Neurite outgrowth and differentiation of rat cortex progenitor cells are sensitive to lithium chloride at non-cytotoxic exposures

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model, where cells were exposed to a chemical during a period of cell differentiation. In control cultures, the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time, indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein, an astrocyte marker, also increased significantly with time. By seeding progenitor cells at varying densities, we demonstrated that neurite length was influenced by cell-cell spacing. After ten days, cultures seeded at densities of 1000 cells/mm2 or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm2 or higher. Progenitor cells were exposed to lithium, a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures, as reported by adenosine triphosphate content, and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L, 1 mmol/L, or 0.3 mmol/L LiCl, which encompass lithium's therapeutic range, had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust, regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions.

► Rat cortex progenitor cells differentiate into a population with 5% neurons and 36% astrocytes after ten days in culture. ► Neurite length is affected by seeding density after ten days in culture. ► Adenosine triphosphate levels indicate that exposure to 0.3-3 mmol/L lithium chloride is not cytotoxic. ► Non-cytotoxic exposure to lithium chloride leads to concentration-dependent decreases in neurite outgrowth after ten days in culture.