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1.
J Neurosci ; 42(10): 1930-1944, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35058371

RESUMO

We aimed to investigate a sexually dimorphic role of calcitonin gene-related peptide (CGRP) in rodent models of pain. Based on findings in migraine where CGRP has a preferential pain-promoting effect in female rodents, we hypothesized that CGRP antagonists and antibodies would attenuate pain sensitization more efficaciously in female than male mice and rats. In hyperalgesic priming induced by activation of interleukin 6 signaling, CGRP receptor antagonists olcegepant and CGRP8-37 both given intrathecally, blocked, and reversed hyperalgesic priming only in females. A monoclonal antibody against CGRP, given systemically, blocked priming specifically in female rodents but failed to reverse it. In the spared nerve injury model, there was a transient effect of both CGRP antagonists, given intrathecally, on mechanical hypersensitivity in female mice only. Consistent with these findings, intrathecally applied CGRP caused a long-lasting, dose-dependent mechanical hypersensitivity in female mice but more transient effects in males. This CGRP-induced mechanical hypersensitivity was reversed by olcegepant and the KCC2 enhancer CLP257, suggesting a role for anionic plasticity in the dorsal horn in the pain-promoting effects of CGRP in females. In spinal dorsal horn slices, CGRP shifted GABAA reversal potentials to significantly more positive values, but, again, only in female mice. Therefore, CGRP may regulate KCC2 expression and/or activity downstream of CGRP receptors specifically in females. However, KCC2 hypofunction promotes mechanical pain hypersensitivity in both sexes because CLP257 alleviated hyperalgesic priming in male and female mice. We conclude that CGRP promotes pain plasticity in female rodents but has a limited impact in males.SIGNIFICANCE STATEMENT The majority of patients impacted by chronic pain are women. Mechanistic studies in rodents are creating a clear picture that molecular events promoting chronic pain are different in male and female animals. We sought to build on evidence showing that CGRP is a more potent and efficacious promoter of headache in female than in male rodents. To test this, we used hyperalgesic priming and the spared nerve injury neuropathic pain models in mice. Our findings show a clear sex dimorphism wherein CGRP promotes pain in female but not male mice, likely via a centrally mediated mechanism of action. Our work suggests that CGRP receptor antagonists could be tested for efficacy in women for a broader variety of pain conditions.


Assuntos
Dor Crônica , Simportadores , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/efeitos adversos , Feminino , Humanos , Hiperalgesia/metabolismo , Masculino , Camundongos , Ratos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Roedores
2.
Brain ; 145(3): 1124-1138, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35323848

RESUMO

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.


Assuntos
Dor Crônica , Simportadores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Humanos , Masculino , Neurônios/metabolismo , Ratos , Caracteres Sexuais
3.
Brain ; 142(6): 1535-1546, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31135041

RESUMO

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.


Assuntos
Neuralgia/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Adolescente , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuralgia/fisiopatologia , Fosforilação , Ratos , Receptores de N-Metil-D-Aspartato/genética , Sinapses/metabolismo , Adulto Jovem
4.
J Neurosci Res ; 95(6): 1307-1318, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27792253

RESUMO

Neuronal inhibition mediated by GABAA receptors constrains nociceptive processing in the spinal cord, and loss of GABAergic inhibition can produce allodynia and hyperalgesia. Extrasynaptic α5 subunit-containing GABAA receptors (α5GABAA Rs) generate a tonic conductance that inhibits neuronal activity and constrains learning and memory; however, it is unclear whether α5GABAA Rs similarly generate a tonic conductance in the spinal cord dorsal horn to constrain nociception. We assessed the distribution of α5GABAA Rs in the spinal cord dorsal horn by immunohistochemical analysis, and the activity and function of α5GABAA Rs in neurons of the superficial dorsal horn using electrophysiological and behavioral approaches in male, null-mutant mice lacking the GABAA R α5 subunit (Gabra5-/-) and wild-type mice (WT). The expression of α5GABAA Rs in the superficial dorsal horn followed a laminar pattern of distribution, with a higher expression in lamina II than lamina I. Similarly, the tonic GABAA current in lamina II neurons had a larger contribution from α5GABAA Rs than in lamina I, with no significant contribution of these receptors to synaptic GABAA current. In behavioural tests, WT and Gabra5-/- mice exhibited similar acute thermal and mechanical nociception, and similar mechanical sensitization immediately following intraplantar capsaicin or Complete Freund's Adjuvant (CFA). However, Gabra5-/- mice showed prolonged recovery from sensitization in these models, and increased responses in the late phase of the formalin test. Overall, our data suggest that tonically-active α5GABAA Rs in the spinal cord dorsal horn accelerate the resolution of hyperalgesia and may therefore serve as a novel therapeutic target to promote recovery from pathological pain. © 2016 Wiley Periodicals, Inc.


