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Cellular aspects of molluskan biochronometry
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CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 781–789
Cellular aspects of molluskan biochronometry David Whitmore and Gene D. Block
The eyes of the marine gastropods Aplysia californica and Bulla gouldiana contain circadian clocks. In both organisms retinal circadian rhythms are generated by individual neurons that express a circadian rhythm in impulse frequency. Phase control of the rhythm, either by light or by serotonin, is mediated by changes in membrane potential affecting a transmembrane calcium flux. Whereas transmembrane ionic fluxes underlie pacemaker entrainment and expression, the generation of the circadian rhythm involves molecular transcription and translation. Both processes appear to be elements in a feedback loop generating the circadian cycle.
nucleus (SCN) also contain competent pacemakers (refs 5-8; see also Zatz, this issue). These studies clearly indicate that the investigation of circadian rhythm generation must focus on intracellular processes rather than on the interactions between neurons or other cells within tissues. Nonetheless, circadian systems exhibit many highly adaptive attributes such as history-dependent period and waveform lability (i.e. ‘after-effects’); and it is not at all certain whether individual pacemaker cells, isolated from tissue interactions and competent to generate a circadian periodicity, will be capable of exhibiting the full range of circadian attributes. Ultimately we may discover that several levels of organization contribute to the sophisticated chronometry exhibited by multicellular organisms.
Key words: Aplysia / Bulla / circadian / entrainment / retina ©1996 Academic Press Ltd
THE EYES OF A NUMBER of opisthobranch mollusks express circadian rhythms in retinal electrical activity.1,2 The most intensively studied of the mollusks, Aplysia californica and Bulla gouldiana, have become important model systems for the cellular and molecular study of circadian rhythmicity. The ability to study rhythmicity in vitro, along with the many technical opportunities afforded by the gastropod nervous system, has provided an unparalleled opportunity to study circadian organization at many levels, from molecular events involved in rhythm generation to multi-oscillator organization and its control of rhythmic behaviors. In Bulla gouldiana single neurons located at the base of the eye are capable of generating a circadian periodicity in membrane conductance when isolated in culture.3 The same appears to be the case in Aplysia californica in which dispersed retinal neurons have been reported to undergo circadian cycling.4 In addition, it is likely, although not completely proven, that single pinealocytes from the chick and single neurons from the mammalian suprachiasmatic
The circadian system of Bulla and Aplysia What we presently understand about the circadian system within the opisthobranch retina is summarized in Figure 1. Conceptually, the system can be divided into three sections: (1) an input pathway which provides synchronizing information from the environment and from other intrinsic circadian oscillators, (2) the oscillator, a feed-back loop (or loops) that generates the periodicity, and (3) an output pathway that couples the oscillatory signals to target sites. The cellular and molecular mechanisms subserving these functions are discussed below.
Pacemaker entrainment The molluskan retina has provided important information regarding the physiology of entrainment. In the case of light-induced phase shifts, the pathway appears similar in both Bulla and Aplysia. Light pulses lead to an increase in cGMP levels in the Aplysia retina, and analogs of cGMP cause phase shifts that mimic those of light.9 cGMP does not have the same effect in Bulla, but differences in membrane permeability to the analog are the most likely explanation. In Aplysia cGMP increases the number of compound action potentials (CAPs) recorded extracellularly in
From the NSF Center for Biological Timing, Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA ©1996 Academic Press Ltd 1084-9521/96/060781 + 09 $25.00/0
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Figure 1. A model of the cellular events within a molluskan pacemaker cell. Subjective day: Bulla BRNs are relatively depolarized due to a reduction in membrane conductance which occurs at projected dawn. This conductance decrease is due to the closure of potassium channels, which we propose is caused by tyrosine kinase-mediated phosphorylation. As a consequence of persistent depolarization and impulse production we suspect that intracellular calcium levels are higher during the subject day than the subjective night. During the whole subjective day (CT 0–12) transcription is critical for the motion of the circadian pacemaker. Translation appears to be required for a shorter duration of time around projected dawn. Protein synthesis is required for phase shifts during the subjective day. Subjective night: During the subjective night many cellular conditions are reversed. Membrane conductance is high, resulting in a relatively hyperpolarized state. This is due to the opening of potassium channels at dusk, possibly by a tyrosine phosphatase and dephosphorylation. Intracellular calcium levels should be relatively low. Active transcription and translation are not critical events for the continued motion of the pacemaker until a few hours before dawn. Phase shifts are produced by light and other treatments which depolarize the pacemaker cell and lead to an increase in intracellular calcium. The mechanism of action of calcium on the circadian pacemaker is unknown; it does not appear to act via increased protein synthesis or transcription.
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Cellular aspects of molluskan biochronometry W-7 and calmidazolium, fail to block light-induced phase shifts.15. Subjective-day phase shifts in the electrical rhythm can be produced in both molluskan preparations. In Bulla the neurotransmitter FMRFamide,16 cAMP analogs,17 hyperpolarizing treatments,10,11 as well as low calcium treatments all lead to phase shifts primarily during the subjective day.18 A conclusion drawn from these results is that treatments that reduce a transmembrane calcium flux during the subjective day produce similar phase shifts.2,10 In general there appear to be two classes of phase response curves (PRCs). The most common PRC, typical for those obtained to light pulses (‘light-pulse PRC’), is one with the break-point between delays and advances in the mid subjective night. A second class of PRCs, where the breakpoint between delays and advances lies in the mid subjective day, is displaced approximately 180° on the time axis from light pulse PRCs; these are sometimes referred to as ‘dark-pulse PRCs’, because rodents maintained in continuous light but given interruptions of darkness generate this type of response.19 A model has been proposed for Bulla in which the two families of phase response curves derive from modulation of a single common process, a transmembrane calcium flux.2,10,18 In this scheme, during the subjective night — a time when the membrane of pacemaker neurons is hyperpolarized — phase shifts to light and other depolarizing agents are due to an increase in intracellular calcium.10 In contrast, phase shifts during the subjective day are generated by reducing a transmembrane calcium flux. During the subjective day the membrane is depolarized by approximately 13 mV compared to the subjective night.20 Treatments which hyperpolarize the membrane during the subjective day, leading to a cessation of impulse activity (thus presumably reducing a transmembrane calcium flux) generate a family of PRCs of similar shape with the transition between delays and advances occurring near Circadian Time 6 (in mid subjective-day).2 In Aplysia phase shifts occurring during the subjective day have been extensively studied by Eskin and colleagues.21 Pulses of the neurotransmitter serotonin (5 HT) phase shift the Aplysia pacemaker primarily during the subjective day.22 Serotonin is present in efferent termini that synapse in the base of the retina.23 The application of serotonin pulses leads to an increase in intracellular cAMP levels, activating adenylate cyclase, presumably through a G-protein dependent mechanism.24,25 Analogs of cAMP, in
the optic nerve, suggesting that cGMP generates a membrane depolarization. Depolarizing treatments produce phase shifts in Bulla that mimic those of light, and hyperpolarization applied concurrently with light blocks the light-induced phase shift.10 The light-induced membrane depolarization leads to a transmembrane calcium flux that is required for the phase shift. Simultaneous application of low calcium/ EGTA seawater and light blocks the light-induced phase shift, as does the application of the Ca2 + channel blocker nickel chloride, suggesting a role for calcium channels in this pathway.10,11,28 Geusz and coworkers have obtained direct measurements of a sustained increase in intracellular calcium following the depolarization of basal retinal neurons (BRNs) by use of the calcium indicator dye, fura-2.12 Intracellular calcium levels remain high for the duration of the phase shifting treatment, suggesting that raised intracellular calcium can encode for the duration of the environmental stimulus. It has yet to be demonstrated, however, that solely increasing intracellular calcium, without membrane depolarization, is sufficient to generate a phase shift. The ‘step’ following an increase in intracellular calcium is presently unclear. It appears unlikely that either protein synthesis or transcription are components of the light-entrainment pathway. The application of anisomycin or cycloheximide, two commonly used protein synthesis inhibitors, do not appear to block light-induced phase delays in the electrical rhythm in either molluskan species.13,14 The issue of the role of protein synthesis in light-induced phase advances is complicated by the impact of these inhibitors on the motion of the pacemaker in the late subjective night (see later). Changes in protein synthesis, as measured by radiolabelling proteins followed by two-dimensional SDS polyacrylamide protein gel electrophoresis, have been reported in the Aplysia retina.13 However, a search for light-induced proteins by the same methods in Bulla has failed to reveal any large amplitude and reliable changes (D. Whitmore, unpublished observations). The reversible transcription inhibitor 5,6-dichlorobenzimidazole riboside (DRB), when applied concurrently with light, does not block light-induced phase delays in Bulla; this suggests that an initiation of transcription is also not required for light-induced phase shifts (B. Bogart et al, unpublished observations). The role of specific kinases has not been extensively examined. There is a report, however, that calcium calmodulin-dependent protein kinase is not involved in entrainment as inhibitors of calmodulin, 783
