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SummarySocial grouping increases survival in many species, including humans1,2. By contrast, social isolation generates an aversive state (loneliness) that motivates social seeking and heightens social interaction upon reunion3-5. The observed rebound in social interaction triggered by isolation suggests a homeostatic process underlying the control of social drive, similar to that observed for physiological needs such as hunger, thirst or sleep3,6. In this study, we assessed social responses in multiple mouse strains and identified the FVB/NJ line as exquisitely sensitive to social isolation. Using FVB/NJ mice, we uncovered two previously uncharacterized neuronal populations in the hypothalamic preoptic nucleus that are activated during social isolation and social rebound and that orchestrate the behavior display of social need and social satiety, respectively. We identified direct connectivity between these two populations of opposite function and with brain areas associated with social behavior, emotional state, reward, and physiological needs, and showed that animals require touch to assess the presence of others and fulfill their social need, thus revealing a brain-wide neural system underlying social homeostasis. These findings offer mechanistic insight into the nature and function of circuits controlling instinctive social need and for the understanding of healthy and diseased brain states associated with social context.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we show that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription defines spatially constrained OR repertoires, among which one OR allele may be selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization preferentially eliminate from this “privileged” repertoire ORs with more dorsal expression destinations, which are ectopically transcribed in neuronal progenitors throughout the olfactory epithelium. Our experiments identify early transcription as an “epigenetic” contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Injury of the tooth pulp is excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both the clinic and preclinical rodent models. Easily quantifiable behavioral assessment in the mouse orofacial area remains a major bottleneck in uncovering molecular mechanisms that govern inflammatory pain in the tooth. In this study we sought to address this problem using the Mouse Grimace Scale and a novel approach to the application of mechanical Von Frey hair stimuli. We use a dental pulp injury model that exposes the pulp to the outside environment, a procedure we have previously shown produces inflammation. Using RNAscope technology, we demonstrate an upregulation of genes that contribute to the pain state in the trigeminal ganglia of injured mice. We found that mice with dental pulp injury have greater Mouse Grimace Scores than sham within 24 hours of injury, suggestive of spontaneous pain. We developed a scoring system of mouse refusal to determine thresholds for mechanical stimulation of the face with Von Frey filaments. This method revealed that mice with a unilateral dental injury develop bilateral mechanical allodynia that is delayed relative to the onset of spontaneous pain. This work demonstrates that tooth pain can be quantified in freely behaving mice using approaches common for other types of pain assessment. Harnessing these assays in the orofacial area during gene manipulation should assist in uncovering mechanisms for tooth pulp inflammatory pain and other forms of trigeminal pain.

Breathing can be heavily influenced by pain or internal emotional states, but the neural circuitry underlying this tight coordination is unknown. Here we report that Oprm1 (μ-opioid receptor)-expressing neurons in the lateral parabrachial nucleus (PBL) are crucial for coordinating breathing with affective pain in mice. Individual PBL(Oprm1) neuronal activity synchronizes with breathing rhythm and responds to noxious stimuli. Manipulating PBL(Oprm1) activity directly changes breathing rate, affective pain perception, and anxiety. Furthermore, PBL(Oprm1) neurons constitute two distinct subpopulations in a “core-shell” configuration that divergently projects to the forebrain and hindbrain. Through non-overlapping projections to the central amygdala and pre-Bötzinger complex, these two subpopulations differentially regulate breathing, affective pain, and negative emotions. Moreover, these subsets form recurrent excitatory networks through reciprocal glutamatergic projections. Together, our data define the divergent parabrachial opioidergic circuits as a common neural substrate that coordinates breathing with various sensations and behaviors such as pain and emotional processing.

