GABA Receptor Research Articles

Hypothalamus-sympathetic-liver axis mediates the early phase of stress-induced hyperglycemia in the male mice

Rapid glucose supply is crucial for animal survival during stress response. How the timescale of stress-induced glucose release precisely controlled by hypothalamic corticotropin-releasing hormone (CRH) neurons remains unclear. Here, we show that stress-induced hyperglycemia can be divided into at least two stages in male mice: the first fast stage is mediated by hypothalamus (paraventricular to ventromedial hypothalamus)-sympathetic (raphe pallidus nucleus to intermediolateral nucleus)-liver (HSL) axis activity; the second delayed stage is mediated by adrenal activity. Blocking the activity of HSL axis impairs predatory evoked flight responses, indicating that the HSL pathway activity is necessary for stress coping. We further reveal the intracellular signal cascade for CRH signal in the hypothalamus, which is mediated by GABAA receptor β3 subunit phosphorylation at S408/409, results in prevention of GABAA receptor membrane recruitment. Thus, we uncovered the precise timescale of glucose supply during stress which is mediated by adrenal independent HSL and adrenal dependent pathway respectively.

12419 Single-nuclei Sequencing Of Normal Donor Cadaveric Parathyroid Glands Reveals Distinct Cellular Subpopulations With Key Differences In Gland Anatomical Location And Suppressed Vs Non-suppressed Physiological Status

Abstract Disclosure: T. Glinin: None. E. Khanafshar: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly & Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly & Company. J. Koh: None. Despite the significant clinical burden of parathyroid disorders, little is known at the molecular level about the cellular composition of normal parathyroid glands (PTGs). We performed a single-nuclei RNAseq study of six cadaveric PTGs (three from the superior anatomical location, three inferior) from four donors (two female). A board-certified pathologist confirmed that the sequenced PTGs were histologically normal. Using the split-pool method, we sequenced 83,693 nuclei, generating 1.7B reads. Data integration was performed with the Harmony algorithm in Seurat 5.0.1. Cluster analysis revealed 14 discrete populations of PTG origin. Gene set enrichment identified clusters corresponding to endothelial cells, neurons, microglia, mast cells, dendritic cells, stromal fibroblasts, macrophages, and smooth muscle. The remaining six clusters were closely related but distinct populations sharing markers including PTH, CaSR, SPOCK3, PRKCE, and PLCB1 (p<2e-300 for group specificity) but with differentially expressed gene (DEG) patterns between clusters suggestive of specialized functions in VEGF signaling (KIAA1549L), GPCR downstream signaling (GRM7, ADGRL3), and subcellular spatial organization (SYNE1) (p<2e-24 for cluster specificity). Superior and inferior PTGs were distinguished by 8 superior-specific DEGs (p<2e-245) including HOXB3, expressed in 71% of superior PTG cells vs 6% of inferior PTG cells. Enhanced expression of HOXB3, a homeobox transcription factor found to inhibit tumorigenesis in multiple tissues, could explain in part why adenomas are three times less likely to arise in superior PTGs. During procurement, one of the presumptive normal donors was found to have a single grossly enlarged, hypercellular PTG, elevated PTH (140 pg/ml), and hypercalcemia (11.2 mg/dL), consistent with primary hyperparathyroidism (PHPT). In PHPT patients with a single tumor, contralateral PTGs are functionally suppressed by the dominant adenoma. We compared the histologically normal suppressed and unsuppressed PTGs. Fifteen genes were more highly expressed in suppressed PTGs, including LRP2, a multi-ligand receptor known to bind PTH (p = 2e-300). Fourteen genes were more highly expressed in unsuppressed PTGs (p<2.14e-296), including PDE11A, NRG2, NRXN3, and PRLR, which are all associated with GABA receptor signaling (q = 2.8e-5). These data independently support our group’s prior work proposing a role for the GABA receptor in PHPT. Differential expression in endothelial cells, neurons, and fibroblasts from suppressed PTGs suggests that non-endocrine cells may also be responsive to changes in PTG activity. Finally, the different proportions of cells with suppressed or unsuppressed gene expression signatures observed between clusters suggests that PTGs incorporate distinct populations of cells reflective of physiological activity or functional suppression. Presentation: 6/2/2024

