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SUMMARYImprovements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies1,2support decades of work on spinal cord lineage studies3–5, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons6–8, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparingHoxb8FlpOmouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenousHoxb8gene expression9, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below.Hoxb8FlpOheterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals10,11, suggesting normal developmental function of the Hoxb8 gene and protein inHoxb8FlpOmice. Compared to the variability of brain recombination in available caudal Cre and Flp lines12,13Hoxb8FlpOactivity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine theHoxb8FlpOmouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses14and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same RORβlineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.In briefWe describe the generation of aHoxb8FlpOmouse line that targets Flp-recombinase expression to the spinal cord, dorsal root ganglia, and caudal viscera. This line can be used in intersectional Cre/Flp strategies to restrict manipulations to the caudal nervous system. Additionally, we describe an intersectional genetics+viral strategy to convert developmental GFP expression into adult Cre expression, allowing for modular incorporation of viral tools into intersectional genetics. This approach allows for manipulation of a developmentally determined lineage in the adult. This strategy is also more accessible than traditional intersectional genetics, and can adapt to the constantly evolving available viral repertoire.Highlights-A newHoxb8FlpOmouse line allows Flp-dependent recombination in the spinal cord, dorsal root ganglia, and caudal viscera.-We observed no ectopic brain expression across mouse genetic backgrounds with theHoxb8FlpOmouse line.-Combining this new mouse line for intersectional genetics and a viral approach, we provide a novel pipeline to target and manipulate developmentally defined adult spinal circuits.

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.

Patients with bi-allelic loss of function mutations in the voltage-gated sodium channel Nav1.7 present with congenital insensitivity to pain (CIP), whilst low threshold mechanosensation is reportedly normal. Using psychophysics (n = 6 CIP participants and n = 86 healthy controls) and facial electromyography (n = 3 CIP participants and n = 8 healthy controls), we found that these patients also have abnormalities in the encoding of affective touch, which is mediated by the specialized afferents C-low threshold mechanoreceptors (C-LTMRs). In the mouse, we found that C-LTMRs express high levels of Nav1.7. Genetic loss or selective pharmacological inhibition of Nav1.7 in C-LTMRs resulted in a significant reduction in the total sodium current density, an increased mechanical threshold and reduced sensitivity to non-noxious cooling. The behavioural consequence of loss of Nav1.7 in C-LTMRs in mice was an elevation in the von Frey mechanical threshold and less sensitivity to cooling on a thermal gradient. Nav1.7 is therefore not only essential for normal pain perception but also for normal C-LTMR function, cool sensitivity and affective touch.

Inflammatory pain represents a complex state involving sensitization of peripheral and central neuronal signaling. Resolving this high-dimensional interplay at the cellular and behavioral level is key to effective therapeutic development. Here, using the carrageenan model of local inflammation of the hind paw, we determine how carrageenan alters both the physiological state of sensory neurons and behaviors at rapid and continuous timescales. We identify higher excitability of sensory neurons innervating the site of inflammation by profiling their physiological state at different time points. To identify millisecond-resolved sensory-reflexive signatures evoked by inflammatory pain, we used a combination of supervised and unsupervised algorithms, and uncovered abnormal paw placement as a defining behavioral feature. For long-term detection and characterization of spontaneous behavioral signatures representative of affective-motivational pain states, we use computer vision coupled to unsupervised machine learning in an open arena. Using the non-steroidal anti-inflammatory drug meloxicam to characterize analgesic states during rapid and ongoing timescales, we identify a return to pre-injury states of some sensory-reflexive behaviors, but by and large, many spontaneous, affective-motivational pain behaviors remain unaffected. Taken together, this comprehensive exploration across cellular and behavioral dimensions reveals peripheral versus centrally mediated pain signatures that define the inflamed state, providing a framework for scaling the pain experience at unprecedented resolution.

Summary: C-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABAA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation. : Bohic et al. demonstrate that mice lacking the transcription factor BHLHA9 exhibit impaired thermotaxis and formalin-evoked pain. BHLHA9 mediates these effects by modulating spinal GABAergic signaling. The data consolidate the role of C-LTRMs in pain sensation and provide in vivo evidence of C-LTRMs in the discriminative aspects of temperature sensation. Keywords: C-LTMRs, Bhhlha9, sexual dimorphism, ionotropic GABAergic signaling

Primary sensory neurons are heterogeneous by myriad of molecular criteria. However, the functional significance of this remarkable heterogeneity is just emerging. We precedently described the GINIP+ neurons as a new subpopulation of non peptidergic C-fibers encompassing the free nerve ending cutaneous MRGPRD+ neurons and C-LTMRs. Using our recently generated ginip mouse model, we have been able to selectively ablate the GINIP+ neurons and assess their functional role in the somatosensation. We found that ablation of GINIP+ neurons affected neither the molecular contents nor the central projections of the spared neurons. GINIP-DTR mice exhibited impaired sensation to gentle mechanical stimuli applied to their hairy skin and had normal responses to noxious mechanical stimuli applied to their glabrous skin, under acute and injury-induced conditions. Importantly, loss of GINIP+ neurons significantly altered formalin-evoked first pain and drastically suppressed the second pain response. Given that MRGPRD+ neurons have been shown to be dispensable for formalin-evoked pain, our study suggest that C-LTMRs play a critical role in the modulation of formalin-evoked pain.

International audience; The heparan sulfate proteoglycan Glypican 4 (Gpc4) is strongly expressed in mouse embryonic stem (ES) cells where it controls the maintenance of self-renewal by modulating Wnt/β-catenin signaling activities. Here we show that mouse ES cells carrying a hypomorphic Gpc4 allele, in a single-step neuronal differentiation protocol, show increased differentiation into dopaminergic neurons expressing tyrosine hydroxylase (TH) and nuclear receptor related-1 protein (Nurr1) 1. In contrast to wild-type cells, these differentiating Gpc4-mutant cells expressed high levels of DOPA decarboxylase and the dopamine transporter, two markers expressed by fully mature dopaminergic neurons. Intrastriatal transplantation of Gpc4 hypomorphic cells into a 6-OHDA rat model for Parkinson's disease improved motor behavior in the cylinder test and amphetamine-induced rotations at a higher level than transplanted wild-type cells. Importantly, Gpc4 hypomorphic cell grafts, in contrast to wild-type cells, did not generate teratomas in the host brains, leading to strongly enhanced animal survival. Therefore, control of Gpc4 activity level represents a new potential strategy to reduce ES cell tumorigenic features while at the same time increasing neuronal differentiation and integration.