Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition

Abstract

Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an α and γ subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating α1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment.

Fig. 1.

Acute BZ treatment decreases surface and total GABA_AR levels. (A) Neurons expressing α2^pHGFP or α1^pHGFP GABA_AR subunits were incubated with or without 250 nM flurazepam (Flz) for 24 h followed by live confocal microscopy experiments (37 °C in HBS). (Right) Enlargements of dendrites in boxed areas. (B) Mean ± SEM cluster size, fluorescence intensity, and density of surface GABA_AR clusters normalized to control (*significantly different from control, P < 0.05, t test; n = 15–20 neurons, 3 independent cultures). (C) Neurons were perfused at a rate of 1 mL/min with pH 7.4 HBS followed by HBS with 50 mM NH₄Cl (+NH₄Cl) to collapse the intracellular pH gradients and reveal intracellular GABA_AR. The center panels are enlargement of cell bodies, and the right panels are dendrites in boxed areas. (D) The ratio of α2^pHGFP total/surface levels was normalized to Flz-treated neurons (*P < 0.05, t test; n = 6–8 neurons per culture, 3 independent cultures; error bars represent ±SEM). (Scale bars, 10 μm.)

Fig. 2.

Decreases in GABA_AR surface levels and GABAergic mIPSC amplitude occur via a BZ binding site-dependent mechanism. (A) Neurons expressing α2^pHGFP were incubated with or without 250 nM Flz for 24 h and then treated for 1 h with 5 μM Ro 15-1788 (BZ antagonist) immediately before live imaging confocal microscopy experiments (37 °C in HBS). (Right) Enlargements of dendrites in boxed areas. (Scale bars, 10 μm.) (B) Surface cluster histograms showing mean area and cumulative α2^pHGFP fluorescence in neurons with 24-h control (Con) or 250 nM Flz conditions with or without 1-h 5 μM Ro 15-1788 treatment (*P < 0.05, one-way ANOVA analysis and Bonferroni’s multiple-comparison test; 22–30 neurons; n = 3 cultures; error bars represent ±SEM). (C) Sample traces of mIPSCs in control (Con) neurons and those treated with 250 nM Flz for 24 h with or without 1-h 5 μM Ro 15-1788 treatment. (D) Bar graph of the mean ± SEM mIPSC amplitudes and frequency (*P < 0.05, Kolmogorov–Smirnov test; n = 7–10 cells for each condition).

Fig. 3.

BZ treatment does not alter the insertion rate of α2 subunit-containing GABA_AR. (A) Confocal imaging of GABA_AR insertion in control and Flz-treated neurons. Neurons expressing α2^pHBBS were treated with or without 250 nM Flz for 24 h, and then incubated with Alexa594::Bgt (red) at 37 °C for 5, 10, and 15 min to label newly inserted GABA_AR, followed by fixation. Total receptor number was determined by permeabilization and staining with anti-GFP antibody (green). Panels below are enlargements of boxed dendrites. (Scale bars, 10 μm.) (B) Analysis of total receptor signal in control and Flz-treated α2^pHBBS neurons. (*P < 0.01, paired t test; 10–12 neurons per condition for each culture, n = 4 cultures; error bars represent ±SEM). (C) Graph shows Alexa594::Bgt fluorescence intensity of newly inserted α2^pHBBS over time in control (Con) or Flz conditions (normalized to initial fluorescence count at 5 min) (10–12 neurons per condition for each culture; n = 4 cultures; error bars represent ±SEM).

Fig. 4.

GABA_AR endocytosis rate is not increased by BZ treatment. (A) Confocal imaging of GABA_AR endocytosis in control and Flz-treated neurons. Surface GABA_AR in α2^pHBBS neurons were live-labeled with Alexa594::Bgt (red), and then unbound Alexa594::Bgt was removed by washing, followed by incubation at 37 °C. At t = 0, 7.5, and 15 min, samples were removed and fixed. Total receptor number was assayed by permeabilization and staining with anti-GFP antibody (green). Panels below are enlargements of boxed dendrites. (Scale bars, 10 μm.) (B) Graph represents the surface GABA_AR Alexa594::Bgt fluorescence loss over time with endocytosis in control (Con) and Flz-treated neurons (normalized to the t = 0 Alexa594::Bgt surface signal for each treatment, respectively) (10–12 neurons per condition; n = 3 cultures; error bars represent ±SEM).

Fig. 5.

GABA_AR surface levels are restored in BZ-treated neurons by leupeptin treatment. (A) Live confocal imaging of neurons treated with or without 250 nM Flz for 24 h followed by control (Con) or 2-h 200 μM treatment with the lysosomal inhibitor leupeptin (Leu). (Right) Enlargements of boxed dendrites. (Scale bars, 10 μm.) (B and C) Mean ± SEM surface receptor α2^pHGFP fluorescence and cluster area [*P < 0.01, one-way ANOVA (Bonferonni post test); 10–20 neurons per condition; n = 3 cultures].