Role for the NR2B subunit of the N-methyl-D-aspartate receptor in mediating light input to the circadian system

Abstract

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N-methyl-D-aspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system of hamsters and mice. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region reduces the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B vary with time of day in SCN tissue using semiquantitative real-time polymerase chain reaction and western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced Ca2+ changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor-mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system.

Fig. 1

Microinjection of ifenprodil inhibits the magnitude of light-induced phase shifts in the circadian rhythm in wheel-running activity. Histograms plot the mean phase shift of hamsters in DD that received a treatment of either vehicle (200nl) plus light or ifenprodil (200nl, 2mg/ml) plus light. Other groups included hamsters treated with vehicle or ifenprodil alone. Light treatments (15min, 150 lux) were delivered at either CT 13.5 (left panel) or CT 19. The vehicle or drug treatments were delivered immediately prior to light. The placement of the canula into the SCN region was histologically confirmed. N = 6-8 for all points; error bars represent SEM. Values were analyzed with t-test and asterisks indicates significance at P < 0.05.

Fig. 2

Locomotor activity records from experimental and control animals maintained in DD. Each horizontal line represents the activity record for a 24 hr day, and successive days are plotted from top to bottom. Grey arrows represent the time of light and/or drug treatment. A) Activity record illustrating the inhibition of the phase-advancing effects of light by an intra-SCN injection of ifenprodil at CT 19. B) Activity record illustrating light-induced phase shift of locomotor activity. Hamsters were exposed to light at CT 19 with vehicle delivered immediately prior to light. C) Activity record illustrating the lack of effect of an injection of ifenprodil at CT 19 on the phase of the circadian rhythm in locomotor activity.

Fig. 3

NR2B transcripts are rhythmically expressed in SCN tissue. Semi-quantitative RT-PCR was used to measure levels of the transcript. The SQ values shown are determined by a standard curve and normalized to the housekeeping gene beta-2 microglobulin. The histogram plots the mean results of 3 independent experiments with error bars representing SEM. For each experiment, SCN tissue was collected from 15 mice at 5 time points (ZT 2, 6, 10, 16, 23) with mRNA from each time point pooled from 3 mice. Values were analyzed with ANOVA followed by Tukey test for pairwise comparisons. Astericks indicate significance at P < 0.05 compared to sample at ZT 2.

Fig. 4

Phospho-NR2B levels vary with time of day in SCN tissue. Western blots were used to measure levels of phospho-NR2B and total NR2B. A) Top panels show examples of the blots. B) Histograms plot the mean results of 3 independent experiments with error bars representing SEM. Values are shown normalized to tubulin. For each experiment, SCN tissue was collect from mice at 3 time points (ZT 6, 16, 22) with protein extracts from each time point pooled from 3 mice. Values were analyzed with ANOVA followed by Tukey test for pairwise comparisons. Asterick indicates significance at P < 0.05 compared to sample at ZT 6.

Fig. 5

Ifenprodil inhibits the magnitude of NMDA-evoked currents in SCN neurons. Whole-cell patch clamp recording methods were used to measure NMDA currents in ventral SCN during the night in a zero Mg²⁺ solution. A) The current-voltage relationship of SCN neuron under baseline, NMDA (25μM), and NMDA plus ifenprodil (3μM). In this example, the drugs were applied in the bath and the neuron moved to a series of voltage steps. B) The inhibitory effects of ifenprodil (3μM) on the inward current generated in response to the focal application of NMDA (100μM). The neuron was held at -70mV during this recording.