Pain hypersensitivity in a pharmacological mouse model of attention-deficit/hyperactivity disorder.

Clinical evidence suggests that pain hypersensitivity develops in patients with attention-deficit/hyperactivity disorder (ADHD). However, the mechanisms and neural circuits involved in these interactions remain unknown because of the paucity of studies in animal models. We previously validated a mouse model of ADHD obtained by neonatal 6-hydroxydopamine (6-OHDA) injection. Here, we have demonstrated that 6-OHDA mice exhibit a marked sensitization to thermal and mechanical stimuli, suggesting that phenotypes associated with ADHD include increased nociception. Moreover, sensitization to pathological inflammatory stimulus is amplified in 6-OHDA mice as compared to shams. In this ADHD model, spinal dorsal horn neuron hyperexcitability was observed. Furthermore, ADHD-related hyperactivity and anxiety, but not inattention and impulsivity, are worsened in persistent inflammatory conditions. By combining in vivo electrophysiology, optogenetics, and behavioral analyses, we demonstrated that anterior cingulate cortex (ACC) hyperactivity alters the ACC-posterior insula circuit and triggers changes in spinal networks that underlie nociceptive sensitization. Altogether, our results point to shared mechanisms underlying the comorbidity between ADHD and nociceptive sensitization. This interaction reinforces nociceptive sensitization and hyperactivity, suggesting that overlapping ACC circuits may be targeted to develop better treatments.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.

Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell-autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1-deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell-autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.

Mindful Kangaroo Care: mindfulness intervention for mothers during skin-to-skin care: a randomized control pilot study.

BACKGROUND: Parents of babies admitted to the Newborn Intensive Care Unit (NICU) undergo considerable stress. There is evidence that mindfulness reduces stress in these parents. Kangaroo Care (KC) is practiced in NICUs across the world and is stress-relieving. Whether mindfulness practiced during KC in the NICU reduces parental distress has not yet been studied. The objective was to explore the feasibility and acceptability of teaching and practicing mindfulness during KC for mothers of premature babies. The objective was also to document preliminary outcomes of Mindful Kangaroo Care (MKC) on maternal stress, anxiety, depression, and mindful awareness. METHODS: In this pilot randomized controlled study, mothers of premature babies who were expected to stay in the NICU for at least four weeks were taught two mindfulness exercises to practice during KC and compared to mothers who received standard care with no mindfulness teaching. Mothers filled out stress, anxiety, depression and mindful awareness scales at recruitment and after four weeks. Acceptability and feasibility questionnaires were also completed. RESULTS: Fifteen mothers per group completed the study. The MKC group demonstrated a significant within-group reduction in anxiety (p = 0.003), depression (p = 0.02) and stress (p = 0.002), and a significant increase in both the curiosity (p = 0.008) and decentering (p = 0.01) scores of the Toronto Mindfulness Scale, all of which had medium to large effect sizes. Only the increases in curiosity and decentering were significant between groups. Fourteen mothers found the intervention acceptable, one neutral. CONCLUSION: MKC was acceptable, feasible and led to a reduction in stress, anxiety and depression in mothers who practiced mindfulness exercises during KC.

A secretagogin locus of the mammalian hypothalamus controls stress hormone release.

A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

Windup of Nociceptive Flexion Reflex Depends on Synaptic and Intrinsic Properties of Dorsal Horn Neurons in Adult Rats.

Windup, a progressive increase in spinal response to repetitive stimulations of nociceptive peripheral fibers, is a useful model to study central sensitization to pain. Windup is expressed by neurons in both the dorsal and ventral horn of the spinal cord. In juvenile rats, it has been demonstrated both in vivo and in vitro that windup depends on calcium-dependent intrinsic properties and their modulation by synaptic components. However, the involvement of these two components in the adults remains controversial. In the present study, by means of electromyographic and extracellular recordings, we show that windup in adults, in vivo, depends on a synaptic balance between excitatory N-methyl-D-aspartate (NMDA) receptors and inhibitory glycinergic receptors. We also demonstrate the involvement of L-type calcium channels in both the dorsal and ventral horn of the spinal cord. These results indicate that windup in adults is similar to juvenile rats and that windup properties are the same regardless of the spinal network, i.e., sensory or motor.

Alteration of nociceptive integration in the spinal cord of a rat model of Parkinson's disease.