Assuntos
Hiperalgesia/genética , Hiperalgesia/patologia , Inibição Neural/genética , Receptores de GABA-A/metabolismo , Corno Dorsal da Medula Espinal/fisiologia , Animais , Bicuculina/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/toxicidade , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , GABAérgicos/farmacologia , Hiperalgesia/induzido quimicamente , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Lectinas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Medição da Dor , Estimulação Física/efeitos adversos , Receptores de GABA-A/genética , Corno Dorsal da Medula Espinal/metabolismo
5.
J Neurosci ; 34(24): 8300-17, 2014 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-24920633

RESUMO

Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and ß3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R ß3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios Aferentes/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA-A/metabolismo , Medula Espinal/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Lectinas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Glicina/metabolismo
6.
Mol Pain ; 10: 57, 2014 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-25189404

RESUMO

Inhibitory interneurons are an important component of dorsal horn circuitry where they serve to modulate spinal nociception. There is now considerable evidence indicating that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain. A loss of these inhibitory neurons after nerve injury is one of the mechanisms being proposed to account for reduced inhibition; however, this remains controversial. This is in part because previous studies have focused on global measurements of inhibitory neurons without assessing the number of inhibitory synapses. To address this, we conducted a quantitative analysis of the spatial and temporal changes in the number of inhibitory terminals, as detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity, in the superficial dorsal horn of the spinal cord following a chronic constriction injury (CCI) to the sciatic nerve in rats. Isolectin B4 (IB4) labelling was used to define the location within the dorsal horn directly affected by the injury to the peripheral nerve. The density of GAD65 inhibitory terminals was reduced in lamina I (LI) and lamina II (LII) of the spinal cord after injury. The loss of GAD65 terminals was greatest in LII with the highest drop occurring around 3-4 weeks and a partial recovery by 56 days. The time course of changes in the number of GAD65 terminals correlated well with both the loss of IB4 labeling and with the altered thresholds to mechanical and thermal stimuli. Our detailed analysis of GAD65+ inhibitory terminals clearly revealed that nerve injury induced a transient loss of GAD65 immunoreactive terminals and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour.


Assuntos
Glutamato Descarboxilase/metabolismo , Inibição Neural/fisiologia , Células do Corno Posterior/enzimologia , Neuropatia Ciática/patologia , Corno Dorsal da Medula Espinal/patologia , Análise de Variância , Animais , Modelos Animais de Doenças , Lateralidade Funcional/fisiologia , Hiperalgesia/etiologia , Lectinas/metabolismo , Masculino , Ratos , Ratos Wistar , Neuropatia Ciática/complicações , Fatores de Tempo
7.
Proc Natl Acad Sci U S A ; 108(17): 7010-5, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21482753

RESUMO

Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA(B) receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 µm(2)) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.


Assuntos
Simulação por Computador , Multimerização Proteica/fisiologia , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo
8.
Sci Adv ; 9(40): eadi8750, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37792939

RESUMO

Early-life adversities are associated with altered defensive responses. Here, we demonstrate that the repeated cross-fostering (RCF) paradigm of early maternal separation is associated with enhancements of distinct homeostatic reactions: hyperventilation in response to hypercapnia and nociceptive sensitivity, among the first generation of RCF-exposed animals, as well as among two successive generations of their normally reared offspring, through matrilineal transmission. Parallel enhancements of acid-sensing ion channel 1 (ASIC1), ASIC2, and ASIC3 messenger RNA transcripts were detected transgenerationally in central neurons, in the medulla oblongata, and in periaqueductal gray matter of RCF-lineage animals. A single, nebulized dose of the ASIC-antagonist amiloride renormalized respiratory and nociceptive responsiveness across the entire RCF lineage. These findings reveal how, following an early-life adversity, a biological memory reducible to a molecular sensor unfolds, shaping adaptation mechanisms over three generations. Our findings are entwined with multiple correlates of human anxiety and pain conditions and suggest nebulized amiloride as a therapeutic avenue.


