Cyclic nucleotides and circadian rhythm generation in Bulla gouldiana

Camp. Eiochem. Physiol. Vol. IOIA,No. 4, pp. 813-817, 1992 F’rintcdin Great Britain

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0300-9629192 $5.00+ 0.00 0 1992Pergamon Press plc

NUCLEOTIDES AND CIRCADIAN RHYTHM GENERATION IN BULLA GOULDIANA MARTIN R. RALPH,* SAT BIR S. KHALSAand GENED. BLCICK

Department of Biology, University of Virginia, Charlottesville, VA 22901, U.S.A. 1 July 1991)

(Received

Abstract-l. Phase shifts of the ocular circadian rhythm in isolated eyes of the marine snail, Bulla gouldiana were produced by 3- and 6-hr pulses of agents that increase or mimic cyclic AMP (CAMP) activity. 2. The magnitude and direction of phase shifts depended upon the circadian phase of the rhythm, and the phase response curve was similar to that produced by membrane hyperpolarizing treatments in this species. 3. Phase shifts induced by CAMP were eliminated by a depolarizing agent, tetraethylammonium, applied simultaneously to experimental and control eyes. 4. As in other species, CAMP may regulate circadian rhythms in Bulla, possibly through effects on membrane potential.

INTRODUCIION eyes of Bulla gouldiana, like those of other opisthobranch mollusks, contain circadian pacemakers (Block and Wallace, 1982; for review, see Jacklet, 1979). Pacemaker activity is thought to be a property of a group of about 100 electrically coupled cells called basal retinal neurons (BRNs) located in an anatomically distinct region beneath the retina (Block and Wallace, 1982; Block and McMahon, 1984). The synchronous firing of these cells produces compound action potentials (CAPS) in the optic nerve whose frequency varies over the circadian cycle. In constant conditions, the abrupt onset of CAP activity marks the beginning of the subjective day. CAP rate is highest at subjective dawn, remains high during the subjective day and is lowest during the subjective night. A circadian rhythm in BRN membrane potential appears to drive the rhythm in CAP frequency (Block et al., 1984; McMahon et al., 1984). In vitro analysis of ocular circadian rhythms in two opisthobranchs, Aplysia californica and Bulla gouldiam, has provided important information regarding the ionic and biochemical mechanisms that underlie the generation and control of rhythmicity. In both organisms, membrane depolarization appears to be necessary for light induced phase shifts (Eskin, 1977; McMahon and Block, 1987) and at least in Bulla, these responses require extracellular calcium acting apparently via a depolarization induced calcium flux (McMahon and Block, 1987; Khalsa and Block, 1988). Roles for cyclic nucleotides have been indicated in the regulation of rhythmicity in various, unrelated species. In Aplysiu, cGMP may be involved in the phase shifting effects of light (Eskin et al., 1984), and CAMP may mediate the phase shift response to The

*To whom all correspondence should be addressed. Current address: Department of Psychology, University of Toronto, Toronto, Canada, M5S lA1.

serotonin (Eskin et al., 1982; Eskin and Takahashi, 1983). In addition, manipulation of cyclic nucleotide activity has been reported to alter rhythmicity in rodents, plants and fungi (for review, see Edmunds, 1988). These results raise the possibility that cyclic nucleotides are ubiquitous factors among circadian systems. To further elucidate the general biochemical mechanisms underlying rhythm generation and control, it is important to know whether circadian systems in different species share common elements. Phase dependent effects of cyclic nucleotides have not been demonstrated for the ocular rhythm of Bulla, and unlike the case for Aplysiu (Corrent et al., 1978) the rhythm does not respond to serotonin with predictable phase shifts (McMahon, 1986). Furthermore, serotonergic neurons which may be involved in central control of ocular rhythmicity in Aplysiu, have not been identified in the Bulla eye (Takahashi and Eskin, 1985). However, these findings do not preclude a role for CAMP in the circadian system of this species. For this reason, we have examined the involvement of CAMP in the regulation of ocular rhythms in Bulla. MATERIALS AND METHODS Bulla gouldiana were obtained from Alacrity Marine Supply, Redondo Beach, CA and from Marinus, Long Beach, CA, and were maintained in a temperature controlled seawater tank at 15°C. Animals were exposed to a light cycle of 12 hr light and 12 hr dark (LD 12:12) for at least 1 week prior to the start of an experiment. Prior to dissection, animals were immobilized with an injection of 10 ml of isotonic MgCl,, and then placed on ice. The eyes, including the optic nerves, were removed from each animal and placed in a dish of artificial seawater (ASW, 20ml per dish). The composition of ASW was 395 mM NaCl, 1OmM KCI, 1OmM CaCl,, 50mM MgCl,, 28mM Na, S04, 30 mM Hepes buffer, 100,000 units penicillin/l and 100,OOOyg streptomycin/l. All eyes were prepared in the light during the last 2 hr of the preceding lightdark cycle.