BACKGROUND: Pain is a major non motor symptom that contributes to impaired quality of life in PD. However, its mechanism is unknown. OBJECTIVES AND METHODS: We sought to identify the pain phenotypes and parallel changes in spinal integration of peripheral stimuli in a rat model of PD induced by lesions of SN dopamine neurons, using behavioral plantar and von Frey tests as well as electrophysiology of the dorsal horn. RESULTS: We show that dopamine depletion by 6-OHDA induced hypersensitivity to mechanical and thermal stimuli. These abnormal behaviors were paralleled by increased neuronal responses and hyperexcitability of wide dynamic range neurons of lamina V of the dorsal horn of the spinal cord in response to electrical stimulation of the sciatic nerve in the 6-OHDA model as compared to sham rats. CONCLUSIONS: These results provide evidence for alteration of nociceptive integration in the spinal dorsal horn neurons in 6-OHDA rats that can reflect changes in pain behavior. © 2018 International Parkinson and Movement Disorder Society.

Group I metabotropic glutamate receptor plasticity after peripheral inflammation alters nociceptive transmission in the dorsal of the spinal cord in adult rats.

Abstract: The dorsal horn of the spinal cord is a crucial site for pain transmission and modulation. Dorsal horn neurons of the spinal cord express group I metabotropic glutamate receptors (group I mGluRs) that exert a complex role in nociceptive transmission. In particular, group I mGluRs promote the activation of L-type calcium channels, voltage-gated channels involved in short- and long-term sensitization to pain. In this study, we analyzed the role of group I mGluRs in spinal nociceptive transmission and the possible cooperation between these receptors and L-type calcium channels in the pathophysiology of pain transmission in the dorsal horn of the spinal cord. We demonstrate that the activation of group I mGluRs induces allodynia and L-type calcium channel-dependent increase in nociceptive field potentials following sciatic nerve stimulation. Surprisingly, in a model of persistent inflammation induced by complete Freund's adjuvant, the activation of group I mGluRs induced an analgesia and a decrease in nociceptive field potentials. Among the group I mGluRs, mGluR1 promotes the activation of L-type calcium channels and increased nociceptive transmission while mGluR5 induces the opposite through the inhibitory network. These results suggest a functional switch exists in pathological conditions that can change the action of group I mGluR agonists into possible analgesic molecules, thereby suggesting new therapeutic perspectives to treat persistent pain in inflammatory settings.

Cav1.2 and Cav1.3 L-type calcium channels independently control short- and long-term sensitization to pain.

KEY POINTS: L-type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short- and long-term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind-up. These results identify Cav1.3 as a key conductance responsible for short-term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long-term plasticity associated with neuropathic pain. Up-regulation of Cav1.2 and down-regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind-up may be not relevant in the treatment of neuropathy. ABSTRACT: Short-term central sensitization to pain temporarily increases the responsiveness of nociceptive pathways after peripheral injury. In dorsal horn neurons (DHNs), short-term sensitization can be monitored through the study of wind-up. Wind-up, a progressive increase in DHNs response following repetitive peripheral stimulations, depends on the post-synaptic L-type calcium channels. In the dorsal horn of the spinal cord, two L-type calcium channels are present, Cav1.2 and Cav1.3, each displaying specific kinetics and spatial distribution. In the present study, we used a mathematical model of DHNs in which we integrated the specific patterns of expression of each Cav subunits. This mathematical approach reveals that Cav1.3 is necessary for the onset of wind-up, whereas Cav1.2 is not and that synaptically triggered wind-up requires NMDA receptor activation. We then switched to a biological preparation in which we knocked down Cav subunits and confirmed the prominent role of Cav1.3 in both naive and spinal nerve ligation model of neuropathy (SNL). Interestingly, although a clear mechanical allodynia dependent on Cav1.2 expression was observed after SNL, the amplitude of wind-up was decreased. These results were confirmed with our model when adapting Cav1.3 conductance to the changes observed after SNL. Finally, our mathematical approach predicts that, although wind-up amplitude is decreased in SNL, plateau potentials are not altered, suggesting that plateau and wind-up are not fully equivalent. Wind-up and long-term hyperexcitability of DHNs are differentially controlled by Cav1.2 and Cav1.3, therefore confirming that short- and long-term sensitization are two different phenomena triggered by distinct mechanisms.

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.

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain.

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

Effect of atomoxetine on ADHD-pain hypersensitization comorbidity in 6-OHDA lesioned mice.

