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.     

TheBulla ocular circadian pacemaker

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.     

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     

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)     

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 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.     

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.     

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)     

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

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. 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 serotonininduced 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.Abbreviations ASW artificial seawater - Ba^{+ +} barium - CAP compound action potential - CT circadian time - 5-HT serotonin - TEA tetraethylammonium     

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.     

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.     

Reduced light response of neuronal firing activity in the suprachiasmatic nucleus and optic nerve of cryptochrome-deficient mice

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day-night difference was not observed in Cry-deficient mice. In addition, we observed day-night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals.     

Serotonin increases excitability of rabbit C-fiber neurons by two distinct mechanisms

Serotonin (5-HT) increases impulse activity in visceral afferent C-fibers in vivo. A 5-HT-induced membrane depolarization may partially account for this effect. Here, we examined the potential contribution of an additional mechanism to the 5-HT-mediated increase in impulse activity. Approximately 40% of rabbit visceral C-fiber neurons exhibit a protracted (greater than 3 s) spike afterhyperpolarization (AHPslow) that is a major determinant of repetitive firing properties in these neurons. Intracellular recording methods were applied to rabbit nodose ganglion neurons in vitro to assess whether 5-HT could increase excitability through effects on the AHPslow. Results revealed a concentration-dependent 5-HT-mediated depression of the AHPslow amplitude and duration that was accompanied by decreased accommodation of action potential firing. Experiments with 5-HT receptor antagonists further showed that this autacoid depressed the AHPslow through a different 5-HT receptor subtype than that subserving the 5-HT-induced depolarization. Thus the AHPslow represents a distinct locus where 5-HT can increase the impulse activity of these visceral C-fiber afferents.     

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity. © 1996 John Wiley & Sons, Inc.     

Serotonin induced protein phosphorylation in the Aplysia eye

Serotonin (5-HT) increases the phosphorylation of two low molecular weight phosphoproteins of 23,000 and 15,000 daltons molecular weight and decreases the phosphorylation of a 20,000 dalton phosphoprotein in the isolated Aplysia eye. The cAMP analog 8-benzylthio cAMP increases and decreases the phosphorylation of the 23,000 and 20,000 dalton 5-HT sensitive phosphoproteins, respectively. The effect of 5-HT on protein phosphorylation is not affected by the phase of the circadian rhythm of spontaneous compound action potentials generated in the eye.     

Antiserum to an eye-specific protein identifies photoreceptor and circadian pacemaker neuron projections in Aplysia

The marine gastropod Aplysia has a circadian clock in each eye that generates a circadian rhythm of optic nerve activity. The axons of pacemaker neurons carry the rhythmic activity to the brain where it can be recorded from various ganglionic connectives as it is distributed throughout the CNS. We had previously identified an eye-specific 48-kD protein using an antiserum, anti-S, that recognizes the period gene product of Drosophila. We have now obtained two partial amino acid sequences of the 48-kD protein and raised a polyclonal antiserum using a synthetic peptide with the amino acid sequence of one of them. The antiserum recognizes a family of spots of M_r 47–48 kD and P_i 5.9–6.0 on 2D immunoblots of eye proteins. The immunoblot staining intensity does not exhibit a circadian rhythm. Used in immunocytochemistry, the antiserum recognizes fibers in the optic nerve and retinal neuropil, pacemaker neurons, certain photoreceptors, and the photoreceptor rhabdom layer. It stains the optic nerve fibers and optic fiber terminals in the cerebral optic ganglion and recognizes the cerebral optic tracts, putative synaptic exchange areas, and optic tract projections from the cerebral ganglion into various head nerves and interganglionic connectives. The function of the 48-kD protein is not known but it could be involved in the maintenance or regulation of the retinal afferent pathways, including the pacemaker neuron axons, known from previous axonal transport and electrical recording studies to be the circadian output pathway. © 1993 John Wiley & Sons, Inc.     

Membrane electrical excitability is necessary for the free‐running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free‐running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell‐autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K⁺ channel stops the free‐running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free‐running larval pacemaker clock—like that of the adult pacemaker neurons they give rise to—requires membrane electrical activity to oscillate. In contrast to the free‐running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light–dark) conditions. This specific disruption of the free‐running clock caused by targeted K⁺ channel expression likely reflects a specific cell‐autonomous clock‐membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005