A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.

Calcium changes in isolated peptidergic neurons during activation by a cAMP analog.

The bag cell neurons of intact Aplysia control egg-laying by producing an approximately 30 min afterdischarge of action potentials, during which the neuropeptide egg-laying hormone is released. Previous studies have shown that cAMP, protein phosphorylation and Ca2+ are involved in the mechanisms of the afterdischarge. Spontaneous discharge and afterdischarge can be produced in dissociated bag cells by treatment with cAMP analogs. While pharmacological evidence exists for a large calcium component in the action potentials of bag cells, direct evidence by measuring (Ca2+)i has not appeared, nor has Ca-buffering during cAMP activation been examined. Our studies were directed at measuring (Ca2+)i changes in isolated bag cells in attached cell culture using the metallochromic indicator, arsenazo III, simultaneously with voltage recordings across the cell membrane. It was found that a single induced action potential causes a negligible rise in (Ca2+)i while a train of 35 spikes causes a significant rise (about 3 X 10(-4) absorbance units). Increasing external (Ca2+) enhances the action potential and Ca2+ signal while cobaltous ions block both the action potentials and the rise in (Ca2+)i. Lengthening the duration of spikes by TEA (50 mM) or 8-benzylthio cAMP (8-BTcAMP; 0.5 mM) enhances Ca2+ influx and allows the detection of Ca2+ transients due to single spikes. During spontaneous groups of action potentials in either TEA or 8-BTcAMP, (Ca2+)i increases but the bag cell's Ca-buffering systems are able to restore the Ca2+ levels with a half-time of about 20 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity of the Ca2+-activated and light-dependent K+ channels for monovalent cations.

The ionic selectivity of the Ca(2+)-activated K(+) channel of Aplysia neurons and of the light-dependent K(+) channel of Pecten photoreceptors to metal and organic cations was studied. The selectivity sequence determined from reversal potential measurements is T1(+) K(+) > Rb(+) > NH(+) (4) > Cs(+) > Na(+), Li(+) and is identical to the sequence determined previously for voltage-dependent K(+) channels in a variety of tissues. Our results suggest that some physical aspect of the K(+) channel is conserved in phyllogenetically different tissues and cells.

Time dependence of the calcium-activated potassium current.

We investigated the dependence of the kinetics of the Ca2+-activated K+ current of the molluscan neuron soma upon membrane potential. The K+ current was activated by intracellular Ca2+ ion injection in neurons with blocked inward Na+ and Ca2+ currents. The difference between currents was measured with brief pulses (less than 100 ms) before and immediately after Ca2+ injection and was used as the Ca2+ activated K+ current at difference membrane potentials. The results in normal (10 mM) and in high (200 nM) external K+ show that the time-course of the Ca2+-activated K+ current depends upon membrane voltage and that the current activates more rapidly with membrane depolarization.

The differential effects of ionizing radiation on the circadian oscillator and other functions in the eye of Aplysia.

Ionizing radiation has been used to selectively separate the circadian oscillator function of the eye of Aplysia from some of its other functions--synchronous compound action potential (CAP) generation, the light response, synaptic transmission between photoreceptors and output neurons, and the bursting pacemaker mechanism. Doses of 4-krad (50 kV peak) x-rays have a minimal effect on the circadian rhythm of CAP frequency, measured from the otpic nerve, whereas irradiation with a 40-krad dose abolishes the rhythm without affecting any of the four other functions of this eye (1 rad = 0.01 J/kg = 0.01/Gy). We estimate a 50% survival of the oscillator function at doses of about 6 krad. The oscillators of irradiated eyes are not merely desynchronized when the rhythm is abolished, because in vitro light-dark entrainment does not restore free-running rhythmicity. The results, including those from selective irradiation of the anterior or posterior poles of the eye, suggest that there are a number of circadian oscillators in the eye--most of them in the posterior portion near the optic nerve. An approximate target size has been obtained from target theory approximately equal to 10(8) A3, which is somewhat larger than the target size for viral infectivity function, as one example. There are reservations about estimating target size in a complex organ such as the eye. However, this approximate target size and the fact that recovery or repair can occur in vivo suggest that the oscillator may involve nucleic acid molecules.

Membrane-potential-sensitive dyes for optical monitoring of activity in Aplysia neurons.

Two membrane-associated dyes (WW375 and NK2367) which change their absorption of light when the membrane potential changes have been studied using several preparations form Aplysia. Action potentials are easily observed in nerve trunks (from a number of axons), in bag cell clusters, in some of the larger single cells of the parietovisceral ganglion, and in the optic nerve. Physiological effects of the dyes on the circadian rhythm of activity in the eye are described.

Electron spin resonance signals in injured nerve.

Under certain conditions nerves (such as the frog sciatic) exhibit electron spin resonance signals with several unusual properties: (i) variable g value and linewidth, (ii) anisotropic g tensor, and (iii) g value dependence on temperature. Such a signal must be due to a small ferromagnetic crystal formed when the nerve is subjected to pressure, such as that due to mechanical injury.