GABA Transporter Research Articles

Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma-aminobutyric acid.

Corynebacterium glutamicum has been used as a sustainable microbial producer for various bioproducts using cheap biomass resources. In this study, a high GABA-producing C. glutamicum strain was constructed by chromosomal editing. Lactobacillus brevis-derived gadB2 was introduced into the chromosome of C. glutamicum ATCC 13032 to produce gamma-aminobutyric acid and simultaneously blocked the biosynthesis of lactate and acetate. GABA transport and degradation in C. glutamicum were also blocked to improve GABA production. As precursor of GABA, l-glutamic acid synthesis in C. glutamicum was enhanced by introducing E. coli gdhA encoding glutamic dehydrogenase, and the copy numbers of gdhA and gadB2 were also optimized for higher GABA production. The final C. glutamicum strain CGY705 could produce 33.17 g/L GABA from glucose in a 2.4-L bioreactor after 78 h fed-batch fermentation. Since all deletion and expression of genes were performed using chromosomal editing, fermentation of the GABA-producing strains constructed in this study does not need supplementation of any antibiotics and inducers. The strategy used in this study has potential value in the development of GABA-producing bacteria.

Postpartum State, but Not Maternal Caregiving or Level of Anxiety, Increases Medial Prefrontal Cortex GAD65 and vGAT in Female Rats.

Upregulation of the inhibitory neurotransmitter, GABA, is involved in many of the behavioral differences between postpartum and nulliparous female rodents. This is evidenced by studies showing that pharmacological blockade of GABAergic activity impairs maternal caregiving and postpartum affective behaviors. However, the influence of motherhood on the capacity for GABA synthesis or release in the medial prefrontal cortex (mPFC; brain region involved in many social and affective behaviors) is not well-understood. Western blotting was used to compare postpartum and nulliparous rats in protein levels of the 65-kD isoform of glutamic acid decarboxylase (GAD65; synthesizes most GABA released from terminals) and vesicular GABA transporter (vGAT; accumulates GABA into synaptic vesicles for release) in the mPFC. We found that postpartum mothers had higher GAD65 and vGAT compared to virgins, but such differences were not found between maternally sensitized and non-sensitized virgins, indicating that reproduction rather than just the display of maternal caregiving is required. To test whether GAD65 and vGAT levels in the mPFC were more specifically related to anxiety-related behavior within postpartum mothers, we selected 8 low-anxiety and 8 high-anxiety dams based on their time spent in the open arms of an elevated plus maze on postpartum day 7. There were no significant differences between the anxiety groups in either GAD65 or vGAT levels. These data further indicate that frontal cortical GABA is affected by female reproduction and more likely contributes to differences in the display of socioemotional behaviors across, but not within, female reproductive state.

Alzheimer's Disease Enhanced Tonic Inhibition is Correlated With Upregulated Astrocyte GABA Transporter-3/4 in a Knock-In APP Mouse Model.

Cognitive decline is a major symptom in Alzheimer’s disease (AD), which is strongly associated with synaptic excitatory-inhibitory imbalance. Here, we investigated whether astrocyte-specific GABA transporter 3/4 (GAT3/4) is altered in APP knock-in mouse model of AD and whether this is correlated with changes in principal cell excitability. Using the APP NL-F/NL-F knock-in mouse model of AD, aged-matched to wild-type mice, we performed in vitro electrophysiological whole-cell recordings combined with immunohistochemistry in the CA1 and dentate gyrus (DG) regions of the hippocampus. We observed a higher expression of GAD67, an enzyme that catalyses GABA production, and GAT3/4 in reactive astrocytes labelled with GFAP, which correlated with an enhanced tonic inhibition in the CA1 and DG of 12–16 month-old APP NL-F/NL-F mice compared to the age-matched wild-type animals. Comparative neuroanatomy experiments performed using post-mortem brain tissue from human AD patients, age-matched to healthy controls, mirrored the results obtained using mice tissue. Blocking GAT3/4 associated tonic inhibition recorded in CA1 and DG principal cells resulted in an increased membrane input resistance, enhanced firing frequency and synaptic excitation in both wild-type and APP NL-F/NL-F mice. These effects exacerbated synaptic hyperactivity reported previously in the APP NL-F/NL-F mice model. Our data suggest that an alteration in astrocyte GABA homeostasis is correlated with increased tonic inhibition in the hippocampus, which probably plays an important compensatory role in restoring AD-associated synaptic hyperactivity. Therefore, reducing tonic inhibition through GAT3/4 may not be a good therapeutic strategy for AD

Paracrine Interleukin 6 Induces Cerebral Remodeling at Early Stages After Unilateral Common Carotid Artery Occlusion in Mice.

