Gastrin-releasing peptide mediates light-like resetting of the suprachiasmatic nucleus circadian pacemaker through cAMP response element-binding protein and Per1 activation

Abstract

Circadian rhythmicity in the primary mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus, is maintained by transcriptional and translational feedback loops among circadian clock genes. Photic resetting of the SCN pacemaker involves induction of the clock genes Period1 (Per1) and Period2 (Per2) and communication among distinct cell populations. Gastrin-releasing peptide (GRP) is localized to the SCN ventral retinorecipient zone, from where it may communicate photic resetting signals within the SCN network. Here, we tested the putative role of GRP as an intra-SCN light signal at the behavioral and cellular levels, and we also tested whether GRP actions are dependent on activation of the cAMP response element-binding protein (CREB) pathway and Per1. In vivo microinjections of GRP to the SCN regions of Per1::green fluorescent protein (GFP) mice during the late night induced Per1::GFP throughout the SCN, including a limited population of arginine vasopressin-immunoreactive (AVP-IR) neurons. Blocking spike-mediated communication with tetrodotoxin did not disrupt overall Per1::GFP induction but did reduce induction within AVP-IR neurons. In vitro GRP application resulted in persistent increases in the spike frequency of Per1::GFP-induced neurons. Blocking endogenous Per1 with antisense oligodeoxynucleotides inhibited GRP-induced increases in spike frequency. Furthermore, inhibition of CREB-mediated gene activation with decoy oligonucleotides blocked GRP-induced phase shifts of PER2::luciferase rhythms in SCN slices. Altogether, these results indicate that GRP communicates phase resetting signals within the SCN network via both spike-dependent and spike-independent mechanisms, and that activation of the CREB pathway and Per1 are key steps in mediating downstream events in GRP resetting of SCN neurons.

Figure 1.

Representative actograms of the wheel-running activity of mice injected with vehicle (A; Veh) or GRP (B) at ZT 21, as indicated by the asterisks. Black tick marks represent the number of wheel revolutions, and the shaded areas indicate lights off. The data are double plotted so that each line represents 48 h. The phase shift (ΔΦ) for each animal is indicated under each actogram and was determined from the difference between the average activity onset for the days before injection and a regression line calculated from the daily activity onsets for days 3–9 after the injection. The activity onsets for the days included in the analysis are indicated by the gray-shaded lines.

Figure 2.

Representative confocal images of Per1::GFP mice injected with vehicle or GRP (top) and GRP plus TTX or TTX (bottom) into the SCN region at ZT 21. Mice were perfused at ZT 24. Alternating sections (40 μm thick) were double labeled for VIP–GFP (left column of each treatment group) or AVP–GFP (right column of each treatment group). Top row, Single-labeled images for AVP immunoreactivity or VIP immunoreactivity (red); middle row, single-labeled images for GFP immunoreactivity (green); bottom row, double-labeled composite of AVP–GFP or VIP–GFP (yellow cells are double labeled).

Figure 3.

Microinjection of GRP into the mouse SCN region at ZT 21 induces Per1::GFP expression. Mean number of GFP-IR cells per animal (A), bright GFP-IR cells per animal (B), and percentage AVP–GFP double-labeled cells (as a percentage of AVP-IR cells) per animal (C) after microinjection of vehicle, GRP, a mixture of GRP plus TTX, or TTX; *p < 0.05.

Figure 4.

Per1 gene activity is necessary for GRP-induced increases in neuronal activity of Per1-expressing neurons. A, Examples of individual, extracellular recordings from slices that were treated with either vehicle (top trace) or GRP (bottom trace) at projected ZT 21. Calibration: 40 pA, 1.0 s. B, Bar graph indicating the mean spontaneous action potential (A.P.) frequency of Per1 fluorescent neurons after treatment with either vehicle or GRP at projected ZT 21. *p < 0.05. C, Examples of individual extracellular recordings from slices that were treated with either random ODN followed by GRP (top trace) or mPer1 antisense ODN followed by GRP (bottom trace) at projected ZT 21. Calibration: 40 pA, 1.0 s. D, Bar graph indicating the mean spontaneous action potential frequency of Per1 fluorescent neurons after treatment with mPer1 antisense ODN followed by GRP (AntiPer1 + GRP), random ODN followed by GRP (RandPer1 + GRP), or mPer1 antisense ODN followed by vehicle (AntiPer1 + Veh). Brain slices were prepared during the day (ZT 1–ZT 4), cultured overnight, and treated with ODN at projected ZT 20 (C, D) and GRP or vehicle at projected ZT 21. Slices were transferred to the recording chamber at projected ZT 22, and recordings were made from projected ZT 0–ZT 1. *p < 0.05, significantly different from the other two groups.

Figure 5.

A, B, Bioluminescence recorded from SCN explants cultured from PER2::LUC mice after treatment with CRE-mis (A) or CRE-decoy (B) at CT 15–CT 16. Baseline-subtracted traces (see Materials and Methods) within each graph are from the same culture treated with GRP (black) or culture medium (med; red). The luminescence amplitude (y-axis) was normalized to the maximum luminescence for that trace. The x-axis represents time in cycles (where 1 cycle = 24/free-running period). The two traces in each graph were aligned to the peak just before treatment (defined as time 0) to allow phase-shift comparison. The solid lines indicate the fitted sine wave for the luminescence data points from cultures treated with GRP (black) or media (red). For the CRE-mis culture (A), the phase difference between GRP and medium is 0.098 cycles, which equals 2.34 h. For the CRE-decoy culture (B), the phase difference between GRP and medium is 0.014 cycles, which equals 0.34 h. C, Line graph of average ± SEM phase delays: filled circles represent cultures that received CRE-mis, and open circles represent cultures that received CRE-decoy. Each culture was treated with GRP or medium for 1 h at CT 15–CT 16 (in which CT 12 is defined as peak PER2::LUC luminescence). Every culture received both conditions (GRP and medium in a counterbalanced order), which were separated by two rinses and a medium change. D, E, Enlarged bioluminescence traces of the first cycle after GRP (black) or culture medium (red) treatment at CT 15–CT 16 for the CRE-mis-treated (D) and a CRE-decoy-treated (E) cultures in A and B above.

Figure 6.

Model of GRP mediation of photic phase resetting in the SCN. During photic phase resetting, we propose that GRP cells and GRP are involved in the following ways. “Transduction of the photic signal in the SCN,” Ventral SCN cells in the night phase expressing GRP and/or VIP (top cell) show a persistent increase in the frequency of spontaneous action potentials (far right, top), as well as the induction of Per1 in the presence of light (represented by the light bulb) (Kuhlman et al., 2003). “Communication of the intra-SCN photic signal,” GRP is released from ventral cells and activates BB₂ receptors (BB2R) on cells concentrated in the dorsal and medial SCN regions (Karatsoreos et al., 2006). One should note that other neurotransmitters and peptides such as VIP may also be released and involved in this process. “Initiation of SCN phase resetting,” MAPK, ERK, and CREB intracellular pathways are activated (Butcher et al., 2002; Antle et al., 2005; this study), resulting in activation of Per1 that is necessary for a persistent increase in the frequency of spontaneous action potentials to be induced (this study). “Phase shift of SCN rhythms and behavior,” Phase advance of the electrical activity rhythm, behavior, and gene expression.