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

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.

Oxytocin signaling in the posterior hypothalamus prevents hyperphagic obesity in mice.

Decades of studies have revealed molecular and neural circuit bases for body weight homeostasis. Neural hormone oxytocin (Oxt) has received attention in this context because it is produced by neurons in the paraventricular hypothalamic nucleus (PVH), a known output center of hypothalamic regulation of appetite. Oxt has an anorexigenic effect, as shown in human studies, and can mediate satiety signals in rodents. However, the function of Oxt signaling in the physiological regulation of appetite has remained in question, because whole-body knockout (KO) of Oxt or Oxt receptor (Oxtr) has little effect on food intake. We herein show that acute conditional KO (cKO) of Oxt selectively in the adult PVH, but not in the supraoptic nucleus, markedly increases body weight and food intake, with an elevated level of plasma triglyceride and leptin. Intraperitoneal administration of Oxt rescues the hyperphagic phenotype of the PVH Oxt cKO model. Furthermore, we show that cKO of Oxtr selectively in the posterior hypothalamic regions, especially the arcuate hypothalamic nucleus, a primary center for appetite regulations, phenocopies hyperphagic obesity. Collectively, these data reveal that Oxt signaling in the arcuate nucleus suppresses excessive food intake.

Recording and manipulation of the maternal oxytocin neural activities in mice.

Pulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation.1-3 These pulses are generated by the unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits.1,4-10 Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11,12 and make better use of cell-type-specific neuroscience toolkits,13 a mouse model needs to be developed for the studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. A sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found the suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory inputs.

A single vomeronasal receptor promotes intermale aggression through dedicated hypothalamic neurons.

The pheromonal information received by the vomeronasal system plays a crucial role in regulating social behaviors such as aggression in mice. Despite accumulating knowledge of the brain regions involved in aggression, the specific vomeronasal receptors and the exact neural circuits responsible for pheromone-mediated aggression remain unknown. Here, we identified one murine vomeronasal receptor, Vmn2r53, that is activated by urine from males of various strains and is responsible for evoking intermale aggression. We prepared a purified pheromonal fraction and Vmn2r53 knockout mice and applied genetic tools for neuronal activity recording, manipulation, and circuit tracing to decipher the neural mechanisms underlying Vmn2r53-mediated aggression. We found that Vmn2r53-mediated aggression is regulated by specific neuronal populations in the ventral premammillary nucleus and the ventromedial hypothalamic nucleus. Together, our results shed light on the hypothalamic regulation of male aggression mediated by a single vomeronasal receptor.

Plasticity of neural connections underlying oxytocin-mediated parental behaviors of male mice.

The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naive male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Here, using viral-genetic approaches, we establish hypothalamic oxytocin neurons as the key regulators of the parental caregiving behaviors of male mice. We then use rabies-virus-mediated unbiased screening to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region that is classically assumed to be hard-wired.

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents.

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species.

A Labeled-Line Neural Circuit for Pheromone-Mediated Sexual Behaviors in Mice.

In mice, various instinctive behaviors can be triggered by olfactory input. Despite growing knowledge of the brain regions involved in such behaviors, the organization of the neural circuits that convert olfactory input into stereotyped behavioral output remains poorly understood. Here, we mapped the neural circuit responsible for enhancing sexual receptivity of female mice by a male pheromone, exocrine gland-secreting peptide 1 (ESP1). We revealed specific neural types and pathways by which ESP1 information is conveyed from the peripheral receptive organ to the motor-regulating midbrain via the amygdala-hypothalamus axis. In the medial amygdala, a specific type of projection neurons gated ESP1 signals to the ventromedial hypothalamus (VMH) in a sex-dependent manner. In the dorsal VMH, which has been associated with defensive behaviors, a selective neural subpopulation discriminately mediated ESP1 information from a predator cue. Together, our data illuminate a labeled-line organization for controlling pheromone-mediated sexual behavioral output in female mice.