Analysis of Aplysia Neuronal Network by Fluorescent Voltage Imaging

Voltage sensitive dye (VSD) is a potential tool for tracing signals in neuronal network. Aplysia, a kind of sea snail, is an ideal model animal for study of neuronal network due to its identifiable large neuron. Dramatical progress in neuroscience can be expected by analyzing network in central nerve system (CNS) of the Aplysia using the VSD and imaging technology. However, the method of staining Aplysia neurons with VSD is yet to be established. We have found the successful method for staining Aplysia neurons by Di-4-ANEPPS which is the most popular fluorescence VSD using Aplysia hemolymph and appropriate solubilizer. Change in fluorescent intensity responding to change in membrane potential of the neurons was detected by a CCD camera. Then, we succeeded in detecting output from central pattern generator (CPG), which controls the buccal mass for chewing, induced by electric stimulation of esophageal nerve bundle.

However, fluorescent intensity drifted due to expansion and contraction of muscle in the ganglion by this stimulation. The drift could be inhibited by pretreatment with protease before the staining. The protease may be potential tool for stable voltage imaging of Aplysia CNS, as far as it does not affect on the neuronal network.