A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits

Abstract

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of mammals, coordinating daily rhythms of behavior and metabolism. Circadian timekeeping in SCN neurons revolves around transcriptional/posttranslational feedback loops, in which Period (Per) and Cryptochrome (Cry) genes are negatively regulated by their protein products. Recent studies have revealed, however, that these "core loops" also rely upon cytosolic and circuit-level properties for sustained oscillation. To characterize interneuronal signals responsible for robust pacemaking in SCN cells and circuits, we have developed a unique coculture technique using wild-type (WT) "graft" SCN to drive pacemaking (reported by PER2::LUCIFERASE bioluminescence) in "host" SCN deficient either in elements of neuropeptidergic signaling or in elements of the core feedback loop. We demonstrate that paracrine signaling is sufficient to restore cellular synchrony and amplitude of pacemaking in SCN circuits lacking vasoactive intestinal peptide (VIP). By using grafts with mutant circadian periods we show that pacemaking in the host SCN is specified by the genotype of the graft, confirming graft-derived factors as determinants of the host rhythm. By combining pharmacological with genetic manipulations, we show that a hierarchy of neuropeptidergic signals underpins this paracrine regulation, with a preeminent role for VIP augmented by contributions from arginine vasopressin (AVP) and gastrin-releasing peptide (GRP). Finally, we show that interneuronal signaling is sufficiently powerful to maintain circadian pacemaking in arrhythmic Cry-null SCN, deficient in essential elements of the transcriptional negative feedback loops. Thus, a hierarchy of paracrine neuropeptidergic signals determines cell- and circuit-level circadian pacemaking in the SCN.

Fig. 1.

Restoration of SCN circadian gene expression by coculture. (A) Representative bioluminescence rhythm from VIP-null SCN grafted with nonreporter WT SCN after 10 d. Note damping followed by immediate restoration of rhythms by graft. (B) Relative amplitude (percentage of initial peak value) of successive peaks of circadian bioluminescence of VIP-null SCN recorded for 10 d and then given grafts of SCN, PVN, or cerebral cortex (plotted as mean ± SEM, n ≥ 5). (C) Period of bioluminescence rhythm of VIP-null SCN (mean ± SEM) grafted with WT, Tau, or Afh mutant SCN (n ≥ 4). **P = 0.01 vs. WT treatment (ANOVA, F = 163.8). (D) Representative raster plot of circadian gene expression in 50 cells from WT SCN with accompanying Raleigh plot. (E and F) Representative raster plot from VIP-null SCN before (E) and after (F) grafting with WT SCN. Progressive loss of synchrony and its restoration are reflected in Rayleigh plots on days −9, −1, and +3 relative to grafting.

Fig. 2.

Paracrine signaling of circadian cues between SCN. (A) Confocal reconstruction of representative VIP-null SCN with WT graft. (Left) Low power (10×) phase and bioluminescence images identifying boxed areas examined at higher power. (Scale bar, 1 mm.) (Right) Confocal images (60×) for AVP- and VIP-ir in graft and host tissues. (Scale bar, 50 μm.) (B) Representative bioluminescence rhythm from VIP-null SCN grafted after 10 d with WT SCN separated by 2-kDa (red) or 10-kDa (blue) cutoff membranes. After 3 d the grafts were placed directly on the host.

Fig. 3.

Restoration of SCN circadian gene expression by signals other than VIP. (A) Representative bioluminescence emission from VPAC2-null SCN grafted with nonreporter WT SCN after 10 d. Note damping of rhythm before grafting, followed by progressive restoration of rhythm. (B) Relative amplitude (percentage of initial peak value) of successive peaks of circadian bioluminescence of WT SCN (green, n = 4) recorded for 20 d (mean ± SEM) or of VPAC2-null (red, n = 6) or VIP-null (blue, n = 6) SCN recorded for 10 d and then given grafts of WT SCN. (C) Representative raster plot of circadian bioluminescence in 50 cells from VPAC2-null SCN with accompanying Raleigh plots from days −9, −1, +3, and +6 relative to grafting. Note progressive loss of synchrony and its restoration.

Fig. 4.

Absence of VIP/VPAC2 signaling reveals dependence of SCN circadian gene expression on AVP- and GRP-mediated signaling. (A) Relative amplitude (percentage of initial peak value) of successive peaks of circadian bioluminescence of VIP-null SCN grafted with WT SCN and treated with vehicle (n = 3) or GRP receptor antagonist (n = 4, mean ± SEM). (B) As in A but with AVP V1a and V1b antagonists (vehicle, n = 4; antagonists, n = 5). (C) Representative bioluminescence recordings of VPAC2-null SCN grafted with WT SCN and treated with vehicle or GRP antagonist. (D) As in C but treatment with vehicle or AVP V1a and V1b receptor antagonists. (E) Relative amplitude (percentage of initial peak value) of successive peaks of circadian bioluminescence of VPAC2-null SCN grafted with WT SCN and treated with vehicle (n = 5) or GRP receptor blocker (n = 6, mean ± SEM). (F) As in E but with vehicle (n = 5) or AVP V1a and V1b receptor antagonists (n = 7).

Fig. 5.

Residual circadian pacemaking in Cry-null SCN slices and cells and its coordination by interneuronal signaling. (A) Representative coherent circadian bioluminescence rhythms of Cry-null SCN. (B) Aggregate bioluminescence emission of representative nonrhythmic Cry-null SCN and individual raster plots from 50 cells. (C) Representative bioluminescence emission from (nonrhythmic) Cry-null (black) and VIP-null (blue) SCN grafted with WT SCN. Note immediate restoration of rhythms in VIP-null and progressive effect in Cry-null SCN. (D) Raster and Rayleigh plots (with accompanying mean vector) of bioluminescence of 50 cells in representative Cry-null SCN that received a WT graft.