Parental history of opioid exposure is seldom considered when prescribing opioids for pain relief. To explore whether parental opioid exposure may affect sensitivity to morphine in offspring, we developed a “rat pain scale” with high-speed imaging, machine learning, and mathematical modeling in a multigenerational model of paternal morphine self-administration. We find that the most commonly used tool to measure mechanical sensitivity in rodents, the von Frey hair, is not painful in rats during baseline conditions. We also find that male progeny of morphine-treated sires had no baseline changes in mechanical pain sensitivity but were more sensitive to the pain-relieving effects of morphine. Using RNA sequencing across pain-relevant brain regions, we identify gene expression changes within the regulator of G protein signaling family of proteins that may underlie this multigenerational phenotype. Together, this rat pain scale revealed that paternal opioid exposure increases sensitivity to morphine’s pain-relieving effects in male offspring.

Objectively measuring and interpreting an animal's sensory experience remains a challenging task. This is particularly true when using preclinical rodent models to study pain mechanisms and screen for potential new pain treatment reagents. How to determine their pain states in a precise and unbiased manner is a hurdle that the field will need to overcome. Here, we describe our efforts to measure mouse somatosensory reflexive behaviors with greatly improved precision by high-speed video imaging. We describe how coupling sub-second ethograms of reflexive behaviors with a statistical reduction method and supervised machine learning can be used to create a more objective quantitative mouse "pain scale." Our goal is to provide the readers with a protocol of how to integrate some of the new tools described here with currently used mechanical somatosensory assays, while discussing the advantages and limitations of this new approach.

Social touch can act as a stress buffer, reducing behavioral and physiological responses to stressful scenarios. However, skin-brain touch pathways that promote stress resilience remain unknown. Here, we show that mice with an early life genetic ablation of Mrgprb4-lineage touch neurons display stress vulnerability behaviors in adulthood. Chemogenetic activation of these touch neurons reduced corticosterone levels under mild acute stress conditions. In addition, whole-brain c-Fos activity mapping while chemogenetically turning on these neurons uncovered differential neural activity patterns in brain areas relevant to somatosensation, reward, and affect. To gain mechanistic insight into this skin-brain touch pathway for stress susceptibility, we used multi-circuit neurophysiological recordings across seven brain regions at baseline and after stress in mice that had Mrgprb4-lineage touch neurons ablated in early life. Interestingly, the Mrgprb4-lineage neuron-ablated mice have alterations in local field potential phase directionality and power in the theta frequencies in mesolimbic reward regions, which may underlie our observed stress susceptibility phenotype. Together, these studies revealed that sensory neurons in the skin engage networks across the brain to promote stress resilience.

Pleasurable touch during social behavior is the key to building familial bonds and meaningful connections. One form of social touch occurs during sexual encounters. Although sexual behavior is initiated in part by touch, and touch is ongoing throughout copulation, the identity and role of sensory neurons that transduce sexual touch remain unknown. A population of sensory neurons labeled by the G-protein coupled receptor Mrgprb4 detect stroking touch in mice, however, these neurons have never been implicated in any natural social behaviors. Here, we study the social relevance of Mrgprb4-lineage neurons by genetically engineering mice to allow activation or ablation of this population and reveal that these neurons are required for sexual receptivity and sufficient to induce dopamine release in the brain. Even in social isolation, optogenetic stimulation of Mrgprb4-lineage neurons through the back skin is sufficient to induce a conditioned place preference and a striking dorsiflexion resembling the lordotic copulatory posture in females. In the absence of Mrgprb4-lineage neurons, female mice no longer find male mounts rewarding: sexual receptivity is supplanted by aggression and a coincident decline in dopaminergic release in the mesolimbic reward pathway. In addition to sexual behavior, Mrgprb4-lineage neurons are also required for social postures induced by female-to-female back touch. Together, these findings establish that Mrgprb4-lineage neurons are the first neurons of a skin-to-brain circuit encoding the rewarding quality of social touch.