BACKGROUND: Methylphenidate and atomoxetine are used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Our previous studies established the validity of the 6-hydroxydopamine (6-OHDA) mouse model of ADHD and demonstrated hypersensitivity to pain, in line with clinical reports in ADHD patients. Acute methylphenidate treatment reduces hyperactivity and increases attention, but does not affect pain behaviors in this mouse model. Whereas atomoxetine has been shown to be effective against some symptoms of ADHD, nothing is known about its possible action on comorbid pain hypersensitivity. The objectives of the present research are (1) to investigate the effects of acute and chronic treatment with atomoxetine on ADHD-like symptoms and nociceptive thresholds, and (2) to explore the catecholaminergic systems underlying these effects. METHODS: Sham and 6-OHDA cohorts of male mice were tested for hyperactivity (open field), attention and impulsivity (5-choice serial reaction time task test), and thermal (hot plate test) and mechanical (von Frey test) thresholds after acute or repeated treatment with vehicle or atomoxetine (1, 3 or 10 mg/kg). RESULTS: Acute administration of atomoxetine (10 mg/kg) reduced the hyperactivity and impulsivity displayed by 6-OHDA mice, without affecting attention or nociception. However, atomoxetine administered at 3 mg/kg/day for 7 days alleviated the ADHD-like core symptoms and attenuated the hyperalgesic responses. Furthermore, hyperlocomotion and anti-hyperalgesic activity were antagonized with phentolamine, propranolol, and sulpiride pre-treatments. CONCLUSION: These findings demonstrated that when administered chronically, atomoxetine has a significant effect on ADHD-associated pain hypersensitization, likely mediated by both α- and ß-adrenergic and D2/D3 dopaminergic receptors, and suggest new indications for atomoxetine that will need to be confirmed by well-designed clinical trials.

Targeting Neuroinflammation with Abscisic Acid Reduces Pain Sensitivity in Females and Hyperactivity in Males of an ADHD Mice Model.

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental syndrome characterized by dopaminergic dysfunction. In this study, we aimed to demonstrate that there is a link between dopaminergic deficit and neuroinflammation that underlies ADHD symptoms. We used a validated ADHD mice model involving perinatal 6-OHDA lesions. The animals received abscisic acid (ABA), an anti-inflammatory phytohormone, at a concentration of 20 mg/L (drinking water) for one month. We tested a battery of behavior tests, learning and memory, anxiety, social interactions, and pain thresholds in female and male mice (control and lesioned, with or without ABA treatment). Postmortem, we analyzed microglia morphology and Ape1 expression in specific brain areas related to the descending pain inhibitory pathway. In females, the dopaminergic deficit increased pain sensitivity but not hyperactivity. In contrast, males displayed hyperactivity but showed no increased pain sensitivity. In females, pain sensitivity was associated with inflammatory microglia and lower Ape1 levels in the anterior cingulate cortex (ACC) and posterior insula cortex (IC). In addition, ABA treatment alleviated pain sensitivity concomitant with reduced inflammation and normalized APE1. In males, ABA reduced hyperactivity but had no significant effect on inflammation in these areas. This is the first study proving a sex-dependent association between dopamine dysfunction and inflammation in specific brain areas, hence leading to different behavioral outcomes in a mouse model of ADHD. These findings provide new clues for potential treatments for ADHD.

P2X4 signalling contributes to hyperactivity but not pain sensitization comorbidity in a mouse model of attention deficit/hyperactivity disorder.

Introduction: Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by hyperactivity, inattention, and impulsivity that often persist until adulthood. Frequent comorbid disorders accompany ADHD and two thirds of children diagnosed with ADHD also suffer from behavioural disorders and from alteration of sensory processing. We recently characterized the comorbidity between ADHD-like symptoms and pain sensitisation in a pharmacological mouse model of ADHD, and we demonstrated the implication of the anterior cingulate cortex and posterior insula. However, few studies have explored the causal mechanisms underlying the interactions between ADHD and pain. The implication of inflammatory mechanisms has been suggested but the signalling pathways involved have not been explored. Methods: We investigated the roles of purinergic signalling, at the crossroad of pain and neuroinflammatory pathways, by using a transgenic mouse line that carries a total deletion of the P2X4 receptor. Results: We demonstrated that P2X4 deletion prevents hyperactivity in the mouse model of ADHD. In contrast, the absence of P2X4 lowered thermal pain thresholds in sham conditions and did not affect pain sensitization in ADHD-like conditions. We further analysed microglia reactivity and the expression of inflammatory markers in wild type and P2X4KO mice. Our results revealed that P2X4 deletion limits microglia reactivity but at the same time exerts proinflammatory effects in the anterior cingulate cortex and posterior insula. Conclusion: This dual role of P2X4 could be responsible for the differential effects noted on ADHD-like symptoms and pain sensitization and calls for further studies to investigate the therapeutic benefit of targeting the P2X4 receptor in ADHD patients.

