Circadian pacemaker neurons: membranes and molecules

1. A circadian pacemaker is located in the eyes of a variety of marine gastropods, including Aplysia and Bulla. It produces a circadian rhythm in the frequency of spontaneously occurring optic nerve (ON) compound action potentials (CAPs). The circadian pacemaker in Bulla includes a population of 100 retinal pacemaker neurons, that produce the rhythmic CAP output. Intracellular recording from the Bulla pacemaker neurons has yielded new insight into their time-keeping ability. 2. Intracellular injection of Lucifer yellow dye into a single pacemaker neuron results in the spread of dye to several neurons. This dye coupling is presumably mediated by the gap junctions among neurons that are responsible for the synchronous firing of the population of pacemaker neurons and the generation of ON CAPs. 3. The circadian pacemaker in each eye interacts with the paired pacemaker in the contralateral eye. The interaction results in the coordinating firing of CAPs from each eye and in the coordinated phasing of the circadian rhythms of CAP activity generated in each eye. This interaction occurs by reciprocal excitatory chemical synapses. These synaptic receptors occur in the ON as well as in the retinal neuropil and CAP synchrony occurs in the ON as well as in the basal retina. 4. Pacemaker neurons are depolarized by 5-HT and membrane permeable cAMP analogues. The membrane resistance increases during the depolarization suggesting a background potassium current is decreased. 5. The tetrapeptide FMRF-HN2 hyperpolarizes the pacemaker neurons. It reverses the effect of 5-HT and cAMP, suggesting 5-HT and FMRF-NH2 may be acting on the same membrane channel, the S channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.     

TheBulla ocular circadian pacemaker

We have used intracellular recording to directly measure the effects of three experimental agents, light, elevated potassium seawater, and lowered sodium seawater on the membrane potential of the putative circadian pacemaker neurons of the Bulla eye. These agents were subsequently tested for effects on the free running period of the circadian pacemaker. We report that: 1. When applied to the eye, light and elevated potassium seawater depolarized the putative pacemaker neurons, while lowered sodium seawater hyperpolarized them. The membrane potential changes induced by these agents are sustained for at least one hour, suggesting that they produce persistent changes in the average membrane potential of the putative pacemaker neurons. 2. The amplitude of the membrane potential response to the depolarizing agents varies with the phase of the circadian cycle. Depolarizations induced by light and elevated potassium seawater are twice as large during the subjective night than they are during the subjective day. No significant difference was found in the response to lowered sodium seawater at different phases. 3. Continuous application of each of these agents caused a lengthening of the free running period of the Bulla eye. Constant light increased the period by 0.9 h, while the other depolarizing treatment (elevated potassium seawater) increased the free running period by 0.6 h. Both treatments increased the mean peak impulse frequency of treated eyes. The hyperpolarizing treatment also increased the period of the ocular pacemaker (+0.8 h), but had little effect on peak impulse frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

FMRF-amide-like immunoreactive efferent fibers and FMRF-amide suppression of pacemaker neurons in eyes of Bulla

The eyes of certain marine gastropods including Aplysia and Bulla, contain circadian pacemakers, which produce a circadian rhythm of autogenous compound action potential (CAP) activity. The CAPs are generated by the synchronous spike discharge of a distinctive population of retinal pacemaker neurons whose axons convey the CAP activity to the CNS. When CAP activity is recorded from a preparation with eyes attached to the CNS, the CAP activity is modulated by efferent activity. In this study we have identified FMRF-amide-like immunoreactive efferent axons in the optic nerves of Bulla. These axons arborize in the basal retinal neuropil adjacent to the pacemaker neurons and are in a position to make synaptic contacts with their dendrites. Similar immunoreactive fibers are not observed in Aplysia eyes. Exogenous FMRF-amide at micromolar concentrations suppresses ongoing CAP activity in isolated eyes but does not suppress the ERG or phase shift the circadian rhythm of CAP activity. Intracellular recordings from the retinal pacemaker neurons reveal that FMRF-amide hyperpolarizes the membrane potential, suppresses spike discharge, and decreases the input resistance, suggesting that a K conductance is increased. Electrical stimulation of the region of the cerebral ganglion that contains FMRF-amide immunoreactive neurons suppresses ongoing CAP activity. All these results are consistent with the idea that the FMRF-amide immunoreactive central neurons and their axons provide a pathway for efferent modulation of the CAP rhythm generated by the retinal pacemaker neurons.     

