Emergence of functional neuromuscular junctions in an engineered, multicellular spinal cord-muscle bioactuator.

Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like "feet." We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a "spinobot" by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.

The hypothalamic-neurohypophyseal system: from genome to physiology.

The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22 333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed as a result of genomic studies. This complexity is evident at a number of levels: (i) cis-regulatory sequences; (ii) noncoding and antisense mRNAs, most of which have no known function; (iii) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; and (iv) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamic-neurohypophyseal system (HNS). Five unique stories explain: (i) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; (ii) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (iii) the use of transgenic lines that express fusion transgenes enabling (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, as well as their neuroanatomy, electrophysiology and activation upon exposure to any given stimulus; (iv) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (v) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours.

Different patterns of circadian oscillation in the suprachiasmatic nucleus of hamster, mouse, and rat.

Although spontaneous neural firing in the mammalian suprachiasmatic nucleus is accepted to peak once during mid-subjective day, dual activity peaks have been reported in horizontal brain slices taken from hamsters. These two peaks were interpreted as new evidence for the theory of dual circadian oscillators and raised the expectation that such activity would be found in other circadian model systems. We examined hamster, mouse, and rat slices in both coronal and horizontal planes and found a second peak of activity only in hamster horizontal preparations. This raises interesting questions about the relative circadian physiology of these important experimental animals.

Carbon monoxide and nitric oxide: interacting messengers in muscarinic signaling to the brain's circadian clock.

Within the central nervous system, acetylcholine (ACh) functions as a state-dependent modulator at a range of sites, but its signaling mechanisms are yet unclear. Cholinergic projections from the brain stem and basal forebrain innervate the suprachiasmatic nucleus (SCN), the master circadian clock in mammals, and cholinergic stimuli adjust clock timing. Cholinergic effects on clock state require muscarinic receptor-mediated activation of guanylyl cyclase and cGMP synthesis, although the effect is indirect. Here we evaluate the roles of carbon monoxide (CO) and nitric oxide (NO), major activators of cGMP synthesis. Both heme oxygenase 2 (HO-2) and neuronal nitric oxide synthase (nNOS), enzymes that synthesize CO and NO, respectively, are expressed in rat SCN, with HO-2 localized to the central core of the SCN, whereas nNOS is a punctate plexus. Hemin, an activator of HO-2, but not the NO donor, SNAP, mimicked cholinergic effects on circadian timing. Selective inhibitors of HO fully blocked cholinergic clock resetting, whereas NOS inhibition partially attenuated this effect. Hemoglobin, an extracellular scavenger of both NO and CO, blocked cholinergic stimulation of cGMP synthesis, whereas l-NAME, a specific inhibitor of NOS, had no effect on cholinergic stimulation of cGMP, but decreased the cGMP basal level. We conclude that basal NO production generates cGMP tone that primes the clock for cholinergic signaling, whereas HO/CO transmit muscarinic receptor activation to the cGMP-signaling pathway that modulates clock state. In light of the recently reported inhibitory interaction between HO-2/CO and amyloid-beta, a marker of Alzheimer's disease (AD), we speculate that HO-2/CO signaling may be a defective component of cholinergic neurotransmission in the pathophysiology of AD, whose manifestations include disintegration of circadian timing.

Role of the M1 receptor in regulating circadian rhythms.

Cholinergic stimuli are potent regulators of the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Using a brain slice model, we have found that the SCN clock is subject to muscarinic regulation, a sensitivity expressed only during the night of the clock's 24-h cycle. Pharmacological and signal transduction characteristics are compatible with a response mediated by an M1-like receptor. Molecular manipulation of muscarinic receptors will provide important insights as to the receptor subtype(s) regulating circadian rhythms.

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock.

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.

Differential cAMP gating of glutamatergic signaling regulates long-term state changes in the suprachiasmatic circadian clock.

