Immunosensation: Neuroimmune Cross Talk in the Skin.

Classically, skin was considered a mere structural barrier protecting organisms from a diversity of environmental insults. In recent decades, the cutaneous immune system has become recognized as a complex immunologic barrier involved in both antimicrobial immunity and homeostatic processes like wound healing. To sense a variety of chemical, mechanical, and thermal stimuli, the skin harbors one of the most sophisticated sensory networks in the body. However, recent studies suggest that the cutaneous nervous system is highly integrated with the immune system to encode specific sensations into evolutionarily conserved protective behaviors. In addition to directly sensing pathogens, neurons employ novel neuroimmune mechanisms to provide host immunity. Therefore, given that sensation underlies various physiologies through increasingly complex reflex arcs, a much more dynamic picture is emerging of the skin as a truly systemic organ with highly coordinated physical, immunologic, and neural functions in barrier immunology.

Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Sneezing reflex is mediated by a peptidergic pathway from nose to brainstem.

Sneezing is a vital respiratory reflex frequently associated with allergic rhinitis and viral respiratory infections. However, its neural circuit remains largely unknown. A sneeze-evoking region was discovered in both cat and human brainstems, corresponding anatomically to the central recipient zone of nasal sensory neurons. Therefore, we hypothesized that a neuronal population postsynaptic to nasal sensory neurons mediates sneezing in this region. By screening major presynaptic neurotransmitters/neuropeptides released by nasal sensory neurons, we found that neuromedin B (NMB) peptide is essential for signaling sneezing. Ablation of NMB-sensitive postsynaptic neurons in the sneeze-evoking region or deficiency in NMB receptor abolished the sneezing reflex. Remarkably, NMB-sensitive neurons further project to the caudal ventral respiratory group (cVRG). Chemical activation of NMB-sensitive neurons elicits action potentials in cVRG neurons and leads to sneezing behavior. Our study delineates a peptidergic pathway mediating sneezing, providing molecular insights into the sneezing reflex arc.

A genetically tractable jellyfish model for systems and evolutionary neuroscience.

Jellyfish are radially symmetric organisms without a brain that arose more than 500 million years ago. They achieve organismal behaviors through coordinated interactions between autonomously functioning body parts. Jellyfish neurons have been studied electrophysiologically, but not at the systems level. We introduce Clytia hemisphaerica as a transparent, genetically tractable jellyfish model for systems and evolutionary neuroscience. We generate stable F1 transgenic lines for cell-type-specific conditional ablation and whole-organism GCaMP imaging. Using these tools and computational analyses, we find that an apparently diffuse network of RFamide-expressing umbrellar neurons is functionally subdivided into a series of spatially localized subassemblies whose synchronous activation controls directional food transfer from the tentacles to the mouth. These data reveal an unanticipated degree of structured neural organization in this species. Clytia affords a platform for systems-level studies of neural function, behavior, and evolution within a clade of marine organisms with growing ecological and economic importance.

A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Small-Molecule Agonists of Ae. aegypti Neuropeptide Y Receptor Block Mosquito Biting.

Female Aedes aegypti mosquitoes bite humans to obtain blood to develop their eggs. Remarkably, their strong attraction to humans is suppressed for days after the blood meal by an unknown mechanism. We investigated a role for neuropeptide Y (NPY)-related signaling in long-term behavioral suppression and discovered that drugs targeting human NPY receptors modulate mosquito host-seeking. In a screen of all 49 predicted Ae. aegypti peptide receptors, we identified NPY-like receptor 7 (NPYLR7) as the sole target of these drugs. To obtain small-molecule agonists selective for NPYLR7, we performed a high-throughput cell-based assay of 265,211 compounds and isolated six highly selective NPYLR7 agonists that inhibit mosquito attraction to humans. NPYLR7 CRISPR-Cas9 null mutants are defective in behavioral suppression and resistant to these drugs. Finally, we show that these drugs can inhibit biting and blood-feeding on a live host, suggesting a novel approach to control infectious disease transmission by controlling mosquito behavior. VIDEO ABSTRACT.

A GPCR-neuropeptide axis dampens hyperactive neutrophils by promoting an alternative-like polarization during bacterial infection.

