Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock

Dong, Chen; Hurst, William J.; Ding, Jian; Faiman, Lia E.; Mayer, Bernd; Gillette, Martha U.

doi:10.1046/j.1471-4159.1997.68020855.x

Cited by 28 publications

(23 citation statements)

References 39 publications

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“…Bands against neuronal NOS, a mediator of nighttime phase shifts induced by light (31), were evident at 155 kDa in SCN 2.2 extracts, consistent with SCN tissue and previous reports (Fig. 4A) (48). During the circadian night-phase, activation of heme oxygenase-2 (HO-2) mimics cGMP phase shifting and is downstream of acetylcholine signaling (49).…”

Section: Resultssupporting

confidence: 90%

Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

Hurst

Earnest

Gillette

2002

Biochemical and Biophysical Research Communications

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We undertook an extensive antigenic characterization of the SCN 2.2 cell line in order to further evaluate whether the line expresses components of circadian regulatory pathways common to the hypothalamic suprachiasmatic nucleus (SCN), the central circadian clock in mammals. We found that differentiated SCN 2.2 cultures expressed a broad range of putative clock genes, as well as components of daytime, nighttime, and crepuscular circadian regulatory pathways found within the SCN in vivo. The line also exhibits several antigens that are highly expressed in a circadian pattern and/or differentially localized in the SCN relative to other hypothalamic regions. Expression of a broad complement of circadian regulatory proteins and putative clock genes further support growing evidence in recent reports that the SCN 2.2 cell line is an appropriate model for investigating the regulation of central mammalian pacemaker. © 2002 Elsevier Science (USA) Key Words: period; cryptochrome; casein kinase; PACAP; PKA; PKC; PKG; NOS; I1; VGF.The central biological pacemaker in mammals lies in the hypothalamic suprachiasmatic nucleus (SCN). The SCN drives circadian behavior, expresses rhythmic gene expression and integrates external stimuli in order to synchronize molecular timekeeping mechanisms with changing environmental conditions. Receptivity to phase shifting stimuli is gated by the circadian state of the central pacemaker. This gating behavior is most clearly demonstrated by the sensitivity of the SCN to light during the night phase, but not during the day phase. All photic, social and hormonal influences on the circadian clock are integrated at the cellular level through multiple signaling pathways within the SCN.The complexity of interacting signaling pathways involved in regulation of the SCN has encouraged researchers to seek cell culture systems to complement in vivo analysis of the mammalian circadian clock. Whether a cell line exhibits central or peripheral circadian clock characteristics complicates selection of an appropriate model system. To provide useful applications for central mammalian pacemaker research, cell line models must exhibit persistent self-sustained oscillations in circadian gene products, restore animal circadian rhythms in SCN-lesioned hosts and exhibit time-dependent responses to stimuli through regulatory pathways that characterize SCN function. Rhythmic gene expression has been demonstrated in NIH/ 3T3 fibroblasts, Rat-1 fibroblasts and spontaneously immortalized embryonic mouse fibroblasts after synchronizing signals (1-5). However, there is no published evidence that these lines express spontaneous, self-sustained rhythms or rescue rhythms in SCNlesioned animals (5, 6). In contrast, another cell line derived from fetal rat SCN, the SCN 2.2 cell line, exhibits endogenous spontaneous circadian rhythmicity in vitro (5, 7). We have focused on the SCN 2.2 line to further assess its usefulness as a model system for studying the mammalian central circadian pacemaker.The SCN 2.2 cell line is a pluripoten...

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Section: Resultssupporting

confidence: 90%

Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

Hurst

Earnest

Gillette

2002

Biochemical and Biophysical Research Communications

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“…Localization of the rat's M 1 •R expression by immunocytochemistry and in situ hybridization analysis includes neurons in the central area of the SCN (19). Thus, both proteins, M 1 •R and HO-2, are expressed in central SCN neurons, whereas nNOS expression in the central core area of the SCN is largely absent (5).…”

Section: Figmentioning

confidence: 98%

Carbon Monoxide and Nitric Oxide: Interacting Messengers in Muscarinic Signaling to the Brain's Circadian Clock

Artinian

Ding

Gillette

2001

Experimental Neurology

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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-␤, 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.

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“…The distance this signal travels is apparently limited by the half-life of NO. Several studies have now shown that the enzyme responsible for the production of this gas, nitric oxide synthase (NOS), is present in the SCN and co-localized with VIP (70)(71)(72). Another population of NOS-immunoreactive cells is found among the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes (73).…”

Section: Gases: Nitric Oxidementioning

confidence: 99%

Cellular Communication and Coupling Within the Suprachiasmatic Nucleus

Michel

Colwell

2001

Chronobiology International

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In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Importantly, when SCN neurons are removed from the organism and maintained in a brain slice preparation, they continue to generate 24h rhythms in electrical activity, secretion, and gene expression. Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells in the SCN. If we assume that individual cells in the SCN are competent circadian oscillators, it is obviously important to understand how these cells communicate and remain synchronized with each other. Cell-to-cell communication is clearly necessary for conveying inputs to and outputs from the SCN and may be involved in ensuring the high precision of the observed rhythm. In addition, there is a growing body of evidence that a number of systems-level phenomena could be dependent on the cellular communication between circadian pacemaker neurons. It is not yet known how this cellular synchronization occurs, but it is likely that more than one of the already proposed mechanisms is utilized. The purpose of this review is to summarize briefly the possible mechanisms by which the oscillatory cells in the SCN communicate with each other.

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Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock

Cited by 28 publications

References 39 publications

Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

Carbon Monoxide and Nitric Oxide: Interacting Messengers in Muscarinic Signaling to the Brain's Circadian Clock

Cellular Communication and Coupling Within the Suprachiasmatic Nucleus

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