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Figure 2. Role of transcription and translation in circadian cycle. Figure 2A shows the regions of the circadian cycle in Bulla where transcription and translation inhibitors arrest the motion of the circadian pacemaker. Translation is required for pacemaker motion in a relatively narrow region just prior to dawn and until approximately CT4. Transcription is required for the majority of the subjective day. Figure 2B shows the result of applying the transcription inhibitor DRB to the Bulla eye from CT 6–12. A six hour pulse of DRB produces an approximately 7-hour phase delay. At the same phase a pulse of the translation inhibitor cycloheximide does not produce a significant phase shift. The DRB phase shift is not affected by concurrent inhibition of protein synthesis with cycloheximide (from Khalsa et al, 1996). Figure 2C shows three simple schemes detailing possible roles for transcription and translation in circadian rhythm generation. The series of processes that are responsible for generating the circadian rhythm are represented by the circle of letters. In the upper panel neither transcription nor translation are central components of the timing mechanism. Their rates of synthesis are not timing elements within the feedback loop. However, inhibition of either process may stop the motion of the pacemaker. In this example a protein is required at event ‘D’ in order to move on to event ‘E’. In the middle panel only the translation process is part of the timing loop whereas in the lower panel both transcription and translation are oscillating components of the pacemaker mechanism. Stopping the motion of the circadian pacemaker with inhibitors will not distinguish between these three models.
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particular 8-bromo-cAMP, and the addition of forskolin, which causes the direct activation of adenylate cyclase, produce phase shifts that mimic those of serotonin. The simultaneous application of depolarizing treatments, i.e. high potassium artificial sea water, along with serotonin block the serotonin-induced phase shift.26 These results suggest that serotonin acts through hyperpolarization of the cell membrane potential, a hypothesis that is further supported by the fact that a broad-spectrum potassium-channel blocker, barium chloride, also blocks the serotonin-induced phase shift.27 The most likely action of membrane hyperpolarization is the reduction of a transmembrane calcium flux. Low calcium treatments also phase shift the Aplysia retina suggesting, similar to the situation in Bulla, a central role for modulation of a transmembrane calcium flux in phase control.28 Serotonin-induced phase shifts can be blocked by the simultaneous application of the protein synthesis inhibitor anisomycin.29 At Circadian Time 6–12 (CT 6–12), when serotonin generates phase advances, anisomycin has no effect on the phase of the rhythm and yet it blocks the serotonin-induced phase shift. The fact that the serotonin phase shift is blocked by anisomycin is most easily explained by protein synthesis being a critical component of the entrainment pathway. Investigators using preparative two-dimensional SDS polyacrylamide gel electrophoresis and micro-sequencing techniques have sequenced several of the proteins that are changed by serotonin as well as those altered by other phase-shifting treatments such as light pulses.30,31 This ‘matrix’ of altered proteins includes heat-shock protein 70 (HSP 70), binding protein (BiP), and glucose-regulated protein 78 (GRP 78); all of these can play roles as protein chaperones and in protein folding.31 Low calcium treatments in Aplysia and low calcium and hyperpolarizing treatments in Bulla are also blocked by the paired application of protein-synthesis inhibitors.28,32,33 In both Aplysia and Bulla it appears that phase shifts during the subjective day require protein synthesis. It is worth noting that it is a reduction of electrical activity through membrane hyperpolarization that activates the signal transduction cascade responsible for protein synthesis-dependent phase shifting. Such a process of hyperpolarization-activated signaling may be of general significance to neurobiology: many of the synaptic interactions in the brain are of an inhibitory nature.
The output pathway of circadian systems has in general received much less attention than the input pathway, the most likely reason being that most identified (and localized) circadian pacemakers appear to be closely associated with photic pathways, thus residing on the ‘sensory side’ of sensory/motor organization. However, the development of several invitro pacemaker models, for which the output being measured is likely to be more intimately associated with the pacemaker, have made it reasonable to begin to ‘work backwards’ from the overtly measured rhythm to the identification of processes in the output pathway; perhaps this would permit components of the pacemaker ultimately to be identified. In the Bulla retina, reduction in a K + conductance occurs near projected dawn (ca CTO), and this diminished conductance is likely to be responsible for the circadian modulation in membrane potential and firing rate.3,34 The membrane conductance increases again at dusk as the membrane hyperpolarizes and spike frequency falls. The application of the potassium channel blocker tetraethylammonium chloride (TEA) reduces membrane conductance prior to dawn but has no effect following dawn, presumably because TEA-sensitive channels have been closed by the clock. A preliminary report by Jacklet and Barnes4 reveals a similar circadian rhythm in membrane conductance in Aplysia: lower conductance during the subjective day than during the subjective night.4 How the mechanisms underlying this change in channel conductance are regulated is currently unknown, but alterations in either channel number or channel phosphorylation state are possibilities. There is a suggestion that phosphorylation on tyrosine residues may be involved in rhythm expression. Roberts and co-workers35 report that genistein, a tyrosine kinase inhibitor, leads to an inhibition of spontaneous circadian optic nerve activity, although the optic nerve light response is unaffected.35 Recent unpublished experiments in our laboratory (D. Whitmore et al) confirm this observation. In contrast, serine-threonine kinase and phosphatase inhibitors had no acute effect on spike frequency. This leads us to suspect that rhythmic channel phosphorylation on tyrosine residues drives the conductance rhythm, leading to the circadian rhythm in impulse activity. There appears to be a close association between the circadian pacemaker and the regulation of membrane potential. The inhibition of transcription during the subjective day produces rapid phase shifts of the 785
D. Whitmore and G. D. Block day (Figure 2A). The application of DRB just prior to dawn (CT 0) through to dusk (CT12) stopped the pacemaker for the duration of the inhibitor pulse. DRB at similar concentrations had no effect when applied during the subjective night. Figure 2B shows the phase shift produced by a DRB pulse applied in the late subjective day and the failure of cycloheximide to produce a shift at the same phase. The DRB phase shift was not blocked by the simultaneous application of cycloheximide. The transcription-translation sensitive phases in Aplysia, revealed by PRCs generated with short pulses,42 are remarkably similar to the critical regions shown by long-duration inhibitor pulse experiments in Bulla. The fact that transcription and translation are required for continued pacemaker motion does not prove that these processes are intrinsic elements in a feedback loop, i.e. ‘state variables’ involved in generating the circadian rhythm. Three simple schemes (Figure 2C) can be envisioned in which transcription and translation are essential for sustained oscillations: (a) both transcription and translation are outside of the pacemaker loop (i.e. are not state variables); (b) transcription resides outside of the loop, but translation is a state variable in the loop — and therefore unlike transcription, must oscillate; (c) both transcription and translation are within the loop, and both processes must oscillate. In an attempt to distinguish which model best characterizes the role of transcription and translation in the molluskan retina, a series of double-pulse experiments was devised to evaluate the speed at which a phase shift to DRB occurs36 (see Figure 3). As shown in Figure 2B, transcription inhibition by DRB from CT 6–12 caused a 6-hour delay of the rhythm, but translation inhibition at this same phase produced no shift. If transcription is acting through translation, but resides outside of the oscillator loop, then the phase shift to DRB cannot occur until dawn, when clock-required translation occurs. If inhibiting transcription leads to an immediate phase shift, prior to the translation-dependent phase, then transcription itself must be part of the feedback loop and therefore, presumably, so must translation. DRB pulses were given to control and experimental eyes from CT 6–12 (Figure 3). A light pulse was then given to the experimental eye at one of three phases. In this experimental paradigm, a light pulse is used to probe the phase of the pacemaker. If light produces phase shifts following the transcription inhibitor pulse, of a magnitude and sign that would be predicted by the unshifted PRC, then one can
circadian pacemaker (see later). If a transcription inhibitor is applied in the early subjective day, the phase shift can be seen in the falling phase of the electrical rhythm; in Aplysia this is evident within an hour of the transcription inhibitor pulse.36,37 Thus, not only is the pacemaker itself shifted quickly, but the output rapidly also reflects this change. This suggests that the protein(s) responsible for mediating the electrical rhythm must have a short half-life to allow for the dynamic association between pacemaker transcription and spike frequency.
