Impairment of cortical GABAergic synaptic transmission in an environmental rat model of autism

Abstract

The biological mechanisms of autism spectrum disorders (ASDs) are largely unknown in spite of extensive research. ASD is characterized by altered function of multiple brain areas including the temporal cortex and by an increased synaptic excitation:inhibition ratio. While numerous studies searched for evidence of increased excitation in ASD, fewer have investigated the possibility of reduced inhibition. We characterized the cortical γ-amino butyric acid (GABA)ergic system in the rat temporal cortex of an ASD model [offspring of mothers prenatally injected with valproic acid (VPA)], by monitoring inhibitory post-synaptic currents (IPSCs) with patch-clamp. We found that numerous features of inhibition were severely altered in VPA animals compared to controls. Among them were the frequency of miniature IPSCs, the rise time and decay time of electrically-evoked IPSCs, the slope and saturation of their input/output curves, as well as their modulation by adrenergic and muscarinic agonists and by the synaptic GABAA receptor allosteric modulator zolpidem (but not by the extra-synaptic modulator gaboxadol). Our data suggest that both pre- and post-synaptic, but not extra-synaptic, inhibitory transmission is impaired in the offspring of VPA-injected mothers. We speculate that impairment in the GABAergic system critically contributes to an increase in the ratio between synaptic excitation and inhibition, which in genetically predisposed individuals may alter cortical circuits responsible for emotional, communication and social impairments at the core of ASD.

Fig. 1

Miniature inhibitory post-synaptic current (mIPSCs) in control and valproic acid (VPA) animals. (a) Representative traces of mIPSCs from layer II/III pyramidal cells of control and VPA animals in the presence of tetrodotoxin (0.5 μm). (b) Average of 20 aligned mIPSCs in control or VPA animals (expanded scaling; control, black; VPA, grey). (c and d) Average frequency and amplitude, respectively, of mIPSCs in control and VPA (n=10 each). (e and f) The mIPSC rise time and decay time in VPA and controls. VPA mIPSC displayed a lower frequency and slower kinetics compared to control animals. * p<0.05.

Fig. 2

Basal inhibitory synaptic transmission in control and valproic acid (VPA) animals. (a) Example of input/output (I/O) curve. Each trace corresponds to the averaged responses over at least four extracellular electrical pulses delivered at the same intensity. (b–d) I/O curve threshold, slope, and saturation level, respectively, in control and VPA animals. (e) Representative traces of 10 evoked inhibitory post-synaptic current (eIPSCs) in control (black) and VPA (grey) animals. (f) Paired pulse ratio (PPR) A₂/A₁ in control and VPA animals. (g) Coefficient of variation (1/CV²) analysis, averages for control (black) and VPA (grey). (h) Decay time in control (black) and VPA (grey) animals. * p<0.05, ** p<0.02.

Fig. 3

Different modulation of synaptic and extra-synaptic GABA_A receptors (GABA_AR)-mediated currents: (a) Bath-application of zolpidem (500 nm) showing a decrease of eIPSC amplitude in control animals. The insert shows an example of an evoked inhibitory post-synaptic current (eIPSC) at an expanded scaling control (black) zolpidem (grey). The numbers 1 and 2 in the insert refer to the average of four traces in the amplitude time-course. (b) Same as (a) but in valproic acid (VPA) animals; time-course and traces (insert) showing the effect of zolpidem (500 nm) in eIPSC; control (black), zolpidem (grey). (c) Shows normalized zolpidem-induced change of eIPSC amplitude in control and VPA animals (n=9). (d and e) Change in GABA_AR-mediated tonic currents (in 10 μm 6,7-dinitroquinoxaline-2,3-dione and 2 mm kynurenic acid). Examples of time-course of the bath-application of gaboxadol (5 μm), a specific agonist for the extrasynaptic δ subunit of GABA_AR, which caused an increase of tonic conductance, revealed by increased holding current blocked by picrotoxin (100 μm), in control and VPA animals, respectively. (f) Mean of picrotoxin sensitive current in gaboxadol normalized to the cell capacitance, control and VPA animals (n=11). * p<0.05.

Fig. 4

Differential norepinephrine (NE) and oxotremorine (Oxo) on modulation of evoked inhibitory post-synaptic currents (eIPSCs): (a) Time-course and traces (in the inserts) showing the effect of bath application of NE (20 μm) on eIPSC amplitude in control and valproic acid (VPA) animals, respectively (insert: baseline black; NE grey). The numbers 1 and 2 in the insert refer to the average of four traces in the amplitude time-course. (b) Average NE-induced change in eIPSC amplitude in control and VPA animals, respectively. (c) Average NE-induced change in paired pulse ratio (PPR) in control and VPA animals, respectively (d) Time-course and traces (in the inserts) showing the effect of bath application of Oxo (10 μm) on eIPSC amplitude in control and VPA animals, respectively (insert: baseline, black, Oxo, grey). (e) Average Oxo-induced change in eIPSC amplitude in control and VPA animals, respectively. (f) Average Oxo-induced change in PPR in control and VPA animals, respectively. * p<0.05.