Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis

This study describes a preparation that will enable us to study, using voltage clamp techniques, ionic currents from dissociated cardiovascular neurons that have retained their anatomical and functional identity of the intact animal. To identify dispersed preganglionic cardiac motoneurons various fluorescent dyes (rhodamine, fluorogold, microspheres, bizbenzimide and dextrans) were examined to determine which can be absorbed by preganglionic cardiac motorneuron nerve terminals (without surgical penetration of cardiac tissue), transported retrogradely to their soma in the medulla and retained during dissociation of the neurons. Rhodamine fulfilled these criteria. Dissociated preganglionic cardiac motorneurons had resting membrane potentials of -52.4 +/- 3 mV and input resistances of 236 +/- 71 M omega (mean +/- S.E.M., n = 10). Depolarizing voltage steps to -50 mV or above evoked a tetrodotoxin (TTX) sensitive inward sodium current followed by a biphasic outward current.