Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu Hypocretin (orexin) immunoreactivity in the feline midbrain: Relevance for the generation of wakefulness Alicia Costa a , Jaime Monti b , Pablo Torterolo a, * a Laboratorio de Neurobiología del Sueño, Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay b Departamento de Farmacología y Terapéutica, Hospital de Clínicas, Universidad de la República, Montevideo, Uruguay ARTICLE INFO Keywords: Hypothalamus Arousal Mood Peptide Sleep REM Sleep ABSTRACT Hypocretins (Hcrt) 1 and 2 are two neuropeptides synthesized from neurons that are located in the perifornical area of the lateral hypothalamus. These neurons project diffusely throughout the central nervous system, and have been implicated in the generation and maintenance of wakefulness, as well as in critical physiological processes that occur during this behavioral state, such as motivation. The hypocretinergic projections towards the feline midbrain have not been studied before. Therefore, the aim of the present study was to analyze their relationship to the midbrain neurons, that are critically involved in the control of sleep and wakefulness. With this purpose, we examined the distribution of Hcrt1-positive fibers in the midbrain and pontomesencephalic area of the domestic cat (Felis catus), and their relationship with catechola- minergic and cholinergic neurons by means of single and double immunohistochemistry. Hcrtergic axons with distinctive varicosities and buttons were heterogeneously distributed, exhibiting different densities in distinct regions of the midbrain. High Hcrtergic fiber densities were observed in the periaqueductal gray, inter- peduncular nucleus, locus coeruleus and cholinergic mesopontine regions. In addition, we studied in detail the Hcrtergic projection towards the dopaminergic nuclei of the midbrain. While very few Hcrt + fibers were ob- served in the substantia nigra pars compacta, the highest density of Hcrtergic fibers was found in the dopaminergic ventral periaqueductal gray area (also called A10dc area); appositions between Hcrtergic terminals and dopa- minergic somata and dendrites were observed within this area. Because this dopaminergic area has been in- volved in the control of wakefulness, the present anatomical data provides relevant support about the role of the Hcrtergic system in the generation of this behavioral state. 1. Introduction Hypocretin (Hcrt) 1 and 2 (also known as orexin A and B, respec- tively) are excitatory hypothalamic neuropeptides derived from a common precursor molecule (pre-pro-hypocretin) (de Lecea et al., 1998; Sakurai et al., 1998). Both peptides bind to the Hcrt receptors type 1 and 2 (HcrtR-1 and HcrtR-2) (Kukkonen, 2017; Wang et al., 2018). Hcrtergic neurons are located in the postero-lateral hypotha- lamic area, mainly at the perifornical region, and project throughout the central nervous system (de Lecea et al., 1998; Peyron et al., 1998). The Hcrtergic neurons receive and integrate internal and external in- formation and regulate autonomic and neuroendocrine systems, arousal and behavior. Hence, this system has been implicated in an array of functions ranging from wakefulness (W), mood regulation, feeding and analgesia (Burdakov, 2019; Chase, 2013; Mahler et al., 2014; Nevarez and de Lecea, 2018; Torterolo and Chase, 2014; Torterolo et al., 2015; Tyree et al., 2018). Mahler et al. (2014) suggest that the Hcrtergic system is involved in the motivational processes underlying adaptive, goal-oriented behaviors. Chase (2013) emphasizes its role for survival: “the Hcrtergic system initiates, coordinates and maintains survival be- haviors and survival-related processes”, according to his “unified sur- vival theory of the functioning of the Hcrtergic system”. Of note, several studies tend to indicate that Hcrtergic neurons participate also in the regulation of reinforcement and addiction processes. ln this respect, it has been shown that the Hcrt-1 receptor antagonist S8-334867 disrupts dopamine (DA) signaling and reduces self-administration of cocaine in rats (Espana et al., 2011). Hcrt-1 administered into the ventral teg- mental area (VTA) enhances DA responses to cocaine and promotes cocaine self-administration. Interestingly, Hcrtergic projections towards DAergic neurons of the human VTA and substantia nigra (SN) have been described by Hrabovszky et al. (2013). This is an important issue since tends to support a role of the Hcrtergic system in humans, in reward https://doi.org/10.1016/j.jchemneu.2020.101769 Received 3 December 2019; Received in revised form 19 February 2020; Accepted 19 February 2020 ⁎ Corresponding author. E-mail address: ptortero@fmed.edu.uy (P. Torterolo). Journal of Chemical Neuroanatomy 105 (2020) 101769 Available online 04 March 2020 0891-0618/ © 2020 Elsevier B.V. All rights reserved. T