Circadian rhythm of optic nerve impulses recorded in darkness from isolated eye of Aplysia.

The isolated eye of the sea hare Aplysia califomica shows a circadian rhythm of optic nerve impulses when kept in total darkness. Peak activity on the first day of isolation occurs during the projected " dawn" of the light-dark cycle to which the whole animal had previously been entrained. Eyes from animals previously exposed to constant light show a free-running rhythm. This simple photoreceptor provides a quantized output with an ideal control (the other eye) for studies on rhythms.

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: 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.

Habituation and dishabituation: interactions between peripheral and central nervous systems in Aplysia.

The withdrawal response of the isolated siphon of Aplysia habituates to a light stimulus and dishabituates to a tactile stimulus, and vice versa, with or without connections to the central nervous system. The peripheral nervous system can dishabituate or enhance the response mediated by the central nervous system and vice versa. Normally the adaptive siphon withdrawal response of the intact animal must be mediated by the integrated activity of the peripheral and central nervous systems.

Synaptic connections between a transplanted insect ganglion and muscles of the host.

When a metathoracic ganglion from one cockroach (Periplaneta americana) is transplanted into the coxa of another cockroach, it innervates only those leg muscles that have been previously denervated. The transplanted ganglion evokes hyperpolarizing synaptic potentials in the host muscles that it innervates. These potentials are correlated with twitching of the host limb.

Nerve regeneration: correlation of electrical, histological, and behavioral events.

Wihtin 5 days after the leg nerves of a cockroach are injured, miniature end-plate potentials have disappeared. and the muscle is unresponsive to electrical stimulation. The soma of the injured neutron has a dense perinuclear ring of RNA. By 40 days after the injury, locomotor activity has returned, and the miniature end-plate potentials and evoked electrical responses have reappeared in the muscle. The RNA ring has disappeared, and the nucleus of the regenerating neuron has shifted to an eccentric position.

Habituation of reflexes in Aplysia: contribution of the peripheral and central nervous systems.

We studied the contribution of the Aplysia peripheral nervous system, in the siphon and gill, to habituation of the gill withdrawal reflex. After removal of one central ganglion, the parietovisceral, repeated stimulation of the siphon caused habituation of the reflex as it had with the ganglion intact, showing that there is a peripheral pathway between the siphon and gill with competence to mediate habituation. Repeated electrical stimulation of two efferent nerves to the gill, after removal of the parietovisceral ganglion, resulted in habituation of withdrawal movements, which shows that the terminals of the ganglion neurons in the gill are a site of habituation. Also, stimulation of one nerve dishabituates the withdrawal movements elicited by the other. These identify two sites of habituation in the gill in addition to sites in the parietovisceral ganglion.

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.

Nitric oxide stimulates cGMP production and mimics synaptic responses in metacerebral neurons of Aplysia.

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous systems of many vertebrates and invertebrates. We investigated the mechanism of NO action at an identified synapse between a mechanoafferent neuron, C2, and the serotonergic metacerebral cell (MCC) in the cerebral ganglion of the mollusc Aplysia californica. Stimulation of C2 produces a decreasing conductance, very slow EPSP in the MCC. C2 is thought to use histamine and NO as cotransmitters at this synapse, because both agents mimic the membrane responses. Now we provide evidence that treatment with NO donors stimulates soluble guanylyl cyclase (sGC) in the MCC, and as a result cGMP increases. S-Nitrosocysteine (SNC, an NO donor) and 8-bromo-cGMP (8-Br-cGMP) both induced the membrane depolarization and increase in input resistance that are characteristic of the very slow EPSP. Two inhibitors of sGC, 6-anilino-5,8-quinolinequinone (LY83583) and 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ), suppressed both the very slow EPSP and the membrane responses to SNC but not the histamine membrane responses. NO-induced cGMP production was determined in the MCC using cGMP immunocytochemistry (cGMP-IR). In the presence of 3-isobutyl-1-methylxanthine (IBMX), 10 microM SNC was sufficient to induce cGMP-IR, and the staining intensity increased as the SNC dose was increased. This cGMP-IR was suppressed by ODQ in a dose-dependent manner and completely blocked by 10 microM ODQ. Histamine did not induce cGMP-IR. The results suggest that NO stimulates sGC-dependent cGMP synthesis in the MCC and that cGMP mediates the membrane responses. The cotransmitter histamine induces essentially the same membrane responses but seems to use a separate and distinct second messenger pathway.

Electrophysiological organization of the eye of Aplysia.

