Eye Of Aplysia Research Articles

Effects of efferent activity on entrainment of the Aplysia eye

1. 1. The effects of efferent optic nerve activity on the speed of entrainment of the ocular oscillators in Aplysia were investigated by severing one optic nerve in 21 animals and evaluating reentrainment rates to a light cycle phase advanced 10hr in relation to a prior light schedule. 2. 2. Reentrainment to this phase advanced light cycle was essentially complete in both the intact and denervated eyes within 3 days; and there was no indication that the time course of entrainment was significantly different in the two eyes. 3. 3. The phase angle of the rhythm of neurally isolated eyes, however, consistently phase led the intact eyes by approx 1 hr. Thus, the results indicate that while efferent activity does not significantly effect the speed of entrainment, it appears to influence steady-state phase angle.

Serotonin Shifts the Phase of the Circadian Rhythm from the Aplysia Eye

A putative neurotransmitter, serotonin, may be used to transmit temporal information in the eye of Aplysia, because it can shift the phase of the circadian rhythm of spontaneous optic nerve impulses from the eye and the eye contains a significant quantity of serotonin. Serotonin acts either directly on the cell, or cells, containing the circadian pacemaker or on cells electronically coupled to the pacemaker cells.

Neuronal circadian rhythm: phase shifting by a protein synthesis inhibitor.

A potent inhibitor of protein synthesis, anisomycin, was applied (10(-6)M) in 6-hour pulses at specific phases in the circadian rhythm of endogenous compound action potential (CAP) activity recorded from the eye of Aplysia in vitro. The phase of the circadian rhythm was systematically advanced or delayed (up to 15 hours) depending on the specific phase at which the pulse was applied. The resultant phase response curve implicates protein synthesis on the eukaryotic ribosome as a fundamental part of the controlling processes that constitutes the circadian clock.

Circadian Rhythm of Output From Neurones in the Eye Of Aplysia

ABSTRACT A relaxation oscillator, feed-back model for the circadian clock in the eye of Aplysia is proposed to account for the experimental findings described earlier. Further data on the effects of light pulses and temperature pulses are reported here to test the hypothesis that light and temperature perturb the clock oscillation at different points in the feed-back loop. The rising phase of the CAP frequency rhythm is postulated to be due to an energy-requiring, synthesis process, and the falling phase to a passive, diffusional process. Synthesis produces a substance, C, which controls CAP frequency, and the level of which oscillates about a reference level, R. The synthesis phase of the oscillation is suggested to be temperature compensated from about 9 °C to at least 22.5 °C. Cooling the eye to 6 °C for long periods therefore inhibits synthesis so that the clock eventually stops at its lowest phase point. 12 h cold pulses of 4 °C applied during the rising phase of the rhythm (i.e. during synthesis) cause large phase delays (9 h), while similar cold pulses applied during the falling phase (i.e. during diffusion} cause only small phase delays (2 h). The action of light is to lower the value of the reference level, R, so that the constant illumination damps the oscillation until the clock is stopped at its lowest phase point. The model predicts that light pulses applied during the rising phase will effectively accelerate the increase in level of C, thus causing phase advances, while phase delays will result from light pulses applied during the falling phase. A phase response curve for 2 h, 1100 lux light pulses confirms this. A rhythm splitting effect due to an appropriately timed light pulse is predicted and tested. Possibilities of clock control of the CAP generating mechanism are discussed with reference to recent findings on the regulation of membrane potential oscillations in molluscan bursting pacemaker neurones.

Circadian Rhythm of Output from Neurones in the Eye of Aplysia

ABSTRACT The compound action potential (CAP) output from the isolated eye of Aplysia, in darkness at constant temperature, exhibits a circadian rhythm of CAP frequency and a circadian rhythm of CAP amplitude when recorded in culture medium for up to two weeks. Deuterated culture medium lengthened the period of the CAP frequency rhythm linearly from 26-7 h in normal culture medium to 33·7 h in 50% D2O culture medium. In 60 % D2O culture medium, the rhythm of CAP frequency was abolished after a single cycle. Lowering temperature had a direct inhibitory effect on the CAP generating mechanism, so that CAP production was virtually abolished below 7 °C. Lowering temperature decreased the amplitude and increased the period of the rhythm, and in eyes maintained at 9·5 °C the rhythm damped out after several cycles. Between 15 and 22·5 °C there was almost complete temperature compensation (Q10 1·07). It is suggested that the CAP generating cells of the eye may be similar in mechanism to central endogenous bursters in Aplysia and other gastropods, and that the circadian rhythm of CAP frequency and CAP burst frequency may be due to a clock-controlled change in frequency of a membrane potential oscillation in the electrotonically coupled secondary neurones.

