The Excitatory Effects of GABA within the Suprachiasmatic Nucleus: Regulation of Na-K-2Cl Cotransporters (NKCCs) by Environmental Lighting Conditions

Abstract

The suprachiasmatic nucleus (SCN) contains a pacemaker that generates circadian rhythms and entrains them with the 24-h light-dark cycle (LD). The SCN is composed of 16,000 to 20,000 heterogeneous neurons in bilaterally paired nuclei. γ-amino butyric acid (GABA) is the primary neurochemical signal within the SCN and plays a key role in regulating circadian function. While GABA is the primary inhibitory neurotransmitter in the brain, there is now evidence that GABA can also exert excitatory effects in the adult brain. Cation chloride cotransporters determine the effects of GABA on chloride equilibrium, thereby determining whether GABA produces hyperpolarizing or depolarizing actions following activation of GABA_A receptors. The activity of Na-K-2Cl cotransporter1 (NKCC1), the most prevalent chloride influx cotransporter isoform in the brain, plays a critical role in determining whether GABA has depolarizing effects. In the present study, we tested the hypothesis that NKCC1 protein expression in the SCN is regulated by environmental lighting and displays daily and circadian changes in the intact circadian system of the Syrian hamster. In hamsters housed in constant light (LL), the overall NKCC1 immunoreactivity (NKCC1-ir) in the SCN was significantly greater than in hamsters housed in LD or constant darkness (DD), although NKCC1 protein levels in the SCN were not different between hamsters housed in LD and DD. In hamsters housed in LD cycles, no differences in NKCC1-ir within the SCN were observed over the 24-h cycle. NKCC1 protein in the SCN was found to vary significantly over the circadian cycle in hamsters housed in free-running conditions. Overall, NKCC1 protein was greater in the ventral SCN than in the dorsal SCN, although no significant differences were observed across lighting conditions or time of day in either subregion. These data support the hypothesis that NKCC1 protein expression can be regulated by environmental lighting and circadian mechanisms within the SCN.

Keywords: GABAA receptors; chloride; circadian; entrainment; phase shifting.

Figure 1.

Effect of lighting condition on NKCC1 protein. lighting condition changed NKCC1 protein immunoreactivity in the SCN, as analyzed by one-way analysis of variance (F_2,56 = 15.988, p = 0.000006). Means represent all hamsters within the lighting condition, regardless of the time of sacrifice (LL/DD = CT 1, 6, 13.5, and 19; LD = ZT 1, 6, 13.5, and 19). Tukey’s honestly significant difference post hoc testing revealed that LL increased protein immunoreactivity as compared with both LD and DD light cycles (p = 0.0001 and p = 0.001, respectively). *p < 0.05. Scale bar: 200 μm. Group sizes by light cycle ad zeitgeber/circadian time can be seen in Table 1.

Figure 2.

NKCC1 expression in LD 14:10. When hamsters were housed in LD 14:10, one-way analysis of variance revealed that NKCC1 protein immunoreactivity in the SCN did not differ across zeitgeber time (F_3,15 = 0.075, p = 0.972). Scale bar: 200 μm. Color versions are available online.

Figure 3.

NKCC1 protein expression in constant dark (DD). In hamsters housed in constant dark, one-way analysis of variance of NKCC1 protein immunoreactivity (ir) showed changes over circadian time (CT) (F_3,15 = 4.542, p = 0.017). Tukey’s honestly significant difference post hoc testing revealed that NKCC1 protein in the early subjective night (CT 13.5) was lower than both early subjective day (CT 1) and late subjective night (CT 19) (p = 0.040 and 0.033, respectively). NKCC1 protein-ir in the middle of the subjective day (CT 6) did not differ from any other circadian time (CT 1, CT 13.5, CT 19; p = 0.227, 0.628, and 0.192, respectively). *p < 0.05. Scale bar: 200 μm. Color versions are available online.

Figure 4.

NKCC1 protein expression in constant light (LL). In hamsters housed in constant light (200 lux), a one-way analysis of variance revealed that NKCC1 protein immunoreactivity (ir) varied over circadian time, although the pattern of NKCC1 expression was different than in DD (Fig. 3; F_3,16 = 3.982, p = 0.027). Tukey’s honestly significant difference post hoc testing revealed that NKCC1 protein-ir was elevated in the early subjective day (CT 1) as compared with the late subjective night (CT 19; p = 0.026). NKCC1 protein-ir in the middle of the subjective day (CT 6) and the early subjective night (CT 13.5) did not differ from other circadian times. Scale bar: 200 μm. Color versions are available online.

Figure 5.

NKCC1 expression in LD; vSCN and dSCN. A repeated-measures analysis of variance revealed a significant main effect in the amount of NKCC1 protein expression between SCN subregions (F_1,55 = 14.551, p = 0.00035). Circles indicate the areas of the SCN where NKCC1 protein immunoreactivity was compared (diameter = 150 μm). There was no dorsal/ventral interaction within light cycle (F_2,55 = 0.272, p = 0.763).

Figure 6.

NKCC1 mRNA in LD and DD. One-way analysis of variance showed that nkcc1 mRNA did not differ across the day in hamsters housed in entrained (LD: F_3,23 = 0.855, p = 0.478) or in free-running conditions (DD: F_3,17 = 1.035, p = 0.402).