Rhythm generation in Aplysia and Bulla Although a transmembrane Ca2 + flux mediates pacemaker entrainment, and a K + conductance rhythm appears to comprise part of the output pathway, there is no evidence in mollusks that plasma membrane ionic fluxes are involved in the generation of the circadian oscillation. Removal of bath Ca2 + ,38 Cl–,39 Mg2 + or Na + 40 do not prevent circadian oscillations. In contrast, the molecular processes of transcription and translation appear critical for generation of a circadian periodicity. In Aplysia, pulses of both transcription and translation inhibitors produce phase shifts at specific phases of the circadian cycle.41,42 In Bulla systematically increased doses of the protein synthesis inhibitor cycloheximide led to dramatic period lengthening of the ocular rhythm.43 To address the question of whether or not high concentrations of proteinsynthesis inhibitor stop the Bulla circadian pacemaker, cycloheximide was applied in a series of longduration pulses, from 6 hours up to 44 hours. Although the electrical rhythm cannot be observed during the pulse, the effect of the treatment can be determined by examining the resultant phase of the pacemaker on subsequent cycles following drug removal. By varying the starting phase for these pulses it is possible to ‘scan’ across the whole circadian cycle and determine regions of sensitivity. The results from this series of long-duration pulses reveals a critical phase at which the application of protein synthesis inhibitors appeared to stop the circadian pacemaker, and to do so for the duration of the inhibitor pulse. The critical window for translation appears to start just prior to dawn and to extend 3–4 hours into the early subjective day (Figure 2A). A similar series of experiments was also performed with the reversible transcription inhibitor DRB.36 In contrast to protein synthesis inhibitors, it appears that transcription is required during most of the subjective 786
Cellular aspects of molluskan biochronometry conclude that the pacemaker has not been immediately phase shifted. If, however, the phase response curve (PRC) to light is delayed 6 hours (the size of the DRB phase shift), then the transcription-induced phase shift must have occurred immediately and prior
to the requirement for protein synthesis. The results of the experiment revealed that the PRC to light was shifted by 6 hours and thus the DRB-induced phase shift must have occurred rapidly and prior to clock-required protein synthesis. These data strongly suggest that transcription, and thus translation, are both intrinsic components of a feedback loop that generates the circadian rhythm within the Bulla eye.
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There is accumulating evidence that the mammalian and molluskan circadian systems share many common features. At a fundamental level in both systems it appears that rhythmicity emerges at the cellular rather than a tissue level of organization. As mentioned, individual neurons at the base of the Bulla retina are competent circadian pacemakers. In the mammalian SCN, dispersed cells continue to produce a rhythm in electrical activity with different neurons within the culture exhibiting differing phases and free-running periods.46 Although these observations suggest cellular autonomy of circadian rhythm genesis, it will be important to fully isolate SCN neurons to confirm their ability to independently generate a rhythm. With regard to entrainment pathways, many similarities exist, especially in the second messengers that appear to be involved. For example, the application of the neurotransmitter serotonin to the SCN in a brain slice preparation produced phase shifts during the subjective day, as is the case for the Aplysia retinal pacemaker47. These phase shifts are mimicked by the application of cAMP analogs, suggesting a conserved ‘daytime’ role for the cAMP signaling pathway.48,49 The role of cyclic nucleotides in phase shifting may also be conserved, as it has been observed that the application of cGMP phase shifts both the Aplysia and mammalian pacemakers primarily during the subjective night.50 A critical role for intracellular calcium changes in the mammalian phase shifting pathway is likely, but has not been explicitly examined. However, a role for the excitatory amino acid neurotransmitter NMDA in light-dependent phase shifting in the hamster has been suggested.51 The application of NMDA to SCN neurons in a brain slice preparation loaded with the calcium-indicator dye Fura 2 has been shown to increase intracellular calcium levels, but whether or not these cells contain a functional circadian pacemaker is unknown.52 The requirement for protein synthesis in mediating
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Figure 3. Evaluation of light pulse PRC following DRB pulse. When a pulse of DRB is applied from CT 6–12 the phase shift it produces cannot be measured for at least another 18 hours until the eye becomes electrically active again at subjective dawn. In order to determine if this DRB phase shift occurs rapidly during the DRB pulse or does not occur until many hours later at dawn when protein synthesis is important, a series of double-pulse experiments were performed where light was used to probe the phase of the pacemaker. Panel A details the experiment. (1) Control eyes receive a light pulse at a number of phases to produce a standard light phase response curve. (2) The experimental eyes receive the same light pulses, but after receiving a transcription inhibitor (DRB) pulse from CT 6-12. Panel B shows the light PRC produced in the untreated control eyes compared to that produced by DRB treated eyes. The light PRC generated after a 6-hour DRB pulse is delayed relative to that produced in control eyes. Panel C compares the PRCs after the DRB treated PRC is shifted back by 6 hours. The fact that the phase-response curve to light is shifted following a DRB pulse demonstrates that the DRB phase shift occurs rapidly and prior to the critical phase for protein synthesis on the following dawn. (Redrawn from Khalsa et al, Am J Physiol 1996.)
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D. Whitmore and G. D. Block Center for Biological Timing. We thank Richard Day for many useful discussions, and Sat Bir Khalsa for help preparing the figures.
subjective-day phase shifts in the SCN has not been examined in detail, and so it is not clear whether or not this is a common feature between mollusks and mammals. However, a discrepancy does seem to exist between these systems with respect to light-dependent phase shifting. In the mollusks, transcription and translation inhibitors fail to block light-induced phase delays. The analysis of light-induced phase advances is complicated for reasons already discussed. In the mammalian SCN it is clear that the immediate early gene, c-fos, is induced by light pulses only when light produces phase shifts (refs 53-55; see also Ralph, this issue). The application of antisense for c-fos and junB supports the idea of a critical role for this gene in light-dependent phase shifting.56 Transcriptional and translational processes must, therefore, be involved in mammalian light-induced phase shifting, and these phase shifts should be blocked by the parallel application of transcription and translation inhibitors.
