Chronic pain is associated with anxiety and depression episodes. The amygdala plays a key role in the relationship between emotional responses and chronic pain. Here, we investigated the role of Acid-Sensing Ion Channels 1a within the basolateral amygdala (BLA), in pain and associated anxiety in a rat model of monoarthritis (MoAr). Administration within the BLA of PcTx1 or mambalgin-1, two specific inhibitors of ASIC1a-containing channels significantly inhibited pain and anxiety-related behaviours in MoAr rats. The effect of PcTx1 was correlated with a reduction of c-Fos expression in the BLA. We examined the expression profile of ASICs and other genes in the amygdala in MoAr and sham animals, and found no variation of the expression of ASIC1a, which was confirmed at the protein level. However, an increase in the BLA of MoAr rats of both PI3Kinase mRNA and the phosphorylated form of Akt, along with Bdnf mRNA, suggest that the BDNF/PI3-kinase/Akt pathway might regulate ASIC1a in BLA neurons as demonstrated in spinal sensitisation phenomenon. We also observed changes in several kinase mRNAs expression (PICK1, Sgk1) that are potentially involved in ASIC1a regulation. These results show a crucial role of ASIC1a channels in the BLA in pain and anxiety-related behaviours during arthritis.

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change >= 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (p< 2.26 10(-7)), -24.1 (p< 5.6 10(-9)) and -17.7 (p< 1.2 10(-7)). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram.

Antidepressants remain one of the first line treatments prescribed to neuropathic pain patients despite their limited efficacy and/or their numerous side effects. More and more, pharmacotherapy for neuropathic pain has evolved towards the use of therapeutic combinations. The goal of the present study was to assess the efficacy of the combination of antidepressants - selective serotonin reuptake inhibitors and serotonin-noradrenaline reuptake inhibitors-with a peptide (TAT-2ASCV) able to disrupt the interaction between serotonin type 2A (5-HT2A) receptors and associated PDZ proteins. Mechanical hypersensitivity was assessed in sciatic nerve ligation-induced neuropathic pain in rats using paw pressure test after acute treatment with TAT-2ASCV alone or in combination with repeated treatment with fluoxetine or duloxetine or clomipramine. First, we validated the anti-hyperalgesic effect of TAT-2ASCV on mechanical hypersensitivity at the dose of 100 ng/rat (single i.t. injection). Second, using selective receptor antagonists, we found that the effect of TAT-2ASCV on mechanical hypersensitivity involves 5-HT2A as well as GABAA receptors. Finally, we showed that the association of TAT-2ASCV (100 ng, single i.t. injection) with fluoxetine (10 mg/kg, five i.p. injections) reveals its anti-hyperalgesic effect, while the association with duloxetine (1 mg/kg, five i.p. injections) or clomipramine (2.5 mg/kg, five i.p. injections) is only additive. Those results further accentuate the interest to develop small molecules acting like TAT-2ASCV in order to treat neuropathic pain as a monotherapy or in combination with antidepressants.

Histamine H3 receptors are mainly expressed on CNS neurons, particularly along the nociceptive pathways. The potential involvement of these receptors in pain processing has been suggested using H3 receptor inverse agonists.