12237 Monosynaptic Gabaergic Signaling From Suprachiasmatic Nucleus Neuromedin S Neurons To Preoptic Area Kisspeptin Neurons In Mice

Abstract Disclosure: W.I. Abdulmajeed: None. B.B. Jamieson: None. S.X. Thomas: None. Y. Rim: None. A.G. Novak: None. R.E. Campbell: None. R. Piet: None. In female rodents, projections from suprachiasmatic nucleus (SCN) neurons relay timing cues to the gonadotropin-releasing hormone (GnRH) neuronal network for the preovulatory surge. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) are thought to integrate these cues with estradiol positive feedback to drive GnRH neuron activity, the gonadotropin surge and ovulation. We recently reported that SCN neuron projections release arginine vasopressin (AVP), thereby stimulating female RP3VKiss[1] neuron action potential firing in an estrous cycle stage-dependent manner. In the SCN, AVP is expressed in a subset of neuromedin S (NMS) neurons, a neuronal population essential for circadian rhythms. Moreover, a previous report indicates that exogenous NMS stimulates LH secretion in female rodents. SCNNMS neurons might, therefore, play a role in timing the preovulatory surge. However, whether SCNNMS neurons project to and regulate the activity of RP3VKISS[1] neurons is currently unknown. To address this question, we combined anterograde viral tract-tracing and channelrhodopsin-2 (ChR2)- assisted circuit mapping. Female mice expressing Cre recombinase in NMS cells (NMS-Cre) received stereotaxic injections in the SCN of adeno-associated viral vectors (AAVs) carrying the Cre-dependent sequence for mCherry. Fluorescence immunohistochemistry revealed that mCherry-expressing fibers come in close apposition with the great majority (>80%) of KISS1-immunoreactive neurons in the RP3V, suggesting a SCNNMS-to-RP3VKISS[1] neuron circuit. To interrogate this circuit, we next injected AAVs carrying Cre-dependent channelrhodopsin (ChR2) in the SCN of female NMS-Cre mice that also express the green fluorescent protein in KISS1 cells. Blue LED light (460-487 nm) activation of ChR2-expressing SCNNMS neuronal fibers in the RP3V evoked postsynaptic currents (PSCs) in most KISS1 neurons recorded in whole-cell voltage clamp (-60 mV) in brain slices. Evoked PSCs had short latencies and were blocked by bath application of 0.5 µM tetrodotoxin (TTX), indicating that they were generated by action potential-dependent release. Addition of 100 µM 4-Aminopyridine (4-AP, a potassium channel blocker) to TTX rescued these responses, indicating the existence of a monosynaptic SCNNMS-to-RP3VKISS[1] connection. In addition, evoked PSCs were suppressed by 5 µM gabazine, a GABAA receptor antagonist, but not by 20 µM NBQX and 50 µM D-AP5, AMPA- and NMDA-type glutamate receptor antagonists, respectively, revealing that SCNNMS projections release GABA onto RP3VKISS[1] neurons. Together, our findings are consistent with SCNNMS neurons extending monosynaptic GABAergic projections to RP3VKISS[1] neurons. Further studies are needed to determine the impact of these projections on RP3VKISS[1] neuron activity and the role they might play in the preovulatory surge. Presentation: 6/1/2024

PET imaging identifies anti-inflammatory effects of fluoxetine and a correlation of glucose metabolism during epileptogenesis with chronic seizure frequency

The serotonergic system has shown to be altered during epileptogenesis and in chronic epilepsy, making selective serotonin reuptake inhibitors interesting candidates for antiepileptogenic therapy. In this study, we aimed to evaluate disease-modifying effects of fluoxetine during experimental epileptogenesis.Status epilepticus (SE) was induced by lithium-pilocarpine, and female rats were treated either with vehicle or fluoxetine over 15 days. Animals were subjected to 18F-FDG (7 days post-SE), 18F-GE180 (15 days post-SE) and 18F-flumazenil positron emission tomography (PET, 21 days post-SE). Uptake (18F-FDG), volume of distribution (18F-GE180) and binding potential (18F-flumazenil) were calculated. In addition, hyperexcitability testing and video-EEG monitoring were performed.Fluoxetine treatment did not alter brain glucose metabolism. 18F-GE180 PET indicated lower neuroinflammation in the hippocampus of treated animals (−22.6%, p = 0.042), but no differences were found in GABAA receptor density. Video-EEG monitoring did not reveal a treatment effect on seizure frequency. However, independently of the treatment, hippocampal FDG uptake 7 days after SE correlated with seizure frequency during the chronic phase (r = −0.58; p = 0.015).Fluoxetine treatment exerted anti-inflammatory effects in rats during epileptogenesis. However, this effect did not alter disease outcome. Importantly, FDG-PET in early epileptogenesis showed biomarker potential as higher glucose metabolism correlated to lower seizure frequency in the chronic phase.