References 1. Jacklet J (1989) Circadian neuronal oscillators, in Neuronal and Cellular Oscillators (Jacklet J, ed.) pp 483-527. Marcel Dekker Inc., New York 2. Block GD, Khalsa SBS, McMahon DG, Michel S, Geusz M (1993) Biological clocks in the retina: cellular mechanisms of biological timekeeping. Int Rev Cytol 146:83-143 3. Michel S, Geusz ME, Zaritsky JJ, Block GD (1993) Circadian rhythm in membrane conductance expressed in isolated neurons. Science 259:239-241 4. Jacklet J, Barnes S (1995) Circadian phase differences in a retinal pacemaker neuron delayed rectifier K + current and increased current induced by a phase-shifting neurotransmitter, serotonin. Physiologist 38:A-22 5. Deguchi T (1979) A circadian oscillator in cultured cells of chicken pineal gland. Nature 282:94-96 6. Robertson LM, Takahashi JS (1988) Circadian clock in cell culture: I. Oscillation of melatonin release from dissociated chick pineal cells in flow through microcarrier culture. J Neurosci 8:12-21 7. Robertson LM, Takahashi JS (1988b) Circadian clock in cell culture: II. In vitro entrainment to light of melatonin oscillation from dissociated chick pineal cells. J Neurosci 8:22-30 8. Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14:697-706 9. Eskin A, Takahashi JS, Zatz M, Block GD (1984) Cyclic guanosine 3':5'-monophosphate mimics the effects of light on a circadian pacemaker in the eye of Aplysia. J Neurosci 4:2466-2471 10. McMahon DG, Block GD (1987) The Bulla ocular circadian pacemaker I: Pacemaker neuron membrane potential controls phase through a calcium dependent mechanism. J Comp Physiol 161A:335-346 11. Khalsa SBS, Block GD (1988) Calcium channels mediate phase shifts of the Bulla circadian pacemaker. J Comp Physiol 164A:195-206 12. Geusz ME, Michel S, Block GD (1994) Intracellular calcium responses of circadian pacemaker neurons measured with fura2. Brain Res 638:109-116 13. Raju U, Yeung SJ, Eskin A (1990) Involvement of proteins in light resetting ocular circadian oscillators of Aplysia. Am J Physiol 258:256-262 14. Bogart B, Block G (1990) The effects of temperature on the circadian ocular rhythm of Bulla gouldiana. Neuro Abstr 270.5 15. Khalsa SB, Block GD (1988) Phase-shifts of the Bulla ocular pacemaker in the presence of calmodulin antagonists. Life Sci 43:1551-1556 16. Colwell CS, Khalsa SB, Block GD (1992) FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla. J Comp Physiol A 170:211-215 17. Ralph MR, Khalsa SB, Block GD (1992) Cyclic nucleotides and circadian rhythm generation in Bulla gouldiana. Comp Biochem Physiol A 101:813-817 18. Khalsa SBS, Block GD (1990) Calcium in phase control of the Bulla circadian pacemaker. Brain Res 506:40-45 19. Mrosovsky N, Salmon PA (1987) A behavioral method for accelerating re-entrainment of rhythms to new light–dark cycles. Nature 330:372-373
Conclusions Much remains to be learned about circadian organization within the molluskan retina. Importantly, the specific proteins and transcripts involved in the generation of circadian periodicity require identification. The difficulty of this task raises the issue of whether it is appropriate to invest effort in a preparation, such as the molluskan eye, for which molecular genetic approaches are not feasible. Certainly the identification and characterization of mutants in Drosophila and Neurospora has greatly facilitated the identification of specific genes and gene products involved in circadian timing. Nonetheless, the molluskan retina exhibits properties not currently enjoyed by other model systems. The capacity to measure and manipulate the physiology of individual neurons, the precision and ease in recording the circadian rhythm of optic nerve activity, and the ability to study multioscillator organization in vitro places the molluskan retina in a favorable position to address many important issues regarding the physiology of circadian timing systems. We suspect that the molluskan eye will continue to make an important contribution to our understanding of biological timing.
Acknowledgements Support provided by NIH Grant NS12564 and the US NSF
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Cellular aspects of molluskan biochronometry 39. Khalsa SBS, Ralph MR, Block GD (1990) Chloride conductance contributes to period determination of a neuronal circadian pacemaker. Brain Res 520:166-169 40. Khalsa SBS, Michel S, Block GD (1996) The role of extracellular sodium in the mechanism of a neuronal in vitro circadian pacemaker. Chron Int (in press) 41. Lotshaw DP, Jacklet JW (1986) Involvement of protein synthesis in circadian clock of Aplysia eye. Am J Physiol 250:R5-17 42. Raju U, Koumenis C, Nunez-Regueiro M, Eskin A (1991) Alteration of the phase and period of a circadian oscillator by a reversible transcription inhibitor. Science 253:673-675 43. Khalsa SBS, Whitmore D, Block GD (1992) Stopping the circadian pacemaker with inhibitors of protein synthesis. Proc Natl Acad Sci USA 89:10862-10866 44. Ralph MR, Menaker M (1988) A mutation of the circadian system in golden hamsters. Science 241:1225-1227 45. Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey JD, McDonald JD, Dove WF, Pinto LH, Turek FW, Takahashi JS (1994) Mutagenesis and mapping of a mouse gene, clock, essential for circadian behavior. Science 264:719-725 46. Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14:697-706 47. Medanic M, Gillette MU (1992) Serotonin regulated the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day. J Physiol (Lond) 450:629-642 48. Prosser MA, Gillette MU (1987) The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP. J Neurosci 9:1073-1081 49. Gillette MU, Prosser RA (1988) Circadian rhythm of the rat suprachiasmatic brain slice is rapidly reset by daytime application of cAMP analogs. Brain Res 474:348-352 50. Prosser RA, McArthur AJ, Gillette MU (1989) cGMP induces phase shifts of a mammalian circadian pacemaker at night in antiphase to cAMP effects. PNAS 80:812-815 51. Colwell CS, Menaker M (1992) NMDA as well as non-NMDA receptor antagonists can prevent the phase shifting effects of light on the circadian system of the golden hamster. J Biol Rhythms 7:125-136 52. Tominaga K, Geusz ME, Michel S, Inouye ST (1994) Calcium imaging in organotypic cultures of the rat suprachiasmatic nucleus. Neuroreport 5:1901-1905 53. Rusak B, Robertson HA, Wisden W, Hunt SP (1990) Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus. Science 248:1237-1240 54. Aronin N, Sagar SM, Sharp FR, Schwartz WJ (1990) Light regulates expression of a fos-related protein in rat suprachiasmatic nuclei. PNAS 87:5959-5962 55. Kornhauser JM, Nelson DE, Mayo KE, Takahashi JS (1990) Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus. Neuron 5:127-134 56. Wollnik F, Brysch W, Uhlmann E, Gillardon F, Bravo R, Zimmermann N, Schlingensiepen KH, Herdegen T (1995) Block of c-fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock. Eur J Neurosci 7:288-393
