The regulatory functions of the orexin system are significantly correlated with pain. Parvalbumin (PV) interneurons, the main subtype of inhibitory gamma-aminobutyric acid (GABA) interneurons, have also been shown to be involved in the regulation of pain. To provide evidence for neural connections between these two types of neurons in pain-related nuclei, a double-label immunohistochemical staining technique was used to reveal the overlap between PV and orexin immunoreactivity in the mouse brain. At the forebrain level, the medial prefrontal cortex (mPFC) mainly consists of the cingulate cortex, area 1 (Cg1), prelimbic cortex (PrL), infralimbic cortex (IL), and dorsal peduncular cortex (DP). These regions all exhibited a similar, clear overlap between orexin-positive fibers and PV-positive neuronal somata and their axons. In the basal forebrain system, specific overlapping patterns were observed in the core of the nucleus accumbens (AcbC) and the shell of the nucleus accumbens (AcbSh). In the diencephalon, close overlap was found between orexinergic fibers and PV-positive neuronal structures in the basolateral amygdala (BLA) and lateral hypothalamic area (LH). In the brainstem, the density of intermingled orexin and PV fibers decreased in the order of the dorsal raphe nucleus (DR) and periaqueductal gray (PAG), with orexin boutons embedded within PV fibers. Additionally, in the ventral tegmental area (VTA) and lateral parabrachial nucleus (LPB), co-localization was observed between PV-positive somata with axons and orexin fibers. The PV and orexinergic systems anatomically converge by jointly targeting brain regions involved in pain regulation. These findings provide key morphological evidence for elucidating the synergistic mechanisms of the two systems in the pathophysiology of pain.

KEY WORDS: Pain; Parvalbumin; Orexin; Immunohistochemistry; Neural Circuit.