Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa.

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.

ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function.

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

Insulin differentially modulates GABA signalling in hippocampal neurons and, in an age-dependent manner, normalizes GABA-activated currents in the tg-APPSwe mouse model of Alzheimer's disease.

AIM: We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. METHODS: GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10 weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. RESULTS: In young mice, insulin (1 nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6 months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. CONCLUSION: Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.

Endogenous Levels of Gamma Amino-Butyric Acid Are Correlated to Glutamic-Acid Decarboxylase Antibody Levels in Type 1 Diabetes.

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system (CNS) and outside of the CNS, found in the highest concentrations in immune cells and pancreatic beta-cells. GABA is gaining increasing interest in diabetes research due to its immune-modulatory and beta-cell stimulatory effects and is a highly interesting drug candidate for the treatment of type 1 diabetes (T1D). GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD), one of the targets for autoantibodies linked to T1D. Using mass spectrometry, we have quantified the endogenous circulating levels of GABA in patients with new-onset and long-standing T1D and found that the levels are unaltered when compared to healthy controls, i.e., T1D patients do not have a deficit of systemic GABA levels. In T1D, GABA levels were negatively correlated with IL-1 beta, IL-12, and IL-15 15 and positively correlated to levels of IL-36 beta and IL-37. Interestingly, GABA levels were also correlated to the levels of GAD-autoantibodies. The unaltered levels of GABA in T1D patients suggest that the GABA secretion from beta-cells only has a minor impact on the circulating systemic levels. However, the local levels of GABA could be altered within pancreatic islets in the presence of GAD-autoantibodies.

Tonic GABA-activated synaptic and extrasynaptic currents in dentate gyrus granule cells and CA3 pyramidal neurons along the mouse hippocampal dorsoventral axis.

The hippocampus is a medial temporal lobe structure in the brain and is widely studied for its role in memory and learning, in particular, spacial memory and emotional responses. It was thought to be a homogenous structure but emerging evidence shows differentiation along the dorsoventral axis and even microdomains for functional and cellular markers. We have examined in two cell-types of the hippocampal projection neurons, the dentate gyrus (DG) granule cells and CA3 pyramidal neurons, if the GABA-activated tonic current density varied between the dorsal (septal) and the ventral (temporal) poles of the male mouse hippocampus. Tonic synaptic currents, arising from spontaneous and miniature inhibitory postsynaptic currents (sIPSC, mIPSC), and extrasynaptic tonic currents were evaluated. The results revealed different levels of sIPSC but not mIPSC density between the dorsal and ventral hippocampal neurons for both the DG granule cells and the CA3 pyramidal neurons. The extrasynaptic tonic current density was larger in the DG granule cells as compared to the CA3 pyramidal neurons but did not vary between the dorsal and ventral regions. IPSC bursting was observed in both cell-types in the ventral hippocampus but was more common in the CA3 pyramidal neurons. Only in the dorsal DG granule cells was the level of the sIPSC and mIPSC density similar. The results indicate that the tonic GABAergic inhibition is particularly strong in the ventral hippocampal DG granule cells and enhanced in the dorsal as compared to the ventral hippocampal CA3 pyramidal neurons.

GABAA Receptor-Mediated Currents and Hormone mRNAs in Cells Expressing More Than One Hormone Transcript in Intact Human Pancreatic Islets.

In pancreatic islets, the major cell-types are α, ß and δ cells. The γ-aminobutyric acid (GABA) signalling system is expressed in human pancreatic islets. In single hormone transcript-expressing cells, we have previously characterized the functional properties of islet GABAA receptors (iGABAARs). Here, we extended these studies to islet cells expressing mRNAs for more than one hormone and sought for correlation between iGABAAR activity level and relative mRNA expression ratio. The single-cell RT-PCR in combination with the patch-clamp current recordings was used to examine functional properties of iGABAARs in the multiple hormone mRNA-expressing cells. We detected cells expressing double (α/ß, α/δ, ß/δ cell-types) and triple (α/ß/δ cell-type) hormone transcripts. The most common mixed-identity cell-type was the α/ß group where the cells could be grouped into ß- and α-like subgroups. The ß-like cells had low GCG/INS expression ratio (<0.6) and significantly higher frequency of iGABAAR single-channel openings than the α-like cells where the GCG/INS expression ratio was high (>1.2). The hormone expression levels and iGABAAR single-channel characteristics varied in the α/ß/δ cell-type. Clearly, multiple hormone transcripts can be expressed in islet cells whereas iGABAAR single-channel functional properties appear to be α or ß cell specific.

