Sustained Inhibition of GABA-AT by OV329 Enhances Neuronal Inhibition and Prevents Development of Benzodiazepine Refractory Seizures.

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain which mediates its rapid effects on neuronal excitability via ionotropic GABAA receptors. GABA levels in the brain are critically dependent upon GABA-aminotransferase (GABA-AT) which promotes its degradation. Vigabatrin, a low-affinity GABA-AT inhibitor, exhibits anticonvulsant efficacy, but its use is limited due to cumulative ocular toxicity. OV329 is a rationally designed, next-generation GABA-AT inhibitor with enhanced potency. We demonstrate that sustained exposure to OV329 in mice reduces GABA-AT activity and subsequently elevates GABA levels in the brain. Parallel increases in the efficacy of GABAergic inhibition were evident, together with elevations in electroencephalographic delta power. Consistent with this, OV329 exposure reduced the severity of status epilepticus and the development of benzodiazepine refractory seizures. Thus, OV329 may be of utility in treating seizure disorders and associated pathologies that result from neuronal hyperexcitability.

Baicalin and baicalein from Scutellaria baicalensis Georgi alleviate aberrant neuronal suppression mediated by GABA from reactive astrocytes.

AIMS: γ-aminobutyric acid (GABA) from reactive astrocytes is critical for the dysregulation of neuronal activity in various neuroinflammatory conditions. While Scutellaria baicalensis Georgi (S. baicalensis) is known for its efficacy in addressing neurological symptoms, its potential to reduce GABA synthesis in reactive astrocytes and the associated neuronal suppression remains unclear. This study focuses on the inhibitory action of monoamine oxidase B (MAO-B), the key enzyme for astrocytic GABA synthesis. METHODS: Using a lipopolysaccharide (LPS)-induced neuroinflammation mouse model, we conducted immunohistochemistry to assess the effect of S. baicalensis on astrocyte reactivity and its GABA synthesis. High-performance liquid chromatography was performed to reveal the major compounds of S. baicalensis, the effects of which on MAO-B inhibition, astrocyte reactivity, and tonic inhibition in hippocampal neurons were validated by MAO-B activity assay, qRT-PCR, and whole-cell patch-clamp. RESULTS: The ethanolic extract of S. baicalensis ameliorated astrocyte reactivity and reduced excessive astrocytic GABA content in the CA1 hippocampus. Baicalin and baicalein exhibited significant MAO-B inhibition potential. These two compounds downregulate the mRNA levels of genes associated with reactive astrogliosis or astrocytic GABA synthesis. Additionally, LPS-induced aberrant tonic inhibition was reversed by both S. baicalensis extract and its key compounds. CONCLUSIONS: In summary, baicalin and baicalein isolated from S. baicalensis reduce astrocyte reactivity and alleviate aberrant tonic inhibition of hippocampal neurons during neuroinflammation.

Neurosteroid Modulation of Synaptic and Extrasynaptic GABAA Receptors of the Mouse Nucleus Accumbens.

The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2ßγ2) and extrasynaptic (α4ßδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed.

Phasic/tonic glial GABA differentially transduce for olfactory adaptation and neuronal aging.

Phasic (fast) and tonic (sustained) inhibition of γ-aminobutyric acid (GABA) are fundamental for regulating day-to-day activities, neuronal excitability, and plasticity. However, the mechanisms and physiological functions of glial GABA transductions remain poorly understood. Here, we report that the AMsh glia in Caenorhabditis elegans exhibit both phasic and tonic GABAergic signaling, which distinctively regulate olfactory adaptation and neuronal aging. Through genetic screening, we find that GABA permeates through bestrophin-9/-13/-14 anion channels from AMsh glia, which primarily activate the metabolic GABAB receptor GBB-1 in the neighboring ASH sensory neurons. This tonic action of glial GABA regulates the age-associated changes of ASH neurons and olfactory responses via a conserved signaling pathway, inducing neuroprotection. In addition, the calcium-evoked, vesicular glial GABA release acts upon the ionotropic GABAA receptor LGC-38 in ASH neurons to regulate olfactory adaptation. These findings underscore the fundamental significance of glial GABA in maintaining healthy aging and neuronal stability.

