Mapping responses to focal injections of bicuculline in the lateral parafacial region identifies core regions for maximal generation of active expiration.

The lateral parafacial area (pFL) is a crucial region involved in respiratory control, particularly in generating active expiration through an expiratory oscillatory network. Active expiration involves rhythmic abdominal (ABD) muscle contractions during late-expiration, increasing ventilation during elevated respiratory demands. The precise anatomical location of the expiratory oscillator within the ventral medulla's rostro-caudal axis is debated. While some studies point to the caudal tip of the facial nucleus (VIIc) as the oscillator's core, others suggest more rostral areas. Our study employed bicuculline (a γ-aminobutyric acid type A [GABA-A] receptor antagonist) injections at various pFL sites (-0.2 mm to +0.8 mm from VIIc) to investigate the impact of GABAergic disinhibition on respiration. These injections consistently elicited ABD recruitment, but the response strength varied along the rostro-caudal zone. Remarkably, the most robust and enduring changes in tidal volume, minute ventilation, and combined respiratory responses occurred at more rostral pFL locations (+0.6/+0.8 mm from VIIc). Multivariate analysis of the respiratory cycle further differentiated between locations, revealing the core site for active expiration generation with this experimental approach. Our study advances our understanding of neural mechanisms governing active expiration and emphasizes the significance of investigating the rostral pFL region.

Optimizing GABAA receptor antagonism for the induction of long-term potentiation in the mouse and rat dentate gyrus in vitro.

The reliable induction of long-term potentiation (LTP) in the dentate gyrus (DG) in vitro requires the blockade of the γ-aminobutyric acid A (GABAA) receptor. In these studies we examined the effectiveness of the specific GABAA receptor antagonist bicuculline methiodide (BMI) in facilitating LTP in the DG from hippocampal slices obtained from either C57Bl/6 mice or Sprague-Dawley rats, two species commonly used for electrophysiology. In the C57Bl/6 mice, maximal short-term potentiation and LTP in the DG were produced with a concentration of 5 µM BMI. In contrast, a concentration of 10 µM BMI was required to produce maximal short-term potentiation and LTP in the DG of Sprague-Dawley rats. These results reveal that there are species differences in the optimal amount of BMI required to produce robust and reliable LTP in the rodent DG in vitro and highlight the need to take consideration of the species being used when choosing concentrations of pharmacological agents to employ for electrophysiological use.NEW & NOTEWORTHY In this report we provide specific neurophysiological evidence for concentrations of GABAA antagonist required to study long-term potentiation in the medial perforant pathway of the dentate gyrus. Two commonly used species, Sprague-Dawley rats and C57Bl/6 mice, require different concentrations of bicuculline methiodide to induce optimal short-term and long-term potentiation.

Allopregnanolone and its antagonist modulate neuroinflammation and neurological impairment.

Neuroinflammation accompanies several brain disorders, either as a secondary consequence or as a primary cause and may contribute importantly to disease pathogenesis. Neurosteroids which act as Positive Steroid Allosteric GABA-A receptor Modulators (Steroid-PAM) appear to modulate neuroinflammation and their levels in the brain may vary because of increased or decreased local production or import from the systemic circulation. The increased synthesis of steroid-PAMs is possibly due to increased expression of the mitochondrial cholesterol transporting protein (TSPO) in neuroinflammatory tissue, and reduced production may be due to changes in the enzymatic activity. Microglia and astrocytes play an important role in neuroinflammation, and their production of inflammatory mediators can be both activated and inhibited by steroid-PAMs and GABA. What is surprising is the finding that both allopregnanolone, a steroid-PAM, and golexanolone, a novel GABA-A receptor modulating steroid antagonist (GAMSA), can inhibit microglia and astrocyte activation and normalize their function. This review focuses on the role of steroid-PAMs in neuroinflammation and their importance in new therapeutic approaches to CNS and liver disease.

Chemogenetic attenuation of cortical seizures in nonhuman primates.

Epilepsy is a disorder in which abnormal neuronal hyperexcitation causes several types of seizures. Because pharmacological and surgical treatments occasionally interfere with normal brain function, a more focused and on-demand approach is desirable. Here we examined the efficacy of a chemogenetic tool-designer receptors exclusively activated by designer drugs (DREADDs)-for treating focal seizure in a nonhuman primate model. Acute infusion of the GABAA receptor antagonist bicuculline into the forelimb region of unilateral primary motor cortex caused paroxysmal discharges with twitching and stiffening of the contralateral arm, followed by recurrent cortical discharges with hemi- and whole-body clonic seizures in two male macaque monkeys. Expression of an inhibitory DREADD (hM4Di) throughout the seizure focus, and subsequent on-demand administration of a DREADD-selective agonist, rapidly suppressed the wide-spread seizures. These results demonstrate the efficacy of DREADDs for attenuating cortical seizure in a nonhuman primate model.

