Α5 Subunit-containing GABAA Receptors Research Articles

In-silico testing of new pharmacology for restoring inhibition and human cortical function in depression

Reduced inhibition by somatostatin-expressing interneurons is associated with depression. Administration of positive allosteric modulators of α5 subunit-containing GABAA receptor (α5-PAM) that selectively target this lost inhibition exhibit antidepressant and pro-cognitive effects in rodent models of chronic stress. However, the functional effects of α5-PAM on the human brain in vivo are unknown, and currently cannot be assessed experimentally. We modeled the effects of α5-PAM on tonic inhibition as measured in human neurons, and tested in silico α5-PAM effects on detailed models of human cortical microcircuits in health and depression. We found that α5-PAM effectively recovered impaired cortical processing as quantified by stimulus detection metrics, and also recovered the power spectral density profile of the microcircuit EEG signals. We performed an α5-PAM dose-response and identified simulated EEG biomarker candidates. Our results serve to de-risk and facilitate α5-PAM translation and provide biomarkers in non-invasive brain signals for monitoring target engagement and drug efficacy.

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors

α5 subunit-containing γ-aminobutyric acid type A (GABAA) 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.

Pharmaco-EEG analysis of ligands varying in selectivity for α1 subunit-containing GABAA receptors during the active phase in rats.

Benzodiazepines are known to evoke changes in cortical electrophysiological activity that can be correlated with action at distinct γ-aminobutyric acid type A (GABAA) receptorsubtypes. We used electroencephalography (EEG) paired with electromyography (EMG) to evaluate the role of α1 subunit-containing GABAA receptors (α1GABAARs) in benzodiazepine-induced sedation and changes in EEG band frequencies during the active phase of the light/dark cycle. Male Sprague-Dawley rats (N = 4/drug) were surgically instrumented with EEG/EMG electrodes. The rats were injected i.p. with zolpidem, an α1GABAAR-preferring compound, or L-838,417, which has selective efficacy for α2/3/5 subunit-containing GABAARs (i.e., α1GABAAR-sparing compound), in comparison with the non-selective benzodiazepine, triazolam. All ligands evaluated induced changes in sleep-wake states during the active phase consistent with an increase in slow-wave sleep (SWS). The degree of SWS increase appeared to be related to the magnitude of delta power band changes induced by the ligands, with the strongest effects engendered by the α1GABAAR-preferring drug zolpidem and the weakest effects by the α1GABAAR-sparing compound, L-838,417. Consistent with other research, a selective increase in beta band power was observed with L-838,417, which may be associated with α2GABAAR-mediated anxiolysis. Overall, these findings support the establishment of pharmaco-EEG "signatures" for identifying subtype-selective GABAA modulators in vivo.

Identification of Triazolopyridines as Selective α5-GABAA Receptor Negative Allosteric Modulators by a Hybridization Approach.

The identification and characterization of novel triazolopyridine derivatives with selective α5 subunit-containing GABAA receptor negative allosteric modulator (NAM) activity are disclosed. As a result of in silico screening of our corporate compound deck, we identified a moderately potent hit that was converted to an advanced hit bearing better physicochemical and pharmacological properties using a hybridization approach. Subsequent optimization led to the identification of in vitro potent and subtype-selective α5-GABAA receptor NAMs representing a new chemotype in this area.

BDNF controls GABAAR trafficking and related cognitive processes via autophagic regulation of p62.

Reduced brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid (GABA) neurotransmission co-occur in brain conditions (depression, schizophrenia and age-related disorders) and are associated with symptomatology. Rodent studies show they are causally linked, suggesting the presence of biological pathways mediating this link. Here we first show that reduced BDNF and GABA also co-occur with attenuated autophagy in human depression. Using mice, we then show that reducing Bdnf levels (Bdnf+/-) leads to upregulated sequestosome-1/p62, a key autophagy-associated adaptor protein, whose levels are inversely correlated with autophagic activity. Reduced Bdnf levels also caused reduced surface presentation of α5 subunit-containing GABAA receptor (α5-GABAAR) in prefrontal cortex (PFC) pyramidal neurons. Reducing p62 gene dosage restored α5-GABAAR surface expression and rescued PFC-relevant behavioral deficits of Bdnf+/- mice, including cognitive inflexibility and reduced sensorimotor gating. Increasing p62 levels was sufficient to recreate the molecular and behavioral profiles of Bdnf+/- mice. Collectively, the data reveal a novel mechanism by which deficient BDNF leads to targeted reduced GABAergic signaling through autophagic dysregulation of p62, potentially underlying cognitive impairment across brain conditions.

Specific inhibition of α5 subunit-containing GABAA receptors enhances locomotor activity and neuronal activity in the motor cortex.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system (CNS). This study examined the effect of specific inhibition of α5 subunit-containing GABAA receptors (α5GABAAR) on the behavioral profile and neuronal activity of the CNS using a compound called L-655,708, which is a selective negative allosteric modulator of α5GABAAR. L-655,708 administration significantly increased locomotor activity without anxiety-related behavior. Furthermore, L-655,708 administration significantly increased c-Fos mRNA expression (a neuronal activity marker) in motor area of the cerebral cortex, whereas it hardly altered c-Fos mRNA expression in the sensory cortex, hippocampus, and spinal cord. This study revealed for the first time that alteration of neuronal activity with specific inhibition of α5GABAAR differs depending on each CNS region. α5GABAAR could be a potential target for modulating CNS excitability and behavioral activity.

Scaling of Network Excitability and Inhibition may Contribute to the Septotemporal Differentiation of Sharp Waves–Ripples in Rat Hippocampus In Vitro

The functional organization of the hippocampus along its longitudinal (septotemporal or dorsoventral) axis is conspicuously heterogeneous. This functional diversification includes the activity of sharp wave and ripples (SPW–Rs), a complex intrinsic network pattern involved in memory consolidation. In this study, using transverse slices from the ventral and the dorsal rat hippocampus and recordings of CA1 field potentials we studied the development of SPW–Rs and possible changes in local network excitability and inhibition, during in vitro maintenance of the hippocampal tissue. We found that SPW–Rs develop gradually in terms of magnitude and rate of occurrence in the ventral hippocampus. On the contrary, neither the magnitude nor the rate of occurrence significantly changed in dorsal hippocampal slices during their in vitro maintenance. The development of SPW–Rs was accompanied by an increase in local network excitability more in the ventral than in the dorsal hippocampus, and an increase in local network inhibition in the ventral hippocampus only. Furthermore, the amplitude of SPWs positively correlated with the level of maximum excitation of the local neuronal network in both segments of the hippocampus, and the local network excitability and inhibition in the ventral but not the dorsal hippocampus. Blockade of α5 subunit-containing GABAA receptor by L-655,708 significantly reduced the rate of occurrence of SPWs and enhanced the probability of their generation in the form of clusters in the ventral hippocampus without affecting activity in the dorsal hippocampus. The present evidence suggests that a dynamic upregulation of excitation and inhibition in the local neuronal network may significantly contribute to the generation of SPW–Rs, particularly in the ventral hippocampus.

Selective Modulation of α5 GABAA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in APP Mouse Model of Alzheimer's Disease.

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABAA receptors (GABAARs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer’s disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABAARs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABAAR subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD (APPNL−F/NL−F), which expresses a mutant form of human amyloid-β (Aβ), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged APPNL−F/NL−F mice, selective preservation of α5 GABAARs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the APPNL−F/NL−F mouse model, here we show that this excessive inhibition was “normalized” to control values with bath-applied α5-SOP002 (1 μM). However, α5-SOP002, further impaired inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a deficit of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABAAR NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABAAR is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABAARs.