Enhancing GABAergic Tonic Inhibition Reduces Seizure-Like Activity in the Neonatal Mouse Hippocampus and Neocortex.

Approximately one-third of neonatal seizures do not respond to first-line anticonvulsants, including phenobarbital, which enhances phasic inhibition. Whether enhancing tonic inhibition decreases seizure-like activity in the neonate when GABA is mainly depolarizing at this age is unknown. We evaluated if increasing tonic inhibition using THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, gaboxadol], a δ-subunit-selective GABAA receptor agonist, decreases seizure-like activity in neonatal C57BL/6J mice (postnatal day P5-8, both sexes) using acute brain slices. Whole-cell patch-clamp recordings showed that THIP enhanced GABAergic tonic inhibitory conductances in layer V neocortical and CA1 pyramidal neurons and increased their rheobase without altering sEPSC characteristics. Two-photon calcium imaging demonstrated that enhancing the activity of extrasynaptic GABAARs decreased neuronal firing in both brain regions. In the 4-aminopyridine and the low-Mg2+ model of pharmacoresistant seizures, THIP reduced epileptiform activity in the neocortex and CA1 hippocampal region of neonatal and adult brain slices in a dose-dependent manner. We conclude that neocortical layer V and CA1 pyramidal neurons have tonic inhibitory conductances, and when enhanced, they reduce neuronal firing and decrease seizure-like activity. Therefore, augmenting tonic inhibition could be a viable approach for treating neonatal seizures.

Cortical Tonic Inhibition Gates the Expression of Spike-and-Wave Discharges Associated with Absence Epilepsy.

OBJECTIVE: Absence seizures result from aberrant thalamocortical processing that confers synchronous, bilateral spike-and-wave discharges (SWDs) and behavioral arrest. Previous work has demonstrated that SWDs can result from enhanced thalamic tonic inhibition, consistent with the mechanism of first-line antiabsence drugs that target thalamic low-voltage-activated calcium channels. However, nearly half of patients with absence epilepsy are unresponsive to first-line medications. In this study we evaluated the role of cortical tonic inhibition and its manipulation on absence seizure expression. METHODS: We used video-electroencephalogram (EEG) monitoring to show that mice with a γ-aminobutyric acid type A (GABAA) receptor mutation (γ2R43Q) display absence seizures. Voltage-clamp recordings in brain slices from wild type and γ2R43Q mice were used to evaluate the amount of tonic inhibition and its selective pharmacological modulation. Finally, we determined whether modulating tonic inhibition controls seizure expression. RESULTS: γ2R43Q mice completely lack tonic inhibition in principal neurons of both layer 2/3 cortex and ventrobasal thalamus. Blocking cortical tonic inhibition in wild type mice is sufficient to elicit SWDs. Tonic inhibition in slices from γ2R43Q mice could be rescued in a dose-dependent fashion by the synthetic neurosteroid ganaxolone. Low-dose ganaxolone suppressed seizures in γ2R43Q mice. CONCLUSIONS: Our data suggest that reduced cortical tonic inhibition promotes absence seizures and that normal function can be restored via selective pharmacological rescue. These results, together with previous findings, suggest that deviations of tonic inhibition either above or below an optimal set point can contribute to absence epilepsy. Returning the thalamocortical system to this set point may provide a novel treatment for refractory absence epilepsy.

Disinhibition Is an Essential Network Motif Coordinated by GABA Levels and GABA B Receptors.

