Allosteric Modulators Of GABAA Receptors Research Articles

An Update on Stiripentol Mechanisms of Action: A Narrative Review.

Stiripentol (Diacomit®) (STP) is an orally active antiseizure medication (ASM) indicated as adjunctive therapy, for the treatment of seizures associated with Dravet syndrome (DS), a severe form of childhood epilepsy, in conjunction with clobazam and, in some regions valproic acid. Since the discovery of STP, several mechanisms of action (MoA) have been described that may explain its specific effect on seizures associated with DS. STP is mainly considered as a potentiator of gamma-aminobutyric acid (GABA) neurotransmission: (i) via uptake blockade, (ii) inhibition of degradation, but also (iii) as a positive allosteric modulator of GABAA receptors, especially those containing α3 and δ subunits. Blockade of voltage-gated sodium and T-type calcium channels, which is classically associated with anticonvulsant and neuroprotective properties, has also been demonstrated for STP. Finally, several studies indicate that STP could regulate glucose energy metabolism and inhibit lactate dehydrogenase. STP is also an inhibitor of several cytochrome P450 enzymes involved in the metabolism of other ASMs, contributing to boost their anticonvulsant efficacy as add-on therapy. These different MoAs involved in treatment of DS and recent data suggest a potential for STP to treat other neurological or non-neurological diseases.

Ivermectin Exerts Anticonvulsant Effects Against Status Epilepticus Induced by Lithium-Pilocarpine in Rats via GABAA Receptor and Neuroinflammation Modulation: Possible Interaction of Opioidergic Pathways and KATP Channel with Nitrergic System.

Status epilepticus (SE) is a critical medical emergency marked by persistent or rapidly repeating seizures, posing a threat to life. Using the lithium-pilocarpine-induced SE model, we decide to evaluate the anti-seizure effects of ivermectin as a positive allosteric modulator of GABAA receptor and the underlying mechanisms involved. Lithium chloride was injected intraperitoneally at a dose of 127 mg/kg, followed by the administration of pilocarpine at a dose of 60 mg/kg after a 20-h interval in order to induce SE. Subsequently, the rats received varying amounts of ivermectin (0.3, 1, 3, 5, and 10 mg/kg, i.p.) 30 min before the onset of SE. To study the underlying molecular mechanisms, we had pharmacological interventions of diazepam (1 mg/kg), glibenclamide and nicorandil as ATP-sensitive potassium channel blocker and opener (both 1 mg/kg, i.p.), naltrexone and morphine, as opioid receptor antagonist and agonist (1 mg/kg and 0.5 mg/kg, i.p., respectively). In addition, three nitric oxide inhibitors, namely, L-NAME (10 mg/kg, i.p.), 7-NI (30 mg/kg, i.p.), and aminoguanidine (100 mg/kg, i.p.), were administered to the rats in the experiment. Finally, we use ELISA and western blotting, respectively, to examine the amounts of pro-inflammatory cytokines (TNF-α and IL-1β), nitrite, and GABAA receptors in the rat hippocampal tissue. The study found that ivermectin, at doses of 3, 5, and 10 mg/kg, exerts anti-seizure effects and decrease Racine's scale SE score. Interestingly glibenclamide and naltrexone reduced the anti-seizure effects of ivermectin, and from other hand diazepam, nicorandil, morphine, L-NAME, 7-NI, and aminoguanidine, enhance the effects when co-administrated with subeffective dose of ivermectin. Additionally, the study found that ivermectin decreased the elevated levels of TNF-α and IL-1β following SE, while increased the reduced expression of GABAA receptors. Overall, these findings suggest that ivermectin has anti-seizure effects in a SE seizure which may be mediated by the modulation of GABAergic, opioidergic, and nitrergic pathways and KATP channels.

A Randomized Phase 2 KINETIC Trial Evaluating SAGE-324/BIIB124 in Individuals with Essential Tremor.

