GABAA Activation Research Articles

Chronic Intermittent Hypoxia Alters GABAergic Inhibition in Median Preoptic Nucleus (MnPO) Neurons of Rats

Chronic intermittent hypoxia (CIH) is an animal model simulating the hypoxemia associated with obstructive sleep apnea (OSA) in humans. Male rats exposed to CIH exhibit increased sympathetic tone and persistent elevations in blood pressure similar to that observed in patients with. In MnPO neurons, CIH reduces GABAa inhibition and, in some neurons, GABAa activation produces excitation. Here, loose patch and perforated patch recordings were conducted in MnPO neurons in response to optogenetic stimulation of GABAergic SFO (efferent) and MnPO (local) neurons to assess the role of GABAergic signaling in these nuclei on MnPO excitability. Our working hypothesis was that CIH would diminish the inhibitory effects of optogenetic stimulation of GABA neurons. This hypothesis was tested using an AAV vector that selectively expresses channel rhodopsin in GABA neurons Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received infusions (0.2 μL) of pAAV-mDlx-ChR2-mCherry-Fishell-3 in the SFO or MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hr) or normoxia (Norm). CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For in vitro recordings, rats were anesthetized with isoflurane (2-3%), and sagittal or coronal slices (300 μm) containing MnPO were cut using standard slice procedures. Voltage-clamp loose patch or perforated patch recordings using gramicidin (100 ug/ml in 140 mM K-gluconate) were collected in MnPO neurons in response to optogenetic stimulation of GABAergic neurons (2 ms pulse, 20 Hz, 1 sec). MnPO neurons from CIH-treated rats showed differing responses to GABAergic stimulation while MnPO neurons from Norm-treated rats predominantly exhibited GABAergic inhibition. MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH-treated rats show variable mIPSC frequencies and heterogeneous responses to GABAergic neuronal activation in the SFO and MnPO. The results demonstrate that CIH alters the Cl− homeostasis in some MnPO neurons altering their response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH. Supported by R01 HL155977. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phenols and GABAA receptors: from structure and molecular mechanisms action to neuropsychiatric sequelae.

γ-Aminobutyric acid type A receptors (GABAARs) are members of the pentameric ligand-gated ion channel (pLGIC) family, which are widespread throughout the invertebrate and vertebrate central nervous system. GABAARs are engaged in short-term changes of the neuronal concentrations of chloride (Cl-) and bicarbonate (HCO3 -) ions by their passive permeability through the ion channel pore. GABAARs are regulated by various structurally diverse phenolic substances ranging from simple phenols to complex polyphenols. The wide chemical and structural variability of phenols suggest similar and different binding sites on GABAARs, allowing them to manifest themselves as activators, inhibitors, or allosteric ligands of GABAAR function. Interest in phenols is associated with their great potential for GABAAR modulation, but also with their subsequent negative or positive role in neurological and psychiatric disorders. This review focuses on the GABAergic deficit hypotheses during neurological and psychiatric disorders induced by various phenols. We summarize the structure-activity relationship of general phenol groups concerning their differential roles in the manifestation of neuropsychiatric symptoms. We describe and analyze the role of GABAAR subunits in manifesting various neuropathologies and the molecular mechanisms underlying their modulation by phenols. Finally, we discuss how phenol drugs can modulate GABAAR activity via desensitization and resensitization. We also demonstrate a novel pharmacological approach to treat neuropsychiatric disorders via regulation of receptor phosphorylation/dephosphorylation.

Molecular Mechanistic Insight Spirulina as Anti-Stress Agent

When under stress, our body reacts by releasing hormones and triggering the nervous system. The hypothalamic-pituitary-adrenal axis, which transmits the stress response, is in charge of the neuroendocrine adaptation of the stress response. Corticotrophin releasing hormone neurons control hypothalamic-pituitary-adrenal function. Increased. Many metabolic and neurological problems linked to stress are associated with the synthesis of corticotrophin releasing hormone. Gamma-aminobutyric acid (GABA) and GABAergic inhibition both play a significant role in controlling the activity of CRH neurons. Several synthetic medicines that target the GABAa receptor and activate GABAa activity are used to lessen the effects of stress. These treatments have relaxing effects but also come with a variety of negative effects. As they have no adverse effects and are harmless, anti-stress herbs are utilised as an alternative therapy to help the adrenal system. Spirulina platensis (commonly referred to as ‘Spirulina’), are well for their Immune system modulation; anti-viral activity; cancer preventive properties, and cardiovascular benefits. In present work an attempt had been made to evaluate the anti-stress potential of active constituent of spirulina by molecular docking.