Conservation of antigen 3G6: A crystalline cone constitutent in the compound eye of arthropods

A monoclonal antibody (MAb), 3G6, highly selective for neuropil glia in the CNS of the house cricket Acheta domesticus, also demonstrates remarkable selectivity for the nonneuronal crystalline cone cells of the compound eye. MAb 3G6 labels cone cells in eucone eyes throughout Insecta, from ancestral forms such as the bristle tail to the more recent honeybee; eucone structures are also recognized in Crustacea. Analogous nonneural structures found in pseudocone or acone eyes also express detectable 3G6 immunoreactivity. Immunoblot analysis demonstrates that MAb 3G6 binds to similar M_r 85 kDa glycoproteins in the cricket CNS and retina, corresponding to the glial and crystalline cone forms of the antigen. Further, polypeptides of similar relative mass are also recognized in the eucone eye of the butterfly Pieris and the pseudocone eye of the fly Calliphora. The properties and function of glycoprotein 3G6 in the CNS and retina are yet to be explored. However, the finding that a unique antigen is highly conserved within the crystalline cone or analogous regions of the retina throughout the Arthropoda lends support at the molecular level to the notion that the arthropod compound eye has a monophyletic origin.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.     

Monoclonal antibodies against species-specific antigens in the chick central nervous system: putative application as transplantation markers in the chick-quail chimera

With the recent progress in transplantation of neuronal tissues, cellular markers are needed to distinguish the grafted cells from the host. To generate monoclonal antibodies (MAb) recognizing species-specific antigens in the chick nervous system, we immunized mice with chick optic nerves and obtained 2 MAb which bind to chick but not to quail neural tissues. MAb-39B11 recognizes the cell surface antigen on the nerve fibers. MAb-37F5 recognizes the cytoplasmic components in several cell types, including ependymal cells and some large neurons. The utility of these MAb as markers for chick cells in the chick-quail chimeric brain and their advantages over conventional markers are discussed.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Properties of mutual coupling between the two circadian pacemakers in the eyes of the mollusc Bulla gouldiana.

We examined, in vitro, the effects of changing the free-running period (tau) of one oscillator on the phase relationship between the circadian rhythms of impulse activity in the optic nerves that are driven by the bilaterally paired ocular pacemakers in Bulla gouldiana. One eye of the coupled pair was treated either with lithium artificial seawater (to lengthen tau) or with low-chloride artificial seawater (to shorten tau). The results suggested that the coupling is relatively weak, since the majority (9 to 16) of eyes were unable to maintain a stable phase relationship when tau differences between the eyes were only about 1 hr. When stable phase differences were achieved, the tau of the coupled system was intermediate between the tau's of the individual oscillators, and the eye with the shorter intrinsic tau would invariably phase-lead the pair. Interestingly, in a few instances, pairs of eyes that had desynchronized by 9.5-10.5 hr resynchronized within a single cycle via a massive phase advance in the rhythm from the phase-lagging eye. The result suggests the existence of a novel phase-shifting mechanism that is part of the mutual coupling pathway. We found evidence that connection of the eye with the cerebral ganglion increases the tau of the ocular pacemaker, suggesting that efferent signals from the central nervous system influence tau. These signals may also modulate the phase-shifting response.     

Parallel processing in the mammalian retina

Our eyes send different 'images' of the outside world to the brain - an image of contours (line drawing), a colour image (watercolour painting) or an image of moving objects (movie). This is commonly referred to as parallel processing, and starts as early as the first synapse of the retina, the cone pedicle. Here, the molecular composition of the transmitter receptors of the postsynaptic neurons defines which images are transferred to the inner retina. Within the second synaptic layer - the inner plexiform layer - circuits that involve complex inhibitory and excitatory interactions represent filters that select 'what the eye tells the brain'.     

Analysis of mutual circadian pacemaker coupling between the two eyes of Bulla

The eyes of Bulla, a marine snail, express a circadian rhythm in the frequency of optic nerve compound action potentials (CAPs). The two ocular pacemakers are mutually coupled, and their interaction can be observed in vitro. The evidence for mutual coupling, as demonstrated in the present experiments, was as follows: (1) When intact Bulla were placed into darkness for up to 72 days, the two pacemakers did not desynchronize. (2) The free-running period of the ocular rhythm in the intact system (24.4 hr) was longer than the free-running period of the rhythm recorded from isolated eyes (23.7 hr). (3) When the two ocular pacemakers were experimentally desynchronized in vitro, resynchronization occurred if the pacemakers were allowed to interact for 48 hr. The coupling signals are most likely the CAPs. These impulses are conducted through the central ganglia and emerge as efferent impulses in the opposite optic nerve. Ocular-derived efferent impulse activity affects spontaneous impulse production in the target eye and alters the waveform of the circadian rhythm. The coupling pathway mediating syncrhonization consists of the two optic nerves, the cerebral ganglia, and the cerebral commissure. The demonstration of coupling in vitro provides a new opportunity for studying the cellular mechanisms underlying mutual pacemaker entrainment.     

The circadian clock of fruit flies is blind after elimination of all known photoreceptors

Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.     

