Brain activity is highly regulated by GABAergic activity, which can suppress neuronal excitability and synaptic integration. Tonic GABAergic conductances mediated by distinct receptor subtypes can also inhibit neural activity, although the consequences for dendritic calcium signaling are unclear. Here, we use 2-photon calcium imaging both ex vivo and in awake mice to show that α5-GABAARs mediate tonic currents in cortical pyramidal neurons that paradoxically enhance action potential-evoked dendritic calcium influx. Experimental and computational data indicate that the increased calcium influx arises via deinactivation of low-threshold voltage-gated channels. Tonic α5-mediated GABAergic currents and dendritic calcium signals are both enhanced by optogenetic activation of somatostatin-expressing interneurons (SST-INs). In addition, α5-mediated GABAergic facilitation of postsynaptic calcium signaling modulates the short-term plasticity of GABAergic transmission at SST-IN synapses. Our results demonstrate unexpected diversity in the function of both SST-INs and GABAergic signaling to influence dendritic activity and synaptic transmission in the cortex.

We investigated the correlation between increased gene copy number of gamma aminobutyric acid type A (GABAA) receptor α5-containing subunits and electrophysiological and behavioral phenotypes in a mouse model of Dup15q syndrome (15q dup) and tested the hypothesis that selectively inhibiting the activity of GABAA-α5 receptors may have therapeutic effects. Dup15q syndrome is a rare neurodevelopmental disorder caused by copy number gains of the 15q11.2-q13.1 chromosomal region, which includes UBE3A and a cluster of three genes (GABRA5, GABRB3, and GABRG3) encoding GABAA receptor subunits, all of which are critical for neural development and function. Most affected children display hypotonia, motor delays, intellectual disability, and epilepsy, as well as a characteristic electroencephalography (EEG) beta-band phenotype. There is no disease-modifying therapy available. Autoradiography showed increased density of GABAA-α5 receptors in the brains of 15q dup mice, while electrophysiology revealed enhanced GABAergic transmission in hippocampal slices from these mice. A GABAA-α5 negative allosteric modulator, RO4938581, decreased inhibitory synaptic charge transfer in 15q dup hippocampal slices. The behavioral analyses confirmed inflexibility in learning and abnormal social behaviors in 15q dup mice, and both phenotypes were normalized following chronic treatment with RO4938581. EEG recordings showed increased beta-power in 15q dup mice – which resembled the spectral signature of subjects with Dup15q – and was partially normalized following RO4938581 treatment. Our results suggest that excessive expression and function of the GABAA-α5 receptor subtype plays a key role in the pathophysiology of Dup15q and GABAA-α5 NAMs may represent a potential precision medicine therapeutic option.

Background:Alterations in the GABAergic system contribute to the pathophysiology of neurodevelopmental disorders, including autism spectrum disorder (ASD) and Angelman syndrome (AS), particularly in cases involving large deletions in the 15q11-13 region. Positive modulation of GABAA-α5 receptors may provide a novel therapeutic approach without the typical side effects associated with non-selective GABAA positive allosteric modulators such as diazepam. Methods:Alogabat was assessed for binding and functional activity at GABAA-α5β3γ2 receptors in vitro and in electrophysiological studies using hippocampal slices. In vivo studies in rodents included receptor occupancy (RO) using a selective GABAA-α5 tracer (autoradiography), pharmacological MRI, and electroencephalography (EEG). Alogabat was evaluated for its effects on the repetitive behavior phenotype in BTBR and contactin-associated protein-like 2 (Cntnap2-/-) knockout mice, seizure models, cognitive performance in rats, and rotarod performance following combination treatment with diazepam. Results:Alogabat is a potent positive allosteric modulator of GABAA-α5 receptors, with binding and functional selectivity. Receptor occupancy studies provided direct proof of dose-dependent target engagement. Functional circuit modulation was demonstrated by dose-dependent regional perfusion changes in pharmacological MRI and changes in EEG theta- and beta-band power in rats. At >50% GABAA-α5 receptor occupancy, alogabat normalized elevated self-grooming behavior in both Cntnap2-/- and BTBR mice and exhibited antiepileptic activity in rats. Alogabat did not impair cognition in wildtype rats at GABAA-α5 receptor occupancy up to 75%, although impairment occurred at higher doses, probably due to increased activity at other receptor subtypes and/or saturation of α5 receptors. Alogabat did not worsen diazepam-induced impairment on the rotarod test. Conclusion:Alogabat showed beneficial effects in mouse models relevant to neurodevelopmental disorders and anti-seizure activity at doses that did not produce cognitive, sedative, or motoric side effects.

