Alterations in membrane potential after axotomy at different distances from the soma of an identified neuron and the effect of depolarization on neurite outgrowth and calcium channel expression

Abstract

1. Intracellular recordings were made from the soma of an identified neuron B5 within the buccal ganglion of the mollusc, Helisoma trivolvis, during axotomy induced by crushing or cutting the esophageal nerve. Axotomy was associated with a rapid depolarization and occasionally a burst of action potentials (injury discharge). The magnitude of the membrane depolarization in the soma in response to axotomy decayed exponentially when the distance between the soma and site of injury was increased. Input resistance measurements taken during axotomy showed that a barrier to current flow formed rapidly and gradually recovered within 2 h. A barrier to the diffusion of intracellularly injected carboxyfluorescein formed at the site of injury within 15 min of axotomy. 2. To examine the effect of chronic depolarization on neurite outgrowth, the extracellular potassium ion concentration [K+]o was manipulated. The membrane potential of neurons B5 exhibited a 51.8 mV/decade potassium dependence between 20 and 150 mM [K+]o. The initiation of neurite outgrowth from axons crushed 800 microns from the soma and bathed in different concentrations of [K+]o was examined by fluorescence microscopy after filling neurons with Lucifer yellow. We compared the percentage of axons with sprouts 9 and 24 h after organ culture in saline containing [K+]o ranging from 0.1 to 50 mM. Sprouting occurred from 33% of neurons B5 in normal saline (1.7 mM [K+]o) after 9 h and from 100% of neurons after 24 h. No sprouting was observed from neurons B5 9 or 24 h after axotomy when bathed in saline containing reduced or elevated concentrations of [K+]o. 3. To examine the effects of chronic depolarizatin on neurite outgrowth over several days, neurons B5 were axotomized close to the soma and maintained in organ culture in Liebovitz medium (defined medium or medium conditioned with central ganglia). Neurite outgrowth was ranked from 0 to 5 after filling neurons with Lucifer yellow, and our analysis indicated that a small increase in neurite outgrowth occurred in medium supplemented with 10 mM potassium. 4. Elevated potassium did not trigger neurite outgrowth from isolated neurons B5 in cell culture within defined medium, but whole-cell patch-clamp analysis revealed that chronic depolarization associated with elevated potassium altered the expression of calcium currents. Low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium currents were detected in acutely isolated neurons B5.(ABSTRACT TRUNCATED AT 400 WORDS)