Neurons of the parabrachial nucleus (PB) receive nociceptive input from the dorsal horn (DH) of the spinal cord and caudal part of the spinal trigeminal nucleus (Vc). Previously, we demonstrated that glutamatergic lateral PB neurons innervate orexin (ORX) neurons in the perifornical area (PeF) of the hypothalamus. However, the neural circuit via which ORX neurons receive nociceptive input from the DH and brainstem remains to be determined. In the present study, we aimed to clarify the potential nociceptive circuit from DH/Vc to PeF via lateral PB. We first examined the neuronal activity of fluorogold (FG)-labeled, PeF-projecting lateral PB neurons in Wistar rats following either saline or formalin injection to the forepaw or lips. We clearly detected more abundant c-Fos-positive, FG-labeled neurons in the PB nucleus. To investigate the relay from the DH/Vc to the PeF via the lateral PB, we injected FG into the PeF and biotinylated dextranamine (BDA) into the contralateral DH or ipsilateral Vc. We observed the most prominent overlap between BDA-labeled axon terminals and FG-labeled neurons in the dorsal lateral and central lateral subnuclei. Furthermore, we found that FG-labeled neurons formed close contact sites with BDA-labeled axons with synaptophysin immunoreactivity. Using electron microscopy, we confirmed that these contact sites were truly synapses. Taken together, our results indicate that the DH/Vc transmits nociceptive information to the PeF via the lateral PB, suggesting the involvement of ORX neurons in the pain pathway. (C) 2019 Elsevier Inc. All rights reserved.