Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1

Abstract

GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl(-)](i) in immature neocortical neurones causes GABA(A) receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl(-) accumulation remain controversial. Therefore, the GABA reversal potential (E(GABA)) or [Cl(-)](i) in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation-Cl(-) cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl(-)](i). The mRNA expression levels were positively (the Cl(-) accumulating Na(+),K(+)-2Cl(-) cotransporter NKCC1) or negatively (the Cl(-) extruding K(+)-Cl(-) cotransporter KCC2) correlated with [Cl(-)](i). NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl(-)](i) and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on E(GABA) were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex.

Figure 1. Typical gramicidin-perforated patch-clamp recordings, with subsequent single-cell multiplex RT-PCR, in cortical plate (CP) and layer II/III pyramidal neurones

A, CP neurone at P2. B, layer II/III pyramidal neurone at P17. a, voltage responses to current injections recorded in current-clamp mode reveal strong adaptation of action potentials in P2 (Aa), but not in P17 (Ba) neurones. b, GABA (50 μm)-induced currents at different command potentials reveal more negative reversal potential at P17 (Bb) than at P2 (Ab). c, current–voltage relationship estimated from the peak current measured during the responses shown in b shows that the E_GABA values determined by fitting the I–V curve to a second-order polynomial function were −38.2 mV at P2 (Ac) and −68.2 mV at P17 (Bc). d, single-cell mRNA expression profiles for NKCC1, KCC2 and β-actin. Note that the P17 cell was lacking in NKCC1 (Bd), while the P2 cell had a weaker expression of KCC2 (Ad) than the P17 cell (Bd). All P2 data (A) were obtained from the same cell, as were the P17 data (B).

Figure 2. Negative shift in E_GABA during development

A, averaged GABA-induced current plotted as a function of membrane potential (I–V relationship) for P1–3 (a, n = 15) and P11−20 (b, n = 10) neurones. E_GABA was significantly more positive at P1−3 than at P11–20. Error bars indicate s.e.m. B, expressions of NKCC1, KCC2 and β-actin mRNAs were detected by means of single-cell multiplex RT-PCR from the cells used for A. N: negative control using RNase- and DNase-free water. Note that NKCC1 mRNA was not detected at all in P11–20 cells.

Figure 3. Changes in [Cl⁻]_i and expression levels of NKCC1 and KCC2 mRNAs during development

A, the calculated [Cl⁻]_I values were plotted for P1–3 (n = 15) P11–20 neurones (n = 10) (♦: mean ± s.d.). The expression levels of NKCC1 and KCC2 mRNAs (harvested from the same cells as in A) are shown in B and C, respectively. The intensity of the PCR products for NKCC1 and KCC2 mRNAs were normalized with respect to that for β-actin. The relative intensity for NKCC1 mRNA decreased significantly, from 0.8 ± 0.6 to 0, whereas that for KCC2 mRNA increased significantly, from 0.6 ± 0.3 to 1.2 ± 0.4.

Figure 4. Regional differences in [Cl⁻]_i and Cl⁻ transporter mRNA expression in the same slice

A, IR-DIC images and GABA-evoked currents for cells in CP (a) and layer V/VI (b) of a P4 rat. B, current–voltage relationships for the GABA-induced currents in the CP (a) and layer V/VI (b) neurones shown in Aa and Ab. C, [Cl⁻]_i was significantly higher in CP cells than in layer V/VI neurones at either P1–3 (n = 15 and 4, respectively) or P5–7 (n = 5 and 5, respectively). D, normalized NKCC1 mRNA expression (as estimated by single-cell RT-PCR) was significantly higher in CP than in layer V/VI neurones (in the same slices) at P1–3, but not at P5-7. E, normalized KCC2 mRNA expression was not significantly different between CP and layer V/VI at either P1–3 or P5–7.

Figure 5. Differential effects of a Na⁺,K⁺–2Cl⁻ cotransporter inhibitor on NKCC1-expressing and NKCC1-negative neurones

Bumetanide (20 μm) shifted E_GABA in the negative direction in NKCC1-expressing P2 CP neurones (A), but not in NKCC1-negative P5 layer V/VI neurones (B). The expression profiles of the NKCC1 and KCC2 mRNAs were confirmed by single-cell multiplex RT-PCR (b). C, E_GABA was significantly more positive in NKCC1-expressing (+) cells (n = 5) than in NKCC1-negative (−) cells (n = 4). D, the bumetanide-induced negative shift in E_GABA was significantly larger in NKCC1-expressing than in NKCC1-negative neurones.

Figure 6. Differences in Cl⁻ homeostasis as a function of K⁺–Cl⁻ cotransporter expression level

The K⁺–Cl⁻ cotransporter inhibitor DIOA (50 μm) did not alter E_GABA in immature neurones (P3 CP, A), but it shifted E_GABA in the positive direction in mature neurones (P19 layer II/III, B). C, E_GABA was significantly more negative in DIOA-sensitive (+) neurones (n = 6) than in DIOA-insensitive (−) neurones (n = 4). D, plot of the effect of DIOA on E_GABA against the KCC2 mRNA expression level (n = 7). The line was fitted by eye.

Figure 7. Correlation of expressions of NKCC1 and KCC2 mRNAs with [Cl⁻]_i

[Cl⁻]_i is plotted against the relative expression levels of the mRNAs for both NKCC1 and KCC2 (normalized with respect to β-actin) in the same cell (n = 42). The bivariate linear regression was fitted to the data points by the least-squares method (z = 19.2 + 10x − 6.2y; where z is [Cl⁻]_I, x is normalized expression level for NKCC1 mRNA, and y is normalized expression level for KCC2 mRNA). The above equation indicates that if neither NKCC1 nor KCC2 was operating, [Cl⁻]_i would be 19.2 mm(E_Cl = −50.5 mV). Note that the cubic graphs in A and B are identical ones viewed from different angles. A, in this view of the bivariate linear regression, the positive slope for x indicates a significant positive correlation between NKCC1 mRNA and [Cl⁻]_i(r_NKCC1,[Cl]i = 0.65). B, in this view of the regression, the negative slope for y indicates a significant negative correlation between KCC2 mRNA and [Cl⁻]_i(r_KCC2,[Cl]i = −0.32). Thus, NKCC1 and KCC2 had opposite correlations with [Cl⁻]_i.

Figure 8. Changes in [Ca²⁺]_i response to GABA application in developing cortical neurones

A, CP neurones (P2) showed [Ca²⁺]_i elevations in response to GABA (10 μm). Picrotoxin (50 μm) reversibly abolished these responses (a). Even in the presence of TTX (3 μm), GABA still evoked equivalent [Ca²⁺]_i increases (b). However, nifedipine (100 μm) attenuated the GABA-induced response (c). After 20 min of bumetanide (20 μm) perfusion, the GABA-induced [Ca²⁺]_i elevations were completely abolished (d), whereas DIOA had no effect on such elevations (e). f, summary of effects of drugs on GABA-induced [Ca²⁺]_i elevations (***P < 0.001, ANOVA followed by Dunnett's test). B, in P1 CP neurones, the GABA (10 μm)-evoked [Ca²⁺]_i elevations were significantly (P < 0.001) greater than the glutamate (10 μm)-induced elevations. C, the GABA-induced Ca²⁺ transients were significantly larger in CP than in layer V/VI neurones at P2 (a). The difference gradually decreased as development proceeded, with no significant difference being observed between layer II/III and layer V/VI neurones at P6 (b) (***P < 0.001). Responses in three neighbouring cells in the same region of a single slice are shown in this figure. Error bars indicate s.d.