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.

The 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 Ca²⁺, 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 deficient in components of the core feedback loop (8, 9). Finally, in Bmal1-null SCN, devoid of transcriptional activation of the core loop, intercellular coupling of an unidentified nature is sufficient to drive stochastic transcriptional/translational rhythms with quasi-circadian periods (10).

Coherent pacemaking in the SCN is reliant, therefore, upon mutually dependent intra- and intercellular processes. Isolated SCN neurons are less reliable oscillators (11) and “neuronal network properties are integral to normal function of the SCN” (ref. 9, p. 551). Identifying how circadian information is communicated across SCN circuits is therefore an important goal for circadian neurobiology. Various mechanisms, including gap junctions, synaptic signaling, and GABA-ergic and neuropeptidergic cues (reviewed in ref. 5) have been proposed as synchronizers, although these factors may act over different temporal and spatial frameworks. For example, the SCN is divided into core and shell subregions (12), and both peptidergic and GABA-ergic cues have been implicated in core-shell signaling. Analysis of circadian communication between SCN neurons is thus best studied in an organotypic slice preparation where local and regional circuitry is preserved. We therefore developed a unique coculture procedure in which a circadian deficient “host” SCN carrying a bioluminescent reporter (PER2::LUC) receives a second nonreporter “graft” SCN. By varying the genetic condition of host and graft, in combination with pharmacological manipulations, this preparation provides a unique opportunity to examine how both presynaptic (graft) and postsynaptic (host) mechanisms coordinate SCN cellular and circuit pacemaking.

Results

Paracrine Signaling Restores Circadian Pacemaking to VIP-Deficient SCN.

Wild-type (WT), PER2::LUC SCN slices exhibited stable and precise bioluminescent rhythms for >20 d without medium change (Fig. S1A). Although utilization of the luciferin substrate caused a progressive decline in peak amplitude, the precision of the oscillation remained high, as evidenced by the low relative amplitude error (RAE) in Brass analysis (days 1–10, RAE = 0.05 ± 0.01; days 11–20, RAE = 0.02 ± 0.002; n = 4, mean ± SEM). In contrast, the bioluminescence rhythm of Vip^−/− SCN slices damped out after ∼10 d (Fig. 1A) (RAE = 0.14 ± 0.01, n = 27). Placement of a freshly prepared, WT nonreporter graft SCN onto the VIP-null host immediately (<2 d) and reliably (26/27) restored and maintained high-amplitude, persistent (≥10 d) bioluminescence rhythms in the mutant SCNs (Fig. 1 A and B). The restored cycles were significantly (P < 0.01) more precise than before grafting (RAE postgraft = 0.04 ± 0.01, n = 26). Neonatal and adult SCN slices were equally effective in their ability to restore rhythms in the VIP-null SCN (Fig. S1B). In contrast, cerebral cortex or hypothalamic paraventricular nucleus (PVN) tissues did not restore coherent rhythmic expression to the VIP-null host, indicating a specific action of SCN-derived factors (Fig. 1B and Fig. S1C).

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.

Restoration of circadian gene expression does not, of itself, prove that the observed rhythm is dependent upon rhythmic cues from the graft. The graft may simply provide a limiting factor that, once replenished, activates the intrinsic pacemaker of the host. To exclude this, VIP-null hosts received grafts from either short period (20 h, CSK1e^Tau/Tau) or long period (28 h, Fbxl3^Afh/Afh) Vip^+/+ nonreporter mice. Both types of mutant SCN restored bioluminescence rhythms to their hosts. Importantly, the period of the restored rhythms was determined by the genotype-specific period of the grafts (Fig. 1C). Thus, Tau mutant grafts drove coherent short period rhythms whereas the Afh mutants restored coherent long period rhythms (RAE mean + SEM, WT graft before = 0.13 + 0.02, after = 0.03 + 0.01; Tau graft before = 0.15 + 0.03, after = 0.06 + 0.01; Afh graft before = 0.19 + 0.04, after = 0.05 + 0.01). Genotypically specific restoration of period confirms that rhythmic signals emanating from the graft confer specific circadian information to the host SCN.

