MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

Modulation of host immunity by sensory neurons.

Recent studies have uncovered a new role for sensory neurons in influencing mammalian host immunity, challenging conventional notions of the nervous and immune systems as separate entities. In this review we delve into this groundbreaking paradigm of neuroimmunology and discuss recent scientific evidence for the impact of sensory neurons on host responses against a wide range of pathogens and diseases, encompassing microbial infections and cancers. These valuable insights enhance our understanding of the interactions between the nervous and immune systems, and also pave the way for developing candidate innovative therapeutic interventions in immune-mediated diseases highlighting the importance of this interdisciplinary research field.

Sensory neuronal control of skin barrier immunity.

Peripheral sensory neurons recognize diverse noxious stimuli, including microbial products and allergens traditionally thought to be targets of the mammalian immune system. Activation of sensory neurons by these stimuli leads to pain and itch responses as well as the release of neuropeptides that interact with their cognate receptors expressed on immune cells, such as dendritic cells (DCs). Neuronal control of immune cell function through neuropeptide release not only affects local inflammatory responses but can impact adaptive immune responses through downstream effects on T cell priming. Numerous neuropeptide receptors are expressed by DCs but only a few have been characterized, presenting opportunities for further investigation of the pathways by which cutaneous neuroimmune interactions modulate host immunity.

Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

Satellite Glial Cells in Human Disease.

Satellite glial cells (SGCs) are the main type of glial cells in sensory ganglia. Animal studies have shown that these cells play essential roles in both normal and disease states. In a large number of pain models, SGCs were activated and contributed to the pain behavior. Much less is known about SGCs in humans, but there is emerging recognition that SGCs in humans are altered in a variety of clinical states. The available data show that human SGCs share some essential features with SGCs in rodents, but many differences do exist. SGCs in DRG from patients suffering from common painful diseases, such as rheumatoid arthritis and fibromyalgia, may contribute to the pain phenotype. It was found that immunoglobulins G (IgG) from fibromyalgia patients can induce pain-like behavior in mice. Moreover, these IgGs bind preferentially to SGCs and activate them, which can sensitize the sensory neurons, causing nociception. In other human diseases, the evidence is not as direct as in fibromyalgia, but it has been found that an antibody from a patient with rheumatoid arthritis binds to mouse SGCs, which leads to the release of pronociceptive factors from them. Herpes zoster is another painful disease, and it appears that the zoster virus resides in SGCs, which acquire an abnormal morphology and may participate in the infection and pain generation. More work needs to be undertaken on SGCs in humans, and this review points to several promising avenues for better understanding disease mechanisms and developing effective pain therapies.

Mechanisms of Action of Dorsal Root Ganglion Stimulation.

The dorsal root ganglion (DRG) serves as a pivotal site for managing chronic pain through dorsal root ganglion stimulation (DRG-S). In recent years, the DRG-S has emerged as an attractive modality in the armamentarium of neuromodulation therapy due to its accessibility and efficacy in alleviating chronic pain refractory to conventional treatments. Despite its therapeutic advantages, the precise mechanisms underlying DRG-S-induced analgesia remain elusive, attributed in part to the diverse sensory neuron population within the DRG and its modulation of both peripheral and central sensory processing pathways. Emerging evidence suggests that DRG-S may alleviate pain by several mechanisms, including the reduction of nociceptive signals at the T-junction of sensory neurons, modulation of pain gating pathways within the dorsal horn, and regulation of neuronal excitability within the DRG itself. However, elucidating the full extent of DRG-S mechanisms necessitates further exploration, particularly regarding its supraspinal effects and its interactions with cognitive and affective networks. Understanding these mechanisms is crucial for optimizing neurostimulation technologies and improving clinical outcomes of DRG-S for chronic pain management. This review provides a comprehensive overview of the DRG anatomy, mechanisms of action of the DRG-S, and its significance in neuromodulation therapy for chronic pain.

Mechanisms underlying the gut-brain communication: How enterochromaffin (EC) cells activate vagal afferent nerve endings in the small intestine.

How the gastrointestinal tract communicates with the brain, via sensory nerves, is of significant interest for our understanding of human health and disease. Enterochromaffin (EC) cells in the gut mucosa release a variety of neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT) in the body. How 5-HT and other substances released from EC cells activate sensory nerve endings in the gut wall remains a major unresolved mystery. We used in vivo anterograde tracing from nodose ganglia to determine the spatial relationship between 5-HT synthesizing and peptide-YY (PYY)-synthesizing EC cells and their proximity to vagal afferent nerve endings that project to the mucosa of mouse small intestine. The shortest mean distances between single 5-HT- and PYY-synthesizing EC cells and the nearest vagal afferent nerve endings in the mucosa were 33.1 ± 14.4 µm (n = 56; N = 6) and 70.3 ± 32.3 µm (n = 16; N = 6). No morphological evidence was found to suggest that 5-HT- or PYY-containing EC cells form close morphological associations with vagal afferents endings, or varicose axons of passage. The large distances between EC cells and vagal afferent endings are many hundreds of times greater than those known to underlie synaptic transmission in the nervous system (typically 10-15 nm). Taken together, the findings lead to the inescapable conclusion that communication between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa of the mouse small intestinal occurs in a paracrine fashion, via diffusion. New and Noteworthy None of the findings here are consistent with a view that close physical contacts occur between 5-HT-containing EC cells and vagal afferent nerve endings in mouse small intestine. Rather, the findings suggest that gut-brain communication between EC cells and vagal afferent endings occurs via passive diffusion. The morphological data presented do not support the view that EC cells are physically close enough to vagal afferent endings to communicate via fast synaptic transmission.