Molecular Mechanistic Insight Spirulina as Anti-stress Agent

When under stress, our body reacts by releasing hormones and triggering the nervous system. The hypothalamic-pituitary-adrenal axis, which transmits the stress response, is in charge of the neuroendocrine adaptation of the stress response. Corticotrophin releasing hormone neurons control hypothalamic-pituitary-adrenal function Increased. Many metabolic and neurological problems linked to stress are associated with the synthesis of corticotrophin releasing hormone. Gamma-aminobutyric acid (GABA) and GABAergic inhibition both play a significant role in controlling the activity of CRH neurons. Several synthetic medicines that target the GABAa receptor and activate GABAa activity are used to lessen the effects of stress. These treatments have relaxing effects but also come with a variety of negative effects. As they have no adverse effects and are harmless, anti-stress herbs are utilised as an alternative therapy to help the adrenal system. Spirulina platensis (commonly referred to as ‘Spirulina’), are well for their Immune system modulation; anti-viral activity; cancer preventive properties, and cardiovascular benefits. In present work an attempt had been made to evaluate the anti-stress potential of active constituent of spirulina by molecular docking.

Commonly Used Therapeutics Associated with Changes in Arousal Inhibit GABAAR Activation.

GABAA receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABAA receptor negative allosteric modulators (GABAARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABAARNAMs can restore the Excitation/Inhibition (E/I) balance in vitro and arousal in vivo, we chose to screen 11 compounds that have been reported to modulate arousal, to see if they shared a GABA-related mechanism. We determined modulation with both conventional and microfluidic patch clamp methods. We found that receptor activation was variably modulated by all 11 compounds: Rifampicin (RIF), Metronidazole (MET), Minocycline (MIN), Erythromycin (ERY), Ofloxacin (OFX), Chloroquine (CQ), Hydroxychloroquine sulfate (HCQ), Flumazenil (FLZ), Pentylenetetrazol (PTZ), (-)-Epigallocatechin Gallate (EGCG), and clarithromycin (CLR). The computational modeling of modulator-receptor interactions predicted drug action at canonical binding sites and novel orphan sites on the receptor. Our findings suggest that multiple avenues of investigation are now open to investigate large and brain-penetrant molecules for the treatment of patients with diminished CNS E/I balance.

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.

Repeated Sevoflurane Exposure in Neonatal Rats Enhances the Sensitivity to Pain and Traumatic Stress Later in Juvenile Life.

PurposeːSevoflurane exposure in the neonatal period of rodent animals was reported to be associated with neuroendocrine dysregulations later in life. We tested the hypothesis that repeated sevoflurane exposure in neonatal rats enhances the sensitivity to pain and acute traumatic stress response later in juvenile life and investigated whether the neonatal brain depolarizing γ-aminobutyric acid type A receptor (GABAAR) activity is involved in mediating these abnormalities.MethodsːThe postnatal 6 days (P6) Sprague-Dawley male rat pups pretreated with vehicle or the NKCC1 inhibitor, bumetanide, received sequential exposures to 2.1% sevoflurane exposure for 2 hours daily in 3 consecutive days.ResultsːThe results showed that repeated exposures to sevoflurane in neonatal rats significantly reduced the paw withdrawal thermal latency (PWTL) at P9, P45. Repeated exposures to sevoflurane in neonatal rats did not significantly affect the basal secretion of serum corticosterone at juvenile period P45, whereas the level of corticosterone for neonatal sevoflurane-exposed rats at P45 was significantly higher than the CON group after subject to conditioned fear traumatic stress (CFTS). The resulting NKCC1/KCC2 mRNA ratio was significantly increased immediately after the neonatal rats received the last sevoflurane exposure, which was alleviated by pretreated with the NKCC1 inhibitor bumetanide.ConclusionːRepeated exposures to sevoflurane in neonatal rats enhanced the sensitivity to pain and acute traumatic stress response in juvenile life. The neonatal brain depolarizing GABAAR activity is involved in mediating these abnormalities.