Neostriatum neuronal TRPV1-signalling mediates striatal anandamide at high concentration facilitatory influence on neostriato-nigral dishinhibitory GABAergic connections.

RATIONALE: Several lines of evidence have demonstrated that the cannabinoid type 1 receptor (CB1) is found in the caudate nucleus and putamen (CPu) in addition to the substantia nigra pars reticulata (SNpr). Here, we investigated the role of endocannabinoid neuromodulation of striato-nigral disinhibitory projections on the activity of nigro-collicular GABAergic pathways that control the expression of unconditioned fear-related behavioural responses elicited by microinjections of the GABAA receptor selective antagonist bicuculline (BIC) in the deep layers of the superior colliculus (dlSC). METHODS: Fluorescent neural tract tracers were deposited in either CPu or in SNpr. Wistar rats received injection of vehicle, anandamide (AEA), either at low (50 pmol) or high (100 pmol) concentrations in CPu followed by bicuculline microinjections in dlSC. RESULTS: Connections between CPu, the SNpr and dlSC were demonstrated. The GABAA receptor blockade in dlSC elicited panic-like behaviour. AEA at the lowest concentration caused a panicolytic-like effect that was antagonised by the CPu pretreatment with AM251 at 100 pmol. AEA at the highest concentration caused a panicogenic-like effect that was antagonised by the CPu pretreatment with 6-iodonordihydrocapsaicin (6-I-CPS) at different concentrations (0.6, 6, 60 nmol). CONCLUSION: These findings suggest that while pre-synaptic CB1-signalling subserves an indirect facilitatory effect of AEA on striato-nigral pathways causing panicolytic-like responses through midbrain tectum enhanced activity, post-synaptic TRPV1-signalling in CPu mediates AEA direct activation of striato-nigral disinhibitory pathways resulting in increasing dlSC neurons activity and a panicogenic-like response. All these actions seem to depend on the interface with the nigro-collicular inhibitory GABAergic pathways.

Reduced mechanical hypersensitivity by inhibition of the amygdala in experimental neuropathy: Sexually dimorphic contribution of spinal neurotransmitter receptors.

Here we studied spinal neurotransmitter mechanisms involved in the reduction of mechanical hypersensitivity by inhibition of the amygdaloid central nucleus (CeA) in male and female rats with spared nerve injury (SNI) model of neuropathy. SNI induced mechanical hypersensitivity that was stronger in females. Reversible blocking of the CeA with muscimol (GABAA receptor agonist) induced a reduction of mechanical hypersensitivity that did not differ between males and females. Following spinal co-administration of atipamezole (α2-adrenoceptor antagonist), the reduction of mechanical hypersensitivity by CeA muscimol was attenuated more in males than females. In contrast, following spinal co-administration of raclopride (dopamine D2 receptor antagonist) the reduction of hypersensitivity by CeA muscimol was attenuated more in females than males. The reduction of mechanical hypersensitivity by CeA muscimol was equally attenuated in males and females by spinal co-administration of WAY-100635 (5-HT1A receptor antagonist) or bicuculline (GABAA receptor antagonist). The CeA muscimol induced attenuation of ongoing pain-like behavior (conditioned place preference test) that was reversed by spinal co-administration of atipamezole in both sexes. The results support the hypothesis that CeA contributes to mechanical hypersensitivity and ongoing pain-like behavior in SNI males and females. Disinhibition of descending controls acting on spinal α2-adrenoceptors, 5-HT1A, dopamine D2 and GABAA receptors provides a plausible explanation for the reduction of mechanical hypersensitivity by CeA block in SNI. The involvement of spinal dopamine D2 receptors and α2-adrenoceptors in the CeA muscimol-induced reduction of mechanical hypersensitivity is sexually dimorphic, unlike that of spinal α2-adrenoceptors in the reduction of ongoing neuropathic pain.

The flavonoid kaempferol protects the fruit fly Drosophila melanogaster against the motor impairment produced by exposure to the insecticide fipronil.