Network dynamics are crucial for action and sensation. Changes in synaptic physiology lead to the reorganization of local microcircuits. Consequently, the functional state of the network impacts the output signal depending on the firing patterns of its units. Networks exhibit steady states in which neurons show various activities, producing many networks with diverse properties. Transitions between network states determine the output signal generated and its functional results. The temporal dynamics of excitation/inhibition allow a shift between states in an operational network. Therefore, a process capable of modulating the dynamics of excitation/inhibition may be functionally important. This process is known as disinhibition. In this review, we describe the effect of GABA levels and GABAB receptors on tonic inhibition, which causes changes (due to disinhibition) in network dynamics, leading to synchronous functional oscillations.

GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex.

Extrasynaptic GABAA receptors (GABAARs) mediating tonic inhibition are thought to play an important role in the regulation of neuronal excitability. However, little is known about a cell type-specific tonic inhibition in molecularly distinctive types of GABAergic interneurons in the mammalian neocortex. Here, we used whole-cell patch-clamp techniques in brain slices prepared from transgenic mice expressing red fluorescent protein (TdTomato) in vasoactive intestinal polypeptide- or somatostatin- positive interneurons (VIP-INs and SST-INs, respectively) to investigate tonic and phasic GABAAR-mediated inhibition as well as effects of GABAA inhibition on intrinsic excitability of these interneurons in layers 2/3 (L2/3) of the somatosensory (barrel) cortex. We found that tonic inhibition was stronger in VIP-INs compared to SST-INs. Contrary to the literature data, tonic inhibition in SST-INs was comparable to pyramidal (Pyr) neurons. Next, tonic inhibition in both interneuron types was dependent on the activity of delta subunit-containing GABAARs. Finally, the GABAAR activity decreased intrinsic excitability of VIP-INs but not SST-INs. Altogether, our data indicate that GABAAR-mediated inhibition modulates neocortical interneurons in a type-specific manner. In contrast to L2/3 VIP-INs, intrinsic excitability of L2/3 SST-INs is immune to the GABAAR-mediated inhibition.

Differential expression of GABAA receptor subunits δ and α6 mediates tonic inhibition in parvalbumin and somatostatin interneurons in the mouse hippocampus.

Inhibitory γ-aminobutyric acid (GABA)-ergic interneurons mediate inhibition in neuronal circuitry and support normal brain function. Consequently, dysregulation of inhibition is implicated in various brain disorders. Parvalbumin (PV) and somatostatin (SST) interneurons, the two major types of GABAergic inhibitory interneurons in the hippocampus, exhibit distinct morpho-physiological properties and coordinate information processing and memory formation. However, the molecular mechanisms underlying the specialized properties of PV and SST interneurons remain unclear. This study aimed to compare the transcriptomic differences between these two classes of interneurons in the hippocampus using the ribosome tagging approach. The results revealed distinct expressions of genes such as voltage-gated ion channels and GABAA receptor subunits between PV and SST interneurons. Gabrd and Gabra6 were identified as contributors to the contrasting tonic GABAergic inhibition observed in PV and SST interneurons. Moreover, some of the differentially expressed genes were associated with schizophrenia and epilepsy. In conclusion, our results provide molecular insights into the distinct roles of PV and SST interneurons in health and disease.

Neuroendocrine insights into neurosteroid therapy for postpartum depression.

Postpartum depression (PPD) is associated with a decline in progesterone-derived anxiolytic-antidepressant neurosteroids after delivery. Neurosteroid replacement therapy (NRT) with GABA-A receptor-modulating allopregnanolone (brexanolone) shows promise as the first drug treatment for PPD. Here we describe the molecular insights of the neurosteroid approach for rapid relief of PPD symptoms compared with traditional antidepressants.

Bidirectional plasticity of GABAergic tonic inhibition in hippocampal somatostatin- and parvalbumin-containing interneurons.

