A novel in vitro system for studying the interactions between Merkel cells, sensory neurons, and sympathetic neurons

A method was developed for growing dissociated cultures of rat buccal pad epidermis enriched for Merkel cells. Quinacrine, a marker for Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. These cells had large, phase bright nuclei, with prominent nucleoli, surrounded by phase dark perinuclear collars. The same population of cells was labeled whether quinacrine was applied in utero or in vitro, and comparable numbers of quinacrine fluorescent cells (QFC) were obtained with either method of application. Antibodies directed against neuron specific enolase (NSE), another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of QFC. Electron microscopic examination demonstrated that cells with ultrastructural features of Merkel cells were present in our cultures. Merkel cells supported survival and outgrowth of both sensory and sympathetic neurons in serum-free medium lacking exogenous nerve growth factor (NGF). NGF-like immunoreactivity was colocalized with quinacrine fluorescence in situ and in vitro. Anti-NGF added to co-cultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and survival of sensory neurons by 60%. These results suggest that Merkel cells produce NGF. Selective recognition of Merkel cells by sensory and sympathetic neurons was characterized using time-lapse videomicroscopy and standard morphometry techniques. Approximately 60% of the trigeminal ganglion sensory neurons extended neurites onto clusters of Merkel cells. Sensory neurites growing on Merkel cells were more highly branched than those growing on collagen. In contrast, greater than 90% of sympathetic neurons failed to grow onto Merkel cells. Fixation of Merkel cells prior to co-culture did not affect this differential response to Merkel cells by sensory and sympathetic neurons; However, it eradicated the target-induced branching of sensory neurites. These results suggest that innervation of Merkel cells in vivo may be limited to sensory neurons, in part, by molecules present on the surface of the target cells. Furthermore, a target-derived agent may promote arborization of sensory neurons