In vitro adrenergic and cholinergic innervation of the developing rat myocyte

Abstract

We studied the development of selective adrenergic and cholinergic neuroeffector transmission in primary cultures of isolated ventricular muscle cells. Explants of either thoraco-lumbar sympathetic ganglia or sacrococcygeal spinal cord were added to newborn rat ventricular cultures harvested prior to the onset of in vivo autonomic innervation. Neuronal growth, migration, and the formation of neuromuscular junctions were observed with light and scanning electron microscopy. Glycoxylic acid histofluorescence, reflecting catecholamine synthesis, was found in only the sympathetic neuromuscular cultures. Choline acetyltransferase activity was detected in both spinal cord and sympathetic neuromuscular cultures, but was significantly higher in the spinal cord neuromuscular cultures. The isolated ventricular muscle cells remained at a constant spontaneous contraction frequency, regardless of the type of culture preparation. Guanethidine sulfate application produced a positive chronotropic response, blocked by propranolol, in the sympathetic neuromuscular cultures, but not in the spinal cord neuromuscular cultures. Bethanechol sulfate produced a negative chronotropic response, blocked by atropine, in the spinal cord neuromuscular cultures, but not in the sympathetic neuromuscular cultures. Isolated ventricular muscle cells in the absence of neurons failed to respond to either agent. Direct microelectrode stimulation of adrenergic or cholinergic neurons likewise respectively produced either a positive or negative ventricular muscle cell chronotropic response. These studies are the first to establish the selective production of functional cholinergic an adrenergic innervation of isolated cardiac muscle cells in vitro.