Neuroendocrine cells initiate protective upper airway reflexes.

Airway neuroendocrine (NE) cells have been proposed to serve as specialized sensory epithelial cells that modulate respiratory behavior by communicating with nearby nerve endings. However, their functional properties and physiological roles in the healthy lung, trachea, and larynx remain largely unknown. In this work, we show that murine NE cells in these compartments have distinct biophysical properties but share sensitivity to two commonly aspirated noxious stimuli, water and acid. Moreover, we found that tracheal and laryngeal NE cells protect the airways by releasing adenosine 5'-triphosphate (ATP) to activate purinoreceptive sensory neurons that initiate swallowing and expiratory reflexes. Our work uncovers the broad molecular and biophysical diversity of NE cells across the airways and reveals mechanisms by which these specialized excitable cells serve as sentinels for activating protective responses.

Interleukin-1α links peripheral CaV2.2 channel activation to rapid adaptive increases in heat sensitivity in skin.

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated CaV2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1α (IL-1α), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1α was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1α levels were also dependent on peripheral CaV2.2 channel activity. Neutralizing IL-1α in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1α enhances behavioral responses to stimuli and, in culture, IL-1α enhances the responsiveness of Trpv1-expressing sensory neurons. Together, our data suggest that IL-1α is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin.

Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

Type 2 vomeronasal receptor-A4 subfamily: Potential predator sensors in mice.

Sensory signals detected by olfactory sensory organs are critical regulators of animal behavior. An accessory olfactory organ, the vomeronasal organ, detects cues from other animals and plays a pivotal role in intra- and inter-species interactions in mice. However, how ethologically relevant cues control mouse behavior through approximately 350 vomeronasal sensory receptor proteins largely remains elusive. The type 2 vomeronasal receptor-A4 (V2R-A4) subfamily members have been repeatedly detected from vomeronasal sensory neurons responsive to predator cues, suggesting a potential role of this receptor subfamily as a sensor for predators. This review focuses on this intriguing subfamily, delving into its receptor functions and genetic characteristics.

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

TMC7 functions as a suppressor of Piezo2 in primary sensory neurons blunting peripheral mechanotransduction.

The transmembrane channel-like (TMC) protein family comprises eight members, with TMC1 and TMC2 being extensively studied. This study demonstrates substantial co-expression of TMC7 with the mechanosensitive channel Piezo2 in somatosensory neurons. Genetic deletion of TMC7 in primary sensory ganglia neurons in vivo enhances sensitivity in both physiological and pathological mechanosensory transduction. This deletion leads to an increase in proportion of rapidly adapting (RA) currents conducted by Piezo2 in dorsal root ganglion (DRG) neurons and accelerates RA deactivation kinetics. In HEK293 cells expressing both proteins, TMC7 significantly suppresses the current amplitudes of co-expressed Piezo2. Our findings reveal that TMC7 and Piezo2 exhibit physical interactions, and both proteins also physically interact with cytoskeletal ß-actin. We hypothesize that TMC7 functions as an inhibitory modulator of Piezo2 in DRG neurons, either through direct inhibition or by disrupting the transmission of mechanical forces from the cytoskeleton to the channel.

The nociceptive activity of peripheral sensory neurons is modulated by the neuronal membrane proteasome.

Proteasomes are critical for peripheral nervous system (PNS) function. Here, we investigate mammalian PNS proteasomes and reveal the presence of the neuronal membrane proteasome (NMP). We show that specific inhibition of the NMP on distal nerve fibers innervating the mouse hind paw leads to reduction in mechanical and pain sensitivity. Through investigating PNS NMPs, we demonstrate their presence on the somata and proximal and distal axons of a subset of dorsal root ganglion (DRG) neurons. Single-cell RNA sequencing experiments reveal that the NMP-expressing DRGs are primarily MrgprA3+ and Cysltr2+. NMP inhibition in DRG cultures leads to cell-autonomous and non-cell-autonomous changes in Ca2+ signaling induced by KCl depolarization, αß-meATP, or the pruritogen histamine. Taken together, these data support a model whereby NMPs are expressed on a subset of somatosensory DRGs to modulate signaling between neurons of distinct sensory modalities and indicate the NMP as a potential target for controlling pain.

Sensory neurons: An integrated component of innate immunity.

