IN SEARCH OF THE CENTRAL RESPIRATORY NEURONS .1. DISSOCIATED CELL-CULTURES OF RESPIRATORY AREAS FROM THE UPPER MEDULLA

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen-coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex- or medulla-derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5-1.0 x 10(6) cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen-coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590-597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron-specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies.