RESUMO
Spinal cord dorsal horn inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain are poorly understood. Here, we show that a critical step in the loss of inhibitory tone is the change in the firing pattern of inhibitory parvalbumin (PV)-expressing neurons (PVNs). Our results show that PV, a calcium-binding protein, controls the firing activity of PVNs by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient for the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of the firing pattern is due to the recruitment of calcium-activated potassium (SK) channels, and blocking them during chronic pain restores normal tonic firing and alleviates chronic pain. Our findings indicate that PV is essential for controlling the firing pattern of PVNs and for preventing allodynia. Developing approaches to manipulate these mechanisms may lead to different strategies for chronic pain relief.
Assuntos
Dor Crônica , Parvalbuminas , Parvalbuminas/metabolismo , Animais , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Potenciais de Ação/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
Parvalbumin-expressing interneurons (PVINs) play a crucial role within the dorsal horn of the spinal cord by preventing touch inputs from activating pain circuits. In both male and female mice, nerve injury decreases PVINs' output via mechanisms that are not fully understood. In this study, we show that PVINs from nerve-injured male mice change their firing pattern from tonic to adaptive. To examine the ionic mechanisms responsible for this decreased output, we used a reparametrized Hodgkin-Huxley type model of PVINs, which predicted (1) the firing pattern transition is because of an increased contribution of small conductance calcium-activated potassium (SK) channels, enabled by (2) impairment in intracellular calcium buffering systems. Analyzing the dynamics of the Hodgkin-Huxley type model further demonstrated that a generalized Hopf bifurcation differentiates the two types of state transitions observed in the transient firing of PVINs. Importantly, this predicted mechanism holds true when we embed the PVIN model within the neuronal circuit model of the spinal dorsal horn. To experimentally validate this hypothesized mechanism, we used pharmacological modulators of SK channels and demonstrated that (1) tonic firing PVINs from naive male mice become adaptive when exposed to an SK channel activator, and (2) adapting PVINs from nerve-injured male mice return to tonic firing on SK channel blockade. Our work provides important insights into the cellular mechanism underlying the decreased output of PVINs in the spinal dorsal horn after nerve injury and highlights potential pharmacological targets for new and effective treatment approaches to neuropathic pain.SIGNIFICANCE STATEMENT Parvalbumin-expressing interneurons (PVINs) exert crucial inhibitory control over Aß fiber-mediated nociceptive pathways at the spinal dorsal horn. The loss of their inhibitory tone leads to neuropathic symptoms, such as mechanical allodynia, via mechanisms that are not fully understood. This study identifies the reduced intrinsic excitability of PVINs as a potential cause for their decreased inhibitory output in nerve-injured condition. Combining computational and experimental approaches, we predict a calcium-dependent mechanism that modulates PVINs' electrical activity following nerve injury: a depletion of cytosolic calcium buffer allows for the rapid accumulation of intracellular calcium through the active membranes, which in turn potentiates SK channels and impedes spike generation. Our results therefore pinpoint SK channels as potential therapeutic targets for treating neuropathic symptoms.