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An experimental eye was always paired with a control from the same animal. For extracellular recordings, the optic nerve was pulled up into a Seawater-filled suction electrode mounted on to the side of the dish. Each dish was fitted with a fill/drain tube for fluid exchanges and was placed in constant dark in a light-tight recording chamber for the duration of the experiment. Fluid exchanges were accomplished in total darkness, and control eyes always received a pulse of ASW vehicle solution. Compound action potentials (CAPS) were displayed on a polygraph and were counted by computer in ISmin bins for analysis. The reference point used in these studies was the half-maximum point during the rising phase of the daily CAP cycle. phase shifts were determined as the change in the timing of

the rhythm of the experimental eye with respect to its genetically matched control, on the second cycle following an experimental manipulation (see Khalsa and Block, 1988).

Application of the CAMP anafog, 8-bromo-camp (&Br-CAMP) (2mM), during the subjective day resuited in a reduction in the frequency of CAPS produced by each eye. CAPS were completely suppressed at the beginning of the pulse and sometimes were eliminated for the entire pulse; however, in most cases, CAPS resumed at a low rate after a short period of inactivity (20-60 min). CAP frequency returned to control bvels within 30min following the end of a pulse. When applied in 3-hr pulses, 8-Br-CAMP produced phase shifts of the circadian rhythm (Fig. 1) whose magnitude and direction were dependent upon the phase of the circadian cycle. The phase response curve (PRC) for 3-hr pulses is shown in Fig. 2. Phase delays of the rhythm were produced in the early subjective day (circadian time: CT O-6) and phase advances were produced in the early subjective night (CT 12-18). Longer pulses (6 hr) of 8-Br-CAMP produced phase delays when applied at CT 23-S or CT 2-8 and phase advances when applied at CT

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Fig. 2. Phase dependent effects of cyclic AMP pulses on the phase of the CAP rhythm. Closed circles: 3 hr pulses of 8-Br-CAMP, open circles: 6 hr pulses of S-Br-CAMP, triangles: 6 hr pulses of forskolin. Each point represents the beginning of a pulse.

1l-17 (Fig. 2). These shifts were no larger than those induced by the 3-hr pulses, and for these circadian times, were similar in magnitude and direction to shifts induced by hyperpolarizing agents (Khalsa and Block, 1990). Effects of forskolin and caffeine The adenylate cyclase activator, forskohn (1 PM), produced both phase delays and advances that were similar in ma~itude, direction and circadian timing to those produced by 3- and 6-hr pulses of 8-BrCAMP (Fig. 2). In addition, forskolin had similar acute effects on CAP frequency. With the exception of a 2-hr interval near projected dawn, the effects of caffeine on phase (Fig. 3) were essentially identical to those of forskolin and 8-BrCAMP. Phase delays occurred in the early subjective day, the delays were reduced in the late subjective day and phase advances occurred in the early subjective night. Around subjective dawn, however, the responses to caffeine were not similar to the CAMP analog. When pulses ended just prior to the projected onset of CAP activity, large phase advances were produced. The magnitude of the advances were reduced if pulses ended after CAP activity (in the control eye) had commenced. The largest phase delays were obtained if the caffeine pulse began just

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TIME (days) Fig. 1. Phase shift of the ocular rhythm induced by a 3 hr pulse of 2mM 8-Br-CAMP. Dotted line represents the experimental eye; solid line represents the paired ASW control. Beginning and end of the 8&-CAMP pulse are indicated by closed and open triangles, respectively. The pulse, given at CT 9-12 caused a 5%min phase advance.