Ultrastructure of the secondary cells in the Aplysia eye

The ultrastructure of nonreceptor secondary cells (SC) in the Aplysia eye was studied. SC are found along the periphery of the retina, possess a single axon whose branches are found in the neuropile and the optic nerve, and contain dense core vesicles with a mean diameter of 110 nm. Results of electron microscope histochemistry suggest the dense cores contain a catecholamine. Intercellular junctions are found between SC and between SC and receptor cells. Synapses, in which presynaptic processes contain small clear vesicles and postsynaptic processes contain dense vesicles similar to those in the SC, occur in the neuropile. Other vesicles with a mean diameter of 150 nm and granular cores of varying electron density were observed in axons in the neuropile and the optic nerve but not in SC. These findings are correlated with previous studies of SC physiology and the previous demonstration that SC contain a catecholamine, probably dopamine.

Neurophysiological mechanisms involved in photo-entrainment of the circadian rhythm from the Aplysia eye.

AbstractAn attempt was made to identify the neurophysiological processes involved in entrainment of the circadian rhythm of spontaneous optic nerve potentials from the Aplysia eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information.A secretory step does not appear to be involved in the flow of entrainment information from the environment to the circadian oscillator. A treatment (HiMg LoCa) capable of inhibiting secretion did not interfere with phase shifting by light. Furthermore, treating eyes with putative transmitters or extracts of eyes did not phase shift the free running rhythm. Also, the phase shifting information is not translated into action potentials before reaching the oscillator since TTX–HiMg LoCa solutions did not block the light‐induced phase shift. The photoreceptor potential does seem to be important for light‐induced phase shifts. A correlation was found between the effects of treatments on the ERG and their effects on the light‐induced phase shift. Solutions which decreased the ERG by 90% or more blocked phase shifting whereas solutions which decreased the ERG by less than 74% had no effect on phase shifting by light.The results from these studies are consistent with two pathways for the flow of phase shifting information to the circadian oscillator. The circadian oscillator may be associated with receptor cells and the entrainment pathway would include a step involving the photoreceptor potential. Alternatively, the circadian oscillator may be associated with secondary cells and receive entrainment information via the photoreceptor potential and passive spread of current through a gap junction. Higher order cells than second‐order ones are probably not involved in the entrainment pathway.

Eye of Navanax: Optic activity, circadian rhythm and morphology

1. 1. Navanax eyes display a circadian rhythm of spontaneous neural activity. This rhythm re-entrains to a phase shifted light-dark cycle. 2. 2. The eye spontaneously generates large and small amplitude nerve impulses and the eye receives centrifugal neural activity from the cerebral ganglion. 3. 3. The eye is a vesicular type with the typical gastropod stratified retina of villous, pigmented, somatic, and neural layers. The receptor cells of Navanax are relatively large in size and there are relatively few of these in the eye. 4. 4. The eye has a unique bi-lobed lens and associated retina which contrasts to the more usual spherical arrangement in gastropods. 5. 5. Similarities and differences between the Navanax and the Aplysia eyes are discussed. 6. 6. The features of the Navanax eye indicate that it is an interesting preparation for both circadian rhythm and photoreceptor physiology studies .

Effects of divalent cations and metabolic poisons on the circadian rhythm from theAplysia eye

1. The isolated eyeof Aplysia has a circadian rhythm of compound optic nerve potentials. An attempt to inhibit entrainment of this rhythm by blocking synaptic transmission led to the discovery that one such inhibitor, manganese, produced phase shifts in the rhythm. Treatments of manganese (10 mM) of 6 h duration at different phases of the rhythm resulted in a phase response curve composed of only delay phase shifts (Fig. 1). 2. Many biological effects of Mn++ involve Ca++ dependent processes and this appears to be the case here since the phase shifting effect of Mn++ was antagonized by increasing external Ca++ concentrations (Fig. 3). 3. Mn++ does not appear to be producing phase shifts by disrupting the flux of Ca++ across the plasma membrane since changing such fluxes by either raising extracellular Ca++ (50 mM) or lowering extracellular Ca++ with EGTA (Ca++ <10−7 M) did not cause phase shifts in the rhythm. 4. A Ca++/Mg++ ionophore, A23187, was used to affect the fluxes of these ions across intracellular membranes. Treatments of A23187 (7.5×10−6 M) produced delay phase shifts in the rhythm whether or not a Ca++ or Mg++ concentration gradient existed across the plasma membrane (Figs. 4, 5). In addition, an all-delay response curve resulted when eyes were treated at different phases with A23187 plus EGTA (Fig. 6). 5. Our results, and the known effects of A23187, led to the hypothesis that phase shifting by A23187 resulted either from the transport of Ca++ and Mg++ out of mitochondria or from oxidative phosphorylation becoming uncoupled as a result of the reuptake of these ions. 6. DNP (0.2 mM) and NaCN (2 mM) which both inhibit oxidative phosphorylation and cause release of Ca++ and Mg++ from mitochondria, also produced phase response curves with only delays (Fig. 9). 7. Mn++, A23187, DNP, and NaCN have a common effect on oxidative phosphorylation and on the ability of mitochondria to regulate Ca++ and Mg++. The correlation between the similar phase response curves of these treatments (Fig. 10) and their common effects on mitochondria suggests that they are all affecting the rhythm by blocking energy production and/or causing the release of mitochondrial Ca++ and Mg++. The manner in which changes in energy production or in the concentrations of Ca++/Mg++ might affect the rhythm is discussed.