We investigated a role for cAMP/protein kinase A (PKA) in light/glutamate (GLU)-stimulated state changes of the mammalian circadian clock in the suprachiasmatic nucleus (SCN). Nocturnal GLU treatment elevated [cAMP]; however, agonists of cAMP/PKA did not mimic the effects of light/GLU. Coincident activation of cAMP/PKA enhanced GLU-stimulated state changes in early night but blocked light/GLU-induced state changes in the late night, whereas inhibition of cAMP/PKA reversed these effects. These responses are distinct from those mediated by mitogen-activated protein kinase (MAPK). MAPK inhibitors attenuated both GLU-induced state changes. Although GLU induced mPer1 mRNA in both early and late night, inhibition of PKA blocked this event only in early night, suggesting that cellular mechanisms regulating mPer1 are gated by the suprachiasmatic circadian clock. These data support a diametric gating role for cAMP/PKA in light/GLU-induced SCN state changes: cAMP/PKA promotes the effects of light/GLU in early night, but opposes them in late night.

Suprachiasmatic nucleus: the brain's circadian clock.

The tiny suprachiasmatic nucleus (SCN) of the hypothalamus plays a central role in the daily programming of organismic functions by regulating day-to-day oscillations of the internal milieu and synchronizing them to the changing cycles of day and night and of body state. This biological clock drives the daily expression of vital homeostatic functions as diverse as feeding, drinking, body temperature, and neurohormone secretion. It adaptively organizes these body functions into near-24-hour oscillations termed circadian rhythms. The SCN imposes temporal order 1) through generating output signals that relay time-of-day information, and 2) through gating its own sensitivity to incoming signals that adjust clock timing. Each of these properties, derived from the timebase of the SCN's endogenous near-24-hour pacemaker, persists when the SCN is maintained in a hypothalamic brain slice in vitro. Single-unit recording experiments demonstrate a spontaneous peak in the electrical activity of the ensemble of SCN neurons near midday. By utilizing this time of peak as a "pulse" of the clock, we have characterized a series of time domains, or windows of sensitivity, in which the SCN restricts its own sensitivity to stimuli that are capable of adjusting clock phase. Pituitary adenylyl cyclase-activating peptide (PACAP) and cAMP comprise agents that reset clock phase during the day time domain; both PACAP and membrane-permeable cAMP analogs cause phase advances only when applied during the day. In direct contrast to PACAP and cAMP, acetylcholine and cGMP analogs phase advance the clock only when applied during the night. Sensitivity to light and glutamate arises concomitant with sensitivity to acetylcholine and cGMP. Light and glutamate cause phase delays in the early night, by acting through elevation of intracellular Ca2+, mediated by activation of a neuronal ryanodine receptor. In late night, light and glutamate utilize a cGMP-mediated mechanism to induce phase advances. Finally, crepuscular domains, or dusk and dawn, are characterized by sensitivity to phase resetting by the pineal hormone, melatonin, acting through protein kinase C. Our findings indicate that the gates to both daytime and nighttime phase resetting lie beyond the level of membrane receptors; they point to critical gating within the cell, downstream from second messengers. The changing patterns of sensitivities in vitro demonstrate that the circadian clock controls multiple molecular gates at the intracellular level, to assure that they are selectively opened in a permissive fashion only at specific points in the circadian cycle. Discerning the molecular mechanisms that generate these changes is fundamental to understanding the integrative and regulatory role of the SCN in hypothalamic control of organismic rhythms.

Pituitary adenylyl cyclase-activating peptide: a pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock.

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep-wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6-38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6-38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.

Nitric oxide synthase imunolabeling in the molluscan CNS and peripheral tissues.

