RESUMO
BACKGROUND: Recent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception. RESULTS: We show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Adelta and C primary afferent-mediated sensory messages. CONCLUSION: Our results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.
Assuntos
Analgesia/métodos , Ácido Glutâmico , Neurônios/fisiologia , Ocitocina/fisiologia , Medula Espinal/citologia , Ácido gama-Aminobutírico , Animais , Eletrofisiologia , Antagonistas GABAérgicos , Ocitocina/metabolismo , Dor/tratamento farmacológico , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos WistarRESUMO
Substance P (SP) is a well-established pain messenger in the spinal cord, although its role in substantia gelatinosa (lamina II) still remains elusive. We carried out patch-clamp recordings on lamina II neurons from transverse mouse spinal cord slices (P8-12), using the selective NK1 receptor agonist [Sar9,Met(O2)11]-SP (SM-SP, 3-5 microM) in the presence of NBQX. Activation of NK1 receptors was confirmed after pre-incubation with selective NK1 antagonist L732,138 (4 microM) that consistently blocked the effects of SM-SP (nine neurons). After SM-SP challenge and spontaneous inhibitory post-synaptic current (sIPSC) analysis, 50% of recorded neurons (15 out of 30) were found to display a transient increase in frequency; in five neurons this was also associated with increase of peak amplitude. Five out of eight neurons displayed pure GABAA microM) receptor-mediated sIPSCs, whereas the remaining ones showed mixed GABAergic/glycinergic events. After miniature IPSC analysis, a significant increase in frequency was observed in three out of 14 SM-SP responsive neurons. At least four different morphological types were apparent among NK1-responsive neurons after filling with Lucifer Yellow/biocytin: fusiform with dorso-ventral dendritic arbors (i); round-to-oval with dendritic arborization mainly directed to lamina I (ii) or III (iii), and round-to-oval with dendrites sparsely distributed all around the cell body (iv). Thus, there was no correlation between morphology and electrophysiological properties of responsive neurons. Our observations provide new insights on the processing of sensory neurotransmission in spinal cord, and indicate that activation of NK1 receptors is involved in the maintenance of the inhibitory tone of substantia gelatinosa interneurons.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Receptores da Neurocinina-1/metabolismo , Substância Gelatinosa/fisiologia , Transmissão Sináptica/fisiologia , Animais , Bicuculina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Quinoxalinas/farmacologia , Substância Gelatinosa/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Neurotrophins are a well-known family of growth factors for the central and peripheral nervous systems. In the course of the last years, several lines of evidence converged to indicate that some members of the family, particularly NGF and BDNF, also participate in structural and functional plasticity of nociceptive pathways within the dorsal root ganglia and spinal cord. A subpopulation of small-sized dorsal root ganglion neurons is sensitive to NGF and responds to peripheral NGF stimulation with upregulation of BDNF synthesis and increased anterograde transport to the dorsal horn. In the latter, release of BDNF appears to modulate or even mediate nociceptive sensory inputs and pain hypersensitivity. We summarize here the status of the art on the role of neurotrophins in nociceptive pathways, with special emphasis on short-term synaptic and intracellular events that are mediated by this novel class of neuromessengers in the dorsal horn. Under this perspective we review the findings obtained through an array of techniques in naïve and transgenic animals that provide insight into the modulatory mechanisms of BDNF at central synapses. We also report on the results obtained after immunocytochemistry, in situ hybridization, and monitoring intracellular calcium levels by confocal microscopy, that led to hypothesize that also NGF might have a direct central effect in pain modulation. Although it is unclear whether or not NGF may be released at dorsal horn endings of certain nociceptors in vivo, we believe that these findings offer a clue for further studies aiming to elucidate the putative central effects of NGF and other neurotrophins in nociceptive pathways.
Assuntos
Vias Aferentes/fisiologia , Fatores de Crescimento Neural/fisiologia , Dor/metabolismo , Medula Espinal/fisiologia , Animais , Cálcio/metabolismo , Humanos , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Fatores de Crescimento Neural/química , Neurônios/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/anatomia & histologiaRESUMO
Decades after the discovery that ionic zinc is present at high levels in glutamatergic synaptic vesicles, where, when, and how much zinc is released during synaptic activity remains highly controversial. Here we provide a quantitative assessment of zinc dynamics in the synaptic cleft and clarify its role in the regulation of excitatory neurotransmission by combining synaptic recordings from mice deficient for zinc signaling with Monte Carlo simulations. Ambient extracellular zinc levels are too low for tonic occupation of the GluN2A-specific nanomolar zinc sites on NMDA receptors (NMDARs). However, following short trains of physiologically relevant synaptic stimuli, zinc transiently rises in the cleft and selectively inhibits postsynaptic GluN2A-NMDARs, causing changes in synaptic integration and plasticity. Our work establishes the rules of zinc action and reveals that zinc modulation extends beyond hippocampal mossy fibers to excitatory SC-CA1 synapses. By specifically moderating GluN2A-NMDAR signaling, zinc acts as a widespread activity-dependent regulator of neuronal circuits.
