Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as an important regulator of nervous system’s health and disease states. Yet, its contribution to the pathogenesis of chronic somatic pain remains poorly documented. Here, we report that male but not female mice lacking Myosin1a (KO) raised under single genotype housing conditions (KO-SGH) are predisposed to develop chronic pain in response to a peripheral tissue injury. We further underscore the potential of MYO1A loss-of-function to alter the composition of the gut microbiota and uncover a functional connection between the vulnerability to chronic pain and the dysbiotic gut microbiota of KO-SGH males. As such, parental antibiotic treatment modifies gut microbiota composition and completely rescues the injury-induced pain chronicity in male KO-SGH offspring. Furthermore, in KO-SGH males, this dysbiosis is accompanied by a transcriptomic activation signature in the dorsal root ganglia (DRG) macrophage compartment, in response to tissue injury. We identify CD206+CD163- and CD206+CD163+ as the main subsets of DRG resident macrophages and show that both are long-lived and self-maintained and exhibit the capacity to monitor the vasculature. Consistently, in vivo depletion of DRG macrophages rescues KO-SGH males from injury-induced chronic pain underscoring a deleterious role for DRG macrophages in a Myo1a-loss-of function context. Together, our findings reveal gene-sex-microbiota interactions in determining the predisposition to injury-induced chronic pain and point-out DRG macrophages as potential effector cells.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

Oxytocin is an important regulator of the social brain. In some animal models of autism, notably in Magel2 tm1.1Mus -deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms, focusing on social memory performance. Using in situ hybridization (RNAscope), we have established that Magel2 and oxytocin receptor are co-expressed in the dentate gyrus and CA2/CA3 hippocampal regions involved in the circuitry underlying social memory. Then, we have shown that Magel2 tm1.1Mus - deficient mice, evaluated in a three-chamber test, present a deficit in social memory. Next, in hippocampus, we conducted neuroanatomical and functional studies using immunostaining, oxytocin-binding experiments, ex vivo electrophysiological recordings, calcium imaging and biochemical studies. We demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin receptors and of somatostatin interneurons in both DG and CA2/CA3 regions. We also revealed a delay in the GABAergic development sequence in Magel2 tm1.1Mus -deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring hippocampal alterations and social memory at adulthood. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.

Pain, whether acute or persistent, is a serious medical problem worldwide. However, its management remains unsatisfactory, and new analgesic molecules are required. We show here that TAFA4 reverses inflammatory, postoperative, and spared nerve injury (SNI)-induced mechanical hypersensitivity in male and female mice. TAFA4 requires functional low-density lipoprotein receptor-related proteins (LRPs) because their inhibition by RAP (receptor-associated protein) dose-dependently abolishes its antihypersensitive actions. SNI selectively decreases A-type K+ current (IA) in spinal lamina II outer excitatory interneurons (L-IIo ExINs) and induces a concomitant increase in IA and decrease in hyperpolarization-activated current (Ih) in lamina II inner inhibitory interneurons (L-IIi InhINs). Remarkably, SNI-induced ion current alterations in both IN subtypes were rescued by TAFA4 in an LRP-dependent manner. We provide insights into the mechanism by which TAFA4 reverses injury-induced mechanical hypersensitivity by restoring normal spinal neuron activity and highlight the considerable potential of TAFA4 as a treatment for injury-induced mechanical pain.

Several studies on rodent models with an Autism Spectrum Disorders-like (ASD) phenotype, notably Magel2-deficient mice, have shown a rescue of deficits in adult social behavior after neonatal administration of oxytocin. However, the neurobiological alterations responsible for the social deficits and the mechanism by which oxytocin-administration in infancy has a rescue effect in adulthood remain unclear. Here we show that Magel2-deficient adult mice exhibit a deficit in social memory that is corrected by neonatal oxytocin administration. We studied hippocampal regions known to be associated with social memory engrams involving the OT-system. At critical stages of development, we characterized cellular, physiological and biochemical alterations of these hippocampal regions in Magel2-deficient mice, alterations present in several ASD models. Overall we demonstrate a strong impact of oxytocin-administration at key stages of postnatal hippocampal neurodevelopment, shedding light on the role for oxytocin in treating neurodevelopmental disorders characterized by deficits in social memory.