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Fig. 3. Phase response curve for 3 hr pulses of 5mM caffeine. Each point represents the beginning of a pulse.

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prior to the onset of CAPS and extended through projected dawn (Fig. 4). The acute effect of caffeine on CAP activity during the subjective day was highly variable and ranged from complete suppression of activity to production of slow oscillations in CAP frequency. Like the response to %Br-CAMP, CAPS were suppressed initially but usually resumed during the pulse. Pulses that extended through subjective dawn caused a delay in the onset of CAPS on that cycle. A strong activity onset was never eliminated by caffeine at this circadian time point. Furthermore, the onset of CAPS during dawn pulses was abrupt and of high amplitude, unlike the relatively slow resumption of CAP activity observed during pulses given at other times during the subjective day. None of the treatments described here induced CAP activity during the subjective night when the eye was quiescent, nor did the return to normal seawater following a pulse induce CAP activity at this time. Interactions between CAMP and a depolarizing treatment Because CAMP analogs cause membrane hyperpolarization (McMahon and Block, 1987) that might account for some of these results, we asked whether the phase shifting effects of 8-Br-CAMP could be blocked by TEA, an agent known to cause membrane depolarization. Unfortunately, the PRCs for 8-BrCAMP and for depolarization (see Eskin, 1977) overlap to the extent that a block of one response by the other treatment could always be considered additive. In these experiments, therefore, TEA was added to both experimental and control eyes at CT 2-8 where 8-Br-CAMP had been found to induce delays and TEA (200 mM) to induce advances (shift = + 84 f 55 min; N = 6). Phase delays induced by 8-Br-CAMP were reduced to insignificance by TEA at 100 and 200mM (Fig. 5). Both TEA alone and TEA plus 8-Br-CAMP caused an increase in the frequency of

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Fig. 4. Phase shift of the ocular rhythm induced by 5 mM caffeine. Dotted line represents the experimental eye; solid line represents the paired ASW control. Beginning and end of the caffeine pulse are indicated by closed and open triangles, respectively. The pulse, given at subjective dawn, CT 23-02, caused a 188 min phase delay.

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bathing medium. DISCUSSION

The data presented here suggest that CAMP may play a role in the ocular pacemaker system in Bulla. In Aplysia, CAMP is thought to mediate the phase shift response to exogenously applied serotonin (Eskin et al., 1982; Eskin and Takahashi, 1983). In these animals, serotonergic neurons that innervate the eyes may be involved in modulatory control of pacemaker activity. While a CAMP-mediated input to the pacemaker, like serotonin in Aplysia, has not been identified in Bulla, CAMP appears to have access to the pacemaker system. Therefore, any perturbation that results in a change in CAMP metabolism or activity has the potential of altering pacemaker activity. The phase dependency of the responses to 8-BrCAMP and to forskolin suggest that CAMP might occupy a similar position in the circadian systems of Bulla and Aplysia. In both of these organisms, the PRC for CAMP is approximately 180” out of phase with the PRC to light. Furthermore, in both species, the PRCs to CAMP are similar in magnitude, direction and circadian timing to those for hyperpolarizing treatments (0 K+ in Aplysia, Eskin et al., 1982; low Nat and direct current injection in Bulla, McMahon and Block, 1987) and for pulses of low Ca2+ seawater in Bulla (Khalsa and Block, 1990). It is possible, therefore, that all three treatments, CAMP, hyperpolarization and low Ca2+, affect the same or convergent pathways to the pacemaker. The similarities in the effects of hyperpolarization and increased CAMP activity support the suggestion of McMahon and Block (1987) that CAMP-induced phase shifts are mediated via membrane hyperpolarization. This interpretation is strengthened by the finding that TEA, a depolarizing agent that acts by blocking K+ channels, eliminates phase delays induced by 8-Br-CAMP. This does not appear to be a simple addition of two opposite phase shift responses since the effects of the CAMP analog were blocked when TEA was applied to both eyes during the pulse. Nor is it likely that the reduction is due to the two agents effectively acting at different circadian phases:

