Early Subjective Day Research Articles

Spontaneous circadian and light-induced expression of junB mRNA in the hamster suprachiasmatic nucleus

We examined both spontaneous and light-evoked expression of junB mRNA in the hamster suprachiasmatic nucleus (SCN), an endogenous circadian pacemaker. junB was expressed in the SCN in response to a light pulse during the subjective night and early subjective day as well as spontaneously during the transition from subjective night to subjective day. Light-evoked expression was strongest in the ventral SCN, while spontaneous expression was stronger in the dorsal SCN. Spontaneous expression began around subjective dawn and persisted for at least 4 h into the subjective day. The expression of junB mRNA may play a role in both phase-shifting responses to light and in spontaneous oscillation of the SCN pacemaker.

Calmodulin inhibitors produce phase shifts of circadian rhythms in vivo and in vitro.

The effect of calmodulin inhibitors on the circadian rhythm of locomotor activity and on the rhythm of suprachiasmatic nuclear (SCN) neuron firing rate recorded in vitro from hypothalamic slices was examined. Trifluoperazine produces changes in a dose-dependent manner in the phase of the activity rhythm, with phase advances throughout most of the subjective day extending into the subjective night. These phase changes in the activity rhythm occur rapidly and without induction of locomotor activity at the time of treatment. Similarly, trifluoperazine and the naphthalenesulfonamide W-7 produce changes in phase delays in the subjective night extending into early subjective day. The effects are greater with respect to amplitude when measured acutely after treatment than in the next cycle, and both the acute and next-day effects are greater than those observed in vivo, indicating that data from in vitro studies need to be interpreted with caution. These observations indicate that calmodulin inhibitors affect rhythms directly in vivo by altering SCN neuron pacemaker function, as this reflects involvement of calcium-calmodulin binding with activation of a calmodulin-dependent kinase, either to alter intracellular cAMP levels or to alter gene expression directly to modulate the phase of the SCN clock.

Temporal regulation of light-induced fos and fos-like protein expression in the ventrolateral subdivision of the rat suprachiasmatic nucleus

We measured c- fos messenger RNA levels and Fos protein immunoreactivity in the suprachiasmatic nucleus of rats as a function of light and time of day. Immunohistochemistry demonstrated a daily rhythm of immunoreactive Fos in the ventrolateral subdivision of the suprachiasmatic nucleus of animals entrained to a 12 h/12 h light-dark cycle; expression was low during the dark phase, peaked about 2 h after light onset at dawn, and remained elevated at an intermediate level for the remainder of the light phase. Immunoblots of nuclear extracts showed a 54,000 mol. wt band that increased in density from the dark phase to the early light phase and decreased again during the late light phase. In situ hybridization using a radiolabeled cDNA probe revealed a c- fos messenger RNA signal that was detected as early as 15 min after dawn, prominent at 30 min, and absent by 2 h. The expression of c- fos messenger RNA and Fos immunoreactivity in the suprachiasmatic nucleus depended on the presence of ambient light. In rats entrained to two daily 1-h light pulses corresponding to dawn and dusk (“skeleton” photoperiod) instead of the complete light-dark cycle, immunoreactive Fos was elicited by the dawn pulse alone and was less persistent than during the complete photoperiod. In rats free-running in constant darkness, c- fos messenger RNA and Fos immunoreactivity were stimulated by 2-h light pulses administered only during the subjective night and early subjective day, but not by light pulses during the middle or late subjective day or in the absence of light pulses.

Diazepam phase shifts the circadian clock of the field mouseMus booduga

Single 2h administration of diazepam (benzodiazepine) in 3.5% ethanol solution was found to evoke advance and delay phase shifts in the locomotor activity rhythm in the field mouseMus booduga. Through such pulsed administration of diazepam at various phases of circadian rhythm a phase response curve could be constructed. Phase advance occurred during early subjective day (CT 2) and phase delays were observed in the remaining phases. The shape of the diazepam phase response curve is similar to the general shape of the phase response curves generated by intraperitoneal injections of other benzodiazepines in hamsters. The phase shifting action of diazepam may be explained by its agonistic action on the neurotransmitter gamma-aminobutyric acid.