Exposure to pesticides across species has been associated with cognitive and motor impairments. As the problem impacts ecosystem stability, food production and public health, it is urgent to develop multifactorial solutions, from regulatory legislation to pharmacological alternatives that ameliorate the impairments. Fipronil, a commonly used insecticide, acts as a GABAA receptor (GABAAR) antagonist and induces motor impairments in vertebrates and invertebrates. Here, we hypothesized that kaempferol, a secondary metabolite derived from plants, acting as an allosteric modulator of GABAARs, would protect against the negative effects induced by the administration of fipronil in adults of the fruit fly Drosophila melanogaster. We further evaluated our hypothesis via co-administration of flumazenil, a competitive antagonist on the GABAAR, and through in silico analyses. We administered kaempferol prophylactically at three concentrations (10, 30 and 50 µmol l-1) and evaluated its protective effects against motor impairments induced by fipronil. We then used a single dose of kaempferol (50 µmol l-1) to evaluate its protective effect while administering flumazenil. We found that oral administration of fipronil impaired motor control and walking ability. In contrast, kaempferol was innocuous and protected flies from developing the motor-impaired phenotype, whereas the co-administration of flumazenil counteracted these protective effects. These results are supported by the binding of the ligands with the receptor. Together, our results suggest that kaempferol exerts a protective effect against fipronil via positive allosteric modulation of GABAARs, probably within brain areas such as the central complex and the mushroom bodies. These findings further support current attempts to use metabolites derived from plants as protectors against impairments produced by pesticides.

Positive allosteric modulators of GABAA receptor restore chloride current from blockade by competitive antagonists in a ligand-dependent manner.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter. GABA receptor type A (GABAAR) possesses binding sites for a large group of pharmacological agents which are supposed to interact allosterically with each other. The aim of this work was to study the interaction between the positive allosteric modulators (PAMs) and the competitive antagonists of GABAARs. The GABA-induced chloride current (IGABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique. PAMs, neurosteroid allopregnanolone (Allo) and zolpidem (Zolp), a drug that positively modulates the GABAAR through interaction with the benzodiazepine (BDZ) site, doubled the IGABA amplitude in the control solution. Competitive antagonist of GABAARs, bicuculline (Bic, 5 µM) blocked the IGABA by 90%. The addition of 1 µM Allo or 0.5 µM Zolp to the Bic solution caused an unblocking effect, so that the IGABA amplitude increased 10 and 4 times from control value, correspondingly. This unblocking effect developed slowly, as evidenced by a threefold increase in the current rise time. Competitive antagonist of GABAARs, gabazine (GBZ, 0.5 µM) blocked the IGABA by 87%. The addition of 1 µM Allo to the GBZ solution caused an unblocking effect, so that the IGABA amplitude increased 7-fold. However, the addition of 0.5 µM Zolp to the GBZ solution did not cause an unblocking effect. So, Allo appeared to have a stronger unblocking potential than Zolp, and Bic binding site showed a higher sensitivity to the action of unblocking PAMs than GBZ binding site. The results indicate for the first time the existence of an allosteric relationship between the sites binding PAMs and the competitive antagonists of GABAAR.

Golexanolone, a GABAA receptor modulating steroid antagonist, restores motor coordination and cognitive function in hyperammonemic rats by dual effects on peripheral inflammation and neuroinflammation.

AIMS: Hyperammonemic rats show peripheral inflammation, increased GABAergic neurotransmission and neuroinflammation in cerebellum and hippocampus which induce motor incoordination and cognitive impairment. Neuroinflammation enhances GABAergic neurotransmission in cerebellum by enhancing the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. Golexanolone reduces GABAA receptors potentiation by allopregnanolone. This work aimed to assess if treatment of hyperammonemic rats with golexanolone reduces peripheral inflammation and neuroinflammation and restores cognitive and motor function and to analyze underlying mechanisms. METHODS: Rats were treated with golexanolone and effects on peripheral inflammation, neuroinflammation, TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways, and cognitive and motor function were analyzed. RESULTS: Hyperammonemic rats show increased TNFα and reduced IL-10 in plasma, microglia and astrocytes activation in cerebellum and hippocampus, and impaired motor coordination and spatial and short-term memories. Treating hyperammonemic rats with golexanolone reversed changes in peripheral inflammation, microglia and astrocytes activation and restored motor coordination and spatial and short-term memory. This was associated with reversal of the hyperammonemia-enhanced activation in cerebellum of the TNFR1-glutaminase-GAT3 and TNFR1-CCL2-TrkB-KCC2 pathways. CONCLUSION: Reducing GABAA receptors activation with golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in hyperammonemic rats. The effects identified would also occur in patients with hepatic encephalopathy and, likely, in other pathologies associated with neuroinflammation.

Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus.

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

A double-blind randomised crossover trial of low-dose flumazenil for benzodiazepine withdrawal: A proof of concept.

INTRODUCTION: Benzodiazepines (BZD) are a class of anxiolytics with varying uses, which primarily act on the GABAA receptor resulting in hyperpolarisation. BZDs are often a difficult drug class to cease once neuroadaptation has occurred; recommendations usually involve gradual dose reductions at variable rates. A growing body of evidence has suggested that low-dose flumazenil, a GABAA receptor antagonist, may be a useful agent to allow for rapid detoxification. AIM: To collect pilot data on the safety and efficacy of low-dose subcutaneous flumazenil to reduce BZD use, withdrawal symptoms, and craving in participants taking above and below the therapeutic maximum diazepam equivalent of 30 mg to inform on sample size for future trials. METHOD: In a randomised double-blinded crossover study design, participants received low-dose flumazenil first (4 mg/24 h for approximately eight days) or placebo first. Groups were divided into those taking < 30 mg diazepam equivalent and ≥ 30 mg diazepam equivalent at baseline. Main outcome measures were percentage reduction in daily diazepam use, withdrawal symptoms, and craving scores from baseline, difference in diazepam use across the placebo first group, and flumazenil related adverse events. RESULTS: Twenty-eight participants were recruited and randomised to flumazenil first (n = 14) and placebo first (n = 14). In participants taking ≥ 30 mg diazepam equivalent at baseline (n = 15), flumazenil significantly reduced diazepam use by 30.5% (p = 0.024) compared to placebo. CONCLUSION: Low-dose flumazenil may aid in BZD detoxification in participants taking daily diazepam equivalent doses greater than or equal to the therapeutic maximum (≥30 mg) by reducing the need for diazepam.

Early life GABAA blockade alters the synaptic plasticity and cognitive functions in male and female rats.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in adults, has a critical contribution to balanced excitatory-inhibitory networks in the brain. Alteration in depolarizing action of GABA during early life is connected to a wide variety of neurodevelopmental disorders. Additionally, the effects of postnatal GABA blockade on neuronal synaptic plasticity are not known and therefore, we set out to determine whether postnatal exposure to bicuculline, a competitive antagonist of GABAA receptors, affects electrophysiologic changes in hippocampal CA1 neurons later on. To this end, male and female Wistar rats received vehicle or bicuculline (300 µg/kg) on postnatal days (PNDs) 7, 9 and 11, and then underwent different behavioral and electrophysiological examinations in adulthood. Postnatal exposure to bicuculline did not affect basic synaptic transmission but led to a pronounced decrease in paired-pulse facilitation (PPF) in CA1 pyramidal neurons. Bicuculline treatment also attenuated the long-term potentiation (LTP) and long-term depression (LTD) of CA1 neurons accompanied by decreased theta-burst responses in male and female adult rats. These electrophysiology findings together with the reduced brain-derived neurotrophic factor (BDNF) levels in the hippocampus and prefrontal cortex reliably explain the disturbance in spatial reference and working memories of bicuculline-treated animals. This study suggests that postnatal GABAA blockade deteriorates short- and long-term synaptic plasticity of hippocampal CA1 neurons and related encoding of spatial memory in adulthood.

Upregulation of the Endogenous Peptide Antisecretory Factor Enhances Hippocampal Long-term Potentiation and Promotes Learning in Wistar Rats.

Antisecretory Factor (AF) is an endogenous peptide known for its powerful antisecretory and anti-inflammatory properties. We have previously shown that AF also acts as a neuromodulator of GABAergic synaptic transmission in rat hippocampus in a way that results in disinhibition of CA1 pyramidal neurons. Disinhibition is expected to facilitate the induction of long-term potentiation (LTP), and LTP is known to play a crucial role in learning and memory acquisition. In the present study we investigated the effect of AF on LTP in CA3-CA1 synapses in rat hippocampus. In addition, endogenous AF plasma activity was upregulated by feeding the rats with specially processed cereals (SPC) and spatial learning and memory was studied in the Morris Water Maze (MWM). We found that LTP was significantly enhanced in the presence of AF, both when added exogenously in vitro as well as when upregulated endogenously by SPC-feeding. In the presence of the GABAA-receptor antagonist picrotoxin (PTX) there was however no significant enhancement of LTP. Moreover, rats fed with SPC demonstrated enhanced spatial learning and short-term memory, compared with control animals. These results show that the disinhibition of GABAergic transmission in the hippocampus by the endogenous peptide AF enhances LTP as well as spatial learning and memory.

