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
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.