We identified seven families associating NEUROD2 pathogenic mutations with ASD and intellectual disability. To get insight into the pathophysiological mechanisms, we analyzed cortical development in Neurod2 KO mice. Cortical projection neurons (CPNs) over-migrated during embryogenesis, inducing abnormal thickness and laminar positioning of cortical layers. At juvenile ages, dendritic spine turnover and intrinsic excitability were increased in L5 CPNs. Differentially expressed genes in Neurod2 KO mice were enriched for voltage-gated ion channels, and the human orthologs of these genes were strongly associated with ASD. Furthermore, adult Neurod2 KO mice exhibited core ASD-like behavioral abnormalities. Finally, by generating Neurod2 conditional mutant mice we demonstrate that forebrain excitatory neuron-specific Neurod2 deletion recapitulates cellular and behavioral ASD phenotypes found in full KO mice. Our findings demonstrate crucial roles for Neurod2 in cortical development and function, whose alterations likely account for ASD and related symptoms in the newly defined NEUROD2 mutation syndrome.

Karen Runge 1 *, Rémi Mathieu 1 *, Stéphane Bugeon 2 *, Sahra Lafi 1,2, Corinne Beurrier 2 &, Surajit Sahu 1&, Fabienne Schaller 1, Arthur Loubat 1, Leonard Herault 3, Stéphane Gaillard 4, Mélanie Cahuc 1, Emilie Pallesi-Pocachard 1, Aurélie Montheil 1, Andreas Bosio 5, Jill A Rosenfeld 6, Eva Hudson 7, Kristin Lindstrom 8, Saadet Mercimek-Andrews 9, Lauren Jeffries 10, Arie van Haeringen 11, Olivier Vanakker 12, Bruno Pichon 13, Audrey Van Hecke 14, Dina Amrom 14, Sebastien Küry 15, Candace Gamble 7, Alfonso Represa 1, Carlos Cardoso 1 *, Harold Cremer 2 * and Antoine de Chevigny 1,£

The transcription factor NeuroD2 is a recent candidate for neuropsychiatric disorders but its impact in cortical networks and associated behaviors remains unknown. Here we show that in the mouse neocortex, NeuroD2 is restricted to pyramidal neurons, from development to adulthood. In NeuroD2 deficient mice, layer 5 pyramidal neurons of motor area displayed reduced dendritic complexity and reduced spine density. In contrast, production, radial migration, laminar organization and axonal target specificity of pyramidal neurons were normal, revealing a synaptopathy phenotype. Electrophysiologically, intrinsic excitability and inhibitory inputs onto pyramidal neurons were increased. Behaviorally, NeuroD2 homozygous and heterozygous mice exhibited normal interest and memory for objects but altered sociability and social memory, stereotypies, spontaneous epilepsy and hyperactivity. RNA sequencing from microdissected neocortex revealed that NeuroD2 target genes are highly associated with in cell intrinsic excitability, synaptic regulation, autism and schizophrenia. These results strongly reinforce the potential implication of NeuroD2 mutations in human neuropsychiatric disorders.

AbstractWe identified seven families associatingNEUROD2pathogenic mutations with ASD and intellectual disability. To get insight into the pathophysiological mechanisms, we analyzed cortical development inNeurod2KO mice. Cortical projection neurons (CPNs) over-migrated during embryogenesis, inducing abnormal thickness and laminar positioning of cortical layers. At juvenile ages, dendritic spine turnover and intrinsic excitability were increased in L5 CPNs. Differentially expressed genes inNeurod2KO mice were enriched for voltage-gated ion channels, and the human orthologs of these genes were strongly associated with ASD. Furthermore, adultNeurod2KO mice exhibited core ASD-like behavioral abnormalities. Finally, by generatingNeurod2conditional mutant mice we demonstrate that forebrain excitatory neuron-specificNeurod2deletion recapitulates cellular and behavioral ASD phenotypes found in full KO mice. Our findings demonstrate crucial roles forNeurod2in cortical development and function, whose alterations likely account for ASD and related symptoms in the newly definedNEUROD2mutation syndrome.

