**2. Fluorescent-imaging technique**

In fluorescent-imaging technique, the dyes, which change their fluorescence intensities due to the voltage or calcium ion concentration change [1], are loaded into cells, and their fluorescence changes are acquired into a computer as a series of images. However, the voltage-sensitive dyes generally exhibit a relatively small change in the fluorescence intensities, resulting in a low signal-to-noise (S/N) ratio. On the other hand, the calcium-sensitive dyes exhibit a larger change in the fluorescence intensities than that of the voltage-sensitive dyes. Therefore, their fluorescence changes can be detected easily, which enables us to indirectly measure neural activities because the intracellular calcium concentration often increases with neural activities.

As the calcium-sensitive dyes, those bonded with the acetoxymethyl (AM) group are commonly used because they are permeable into cells. After the AM-bonded dyes permeate into cells, the AM group is dissociated by intracellular esterase, and then the dyes can be nonpermeable and loaded into cells. Vertebrate neurons are well known to be easily loaded with the AM-bonded calcium-sensitive dyes. In invertebrates, however, the AM-bonded dyes cannot be easily loaded into neurons because the AM group is difficult to be dissociated due to their weak activity of intracellular esterase.

For these reasons, it has not been determined yet which type of dyes is better for fluorescent imaging of neural activities in invertebrates. In Section 4, we introduce some studies on the spatiotemporal neural activities of the land slug *Limax* using fluorescent-imaging technique with both types of dyes.