NOS immunoreactivity was assayed in CNS and peripheral tissues of the sea slugs Pleurobranchaea californica, Tritonia diomedea and Aplysia californica using different antisera against mammalian nitric oxide synthase in Western blots. Polyclonal anti-nNOS labeled at 250, 185, 170, 155, 100, 75, and 65 kD in extracts of Pleurobranchaea CNS, salivary gland and esophagus but not of gills or muscle. The labeling pattern for Tritonia in bands at 250, 200, 120/110, 100, 69, 65, and 60 kD differed somewhat. Anti-nNOS labeling in Aplysia was markedly different, with bands labeled only at 69 and 60 kD in CNS extracts, and at 200, 190, 69 and 60 kD in salivary and esophagus extracts. The wide variation in NOS immunoreactivity is consistent with species differences in tissue localization and biochemical properties of molluscan NOS isoforms.

Oscillation and light induction of timeless mRNA in the mammalian circadian clock.

Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1.

A neuronal ryanodine receptor mediates light-induced phase delays of the circadian clock.

Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.

Pituitary adenylate cyclase activating peptide (PACAP) in the retinohypothalamic tract: a daytime regulator of the biological clock.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the brain biological clock in the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing to light exposure at night. Here we report a new signaling pathway by which the RHT regulates circadian timing in the daytime as well. Using dual-immunocytochemistry for PACAP and the in vivo tracer Cholera toxin subunit B (ChB), intense PACAP immunoreactivity (PACAP-IR) was observed in retinal afferents at the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR was nearly lost upon bilateral eye enucleation. PACAP afferents originated from ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced by application of PACAP-38 during the subjective day, but not at night. The effect is channelled to the clock via a PACAP 1 receptor-cAMP signaling mechanism. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT can adjust the biological clock by a PACAP-cAMP-dependent mechanism during the daytime.

Pituitary adenylate cyclase-activating peptide (PACAP) in the retinohypothalamic tract: a potential daytime regulator of the biological clock.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing via a glutamatergic pathway at night. Here we report a new signaling pathway by which the RHT may regulate circadian timing in the daytime as well. We used dual immunocytochemistry for pituitary adenylate cyclase-activating peptide (PACAP) and the in vivo tracer cholera toxin subunit B and observed intense PACAP-immunoreactivity (PACAP-IR) in retinal afferents in the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR in the SCN as well as in the IGL was nearly lost after bilateral eye enucleation. PACAP afferents originated from small ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced (3.5 +/- 0.4 hr) by application of 1 x 10(-6) M PACAP-38 during the subjective day [circadian time (CT)-6] but not at night (CT14 and CT19). The phase-shifting effect is channeled to the clock via a PACAP-R1 receptor, because mRNA from this receptor was demonstrated in the ventral SCN by in situ hybridization. Furthermore, vasoactive intestinal peptide was nearly 1000-fold less potent in stimulating a phase advance at CT6. The signaling mechanism was through a cAMP-dependent pathway, which could be blocked by a specific cAMP antagonist, Rp-cAMPS. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT may adjust the biological clock by a PACAP/cAMP-dependent mechanism during the daytime.

Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn.

Nocturnal synthesis of the pineal hormone melatonin (MEL) is regulated by the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. We examined the hypothesis that MEL can feed back to regulate the SCN using a brain slice preparation from rat. We monitored the SCN ensemble firing rate and found that MEL advanced the time of peak firing rate by more than 3 h at restricted circadian times (CTs) near subjective dusk [CT 10-14 (10-14 h after lights on)] and dawn (CT 23-0) on days 2 and 3 after treatment. The effect of MEL at CT 10 was blocked by pertussis toxin. The protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate, reset the SCN firing rate rhythm with a profile of temporal sensitivity congruent with that of MEL. Two specific PKC inhibitors, calphostin C and chelerythrine chloride, independently blocked MEL-induced phase advances at each sensitive period. Furthermore, MEL administration increased PKC phosphotransferase activity transiently to 200% at CT 10 and CT 23, but not at CT 6. These data demonstrate that 1) MEL can directly modulate the circadian timing of the SCN within two windows of sensitivity corresponding to dusk and dawn; and 2) MEL alters SCN cellular function via a pertussis toxin-sensitive G protein pathway that activates PKC.

Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock.

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.

Resetting the biological clock: mediation of nocturnal CREB phosphorylation via light, glutamate, and nitric oxide.

Synchronization between the environmental lighting cycle and the biological clock in the suprachiasmatic nucleus (SCN) is correlated with phosphorylation of the Ca2+/cAMP response element binding protein (CREB) at the transcriptional activating site Ser133. Mechanisms mediating the formation of phospho-CREB (P-CREB) and their relation to clock resetting are unknown. To address these issues, we probed the signaling pathway between light and P-CREB. Nocturnal light rapidly and transiently induced P-CREB-like immunoreactivity (P-CREB-lir) in the rat SCN. Glutamate (Glu) or nitric oxide (NO) donor administration in vitro also induced P-CREB-lir in SCN neurons only during subjective night. Clock-controlled sensitivity to phase resetting by light. Glu, and NO is similarly restricted to subjective night. The effects of NMDA and nitric oxide synthase (NOS) antagonists on Glu-mediated induction of P-CREB-lir paralleled their inhibition of phase shifting. Significantly, among neurons in which P-CREB-lir was induced by light were NADPH-diaphorase-positive neurons of the SCN's retinorecipient area. Glu treatment increased the intensity of a 43 kDa band recognized by anti-P-CREB antibodies in subjective night but not day, whereas anti-alpha CREB-lir of this band remained constant between night and day. Inhibition of NOS during Glu stimulation diminished the anti-P-CREB-lir of this 43 kDa band. Together, these data couple nocturnal light, Glu, NMDA receptor activation and NO signaling to CREB phosphorylation in the transduction of brief environmental light stimulation of the retina into molecular changes in the SCN resulting in phase resetting of the biological clock.

Coupling of muscarinic cholinergic receptors and cGMP in nocturnal regulation of the suprachiasmatic circadian clock.

Acetylcholine has long been implicated in nocturnal phase adjustment of circadian rhythms, yet the subject remains controversial. Although the suprachiasmatic nucleus (SCN), site of the circadian clock, contains no intrinsic cholinergic somata, it receives choline acetyltransferase-immunopositive projections from basal forebrain and mesopontine tegmental nuclei that contribute to sleep and wakefulness. We have demonstrated that the SCN of inbred rats in a hypothalamic brain slice is sensitive to cholinergic phase adjustment via muscarinic receptors (mAChRs) only at night. We used this paradigm to probe the muscarinic signal transduction mechanism and the site(s) gating nocturnal responsiveness. The cholinergic agonist carbachol altered the circadian rhythm of SCN neuronal activity in a pattern closely resembling that for analogs of cGMP; nocturnal gating of clock sensitivity of each is preserved in vitro. Specific inhibitors of guanylyl cyclase (GC) and cGMP-dependent protein kinase (PKG), key elements in the cGMP signal transduction cascade, blocked phase shifts induced by carbachol. Further, carbachol administration to the SCN at night increased cGMP production and PKG activity. The carbachol-induced increase in cGMP was blocked both by atropine, an mAChR antagonist, and by LY83583, a GC inhibitor. We conclude that (1) mAChR regulation of the SCN is mediated via GC-->cGMP-->PKG, (2) nocturnal gating of this pathway is controlled by the circadian clock, and (3) a gating site is positioned downstream from cGMP. This study is among the first to identify a functional context for mAChR-cGMP coupling in the CNS.

Cellular and biochemical mechanisms underlying circadian rhythms in vertebrates.

Circadian clocks organize neural processes, such as motor activities, into near 24-hour oscillations and adaptively synchronize these rhythms to the solar cycle. Recently, the first mammalian clock genes have been found. Unpredicted diversity in signaling pathways and clock-controlled gating of signals that modulate timekeeping has been discovered. A diffusible clock output has been found to control some behavioral rhythms. Consensus is emerging that circadian mechanisms are conserved across phylogeny, but that mammals have developed a great complexity of controls.