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.

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10(-9) M and 10(-7) M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca(2+) concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore-independent mechanism. Pharmacological investigations revealed that the Ca(2+) oscillations are caused by the ET-induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET-induced Ca(2+) signals. Activation of the N-methyl-D-aspartate receptor (NMDA-R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24 h. As this effect was suppressed by antagonizing mGluR1 and NMDA-R, demyelination is therefore caused by the initial ET-induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor-mediated pathway.

The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.

One feature of neuropathic pain is a reduced GABAergic inhibitory function. Nociceptors have been suggested to play a key role in this process. However, the mechanisms behind nociceptor-mediated modulation of GABA signaling remain to be elucidated. Here we describe the identification of GINIP, a Gαi-interacting protein expressed in two distinct subsets of nonpeptidergic nociceptors. GINIP null mice develop a selective and prolonged mechanical hypersensitivity in models of inflammation and neuropathy. GINIP null mice show impaired responsiveness to GABAB, but not to delta or mu opioid receptor agonist-mediated analgesia specifically in the spared nerve injury (SNI) model. Consistently, GINIP-deficient dorsal root ganglia neurons had lower baclofen-evoked inhibition of high-voltage-activated calcium channels and a defective presynaptic inhibition of lamina IIi interneurons. These results further support the role of unmyelinated C fibers in injury-induced modulation of spinal GABAergic inhibition and identify GINIP as a key modulator of peripherally evoked GABAB-receptors signaling.

Neurotrophins and their receptors control a number of cellular processes, such as survival, gene expression and axonal growth, by activating multiple signalling pathways in peripheral neurons. Whether each of these pathways controls a distinct developmental process remains unknown. Here we describe a novel knock-in mouse model expressing a chimeric TrkA/TrkC (TrkAC) receptor from TrkA locus. In these mice, prospective nociceptors survived, segregated into appropriate peptidergic and nonpeptidergic subsets, projected normally to distinct laminae of the dorsal spinal cord, but displayed aberrant peripheral target innervation. This study provides the first in vivo evidence that intracellular parts of different Trk receptors are interchangeable to promote survival and maturation of nociceptors and shows that these developmental processes can be uncoupled from peripheral target innervation. Moreover, adult homozygous TrkAC knock-in mice displayed severe deficits in acute and tissue injury-induced pain, representing the first viable adult Trk mouse mutant with a pain phenotype.

Endogenous acetylcholine (ACh) is a well-known modulator of nociceptive transmission in the spinal cord of rodents. It arises mainly from a sparse population of cholinergic interneurons located in the dorsal horn of the spinal cord. This population was thought to be absent from the spinal cord of monkey, what might suggest that spinal ACh would not be a relevant clinical target for pain therapy. In humans, however, pain responses can be modulated by spinal ACh, as evidenced by the increasingly used analgesic procedure (for postoperative and labor patients) consisting of the epidural injection of the acetylcholinesterase inhibitor neostigmine. The source and target of this ACh remain yet to be elucidated. In this study, we used an immunolabeling for choline acetyltransferase to demonstrate, for the first time, the presence of a plexus of cholinergic fibers in laminae II-III of the dorsal horn of the macaque monkey. Moreover, we show the presence of numerous cholinergic cell bodies within the same laminae and compared their density and morphological properties with those previously described in rodents. An electron microscopy analysis demonstrates that cholinergic boutons are presynaptic to dorsal horn neurons as well as to the terminals of sensory primary afferents, suggesting that they are likely to modulate incoming somatosensory information. Our data suggest that this newly identified dorsal horn cholinergic system in monkeys is the source of the ACh involved in the analgesic effects of epidural neostigmine and could be more specifically targeted for novel therapeutic strategies for pain management in humans.

