Presumptive insect circadian pacemakers in vitro: immunocytochemical characterization of cultured pigment-dispersing hormone-immunoreactive neurons of Leucophaea maderae

The accessory medulla with its associated pigment-dispersing hormone-immuno reactive neurons appears to be the pacemaker that controls the circadian lo comotor activity rhythm of the cockroach Leucophaea maderae. To permit stud ies at the level of individual, identified, pacemaker neurons, we developed specific long-term primary cell cultures of fully differentiated adult neu rons of the accessory medulla. As judged from soma diameter distribution, t he cultures contain an unbiased representation of apparently all neuronal t ypes of the accessory medulla. The cultured cells survive and grow processe s for more than 2 months with or without additional hemocyte coculturing. H owever, a strong positive effect on initial outgrowth was observed with hem ocyte coculturing. At least six different morphological cell types of the a ccessory medulla could be distinguished in vitro. Among these only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiabl e monopolar type C cells are candidates for circadian pacemaker neurons and will be the focus of further physiological characterizations.