Analysis of in vitro glucose utilization in a circadian pacemaker model

An in vitro glucose utilization method, based upon 14C-2-deoxyglucose kinetics in brain slices, has been used to study circadian rhythms in hypothalamic slices containing the suprachiasmatic nucleus (SCN). Spontaneous SCN metabolic activity in vitro is similar to that observed in vivo with higher metabolic rates in subjective daytime and lower rates during subjective night. However, in vitro SCN metabolic activity during late subjective day is above that seen when glucose utilization is measured in vivo, suggesting that an inhibitory influence normally active in vivo is lost during slice isolation. Incubation of slices containing SCN in the presence of TTX exposes a TTX-insensitive component of metabolic activity in early subjective day, supporting prior suggestions that glucose utilization by the circadian oscillator continues in the absence of Na(+)-dependent action potentials. Studies with high Mg2+ concentrations are consistent with the hypothesis that most metabolic activity above the basal level observed with the glucose utilization method is related to synaptic activity. Pharmacological studies of the SCN brain slice model with radiotracers offer potential for analysis of both circadian rhythmicity and neural regulation.

Circadian rhythms of somatostatin-immunoreactivity in the suprachiasmatic nucleus of the rat

Twenty-four hour patterns in somatostatin (SS)-like immunoreactivity (LI) within the suprachiasmatic nucleus (SCN) were determined by enzyme immunoassay in rats blinded by bilateral orbital enucleation or kept sighted under light-dark conditions. A remarkable circadian rhythm was found in the concentration of SS-LI in the SCN under blinded conditions. The peak time appeared at about circadian time (CT) 4 in the early subjective day and the trough at around CT 16 or 20 in the subjective night. Light-dark cycles did not alter the circadian patterns of SS-LI observed in the blinded rat SCN. These results indicate the presence of an endogenous circadian rhythm in SS-LI in the rat SCN, independent of environmental lighting cycles.

Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract

The suprachiasmatic nuclei (SCN) contain the major pacemaker for mammalian circadian rhythms. The SCN receive photic input both directly, via the retinohypothalamic tract (RHT), and indirectly, via the geniculohypothalamic tract (GHT), which originates in cells in the intergeniculate leaflet (IGL) and anterior portions of the ventral lateral geniculate nucleus (vLGN). We tested whether electrical stimulation of the GHT would induce phase shifts in wheel-running activity rhythms of Syrian hamsters housed in continuous darkness or continuous illumination. In both lighting conditions, electrical stimulation of the GHT induced mainly phase advances when given during the late subjective day and small phase delays when given during the late subjective night and early subjective day. Stimulation in the thalamus outside the GHT failed to produce similar phase shifts. Repeated daily stimulation had only a weak entraining effect on the activity rhythm. Activation of GHT neurons appears to influence the pacemaker for activity rhythms in a phase-dependent manner.

The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP

A circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus controls the daily behavioral and physiological rhythms of mammals. While the mammalian circadian system has been the focus of research for many years, very little work has been directed at understanding its underlying biochemical mechanisms. In these experiments we used the hypothalamic brain slice technique to investigate these mechanisms, focusing specifically on the intrinsic resetting properties of the circadian clock of the rat. We monitored a primary expression of the clock or pacemaker, the circadian rhythm of electrical activity of SCN neurons. This rhythm continues the oscillatory pattern seen in vivo for up to 60 hr in vitro, with an activity peak near midday that shows very little variation among SCN from different rats. The stability of the rhythm in vitro enabled us to use the time of peak activity to monitor the phase of the underlying pacemaker. Bath application of membrane-soluble cAMP analogs in 1 hr pulses induced robust advances in the phase of the rhythm that remained stable for 2 cycles. This effect depended on the phase of the pacemaker at the time of treatment: the peak was maximally advanced (4-6 hr) by treatments during the middle of the subjective day (projected from the donor's cycle); treatments during most of the subjective night and early subjective day induced no phase changes. Half-maximal phase resetting was induced at 1 x 10(-10) M concentrations of active cAMP analog.(ABSTRACT TRUNCATED AT 250 WORDS)