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the magnitude of the shift induced by TEA would not have moved the pacemaker to a CAMP-insensitive phase. Although the most parsimonious explanation for these results therefore, is that the effects of CAMP activators are mediated through membrane hyperpolarization, the case for hyperpolarization alone is still not certain. It is possible that CAMP and hyperpolarization are on different but convergent pathways to the pacemaker, and that CAMP either incidentally or indirectly influences membrane potential. TEA could act by cancelling out the hyperpolarization induced by CAMP as well as acting directly on a CAMP sensitive mechanism. The latter possibility implies that in this case, shifts induced by membrane hyperpolarization are secondary effects of the CAMP analog. One possible alternative is that all of the treatments used have some effect on protein synthesis. Protein synthesis inhibitors can cause phase shifts of the rhythm in Aplysia (Rothman and Strumwasser, 1976; Jacklet, 1977; Lotshaw and Jacklet, 1986), and can block serotonin-induced phase shifts (Eskin et al., 1984). It is possible that membrane depolarization also leads to an inhibition of protein synthesis in pacemaker neurons, thereby reducing the phase shifting response to CAMP. Therefore, although a direct effect of CAMP on membrane potential appears to be the most likely explanation for all of our results, separate mechanisms for CAMP and membrane potential cannot be ruled out entirely. For the most part, the effects of caffeine on the pacemaker strengthen the interpretation that CAMP plays a role in the regulation of rhythmicity in Bulla. Nearly all of the phase shifting effects of caffeine can be explained by its anti-phosphodiesterase activity. Except for a few hours before and after projected dawn, the PRCs for caffeine and 8-Br-CAMP are essentially identical. At subjective dawn, however, the two response curves are dissimilar: caffeine produced large phase advances prior to dawn when &Br-CAMP had little effect; caffeine produced very large phase delays if the pulse coincided with subjective dawn, when %Br-CAMP produced only small delays. This is probably not a trivial difference between the potency of the two agents since (1) the concentration of the analog used for the PRC (2 mM) appears to be saturating, 5 mM pulses had no additional effect at CT 13-16 (shift = +45 min, N = 2); (2) the size of the shifts induced by forskolin were similar to 8-Br-CAMP; and (3) the effects of 8-Br-CAMP and caffeine are similar throughout most of the circadian cycle and the maximum difference occurs at a point where the system is least responsive to 8-Br-CAMP. A more likely explanation is that caffeine has more than one effect on the pacemaker. One documented effect of caffeine is the release of Ca2+ from intracellular stores (Thayer et al., 1988). Calcium regulation is important for normal circadian timekeeping in Bulla, and agents that are known to interfere with intracellular Ca2+ regulation can have profound effects on pacemaker activity (Woolum and Strumwasser, 1983). Thus, it is reasonable to assume that caffeine might exert dual effects on the pacemaker; one,

RALPH et al.

through its anti-phosphodiesterase activity, and a second, through its effect on Ca2+ stores. Involvement of cyclic nucleotides, especially CAMP, has been suggested in the regulation of circadian timekeeping in a variety of species. Like circadian responses to light, the responses of different circadian systems to CAMP perturbations differ considerably in amplitude but show similarities in the timing and direction of phase shifts. This suggests the possibility of a general role for cyclic nucleotides among circadian timekeeping systems. Variations in responsiveness among diverse species, rather than indicating fundamental differences in the organization of each pacemaker, may reflect changes in the way that cyclic nucleotides are used to transduce endogenous and environmental signals.