Assuntos
Cálcio , Neuralgia , Camundongos , Masculino , Feminino , Animais , Parvalbuminas/metabolismo , Neuralgia/metabolismo , Interneurônios/fisiologia , Corno Dorsal da Medula Espinal/metabolismoRESUMO
The NMDAR is thought to play a key role in the refinement of connectivity in developing neural circuits. Pharmacological blockade or genetic loss-of-function manipulations that prevent NMDAR function during development result in the disorganization of topographic axonal projections. However, because NMDARs contribute to overall glutamatergic neurotransmission, such loss-of-function experiments fail to adequately distinguish between the roles played by NMDARs and neural activity in general. The gliotransmitter d-serine is a coagonist of the NMDAR that is required for NMDAR channel opening, but which cannot mediate neurotransmission on its own. Here we demonstrate that acute administration of d-serine has no immediate effect on glutamate release or AMPA-mediated neurotransmission. We show that endogenous d-serine is normally present below saturating levels in the developing visual system of the Xenopus tadpole. Using an amperometric enzymatic biosensor, we demonstrate that glutamatergic activation elevates ambient endogenous d-serine levels in the optic tectum. Chronically elevating levels of d-serine promoted synaptic maturation and resulted in the hyperstabilization of developing axon branches in the tadpole visual system. Conversely, treatment with an enzyme that degrades endogenous d-serine resulted in impaired synaptic maturation. Despite the reduction in axon arbor complexity seen in d-serine-treated animals, tectal neuron visual receptive fields were expanded, suggesting a failure to prune divergent retinal inputs. Together, these findings positively implicate NMDAR-mediated neurotransmission in developmental synapse maturation and the stabilization of axonal inputs and reveal a potential role for d-serine as an endogenous modulator of circuit refinement.SIGNIFICANCE STATEMENT Activation of NMDARs is critical for the activity-dependent development and maintenance of highly organized topographic maps. d-Serine, a coagonist of the NMDAR, plays a significant role in modulating NMDAR-mediated synaptic transmission and plasticity in many brain areas. However, it remains unknown whether d-serine participates in the establishment of precise neuronal connections during development. Using an in vivo model, we show that glutamate receptor activation can evoke endogenous d-serine release, which promotes glutamatergic synapse maturation and stabilizes axonal structural and functional inputs. These results reveal a pivotal modulatory role for d-serine in neurodevelopment.
Assuntos
Axônios/fisiologia , Neurogênese/fisiologia , Neuroglia/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Sinapses/fisiologia , Animais , Neurotransmissores/metabolismo , Xenopus laevisRESUMO
Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening.
Assuntos
Cerebelo/citologia , Hipoglicemiantes/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Insulina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de GABA-A/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Animais Recém-Nascidos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Furosemida/farmacologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA-A/genética , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Fatores de Tempo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Characterizing neurons by their electrophysiological phenotypes is essential for understanding the neural basis of behavioral and cognitive functions. Technological developments have enabled the collection of hundreds of neural recordings; this calls for new tools capable of performing feature extraction efficiently. To address the urgent need for a powerful and accessible tool, we developed ElecFeX, an open-source MATLAB-based toolbox that (1) has an intuitive graphical user interface, (2) provides customizable measurements for a wide range of electrophysiological features, (3) processes large-size datasets effortlessly via batch analysis, and (4) yields formatted output for further analysis. We implemented ElecFeX on a diverse set of neural recordings; demonstrated its functionality, versatility, and efficiency in capturing electrical features; and established its significance in distinguishing neuronal subgroups across brain regions and species. ElecFeX is thus presented as a user-friendly toolbox to benefit the neuroscience community by minimizing the time required for extracting features from their electrophysiological datasets.
Assuntos
Fenômenos Eletrofisiológicos , Análise de Célula Única , Software , Fenômenos Eletrofisiológicos/fisiologia , Animais , Análise de Célula Única/métodos , Neurônios/fisiologia , Humanos , Encéfalo/fisiologia , Camundongos , RatosRESUMO
INTRODUCTION: Characterization of the incidence of spontaneous arrhythmias to identify possible drug-related effects is often an important part of the analysis in safety pharmacology studies using telemetry. METHODS: A retrospective analysis in non-clinical species with and without telemetry transmitters was conducted. Electrocardiograms (24 h) from male and female beagle dogs (n = 131), Göttingen minipigs (n = 108) and cynomolgus non-human primates (NHP; n = 78) were analyzed. RESULTS: Ventricular tachycardia (VT) was observed in 3% of the dogs but was absent in minipigs and NHPs. Ventricular fibrillation (VF) was not observed in the 3 species. Ventricular premature beats (VPBs) were more frequent during daytime and atrioventricular blocks (AVBs) were more frequent at night in all species. A limited number of animals exhibited a high arrhythmia frequency and there was no correlation between animals with higher frequency of an arrhythmia type and the frequency of other arrythmias in the same animals. Clinical chemistry or hematology parameters were not different with or without telemetry devices. NHP with a transmural left ventricular pressure (LVP) catheter exhibited a greater incidence of VPBs and PJCs compared to telemetry animals without LVP. DISCUSSION: All species were similar with regards to the frequency of ventricular ectopic beats (26-46%) while the dog seemed to have more frequent junctional complexes and AVB compared to NHP and minipigs. Arrhythmia screening may be considered during pre-study evaluations, to exclude animals with abnormally high arrhythmia incidence.
