Functional Ontogeny of Hypothalamic Agrp Neurons in Neonatal Mouse Behaviors.

Hypothalamic Agrp neurons regulate food ingestion in adult mice. Whether these neurons are functional before animals start to ingest food is unknown. Here, we studied the functional ontogeny of Agrp neurons during breastfeeding using postnatal day 10 mice. In contrast to adult mice, we show that isolation from the nursing nest, not milk deprivation or ingestion, activated Agrp neurons. Non-nutritive suckling and warm temperatures blunted this effect. Using in vivo fiber photometry, neonatal Agrp neurons showed a rapid increase in activity upon isolation from the nest, an effect rapidly diminished following reunion with littermates. Neonates unable to release GABA from Agrp neurons expressed blunted emission of isolation-induced ultrasonic vocalizations. Chemogenetic overactivation of these neurons further increased emission of these ultrasonic vocalizations, but not milk ingestion. We uncovered important functional properties of hypothalamic Agrp neurons during mouse development, suggesting these neurons facilitate offspring-to-caregiver bonding.

Hypothalamic Agrp neurons drive stereotypic behaviors beyond feeding.

The nervous system evolved to coordinate flexible goal-directed behaviors by integrating interoceptive and sensory information. Hypothalamic Agrp neurons are known to be crucial for feeding behavior. Here, however, we show that these neurons also orchestrate other complex behaviors in adult mice. Activation of Agrp neurons in the absence of food triggers foraging and repetitive behaviors, which are reverted by food consumption. These stereotypic behaviors that are triggered by Agrp neurons are coupled with decreased anxiety. NPY5 receptor signaling is necessary to mediate the repetitive behaviors after Agrp neuron activation while having minor effects on feeding. Thus, we have unmasked a functional role for Agrp neurons in controlling repetitive behaviors mediated, at least in part, by neuropeptidergic signaling. The findings reveal a new set of behaviors coupled to the energy homeostasis circuit and suggest potential therapeutic avenues for diseases with stereotypic behaviors.

O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat.

Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here, we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting.

Immune sensing of food allergens promotes avoidance behaviour.

In addition to its canonical function of protection from pathogens, the immune system can also alter behaviour1,2. The scope and mechanisms of behavioural modifications by the immune system are not yet well understood. Here, using mouse models of food allergy, we show that allergic sensitization drives antigen-specific avoidance behaviour. Allergen ingestion activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus solitarius, parabrachial nucleus and central amygdala. Allergen avoidance requires immunoglobulin E (IgE) antibodies and mast cells but precedes the development of gut allergic inflammation. The ability of allergen-specific IgE and mast cells to promote avoidance requires cysteinyl leukotrienes and growth and differentiation factor 15. Finally, a comparison of C57BL/6 and BALB/c mouse strains revealed a strong effect of the genetic background on the avoidance behaviour. These findings thus point to antigen-specific behavioural modifications that probably evolved to promote niche selection to avoid unfavourable environments.

Mitochondrial dynamics controlled by mitofusins regulate Agrp neuronal activity and diet-induced obesity.

Mitochondria are key organelles in the maintenance of cellular energy metabolism and integrity. Here, we show that mitochondria number decrease but their size increase in orexigenic agouti-related protein (Agrp) neurons during the transition from fasted to fed to overfed state. These fusion-like dynamic changes were cell-type specific, as they occurred in the opposite direction in anorexigenic pro-opiomelanocortin (POMC) neurons. Interfering with mitochondrial fusion mechanisms in Agrp neurons by cell-selectively knocking down mitofusin 1 (Mfn1) or mitofusin 2 (Mfn2) resulted in altered mitochondria size and density in these cells. Deficiency in mitofusins impaired the electric activity of Agrp neurons during high-fat diet (HFD), an event reversed by cell-selective administration of ATP. Agrp-specific Mfn1 or Mfn2 knockout mice gained less weight when fed a HFD due to decreased fat mass. Overall, our data unmask an important role for mitochondrial dynamics governed by Mfn1 and Mfn2 in Agrp neurons in central regulation of whole-body energy metabolism.

Mitofusin 2 in POMC neurons connects ER stress with leptin resistance and energy imbalance.

