Evidence for Modulation of DoDamine-neuronal Function by Tachykinin NK3 Receptor Stimulation in Gerbil Mesencephalic Cell Cultures

Primary cultures of gerbil mesencephalon were used for studying the modulation exerted by tachykinin NK₃ receptor activation on the activity of dopamine (DA) neurons. Dopamine neurons were identified by their ability to take up [³H]DA in a nomifensine-dependent manner. Moreover, tyrosine hydroxylase immunohistochemistry revealed that these neurons accounted for 5–7% of the total cell population. The NK₃ receptor agonists, senktide (EC₅₀= 0.58 nM) and [MePhe⁷]neurokinin B (EC₅₀= 3 nM), increased spontaneous [³H]DA release in a concentration-dependent manner. In contrast, tested at a supramaximal concentration (10-⁷ M), neither septide nor substance P were found to affect [³H]DA release. The senktide-evoked [³H]DA release was not observed when extracellular Ca²⁺ was chelated, but was unaffected by nomifensine. This indicates that this increase in [³H]DA outflow resulted more from an exocytotic process than from reversal of carrier-mediated DA uptake. Moreover, the senktide effect was unaffected by the Na⁺ channel blocker tetrodotoxin, a result suggesting a direct action of senktide on DA neurons. The non-peptide NK₃ receptor antagonist, SR 142801, shifted or blocked (ICs0 = 0.89 nM) the senktide-evoked [³H]DA release, while its (-)-antipode, SR 142806, was 80-fold less potent, in agreement with binding data. Selective antagonists for NK, (SR 140333) or NK₂ (SR 48968) receptors failed to reduce the senktide effect. Light scanning microscopic analysis of mesencephalic cells loaded with the Ca²⁺ sensitive dye, fluo-3, showed that senktide induced a rise in cytosolic Ca²⁺ in 8-10% of the cell population. The senktide-induced elevation in intracellular Ca²⁺ was rapid in onset and transient (at lo⁴ M) or more sustained with no further increase in fluorescence intensity (at 10^-7 M). The proportion of senktide-responsive cells was not significantly modified when extracellular Ca²⁺ was chelated, but was reduced by 87% in the presence of SR 142801 and by 75% in cultures that were pre-treated with the DA neurotoxin l-methyl-4-phenylpyridinium. The present study shows that enhancement of spontaneous [³H]DA release and intracellular Ca²⁺ mobilization may be observed after NK₃ receptor stimulation and that both biochemical events are likely to occur in DA neurons.