(A) Left panel shows confocal image of N/OFQ-immunoreactive fibers in the vicinity of, and in putative contact with, Hcrt-immunoreactive neurons in the PLH of WT mice. N/OFQ (green) fibers are in close proximity to Hcrt-immunoreactive (red) cells. The arrow indicates the N/OFQ innervation of an Hcrt cell body. Middle panel shows light micrograph of a light brown immunolabeled Hcrt neuron contacted by a dark black bouton (arrow) representing immunolabeling for N/OFQ. Right panel shows electron micrograph taken from ultrathin sections of the same labeled terminal and dendrite shown in the light micrograph in the middle panel. Black arrow indicates the N/OFQ-immunolabeled axon terminal in synaptic contact (red arrowhead) with the dendrite of the Hcrt cell. Scale bars: 10 μm (left and middle panels); 1 μm (right panel). (B) Under current clamp, bath application of N/OFQ (1 μM) hyperpolarized Hcrt neurons, decreased input resistance, and blocked spontaneous firing of action potentials. The resting potential of this cell was –54 mV and was manually adjusted to –60 mV with DC current. Membrane resistance was monitored using hyperpolarizing current pulses (–0.3 nA, 800 ms) delivered every 5 seconds throughout the experiment. (C) Under voltage-clamp mode at a V_h of –60 mV, N/OFQ (1 μM) induced an outward current (–53 pA) in an Hcrt neuron. Notice that the frequency but not the amplitude of the miniature synaptic currents (inward currents) recorded in the presence of TTX (0.5 μM) was also reduced.