Assuntos
Amilorida , Privação Materna , Animais , Humanos , Amilorida/farmacologia , RNA Mensageiro , Ansiedade
9.
Sci Adv ; 8(30): eabo0689, 2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35895817

RESUMO

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.

10.
Nat Commun ; 11(1): 3935, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769979

RESUMO

GABAA/glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl-) concentration which is mainly regulated by the K+-Cl- co-transporter 2 (KCC2) in the adult central nervous system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl- extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl- load. Under high Cl- load or heightened synaptic drive, lower Cl- extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl- lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl- extrusion across laminae critically shapes plasticity for selective nociceptive modalities.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Cloretos/metabolismo , Plasticidade Neuronal/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/fisiologia , Animais , Células Cultivadas , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Neurológicos , Optogenética , Cultura Primária de Células , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptor trkB/antagonistas & inibidores , Receptor trkB/metabolismo , Simportadores/metabolismo , Cotransportadores de K e Cl-
11.
Nat Commun ; 11(1): 869, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054836

RESUMO

Spinal disinhibition has been hypothesized to underlie pain hypersensitivity in neuropathic pain. Apparently contradictory mechanisms have been reported, raising questions on the best target to produce analgesia. Here, we show that nerve injury is associated with a reduction in the number of inhibitory synapses in the spinal dorsal horn. Paradoxically, this is accompanied by a BDNF-TrkB-mediated upregulation of synaptic GABAARs and by an α1-to-α2GABAAR subunit switch, providing a mechanistic rationale for the analgesic action of the α2,3GABAAR benzodiazepine-site ligand L838,417 after nerve injury. Yet, we demonstrate that impaired Cl- extrusion underlies the failure of L838,417 to induce analgesia at high doses due to a resulting collapse in Cl- gradient, dramatically limiting the benzodiazepine therapeutic window. In turn, enhancing KCC2 activity not only potentiated L838,417-induced analgesia, it rescued its analgesic potential at high doses, revealing a novel strategy for analgesia in pathological pain, by combined targeting of the appropriate GABAAR-subtypes and restoring Cl- homeostasis.


Assuntos
Analgésicos/farmacologia , Cloretos/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Receptores de GABA-A/fisiologia , Analgesia/métodos , Analgésicos/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Fluorbenzenos/metabolismo , Fluorbenzenos/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Transporte de Íons/efeitos dos fármacos , Ligantes , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptor trkB/metabolismo , Simportadores/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Triazóis/metabolismo , Triazóis/farmacologia , Cotransportadores de K e Cl-
12.
iScience ; 23(10): 101552, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33083721

RESUMO

Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated de novo mutation (p.Cys188Trp) in the GABAA receptor Cl- channel γ-1 subunit (GABRG1) exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na+ and Ca+ channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca2+ channel Cav3.2 (CACNA1H). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis.

13.
Mol Pain ; 5: 24, 2009 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-19432997

RESUMO

Local inhibitory interneurons in the dorsal horn play an important role in the control of excitability at the segmental level and thus determine how nociceptive information is relayed to higher structures. Regulation of inhibitory interneuron activity may therefore have critical consequences on pain perception. Indeed, disinhibition of dorsal horn neuronal networks disrupts the balance between excitation and inhibition and is believed to be a key mechanism underlying different forms of pain hypersensitivity and chronic pain states. In this context, studying the source and the synaptic properties of the inhibitory inputs that the inhibitory interneurons receive is important in order to predict the impact of drug action at the network level. To address this, we studied inhibitory synaptic transmission in lamina II inhibitory interneurons identified under visual guidance in spinal slices taken from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the GAD promoter. The majority of these cells fired tonically to a long depolarizing current pulse. Monosynaptically evoked inhibitory postsynaptic currents (eIPSCs) in these cells were mediated by both GABAA and glycine receptors. Consistent with this, both GABAA and glycine receptor-mediated miniature IPSCs were recorded in all of the cells. These inhibitory inputs originated at least in part from local lamina II interneurons as verified by simultaneous recordings from pairs of EGFP+ cells. These synapses appeared to have low release probability and displayed potentiation and asynchronous release upon repeated activation. In summary, we report on a previously unexamined component of the dorsal horn circuitry that likely constitutes an essential element of the fine tuning of nociception.