Parental exposure to drugs of abuse such as opioids can have profound and long-lasting effects on reward processing and drug sensitivity across generations. However, little is known about the impact of long-term paternal exposure to morphine on offspring sensitivity to morphine-derived antinociception during painful experiences. To address this question, we constructed a rat pain scale at millisecond timescales to measure mechanical nociception in a multigenerational morphine exposure paradigm. Surprisingly, while developing the pain scale, we found that von Frey hair filaments (VFHs), the most common stimuli used in pain research, are not painful to rats and morphine did not change the touch-like responses elicited by VFHs. We next deployed this novel pain scale to determine whether chronic morphine exposure in sires impacted pain sensitivity in the next generation. Offspring produced from a cross of morphine-treated sires and drug-naïve dams, did not show any baseline changes in sensitivity to mechanical nociception. However, morphine-sired male progeny displayed a higher sensitivity to the antinociceptive properties of morphine, as measured by our pain scale. These findings demonstrate that long-term paternal exposure to morphine increases sensitivity to morphine-derived analgesia in the subsequent generation.

Social touch-the affiliative skin-to-skin contact between individuals-can rapidly evoke emotions of comfort, pleasure, or calm, and is essential for mental and physical well-being. Physical isolation from social support can be devastating. During the COVID-19 pandemic, we observed a global increase in suicidal ideation, anxiety, domestic violence, and worsening of pre-existing physical conditions, alerting society to our need to understand the neurobiology of social touch and how it promotes normal health. Gaining a mechanistic understanding of how sensory neuron stimulation induces pleasure, calm, and analgesia may reveal untapped therapeutic targets in the periphery for treatment of anxiety and depression, as well as social disorders and traumas in which social touch becomes aversive. Bridging the gap between stimulation in the skin and positive affect in the brain-especially during naturally occurring social touch behaviors-remains a challenge to the field. However, with advances in mouse genetics, behavioral quantification, and brain imaging approaches to measure neuronal firing and neurochemical release, completing this mechanistic picture may be on the horizon. Here, we summarize some exciting new findings about social touch in mammals, emphasizing both the peripheral and central nervous systems, with attempts to bridge the gap between external stimulation and internal representations in the brain.

Objective and automatic measurement of pain in mice remains a barrier for discovery in neuroscience. Here, we capture paw kinematics during pain behavior in mice with high-speed videography and automated paw tracking with machine and deep learning approaches. Our statistical software platform, PAWS (Pain Assessment at Withdrawal Speeds), uses a univariate projection of paw position over time to automatically quantify seven behavioral features that are combined into a single, univariate pain score. Automated paw tracking combined with PAWS reveals a behaviorally divergent mouse strain that displays hypersensitivity to mechanical stimuli. To demonstrate the efficacy of PAWS for detecting spinally versus centrally mediated behavioral responses, we chemogenetically activated nociceptive neurons in the amygdala, which further separated the pain-related behavioral features and the resulting pain score. Taken together, this automated pain quantification approach will increase objectivity in collecting rigorous behavioral data, and it is compatible with other neural circuit dissection tools for determining the mouse pain state.

Objective and automatic measurement of pain in mice remains a barrier for discovery in both basic and translational neuroscience. Here we capture rapid paw kinematics during pain behavior in mice with high-speed videography and automated paw tracking with machine and deep learning approaches. Our statistical software platform, PAWS (Pain Assessment at Withdrawal Speeds), uses a univariate projection of paw position over time to automatically quantify fast paw dynamics at the onset of paw withdrawal and also lingering pain-related behaviors such as paw guarding and shaking. Applied to innocuous and noxious stimuli across six inbred mouse strains, a linear discriminant analysis reveals a two-dimensional subspace that separates painful from non-painful stimuli on one axis, and further distinguishes the severity of pain on the second axis. Automated paw tracking combined with PAWS reveals behaviorally-divergent mouse strains that display hypo- and hyper-sensitivity to mechanical stimuli. To demonstrate the efficacy of PAWS for detecting hypersensitivity to noxious stimuli, we chemogenetically activated pain-aversion neurons in the amygdala, which further separated the behavioral representation of pain-related behaviors along a low-dimensional path. Taken together, this automated pain quantification approach should increase the ease and objectivity of collecting rigorous behavioral data, and it is compatible with other neural circuit dissection tools for determining the mouse pain state.