Antidepressants are one of the first line treatments for neuropathic pain but their use is limited by the incidence and severity of side effects of tricyclics and the weak effectiveness of selective serotonin reuptake inhibitors (SSRIs). Serotonin type 2A (5-HT2A) receptors interact with PDZ proteins that regulate their functionality and SSRI efficacy to alleviate pain. We investigated whether an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT2A receptors and associated PDZ proteins would improve the treatment of traumatic neuropathic allodynia. Tactile allodynia was assessed in spinal nerve ligation-induced neuropathic pain in rats using von Frey filaments after acute treatment with TAT-2ASCV and/or 5-HT2A receptor agonist, alone or in combination with repeated treatment with fluoxetine. In vivo microdialysis was performed in order to examine the involvement of GABA in TAT-2ASCV/fluoxetine treatment-associated analgesia. TAT-2ASCV (100 ng, single i.t. injection) improved SNL-induced tactile allodynia by increasing 5-HT2A receptor responsiveness to endogenous 5-HT. Fluoxetine alone (10 mg/kg, five i.p. injections) slightly increased tactile thresholds and its co-administration with TAT-2ASCV (100 ng, single i.t. injection) further enhanced the anti-allodynic effect. This effect depends on the integrity of descending serotonergic bulbospinal pathways and spinal release of GABA. The anti-allodynic effect of fluoxetine can be enhanced by disrupting 5-HT2A receptor-PDZ protein interactions. This enhancement depends on 5-HT2A receptor activation, spinal GABA release and GABAA receptor activation.

Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS 1H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT Our study describes a decrease in cholinergic neurotransmission in the posterior insular cortex in neuropathic pain condition and the involvement of M2 receptors. Targeting these cortical muscarinic M2 receptors using central cholinomimetics could be an effective therapy for neuropathic pain treatment.

N‐methyl‐ D ‐aspartate receptor ( NMDAR ) antagonists may be given in persistent neuropathic pain, but adverse events especially with ketamine may limit their clinical use. Less central and cognitive adverse events are described with dextromethorphan and memantine. These molecules have been explored in many preclinical and clinical studies, but data are conflicting as regards neuropathic pain alleviation. Dextromethorphan and memantine have been administered to animals after spinal nerve ligation ( SNL ) to evaluate their antinociceptive/cognitive effects and associated molecular events, including the phosphorylation of several tyrosine (p T yr 1336 , p T yr 1472 ) residues in the NR 2 B NMDAR subunit. Spinal nerve ligation and sham animals received dextromethorphan (10 mg/kg, i.p.), memantine (20 mg/kg, i.p.) or saline (1 mL/kg, i.p.). These drugs were administered once symptoms of allodynia and hyperalgesia had developed. Tests were carried out before and after surgery. Tactile allodynia, mechanical hyperalgesia and spatial memory were, respectively, evaluated by von Frey, Randall & Selitto and Y‐maze tests and molecular events by Western blot analysis. Spinal nerve‐ligated animals displayed nociception and impaired spatial memory. Dextromethorphan, but not memantine, reversed neuropathic pain ( NP ) symptoms, restored spatial memory integrity and decreased the expression of p T yr 1336 NR 2 B . Following postoperative administration of dextromethorphan, this study has demonstrated for the first time a concordance between behaviour, cognitive function and molecular events via p T yr 1336 NR 2 B for neuropathic pain alleviation. Confirmation of these findings in patients would constitute a major step forward in the treatment of neuropathic pain and in the improvement of cognitive function and quality of life.

Despite common pathophysiological mechanisms, inflammatory and neuropathic pain do not respond equally to the analgesic effect of antidepressants, except for selective serotonin reuptake inhibitors (SSRIs), which show a limited efficacy in both conditions. We previously demonstrated that an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT2A receptors and its associated PDZ proteins (e.g. PSD-95) reveals a 5-HT2A receptor-mediated anti-hyperalgesic effect and enhances the efficacy of fluoxetine (a SSRI) in diabetic neuropathic pain conditions in rats. Here, we have examined whether the same strategy would be useful to treat inflammatory pain. Sub-chronic inflammatory pain was induced by injecting λ-carrageenan (100 µl, 2%) into the left hind paw of the rat. Mechanical hyperalgesia was assessed after acute treatment with TAT-2ASCV or/and fluoxetine (SSRI) 2.5 h after λ-carrageenan injection. Possible changes in the level of 5-HT2A receptors and its associated PDZ protein PSD-95 upon inflammation induction were quantified by Western blotting in dorsal horn spinal cord. Administration of TAT-2ASCV peptide (100 ng/rat, intrathecally) but not fluoxetine (10 mg/kg, intraperitoneally) relieves mechanical hyperalgesia (paw pressure test) in inflamed rats. This anti-hyperalgesic effect involves spinal 5-HT2A receptors and GABAergic interneurons as it is abolished by a 5-HT2A antagonist (M100907, 150 ng/rat, intrathecally) and a GABAA antagonist, (bicuculline, 3 µg/rat, intrathecally). We also found a decreased expression of 5-HT2A receptors in the dorsal spinal cord of inflamed animals which could not be rescued by TAT-2ASCV injection, while the amount of PSD-95 was not affected by inflammatory pain. Finally, the coadministration of fluoxetine does not further enhance the anti-hyperalgesic effect of TAT-2ASCV peptide. This study reveals a role of the interactions between 5-HT2A receptors and PDZ proteins in the pathophysiological pathways of inflammatory pain and opens new perspectives in its control thanks to molecules disrupting 5-HT2A receptor/PDZ protein interactions.

