176 Brain Resc~m t:. 5 7t) ( 1 t)8¢~ ] [~ --~ ~, BRE 21676 The suprachiasmatic nuclei: circadian phase-shifts induced at the time of hypothalamic slice preparation are preserved in vitro MARTHA ULBRICK GILLETTE Department of Physiology and Biophysics. University of Illinois at Urbana-Champaign, Urbana. IL 61801 ( U. S. A. p (Accepted April 1st. 19861 Key words: brain slice circadian rhythm hypothalamus-neuronal oscillator - - phase shift single unit suprachiasmatic nucleus Neurons of the suprachiasmatic nuclei [SCN) of the hypothalamus compose a primary oscillator which organizes circadian rhythms in mammals. In cultured hypothalamic slices from rat brain, the SCN diurnal oscillation m neuronal firing rate continued unperturbed when slices were prepared during the light phase of the donor's light/dark cycle. However. when slices were prepared during the uo- nor's dark period, the rhythm was phase-shifted. The sign and shape of the phase-response relationship for resetting in the isolated os- cillator is very similar to that for intact animals, except that in isolation the SCN oscillator undergoes large shifts during the first cycle. The finding that a phase-shifting stimulus at the time of brain slice preparation causes normal phase readjustment in vitro demon- strates that the underlying mechanism is endogenous to the SCN and can be probed in the brain slice The suprachiasmatic nuclei (SCN) at the base of the mammalian brain orchestrate many daily oscilla- tions of physiological, metabolic and behavioral r h y t h m s 1°,14,16A7,22"23"25"28-31. T h e n e u r o n s of these nuclei exhibit a circadian oscillation in firing rate. which peaks near midday in nocturnal rodents ~3. They can be reset when light stimuli appear during the animal's subjective night so that the oscillation rises and falls at a new phase. They also continue to maintain their characteristic circadian pattern of fir- ing rate whether semi-isolated by knife cuts in vivo 12'13 or removed from the animal and cultured in the hypothalamic slice in vitro 4'5~27. Oscillations in the activity of adjacent hypothalamic nuclei, which control such circadian functions as locomotion, eat- ing, drinking, sleep/wakefulness and hormone re- lease, disintegrate under each condition where inputs from the SCN are removed 12. Thus. the neurons of the SCN form an endogenous oscillator which trans- mits time information to other parts of the brain The discovery that the SCN oscillator survives iso- lation using the hypothalamic brain slice technique 6 and continues apparently unperturbed 4"5. provides compelling evidence that the neurons in the slice pos- sess the information to keep 24 h time. However. no studies have yet focused on the mechanism which drives this neuronal oscillator in isolation. In the course of preliminary experiments aimed at examin- ing the phase-shifting mechanism of the SCN in vitro. I found that the circadian clock is reset at the time the animal is killed and the brain slice prepared if this oc- curs during the dark part of the tight-dark cycle. Fur- ther. the adjustment of the phase of the oscillator takes place apparently normally during the first cir- cadian cycle in vitro. These studies were performed on 2-5-month-old Long-Evans rats which had been bred and reared in our laboratory to reduce individual differences. Ani- mals were housed on 12:12 h light-dark cycle, They had been handled at random intervals to reduce stress at the time of decapitation. Each rat was main- tained on its entrained lighting condition until initia- tion of the brain slice procedure. At that moment, the donor was taken from the cage. into laboratory light- Correspondence: M.U. Gillette. Department of Physiology and Biophysics and the Neural and Behavioral Biology Program. 524 Burrill Hall. University of Illinois at Urbana-Champaign. 407 Goodwin Avenue. Urbana. IL 61801. U.S. A 0006-8993/86/$03.50© 1986 Elsevier Science Publishers B.V. (Biomedical Division)