Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator

Abstract

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

Keywords: GABA; GABAA receptor; amygdala; fragile X syndrome; positive allosteric modulator.

Fig. 1

SGE-872 is a more potent and selective positive allosteric modulator than allopregnanolone. A: Representative current responses from LTK cells transfected with α4β3δ GABA_A receptors in response to 2 sec of stimulus with either GABA (2 µM) or GABA plus each modulator at increasing concentrations. B: Dose–response curves for allopregnanolone (at left) and SGE-872 (right) indicate that SGE-872 potentiates α4β3δ-mediated GABAergic currents more than allopregnanolone at concentrations greater than 0.1 µM. In addition, SGE-872 is more selective for α4β3δ-containing receptors vs. α1β2γ2 receptors compared with allopregnanolone. C: The highest concentration tested (10 µM) minimally directly activates α4β3δ GABA_A receptors in the absence of GABA.

Fig. 2

Allopregnanolone and SGE-872 significantly potentiate tonic currents in principal neurons of the BLA. A,B: Representative whole-cell voltage clamp traces recorded from control (A) and Fmr1^−/y (B) principal neurons show 10-sec samples before (baseline) and after (SGE-872) direct application of SGE-872 1 µM in the presence of 2 µM exogenous GABA, followed by application of gabazine 50 µM (V_hold = –70 mV). Gaussian distributions (right) for each sample indicate the differences in mean holding current at each condition. C: Baseline tonic density is decreased in Fmr1^−/y cells in the presence of 2 µM GABA (left). Pairwise comparisons show that allopregnanolone and SGE-872 significantly potentiate baseline tonic current density in control and Fmr1^−/y cells. However, Fmr1^−/y cells require a higher concentration of SGE-872 (1 µM vs. 100 nM) to have a significant effect. Allopregnanolone also significantly potentiates tonic current density in both genotypes. However, in wild-type cells, SGE-872 (1 µM) has a stronger potentiation than allopregnanolone (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

SGE-872 is selective for tonic vs. phasic GABA_A receptors at low concentrations. A: Representative voltage clamp traces of sIPSCs recorded in a principal neuron in the BLA (all measurements taken from control brain slices) before (top) and after (bottom) application of SGE-872 (10 µM) in the absence of exogenous GABA (V_hold = –70 mV). B,C: Quantification of changes in sIPSC properties of cells exposed to SGE-872 at 1 µM (B) and 10 µM (C) indicates average sIPSC frequency, amplitude, weighted decay, and overall GABAergic synaptic efficacy before and after SGE-872 treatment. Representative average sIPSC waveforms (far right, scaled for amplitude) demonstrate changes in weighted decay constant (tau_ω) after SGE-872 application. At 1 µM, SGE-872 had no significant effect on any sIPSC properties (B). In contrast, at 10 µM, SGE-872 significantly increased both the weighted decay constant (tau_ω) of sIPSCs and the total GABAergic synaptic efficacy (C). *P < 0.05, paired t-test.