AimsCarotid artery disease is frequent and can result in chronic modest hypoperfusion of the brain. If no transient ischemic attack or stroke occur, it is classified asymptomatic. In the long-term, though, it can lead to cognitive impairment. Fostering cerebral remodeling after carotid artery occlusion might be a new concept of treatment. Paracrine Interleukin 6 (IL-6) can induce such remodeling processes at early stages. However, it has neurodegenerative long-term effects. With this exploratory study, we investigated the effect of paracrine IL-6 on cerebral remodeling in early stages after asymptomatic carotid artery occlusion to identify new treatment targets.Methods and ResultsTo mimic a human asymptomatic carotid artery disease, we used a mouse model of unilateral common carotid artery (CCA) occlusion. We developed a mouse model for inducible paracrine cerebral IL-6 expression (Cx30-Cre-ERT2;FLEX-IL6) and induced IL-6 2 days after CCA occlusion. We studied the effects of paracrine IL-6 after CCA occlusion on neuronal connectivity using diffusion tensor imaging and on local proteome regulations of the hypo-perfused striatum and contralateral motor cortex using mass spectrometry of laser capture micro-dissected tissues. Paracrine IL-6 induced cerebral remodeling leading to increased inter-hemispheric connectivity and changes in motor system connectivity. We identified changes in local protein abundance which might have adverse effects on functional outcome such as upregulation of Synuclein gamma (Sncg) or downregulation of Proline Dehydrogenase 1 (Prodh). However, we also identified changes in local protein abundance having potentially beneficial effects such as upregulation of Caprin1 or downregulation of GABA transporter 1 (Gat1).ConclusionsParacrine cerebral IL-6 at early stages induces changes in motor system connectivity and the proteome after asymptomatic CCA occlusion. Our results may help to distinguish unfavorable from beneficial IL-6 dependent protein regulations. Focusing on these targets might generate new treatments to improve long-term outcome in patients with carotid artery disease.

Neural Differentiation of Human Dental Mesenchymal Stem Cells Induced by ATRA and UDP-4: A Comparative Study.

Human mesenchymal stem cells (MSC) are multipotent stem cells, which are isolated from various sources. Currently, there is a worldwide interest for dental MSC to be used against neurodegenerative diseases, since they derive from the neural crest and express embryonic stem cell markers. This fact prompted us to explore their potential for neural trans-differentiation in culture. We employed all-trans-retinoic acid (ATRA) and 2-(3-ethylureido)-6-methylpyridine (UDP-4) to induce neural differentiation of human MSC from the dental apical papilla (SCAP). The SCAP were exposed to either agent separately and assessed for proliferation, viability, morphology, and gene expression of the following neural-specific markers: neuron-specific enolase (ENO2), neurofibromin 1 (NF1), choline acetyltransferase (CHAT), tyrosine hydroxylase (TH), and the vesicular GABA transporter (SLC32A1). They were also assessed for the expression of glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) by immunofluorescence. ATRA or UDP-4 treatment inhibited the cell growth and promoted limited cell death, but to a different extent. The addition of the neuroprotective agent recombinant human erythropoietin-alpha (rhEPO-α) enhanced the UDP-4-inducing capacity for more than three weeks. ATRA or UDP-4 treatment significantly upregulated ENO2 and NF1 expression, indicating neuronal differentiation. Moreover, the ATRA treatment significantly induced the upregulation of the GABAergic-specific SLC32A1, while the UDP-4 treatment led to the significant upregulation of the adrenergic-specific TH. The UDP-4 treatment induced the expression of NeuN and GFAP after four and three weeks, respectively, while the ATRA-treatment did not. Our findings indicate that SCAP can be differentiated into neural-like cells after treatment with ATRA or UDP-4 by exhibiting a disparate pattern of differentiation. Therefore, UDP-4 is suggested here as a new potent neural-differentiation-inducing compound, which, when combined with rhEPO-α, could lay the foundation for robust stem-cell-based therapies of neurodegeneration.

Gamma-Aminobutyric Acid Transporters in the Nucleus Tractus Solitarii Regulate Inhibitory and Excitatory Synaptic Currents That Influence Cardiorespiratory Function.