20. McMahon DG, Wallace S, Block G (1984) Cellular analysis of the Bulla ocular circadian pacemaker system II: Neurophysiological basis of circadian rhythmicity. J Comp Physiol 155:379-385 21. Koumenis C, Eskin A (1992) The hunt for mechanisms of circadian timing in the eye of Aplysia. Chronobiol Int 9:201-221 22. Corrent G, McAdoo DJ, Eskin A (1978) Serotonin shifts the phase of the circadian rhythm from the Aplysia eye. Science 202:977-979 23. Takahashi JS, Nelson DE, Eskin A (1989) Immunocytochemical localization of serotonergic fibers innervating the ocular circadian system of Aplysia. Neuroscience 28:139-147 24. Eskin A, Corrent G, Lin C, McAdoo DJ (1982a) Mechanism for shifting the phase of a circadian rhythm by serotonin: involvement of cAMP. Proc Natl Acad Sci USA 79:660-664 25. Eskin A, Takahashi JS (1983) Adenylate cyclase activation shifts the phase of a circadian pacemaker. Science 220:82-84 26. Eskin A (1982) Increasing external K + blocks phase shifts in a circadian rhythm produced by serotonin or cAMP. J Neurobiol 13:241-249 27. Colwell CS, Michel S, Block GD (1992) Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia. J Comp Physiol A 171:651-656 28. Colwell CS, Whitmore D, Michel S, Block GD (1994) Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia. J Comp Physiol A 175:415-423 29. Eskin A, Yeung SJ, Klass MR (1984) Requirement for protein synthesis in the regulation of a circadian rhythm by serotonin. Proc Natl Acad Sci USA 81:7637-7641 30. Raju U, Nunez-Regueiro M, Cook R, Kaetzel MA, Yeung SC, Eskin A (1993) Identification of an annexin-like protein and its possible role in the Aplysia eye circadian system. J Neurochem 61:1236-1245 31. Kuomenis C, Nunez-Regueiro M, Raju U, Cook R, Eskin A (1995) Identification of three proteins in the eye of Aplysia, whose synthesis is altered by serotonin (5-HT). J Biol Chem 270:14619-14627 32. Khalsa SBS, Block GD (1992) Anisomycin blocks phase shifts of the Bulla circadian pacemaker to low extracellular calcium pulses. Neurosci Abstr 18:12251 33. Whitmore D, Khalsa SBS, Block GD (in preparation) Protein synthesis is required for subjective day phase shifting of the circadian pacemaker in the mollusk, Bulla gouldiana 34. Ralph MR, Block GD (1990) Circadian and light-induced conductance changes in putative pacemaker cells of Bulla gouldiana J Comp Physiol A 166:589-595 35. Roberts MH, Towles JA, Leader NK (1992) Tyrosine kinase regulation of a molluskan circadian clock. Brain Res 592:170-174 36. Khalsa SBS, Whitmore D, Bogart B, Block GD (1996) Evidence for a central role of transcription in the timing mechanism of a circadian clock. Am J Physiol (in press) 37. Koumenis C, Tran Q, Eskin A (1996) The use of a reversible transcription inhibitor, DRB, to investigate the involvement of specific proteins in the ocular circadian system of Aplysia. J Biol Rhythms 11:45-56 38. Khalsa SB, Ralph MR, Block GD (1993) The role of extracellular calcium in generating and in phase-shifting the Bulla ocular circadian rhythm. J Biol Rhythms 2:125-139
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CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 781–789
Cellular aspects of molluskan biochronometry David Whitmore and Gene D. Block
The eyes of the marine gastropods Aplysia californica and Bulla gouldiana contain circadian clocks. In both organisms retinal circadian rhythms are generated by individual neurons that express a circadian rhythm in impulse frequency. Phase control of the rhythm, either by light or by serotonin, is mediated by changes in membrane potential affecting a transmembrane calcium flux. Whereas transmembrane ionic fluxes underlie pacemaker entrainment and expression, the generation of the circadian rhythm involves molecular transcription and translation. Both processes appear to be elements in a feedback loop generating the circadian cycle.
nucleus (SCN) also contain competent pacemakers (refs 5-8; see also Zatz, this issue). These studies clearly indicate that the investigation of circadian rhythm generation must focus on intracellular processes rather than on the interactions between neurons or other cells within tissues. Nonetheless, circadian systems exhibit many highly adaptive attributes such as history-dependent period and waveform lability (i.e. ‘after-effects’); and it is not at all certain whether individual pacemaker cells, isolated from tissue interactions and competent to generate a circadian periodicity, will be capable of exhibiting the full range of circadian attributes. Ultimately we may discover that several levels of organization contribute to the sophisticated chronometry exhibited by multicellular organisms.
Key words: Aplysia / Bulla / circadian / entrainment / retina ©1996 Academic Press Ltd
THE EYES OF A NUMBER of opisthobranch mollusks express circadian rhythms in retinal electrical activity.1,2 The most intensively studied of the mollusks, Aplysia californica and Bulla gouldiana, have become important model systems for the cellular and molecular study of circadian rhythmicity. The ability to study rhythmicity in vitro, along with the many technical opportunities afforded by the gastropod nervous system, has provided an unparalleled opportunity to study circadian organization at many levels, from molecular events involved in rhythm generation to multi-oscillator organization and its control of rhythmic behaviors. In Bulla gouldiana single neurons located at the base of the eye are capable of generating a circadian periodicity in membrane conductance when isolated in culture.3 The same appears to be the case in Aplysia californica in which dispersed retinal neurons have been reported to undergo circadian cycling.4 In addition, it is likely, although not completely proven, that single pinealocytes from the chick and single neurons from the mammalian suprachiasmatic
The circadian system of Bulla and Aplysia What we presently understand about the circadian system within the opisthobranch retina is summarized in Figure 1. Conceptually, the system can be divided into three sections: (1) an input pathway which provides synchronizing information from the environment and from other intrinsic circadian oscillators, (2) the oscillator, a feed-back loop (or loops) that generates the periodicity, and (3) an output pathway that couples the oscillatory signals to target sites. The cellular and molecular mechanisms subserving these functions are discussed below.
Pacemaker entrainment The molluskan retina has provided important information regarding the physiology of entrainment. In the case of light-induced phase shifts, the pathway appears similar in both Bulla and Aplysia. Light pulses lead to an increase in cGMP levels in the Aplysia retina, and analogs of cGMP cause phase shifts that mimic those of light.9 cGMP does not have the same effect in Bulla, but differences in membrane permeability to the analog are the most likely explanation. In Aplysia cGMP increases the number of compound action potentials (CAPs) recorded extracellularly in
From the NSF Center for Biological Timing, Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA ©1996 Academic Press Ltd 1084-9521/96/060781 + 09 $25.00/0
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Figure 1. A model of the cellular events within a molluskan pacemaker cell. Subjective day: Bulla BRNs are relatively depolarized due to a reduction in membrane conductance which occurs at projected dawn. This conductance decrease is due to the closure of potassium channels, which we propose is caused by tyrosine kinase-mediated phosphorylation. As a consequence of persistent depolarization and impulse production we suspect that intracellular calcium levels are higher during the subject day than the subjective night. During the whole subjective day (CT 0–12) transcription is critical for the motion of the circadian pacemaker. Translation appears to be required for a shorter duration of time around projected dawn. Protein synthesis is required for phase shifts during the subjective day. Subjective night: During the subjective night many cellular conditions are reversed. Membrane conductance is high, resulting in a relatively hyperpolarized state. This is due to the opening of potassium channels at dusk, possibly by a tyrosine phosphatase and dephosphorylation. Intracellular calcium levels should be relatively low. Active transcription and translation are not critical events for the continued motion of the pacemaker until a few hours before dawn. Phase shifts are produced by light and other treatments which depolarize the pacemaker cell and lead to an increase in intracellular calcium. The mechanism of action of calcium on the circadian pacemaker is unknown; it does not appear to act via increased protein synthesis or transcription.