N-methyl-D-aspartate (NMDA) receptor antagonists are used for post-surgery neuropathic pain but severe side-effects limit their clinical use. Memantine, when given after surgery, shows conflicting results as regard to neuropathic pain alleviation. Here memantine is administered in animals before or after spinal nerve ligation (SNL) in order to evaluate the induced antinociceptive/cognitive effects and associated molecular events, including the phosphorylation of several tyrosine (pTyr(1336), pTyr(1472)) and serine (pSer(1303)) residues in the NR2B subunit of the NMDA receptor. Spinal nerve ligated and sham animals received memantine (20mg/kg/day) or vehicle (1ml/kg/day) by intraperitoneal route. Pre-emptive protocol started 4 days before surgery and continued for 2 days post-surgery. In the post-operative protocol, the 7 day-treatment began on the day of surgery. Tests were done before and after surgery. Tactile allodynia, mechanical hyperalgesia and spatial memory were evaluated by von Frey, Randall & Selitto and Y-maze-tests respectively, and molecular events by western-blot analysis. Spinal nerve ligated animals displayed nociception, impaired memory and increased expression of the 3 phosphorylated residues. Post-operative memantine had no beneficial effect. Pre-emptive memantine prevented the development of post-surgical nociception, impairment of spatial memory and did not increase the expression of pTyr(1472)NR2B at spinal, insular and hippocampal levels. Memantine administered a few days before surgery is a promising strategy to alleviate neuropathic pain development and impairment of cognitive function in animals. The pivotal role of pTyr(1472)NR2B must be studied further, and these findings will now be challenged in patients for the prevention of postsurgical neuropathic pain.

European Journal of Pain SupplementsVolume 5, Issue S1 p. 52-53 T258 FLUOXETINE-MEDIATED ANTI-ALLODYNIC EFFECT NEEDS UNALTERED DESCENDING SEROTONERGIC PATHWAYS AND FUNCTIONAL 5-HT2A RECEPTORS IN TRAUMA-INDUCED NEUROPATHIC PAIN A. Dupuis, A. Dupuis Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorF. Libert, F. Libert Pharmacologie, Inserm U 766, Université d'Auvergne CHU Clermont-Ferrand, Clermont-Ferrand, FranceSearch for more papers by this authorA.-S. Wattiez, A.-S. Wattiez Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorA.-M. Privat, A.-M. Privat Pharmacologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorM. Chalus, M. Chalus Pharmacologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorA. Eschalier, A. Eschalier Pharmacologie, Inserm U 766, Université d'Auvergne Pharmacologie, CHU Clermont-Ferrand, Clermont-Ferrand, FranceSearch for more papers by this authorC. Courteix, C. Courteix Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this author A. Dupuis, A. Dupuis Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorF. Libert, F. Libert Pharmacologie, Inserm U 766, Université d'Auvergne CHU Clermont-Ferrand, Clermont-Ferrand, FranceSearch for more papers by this authorA.-S. Wattiez, A.-S. Wattiez Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorA.-M. Privat, A.-M. Privat Pharmacologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorM. Chalus, M. Chalus Pharmacologie, Inserm U 766, Université d'AuvergneSearch for more papers by this authorA. Eschalier, A. Eschalier Pharmacologie, Inserm U 766, Université d'Auvergne Pharmacologie, CHU Clermont-Ferrand, Clermont-Ferrand, FranceSearch for more papers by this authorC. Courteix, C. Courteix Physiologie, Inserm U 766, Université d'AuvergneSearch for more papers by this author First published: 30 January 2012 https://doi.org/10.1016/S1754-3207(11)70176-6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume5, IssueS1September 2011Pages 52-53 RelatedInformation