Depression, GABA, and Age Correlate with Plasma Levels of Inflammatory Markers.

Immunomodulation is increasingly being recognised as a part of mental diseases. Here, we examined whether levels of immunological protein markers changed with depression, age, or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). An analysis of plasma samples from patients with a major depressive episode and control blood donors (CBD) revealed the expression of 67 inflammatory markers. Thirteen of these markers displayed augmented levels in patients compared to CBD. Twenty-one markers correlated with the age of the patients, whereas 10 markers correlated with the age of CBD. Interestingly, CST5 and CDCP1 showed the strongest correlation with age in the patients and CBD, respectively. IL-18 was the only marker that correlated with the MADRS-S scores of the patients. Neuronal growth factors (NGFs) were significantly enhanced in plasma from the patients, as was the average plasma GABA concentration. GABA modulated the release of seven cytokines in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) from the patients. The study reveals significant changes in the plasma composition of small molecules during depression and identifies potential peripheral biomarkers of the disease.

Ataxia in Patients With Bi-Allelic NFASC Mutations and Absence of Full-Length NF186.

The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.

Interferon-γ potentiates GABAA receptor-mediated inhibitory currents in rat hippocampal CA1 pyramidal neurons.

The neural transmission and plasticity can be differentially modulated by various elements of the immune system. Interferon-γ (IFN-γ) is a "pro-inflammatory" cytokine mainly produced by T lymphocytes, activates its corresponding receptor and plays important roles under both homeostatic and inflammatory conditions. However, the impact of IFN-γ on the γ-aminobutyric acid (GABA)-mediated currents in the hippocampus, a major brain region involved in the cognitive function, has not been investigated. Here we detected abundant expression of both IFN-γ receptor subunit gene transcripts (Ifngr1 and Ifngr2) in the rat hippocampus by quantitative PCR. In addition, we pre-incubated rat hippocampal slices with IFN-γ (100 ng/ml) and recorded GABA-activated spontaneous and miniature postsynaptic inhibitory currents (sIPSCs and mIPSCs) and tonic currents in hippocampal CA1 pyramidal neurons by the whole-cell patch-clamp method. The pre-incubation with IFN-γ increased the frequency but not the mean amplitude, rise time or decay time of both sIPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, suggesting a presynaptic effect of IFN-γ. Moreover, the GABA-activated tonic currents were enhanced by IFN-γ. In conclusion, the potentiation of GABAergic currents in hippocampal neurons by IFN-γ may contribute to the disturbed neuronal excitability and cognitive dysfunction during neuroinflammation.

Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment.

Dravet syndrome (DS) is an early onset refractory epilepsy typically caused by de novo heterozygous variants in SCN1A encoding the α-subunit of the neuronal sodium channel Nav1.1. The syndrome is characterized by age-related progression of seizures, cognitive decline and movement disorders. We hypothesized that the distinct neurodevelopmental features in DS are caused by the disruption of molecular pathways in Nav1.1 haploinsufficient cells resulting in perturbed neural differentiation and maturation. Here, we established DS-patient and control induced pluripotent stem cell derived neural progenitor cells (iPSC NPC) and GABAergic inter-neuronal (iPSC GABA) cells. The DS-patient iPSC GABA cells showed a shift in sodium current activation and a perturbed response to induced oxidative stress. Transcriptome analysis revealed specific dysregulations of genes for chromatin structure, mitotic progression, neural plasticity and excitability in DS-patient iPSC NPCs and DS-patient iPSC GABA cells versus controls. The transcription factors FOXM1 and E2F1, positive regulators of the disrupted pathways for histone modification and cell cycle regulation, were markedly up-regulated in DS-iPSC GABA lines. Our study highlights transcriptional changes and disrupted pathways of chromatin remodeling in Nav1.1 haploinsufficient GABAergic cells, providing a molecular framework that overlaps with that of neurodevelopmental disorders and other epilepsies.

Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes.

Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca2+ channels in the plasma membrane are the voltage-gated Ca2+ (CaV) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and CaV gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the CaV2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the CaV 2.1 and CaV3.2 genes were up-regulated only in pregnancy and the CaV1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and CaV genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.

GABA Regulates Release of Inflammatory Cytokines From Peripheral Blood Mononuclear Cells and CD4+ T Cells and Is Immunosuppressive in Type 1 Diabetes.

The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100nM, 500nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion.

Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic ß Cells.

In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting ß cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet ß cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets ß cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.