The eye of Aplysia californica was studied by electrophysiological and histological methods. It has a central spheroidal lens which is surrounded by a retina composed of several thousand receptor cells which are replete with clear vesicles, pigmented support cells, neurons which contain secretory granules, and glial cells. The thin optic nerve that connects the eye to the cerebral ganglion gives a simple "on" response of synchronized action potentials. Tonic activity occurs in the optic nerve in the dark and is dependent on previous dark adaptation. Micropipette recordings indicate that the ERG is positive (relative to a bathelectrode) on the outer surface of the eye and negative in the region of the distal segments of the receptors. Intracellular recordings show that receptor cells have resting potentials of 40-50 mv and respond to illumination with graded potentials of up to 55 mv. Dark-adapted receptors exhibit discrete bumps on the graded response to brief light flashes. Other elements in the retina that do not give large graded responses fall into two classes. One class responds to illumination with action potentials that are in synchrony with the extracellularly recorded compound optic nerve potentials. The other class is tonically active and is depolarized or hyperpolarized and inhibited upon illumination. It is apparent that complex excitatory and lateral inhibitory interactions occur among the elements of the retina.

Localization of catecholamines in the eyes and other tissues of Aplysia.

A green fluorescence indicative of catecholamines (CA) was localized in the secondary cells (nonreceptor neurons), neuropile and optic nerve of the eye and other tissues in Aplysia by using the formaldehyde-induced fluorescence method for the demonstration of biogenic amines. The specificity of the induced fluorescence was confirmed by its absence in tissue not exposed to formaldehyde vapor, relatively rapid decay upon exposure to UV light and its chemical reduction by sodium borohydride. The fluorescence was greatly reduced in eyes treated with reserpine (depletes serotonin and catecholamines). Furhter confirmation that the green fluorescence in the eye was due to a CA and not to serotonin was obtained by showing that it was decreased or eliminated by alpha-methyl-para-tyrosine (an inhibitor of catecholamine synthesis), increased by incubation in dopamine and exhibited a peak emission (470 nm) characteristic of CA fluroescence. CA fluorescence was also observed in the neuropiles of the cerebral, pedal, pleural and parieto-visceral ganglia and in cells in the pedal ganglion, statocyst, mantle, anterior tentacles and siphon. The finding of CA in secondary neurons of the eye was unusual since CA-containing cells have not been observed in other gastropod eyes. The distribution of CA in Aplysia, in tissue other than the eye, is similar to that of other gastropod molluscs.

Photoresponsive pacemaker neurons from the dissociated retina of Aplysia.

Isolated retinal pacemaker neurons of Aplysia devoid of any morphologically specialized photoreceptive processes responded to illumination. Neurons were dissociated from other retinal cells and maintained in primary culture for whole cell recording. The membrane potential was depolarized and action potentials were evoked by illumination. Light adaptation diminished the response and prolonged the latency. Under voltage clamp neurons responded with increased inward current. Responses were slower and smaller than specialized photoreceptor responses. Pairs and small clusters of monopolar neurons also responded to light. These photoresponsive monopolar neurons are likely to be the output neurons of the ocular circadian clock. Although photoresponsive central neurons have been described in Aplysia, this is the first report of photoresponses from isolated retinal neurons.

Co-localization of NADPH-diaphorase and myomodulin in synaptic glomeruli of Aplysia.

We used NADPH-diaphorase staining as a marker for nitric oxide synthase to identify neurons and synapses in the nervous system of the mollusc Aplysia californica in which nitric oxide may be used as a transmitter. About 30 bilaterally paired neurons in the cerebral ganglion and a few neurons in other major ganglia were stained, as well as specific fiber tracts, neuropil and the lateral terminus, a synaptic glomerulus of the optic tract. The glomerulus was also stained by antisera to myomodulin, a peptide co-transmitter. The co-localization of myomodulin immunoreactivity and NADPH-diaphorase staining in the synaptic glomerulus, and the staining of select neurons and synaptic structures strongly suggests that nitric oxide functions in interneuronal communication.

The exponent of the calcium power function is reduced during steady-state facilitation in neuron R15 of Aplysia.

Stimulation of the right pleurovisceral connective at frequencies greater than 0.2 Hz produces a steady-state facilitation of the Input 1 excitatory postsynaptic potential (EPSP) in neuron R15 of Aplysia. The magnitude of facilitation is reduced by increases in the concentration of extracellular calcium. Unfacilitated EPSPs (EPSP1) exhibited a power function dependence on the concentration of extracellular calcium with an exponent (XEPSP1) of approximately 3. The exponent for facilitated release (XEPSPss) was examined by comparing steady-state EPSP amplitudes obtained in different static concentrations of calcium (Method A), or by changing the calcium concentration during continuous stimulation and determining changes in steady state EPSP amplitudes (Method B). Method A failed to reveal any dependence of XEPSPss on stimulation frequency, while Method B revealed frequency-dependent changes in XEPSPss consistent with the hypothesis that presynaptic calcium accumulation is at least partly responsible for steady-state facilitation. At a stimulation frequency of 0.5 Hz, the XEPSPss value was significantly reduced to 2/3 of the XEPSP1 value.

