An immunochemical and biochemical study of catecholaminergic activity in dissociated hypothalamic cultures

Dopamine (DA)-containing cells of the medial basal hypothalamus (MBH) were dissociated and maintained in culture for up to 9 days. Cultures were evaluated both biochemically and immunochemically for DA activity. DA biosynthesis was determined using incorporation of [3H]tyrosine and was analyzed by HPLC with electrochemical detection. Immunochemical studies were performed to identify tyrosine hydroxylase (TH)-positive and neuron-specific enolase (NSE)-positive cells. Morphometric analyses determined the cell size, density, process length and the percent of neurons which were catecholaminergic. TH-positive neurons ranged from 6 to 8% of the total neuronal population when examined over days 3-9 of culture and the length of TH-positive neurites was significantly greater than that of NSE-positive cells. There was incorporation of [3H]tyrosine into DA as evidenced by the presence of [3H]DA in both the media and tissue and the inhibition of synthesis with alpha-methyl-p-tyrosine. There was a greater amount of labeled DA in the media than in the tissue at every time point examined. On the other hand, biosynthesis of DA in fresh brain tissue revealed approximately equal levels of DA in the media and tissue. These studies indicate that DA continues to be synthesized in dissociated cultures of MBH as evidenced by both the biochemical and immunochemical analyses and that there appears to be some alteration in the ability of these neurons to store the newly synthesized amine.