The sensory nervous system possesses the ability to integrate exogenous threats and endogenous signals to mediate downstream effector functions. Sensory neurons have been shown to activate or suppress host defense and immunity against pathogens, depending on the tissue and disease state. Through this lens, pro- and anti-inflammatory neuroimmune effector functions can be interpreted as evolutionary adaptations by host or pathogen. Here, we discuss recent and impactful examples of neuroimmune circuitry that regulate tissue homeostasis, autoinflammation, and host defense. Apparently paradoxical or conflicting reports in the literature also highlight the complexity of neuroimmune interactions that may depend on tissue- and microbe-specific cues. These findings expand our understanding of the nuanced mechanisms and the greater context of sensory neurons in innate immunity.

PDX1, a transcription factor essential for organ differentiation, regulates SERCA-dependent Ca2+ homeostasis in sensory neurons.

Pancreatic and duodenal homeobox 1 (PDX1) is a transcription factor required for the development and differentiation of the pancreas. Previous studies indicated that PDX1 expression was restricted to the gastrointestinal tract. Using a cre-dependent reporter, we observed PDX1-dependent expression of tdtomato (PDX1-tom) in a subpopulation of sensory nerves. Many of these PDX1-tom afferents expressed the neurofilament 200 protein and projected to the skin. Tdtomato-labeled terminals were associated with hair follicles in the form of longitudinal and circumferential lanceolate endings suggesting a role in tactile and proprioceptive perception. To begin to examine the functional significance of PDX1 in afferents, we used Fura-2 imaging to examine calcium (Ca2+) handling under naïve and nerve injury conditions. Neuropathic injury is associated with increased intracellular Ca2+ signaling that in part results from dysregulation of the sarco/endoplasmic reticulum calcium transport ATPase (SERCA). Here we demonstrate that under naïve conditions, PDX1 regulates expression of the SERCA2B isoform in sensory neurons. In response to infraorbital nerve injury, a significant reduction of PDX1 and SERCA2B expression and dysregulation of Ca2+ handling occurs in PDX1-tom trigeminal ganglia neurons. The identification of PDX1 expression in the somatosensory system and its regulation of SERCA2B and Ca2+ handling provide a new mechanism to explain pathological changes in primary afferents that may contribute to pain associated with nerve injury.

Electrophysiological Properties of Proprioception-Related Neurons in the Intermediate Thoracolumbar Spinal Cord.

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

Neuronal NPR-15 modulates molecular and behavioral immune responses via the amphid sensory neuron-intestinal axis in C. elegans.

The survival of hosts during infections relies on their ability to mount effective molecular and behavioral immune responses. Despite extensive research on these defense strategies in various species, including the model organism Caenorhabditis elegans, the neural mechanisms underlying their interaction remain poorly understood. Previous studies have highlighted the role of neural G-protein-coupled receptors (GPCRs) in regulating both immunity and pathogen avoidance, which is particularly dependent on aerotaxis. To address this knowledge gap, we conducted a screen of mutants in neuropeptide receptor family genes. We found that loss-of-function mutations in npr-15 activated immunity while suppressing pathogen avoidance behavior. Through further analysis, NPR-15 was found to regulate immunity by modulating the activity of key transcription factors, namely GATA/ELT-2 and TFEB/HLH-30. Surprisingly, the lack of pathogen avoidance of npr-15 mutant animals was not influenced by oxygen levels. Moreover, our studies revealed that the amphid sensory neuron ASJ is involved in mediating the immune and behavioral responses orchestrated by NPR-15. Additionally, NPR-15 was found to regulate avoidance behavior via the TRPM (transient receptor potential melastatin) gene, GON-2, which may sense the intestinal distension caused by bacterial colonization to elicit pathogen avoidance. Our study contributes to a broader understanding of host defense strategies and mechanisms underlining the interaction between molecular and behavioral immune responses.

C. elegans males optimize mate-preference decisions via sex-specific responses to multimodal sensory cues.

For sexually reproducing animals, selecting optimal mates is important for maximizing reproductive fitness. In the nematode C. elegans, populations reproduce largely by hermaphrodite self-fertilization, but the cross-fertilization of hermaphrodites by males also occurs. Males' ability to recognize hermaphrodites involves several sensory cues, but an integrated view of the ways males use these cues in their native context to assess characteristics of potential mates has been elusive. Here, we examine the mate-preference behavior of C. elegans males evoked by natively produced cues. We find that males use a combination of volatile sex pheromones (VSPs), ascaroside sex pheromones, surface-associated cues, and other signals to assess multiple features of potential mates. Specific aspects of mate preference are communicated by distinct signals: developmental stage and sex are signaled by ascaroside pheromones and surface cues, whereas the presence of a self-sperm-depleted hermaphrodite is likely signaled by VSPs. Furthermore, males prefer to interact with virgin over mated, and well-fed over food-deprived, hermaphrodites; these preferences are likely adaptive and are also mediated by ascarosides and other cues. Sex-typical mate-preference behavior depends on the sexual state of the nervous system, such that pan-neuronal genetic masculinization in hermaphrodites generates male-typical social behavior. We also identify an unexpected role for the sex-shared ASH sensory neurons in male attraction to ascaroside sex pheromones. Our findings lead to an integrated view in which the distinct physical properties of various mate-preference cues guide a flexible, stepwise behavioral program by which males assess multiple features of potential mates to optimize mate preference.