Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.