Analysis of aplysia neuronal network by fluorescent voltage imaging

Aplysia has been used as an ideal experimental model for studying neuronal networks involved in learning and memory because large and identifiable neurons present in its central nervous system (CNS). Fluorescent voltage imaging with a voltage-sensitive dye (VSD) is a potential tool for multiple-site monitoring of neuronal activity. Fluorescent voltage imaging of Aplysia CNS would contribute greatly toward basic research on neuronal network; however, this has not yet been implemented because of the difficulty in staining of Aplysia neurons with a VSD. In the present study, we developed a variant of voltage imaging using Aplysia ganglion neurons and di-4-ANEPPS, which is a commonly used fluorescent VSD. An Aplysia abdominal ganglion was digested using protease to facilitate the removal of fibrous sheath with microscissors. This treated ganglion was soaked in di-4-ANEPPS solution containing Aplysia hemolymph. The ganglionic neurons were stained well with the VSD, which emitted a detectable fluorescence. The change in fluorescent intensity agreed with the spontaneous firing in membrane potentials of the neurons. Results showed that fluorescent voltage imaging of Aplysia CNS neurons was possible after appropriate pretreatment of the ganglion. However, the sensitivity of the fluorescence to the membrane potential vanished in approximately 2 h. Despite the time limit, the voltage imaging method offers a great breakthrough for basic studies on neuronal network in learning and memory of animals.