Assuntos
Arritmias Cardíacas , Telemetria , Animais , Cães , Suínos , Masculino , Feminino , Porco Miniatura , Incidência , Estudos Retrospectivos , EletrocardiografiaRESUMO
Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. We recently provided a novel perspective on the mechanisms of this symptom by showing that a simple switch in trigeminal glycine synaptic inhibition can turn touch into pain by unmasking innocuous input to superficial dorsal horn nociceptive specific neurons through a local excitatory, NMDA-dependent neural circuit involving neurons expressing the gamma isoform of protein kinase C. Here, we further investigated the clinical relevance and processing of glycine disinhibition. First, we showed that glycine disinhibition with strychnine selectively induced dynamic but not static mechanical allodynia. The induced allodynia was resistant to morphine. Second, morphine did not prevent the activation of the neural circuit underlying allodynia as shown by study of Fos expression and extracellular-signal regulated kinase phosphorylation in dorsal horn neurons. Third, in contrast to intradermal capsaicin injections, light, dynamic mechanical stimuli applied under disinhibition did not produce neurokinin 1 (NK1) receptor internalization in dorsal horn neurons. Finally, light, dynamic mechanical stimuli applied under disinhibition induced Fos expression only in neurons that did not express NK1 receptor. To summarize, the selectivity and morphine resistance of the glycine-disinhibition paradigm adequately reflect the clinical characteristics of dynamic mechanical allodynia. The present findings thus reveal the involvement of a selective dorsal horn circuit in dynamic mechanical allodynia, which operates through superficial lamina nociceptive-specific neurons that do not bear NK1 receptor and provide an explanation for the differences in the pharmacological sensitivity of neuropathic pain symptoms.
Assuntos
Analgésicos Opioides/administração & dosagem , Glicina/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Morfina/administração & dosagem , Limiar da Dor/fisiologia , Receptores da Neurocinina-1/fisiologia , Animais , Bicuculina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Capsaicina/farmacologia , Modelos Animais de Doenças , Resistência a Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Antagonistas GABAérgicos/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Dinâmica não Linear , Proteínas Oncogênicas v-fos/metabolismo , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Estimulação Física/métodos , Células do Corno Posterior/metabolismo , Ratos , Ratos Sprague-Dawley , EstricninaRESUMO
INTRODUCTION: Pre-clinically, safety risk assessment of a drug is primarily tested in vivo using functional evaluation of adult animals while the mechanistic etiology of drug-induced CNS adverse effects is often uncharacterized. In vitro electrophysiology may provide a better understanding of drug effects without additional animal use. However, in vitro protocols are typically designed for using embryonic or juvenile animals. METHODS: We examined whether brain tissue isolated from adult rats (3-5 months old) and adult non-human primates (NHPs) (2-8 years old) can generate qualitatively equivalent readouts for electrophysiology to characterize AMPAR synaptic and single channel currents. We used a known positive AMPAR allosteric modulator (LY451395) to template a response profile and provide proof-of-concept data to assess responses of these native AMPARs in a drug context. RESULTS: Brain slices from adult animals provided a support to measure AMPAR-driven excitatory post-synaptic currents (EPSCs), and can be dissociated into primary neuronal cultures for AMPAR single channel characterization. Additionally, similarities and differences in AMPAR basal kinetics and responses to LY451395 were seen between the two animal species. DISCUSSION: Glutamatergic synaptic activity and AMPAR biophysical properties in adult animals may be used to characterize test-article-mediated alterations in CNS responses. The use of older animals opens the possibility for in vivo test-article administration, either acutely or repeatedly, before in vitro electrophysiological assessment in order to reveal cumulative or delayed-onset effects, adding versatility to safety pharmacology assessment of the CNS.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Primatas/metabolismo , Primatas/fisiologia , Animais , Células Cultivadas , Eletrofisiologia/métodos , Feminino , Masculino , Técnicas de Patch-Clamp/métodos , Ratos , Receptores de AMPARESUMO
Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl(-) levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl(-) regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Sensibilidades de Contraste/fisiologia , Endocanabinoides/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Células Ganglionares da Retina/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Cloretos/metabolismo , Regulação da Expressão Gênica , Transporte de Íons , Larva/fisiologia , Potenciais da Membrana/fisiologia , Imagem Molecular , Técnicas de Patch-Clamp , Receptor CB1 de Canabinoide/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Ganglionares da Retina/citologia , Transdução de Sinais , Membro 2 da Família 12 de Carreador de Soluto/genética , Visão Ocular/fisiologia , Xenopus laevis/fisiologiaRESUMO
The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.