Pain mechanisms in carpal tunnel syndrome: a systematic review and meta-analysis of quantitative sensory testing outcomes.

ABSTRACT: Carpal tunnel syndrome (CTS) is the most common nerve compression in the arm. A mix of peripheral and central contributions on quantitative sensory testing (QST) has been reported in the literature. Thus, this systematic review or meta-analysis aimed to identify the dominant sensory phenotype and draw conclusive evidence about the presence of central sensitization (CS) in CTS. Based on an a priori published protocol and using PRISMA guidelines, 7 databases were searched (Embase, Web of Science, Scopus, PubMed, SAGE, EBSCOhost, and ProQuest). Eligible studies compared the QST findings of individuals with subacute and chronic CTS with those of healthy controls through thermal, mechanical, and vibration detection thresholds; thermal, pressure, and mechanical pain thresholds; mechanical pain sensitivity; presence of allodynia; wind-up ratio; and conditioned pain modulation. Thirty-seven studies were included in the qualitative analysis. Results showed a significant loss of all detection thresholds of hand median nerve territories and hand extramedian areas (little finger and hand dorsum) in CTS ( P < 0.05) but no significant difference ( P > 0.05) in wind-up ratio, cold, heat, or mechanical pain thresholds of the median nerve territories. Furthermore, there was a significant increase in mechanical pain sensitivity in median nerve territories and remotely in the forearm ( P < 0.05) and a significant gain in pressure and heat pain thresholds in the carpal area ( P < 0.05). Conditioned pain modulation was impaired in CTS. Hypoesthesia and increased thermal and mechanical pain ratings are the dominant sensory phenotype with inconclusive evidence about CS in CTS due to the heterogenous results of thermal and mechanical pain thresholds.

Detecting fine and elaborate movements with piezo sensors provides non-invasive access to overlooked behavioral components.

Behavioral phenotyping devices have been successfully used to build ethograms, but many aspects of behavior remain out of reach of available phenotyping systems. We now report on a novel device, which consists in an open-field platform resting on highly sensitive piezoelectric (electromechanical) pressure-sensors, with which we could detect the slightest movements (up to individual heart beats during rest) from freely moving rats and mice. The combination with video recordings and signal analysis based on time-frequency decomposition, clustering, and machine learning algorithms provided non-invasive access to previously overlooked behavioral components. The detection of shaking/shivering provided an original readout of fear, distinct from but complementary to behavioral freezing. Analyzing the dynamics of momentum in locomotion and grooming allowed to identify the signature of gait and neurodevelopmental pathological phenotypes. We believe that this device represents a significant progress and offers new opportunities for the awaited advance of behavioral phenotyping.

Switch of serotonergic descending inhibition into facilitation by a spinal chloride imbalance in neuropathic pain.

Descending control from the brain to the spinal cord shapes our pain experience, ranging from powerful analgesia to extreme sensitivity. Increasing evidence from both preclinical and clinical studies points to an imbalance toward descending facilitation as a substrate of pathological pain, but the underlying mechanisms remain unknown. We used an optogenetic approach to manipulate serotonin (5-HT) neurons of the nucleus raphe magnus that project to the dorsal horn of the spinal cord. We found that 5-HT neurons exert an analgesic action in naïve mice that becomes proalgesic in an experimental model of neuropathic pain. We show that spinal KCC2 hypofunction turns this descending inhibitory control into paradoxical facilitation; KCC2 enhancers restored 5-HT-mediated descending inhibition and analgesia. Last, combining selective serotonin reuptake inhibitors (SSRIs) with a KCC2 enhancer yields effective analgesia against nerve injury-induced pain hypersensitivity. This uncovers a previously unidentified therapeutic path for SSRIs against neuropathic pain.

Knockdown of L calcium channel subtypes: differential effects in neuropathic pain.

The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation-transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes Ca(V)1.2 and Ca(V)1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of Ca(V)1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-Ca(V)1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of Ca(V)1.2 mRNA and protein in neuropathic animals concomitant to specific Ca(V)1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on Ca(V)1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising Ca(V)1.2.