The brainstem nucleus tractus solitarii (nTS) processes and modulates the afferent arc of critical peripheral cardiorespiratory reflexes. Sensory afferents release glutamate to initiate the central component of these reflexes, and glutamate concentration is critically controlled by its removal via astrocytic neurotransmitter transporters. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the nTS providing tonic and phasic modulation of neuronal activity. GABA is removed from the extracellular space through GABA transporters (GATs), however, the role of GATs in nTS synaptic transmission and their influence on cardiorespiratory function is unknown. We hypothesized that GATs tonically restrain nTS inhibitory signaling and given the considerable nTS GABA-glutamate cross-talk, modify excitatory signaling and thus cardiorespiratory function. Reverse transcription real-time polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry showed expression of GAT-1 and GAT-3 mRNA and protein within the rat nTS, with GAT-3 greater than GAT-1, and GAT-3 colocalizing with astrocyte S100B. Recordings in rat nTS slices demonstrated GAT-3 block decreased spontaneous inhibitory postsynaptic current (IPSC) frequency and reduced IPSC amplitude evoked from electrical stimulation of the medial nTS. Block of GAT-3 also increased spontaneous excitatory postsynaptic current (EPSC) frequency yet did not alter sensory afferent-evoked EPSC amplitude. Block of GAT-3 in the nTS of anesthetized rats increased mean arterial pressure, heart rate, sympathetic nerve activity, and minute phrenic nerve activity. These results demonstrate inhibitory and excitatory neurotransmission in the nTS is significantly modulated by endogenous GAT-3 to influence basal cardiorespiratory function.

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.

Evaluation of suprachiasmatic nucleus in Alzheimer’s disease with non-invasive magnetic resonance methods

Evaluation of suprachiasmatic nucleus in Alzheimer’s disease with non-invasive magnetic resonance methods

Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina.

The amacrine neurons in the mammalian retina comprise a large variety of cell types with distinct properties and functions that serve to integrate and modulate signals presented to output neurons. The majority of them use either glycine or GABA as inhibitory neurotransmitters and express the glycine transporter 1 (GlyT1) or glutamic acid decarboxylase (GAD67) and GABA transporters (GAT1 and GAT3), as a glycinergic or GABAergic marker respectively. We report here a novel subpopulation of amacrine neurons expressing both, GABAergic and glycinergic markers, in retinas from wild-type C57BL/6J mice and two transgenic lines. In retinal sections from the transgenic line expressing eGFP under the control of the glycine transporter 2, eGFP expression was exclusively found in cell bodies and dendrites of inhibitory amacrine neurons, identified for their immunoreactivity to syntaxin 1A. All of the glycinergic and a large portion of the GABAergic amacrine neurons contained eGFP; of these, 8-10% of GlyT1 positive neurons were also labeled either with GAD67, GAT1 or GAT3. These findings were confirmed in retinas from a wild-type and a mouse line expressing eGFP under the GAD67 promoter and two different anti-GlyT1 antibodies, showing the presence of a subpopulation with a dual phenotype. Moreover, eGFP-positive dendrites on both mouse lines were found juxtaposed to GlyR subunits and the scaffold protein gephyrin in several areas of the inner plexiform layer, demonstrating the glycinergic character of these neurons. This dual phenotype was also demonstrated in primary retina cultures, in which isolated neurons were positive for GlyT1 and GAD67 or GAT1/3. Altogether, these data provide compelling evidence of a subpopulation of dual inhibitory, glycinergic/GABAergic amacrine neurons. The co-release of both neurotransmitters may serve to strengthen the inhibition on ganglion cells under synaptic hyperexcitability.

Comprehensive Genome-Wide Identification and Transcript Profiling of GABA Pathway Gene Family in Apple (Malus domestica).

γ-Aminobutyric Acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. GABA is involved in pH regulation, maintaining C/N balance, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization via anion flux. Glutamate decarboxylase (GAD, EC 4.1.1.15) and GABA transaminase (GABA-T, EC 2.6.1.19) are two key enzymes involved in the synthesis and metabolism of GABA. Recently, GABA transporters (GATs), protein and aluminium-activated malate transporter (ALMT) proteins which function as GABA receptors, have been shown to be involved in GABA regulation. However, there is no report on the characterization of apple GABA pathway genes. In this study, we performed a genome-wide analysis and expression profiling of the GABA pathway gene family in the apple genome. A total of 24 genes were identified including five GAD genes (namely MdGAD 1–5), two GABA-T genes (namely MdGABA-T 1,2), 10 GAT genes (namely GAT 1–10) and seven ALMT genes (namely MdALMT 1–7). These genes were randomly distributed on 12 chromosomes. Phylogenetic analyses grouped GABA shunt genes into three clusters—cluster I, cluster II, and cluster III—which had three, four, and five genes, respectively. The expression profile analysis revealed significant MdGAD4 expression levels in both fruit and flower organs, except pollen. However, there were no significant differences in the expression of other GABA shunt genes in different tissues. This work provides the first characterization of the GABA shunt gene family in apple and suggests their importance in apple response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of these gene families.

