Abstract
Confocal laser scanning microscopy and conventional
digital-imaging microscopy were used to
record changes in intracellular free Ca induced by
electric stimuli, or by the action of caffeine, in giant
neuronal cells of circumaesophageal ganglia dissected
from a land slug, Incilaria bilineata. Recording
of fluorescence changes of a Ca probe, indo-1,
with a confocal laser scanning microscope (CLSM)
equipped with a UV laser (325 nm) demonstrated
clear distinction in the mode of Ca changes in
cytoplasm and nucleoplasm. Injection of outward
current caused repetitive action potentials and Ca
influx, resulting in a rapid Ca rise in cytoplasm
followed by a slower rise in nucleoplasm. The nuclear
envelope appeared to serve as a barrier to Ca
diffusion from cytosol to nucleoplasm. When the
cell was tetanically stimulated in the presence of 10
mM caffeine, the rate of the Ca rise in the nucleoplasm
became more rapid, thereby suggesting participation
of a Ca-induced Ca release from caffeine-sensitive
intracellular Ca stores. Bath application of
caffeine to a resting cell caused a calcium transient
in most cells. Caffeine also induced in some cells
repeated bursts of spontaneous action potentials
and corresponding Ca oscillation. Cytoplasmic phasic
rise in Ca corresponding to each action potential
was clearly visualized when fluo-3 as a Ca indicator
dye was used for CLSM. Calcium oscillation in this
type of cell may be caused by a membranous oscillator
involving voltage-dependent Ca and K channels
and Ca-induced Ca release from caffeine-sensitive
intracellular Ca stores.
