Loss of bhlha9 Impairs Thermotaxis and Formalin-Evoked Pain in a Sexually Dimorphic Manner

International audienceC-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

Spécialisation fonctionnelle des C-low threshold mechanoreceptors C-LTMRs

Les C-low threshold mechanoreceptors ou C-LTMRs forment une sous-classe bien particulière de fibres C non-nociceptives innervant exclusivement la peau poilue. En temps normal, leur rôle est de transduire la caresse et détecter le refroidissement. Mais de nouvelles données montrent que les C-LTMRs jouent aussi un rôle important dans la modulation de la douleur, aussi bien inflammatoire que neuropathique. Cependant, les mécanismes moléculaires et cellulaires qui sous-tendent cette dualité sont encore méconnus.Notre équipe a récemment publié des données, issues d'une expérience de séquençage ARN à haut débit, qui précisent l'identité moléculaire des C-LTMRs. A ajouter au trois marqueurs moléculaires déjà connus Tafa4, Tyrosine Hydroxylase et Vglut3, nous avons découvert neuf autres gènes dont l'expression est fortement enrichie dans ces neurones. L'un d'eux est le facteur de transcription bhlha9. Le but de ma thèse étant d'améliorer notre connaissance des C-LTMRs, j'ai notamment étudié le rôle de ce gène dans la somatosensation et la physiologie des C-LTMRs en effectuant un grand nombre de tests comportementaux sur des souris chez qui bhlha9 a été inactivé. De façon intéressante, l'inactivation de bhlha9 entraine un large défaut d'adaptation aux variations de température. De plus, ces souris mutantes présentent une réponse exacerbée à la douleur inflammatoire induite par la formaline. Enfin, cette réponse exacerbée est insensible à l'effet normalement analgésique d'un modulateur positif du système GABAergique ionotropique.En conclusion, bhlha9 est un nouveau marqueur des C-LTMRs nécessaire pour une perception normale de la température et de la douleur inflammatoire.C-Low Threshold MechanoReceptors (C-LTMRs) are a unique subset of non-nociceptive C-fibers that innervate exclusively hairy skin. At steady-state, these fibers convey low intensity mechanical stimuli (caress and gentle touch) and detect cooling temperatures.Recent data showed C-LTMRs have an important modulatory role in pain processing following tissue injury, both of inflammatory and neuropathic origin. Yet, their molecular and cellular modes of action are for the most part unknown.To provide a better understanding of these C-LTMRs, our team recently published RNA-seq based data that considerably broadens their molecular repertoire. Besides Tyrosine Hydroxylase, V-GluT3 and Tafa4, already known as restricted to C-LTMRs in DRG, we found nine other genes highly enriched in this subset. One of them encodes for the basic Helix-Loop-Helix (bHLH) family transcription factor bHLHa9.To understand the role of bHLHa9 in somatic sensory biology and in C-LTMRs in particular, I have performed a large panel of behavioural tests on mice lacking its expression, from mechanical to thermal stimulation under acute conditions. Interestingly, bHLHa9 knock-out (KO) mice exhibit a broad defect in temperature sensation. Furthermore, these mice present an enhanced inflammatory pain following hindpaw injection of formalin, along with an impaired analgesic response to GABAA receptor positive modulation.In conclusion, bHLHa9 is highly enriched in C-LTMRs and it is required for proper temperature and inflammatory pain perception

Genetic ablation of GINIP-expressing primary sensory neurons strongly impairs Formalin-evoked pain

International audiencePrimary 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

Mapping the neuroethological signatures of pain, analgesia, and recovery in mice.

Ongoing pain is driven by the activation and modulation of pain-sensing neurons, affecting physiology, motor function, and motivation to engage in certain behaviors. The complexity of the pain state has evaded a comprehensive definition, especially in non-verbal animals. Here, in mice, we used site-specific electrophysiology to define key time points corresponding to peripheral sensitivity in acute paw inflammation and chronic knee pain models. Using supervised and unsupervised machine learning tools, we uncovered sensory-evoked coping postures unique to each model. Through 3D pose analytics, we identified movement sequences that robustly represent different pain states and found that commonly used analgesics do not return an animal's behavior to a pre-injury state. Instead, these analgesics induce a novel set of spontaneous behaviors that are maintained even after resolution of evoked pain behaviors. Together, these findings reveal previously unidentified neuroethological signatures of pain and analgesia at heightened pain states and during recovery

Nav1.7 is required for normal C-low threshold mechanoreceptor function in humans and mice.

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 EMG (n = 3 CIP participants and n = 8 healthy controls) we have found that these patients also have abnormalities in the encoding of affective touch which is mediated by the specialised 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