SAGE-324/BIIB124 is an investigational positive allosteric modulator of GABAA receptors. KINETIC (NCT04305275), a double-blind, randomized, placebo-controlled, phase 2 study, evaluated SAGE-324/BIIB124 in individuals with essential tremor (ET). Individuals aged 18 to 80 years were randomly assigned 1:1 to orally receive 60 mg of SAGE-324/BIIB124 or placebo once daily for 28 days. The primary endpoint was change from baseline in The Essential Tremor Rating Assessment Scale-Performance Subscale (TETRAS-PS) Item 4 (upper-limb tremor) at day 29 with SAGE-324/BIIB124 versus placebo. Between May 2020 and February 2021, 69 U.S. participants were randomly assigned to receive SAGE-324/BIIB124 (n = 34) or placebo (n = 35). There was a significant reduction from baseline in TETRAS-PS Item 4 at day 29 with SAGE-324/BIIB124 versus placebo (least squares mean [standard error]: -2.31 [0.401] vs. -1.24 [0.349], P = 0.0491). The most common treatment-emergent adverse events included somnolence, dizziness, fatigue, and balance disorder. These results support further development of SAGE-324/BIIB124 for potential ET treatment. © 2024 Sage Therapeutics, Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Integrated workflow for the identification of new GABAA R positive allosteric modulators based on the in silico screening with further in vitro validation. Case study using Enamine's stock chemical space.

Numerous studies reported an association between GABAA R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAA R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1β2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1β2γ2 GABAA receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon.

Ivermectin ameliorates bleomycin-induced lung fibrosis in male rats by inhibiting the inflammation and oxidative stress

Background Idiopathic pulmonary fibrosis (IPF) is a pulmonary fibrotic disease characterized by a poor prognosis, which its pathogenesis involves the accumulation of abnormal fibrous tissue, inflammation, and oxidative stress. Ivermectin, a positive allosteric modulator of GABAA receptor, exerts anti-inflammatory and antioxidant properties in preclinical studies. The present study investigates the potential protective effects of ivermectin treatment in rats against bleomycin-induced IPF. Materials and Methods The present study involved 42 male Wistar rats, which were divided into five groups: control (without induction of IPF), bleomycin (IPF-induced by bleomycin 2.5 mg/kg, by intratracheal administration), and three fibrosis groups receiving ivermectin (0.5, 1, and 3 mg/kg). lung tissues were harvested for measurement of oxidative stress [via myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione (GSH)] and inflammatory markers (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and transforming growth factor-β [TGF-β]). Histological assessments of tissue damage were performed using hematoxylin–eosin (H&E) and Masson’s trichrome staining methods. Results The induction of fibrosis via bleomycin was found to increase levels of MPO as well as TNF-α, IL-1β, and TGF-β while decrease SOD activity and GSH level. Treatment with ivermectin at a dosage of 3 mg/kg was able to reverse the effects of bleomycin-induced fibrosis on these markers. In addition, results from H&E and Masson’s trichrome staining showed that ivermectin treatment at this same dose reduced tissue damage and pulmonary fibrosis. Conclusion The data obtained from this study indicate that ivermectin may have therapeutic benefits for IPF, likely due to its ability to reduce inflammation and mitigate oxidative stress-induced toxicity.

β-carbolines that enhance GABAA receptor response expressed in oligodendrocytes promote remyelination in an in vivo rat model of focal demyelination.

Demyelination is typically followed by a remyelination process through mature oligodendrocytes (OLs) differentiated from precursor cells (OPCs) recruited into the lesioned areas, however, this event usually results in uncompleted myelination. Potentiation of the remyelination process is an important target for designing effective therapeutic strategies against white matter loss. Here, it was evaluated the remyelinating effect of different β-carbolines that present differential allosteric modulation on the GABAA receptor expressed in OLs. For this, we used a focalized demyelination model in the inferior cerebellar peduncle (i.c.p.) of rats (DRICP model), in which, demyelination by ethidium bromide (0.05%) stereotaxic injection was confirmed histologically by staining with Black-Gold II (BGII) and toluidine blue. In addition, a longitudinal analysis with diffusion-weighted magnetic resonance imaging (dMRI) was made by computing fractional anisotropy (FA), apparent diffusion coefficient (ADC) and diffusivity parameters to infer i.c.p. microstructural changes. First, dMRI analysis revealed FA decreases together with ADC and radial diffusivity (RD) increases after demyelination, which correlates with histological BGII observations. Then, we evaluated the effect produced by three allosteric GABAA receptor modulators, the N-butyl-β-carboline-3-carboxylate (β-CCB), ethyl 9H-pyrido [3,4-b]indole-3-carboxylate (β-CCE), and 4-ethyl-6,7-dimethoxy-9H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester (DMCM). The results indicated that daily systemic β-CCB (1 mg/Kg) or β-CCE (1 mg/Kg) administration for 2 weeks, but not DMCM (0.35 mg/Kg), in lesioned animals increased FA and decreased ADC or RD, suggesting myelination improvement. This was supported by BGII staining analysis that showed a recovery of myelin content. Also, it was quantified by immunohistochemistry both NG2+ and CC1+ cellular population in the different experimental sceneries. Data indicated that either β-CCB or β-CCE, but not DMCM, produced an increase in the population of CC1+ cells in the lesioned area. Finally, it was also calculated the g-ratio of myelinated axons and observed a similar value in those lesioned animals treated with β-CCB or β-CCE compared to controls. Thus, using the DRICP model, it was observed that either β-CCB or β-CCE, positive modulators of the GABAA receptor in OLs, had a potent promyelinating effect.