GABAA Receptor Subtypes and the Reinforcing Effects of Benzodiazepines in Remifentanil-Experienced Rhesus Monkeys

BackgroundOpioid-use disorder is associated with a high degree of co-abuse with benzodiazepines. While the mechanisms underlying the co-abuse of opioids and benzodiazepines remain unknown, α1 subunit-containing GABAA receptors may play a critical role in the reinforcing effects of benzodiazepine-type compounds, depending on whether the monkeys have a history of benzodiazepine or stimulant self-administration. The present study extended our prior research by comparing the reinforcing effects of a compound lacking activity at α1 subunit-containing GABAA receptors with the reinforcing effects of non-selective GABAA receptor positive allosteric modulators in monkeys with a history of opioid self-administration. MethodsThe reinforcing effects of L-838,417 (partial intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors, but no efficacy at α1 subunit-containing GABAA receptors, i.e., “α1-sparing compound”) were compared with those of the non-selective GABAA receptor partial modulator MRK-696, and non-selective GABAA receptor full modulators, triazolam and lorazepam, in rhesus monkeys (n = 3) experienced in remifentanil self-administration under a progressive-ratio schedule of intravenous drug injection. ResultsNeither the partial modulator nor the α1-sparing compound were self-administered above vehicle levels. The full modulators triazolam and lorazepam were self-administered significantly above vehicle levels, albeit at lower levels than remifentanil. ConclusionsOur findings suggest that relatively high efficacy at one or more GABAA receptor subtypes is required for a compound to have reinforcing effects in monkeys with a history of remifentanil self-administration, in contrast to monkeys with benzodiazepine or stimulant self-administration histories.

T180. REDUCED [3H]RO15-4513 RECEPTOR BINDING IN THE VENTRAL HIPPOCAMPUS IN THE MAM DEVELOPMENTAL DISRUPTION MODEL OF SCHIZOPHRENIA

BackgroundPost-mortem studies in schizophrenia patients have repeatedly found abnormalities in the γ-aminobutyric acid (GABA) inhibitory neurotransmission system in the form of parvalbumin (PV)-expressing GABAergic interneuron reduction and aberrant glutamic acid decarboxylase mRNA expression and receptor distribution. However, these findings cannot be separated from medication effects. Evidence from the methylazoxymethanol acetate (MAM) animal model of schizophrenia has suggested a reduction of hippocampal PV interneurons in MAM-treated rats results in hippocampal dysfunction in the form of increased pyramidal neuron activity, which contributes to dysregulation of the stress response and concurrent disinhibition of dopaminergic neurons in the midbrain. Administration of a positive allosteric modulator of the α5 subunit-containing GABA-A receptor (α5 GABAAR) reversed dopamine neuron hyperactivation in the ventral tegmental area and attenuated schizophrenia-like behaviour in MAM-treated rats, suggesting this receptor subtype’s involvement in the development of psychosis. Conflictingly, an in vivo PET imaging study found no difference in α5 GABAAR receptor binding compared to controls but demonstrated a negative correlation between binding in the hippocampus and negative symptoms of schizophrenia patients. However, this study included previously medicated patients. To understand the involvement of α5 GABAAR in hippocampal dysfunction without a confound of antipsychotic medication, we investigated whether these receptors are differentially expressed in MAM-treated compared to control rats using autoradiography.MethodsMAM-treated (n=21) and saline (vehicle)-treated (n=22) male rats were assessed for schizophrenia- and anxiety-like phenotypes via amphetamine-induced hyperlocomotion (AIH) and the elevated plus maze assays (EPM), respectively. To quantify the density of α5 GABAAR, we used [3H]Ro15-4513, a selective radioligand binding to the benzodiazepine site of this subunit. [3H]Flumazenil, an antagonist of the benzodiazepine-binding site of α1-3,5 GABAAR, was used on adjacent brain slices of the same animals to investigate more general binding to GABAAR. Brain sections were treated with the radioligands and x-ray sensitive films were exposed to the brain sections and radioactivity standards for 4 (flumazenil) or 8 (Ro15-4513) weeks. Developed films were imaged and normalised to background, radioactive binding in images was calculated based on the standard curve using the robust regression method in Graphpad Prism 8. Subsequently, quantified images were sampled for the CA1 of the ventral hippocampus using MCID software. Independent t-tests were used to investigate group differences in time spent in open arms during the EPM, total distance moved during AIH, and receptor binding. The significance threshold was set to p=0.05.ResultsMAM-treatment induced a schizophrenia-like phenotype (AIH: t(39)=‒3.022, p=0.004) and an anxiety-like phenotype (EPM: t(41)=2.810, p=0.008) compared to saline. We found reduced α5 GABAAR binding in the ventral hippocampus CA1 region of MAM-treated rats compared to saline-treated rats (t(41)=2.563, p=0.014), but no differences in flumazenil binding to α1-3,5 GABAAR (t(41)=1.333, p=0.190).DiscussionMAM treatment induced schizophrenia- and anxiety-like phenotypes, accompanied by an α5 GABAAR density decrease in the CA1 region of the ventral hippocampus. Specific reduced binding to α5 was corroborated by the absence of any binding changes in the flumazenil treated sections. These findings reveal part of the molecular mechanisms behind hippocampal dysfunction and implicate the α5 subunit as a potential novel target for treatment of schizophrenia.

