Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice

PLOS ONE RESEARCH ARTICLE Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice Kasane Yaguchi1,2, Mitsue Hagihara ID1, Ayumu Konno3,4, Hirokazu Hirai3,4, Hiroko Yukinaga1, Kazunari Miyamichi ID1* 1 Laboratory for Comparative Connectomics, Riken Center for Biosystems Dynamics Research, Kobe, Hyogo, Japan, 2 Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto, Japan, 3 Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, 4 Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR), Maebashi, Gunma, Japan * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Yaguchi K, Hagihara M, Konno A, Hirai H, Yukinaga H, Miyamichi K (2023) Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice. PLoS ONE 18(5): e0285589. https://doi.org/10.1371/journal. pone.0285589 Editor: Julieta Alfonso, University Hospital Heidelberg: UniversitatsKlinikum Heidelberg, GERMANY Received: October 21, 2022 Accepted: April 27, 2023 Published: May 10, 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.0285589 Copyright: © 2023 Yaguchi 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. Data Availability Statement: All relevant data are within the paper. Abstract Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother–pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother–pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions. Introduction Oxytocin (OT) is a nine-amino-acid peptide hormone known to mediate uterine contractions during parturition and milk ejection during lactation [1–3]. This hormone is predominantly produced by the OT neurons located in the paraventricular (PVH) and supraoptic (SO) hypothalamus and secreted into the circulation via the posterior pituitary. The milk ejection reflex, that is, the active transfer of milk from alveolar storage to mammary ducts in response to a PLOS ONE | https://doi.org/10.1371/journal.pone.0285589 May 10, 2023 1 / 19 PLOS ONE Funding: This study was supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/ MINDS, JP21dm027111) from Japan Agency for Medical Research and Development (AMED, https://brainminds.jp/en/) to H.H. and by KAKENHI (20K20589 and 21H02587) from the Japan Society for the Promotion of Science (JSPS, https://www. jsps.go.jp/english/e-grants/index.html) to K.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Oxytocin pulses of wild-type mice transient increase in plasma OT, is disabled in OT or OT receptor (OTR) knockout postpartum mice [4–7]. Therefore, OT is indispensable for breastfeeding in mice. The secretion of OT is thought to be mediated by the synchronous burst activities of PVH and SO OT neurons [5]. Extracellular recording studies have characterized maternal OT neural activities [8–12] and described afferent circuitry that conveys nipple sensory stimuli to the OT neurons in the hypothalamus [13–15]. Intracerebroventricular injection of OT is known to facilitate the milk ejection reflex, whereas that of OTR antagonist can block the ongoing milk ejection [16, 17]. Despite these classical studies, which were conducted in rats and rabbits, the detailed molecular and neural circuit mechanisms by which OT neurons shape burst synchronous activities during the milk ejection reflex remain poorly understood, in part because it has been difficult to utilize cell-type-specific toolkits for the manipulation of gene functions and neural activities in these species. Our recent study demonstrated cell type-specific calcium ion (Ca2+) imaging of PVH OT neurons in parturient and lactating mother mice by fiber photometry [18]. In that study, we utilized OT-Cre mice [19] combined with the Cre-dependent GCaMP6s driver line, Ai162 [20]. Although these double knock-in mice allowed cell type-specific and intensive expression of GCaMP6s, this strategy compromises the use of various Cre-dependent neuroscience toolkits. Applying a simple Cre-free method for monitoring OT neural activities [21] to mice could overcome this problem. In addition, previous studies [18, 21] have analyzed only the early stages of lactation; therefore, the dynamics of the pulsatile activities of OT neurons throughout the different stages of lactation remain unknown. In the present study, we first aimed to develop an adeno-associated virus (AAV) vector to drive GCaMP6s selectively into the OT neurons via a mouse OT mini-promoter [22]. Then, after validating this system, we aimed to characterize the pulsatile activities of OT neurons quantitatively in different mouse strains and multiple lactation stages, including weaning stages. In addition, we aimed to investigate whether direct physical contact between mothers and pups is always required for the milk ejection reflex in mice. As plasma OT (...truncated)