Schisandrin B, a dual positive allosteric modulator of GABAA and glycine receptors, alleviates seizures in multiple mouse models.

Epilepsy is a prevalent and severe neurological disorder and approximately 30% of patients are resistant to existing medications. It is of utmost importance to develop alternative therapies to treat epilepsy. Schisandrin B (SchB) isa major bioactive constituent of Schisandra chinensis (Turcz.) Bailland has multiple neuroprotective effects, sedative and hypnotic activities.In this study, we investigated the antiseizure effect of SchB in various mouse models of seizure and explored the underlying mechanisms. Pentylenetetrazole (PTZ), strychnine (STR), and pilocarpine-induced mouse seizure models were established.We showed that injection ofSchB (10, 30, 60 mg/kg, i.p.) dose-dependentlydelayed the onset of generalized tonic-clonic seizures (GTCS), reduced the incidence of GTCS and mortality in PTZ and STR models. Meanwhile, injection ofSchB (30 mg/kg, i.p.) exhibited therapeutic potential in pilocarpine-induced status epilepticus model, which was considered as a drug-resistant model. In whole-cell recording from CHO/HEK-239 cells stably expressing recombinant human GABAA receptors (GABAARs) and glycine receptors (GlyRs) and cultured hippocampal neurons, co-application of SchB dose-dependently enhanced GABA or glycine-induced current with EC50 values at around 5 μM, and application of SchB (10 μM) alone did not activate the channels in the absence of GABA or glycine.Furthermore, SchB (10 μM) eliminated both PTZ-induced inhibition on GABA-induced current (IGABA) and strychnine (STR)-induced inhibition on glycine-induced current (Iglycine).Moreover, SchB(10 μM) efficiently rescued the impaired GABAARs associated with genetic epilepsies. In addition,the homologous mutants in both GlyRs-α1(S267Q) and GABAARs-α1(S297Q)β2(N289S)γ2L receptors by site-directed mutagenesis testsabolishedSchB-induced potentiation of IGABA and Iglycine. In conclusion, we have identified SchB as a natural positive allosteric modulator of GABAARs and GlyRs, supporting its potential as alternative therapies for epilepsy.

New GABA-Targeting Therapies for the Treatment of Seizures and Epilepsy: II. Treatments in Clinical Development.

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) plays an important role in the modulation of neuronal excitability, and a disruption of GABAergic transmission contributes to the pathogenesis of some seizure disorders. Although many currently available antiseizure medications do act at least in part by potentiating GABAergic transmission, there is an opportunity for further research aimed at developing more innovative GABA-targeting therapies. The present article summarises available evidence on a number of such treatments in clinical development. These can be broadly divided into three groups. The first group consists of positive allosteric modulators of GABAA receptors and includes Staccato® alprazolam (an already marketed benzodiazepine being repurposed in epilepsy as a potential rescue inhalation treatment for prolonged and repetitive seizures), the α2/3/5 subtype-selective agents darigabat and ENX-101, and the orally active neurosteroids ETX155 and LPCN 2101. A second group comprises two drugs already marketed for non-neurological indications, which could be repurposed as treatments for seizure disorders. These include bumetanide, a diuretic agent that has undergone clinical trials in phenobarbital-resistant neonatal seizures and for which the rationale for further development in this indication is under debate, and ivermectin, an antiparasitic drug currently investigated in a randomised double-blind trial in focal epilepsy. The last group comprises a series of highly innovative therapies, namely GABAergic interneurons (NRTX-001) delivered via stereotactic cerebral implantation as a treatment for mesial temporal lobe epilepsy, an antisense oligonucleotide (STK-001) aimed at upregulating NaV1.1 currents and restoring the function of GABAergic interneurons, currently tested in a trial in patients with Dravet syndrome, and an adenoviral vector-based gene therapy (ETX-101) scheduled for investigation in Dravet syndrome. Another agent, a subcutaneously administered neuroactive peptide (NRP2945) that reportedly upregulates the expression of GABAA receptor α and β subunits is being investigated, with Lennox-Gastaut syndrome and other epilepsies as proposed indications. The diversity of the current pipeline underscores a strong interest in the GABA system as a target for new treatment development in epilepsy. To date, limited clinical data are available for these investigational treatments and further studies are required to assess their potential value in addressing unmet needs in epilepsy management.