Abstract Background There is compelling evidence that dysfunction of the GABAergic system, the main inhibitory neurotransmitter system in the brain, contributes to the pathophysiology of neurodevelopmental disorders including autism spectrum disorder and deletion Angelman syndrome (1, 2, 3, 4). Genetic studies highlight the critical role of GABAA receptor gene dosage and function within the 15q11-13 chromosomal region in the etiology of these conditions (5, 6, 7, 8). This region encompasses several genes including UBE3A and GABRB3, GABRA5, GABRG3 encoding the β3, α5, and g3 GABAA receptor subunits, respectively. These subunits together with the g2 subunit co-assemble to form the GABAA- α5 receptor subtype (9). Therefore, positive modulation of GABAA-α5 receptors may provide a novel therapeutic approach to restore deficient GABAergic signaling without the typical side effects of non- selective GABAA positive allosteric modulators (PAM) i.e., diazepam. Aims & Objectives Assess the in vitro and in vivo pharmacological profile of alogabat, a novel small molecule, selective GABAA-α5 receptor positive allosteric modulator. Provide preclinical proof of concept data to support clinical studies in ASD and Angelman syndrome. Methods Alogabat was evaluated for selective binding and functional activity in vitro at GABAA-α5β3g2 receptors, including electrophysiological studies in hippocampal slices. In vivo studies included receptor occupancy (RO) using a selective GABAA-α5 tracer (autoradiography), pharmacological magnetic resonance imaging (phMRI) and EEG in rodents. Alogabat was assessed on the repetitive behavior phenotype in BTBR and contactin-associated protein-like 2 knockout (Cntnap2-/-) mice, on seizure models and cognitive performance in rats, and on rotarod performance following a combination treatment with diazepam. Results Alogabat is a potent PAM of the GABAA-α5 receptor with binding and functional selectivity. RO studies provided direct proof of dose-dependent target engagement. Functional circuit modulation was demonstrated by dose-dependent regional perfusion changes in phMRI and change in EEG theta and beta band power. Alogabat at >50% RO at GABAA-α5 normalized elevated self-grooming in both Cntnap2-/- and BTBR mice and exhibited antiepileptic activity in rats. Alogabat did not impair cognition in rats at RO up to 88%, although impairment occurred at higher doses probably due to decreased α-subunit selectivity. Alogabat did not worsen diazepam-induced rotarod impairment. Conclusions Alogabat showed beneficial effects in mouse models relevant for NDD, as well as anti- seizure activity, at doses without cognitive, sedative, and motoric side effects. In addition, translational biomarkers were identified to guide clinical studies: PET, phMRI, EEG. References 1. Braat S, Kooy RF. The GABAA receptor as a therapeutic target for neurodevelopmental disorders. Neuron 2015;86:1119-30. 2. Ali Rodriguez R, Joya C, Hines RM. Ribs of inhibitory synaptic dysfunction in the umbrella of neurodevelopmental disorders. Front Mol Neurosci. 2018;11:132. 3. Tang X, Jaenisch R, Sur M. The role of GABAergic signalling in neurodevelopmental disorders. Nature Reviews Neuroscience. 2021 (5):290-307. 4. Zhao H, Mao X, Zhu C, et al. GABAergic system dysfunction in autism spectrum disorders. Front Cell Dev Biol. 2022; 9:781327. 5. Warrier V, Baron-Cohen S, Chakrabarti B. Genetic variation in GABRB3 is associated with Asperger syndrome and multiple endophenotypes relevant to autism. Mol Autism 2013;4:48. 6. Zurek AA, Kemp SW, Aga Z, et al. a5GABAA receptor deficiency causes autism-like behaviors. Ann Clin Transl Neurol 2016;3:392-8. 7. Hogart A, Wu D, LaSalle JM, et al. The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 2010;38:181-91. 8. Frohlich J, Miller MT, Bird LM, et al. Electrophysiological phenotype in Angelman syndrome differs between genotypes. Biol Psychiatry 2019; 85(9):752-9. 9. Sur C, Fresu L, Howell O, et al. Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain. Brain Res 1999; 822:265-70.

IntroductionSocial behavioral changes are a hallmark of several neurodevelopmental and neuropsychiatric conditions, nevertheless the underlying neural substrates of such dysfunction remain poorly understood. Building evidence points to the prefrontal cortex (PFC) as one of the key brain regions that orchestrates social behavior. We used this concept with the aim to develop a translational rat model of social-circuit dysfunction, the chronic PFC activation model (CPA).MethodsChemogenetic designer receptor hM3Dq was used to induce chronic activation of the PFC over 10 days, and the behavioral and electrophysiological signatures of prolonged PFC hyperactivity were evaluated. To test the sensitivity of this model to pharmacological interventions on longer timescales, and validate its translational potential, the rats were treated with our novel highly selective oxytocin receptor (OXTR) agonist RO6958375, which is not activating the related vasopressin V1a receptor.ResultsCPA rats showed reduced sociability in the three-chamber sociability test, and a concomitant decrease in neuronal excitability and synaptic transmission within the PFC as measured by electrophysiological recordings in acute slice preparation. Sub-chronic treatment with a low dose of the novel OXTR agonist following CPA interferes with the emergence of PFC circuit dysfunction, abnormal social behavior and specific transcriptomic changes.DiscussionThese results demonstrate that sustained PFC hyperactivity modifies circuit characteristics and social behaviors in ways that can be modulated by selective OXTR activation and that this model may be used to understand the circuit recruitment of prosocial therapies in drug discovery.