In WT slices, CCD imaging of individual neurons showed that bioluminescence rhythms were highly synchronized, as evidenced by the mean vector of Rayleigh plots (0.96 ± 0.01, n = 3) (Fig. 1D). In contrast, damping of the rhythm in VIP-null host SCN arose from both a decline in the amplitude of peak bioluminescence in cells and a progressive loss of synchrony between cells, evidenced as a reduction in the Rayleigh mean vector (Fig. 1E and Movie S1). Following addition of the graft, cellular PER2::LUC expression levels were increased and the oscillations of individual neurons were rapidly (within two cycles) brought back into synchrony (Movie S2), such that synchrony among grafted VIP-null SCN was comparable to that of WT slices (mean vector pregraft, 0.40 ± 0.20; 3 d postgraft, 0.99 ± 0.01, n = 3) (Fig. 1F).

The rapid (<6 h) activation of bioluminescence and neuronal resynchronization (evident by 24 h and complete within 48 h) occurred well before precise synaptic communication could be established between graft and host. The spatial orientation of the graft and host did not compromise effective communication of circadian time. Placing the mutant SCN above the wild-type SCN (Fig. S2 A and B) or rotating the dorso-ventral alignment between graft and host did not affect the restoration of rhythmicity. SCN grafts deliberately aligned to the host (i.e., ventral to ventral) or 180° out of alignment generated rhythms with RAEs of 0.03 ± 0.01 and 0.04 ± 0.01, respectively (RAEs of hosts before grafting: 0.11 ± 0.02 and 0.11 ± 0.01, respectively, n = 6 and 6). There was, therefore, no evident requirement for point-to-point communication for restoration of host rhythms, and a graft can readily drive a target ∼500 μm, possibly 1 mm, distant. To understand better the neural basis of circadian communication, seven cocultures in which circadian bioluminescence rhythms had been restored for >10 d were reconstructed by confocal microscopic imaging. This process confirmed the absence of VIP-immunoreactive (−ir) cell bodies from the host SCN, which was identified by arginine vasopressin (AVP)-ir cells in spatial register with the bioluminescence signal (Fig. 2A and Fig. S3 A and B). VIP-ir and AVP-ir cell bodies identified the graft SCN, and VIP-ir varicosities could be seen sporadically across host tissue.

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.

The restorative effects of the grafts were rapid and independent of anatomical position, indicating that graft-derived VIP may act in a paracrine fashion before synaptic communication could be established. To demonstrate definitively a nonsynaptic, paracrine effect, a semipermeable molecular weight cutoff (MWCO) membrane was placed onto the Vip^−/− host immediately before coculture. This action did not affect measurement of bioluminescence emission (Fig. S3C). The molecular mass of VIP is 3.3 kDa and with a 10-kDa cutoff membrane between SCN, the graft rapidly reinstated circadian bioluminescence rhythms in the host (Fig. 2B). When the graft and membrane were inverted after 4 d, to bring the slices into immediate contact, the amplitude and coherence of the bioluminescence rhythm were increased further [RAE (mean ± SEM) before = 0.12 ± 0.03, after = 0.05 ± 0.02, n = 3]. Thus, paracrine signals were effective in restoring circadian function to the VIP-null SCN. When VIP-null host and WT graft were separated by a 2-kDa MWCO for 3 d, the graft failed to reinstate rhythms in the host (RAE before = 0.17 ± 0.03; after, no detectable rhythm; n = 3). When the graft and membrane were inverted to allow unimpeded graft-to-host communication, the host rapidly resumed coherent rhythmicity (<2 d, RAE = 0.06 ± 0.02) (Fig. 2B). This result confirms that a nonsynaptic, paracrine factor(s) between ∼2 and 10 kDa, most likely VIP, is responsible for rapid (<2 d) activation of the VIP-deficient SCN. To confirm the essential role of VIP, VIP-null hosts received VIP-null SCN grafts. In the absence of VIP from the graft, circadian bioluminescence rhythms were not restored in the host SCN (Fig. S4 A and B).

