Yumiko Katsuno

Circadian periods of single suprachiasmatic neurons in rats

Neuronal activity of a single neuron was monitored continuously for more than 5 days by means of a multi-electrode dish in dispersed cell culture of the rat suprachiasmatic nucleus (SCN). Sixty-seven out of 88 neurons showed a robust circadian rhythm in firing rate. The mean circadian period was 24.2 h, which was almost identical to that of the locomotor activity rhythm in 114 weanling rats blinded on the day of birth. However, the circadian period in individual SCN neurons was scattered from 20.0 to 28.3 h (SD, 1.4 h), while the period of activity rhythm clustered from 24.0 to 24.8 h (SD, 0.2 h). It is concluded that a large number of SCN neurons contain the circadian oscillator, the period of which is more variable than the circadian period of the SCN as a whole. It is suggested that the circadian rhythms in individual SCN neurons are capable of synchronizing to each other and are integrated to constitute a multiple oscillator system(s) within the SCN.

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons.

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 γ‐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.

Circadian rhythms in the release of vasoactive intestinal polypeptide and arginine-vasopressin in organotypic slice culture of rat suprachiasmatic nucleus.

Temporal profiles of the amount of vasoactive intestinal polypeptide (VIP) were examined in the medium of organotypic suprachiasmatic nucleus (SCN) slice cultures over a 2-day period. Arginine-vasopressin (AVP) level was also measured in the same medium. The slices of the SCN were obtained from 7-8-day-old rats and cultured individually in tubes on a roller drum for 14 days. The VIP amount in the medium of SCN culture showed a circadian rhythm with a approximately 22-h period. Circadian rhythms with identical periods were also observed in AVP amount of the same culture. However, the peak time of the VIP rhythm was slightly ahead of that of the AVP rhythm. Furthermore, the total VIP amount in the medium over a 24-h period was six times as large as that of AVP. These results suggest that there is a circadian rhythm of VIP which is released from the ventrolateral SCN.

Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus.

Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium. Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation. The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h. The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms. Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide. Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed. The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31. Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide. Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.

Light suppression of nocturnal pineal and plasma melatonin in rats depends on wavelength and time of day

Effects of light on the pineal and plasma melatonin were examined in Wistar and Long-Evans rats at two different times in the dark phase (light off from 18.00 h to 06.00 h) using lights of two different monochromatic wavelengths but with the same irradiance. The green light pulse (520 nm) given at 24.00 h suppressed the pineal and plasma melatonin to the day-time level for at least 2 h, while the red light (660 nm) pulse given at the same time of the day suppressed pineal melatonin only transiently and did not suppress the plasma melatonin at all. Both green and red lights given at 4.00 h suppressed the pineal and plasma melatonin to a similar extent. The results demonstrated that the suppression of melatonin by light depends on the wavelength of light and the circadian phase.

Circadian release of amino acids in the suprachiasmatic nucleus in vitro

TEMPORAL patterns of release of aspartate, glutamate and glycine, which are related to excitatory amino acidergic transmission, were examined in organotypic slice cultures of rat suprachiasmatic nucleus over a 60 h period. Vasopressin release in the same culture was measured simultaneously to compare the temporal pattern with that of the amino acids. Amino acids and vasopressin were measured by high performance liquid chromatography and enzyme immunoassay, respectively. Robust circadian rhythms were detected in release of aspartate, glutamate and glycine. Glycine levels were about 10 times higher than those of aspartate and glutamate in the culture. Vasopressin also showed a clear circadian rhythm and the phase angle difference between each amino acid and AVP was not significantly different. The results indicate that cultured SCN cells release these amino acids and the release is under the control of the circadian pacemaker.

Circadian release of excitatory amino acids in the suprachiasmatic nucleus culture is Ca2+-independent

We have previously reported that spontaneous release of excitatory amino acids (aspartate and glutamate) show remarkable circadian rhythms in the organotypic slice culture of rat suprachiasmatic nucleus (SCN). Here we showed effects of extracellular Ca(2+) removal and of L-trans-pyrrolidine-2,4-dicarboxylic acid, a glutamate/aspartate uptake inhibitor on the circadian release of excitatory amino acids in the SCN culture. Amino acids were measured by high-performance-liquid-chromatography. Removal of extracellular Ca(2+) exerted no effect on the spontaneous release of the excitatory amino acids, while it blocked high K(+)-evoked release of the amino acids. Neither the period nor the amplitude of the spontaneous circadian release of amino acids in Ca(2+)-free medium was different from those in the Ca(2+)-containing medium. On the other hand, L-trans-pyrrolidine-2,4-dicarboxylic acid increased the excitatory amino acid levels without affecting the amplitude of excitatory amino acid rhythms. These results indicated that the circadian release of excitatory amino acids in the SCN is Ca(2+)-independent and L-trans-pyrrolidine-2,4-dicarboxylic acid- insensitive. Therefore, Ca(2+)-dependent chemical synaptic transmission may not be involved in the circadian rhythm generation in the SCN.

Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continuous light in the rat.

In order to examine the mechanism for a loss of circadian rhythms in several functions under prolonged continuous light (LL), rats were blinded following LL over 5 months, and the mode of reappearance of circadian rhythms were analyzed in locomotor activity and plasma melatonin levels. Locomotor activity and plasma melatonin levels in individual rats became aperiodic after the exposure to LL. On the day of blinding, plasma melatonin levels showed circadian rhythms having a peak coincided with the activity time of locomotor rhythm which was restored after blinding. The time of melatonin peak was not related to the time of blinding (onset of darkness) nor to the initial time of blood sampling. Circadian rhythm in plasma melatonin levels reappeared faster than those in locomotor activity. The findings suggest that aperiodism developed in these functions under prolonged LL is not due to disruption of the circadian oscillation but to uncoupling of overt functions from the circadian pacemaker.

Food ingestion is more important to plasma corticosterone dynamics than water intake in rats under restricted daily feeding

The roles of food and/or water ingestion in the regulation of plasma corticosterone level were examined in rats under restricted daily feeding. When the time of food-pellets and water supply was restricted to 2 hours in the early light period (meal feeding) for 2 weeks, the corticosterone level increased prior to meal (prefeeding peak). A similar prefeeding hormone peak was observed when supply of food-pellets was restricted to 2 hours with free-access to water (food restriction). In contrast, when water supply was restricted to 2 hours with free-access to food-pellets (water restriction), the hormone level before water supply did not increase as much as that under meal feeding or food restriction. Shortening of an available time for water under water restriction or prolongation of the restriction schedule failed to elevate the hormone level furthermore. On the other hand, the high prefeeding corticosterone level before meal decreased subsequently to meal feeding (prandial fall), which was not observed when rats were kept fasting during the meal time. This prandial fall of the hormone level was not observed by water intake alone, and closely related to food-pellets ingestion. It is concluded that food ingestion is more important than water intake to the formation of the prefeeding corticosterone peak and to the prandial fall of the hormone level under restricted daily feeding.

Effects of Menstrual Cycle on Plasma Melatonin Level and Sleep Characteristics

It is well known that some clinical symptoms (e.g. psychotic symptoms and epileptic seizures) often aggravate around the time of menstruation. Moreover, the change of subjective sleep feeling throughout the menstrual cycle was reported. Therefore, the menstrual cycle is presumed to affect sleep characteristics and biological rhythm. We investigated a circadian pattern of plasma melatonin and body temperature, and sleep characteristics in the different phases of the menstrual cycle under controlled environmental conditions. In this paper, a part of the results is discussed.