α5 subunit-containing γ-aminobutyric acid type A (GABA A ) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Ligand-gated ion channels are integral membrane proteins that activate through a change in conformation upon transmitter binding and were identified as key players of brain function. GABAA receptors are major inhibitory ligand-gated ion channels of this protein family. They are the target of many therapeutic compounds widely used in the clinic and continue to attract the attention of academic and pharmaceutical laboratories. Advances in the knowledge of the structure of GABAA receptors at the molecular level with unprecedented resolution enabled the determination of the binding sites of many allosteric modulators revealing the nature of their interactions with the receptors. Herein, we review the latest findings on allosteric modulation of GABAA receptors and their relevance to drug discovery.

Brain activity is highly regulated by GABAergic activity, which acts via GABARs to suppress somatic spike generation as well as dendritic synaptic integration and calcium signaling. Tonic GABAergic conductances mediated by distinct receptor subtypes can also inhibit neuronal excitability and spike output, though the consequences for dendritic calcium signaling are unclear. Here, we use 2-photon calcium imaging in cortical pyramidal neurons and computational modeling to show that low affinity GABARs containing an α5 subunit mediate a tonic hyperpolarization of the dendritic membrane potential, resulting in deinactivation of voltage-gated calcium channels and a paradoxical boosting of action potential-evoked calcium influx. We also find that GABAergic enhancement of calcium signaling modulates short-term synaptic plasticity, augmenting depolarization-induced suppression of inhibition. These results demonstrate a novel role for GABA in the control of dendritic activity and suggest a mechanism for differential modulation of electrical and biochemical signaling.

Down syndrome (DS) or Trisomy 21 is a developmental disorder leading to cognitive deficits, including disruption of hippocampus-dependent learning and memory. Enhanced inhibition has been suggested to underlie these deficits in DS based on studies using the Ts65Dn mouse model. Here we show that, in this mouse model, GABAergic synaptic inhibition onto dendrites of hippocampal pyramidal cells is increased. By contrast, somatic inhibition was not altered. In addition, synaptic NMDAR currents were reduced. Furthermore, dendritic inhibition was mediated via nonlinear α5-subunit containing GABAARs that closely matched the kinetics and voltage dependence of NMDARs. Thus, enhanced dendritic inhibition and reduced NMDA currents strongly decreased burst-induced NMDAR-mediated depolarization and impaired LTP induction. Finally, selective reduction of α5-GABAAR-mediated inhibition rescued both burst-induced synaptic NMDAR activation and synaptic plasticity. These results demonstrate that reduced synaptic NMDAR activation and synaptic plasticity in the Ts65Dn mouse model of DS can be corrected by specifically targeting nonlinear dendritic inhibition.SIGNIFICANCE STATEMENT Mild to moderate intellectual disability is a prominent feature of Down syndrome. Previous studies in mouse models suggest that increased synaptic inhibition is a main factor for decreased synaptic plasticity, the cellular phenomenon underlying memory. The present study shows that increased inhibition specifically onto dendrites together with reduced NMDAR content in excitatory synapses may be the cause. Reducing a slow nonlinear component that is specific to dendritic inhibitory inputs and mediated by α5 subunit-containing GABAA receptors rescues both NMDAR activation and synaptic plasticity.

Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Dysfunction of the N-methyl-d-aspartate receptor (NMDAR) is thought to play a role in the pathophysiology of neurodevelopmental diseases like schizophrenia. To study the effects of NMDAR dysfunction on synaptic transmission and network oscillations, we used hippocampal tissue of NMDAR subunit GluN2A knockout (KO) mice. Field excitatory postsynaptic potentials were recorded in acute hippocampal slices of adult animals. Synaptic transmission was impaired in GluN2A KO slices compared to wild-type (WT) slices. Further, to investigate whether NMDAR dysfunction would alter neurodevelopment in vitro, we used organotypic hippocampal slice cultures of WT and GluN2A KO mice. Immunostaining performed with cultures kept two, seven, 14, 25 days in vitro (DIV) revealed an increasing expression of parvalbumin (PV) over time. As a functional readout, oscillatory activity induced by the cholinergic agonist carbachol was recorded in cultures kept seven, 13, and 26 DIV using microelectrode arrays. Initial analysis focused on the occurrence of delta, theta, beta and gamma oscillations over genotype, DIV and hippocampal area (CA1, CA3, dentate gyrus (DG)). In a follow-up analysis, we studied the peak frequency and the peak power of each of the four oscillation bands per condition. The occurrence of gamma oscillations displayed an increase by DIV similar to the PV immunostaining. Unlike gamma occurrence, delta, theta, and beta occurrence did not change over time in culture. The peak frequency and peak power in the different bands of the oscillations were not different in slices of WT and GluN2A KO mice. However, the level of PV expression was lower in GluN2A KO compared to WT mice. Given the role of PV-containing fast-spiking basket cells in generation of oscillations and the decreased PV expression in subjects with schizophrenia, the study of gamma oscillations in organotypic hippocampal slices represents a potentially valuable tool for the characterization of novel therapeutic drugs.

Dendrite-targeting GABAergic interneurons powerfully control postsynaptic integration, synaptic plasticity, and learning. However, the mechanisms underlying the efficient GABAergic control of dendritic electrogenesis are not well understood. Using subtype-selective blockers for GABA A receptors, we show that dendrite-targeting somatostatin interneurons and NO-synthase-positive neurogliaform cells preferentially activate α5-subunit- containing GABA A receptors (α5-GABA A Rs), generating slow inhibitory postsynaptic currents (IPSCs) in hippocampal CA1 pyramidal cells. By contrast, only negligible contribution of these receptors could be found in perisomatic IPSCs, generated by fast-spiking parvalbumin interneurons. Remarkably, α5-GABA A R-mediated IPSCs were strongly outward-rectifying generating 4-fold larger conductances above –50 mV than at rest. Experiments and modeling show that synaptic activation of these receptors can very effectively control voltage-dependent NMDA-receptor activation as well as Schaffer-collateral evoked burst firing in pyramidal cells. Taken together, nonlinear-rectifying α5-GABA A Rs with slow kinetics match functional NMDA-receptor properties and thereby mediate powerful control of dendritic postsynaptic integration and action potential firing by dendrite-targeting interneurons.

BACKGROUND:The prefrontal cortex (PFC) has been implicated in the pathophysiology of social dysfunction, but the specific circuit partners mediating PFC function in health and disease are unclear.METHODS:The excitatory designer receptor exclusively activated by designer drugs (DREADD) hM3Dq was used to induce PFC activation during social behavior measured in the three-chamber sociability assay (rats/mice). Functional magnetic resonance imaging was combined with hM3Dq-mediated PFC activation to identify novel nodes in the "social brain" in a hypothesis-free manner. In multiplexed DREADD experiments, hM3Dq and the inhibitory KORDi were used to bidirectionally modulate PFC activity and measure social behavior and global functional magnetic resonance imaging signature. To characterize the functional role of specific nodes identified in this functional magnetic resonance imaging screen, we used anterograde and retrograde tracers, optogenetic and DREADD-assisted circuit mapping, and circuit behavioral experiments.RESULTS:PFC activation suppressed social behavior and modulated activity in a number of regions involved in emotional behavior. Bidirectional modulation of PFC activity further refined this subset of brain regions and identified the habenula as a node robustly correlated with PFC activity. Furthermore, we showed that the lateral habenula (LHb) receives direct synaptic input from the PFC and that activation of LHb neurons or the PFC inputs to the LHb suppresses social preference. Finally, we demonstrated that LHb inhibition can prevent the social deficits induced by PFC activation.CONCLUSIONS:The LHb is thought to provide reward-related contextual information to the mesolimbic reward system known to be involved in social behavior. Thus, PFC projections to the LHb may represent an important part of descending PFC pathways that control social behavior.

GABAA receptors are ligand-gated ion channels that form a fundamental component of inhibitory neurotransmission in the central and peripheral nervous systems. However, since the initial recordings of inhibitory electrical activity of neurons in response to GABA, these receptors have been found to play a more complex role and can, under some circumstances, function in an excitatory manner. This has been demonstrated via electrophysiological recordings conducted in both mature and developing neurons from different brain regions, as well as in various subcellular locations such as dendritic and axonal membranes. The balance between the inhibitory and excitatory effects mediated by GABAA receptor activation depends not only on multiple factors that govern the equilibrium of the transmembrane chloride gradient, but also on bicarbonate concentration. Moreover, electrophysiological and fluorescence measurements have revealed that a spatial distribution of the chloride gradient exists within neurons, which locally influences the effects mediated by GABAA receptor activation. In recent years, it has also become apparent that intra-neuronal chloride concentration is partially regulated by cation-chloride co-transporters (CCCs), in particular NKCC1 and KCC2. The aim of the present commentary is to discuss, in light of the latest findings, potential implications of the tight spatial and temporal regulation of chloride equilibrium in health and disease, as well as its relevance for the therapeutic effects of molecules acting at GABAA receptors.

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2+/− and TSC2−/− neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.