Purpose of Review This review aims to summarize the current body of behavioral, physiological, and molecular knowledge concerning tactile sensitivity in autism spectrum disorder (ASD), with a focus on recent studies utilizing rodent models. Recent Findings Mice with mutations in the ASD-related genes, Shank3, Fmr1, UBE3A, and Mecp2, display tactile abnormalities. Some of these abnormalities appear to be caused by mutation-related changes in the PNS, as opposed to changes in the processing of touch stimuli in the CNS, as previously thought. There is also growing evidence suggesting that peripheral mechanisms may contribute to some of the core symptoms and common comorbidities of ASD. Researchers are therefore beginning to assess the therapeutic potential of targeting the PNS in treating some of the core symptoms of ASD. Summary Sensory abnormalities are common in rodent models of ASD. There is growing evidence that sensory hypersensitivity, especially tactile sensitivity, may contribute to social deficits and other autism-related behaviors.

The gate control theory (GCT) of pain proposes that pain- and touch-sensing neurons antagonize each other through spinal cord dorsal horn (DH) gating neurons. However, the exact neural circuits underlying the GCT remain largely elusive. Here, we identified a new population of deep layer DH (dDH) inhibitory interneurons that express the receptor tyrosine kinase Ret neonatally. These early RET+ dDH neurons receive excitatory as well as polysynaptic inhibitory inputs from touch- and/or pain-sensing afferents. In addition, they negatively regulate DH pain and touch pathways through both pre- and postsynaptic inhibition. Finally, specific ablation of early RET+ dDH neurons increases basal and chronic pain, whereas their acute activation reduces basal pain perception and relieves inflammatory and neuropathic pain. Taken together, our findings uncover a novel spinal circuit that mediates crosstalk between touch and pain pathways and suggest that some early RET+ dDH neurons could function as pain "gating" neurons.

Odorant receptor (OR) gene choice in mammals is a paradigmatic example of monogenic and monoallelic transcriptional selection, in which each olfactory sensory neuron (OSN) chooses to express one OR allele from over 1,000 encoded in the genome [1-3]. This process, critical for generation of the circuit from nose to brain [4-6], is thought to occur in two steps: a slow initial phase that randomly activates a single OR allele, followed by a rapid feedback that halts subsequent expression [7-14]. Inherent in this model is a finite failure rate wherein multiple OR alleles may be activated prior to feedback suppression [15, 16]. Confronted with more than one receptor, the neuron would need to activate a refinement mechanism to eliminate multigenic OR expression and resolve unique neuronal identity [16], critical to the generation of the circuit from nose to olfactory bulb. Here we used a genetic approach in mice to reveal a new facet of OR regulation that corrects adventitious activation of multiple OR alleles, restoring monogenic OR expression and unique neuronal identity. Using the tetM71tg model system, in which the M71 OR is expressed in >95% of mature OSNs and potently suppresses the expression of the endogenous OR repertoire [10], we provide clear evidence of a post-selection refinement (PSR) process that winnows down the number of ORs. We further demonstrate that PSR efficiency is linked to OR expression level, suggesting an underlying competitive process and shedding light on OR gene switching and the fundamental mechanism of singular OR choice.

Rodents are often used for studying chronic pain mechanisms and developing new pain therapeutics, but objectively determining the animal’s pain state is a major challenge. To improve the precision of using reflexive withdrawal behaviors for interpreting the mouse pain state, we adopted high-speed videography to capture sub-second movement features of mice upon hind paw stimulation. We identified several parameters that are significantly different between behaviors evoked by innocuous and noxious stimuli, and combined them to map the mouse pain state through statistical modeling and machine learning. To test the utility of this approach, we determined the pain state triggered by von Frey hairs (VFHs) and optogenetic activation of two nociceptor populations. Our method reliably assesses the “pain-like” probability for each mouse paw withdrawal reflex under all scenarios, highlighting the improved precision of using this high resolution behavior-centered composite methodology to determine the mouse pain state from reflexive withdrawal assays.