Assuntos
Interneurônios/fisiologia , Inibição Neural/fisiologia , Células do Corno Posterior/metabolismo , Medula Espinal/metabolismo , Animais , Proteínas de Fluorescência Verde/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transmissão Sináptica/fisiologia
14.
Cell Rep ; 28(3): 590-596.e4, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315039

RESUMO

The behavioral features of neuropathic pain are not sexually dimorphic despite sex differences in the underlying neuroimmune signaling. This raises questions about whether neural processing is comparably altered. Here, we test whether the K+-Cl- co-transporter KCC2, which regulates synaptic inhibition, plays an equally important role in development of neuropathic pain in male and female rodents. Past studies on KCC2 tested only males. We find that inhibiting KCC2 in uninjured animals reproduces behavioral and electrophysiological features of neuropathic pain in both sexes and, consistent with equivalent injury-induced downregulation of KCC2, that counteracting chloride dysregulation reverses injury-induced behavioral and electrophysiological changes in both sexes. These findings demonstrate that KCC2 downregulation contributes equally to pain hypersensitivity in males and females. Whereas diverse (and sexually dimorphic) mechanisms regulate KCC2, regulation of intracellular chloride relies almost exclusively on KCC2. Directly targeting KCC2 thus remains a promising strategy for treatment of neuropathic pain in both sexes.


Assuntos
Cloretos/metabolismo , Neuralgia/metabolismo , Células do Corno Posterior/metabolismo , Medula Espinal/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Acetazolamida/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Regulação para Baixo , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/genética , Neuralgia/fisiopatologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/fisiologia , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/cirurgia , Simportadores/genética , Tiazóis/farmacologia , Tioglicolatos/farmacologia , Cotransportadores de K e Cl-
15.
Sci Adv ; 5(6): eaav9946, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31223654

RESUMO

Neuropathic pain is a major incurable clinical problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mechanical allodynia. Using sortilin-deficient mice or receptor inhibition by antibodies or a small-molecule antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clinically relevant target.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neuralgia/metabolismo , Neurotensina/metabolismo , Animais , Regulação para Baixo/fisiologia , Feminino , Humanos , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos dos Nervos Periféricos/metabolismo , Receptores de Neurotensina/metabolismo , Transdução de Sinais/fisiologia
16.
J Comp Neurol ; 504(2): 112-26, 2007 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-17626281

RESUMO

Premotor inhibitory neurons responsible for the decrease in the firing discharge during fast or slow eye movements selectively target the cell bodies and the dendrites of abducens motoneurons. Gamma-aminobutyric acid (GABA) and glycine, the main inhibitory synaptic neurotransmitters in the central nervous system, act via glycine and GABAA receptors, assembled from various types of subunits, which determine the kinetics of the currents mediated. Therefore, our hypothesis was that the expression of the inhibitory receptors on the somatic and the dendritic compartments, involved in different functions, may differ. In this study, we compared the subcellular patterns of expression of the main GABAA receptor subunits (GABAARalpha1, alpha2, alpha3, alpha5), glycine receptors (GlyRalpha1), and gephyrin in the somatic and dendritic compartments of rat abducens motoneurons, using double or triple immunocytochemical experiments with confocal microscopy. Significant differences exist in the patterns of organization and the synaptic expression of the GlyR and GABAAR subunits in the cell bodies and dendrites of abducens motoneurons. In the somata, only the GABAARalpha1 subunit was expressed, whereas both GABAARalpha1 and GABAARalpha3 were present in the dendrites. The GlyRalpha1 to GABAARalpha1 density ratio was reversed in the somatic and dendritic compartments (0.9 vs. 2.3). A quantitative electron microscopy study showed that the modes whereby gephyrin reaches its postsynaptic inhibitory synaptic target differ between the somata and the dendrites. Therefore, our results support the idea that a structure-function adaptation occurs at the single-neuron level.


