Figure 2.

Recordings from OX^−/−₂ slices indicate that OX₁ is sufficient for orexin-mediated excitation and enhancement of voltage-dependent Ca²⁺ transients in LDT, DR, and LC neurons. (A) Whole-cell voltage clamp recording from LDT neurons in OX^−/−₂ slices showed that 300 nM orexin-A (Orx-A) still evoked an inward current with increased sEPSCs [holding potential: −60 mV; (A₁); normal ACSF] and also augmented the Ca²⁺ transients evoked by 5 s voltage-steps from −60 to −30 mV [(A₂); DABST-containing ACSF]. (B) Voltage clamp recordings from DR neurons in OX^−/−₂ slices showed that orexin-A evoked an inward [holding potential: −60 mV; (B₁); normal ACSF] and augmented Ca²⁺ transients [(B₂); DABST-containing ACSF]. (C) Voltage clamp recordings from LC neurons in OX^−/−₂ slices showed that orexin-A evoked an inward current [holding potential: −60 mV; (C₁); normal ACSF] and augmented Ca²⁺ transients [(C₂); DABST-containing ACSF]. In (A₁,B₁,C₁), the horizontal bar indicates time of 300 nM orexin-A superfusion. In (A₂,B₂,C₂), top traces show somatic Ca²⁺-dependent fluorescence (dF/F); Middle traces show whole-cell current; Bottom traces show membrane voltage. Calibration bars indicate 2% dF/F, 200 pA, 30 mV, and 2 s. (D) Neither the orexin-evoked inward current nor the orexin-enhanced voltage-dependent Ca²⁺ transients was different in neurons from OX^−/−₂ slices compared to wild-type slices. Comparison by nuclei of the mean ± SEM of the post-synaptic inward current [(D₁); LDT and DR recorded in low-Ca²⁺ DABST-containing ACSF with Cs; LC recorded in DABST-containing ACSF] and mean ± SEM of the Ca²⁺ transient enhancement [(D₂); DABST-containing ACSF] produced by orexin-A in neurons recorded from OX^−/−₂ slices and C57BL6 slices under the same conditions.