Developmental Regulation of KCC2 Phosphorylation Has Long-Term Impacts on Cognitive Function

Abstract

GABA_A receptor-mediated currents shift from excitatory to inhibitory during postnatal brain development in rodents. A postnatal increase in KCC2 protein expression is considered to be the sole mechanism controlling the developmental onset of hyperpolarizing synaptic transmission, but here we identify a key role for KCC2 phosphorylation in the developmental E_GABA shift. Preventing phosphorylation of KCC2 in vivo at either residue serine 940 (S940), or at residues threonine 906 and threonine 1007 (T906/T1007), delayed or accelerated the postnatal onset of KCC2 function, respectively. Several models of neurodevelopmental disorders including Rett syndrome, Fragile × and Down's syndrome exhibit delayed postnatal onset of hyperpolarizing GABAergic inhibition, but whether the timing of the onset of hyperpolarizing synaptic inhibition during development plays a role in establishing adulthood cognitive function is unknown; we have used the distinct KCC2-S940A and KCC2-T906A/T1007A knock-in mouse models to address this issue. Altering KCC2 function resulted in long-term abnormalities in social behavior and memory retention. Tight regulation of KCC2 phosphorylation is therefore required for the typical timing of the developmental onset of hyperpolarizing synaptic inhibition, and it plays a fundamental role in the regulation of adulthood cognitive function.

Keywords: KCC2; autism; cognition; depolarizing GABA; memory; phosphorylation; social behavior.

Figure 1

KCC2-S940 and T906/T1007 phosphorylation orchestrate the postnatal upregulation of KCC2 function. (A) Western blotting was used to assess developmental changes in KCC2 expression and KCC2-S940 phosphorylation in cultured hippocampal neurons at 5, 10, 15 and 20 DIV. Levels of KCC2 expression and S940 phosphorylation are expressed relative to 20 DIV. KCC2 expression progressively increased between 5 and 20 DIV. Relative to levels of KCC2 expression, S940 phosphorylation decreased between 5 and 10 DIV but was followed by maintenance at this level as the neurons further matured. (B) The polarity of GABA_A currents was recorded from WT, S940A and T906A/T1007A cultured mouse neurons between 4 and 22 DIV by activating GABA_A receptors using brief muscimol application. WT and S940A neurons displayed depolarizing GABA_A currents at the earliest stages of neuronal development but shifted to hyperpolarizing responses by 16 DIV. In contrast, T906A/T1007A neurons displayed hyperpolarizing GABA_A currents as early as 4 DIV. (C) E_GABA measurements were obtained from WT, S940A and T906A/T1007A neurons between 4 and 22 DIV. A progressive negative E_GABA shift was detected in WT neurons as development proceeded. In contrast, this negative E_GABA shift was delayed in the S940A neurons and accelerated in the T906A/T1007A neurons. E_GABA values were also binned into groups of 2–4 DIV to enable statistical analysis. The corresponding [Cl⁻]_i measurements were also calculated (D). *Statistically significant (p < 0.05) for the indicated comparisons, see text and Supplementary Tables 1 and 2 for exact p values.

Figure 2

Both delayed and accelerated developmental onset of hyperpolarizing inhibition impact social behavior. A 3-chamber social interaction assay was used to assess sociability and preference for social novelty in the S940A and T906A/T1007A mice. Heat maps indicate the predominate regions of exploration within the 3 chambers (warm colors indicate longer time spent than cooler colors). (A) Diagram of the sociability assay. Mice were allowed to explore either an unfamiliar mouse or an empty cage. (B) Both S940A mice and their WT littermates spent more time interacting with the mouse vs. the empty cage, but the S940A mice spent less time interacting with the mouse than their WT littermates did, suggesting a mild sociability deficit in the S940A mice. (C) T906A/T1007A mice and their WT littermates both showed preference for interacting with the mouse vs. the empty cage, but the T906A/T1007A mice spent more time with the mouse than the WT mice, suggesting that the T906A/T1007A mice are more social. (D) Diagram of the preference for social novelty assay. Immediately after the sociability test, an additional unfamiliar mouse was placed under the empty cage and mice were allowed to explore both of these mice. (E) S940A mice showed no preference for interacting with the novel mouse, unlike their WT littermates. (F) T906A/T1007A mice showed no preference for interacting with the novel mouse, unlike their WT littermates. *Statistically significant (p < 0.05) for the indicated comparisons, see text for exact p values. ns, no significant change.

Figure 3

KCC2 phosphorylation can impact rate of spatial learning. (A) Diagram of the Barnes maze assay used to assess spatial learning in the mutant mice. Mice were exposed to the maze over six consecutive days, learning to associate the location of an escape hole with a spatial cue (concentric circles). Additional spatial cues were placed around the maze as depicted. (B) Latency to enter the goal hole was measured on days 1–6, and the S940A mice showed no significant differences in time to goal hole compared to WT littermates on any of these days. Rate of learning (day in which there is a significant reduction in latency to goal compared to that of day 1) was also comparable. (C) Latency to enter the goal hole was measured on days 1–6, and the T906A/T1007A mice showed no significant differences in time to goal hole compared to WT littermates on any of these days. However, rate of learning (day in which there is a significant reduction in latency to goal compared to that of day 1) was accelerated in the T906A/T1007A mice. *Statistically significant (p < 0.05) for the indicated comparisons, see Supplementary Tables 4 and 5 for exact p values.

Figure 4

KCC2-S940A and KCC2-T906A/T1007A mutations have bidirectional effects on spatial memory retention. Spatial memory was assessed using a Barnes maze assay. After 6 days of learning the location of an escape hole (see Figure 3) time spent at each of the holes was measured on day 7 and day 14 upon removal of the escape tunnel, and data binned into 45° groups. (A) S940A mice and their WT littermates both show preference for the goal area on day 7, and spent comparable time at the goal area as their WT littermates (B). (C) T906A/T1007A mice and their WT littermates both show preference for the goal area on day 7, but T906A/T1007A mice spent more time at the goal than their WT littermates (D). (E) S940A mice show no preference for the goal hole on day 14, but time spent at the goal was comparable to their WT littermates (F). (G) T906A/T1007A mice show enhanced specificity of the spatial memory on day 14 compared to their WT littermates, and T906A/T1007A mice spent more time at the goal than their WT littermates (H), indicating that the T906A/T1007A mutations enhance spatial memory retention. *Statistically significant (p < 0.05) for the indicated comparisons, see Supplementary Table 6 for exact p values. ns, no significant change.