PC12 cells, derived from rat pheochromocytoma, are widely used to model neuronal differentiation and disease. Upon nerve growth factor (NGF) stimulation, they adopt a neuron-like phenotype, offering insights into neuronal processes. However, their fidelity in mimicking neuronal presynaptic organization has been debated, and their lack of postsynaptic organization is apparent. This study evaluates NGF-differentiated PC12 cells for up to 21 days post-induction, focusing on synaptic vesicle-like vesicle (SVLV) recycling. Using immunofluorescence, correlative light and electron microscopy (CLEM), cryo-electron tomography, and electrophysiology, we assessed presynaptic-like architecture and functionality. By combining ultrastructural and functional modalities, we provide a more integrated evaluation of synaptogenic features in this widely used model. We found that NGF-differentiated PC12 cells exhibit neuron-like features, including electrical excitability, neurite outgrowth, presynaptic-like ultrastructure, and protein clustering (synaptophysin, Rab3a). Additionally, their neurites develop characteristics resembling both axons and dendrites. However, they do not develop synapses. Although endocytosis was observed in neurites, exocytosis of recently endocytosed vesicles was absent, indicating a lack of spatiotemporally coupled SVLV recycling. Our findings suggest that NGF-induced neuronal differentiation of PC12 cells is limited. The cells lack the fully developed presynaptic organization required for sustained synaptic vesicle recycling. Thus, synaptogenesis is incomplete. These findings define the structural and functional boundaries of PC12 cells as a synaptic model. The limited synaptic competence of NGF-differentiated PC12 cells underscores the importance of model selection in synaptic research.
