We examined the spatio-temporal pattern of changes in intracellular Ca²⁺ concentration, [Ca²⁺l_i, in a median giant fiber (MGF) of the ventral nerve cord of the earthworm using confocal laser-scanning microscopy and fluorescent Ca²⁺ indicator. We found that the spatial distribution of [Ca²⁺l_i in unstimulated conditions was heterogeneous along the longitudinal axis of the MGF. Heterogeneity of the resting Ca²⁺ levels, therefore, may be the state of the MGF. Tetanic electrical stimulation of the ventral nerve cord evoked heterogeneous Ca²⁺ increases in the MGF. Depolarization by bath-application of high-K⁺ (62.5 mM) induced the transient and localized elevation of [Ca²⁺]_i in the MGF. These Ca²⁺ rises were inhibited by a mixture of putative nonselective voltage-dependent Ca²⁺ channel (VDCC) blockers (Co²⁺, Ni²⁺ and Cd²⁺). Bath-application of caffeine (1 mM), which could activate the Ca²⁺-induced Ca²⁺-release (CICR), increased [Ca²⁺l_i all over the MGF. However, time-course analysis of the caffeineinduced response indicated that the Ca²⁺-rising velocities and the sustained Ca²⁺ levels were varied in cellular areas of the MGF. These results suggest that the VDCCs are localized on the plasma membrane of the MGF, while the ryanodine receptors mediating CICR are homogeneously distributed over the MGF.

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Glutamate has been known as an excitatory neurotransmitter in both vertebrate and invertebrate neural systems. We found that glutamate depolarized the membrane potential of the ventral nerve cord in the earthworm, Eisenia foetida (Oka et al., 1994). We extensively characterized the role of glutamate from Ca²⁺ and membrane potential responses using the optical recording method on the ventral nerve cord of the earthworm. Glutamate and four types of glutamate receptor subtype agonists, N-methyl-D-aspartate (NMDA), kainate, quisqualate, and trans-1-aminocyclopentane-1,3-dicarboxylic acid (transACPD), increased intracellular calcium concentration ([Ca²⁺]_i in the MGF. Based on the temporal pattern of Ca²⁺ responses, the Ca²⁺ elevations induced by NMDA, kainate, and trans-ACPD were transient, and [Ca²⁺]_i was restored to basal levels during the stimulation, while a Ca²⁺ increase induced by quisqualate was sustained and weak compared with the other tested agonists. From the spatial pattern of Ca²⁺ responses, NMDA and kainate-induced responses were regionally different in contrast to the uniform Ca²⁺ increase by trans-ACPD stimulation. Ionotropic glutamate receptor agonists (NMDA, quisqualate, and kainate) depolarized the membrane potential of neurons in the ventral nerve cord, but a metabotropic glutamate receptor agonist, trans-ACPD, did not affect the membrane potential. Our results indicate that there are at least three types of glutamate receptors, NMDA, non-NMDA, and metabotropic ones, on the earthworm ventral nerve cord.

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We visualized the spread of neural activities evoked in the Schaeffer and/or commissural fibers to pyramidal cells in the CA1 region of rat hippocampal slices stained with a voltage-sensitive styryl dye (NK-3630). Changes in the absorption of the light transmitted through a slice were measured every 0.6 msec by an optical recording system with an area photosensor (128 × 128 pixels). We identified two phases in the propagation of the neural activities along the CA1 region. In the first phase, a focal excitation of the fibers spread along the radiatum and lacunosum-moleculare layers at a conduction velocity of approximately 0.2 m/sec. Postsynaptic action potentials were initiated in the apical dendrites near the soma of pyramidal cells, and spread to the soma and axons and possibly the basal dendrites at a velocity of approximately 0.2—0.3 m/sec. In the second phase, a wave of the neural activity of pyramidal cells propagated at the velocity of 0.1 m/sec along the CA1 region where no action potential of the Schaeffer fibers is recorded, reaching a maximum length of 2.5 mm toward the subiculum. The latter phase of the propagation may be propelled by recurrent collaterals of pyramidal cells or interneurons.

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Antisense phosphorothioate (PS) oligonucleotides are widely used as inhibitors of gene expression in cell cultures and have been proposed as potential therapeutic agents. In the present study, intracellular distributions of both fluorescein isothiocyanate (FITC)-labeled PS oligonucleotides and FITC-labeled cholesterol conjugated phosphorothioate (ChPS) oligonucleotides were determined by confocal laser microscopy. The observation showed that almost all the PS oligonucleotides were accumulated in the cytoplasmic vesicles, though a small number of oligonucleotides were distributed in both cytoplasm and nuclei. In contrast, ChPS oligonucleotides distributed in the cytoplasm and bound to both cell membranes and nuclear membranes. Colocalization analysis using FITC-labeled oligonucleotides and Texas red-labeled dextran showed that they were colocalized in the cytoplasmic vesicles, demonstrating that oligonucleotides accumulated in the endosomes or lysosomes. Our bioimaging method is helpful in predicting the inhibitory activity of gene expression by antisense oligonucleotides.

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We have investigated antigen receptor-mediated calcium signals in B cells and basophils by confocal fluorescence microscopy. Calcium signals were induced in both cells depending on the rigidity of receptor-crossbridges. Fcε-receptors in basophils required more rigid crossbridges than antigen-receptor (mlgM) in B cells for cell signaling.

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