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Cellular aspects of molluskan biochronometry W-7 and calmidazolium, fail to block light-induced phase shifts.15. Subjective-day phase shifts in the electrical rhythm can be produced in both molluskan preparations. In Bulla the neurotransmitter FMRFamide,16 cAMP analogs,17 hyperpolarizing treatments,10,11 as well as low calcium treatments all lead to phase shifts primarily during the subjective day.18 A conclusion drawn from these results is that treatments that reduce a transmembrane calcium flux during the subjective day produce similar phase shifts.2,10 In general there appear to be two classes of phase response curves (PRCs). The most common PRC, typical for those obtained to light pulses (‘light-pulse PRC’), is one with the break-point between delays and advances in the mid subjective night. A second class of PRCs, where the breakpoint between delays and advances lies in the mid subjective day, is displaced approximately 180° on the time axis from light pulse PRCs; these are sometimes referred to as ‘dark-pulse PRCs’, because rodents maintained in continuous light but given interruptions of darkness generate this type of response.19 A model has been proposed for Bulla in which the two families of phase response curves derive from modulation of a single common process, a transmembrane calcium flux.2,10,18 In this scheme, during the subjective night — a time when the membrane of pacemaker neurons is hyperpolarized — phase shifts to light and other depolarizing agents are due to an increase in intracellular calcium.10 In contrast, phase shifts during the subjective day are generated by reducing a transmembrane calcium flux. During the subjective day the membrane is depolarized by approximately 13 mV compared to the subjective night.20 Treatments which hyperpolarize the membrane during the subjective day, leading to a cessation of impulse activity (thus presumably reducing a transmembrane calcium flux) generate a family of PRCs of similar shape with the transition between delays and advances occurring near Circadian Time 6 (in mid subjective-day).2 In Aplysia phase shifts occurring during the subjective day have been extensively studied by Eskin and colleagues.21 Pulses of the neurotransmitter serotonin (5 HT) phase shift the Aplysia pacemaker primarily during the subjective day.22 Serotonin is present in efferent termini that synapse in the base of the retina.23 The application of serotonin pulses leads to an increase in intracellular cAMP levels, activating adenylate cyclase, presumably through a G-protein dependent mechanism.24,25 Analogs of cAMP, in
the optic nerve, suggesting that cGMP generates a membrane depolarization. Depolarizing treatments produce phase shifts in Bulla that mimic those of light, and hyperpolarization applied concurrently with light blocks the light-induced phase shift.10 The light-induced membrane depolarization leads to a transmembrane calcium flux that is required for the phase shift. Simultaneous application of low calcium/ EGTA seawater and light blocks the light-induced phase shift, as does the application of the Ca2 + channel blocker nickel chloride, suggesting a role for calcium channels in this pathway.10,11,28 Geusz and coworkers have obtained direct measurements of a sustained increase in intracellular calcium following the depolarization of basal retinal neurons (BRNs) by use of the calcium indicator dye, fura-2.12 Intracellular calcium levels remain high for the duration of the phase shifting treatment, suggesting that raised intracellular calcium can encode for the duration of the environmental stimulus. It has yet to be demonstrated, however, that solely increasing intracellular calcium, without membrane depolarization, is sufficient to generate a phase shift. The ‘step’ following an increase in intracellular calcium is presently unclear. It appears unlikely that either protein synthesis or transcription are components of the light-entrainment pathway. The application of anisomycin or cycloheximide, two commonly used protein synthesis inhibitors, do not appear to block light-induced phase delays in the electrical rhythm in either molluskan species.13,14 The issue of the role of protein synthesis in light-induced phase advances is complicated by the impact of these inhibitors on the motion of the pacemaker in the late subjective night (see later). Changes in protein synthesis, as measured by radiolabelling proteins followed by two-dimensional SDS polyacrylamide protein gel electrophoresis, have been reported in the Aplysia retina.13 However, a search for light-induced proteins by the same methods in Bulla has failed to reveal any large amplitude and reliable changes (D. Whitmore, unpublished observations). The reversible transcription inhibitor 5,6-dichlorobenzimidazole riboside (DRB), when applied concurrently with light, does not block light-induced phase delays in Bulla; this suggests that an initiation of transcription is also not required for light-induced phase shifts (B. Bogart et al, unpublished observations). The role of specific kinases has not been extensively examined. There is a report, however, that calcium calmodulin-dependent protein kinase is not involved in entrainment as inhibitors of calmodulin, 783
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Figure 2. Role of transcription and translation in circadian cycle. Figure 2A shows the regions of the circadian cycle in Bulla where transcription and translation inhibitors arrest the motion of the circadian pacemaker. Translation is required for pacemaker motion in a relatively narrow region just prior to dawn and until approximately CT4. Transcription is required for the majority of the subjective day. Figure 2B shows the result of applying the transcription inhibitor DRB to the Bulla eye from CT 6–12. A six hour pulse of DRB produces an approximately 7-hour phase delay. At the same phase a pulse of the translation inhibitor cycloheximide does not produce a significant phase shift. The DRB phase shift is not affected by concurrent inhibition of protein synthesis with cycloheximide (from Khalsa et al, 1996). Figure 2C shows three simple schemes detailing possible roles for transcription and translation in circadian rhythm generation. The series of processes that are responsible for generating the circadian rhythm are represented by the circle of letters. In the upper panel neither transcription nor translation are central components of the timing mechanism. Their rates of synthesis are not timing elements within the feedback loop. However, inhibition of either process may stop the motion of the pacemaker. In this example a protein is required at event ‘D’ in order to move on to event ‘E’. In the middle panel only the translation process is part of the timing loop whereas in the lower panel both transcription and translation are oscillating components of the pacemaker mechanism. Stopping the motion of the circadian pacemaker with inhibitors will not distinguish between these three models.
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particular 8-bromo-cAMP, and the addition of forskolin, which causes the direct activation of adenylate cyclase, produce phase shifts that mimic those of serotonin. The simultaneous application of depolarizing treatments, i.e. high potassium artificial sea water, along with serotonin block the serotonin-induced phase shift.26 These results suggest that serotonin acts through hyperpolarization of the cell membrane potential, a hypothesis that is further supported by the fact that a broad-spectrum potassium-channel blocker, barium chloride, also blocks the serotonin-induced phase shift.27 The most likely action of membrane hyperpolarization is the reduction of a transmembrane calcium flux. Low calcium treatments also phase shift the Aplysia retina suggesting, similar to the situation in Bulla, a central role for modulation of a transmembrane calcium flux in phase control.28 Serotonin-induced phase shifts can be blocked by the simultaneous application of the protein synthesis inhibitor anisomycin.29 At Circadian Time 6–12 (CT 6–12), when serotonin generates phase advances, anisomycin has no effect on the phase of the rhythm and yet it blocks the serotonin-induced phase shift. The fact that the serotonin phase shift is blocked by anisomycin is most easily explained by protein synthesis being a critical component of the entrainment pathway. Investigators using preparative two-dimensional SDS polyacrylamide gel electrophoresis and micro-sequencing techniques have sequenced several of the proteins that are changed by serotonin as well as those altered by other phase-shifting treatments such as light pulses.30,31 This ‘matrix’ of altered proteins includes heat-shock protein 70 (HSP 70), binding protein (BiP), and glucose-regulated protein 78 (GRP 78); all of these can play roles as protein chaperones and in protein folding.31 Low calcium treatments in Aplysia and low calcium and hyperpolarizing treatments in Bulla are also blocked by the paired application of protein-synthesis inhibitors.28,32,33 In both Aplysia and Bulla it appears that phase shifts during the subjective day require protein synthesis. It is worth noting that it is a reduction of electrical activity through membrane hyperpolarization that activates the signal transduction cascade responsible for protein synthesis-dependent phase shifting. Such a process of hyperpolarization-activated signaling may be of general significance to neurobiology: many of the synaptic interactions in the brain are of an inhibitory nature.
