Coexistence of neuropeptides and their possible relation to neuritic regeneration in primary cultures of magnocellular neurons isolated from adult rat supraoptic nuclei

The coexistence of vasopressin (VP), oxytocin (OXY), galanin (GAL) and chol ecystokinin (CCK) and the synthesis of GAL and CCK during neuritic regenera tion was investigated in cultured magnocellular neurons, isolated from adul t rat supraoptic nuclei. Double-labelling immunofluorescence was performed after 7 days of culture using primary antibodies for VP, OXY, GAL and CCK ( paired in all possible combinations) and secondary antibodies labelled with either fluorescein or rhodamine. Confocal laser scanning microscopy reveal ed the coexistence of the mentioned peptides in all possible combinations, an unexpected result considering that the only combinations observed in tis sue sections are VP-GAL and OXY-CCK. Freshly dispersed cells were devoid of any neuritic processes and showed a very poor immunocytochemical staining reaction for GAL and CCK. In contrast, neurons cultured for 7, 12 and 21 da ys showed many neurites and a strong immunoreactivity for GAL and CCK indic ative of an increased synthesis of both peptides in the regenerating neuron s. This increased synthetic activity is consistent with transient upregulat ion of these peptides observed in situ after hypophysectomy by other author s. The results suggest that the upregulation of GAL and CCK is functionally related to the neuronal regeneration processes observed during culture and that the 'uncommon' coexistences as well as the prolonged sythesis of GAL and CCK may be due to the lack of environmental inputs, which normally regu late the expression and up- and downregulation of these peptides in vivo.