An 5 GABAA Receptor Inverse Agonist, 5IA, Attenuates Amyloid Beta-Induced Neuronal Death in Mouse Hippocampal Cultures.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder for which no cognition-restoring therapies exist. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. Increasing evidence suggests a remodeling of the GABAergic system in AD, which might represent an important therapeutic target. An inverse agonist of α5 subunit-containing GABAA receptors (α5GABAARs), 3-(5-Methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3–a]phthalazine (α5IA) has cognition-enhancing properties. This study aimed to characterize the effects of α5IA on amyloid beta (Aβ1–42)-induced molecular and cellular changes. Mouse primary hippocampal cultures were exposed to either Aβ1-42 alone, or α5IA alone, α5IA with Aβ1–42 or vehicle alone, and changes in cell viability and mRNA expression of several GABAergic signaling components were assessed. Treatment with 100 nM of α5IA reduced Aβ1–42-induced cell loss by 23.8% (p < 0.0001) after 6 h and by 17.3% after 5 days of treatment (p < 0.0001). Furthermore, we observed an Aβ1-42-induced increase in ambient GABA levels, as well as upregulated mRNA expression of the GABAAR α2,α5,β2/3 subunits and the GABABR R1 and R2 subunits. Such changes in GABARs expression could potentially disrupt inhibitory neurotransmission and normal network activity. Treatment with α5IA restored Aβ1-42-induced changes in the expression of α5GABAARs. In summary, this compound might hold neuroprotective potential and represent a new therapeutic avenue for AD.

Evaluation of the anti-conflict, reinforcing, and sedative effects of YT-III-31, a ligand functionally selective for α3 subunit-containing GABAA receptors.

In recent years, pharmacological strategies have implicated α3 subunit-containing GABAA (α3GABAA) receptor subtypes in the anxiety-reducing effects of benzodiazepines, whereas transgenic mouse approaches have implicated α2 or α5 subunit-containing GABAA receptors. We investigated the role of α3GABAA subtypes in benzodiazepine-induced behaviors by evaluating the anti-conflict, reinforcing, and sedative-motor effects of the novel compound YT-III-31, which has functional selectivity for α3GABAA receptors. Female and male rhesus monkeys were trained under a conflict procedure (n = 3), and a progressive-ratio schedule of reinforcement with midazolam as the training drug (n = 4). Sedative-like behavior was assessed using a quantitative behavioral observation procedure (n = 4). A range of doses of YT-III-31 was administered in all tests using the i.v. route of administration. In the conflict procedure, increasing doses of YT-III-31 resulted only in dose-dependent attenuation of non-suppressed responding. In the progressive-ratio model of self-administration, YT-III-31 maintained average injections/session above vehicle levels at 0.1 and 0.18 mg/kg/injection. In quantitative observation procedures, YT-III-31 engendered mild sedative effects ("rest/sleep posture"), and deep sedation at the highest dose tested (5.6 mg/kg, i.v.), along with a suppression of tactile/oral exploration and increased observable ataxia. In contrast to other benzodiazepine-like ligands, YT-III-31 uniquely engendered a biphasic dose-response function for locomotion and suppressed self-groom. The finding that YT-III-31 lacked anti-conflict properties is in accordance with transgenic mouse research indicating no role for α3GABAA subtypes in benzodiazepine-mediated anxiety reduction. Instead, our results raise the possibility of a role for α3GABAA receptors in the abuse potential and sedative effects of benzodiazepine-type drugs.