Control systems model of the spontaneous and light-evoked patterns of the compound action potentials in the eye of Aplysia.

The spontaneous and light-evoked firing patterns of the compound action potentials (CAPs) in the isolated eye of Aplysia are experimentally observed from a control systems viewpoint. The eye, exhibiting an endogenous bursting activity and a marked circadian rhythm in darkness, is a self-excited system and responds to illumination with a controlled electrical activity. It is represented by the nonlinear systems model predicting the firing-rate variation of the CAPs. Construction of the model is based on the eye's biological model reported in the literature, and involves an experimental analysis of the system characteristics using linear approximation and conventional control systems techniques. The model is simulated and found to adequately conform to the observed behavior of the eye under various experimental conditions.

Aplysia eye: modulation by efferent optic nerve activity

Aplysia eye: modulation by efferent optic nerve activity

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies.

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0-7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.

Dye marking neurons in the eye of Aplysia

1. 1. Intracellular responses from the cells of the eye of Aplysia were of 3 general types: depolarizing receptors (R), hyperpolarizing receptors (H) and neurons (D) that spike in correlation with both dark activity and light evoked CAP in the optic nerve (Fig. 2). 2. 2. These cell types were injected with Procion Yellow to mark their position in the retina (Figs. 3 & 4). 3. 3. Backfilling the retinal cells with Procion Yellow filled receptor cells in the pigmented retina and (D) neurons in the neuropile region showing that probably both of these cell types have axons in the optic nerve (Figs. 5, 6 & 7). 4. 4. These data suggest that autogenic (or circadian) activity in the R receptors or D neurons would be directly represented in the optic nerve by the axons of these cells.

Extraocular photoreceptors can entrain the circadian oscillator in the eye ofAplysia

The eye ofAplysia contains a circadian oscillator which can be entrained by cycles of white light (Jacklet, 1969b). Photoreceptors sufficient for entrainment of this oscillator reside within the eye itself (Eskin, 1971). We now report that extraocular photoreceptors can also entrain the ocular oscillator. Our evidence is: TheAplysia nervous system contains several circadian oscillators and several photoreceptors. Functional integrity of the organism requires that these elements be synchronized with one another. A neural, as opposed to hormonal, link is required for coupling the circadian oscillator in the eye to other neural sources of temporal information.

The effects of constant light and light pulses on the circadian rhythm in the eye ofAplysia

1. A circadian rhythm in the frequency and amplitude of compound action potential (CAP) from the isolated eye ofAplysia persists for a week or morein vitro in constant darkness. This rhythm has a period of about 26 hours (Figs. 1,2) in a specific culture medium and may express several periodic amplitude components (Fig. 3). 2. Constant light (LL) of low intensity shortens the period (Pig. 4) and reduces the range of oscillations. Higher intensity LL results in a further reduction in range, a greater variability of CAP frequency from hour to hour, alterations in the period, and possibly rhythm splits (Fig. 5). 3. Pulses of light given at specific points in the circadian cycle shift the phase of the rhythm (Fig. 7). The resulting phase response curve (Fig. 8) is similar to response curves for the activity of diurnal animals and potassium pulses on the eye rhythm.

