Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice

Abstract

Alterations in the balance between neuronal excitation and inhibition (E:I balance) have been implicated in the neural circuit activity-based processes that contribute to autism phenotypes. We investigated whether acutely reducing E:I balance in mouse brain could correct deficits in social behavior. We used mice lacking the CNTNAP2 gene, which has been implicated in autism, and achieved a temporally precise reduction in E:I balance in the medial prefrontal cortex (mPFC) either by optogenetically increasing the excitability of inhibitory parvalbumin (PV) neurons or decreasing the excitability of excitatory pyramidal neurons. Surprisingly, both of these distinct, real-time, and reversible optogenetic modulations acutely rescued deficits in social behavior and hyperactivity in adult mice lacking CNTNAP2 Using fiber photometry, we discovered that native mPFC PV neuronal activity differed between CNTNAP2 knockout and wild-type mice. During social interactions with other mice, PV neuron activity increased in wild-type mice compared to interactions with a novel object, whereas this difference was not observed in CNTNAP2 knockout mice. Together, these results suggest that real-time modulation of E:I balance in the mouse prefrontal cortex can rescue social behavior deficits reminiscent of autism phenotypes.

Fig. 1. Generation of PV::Cre/CNTNAP2 mice and characterization of SSFO properties in mPFC PV neurons

(A) Breeding strategy to generate PV::Cre/CNTNAP2 mice is shown. Only the combination of genotypes that were chosen for the next breeding step is shown. PV::Cre^+/+ CNTNAP2^+/− mice were bred to generate WT and CNTNAP2 KO mice. (B) AAV5-EF1α::DIO-SSFO-YFP expression in PV neurons in WT mice. Bilateral optical fiber implantation above the mPFC in WT mice is also shown. Scale bar, 100 μm. (C) Representative short–time scale current-clamp trace of a PV neuron [identified by yellow fluorescent protein (YFP) expression] in a brain slice from the mPFC of a PV::Cre mouse, prepared 2 to 3 weeks after injection of a Cre-dependent adeno-associated virus (AAV) encoding the SSFO. Trace shows the minimum amount of electrical current injection (200 pA) required for spike generation. Resting potential, −72 mV. (D) Representative trace of the same PV neuron showing the much lower minimum current injection (50 pA) required for spike generation during SSFO activation by blue light of wavelength 475 nm (28-nm bandpass) (light blue bar). Light power density was 8 mW/mm². SSFO was deactivated using red light of wavelength 632/622 nm (red bar). Light power density was 5 mW/mm². (E) Bar graph summarizing the electrical current needed to induce spiking without or with SSFO activation (n = 8 cells) (table S1). (F) Representative extended–time scale voltage-clamp trace of an SSFO-expressing PV neuron after blue light and red light application. Cell was held at −77 mV. (G) Representative current-clamp trace of same cell after blue and red light application. Note the lack of directly elicited spiking. (H) Voltage-clamp studies showing SSFO-induced initial peak and final steady-state photocurrent amplitude (n = 8 cells). Resting potential of cells held in voltage clamp at −80 mV is shown (table S2). (I) Current-clamp studies showing SSFO-induced initial peak and final steady-state depolarization (n = 8 cells) (table S3). (J) Sample traces showing stable, high-fidelity action potential generation in a PV neuron expressing SSFO after blue light delivery, combined with increasing amounts of electrical current injection (5-ms pulses, 20-Hz frequency), followed by deactivation with red light. Current amplitude was applied in 250-pA steps, beginning at 250 pA. In all cases, spiking persisted for 60 s until experimental termination of the costimulation. (K) Bar graph summary of spike fidelity (spikes per 5-ms current pulse) throughout the entire SSFO activation protocol, in combination with the varying electrical current injection steps, as shown in (J). n = 7 cells from WT mice; P = 0.101 by Kruskal-Wallis H test (table S4). Bar graphs in (E), (H), (I), and (K) are presented as mean ± SEM. *P < 0.05 by paired t test.