Assuntos
Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Zinco/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Transgênicos , Método de Monte CarloRESUMO
Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.
Assuntos
Neurônios/efeitos dos fármacos , Dor/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , Oligoelementos/farmacologia , Estimulação Acústica , Análise de Variância , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Força da Mão/fisiologia , Hipocampo/citologia , Histidina/genética , Técnicas In Vitro , Larva , Locomoção/efeitos dos fármacos , Locomoção/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Dor/etiologia , Dor/fisiopatologia , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Estimulação Física , Ligação Proteica/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Receptores de N-Metil-D-Aspartato/genética , Reflexo/efeitos dos fármacos , Teste de Desempenho do Rota-Rod/métodos , Serina/genética , Olfato/efeitos dos fármacos , Olfato/genética , Medula Espinal/citologia , Estatísticas não Paramétricas , Percepção do Tato/efeitos dos fármacos , Percepção do Tato/genética , Oligoelementos/uso terapêutico , Xenopus , Zinco/farmacologia , Zinco/uso terapêuticoRESUMO
The activity of GABAergic inhibitory interneurones located in lamina II of the spinal cord is of fundamental importance for the processing of peripheral nociceptive messages. We have recently shown that 3alpha-hydroxy ring A-reduced pregnane neurosteroids [3alpha5alpha-neurosteroids (3alpha5alphaNS)], potent allosteric modulators of GABA(A) receptors (GABA(A)Rs), are synthesized in the spinal cord and limit thermal hyperalgesia during inflammatory pain. Because changes in the expression of calcium-dependent protein kinases [protein kinase C (PKC)] are observed during pathological pain in the spinal cord, we examined the possible interactions between PKC and 3alpha5alphaNS at synaptic GABA(A)Rs. Using patch-clamp recordings of lamina II interneurones in the spinal cord of 15-20-day-old rats, we showed that synaptic inhibition mediated by GABA(A)Rs and its modulation by 3alpha5alphaNS in lamina II of the spinal cord largely depend on activation of PKC. Our experimental results suggested that activation of PKC locks synaptic GABA(A)Rs in a functional state precluding further positive allosteric modulation by endogenous and exogenous 3alpha5alphaNS. This effect was fully prevented by coadministration of chelerythrin, an inhibitor of PKC. Furthermore, application of chelerythrin alone rendered synaptic GABA(A)Rs hypersensitive to endogenously produced or exogenously applied 3alpha5alphaNS. These findings confirmed that there was a significant production of endogenous 3alpha5alphaNS in lamina II of the spinal cord but also indicated that PKC-dependent phosphorylation processes were tonically activated to control GABA(A)R-mediated inhibition under resting conditions. We therefore can conclude that PKC activation sets an upper limit to the functional plasticity of GABAergic transmission induced by endogenous neurosteroids.
Assuntos
Desoxicorticosterona/análogos & derivados , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Proteína Quinase C/metabolismo , Esteroides/metabolismo , Ácido gama-Aminobutírico/metabolismo , Alcaloides/farmacologia , Animais , Benzofenantridinas/farmacologia , Desoxicorticosterona/farmacologia , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Finasterida/farmacologia , GABAérgicos/farmacologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/fisiologia , Isoquinolinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Dibutirato de 12,13-Forbol/farmacologia , Ratos , Ratos Wistar , Medula Espinal/citologiaRESUMO
Inhibitory neurotransmission in spinal cord dorsal horn is mainly mediated by gamma-amino butyric acid (GABA) and glycine. By patch clamp recordings and correlative immunocytochemistry, we studied here the effect of 2 microM capsaicin-induced vanilloid receptor-1 (TRPV1) activation on IPSCs in spinal lamina II neurons from post-natal mice. Specificity was confirmed after pre-incubation with the competitive antagonist SB366791 (10 microM). After a single capsaicin pulse, an intense increase of spontaneous IPSC (sIPSC) frequency was observed in the presence of NBQX 10 microM (62/81 neurons; approximately 76%) or NBQX 10 microM + AP-5 20-100 microM (27/42 neurons; approximately 64%). Only a subpopulation (approximately 40%) of responsive neurons showed a significant amplitude increase. Seventy-two percent of the neurons displayed pure GABA(A) receptor-mediated sIPSCs, whereas the remaining ones showed mixed GABAergic/glycinergic events. After two consecutive capsaicin pulses, frequency rises were very similar, and both significantly higher than controls. When the second pulse was given in the presence of 4 microM L732,138, a selective antagonist of the substance P (SP) preferred receptor NK1, we observed a significant loss in frequency increase (63.90% with NBQX and 52.35% with NBQX + AP-5). TTX (1 microM) largely (approximately 81.5%) blocked the effect of capsaicin. These results show that TRPV1 activation on primary afferent fibers releases SP. The peptide then excites inhibitory neurons in laminae I, III and IV, leading to an increased release of GABA/glycine in lamina II via a parallel alternative pathway to glutamate.