Dissociated histaminergic neuron cultures from the tuberomammillary nucleus of rats: culture methods and ghrelin effects.

The tuberomammillary nucleus (TMN) in the hypothalamus is the sole source of histamine in the brain. This nucleus, by innervating various brain regions, plays an important role for vital functions such as arousal and appetite. We have developed dissociated primary histaminergic neuron cultures from TMN of postnatal (3 and 10-day-old) rats. More than 50% of our cultured neurons from the TMN were histaminergic as revealed by adenosine deaminase (AD) as well as histamine immunocytochemistry. Among large neurons (diameter, >22 microm), more than 88% were histaminergic. Such large neurons (mean diameter, 26.5 microm) were used for electrophysiology. Using about 2-month-old TMN cultures, we investigated the effects of ghrelin, a recently discovered appetite-stimulating endogenous peptide. In GTPgammaS-loaded neurons, ghrelin (3 microM) suppressed currents that had previously been activated by an inhibitory neuropeptide, nociceptin. The mean current suppression by ghrelin was 471+/-128 pA (S.E.M., n=7). The I-V relationship revealed that the ghrelin-suppressed current was inwardly rectifying with a reversal potential around E(K). These results suggest that ghrelin inhibits G protein-coupled inward rectifier K+ channels (Kir3, GIRK) of TMN neurons and that our TMN cultures are useful for investigating physiological properties of brain histaminergic neurons.