Responses to pulsed light stimuli in a pulmonate slug (Limax pseudoflavus)

The phase changes in the activity rhythm of the pulmonate slug Limax pseudoflavus caused by light pulses (30 min, 10,000 lux) in otherwise dark conditions were recorded using time lapse cinematography and tipping aktographs. From the timing of the pulse relative to an individual activity cycle a phase response curve was constructed. Phase delays occurred when the pulse was administered in the early subjective night and subjective day, phase advances were recorded during the mid and late subjective night.The effects of two pulses (30 min, 10,000 lux) forming symmetric and asymmetric skeleton photoperiods were also recorded and related to the phase response curve. Both stable and unstable entrained states were found, the condition being dependent upon the relative timing of the two pulses and the previous activity onset. It was also shown that there was a time lag between the light pulse and its expressed phase setting effect. Thus phase setting is not instantaneous as with some insects.

Phase-dependent shift of free-running human circadian rhythms in response to a single bright light pulse.

Responsiveness of free-running human circadian rhythms to a single pulse of bright light was examined in a temporal isolation unit. Bright light (5000 lx) of either 3 or 6 h duration, applied during the early subjective day, produced phase-advance shifts in both the sleep-wake cycle and the rhythm of rectal temperature; the light pulse had essentially no effect on the phase of the circadian rhythms, when it was introduced during the late subjective day or the early subjective night. The results indicate that bright light can reset the human circadian pacemaker.

Phase shift of daily profiles of N-acetyltransferase in the rat pineal gland.

Pineal N-acetyltransferase activity (NAT) has a circadian rhythm with peak values in the dark time and low values in the light time. NAT time profiles were measured in rats exposed to LD 14:10, to constant dark, and to acute (less than 48 hr) light-dark treatments. In all experiments, imposition of light suppressed NAT. The phase of the dark time NAT cycle was altered 2 hr or less by the following treatments: 3 hr light in the early subjective night, 3 hr light in the late subjective night, 2 hr or 6 hr light in the early subjective day, 4 hr early lights-on, 1 day of constant dark, or 1 day of constant light. When light was extended 4 hr into the dark time, NAT rose at lights out but fell again as the time of "expected" dawn approached. In contrast, the phase of the NAT cycle was shifted 12 hr (180 degrees) within 72 hr by reversing the phase of the light-dark cycle. NAT did not rise in the first dark period (coincident with the time of the subjective light time). The amplitude of the first shifted cycle was less than four control NAT profiles measured in rats kept in the original (unshifted) light-dark cycle.

Phase-resetting action of light on the circadian activity rhythm of Rattus exulans.

The phase resetting action of light on the circadian rhythm of locomotor activity has been examined in wild-caught Polynesian rats (Rattus exulans). Phase-response curves to 4-, 8-, and 16-h light pulses have been derived. All three curves conform to the generalization that pulses occurring during late subjective day and early subjective night produce delays, whereas advances occur in response to pulses coinciding with the late subjective night and early subjective day. Weak (type 1) phase resetting is observed in response to 4-h pulses and perhaps 8-h pulses, whereas strong (type 0) resetting apparently occurs in response to 16-h pulses. These data evidently constitute the first report of strong phase resetting in mammalian circadian rhythms. The phase relationship between an entrained activity rhythm and the light-dark cycle is dependent on the photoperiod and, in 24-h cycles, on the period difference between the rhythm and the zeitgeber. In longer zeitgeber cycles, activity onset is delayed by a direct masking effect of light. A primarily nonparametric action of light in natural entrainment is consistent with these data and with field observations.