Repeated Sevoflurane Exposures in Neonatal Rats Increased the Brain Vulnerability to Future Stress Exposure and Resulted in Fear Extinction Deficit.

Sevoflurane anesthesia during neonatal period was reported to sensitize the rodent animals to stress later in life. The authors tested the hypothesis that repeated sevoflurane exposures in neonatal rats increased the brain vulnerability to future stress exposure and resulted in fear extinction deficit and investigated whether the neonatal brain depolarizing γ-aminobutyric acid type A receptor (GABAAR) is involved in mediating these abnormalities. Neonatal Sprague-Dawley male rats, pretreated with vehicle or the NKCC1 inhibitor, bumetanide, received sequential exposures to 3% sevoflurane for 2h on postnatal days (P) 5, P6, and P7 and then were exposed to electric foot shock stress in fear conditioning training at P14. Juvenile rats at different developmental brain stage receiving identical sevoflurane exposures on P25, P26, and P27 were also studied. The results showed repeated sevoflurane exposures in neonatal rats and increased the cation-chloride cotransporters NKCC1/KCC2 ratio in the PFC at P14. Repeated exposures to sevoflurane in neonatal rather than juvenile rats enhanced the stress response and exacerbated neuroapoptosis in the PFC after exposed to electricfoot shock in fear conditioning training. Neonatal rather than juvenile sevoflurane-exposed rats exhibited deficits in fear extinction training and recall. Pretreatment of neonatal rats prior to sevoflurane exposures with bumetanide reduced the NKCC1/KCC2 ratio at P14 and ameliorated most of the subsequent adverse effects. Our study indicates that repeated sevoflurane exposures in neonatal rats might increase the brain vulnerability to future stress exposure and resulted in fear extinction deficit, which might be associated with the neonatal enhanced brain depolarizing GABAAR activity.

Stimulation of GABA Receptors in the Lateral Septum Rapidly Elicits Food Intake and Mediates Natural Feeding

The increasing prevalence of obesity and eating disorders makes identifying neural substrates controlling eating and regulating body weight a priority. Recent studies have highlighted the role of the lateral septum (LS) in eating control mechanisms. The current study explored the roles of gamma-aminobutyric acid (GABA) receptors within the LS in the control of food intake. Experiments with a rat model (n ≥ 11/group) showed that LS microinjection of the GABAA receptor agonist, muscimol, and the GABAB receptor agonist, baclofen hydrochloride (baclofen), elicited intense, dose-dependent feeding. In contrast, LS pretreatment with the GABAA receptor antagonist, picrotoxin, markedly reduced the muscimol-elicited feeding, and pretreatment injections with the GABAB receptor antagonist, 2-hydroxysaclofen (2-OH saclofen), reduced the baclofen evoked response. Next, we showed that picrotoxin injection at the beginning of the dark phase of the light-dark cycle—when rats show a burst of spontaneous eating—reduced naturally occurring feeding, whereas 2-OH saclofen was ineffective. These results indicate that the activation of LS GABAA and GABAB receptors strongly stimulates feeding and suggests potential roles in feeding control neurocircuitry. In particular, our evidence indicates that endogenous LS GABA and GABAA receptors may be involved in mediating naturally occurring nocturnal feeding.

