Influx of calcium through L-type calcium channels in early postnatal regulation of chloride transporters in the rat hippocampus.

Early in neuronal development the neurotransmitter γ-aminobutyric acid (GABA) exerts an excitatory rather than inhibitory effect due to a high concentration of intracellular chloride ions (Cherubini et al., 1991). During the first two postnatal weeks in the hippocampus of rats the internal concentration of chloride ions decreases. This causes a change in the reversal potential of chloride and thus, a shift from an excitatory effect of GABAA receptor activation to an inhibitory effect (Ben-Ari et al., 1989; Leinekugel et al., 1995, 1997, 1998; Khazipov et al., 1997; Rivera et al., 1999; Ganguly et al., 2001). Evidence suggests that the upregulation of the K+Cl- co-transporter (KCC2) and the downregulation of the Na+K+2Cl- co-transporter (NKCC1) are responsible for shifting the chloride reversal potential (Plotkin et al., 1997; Lu et al., 1999; Rivera et al., 1999; Hubner et al., 2001). NKCC1 expression predominates in immature neurons and mediates chloride influx, while KCC2 expression predominates in mature neurons and mediates chloride efflux (for review see Delpire, 2000; Payne et al., 2003). Due to the high intracellular chloride concentration in immature neurons the activation of GABAA receptors depolarizes the cell, which subsequently activates voltage-dependent calcium channels, particularly L-type calcium channels (Yuste and Katz, 1991; Leinekugel et al., 1995; Khazipov et al., 1997; Ganguly et al., 2001). This GABAergic excitation is important for proper neuronal development (for review see Ben-Ari, 2002; Owens and Kriegstein, 2002; Fiumelli and Woodin, 2007; Galanopoulou, 2008; Kahle et al., 2008; Blaesse et al., 2009). As the brain matures, the number of neurons that are excitatory in response to GABA decreases and thus, the magnitude of calcium influx with GABA receptor activation decreases. Once neurons have fully developed, GABA responses are hyperpolarizing and inhibit the cell from reaching threshold. Additionally, the subunit composition of the GABAA receptor changes during development (Kanaumi et al., 2006; Liu and Wong-Riley, 2006; Rissman et al., 2006; Yu et al., 2006). This change in subunit composition should not affect the reversal potential directly since that is dependent on the internal and external chloride concentrations, but it does affect the response to various modulators such as zinc and benzodiapines. Treatment of embryonic rat hippocampal cultures with L-type calcium channel antagonists prohibits the shift in the chloride reversal potential, suggesting that calcium influx through L-type calcium channels is involved in the changes of chloride transporter expression (Ganguly et al., 2001). However, Ganguly and co-workers did not directly look at the effect of calcium influx through L-type channels on chloride transporter expression. Previous experiments in our laboratory have demonstrated facilitation of L-type calcium current by activation of the metabotropic GABAB receptor in acutely cultured hippocampal neurons isolated from 5-7 day old rat pups (Carter and Mynlieff, 2004). Facilitation of L-type calcium current has only been observed in salamander retinal cells (Shen and Slaughter, 1999) and rat dorsal root ganglion cells (Fujikawa et al., 1997), but has not been observed by other investigators studying GABAB receptors using hippocampal tissue from either embryonic or adult rats. The main effect of GABAB receptor activation in adult hippocampus is to decrease N-type calcium current and increase potassium current (Newberry and Nicoll, 1984; Gahwiler and Brown, 1985; Dutar and Nicoll, 1988; Harrison, 1990; Lambert et al., 1991; Scholz and Miller, 1991; Thompson and Gahwiler, 1992; Davies and Collingridge, 1993; Pfrieger et al., 1994; Wu and Saggau, 1995; Takahashi et al., 1998; for review see Bettler et al., 2004). One possibility that L-type calcium current facilitation by GABAB receptor activation has not been previously observed within the rat hippocampus is that it is a phenomenon only present at a specific time point in development and may play a role in the developmental changes in gene expression such as those seen with chloride transporters. The present study explores the potential connection between chloride transporter expression and calcium influx through L-type calcium channels in the early neonatal period. Although changes in reversal potential have been shown to be dependent on calcium influx through L-type calcium channels, this is the first study to directly investigate the effect of calcium influx on the chloride transporter protein levels in hippocampal neurons. Since calcium influx is enhanced in a subset of neonatal hippocampal neurons by activation of GABAB receptors, activation of these receptors may also alter KCC2 and NKCC1 transporter expression. Electrophysiological experiments with the GABAB agonist baclofen were performed on cultured hippocampal neurons obtained from different aged rats to identify the timecourse of L-type calcium current facilitation by GABAB receptor activation. The KCC2 and NKCC1 expression levels throughout early postnatal development were determined by Western blot analysis in the presence and absence of an L-type channel antagonist and a GABAB receptor agonist.