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.     

Analysis of coupling between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between weakly coupled two optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording the locomotor activity, under light-dark cycles with various lengths, after the optic nerve was unilaterally severed. The activity rhythm split into two components under the light cycles different from 24 h: one was readily entrained to the light cycle and the other only loosely entrained or freeran. Additional removal of the optic lobe on the intact side resulted in a loss of the entrained component and that on the blinded side caused the reverse effect, indicating that the entrained component was driven by the pacemaker on the intact side and the other by the one on the blinded side. The synchronization between the two components was achieved only in light cycles with a limited length between 23 and 25 h. Without this range, the desynchronization of the components occurred. In the split rhythm, the phase-dependent modulation of the period of freerunning component and the mutual suppression of locomotor activity during the subjective day phase were clearly observed. The suppression was also evident in the lights-on peak that was the masking effect of light. The light cycle with dim light significantly reduced the ratio of animals with the pacemaker coupling as well as the magnitude of the period modulation. These results suggest (1) that the mutual coupling is achieved only when the difference in the periods between the two pacemakers is within an allowable range, (2) that the photic information is also involved in the mechanism of mutual coupling, and (3) that the suppression of activity occurs at the regulatory center for locomotion.Abbreviations CT circadian time - DD constant darkness - LL constant light - LD light to dark cycle - T length of light to dark cycle - freerunning period     

Neural pathways involved in mutual interactions between optic lobe circadian pacemakers in the cricketGryllus bimaculatus

The circadian locomotor rhythm of the cricketGryllus bimaculatus is primarily generated by a pair of optic lobe circadian pacemakers. The two pacemakers mutually interact to keep a stable temporal structure in the locomotor activity. The interaction has two principal effects on the activity rhythm, i.e., phase-dependent modulation of the freerunning period and phase-dependent suppression of activity driven by the partner pacemaker. Both effects were mediated by neural pathways, since they were immediately abolished after the optic stalk connecting the optic medulla to the lobula was unilaterally severed. The neural pathways were examined by recording locomotor activity, under a 13 h light to 13 h dark cycle, after the optic nerves were unilaterally severed and the contralateral optic stalk was partially destroyed near the lobula. When the dorsal half of the optic stalk was severed, locomotor rhythm mostly split into two components: one was readily entrained to the given light-dark cycle and the other freeran with a marked fluctuation in freerunning period, where the period of the freerunning component was lengthened or shortened when the onset of the entrained component occurred during its subjective night or day, respectively. The phase-dependent modulation of activity was also observed in both components. However, severance of the ventral half of the optic stalk resulted in appearance only of the freerunning component; neither the phase-dependent modulation of its freerunning period nor the change in activity level was observed. These results suggest that neurons driving the mutual interaction and the overt activity rhythm run in the ventral half of the proximal optic stalk that includes axons of large medulla neurons projecting to the cerebral lobe and the contralateral medulla.Abbreviations LD light dark cycle - freerunning period     

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

Summary 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. Both light and serotonin are known to influence the phase of this ocular rhythm. The current study evaluated the effect of FMRFamide on both light and serotonin induced phase shifts of this rhythm. The application of FMRFamide was found to block serotonin induced phase shifts but, by itself, FMRFamide did not cause significant phase shifts. Furthermore, the effects of FMRFamide on light-induced phase shifts appeared to be phase dependent (i.e., the application of FMRFamide inhibited light-induced phase delays but actually enhanced the magnitude of phase advances). As in Aplysia, the eye of Bulla gouldiana also contains a circadian pacemaker. In Bulla, FMRFamide prevented light-induced phase advances and delays. Although FMRFamide alone generated phase dependent phase shifts, it did not cause phase shifts at the phases where it blocked the effects of light. These data demonstrate that FMRFamide can have pronounced modulatory effects on phase shifting inputs to the ocular pacemakers of both Aplysia and Bulla.Abbreviations ASW artificial seawater - CAP compound action potential - CT circadian time - 5-HT serotonin     

Circadian and light-induced conductance changes in putative pacemaker cells of Bulla gouldiana

The ocular circadian rhythm of compound action potential frequency in Bulla gouldiana is driven by rhythmic changes in the membrane potential of putative circadian pacemaker cells. Changes in the membrane potential of these neurons is required for light-induced phase shifts of the rhythm. We have tested the proposition that these changes in membrane potential reflect underlying changes in ionic conductances. We have found that: 1. Membrane conductance in the dark is highest during the subjective night when the cells are hyperpolarized, decreases as the cells depolarize spontaneously near projected dawn and is lowest during the subjective day. The changes in membrane potential and conductance follow a similar time course. 2. Long pulses of light delivered to eyes during their subjective night produce a characteristic response: There is initially a large, phasic depolarization accompanied by a burst of CAPs; this is followed by a repolarizing phase during which CAP activity is reduced to zero; and finally a tonic depolarization develops that is accompanied by a resumption of CAP activity at a steady rate. 3. During the subjective night, the tonic depolarization is accompanied by a decrease in conductance compared to the previous dark value. However, light pulses of similar duration delivered to eyes during their subjective day causes tonic depolarizations and increased CAP activity, but no measurable change in conductance. 4. Membrane responses to light are sensitive to agents that reduce Ca2+ flux. Light pulses during the subjective night produce a phasic depolarization, but the repolarization phase is eliminated in low Ca2+/EGTA seawater and is reduced in 5 mM Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of neuropeptides in efferent terminals of the eye in the marine snail,Bulla gouldiana

