Figure 2. Neurotensin activates orexin neurons.

A, Whole cell current clamp recording of orexin neurons shows that neurotensin (100 nM) depolarized orexin neurons in the absence (top) or presence (bottom) of TTX (1 µM). B, Concentration dependence of neurotensin-induced depolarization (mV) in the presence of TTX. EC₅₀ and Emax were 3.84±0.02 nM and 7.60±0.01 mV, respectively (n = 3–5). C, Effects of NTSR-1 or NTSR-2 antagonist on effects of neurotensin on orexin neurons. Both SR142948 (n = 7, 10 µM), a non-selective antagonist, and levocabastine (n = 7, 1 µM), an NTSR-2 preferential antagonist, almost completely blocked the effects of neurotensin-induced inward current in orexin neurons. Left panel, a typical trace. Right panel, effect of neurotensin-induced current in orexin neurons in the presence of SR142948 or lebocavastine. Extracellular solution was used as vehicle control. D, Double-labeling in situ hybridization shows most neurons expressing orexin mRNA (red) also express Ntsr-2 mRNA (blue). Black arrowheads show colocalization. Scale bar indicates 20 µm. E, Effect of extracellular Na⁺ on neurotensin-induced depolarization. NaCl was replaced by an equimolar concentration of choline chloride in the presence of TTX (1 µM) (Na⁺-free). Neurotensin (10 nM)-induced depolarization was markedly decreased in Na⁺-free solution (n = 4). F, Effect of extracellular Ca²⁺ on neurotensin-induced inward current. Neurotensin (10 nM) induced an inward current in the presence of extracellular calcium. This inward current was markedly increased in Ca²⁺-free extracellular solution (n = 7). Lower panel shows the fold-increase in inward current (pA) in Ca²⁺-free solution. G, Current-voltage relationship obtained by the current step protocol using a CsCl pipette in K⁺- and Ca²⁺-free extracellular solution. The steady state potential, at the end of the current step (marked by circle in left panel), is plotted in the current-voltage relationship in the right panel. I-V curve shows that the reversal potential of the neurotensin (10 nM)-induced current was 9.82 mV (n = 6–10). Neurons were current-clamped and the current was stepped from –240 pA to +280 pA (with increments of 40 pA with a duration of 100 ms). Open circle indicates control and filled circle indicates neurotensin (10 nM) application. H, Effects of non-selective cation channel blocker, SKF96365. Left panel, SKF96365 (10 µM) inhibited neurotensin-induced depolarization. Right panel, Bar graph indicates that SKF96365 inhibited neurotensin-induced depolarization (n = 4). Values are mean±S.E.M. *, p<0.05.