Preparation of primary cultures and acute slices of the nervous system of the moth Manduca sexta

Interactions among receptor neurons, glial cells and neurons intrinsic to the antennal lobe of the moth underlie the formation of olfactory glomeruli. To isolate these interactions, as well as to understand the effect of a variety of humoral agents on differentiation of the neurons and glia, we generate primary cultures of neurons or glia. These methods are described. In addition, we describe a protocol for producing slice preparations of the developing moth brain that we are using to study the biophysical and morphological development of glial cells. This technique allows us to examine a class of glial cells associated with the glomeruli that otherwise are nearly inaccessible using standard intracellular recording techniques. It also preserves the 3-dimensional arrangement of glia that may strongly influence the development of glomeruli.