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The central role of the suprachiasmatic nuclei in regulating mammalian circadian rhythms is well established. We study the temporal organization of neuronal properties in the suprachiasmatic nucleus (SCN) using a rat hypothalamic brain slice preparation. Electrical properties of single neurons are monitored by extra-cellular and whole-cell patch recording techniques. The ensemble of neurons in the SCN undergoes circadian changes in spontaneous activity, membrane properties and sensitivity to phase adjustment. At any point in this cycle, diversity is observed in individual neurons' electrical properties, including firing rate, firing pattern and response to injected current. Nevertheless, the SCN generate stable, near 24 h oscillations in ensemble neuronal firing rate for at least three days in vitro. The rhythm is sinusoidal, with peak activity, a marker of phase, appearing near midday. In addition to these electrophysiological changes, the SCN undergoes sequential changes in vitro in sensitivities to adjustment. During subjective day, the SCN progresses through periods of sensitivity to cyclic AMP, serotonin, neuropeptide Y, and then to melatonin at dusk. During the subjective night, sensitivities to glutamate, cyclic GMP and then neuropeptide Y are followed by a second period of sensitivity to melatonin at dawn. Because the SCN, when maintained in vitro, is under constant conditions and isolated from afferents, these changes must be generated within the clock in the SCN. The changing sensitivities reflect underlying temporal domains that are characterized by specific sets of biochemical and molecular relationships which occur in an ordered sequence over the circadian cycle.

The basis of the decline in circadian rhythms with aging was addressed by comparing the patterns of three behavioral rhythms in young and old rats with the in vitro rhythm of neuronal activity in the suprachiasmatic nuclei (SCN), the primary circadian pacemaker. In some old rats, rhythms of body temperature, drinking, and activity retained significant 24-h periodicities in entraining light-dark cycles; in others, one or two of the rhythms became aperiodic. When these rats were 23-27.5 mo old they were killed, and single-unit firing rates in SCN brain slices were recorded continuously for 30 h. There was significant damping of mean peak neuronal firing rates in old rats compared with young. SCN neuronal activities were analyzed with reference to previous entrained behavioral rhythm patterns of individual rats as well. Neuronal activity from rats with prior aperiodic behavioral rhythms was erratic, as expected. Neuronal activity from rats that were still maintaining significant 24-h behavioral rhythmicity at the time they were killed was erratic in most cases but normally rhythmic in others. Thus there was no more congruence between the behavioral rhythms and the brain slice rhythms than there was among the behavioral rhythms alone. These results, the first to demonstrate aberrant SCN firing patterns and a decrease in amplitude in old rats, imply that aging could either disrupt coupling between SCN pacemaker cells or their output, or cause deterioration of the pacemaking properties of SCN cells.

The geniculohypothalamic tract carries visual information from the intergeniculate leaflet to the suprachiasmatic circadian pacemaker. NPY, found in this projection, has been shown to affect the phase of behavioral rhythms and influence photic entrainment. We now demonstrate that NPY, when briefly applied to the geniculate projection sites of rat SCN in vitro, induces permanent phase-shifts in the rhythm of neuronal electrical activity at two separate phases of the circadian cycle.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

1. The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary pacemaker for circadian rhythms in mammals. The 24 h pacemaker is endogenous to the SCN and persists for multiple cycles in the suprachiasmatic brain slice. 2. While serotonin is not endogenous to the SCN, a major midbrain hypothalamic afferent pathway is serotonergic. Within this tract the dorsal raphe nucleus sends direct projections to the ventrolateral portions of the SCN. We investigated a possible regulatory role for serotonin in the mammalian circadian system by examining its effect, when applied at projection sites, on the circadian rhythm of neuronal activity in rat SCN in vitro. 3. Eight-week-old male rats from our inbred colony, housed on a 12 h light: 12 h dark schedule, were used. Hypothalamic brain slices containing the paired SCN were prepared in the day and maintained in glucose and bicarbonate-supplemented balanced salt solution for up to 53 h. 4. A 10(-11) ml drop of 10(-6) M-serotonin (5-hydroxytryptamine (5-HT) creatinine sulphate complex) in medium was applied to the ventrolateral portion of one of the SCN for 5 min on the first day in vitro. The effect of the treatment at each of seven time points across the circadian cycle was examined. The rhythm of spontaneous neuronal activity was recorded extracellularly on the second and third days in vitro. Phase shifts were determined by comparing the time-of-peak of neuronal activity in serotonin- vs. media-treated slices. 5. Application of serotonin during the subjective day induced significant advances in the phase of the electrical activity rhythm (n = 11). The most sensitive time of treatment was CT 7 (circadian time 7 is 7 h after 'lights on' in the animal colony), when a 7.0 +/- 0.1 h phase advance was observed (n = 3). This phase advance was perpetuated on day 3 in vitro without decrement. Serotonin treatment during the subjective night had no effect on the timing of the electrical activity rhythm (n = 9). 6. The specificity of the serotonin-induced phase change was assessed by treating slices in the same manner with a microdrop of serotonergic agonists, 5-carboxamidotryptamine, that targets the 5-HT1 class of receptors, or 8-hydroxy-dipropylaminotetralin (8-OH DPAT), that acts on the 5-HT1A receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)