Alterations in cortical excitability are implicated in the pathophysiology of migraine. However, the relationship between cortical spreading depression (CSD) and headache has not been fully elucidated. We aimed to identify the corticofugal networks that directly influence meningeal nociception in the brainstem trigeminocervical complex (Sp5C) of the rat. Cortical areas projecting to the brainstem were first identified by retrograde tracing from Sp5C areas that receive direct meningeal inputs. Anterograde tracers were then injected into these cortical areas to determine the precise pattern of descending axonal terminal fields in the Sp5C. Descending cortical projections to brainstem areas innervated by the ophthalmic branch of the trigeminal nerve originate contralaterally from insular (Ins) and primary somatosensory (S1) cortices and terminate in laminae I–II and III–V of the Sp5C, respectively. In another set of experiments, electrophysiological recordings were simultaneously performed in Ins, S1 or primary visual cortex (V1), and Sp5C neurons. KCl was microinjected into such cortical areas to test the effects of CSD on meningeal nociception. CSD initiated in Ins and S1 induced facilitation and inhibition of meningeal-evoked responses, respectively. CSD triggered in V1 affects differently Ins and S1 cortices, enhancing or inhibiting meningeal-evoked responses of Sp5C, without affecting cutaneous-evoked nociceptive responses. Our data suggest that “top-down” influences from lateralized areas within Ins and S1 selectively affect interoceptive (meningeal) over exteroceptive (cutaneous) nociceptive inputs onto Sp5C. Such corticofugal influences could contribute to the development of migraine pain in terms of both topographic localization and pain tuning during an attack.

This study analysed the organization of central nervous system networks involved in the processing of meningeal inputs in the male, Sprague-Dawley rat. We injected the anterograde tracer, biotin dextran, into areas of the medullary trigeminal nucleus caudalis (Sp5C), which receive inputs from the ophthalmic division of the trigeminal nerve. Double-labelling immunohistochemical studies were then performed to compare calcitonin gene-related peptide (CGRP) or serotonin 1D (5HT1(D)) receptor distributions in the areas innervated by Sp5C neurons. Dense, topographically organized intratrigeminal connections were observed. Sp5C neurons projected to the commissural subnucleus of the solitary tract, A5 cell group region/superior salivatory nucleus, lateral periaqueductal grey matter, inferior colliculus and parabrachial nuclei. Trigeminothalamic afferents were restricted to the posterior group and ventroposteromedial thalamic nuclei. Some of these areas are also immunoreactive for 5HT1(D) and CGRP and thus remain potential central targets of triptan molecules and other antimigraine drugs.