Unexpected Effect of IL-1β on the Function of GABAA Receptors in Pediatric Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) type II is an epileptogenic malformation of the neocortex, as well as a leading cause of drug-resistant focal epilepsy in children and young adults. The synaptic dysfunctions leading to intractable seizures in this disease appear to have a tight relationship with the immaturity of GABAergic neurotransmission. The likely outcome would include hyperpolarizing responses upon activation of GABAARs. In addition, it is well-established that neuroinflammation plays a relevant role in the pathogenesis of FCD type II. Here, we investigated whether IL-1β, a prototypical pro-inflammatory cytokine, can influence GABAergic neurotransmission in FCD brain tissues. To this purpose, we carried out electrophysiological recordings on Xenopus oocytes transplanted with human tissues and performed a transcriptomics analysis. We found that IL-1β decreases the GABA currents amplitude in tissue samples from adult individuals, while it potentiates GABA responses in samples from pediatric cases. Interestingly, these cases of pediatric FCD were characterized by a more depolarized EGABA and an altered transcriptomics profile, that revealed an up-regulation of chloride cotransporter NKCC1 and IL-1β. Altogether, these results suggest that the neuroinflammatory processes and altered chloride homeostasis can contribute together to increase the brain excitability underlying the occurrence of seizures in these children.

Postnatal GABAA Receptor Activation Alters Synaptic Plasticity and Cognition in Adult Wistar Rats.

Early life alteration in the activity of gamma-aminobutyric acid (GABA) receptors is associated with long-lasting developmental effects on the brain and behavior. GABAA receptors act as excitatory rather than inhibitory in neonates. Excessive activation of GABAA receptors during the early postnatal period may affect cognitive functions later in life. In this study, we sought to determine whether neonatal activation of GABAA receptors with muscimol can alter the electrophysiology profile of hippocampal CA1 neurons and spatial learning and memory in adult rats. Male and female Wistar rat pups received a subcutaneous injection of either saline or muscimol (500µg/kg) on postnatal days (PND) 7, 9, and 11 and then underwent different electrophysiology and behavioral experiments in adulthood. Early life treatment with muscimol did not alter the basic synaptic transmission but significantly reduced the paired-pulse facilitation (PPF) in the CA1 area. Neonatal application of muscimol led to a pronounced decrease in long-term potentiation (LTP) and long-term depression (LTD) in CA1 neurons along with a declined theta-burst responses in both sexes. We obtained some evidence that neonatal GABAA activation leads to reduced brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex. Our electrophysiology data was supported with spatial reference and working memory deficits in rats. This study provides the first detailed description of altered electrophysiology in hippocampal CA1 neurons in adult rats undergone GABAA activation early in life.

Axonal GABAA stabilizes excitability in unmyelinated sensory axons secondary to NKCC1 activity.

GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA receptor. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to Na-K-Cl cotransporter type 1 (NKCC1) loading of axonal chloride. Activation of axonal GABAA receptor stabilized C-fibre excitability during prolonged low frequency (2.5Hz) firing. NKCC1maintains intra-axonal chloride to provide feed-forward stabilization of C-fibre excitability and thus support sustained firing. GABAA receptor (GABAA R)-mediated depolarization of dorsal root ganglia (DRG) axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. In the present study, we report that the peripheral axons of DRG neurons are also depolarized by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R β3subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5-2.5Hz). However, during Na-K-Cl cotransporter type 1 (NKCC1) blockade, the electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (activity-dependent slowing; ADS) was used to quantify C-fibre excitability loss during a 2.5Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, β3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilizes C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilized by GABAA R to stabilize axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres.

Homosynaptic frequency-dependent depression by release site inactivation at neonatal hippocampal synapses in the stratum lacunosum-moleculare.

