In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.Many lines of evidence indicate that the hypothalamic suprachiasmatic nucleus (SCN) generates mammalian circadian rhythms (1). Arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) are synthesized in neurons within different subdivisions of the SCN (2). In vivo studies have shown that on the 3rd day of constant darkness (DD), circadian rhythms are observed in AVP content (3) and mRNA (4,5) but not in VIP content (6) or mRNA (7). This suggests that AVP and VIP rhythms reflect different aspects of the circadian pacemaking mechanism. In SCN cultures, however, AVP (8-12) and VIP releases (12) undergo circadian changes, which indicates that the VIP is also regulated by the circadian pacemaker as well as the AVP.Several properties of the circadian pacemaker system that underlie the locomotor activity rhythm in mammals are best interpreted in terms of the two-oscillator system. One such property is the phenomenon known as splitting, in which different circadian periods are simultaneously expressed in constant light (13). The two-oscillator system can account for various other rhythm properties in rodents, such as a compression and decompression of activity time (13), aftereffects by previous entrainment (13), and a difference in the immediate and steady-state phase shifts (14). However, two different circadian oscillations have never been found within the SCN (1).The present study was conducted to determine whether peptides in different subdivisions of the SCN are regulated by distinct oscillators, using the organotypic slice culture of the SCN. First, we examined 48-hr patterns of AVP and VIP release in the culture with or without antimitotic treatment, which is generally used to reduce the number of glial cells. Second, to measure the respective period in the same culture, we monitored two peptide rhythms simultaneously for 6 days. Finally, we examined the phase-shifting effect of N-methyl-Daspartate (NMDA), whose receptors are suggested to be involved in the visual input to the SCN (15, 16). MATERIALS AND METHODS Preparation of Organotypic SCN Culture. Rats of theWistar strain were bred and reared in our animal quarters (lights on from 06:00 to 18:00). Organotypic slic...

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The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross-correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)-dependent synaptic transmission in the cellular communication was indicated by dose-dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.

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Effects of local destruction of the brain catecholaminergic neurons were examined on the light- and feeding-associated circadian rhythms in plasma corticosterone in rats. 6-Hydroxydopamine (6-OHDA), a selective and long-lasting neurotoxin of the catecholaminergic neurons, was microinjected into the following discrete areas of the brain: the paraventricular nucleus (PVN), median eminence (ME), suprachiasmatic nucleus (SCN), ventromedial hypothalamic nucleus (VMH), lateral hypothalamic nucleus (LH), and the ascending bundle of noradrenergic neurons (NAB). And the feeding-associated as well as the light-associated circadian rhythms in plasma corticosterone were determined. The light-associated circadian rhythm was assayed under a 24-h light-dark cycle with free access to food, whereas the feeding-associated circadian rhythm was measured under restricted daily feeding in which rats had free access to food at a fixed time of day. 6-OHDA reduced the norepinephrine concentrations in respective regions to 10-30% of the control value, except for the LH. The light-associated circadian rhythm was not affected by 6-OHDA into the SCN or PVN. By contrast, 6-OHDA into the PVN or ventral NAB suppressed the feeding-associated circadian peak. 6-OHDA into the VMH and LH showed some effects on plasma corticosterone level but not on the feeding-associated circadian rhythm. 6-OHDA had no systematic effect on plasma corticosterone level when injected into the SCN, ME, and dorsal NAB. These findings indicate that the catecholaminergic neurons projecting to the PVN are involved in the feeding-associated but not in the light-associated circadian rhythms.