Role of the Kölliker-Fuse/parabrachial complex in the generation of postinspiratory vagal and sympathetic nerve activities and their recruitment by hypoxemic stimuli in the rat.

In the rat, the activity of laryngeal adductor muscles, the crural diaphragm, and sympathetic vasomotor neurons is entrained to the postinspiratory (post-I) phase of the respiratory cycle, a mechanism thought to enhance cardiorespiratory efficiency. The identity of the central neurons responsible for transmitting respiratory activity to these outputs remains unresolved. Here we explore the contribution of the Kölliker-Fuse/parabrachial nuclei (KF-PBN) in the generation of post-I activity in vagal and sympathetic outputs under steady-state conditions and during acute hypoxemia, a condition that potently recruits post-I activity. In artificially ventilated, vagotomized, and urethane-anesthetized rats, bilateral KF-PBN inhibition by microinjection of the GABAA receptor agonist isoguvacine evoked stereotypical responses on respiratory pattern, characterized by a reduction in phrenic nerve burst amplitude, a modest lengthening of inspiratory time, and an increase in breath-to-breath variability, while post-I vagal nerve activity was abolished and post-I sympathetic nerve activity diminished. During acute hypoxemia, KF-PBN inhibition attenuated tachypneic responses and completely abolished post-I vagal activity while preserving respiratory-sympathetic coupling. Furthermore, KF-PBN inhibition disrupted the decline in respiratory frequency that normally follows resumption of oxygenation. These findings suggest that the KF-PBN is a critical hub for the distribution of post-I activities to vagal and sympathetic outputs and is an important contributor to the dynamic adjustments to respiratory patterns that occur in response to acute hypoxia. Although KF-PBN appears essential for post-I vagal activity, it only partially contributes to post-I sympathetic nerve activity, suggesting the contribution of multiple neural pathways to respiratory-sympathetic coupling.NEW & NOTEWORTHY Inhibition of neurons in the pontine Kölliker-Fuse/parabrachial complex (KF-PBN) differentially inhibited postinspiratory (post-I) activity in vagal and sympathetic outputs. The strong recruitment of post-I vagal activity that occurs in response to hypoxemia is selectively abolished by KF-PBN inhibition. This suggests that 1) post-I activity in vagal and sympathetic outputs may be generated by partially independent mechanisms and 2) neurons in the KF-PBN are a preeminent source of drive for the generation of eupneic post-I activity.

Differential chemoproteomic analysis of RRS-1 candidate molecule and molecules of several nonsteroidal anti-inflammatory drugs

Background. To plan effective and safe pharmacotherapy for inflammation and pain, it is important to evaluate the mechanisms and spectrum of action of nonsteroidal anti-inflammatory drugs (NSAIDs), including their effects on human proteome.Objective: to identify and evaluate the most significant specific differences of candidate molecule RRS-1 (N-{(Z)-2-(1-methyl-1H-indol-3-yl)-1-[(propylamino)carbonyl]vinyl}benzamide) from other NSAIDs through differential chemoreactome analysis.Materials and methods. Chemoproteomic modeling of pharmacological effects of RRS-1 molecule and a number of well-known NSAIDs (diclofenac, nimesulide, ketorolac) on human proteome was carried out on the basis of numerical prediction algorithms over the space of heterogeneous feature descriptions, developed in the topological approach to recognition by Yu.I. Zhuravlev and K.V. Rudakov scientific school.Results. Significant differences in the effects of the studied molecules were found for 1232 proteins of human proteome. The features of assessing interactions of the studied molecules with 47 target proteins, which most distinguished the effects of RRS-1 molecule from all others were identified. RRS-1 could activate adenosine and dopamine receptors, cannabinoid receptor 2 and GABAA receptor to a greater extent than other molecules. Activation of these receptors corresponded to anti-inflammatory, anti-nociceptive and neuroprotective effects. RRS-1 could preferably inhibit a number of pro-inflammatory proteins, receptor bradykinin 1, metabotropic glutamate receptor 5, matrix metalloproteinases 8, 9, 12, and blood coagulation factor X. Additionally, RRS-1 molecule showed preferable inhibition of a number of kinases targeted in antitumor and anti-inflammatory therapy. RRS-1, less than other studied molecules, interacted with the receptors of vitamin D3, thyroid hormone, acetylcholine, cannabinoids and opioids, orexin, and various metabolic enzymes, which is important in assessment of the safety of using drugs based on this molecule. RRS-1 characteristically exhibited a moderate profile of antivitamin action: the total score of vitamin and mineral loss (7.4±3.7) was significantly less in comparison to diclofenac (11.7±4.5) and was actually on the same level as nimesulide (6.9±3.7) and ketorolac (6.7±3.6).Conclusion. Chemoreactomic and chemoproteomic profiling of RRS-1 candidate molecule provided pre-experimental assessments of its efficacy and safety through modeling interactions with the human proteome.