GABAA receptors present in extrasynaptic areas mediate tonic inhibition in hippocampal neurons regulating the performance of neural networks. In this study, we investigated the effect of NMDA-induced plasticity on tonic inhibition in somatostatin- and parvalbumin-containing interneurons. Using pharmacological methods and transgenic mice (SST-Cre/PV-Cre x Ai14), we induced the plasticity of GABAergic transmission in somatostatin- and parvalbumin-containing interneurons by a brief (3 min) application of NMDA. In the whole-cell patch-clamp configuration, we measured tonic currents enhanced by specific agonists (etomidate or gaboxadol). Furthermore, in both the control and NMDA-treated groups, we examined to what extent these changes depend on the regulation of distinct subtypes of GABAA receptors. Tonic conductance in the somatostatin-containing (SST+) interneurons is enhanced after NMDA application, and the observed effect is associated with an increased content of α5-containing GABAARs. Both fast-spiking and non-fast-spiking parvalbumin-positive (PV+) cells showed a reduction of tonic inhibition after plasticity induction. This effect was accompanied in both PV+ interneuron types by a strongly reduced proportion of δ-subunit-containing GABAARs and a relatively small increase in currents mediated by α5-containing GABAARs. Both somatostatin- and parvalbumin-containing interneurons show cell type-dependent and opposite sign plasticity of tonic inhibition. The underlying mechanisms depend on the cell-specific balance of plastic changes in the contents of α5 and δ subunit-containing GABAARs.

Functional and structural synaptic remodeling mechanisms underlying somatotopic organization and reorganization in the thalamus.

The somatosensory system organizes the topographic representation of body maps, termed somatotopy, at all levels of an ascending hierarchy. Postnatal maturation of somatotopy establishes optimal somatosensation, whereas deafferentation in adults reorganizes somatotopy, which underlies pathological somatosensation, such as phantom pain and complex regional pain syndrome. Here, we focus on the mouse whisker somatosensory thalamus to study how sensory experience shapes the fine topography of afferent connectivity during the critical period and what mechanisms remodel it and drive a large-scale somatotopic reorganization after peripheral nerve injury. We will review our findings that, following peripheral nerve injury in adults, lemniscal afferent synapses onto thalamic neurons are remodeled back to immature configuration, as if the critical period reopens. The remodeling process is initiated with local activation of microglia in the brainstem somatosensory nucleus downstream to injured nerves and heterosynaptically controlled by input from GABAergic and cortical neurons to thalamic neurons. These fruits of thalamic studies complement well-studied cortical mechanisms of somatotopic organization and reorganization and unveil potential intervention points in treating pathological somatosensation.

Pallidal GABA B receptors: involvement in cortex beta dynamics and thalamic reticular nucleus activity.

The external globus pallidus (GP) firing rate synchronizes the basal ganglia-thalamus-cortex network controlling GABAergic output to different nuclei. In this context, two findings are significant: the activity and GABAergic transmission of the GP modulated by GABA B receptors and the presence of the GP-thalamic reticular nucleus (RTn) pathway, the functionality of which is unknown. The functional participation of GABA B receptors through this network in cortical dynamics is feasible because the RTn controls transmission between the thalamus and cortex. To analyze this hypothesis, we used single-unit recordings of RTn neurons and electroencephalograms of the motor cortex (MCx) before and after GP injection of the GABA B agonist baclofen and the antagonist saclofen in anesthetized rats. We found that GABA B agonists increase the spiking rate of the RTn and that this response decreases the spectral density of beta frequency bands in the MCx. Additionally, injections of GABA B antagonists decreased the firing activity of the RTn and reversed the effects in the power spectra of beta frequency bands in the MCx. Our results proved that the GP modulates cortical oscillation dynamics through the GP-RTn network via tonic modulation of RTn activity.

Ventral tegmental area astrocytes modulate cocaine reward by tonically releasing GABA.

