Kengo Inada

The mammalian brain orchestrates a broad range of social behaviors, including interactions with conspecific young. Sexually inexperienced male mice typically exhibit pup-directed aggression, whereas fathers display caregiving behaviors. Although this behavioral transition provides a valuable framework for understanding life stage-dependent modulation of social behavior, the underlying mechanisms remain unclear. The posterior bed nucleus of the stria terminalis (BST), particularly its principal subdivision (BSTpr), is a critical node regulating pup-directed aggression. Within this region, neurons expressing estrogen receptor 1 ( Esr1 ) promote pup-directed aggression in female mice; however, the specific neuronal subtypes mediating this behavior in males remain unknown. Here, reanalysis of single-cell transcriptomic datasets shows that neurons expressing (+) cocaine- and amphetamine-regulated transcript prepropeptide ( Cartpt ) constitute a subset of Esr1 + neurons within the BST. Exposure to pups selectively increased c-fos expression, a marker of neuronal activation, in BSTpr Cartpt + neurons in infanticidal virgin males. Chemogenetic activation of these neurons enhanced pup-directed aggression, whereas their ablation suppressed infanticide. Anatomical analyses demonstrated that BSTpr Cartpt + neurons received direct inhibitory inputs from the anterior commissure nucleus (ACN), a region associated with parental behavior. Additionally, inhibitory transmission from the ACN to BSTpr Cartpt + neurons was enhanced in fathers. Together, these findings identify BSTpr Cartpt + neurons as key mediators of pup-directed aggression in male mice, suggesting a neural circuit mechanism underlying the transition from aggression to parental care.

The transition to parenthood brings significant changes in behavior toward offspring. For instance, in anticipation of their offspring, male mice shift from infanticidal to caregiving behaviors. While the release of oxytocin from the paraventricular hypothalamus (PVH) plays a critical role in paternal caregiving, it does not fully account for the entire behavioral shift. The specific downstream neurons and signaling mechanisms involved in this process remain obscure. Here, we demonstrate that PVH vasopressin neurons also essentially contribute to a paternal behavioral shift. This vasopressin signal is partially transmitted through oxytocin receptors (OTRs) expressed in the anterior commissure and medial nuclei of the preoptic area. These OTR-expressing neurons receive inputs from both PVH oxytocin and vasopressin neurons and are responsible for expressing paternal caregiving behaviors. Collectively, this non-canonical vasopressin-to-OTR crosstalk within specific limbic circuits acts as a pivotal regulator of paternal behavioral changes in mice.

Fetal development in endothermic mammals relies on a tightly regulated maternal body temperature. In animal models, deviations from normothermia during gestation cause fetal developmental abnormalities. However, the direct and physiological manipulation of the maternal core temperature has been technologically challenging, as traditional methods involve the imposition of external thermal stress. Recent advances in the identification of thermoregulatory neurons in the mouse preoptic area have allowed precise control of maternal body temperature without altering environmental conditions. Here we show that the activation of excitatory neurons in the medial preoptic area (MPO) induces a torpor-like hypothermic state in pregnant mice. When induced during early gestation, this state resulted in pregnancy loss, likely due to implantation failure. Hypothermia in mid- and late-gestation reduced fetal liver and kidney size and caused severe growth retardation. Similar outcomes were observed following pyroglutamylated RFamide peptide (Qrfp)-expressing neuron activation in the MPO, whereas inhibitory neuron activation had minimal effect. Furthermore, activating MPO excitatory neurons projecting to the dorsomedial hypothalamic nucleus reproduced both the torpor-like state and fetal growth retardation. These results underscore the importance of a stable maternal body temperature in fetal development and establish a new model for studying the effects of altered maternal temperature on embryogenesis.

Pregnancy represents a unique physiological state marked by substantial adaptations influencing both behavior and metabolism ¹ . Alterations have been observed across multiple levels—from changes in cellular morphology, synaptic density, and neuroglial interactions to a brain-wide reduction in gray matter volume ^2–7 . While these adaptations are thought to support offspring survival and promote maternal physiological and psychological well-being ⁸ , their functional significance remains largely unresolved. In particular, linking localized synaptic plasticity to a broader brain-wide circuit dynamics has posed a major challenge. Using virus-mediated screening in mice, we investigated the pregnancy-induced remodeling of the presynaptic landscape of paraventricular hypothalamic (PVH) oxytocin neurons, a central hub of maternal physiology ^9,10 . Here we show a selective and reversible elimination of excitatory synaptic inputs from the medial preoptic nucleus to PVH oxytocin neurons, displaying region-, cell-type-, and pathway-specificity. This reduction in excitatory drive attenuated the anorexigenic activity of oxytocin neurons during feeding, thereby promoting the increased food intake characteristic of pregnancy. These data demonstrate that the selective suppression of anorexigenic PVH oxytocin neuron activity plays a critical role in appetite regulation during pregnancy. More broadly, our data identify a pregnancy-associated remodeling of hypothalamic neural circuits with direct physiological relevance.