Changes in social, sexual, and hedonic behaviors in rats in response to stress and restoration by a negative allosteric modulator of α5-subunit containing GABA receptor

Major depressive disorder (MDD) is a debilitating and costly human condition. Treatment for MDD relies heavily on the use of antidepressants that are slow to produce mood-related changes and are not effective in all patients, such as selective serotonin reuptake inhibitors (SSRIs). Several novel compounds, including negative allosteric modulators of GABA-A receptors containing the α5-subunit (GABA-NAMs), are under investigation for potential fast acting therapeutic use in MDD. Preclinical evidence that these compounds produce a rapid antidepressant-like response comes primarily from simple tests of escape behavior and preference for rewarding stimuli after chronic stress. To increase the ethological relevance of these compounds, we tested the hypothesis that the GABA-NAM, L-655,708, would produce an antidepressant-like response in more complex stress-sensitive social and sex behaviors, which are of relevance to the symptoms of human depression. In male rats subjected to chronic restraint stress, injection of L-655,708 increased reward in a sexual conditioned place preference task, increased male sexual activity with a receptive female, and re-established male social dominance hierarchies within 24 h. We also report increased sucrose preference in the social defeat stress (SDS) model of depression following GABA-NAM administration, demonstrating that its antidepressant-like actions are independent of the type of chronic stress administered. This work extends the impact of GABA-NAMs beyond traditional tests of anhedonia and further supports the development of alpha5 subunit-selective GABA-NAMs as a potential fast-acting therapeutic approach for treating human MDD.

CNSC-01. GABAERGIC NEURON-TO-GLIOMA SYNAPSES IN DIFFUSE MIDLINE GLIOMAS

Abstract High-grade gliomas include clinically and molecularly distinct subtypes that stratify by anatomical location into diffuse midline gliomas (DMG) such as diffuse intrinsic pontine glioma (DIPG) and hemispheric high-grade gliomas. Neuronal activity drives high-grade glioma progression both through paracrine signaling and direct neuron-to-glioma synapses. Glutamatergic, AMPA receptor-dependent synapses between neurons and malignant glioma cells have been demonstrated in both pediatric and adult high-grade gliomas, but neuron-to-glioma synapses mediated by other neurotransmitters remain largely unexplored. Using whole-cell patch clamp electrophysiology, in vivo optogenetics and patient-derived glioma xenograft models, we have now identified functional, tumor-promoting GABAergic neuron-to-glioma synapses mediated by GABAA receptors in DMGs. GABAergic input has a depolarizing effect on DMG cells due to NKCC1 expression and consequently elevated intracellular chloride concentration in DMG tumor cells. As membrane depolarization increases glioma proliferation, we find that the activity of GABAergic interneurons promotes DMG proliferation in vivo. Increasing GABA signaling with the benzodiazepine lorazepam – a positive allosteric modulator of GABAA receptors commonly administered to children with DMG for nausea or anxiety - increases GABAA receptor conductance and increases glioma proliferation in orthotopic xenograft models of DMG. Conversely, levetiracetam, an anti-epileptic drug that attenuates GABAergic neuron-to-glioma synaptic currents, reduces glioma proliferation in patient-derived DMG xenografts and extends survival of mice bearing DMG xenografts. Concordant with gene expression patterns of GABAA receptor subunit genes across subtypes of glioma, depolarizing GABAergic currents were not found in hemispheric high-grade gliomas. Accordingly, neither lorazepam nor levetiracetam influenced the growth rate of hemispheric high-grade glioma patient-derived xenograft models. Retrospective real-world clinical data are consistent with these conclusions and should be replicated in future prospective clinical studies. Taken together, these findings uncover GABAergic synaptic communication between GABAergic interneurons and DMG cells, underscoring a tumor subtype-specific mechanism of brain cancer neurophysiology with important potential implications for commonly used drugs in this disease context.