REFERENCES

Block G. D. and McMahon D. G. (1984) Cellular analysis of the Bulla ocular circadian pacemaker system: III. Localization of the circadian pacemaker. J. camp. Physiol. 155A, 387-395. Block G. D., McMahon D. G., Wallace S. H. and Friesen W. 0. (1984) Cellular analysis of the Bufla ocular circadian pacemaker system: I.-A model for retinal organization. J. camp. Phvsiol. MA, 365-378. Block G. D. and Wallace S. Fl (1982) Localization of a circadian pacemaker in the eye of a mollusc, Bulla. Science 217, 155457. Corrent G., McAdoo D. J. and Eskin A. (1978) Serotonin shifts the phase of the circadian rhythm from the Aplysia eye. Science 202, 977-979.

JZdmunds L. N. (1988) Cellular and Molecular Bases of Biological Clocks, pp. 262-264 and 334-336. Springer, New York. Eskin A. (1977) Neurophysiological mechanisms involved in photoentrainment of the circadian rhythm from the Aplysia eye. J. Neurobiol. 8, 273-299.

Eskm A., Corrent G., Lin C.-Y. and McAdoo D. J. (1982) Mechanism for shifiina the chase of a circadian rhythm by serotonin: involvement of CAMP. Proc. natn. Acad. Sci. U.S.A. 79, 660-664.

Eskin A. and Takahashi J. S. (1983) Adenylate cyclase activation shifts the phase of a circadian pacemaker. Science 220, 82-84.

Eskin A., Takahashi J. S., Zatz M. and Block G. D. (1984) Cyclic guanosine 3’: S-monophosphate mimics the effects of light on a circadian pacemaker in the eye of Aplysia. J. Neurosci. 4, 2466-247 1.

Eskin A., Yeung S. L. and Klass M. R. (1984) Requirement for protein synthesis in the regulation of a circadian rhythm by serotonin. Proc. natn. Acad. Sci. U.S.A. 81, 7637-7641.

Jacklet J. W. (1977) Neuronal circadian rhythm: phase shifting by a protein synthesis inhibitor. Science 7, 69-71. Jacklet J. W. (1979) Circadian neuronal oscillators. In Neuronal and Celhdar Oscillators (Edited by Jacklet J. W.).,. __ DD. 483-527. Marcel Decker, New York. Khalsa S. B. S. and Block G. D. (1988) Calcium channels mediate phase shifts of the BuUa circadian pacemaker. J. camp. Physiol. 164A, 195-206. Khalsa S. B. S. and Block G. D. (1990) Calcium in the phase control of the BuNa circadian pacemaker. Brain Res. 506, 40-45.

Lotshaw D. P. and Jacklet J. W. (1986) Involvement of protein synthesis in circadian clock of Aplysia eye. Am. J. Physiol. 250, R5-Rll.

Cyclic nucleotides and circadian rhythms McMahon D. G. (1986) Cellular mechanisms of circadian pacemaker entrainment in the mollusc, Bulla. PhD Dissertation, Dept Biology, University of Virginia, Charlottesville, VA. McMahon D. G. and Block G. D. (1987) The Bulla ocular circadian pacemaker. I. Pacemaker neuron membrane potential controls phase throuah a calcium-dependent mechanism. J. co& Physiol. lalA, 335-346. _ McMahon D. G.. Wallace S. H. and Block G. D. (1984) Cellular analysis of the BuNa ocular circadian pacemaker system. II. Neurophysiological basis of circadian rhythmicity. J. camp. Physiol. MA, 379-385. Rothman B. S. and Strumwasser F. (1976) Phase shifting the circadian rhythm of neuronal activity in the isolated

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Takahashi J. S. and Eskin A. (1985) Serotonergic innervation of moluscan eyes containing circadian pacemakers. Sot. Neurosci. Abstr. 11, 537. Thayer S. A., Himing L. D. and Miller R. J. (1988) The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro. Mol. Pharmac. 34, 664-673. Woolum J. C. and Strumwasser F. (1983) Is the period of the circadian oscillator in the eye of Aplysia directly homeostatically regulated? J. camp. Physiol. 151A, 253-259.