The Molluscan Cell Culture Techniques

Large molluscan neurons, such as those of Lymnaea (Figure 2.1A; Colorplate 2), are often clearly discernable in the central ring ganglia, even under a dissection microscope. Neuronal identification is facilitated by their size, coloration (white to red), and unique position within the ganglia. Following enzymatic treatment, which softens the connective tissue surrounding the ganglia, and subsequent mechanical removal of the inner sheath that encapsulates the neurons, individual somata can be extracted by applying gentle pressure through a suction pipette attached to a micromanipulator (Figure 2.1B). The isolated neurons (somata with their intact axon stumps) are then plated either on plastic or poly-L-lysine-coated glass coverslips attached to tissue culture dishes (Figure 2.1C). Whereas Lymnaea and Helisoma brain conditioned medium (CM - contains trophic factors), prepared by incubating central ring ganglia (2 brains/ml) in defined medium (DM) over a period of several days, is used to induce neurite outgrowth from their respective neurons, most Aplysia neurons are first cultured in defined medium (DM - does not contain trophic factors), with hemolymph (Aplysia blood) added later to promote sprouting. Within hours of neuronal plating in growth permissive medium, growth cones (Figure 2.1D) emerge either from the axon stump or from the soma itself (Figure 2.1E). Although these molluscan growth cones are much larger in size (50100 ||M), they are structurally and functionally similar to their vertebrate counterparts. When cultured in CM, molluscan neurons exhibit extensive outgrowth (Figure 2.1F) and develop synapses that are similar to those seen in vivo.

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