The Journal of Neuroscience, January 1988, 8(i): 2230 Circadian Clock in Cell Culture: II. /n vitro Photic Entrainment of Melatonin Oscillation from Dissociated Chick Pineal Cells Linda M. Robertson and Joseph S. Takahashi Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60201 The avian pineal gland contains circadian oscillators that regulate the rhythmic synthesisof melatonin (Takahashi et al., regulate the rhythmic synthesis of melatonin. We have de- 1980; Menaker and Wisner, 1983; Takahashi and Menaker, veloped a flow-through cell culture system in order to begin 1984b). Previous work has shown that light exposure in vitro to study the cellular and molecular basis of this vertebrate can modulate N-acetyltransferase activity and melatonin pro- circadian oscillator. Pineal cell cultures express a circadian duction in chick pineal organ cultures (Deguchi, 1979a, 1981; oscillation of melatonin release for at least 5 cycles in con- Wainwright and Wainwright, 1980; Hamm et al., 1983; Taka- stant darkness with a period close to 24 hr. In all circadian hashi and Menaker, 1984b). Although acute exposure to light systems, light regulates the rhythm by the process of en- can suppressmelatonin synthesis, photic entrainment of cir- trainment that involves control of the phase and period of cadian rhythms in the pineal in vitro has not been definitively the circadian oscillator. In chick pineal cell cultures we have demonstrated. Preliminary work hassuggested that entrainment investigated the entraining effects of light in 2 ways: by shift- may occur; however, none of these studies demonstrated that ing the light-dark cycle in vitro and by measuring the phase- the steady-state phase of the oscillator was regulated by light shifting effects of single light pulses. A 6 hr advance or delay (Deguchi, 1979b; Kasal and Perez-Polo, 1980; Takahashi and of a LD 12:12 light-dark cycle produced a corresponding Menaker, 1984b). shift in the melatonin rhythm. The phase shifts of the rhythms In the previous paper we reported that dissociatedchick pineal persisted after transfer to constant darkness, showing that cell cultures expressa circadian oscillation of melatonin release the underlying circadian oscillator was entrained. Photic en- (Robertson and Takahashi, 1988). The rhythm persistsin con- trainment of the oscillator was further characterized by mea- stant darknessfor at least 5 cycles; however, the amplitude of suring the phase-shifting effects of single 6 hr light pulses. the rhythm damps. When maintained in a lightdark cycle the Single pulses of light shifted the phase of the circadian os- cells express a high-amplitude melatonin rhythm. The main- cillator in a phase-dependent manner. Light pulses begin- tenance of a high-amplitude oscillation in a light-dark cycle ning early in the subjective night delayed the phase of the indirectly suggeststhat the dispersedpineal cells are photore- oscillation 6 hr relative to dark controls. Conversely, light ceptive in culture. In addition, the rhythm does not appear to pulses beginning late in the subjective night advanced the be entirely driven by the light-dark cycle, but rather exhibits phase of the oscillation nearly 6 hr. Thus, photoreceptors an anticipatory increaseprior to lights out and a decreaseprior within the cell cultures can mediate entrainment of the pineal to lights on. Furthermore, constant light exposure lengthensthe oscillators. Phase-dependent phase shifts to light pulses (as free-running period of the melatonin rhythm. The effects of light described in a “phase-response curve”) are a fundamental on the period length demonstrate that photic information has characteristic of all circadian systems. The expression of input to the circadian oscillator. Taken together, these results this circadian property in the pineal cell cultures should per- suggestthat light may be capable of entraining the circadian mit a mechanistic analysis of the photic entrainment path- oscillation in pineal cell cultures in vitro. We have tested this way. hypothesis directly by investigating the effects of shifted light- dark cycles and the effects of singlelight pulsesupon the steady- Environmental light cycles regulateor entrain circadian rhythms state phaseof the melatonin rhythm in pineal cell cultures. We by shifting the phase of the circadian oscillator underlying the report here that light exposure in vitro can entrain the circadian rhythm. Although the formal properties of entrainment have oscillation of melatonin releasefrom dissociatedchick pineal been thoroughly studied (Pittendrigh, 198 l), little information cells. exists concerning the cellular and biochemical mechanismsthat regulate circadian oscillators, especially among vertebrates Materials and Methods (Takahashi and Zatz, 1982; Jacket, 1984; Takahashi and Men- Animals aker, 1984a). In birds and reptiles, the isolated pineal gland Newly hatched male chicks (GuNus domesticus,white leghorn) were contains circadian oscillators, with