Summary Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of ¹²⁵I-NGF to solubilized receptors. Analysis of ¹²⁵I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.Abbreviations DRG dorsal root ganglia - NGF nerve growth factor - RA retinoic acid     

Physiology of avian circadian pacemakers.

The pineal gland plays a cental role in the circadian organization of birds, although it is clearly only one component in a system with other components that have not yet been positively identified. The relative importance of the pineal and other components may vary from one group of birds to another. In the most thoroughly studied species, the house sparrow, pineal removal abolishes circadian rhythmicity; rhythmicity is restored by transplantation of a donor bird's pineal and the restored rhythm has the phase of the donor. This, and other evidence, argues convincingly that the pineal is a pacemaker in the sparrow circadian system. The pineal of the chicken has circadian rhythms in several biochemical parameters that result in the rhythmic synthesis of melatonin. The activity of one enzyme in this pathway is rhythmic for at least two cycles in organ culture. In view of this result it is interesting that pineal removal does not abolish circadian rhythmicity in chickens. The fact that lesions of the suprachiasmatic nuclei abolish circadian rhythms in sparrows, several mammalian species, and perhaps Japanese quail and reptiles, suggests that vertebrate circadian organization may be based on differentially weighted interactions between the pineal, the suprachiasmatic nuclei, and perhaps other brain regions.     

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.     

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.     

Mutual interactions between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between the bilaterally paired optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording locomotor activity, under constant light or constant red light, after the optic nerve was unilaterally severed.

Properties of two molecular forms of beta-glucuronidase from the mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.     

A delayed rectifier current is modulated by the circadian pacemaker in Bulla

Basal retinal neurons of the marine mollusc Bulla gouldiana continue to express a circadian modulation of their membrane conductance for at least two cycles in cell culture. Voltage-dependent currents of these pacemaker cells were recorded using the whole-cell perforated patch-clamp technique to characterize outward currents and investigate their putative circadian modulation. Three components of the outward potassium current were identified. A transient outward current (IA) was activated after depolarization from holding potentials greater than -30 mV, inactivated with a time constant of 50 ms, and partially blocked by 4-aminopyridine (1-5 mM). A Ca(2+)-dependent potassium current (IK(Ca)) was activated by depolarization to potentials more positive than -10 mV and was blocked by removing Ca2+ from the bath or by applying the Ca2+ channel blockers Cd2+ (0.1-0.2 mM) and Ni2+ (1-5 mM). A sustained Ca(2+)-independent current component including the delayed rectifier current (IK) was recorded at potentials positive to -20 mV in the absence of extracellular Na+ and Ca2+ and was partially blocked by tetraethylammonium chloride (TEA, 30mM). Whole-cell currents recorded before and after the projected dawn and normalized to the cell capacitance revealed a circadian modulation of the delayed rectifier current (IK). However, the IA and IK(Ca) currents were not affected by the circadian pacemaker.     

Phase-shifts of the Bulla 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.     

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.     

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

The circadian rhythm and photosensitivity of small impulses of the Bulla eye

Summary The eye of the mollusk Bulla gouldiana contains a pacemaker that generates a circadian rhythm in compound action potentials (CAPs) in the optic nerve. In this paper, we present evidence of a second circadian rhythm in the optic nerve of the eye maintained in darkness at 15 °C. This is a rhythm in the frequency of small (10–40 V) neural impulses that occurs about 12 h out-of-phase with the rhythm in CAPs. Typically, the small-spike frequency is at a minimum within an hour of the peak in CAP frequency and is maximal during the subjective night. Like the CAP rhythm, the phase of the small-spike rhythm is determined by the prior light/dark cycle. A rebound in small-spike activity following the end of a light pulse and the presence of photoinhibited impulses in surgically reduced eyes suggests that the cells that generate the small-spikes may be photoreceptors that are inhibited by light. In addition, by using isolated nervous system preparations, we have found that smallspikes occur in the two optic nerves in a one-for-one relationship immediately following a light-to-dark transition. This inter-eye communication may be involved in the coupling of the ocular pacemakers.Abbreviations ASW artificial sea water - BRN basal retinal neuron - CAP compound action potential