Nitric oxide signaling in invertebrates.

Nitric oxide (NO) is an unconventional neurotransmitter and neuromodulator molecule that is increasingly found to have important signaling functions in animals from nematodes to mammals. NO signaling mechanisms in the past were identified largely through experiments on mammals, after the discovery of NO's vasodilatory functions. The use of gene knock out mice has been particularly important in revealing the functions of the several isoforms of nitric oxide synthase (NOS), the enzyme that produces NO. Recent studies have revealed rich diversity in NO signaling. In addition to the well-established pathway in which NO activates guanylyl cyclase and cGMP production, redox mechanisms involving protein nitrosylation are important contributors to modulation of neurotransmitter release and reception. NO signaling studies in invertebrates are now generating a wealth of comparative information. Invertebrate NOS isoforms have been identified in insects and molluscs, and the conserved and variable amino acid sequences evaluated. Calcium-calmodulin dependence and cofactor requirements are conserved. NADPH diaphorase studies show that NOS is found in echinoderms, coelenterates, nematodes, annelids, insects, crustaceans and molluscs. Accumulating evidence reveals that NO is used as an orthograde transmitter and cotransmitter, and as a modulator of conventional transmitter release. NO appears to be used in diverse animals for certain neuronal functions, such as chemosensory signaling, learning, and development, suggesting that these NO functions have been conserved during evolution. The discovery of NO's diverse and unconventional signaling functions has stimulated a plethora of enthusiastic investigations into its uses. We can anticipate the discovery of many more interesting and some surprising NO signaling functions.

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.

Protein synthesis requirement of the Aplysia circadian clock. Tested by active and inactive derivatives of the inhibitor anisomycin.

1. The circadian rhythm of compound action potential frequency recorded from the isolated eye of Aplysia in culture medium and darkness was subjected to 6 h pulse treatments with either anisomycin, a protein synthesis inhibitor, or inactive derivatives of anisomycin. 2. Anisomycin caused phase-dependent phase shifts of the rhythm as expected from previous experiments, but none of the derivative molecules caused phase shifts or perturbed the rhythm. 3. Anisomycin inhibited eye-protein synthesis by 75% at the concentrations (10(-6) M) used in the phase shifting experiments but none of the derivatives inhibited synthesis. 4. Only those molecules that actually inhibited protein synthesis caused phase shifts of the clock, although the inactive derivatives differed from anisomycin by only an acetyl group. 5. The results strengthen the conclusion that the inhibition of protein synthesis caused by anisomycin is important in perturbing the timing of the circadian clock and not some other characteristic effect of the inhibitor molecule. Together with the results from other systems, these findings imply that the daily synthesis of protein is a general requirement for circadian clocks.

Circadian rhythm from the eye of Aplysia: temperature compensation of the effects of protein synthesis inhibitors.

1. The circadian rhythm of compound action potentials (CAP) frequency recorded from the isolated eye of Aplysia in culture medium and darkness was subjected to step and pulse treatments with anisomycin, a protein synthesis inhibitor. 2. The step application of anisomycin and its continued presence in the culture medium lengthened the period of the rhythm in a dose-dependent manner. At 10(-8) M the period was increased from the normal 26.5 h to about 28 h and at 10(-7) M the period was lengthened to 31 h or longer. At 10(-6) M the rhythm was suppressed but the CAP activity continued without the cyclic variations in CAP frequency. 3. Six-hour pulses of anisomycin at 10(-6) M caused phase-dependent phase-shifts of the rhythm. Maximum phase delays of 15 h were obtained at CT (circadian time) 2 and maximum phase advances of 4 h were obtained at CT 6. The phase response curves at 12, 15 and 17 degrees C were essentially identical. 4. Anisomycin appears to act rather selectively on the circadian clock mechanism. It does not alter the CAP amplitude and duration and it does not alter the bursting pacemaker mechanism of the optic nerve CAP or central neurones. 5. The results support the hypothesis that the synthesis of a protein or polypeptide on eucaryotic ribosomes is an essential part of the circadian clock timing mechanism. The sensitivity of the clock to anisomycin is the same at three different temperatures (12, 15 and 17 degrees C) within the physiological range of temperatures for Aplysia, as expected for a clock whose period length is temperature compensated (Q10 1.02) over that same range. 6. At the critical phases of CT 1-4, anisomycin pulses often caused unusual perturbations of the rhythm. These effects are consistent with the hypothesis that the circadian rhythm is a multioscillator system.