Restoration of Circadian Pacemaking in VIP Receptor-Deficient SCN.

To test whether signaling pathways other than VIP can confer circadian organization to SCN circuits, the coculture technique was applied to PER2::LUC slices lacking the VPAC2 receptor for VIP (Vip2r^−/−). As with VIP-null slices, the aggregate bioluminescence rhythm of VPAC2-null SCN damped rapidly (Fig. 3 A and B and Movie S3) due to a decline in cellular emission and phase dispersal of the individual cellular oscillators (Fig. 3C) (RAE = 0.19 ± 0.02, n = 35). The initial response to a WT graft was less pronounced than in VIP-null slices, and it took several days for a bioluminescence rhythm to develop, reaching peak amplitude after ∼7 d (Fig. 3B, Fig. S5A, and Movie S4) (RAE = 0.05 ± 0.01, n = 30/35 grafts measurable). The resynchronization of neurons within the host SCN was evident in the Rayleigh mean vector (Fig. 3C and Fig. S5B).

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.

The response of VPAC2-null slices differed from that of VIP-null in another important way. Whereas Vip^−/− SCN could express rhythms over a wide range of periods when driven by circadian mutant grafts, VPAC2-null SCN were unable to respond effectively to mutant SCN (Tau n = 5/7, Afh 4/5). Hence, when rhythms were reestablished, the period of the restored rhythm was essentially WT and not significantly different (ANOVA: F = 2.6, not significant) between the grafts of different genotypes (Fig. S5C); i.e., the period of the host did not reflect the genotype of the graft. Moreover, where the rhythms were detected in Vip2r^−/− SCN with circadian mutant grafts, they were highly variable in their interpeak period and their quality (RAE) was low. Whereas both Tau and Afh mutant SCN significantly reduced the RAE error of VIP-null slices, this was not the case for VPAC2-null slices (Fig. S5 D and E). Only WT grafts were able to reduce RAE measures in VPAC2-null slices. Together, the data with VPAC2-null hosts support several conclusions. First, factors other than VIP/VPAC2 signaling can reliably and sustainably synchronize SCN cellular pacemakers. Second, such factors are less effective as mediators between circuits that do not share a common intrinsic period. Third, interneuronal signaling via VIP/VPAC2 appears to make a preeminent contribution to circadian coordination in the SCN and may have a particular role in setting pacemaker period.

A Hierarchy of Factors Drives SCN Paracrine Circadian Coordination.

Having established that paracrine factors other than VIP can restore bioluminescence rhythms, we tested specifically the roles of two other SCN neuropeptides, gastrin-releasing peptide (GRP) and AVP. GRP receptors (BB2r) are expressed in the dorsal SCN but, compared with vehicle-treated slices, administration of the BB2r antagonist PD176252 (5 μM; n = 3) had no effect on the rate of damping or the amplitude of rhythmicity over 10 d (Fig. S6). BB2r signaling is not, therefore, essential to the WT SCN clockwork. Equally, administration of 5 μM PD176252 to Vip^−/− SCN slices at the time of grafting with WT SCN had no effect on the ability of the graft to drive rhythms in the host (Fig. 4A). In contrast, concomitant administration of the BB2r antagonist at the time of grafting onto Vip2r^−/− SCN slices significantly suppressed the induced rhythms (Fig. 4 C and E). Although the graft appeared to be initially effective, after about three cycles the amplitude of the restored bioluminescence rhythms decreased in comparison with vehicle-treated, grafted VPAC2-null slices. Thus, in the absence of VIP-mediated signaling, GRP/BB2r becomes a limiting component of the paracrine signals driving the SCN pacemaker.

