A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We show here that hyperalgesia-inducing treatment with morphine causes downregulation of the K+-Cl− cotransporter KCC2, impairing Cl− homeostasis in spinal lamina l neurons. Restoring Eanion reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor-dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signalling preserved Cl− homeostasis and reversed the hyperalgesia. Gene-targeted mice in which BDNF was deleted from microglia did not develop hyperalgesia to morphine. Yet, neither morphine antinociception nor tolerance was affected in these animals. Our findings dissociate morphine-induced hyperalgesia from tolerance and unveil the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target to prevent hyperalgesia without affecting morphine analgesia.

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, but its expression pattern within primary afferent neurons and its contribution to modality-specific signaling remain obscure. Here, we elucidate this issue using a unique knockin/flox mouse strain wherein Cav3.2 is replaced by a functional Cav3.2-surface-ecliptic GFP fusion. We demonstrate that Cav3.2 is a selective marker of two major low-threshold mechanoreceptors (LTMRs), Aδ- and C-LTMRs, innervating the most abundant skin hair follicles. The presence of Cav3.2 along LTMR-fiber trajectories is consistent with critical roles at multiple sites, setting their strong excitability. Strikingly, the C-LTMR-specific knockout uncovers that Cav3.2 regulates light-touch perception and noxious mechanical cold and chemical sensations and is essential to build up that debilitates allodynic symptoms of neuropathic pain, a mechanism thought to be entirely A-LTMR specific. Collectively, our findings support a fundamental role for Cav3.2 in touch/pain pathophysiology, validating their critic pharmacological relevance to relieve mechanical and cold allodynia.

Pain is a quite frequent complaint accompanying numerous pathologies. Among these pathological cases, numerous neuropathies are retrieved with identified etiologies (chemotherapies, diabetes, surgeries…) and also more diffuse syndromes such as fibromyalgia. More broadly, pain is one of the first consequences of most inherited diseases. Despite its importance for the quality of life, current pain management is limited to drugs that are either old or with a limited efficacy or that possess a bad risk benefit ratio. As no new pharmacological concept has led to new analgesics in the last decades, the discovery of new medications is needed, and to this aim, the identification of new druggable targets in pain transmission is a first step. Therefore, studies of ion channels in pain pathways are extremely active. This is particularly true with ion channels in peripheral sensory neurons in dorsal root ganglia known how to express unique sets of these channels. Moreover, both spinal and supraspinal levels are clearly important in pain modulation. Among these ion channels, we and others revealed the important role of low voltage-gated calcium channels in cellular excitability in different steps of the pain pathways. These channels, by being activated nearby resting membrane potential, have biophysical characteristics suited to facilitate action potential generation and rhythmicity. In this review, we will present the current knowledge on the role of these channels in the perception and modulation of pain.

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain.

The use of coherent anti-Stokes Raman scattering microscopy tuned to the lipid vibration for quantitative myelin imaging suffers from the excitation polarization dependence of this third-order nonlinear optical effect. The contrast obtained depends on the orientation of the myelin membrane, which in turn affects the morphometric parameters that can be extracted with image analysis. We show how circularly polarized laser beams can be used to avoid this complication, leading to images free of excitation polarization dependence. The technique promises to be optimal for in vivo imaging and the resulting images can be used for coherent anti-Stokes Raman scattering optical histology on native state tissue.

The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations.

SummaryCutaneous C-unmyelinated MRGPRD+ free nerve endings and C-LTMRs innervating hair follicles convey two opposite aspects of touch sensation: a sensation of pain and a sensation of pleasant touch. The molecular mechanisms underlying these diametrically opposite functions are unknown. Here, we used a mouse model that genetically marks C-LTMRs and MRGPRD+ neurons in combination with fluorescent cell surface labeling, flow cytometry, and RNA deep-sequencing technology (RNA-seq). Cluster analysis of RNA-seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in MRGPRD-expressing neurons and C-LTMRs, respectively. We validated 48 MRGPD- and 68 C-LTMRs-enriched genes using a triple-staining approach, and the Cav3.3 channel, found to be exclusively expressed in C-LTMRs, was validated using electrophysiology. Our study greatly expands the molecular characterization of C-LTMRs and suggests that this particular population of neurons shares some molecular features with Aβ and Aδ low-threshold mechanoreceptors.