Anisomycin induces phase shifts of circadian pacemaker in primary cultures of rat suprachiasmatic nucleus

doi:10.1016/0006-8993(95)00414-L

Brain Research

Volume 684, Issue 2, 3 July 1995, Pages 179-184

https://doi.org/10.1016/0006-8993(95)00414-L Get rights and content

Abstract

We have developed a cell culture system for the rat suprachiasmatic nucleus, in which a clear circadian oscillation of vasopressin release was observed. Using this culture system, the effect of anisomycin, an inhibitor of protein synthesis, on the circadian rhythm was studied. A phase-delay of more than 15 h could be produced by a 6-h anisomycin pulse. The magnitude of the phase-shift was dependent on the circadian time of the drug treatment and on its dose. The phase-response curve was similar to the response curves that have been measured for protein synthesis inhibitors in other organisms. These results strongly suggest that protein synthesis may be involved in the generation of circadian rhythms in mammals.

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Genetics of Circadian Rhythms
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In 1961, Colin Pittendrigh11 noted that the clock mechanism could be contained in single cells and was considered unlikely to be simply a chemical reaction because it was relatively insensitive to being sped up or slowed down by changes in temperature. Further evidence that gene transcription and translation were involved included demonstration that inhibition of protein synthesis could reset, or temporarily stop, the circadian clock.12–14 To identify what the genes and proteins comprising the circadian clock were, induction of mutations and screening for abnormal circadian rhythms proved the most effective approach, in both Drosophila melanogaster15 and in mice.16
Molecular neurobiology of circadian rhythms
2011, Handbook of Clinical Neurology
Citation Excerpt :
The initial evidence for this is that application of protein synthesis inhibitors in the region of the SCN shifts the circadian phase of activity of animals by an amount and in a direction that depends upon the time at which the inhibition is imposed (Inouye et al., 1988; Takahashi, 1995). A similar shift in the phase of vasopressin release from explanted SCN also results from inhibition of protein synthesis (Watanabe et al., 1995). Thus, even before circadian clock genes were discovered in mammals, gene expression and protein synthesis were identified as being central to the generation of circadian oscillations.
This chapter describes the genes expressed in the suprachiasmatic nuclei (SCN) and the proteins they encode that are responsible for this daily rhythmicity. The core circadian oscillator is autonomous to individual neurons of the SCN and is the result of the daily oscillation in the levels of several clock component proteins. The basis for this oscillation in mammals, as in other organisms, lies in rhythmic feedback regulation of transcription of the genes encoding these proteins. The levels of the period (PER) and cryptochrome (CRY) proteins alter the rate of transcription of their own genes. This alteration is achieved by inhibition of the enhancement of transcription that results from binding of the CLOCK–BMAL 1 heterodimer to the E-box element of the promoter region of the Per and Cry genes. Rhythmic transcription of Bmal1 appears to result from regulation via an orphan nuclear receptor (REV-ERB α), itself regulated by E-box elements. Rhythms in histone acetylation contribute to the circadian expression pattern of some core circadian genes. Additional genes have been identified based on altered circadian rhythms in mutants, although the roles of these genes in the circadian system remain to be determined. It is fortuitous that the unraveling of the molecular basis for circadian rhythmicity is occurring at a time when the biomedical research community and the general public are becoming aware of the importance of normal circadian time keeping for human health, safety, performance, and productivity.
Circadian Clock Genes
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Regional differences in circadian period within the suprachiasmatic nucleus
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Citation Excerpt :
Our recordings of single-cell firing rhythm have also shown that the period ranges from 21.5 h to 25.6 h (23.7 ± 2.3 h, mean ± SD) (unpublished data). When cells are dissociated and cultured at high density, however, they show one integrated AVP rhythm, and the variance in period among cultures is much smaller than that of a single-cell period (23.7 ± 0.3 h) (Watanabe et al., 1995). The AVP rhythm of slice culture and high-density cell culture would be the results of the synchronization of individual cells.
In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN), which is composed of multiple, single-cell oscillators. Isolated SCN tissue shows clear circadian oscillation in release of arginine vasopressin (AVP) in organotypic slice cultures. Previously, we reported that the oscillators in the dorsal SCN have shorter periods than those in the ventral part. Here, we examined whether a correlation between the period and the rostral–caudal co-ordination could exist. The rostral, central and caudal SCN were cultured separately and the periods of circadian rhythms of AVP release were measured. The rostral and caudal parts of the SCN showed shorter periods than the central SCN. Together with previous findings, it is suggested that the shorter period region originates from AVP containing areas, while the longer period region corresponds with vasoactive intestinal polypeptide (VIP) containing cells. In our VIP-immunoreactive slices, the application of VIP antagonists shortened the periods of the AVP-releasing rhythm. These data indicate that the oscillators in AVP cells have short periods and are entrained by VIP cells to form a single integrated rhythm.
Molecular Genetic Basis for Mammalian Circadian Rhythms
2005, Principles and Practice of Sleep Medicine
Primary cell culture of suprachiasmatic nucleus
2003, Brain Research Bulletin
In mammals, the suprachiasmatic nucleus (SCN) contains a biological clock that drives circadian rhythms in vivo and in vitro. Primary dissociated neuronal culture is a useful research tool, which allows cell-by-cell morphological and physiological study of the SCN. A long-term primary dissociated SCN neuron culture is the prerequisite to understanding how neural activity and morphology interact in the SCN. The essential details of recent effective SCN culture methods are reviewed, including preparation of cells, medium and substrate, maintenance of cultures, and characterization of cultured SCN neurons. This technique is growing in importance, especially with the advent of multi-electrode array (MEA) recording.

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Abstract

References (19)

Long-term cultured neurons from rat suprachiasmatic nucleus retain the capacity for circadian oscillation of vasopressin release

Brain Res.

The avian pineal, a vertebrate model system of the circadian oscillator: cellular regulation of circadian rhythms by light, second messengers, and macromolecular synthesis

Rec. Prog. Horm. Res.

Anisomycin, an inhibitor of protein synthesis, perturbs the phase of a mammalian circadian pacemaker

Brain Res.

Circadian rhythm of vasopressin release in primary cultures of rat suprachiasmatic nucleus

Brain Res.

Phase shifting the circadian clock with cycloheximide: response of hamsters with an intact or split rhythm of locomotor activity

Brain Res.

On the role of protein synthesis in the circadian clock of Neurospora crassa

Cellular and Molecular Basis of Biological Clocks

Effects of divalent cations and metabolic poisons on the circadian rhythm from the Aplysia eye

J. Comp. Physiol.

Lengthening the period of a biological clock in Euglena by cycloheximide, an inhibitor of protein synthesis

Cited by (16)

Genetics of Circadian Rhythms

Molecular neurobiology of circadian rhythms

Circadian Clock Genes

Regional differences in circadian period within the suprachiasmatic nucleus

Molecular Genetic Basis for Mammalian Circadian Rhythms

Primary cell culture of suprachiasmatic nucleus