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).

Both AVP1a and -1b receptors are expressed in the SCN, and so a combination of AVP1a (SR49059; 10 nM) and AVP1b receptor antagonists (SSR149415; 10 nM) was applied to WT slices (n = 3). This action had no effect on the rate of damping or the amplitude of rhythms compared with vehicle (Fig. S6). When Vip^−/− SCN received both AVP receptor antagonists at the time of grafting, the graft initially restored bioluminescence levels but the amplitude of the restored cycles decreased over time (Fig. 4B). In the absence of VIP, therefore, AVP-mediated signaling became an essential feature of the circuit. The contribution of AVP-mediated signals was further emphasized in Vip2r^−/− SCN slices, where treatment with the AVP antagonists completely prevented the restoration of bioluminescence rhythms (Fig. 4 D and F). Thus, synchronized cellular pacemaking in the SCN is primarily dependent upon VIP-ergic signaling. Nevertheless, in its absence, cues delivered by AVP and to a lesser degree GRP can contribute.

Cellular Pacemaking in Cry1/Cry2-Deficient SCN Slices and Its Coordination by Paracrine Signaling.

Thus, paracrine signaling can drive circadian pacemaking in SCN circuits defective in VIP signaling. In such slices, however, the apparatus of the core cellular feedback loops remains intact. It has been suggested that interneuronal, circuit-level signaling can defend the SCN clockwork from perturbation of the core feedback loop arising from deletion of Cry1 or Per1 genes (9). To test whether paracrine signals are sufficiently powerful to sustain molecular pacemaking in the absence of multiple core feedback elements, Cry1^−/− and Cry2^−/− double-null slices were used as hosts. At the behavioral level, Cry-null, Per2^Luc mice became arrhythmic immediately on transfer to continuous dim red light (Fig. S7A). Consistent with this result, a number of Cry-null SCN slices recorded by photomultiplier (PMT) failed to exhibit circadian bioluminescence rhythms in culture (Fig. S7B). Remarkably, and contrary to expectations however, the majority (23/39, 59%) of Cry-null slices continued to oscillate for at least four cycles and some continued for up to eight cycles (Fig. 5A). The period of these rhythmic Cry-null slices (19.5 ± 0.9 h, n = 23) was significantly (P < 0.01) shorter than that of WT and single mutant Cry1- and Cry2-null SCN (24.2 ± 0.1 h, 22.58 ± 0.09 h, and 26.16 ± 0.12 h, respectively, n = 5–8). The rhythms were also less well defined, with a higher relative amplitude error (RAE: WT, 0.04 ± 0.01; Cry double-null, 0.20 ± 0.02) and lower overall amplitude. CCD imaging revealed that individual cells in nonrhythmic, Cry-null SCN slices continued to exhibit bioluminescence rhythms (Fig. 5B and Movie S5). The frequency of rhythmic cells was lower and more variable than in WT slices (100% vs. 67.6 ± 18.1%, 50 cells analyzed in three and seven SCN, respectively) and their rhythms were of lower amplitude (1,211 ± 276 vs. 175 ± 51) and higher RAE (0.08 ± 0.01 vs. 0.47 ± 0.08). Nevertheless, these data show that in the absence of Cry-mediated transcriptional feedback, SCN neurons retain some competence as circadian pacemakers, consistent with the view that interneuronal and cytosolic events outside the core feedback loops contribute to pacemaking.

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.