Blockade of spinal α5-GABAA receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats

The role of spinal α5 subunit-containing GABAA (α5-GABAA) receptors in chronic pain is controversial. The purpose of this study was to investigate the participation of spinal α5-GABAA receptors in the reserpine-induced pain model. Reserpine administration induced tactile allodynia and muscle hyperalgesia in female and male rats. Intrathecal injection of L-655,708 and TB 21007 (7 days after the last reserpine injection) decreased tactile allodynia and, at a lesser extent, muscle hyperalgesia in female rats. The effects of these drugs produced a lower antiallodynic and antihyperalgesic effect in male than in female rats. Contrariwise, these drugs produced tactile allodynia and muscle hyperalgesia in naïve rats and these effects were lower in naïve male than female rats. Intrathecal L-838,417 prevented or reversed L-655,708-induced antiallodynia in reserpine-treated female rats. Repeated treatment with α5-GABAA receptor small interfering RNA (siRNA), but not scramble siRNA, reduced reserpine-induced allodynia in female rats. Accordingly, α5-GABAA receptor siRNA induced nociceptive hypersensitivity in naïve female rats. Reserpine enhanced α5-GABAA receptors expression in spinal cord and dorsal root ganglia (DRG), while it increased CD11b (OX-42) and glial fibrillary acidic protein (GFAP) fluorescence intensity in the lumbar spinal cord. In contrast, reserpine diminished K+-Cl- co-transporter 2 (KCC2) protein in the lumbar spinal cord. Data suggest that spinal α5-GABAA receptors play a sex-dependent proallodynic effect in reserpine-treated rats. In contrast, these receptors have a sex-dependent antiallodynic role in naïve rats.

Enhanced Dendritic Inhibition and Impaired NMDAR Activation in a Mouse Model of Down Syndrome.

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.

α5GABAA receptors play a pronociceptive role and avoid the rate-dependent depression of the Hoffmann reflex in diabetic neuropathic pain and reduce primary afferent excitability.

Diabetic neuropathy is an incapacitating complication in diabetic patients. The cellular and molecular mechanisms involved in this pathology are poorly understood. Previous studies have suggested that the loss of spinal GABAergic inhibition participate in painful diabetic neuropathy. However, the role of extrasynaptic α5 subunit-containing GABAA (α5GABAA) receptors in this process is not known. The purpose of this study was to investigate the role of α5GABAA receptors in diabetes-induced tactile allodynia, loss of rate-dependent depression (RDD) of the Hoffmann reflex (HR), and modulation of primary afferent excitability. Intraperitoneal administration of streptozotocin induced tactile allodynia. Intrathecal injection of α5GABAA receptor inverse agonist, L-655,708, produced tactile allodynia in naive rats, whereas it reduced allodynia in diabetic rats. In healthy rats, electrical stimulation of the tibial nerve at 5 Hz induced RDD of the HR, although intrathecal treatment with L-655,708 (15 nmol) abolished RDD of the HR. Streptozotocin induced the loss of RDD of the HR, while intrathecal L-655,708 (15 nmol) restored RDD of the HR. L-655,708 (15 nmol) increased tonic excitability of the primary afferents without affecting the phasic excitability produced by the primary afferent depolarization. α5GABAA receptors were immunolocalized in superficial laminae of the dorsal horn and L4 to L6 dorsal root ganglion. Streptozotocin increased mean fluorescence intensity and percentage of neurons expressing α5GABAA receptors in dorsal horn and L4 to L6 dorsal root ganglia in 10-week diabetic rats. Our results suggest that spinal α5GABAA receptors modulate the HR, play an antinociceptive and pronociceptive role in healthy and diabetic rats, respectively, and are tonically active in primary afferents.

Input-Specific Synaptic Location and Function of the α5 GABAA Receptor Subunit in the Mouse CA1 Hippocampal Neurons.

