Ultrastructural characterization of rat neurons in primary culture

Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal™ medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14–21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1–10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30–50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission.

Graphical Abstract••• Figure optionsDownload full-size imageDownload high-quality image (184 K)Download as PowerPoint slideHighlights▶Rat neocortical neurons in primary pure culture divided and survived for a long time-duration. ▶They contained organelles, and mitochondria along neurites illustrated intracellular traffic. ▶Synapses of all kinds might be signs of a sustained neurotransmission in cultures. ▶Abnormal mitochondria and vesicles developed, which were enhanced by antibiotics. ▶Cultured neurons are structurally similar to in vivo neurons and are valuable cellular models.