Crustaceans exhibit a variety of overt circadian rhythms. Observations on intact animals suggest the existence of more than one circadian pacemaker in the nervous system. Ablation experiments so far have been inconclusive in pin-pointing the location of putative pacemakers. However, various structures, most notably the optic peduncle, have been shown to sustain circadian rhythmicity in vitro. Retinal sensitivity and neurosecretory activity display circadian rhythms in the isolated optic peduncle, but they are also responsive to synchronizing influences from other regions of the central nervous system, most notably the supraesophageal ganglion. A model based on a number of circadian pacemakers distributed in the central nervous system best fits the experimental results at present. Coupling of rhythmicity between independent circadian pacemakers is likely to occur, and a neuroendocrine stage of integration has been proposed for several rhythms. Various entraining agents have been identified, and more than one may play a part in the synchronization of a given rhythm.

Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1–2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.

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Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

SUMMARYMetal microelectrodes were implanted in the optic nerve of the crayfish, Procambarus czarhi, and the activity of the sustaining fibers (units which react tonically to changes in illumination) was continuously recorded over periods up to 8 weeks. In animals kept in darkness, the response to periodic test light pulses was found to vary in a circadian manner. The duration of the cycles varied between 22-24 hr, with a phase of high responsiveness during the subjective night, which abruptly changed to one of low responsiveness at subjective dawn. The phase, amplitude, and shape of the cycles were modifiable by light pulses. The spontaneous activity of these units in darkness showed a similar fluctuation, and so did the electroretinogram amplitude, the spontaneous motor activity of the animal, as indicated by records from mechanoreceptive and activity fibers (units which are activated, directly or indirectly by body movements), and the motor responsiveness to light. The time course of these cycles suggests that they are driven by a common "clock," through humoral mechanisms.