Regulation of light's action in the mammalian circadian clock: role of the extrasynaptic GABAA receptor

Abstract

GABA(A) receptor agonists act in the suprachiasmatic nucleus (SCN) to reset circadian rhythms during the day but inhibit the ability of light to reset rhythms during the night. In the present study, we examined whether these paradoxical differences in the effect of GABA(A) receptor stimulation on the circadian system are mediated by separate GABA(A) receptor subtypes. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a GABA(A) receptor agonist, preferentially activates GABA(A) receptors in extrasynaptic locations. THIP, muscimol (a GABA(A) agonist), or vehicle were microinjected into the SCN region of Syrian hamsters free-running in constant darkness during the mid-subjective day, early subjective night, or late subjective night. The subjective night injections were followed by a light pulse or sham control. Behavioral phase shifts of wheel running rhythms and both Period1 (Per1) and Per2 mRNA levels in the SCN were assessed. Animals that received THIP during the subjective day did not exhibit significant phase alterations. During the early and late subjective night, however, THIP abolished the phase-shifting effects of light and the ability of light to increase Per1 and Per2 mRNA levels. The ability of N-methyl-d-aspartic acid to phase-shift wheel running rhythms was also attenuated by THIP. Together these data demonstrate that THIP does not produce phase shifts during the subjective day, but does inhibit the ability of light to produce phase shifts. Thus, extrasynaptic GABA(A) receptors appear to play a role in regulating light input to the SCN, while a different population of GABA(A) receptors appears to be responsible for daytime effects of GABA.

Fig. 1.

Representative actograms depicting the actions of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and muscimol. During the mid-subjective day circadian time 6 (CT 6) THIP (22 nmol) did not result in any significant phase alterations (A), while muscimol (4.4 nmol) resulted in large-phase advances of wheel-running activity (B; arrows denote time of treatment). THIP (22 nmol) did abolish the phase-delaying (C) and phase-advancing (D) effects of light (each animal received a microinjection of THIP or saline followed by a light pulse in counterbalanced order; stars with arrows denote time of treatment; THIP top arrows; saline, bottom arrows). Each horizontal line represents 1 day; black bars are the number of wheel revolutions recorded in 5-min bins.

Fig. 2.

THIP inhibits light-induced behavioral phase shifts in the early and late subjective night. THIP (22 nmol) delivered to the suprachiasmatic nucleus (SCN) region just prior to a light pulse during the early subjective night (CT 13.5) inhibited phase delays so that they were not different from saline-treated control animals. THIP administered just prior to a light pulse in the late subjective night (CT 20) significantly inhibited the resulting phase advances. In both the early and late night, the effect of light in saline-treated animals was significantly greater than all other groups. Bars represent means ± SE; *P < 0.05.

Fig. 3.

Dose response for the inhibition of light phase shifts by THIP. THIP delivered to the SCN region in the early night (CT 13.5) inhibited light-induced phase delays in wheel-running activity. A complete block of light's effect was produced by the three highest doses of THIP investigated. The y-axis shows phase shifts in the circadian rhythm of wheel-running activity following a light pulse and THIP ^A,BDifferent letters indicate statistically significant differences, P < 0.05.

Fig. 4.

THIP inhibits light-induced period mRNA expression during the subjective night. A: representative autoradiograms. Per1 and Per2 mRNA increases induced by light in the early night (CT 13.5; A) were abolished in animals pretreated with THIP (B). Arrows indicate the SCN. B: THIP (22 nmol) delivered to the SCN region just prior to a light pulse during the early night (CT 13.5) and late night (CT 20) inhibited light-induced increases in Per1 mRNA. THIP delivered to the SCN region just prior to a light pulse during the early subjective night (CT 13.5) also inhibited light-induced increases in Per2 mRNA. The effect of light in saline-treated animals for Per1 at both time points and Per2 in the early night was significantly greater than all other groups. There were no significant differences between any of the Per2 mRNA groups in the late night. Bars represent means ± SE; *P < 0.05.