C-low-threshold mechanoreceptors (C-LTMRs) are unique among C-unmyelinated primary sensory neurons. These neurons convey two opposite aspects of touch sensation: a sensation of pleasantness, and a sensation of injury-induced mechanical pain. Here, we show that TAFA4 is a specific marker of C-LTMRs. Genetic labeling in combination with electrophysiological recordings show that TAFA4+ neurons have intrinsic properties of mechano-nociceptors. TAFA4-null mice exhibit enhanced mechanical and chemical hypersensitivity following inflammation and nerve injury as well as increased excitability of spinal cord lamina IIi neurons, which could be reversed by intrathecal or bath application of recombinant TAFA4 protein. In wild-type C57/Bl6 mice, intrathecal administration of TAFA4 strongly reversed carrageenan-induced mechanical hypersensitivity, suggesting a potent analgesic role of TAFA4 in pain relief. Our data provide insights into how C-LTMR-derived TAFA4 modulates neuronal excitability and controls the threshold of somatic sensation.

Endogenous acetylcholine is an important modulator of sensory processing, especially at the spinal level, where nociceptive (pain‐related) stimuli enter the central nervous system and are integrated before being relayed to the brain. To decipher the organization of the local cholinergic circuitry in the spinal dorsal horn, we used transgenic mice expressing enchanced green fluorescent protein specifically in cholinergic neurons (ChAT::EGFP) and characterized the morphology, neurochemistry, and firing properties of the sparse population of cholinergic interneurons in this area. Three‐dimensional reconstruction of lamina III ChAT::EGFP neurons based either on their intrinsic fluorescence or on intracellular labeling in live tissue demonstrated that these neurons have long and thin processes that grow preferentially in the dorsal direction. Their dendrites and axon are highly elongated in the rostrocaudal direction, beyond the limits of a single spinal segment. These unique morphological features suggest that dorsal horn cholinergic interneurons are the main contributors to the plexus of cholinergic processes located in lamina IIi, just dorsal to their cell bodies. In addition, immunostainings demonstrated that dorsal horn cholinergic interneurons in the mouse are γ‐aminobutyric acidergic and express nitric oxide synthase, as in rats. Finally, electrophysiological recordings from these neurons in spinal cord slices demonstrate that two‐thirds of them have a repetitive spiking pattern with frequent rebound spikes following hyperpolarization. Altogether our results indicate that, although they are rare, the morphological and functional features of cholinergic neurons enable them to collect segmental information in superficial layers of the dorsal horn and to modulate it over several segments. J. Comp. Neurol. 519:3139–3158, 2011. © 2011 Wiley‐Liss, Inc.

Deciphering the precise in vivo function of a particular neuronal subpopulation is one of the most challenging issues in neurobiology. Dorsal root ganglia (DRG) neurons represent a powerful model system to address this fundamental question. These neurons display many morphological, anatomical and few molecular characteristics. With the aim of expanding the molecular description of the primary sensory neurons, we used Affimetrix microarrays to compare global gene expression profiles of DRG of wild type and trkAtrkC/trkC knock-in mice at birth and identified several hundred potential markers of nociceptive neurons and few markers of proprioceptive neurons. Here, we describe the identification of two members of a family of putative adapter proteins STAC1 and STAC2. We found STAC1 and STAC2 being expressed in a mutually exclusive fashion in adult DRG neurons. STAC1 mainly marks peptidergic nociceptive neurons while STAC2 is expressed in a subset of nonpeptidergic nociceptors, in all trkB+ neurons and in a subpopulation of proprioceptive neurons. Our expression data demonstrate that STAC proteins identify four categories of primary sensory neurons; one class of peptidergic neurons, a subset of nonpeptidergic neurons, all TrkB+neurons and a subset of proprioceptive neurons. Genetic marking of STACs-expressing sensory neurons will lend significant advance into our understanding of DRG neuronal functional diversity.

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

The limited choice and poor performance of red-emitting calcium (Ca2+) indicators have hampered microfluorometric measurements of the intracellular free Ca2+ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca2+ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH2 linker arm. The low-affinity variant (KD,Ca ∼30 μM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca2+ transients. While Calcium Ruby is a mitochondrial Ca2+probe, its conjugation, via the NH2 tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca2+ probe with an affinity matched to microdomain Ca2+ signals. As an example, we show depolarization-evoked Ca2+ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca2+ measurements.