Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca²⁺ concentration (Ca²⁺]_i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input 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 body. 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 of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, Ca²⁺]_i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a Ca²⁺]_i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating Ca²⁺]_i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, Ca²⁺]_i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The Ca²⁺]_i recovers to the control level 7 – 10 min post-axotomy. In Ca²⁺-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased Ca²⁺]_i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, Ca²⁺]_i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca²⁺, Mg²⁺ and Na⁺ into intact axons do not induce the release of Ca²⁺ from intracellular stores, indicates that Ca²⁺ influx through voltage gated Ca²⁺ channels and through the cut end are the primary sources of Ca²⁺]_i following axotomy. However, examination of the spatiotemporal distribution pattern of Ca²⁺]_i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the Ca²⁺]_i gradients. Other Ca²⁺ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.