Comprehensive voltage sensitive dye imaging in the leech nervous system using double-sided microscopy

Abstract

In this note, we introduce comprehensive voltage-sensitive dye (VSD) imaging using a double-sided microscope. We performed this imaging technique on the nervous system of the medicinal leech (Hirudo verbana). In the leech segmental ganglion, there are two neuronal layers: ventral and dorsal surfaces, where cell bodies of approximately 400 neurons, including sensory, motor, and interneurons, distribute. We applied a new generation VSD (VoltageFluor) to both ventral and dorsal surfaces of the ganglion to achieve simultaneous VSD imaging in a whole ganglion. We could detect both small synaptic potentials and action potentials in imaged neurons with a high S/N ratio. Future issues are to develop long-time imaging in a low photo-bleaching condition and its application to semi-intact leech preparations.