Ionic currents of isolated retinal pacemaker neurons: projected daily phase differences and selective enhancement by a phase-shifting neurotransmitter.

Abstract

The eye of Aplysia expresses a robust circadian rhythm of neuronal activity. We dissociated the retinal cells in primary culture and studied isolated pacemaker neurons to identify ionic currents that may have roles in the circadian clock mechanism. Individual neurons were studied with perforated-patch whole cell recording techniques in current- and voltage-clamp modes. Pacemaker neurons had resting potentials near -40 mV and, if neurites had grown out, produced spontaneous action potentials in darkness at <1 Hz. Depolarizing current injections increased the rate of action potential firing. Hyperpolarizing current injections were followed by slowly decaying (1-3 s) afterhyperpolarizations. Four ionic currents were characterized under voltage-clamp, including a Ca current (I(Ca)), a voltage-gated potassium current (I(KV)), an A current (I(A)), and a hyperpolarization-activated Cl current (I(Cl)). I(Cl) was only seen using Cl(-)-filled electrodes when high concentrations of Cl- diffused from the electrode and is therefore unlikely to be important under physiological conditions. The magnitude of I(KV) was significantly larger during the projected zeitgeber predawn phase than during the postdawn phase, whereas the magnitude of I(A) was constant at these circadian phases, suggesting that only I(KV) is controlled by the circadian clock. Serotonin increased I(KV) by 29%, consistent with reports that serotonin suppresses optic nerve activity and phase shifts the circadian rhythm recorded from the intact eye. The enhancement of I(KV) likely contributes to membrane hyperpolarization, and it may be required for phase shifting. The phase-dependent changes in I(KV) provide evidence that each retinal pacemaker neuron contains a circadian clock, but confirmation must await further recordings made from individual pacemaker neurons that are isolated completely from all other cells in primary culture. From the present experiments, it appears that I(KV) is controlled by the circadian clock, in part, and it may be a required element in the pathway that is activated during serotonin-induced phase shifts.