Enhancement of inhibitory neurotransmission by GABAA receptors having α2,3-subunits ameliorates behavioral deficits in a mouse model of autism

Abstract

Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T(+)Itpr3(tf)/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low nonsedating/nonanxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABAA receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABAA receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting that reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit specific-the α2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α1-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α2,3-subunit-selective positive GABAA receptor modulation may be an effective treatment.

Figure 1. Reduced GABAergic neurotransmission in BTBR mice and enhancement by clonazepam.<

br>Spontaneous IPSC (sIPSC) and sEPSC were recorded in the hippocampal slices from 3-week old male BTBR and C57Bl/6J mice. (A and B) Example traces of sIPSC (A) and cumulative plot and average values (inset) of sIPSC frequency in BTBR and C57BL/6J hippocampal CA1 neurons (B). (C and D) Example traces of sEPSC (C) and cumulative plot and average values (inset) of sEPSC frequency in BTBR and C57BL/6J hippocampal CA1 neurons (D). (E and F) Example traces of sIPSC (E) and cumulative plot and average value (inset) of sIPSC amplitude (F) in clonazepam and vehicle treated BTBR CA1 slices. (G and H) Example traces of sEPSC (G) and cumulative plot and average value (inset) of sIPSC frequency (H) in clonazepam and vehicle treated BTBR CA1 slices. CON, Control. CLZ, Clonazepam. All data shown are means ± s.e.m. from 15 – 19 recordings per strain. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Effects of low-dose clonazepam on social interaction and cognitive deficits in BTBR mice

(A–D) Age-matched male C57BL/6J (n = 6; each group) and BTBR mice (n = 9; each group) were treated with a single intraperitoneal dose of clonazepam at the indicated level and subjected to the three-chamber social interaction test. Test mice were not reused; different groups of mice were used for each dose of clonazepam treatment. (A, B) Time in chambers. (C, D) Ratio of time in mouse chamber to time in object chamber. (E–G) The effects of low-dose clonazepam (CLZ; 0.05 mg/kg) on open field activity were measured in C57BL/6J (n = 8) and BTBR mice (n = 10). (H) Time in open arms in elevated plus maze for BTBR mice (n = 10) and C57BL/6J mice (n = 10). (I) In the three-chamber test, BTBR (blue) and C57BL/6J (black) mice were treated with vehicle (Pre, Post) or low-dose clonazepam (CLZ) 30 min before testing social interactions on Day 0 (Pre), Day 7 (CLZ), or Day 14 (Post). The ratio of interaction time with the stranger mouse or object is plotted. (J and K) In the open-field reciprocal social interaction test, BTBR (blue) and C57BL/6J (black) mice were treated with vehicle (Pre, Post) or low-dose clonazepam (CLZ) 30 min before testing social interactions on Day 0 (Pre), Day 7 (CLZ), or Day 14 (Post). The ratio of interaction time with the stranger mouse vs. object is plotted for BTBR mice (n = 9) or C57BL/6J mice (n = 9) for total interaction time (J) and nose-to-nose interaction time (K). (L and M) To test tolerance to the effects of low-dose clonazepam on locomotor and social behaviors, BTBR mice (n = 10; each group) were treated with low-dose (0.05 mg/kg), and high-dose (1 mg/kg) clonazepam for 14 days. (L) Total distance moved during the open field test after drug treatment on Day 1 and Day 14 was measured, and % activity change was calculated by comparing the activity on Day 1 and Day 14 of treatment with the indicated doses of clonazepam. (M) Social interaction behavior was compared after treatment with low-dose clonazepam (n = 10 for each group). CON, Control. CLZ, Clonazepam. All data shown are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Effects of low-dose clonazepam on context-dependent spatial learning and memory deficits in BTBR mice

(A and B) Increasing doses (0, 0.0125, 0.05, and 0.1 mg/kg) of clonazepam were administered 30 min prior to context-dependent fear conditioning. (A) C57BL/6J (n=5–7). (B) BTBR (n = 5 – 7) (C and D) Barnes circular maze. Spatial learning was measured for C57BL/6J and BTBR mice (n = 10 each) by measurement of the latency (C) and number of errors (D) for mice to find safety without and with treatment with 0.05 mg/kg clonazepam as indicated. Note that treatment with clonazepam significantly improved the performance of BTBR mice but, in contrast, significantly worsened the performance of C57BL/6J mice. (E–G) On Day 5 of the Barnes maze test, a single injection of low-dose clonazepam was given 30 min prior to the trial for BTBR and C57BL/6J mice: (E), latency to target; (F), % correct pokes; (G), % time in target. (H and I) BTBR mice (n = 10) were treated with 0.05 mg/kg clonazepam for 14 days. Contextual fear conditioning was performed 30 min after injection on Day 1 (H) and Day 14 (I). CON, Control. CLZ, Clonazepam. All data shown are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Effects of positive and negative GABA_A receptor allosteric modulators on social behaviors and cognitive deficit

(A and B) Effect of clobazam (0.05 mg/kg) on social interaction behavior of BTBR mice in the three-chamber test (n = 7 – 8). (C and D) Effect of clobazam (0.05 mg/kg) on BTBR mice (n=7–8) in the open field test on total distance moved (C) and time spent in center (D). (E and F) Effect of DMCM (0.2 mg/kg) on C57BL/6J mice (n=7–8) in the three-chamber test. (G and H) Effect of DMCM (0.2 mg/kg) on overall exploratory behavior of C57BL/6J mice (n=8) in the open field test, measured as distance moved (G). Anxiety-like behavior of C57BL/6J mice (n=8) in the elevated plus maze test, measured as time in the open arms (H). (I and J) Social interaction behavior of BTBR mice (n=6–8) in the 3-chamber test following treatment with the indicated doses of L838417 (I) or zolpidem (J). Test mice were not reused; different groups of mice were used for each dose. (K and L) Contextual fear conditioning test of BTBR mice (n=5) following treatment with 0.05 mg/kg of L-838,417 (K) or zolpidem (L). Control data were replotted from Figure 2B. (M) Effect of DMCM (0.2 mg/kg) on 129SvJ mice (n=8) in the three-chamber test. (N and O) Effects of L838,417 (N) and zolpidem (O) on Scn1a^+/− mice in the three-chamber test. CON, Control. CBZ, Clobazam. CLZ, Clonazepam. All data shown are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.