The output pathway of circadian systems has in general received much less attention than the input pathway, the most likely reason being that most identified (and localized) circadian pacemakers appear to be closely associated with photic pathways, thus residing on the ‘sensory side’ of sensory/motor organization. However, the development of several invitro pacemaker models, for which the output being measured is likely to be more intimately associated with the pacemaker, have made it reasonable to begin to ‘work backwards’ from the overtly measured rhythm to the identification of processes in the output pathway; perhaps this would permit components of the pacemaker ultimately to be identified. In the Bulla retina, reduction in a K + conductance occurs near projected dawn (ca CTO), and this diminished conductance is likely to be responsible for the circadian modulation in membrane potential and firing rate.3,34 The membrane conductance increases again at dusk as the membrane hyperpolarizes and spike frequency falls. The application of the potassium channel blocker tetraethylammonium chloride (TEA) reduces membrane conductance prior to dawn but has no effect following dawn, presumably because TEA-sensitive channels have been closed by the clock. A preliminary report by Jacklet and Barnes4 reveals a similar circadian rhythm in membrane conductance in Aplysia: lower conductance during the subjective day than during the subjective night.4 How the mechanisms underlying this change in channel conductance are regulated is currently unknown, but alterations in either channel number or channel phosphorylation state are possibilities. There is a suggestion that phosphorylation on tyrosine residues may be involved in rhythm expression. Roberts and co-workers35 report that genistein, a tyrosine kinase inhibitor, leads to an inhibition of spontaneous circadian optic nerve activity, although the optic nerve light response is unaffected.35 Recent unpublished experiments in our laboratory (D. Whitmore et al) confirm this observation. In contrast, serine-threonine kinase and phosphatase inhibitors had no acute effect on spike frequency. This leads us to suspect that rhythmic channel phosphorylation on tyrosine residues drives the conductance rhythm, leading to the circadian rhythm in impulse activity. There appears to be a close association between the circadian pacemaker and the regulation of membrane potential. The inhibition of transcription during the subjective day produces rapid phase shifts of the 785
D. Whitmore and G. D. Block day (Figure 2A). The application of DRB just prior to dawn (CT 0) through to dusk (CT12) stopped the pacemaker for the duration of the inhibitor pulse. DRB at similar concentrations had no effect when applied during the subjective night. Figure 2B shows the phase shift produced by a DRB pulse applied in the late subjective day and the failure of cycloheximide to produce a shift at the same phase. The DRB phase shift was not blocked by the simultaneous application of cycloheximide. The transcription-translation sensitive phases in Aplysia, revealed by PRCs generated with short pulses,42 are remarkably similar to the critical regions shown by long-duration inhibitor pulse experiments in Bulla. The fact that transcription and translation are required for continued pacemaker motion does not prove that these processes are intrinsic elements in a feedback loop, i.e. ‘state variables’ involved in generating the circadian rhythm. Three simple schemes (Figure 2C) can be envisioned in which transcription and translation are essential for sustained oscillations: (a) both transcription and translation are outside of the pacemaker loop (i.e. are not state variables); (b) transcription resides outside of the loop, but translation is a state variable in the loop — and therefore unlike transcription, must oscillate; (c) both transcription and translation are within the loop, and both processes must oscillate. In an attempt to distinguish which model best characterizes the role of transcription and translation in the molluskan retina, a series of double-pulse experiments was devised to evaluate the speed at which a phase shift to DRB occurs36 (see Figure 3). As shown in Figure 2B, transcription inhibition by DRB from CT 6–12 caused a 6-hour delay of the rhythm, but translation inhibition at this same phase produced no shift. If transcription is acting through translation, but resides outside of the oscillator loop, then the phase shift to DRB cannot occur until dawn, when clock-required translation occurs. If inhibiting transcription leads to an immediate phase shift, prior to the translation-dependent phase, then transcription itself must be part of the feedback loop and therefore, presumably, so must translation. DRB pulses were given to control and experimental eyes from CT 6–12 (Figure 3). A light pulse was then given to the experimental eye at one of three phases. In this experimental paradigm, a light pulse is used to probe the phase of the pacemaker. If light produces phase shifts following the transcription inhibitor pulse, of a magnitude and sign that would be predicted by the unshifted PRC, then one can
circadian pacemaker (see later). If a transcription inhibitor is applied in the early subjective day, the phase shift can be seen in the falling phase of the electrical rhythm; in Aplysia this is evident within an hour of the transcription inhibitor pulse.36,37 Thus, not only is the pacemaker itself shifted quickly, but the output rapidly also reflects this change. This suggests that the protein(s) responsible for mediating the electrical rhythm must have a short half-life to allow for the dynamic association between pacemaker transcription and spike frequency.
Rhythm generation in Aplysia and Bulla Although a transmembrane Ca2 + flux mediates pacemaker entrainment, and a K + conductance rhythm appears to comprise part of the output pathway, there is no evidence in mollusks that plasma membrane ionic fluxes are involved in the generation of the circadian oscillation. Removal of bath Ca2 + ,38 Cl–,39 Mg2 + or Na + 40 do not prevent circadian oscillations. In contrast, the molecular processes of transcription and translation appear critical for generation of a circadian periodicity. In Aplysia, pulses of both transcription and translation inhibitors produce phase shifts at specific phases of the circadian cycle.41,42 In Bulla systematically increased doses of the protein synthesis inhibitor cycloheximide led to dramatic period lengthening of the ocular rhythm.43 To address the question of whether or not high concentrations of proteinsynthesis inhibitor stop the Bulla circadian pacemaker, cycloheximide was applied in a series of longduration pulses, from 6 hours up to 44 hours. Although the electrical rhythm cannot be observed during the pulse, the effect of the treatment can be determined by examining the resultant phase of the pacemaker on subsequent cycles following drug removal. By varying the starting phase for these pulses it is possible to ‘scan’ across the whole circadian cycle and determine regions of sensitivity. The results from this series of long-duration pulses reveals a critical phase at which the application of protein synthesis inhibitors appeared to stop the circadian pacemaker, and to do so for the duration of the inhibitor pulse. The critical window for translation appears to start just prior to dawn and to extend 3–4 hours into the early subjective day (Figure 2A). A similar series of experiments was also performed with the reversible transcription inhibitor DRB.36 In contrast to protein synthesis inhibitors, it appears that transcription is required during most of the subjective 786
Cellular aspects of molluskan biochronometry conclude that the pacemaker has not been immediately phase shifted. If, however, the phase response curve (PRC) to light is delayed 6 hours (the size of the DRB phase shift), then the transcription-induced phase shift must have occurred immediately and prior
to the requirement for protein synthesis. The results of the experiment revealed that the PRC to light was shifted by 6 hours and thus the DRB-induced phase shift must have occurred rapidly and prior to clock-required protein synthesis. These data strongly suggest that transcription, and thus translation, are both intrinsic components of a feedback loop that generates the circadian rhythm within the Bulla eye.