When activated at low frequencies (0.1-1 Hz), second postnatal week synapses onto the most distal part of the apical dendritic tree (stratum lacunosum-moleculare) of rat hippocampal CA1 pyramidal cells display a frequency-dependent synaptic depression not observed for the more proximal (stratum radiatum) synapses. Depression in this frequency range is thought of as a possible contributor to behavioural habituation. In fact, in contrast to the proximal synapses, the distal synapses provide more direct sensory information from the entorhinal cortex as well as from thalamic nuclei. The use of antagonists showed that the activation of GABAA , GABAB , NMDA, mGlu, kainate, adenosine, or endocannabinoid receptors was not directly involved in the depression, indicating it to be intrinsic to the synapses themselves. While the depression affected paired-pulse plasticity in a manner indicating a decrease in vesicle release probability, the depression could not be explained by a stimulus-dependent decrease in calcium influx. Despite affecting the synaptic response evoked by brief high-frequency stimulation (10 impulses, 20 Hz) in a manner indicating vesicle depletion, the depression was unaffected by large variations in release probability. The depression was found not only to affect the synaptic transmission at low frequencies (0.1-1 Hz) but also to contribute to the depression evolving during brief high-frequency stimulation (10 impulses, 20 Hz). We propose that a release-independent process directly inactivating release sites with a fast onset (ms) and long duration (up to 20 s) underlies this synaptic depression.

059 Daridorexant: A dual, equipotent, and insurmountable antagonist of both orexin-1 and orexin-2 receptors

Abstract Introduction The orexin neuropeptide–receptor system is a central sleep and wake regulator in the brain. The two orexin receptor subtypes, OX1R and OX2R, are expressed either alone or together in all major wake-promoting brain areas. OX1R and OX2R activation by orexins causes elevation of intracellular calcium, which enhances synaptic transmission in secondary, monoaminergic wake- and arousal-promoting neurotransmitter circuits. Orexin receptor antagonists represent a novel and specific treatment of insomnia, which is different from classical therapy that more broadly inhibits brain activity via GABAA activation. Here we describe the molecular pharmacology of daridorexant, an orexin receptor antagonist which has proven highly effective in improving sleep and daytime functioning in insomnia patients. Methods Orexin-A(OxA)-induced calcium release assays in OX1R- and OX2R-expressing recombinant cell lines were applied to measure the antagonistic potency and kinetic properties of daridorexant in functional assays. Whole-cell competitive binding assays, using an orthosteric tracer were employed to determine the Ki of daridorexant. Comparisons were made with suvorexant and lemborexant. Results In OxA-induced calcium release assays with 2-h pre-incubation time, daridorexant displayed apparent Kb values of 0.5 nM (OX1R) and 0.8 nM (OX2R) with insurmountable antagonism on both receptors, demonstrating equipotent and highly effective functional inhibition of both receptor subtypes. On-target residence times of daridorexant (37oC) expressed as receptor occupancy half-lives (ROt1/2) were 4 min (OX1R) and 8 min (OX2R). In binding assays, daridorexant behaved as highly potent orthosteric antagonist. Also suvorexant behaved as dual insurmountable antagonist at OX1R/OX2R (appKb=0.7nM/1.0nM; ROt1/2=9 min/6 min) and as potent orthosteric antagonist in binding assays. Interestingly, lemborexant displayed a different interaction profile at OX1R/OX2R (appKb=13nM/0.4nM, ROt1/2<2min/<2min), i.e. it behaved as preferential OX2R antagonist with a very short on-target residence time and little insurmountability. Conclusion Daridorexant displays the desired target interaction profile of a dual, equipotent, and insurmountable antagonist of both OX1R and OX2R, which ensures equally efficient inhibition of both arousal-/wake-promoting receptor subtypes. Daridorexant′s on-target residence times are long enough to cause insurmountable inhibition, but short enough to avoid pharmacodynamic effects after drug elimination. Support (if any) Funded by Idorsia Pharmaceuticals Ltd.

Diminished inhibitory efficacy of GABA on PVN neurons in AngII‐salt hypertension reflects a KCC2‐independent increase of intracellular Cl‐ concentration

