Haruto Kuroda

LONG-TERM CULTURED NEURONS FROM RAT SUPRACHIASMATIC NUCLEUS RETAIN THE CAPACITY FOR CIRCADIAN OSCILLATION OF VASOPRESSIN RELEASE

We have developed a dissociated suprachiasmatic nucleus (SCN) cell culture system in order to begin a cellular analysis of the mammalian circadian oscillator. Monolayer-cultured neurons prepared from the SCN regions of 1-day-old rats were maintained in serum-free culture medium for 3-4 weeks and then perfused. In 12 out of 40 wells, a circadian oscillation of vasopressin release persisted for at least 4-5 cycles until the end of the perfusion period with no damping of the amplitude. This finding suggests that cultured SCN neurons retain the capacity for circadian oscillation for a long time, and should provide a useful model for the analysis of a mammalian circadian system at the cellular level.

Individual pineal cells in chick possess photoreceptive, circadian clock and melatonin-synthesizing capacities in vitro

Chick pineal cells express a circadian rhythm of melatonin release under light-dark (LD) cycles, with an increase during the dark period and a decrease during the light period, and this rhythm persists under constant darkness (DD). We cultured individual single pineal cells with 15 microl of medium per well in a Terasaki plate and measured melatonin secretion every 12 h under LD, DL and DD. Individual cells secreted more melatonin during the dark period than during the light period under both LD and DL conditions, and those rhythmic secretions persisted under DD. These results suggest that individual pineal cells in chick have photoreceptive, circadian clock and melatonin-synthesizing capacities.

Rapid reentrainment of the circadian clock itself, but not the measurable activity rhythms, to a new light-dark cycle in the rat

Experiments were performed to determine if the circadian clock reentrains more quickly to an 8-hour phase shift in light-dark (LD) cycles than does the overt rhythm of activity. To investigate the reentrainment of the clock itself to an 8-hour advance or delay in the LD cycle, the rats were released into constant darkness only two or three days after a shift in LD cycle, and the amount of the phase shift of the clock itself was estimated from where free-running rhythm started by backward extrapolation. If the circadian clock could rapidly reset itself to the new LD cycle, it was predicted that the free-running rhythm of activity would start from near the dark period of the new LD cycle rather than the preceding one. When rats were released into constant darkness three days after the LD cycle was advanced by 8 hours, the activity of the free-running rhythm started near time of dark period of the new LD cycle in all rats (n = 16). When rats (n = 24) were released into constant darkness two days after the LD cycle was advanced by 8 hours, 12 rats started the activity near time of dark period of the new LD cycle, while 9 rats started the activity near time of dark period of the preceding LD cycle. The remaining 3 rats showed the activity of the free-running rhythm near intermediate phase (transient phase). On the other hand, when the rats were not released into constant darkness after LD cycle was advanced by 8 hours, it took 6.4 days for activity rhythm to reentrain to the advanced LD cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Daily Wheel Running Activity Modifies the Period of Free-Running Rhythm in Rats Via Intergeniculate Leaflet

The period of free-running rhythms (tau) in rats, as measured using a running wheel, is different from that measured using an Automex. The aim of this work was to examine the effects of lesions of the intergeniculate leaflet (IGL) on the tau of these two activity rhythms. When blind rats were transferred from a cage with a running wheel to a cage without a running wheel, the tau lengthened. The tau of the wheel-running activity was associated with the number of wheel revolutions per day. A complete lesion of the IGL lengthened the tau of the wheel-running activity, and caused a reduction in the number of wheel revolutions per day in all rats. In rats housed in cages without a running wheel, locomotor activity was reduced by IGL lesions, although the tau was unaffected. When IGL-lesioned rats were transferred from a cage with a running wheel to a cage without a running wheel, no further change was observed. These results indicate that the tau is modified by the daily activity of wheel-running, but not by general locomotor activity, and that the IGL may be involved in this modification.

Effects of daily injections of melatonin on locomotor activity rhythms in rats maintained under constant bright or dim light

It has been demonstrated that daily melatonin injections entrain free-running locomotor activity rhythms in rats kept in constant darkness, and synchronize disrupted circadian patterns of wheel-running activity under constant light. Contrary to these previous observations, our result did not show that daily injections of melatonin synchronize disrupted locomotor activity in rats maintained under constant bright light. On the other hand, daily treatment with melatonin entrained the intact free-running rhythm in rats kept in constant dim light. This entrainment took place only when the time of injection corresponded to the activity onset time, and similar results were obtained in blinded rats. Pinealectomy had no influences on either the free-running rhythm or melatonin-induced entrainment. To examine whether a behavioral feedback mechanism is involved in melatonin-induced entrainment, rats were immobilized for 3 h after each daily melatonin injections. This did not interfere with melatonin-induced entrainment. These results suggest that the mechanism underlying melatonin-induced entrainment of activity rhythms may be different from those in photic and behavioral entrainment.

Involvement of protein kinase A in the subjective nocturnal rise of melatonin release by chick pineal cells in constant darkness

Nakahara K, Murakami N, Nasu T, Kuroda H, Murakami T. Involvement of protein kinase A in the subjective nocturnal rise of melatonin release by chick pineal cells in constant darkness. J. Pineal Res. 1997; 23:221–229.

Studies on the storage of dog semen

イヌ精子の保存に関して稀釈液の組成について実験を行なった。1. 実験Iでは塩化マグネシウム,塩化カリウム,第2燐酸ナトリウムおよび塩化ナトリウムから成る基本液に,クエン酸ナトリウムおよび酒石酸カリウム•ナトリウムをそれぞれ加えた緩衝液に対して,卵黄(20%)および牛乳(50%)を加えた稀釈液でイヌ精子の保存試験を行なった。その結果,基本液に卵黄および牛乳を加えた場合は,稀釈直後の生存指数は74から66の間で,実験Iでは最もよい成績を示したが,生存日数は3日以内であった。基本液にクエン酸ナトリウムを加えると,稀釈直後の生存率は低下したが,生存日数は4日であった。酒石酸カリウム•ナトリウムはイヌ精子の保存に有効でなかった。2. 実験IIでは塩化カリウム,第2燐酸ナトリウムおよびクエン酸ナトリウムから成る基本液にグリシソ,ペプトソ,卵黄および牛乳をそれぞれ加えた稀釈液を用いてイヌ精子の保存効果を検討した。基本液にペプトンとグリシンを添加した場合,ペプトンを1g/100 mlの割合に加えると,稀釈ショックが防止され,生存日数は16日に大幅に延長された。しかし,保存第1日目に生存指数は半減するので,さらに組成を改良する必要がある。イヌ精子に対する保護効果は牛乳よりも卵黄の方が優れている。3. 実験IIに用いた稀釈液でイヌ精子に対するクロールプロマジンの保存効果を検討した。クロールプロマジンを稀釈液に5mg/100 mIの割合に添加した場合,基本液にペプトン,グリシンおよび卵黄を加えた稀釈液において,実験IIの成績よりも若干生存率がよくなった。