Abstracts

Rationale: Excitatory GABA is a crucial developmental cue that guides perinatal neuronal migration, synaptogenesis, and circuit formation. Maturing neurons typically switch to hyperpolarizing responses through onset of KCC2 expression. We investigated how indefinitely-prolonged excitatory GABA responses in cortical interneurons (INs) adversely affect cortical circuit development using an IN-specific KCC2 knockout (KO).

Methods: We created the KO by crossing the IN-specific Dlx5:cre-IRES-eGFP and the KCC2^flox lines, and Ai14 reporter. For studies of early IN migration, we injected pregnant dams with EdU to label E13.5-born INs. We measured seizure susceptibility by latency to onset of flurothyl-induced generalized convulsions. Spontaneous seizures were captured with video monitoring. We recorded sIPSCs using whole-cell voltage clamp in layer 5 (L5) pyramidal neurons in acute brain slices. We used immunohistochemistry and epifluorescence/confocal imaging to visualize expression of KCC2 and GABA_A receptor subunits α1-5, and IN distribution by reporters and somatostatin (SST)/parvalbumin (PV) antibodies. Most experiments focused on P12-14 barrel cortex.

Results: We first observed cortical KCC2 expression in L5 INs, as early as E16-18 in WT mice. Therefore, we hypothesized that loss of KCC2 in INs would adversely affect early IN development, but surprisingly found a normal distribution of INs in cortex from P0 KO mice. Moreover, we probed for a mistimed migration by injecting pregnant dams with EdU and tracking a single cohort of E13.5-born INs. Surprisingly, EdU+ INs were similarly distributed in cortices of P0 KO and WT mice, showing no migratory delay in the marginal zone and normal destinations throughout the underlying cortex. However, KO mice exhibit abnormal neurological development, including susceptibility to seizure induction, spontaneous seizures, and late postnatal mortality with preceding seizures, but normal gross and histological anatomy of organs, including feeding structures and the brain. When we investigated INs in P12-14 cortex, we found a 30% increase in density of L5 INs, but no change in IPSC frequency in L5 pyramidal cells. We postulated that the effects of KCC2 loss might be specific to IN subtypes, which in L5 consist mainly of SST and PV INs with distinctly different circuit functions and developmental sequences. We found opposing changes in densities of these subtypes: a 12% increase in L5 density of SST INs in KO, but a decrease in PV IN density in L2-4 and L6.

Conclusions: Excitatory/Inhibitory GABA are important regulators of neuronal development. Contrary to previous literature, we found that indefinitely prolonged depolarizing GABA did not initially disrupt IN migration, but instead produced an imbalance in IN subtypes, seizures and premature death in the second and third weeks of postnatal life.

Funding: Please list any funding that was received in support of this abstract.: VA merit award 1I01BX001189.