Ocular Circadian Pacemaker Research Articles

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia.

The eye of the marine mollusk Aplysia californica contains an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [35S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.

Stopping the circadian pacemaker with inhibitors of protein synthesis.

The requirement for protein synthesis in the mechanism of a circadian pacemaker was investigated by using inhibitors of protein synthesis. Continuous treatment of the ocular circadian pacemaker of the mollusc Bulla gouldiana with anisomycin or cycloheximide substantially lengthened (up to 39 and 52 hr, respectively) the free-running period of the rhythm. To determine whether high concentrations of inhibitor could stop the pacemaker, long pulse treatments of various durations (up to 44 hr) were applied and the subsequent phase of the rhythm was assayed. The observed phases of the rhythm after the treatments were a function of the time of the end of the treatment pulse, but only for treatments which spanned subjective dawn. The results provide evidence that protein synthesis is required in a phase-dependent manner for motion of the circadian pacemaker to continue.

Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia.

The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Serotonin is known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate whether potassium channels are involved in effects on the ocular circadian rhythm. Our experimental approach was to study the effect of the potassium channel antagonist barium on serotonin-induced phase shifts of this rhythm. The application of barium was found to block serotonin-induced phase shifts whereas barium alone did not cause significant phase shifts. The effects of barium were found to be dose dependent. In addition, barium blocked forskolin-induced phase advances but did not interfere with serotonin-induced increases in cAMP content. Finally, barium antagonized serotonin-induced suppression of compound action potential activity. These results are consistent with a model in which the application of serotonin phase shifts the ocular pacemaker by causing a membrane hyperpolarization which is mediated by a cAMP-dependent potassium conductance.

Photoresponsiveness of Aplysia Eye is Modulated by the Ocular Circadian Pacemaker and Serotonin.

The eye of the sea hare, Aplysia, contains a circadian pacemaker that controls rhythmic behaviors of the animal. This report shows that the pacemaker controls the photoresponsiveness of the eye as well. The electro-retinogram (ERG) of the isolated eye-optic nerve preparation, evoked by brief green light pulses in otherwise dark conditions, was recorded regularly, while the circadian rhythm of compound action potential activity was continuously recorded from the optic nerve. The waveform of the ERG changed systematically and rhythmically during the circadian cycle. One wave component of the ERG was prominent during the subjective night phase of the rhythm when the compound action potential frequency was minimal; and it was inconspicuous during the subjective day phase of the rhythm when the compound action potential frequency was maximal. Because eyes attached to the central nervous system and isolated eyes both exhibited the same rhythmic ERG changes, the circadian pacemaker in the eye is responsible for modulation of the ERG. Addition of serotonin, a putative efferent transmitter, to the bathing saline induced the ERG wave component characteristic of the subjective night phase of the rhythm. The threshold serotonin concentration was 10-7 M, and serotonin had a long lasting effect.

Monoclonal antibodies recognize localized antigens in the eye and central nervous system of the marine snail Bulla gouldiana.

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.

Calcium channels mediate phase shifts of the Bulla circadian pacemaker.

1. Light-induced phase advances of the activity rhythm of the Bulla ocular circadian pacemaker are blocked when the extracellular calcium concentration is reduced with EGTA to 0.13 microM. Phase advances are also blocked in low calcium solutions without EGTA [( Ca] less than 50 microM). 2. The dependence of light-induced phase delays on extracellular calcium concentration in EGTA-free seawater was determined. Phase delays are blocked at calcium concentrations below 400 microM, and reduced at concentrations of 1 mM and 3.5 mM (relative to shifts in normal ASW, [Ca] = 10 mM). Phase delays are also reduced and blocked at calcium concentrations higher than normal (60 mM and 110 mM, respectively). 3. Low calcium EGTA also blocked both phase delays and phase advances induced by pulses of depolarizing high K+ seawater. Low calcium EGTA pulses presented alone at the same times did not generate significant phase shifts. 4. The organic calcium channel antagonists verapamil, diltiazem and nitrendipine as well as the inorganic calcium channel antagonists La3+, Co2+, Cd2+, and Mn2+ were applied along with light pulses, however, the treated eyes were either phase shifted by these substances, or these substances were found to be toxic. 5. The inorganic calcium channel antagonist Ni2+ blocked both light-induced phase delays and advances at a concentration of 5 mM. Ni2+ applied alone did not generate significant phase shifts. Phase delays induced by high K+ seawater were blocked in the presence of 50 mM Ni2+ but not in 5 mM Ni2+. The light-induced CAP activity of the putative pacemaker cells was not inhibited by Ni2+, suggesting that its blocking action was probably via its known role as a calcium channel antagonist.

Phase-shifts of the [formula omitted] ocular circadian pacemaker in the presence of calmodulin antagonists

Previous work has shown that light-induced phase shifts of the Bulla ocular circadian pacemaker require extracellular calcium, suggesting the possibility that the action of calcium as a second messenger via calmodulin is an element in the phase shifting mechanism. The calmodulin antagonists calmidazolium, trifluoperazine (TFP) and W7 were applied with phase shifting light pulses. Light phase shifts were not blocked by calmidazolium or TFP, suggesting that calmodulin does not mediate light-induced phase shifts. Period changes were observed with treatments of both TFP and W7, but not with calmidazolium and are probably not calmodulin-mediated.

The Bulla ocular circadian pacemaker. I. Pacemaker neuron membrane potential controls phase through a calcium-dependent mechanism.

In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker of Bulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that: 1. Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization. 2. Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180 degrees in circadian time to depolarization. 3. Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection. 4. The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts. 5. The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3 X 10(-7) M inhibits light-induced phase shifts without blocking the photic response of the BRNs. The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.

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.

Cellular analysis of theBulla ocular circadian pacemaker system

Cellular analysis of theBulla ocular circadian pacemaker system

Cellular analysis of theBulla ocular circadian pacemaker system

Cellular analysis of theBulla ocular circadian pacemaker system

Cellular analysis of theBulla ocular circadian pacemaker system

The isolatedBulla eye expresses a circadian rhythm in optic nerve impulse frequency. In an effort to determine the anatomical location of the circadian pacemaking system within the retina we surgically reduced the eye. We report that: