Mitsue Hagihara

Mammalian reproductive function is driven by arcuate kisspeptin neurons, pacemakers of gonadotropin secretion. During energy shortages, animals reallocate resources from reproduction to survival; however, the underlying neural mechanisms remain elusive. Here we used fiber photometry to chronically monitor synchronized episodes of arcuate kisspeptin neuron activity (SEskiss) in adult mice under various energy-saving conditions. In both sexes, SEskiss frequency was markedly suppressed during fasting-induced torpor and pharmacologically induced hypothermia, whereas hypometabolism alone had no discernible effect. A Q neuron-induced hypothermic state (QIH) robustly suppressed SEskiss, leading to impaired gamete maturation, whereas warming the body temperature during QIH fully restored SEskiss frequency. These findings demonstrate that hypothermia, rather than hypometabolism, is the primary driver of suppression of the hypothalamic reproductive axis during energy-saving conditions. This study provides insights into how thermal signals act as critical gatekeepers in the mammalian reproductive system.

Summary: Atypical sociability is a hallmark of neurodevelopmental disorders arising from genetic susceptibility and prenatal environmental perturbations affecting diverse brain cell types. Using single-cell transcriptomics, we previously identified selective vulnerability of parvocellular oxytocin (OT) neurons in the paraventricular hypothalamus (PVH) following embryonic exposure to valproic acid (VPA), a teratogen that induces social deficits. Neonatal chemogenetic activation of OT neurons rescued these behavioral abnormalities and partially restored dysregulated gene expression. However, the effects of VPA exposure and OT neuron stimulation on non-neuronal PVH cells remained unclear. Here, we show that VPA induces transcriptional abnormalities in PVH microglia. Spatial transcriptomics revealed altered distributions of PVH microglial subtypes. Notably, neonatal OT neuron stimulation reversed a subset of VPA-induced microglial gene downregulation, while pharmacological manipulation of microglia normalized aberrant OT gene expression in putative parvocellular OT neurons. These findings support bidirectional OT neuron–microglia interactions that may underlie social dysfunction following embryonic VPA exposure. Highlights: Embryonic VPA exposure induces potent transcriptional changes in PVH microglia PVH microglia comprise two spatially organized subtypes disrupted by VPA Neonatal OT neuron stimulation restores gene expression in a microglial subtype Microglial manipulation rescues OT ligand in parvocellular PVH neurons

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.

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 sympathetic nervous system is crucial for responding to environmental changes. This regulation is coordinated by the spinal sympathetic preganglionic neurons (SPNs), innervating both postganglionic neurons and the adrenal gland. Despite decades of research supporting the concept of selective control within this system, the neural circuit organization responsible for the output specificity remains poorly understood. Here, by combining recent single-cell transcriptome data with viral-genetic toolkits in mice, we identify two subtypes of SPNs in the lower thoracic spinal cord, defined at the molecular level, exhibiting nonoverlapping patterns of innervation: one specifically projecting to the celiac/superior mesenteric ganglia, and the other targeting the adrenal grand. Chemogenetic manipulations on these distinct SPN subtypes revealed selective impacts on the motility of the gastrointestinal tracts or glucose metabolism mediated by the adrenal gland, respectively. This molecularly delineated parallel labeled-line organization in sympathetic outflows presents a potential avenue for selectively manipulating organ functions.

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.