SPATIOTEMPORAL DISTRIBUTION OF CA2+ FOLLOWING AXOTOMY AND THROUGHOUT THE RECOVERY PROCESS OF CULTURED APLYSIA NEURONS

This study investigates the alterations in the spatiotemporal distribu tion pattern of the free intracellular Ca2+ concentration ([Ca2+]i) du ring axotomy and throughout the recovery process of cultured Aplysia n eurons, and correlates these alterations with changes in the neurons i nput resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura- 2, and monitoring the neurons' resting potential and input resistance (R(i)) with an intracellular microelectrode inserted into the cell bod y. The alterations in the spatiotemporal distribution pattern of [Ca2]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing o f the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.0 5 - 0.1 muM to 1 - 1.5 muM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11 - 16 mu/s from the point of transection towards the intact ends, elevat ing [Ca2+]i to 3 - 18 muM. Following the resealing of the cut end 0.5- 2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreat ing front, travelling from the intact ends towards the cut regions. Th e [Ca2+]i recovers to the control level 7 - 10 min post-axotomy. In Ca 2+-free artificial sea water (2.5 mM EGTA) axotomy does not lead to in creased [Ca2+]i and a membrane seal is not formed over the cut end. Up on reperfusion with normal artificial sea water, [Ca2+]i is elevated a t the tip of the cut axon and a membrane seal is formed. This experime nt, together with the observations that injections of Ca2+, Mg2+ and N a+ into intact axons do not induce the release of Ca2+ from intracellu lar stores, indicates that Ca2+ influx through voltage gated Ca2+ chan nels and through the cut end are the primary sources of [Ca2+]i follow ing axotomy. However, examination of the spatiotemporal distribution p attern of [Ca2+]i following axotomy and during the recovery process in dicates that diffusion is not the dominating process in shaping the [C a2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effe ctive in limiting these gradients, thus enabling the neuron to survive the injury.