Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain.

Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supraspinal level is almost unknown. In the anterior pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a subpopulation of GABAergic neurons coexpressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high-frequency-bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology.

Cav3.2 T-type calcium channels shape electrical firing in mouse Lamina II neurons.

The T-type calcium channel, Cav3.2, is necessary for acute pain perception, as well as mechanical and cold allodynia in mice. Being found throughout sensory pathways, from excitatory primary afferent neurons up to pain matrix structures, it is a promising target for analgesics. In our study, Cav3.2 was detected in ~60% of the lamina II (LII) neurons of the spinal cord, a site for integration of sensory processing. It was co-expressed with Tlx3 and Pax2, markers of excitatory and inhibitory interneurons, as well as nNOS, calretinin, calbindin, PKCγ and not parvalbumin. Non-selective T-type channel blockers slowed the inhibitory but not the excitatory transmission in LII neurons. Furthermore, T-type channel blockers modified the intrinsic properties of LII neurons, abolishing low-threshold activated currents, rebound depolarizations, and blunting excitability. The recording of Cav3.2-positive LII neurons, after intraspinal injection of AAV-DJ-Cav3.2-mcherry, showed that their intrinsic properties resembled those of the global population. However, Cav3.2 ablation in the dorsal horn of Cav3.2GFP-Flox KI mice after intraspinal injection of AAV-DJ-Cav3.2-Cre-IRES-mcherry, had drastic effects. Indeed, it (1) blunted the likelihood of transient firing patterns; (2) blunted the likelihood and the amplitude of rebound depolarizations, (3) eliminated action potential pairing, and (4) remodeled the kinetics of the action potentials. In contrast, the properties of Cav3.2-positive neurons were only marginally modified in Cav3.1 knockout mice. Overall, in addition to their previously established roles in the superficial spinal cord and in primary afferent neurons, Cav3.2 channel appear to be necessary for specific, significant and multiple controls of LII neuron excitability.

Fxyd2 regulates Aδ- and C-fiber mechanosensitivity and is required for the maintenance of neuropathic pain.

Identification of the molecular mechanisms governing sensory neuron subtype excitability is a key requisite for the development of treatments for somatic sensory disorders. Here, we show that the Na,K-ATPase modulator Fxyd2 is specifically required for setting the mechanosensitivity of Aδ-fiber low-threshold mechanoreceptors and sub-populations of C-fiber nociceptors, a role consistent with its restricted expression profile in the spinal somatosensory system. We also establish using the spared nerve injury model of neuropathic pain, that loss of Fxyd2 function, either constitutively in Fxyd2-/- mice or acutely in neuropathic rats, efficiently alleviates mechanical hypersensitivity induced by peripheral nerve lesions. The role of Fxyd2 in modulating Aδ- and C-fibers mechanosensitivity likely accounts for the anti-allodynic effect of Fxyd2 knockdown. Finally, we uncover the evolutionarily conserved restricted expression pattern of FXYD2 in human dorsal root ganglia, thus identifying this molecule as a potentially promising therapeutic target for peripheral neuropathic pain management.

T-type calcium channels in neuropathic pain.

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.

Transcriptional Profiling of Cutaneous MRGPRD Free Nerve Endings and C-LTMRs.

Cutaneous 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 Aß and Aδ low-threshold mechanoreceptors.

The Low-Threshold Calcium Channel Cav3.2 Determines Low-Threshold Mechanoreceptor Function.

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.

T-type calcium channels in chronic pain: mouse models and specific blockers.

Pain is a quite frequent complaint accompanying numerous pathologies. Among these pathological cases, 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 the majority of 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 benefit/risk ratio. As no new pharmacological concept has led to new analgesics in the last decades, the discovery of 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 (DRG) known now 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 review the current knowledge on the role of these channels in the perception and modulation of pain.

T-type calcium channel Cav3.2 deficient mice show elevated anxiety, impaired memory and reduced sensitivity to psychostimulants.

The fine-tuning of neuronal excitability relies on a tight control of Ca(2+) 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 Ca(2+) 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.

Automated method for the segmentation and morphometry of nerve fibers in large-scale CARS images of spinal cord tissue.

A fully automated method for large-scale segmentation of nerve fibers from coherent anti-Stokes Raman scattering (CARS) microscopy images is presented. The method is specifically designed for CARS images of transverse cross sections of nervous tissue but is also suitable for use with standard light microscopy images. After a detailed description of the two-part segmentation algorithm, its accuracy is quantified by comparing the resulting binary images to manually segmented images. We then demonstrate the ability of our method to retrieve morphological data from CARS images of nerve tissue. Finally, we present the segmentation of a large mosaic of CARS images covering more than half the area of a mouse spinal cord cross section and show evidence of clusters of neurons with similar g-ratios throughout the spinal cord.

Alleviating pain hypersensitivity through activation of type 4 metabotropic glutamate receptor.

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.

Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach.

We present an automated two-dimensional Fourier transform (2D-FT) approach to analyze the local organization of myelinated axons in the spinal cord. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to observe lesions in a commonly used animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). A 2D-FT was applied on the CARS images to find the average orientation and directional anisotropy of the fibers within contiguous image domains. We introduce the corrected correlation parameter (CCP), a measure of the correlation between orientations of adjacent domains. We show that in the EAE animal model of MS, the CCP can be used to quantify the degree of organization/disorganization in the myelin structure. This analysis was applied to a large image dataset from animals at different clinical scores and we show that some descriptors of the CCP probability density function are strongly correlated with the clinical scores. This procedure, compatible with live animal imaging, has been developed to perform local in situ evaluation of myelinated axons afflicted by EAE.

Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl⁻ homeostasis.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K(+)-Cl(-) co-transporter KCC2, impairing Cl(-) homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential 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 signaling preserved Cl(-) homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions.

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.

Live animal myelin histomorphometry of the spinal cord with video-rate multimodal nonlinear microendoscopy.

In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.

Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.

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 Ca(2+) 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.

In vivo optical monitoring of tissue pathologies and diseases with vibrational contrast.

Studies of tissue remodeling require in vivo imaging techniques that are as minimally invasive as possible to avoid microenvironment perturbations. To this end, spontaneous Raman techniques have been used but low signals have limited their application mostly to point spectroscopy measurements. Novel Raman-based techniques such as coherent and stimulated Raman scattering can overcome this limitation. This manuscript discusses imaging and spectroscopy applications with Raman-based contrast for in vivo tissue monitoring, and how these can be combined into spectral imaging.

Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation.

Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.