Synaptic vesicle cytochemistry changes when cultured sympathetic neurones develop cholinergic interactions

THE principal neurones of the rat superior cervical ganglion (SCG) can be established as dissociated cells in long term cultures¹ without the survival of the small intensely fluorescent "interneurone" normally found in the animal. These isolated sympathetic neurones (SCGN) exhibit certain expected adrenergic characteristics such as the synthesis and accumulation of catecholamines^2-4. They also specifically take up exogenous noradrenaline and demonstrate Ca²⁺-dependent release of this transmitter during induced depolarisation⁵. When these neurones are cocultured with thoracic spinal cord explants, they receive a typical cholinergic preganglionic input⁶. When cultured with the appropriate target of brown fat, the SCGN will provide characteristic noradrenergic contacts with the fat cells⁷. The interpretation of these generally expected results is confounded by the following observations: (1) cultures of dissociated sympathetic neurones form numerous synaptic contacts with each other ^8-10; (2) cytochemical studies of these synapses show that they contain predominantly dense-cored vesicles and thus appear to be noradrenergic^8-11; and (3) physiological studies clearly indicate that synaptic signals between these cultured neurones are nicotinic-cholin- ergic^7,11. In addition, dissociated SCGN will provide cholinergic innervation to striated muscle in culture¹². Only one type of neurone, however, has been described in these cultures. We have investigated whether synaptic endings with the morphological characteristics of noradrenergic terminals were the only synaptic type to be found in cultures where cholinergic interactions between neurones were readily demonstrable. If, as previously reported, only synapses with noradrenergic cytochemical characteristics were found, it would strongly suggest that one synaptic type was handling both noradrenaline (NA) and acetylcholine (ACh). If two synaptic types were found it would suggest that two types of neurones were present in these cultured networks, thus providing a possible explanation for the paradoxical results discussed above.