Addictive drugs increase ventral tegmental area (VTA) dopamine (DA) neuron activity through distinct cellular mechanisms, one of which involves disinhibition of DA neurons by inhibiting local GABA neurons. How drugs regulate VTA GABA neuron activity and drive addictive behaviors remains poorly understood. Here, we show that astrocytes control VTA GABA neuron activity in cocaine reward via tonic inhibition in mice. Repeated cocaine exposure potentiates astrocytic tonic GABA release through volume-regulated anion channels (VRACs) and augments tonic inhibition of VTA GABA neurons, thus downregulating their activities and disinhibiting nucleus accumbens (NAc) projecting DA neurons. Attenuation of tonic inhibition by either deleting Swell1 (Lrrc8a), the obligatory subunit of VRACs, in VTA astrocytes or disrupting δ subunit of GABAA receptors in VTA GABA neurons reduces cocaine-evoked changes in neuron activity, locomotion, and reward behaviors in mice. Together, our findings reveal the critical role of astrocytes in regulating the VTA local circuit and cocaine reward.

GABAA(δ) receptor hypofunction in the amygdala-hippocampal circuit underlies stress-induced anxiety.

Dysregulated GABAergic inhibition in the amygdala has long been implicated in stress-related neuropsychiatric disorders. However, the molecular and circuit mechanisms underlying the dysregulation remain elusive. Here, by using a mouse model of chronic social defeat stress (CSDS), we observed that the dysregulation varied drastically across individual projection neurons (PNs) in the basolateral amygdala (BLA), one of the kernel amygdala subregions critical for stress coping. While persistently reducing the extrasynaptic GABAA receptor (GABAAR)-mediated tonic current in the BLA PNs projecting to the ventral hippocampus (BLA â†’ vHPC PNs), CSDS increased the current in those projecting to the anterodorsal bed nucleus of stria terminalis (BLA â†’ adBNST PNs), suggesting projection-based dysregulation of tonic inhibition in BLA PNs by CSDS. Transcriptional and electrophysiological analysis revealed that the opposite CSDS influences were mediated by loss- and gain-of-function of δ-containing GABAARs (GABAA(δ)Rs) in BLA â†’ vHPC and BLA â†’ adBNST PNs, respectively. Importantly, it was the lost inhibition in the former population but not the augmentation in the latter population that correlated with the increased anxiety-like behavior in CSDS mice. Virally mediated maintenance of GABAA(δ)R currents in BLA â†’ vHPC PNs occluded CSDS-induced anxiety-like behavior. These findings clarify the molecular substrate for the dysregulated GABAergic inhibition in amygdala circuits for stress-associated psychopathology.

Virally-induced expression of GABAA receptor δ subunits following their pathological loss reveals their role in regulating GABAA receptor assembly.

Decreased expression of the δ subunit of the GABAA receptor (GABAAR) has been found in the dentate gyrus in several animal models of epilepsy and other disorders with increased excitability and is associated with altered modulation of tonic inhibition in dentate granule cells (GCs). In contrast, other GABAAR subunits, including α4 and γ2 subunits, are increased, but the relationship between these changes is unclear. The goals of this study were to determine if viral transfection of δ subunits in dentate GCs could increase δ subunit expression, alter expression of potentially-related GABAAR subunits, and restore more normal network excitability in the dentate gyrus in a mouse model of epilepsy. Pilocarpine-induced seizures were elicited in DOCK10-Cre mice that express Cre selectively in dentate GCs, and two weeks later the mice were injected unilaterally with a Cre-dependent δ-GABAAR viral vector. At 4-6 weeks following transfection, δ subunit immunolabeling was substantially increased in dentate GCs on the transfected side compared to the nontransfected side. Importantly, α4 and γ2 subunit labeling was downregulated on the transfected side. Electrophysiological studies revealed enhanced tonic inhibition, decreased network excitability, and increased neurosteroid sensitivity in slices from the δ subunit-transfected side compared to those from the nontransfected side of the same pilocarpine-treated animal, consistent with the formation of δ subunit-containing GABAARs. No differences were observed between sides of eYFP-transfected animals. These findings are consistent with the idea that altering expression of key subunits, such as the δ subunit, regulates GABAAR subunit assemblies, resulting in substantial effects on network excitability.

Elevation of GABA levels in the globus pallidus disinhibits the thalamic reticular nucleus and desynchronized cortical beta oscillations.

