Three-Dimensional Imaging and Electrophysiology of Live Aplysia Neurons during Volume Pertubation: Confocal Light and High-Voltage Electron Microscopy

We have used confocal laser scanning microscopy (CLSM), high-voltage electron microscopy (HVEM), and intracellular recording techniques to study volume changes in cultured Aplysia pacemaker neurons. Hyper- and hypo-tonic artificial sea water (ASW) decreased the pacemaker frequency and led to depolarization and hyperpolarization, respectively. However, when negative or positive current was injected into neurons in normal ASW, the frequency decreased with hyperpolarization but increased with depolarization. This suggests that the membrane potential is not the only factor underlying the reduction of the pacemaker activity. Changes in cell volume were monitored with a CLSM and paralleled progressive changes in osmolarity. The neurons swelled and shrank nonuniformly, and, although an optical section through the middle of the cell was monitored every 4 s for as long as 14 min, a regulatory volume decrease or increase was never observed, indicating an osmometer-like behavior. The time course of shrinkage was faster than swelling after returning to control ASW after a hypotonic shock, reflecting a possible mechanical stress on the cytoskeleton. Thick sections observed in the HVEM confirmed that membrane infoldings were present in our cultured Aplysia neurons. We hypothesize that a change in length of the latter in shrunken and swollen neurons could provide an explanation on the ultrastructural level for the increase and decrease in membrane surface area observed by CLSM. We conclude that by combining a confocal microscope with an electrophysiological set-up, three-dimensional morphology and physiological properties can be studied in living cells in real-time. This approach provides the means to correlate cell volume-related alterations and physiology.