Fetal development in endothermic mammals relies on a tightly regulated maternal body temperature. In animal models, deviations from normothermia during gestation cause fetal developmental abnormalities. However, the direct and physiological manipulation of the maternal core temperature has been technologically challenging, as traditional methods involve the imposition of external thermal stress. Recent advances in the identification of thermoregulatory neurons in the mouse preoptic area have allowed precise control of maternal body temperature without altering environmental conditions. Here we show that the activation of excitatory neurons in the medial preoptic area (MPO) induces a torpor-like hypothermic state in pregnant mice. When induced during early gestation, this state resulted in pregnancy loss, likely due to implantation failure. Hypothermia in mid- and late-gestation reduced fetal liver and kidney size and caused severe growth retardation. Similar outcomes were observed following pyroglutamylated RFamide peptide (Qrfp)-expressing neuron activation in the MPO, whereas inhibitory neuron activation had minimal effect. Furthermore, activating MPO excitatory neurons projecting to the dorsomedial hypothalamic nucleus reproduced both the torpor-like state and fetal growth retardation. These results underscore the importance of a stable maternal body temperature in fetal development and establish a new model for studying the effects of altered maternal temperature on embryogenesis.

Maternal behaviors, which are crucial for the survival of mammalian infants, require the coordinated operation of multiple brain regions to process infant cues, make decisions, and execute motor plans. Although these processes likely demand higher cognitive functions, the prefrontal areas that regulate limbic parental programs remains poorly understood. Here, we show that the orbitofrontal cortex (OFC) excitatory projection neurons promote alloparental caregiving behaviors in female mice. By chronic microendoscopy, we observed robust yet adaptable representations of pup-directed anticipatory and motor-related activities within the OFC. Some of these plastic responses were significantly overlapped with those related to nonsocial reward signals. The inactivation of OFC output reduced the phasic activities of midbrain dopamine (DA) neurons specifically tied to pup retrieval and impaired the modulation of DA release to the ventral striatum during the acquisition of alloparental behaviors. These findings suggest that the OFC transiently boosts DA activity during the acquisition phase, thereby facilitating the manifestation of alloparental behaviors.

Neural circuits are composed of numerous neurons that perform diverse functions. Understanding the mechanisms of neural processing requires elucidating the connections among individual neurons. Rabies virus (RV)-mediated trans-synaptic tracing enables the visualization of direct presynaptic neurons of a defined neural population, facilitating the precise mapping of neural circuits across various brain regions. This method relies on RV mutants that require the expression of the TVA receptor and rabies glycoprotein to infect and spread to presynaptic neurons. Traditionally, adeno-associated virus (AAV) has been used to express these proteins. However, because AAV requires several weeks to achieve sufficient gene expression, it is challenging to use this approach for studying neural connections during postnatal development. To address this limitation, we generated a transgenic mouse line, termed Ai162-nCTG, which expresses nuclear-localized mCherry, the TVA receptor, and rabies glycoprotein in a Cre-dependent manner. As a proof-of-principle, we crossed the Ai162-nCTG line with the vasopressin-Cre line. In the paraventricular hypothalamic nucleus, where a major cluster of vasopressin neurons exists, mCherry expression was highly specific to vasopressin neurons, although not all vasopressin neurons co-expressed mCherry. We injected RV into the paraventricular hypothalamic nucleus and compared the labeling patterns with those of the conventional AAV-based approach. Although both methods labeled input cells in similar brain regions, the AAV-based approach was superior in terms of labeling efficiency. We also demonstrated that the Ai162-nCTG-based method enables rabies virus-mediated trans-synaptic tracing in mice at postnatal day 7 and 30. The distribution of presynaptic neurons was largely similar in the juvenile and adult stages, suggesting that paraventricular vasopressin neurons do not significantly change their presynaptic inputs during post-weaning development. Taken together, these findings suggest that the Ai162-nCTG line can be used for rabies virus-mediated trans-synaptic tracing when AAV administration is challenging. We also acknowledge and discuss the technical constraints associated with this mouse line.

The transition to parenthood brings significant changes in behavior toward offspring. For instance, in anticipation of their offspring, male mice shift from infanticidal to caregiving behaviors. While the release of oxytocin from the paraventricular hypothalamus (PVH) plays a critical role in paternal caregiving, it does not fully account for the entire behavioral shift. The specific downstream neurons and signaling mechanisms involved in this process remain obscure. Here, we demonstrate that PVH vasopressin neurons also essentially contribute to a paternal behavioral shift. This vasopressin signal is partially transmitted through oxytocin receptors (OTRs) expressed in the anterior commissure and medial nuclei of the preoptic area. These OTR-expressing neurons receive inputs from both PVH oxytocin and vasopressin neurons and are responsible for expressing paternal caregiving behaviors. Collectively, this non-canonical vasopressin-to-OTR crosstalk within specific limbic circuits acts as a pivotal regulator of paternal behavioral changes in mice. Highlights PVH vasopressin neurons are required for and can trigger paternal caregiving behaviors. Vasopressin-induced paternal behaviors are mediated in part by OTRs in the preoptic area (POA). POA OTR neurons receive inputs from both PVH oxytocin and vasopressin neurons. POA OTR neurons play a critical and facilitative role in promoting paternal caregiving behaviors.