The importance of oxytocin neurons in the supraoptic nucleus for breastfeeding in mice

PLOS ONE RESEARCH ARTICLE The importance of oxytocin neurons in the supraoptic nucleus for breastfeeding in mice Mitsue Hagihara☯, Kazunari Miyamichi ID*, Kengo Inada ID*☯ RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Hyogo, Japan ☯ These authors contributed equally to this work. * (KI); (KM) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Hagihara M, Miyamichi K, Inada K (2023) The importance of oxytocin neurons in the supraoptic nucleus for breastfeeding in mice. PLoS ONE 18(3): e0283152. https://doi.org/10.1371/ journal.pone.0283152 Editor: Michael Schubert, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, FRANCE Received: September 21, 2022 Accepted: March 2, 2023 Published: March 17, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0283152 Copyright: © 2023 Hagihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The hormone oxytocin, secreted from oxytocin neurons in the paraventricular (PVH) and supraoptic (SO) hypothalamic nuclei, promotes parturition, milk ejection, and maternal caregiving behaviors. Previous experiments with whole-body oxytocin knockout mice showed that milk ejection was the unequivocal function of oxytocin, whereas parturition and maternal behaviors were less dependent on oxytocin. Whole-body knockout, however, could induce the enhancement of expression of related gene(s), a phenomenon called genetic compensation, which may hide the actual functions of oxytocin. In addition, the relative contributions of oxytocin neurons in the PVH and SO have not been well documented. Here, we show that females with conditional knockout of oxytocin gene in both the PVH and SO undergo grossly normal parturition and maternal caregiving behaviors, while dams with a smaller number of remaining oxytocin-expressing neurons exhibit severe impairments in breastfeeding, leading to the death of their pups within 24 hours after birth. We also found that the growth of pups is normal even under oxytocin conditional knockout in PVH and SO as long as pups survive the next day of delivery, suggesting that the reduced oxytocin release affects the onset of lactation most severely. These phenotypes are largely recapitulated by SO-specific oxytocin conditional knockout, indicating the unequivocal role of oxytocin neurons in the SO in successful breastfeeding. Given that oxytocin neurons not only secrete oxytocin but also non-oxytocin neurotransmitters or neuropeptides, we further performed cell ablation of oxytocin neurons in the PVH and SO. We found that cell ablation of oxytocin neurons leads to no additional abnormalities over the oxytocin conditional knockout, suggesting that non-oxytocin ligands expressed by oxytocin neurons have negligible functions on the responses measured in this study. Collectively, our findings confirm the dispensability of oxytocin for parturition or maternal behaviors, as well as the importance of SO-derived oxytocin for breastfeeding. Data Availability Statement: All relevant data are within the paper. Introduction Funding: K.I. was supported by the RIKEN Special Postdoctoral Researchers Program (https://www. Oxytocin (OT) is a nonapeptide hormone produced by OT neurons in the paraventricular (PVH) and supraoptic (SO) hypothalamic nuclei. Recent studies have reported that OT plays PLOS ONE | https://doi.org/10.1371/journal.pone.0283152 March 17, 2023 1 / 16 PLOS ONE riken.jp/en/careers/programs/spdr/), a grant from the Kao Foundation for Arts and Sciences (https:// www.kao-foundation.or.jp/english.html), and Japan society promotion science KAKENHI (19J00403 and 19K16303) (https://www.jsps.go. jp/english/) K.M. was supported by Japan society promotion science KAKENHI (20K20589 and 21H02587) (https://www.jsps.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder (RIKEN Special Postdoctoral Researchers Program) provided support in the form of salaries for the last author [KI], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the author are articulated in the ‘author contributions’ section. Competing interests: The authors have declared that no competing interests exist. Conditional knockout of oxytocin gene for maternal functions important roles in sexual, maternal, and social behaviors [1–3], in addition to the functions documented in classical studies such as the induction of labor and milk ejection. However, studies on whole-body OT knockout (KO) mice have shown that milk ejection is a specific and essential function of OT, but dispensable for parturition [4–6]. Similarly, the expression of maternal caregiving behaviors does not require OT, given that the performance of parental behaviors by OT KO dams is largely similar to that by controls [5, 7], except in food-limited stressful environments [8]. Despite the clear consistencies across studies, the significance of OT signaling in the regulation of labor, milk ejection, and parental behaviors remains unclear, given that the phenotypes of a whole-body KO might be genetically compensated by the upregulation of related gene(s) [9, 10]. For example, a recent study analyzing a different function of OT neurons, weight homeostasis [11–13], reported that PVH-specific OT conditional KO (cKO) mice showed a hyperphagic obesity phenotype that was not apparent in the whole-body OT KO [14]. In addition, although OT neurons can release not only OT, but also other neurotransmitters or neuropeptides, such as glutamate [15], to our knowledge, the functional roles of such non-OT ligands in the regulation of labor, milk ejection, and parental behaviors have not been described. Here, we show the relevance of OT secretion on labor, milk ejection, and parental behaviors using an OT cKO mouse line described previously [16]. Our approach offers a better temporal resolution, which allows us to avoid the influence of possible developmental or genetic compensation [9, 10]. We also test the relative contributions of OT secretion from the PVH and SO nuclei to maternal physiology and behaviors by restricting the manipulation to a single hypothalamic nucleus. This improved spatial resolution may reveal distinct functions of OT neurons in the PVH and SO, which show distinct input–output organizations [17]. Furthermore, we compare the phenotype of OT cKO with tha (...truncated)