Calbindin 1 identifies magnocellular oxytocin neurons, Reelin marks parvocellular oxytocin neurons in male mice

Oxytocin (OXT) and arginine vasopressin (AVP) neurons are crucial for diverse physiological functions, traditionally categorized as magnocellular or parvocellular based on their projection targets. However, reliable molecular markers to distinguish these subtypes across various hypothalamic regions have remained elusive. This lack of specific markers hinders precise investigation into the distinct roles and regulatory mechanisms of these neuronal populations. Recent transcriptomic data suggested Calb1 and Reln as potential markers for OXT neurons in the hypothalamic paraventricular nucleus (PVN), but their broader applicability to OXT neurons elsewhere or to AVP neurons was unconfirmed.

Researchers administered Fluorogold (FG) via tail vein injection to adult male C55BL/6J mice to retrogradely label magnocellular neurons projecting to the posterior pituitary. They then used immunocytochemistry to examine the expression of Calbindin 1 and Reelin in OXT- and AVP-synthesizing neurons across key hypothalamic regions, including the bed nucleus of the stria terminalis (BNST), paraventricular nucleus (PVN), supraoptic nucleus (SON), and retrochiasmatic supraoptic nucleus (SOR). Additionally, CRH-Venus knock-in mice were utilized to investigate colocalization of AVP and CRH within the PVN and median eminence.

Fluorogold (FG)-labeled neurons, indicating posterior pituitary projection, were primarily distributed in the PVN, SON, and SOR. Most OXT-immunoreactive (OXT-ir) neurons in the anterior PVN, SON, and SOR were FG-positive, consistent with a magnocellular phenotype. In contrast, many OXT-ir neurons in the posterior PVN were FG-negative, suggesting a parvocellular nature. Crucially, most FG-positive OXT-ir neurons showed calbindin 1 immunoreactivity, establishing it as a marker for magnocellular OXT neurons. Conversely, two-thirds of FG-negative OXT-ir neurons in the posterior PVN expressed reelin, with almost all Reln-positive OXT-ir neurons being FG-negative, identifying Reelin with a subset of parvocellular OXT neurons. For AVP neurons, nearly all AVP-ir neurons were FG-positive, and more than half expressed calbindin 1, while reelin expression was not detected. Studies in CRH-Venus knock-in mice confirmed that AVP-ir neurons and CRH-positive neurons were not colocalized within the PVN, and their fibers occupied distinct layers of the median eminence, suggesting AVP expression in parvocellular CRH neurons was below detection limits.