Mitofusin 2 (MFN2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here, we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted in loss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure, and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establish MFN2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for MFN2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

Phosphoribosomes for fingerprinting neurons.

The mammalian brain comprises a diverse variety of cell types. Fine characterization of specific subpopulations of neurons, however, has been a technical challenge. Here, Knight et al. describe an elegant technique for high throughput of molecular profiling of activated hypothalamic neurons.

Synaptic plasticity of feeding circuits: hormones and hysteresis.

The drive to eat is controlled by neuronal circuits in the hypothalamus that respond to hormones signaling hunger or satiety. In this issue of Cell, Yang et al. (2011) reveal an AMPK-dependent synaptic pathway that sustains excitatory stimulation of the NPY/AgRP neurons that promote feeding behavior until satiety signals kick in.

GABA keeps up an appetite for life.

In the arcuate nucleus of the hypothalamus, neurons that produce the neuropeptides NPY and AgRP play a vital role in the maintenance of energy homeostasis. In this issue, Wu et al. (2009) show that these neurons modulate feeding behavior in mice by providing GABAergic input to the parabrachial nucleus in the brainstem.

AgRP neurons control structure and function of the medial prefrontal cortex.

Hypothalamic agouti-related peptide and neuropeptide Y-expressing (AgRP) neurons have a critical role in both feeding and non-feeding behaviors of newborn, adolescent, and adult mice, suggesting their broad modulatory impact on brain functions. Here we show that constitutive impairment of AgRP neurons or their peripubertal chemogenetic inhibition resulted in both a numerical and functional reduction of neurons in the medial prefrontal cortex (mPFC) of mice. These changes were accompanied by alteration of oscillatory network activity in mPFC, impaired sensorimotor gating, and altered ambulatory behavior that could be reversed by the administration of clozapine, a non-selective dopamine receptor antagonist. The observed AgRP effects are transduced to mPFC in part via dopaminergic neurons in the ventral tegmental area and may also be conveyed by medial thalamic neurons. Our results unmasked a previously unsuspected role for hypothalamic AgRP neurons in control of neuronal pathways that regulate higher-order brain functions during development and in adulthood.

Hypothalamic POMC neurons promote cannabinoid-induced feeding.

Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide ß-endorphin. CB1R activation selectively increases ß-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Sirtuin 1 and sirtuin 3: physiological modulators of metabolism.

The sirtuins are a family of highly conserved NAD(+)-dependent deacetylases that act as cellular sensors to detect energy availability and modulate metabolic processes. Two sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are activated by high NAD(+) levels, a condition caused by low cellular energy status. By deacetylating a variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease.

Activation of Agrp neurons modulates memory-related cognitive processes in mice.

Hypothalamic Agrp neurons are critical regulators of food intake in adult mice. In addition to food intake, these neurons have been involved in other cognitive processes, such as the manifestation of stereotyped behaviors. Here, we evaluated the extent to which Agrp neurons modulate mouse behavior in spatial memory-related tasks. We found that activation of Agrp neurons did not affect spatial learning but altered behavioral flexibility using a modified version of the Barnes Maze task. Furthermore, using the Y-maze test to probe working memory, we found that chemogenetic activation of Agrp neurons reduced spontaneous alternation behavior mediated by the neuropeptide Y receptor-5 signaling. These findings suggest novel functional properties of Agrp neurons in memory-related cognitive processes.

Hunger-promoting hypothalamic neurons modulate effector and regulatory T-cell responses.

Whole-body energy metabolism is regulated by the hypothalamus and has an impact on diverse tissue functions. Here we show that selective knockdown of Sirtuin 1 Sirt1 in hypothalamic Agouti-related peptide-expressing neurons, which renders these cells less responsive to cues of low energy availability, significantly promotes CD4(+) T-cell activation by increasing production of T helper 1 and 17 proinflammatory cytokines via mediation of the sympathetic nervous system. These phenomena were associated with an impaired thymic generation of forkhead box P3 (FoxP3(+)) naturally occurring regulatory T cells and their reduced suppressive capacity in the periphery, which resulted in increased delayed-type hypersensitivity responses and autoimmune disease susceptibility in mice. These observations unmask a previously unsuspected role of hypothalamic feeding circuits in the regulation of adaptive immune response.