The notion that neutrophils exist as a homogeneous population is being replaced with the knowledge that neutrophils adopt different functional states. Neutrophils can have a pro-inflammatory phenotype or an anti-inflammatory state, but how these states are regulated remains unclear. Here, we demonstrated that the neutrophil-expressed G-protein-coupled receptor (GPCR) Mrgpra1 is a negative regulator of neutrophil bactericidal functions. Mrgpra1-mediated signaling was driven by its ligand, neuropeptide FF (NPFF), which dictated the balance between pro- and anti-inflammatory programming. Specifically, the Mrgpra1-NPFF axis counter-regulated interferon (IFN) γ-mediated neutrophil polarization during acute lung infection by favoring an alternative-like polarization, suggesting that it may act to balance overzealous neutrophilic responses. Distinct, cross-regulated populations of neutrophils were the primary source of NPFF and IFNγ during infection. As a subset of neutrophils at steady state expressed NPFF, these findings could have broad implications in various infectious and inflammatory diseases. Therefore, a neutrophil-intrinsic pathway determines their cellular fate, function, and magnitude of infection.

A Neural Circuit for the Suppression of Pain by a Competing Need State.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

GABAergic signaling between enteric neurons and intestinal smooth muscle promotes innate immunity and gut defense in Caenorhabditis elegans.

The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-ß-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.

CGRP physiology, pharmacology, and therapeutic targets: migraine and beyond.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse physiological functions. Its two isoforms (α and ß) are widely expressed throughout the body in sensory neurons as well as in other cell types, such as motor neurons and neuroendocrine cells. CGRP acts via at least two G protein-coupled receptors that form unusual complexes with receptor activity-modifying proteins. These are the CGRP receptor and the AMY1 receptor; in rodents, additional receptors come into play. Although CGRP is known to produce many effects, the precise molecular identity of the receptor(s) that mediates CGRP effects is seldom clear. Despite the many enigmas still in CGRP biology, therapeutics that target the CGRP axis to treat or prevent migraine are a bench-to-bedside success story. This review provides a contextual background on the regulation and sites of CGRP expression and CGRP receptor pharmacology. The physiological actions of CGRP in the nervous system are discussed, along with updates on CGRP actions in the cardiovascular, pulmonary, gastrointestinal, immune, hematopoietic, and reproductive systems and metabolic effects of CGRP in muscle and adipose tissues. We cover how CGRP in these systems is associated with disease states, most notably migraine. In this context, we discuss how CGRP actions in both the peripheral and central nervous systems provide a basis for therapeutic targeting of CGRP in migraine. Finally, we highlight potentially fertile ground for the development of additional therapeutics and combinatorial strategies that could be designed to modulate CGRP signaling for migraine and other diseases.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Sympathetic Neuronal Activation Triggers Myeloid Progenitor Proliferation and Differentiation.

There is a growing body of research on the neural control of immunity and inflammation. However, it is not known whether the nervous system can regulate the production of inflammatory myeloid cells from hematopoietic progenitor cells in disease conditions. Myeloid cell numbers in diabetic patients were strongly correlated with plasma concentrations of norepinephrine, suggesting the role of sympathetic neuronal activation in myeloid cell production. The spleens of diabetic patients and mice contained higher numbers of tyrosine hydroxylase (TH)-expressing leukocytes that produced catecholamines. Granulocyte macrophage progenitors (GMPs) expressed the ß2 adrenergic receptor, a target of catecholamines. Ablation of splenic sympathetic neuronal signaling using surgical, chemical, and genetic approaches diminished GMP proliferation and myeloid cell development. Finally, mice lacking TH-producing leukocytes had reduced GMP proliferation, resulting in diminished myelopoiesis. Taken together, our study demonstrates that catecholamines produced by leukocytes and sympathetic nerve termini promote GMP proliferation and myeloid cell development.

Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila.

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Social stress-induced serotonin dysfunction activates spexin in male Nile tilapia (Oreochromis Niloticus).