Median raphe region GABAergic neurons contribute to social interest in mouse

Gamma-aminobutyric acid (GABA) is a well-known inhibitory neurotransmitter implicated in numerous physiological and pathological behaviors including social interest. Dysregulation of the median raphe region (MRR), a main serotoninergic nucleus, is also characterized by increased social problems. As the majority of MRR cells are GABAergic, we aimed to reveal the social role of these cells.Chemogenetic techniques were used in vesicular GABA transporter Cre mice and with the help of adeno-associated virus vectors artificial receptors (DREADDs, stimulatory, inhibitory or control, containing only a fluorophore) were expressed in MRR GABAergic cells confirmed by immunohistochemistry. Four weeks after viral injection a behavioral test battery (sociability; social interaction; resident-intruder) was conducted. The artificial ligand (clozapine-N-oxide, 1 mg/10 ml/kg) was administrated 30 min before the tests. As possible confounding factors, locomotion (open field/OF), anxiety-like behavior (elevated plus maze/EPM), and short-term memory (Y-maze) were also evaluated.Stimulation of the GABAergic cells in MRR had no effect on locomotion or working and social memory; however, it increased social interest during sociability and social interaction but not in resident-intruder tests. Accordingly, c-Fos elevation in MRR-GABAergic cells was detected after sociability, but not resident-intruder tests. In the EPM test, the inhibitory group entered into the open arms later, suggesting an anxiogenic-like tendency.We confirmed the role of MRR-GABAergic cells in promoting social interest. However, different subpopulations (e.g. long vs short projecting, various neuropeptide containing) might have divergent roles, which might remain hidden and requires further studies.

Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

P.0608 The influence of zaleplon on GABA and glutamate transporter in the hippocampal synapses in rats

P.0608 The influence of zaleplon on GABA and glutamate transporter in the hippocampal synapses in rats

Microglia sense neuronal activity via GABA in the early postnatal hippocampus.

Microglia, the resident macrophages in the central nervous system, express receptors for classical neurotransmitters, such as γ-aminobutyric acid (GABA) and glutamate, suggesting that they sense synaptic activity. To detect microglial Ca2+ responses to neuronal activity, we generate transgenic mouse lines expressing the fluorescent Ca2+ indicator GCaMP6m, specifically in microglia and demonstrate that electrical stimulation of the Schaffer collateral pathway results in microglial Ca2+ responses in early postnatal but not adult hippocampus. Preceding the microglial responses, we also observe similar Ca2+ responses in astrocytes, and both are sensitive to tetrodotoxin. Blocking astrocytic glutamate uptake or GABA transport abolishes stimulation-induced microglial responses as well as antagonizing the microglial GABAB receptor. Our data, therefore, suggest that the neuronal activity-induced glutamate uptake and the release of GABA by astrocytes trigger the activation of GABAB receptors in microglia. This neuron, astrocyte, and microglia communication pathway might modulate microglial activity in developing neuronal networks.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Identification and characterization of rostral ventromedial medulla neurons synaptically connected to the urinary bladder afferents in female rats with or without neonatal cystitis.

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96h postinoculation for serotonergic, GABAergic, and enkephalinergic expressions using in situ hybridization and/or immunohistochemistry techniques. The results revealed that>50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker tryptophan hydroxylase 2 (TpH2). Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated vesicular GABA transporter (VGAT) with a concomitant increase in TpH2 expression in bladder-linked RVM neurons, suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity.