The human distal limbs have a high spatial acuity for noxious stimuli but a low density of pain-sensing neurites. To elucidate mechanisms underlying the ‘pain fovea’, we sparsely traced non-peptidergic nociceptors across the body using a newly generatedMrgprDCreERT2mouse line. We found that mouse plantar paw skin also has a low density of MrgprD+neurites, and individual arbors in different locations are comparable in size. Surprisingly, the central arbors of plantar paw and trunk innervating nociceptors have distinct morphologies in the spinal cord. This regional difference is well correlated with a heightened signal transmission for plantar paw circuits, as revealed by both spinal cord slice recordings and behavior assays. Taken together, our results elucidate a novel somatotopic functional organization of the mammalian pain system and suggest that regional central arbor structure could facilitate the magnification of plantar paw regions to contribute to the ‘pain fovea’.

SUMMARY Rodents are the main model systems for pain research, but determining their pain state is challenging. To develop an objective method to assess pain sensation in mice, we adopt high-speed videography to capture sub-second behavioral features following hind paw stimulation with both noxious and innocuous stimuli and identify several differentiating parameters indicating the affective and reflexive aspects of nociception. Using statistical modeling and machine learning, we integrate these parameters into a single index and create a “mouse pain scale,” which allows us to assess pain sensation in a graded manner for each withdrawal. We demonstrate the utility of this method by determining sensations triggered by three different von Frey hairs and optogenetic activation of two different nociceptor populations. Our behavior-based “pain scale” approach will help improve the rigor and reproducibility of using withdrawal reflex assays to assess pain sensation in mice., Graphical Abstract, In Brief Abdus-Saboor et al. develop a behavior-centered “mouse pain scale” using high-speed videography, statistical modeling, and machine learning. With this method, they assess the sensation induced by noxious, innocuous, and optogenetic stimuli. This method will improve the reliability of using the mouse hind paw withdrawal to measure pain.

Cells perform wide varieties of functions that are facilitated, in part, by adopting unique shapes. Many of the genes and pathways that promote cell fate specification have been elucidated. However, relatively few transcription factors have been identified that promote shape acquisition after fate specification. Here we show that the Nkx5/HMX homeodomain protein MLS-2 is required for cellular elongation and shape maintenance of two tubular epithelial cells in the C. elegans excretory system, the duct and pore cells. The Nkx5/HMX family is highly conserved from sea urchins to humans, with known roles in neuronal and glial development. MLS-2 is expressed in the duct and pore, and defects in mls-2 mutants first arise when the duct and pore normally adopt unique shapes. MLS-2 cooperates with the EGF-Ras-ERK pathway to turn on the LIN-48/Ovo transcription factor in the duct cell during morphogenesis. These results reveal a novel interaction between the Nkx5/HMX family and the EGF-Ras pathway and implicate a transcription factor, MLS-2, as a regulator of cell shape.

Receptor tyrosine kinases and Notch are crucial for tube formation and branching morphogenesis in many systems, but the specific cellular processes that require signaling are poorly understood. Here we describe sequential roles for Notch and Epidermal growth factor (EGF)-Ras-ERK signaling in the development of epithelial tube cells in the C. elegans excretory (renal-like) organ. This simple organ consists of three tandemly connected unicellular tubes: the excretory canal cell, duct and G1 pore. lin-12 and glp-1/Notch are required to generate the canal cell, which is a source of LIN-3/EGF ligand and physically attaches to the duct during de novo epithelialization and tubulogenesis. Canal cell asymmetry and let-60/Ras signaling influence which of two equivalent precursors will attach to the canal cell. Ras then specifies duct identity, inducing auto-fusion and a permanent epithelial character; the remaining precursor becomes the G1 pore, which eventually loses epithelial character and withdraws from the organ to become a neuroblast. Ras continues to promote subsequent aspects of duct morphogenesis and differentiation, and acts primarily through Raf-ERK and the transcriptional effectors LIN-1/Ets and EOR-1. These results reveal multiple genetically separable roles for Ras signaling in tube development, as well as similarities to Ras-mediated control of branching morphogenesis in more complex organs, including the mammalian kidney. The relative simplicity of the excretory system makes it an attractive model for addressing basic questions about how cells gain or lose epithelial character and organize into tubular networks.