Assuntos
Nervo Abducente/metabolismo , Neurônios Motores/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Nervo Abducente/citologia , Animais , Transporte Biológico , Dendritos/metabolismo , Espaço Intracelular/metabolismo , Masculino , Ratos , Ratos Wistar , Distribuição Tecidual
17.
Sci Rep ; 7(1): 3870, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28634406

RESUMO

Morphine-induced hyperalgesia (MIH) is a severe adverse effect accompanying repeated morphine treatment, causing a paradoxical decrease in nociceptive threshold. Previous reports associated MIH with a decreased expression of the Cl- extruder KCC2 in the superficial dorsal horn (SDH) of the spinal cord, weakening spinal GABAA/glycine-mediated postsynaptic inhibition. Here, we tested whether the administration of small molecules enhancing KCC2, CLP257 and its pro-drug CLP290, may counteract MIH. MIH was typically expressed within 6-8 days of morphine treatment. Morphine-treated rats exhibited decreased withdrawal threshold to mechanical stimulation and increased vocalizing behavior to subcutaneous injections. Chloride extrusion was impaired in SDH neurons measured as a depolarizing shift in E GABA under Cl- load. Delivering CLP257 to spinal cord slices obtained from morphine-treated rats was sufficient to restore Cl- extrusion capacity in SDH neurons. In vivo co-treatment with morphine and oral CLP290 prevented membrane KCC2 downregulation in SDH neurons. Concurrently, co-treatment with CLP290 significantly mitigated MIH and acute administration of CLP257 in established MIH restored normal nociceptive behavior. Our data indicate that enhancing KCC2 activity is a viable therapeutic approach for counteracting MIH. Chloride extrusion enhancers may represent an effective co-adjuvant therapy to improve morphine analgesia by preventing and reversing MIH.


Assuntos
Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Morfina/efeitos adversos , Simportadores/metabolismo , Animais , Fenômenos Eletrofisiológicos , Expressão Gênica , Masculino , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Ratos , Simportadores/genética , Tiazolidinas/farmacologia
18.
Pain ; 158(9): 1792-1801, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28746078

RESUMO

While spinal microglia play a role in early stages of neuropathic pain etiology, whether they are useful targets to reverse chronic pain at late stages remains unknown. Here, we show that microglia activation in the spinal cord persists for >3 months following nerve injury in rodents, beyond involvement of proinflammatory cytokine and chemokine signalling. In this chronic phase, selective depletion of spinal microglia in male rats with the targeted immunotoxin Mac1-saporin and blockade of brain-derived neurotrophic factor-TrkB signalling with intrathecal TrkB Fc chimera, but not cytokine inhibition, almost completely reversed pain hypersensitivity. By contrast, local spinal administration of Mac1-saporin did not affect nociceptive withdrawal threshold in control animals nor did it affect the strength of afferent-evoked synaptic activity in the spinal dorsal horn in normal conditions. These findings show that the long-term, chronic phase of nerve injury-induced pain hypersensitivity is maintained by microglia-neuron interactions. The findings also effectively separate the central signalling pathways underlying the maintenance phase of the pathology from the early and peripheral inflammatory reactions to injury, pointing to different targets for the treatment of acute vs chronic injury-induced pain.


Assuntos
Citocinas/metabolismo , Microglia/fisiologia , Neuralgia/patologia , Transdução de Sinais/fisiologia , Medula Espinal/patologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cicloexanóis/farmacologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Oximas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Transdução de Sinais/efeitos dos fármacos
19.
Nat Commun ; 7: 11636, 2016 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-27198043

RESUMO

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.


Assuntos
Fotodegradação , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Animais , Cães , Genômica/métodos , Humanos , Lasers , Células Madin Darby de Rim Canino
20.
Nat Neurosci ; 16(2): 183-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23292683

RESUMO

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.


Assuntos
Cloretos/metabolismo , Homeostase/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Microglia/efeitos dos fármacos , Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Neurônios/efeitos dos fármacos , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Temperatura Alta/efeitos adversos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/fisiologia , Atividade Motora/efeitos dos fármacos , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Limiar da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Teste de Desempenho do Rota-Rod , Saporinas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Medula Espinal/citologia , Simportadores/metabolismo , Fatores de Tempo , Tato , Vocalização Animal/efeitos dos fármacos , Cotransportadores de K e Cl-
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