The hypothalamic PVN receives glutamatergic excitation from sodium and AngII sensing regions of the forebrain along with dense GABAergic inhibition from the surrounding peri-nuclear zone (PNZ). Previously, we found that synaptic glutamate release in PVN enhances GABA inhibition, but whether forebrain inputs can do so in the setting of hypertension (HTN) is unknown. Studies indicate that a high salt diet alone does not raise mean arterial pressure (MAP), while AngII treatment raises MAP in proportion to the level of salt intake. This suggests that whereas excitatory inputs to PVN driven by high salt intake alone cannot overcome PVN GABA inhibition, AngII driven inputs can. The notion that GABA inhibition of PVN is strengthened forebrain inputs in AngII-salt HTN is contrary to a prior report that GABA inhibition in AngII-salt HTN is reduced. To reconcile these seemingly disparate findings, we compared mechanisms regulating PVN GABA inhibition in mice consuming a normal salt (0.4% NaCl) diet (NSD), a high salt (4% NaCl) diet (HSD) or a HSD with AngII (600 ng/kg/min, s.c.) infusion (AngII-salt HTN). Using gramicidin perforated patch recordings, we determined that the GABA equilibrium potential (EGABA) of PVN neurons was not different between NSD (-67.2 ± 6.3 mV, n=3 cells) and HSD (-53.7 ± 15.6 mV, n=4 cells) mice, but was significantly depolarized in mice with AngII-salt HTN (-32.9 ± 8.8 mV, n=6 cells)(P=0.0033). This indicates that in AngII-salt HTN the inhibitory effect of GABA on PVN neurons decreases as [Cl-]i increases. Normally, [Cl-]i is held low by activities of KCC2 and NKCC1, which promote neuronal Cl- efflux and influx, respectively. In brain slices, KCC2 inhibition (VU0463271, 10 µM) rapidly depolarized PVN EGABA, consistent with blunting of GABA inhibition. Accordingly, PVN inhibition of KCC2 (VU0463271, 100 pmol, 25 nL) in vivo increased renal SNA, suggesting GABA was switched from an inhibitory to an excitatory transmitter. Consistent with this, PVN GABA-AR activation (muscimol, 50 pmol, 25 nL) during KCC2 inhibition increased renal SNA. Thus, KCC2 appears critical for maintaining low [Cl-]i and a hyperpolarized EGABA in PVN. In light of this, it would seem likely that elevated PVN [Cl-]i in AngII-salt HTN reflects a relative loss of KCC2 Cl- efflux. To investigate this, western blot analysis of PVN punches was performed and revealed that expression of KCC2 and NKCC1 was increased and decreased, respectively, both in mice consuming a HSD alone and mice with AngII-salt HTN. These changes were unexpected as they are predicted to drive [Cl-]i low, not high. Therefore, expression changes appear aimed at buffering elevated [Cl-]i caused by an alternative mechanism, possibly excess Cl- influx through GABA-AR, reflecting increased, not decreased, GABA release in AngII-salt HTN. Similar expression changes in HSD mice suggests that high salt intake generates a permissive state of Cl- ionic plasticity that blunts PVN GABA inhibition and facilitates AngII-salt HTN.

Phytohormone abscisic acid boosts pentobarbital-induced sleep through activation of GABA-A, PPARβ and PPARγ receptor signaling.

Sleep disorders induce anxiety and forgetfulness and change habits. The chemical hypnotic drugs currently used have serious side effects and, therefore, people are drawn towards using natural compounds such as plant-based healing agents. Abscisic acid (ABA) is produced in a variety of mammalian tissues and it is involved in many neurophysiological functions. To investigate the possible effect of ABA on pentobarbital-induced sleep and its possible signaling through GABA-A and PPAR (γ and β) receptors, in male Wistar rats. The possible effect of ABA (5 and 10 µg/rat, intracerebroventricularly) on sleep onset latency time and duration was evaluated in a V-maze model of sleep. Pentobarbital sodium (40 mg/kg, intraperitoneally) was injected to induce sleep 30 min after administration of ABA. PPARβ (GSK0660, 80 nM/rat), PPARγ (GW9662, 3 nM/rat) or GABA-A receptor (bicuculline, 6 µg/rat) antagonists were given 15 min before ABA injection. Diazepam (2 mg/kg, intraperitoneally) was used as a positive control group. ABA at 5 µg significantly boosted the pentobarbital-induced subhypnotic effects and promoted induction of sleep onset in a manner comparable to diazepam treatment. Furthermore, pretreatment with bicuculline significantly abolished the ABA effects on sleep parameters, while the amplifying effects of ABA on the induction of sleep onset was not significantly affected by PPARβ or PPARγ antagonists. The sleep prolonging effect of ABA was significantly prevented by both PPAR antagonists. The data showed that ABA boosts pentobarbital-induced sleep and that GABA-A, PPARβ and PPARγ receptors are, at least in part, involved in ABA signaling.