Hippocampus-dependent learning processes are coordinated via a large diversity of GABAergic inhibitory mechanisms. The α5 subunit-containing GABAA receptor (α5-GABAAR) is abundantly expressed in the hippocampus populating primarily the extrasynaptic domain of CA1 pyramidal cells, where it mediates tonic inhibitory conductance and may cause functional deficits in synaptic plasticity and hippocampus-dependent memory. However, little is known about synaptic expression of the α5-GABAAR and, accordingly, its location site-specific function. We examined the cell- and synapse-specific distribution of the α5-GABAAR in the CA1 stratum oriens/alveus (O/A) using a combination of immunohistochemistry, whole-cell patch-clamp recordings and optogenetic stimulation in hippocampal slices obtained from mice of either sex. In addition, the input-specific role of the α5-GABAAR in spatial learning and anxiety-related behavior was studied using behavioral testing and chemogenetic manipulations. We demonstrate that α5-GABAAR is preferentially targeted to the inhibitory synapses made by the vasoactive intestinal peptide (VIP)- and calretinin-positive terminals onto dendrites of somatostatin-expressing interneurons. In contrast, synapses made by the parvalbumin-positive inhibitory inputs to O/A interneurons showed no or little α5-GABAAR. Inhibiting the α5-GABAAR in control mice in vivo improved spatial learning but also induced anxiety-like behavior. Inhibiting the α5-GABAAR in mice with inactivated CA1 VIP input could still improve spatial learning and was not associated with anxiety. Together, these data indicate that the α5-GABAAR-mediated phasic inhibition via VIP input to interneurons plays a predominant role in the regulation of anxiety while the α5-GABAAR tonic inhibition via this subunit may control spatial learning.SIGNIFICANCE STATEMENT The α5-GABAAR subunit exhibits high expression in the hippocampus, and regulates the induction of synaptic plasticity and the hippocampus-dependent mnemonic processes. In CA1 principal cells, this subunit occupies mostly extrasynaptic sites and mediates tonic inhibition. Here, we provide evidence that, in CA1 somatostatin-expressing interneurons, the α5-GABAAR subunit is targeted to synapses formed by the VIP- and calretinin-expressing inputs, and plays a specific role in the regulation of anxiety-like behavior.

Enhanced GABAergic Tonic Inhibition Reduces Intrinsic Excitability of Hippocampal CA1 Pyramidal Cells in Experimental Autoimmune Encephalomyelitis

Cognitive impairment (CI), a debilitating and pervasive feature of multiple sclerosis (MS), is correlated with hippocampal atrophy. Findings from postmortem MS hippocampi indicate that expression of genes involved in both excitatory and inhibitory neurotransmission are altered in MS, and although deficits in excitatory neurotransmission have been reported in the MS model experimental autoimmune encephalomyelitis (EAE), the functional consequence of altered inhibitory neurotransmission remains poorly understood. In this study, we used electrophysiological and biochemical techniques to examine inhibitory neurotransmission in the CA1 region of the hippocampus in EAE. We find that tonic, GABAergic inhibition is enhanced in CA1 pyramidal cells from EAE mice. Although plasma membrane expression of the GABA transporter GAT-3 was decreased in the EAE hippocampus, an increased surface expression of α5 subunit-containing GABAA receptors appears to be primarily responsible for the increase in tonic inhibition during EAE. Enhanced tonic inhibition during EAE was associated with decreased CA1 pyramidal cell excitability and inhibition of α5 subunit-containing GABAA receptors with the negative allosteric modulator L-655,708 enhanced pyramidal cell excitability in EAE mice. Together, our results suggest that altered GABAergic neurotransmission may underlie deficits in hippocampus-dependent cognitive function in EAE and MS.

Comparison of rapid and long-lasting antidepressant effects of negative modulators of α5-containing GABAA receptors and (R)‑ketamine in a chronic social defeat stress model

