whether prenatal sevoflurane exposure (PSE) disturbs cortical neuronal development and brain function. Pregnant rats at the gestational day 14.5 were subjected to sevoflurane exposure at 3.0% for 3 h and their offspring were studied thereafter. We found a significant increase of parvalbumin-positive neurons, vesicular GABA transporter (VGAT) and GAD67 expression, and GABA neurotransmitter, and a significant decrease of vesicular glutamate transporter 1 (VGLUT1) expression and glutamate in the medial prefrontal cortex (mPFC) of offspring. Pyramidal neurons showed atrophy with shorter dendrites, less branches and lower spine density visualized by Golgi stain and a decrease of excitability with the increased miniature inhibitory postsynaptic current (mIPSC) frequency and amplitude, the decreased miniature excitatory postsynaptic current (mEPSC) frequency and excitation/inhibition (E/I) ratio using whole-cell recording in offspring. There was a significant increase of inhibitory synapse in the mPFC detected by electron microscopy. Furthermore, PSE animals showed hypo-excitatory phenotype including depression-like behaviors and learning deficits. Thus, our studies provide novel evidence that PSE causes the persisted imbalance of excitatory and inhibitory neurons in the mPFC, and this is very likely the mechanisms of the sevoflurane-induced brain functional abnormalities.

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.

Essential and sex-specific effects of mGluR5 in ventromedial hypothalamus regulating estrogen signaling and glucose balance

The ventromedial hypothalamus (VMH) plays chief roles regulating energy and glucose homeostasis and is sexually dimorphic. We discovered that expression of metabotropic glutamate receptor subtype 5 (mGluR5) in the VMH is regulated by caloric status in normal mice and reduced in brain-derived neurotrophic factor (BDNF) mutants, which are severely obese and have diminished glucose balance control. These findings led us to investigate whether mGluR5 might act downstream of BDNF to critically regulate VMH neuronal activity and metabolic function. We found that mGluR5 depletion in VMH SF1 neurons did not affect energy balance regulation. However, it significantly impaired insulin sensitivity, glycemic control, lipid metabolism, and sympathetic output in females but not in males. These sex-specific deficits are linked to reductions in intrinsic excitability and firing rate of SF1 neurons. Abnormal excitatory and inhibitory synapse assembly and elevated expression of the GABAergic synthetic enzyme GAD67 also cooperate to decrease and potentiate the synaptic excitatory and inhibitory tone onto mutant SF1 neurons, respectively. Notably, these alterations arise from disrupted functional interactions of mGluR5 with estrogen receptors that switch the normally positive effects of estrogen on SF1 neuronal activity and glucose balance control to paradoxical and detrimental. The collective data inform an essential central mechanism regulating metabolic function in females and underlying the protective effects of estrogen against metabolic disease.

Short-Term Effect of MgSO4 on the Expression of NRG-ErbB, Dopamine, GABA, and Glutamate Systems in the Fetal Rat Brain.

MgSO4 has been used for the past two decades as neuroprotective treatment in a variety of preterm conditions. Despite the putative advantages of MgSO4 as a neuroprotective agent in the preterm brain, the short- and long-term molecular function of MgSO4 as a neuroprotective agent has not been fully elucidated. Neuregulin (NRG1)-ErbB4 signaling plays a critical role in embryonic brain development, in the biology of dopaminergic, GABAergic, and glutamatergic systems. We hypothesize that this pathway may be associated with the neuroprotective role of MgSO4. The current study aims to investigate the ability of MgSO4 to modulate the normal developing expression pattern of selected genes related to the NRG1-ErbB, dopaminergic, GABAergic, and glutamatergic systems. We demonstrate that overall short-term treatment of dam rats with MgSO4 affects the expression of fetal brain NRG1, NRG3, ErbB4, GAD67, tyrosine hydroxylase (TH), dopamine D2 and D1 receptors, GluN1, and GluN2B. More specifically, the administration of MgSO4 alters the expression of NRG-ErbB, GAD67, TH, and D2R at early gestation day 16 (GD16) regardless of the activation of the maternal immune system by lipopolysaccharide (LPS). Our data suggest that MgSO4 treatment may affect the expression of major neuronal systems and pathways mostly at an early gestation day. These changes might be an initial clue (foundation stone) in the molecular mechanism that underlies the beneficial effect of MgSO4 as a neuroprotective agent for the developmental brain.

