Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

Abstract

Objectives: We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.

Methods: Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.

Results: Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (E(gly)) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted E(gly) was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.

Conclusion: The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.

Keywords: Circling mice; Lateral superior olive; Potassium-chloride co-transporter; Sodium-potassium-2 chloride co-transporter.

Fig. 1

The reversal potential of the glycine-evoked currents measured from the amplitudes of the responses at different step potentials. Glycine puffs during the voltage steps from -100 mV to -40 mV with increments of 10 mV elicited transient currents (A). In (C), the baseline current at each step potential was subtracted from the raw traces shown in (A). The amplitude of the currents at the vertical dotted line in (C) was plotted as a function of applied potentials (B). Filled circles represent the amplitudes as a function of the command potential, while hollow circles represent the amplitudes as a function of the corrected potential. The corrected potentials (-56, -58, -60, and -63) at given commanding potentials (-40, -50, -60, and -70) are present in (C).

Fig. 2

The localization of potassium-chloride co-transporter 2 (KCC2) immunoreactivity (IR) in lateral superior olive (LSO) of heterozygous (+/cir) (A, C) and homozygous (cir/cir) mice (B, D). At P16, KCC2 immunoreactivities were observed in LSO of both genotypes (A, B). The selected areas (dashed-line box) in A and B are magnified in C and D, respectively. The arrows indicate KCC2 immunoreactivity confined to the cell membrane (C, D). Scale bars=200 µm in A, B; 50 µm in C, D.

Fig. 3

The localization of sodium-potassium-2 chloride co-transporter 1 (NKCC1) IR in the lateral superior olive (LSO) of heterozygous (+/cir) (A, C) and homozygous (cir/cir) mice (B, D). On P16, NKKC1 immunoreactivities were observed in LSO of both genotypes (A, B). The selected areas (dashed-line box) in A and B are magnified in C and D, respectively. The arrows indicate NKCC1 immunoreactivities (C, D). Scale bars=200 µm in A, B; 50 µm in C, D.

Fig. 4

The reversal potential of the glycine-evoked currents at different step potentials. The amplitudes of the currents obtained from P9-P12 heterozygous (Het) (+/cir) or homozygous (Homo) (cir/cir) were plotted as a function of corrected potentials (A, C). The solid lines through the symbols were obtained from a linear fit to the current amplitudes. Filled circles represent the data obtained from control condition, while filled squares and filled triangles present the data from 50 µM bumetanide (Bum) and 50 µM bumetanide plus 1 mM furosemide (Furo), respectively. The means of the E_gly with standard errors are presented in (B, D) with bar graphs. The statistical data were obtained from different mice groups (B, Het [+/cir] older than P9; D, Homo [cir/cir] older than P9). ^*The statistical significance (P<0.05).