Insulin enhances GABAA receptor-mediated inhibitory currents in rat central amygdala neurons.

Insulin, a pancreatic hormone, can access the central nervous system, activate insulin receptors distributed in selective brain regions and affect various cellular functions such as neurotransmission. We have previously shown that physiologically relevant concentration of insulin potentiates the GABAA receptor-mediated tonic inhibition and reduces excitability of rat hippocampal CA1 neurons. The central nucleus of the amygdala (CeA) comprises heterogeneous neuronal populations that can respond to hormonal stimulus. Using quantitative PCR and immunofluorescent labeling, we report that the mRNA and protein of the insulin receptor are abundantly expressed in the rat CeA. The insulin receptor mRNA is also detected in the CeA from post-mortem human brain samples. Furthermore, our whole-cell patch-clamp recordings show that the application of insulin (5 and 50 nM) selectively enhances the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in rat CeA neurons. Our findings reveal that GABAergic synaptic transmission is a target in the CeA for insulin receptor signaling that may underlie insulin modulation of emotion- and feeding-related behaviors.

Liraglutide modulates GABAergic signaling in rat hippocampal CA3 pyramidal neurons predominantly by presynaptic mechanism.

BACKGROUND: γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain where it regulates activity of neuronal networks. The receptor for glucagon-like peptide-1 (GLP-1) is expressed in the hippocampus, which is the center for memory and learning. In this study we examined effects of liraglutide, a GLP-1 analog, on GABA signaling in CA3 hippocampal pyramidal neurons. METHODS: We used patch-clamp electrophysiology to record synaptic and tonic GABA-activated currents in CA3 pyramidal neurons in rat hippocampal brain slices. RESULTS: We examined the effects of liraglutide on the neurons at concentrations ranging from one nM to one µM. Significant changes of the spontaneous inhibitory postsynaptic currents (sIPSCs) were only recorded with 100 nM liraglutide and then in just ≈50% of the neurons tested at this concentration. In neurons affected by liraglutide both the sIPSC frequency and the most probable amplitudes increased. When the action potential firing was inhibited by tetrodotoxin (TTX) the frequency and amplitude of IPSCs in TTX and in TTX plus 100 nM liraglutide were similar. CONCLUSIONS: The results demonstrate that liraglutide regulation of GABA signaling of CA3 pyramidal neurons is predominantly presynaptic and more limited than has been observed for GLP-1 and exendin-4 in hippocampal neurons.

AMPA, NMDA and kainate glutamate receptor subunits are expressed in human peripheral blood mononuclear cells (PBMCs) where the expression of GluK4 is altered by pregnancy and GluN2D by depression in pregnant women.

The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells.

Characterization of the γ-aminobutyric acid signaling system in the zebrafish (Danio rerio Hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction.

In the vertebrate brain, inhibition is largely mediated by γ-aminobutyric acid (GABA). This neurotransmitter comprises a signaling machinery of GABAA, GABAB receptors, transporters, glutamate decarboxylases (gads) and 4-aminobutyrate aminotransferase (abat), and associated proteins. Chloride is intimately related to GABAA receptor conductance, GABA uptake, and GADs activity. The response of target neurons to GABA stimuli is shaped by chloride-cation co-transporters (CCCs), which strictly control Cl- gradient across plasma membranes. This research profiled the expression of forty genes involved in GABA signaling in the zebrafish (Danio rerio) brain, grouped brain regions and retinas. Primer pairs were developed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The mRNA levels of the zebrafish GABA system share similarities with that of mammals, and confirm previous studies in non-mammalian species. Proposed GABAA receptors are α1ß2γ2, α1ß2δ, α2bß3γ2, α2bß3δ, α4ß2γ2, α4ß2δ, α6bß2γ2 and α6bß2δ. Regional brain differences were documented. Retinal hetero- or homomeric ρ-composed GABAA receptors could exist, accompanying α1ßyγ2, α1ßyδ, α6aßyγ2, α6aßyδ. Expression patterns of α6a and α6b were opposite, with the former being more abundant in retinas, the latter in brains. Given the stoichiometry α6wßyγz, α6a- or α6b-containing receptors likely have different regulatory mechanisms. Different gene isoforms could originate after the rounds of genome duplication during teleost evolution. This research depicts that one isoform is generally more abundantly expressed than the other. Such observations also apply to GABAB receptors, GABA transporters, GABA-related enzymes, CCCs and GABAA receptor-associated proteins, whose presence further strengthens the proof of a GABA system in zebrafish.