Chronic ethanol exposure in mice evokes pre- and postsynaptic deficits in GABAergic transmission in ventral tegmental area GABA neurons.

GABAergic neurons in mouse ventral tegmental area (VTA) exhibit elevated activity during withdrawal following chronic ethanol exposure. While increased glutamatergic input and decreased GABAA receptor sensitivity have been implicated, the impact of inhibitory signaling in VTA GABA neurons has not been fully addressed. We used electrophysiological and ultrastructural approaches to assess the impact of chronic intermittent ethanol vapour exposure in mice on GABAergic transmission in VTA GABA neurons during withdrawal. We used CRISPR/Cas9 ablation to mimic a somatodendritic adaptation involving the GABAB receptor (GABABR) in ethanol-naïve mice to investigate its impact on anxiety-related behaviour. The frequency of spontaneous inhibitory postsynaptic currents was reduced in VTA GABA neurons following chronic ethanol treatment and this was reversed by GABABR inhibition, suggesting chronic ethanol strengthens the GABABR-dependent suppression of GABAergic input to VTA GABA neurons. Similarly, paired-pulse depression of GABAA receptor-dependent responses evoked by optogenetic stimulation of nucleus accumbens inputs from ethanol-treated mice was reversed by GABABR inhibition. Somatodendritic currents evoked in VTA GABA neurons by GABABR activation were reduced following ethanol exposure, attributable to the suppression of GIRK (Kir3) channel activity. Mimicking this adaptation enhanced anxiety-related behaviour in ethanol-naïve mice. Chronic ethanol weakens the GABAergic regulation of VTA GABA neurons in mice via pre- and postsynaptic mechanisms, likely contributing to their elevated activity during withdrawal and expression of anxiety-related behaviour. As anxiety can promote relapse during abstinence, interventions targeting VTA GABA neuron excitability could represent new therapeutic strategies for treatment of alcohol use disorder.

Nonsedating anxiolytics.

Anxiety disorders are the most prevalent psychiatric pathology with substantial cost to society, but the existing treatments are often inadequate. This has rekindled the interest in the GABAA-receptor (GABAAR) positive allosteric modulator (PAM) compounds, which have a long history in treatment of anxiety beginning with diazepam, chlordiazepoxide, and alprazolam. While the GABAAR PAMs possess remarkable anxiolytic efficacy, they have fallen out of favor due to a host of adverse effects including sedation, motor impairment, addictive potential and tolerance development. A substantial effort was thus devoted to the design of GABAAR PAMs as anxiolytics with reduced sedative liabilities. Several non-benzodiazepine (BZD) GABAAPAMs progressed to clinical trials (bretazenil, abecarnil, alpidem, and ocinaplon) with alpidem obtaining regulatory approval as anxiolytic, but later withdrawn from market due to hepatotoxicity. Advances in molecular biology gave birth to a host of subtype selective GABAAR-PAMs which suffered from signs of sedation and motor impairment and only three compounds progressed to proof-of-concept studies (TPA-023, AZD7325 and PF-06372865). TPA-023 was terminated due to toxicity in preclinical species while AZD7325 and PF-06372865 did not achieve efficacy endpoints in patients. We highlight a new compound, KRM-II-81, that is an imidazodiazepine selective for GABAAR containing α2/3 and β3 proteins. In preclinical studies KRM-II-81 produced anxiolytic-like effects but with minimal sedation, respiratory depression, and abuse liability. Thus, KRM-II-81 is a newly discovered, non- BZD anxiolytic compound, which targets a selective population of GABAAR for improved therapeutic gain and reduced side effects.