Spontaneous and light-induced compound action potentials in the isolated eye of Aplysia: Initiation and synchronization

(1) The isolated eye of Aplysia shows a bursting pattern of spontaneous compound action potentials (CAPs) in the dark. The ‘light response’, also composed of CAPs, may be separated into a phasic initial response and a tonic late response similar in form to the dark discharges when the illumination is of low intensity. (2) Blockage of chemical synapses with La 3+ or hi Mg 2+-lo Ca 2+ stops the dark discharge and tonic light response but not the phasic light response. Synchrony of the CAPs is not affected. (3) Ca 2+-free solutions produce continuous rapid firing of CAPs, seldom coordinated into bursts. (4) Replacement of chloride by propionate abolishes all CAPs for several hours, but leaves the ERG intact and the optic nerve electrically excitable. (5) Low sodium levels (about 50% normal) suppress dark discharge and tonic light activity but allow a normal phasic light response. (6) It is concluded that receptors transmit light-induced excitation to a higher order neuron population via electrical junctions, and that synchrony of the CAPs is also due to electrical junctions, interconnecting the higher order population. One or more pacemaker cells are postulated to drive the higher order neurons by chemical synapses, producing the dark discharge and the low intensity tonic light response. The pacemaker mechanism may be sodium dependent.

The circadian rhythm in the eye ofAplysia

1. The frequency of spontaneous compound action potentials (CAP) from the optic nerve of the eye ofAplysia is dramatically increased in zero calcium artificial sea water (ASW) but the circadian rhythm in this activity persists. 2. In high magnesium, >2.7 × normal, the spontaneous CAP activity is blocked. This effect is a result of decreased membrane responsiveness and not due to blocking chemical synapses. 3. In low calcium and high magnesium (at levels where molluscan chemical synapses are blocked) the spontaneous CAP activity and the circadian rhythm are clearly expressed. The average period for the rhythm is 23.3 hours, which is not different from the period in normal ASW. 4. These results suggest the neuronal oscillators that produce the circadian rhythm have axons directly in the optic nerve or they are electrontonically coupled to axons in the optic nerve, and that chemical synapses are not necessary for expression of the circadian rhythm.

Ultrastructure of the eye of Aplysia

The eye and optic nerve of the sea hare, Aplysia californica , were examined with the light and electron microscopes. The eye is a closed vesicle type with a spheroidal lens nearly completely surrounded by the retina. It contains several thousand receptor, pigmented, and other cells. Receptor cells are replete with 500–700 A clear vesicles, pigment granules, and have a diffusely organized microvillus rhabdomere that projects to the lens. The fibers of the receptor cells and other cells form a retinal neuropile where synapses occur between these fibers. Pigmented cells have a short microvillous distal segment and associated rudimentary cilia. Cellular processes which contain dense vesicles (900–1200 A) are prominent in the retinal neuropile. The majority of the processes appear to be the neurites of the receptor and other cells that contribute to the optic nerve. Some of these cellular processes end in microvillous processes on diffuse vascular spaces at the base of the retina near the epineural sheath. Receptor fibers and perhaps others contribute to the several thousand optic nerve fibers (0.3–3.0 μ in diameter).

Phase shifting a circadian rhythm in the eye ofAplysia by high potassium pulses

1. The circadian rhythm of spontaneous optic nerve impulses from the isolated eye ofAplysia californcia was studied. A general hypothesis was investigated: light-dark cycles, which can entrainin vitro the rhythm from the eye, couple to the intracellular clock mechanism through membrane depolarization. This hypothesis was tested by exposing isolated eyes for fixed durations of time to a depolarizing stimulus, namely, elevation of K0+ (hi-K pulses). 2. Depending on the phase of the rhythm in at which the eyes were treated, hi-K pulses produced either advance or delay phase shifts in the rhythm. A phase response curve was thus generated for 107 mM k0+ pulses of 4 hr duration. 3. If the duration of the hi-K pulse was varied, keeping the phase at which the pulse started constant, the magnitude of the phase shift varied in a linear manner with the duration of the pulse. 4. To examine the involvement of transmitter and neurohormone release in the production of the phase shifts, eyes were exposed to hi-K pulses in the presence of a medium (high Mg++, low Ca++) which should inhibit such release. Since only slight differences were observed in the phase shifts produced by hi-K and by hi-K in the presence of high Mg++, low Ca++, transmitter and neurohormone release do not appear to be involved in phase shift production by hi-K. It is likely that membrane depolarization alone is responsible for the phase shifts.

Circadian rhythm: population of interacting neurons.

The circadian rhythm in the requency of compound action potentials recorded from the isolated eye of Aplysia is a consequence of interactions among the cells of the retinal population. As the population number is reduced to a critical 20 percent, progressively shorter circadian periods and ranges are expressed. Below the critical number, the population oscillates at ultradian frequencies.