Calcium excitability and oscillations in suprachiasmatic nucleus neurons and glia in vitro

Abstract

Converging lines of evidence suggest that the hypothalamic suprachiasmatic nucleus (SCN) is the site of the endogenous biological clock controlling mammalian circadian rhythms. To study the calcium responses of the cellular components that make up the clock, computer-controlled digital video and confocal scanning laser microscopy were used with the Ca*+ indicator dye fluo-3 to examine dispersed SCN cells and SCN explants with repeated sampling over time. Ca2+ plays an important second messenger role in a wide variety of cellular mechanisms from gene regulation to electrical activity and neurotransmitter release, and may play a role in clock function and entrainment. SCN neurons and astrocytes showed an intracellular Ca2+ increase in response to glutamate and 5-HT, two major neurotransmitters in afferents to the SCN. Astrocytes showed a marked heterogeneity in their response to the serial perfusion of different transmitters; some responded to both 5-HT and glutamate, some to neither, and others to only one or the other. Under constant conditions, most neurons showed irregular temporal patterns of Ca2+ transients. Expression of regular neuronal oscillations could be blocked by the inhibitory transmitter GABA. Astrocytes, on the other hand, showed very regular rhythms of cytoplasmic Ca2+ concentrations with periods ranging from 7 to 20 sec. This periodic oscillation could be initiated by in vitro application of glutamate, the putative neurotransmitter conveying visual input to the SCN critical for clock entrainment. Long-distance communication between glial cells, seen as waves of fluorescence moving from cell to cell, probably through gap junctions, was induced by glutamate, 5-HT, and ATP. These waves increased the period length of cellular Ca2+ rises to 45-70 sec. Spontaneously oscillating cells were common in culture medium, serum, or rat cerebrospinal fluid, but rare in HEPES buffer. One source for cytoplasmic Ca2+ increases was an influx of extracellular Ca2+, as seen under depolarizing conditions in about 75% of the astroglia studied. All neurotransmitterinduced Ca2+ fluxes were not dependent on voltage changes, as Ca*+ oscillations could be initiated under both normal and

MAMMALIAN CIRCADIAN BIOLOGY: Elucidating Genome-Wide Levels of Temporal Organization

“…Neurons within the intact SCN, however, are coupled to form an ensemble expressing synchronized circadian rhythms of spontaneous electrical activity (155), calcium oscillations (43,92,146,204), humoral output (8,40,98,109), metabolic activity (178), and gene expression (161), with distinct spatial and temporal properties (44,93,123,135,173,221,225). Conclusive evidence that the SCN comprises the master circadian pacemaker came from lesioning studies and from experiments showing that transplantation of SCN tissue from donor animals harboring circadian clock gene mutations into SCN-lesioned wild-type hosts conferred upon the host the mutant circadian phenotype (157,188).…”

Two different constituents of plasma increase cytosolic calcium selectively in neurons or glia of primary rat cerebellar cultures.

“…With regard to the origin of glutamate, both massive activation of glutamatergic synapses and nonsynaptic release of glutamate either from neurons, or from glia by reversal of the glutamate transporter, have been suggested (Szatkowski & Attwell, 1994). Serum-induced increases of [Ca2+] in neurons and glia have been reported previously ( Van den Pol, Finkbeiner & Cornell-Bell, 1992). However, the possible damaging effect of blood factors reaching the brain's extracellular fluid upon disruption of the blood-brain barrier by hypoxia have scarcely been considered.…”

Intracellular Calcium Oscillation in Cultured Rat Hippocampal Neurons: A Model for Glutamatergic Neurotransmission

“…It is widely known that oscillation of intracellular Ca" concentration ([Ca 21],) is observed in several neu ronal preparations in vitro, such as cultured hippocampal (1,2) and cortical neurons (3), and the slice preparation of hypothalamic suprachiasmatic nucleus (4). Such intra cellular Ca2+ dynamics are synchronized to the burst of action potentials and seem to be due to Ca2+ influxes during the spike bursts (5).…”