Deficient tonic GABAergic conductance and synaptic balance in the fragile X syndrome amygdala

Abstract

Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability. Comorbidities of FXS such as autism are increasingly linked to imbalances in excitation and inhibition (E/I) as well as dysfunction in GABAergic transmission in a number of brain regions including the amygdala. However, the link between E/I imbalance and GABAergic transmission deficits in the FXS amygdala is poorly understood. Here we reveal that normal tonic GABAA receptor-mediated neurotransmission in principal neurons (PNs) of the basolateral amygdala (BLA) is comprised of both δ- and α5-subunit-containing GABAA receptors. Furthermore, tonic GABAergic capacity is reduced in these neurons in the Fmr1 knockout (KO) mouse model of FXS (1.5-fold total, 3-fold δ-subunit, and 2-fold α5-subunit mediated) as indicated by application of gabazine (50 μM), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 μM), and α5ia (1.5 μM) in whole cell patch-clamp recordings. Moreover, α5-containing tonic GABAA receptors appear to preferentially modulate nonsomatic compartments of BLA PNs. Examination of evoked feedforward synaptic transmission in these cells surprisingly revealed no differences in overall synaptic conductance or E/I balance between wild-type (WT) and Fmr1 KO mice. Instead, we observed altered feedforward kinetics in Fmr1 KO PNs that supports a subtle yet significant decrease in E/I balance at the peak of excitatory conductance. Blockade of α5-subunit-containing GABAA receptors replicated this condition in WT PNs. Therefore, our data suggest that tonic GABAA receptor-mediated neurotransmission can modulate synaptic E/I balance and timing established by feedforward inhibition and thus may represent a therapeutic target to enhance amygdala function in FXS.

Keywords: GABA; amygdala; fragile X syndrome; tonic inhibition.

Fig. 1.

Fmr1 knockout (KO) principal neurons (PNs) of the basolateral amygdala (BLA) have reduced δ-subunit-mediated tonic GABAergic currents. A and B: representative current-clamp traces showing typical responses of PNs to depolarizing (+150 pA) and hyperpolarizing (−100 pA) current injections (600 ms). Aii and Bii: representative whole cell voltage-clamp traces recorded from wild-type (WT; A) and Fmr1 KO (B) PNs showing 10-s samples before (baseline) and after (THIP) bath application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 μM) [holding potential (V_hold) = −60 mV]. Gaussian distributions (right) for each sample indicate the differences in mean holding current at each condition. C: averaged group data reveal significantly reduced δ-subunit-mediated current (left) and current density (right) in Fmr1 KO cells vs. WT at 1 μM THIP. *P < 0.05.

Fig. 2.

Fmr1 KO PNs in the BLA have diminished α5-subunit-specific and total tonic current capacity compared with WT cells. A and B: representative whole cell voltage-clamp traces recorded from WT (A) and Fmr1 KO (B) PNs showing 10-s samples recorded at baseline (black), after application of α5ia (1.5 μM; red), and after application of gabazine (50 μM; gray) (V_hold = −70 mV). Gaussian distributions (right) for the samples indicate the differences in mean holding current at each condition. C: averaged group data reveal significantly reduced α5-subunit-mediated capacity (left) and current density (right) in Fmr1 KO cells vs. WT. Similarly in D, averaged group data reveal significantly reduced total tonic current capacity (left) and current density (right) in Fmr1 KO cells vs. WT. *P < 0.05.

Fig. 3.

Blockade of α5-GABA_A receptors increases GABAergic inhibitory efficacy as recorded at the soma. A and B: application of α5ia (1.5 μM) increases the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in both WT and Fmr1 KO PNs of the BLA shown here as a decrease in the distribution of the cumulative probability of the interevent interval (B) before and after application of α5ia (V_hold = −60 mV). C: in the presence of α5ia, amplitude also increases in both WT and Fmr1 KO cells. However, both frequency (B) and amplitude (C) changes are reduced in the Fmr1 KO cells vs. WT. D: average event fits from WT and Fmr1 KO sIPSCs [WT: left, baseline (black solid), α5ia (red solid); Fmr1 KO: right, baseline (gray dotted), α5ia (red dotted)] show slight but significant increases in decay constant τ (Table 1). *P < 0.05.

Fig. 4.

Recording IPSCs with CsCl-based pipette solution occludes increases in inhibitory efficacy in response to α5-GABA_A receptor blockade. A: application of α5ia (1.5 μM) decreases rather than increases sIPSC (solid lines) and mIPSC (dotted lines) as observed with K-gluconate (K-Gluc) recordings. B: amplitude increases only occur in the presence of TTX in CsCl recordings [miniature IPSCs (mIPSCs), right], whereas no significant changes in amplitude occur in sIPSC recordings after application of α5ia (left). C: in both sIPSC (left) and mIPSC (right) recordings the decay constant τ_D increases slightly and significantly in response to α5ia application (Table 1). *P < 0.05.

Fig. 5.

The presence of α5-GABA_A receptor activity affects evoked response kinetics and synaptic balance. A and B: representative conductance measurements [total conductance (G_tot), black; excitatory conductance (G_e), blue; inhibitory conductance (G_i), red] derived from current/voltage (I/V) curves taken from evoked responses recorded in voltage clamp at 3 different holding potentials (inset: −20 mV, black; −45 mV, red; −70 mV, green) from WT (A) and KO (B) PNs. Aii and Bii: representative examples of baseline conductance kinetics for WT (Aii) and Fmr1 KO (Bii). Aiii and Biii: representative examples of conductance kinetics in the presence of α5-GABA_A receptor blockade (α5ia, 1.5 μM) in WT (Aiii) and Fmr1 KO (Biii) cells. C: conductance measurements of WT and Fmr1 KO cells reveal no significant differences in G_tot, G_e, or G_i between genotypes or conditions (baseline or α5ia) [conductance density (nS/pF)]. D: in addition, overall E/I balance is similar among genotypes and conditions [Conductance Area: G_e (nS/ms)/G_i (nS/ms)]. E: conductance kinetics demonstrate a significantly longer duration between G_e and G_i peaks in WT baseline cells compared with WT cells in the presence of α5ia or Fmr1 KO cells (*P < 0.05). F: the E/I conductance ratio at the G_e peak is increased in WT baseline compared with WT cells in the presence of α5ia and Fmr1 KO baseline cells (WT baseline vs. WT α5ia and Fmr1 KO baseline, *P < 0.05; vs. Fmr1 KO α5ia, P = 0.06). G: increased G_e-to-G_i peak times in WT baseline cells associate with changes solely in the G_e latency (center) and not the G_e onset (left) or G_i latency (right) compared with other conditions (WT baseline vs. WT α5ia and Fmr1 KO baseline, *P < 0.05; vs. Fmr1 KO α5ia, P = 0.09). H: summary data from all cells indicate that G_e latency (G) and E/I ratio at peak G_e (F) are negatively correlated (linear regression, r = −0.587, P < 0.0001).