Once Cry-null SCN had become arrhythmic, as monitored by PMT for >10 d, they received a WT SCN. The immediate restoration of rhythmicity seen in grafted VIP-null slices was not seen, however. Instead, for 2–3 d there was a progressive increase in bioluminescence from the host, and only then were spontaneous cycles of bioluminescence initiated (Fig. 5 C and D). These rhythms attained their peak amplitude significantly (P < 0.01) later than did VIP-null SCN (Fig. S5A). Nevertheless, once established, circadian rhythms in the Cry-null SCN were sustained for the remainder of the recording (>10 d). As with the VIP-null slices, CCD imaging showed that restored pacemaking arose from changes across the population of individual neurons (Fig. 5D): Not only did the level of bioluminescence increase, but also the cellular rhythms became better defined (RAE pregraft = 0.47 ± 0.08, postgraft = 0.20 ± 0.01) and the cells progressively became synchronized (Fig. 5D and Movie S6), as evidenced by an increase in the mean vector (Fig. S7C). Thus, even though the effect takes longer to become apparent, paracrine signals delivered from the graft were sufficiently powerful to sustain rhythmic gene expression in the absence of essential elements of the core transcriptional feedback loop.

Discussion

Coordination of behavioral and physiological rhythms depends upon synchronization of the ∼10,000 neurons within the SCN circuit (5). By developing a unique coculture technique, combined with genetic and pharmacological manipulations, we have shown how a diversity of neuropeptidergic, paracrine cues sustains amplitude, period, and synchrony of cellular circadian pacemaking in the intact SCN. Preeminent is the VIP/VPAC2 signaling cascade, but in its absence AVP- and GRP-mediated signals can be effective, although the efficiency of these non-VPAC2 mechanisms, in terms of rate of restoration and control over circadian period, is less. These paracrine cues are sufficiently potent to overcome genetic deficiencies within the core transcriptional feedback loop of Cry-null SCN. These findings provide a unique perspective on the relative contributions of intra- and intercellular, circuit-level mechanisms in coordinating the circadian pacemaker.

Previous interest in circadian paracrine/humoral signaling has focused on SCN outputs, rather than how SCN neurons might communicate with each other. For example, SCN grafts can restore genotypically specific in vivo rest activity and (some) physiological cycles in SCN-ablated adult hosts (13–15). Moreover, SCN grafts encapsulated in semipermeable membrane to prevent neuronal outgrowth restore behavioral, but not other, circadian rhythms (16), whereas in vitro, SCN cells (17) and slices (18) can restore gene oscillations in fibroblasts and rhythmic electrical activity in the PVN (19). As for intra-SCN communication, unknown paracrine signals have been inferred in early fetal development, when synaptogenesis is incomplete (20). The MWCO barriers demonstrate that paracrine signals are potent stimuli within the adult SCN. In particular, the ∼2-kDa membrane, which would exclude access to the host of graft-derived VIP and GRP1-27, blocked circadian cues: consistent with the genetic and pharmacological evidence of the circadian roles of these peptides. The current study, therefore, uniquely reveals the potency of diverse paracrine signals within and between intact SCN circuits. Moreover, it shows explicitly that this potency is a property of cues derived from the graft SCN and that such cues can impose long-term circadian function over genetically deficient SCN circuits. Indeed, the host SCN behaves much like a driven oscillator. Of interest, therefore, is the degree to which particular factors (AVP, VIP, and GRP) and second messenger pathways (cAMP and Ca²⁺) contribute to signaling both within the SCN and between the SCN and its targets. It should be noted, however, that both VIP and AVP are expressed in tissues (cerebral cortex and PVN) that were unable to restore circadian cycles in mutant host SCN. Thus, the SCN circuitry as well as its neurochemistry confers specific pacemaking properties.

VIP/VPAC2 signals have a primary role in communicating circadian information across the SCN circuit, conferring period, amplitude, and synchronization. These properties are sensitive to signaling by cAMP (21), the biochemical mediator of VIP/VPAC2 in the SCN (22). The downstream consequences of VPAC2/cAMP activation in SCN neurons will ultimately involve induction of Per genes via their cAMP/calcium-response elements (23). Indeed, acute treatment of VIP-null SCN with exogenous VIP rapidly induces Per-driven bioluminescence. This activation may involve both direct (cAMP/PKA) and indirect biochemical pathways as well as altered patterns of electric firing activity (24). Remarkably, these network properties of VIP are echoed in Drosophila, where the neuropeptide pigment-dispersing factor (PDF) synchronizes fly clock neurons via the fly homolog of the VPAC2 receptor (25). Thus, VIP/PDF/VPAC2 signaling, and its associated regulation of cAMP (21, 25), may be a conserved mechanism in circadian circuits.