Fig. 2. SSFO or SwiChR++ modulation of mPFC neurons and rescue of social behavior deficits in CNTNAP2 KO mice

(A) Testing of social interaction behavior for 10 min in mouse pairs matched by genotype and sex is shown. A single 2-s, 473-nm pulse of blue light was administered immediately before the behavioral test in the stimulation condition. (B) The duration of social explorations during the first 10 interaction bouts with or without SSFO stimulation is shown (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P < 0.0001) (table S5). In (B) to (J), two-way ANOVA was used, followed by Dunnett’s pairwise comparisons; parameter interaction values and n values (at least eight pairs per condition) for each arm are shown in the Supplementary Materials. (C) Duration of social explorations during the total 10 min of the test with and without SSFO stimulation (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P < 0.05) (table S6). (D) Mean social exploration duration per interaction bout with and without SSFO stimulation (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P < 0.01) (table S7). (E) Mean social approach velocity with and without SSFO stimulation (WT_baseline versus KO_baseline, P < 0.001; KO_baseline versus KO_stimulation, P < 0.05) (table S8). (F) Social interaction test after a 1-s intermittent 473-nm pulse of blue light to stimulate the inhibitory channelrhodopsin SwiChR++. (G) Social exploration duration during the first 10 interaction bouts with and without inhibitory SwiChR++ stimulation (WT_stimulation ver sus KO_baseline, P < 0.01; KO_baseline versus KO_stimulation, P < 0.05) (table S9). (H) Social exploration duration during the total length of the test (10 min) with and without inhibitory SwiChR++ stimulation (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P < 0.05) (table S10). (I) Mean social exploration duration per interaction bout with and without inhibitory SwiChR++ stimulation (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P < 0.05) (table S11). (J) Mean social approach velocity with and without inhibitory SwiChR++ stimulation (WT_baseline versus KO_baseline, P < 0.05; KO_baseline versus KO_stimulation, P > 0.05) (table S12). Two separate cohorts were tested and included in the analyses. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***, ***P < 0.001, and ****P < 0.0001.

Fig. 3. Differential mPFC PV neuron activity during exploratory behaviors in CNTNAP2 KO and WT mice

(A) Left: Schematic showing viral targeting of mPFC PV neurons for the purposes of fiber photometry recordings. Right: Neurons expressing the fluorescent activity reporter GCaMP6f adjacent to the optical fiber. Scale bar, 150 mm. (B) Behavioral test during 20 min of continuous fiber photometry recording. Animals were presented with a sex- and age-matched new (non-cagemate) mouse (yellow), a familiar (cagemate) mouse (blue), and a novel object (pink) for 3 or 4 min, with rest periods in between. (C) Photometry traces from WT animals during the behavioral test. Behavioral bouts are represented as colored lines with thickness reflecting duration of the bout. The yellow panel below is a zoomed-in image of the corresponding trace. (D) Representative plot showing the average photometry trace aligned to the start of the social interaction for a WT animal. Black line represents mean dF/F (change in fluorescence divided by baseline fluorescence) of the first 10 bouts. Gray lines correspond to SE. (E) Photometry traces from CNTNAP2 KO animals during the behavioral test. Behavioral bouts are represented as colored lines with thickness reflecting their duration. The panel below is the zoomed-in image of the corresponding trace above. (F) Representative plot showing the average photometry trace aligned to the start of the interaction for a CNTNAP2 KO animal. Black line represents average mean dF/F of the first 10 bouts. Gray lines correspond to SEM. (G) Mean total exploration time during each condition [novel social interaction (yellow), Student’s t test, P = 0.01; WT, n = 6; KO, n = 12; familiar social interaction (blue), P = 0.55; WT, n = 6; KO, n = 8; novel object interaction (pink), P = 0.20; WT, n = 10; KO, n = 11] (table S13). (H) Mean novel social exploration duration during the initial and final 10 bouts of the social interaction behavior test (initial 10 bouts, Student’s t test, P = 0.03; final 10 bouts, P = 0.27; WT, n = 6; KO, n = 9) (table S14). (I) Mean z-scored dF/F during the first 10 bouts. WT mice (left, n = 5; novel social interaction versus novel object interaction, P = 0.003; novel social interaction versus familiar social interaction, P = 0.86; familiar social interaction versus novel object interaction, P = 0.04) and CNTNAP2 KO mice (right, n = 5; novel social interaction versus novel object interaction, P = 0.21; novel social interaction versus familiar social interaction, P = 0.0240; familiar social interaction versus novel object interaction, P = 0.0012) (movies S1 and S2; note social PV recruitment in both cases, but to a greater extent than novel-object PV recruitment only for WT genotype as shown in figure). Wilcoxon rank-sum test was used to compare combined bouts across all mice for each condition. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.