Recent Developments in Pharmacotherapy of Depression: Bench to Bedside.

For the last 70 years, we did not move beyond the monoamine hypothesis of depression until the approval of the S-enantiomer of ketamine, an N-methyl-D-aspartate (NMDA) receptor blocker and the first non-monoaminergic antidepressant characterized by rapid antidepressant and antisuicidal effects. A similar profile has been reported with another NMDA receptor antagonist, dextromethorphan, which has also been approved to manage depression in combination with bupropion. More recently, the approval of a positive allosteric modulator of GABA-A receptors, brexanolone, has added to the list of recent breakthroughs with the relatively rapid onset of antidepressant efficacy. However, multiple factors have compromised the clinical utility of these exciting discoveries in the general population, including high drug acquisition costs, mandatory monitoring requirements, parenteral drug administration, lack of insurance coverage, indirect COVID-19 effects on healthcare systems, and training gaps in psychopharmacology. This narrative review aims to analyze the clinical pharmacology of recently approved antidepressants and discuss potential barriers to the bench-to-bedside transfer of knowledge and clinical application of exciting recent discoveries. Overall, clinically meaningful advances in the treatment of depression have not reached a large proportion of depressed patients, including those with treatment-resistant depression, who might benefit the most from the novel antidepressants.

Saying goodbye to an unwelcome guest: Towards robust methods for controlling multi-sensory confounds in TMS-EEG research.

