Mapping of oxytocin‐ and arginine vasopressin‐expressing neurons with calbindin 1 or reelin in the male mouse brain

Oxytocin (OXT)- and arginine vasopressin (AVP)-synthesizing neurons are traditionally classified into magnocellular and parvocellular types based on their projection targets. However, molecular markers that reliably distinguish these neuronal subtypes across hypothalamic regions remain undefined. Recent single-cell transcriptomic studies suggest that Calb1 and Reln are differentially expressed in magnocellular and parvocellular OXT-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN). Whether this molecular distinction extends to OXT neurons beyond the PVN or to AVP neurons has remained unclear. Fluorogold (FG) was administered to adult male C57BL/6J mice via tail vein injection. Calbindin 1 and reelin expression in OXT- and AVP-synthesizing neurons in the bed nucleus of the stria terminalis (BNST), PVN, supraoptic nucleus (SON), and retrochiasmatic supraoptic nucleus (SOR) was examined by immunocytochemistry. FG-labeled neurons were distributed in the PVN, SON, and SOR. Most OXT-immunoreactive (ir) neurons in the anterior PVN, SON, and SOR were FG-positive, whereas many OXT-ir neurons in the posterior PVN were FG-negative. Most FG-positive OXT-ir neurons showed calbindin 1 immunoreactivity. Two-thirds of FG-negative OXT-ir neurons in the posterior PVN expressed reelin. Almost all Reln-positive OXT-ir neurons were FG-negative. Nearly all AVP-ir neurons were FG-positive, and more than half expressed calbindin 1, whereas reelin expression was not detected. In corticotropin-releasing hormone (CRH)-Venus knock-in mice, AVP-ir neurons and CRH-positive neurons were not colocalized within the PVN, and AVP-ir and CRH-positive fibers were localized to distinct layers of the median eminence. These findings indicate that AVP expression in parvocellular CRH neurons was below the detection threshold of the methods used in this study. Calbindin 1 serves as a marker for magnocellular OXT neurons, whereas reelin identifies a subset of parvocellular OXT neurons. Given that the detected AVP-ir neurons were predominantly magnocellular, calbindin 1 labels a subset of this magnocellular AVP population. This molecular distinction provides a framework for distinguishing OXT- and AVP-expressing neuronal populations and for examining their respective roles in endocrine and behavioral regulation.