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Opioid analgesia is counteracted on chronic use by tolerance and hyperalgesia inducing dose escalation and life-threatening overdoses. Mu opiate receptors (MOR) expressed in primary sensory neurons were recently found to control tolerance and hyperalgesia, but the underlying mechanisms remained elusive. Here we show that genetic inactivation offms-like tyrosine kinase receptor 3 (FLT3) receptor in sensory neurons abrogates morphine tolerance and hyperalgesia by preventing MOR-induced hyperactivation of the cAMP signaling pathway and subsequent excitatory adaptive processes. Moreover, the specific FLT3 inhibitor BDT001 potentiates morphine analgesia in acute and chronic pain models, without aggravating morphine adverse effects, and reverses tolerance and hyperalgesia once installed. Thus, FLT3 appears as a key regulator of the MOR signaling pathway and its pharmacological blockade shows promise to enhance chronic opioid analgesic efficacy.

Acute pain events have been associated with persistent pain sensitization of nociceptive pathways increasing the risk of transition from acute to chronic pain. However, it is unclear whether injuryinduced persistent pain sensitization can promote long-term mood disorders. The receptor tyrosine kinase FLT3 is causally required for pain chronification after peripheral nerve injury, questioning its role in the development of pain-induced mood alterations. Here, we evaluated the emotional and sensorial components of pain after a single (SI) or double paw incision (DI). We then investigated the role of FLT3 either by inhibition using transgenic knock-out mice and functional antibodies or by activation with FLT3 ligand (FL) administrations.DI mice showed significant anxiodepressive-like and spontaneous pain behaviors as opposed to SI mice. DI also promoted and extended mechanical pain hypersensitivity compared to SI. This emotional and sensorial pain exaggeration correlated with spinal changes especially by increased microglia activation after DI versus SI. Intrathecal minocycline, a microglial inhibitor, specifically reversed DI inducedmechanical hypersensitivity in males. Repeated treatment with the microglia proliferation inhibitor GW2580 not only eliminated the exaggerated pain hypersensitivity produced by DI but also prevented anxiodepressive-related behaviors in DI animals. Finally, FL injections in naive animals provoked mechanical allodynia and anxiodepressive-like disorders concomitant with a strong microglial activation while Flt3 silencing in a genetic mouse line or FLT3 blocking via functional antibodies, blunted the development of persistent pain and depression after DI. Altogether our results show that the repetition of peripheral lesions facilitate not only exaggerated nociceptive behaviors but also induced anxiodepressive disorders supported by spinal central changes. The inhibition of FLT3 could thus become a promising therapy in the management of pain sensitization and related mood alterations.

International audience; Dorsal root ganglia (DRG) neurons exhibit a wide molecular heterogeneity in relation to the various sensory modalities (mechanoception, thermoception, nociception) that they subserve. Finding markers of subpopulations is an important step in understanding how these neurons convey specific information. We identified fibroblast growth factor homologous factor 1 (FHF1) in a search for markers of subpopulations of DRG neurons. FHFs constitute a family of four factors that share some structural properties with fibroblast growth factors (FGFs) but are functionally distinct. They are expressed in specific subsets of neurons and are involved in the modulation of sodium channel activity. The pattern of expression of FHF1 in the DRG was determined during development, in the adult and after axotomy. We show that in the adult, FHF1 is expressed in two populations, one composed of nociceptors and another in which no neurotrophic factor receptors were detected (panTrk-/c-Ret-). Interestingly, in the nociceptors, FHF1 expression was restricted to a subset of TrkA+/calcitonin gene-related peptide (CGRP)-positive neurons. Neurofilament 200 (NF-200) and peripherin labeling indicates that 70% of the FHF1-expressing neurons contribute to A-fibers and 30% to C-fibers. FHF1 interacts with the Na(v)1.9 sodium channel isoform, which is strongly expressed in cRet+/isolectin-B4 binding neurons, but we show that FHF1 is not expressed in the cRet+/IB4+ subclass and that it does not colocalize with Na(v)1.9. Our results argue strongly against the possibility that FHF1 has a modulatory effect on this channel in cRet+/IB4+ neurons, but FHF1 could play a role in a distinct subset of TrkA+/CGRP+ nociceptors.

Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between −65 and −45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na+ current that displays most of the biophysical and pharmacological characteristics of neuronal Na+ current. Single-cell RT-PCR reveals that several alpha-subunit isoforms of the Na+ channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na+ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na+ currents decrease dramatically from post-natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na+ channels able to generate Na+-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.

Objective A calcium-activated chloride current (IClCa) has been observed in medium-sized sensory neurons of the dorsal root ganglion (DRG). Axotomy of the sciatic nerve induces a similar current in the majority of medium and large diameter neurons. Our aim is to identify the molecule(s) underlying this current. Methods Using conventional and quantitative RT-PCR, we examined the expression in DRG of members of three families of genes, which have been shown to have IClCa current inducing properties. Results We showed the detection of transcripts representing several members of these families, i.e. chloride channel calcium-activated (CLCA), Bestrophin and Tweety gene families in adult DRG, in the normal state and 3 d after sciatic nerve section, a model for peripheral nerve injury. Conclusion Our analysis revealed that that mBest1 and Tweety2 appear as the best candidates to play a role in the injury-induced IClCa in DRG neurons.

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.

Changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca 2+ stores, via both ryanodine-sensitive and IP 3 -sensitive intracellular Ca 2+ channels control [Ca 2+ ] i homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca 2+ ] i increased progressively from days 1–7 in culture. These responses were blocked by ryanodine and nicardipine but not by ω-conotoxin-GVIA or ω-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca v 1 Ca 2+ channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca 2+ oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca 2+ stores from the endoplasmic reticulum, induced an increase in [Ca 2+ ] i in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5–6 days in vitro. Xestospongin-C, a membrane-permeable IP 3 receptor inhibitor blocked the CPA-induced [Ca 2+ ] i response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP 3 Ca 2+ channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca 2+ ] i homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca 2+ channel responsible for a Ca 2+ -induced Ca 2+ release in embryonic motoneurons following voltage-dependent Ca 2+ entry via L-type Ca 2+ channels.