Cntnap2 -/- Autism Model Mice Display Deficits in Tonic and Phasic Inhibition in Primary Visual Cortex

Background: Investigations into the genetics of individuals with autism suggest that mutations in genes involved in synaptic function are a common factor. Both human studies and animal models suggest that such synaptic dysfunction frequently leads to excitatory/inhibitory (E/I) imbalance in the form of reduced inhibition and/or over-excitation, and that neuronal inhibition may be impacted by both pre- and post-synaptic changes. Cntnap2 -/- mice are a well-established model of ASD. A number of studies have found that these mice exhibit autism-like behaviors including reduced social interaction, hyperactivity, and repetitive/stereotyped behavior. In addition, Cntnap2-/- mice exhibit aberrant neuronal migration, reduced interneuron numbers, seizure activity, and alterations in synaptic spines.

Objectives: Due to reported decreases in PV+ interneurons in Cntnap2 -/- mice, we hypothesized that loss of the neurexin family gene Cntnap2would result in reduced inhibition and increased excitation in pyramidal neurons in the visual cortex. We aimed to test multiple forms of inhibition and excitation including intrinsic membrane excitability and tonic inhibition, which is mediated by extrasynaptic GABA receptors and modulates neuronal excitability.

Methods: We used whole-cell patch clamp electrophysiology to examine membrane properties and tonic and phasic inhibition in L2/3 pyramidal cells of primary visual cortex (V1) of Cntnap2 -/- and +/+ mice at two different time points, 3-4 weeks and 6-8 weeks of age. Tonic inhibitory currents were induced by application of 5 µM GABA or 10 µM THIP, in the presence of blockers of excitatory neurotransmission. GABA_AR subunit protein levels were measured via western blot using tissue lysates prepared from area V1, with antibodies against the γ2 and δ subunits.

Results: We found that L2/3 pyramidal cells from 6-8 week old Cntnap2 -/- mice exhibited significantly smaller GABA-induced inhibitory tonic conductance. Application of THIP, a specific activator of δ-subunit containing GABA_ARs, also induced a smaller tonic current in Cntnap2 -/- mice compared to controls. In 3-4 week old mice we found no significant effect of genotype on tonic inhibition. We also analyzed sIPSCs in both genotypes and age groups. In 3-4 week old mice we found no differences in sIPSC amplitude, frequency, or kinetics, but did observe an age-dependent effect of genotype in 6-8 week old animals, with lower sIPSC frequency in Cntnap2 -/- mice. We did not observe an effect of age or genotype on resting potential or intrinsic excitability of L2/3 pyramidal cells in area V1. Western blot analysis of GABA_AR subunit protein levels also found reduced levels of the γ2 subunit, but not the δ subunit, in Cntnap2-/- mice.

Conclusions : Our findings indicate that network-level GABAergic function is disturbed in Cntnap2 -/- mice in a manner dependent on GABA_ARs containing the δ subunit. This result is consistent with previous reports of reduced interneuron numbers, altered network activity, seizure susceptibility, and reduced interneuron numbers in autism. Our data suggest that reduced tonic inhibition could underlie autism-like behaviors, and future studies should investigate cellular/molecular mechanisms of reduced tonic inhibition as well as the effects of diminished inhibition on cortical function in vivo.