SqueakOut: Autoencoder-based segmentation of mouse ultrasonic vocalizations.

Mice emit ultrasonic vocalizations (USVs) that are important for social communication. Despite great advancements in tools to detect USVs from audio files in the recent years, highly accurate segmentation of USVs from spectrograms (i.e., removing noise) remains a significant challenge. Here, we present a new dataset of 12,954 annotated spectrograms explicitly labeled for mouse USV segmentation. Leveraging this dataset, we developed SqueakOut, a lightweight (4.6M parameters) fully convolutional autoencoder that achieves high accuracy in supervised segmentation of USVs from spectrograms, with a Dice score of 90.22. SqueakOut combines a MobileNetV2 backbone with skip connections and transposed convolutions to precisely segment USVs. Using stochastic data augmentation techniques and a hybrid loss function, SqueakOut learns robust segmentation across varying recording conditions. We evaluate SqueakOut's performance, demonstrating substantial improvements over existing methods like VocalMat (63.82 Dice score). The accurate USV segmentations enabled by SqueakOut will facilitate novel methods for vocalization classification and more accurate analysis of mouse communication. To promote further research, we release the annotated 12,954 spectrogram USV segmentation dataset and the SqueakOut implementation publicly.

Immune sensing of food allergens promotes aversive behaviour.

In addition to its canonical function in protecting from pathogens, the immune system can also promote behavioural alterations 1â€"3 . The scope and mechanisms of behavioural modifications by the immune system are not yet well understood. Using a mouse food allergy model, here we show that allergic sensitization drives antigen-specific behavioural aversion. Allergen ingestion activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus solitarius, parabrachial nucleus, and central amygdala. Food aversion requires IgE antibodies and mast cells but precedes the development of gut allergic inflammation. The ability of allergen-specific IgE and mast cells to promote aversion requires leukotrienes and growth and differentiation factor 15 (GDF15). In addition to allergen-induced aversion, we find that lipopolysaccharide-induced inflammation also resulted in IgE-dependent aversive behaviour. These findings thus point to antigen-specific behavioural modifications that likely evolved to promote niche selection to avoid unfavourable environments.

Preparatory neurons for building a nest.

Mothers build nests in anticipation of the delivery of their offspring. How the brain coordinates this behavior is unknown. Topilko et al. (2022) demonstrate that nest building in pregnant females relies on the activity of peptidergic neurons in the Edinger-Westphal nucleus.

Development of "Hunger Neurons" and the Unanticipated Relationship Between Energy Metabolism and Mother-Infant Interactions.

Over the course of a lifetime, the perinatal period plays an outsized role in the function of physiological systems. Here, we discuss how neurons that regulate energy metabolism contribute to the infant's relationship with the mother. We focus our discussion on Agrp neurons, which are located in the arcuate nucleus of the hypothalamus. These neurons heavily regulate energy metabolism. Because offspring transition from a period of dependence on the caregiver to independence, we discuss the importance of the caregiver-offspring relationship for the function of Agrp neurons. We present evidence that in the adult, Agrp neurons motivate the animal to eat, while in the neonate, they motivate the offspring to seek the proximity of the caregiver. We specifically highlight the peculiarities in the development of Agrp neurons and how they relate to the regulation of metabolism and behavior over the course of a lifetime. In sum, this review considers the unique insights that ontogenetic studies can offer toward our understanding of complex biological systems, such as the regulation of energy metabolism and mother-infant attachment.

Deficiency of the paternally inherited gene Magel2 alters the development of separation-induced vocalization and maternal behavior in mice.

The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited gene Magel2 in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than 1000 USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6-12 (P6-P12), a critical phase of mouse development that covers the peak of vocal behavior in pups. Our analyses show that Magel2 deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features mainly at P8. We also show that dams display altered behavior towards their own Magel2 deficient offspring at this age. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster than Magel2 deficient offspring. These results suggest that the loss of Magel2 impairs the expression of separation-induced vocalization in pups as well as maternal behavior at a specific age of postnatal development, both of which support the pups' growth and development.