Akihiro Tanakadate

Circadian Behavioural Rhythm in Caenorhabditis elegans

We are very grateful to M. Souma for his technical assistance, and Ian G. Gleadall for comments on the manuscript. This work was partly supported by the Project Fund of Graduate School of Medicine, Kitasato University.

Evaluation of Water Intake in Seawater Adaptation in Eels Using a Synchronized Drop Counter and Pulse Injector System

Abstract A new system has been developed for continuous measurement of drinking rate in eels in which drunk water exteriorized via an esophageal fistula was reintroduced into the stomach by a pulse injector synchronized with a drop counter. Using intact fish (controls), esophagus-cannulated fish whose drunk water was drained (drained fish), and esophagus and stomach-cannulated fish whose drunk water was reintroduced into the stomach (reintroduced fish), the validity of this system was examined by monitoring the changes in drinking rate and hydromineral balance after exposure to seawater (SW). In reintroduced fish, the SW exposure was followed by an immediate burst of drinking and a subsequent cyclic pattern of drinking. The drained fish exhibited a similar pattern of drinking but at much higher rate. The plasma Na concentration and osmolality increased linearly for one day and then decreased gradually to a steady SW level in 5–6 days in control and reintroduced fish. However, both parameters increased linearly for 4–5 days in drained fish until they died at plasma osmolality of ca. 500 mOsm. The initial increase in plasma Na and osmolality was steeper for a day in control and reintroduced fish than in drained fish. Hematocrit scarcely changed for one week in control and reintroduced fish, but it increased abruptly from the second day in drained fish, suggesting severe hypovolemia. These results show that the water and electrolyte balance of reintroduced fish were normal as in intact fish after exposure to SW. Thus, the drinking rate measured by the current system may represent actual drinking. The present study also provides first direct evidence to show that drinking plays a key role in SW adaptation in fish.

Circadian rhythm of locomotor behavior in a population of Paramecium multimicronucleatum: its characteristics as derived from circadian changes in the swimming speeds and the frequencies of avoiding response among individual cells

Abstract The locomotor behavior of Paramecium is determined by two components: swimming speed and the frequency of avoiding responses. The circadian oscillations of these two components were examined in order to interpret characteristics of the circadian locomotor rhythm, previously found in a Paramecium population using an originally defined parameter, “traverse frequency” (Hasegawa et al., 1978, 1982). In our present study, a modified version of the previously developed, fully computerized, close‐up video/photoamplifier system was used. Results indicate that individual specimens swam fast and unidirectionally during the day, while at night, in a light–dark cycle, they swam slowly and frequently turned. This oscillatory pattern was sustained in continuous darkness, where fluctuation in the frequency of avoiding responses was a dominant characteristic. The time structure of the “random walk” of Paramecium behavior was also examined by constructing and stochastically testing histograms of the interval times between specimens consecutively traversing beneath an observation point. Statistical analyses of observation data indicated that the circadian organization of the two components by individual specimens resulted in a circadian accumulation/dispersal rhythm of the entire population. It was concluded that the circadian “traverse frequency” rhythm principally represented this circadian accumulation/dispersal rhythm.

Circadian rhythm in the locomotor behavior in a population of Paramecium multimicronucleatum

Abstract The circadian behavioral rhythm in a population of Paramecium multimicronucleatum was examined by means of a computer/video system. A parameter, “traverse frequency”;, was introduced to index a locomotor activity of the specimens. The frequency is the average number of paramecia images per hour which individually traversed underneath photo‐cells placed on the CRT‐screen of the TV monitor. The traverse frequency was entrained and phase‐shifted by light‐dark (LD) cycles. It was highest at about 4 h after the beginning of the dark period and lowest at about 4 h after the beginning of the light period in LD 12:12 (20°C). Its rhythm free‐ran in DD and LL with about a 24 h period, the LL rhythm being relatively less stable than the DD rhythm. The free‐running period of DD rhythm was temperature‐compensated for 15–25°C. The circadian rhythmicity in the traverse frequency implies that the daily locomotor behavior of Paramecium is controlled by its circadian pacemaker.

Microcomputerized measurement of the circadian locomotor rhythm in microorganisms

A computerized close-up video/photoamplifier system was implemented for the study of circadian locomotor rhythm in a population of a ciliate protozoan, Paramecium. This fully microcomputerized system facilitated automatic long-term measurement of three parameters in parallel: (1) numbers of specimens per 10 min traversing beneath a fixed point in an experimental vessel, (2) times taken by specimens to traverse the point, and (3) interval times between subsequent specimens traversing the point. Stochastic analyses using these parameters can derive the circadian fluctuation of physiological variables, such as swimming speed and the frequency of avoiding reaction (abrupt change in swimming direction). The computerized system simultaneously accomplished the acquisition of these three sets of data, their transient storage, and their graphic display according to given format. The system software was constituted so that an experimenter with little computer knowledge, could easily operate the system by answering questions displayed on the computer monitor.

Sapecin B alters kinetic properties of rapidly inactivating K(+) channels in rat pituitary GH(3) cells.

Sapecin B is an antibacterial protein isolated and purified from culture medium of the embryonic cell line derived from the flesh fly (Sarcophaga peregrina). It has structural similarities to the scorpion toxin charybdotoxin (CTX). We have first detailed the effects of the newly described toxin (sapecin B) on the gating kinetics of the 4-aminopyridine-sensitive, rapidly inactivating K+ current in rat pituitary GH3 cells in order to investigate this protein’s site of action, with whole-cell voltage-clamp methods. We have found that sapecin B alters the kinetics of activation and deactivation whereas there was no effect on the inactivation process. None of the effects of sapecin B was voltage dependent. In addition, sapecin B reduced whole-cell conductance. We suggest that the toxin may be ineffective against the voltage-sensitive segment, as well as the N-terminal and C-terminal domains, and CTX and sapecin B probably may have different binding sites.

An inter‐oscillator mechanism modulating circadian clock period in paramecium populations

Abstract In populations of the ciliate protozoan, Paramecium multimicronucleatum, the circadian‐clock‐con‐trolled mating reaction expressed by a limited number of cells among them feeds back to contribute to coherence of their circadian rhythms of motility and mating reaction. This eventually causes a decrease in the period of the rhythms from the entrained 24h period to a steady‐state period of about 22h, with the rate of decrease depending on the strength of the mating reaction. These results suggest that the interaction among oscillators may be one of the factors which modulate the period of a circadian clock composed of nearly identical oscillators. The clock‐controlled mating reaction provides a promising inter‐oscillator pathway for obtaining more insight into the mechanism of modulation of the period of such circadian clocks through inter‐oscillator interaction.