Pharmacological blockade of GRP receptors had no effect on WT SCN slices, consistent with the normal behavioral and SCN gene expression rhythms in BB2r-null mice (26). Similarly, although loss of the AVP V1a receptor may disrupt behavioral rhythms to a variable extent (27), Per gene expression in the SCN is unaffected in AVP V1a-null mice and blockade of AVP-Gq/11-coupled signaling with specific antagonists to both the V1a and V1b subtypes did not affect bioluminescence rhythms in WT SCN. Critically, however, V1a, V1b, and BB2r blockade revealed how both peptidergic pathways are necessary in SCN lacking VIP/VPAC2. This VIP/VPAC2-independent signaling took longer to take effect, however, and failed to drive host SCN at atypical circadian periods. Moreover, there were differences between AVP- and GRP-mediated effects. In VIP-null SCN, blockade of V1a/V1b prevented the long-term restoration of rhythms whereas blockade of BB2r receptors had no effect (perhaps due to competent AVP signals). Equally, in VPAC2-null SCN hosts, V1a/V1b blockade was immediately effective, whereas BB2r blockade took several days to become effective, indicating a more potent role for AVP over GRP. Thus, within the intact SCN circuit, distinct neurochemical pathways may convey specific circadian properties. What is being communicated (amplitude, period, and phase) may vary between intercellular contacts and neurochemical pathways, with a VIP > AVP > GRP hierarchy of effect. In addition to this peptidergic system, GABA appears to modulate peak firing rate and precision of the oscillations whereas additional factors signaling via PTX-sensitive G proteins may contribute to synchronization (28).

Of particular interest is the interdependence between the two principal subregions, the AVP-rich shell and the VIP- and GRP-rich, retinorecipient core. Their surgical separation desynchronizes the dorsomedial shell, presumably due to loss of VIP- and GRP-ergic signals (29), and a simple model is that core and shell contain reciprocally supportive oscillators coupled by peptidergic signals. Thus, the shell receives a dense innervation by VIP and GRP (30) and expresses VPAC2 (31) and BB2r (26) whereas the V1a receptor is widely distributed across SCN (27). This model complements the conclusion that there is no uniquely specialized or anatomically localized class of cell-autonomous pacemakers (11): rather, AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling. It has been argued that intrinsic instability is an adaptive property of cells such that it renders them more responsive to extracellular cues. In the context of synchronization, this result has two aspects: the synchronization of the circuit's spontaneous oscillation and the resynchronization associated with entrainment by environmental cues, e.g., light. The present study reveals a role for VIP, AVP, and GRP in the former, and VIP and GRP also mediate resetting by light. The underlying cellular processes may therefore be common regardless of the context in which synchronization occurs.