The TMEM132B-GABAA receptor complex controls alcohol actions in the brain.

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABAA receptor (GABAAR) auxiliary subunit. Functionally, TMEM132B promotes GABAAR expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABAAR interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABAAR-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABAAR auxiliary subunit, identify the TMEM132B-GABAAR complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors.

Evaluation of thiopyrano[2,3-d]thiazole derivatives as potential anticonvulsant agents.

Anticonvulsant drug discovery has achieved significant progress; however, pharmacotherapy of epilepsy continues to be a challenge for modern medicine and pharmacy. To expand the chemical space of heterocycles as potential antiepileptic agents, herein we report on the synthesis and evaluation of anticonvulsant properties of a series of thiopyrano[2,3-d]thiazoles. The studied heterocycles are characterized by satisfactory drug-likeness and pharmacokinetics properties, calculated in silico using SwissADME. The anticonvulsant activity of thiopyrano[2,3-d]thiazole derivatives was evaluated in vivo using the subcutaneous pentylenetetrazole test. Three hits, that is, compounds 12, 14, and 16, that caused a pronounced anticonvulsant effect were identified. Derivatives 12, 14, and 16 positively affected the latent period of onset of clonic seizures, number of seizures, mortality rate, and duration of the seizure period of animals under experimental conditions. The anticonvulsant properties of compound 14 were equivalent to the effect of the reference drug, sodium valproate. All hit compounds are characterized by satisfying toxicity properties in the human lymphocytes and HEK293 cell line. The most active hit 14 possesses a potential affinity with the GABAA receptor in the molecular docking study and forms a stable complex in the molecular dynamics experiments equal to diazepam. Preliminary SAR results were obtained and discussed based on screening data.

N318L Blocks the Interaction of Fluralaner but Not Broflanilide or Fipronil with the Insect GABA Receptor In Vivo.

Fluralaner is a novel insecticide targeting the ionotropic GABA receptor (GABAR) subunit, RDL. A recent study revealed that N316L, a substitution of asparagine (N) with leucine (L), in the second transmembrane (M2)-spanning region reduced the antagonist action of fluralaner on the housefly Musca domestica RDL (MdRDL) in vitro. To verify the impact of N316L in vivo, the corresponding mutation (N318L) in the fruitfly Drosophila melanogaster RDL (DmRDL) was constructed using CRISPR/Cas9 genome editing. The homozygous DmRDLN318L mutant showed a 9.87-fold resistance to fluralaner compared with w1118 while still being highly sensitive to broflanilide and fipronil, which is consistent with those findings observed in the electrophysiology assays of the homomeric DmRDLWT or DmRDLN318L channel. Moreover, DmRDLN318L led to malformed ovaries, stunted eggs, and sterility in homozygous females. These results highlighted N318 as a molecular site for fluralaner in vivo and in vitro and might elucidate the resistance mechanisms of insects against fluralaner.

Synthesis, characterization, anxiolytic and anticonvulsant activity, DFT, molecular docking, DMPK studies of chalcone derived from maleic anhydride

Anxiety typically doesn't cause convulsions, but intense stress can reduce the threshold for convulsive bouts in predisposed individuals. Chalcones are known to act directly on the central nervous system (CNS) and the presence of electron donor and acceptor groups attached to the aromatic rings in various positions can alter the properties of the molecule. Thus, this work investigated the anxiolytic and anticonvulsant potential of the new chalcone derivative (E)-6-(4-((E)-3-(3-nitrophenyl)acryloyl)phenyl)-5-oxohex-2-enoic acid (CAMEL). 1H and 13C NMR and ATR-FTIR analyses helped to determine the molecular structure of this chalcone. The energy gap analysis and the higher hardness values than the softness values confirm the stability of CAMEL, with CGDRs indicating that it is more electrophilic in nature. In relation to its potential anxiolytic effect, the tested dose of 40 mg kg-1 of the derivative showed behavior like Diazepam, with activity via GABAA. In addition, the derivative also exhibited a possible anticonvulsant effect at the dose of 20 mg kg-1, being like Diazepam, showing involvement in stages 2 and 3 of GABAA receptors in this process. Given the anxiolytic and anticonvulsant activity shown in vivo and in silico tests, CAMEL is a promising candidate for the treatment of diseases that affect the CNS.