The external globus pallidus (GP) is a GABAergic node involved in motor control regulation and coordinates firing and synchronization in the basal ganglia-thalamic-cortical network through inputs and electrical activity. In Parkinson's disease, high GABA levels alter electrical activity in the GP and contribute to motor symptoms. Under normal conditions, GABA levels are regulated by GABA transporters (GATs). GAT type 1 (GAT-1) is highly expressed in the GP, and pharmacological blockade of GAT-1 increases the duration of currents mediated by GABA A receptors and induces tonic inhibition. The functional contribution of the pathway between the GP and the reticular thalamic nucleus (RTn) is unknown. This pathway is important since the RTn controls the flow of information between the thalamus and cortex, suggesting that it contributes to cortical dynamics. In this work, we investigated the effect of increased GABA levels on electrical activity in the RTn by obtaining single-unit extracellular recordings from anesthetized rats and on the motor cortex (MCx) by corticography. Our results show that high GABA levels increase the spontaneous activity rate of RTn neurons and desynchronize oscillations in the beta frequency band in the MCx. Our findings provide evidence that the GP exerts tonic control over RTn activity through the GP-reticular pathway and functionally contributes to cortical oscillation dynamics.

FMRP regulates GABAA receptor channel activity to control signal integration in hippocampal granule cells.

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by loss of fragile X mental retardation protein (FMRP). GABAergic system dysfunction is one of the hallmarks of FXS, yet the underlying mechanisms remain poorly understood. Here, we report that FMRP interacts with GABAA receptor (GABAAR) and modulates its single-channel activity. Specifically, FMRP regulates spontaneous GABAAR opening through modulating its single-channel conductance and open probability in dentate granule cells. FMRP loss reduces spontaneous GABAAR activity underlying tonic inhibition, while N-terminal FMRP fragment (aa 1-297) is sufficient to rapidly normalize tonic inhibition in Fmr1 knockout (KO) granule cells. FMRP-GABAAR interaction is supported by co-immunoprecipitation of FMRP with at least one GABAAR subunit, the α5. Functionally, FMRP-GABAAR interaction ensures accuracy of coincidence detection of granule cells, which is markedly reduced in Fmr1 KOs. Our study reveals a mechanism underlying FMRP regulation of the GABAergic system and information processing in the hippocampus.

Activity-Dependent Modulation of Tonic GABA Currents by Endocannabinoids in Hirudo verbana.

Endocannabinoids are lipid neuromodulators that are synthesized on demand and primarily signal in a retrograde manner to elicit depression of excitatory and inhibitory synapses. Despite the considerable interest in their potential analgesic effects, there is evidence that endocannabinoids can have both pro-nociceptive and anti-nociceptive effects. The mechanisms contributing to the opposing effects of endocannabinoids in nociception need to be better understood before cannabinoid-based therapies can be effectively utilized to treat pain. Using the medicinal leech, Hirudo verbana, this work investigates whether endocannabinoids modulate tonic inhibition onto non-nociceptive afferents. In voltage clamp recordings, we analyzed changes in the tonic inhibition in pressure-sensitive (P) cells following pre-treatment with endocannabinoids, 2-arachidonoylglycerol (2-AG) or anandamide (AEA). We also tested whether high frequency stimulation (HFS) of nociceptive (N) cells could also modulate tonic inhibition. Both endocannabinoid application and N cell HFS depressed tonic inhibition in the P cell. Depression of tonic inhibition by N cell HFS was blocked by SB 366791 (a TRPV1 inhibitor). SB 366791 also prevented 2-AG-and AEA-induced depression of tonic inhibition. HFS-induced depression was not blocked by tetrahydrolipstatin (THL), which prevents 2-AG synthesis, nor AM 251 (a CB1 receptor inverse agonist). These results illustrate a novel activity-dependent modulation of tonic GABA currents that is mediated by endocannabinoid signaling and is likely to play an important role in sensitization of non-nociceptive afferent pathways.