Social disturbance in interpersonal relationships is the primary source of stress in humans. Spexin (SPX, SPX1a in cichlid), an evolutionarily conserved neuropeptide with diverse physiological functions, is up-regulated in the brain during chronic social defeat stress in teleost. On the other hand, repeated exposure to social stress can lead to dysregulation of the monoaminergic system and increase the vulnerability of developing depression. Since dysfunction of the serotonin (5-hydroxytryptamine, 5-HT) system is associated with social stress and the pathophysiology of depression, the present study investigated the regulatory relationship between the central 5-HT system and SPX1a in the male teleost, Nile tilapia (Oreochromis niloticus). To identify stress factors that regulate SPX1a gene expression, cortisol, dexamethasone (DEX), and 5-HT were used to treat tilapia brain primary cultures. Our study shows cortisol and DEX treatment had no effect on SPX1a gene expression, but SPX1a gene expression was down-regulated following 5-HT treatment. Anatomical localization showed a close association between 5-HT immunoreactive projections and SPX1a neurons in the semicircular torus. In addition, 5-HT receptors (5-HT2B) were expressed in SPX1a neurons. SPX1a immunoreactive neurons and SPX1a gene expression were significantly increased in socially defeated tilapia. On the other hand, citalopram (antidepressant, 5-HT antagonist) treatment to socially defeated tilapia normalized SPX1a gene expression to control levels. Taken together, the present study shows that 5-HT is an upstream regulator of SPX1a and that the inhibited 5-HT activates SPX1a during social defeat.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Endogenous l- to d-amino acid residue isomerization modulates selectivity between distinct neuropeptide receptor family members.

The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.

Neuronal diversity of neuropeptide signaling, including galanin, in the mouse locus coeruleus.

The locus coeruleus (LC) is a small nucleus in the pons from which ascending and descending projections innervate major parts of the central nervous system. Its major transmitter is norepinephrine (NE). This system is evolutionarily conserved, including in humans, and its functions are associated with wakefulness and related to disorders, such as depression. Here, we performed single-cell ribonucleic acid-sequencing (RNA-seq) to subdivide neurons in the LC (24 clusters in total) into 3 NE, 17 glutamate, and 5 γ-aminobutyric acid (GABA) subtypes, and to chart their neuropeptide, cotransmitter, and receptor profiles. We found that NE neurons expressed at least 19 neuropeptide transcripts, notably galanin (Gal) but not Npy, and >30 neuropeptide receptors. Among the galanin receptors, Galr1 was expressed in ~19% of NE neurons, as was also confirmed by in situ hybridization. Unexpectedly, Galr1 was highly expressed in GABA neurons surrounding the NE ensemble. Patch-clamp electrophysiology and cell-type-specific Ca2+-imaging using GCaMP6s revealed that a GalR1 agonist inhibits up to ~35% of NE neurons. This effect is direct and does not rely on feed-forward GABA inhibition. Our results define a role for the galanin system in NE functions, and a conceptual framework for the action of many other peptides and their receptors.

Hedonic eating, obesity, and addiction result from increased neuropeptide Y in the nucleus accumbens during human brain evolution.

The nucleus accumbens (NAc) is central to motivation and action, exhibiting one of the highest densities of neuropeptide Y (NPY) in the brain. Within the NAc, NPY plays a role in reward and is involved in emotional behavior and in increasing alcohol and drug addiction and fat intake. Here, we examined NPY innervation and neurons of the NAc in humans and other anthropoid primates in order to determine whether there are differences among these various species that would correspond to behavioral or life history variables. We quantified NPY-immunoreactive axons and neurons in the NAc of 13 primate species, including humans, great apes, and monkeys. Our data show that the human brain is unique among primates in having denser NPY innervation within the NAc, as measured by axon length density to neuron density, even after accounting for brain size. Combined with our previous finding of increased dopaminergic innervation in the same region, our results suggest that the neurochemical profile of the human NAc appears to have rendered our species uniquely susceptible to neurophysiological conditions such as addiction. The increase in NPY specific to the NAc may represent an adaptation that favors fat intake and contributes to an increased vulnerability to eating disorders, obesity, as well as alcohol and drug dependence. Along with our findings for dopamine, these deeply rooted structural attributes of the human brain are likely to have emerged early in the human clade, laying the groundwork for later brain expansion and the development of cognitive and behavioral specializations.