Phytol exerts sedative-like effects and modulates the diazepam and flumazenil’s action, possibly through the GABAA receptor interaction pathway

This study aimed at the evaluation of the sedative effect of phytol (PHY) with possible molecular mechanisms through in vivo and in silico studies. For this, adult male mice were randomly divided into six individual groups, namely control (vehicle), two standards (DZP: diazepam at 2 m/kg, FLU: flumazenil at 0.1 mg/kg), three test groups (PHY at 25, 50, and 75 mg/kg), and three combined groups with the DZP-2 and/or FLU-0.1 with PHY-75 mg/kg. After thirty minutes, each animal was treated with thiopental sodium (TS) at 40 mg/kg to produce sedation and observed for latency and duration of sleep up to 4 h. In silico studies were performed with the 6X3X protein of the GABAA receptor α1 and β2 subunits. The results demonstrate that PHY dose-dependently enhanced sleep duration in animals. However, it produced an insignificant sleep duration compared to the control and standard groups. It also significantly (p < 0.05) decreased the latency and increased the duration of sleep with DZP-2, while reducing these parameters with FLU-0.1. In in silico studies, DZP and FLU exhibited binding affinities with 6X3X by −6.8 and −6.9 kcal/mol, respectively, while PHY exhibited −6.9 kcal/mol. Taken together, PHY may exert a sedative-like effect in TS-induced sleeping mice and modulate the effects of DZP and FLU, possibly through interacting with the 6X3X protein of the GABAA receptor. PHY may be one of the good candidates for the management of sleep disturbances, such as insomnia.

Systemic corticosterone enhances fear memory extinction in rats: Involvement of the infralimbic medial prefrontal cortex GABAA and GABAB receptors.

The infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) regulates the extinction of conditioned fear memory. Glucocorticoid and gamma-aminobutyric acid (GABA) receptors are expressed in the mPFC and are also critical in fear extinction. This study investigated the possible interactive effects of the glucocorticoids and GABAergic system in the IL on the regulation of fear extinction. The rats were trained using an auditory fear conditioning task during which they received three conditioned stimuli (tones, 30s, 4kHz, 80dB), co-terminated with the three unconditioned stimuli (footshock, 0.8mA, 1s). Extinction testing was conducted over 3 days (Ext 1-3). Thirty minutes before the first extinction trial (Ext 1), the rats received bicuculline (BIC, 1mg/kg/2mL, intraperitoneal [i.p.]) as a GABAA receptor antagonist or CGP55845 (CGP, 0.1mg/kg/2ML, i.p.) as a GABAB receptor antagonist followed by systemic injection of corticosterone (CORT, 3mg/kg/2ML, i.p.). Furthermore, separate groups of rats received a bilateral intra-IL injection of BIC (100ng/0.3µL/side) or CGP (10ng/0.3µL/side) followed by a systemic injection of CORT (3mg/kg/2ML, i.p.) before the first extinction trial (Ext 1). The extracellular signal-regulated kinase (ERK1) and cAMP response element-binding (CREB) activity in the IL was examined by Western blot analysis after Ext 1. The results indicated that systemic CORT injection facilitated fear extinction and increased the expression of ERK1 but not CREB in the IL. Both systemic and intra-IL co-injection of BIC or CGP blocked the effects of CORT on fear extinction and ERK1 expression. These findings suggest that glucocorticoids and the GABAergic system may modulate fear extinction through the ERK pathway in the IL.

Identification of the GABARAP binding determinant in PI4K2A.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion. Here we identify a 7-amino acid segment within the PI4K2A catalytic domain that contains the GABARAP interaction motif (GIM). This segment resides in an exposed loop that is not conserved in the other mammalian Type II PI 4-kinase, PI4K2B, explaining the specificity of GABARAP binding to the PI4K2A isoform. Mutation of the PI4K2A GIM inhibits GABARAP binding and PI4K2A-mediated recruitment of cytosolic GABARAP to subcellular organelles. We further show that GABARAP binds to mono-phosphorylated phosphoinositides, PI3P, PI4P, and PI5P, raising the possibility that these lipids contribute to the binding energies that drive GABARAP-protein interactions on membranes.

