Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: An in vitro study

Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep–wakefulness and feeding. ORX neurons project to the pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep. Thus, we examined electrophysiological effects of ORXs on rat PPT neurons with a soma size of more than 30 μm. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarized PPT neurons dose-dependently in normal and/or tetrodotoxin containing artificial cerebrospinal fluids (ACSFs), and the EC50 values for ORX-A and ORX-B were 66 nM and 536 nM, respectively. SB-334867, a selective inhibitor for ORX 1 (OX1) receptors, significantly suppressed the ORX-A-induced depolarization. The ORX-A-induced depolarization was reduced in high K+ ACSF with extracellular K+ concentration of 13.25 mM or N-methyl-d-glucamine (NMDG+)-containing ACSF in which NaCl was replaced with NMDG-Cl, and abolished in high K+-NMDG+ ACSF or in a combination of NMDG+ ACSF and recordings with Cs+-containing pipettes. An inhibitor of Na+/Ca2+ exchanger and chelating intracellular Ca2+ had no effect on the depolarization. Most of PPT neurons studied were characterized by an A-current or both A-current and a low threshold Ca2+ spike, and predominantly cholinergic. These results suggest that ORXs directly depolarize PPT neurons via OX1 receptors and via a dual ionic mechanism including a decrease of K+ conductances and an increase of non-selective cationic conductances, and support the notion that ORX neurons affect the activity of PPT neurons directly and/or indirectly to control sleep–wakefulness, especially REM sleep.