Association between benzodiazepine receptor agonist use and mortality in patients hospitalised for COVID-19: a multicentre observational study.

AIMS: To examine the association between benzodiazepine receptor agonist (BZRA) use and mortality in patients hospitalised for coronavirus disease 2019 (COVID-19). METHODS: A multicentre observational study was performed at Greater Paris University hospitals. The sample involved 14 381 patients hospitalised for COVID-19. A total of 686 (4.8%) inpatients received a BZRA at hospital admission at a mean daily diazepam-equivalent dose of 19.7 mg (standard deviation (s.d.) = 25.4). The study baseline was the date of admission, and the primary endpoint was death. We compared this endpoint between patients who received BZRAs and those who did not in time-to-event analyses adjusted for sociodemographic characteristics, medical comorbidities and other medications. The primary analysis was a Cox regression model with inverse probability weighting (IPW). RESULTS: Over a mean follow-up of 14.5 days (s.d. = 18.1), the primary endpoint occurred in 186 patients (27.1%) who received BZRAs and in 1134 patients (8.3%) who did not. There was a significant association between BZRA use and increased mortality both in the crude analysis (hazard ratio (HR) = 3.20; 95% confidence interval (CI) = 2.74-3.74; p < 0.01) and in the IPW analysis (HR = 1.61; 95% CI = 1.31-1.98, p < 0.01), with a significant dose-dependent relationship (HR = 1.55; 95% CI = 1.08-2.22; p = 0.02). This association remained significant in sensitivity analyses. Exploratory analyses indicate that most BZRAs may be associated with an increased mortality among patients hospitalised for COVID-19, except for diazepam, which may be associated with a reduced mortality compared with any other BZRA treatment. CONCLUSIONS: BZRA use may be associated with an increased mortality among patients hospitalised for COVID-19, suggesting the potential benefit of decreasing dose or tapering off gradually these medications when possible.

Mechanisms of inhibition and activation of extrasynaptic αß GABAA receptors.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.

Chronic neuronal excitation leads to dual metaplasticity in the signaling for structural long-term potentiation.

Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of long-term potentiation (LTP), known as metaplasticity. However, the metaplastic regulation of structural LTP (sLTP) remains unclear. We investigate glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAA receptor antagonists. We find that the neuronal excitation decreases the glutamate uncaging-evoked Ca2+ influx mediated by GluN2B-containing NMDA receptors and suppresses sLTP induction. In addition, single-spine optogenetic stimulation using paCaMKII indicates the suppression of CaMKII signaling. While the inhibition of Ca2+ influx is protein synthesis independent, the paCaMKII-induced sLTP suppression depends on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., dual inhibition of Ca2+ influx and CaMKII signaling). This dual inhibition mechanism may contribute to robust neuronal protection in excitable environments.

The role of neuronal excitation and inhibition in the pre-Bötzinger complex on the cough reflex in the cat.

Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg, iv initially). Neuronal excitation by unilateral microinjection of glutamate analog d,l-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared with effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase, and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate-related neuronal excitation by bilateral microinjections of nonspecific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor-related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.NEW & NOTEWORTHY This study is the first to explore the role of the inspiratory rhythm and pattern generator, the pre-Bötzinger complex (PreBötC), in cough motor pattern formation. In the PreBötC, excitatory glutamatergic neurotransmission affects cough intensity and patterning but not rhythm, and GABAA receptor-related inhibition affects coughing and breathing phase durations similarly to each other. Our data show that the PreBötC is important for cough motor pattern generation, but cough rhythmogenesis appears to be controlled elsewhere.

Chronic ethanol exposure during adolescence impairs simple spike activity of cerebellar Purkinje cells in vivo in mice.

Cerebellar Purkinje cells (PCs) play critical roles in motor coordination and motor learning through their simple spike (SS) activity. Previous studies have shown that chronic ethanol exposure (CEE) in adolescents impairs learning, attention, and behavior, at least in part by impairing the activity of cerebellar PCs. In this study, we investigated the effect of CEE on the SS activity in urethane-anesthetized adolescent mice by in vivo electrophysiological recordings and pharmacological methods. Our results showed that the cerebellar PCs in CEE adolescent mice expressed a significant decrease in the frequency and an increase in the coefficient of variation (CV) of SS than control group. Blockade of ɤ-aminobutyric acid A (GABAA) receptor did not change the frequency and CV of SS firing in control group but produced a significant increase in the frequency and a decrease in the CV of SS firing in CEE mice. The CEE-induced decrease in SS firing rate and increase in CV were abolished by application of an N-methyl-D-aspartate (NMDA) receptor blocker, D-APV, but not by anα-amino-3-hydroxy-5-methyl -4-isoxazolepropionic acid (AMPA) receptor antagonist, NBQX. Notably, the spontaneous spike rate of molecular layer interneurons (MLIs) in CEE mice was significantly higher than control group, which was also abolished by application of D-APV. These results indicate that adolescent CEE enhances the spontaneous spike firing rate of MLIs through activation of NMDA receptor, resulting in a depression in the SS activity of cerebellar PCs in vivo in mice.