Diminished light sensitivities of ON alpha retinal ganglion cells observed in a mouse model of hyperglycemia

This study aimed to investigate potential functional changes in retinal ganglion cells (RGCs) in a mouse model of hyperglycemia and explore possible therapeutic approaches. Hyperglycemia resembling type 1 diabetes mellitus (DM) was induced in C57BL/6 mice through intraperitoneal injection of streptozotocin (STZ). Blood glucose levels were confirmed to be elevated after 1 week and 4 weeks of injection. Mice with blood glucose levels above 350 mg/mL after 4 weeks of one-dose STZ injection were considered hyperglycemic. The light sensitivity of ON alpha (α) retinal ganglion cells (RGCs), not OFF αRGCs, was reduced in the hyperglycemic mouse model. The number of apoptotic cells, RGCs, and amacrine cells (ACs) remained unaffected at this stage. Similarly, the eletroretinogram (ERG) and optokinetic test results showed no significant differences. The application of picrotoxin (PTX) to block GABA receptors could increase the light sensitivity of ON αRGCs by 1 log unit in hyperglycemic mice. The results show that ON αRGCs may be more susceptible to microenvironmental changes caused by hyperglycemia than OFF αRGCs. This decline in light sensitivity may occur before cell apoptosis during the early stages of the hyperglycemic mouse model but has the potential to be reversed.

Shank3 deficiency alters midbrain GABAergic neuron morphology, GABAergic markers and synaptic activity in primary striatal neurons

Abnormalities in gamma-aminobutyric acid (GABA)ergic neurotransmission play a role in the pathogenesis of autism, although the mechanisms responsible for alterations in specific brain regions remain unclear. Deficits in social motivation and interactions are core symptoms of autism, likely due to defects in dopaminergic neural pathways. Therefore, investigating the morphology and functional roles of GABAergic neurons within dopaminergic projection areas could elucidate the underlying etiology of autism. The aim of this study was to (1) compare the morphology and arborization of glutamate decarboxylase (GAD)-positive neurons from the midbrain tegmentum; (2) evaluate synaptic activity in primary neurons from the striatum; and (3) assess GABAergic postsynaptic puncta in the ventral striatum of wild-type (WT) and Shank3-deficient mice. We found a significant decrease in the number of short neurites in GAD positive primary neurons from the midbrain tegmentum in Shank3-deficient mice. The application of a specific blocker of GABAA receptors (GABAAR) revealed significantly increased frequency of spontaneous postsynaptic currents (sPSCs) in Shank3-deficient striatal neurons compared to their WT counterparts. The mean absolute amplitude of the events was significantly higher in striatal neurons from Shank3-deficient compared to WT mice. We also observed a significant reduction in gephyrin/GABAAR γ2 colocalization in the striatum of adult male Shank3-deficient mice. The gene expression of collybistin was significantly lower in the nucleus accumbens while gephyrin and GABAAR γ2 were lower in the ventral tegmental area (VTA) in male Shank3-deficient compared to WT mice. In conclusion, Shank3 deficiency leads to alterations in GABAergic neurons and impaired GABAergic function in dopaminergic brain areas. These changes may underlie autistic symptoms, and potential interventions modulating GABAergic activity in dopaminergic pathways may represent new treatment modality.

Increases in 4-Acetaminobutyric Acid Generated by Phosphomevalonate Kinase Suppress CD8+ T Cell Activation and Allow Tumor Immune Escape.

