?2003). To overcome this problem, we measured the reversal potential of K+ currents in cell-attached mode (assuming 150 mm K+ in the patch solution gives a symmetric K+ concentration) to estimate the resting membrane potential (Fig. 7A; see Wang?et al.?2003). We found that the resting potential was more hyperpolarized (19.5 �� 2.7 mV,?n?= 6) than that measured with perforated patch recordings implying that in most cases GABA depolarizes CC-contacting neurones from rest (Fig. 7B). The depolarizing action via GABAA receptors is due to a high [Cl?]i attained by the co-transporter NKCC1 which is counteracted by the extrusion diglyceride
of Cl? by KCC2 (Ben-Ari, 2002; Spitzer, 2010). Thus, the actual effect of GABA would be determined by the balance of NKCC1 and KCC2 in CC-contacting neurones. Figure 7C shows that in many cells (14 out of 17) the selective NKCC1 antagonist bumetanide (20 ��m) shifted?EGABA to more hyperpolarized potentials (�C56.5 �� 2.4 mV to �C64.7 �� 2.2 mV;?P?< 0.004, Wilcoxon's matched pairs test,?n?= 14; Fig. 7Ca�Cc). To check whether KCC2 could be active in cells with relatively hyperpolarized?EGABA (�ݨC55 mV), we applied the non-selective antagonist of cation chloride co-transporters furosemide (Payne?et al.?2003). In 5 out of 8 cells,?EGABA shifted to depolarized membrane potentials (�C72 �� 3.3 mV to �C59.65 �� 2.8 mV;?P?< 0.03, Wilcoxon's matched pairs test,?n?= 5) in GDC941
the presence of furosemide (50�C200 ��m, Fig. 7Ca�Cc) suggesting a high KCC2:NKCC1 click here
ratio (Woodin?et al.?2003). Immunohistochemistry for these transporters showed a high expression of NKCC1 in most CC-contacting neurones (Fig. 7D) whereas the expression of KCC2 was less robust and mostly confined to cells located dorso-laterally (Fig. 7E, arrowheads). However, the expression of KCC2 in neurones outside the ependyma was conspicuous (Fig. 7Eb?and?c; arrows). As a functional correlate of KCC2 immunohistochemical data, we tested the activity of KCC2 in CC-contacting neurones with different?EGABA using a strategy described by Ben-Ari?et al.?(2011) in the hippocampus. In gramicidin perforated patch recordings we first applied GABA at a holding potential equal to?EGABA and then shifted to a depolarized potential (0 mV, 1 min), where GABA generated large outward currents due to the influx of Cl? via GABAA receptors (Fig. 7Fa?and?b,?n?= 13). After returning to the initial holding potential, GABA generated inward currents because of the shift in?EGABA induced by the load of Cl? (Fig. 7Fa?and?b). The time course of recovery of?EGABA reflects the dynamic removal of Cl? by KCC2 (Ben-Ari?et al.?2011; Nardou?et al.?2011). We found that in cells with depolarized?EGABA the recovery was slow, taking up to 10 min (Fig. 7Fa?and?c). However, in CC-contacting neurones with a hyperpolarized?EGABA (Fig.