Combined treatment with exercise and α5GABAAR inhibitor promotes motor function recovery after intracerebral hemorrhage.

The present study aimed to examine the synergistic effects of exercise and pharmacological inhibition of the α5 subunit-containing gamma-aminobutyric acid (GABA)A receptors (α5GABAAR) on motor function recovery after intracerebral hemorrhage (ICH). Wistar rats were divided into five groups (n = 8 per group): SHAM, ICH, ICH + exercise (ICH + EX), ICH + L-655,708 (ICH + L6), and ICH + L-655,708 and exercise (ICH + L6EX) groups. ICH was induced by microinjection of a collagenase solution. The ICH + EX and ICH + L6EX groups exercised on a treadmill (12 m/min for 30 min/day). L-655,708 (0.5 mg/kg), a negative allosteric modulator of α5GABAAR, was administered intraperitoneally to the ICH + L6 and ICH + L6EX groups. Each intervention was initiated 1 week after the ICH surgery and was performed for 3 weeks, followed by tissue collection, including the motor cortex and spinal cord. At 4 weeks after ICH, significant motor recovery was found in the ICH + L6EX group compared to the ICH group. L-655,708 administration increased brain-derived neurotrophic factor (BDNF) expression in the cortex. Regarding neuroplastic changes in the spinal cord, rats in the ICH + L6EX group showed a significant increase in several neuroplastic markers: 1) BDNF, 2) growth-associated protein 43 as an axonal sprouting marker, 3) synaptophysin as a synaptic marker, and 4) Nogo-A as an axonal growth inhibitor. This study is the first to demonstrate that combined treatment with exercise and α5GABAAR inhibitor effectively promoted motor function recovery after ICH. Regarding the underlying mechanism of post-ICH recovery with the combined treatment, the present study highlights the importance of both growth and inhibitory modification of axonal sprouting in the spinal cord.

Positive GABAA receptor modulating steroids and their antagonists: Implications for clinical treatments.

GABA is the main inhibitory neurotransmitter in the brain and GABAergic transmission has been shown to be of importance for regulation of mood, memory and food intake. The progesterone metabolite allopregnanolone (Allo) is a positive GABAA receptor modulating steroid with potent effects. In humans, disorders such as premenstrual dysphoric disorder (PMDD), hepatic encephalopathy and polycystic ovarian syndrome are associated with elevated Allo levels and increased negative mood, disturbed memory and increased food intake in some individuals. This is surprising because Allo shares many properties with benzodiazepines and is mainly considered to be anxiolytic and anti-depressant. However, it is well established that, in certain individuals, GABAA receptor activating compounds could have paradoxical effects and thus be anxiogenic in low physiological plasma concentrations but anxiolytic at high levels. We have demonstrated that isoallopregnanolone (Isoallo), the 3ß-OH sibling of Allo, functions as a GABAA receptor modulating steroid antagonist (GAMSA) but without any effects of its own on GABAA receptors. The antagonistic effect is noted in most GABAA subtypes investigated in vitro to date. In vivo, Isoallo can inhibit Allo-induced anaesthesia in rats, as well as sedation or saccadic eye velocity in humans. Isoallo treatment has been studied in women with PMDD. In a first phase II study, Isoallo (Sepranolone; Asarina Pharma) injections significantly ameliorated negative mood in women with PMDD compared with placebo. Several GAMSAs for oral administration have also been developed. The GAMSA, UC1011, can inhibit Allo induced memory disturbances in rats and an oral GAMSA, GR3027, has been shown to restore learning and motor coordination in rats with hepatic encephalopathy. In humans, vigilance, cognition and pathological electroencephalogram were improved in patients with hepatic encephalopathy on treatment with GR3027. In conclusion GAMSAs are a new possible treatment for disorders and symptoms caused by hyperactivity in the GABAA system.