Combined transcranial magnetic stimulation and electroencephalography (TMS-EEG) is a multi-modal functional neuroimaging approach that has seen growing uptake across basic, cognitive, and clinical neuroscience as a method for safely interrogating cortical physiology in vivo. TMS uses a coil placed over the scalp to produce a brief, time-varying magnetic field capable of passing unimpeded through the skull to activate underlying neuronal populations (Barker et al., 1985). By combining TMS with EEG, the electrical neuronal response to stimulation can be captured across the cortex with millisecond temporal precision, providing insight into brain responsivity both at the stimulation site and across broader neural networks (Tremblay et al., 2019). As such, TMS-EEG has opened exciting new avenues for understanding healthy brain function and probing neuropathophysiological circuits across several psychiatric, neurological and neurodevelopmental disorders. However, in addition to transcranially stimulating cortical neuronal populations, TMS also exerts effects on brain function via indirect multi-sensory pathways. This includes activation of the auditory system through the ‘click’ sound generated when the TMS coil discharges, as well as via signal propagation through somatosensory pathways caused by stimulation of the scalp and vibration of the TMS coil. These sensory co-activations lead to peripherally evoked potentials (PEPs) on the EEG record, which, if not adequately accounted for, can obscure the TMS-evoked potentials (TEPs) arising from direct transcranial stimulation of the cortex. In this issue of The Journal of Physiology, Gordon et al. (2023) report results from a study aimed at better understanding TEPs and PEPs captured with TMS-EEG. To achieve their aim, the authors used an experimental approach incorporating both an optimised sham TMS protocol and pharmacological challenge. Healthy participants completed two experimental sessions during which TMS-EEG recordings were obtained both before and after administration of either a positive allosteric modulator of GABAA receptors (oral diazepam) or placebo. This pharmaco-TMS-EEG design enabled the authors to investigate changes in brain responses to a drug previously shown to alter cortical reactivity using TMS-EEG (Premoli et al., 2014). Gordon et al. (2023) implement a unique stimulation approach targeting motor cortex that interleaved active and sham stimulation within the same experimental block along with concomitant high-intensity electrical stimulation of the scalp. This sophisticated methodology enabled the authors to closely align the auditory and somatosensory inputs generated across these two conditions. There are, nevertheless, some potential caveats surrounding this approach. For instance, the use of electrical stimulation in close proximity to recording electrodes will unavoidably produce artifacts on the EEG trace – a limitation the authors themselves acknowledge. If not completely removed, this could distort or obscure TEPs, particularly at early time points. Also, given that the authors used high electrical stimulation intensities (average = 25.6 mA) to saturate the PEPs in both the active and sham TMS conditions, this raises interesting questions regarding intensities at which these might begin to induce meaningful physiological effects in the brain via injection of current through the scalp and skull. Although this is perhaps unlikely, given that the E-field induced in the brain would be diminished manyfold in comparison to TMS, it could be a potentially interesting avenue to explore in future research. A central finding by Gordon et al. (2023) was that modulation of GABAA receptor-mediated neurotransmission via diazepam was able to elicit demonstrable changes on the TMS-EEG record following both active and sham TMS. Specifically, diazepam attenuated late responses in both the active (>80 ms) and sham (>85 ms) conditions, while also enhancing the negativity of the P60 potential in the active TMS condition only. This indicates that cortical reactivity to both direct transcranial stimulation and sensory co-activation can be pharmacologically modulated. A further particularly interesting discovery relates to the N100 response. This prominent negative potential has been previously linked to cortical inhibitory processes, including by pharmaco-TMS-EEG studies using GABA receptor agonists (Premoli et al., 2014). However, the N100 is also sensitive to multi-sensory input (Biabani et al., 2019). The results of Gordon et al. (2023) indicate that although diazepam was able to attenuate the N100 amplitude in both the active and sham TMS conditions, after subtracting the sham from the active TMS response this effect disappeared. This raises the intriguing possibility that GABAA receptor-mediated modulation of the N100 via diazepam could in fact be driven by PEPs. The potential significance of this observation is substantial given that the N100 is frequently interpreted as reflecting cortical inhibition in motor cortex. Research is needed to assess the replicability of this result and would likely also benefit from the inclusion of a drug targeting GABAB receptors, given past links between the N100 and GABAB receptor-mediated inhibition (Tremblay et al., 2019). In summary, this timely study by Gordon et al. (2023) significantly contributes to ongoing endeavours to advance mechanistic understanding of neural responses captured using TMS-EEG. The findings corroborate growing evidence supporting the need for adequate control of PEPs on the TMS-EEG record, particularly when analysing later-occurring responses (i.e. >∼80 ms post-TMS). Thus, while continued empirical efforts will no doubt be required to further fine-tune best-practice approaches for preventing contamination of the EEG record by multi-sensory phenomena, the need for adequate sensory controls is growing increasingly apparent. This work also provides a solid platform for future studies aimed at further characterising EEG responses to neural perturbation with TMS. This might include work employing larger sample sizes to also investigate the role of inter-individual variability in TEPs and PEPs, as well as assessment of the influence of additional CNS-active drugs on these responses. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The author reports no conflicts of interest. Sole author. The author reports no external funding. Open access publishing facilitated by Deakin University, as part of the Wiley - Deakin University agreement via the Council of Australian University Librarians.

A neolignan from Connarus tuberosus as an allosteric GABAA receptor modulator at the neurosteroid binding site

In a screening of a small library of extracts from plants of the Amazonian and Cerrado biomes, a hexane extract of Connarus tuberosus roots was found to significantly potentiate the GABA induced fluorescence in a fluorescence (FLIPR) assay in CHO cells stably expressing the α1β2γ2 subtype of human GABAA receptors. With the aid of HPLC-based activity profiling the activity was linked to the neolignan connarin. In CHO cells the activity of connarin was not abolished by increasing concentrations of flumazenil, while the effect of diazepam was increased by increasing concentrations of connarin. The effect of connarin was abolished by pregnenolone sulfate (PREGS) in a concentration-dependent manner, and the effect of allopregnanolone was further increased by increasing concentrations of connarin. In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes transiently expressing GABAA receptors composed of human α1β2γ2S and α1β2 subunits connarin potentiated the GABA-induced currents, with EC50 values of 1.2 ± 0.3 μM (α1β2γ2S) and 1.3 ± 0.4 μM (α1β2), and with a maximum enhancement of currents Emax of 1959 ± 70% (α1β2γ2S) and 185 ± 48% (α1β2). The activation induced by connarin was abolished by increasing concentrations of PREGS.

Structural basis for the allosteric modulation of GABAA receptors by neurosteroids.