Progesterone's Effects on Cognitive Performance of Male Mice Are Independent of Progestin Receptors but Relate to Increases in GABAA Activity in the Hippocampus and Cortex.

Progestogens' (e.g., progesterone and its neuroactive metabolite, allopregnanolone), cognitive effects and mechanisms among males are not well-understood. We hypothesized if progestogen's effects on cognitive performance are through its metabolite allopregnanolone, and not actions via binding to traditional progestin receptors (PRs), then progesterone administration would enhance performance in tasks mediated by the hippocampus and cortex, coincident with increasing allopregnanolone concentrations, brain derived neurotrophic factor (BDNF) and/or muscimol binding of PR knock out (PRKO) and wild-type PR replete mice. Experiment 1: Progesterone (4 mg/kg, subcutaneously (SC; n = 12/grp), or oil vehicle control, was administered to gonadally-intact adult male mice PRKO mice and their wild-type counterparts and cognitive behaviors in object recognition, T-maze and water maze was examined. Progesterone, compared to vehicle, when administered post-training increased time investigating novel objects by the PRKO and wild-type mice in the object recognition task. In the T-maze task, progesterone administration to wild-type and PRKO mice had significantly greater number of spontaneous alternations compared to their vehicle-administered counterparts. In the water maze task, PRKO mice administered vehicle spent significantly fewer seconds in the quadrant associated with the escape platform on testing compared to all other groups. Experiment 2: Progesterone administered to wild-type and PRKO mice increased plasma progesterone and allopregnanolone levels (n = 5/group). PRKO mice had higher allopregnanolone levels in plasma and hippocampus, but not cortex, when administered progesterone and compared to wild-type mice. Experiment 3: Assessment of PR binding revealed progesterone administered wild-type mice had significantly greater levels of PRs in the hippocampus and cortex, compared to all other groups (n = 5/group). Wild-type mice administered progesterone, but not vehicle, had increased BDNF levels in the hippocampus, but not the cortex, compared to PRKOs. Wild-type as well as PRKO mice administered progesterone experienced significant increases in maximal GABAA agonist, muscimol, binding in hippocampus and cortex, compared to their vehicle-administered counterparts. Thus, adult male mice can be responsive to progesterone for cognitive performance, and such effects may be independent of PRs trophic actions of BDNF levels in the hippocampus and/or increases in GABAA activity in the hippocampus and cortex.

Prolactin enhances hippocampal synaptic plasticity in female mice of reproductive age

Dynamic signaling between the endocrine system (ES) and the nervous system (NS) is essential for brain and body homeostasis. In particular, reciprocal interaction occurs during pregnancy and motherhood that may involve changes in some brain plasticity processes. Prolactin (PRL), a hormone with pleiotropic effects on the NS, promotes maternal behavior and has been linked to modifications in brain circuits during motherhood; however, it is unclear whether PRL may regulate synaptic plasticity. Therefore, the main aim of the present work was to determine the cellular and molecular mechanisms triggered by PRL that regulate synaptic plasticity in the hippocampus. By analyzing extracellular recordings in CA3‐CA1 synapses of hippocampal slices, we report that PRL modifies short and long‐term synaptic plasticity in female mice of reproductive age, but not in sexually immature females or adult males. This effect is carried out through mechanisms that include participation of GABAA receptors and activation of the JAK2‐mediated signaling pathway. These findings show for the first time how PRL enhances the synaptic strength in hippocampal circuits and that this effect is sexually dimorphic, which would influence complex brain processes in physiological conditions like pregnancy and lactation.

White matter damage and altered connectivity between primary motor cortices in chronic obstructive pulmonary disease.

White matter damage and altered connectivity between primary motor cortices in chronic obstructive pulmonary disease.

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity

Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. Invivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.