A transgenic approach in mice allows the functional interrogation of an odorant receptor locus in vivo and reveals characteristics of its monogenic and monoallelic expression., The odorant receptor (OR) genes constitute the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion, through an unknown mechanism that likely exploits positive and negative regulation. We devised a genetic strategy in mice to examine OR selection by determining the transcriptional activity of an exogenous promoter homologously integrated into an OR locus. Using the tetracycline-dependent transactivator responsive promoter (teto), we observed that the OR locus imposes spatial and temporal constraints on teto-driven transcription. Conditional expression experiments reveal a developmental change in the permissiveness of the locus. Further, expression of an OR transgene that suppresses endogenous ORs similarly represses the OR-integrated teto. Neurons homozygous for the teto-modified allele demonstrate predominantly monoallelic expression, despite their potential to express both copies. These data reveal multiple axes of regulation, and support a model of initiation of OR choice limited by nonpermissive chromatin and maintained by repression of nonselected alleles., Author Summary Odorant receptor (OR) gene choice is a paradigmatic example of transcriptional regulation in which each olfactory sensory neuron selects a single OR from a repertoire of over 1,000 genes. Two mechanistic models of OR choice have been proposed. One postulates the existence of a specialized transcriptional machinery that selects just one OR allele, while a second, kinetic model proposes that OR chromatin is intrinsically nonpermissive, such that inefficient activation during a critical window of time restricts expression to a single OR allele. Here, we used a transgenic approach in mice in which we inserted a conditionally regulated exogenous promoter into an OR locus by homologous recombination in embryonic stem cells. The resulting novel mouse lines allowed the functional interrogation of the OR locus in vivo during development of the olfactory epithelium, enabling us to directly test models of OR choice. Using this experimental strategy we found that OR loci are indeed slow to activate and that the subsequent phenomenon of spatial restriction of OR expression is accomplished by repression. We also observed a developmental shutdown of OR loci concomitant with expression of the OR repertoire. Together, these experiments provide prima facie evidence for a kinetic model of initiation of OR gene choice, coupled with repression of nonselected OR alleles.

The olfactory receptors (ORs), which are G-protein coupled receptors, number more than 1,000 and comprise the largest gene family in the mammalian genome. ORs are expressed both monogenically and monoallelically in olfactory sensory neurons (OSNs) and the mechanism that controls their regulation is largely unknown. ORs reside in constitutive heterochromatin and selection of one OR and from one allele occurs stochastically. To ensure singular monoallelic OR expression, a negative feedback mechanism is elicited by the first selected OR to suppress non-selected ORs in a given neuron. Here we describe results for mice with a 'monoclonal' nose that express one OR, M71,in 95% of all mature OSNs. The M71 transgene suppresses expression of endogenous ORs, and remaining endogenous expression is mostly restricted to immature neurons in the olfactory epithelium. We show that the endogenous OR repertoire are expressed prior to suppression by M71 transgene expression, contrary to current models. When we introduced a second transgene into M71 mice that expressed another OR in most mature OSNs, OSNs uncharacteristically expressed both of the ORs. We hypothesize that unresolved OR competition compromised the neuron's ability to express only one receptor. We further show that suppression of endogenous ORs by M71 is not reversible, and that M71 does not need to be continuously expressed for endogenous ORs to remain suppressed. In these experiments, we have engineered OSNs to express more than one OR in an OSN at a time, which is normally a low probability event. We believe that these experiments reveal the existence of a backup pathway that ensures only one OR will be expressed per neuron. It is possible that mammals have evolved similar mechanisms in other examples of stochastic, monoallelic gene expression, such as in random X-chromosome inactivation or lymphocyte receptor expression.