GABAA receptor, a pentameric ligand-gated ion channel (pLGIC) is the major inhibitory receptor in the human central nervous system. GABAA receptors could be assembled from 19 different subunits to form several subtypes. Both α1β2γ2 and α1β3γ2 subtypes constitute around 60% of all GABAA receptors in humans. GABAA receptor dysfunction is implicated in several neuropsychiatric abnormalities, including anxiety, depression, and epilepsy. Neurosteroids have been shown to modulate GABAA receptors through transmembrane sites, majorly through those located at the β/α interface. Recent FDA approval of allopregnanolone and ganaxolone for post-partum depression and epilepsy, respectively, demonstrate the increasingly important therapeutic role of neurosteroids. However, how subtle differences in their structures affect their subtype-specific binding and subsequent activation mechanism remain poorly understood. In this study, we examined the structural basis for allosteric modulation of α1β2γ2 and α1β3γ2 subtypes of the GABAA receptor by endogenous (allopregnanolone, THDOC) and synthetic (ganaxolone) neurosteroids. Using classical and enhanced simulation techniques, we studied the membrane partitioning characteristics and elucidated the access pathways of the neurosteroids to the receptor. Furthermore, unbiased simulations of closed and desensitized GABAA receptors in the presence and absence of neurosteroids were investigated for a total simulation time of 16 μs. We monitored the dynamic structural observables including global twisting, tilting, pore geometry, network interactions between orthosteric and allosteric sites, and orientations of important loops. We observed differential polar and hydrophobic interactions of the neurosteroids within the binding site, supported by membrane lipids. Our in-silico analyses provide useful insights into the structural basis for positive allosteric modulation of GABAA receptors, which will guide the design of new allosteric modulators of GABAA receptors.

Neurosteroids and translocator protein 18kDa (TSPO) in depression: implications for synaptic plasticity, cognition, and treatment options.

There is need for novel fast acting treatment options in affective disorders. 3α-reduced neurosteroids such as allopregnanolone are powerful positive allosteric modulators of GABAA receptors and target also extrasynaptic receptors. Their synthesis is mediated by the translocator protein 18kDa (TSPO). TSPO ligands not only promote endogenous neurosteroidogenesis, but also exert a broad spectrum of functions involving modulation of mitochondrial activity and acting as anti-inflammatory and neuroregenerative agents. Besides affective symptoms, in depression cognitive impairment can be frequently observed, which may be ameliorated through targeting of extrasynaptic GABAA receptors either via TSPO ligands or exogenously administered 3α-reduced neurosteroids. Interestingly, recent findings indicate an enhanced activation of the complement system, e.g., enhanced expression of C1q, both in depression and dementia. It is of note that benzodiazepines have been shown to reduce long-term potentiation and to cause cognitive decline. Intriguingly, TSPO may be crucial in mediating the effects of benzodiazepines on synaptic pruning. Here, we discuss how benzodiazepines and TSPO may interfere with synaptic pruning. Moreover, we highlight recent developments of TSPO ligands and 3α-reduced neurosteroids as therapeutic agents. Etifoxine is the only clinically available TSPO ligand so far and has been studied in anxiety disorders. Regarding 3α-reduced neurosteroids, brexanolone, an intravenous formulation of allopregnanolone, has been approved for the treatment of postpartum depression and zuranolone, an orally available 3α-reduced neurosteroid, is currently being studied in major depressive disorder and postpartum depression. As such, 3α-reduced neurosteroids and TSPO ligands may constitute promising treatment approaches for affective disorders.

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.

Rescue therapies for seizure clusters: Pharmacology and target of treatments.

