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::LUCIFER-ASE 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.T he circadian pacemaker of the suprachiasmatic nucleus (SCN) directs, on a daily basis, the most fundamental processes of physiology and metabolism, sleep and wakefulness (1). The established molecular model of the SCN oscillator involves interlocked transcriptional/posttranslational negative feedback loops, centered on rhythmic expression of the transcriptional inhibitors Period (Per) and Cryptochrome (Cry), driven by the positive activators Clock and Bmal1. The period of the daily oscillation is determined by the transcriptional activity of Clock (1) and by the stability of Per and Cry proteins (2, 3). More recently the model of SCN neurons as autonomous transcriptional/translational feedback oscillators has been reappraised. Rhythmic cytosolic signaling pathways, particularly cAMP and Ca 2+ , not only are driven by the core feedback loops, but also support them in a mutually dependent fashion (4, 5). Furthermore, within SCN circuits, loss of signaling by the neuropeptide vasoactive intestinal peptide (VIP) through its VPAC2 receptor (a positive regulator of cAMP) both desynchronizes and damps cellular transcriptional pacemaking (6, 7). Conversely, intercellular coupling maintains robustness within SCN cells deficie...

Na V 1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms

“…Several studies have identified putative neurotransmitters underlying circadian coupling within the SCN neuronal network. Both GABA and VIP have been proposed as critical signals that mediate coupling of neuronal activity (5,11,50,(57)(58)(59). The partial rescue of the lengthened circadian period and light-induced phase shifts in Scn1a +/− mice by GABAergic transmission-enhancing drugs is consistent with a critical role of SCN GABAergic transmission in period determination and decoding of photic information.…”

Cryptochromes are critical for the development of coherent circadian rhythms in the mouse suprachiasmatic nucleus

“…Recently, robust circadian rhythms of PER2HLUC were demonstrated at the single-cell level and at the tissue level of cultured SCN from neonatal Cry1 À / À /Cry2 À / À mice 9 . The tissue-level PER2::LUC rhythms damped out in several cycles but were restored by coculture with a wild-type (WT) SCN, suggesting that a diffusible factor(s) supports synchrony of the SCN 9 .…”

“…The tissue-level PER2::LUC rhythms damped out in several cycles but were restored by coculture with a wild-type (WT) SCN, suggesting that a diffusible factor(s) supports synchrony of the SCN 9 . We also reported that the spontaneous neuronal firing in cultured SCN of neonatal Cry1 À / À /Cry2 À / À mice is rhythmic 10 .…”

Neuropeptides go the distance for circadian synchrony

“…Previous studies had demonstrated that loss of vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2R (encoded by the Vipr2 gene) abolished synchronized circadian rhythms in the SCN and in locomotor behavior (11,12). The new study (2) shows that wild-type SCN can quickly restore rhythms to SCN lacking VIP. By changing the circadian genotype of the graft SCN, the authors cleverly reveal they can drive the host SCN at either much shorter or longer periods.…”