Chemo/optogenetic stimulation and photometry monitoring of neuron activity in mice

Abstract The neuropeptide oxytocin (OXT) is well recognized for eliciting anxiolytic effects and promoting social reward. However, there is emerging evidence to show that OXT increases aversive events. These seemingly inconsistent results may be attributable to the broad OXT receptor (OXTr) expression in the central nervous system. We applied chemo/optogenetic approach to selectively stimulate septal neurons expressing OXT and /or OXTr. We found that activation of septal OXT or OXTr neurons induced anxiety- but not depressive-like behavior. Septal OXTr neurons projected dense fibers to the diagonal band’s horizontal limb (HDB), and selective stimulation of those HDB projections also elicited anxiety-like behavior. We also found that septal OXTr neurons express the vesicular GABA transporter (vGAT) protein, and optogenetic stimulation of septal OXTr projections to the HDB inactivated HDB neurons. Our novel wireless photometry results show that ventral hippocampal neurons and septal OXT or PXTR neurons are stimulated under anxiety conditions. More interestingly, selective optogenetic stimulation of ventral hippocampus or their projections to the septum inhibited the neurons localized within the HDB. Our data collectively reveal that septal OXTr neurons increase anxiety by projecting inhibitory GABAergic inputs to the HDB, and probably partake in the ventral hippocampus-dependent anxiety. Keywords: Chemo/Optogenetic, Electrophysiology, Photometry, Anxiety

GLUTAMAT/GABA-GLUTAMİN ÇEVRİMİNDE GÖREV ALAN TAŞIYICI PROTEİNLER İÇİN TERMODİNAMİĞİN BİRİNCİ YASA ANALİZİ

The glutamine–glutamate/GABA cycle (GGC) is a sequence of events that provides replenishment of the neurotransmitter pool of glutamate in order to maintain neurotransmitter homeostasis. In the GGC, glutamate or GABA molecules are released from neurons and subsequently taken up into astrocytes. Astrocytes convert glutamate or GABA molecules into glutamine and release them into the synapse. Glutamine molecules are taken up by neurons to be used as a precursor for the synthesis of glutamate or GABA. The transport of these molecules across the membranes of neurons and astrocytes is facilitated by transporter proteins. Each of these transporter proteins is a biomolecular machine; they operate on thermodynamic cycles and convert part of the supplied energy input into useful work output. Energy harnessed from the translocation of molecules/ions down their electrochemical gradient is converted into mechanical useful work translocating molecules/ions against their electrochemical gradient. Conservation of energy principle was applied and thermodynamic first law efficiencies, showing how much of the energy input per cycle is converted into useful work, were evaluated for the thermodynamic cycles of EAAT, ASCT2, B0AT2, SA, SN, and GABA transporters involved in the GGC. Neurotransmitter concentrations in the synapse change upon signal arrival and subsequently return to resting levels, causing transporters to operate under various first law efficiencies. Range of first law efficiencies for EAAT (for glutamate transport), ASCT2, B0AT2, SA SN, GABA (forward mode) were calculated as 60-85%, 46-78%, 61-89%, 61-89%, 55-80%, and 54-76%, respectively. Efficiency values obtained for these transporters are much higher than those of the macro-scaled heat engines we encounter in our daily lives. Furthermore, EAAT showed larger thermodynamic first law efficiency for glutamate transport than aspartate transport, which takes place with a maximum efficiency of 45%. Thus, suggesting the possibility that transport of different substrates by the same transporter may take place with different efficiencies.

Metabolomics Analysis of the Effect of GAT-2 Deficiency on Th1 Cells in Mice.

The classic neurotransmitter γ-aminobutyric acid (GABA) has been shown to shape the activation and function of immune cells. There are four high-affinity GABA transporters (GATs, including GAT-1, GAT-2, GAT-3, and GAT-4) responsible for the transmembrane transport of GABA in mice. To explore the effect of GAT-2 on type 1 helper T (Th1) cells, naïve CD4+ T cells were isolated from splenocytes of GAT-2 knockout (KO) and wild-type (WT) mice and cultured for Th1 cell differentiation, and then, metabolomics analysis of Th1 cells was performed via gas chromatography coupled to time-of-flight mass spectrometry added with multivariate analyses. Based on the variable importance projection value > 1 and P < 0.05, a total of nine differentially expressed metabolites (DEMs) were identified between WT and KO. Then, DEMs were mapped to the KEGG database, and five metabolic pathways were significantly enriched, including the cysteine and methionine metabolism, the riboflavin metabolism, the purine metabolism, the glycerolipid metabolism, and the glycerophospholipid metabolism. Collectively, our metabolomics analysis revealed that deficiency of GAT-2 influenced the metabolomics profile of Th1 cells, which will provide insights into T cell response to GAT-2 deficiency in mice. Data are available via MetaboLights with identifier MTBLS3358.

Caffeine Improves GABA Transport in the Striatum of Spontaneously Hypertensive Rats (SHR).

The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker,NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.