photoreceptive input, which purchased from Combelt hatcheries (Forest, IL). The animals were raised under 12 hr light: 12 hr dark (LD 12: 12) lighting regime with lights on at 0700 CST for 3-6 weeks prior to study. Food (Purina Chick Startena) Received Dec. 31, 1986; revised July 16, 1987; accepted July 16, 1987. and water were available continuously. This work was supported by NIMH Grant MH-39552, NSF Presidential Young Investigator Award DCB-845 1642, and Searle Scholars Award 85-H- 107 to J.S.T. Cell culture Correspondence should be addressed to Dr. Takahashi, Department of Neu- robiology and Physiology, Northwestern University, Hogan Hall, Evanston, IL The flow-through cell culture procedure used in these experiments has 6020 1. been described in detail in the preceding paper (Robertson and Taka- Copyright 0 1988 Society for Neuroscience 0270-6474/88/010022-09$02.00/O hashi, 1988). For each experiment 10 pineal glands were enzymatically The Journal of Neuroscience, January 1988, 8(l) 23 -r T T A i B Figure 1. In vitro entrainment of cir- cadian oscillation of melatonin release from dispersed chick pineal cells. Points and bars represent mean k SEM of 3 A replicate cell chambers. Dark bars in- dicate the light-dark cycle. Flow rate was 0.5 ml/hr. All cell chambers were exposed to LD 12:12 to ensure initial synchrony of the oscillation. A, Six-hour I advance of light-dark cycle advanced the melatonin oscillation in cyclic light fL2 72 96 I20 144 I 6E and constant conditions. B, Six-hour delay of light-dark cycle delayed the melatonin oscillation in cyclic light and TIME IN HOURS constant conditions. dispersed with collagenase (1 mg/ml) and collected by differential cen- method for calculating this reference point has been described in detail trifugation. The dispersed cells were maintained in culture medium with (Robertson and Takahashi, 1988). The midpoint phase reference is the following composition: Medium- 199 with Hank’s salts and L-&I- defined as the time midway bewteen the half-rise and half-fall of each tamine (GIBCO, #400-l 200) supplemented with 10 mM HEPES buffer melatonin peak. (US Biochem), 5% fetal bovine serum (Biologos, # 12 10378), 10% heat- Design of perturbation experiments. Measurement of the phase-re- inactivated horse serum (GIBCO, #200-2050), 100 U/ml penicillin G, sponse curve required 3 sets of experiments. One experiment involved 100 pg/ml streptomycin, and 0.9 mg/ml NaHCO, supplemented for 5% 1 experimental group and 1 control group (4 replicates each per con- CO, atmosphere. Cytodex 3, collagen-coated microcarriers (Pharmacia) dition, a total of 8 cell chambers). The remaining 2 experiments involved were used as a substrate for cell attachment. The cells were maintained 4 or 5 experimental groups and 1 control group (4 or 5 replicates per in an incubator at 37°C with 95O’a sir/5% CO, and exposed to LD 12: group, in each experiment). Each group of experimental chambers was 12 lighting conditions. Half the medium was changed every other day. exposed to a single pulse of light. The light intensity at the level of the After 4-5 d in culture, the cells were loaded into the flow-through ap- chambers was 100 pW/cm2. The subsequent rhythm was assayed in paratus described in the previous paper. The cell chambers were housed constant darkness to determine the phase of the oscillation. within light-controlled boxes fitted with a 4 W fluorescent bulb and a Calculations ofphase shifts. The phases of each culture within a given Schott KG1 heat filter. The entire apparatus was maintained at 37°C. experimental group were averaged for each cycle. The mean of the Approximately l-2 x lo5 cells were loaded into each chamber. For experimental group was subtracted from that of a control group to flow-through experiments an air-equilibrated medium was used with determine the magnitude of the phase difference. Significance was de- the following composition: Medium- 199 with Hank’s salts and L- glu- termined using analysis of variance followed by Student’s t test for tamine supplemented with 10 mM HEPES buffer, 5% fetal bovine serum, pairwise comparison of treatment means, and 95% confidence limits 10% heat-inactivated horse serum, and 50 &ml gentamicin. Medium were calculated using the SDS of the 2 groups to calculate a weighted was infused through the cell chambers at a rate of 0.25-0.5 ml/hr de- SD for the difference of the mean values. pending on the experiment. Two-hour fractions were collected and as- Normalization. In order to reduce the differences among cell chambers sayed for melatonin content using a specific radioimmunoassay as pre- in the absolute amplitude of the melatonin rhythm, some of the data viously described (Takahashi et al., 1980; Robertson and Takahashi, are presented in normalized form relative to the mean value of the 1988). record. This was performed by dividing the melatonin values from each channel by the mean value of the time series of that channel and mul- tiplying by 100.