KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses

Abstract

During brain development, GABA and glycine switch from being depolarizing to being hyperpolarizing neurotransmitters. This conversion results from a gradual decrease in the chloride electrochemical equilibrium potential (ECl) of developing neurons, which correlates to an increase in the expression or activity of the potassium chloride cotransporter, KCC2. However, evidence as to whether KCC2 expression is sufficient, in and of itself, to induce this switch is lacking. In order to address this question, we used a gain-of-function approach by over-expressing human KCC2 (hKCC2) in immature cortical neurons, before endogenous up-regulation of KCC2. We found that premature expression of hKCC2 produced a substantial negative shift in the GABA reversal potential and decreased or abolished GABA-elicited calcium responses in cultured neurons. We conclude that KCC2 expression is not only necessary but is also sufficient for ending the depolarizing period of GABA in developing cortical neurons.

Fig. 1

Heterologous expression of hKCC2. (A) pMES vector (control) and hKCC2 expression vector used in this study. (B) Western blotting reveals a protein of approximately 150 kDa molecular weight in hKCC2 transfected COS7 cells but not in vector-transfected cells. β-actin (42 kDa) was detected using a different blot from the same membrane preparation. (C) Embryonic cortical neurons transfected with control vector (pMES) showed high EGFP-expression (green) but only very low levels of KCC2 expression (red) that were detectable only when drastically increasing the gain of the photomultiplier tube (D). (E and F) KCC2-immunoreactivity was high in KCC2 transfected cells. (G and H) Higher magnification shows that KCC2 appeared to be concentrated in or near the cell membrane (H).

Fig. 2

Overexpression of hKCC2 produces a negative shift in E_GABA in immature cortical neurons. Gramicidin-perforated patch clamp recordings from cortical neurons in voltage clamp (A–C) and current clamp (D–F). Voltage–current relationship of membrane potential and GABA-elicited (0.5 mm, 200 ms) currents in a control neuron (A) and a KCC2-transfected neuron (B). (C) Summary data. (D) GABA-elicited membrane potential responses in a control neuron (D) and a hKCC2-transfected neuron. (F) Summary data. *P < 0.001, Student’s t-test. Scale bars, 200 ms, 50 pA (A and B); 1 s, 20 mV (D and E).

Fig. 3

KCC2 expression decreases GABA-elicited calcium responses. (A) Photomicrograph of EGFP fluorescence (488 nm excitation) of an hKCC2-transfected neuron. (B) Same field showing Fura-2 labelling (380 nm excitation). (C) Calcium responses to GABA (1 mm) and KCl (60 mm) of cells numbered in (B). The hKCC2 transfected cell (asterisk) did respond to KCl but not to GABA (arrow). Calibration: 0.4 ΔR/R, 100 s. (D) Percentage of KCl responding cells that also responded to GABA (*P < 0.01, Fisher’s exact test). (E) Calcium responses (ΔR/R normalized to KCl response) of hKCC2-transfected, GABA responding and GABA non-responding cells. Scale bar, 100 μm (A and B).