The primary goal of treatment for seizure clusters is cessation of the cluster to avoid progression to more severe conditions, such as prolonged seizures and status epilepticus. Rescue therapies are key components of treatment plans for patients with seizure clusters. Three rescue therapies are approved in the United States for the treatment of seizure clusters: diazepam rectal gel, midazolam nasal spray, and diazepam nasal spray. This review characterizes the pharmacological function of rescue therapies for seizure clusters, as well as describing γ‐aminobutyric acid A (GABAA) receptor functions. GABAA receptors are heteropentamers, consisting primarily of α1‐6, β1‐3, γ2, and δ subunits in the central nervous system. These subunits can traffic to and from the membrane to regulate membrane potential. Benzodiazepines, such as diazepam and midazolam, are positive allosteric modulators of GABAA receptors, the activation of which leads to an increase in intracellular chloride, hyperpolarization of the cell membrane, and a reduction in excitation. GABAA receptor subunit mutations, dysregulation of trafficking, and degradation are associated with epilepsy. Although benzodiazepines are effective GABAA receptor modulators, individual formulations have unique profiles in practice. Diazepam rectal gel is an effective rescue therapy for seizure clusters; however, adults and adolescents may have social reservations regarding its administration. Intranasal delivery of midazolam or diazepam is a promising alternative to rectal administration because these formulations offer easy, socially acceptable administration and exhibit a rapid onset. Off‐label benzodiazepines, such as orally disintegrating lorazepam and intranasal use of an intravenous formulation of midazolam via nasal atomizer, are less well characterized regarding bioavailability and tolerability compared with approved agents.

Pronounced antiseizure activity of the subtype-selective GABAA positive allosteric modulator darigabat in a mouse model of drug-resistant focal epilepsy.

AimDarigabat is an α2/3/5 subunit‐selective positive allosteric modulator of GABAA receptors that has demonstrated broad‐spectrum activity in several preclinical models of epilepsy as well as in a clinical photoepilepsy trial. The objective here was to assess the acute antiseizure effect of darigabat in the mesial temporal lobe epilepsy (MTLE) mouse model of drug‐resistant focal seizures.MethodsThe MTLE model is generated by single unilateral intrahippocampal injection of low dose (1 nmole) kainic acid in adult mice, and subsequent epileptiform activity is recorded following implantation of a bipolar electrode under general anesthesia. After a period of epileptogenesis (~4 weeks), spontaneous and recurrent hippocampal paroxysmal discharges (HPD; focal seizures) are recorded using intracerebral electroencephalography. The number and cumulated duration of HPDs were recorded following administration of vehicle (PO), darigabat (0.3–10 mg kg−1, PO), and positive control diazepam (2 mg kg−1, IP).RESULTSDarigabat dose‐dependently reduced the expression of HPDs, demonstrating comparable efficacy profile to diazepam at doses of 3 and 10 mg kg−1.CONCLUSIONSDarigabat exhibited a robust efficacy profile in the MTLE model, a preclinical model of drug‐resistant focal epilepsy. A Phase II proof‐of‐concept placebo‐controlled, adjunctive‐therapy trial (NCT04244175) is ongoing to evaluate efficacy and safety of darigabat in patients with drug‐resistant focal seizures.

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.

Preventive Effects of Baclofen but Not Diazepam on Hippocampal Memory and Glucocorticoid Alterations After Prolonged Alcohol Withdrawal in Mice.

Our study aims at comparing in C57/Bl male mice, the impact of repeated injections of baclofen (an agonist of GABAB receptor) or diazepam (a benzodiazepine acting through a positive allosteric modulation of GABAA receptor) administered during the alcohol-withdrawal period on hippocampus-dependent memory impairments and brain regional glucocorticoid dysfunction after a short (1-week) or a long (4-week) abstinence. Hence, mice were submitted to a 6-month alcohol consumption (12%v/v) and were progressively withdrawn to water. Then, after a 1- or 4-weeks abstinence, they were submitted to a contextual memory task followed by measurements of corticosterone concentrations in the dorsal hippocampus (dHPC), the ventral hippocampus (vHPC) and the prefrontal cortex (PFC). Results showed that 1- and 4-week withdrawn mice exhibited a severe memory deficit and a significant abnormal rise of the test-induced increase of corticosterone (TICC) in the dHPC, as compared to water-controls or to mice still under alcohol consumption. Repeated daily systemic administrations of decreasing doses of diazepam (ranged from 0.5 to 0.12 mg/kg) or baclofen (ranged from 1.5 to 0.37 mg/kg) during the last 15 days of the withdrawal period, normalized both memory and TICC scores in the dHPC in 1-week withdrawn animals; in contrast, only baclofen-withdrawn mice showed both normal memory performance and TICC scores in the dHPC after a 4-week withdrawal period. In conclusion, the memory improvement observed in 4-week withdrawn mice administered with baclofen stem from the protracted normalization of glucocorticoid activity in the dHPC, a phenomenon encountered only transitorily in diazepam-treated withdrawn mice.