An unexpected finding was that cells in Cry-null SCN retain oscillatory properties and that ensemble circadian gene expression persisted for several cycles in the majority of SCN. This result contrasts with the immediate loss of circadian behavior in Cry-null mice placed into continuous darkness and the absence of spontaneous circadian rhythms of ensemble electrical activity in acute SCN slices (32). Thus, the Cry-null SCN may continue to oscillate in vivo but may be deficient in peptidergic factors, e.g., prokineticin (33) necessary to coordinate behavior and/or the electrical patterns required to release them at postsynaptic targets. Thus, although Cry1 and Cry2 have been viewed as essential components of the negative limb of the circadian clock feedback loop (34), our results show that oscillatory properties exist in SCN neurons in the absence of Cry. A parallel to this was recently reported from SCN slices lacking the positive circadian regulator Bmal1 (10), which exhibited long-term stochastic oscillations in the circadian domain, dependent on intercellular communication and cAMP signaling. Cry proteins have recently been identified as negative regulators of cAMP signaling (35) and so an up-regulation of cAMP-mediated cues might be anticipated in Cry-null SCN cells and possibly adequate to sustain Per gene expression. The results on Bmal1- and Cry-null SCN are consistent with the view that oscillatory mechanisms involving cytoplasmic signaling and membrane electrical activity are important components of the cellular pacemaker (4, 36). This effect may also underlie the (in vivo) restoration of behavioral rhythms in Per2 mutant mice exposed to continuous bright light that would activate both glutamatergic and peptidergic signaling within the SCN (37). In conclusion, intercellular signals emitted by WT graft SCN were able to restore competent circadian gene expression rhythms in Cry-null SCN. Therefore, loss of intracellular factors (Cry) or intercellular factors (VIP) can confound the SCN circuit, but suitable restoration of intercellular signals can overcome these deficits.

Materials and Methods

Experiments were conducted under the 1986 Home Office Animals (Scientific Procedures) Act (United Kingdom). PER2::LUC, Vip^−/−, Vipr2^−/−, Cry1^−/−:Cry2^−/− CSK1e^Tau/Tau, and Fbxl3^Afh/Afh mice were generated by Joe Takahashi (University of Texas Southwestern Medical Center, Dallas), Chris Colwell (University of California, Los Angeles), Tony Harmar (University of Edinburgh, Edinburgh), G. van der Horst (Erasmus University, Rotterdam), Andrew Loudon (University of Manchester, Manchester, UK), and Pat Nolan (Medical Research Council, Harwell, UK) and subsequently bred at the Medical Research Council, Cambridge, UK, on C57Bl6 backgrounds. Mice were housed under 12 h:12 h light:dim red light (LD) with ad libitum food and water.

Brains were removed from adults or pups of at least 10 d of age and sectioned as reported previously (7). Bioluminescent emissions from PER2::LUC SCN slices were recorded by PMTs and CCD cameras (Hamamatsu) as described previously (7). After at least 10 d of recording from the host SCN, a second, freshly prepared nonreporter organotypic SCN slice (graft) from either WT or circadian period mutant mice (10–14 d old) was placed directly on top of the host PER2::LUC SCN. Rhythms in bioluminescence were recorded for a further 10 d. To assess the specificity of the SCN graft, some PER2::LUC, Vip^−/− slices received a graft of either hypothalamic PVN or cerebral neocortex from nonreporter mice. Waveforms of rhythmic bioluminescence emission from whole slices and individual cells were analyzed in BRASS software (A. Millar, University of Edinburgh, Edinburgh, and M. Straume, University of Virginia, Charlottesville, VA).

Following recording, slices were fixed in 4% paraformaldehyde for 30 min at room temperature, washed in 0.1 M PBS, and then incubated for 1 h in 20% donkey serum in 0.01 M PBS containing 0.3% BSA and 0.1% Triton. They were then incubated overnight at room temperature with primary antisera against AVP (rabbit, 1:50; Abcam), VIP (guinea-pig, 1:5,000; Peninsula Laboratories), and GFAP (chicken, 1:5,000; Abcam); washed; and then incubated for 2 h in secondary antibody (AVP, anti-rabbit Alexa Fluor 488; VIP, anti-guinea pig Alexa Fluor 647; GFAP, anti-chicken Alexa Fluor 568). Slices were washed, mounted on slides using Vectashield-DAPI mounting medium, and imaged with a Bio-Rad Radiance 2100 confocal microscope. BB2r antagonist PD176252 and AVP1a antagonist SR49059 were from Tocris, and AVP1b antagonist SAR149415 was a gift from C. Serradeil Le-Gal, Sanofi, Toulouse, France. Data were compared by two- and one-way ANOVA in GraphPad Prism with a post hoc Bonnferroni t test or Student's t test where applicable.