In vivo labeling and in vitro characterisation of central complex neurons involved in the control of sound production

Injection of muscarine into the central complex of the grasshopper brain can stimulate species-specific sound production through activation of the phospholipase C-initiated transduction pathway. We introduce a strategy, to label central complex interneurons that are directly stimulated by the injected muscarine and to study their physiology in dissociated primary cell culture. Fluorescent dextranes, co-injected to brain sites where muscarine stimulates sound production, are incorporated from the extracellular space by 3-14 central complex neurons. Most labeled neurons are columnar neurons that express muscarinic acetylcholine receptors. An average of 3-4 dextrame-labeled central complex neurons per brain can be recognised by their fluorescence in dissociated cell cultures. Their function as potential direct targets of previous in vivo pharmacological stimulation of the intact brain was supported by expression of muscarinic receptors in cytomembranes of isolated neuronal cell bodies and muscarine-stimulated calcium responses in vitro. Pharmacological inhibition of phospholipase C function and removal of extracellular calcium indicated that release from inositolphosphate-regulated internal stores mediates the increase of cytosolic calcium concentrations. The experimental procedures described in this study can be applied to any preparation in which focal drug application elicits, terminates or modulates behavior in order to label and physiologically analyse those interneurons within the circuit that serve as direct targets of the injected drug. (C) 2009 Elsevier B.V. All rights reserved.