Analysis of the formation of tissue antigens of the retina of chicks in embryogenesis]

The following antigens were found in the chick retina: one organospecific, three interorganic antigens of narrow specificity characteristic of only the tissues of the eye and brain, and seven interorganic antigens of broad specificity characteristic of, besides the tissues of the eye and brain, many other organs of the chick. The interorganic antigens of broad specificity manifest themselves the first in the retina during development. The interorganic antigens of narrow specificity and the organospecific antigen arise during the period of retinal histogenesis. One of the antigens of narrow specificity (retina--iris--brain) arises in the brain at the same time as in the retina.     

Tuning Properties of MT and MSTd and Divisive Interactions for Eye-Movement Compensation

The primate brain intelligently processes visual information from the world as the eyes move constantly. The brain must take into account visual motion induced by eye movements, so that visual information about the outside world can be recovered. Certain neurons in the dorsal part of monkey medial superior temporal area (MSTd) play an important role in integrating information about eye movements and visual motion. When a monkey tracks a moving target with its eyes, these neurons respond to visual motion as well as to smooth pursuit eye movements. Furthermore, the responses of some MSTd neurons to the motion of objects in the world are very similar during pursuit and during fixation, even though the visual information on the retina is altered by the pursuit eye movement. We call these neurons compensatory pursuit neurons. In this study we develop a computational model of MSTd compensatory pursuit neurons based on physiological data from single unit studies. Our model MSTd neurons can simulate the velocity tuning of monkey MSTd neurons. The model MSTd neurons also show the pursuit compensation property. We find that pursuit compensation can be achieved by divisive interaction between signals coding eye movements and signals coding visual motion. The model generates two implications that can be tested in future experiments: (1) compensatory pursuit neurons in MSTd should have the same direction preference for pursuit and retinal visual motion; (2) there should be non-compensatory pursuit neurons that show opposite preferred directions of pursuit and retinal visual motion.     

Cellular analysis of ocular circadian pacemaker coupling in Bulla: role of efferent impulses in phase shifting

The eyes of Bulla gouldiana, a marine snail, contain circadian oscillators that are coupled to each other. Obvious candidates for the coupling signals are the optic nerve compound action potentials (CAPs) that express the circadian rhythm and lead to efferent impulses in the contralateral optic nerve. In the present experiments, the role of the CAPs as coupling signals was evaluated. We found that, following desynchronization of the two ocular oscillators by phase-delaying one eye with manganese, subsequent phase shifts in the initially unshifted ocular rhythm only occurred during the time that efferent optic nerve signals were present. In addition, in the absence of ocular desynchrony, phase shifts of the ocular rhythm could still be effected by activation of the efferent pathway. The influence of efferent impulses on identified retinal cells was also evaluated. No effect of efferent signals on receptor layer cells was detected, while it was found that efferent impulses generated depolarizations in basal retinal neurons (BRNs), the putative circadian oscillator cells. Depolarization of the BRNs has been shown previously to be involved in the light entrainment pathway. Depolarization appears to be similarly involved in the coupling pathway, since membrane depolarizations that mimicked the efferent-induced postsynaptic potentials likewise generated phase shifts of the ocular rhythm.     

Nonvisual photoreceptors of the deep brain, pineal organs and retina

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.     

Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.     

Identification of CDK- and cyclin-like proteins in the eye of Bulla gouldiana

The ocular circadian rhythm in the eye of Bulla gouldiana is generated by a rhythm in membrane potential of retinal neurons that is driven by alterations in potassium conductance. Since potassium conductance may be modulated by the phosphorylation of potassium channels, the circadian rhythm may reflect rhythmic changes in protein kinase activity. Furthermore, the circadian rhythm recorded from the Bulla eye can be phase shifted by agents that affect protein synthesis and protein phosphorylation on tyrosine residues. Interestingly, the eukaryotic cell division residues. Interestingly, the eukaryotic cell division cycle is generated by similar processes. Rhythmic cell division is regulated by periodic synthesis and degradation of a protein, cyclin, and periodic tyrosine phosphorylation of a cyclin-dependent kinase (cdk), p34^cdc2. The interaction between these two proteins results in rhythmic kinase activity of p34^cdc2. Both cyclin and p34^cdc2 are pat of two diverse gene families, some of whose members have been localized to postmitotic cell types with no function yet determined. In the current work, we identify proteins similar to the cdks and cyclin in the eye of Bulla. Neither of these ocular proteins are found in mitotic cells in Bulla, and the cdk-like protein (p40) is specific to the eye. Furthermore, the concentration of the cyclin-like protein (p66) is affected by treatments that phase shift the circadain rhythm. The identification of cdk and cyclin-like proteins in the Bulla eye is consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in Bulla is related to the biochemical mechnism that regulates the eukaryotic cell division cycle. 1994 John Wiley & Sons, Inc.