Sensory maps for pain can be modified by deafferentation or injury, and such plasticity has been attributed mainly to changes in the convergence of projections in "bottom-up" mechanisms. We addressed the possible contribution of "top-down" mechanisms by investigating the functional significance of corticofugal influences from the primary somatosensory cortex (S1) to the ventroposterolateral thalamic nucleus (VPL). The strong convergence of spinal and lemniscal afferents to the VPL and the close correspondence between afferents and efferents within the VPL-S1 network suggest the existence of functionally related thalamocortical circuits that are implicated in the detection of innocuous and noxious inputs. Functional characterization of single nociceptive, wide dynamic range, and non-nociceptive VPL neurons and labeling the axons and terminal fields with the juxtacellular technique showed that all three types of cells project to a restricted area, within S1. The convergence of the terminal trees of axons from VPL neurons activated by innocuous, noxious, or both inputs suggests that their inputs are not segregated into anatomically distinct regions. Microinjections within S1 were performed for pharmacological manipulation of corticofugal modulation. Glutamatergic activation of corticofugal output enhanced noxious-evoked responses and affected in a biphasic way tactile-evoked responses of VPL cells. GABA(A)-mediated depression of corticofugal output concomitantly depressed noxious and enhanced innocuous-evoked responses of VPL neurons. Microinjections of a GABA(A) antagonist on corticofugal cells enhanced noxious-evoked responses of VPL cells. Our findings demonstrate that corticofugal influences from S1 contribute to selectively modulate somatosensory submodalities at the thalamic level.

Recent evidence has been accumulated that not only spinal trigeminal nucleus caudalis (Sp5C) neurons but also spinal trigeminal nucleus oralis (Sp5O) neurons respond to noxious stimuli. It is unknown, however, whether Sp5O neurons project to supratrigeminal structures implicated in the sensory processing of orofacial nociceptive information. This study used retrograde tracing with Fluorogold in rats to investigate and compare the projections from the Sp5O and Sp5C to two major thalamic nuclei that relay ascending somatosensory information to the primary somatic sensory cortex: the ventroposteromedial thalamic nucleus (VPM) and the posterior thalamic nuclear group (Po). Results not only confirmed the existence of contralateral projections from the Sp5C to the VPM and Po, with retrogradely labelled neurons displaying a specific distribution in laminae I, III and V, they also showed consistent and similar numbers of retrogradely labelled cell bodies in the contralateral Sp5O. In addition, a topographic distribution of VPM projections from Sp5C and Sp5O was found: neurons in the dorsomedial parts of Sp5O and Sp5C projected to the medial VPM, neurons in the ventrolateral Sp5O and Sp5C projected to the lateral VPM, and neurons in intermediate parts of Sp5O and Sp5C projected to the intermediate VPM. All together, these data suggest that not only the Sp5C, but also the Sp5O relay somatosensory orofacial information from the brainstem to the thalamus. Furthermore, trigemino-VPM pathways conserve the somatotopic distribution of primary afferents found in each subnucleus. These results thus improve our understanding of trigeminal somatosensory processing and help to direct future electrophysiological investigations.

In the thalamus, noradrenergic output from the pontine nucleus locus coeruleus (LC) may actively shape the response properties of various sensory networks en route to the cortex. Little is known, however, about the involvement of ascending noradrenergic innervation of the somatosensory thalamus in the processing of nociceptive information. To address this question, we combined the study of Fos expression upon nociceptive tooth pulp stimulation in the anaesthetized rat, with the detection of retrogradely traced neurones from the somatosensory thalamus. Cell bodies labelled retrogradely from the left thalamus were observed on both sides of the LC, with an ipsilateral predominance (n=8). Electrical stimulation of the right incisor pulp (n=4) provoked a significantly stronger Fos expression (around twice) than sham surgery (n=4), in both the ipsi- and contralateral LC. Significantly larger numbers of double labelled neurones were counted in the LC of tooth-pulp-stimulated animals (representing around 30% of retrogradely labelled cells in LC) than in the LC of sham animals. They were found bilaterally, but with a clear, significant, ipsilateral (i.e. left) predominance. The present data offer an anatomical framework to understand how the LC is involved in the sensory processing of nociceptive information in the thalamus. For the first time, it is shown that nociceptive stimulation activates LC neurones projecting to the somatosensory thalamus. This suggests a new role for LC in modulating nociception within the thalamus.