Activation of AMPA receptors in the suprachiasmatic nucleus phase-shifts the mouse circadian clock in vivo and in vitro

Abstract

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN.

Figure 1. AMPA microinjection at CT14 induced phase delays and Per1 expressions in vivo.

(A) Topographic analysis of AMPA receptor mRNA expressions (GluR1-4) in the mouse SCN by in situ hybridization using digoxigenin-labeled riboprobes. Scale bar, 200 µm. (B–G) Representative double-plotted actograms of circadian locomotor activity rhythms in mice injected with either (B) vehicle, (C) AMPA, (D) AMPA + NBQX, (E) AMPA + AP5, (F) NBQX or (G) AP5. Mice were maintained in constant darkness and microinjections were given at CT14 (marked by asterisks) under dim red light illumination. The magnitude of the phase delays was calculated by comparing eye-fitted lines drawn according to the onset of the locomotor activity before and after the microinjection. (H) Summary of phase delays (Mean ± SEM) induced by microinjection of drugs at CT14. Minus values mean phase delays. Numbers at the bars denote sample sizes for each condition. ** p<0.01 (one-way ANOVA, followed by Scheffe's multiple comparisons). (I) Acute induction of Per1 mRNA (Mean ± SEM) induced by AMPA or vehicle microinjection, detected by in situ hybridization using [³³P]-labeled riboprobes. The average value of vehicle microinjection was set to 1. * p<0.05 (Student's t-test). Inset panels show representative autoradiograph images of Per1 mRNA expression induced by vehicle (left) or AMPA (right) microinjection at CT14. Scale bar, 500 µm.

Figure 2. AMPA-induced phase shifts of luminescence rhythms in organotypic SCN slice cultures.

Images of the representative results are shown in the upper panels. The corresponding graphs are shown below, defining the second peak values (time 0) as 100%. AMPA application, (A) at 6 hr or (C) at 14 hr after the peak of the luminescence, induced phase delays and advances, respectively. Control medium treatment without AMPA (B) at 6 hr or (D) at 14 hr after the peak had no effect on the phase. p<0.01 (both at 6 hr and at 14 hr, AMPA vs. control, Student's t-test). To calculate the period length, each middle point between peak and trough in the increasing phase was first determined, and the time at the middle point was subtracted by the time at the previous middle point. (E) PRC obtained with SCN slice cultures stimulated by AMPA application. The x axis represents the normalized time after the peak (1 normalized hour  =  free running period/24 hr). The y axis represents the magnitude of phase shifts normalized by multiplying each shift in hour by the factor of 24 hr/free-running period. Plus and minus values mean phase advances and delays, respectively. Each value is the Mean ± SEM. The data obtained from multiple SCN slices during two hours were averaged. Hours shown on the x axis represent the middle of each two hours interval. One-way ANOVA revealed significant differences in PRC amplitudes obtained by AMPA application, but not in that obtained by control application (see Materials and Method). Post-hoc analysis using Scheffe's multiple comparisons revealed that the magnitude of AMPA-induced phase shifts at 6 hr was significantly different from the magnitudes at all other time points except at 2 and 4 hr (p<0.01).