Fos activation in cultured tyrosine hydroxylase and oxytocin immunoreactive neurons

Fos and other immediate early gene products are used as markers for neuronal activity. We identified Fos immunocytochemically after KCI-induced depolarization of cultured hypothalamic neurons. Five-day cultures were treated for 1 h with 50 mM KCI or media and fixed at 0, 0.5, 1, 2, and 4 h posttreatment. Sequential immunocytochemistry was performed to identify Fos immunoreactivity in tyrosine hydroxylase (TH)-immunoreactive (-ir) or oxytocin (OT)-ir neurons. Activated neurons [brown cells (TH-ir or OT-ir) with purple nuclei (Fos-ir)] were counted microscopically. KCI treatment resulted in an increased percentage of Fos-ir TH-ir and OT-ir neurons over control levels over the time course examined. Fos-ir peaked in TH-ir neurons at 0.5 to 1 h posttreatment, with levels returning to baseline at 4 h. Fos-ir in OT-ir neurons peaked at 2 h, and remained elevated at 4 h, showing prolonged activation. These results demonstrate that KCI-induced depolarization of cultured hypothalamic neurons increases Fos with a different time course in TH-ir vs. OT-ir neurons.