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There is accumulating evidence that the mammalian and molluskan circadian systems share many common features. At a fundamental level in both systems it appears that rhythmicity emerges at the cellular rather than a tissue level of organization. As mentioned, individual neurons at the base of the Bulla retina are competent circadian pacemakers. In the mammalian SCN, dispersed cells continue to produce a rhythm in electrical activity with different neurons within the culture exhibiting differing phases and free-running periods.46 Although these observations suggest cellular autonomy of circadian rhythm genesis, it will be important to fully isolate SCN neurons to confirm their ability to independently generate a rhythm. With regard to entrainment pathways, many similarities exist, especially in the second messengers that appear to be involved. For example, the application of the neurotransmitter serotonin to the SCN in a brain slice preparation produced phase shifts during the subjective day, as is the case for the Aplysia retinal pacemaker47. These phase shifts are mimicked by the application of cAMP analogs, suggesting a conserved ‘daytime’ role for the cAMP signaling pathway.48,49 The role of cyclic nucleotides in phase shifting may also be conserved, as it has been observed that the application of cGMP phase shifts both the Aplysia and mammalian pacemakers primarily during the subjective night.50 A critical role for intracellular calcium changes in the mammalian phase shifting pathway is likely, but has not been explicitly examined. However, a role for the excitatory amino acid neurotransmitter NMDA in light-dependent phase shifting in the hamster has been suggested.51 The application of NMDA to SCN neurons in a brain slice preparation loaded with the calcium-indicator dye Fura 2 has been shown to increase intracellular calcium levels, but whether or not these cells contain a functional circadian pacemaker is unknown.52 The requirement for protein synthesis in mediating
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Figure 3. Evaluation of light pulse PRC following DRB pulse. When a pulse of DRB is applied from CT 6–12 the phase shift it produces cannot be measured for at least another 18 hours until the eye becomes electrically active again at subjective dawn. In order to determine if this DRB phase shift occurs rapidly during the DRB pulse or does not occur until many hours later at dawn when protein synthesis is important, a series of double-pulse experiments were performed where light was used to probe the phase of the pacemaker. Panel A details the experiment. (1) Control eyes receive a light pulse at a number of phases to produce a standard light phase response curve. (2) The experimental eyes receive the same light pulses, but after receiving a transcription inhibitor (DRB) pulse from CT 6-12. Panel B shows the light PRC produced in the untreated control eyes compared to that produced by DRB treated eyes. The light PRC generated after a 6-hour DRB pulse is delayed relative to that produced in control eyes. Panel C compares the PRCs after the DRB treated PRC is shifted back by 6 hours. The fact that the phase-response curve to light is shifted following a DRB pulse demonstrates that the DRB phase shift occurs rapidly and prior to the critical phase for protein synthesis on the following dawn. (Redrawn from Khalsa et al, Am J Physiol 1996.)
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D. Whitmore and G. D. Block Center for Biological Timing. We thank Richard Day for many useful discussions, and Sat Bir Khalsa for help preparing the figures.
subjective-day phase shifts in the SCN has not been examined in detail, and so it is not clear whether or not this is a common feature between mollusks and mammals. However, a discrepancy does seem to exist between these systems with respect to light-dependent phase shifting. In the mollusks, transcription and translation inhibitors fail to block light-induced phase delays. The analysis of light-induced phase advances is complicated for reasons already discussed. In the mammalian SCN it is clear that the immediate early gene, c-fos, is induced by light pulses only when light produces phase shifts (refs 53-55; see also Ralph, this issue). The application of antisense for c-fos and junB supports the idea of a critical role for this gene in light-dependent phase shifting.56 Transcriptional and translational processes must, therefore, be involved in mammalian light-induced phase shifting, and these phase shifts should be blocked by the parallel application of transcription and translation inhibitors.
References 1. Jacklet J (1989) Circadian neuronal oscillators, in Neuronal and Cellular Oscillators (Jacklet J, ed.) pp 483-527. Marcel Dekker Inc., New York 2. Block GD, Khalsa SBS, McMahon DG, Michel S, Geusz M (1993) Biological clocks in the retina: cellular mechanisms of biological timekeeping. Int Rev Cytol 146:83-143 3. Michel S, Geusz ME, Zaritsky JJ, Block GD (1993) Circadian rhythm in membrane conductance expressed in isolated neurons. Science 259:239-241 4. Jacklet J, Barnes S (1995) Circadian phase differences in a retinal pacemaker neuron delayed rectifier K + current and increased current induced by a phase-shifting neurotransmitter, serotonin. Physiologist 38:A-22 5. Deguchi T (1979) A circadian oscillator in cultured cells of chicken pineal gland. Nature 282:94-96 6. Robertson LM, Takahashi JS (1988) Circadian clock in cell culture: I. Oscillation of melatonin release from dissociated chick pineal cells in flow through microcarrier culture. J Neurosci 8:12-21 7. Robertson LM, Takahashi JS (1988b) Circadian clock in cell culture: II. In vitro entrainment to light of melatonin oscillation from dissociated chick pineal cells. J Neurosci 8:22-30 8. Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14:697-706 9. Eskin A, Takahashi JS, Zatz M, Block GD (1984) Cyclic guanosine 3':5'-monophosphate mimics the effects of light on a circadian pacemaker in the eye of Aplysia. J Neurosci 4:2466-2471 10. McMahon DG, Block GD (1987) The Bulla ocular circadian pacemaker I: Pacemaker neuron membrane potential controls phase through a calcium dependent mechanism. J Comp Physiol 161A:335-346 11. Khalsa SBS, Block GD (1988) Calcium channels mediate phase shifts of the Bulla circadian pacemaker. J Comp Physiol 164A:195-206 12. Geusz ME, Michel S, Block GD (1994) Intracellular calcium responses of circadian pacemaker neurons measured with fura2. Brain Res 638:109-116 13. Raju U, Yeung SJ, Eskin A (1990) Involvement of proteins in light resetting ocular circadian oscillators of Aplysia. Am J Physiol 258:256-262 14. Bogart B, Block G (1990) The effects of temperature on the circadian ocular rhythm of Bulla gouldiana. Neuro Abstr 270.5 15. Khalsa SB, Block GD (1988) Phase-shifts of the Bulla ocular pacemaker in the presence of calmodulin antagonists. Life Sci 43:1551-1556 16. Colwell CS, Khalsa SB, Block GD (1992) FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla. J Comp Physiol A 170:211-215 17. Ralph MR, Khalsa SB, Block GD (1992) Cyclic nucleotides and circadian rhythm generation in Bulla gouldiana. Comp Biochem Physiol A 101:813-817 18. Khalsa SBS, Block GD (1990) Calcium in phase control of the Bulla circadian pacemaker. Brain Res 506:40-45 19. Mrosovsky N, Salmon PA (1987) A behavioral method for accelerating re-entrainment of rhythms to new light–dark cycles. Nature 330:372-373
Conclusions Much remains to be learned about circadian organization within the molluskan retina. Importantly, the specific proteins and transcripts involved in the generation of circadian periodicity require identification. The difficulty of this task raises the issue of whether it is appropriate to invest effort in a preparation, such as the molluskan eye, for which molecular genetic approaches are not feasible. Certainly the identification and characterization of mutants in Drosophila and Neurospora has greatly facilitated the identification of specific genes and gene products involved in circadian timing. Nonetheless, the molluskan retina exhibits properties not currently enjoyed by other model systems. The capacity to measure and manipulate the physiology of individual neurons, the precision and ease in recording the circadian rhythm of optic nerve activity, and the ability to study multioscillator organization in vitro places the molluskan retina in a favorable position to address many important issues regarding the physiology of circadian timing systems. We suspect that the molluskan eye will continue to make an important contribution to our understanding of biological timing.
Acknowledgements Support provided by NIH Grant NS12564 and the US NSF
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