Citalopram-induced pathways regulation and tentative treatment-outcome-predicting biomarkers in lymphoblastoid cell lines from depression patients

Antidepressant therapy is still associated with delays in symptomatic improvement and low response rates. Incomplete understanding of molecular mechanisms underlying antidepressant effects hampered the identification of objective biomarkers for antidepressant response. In this work, we studied transcriptome-wide expression followed by pathway analysis in lymphoblastoid cell lines (LCLs) derived from 17 patients documented for response to SSRI antidepressants from the Munich Antidepressant Response Signatures (MARS) study upon short-term incubation (24 and 48 h) with citalopram. Candidate transcripts were further validated with qPCR in MARS LCLs from responders (n = 33) vs. non-responders (n = 36) and afterward in an independent cohort of treatment-resistant patients (n = 20) vs. first-line responders (n = 24) from the STAR*D study. In MARS cohort we observed significant associations of GAD1 (glutamate decarboxylase 1; p = 0.045), TBC1D9 (TBC1 Domain Family Member 9; p = 0.014–0.021) and NFIB (nuclear factor I B; p = 0.015–0.025) expression with response status, remission status and improvement in depression scale, respectively. Pathway analysis of citalopram-altered gene expression indicated response-status-dependent transcriptional reactions. Whereas in clinical responders neural function pathways were primarily up- or downregulated after incubation with citalopram, deregulated pathways in non-responders LCLs mainly involved cell adhesion and immune response. Results from the STAR*D study showed a marginal association of treatment-resistant depression with NFIB (p = 0.068) but not with GAD1 (p = 0.23) and TBC1D9 (p = 0.27). Our results propose the existence of distinct pathway regulation mechanisms in responders vs. non-responders and suggest GAD1, TBC1D9, and NFIB as tentative predictors for clinical response, full remission, and improvement in depression scale, respectively, with only a weak overlap in predictors of different therapy outcome phenotypes.

Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae.

Gamma‐aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen‐responsive mitogen‐activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst‐avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst‐avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst‐avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst‐avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.

Anodal and cathodal tDCS modulate neural activity and selectively affect GABA and glutamate syntheses in the visual cortex of cats.

The present study showed that anodal and cathodal transcranial direct current stimulation (tDCS) can respectively increase and decrease the amplitude of visually evoked field potentials in the stimulated visual cortex of cats, with the effect lasting for ∼60-70min. We directly measured tDCS-induced changes in the concentration of inhibitory and excitatory neurotransmitters in the visual cortex using the enzyme-linked immunosorbent assay method and showed that anodal and cathodal tDCS can selectively decrease the concentration of GABA and glutamate in the stimulated cortical area. Anodal and cathodal tDCS can selectively inhibit the synthesis of GABA and glutamate by suppressing the expression of GABA- and glutamate-synthesizing enzymes, respectively. Transcranial direct current stimulation (tDCS) evokes long-lasting neuronal excitability in the target brain region. The underlying neural mechanisms remain poorly understood. The present study examined tDCS-induced alterations in neuronal activities, as well as the concentration and synthesis of GABA and glutamate (GLU), in area 21a (A21a) of cat visual cortex. Our analysis showed that anodal and cathodal tDCS respectively enhanced and suppressed neuronal activities in A21a, as indicated by a significantly increased and decreased amplitude of visually evoked field potentials (VEPs). The tDCS-induced effect lasted for ∼60-70min. By contrast, sham tDCS had no significant impact on the VEPs in A21a. On the other hand, the concentration of GABA, but not that of GLU, in A21a significantly decreased after anodal tDCS relative to sham tDCS, whereas the concentration of GLU, but not that of GABA, in A21a significantly decreased after cathodal tDCS relative to sham tDCS. Furthermore, the expression of GABA-synthesizing enzymes GAD65 and GAD67 in A21a significantly decreased in terms of both mRNA and protein concentrations after anodal tDCS relative to sham tDCS, whereas that of GLU-synthesizing enzyme glutaminase (GLS) did not change significantly after anodal tDCS. By contrast, both mRNA and protein concentrations of GLS in A21a significantly decreased after cathodal tDCS relative to sham tDCS, whereas those of GAD65/GAD67showed no significant change after cathodal tDCS. Taken together, these results indicate that anodal and cathodal tDCS may selectively reduce GABA and GLU syntheses and thus respectively enhance and suppress neuronal excitability in the stimulated brain area.