Dynamic imaging of functional nerve terminals and Schwann cells in presynaptic 'nerve plates' isolated from the skate electric organ

Abstract

The cholinergic innervation and its glial support were isolated in a functional state from the electric organ of the skate (Raja species) using a combined enzymatic and mechanical dissociation technique. Examination using light and electron microscopy showed that this 'nerve plate' is a disc-shaped structure several hundred micrometres in diameter consisting of a dense plexus of nerve terminals attached to branching nerve fibrils with numerous associated myelinating and perisynaptic Schwann cells. In unfixed nerve plates, depolarisation and Ca2+-dependent staining of the nerve terminals was seen with RH-414, a fluorescent marker for functional motor terminals. The components of the nerve plate could be loaded with the Ca2+-sensitive dyes Fluo-3 and Fura-2. Depolarisation of nerve plates loaded with either dye leads to an immediate increase of intracellular Ca2+ levels ([Ca2+]i) in the nerve terminals. This response was blocked by certain Ca2+ channel antagonists from the -conotoxin family. Glial cell responses to depolarisation were in general minimal. However, increases of [Ca2+]i were seen in these cells, but not in nerve terminals, during applications of ATP and acetylcholine. These results show that both nerve terminal and glial elements in this 'nerve plate' preparation are functional. The ease of this preparation and its functional reliability give it considerable potential as a useful model for elucidating presynaptic mechanisms.