Enhanced Social Dominance and Altered Neuronal Excitability in the Prefrontal Cortex of Male KCC2b Mutant Mice

Abstract

The K-Cl cotransporter KCC2 is essential in the development of the "GABA switch" that produces a change in neuronal responses to GABA signaling from excitatory to inhibitory early in brain development, and alterations in this progression have previously been hypothesized to play a causal role in autism spectrum disorder (ASD). We investigated the KCC2b (Slc12a5) heterozygous knockout mouse using a battery of rodent behavioral tests relevant to core and comorbid ASD symptoms. Compared to wild-type littermates, KCC2^+/- mice were normal in standard measures of locomotor activity, grooming and digging behaviors, and social, vocalization, and anxiety-like behaviors. However, KCC2^+/- mice exhibited increased social dominance behaviors and increased amplitude of spontaneous postsynaptic currents in the medial prefrontal cortex (PFC) that were previously implicated in governing social hierarchy and dominance behaviors. Treatment of wild-type mouse brain slices with the KCC2 inhibitor VU0240511 increased the amplitude and frequency of excitatory postsynaptic currents, partially recapitulating the phenotype of KCC2^+/- mice. These findings indicate that the activity of KCC2 plays a role in social dominance, in parallel with effects on PFC signaling, further suggesting that KCC2 function has some relevance to social behavior but without the breadth of impact on autism-like behavior suggested by previous studies. Further testing could assess whether KCC2 alters other circuits and whether additional factors such as environmental insults may precipitate autism-related behavioral phenotypes. Autism Research 2019, 12: 732-743. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: A mouse model of altered chloride transporter expression was used to look for a role in behaviors and brain function relevant to autism. There was an imbalance in signaling in the prefrontal cortex, and increased social dominance behavior, although other autism-related behaviors were not changed. These findings indicate that altered chloride transporter function affects prefrontal cortex function and social dominance without a broader impact on autism-like behaviors.

Keywords: GABA; autism; dominance; excitatory; inhibitory; social.

Figure 1.

No effect of KCC2 deficiency on anxiety-like behavior, sociability, or ultrasonic vocalizations. (A) KCC2^+/− mice (n = 16) did not exhibit any difference compared to WT mice (n = 17) in anxiety-like behavior in the elevated zero maze, as measured by proportion of time spent in open arms (left), differences in number of closed or open arm entries (center left and right), and did not alter general activity in the maze (right). (B) KCC2^+/− mice (n = 15) did not exhibit any difference compared to WT mice (n = 17) in sociability in the three-chamber test, measured by time spent in the chamber (left) or near the cup (right) containing the social stimulus, which was significantly higher than time spent in the non-social chamber or near the non-social cup for both KCC2^+/− and WT mice. (C) USVs emitted in response to maternal separation decreased by postnatal day 12, but there was no interaction with genotype (KCC2^+/− mice n = 33, WT mice n = 37). * significant effect of social stimulus, p<0.0001; ^ns non-significant effect of genotype

Figure 2.

No effect of KCC2 deficiency on activity or other home cage behaviors. Activity level was not different between KCC2^+/− (n = 16) and WT (n = 17) mice in the first 5 (A) or full 90 (B) minutes of the test in the open field. There was no interaction between activity and genotype (KCC2^+/− mice n = 16, WT mice n = 15) in home cage behaviors (C).

Figure 3.

No effect of KCC2 deficiency on repetitive behaviors grooming or digging in a novel cage. (KCC2^+/− mice n = 16, WT mice n = 17)

Figure 4.

KCC2^+/− mice exhibited higher levels of dominance behavior in the tube test. Each mouse completed 12 matches with 6 subjects of the opposite genotype. KCC2^+/− mice (n = 16) achieved an average of 8.25 wins, a significantly greater number than WT mice (n = 17) that averaged 5.41 wins (A). The total number of wins achieved by each genotype across all matches was also significantly higher in KCC2^+/− mice than WT (B). *p<0.05

Figure 5.

No effect of KCC2 deficiency on social interaction behavior in the social reciprocity test with another mouse of the same genotype. (KCC2^+/− pairs n = 7, WT pairs n = 7)

Figure 6.

Excitatory synaptic tone onto L5 mPFC neurons is increased by genetic reduction and pharmacological inhibition of KCC2. (A) Quantitation and sample traces of mEPSC amplitudes and frequencies in WT and KCC2^+/− mice show a significant increase in the amplitude of events in heterozygous neurons. (WT n = 18 cells/4 mice, KCC2^+/− n = 19/4). *p = 0.02. Scale bar = 5 pA/2s (B) Quantitation and sample traces of mIPSC amplitudes and frequencies in WT and KCC2^+/− mice revealed no significant changes in amplitude or frequency. (WT n = 11 cells/2 mice, KCC2^+/− n = 13 cells/2 mice). Scale bar = 5 pA/500 ms (C) Quantitation and sample traces of mEPSC amplitudes and frequencies in WT cells incubated with 3 uM KCC2 inhibitor VU 0240551. KCC2 inhibition caused a significant increase in both the amplitude and frequency of mEPSCs in these neurons (WT n = 14/4, inhibitor n = 13/3). ***p = 0.001, **p = 0.02. Scale bar = 5 pA/2s (D) Quantitation and sample traces of mIPSC amplitudes and frequencies in WT cells incubated with 3 uM KCC2 inhibitor VU 0240551. KCC2 inhibition did not cause a significant change in mIPSC amplitude nor frequency (WT n = 12/2, inhibitor n = 12/3). Scale bar = 20 pA/500ms