Fos activation in cultured tyrosine hydroxylase and oxytocin immunoreactive neurons

Abstract

Fos and other immediate early gene products are used as markers for neuronal activity. We identified Fos immunocytochemically after KCl-induced depolarization of cultured hypothalamic neurons. Five-day cultures were treated for 1 h with 50 mM KCl or media and fixed at 0, 0.5, 1, 2, and 4 h post-treatment. 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. KCl 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 KCl-induced depolarization of cultured hypothalamic neurons increases Fos with a different time course in TH-ir vs. OT-ir neurons.