The negative allosteric modulators (NAMs: L-655,708 and MRK-016) of α5 subunit-containing GABAA receptors are reported to show rapid-acting antidepressant effects in rodents. However, there are no reports comparing these NAMs and (R)‑ketamine, (R)-enantiomer of the rapid-acting antidepressant ketamine, in a chronic social defeat stress (CSDS) model. Here we measured expression of α5 GABAA receptor in the brain regions from CSDS susceptible mice and postmortem brain samples from depressed patients. Expression of α5 GABAA receptor in the prefrontal cortex and hippocampus from CSDS susceptible mice was significantly higher than that of control mice. Furthermore, expression of α5 GABAA receptor in the parietal cortex from depressed patients was also higher than that of control subjects. In the tail suspension and forced swimming tests, (R)‑ketamine and MRK-016 significantly attenuated the increased immobility time in the susceptible mice, compared with the vehicle-treated group. In the sucrose preference test, (R)‑ketamine and MRK-016 significantly enhanced the reduced preference in CSDS susceptible mice two days after a single injection. Unlike (R)‑ketamine, MRK-016 did not attenuate the reduced sucrose preference in susceptible mice 7 days after a single injection. In contrast, L-655,708 did not show antidepressant effects in the same model. In conclusion, this study shows that increased levels of α5 GABAA receptors in the PFC and hippocampus may play a role in depression-like phenotype after CSDS. It is unlikely that MRK-016 has long-lasting antidepressant effects although it elicits rapid-acting antidepressant effects.

Evidence That Sedative Effects of Benzodiazepines Involve Unexpected GABAA Receptor Subtypes: Quantitative Observation Studies in Rhesus Monkeys.

In nonhuman primates we tested a new set of behavioral categories for observable sedative effects using pediatric anesthesiology classifications as a basis. Using quantitative behavioral observation techniques in rhesus monkeys, we examined the effects of alprazolam and diazepam (nonselective benzodiazepines), zolpidem (preferential binding to α1 subunit-containing GABAA receptors), HZ-166 (8-ethynyl-6-(2'-pyridine)-4H-2,5,10b-triaza-benzo[e]azulene-3-carboxylic acid ethyl ester; functionally selective with relatively high intrinsic efficacy for α2 and α3 subunit-containing GABAA receptors), MRK-696 [7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-b)pyridazine; no selectivity but partial intrinsic activity], and TPA023B 6,2'-diflouro-5'-[3-(1-hydroxy-1-methylethyl)imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile; partial intrinsic efficacy and selectivity for α2, α3, α5 subunit-containing GABAA receptors]. We further examined the role of α1 subunit-containing GABAA receptors in benzodiazepine-induced sedative effects by pretreating animals with the α1 subunit-preferring antagonist β-carboline-3-carboxylate-t-butyl ester (βCCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, and moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, and HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by βCCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to βCCT administration. These data suggest that α2/3-containing GABAA receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas α1-containing GABAA receptors may play a role in moderate/deep sedation.

Behavioral Effects of α2/α3 Subtype‐Selective GABAA Receptor Positive Allosteric Modulators

Millions of individuals suffer from chronic pain. Opioids are effective for treating chronic pain but their use is limited due to undesirable side‐effects. Previous studies have shown that different subunits of the GABAA receptor are associated with different actions of the benzodiazepine site. While α2/α3 subunit‐containing GABAA receptors mediate antinociceptive effects, α1 subunit‐containing GABAA receptors mediate sedative and abuse‐related effects. This study sought to examine the behavioral effects of two α2/α3‐subtype selective PAMs: KRM‐II‐81 and NS16085. In a rat model of inflammatory pain (complete Freund's adjuvant) and a rat model of neuropathic pain (chronic constriction injury), both PAMs and the classical benzodiazepine midazolam attenuated mechanical hyperalgesia (as measured by von Frey test). In the procedure of food‐maintained operant responding, KRM‐II‐81 and NS16085 did not significantly decrease the response rate at doses that produced maximal antinociception. Contrastingly, midazolam significantly reduced the response rate at doses that attenuated mechanical hyperalgesia. In a horizontal wire test aimed to measure muscle relaxation, within the dose range that produced antinociception, only midazolam dose‐dependently increased the percentage of rats unable to grasp the wire, indicating muscle‐relaxant activity. These behavioral effects can be attenuated by benzodiazepine receptor antagonist flumazenil, confirming that the behavioral effects of these subtype‐selective GABAA PAMs are mediated through the benzodiazepine site of GABAA receptors. Taken together, while midazolam produced antinociceptive, rate‐suppressing, and muscle‐relaxant activity at similar doses, both subtype‐selective GABAA receptor PAMs selectively produced antinocicpetive effects. Collectively, these data support the notion of α2/α3‐subtype selective GABAA PAMs as novel analgesics.Support or Funding InformationR01DA034806This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.