The suprachiasmatic nuclei contain a tetrodotoxin-resistant circadian pacemaker.

Abstract

Tetrodotoxin was infused into the suprachiasmatic nuclei of unanesthetized and unrestrained rats continuously for 14 days. The internal timekeeping mechanism of the circadian pacemaker in the nuclei continued to oscillate unaffected by this treatment, although the toxin reversibly blocked function of both the input pathway for pacemaker entrainment and an output pathway for expression of the circadian drinking rhythm. Thus, Na+-dependent action potentials appear necessary for entrainment and expression of overt circadian rhythms, but they do not seem necessary for the pacemaker to keep accurate time. The experimental approach presented in this paper is useful because it allows systematic assessment and distinction of the input, pacemaker, and output components of a mammalian circadian timekeeping system in vivo.The suprachiasmatic nuclei (SCN) in the anterior hypothalamus appear to be the site of an endogenous circadian pacemaker in mammals (1). The nuclei receive retinal inputs for entrainment to the environmental light-dark cycle and generate neural outputs for expression of overt, measurable rhythms. Two complementary measures of SCN activity have helped to establish that the nuclei contain a functioning circadian pacemaker. These two properties, in vivo glucose utilization (2, 3) and unit discharge rates (4, 5), exhibit circadian rhythmicity. SCN energy metabolism and electrical activity are both elevated during the day and depressed during the night in nocturnal and diurnal mammals (6, 7).However, the most recent investigations using these two assays have generated some unexpectedly discordant data. On one hand, the rhythm of SCN metabolic activity appears in fetal rats 72 hr before birth (8,9). Such prenatal pacemaker function antedates the postnatal maturation of input and output pathways for photic entrainment and expression of overt circadian rhythms (10). On the other hand, when SCN action potentials are recorded in hypothalamic slices obtained from 7-, 11-, 14-, and 21-day-old rat pups, a circadian rhythm is observed only in those slices from the 14-and 21-day-old animals (11). Although other interpretations are possible, it seems that the circadian rhythm of SCN unit firing rates appears weeks after the rhythm of SCN energy metabolism first begins in utero.To resolve this discrepancy, we propose that the action potentials recorded in the SCN are not a part of the internal timekeeping mechanism of the circadian pacemaker; rather, the electrical impulses function to couple the pacemaker to its input and output pathways. We have tested this idea by chronically infusing tetrodotoxin (TTX) into the SCN of unanesthetized, unrestrained rats. TTX selectively and reversibly blocks voltage-dependent Na+ channels in axons, inhibiting the generation of action potentials without affecting resting membrane potential, K+ currents, Na+ pump mechanism, or local depolarization of postsynaptic membranes (12, 13). Importantly, in vitro recordings in hypothalamic slices demonstrate that SCN action potentials are ...

A Calcium Flux Is Required for Circadian Rhythm Generation in Mammalian Pacemaker Neurons

“…Although previous studies show conflicting conclusions regarding the role of membrane electrical events and ion fluxes in mammalian rhythm generation (Schwartz et al, 1987;Welsh et al, 1995;Shinohara et al, 1998), our data are consistent with recent findings in mice lacking vasoactive intestinal polypeptide receptor subtype 2 (VPAC 2 ) receptors, which are activated by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide. Mice missing the VPAC 2 receptor are unable to maintain normal behavioral rhythmicity and rhythmic expression of Per1, Per2, and Cry1 (Harmar et al, 2002).…”

Functional network inference of the suprachiasmatic nucleus

“…Our strategy involved the application of TTX to disperse single-cell phases through inhibition of intercellular coupling while allowing continued cell-autonomous oscillation (28,29). TTX was then washed out, restoring coupling and allowing reorganization of the SCN over the following 8 d. We applied the maximal information coefficient (MIC) statistic (30) to bioluminescence recordings during resynchronization to identify "functional connections" within the SCN at a single-cell resolution.…”

Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing

“…In the sinoatrial node of the heart, the coupling is electrotonic and mediated by gap junctions. In the suprachiasmatic nuclei of the mammalian circadian pacemaker, the early evidence suggested that neither gap junctions (33) nor synaptic communication (34) were essential for synchrony, leading to the proposal that the coupling was provided instead by diffusion of the inhibitory neurotransmitter ␥-aminobutyric acid (3,35) or some other diffusible substance. More recent experiments, however, seem to indicate a crucial role for actionpotential propagation and synaptic coupling in the suprachiasmatic nuclei (36).…”