Certain metabolites in the tumor microenvironment (TME) can alter innate immunity. Here, it is shown how phosphomevalonate kinase (PMVK) allows hepatocellular carcinoma (HCC) cells to overcome the anti-tumor immunity mediated by CD8+ T cells. In HCCs, depletion of PMVK is required to facilitate CD8+ T cell activation and their subsequent suppression of tumor growth. Mechanistically, PMVK phosphorylates and stabilizes glutamate decarboxylase 1 (GAD1), thus increasing the synthesis of γ-aminobutyric acid (GABA), which normally functions as a neurotransmitter. However, PMVK also recruits acetyl-CoA acetyltransferase 1 (ACAT1) and allows it to convert GABA, to 4-acetaminobutyric acid (4-Ac-GABA), which is released into the TME. There, 4-Ac-GABA activates the GABAA receptor (GABAAR) on CD8+ T cells, which inhibits AKT1 signaling. This in turn suppresses CD8+ T cell activation, intratumoral infiltration, and the anti-tumor response. Inhibiting PMVK or GABAAR in HCC mouse models overcomes resistance to anti-PD-1 immune checkpoint therapy. These findings reveal non-canonical and cooperative functions among the key metabolic enzymes PMVK, GAD1, and ACAT1 that reprogram glutamine metabolism to synthesize a potent CD8+ T cell inhibitor 4-Ac-GABA. Blocking 4-Ac-GABA signaling in CD8+ T cells, particularly when combined with immune checkpoint inhibition, potentially represents a new and potent form of immunotherapy.

The neurotensin receptor 1 agonist PD149163 alleviates visceral hypersensitivity and colonic hyperpermeability in rat irritable bowel syndrome model.

An impaired intestinal barrier with the activation of corticotropin-releasing factor (CRF), Toll-like receptor 4 (TLR4), and proinflammatory cytokine signaling, resulting in visceral hypersensitivity, is a crucial aspect of irritable bowel syndrome (IBS). The gut exhibits abundant expression of neurotensin; however, its role in the pathophysiology of IBS remains uncertain. This study aimed to clarify the effects of PD149163, a specific agonist for neurotensin receptor 1 (NTR1), on visceral sensation and gut barrier in rat IBS models. The visceral pain threshold in response to colonic balloon distention was electrophysiologically determined by monitoring abdominal muscle contractions, while colonic permeability was measured by quantifying absorbed Evans blue in colonic tissue invivo in adult male Sprague-Dawley rats. We employed the rat IBS models, i.e.,lipopolysaccharide (LPS)- and CRF-induced visceral hypersensitivity and colonic hyperpermeability, and explored the effects of PD149163. Intraperitoneal PD149163 (160, 240, 320 μg kg-1) prevented LPS (1 mg kg-1, subcutaneously)-induced visceral hypersensitivity and colonic hyperpermeability dose-dependently. It also prevented the gastrointestinal changes induced by CRF (50 μg kg-1, intraperitoneally). Peripheral atropine, bicuculline (a GABAA receptor antagonist), sulpiride (a dopamine D2 receptor antagonist), astressin2-B (a CRF receptor subtype 2 [CRF2] antagonist), and intracisternal SB-334867 (an orexin 1 receptor antagonist) reversed these effects of PD149163 in the LPS model. PD149163 demonstrated an improvement in visceral hypersensitivity and colonic hyperpermeability in rat IBS models through the dopamine D2, GABAA, orexin, CRF2, and cholinergic pathways. Activation of NTR1 may modulate these gastrointestinal changes, helping to alleviate IBS symptoms.

Zona Incerta GABAergic Neurons Facilitate Emergence from Isoflurane Anesthesia in Mice.

The zona incerta (ZI) predominantly consists of gamma-aminobutyric acid (GABAergic) neurons, located adjacent to the lateral hypothalamus. GABA, acting on GABAA receptors, serves as a crucial neuromodulator in the initiation and maintenance of general anesthesia. In this study, we aimed to investigate the involvement of ZI GABAergic neurons in the general anesthesia process. Utilizing in-vivo calcium signal optical fiber recording, we observed a decrease in the activity of ZI GABAergic neurons during isoflurane anesthesia, followed by a significant increase during the recovery phase. Subsequently, we selectively ablated ZI GABAergic neurons to explore their role in general anesthesia, revealing no impact on the induction of isoflurane anesthesia but a prolonged recovery time, accompanied by a reduction in delta-band power in mice under isoflurane anesthesia. Finally, through optogenetic activation/inhibition of ZI GABAergic neurons during isoflurane anesthesia, we discovered that activation of these neurons facilitated emergence without affecting the induction process, while inhibition delayed emergence, leading to fluctuations in delta band activity. In summary, these findings highlight the involvement of ZI GABAergic neurons in modulating the emergence of isoflurane anesthesia.