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.

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.

Intrinsic neuronal rhythms in the suprachiasmatic nuclei and their adjustment.

The central role of the suprachiasmatic nuclei in regulating mammalian circadian rhythms is well established. We study the temporal organization of neuronal properties in the suprachiasmatic nucleus (SCN) using a rat hypothalamic brain slice preparation. Electrical properties of single neurons are monitored by extra-cellular and whole-cell patch recording techniques. The ensemble of neurons in the SCN undergoes circadian changes in spontaneous activity, membrane properties and sensitivity to phase adjustment. At any point in this cycle, diversity is observed in individual neurons' electrical properties, including firing rate, firing pattern and response to injected current. Nevertheless, the SCN generate stable, near 24 h oscillations in ensemble neuronal firing rate for at least three days in vitro. The rhythm is sinusoidal, with peak activity, a marker of phase, appearing near midday. In addition to these electrophysiological changes, the SCN undergoes sequential changes in vitro in sensitivities to adjustment. During subjective day, the SCN progresses through periods of sensitivity to cyclic AMP, serotonin, neuropeptide Y, and then to melatonin at dusk. During the subjective night, sensitivities to glutamate, cyclic GMP and then neuropeptide Y are followed by a second period of sensitivity to melatonin at dawn. Because the SCN, when maintained in vitro, is under constant conditions and isolated from afferents, these changes must be generated within the clock in the SCN. The changing sensitivities reflect underlying temporal domains that are characterized by specific sets of biochemical and molecular relationships which occur in an ordered sequence over the circadian cycle.

Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO.

Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produced lightlike phase shifts of circadian rhythms. The SCN exhibited calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways blocked Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo blocked the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN mediating the phase resetting of the biological clock.

The organization of the suprachiasmatic circadian pacemaker of the rat and its regulation by neurotransmitters and modulators.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells.

H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle.

Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase.

Böck, August (Purdue University, Lafayette, Ind.), Lia Eidlic Faiman, and Frederick C. Neidhardt. Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase. J. Bacteriol. 92:1076-1082. 1966.-To test our conclusion that Escherichia coli mutant I-9 possesses a valyl soluble ribonucleic acid (sRNA) synthetase that functions in vivo at 30 C but not at 37 C, measurements were made by use of the periodate method, of the level of charged valyl sRNA in this strain. A shift of temperature from 30 to 40 C resulted in a rapid discharging of valyl sRNA coordinate with the cessation of protein synthesis; at the same time, other species of sRNA, such as those for leucine, became fully charged. Identical results were obtained with a derivative of I-9 with relaxed ribonucleic acid (RNA) control. When P1 phage were grown on wild cells and then used at low multiplicities of infection to transduce temperature-resistant growth into I-9, complete cotransduction of normal valyl sRNA synthetase occurred. By means of the interrupted-mating technique, the structural gene for valyl sRNA synthetase was located on the E. coli chromosome map and found to be near thr, one-fifth of the length of the chromosome removed from the structural genes for the isoleucine-valine biosynthetic enzymes. Therefore, (i) the major valyl sRNA synthetase activity of I-9 appears to be temperature-sensitive in vivo, (ii) relaxed amino acid control over RNA synthesis does not appear to be a consequence of a normal charging of sRNA with a substitute molecule, and (iii) one structural gene for valyl sRNA synthetase is located on the E. coli chromosome not closely linked to the cistrons for the valine-biosynthetic enzymes.