Assuntos
Cálcio/análise , Proteínas Luminescentes/química , Imagem Óptica , Engenharia de Proteínas , Prótons , Animais , Células HeLa , Humanos , Indicadores e Reagentes , Microscopia Confocal , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Proteína Vermelha FluorescenteRESUMO
Bone cancer pain is a common and disruptive symptom in cancer patients. In cancer pain animal models, massive reactive astrogliosis in the dorsal horn of the spinal cord has been reported. Because astrocytes may behave as driving partners for pathological pain, we investigated the temporal development of pain behavior and reactive astrogliosis in a rat bone cancer pain model induced by injecting MRMT-1 rat mammary gland carcinoma cells into the tibia. Along with the development of bone lesions, a gradual mechanical and thermal allodynia and hyperalgesia as well as a reduced use of the affected limb developed in bone cancer-bearing animals, but not in sham-treated animals. Dorsal horn Fos expression after nonpainful palpation of the injected limb was also increased in bone cancer-bearing animals. However, at any time during the evolution of tumor, there was no increase in glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal horn. Further analysis at 21days after injection of the tumor showed no increase in GFAP and interleukin (IL) 1ß transcripts, number of superficial dorsal horn S100ß protein immunoreactive astrocytes, or immunoreactivity for microglial markers (OX-42 and Iba-1). In contrast, all these parameters were increased in the dorsal horn of rats 2weeks after sciatic nerve ligation. This suggests that in some cases, bone cancer pain may not be correlated with spinal overexpression of reactive glia markers, whereas neuropathic pain is. Glia may thus play different roles in the development and maintenance of chronic pain in these 2 situations.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Neuroglia/metabolismo , Medição da Dor/métodos , Dor/metabolismo , Medula Espinal/metabolismo , Animais , Neoplasias Ósseas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Neuroglia/patologia , Dor/patologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/patologia , Células Tumorais CultivadasRESUMO
The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function.
Assuntos
Sensação/fisiologia , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Neurônios GABAérgicos/metabolismo , Larva/citologia , Larva/metabolismo , Estimulação Luminosa , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Colículos Superiores/crescimento & desenvolvimento , Colículos Superiores/metabolismo , Vias Visuais/citologia , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
Glycine inhibitory dysfunction provides a useful experimental model for studying the mechanism of dynamic mechanical allodynia, a widespread and intractable symptom of neuropathic pain. In this model, allodynia expression relies on N-methyl-d-aspartate receptors (NMDARs), and it has been shown that astrocytes can regulate their activation through the release of the NMDAR coagonist d-serine. Recent studies also suggest that astrocytes potentially contribute to neuropathic pain. However, the involvement of astrocytes in dynamic mechanical allodynia remains unknown. Here, we show that after blockade of glycine inhibition, orofacial tactile stimuli activated medullary dorsal horn (MDH) astrocytes, but not microglia. Accordingly, the glia inhibitor fluorocitrate, but not the microglia inhibitor minocycline, prevented allodynia. Fluorocitrate also impeded activation of astrocytes and blocked activation of the superficial MDH neural circuit underlying allodynia, as revealed by study of Fos expression. MDH astrocytes are thus required for allodynia. They may also produce d-serine because astrocytic processes were selectively immunolabeled for serine racemase, the d-serine synthesizing enzyme. Accordingly, selective degradation of d-serine with d-amino acid oxidase applied in vivo prevented allodynia and activation of the underlying neural circuit. Conversely, allodynia blockade by fluorocitrate was reversed by exogenous d-serine. These results suggest the following scenario: removal of glycine inhibition makes tactile stimuli able to activate astrocytes; activated astrocytes may provide d-serine to enable NMDAR activation and thus allodynia. Such a contribution of astrocytes to pathological pain fuels the emerging concept that astrocytes are critical players in pain signaling. Glycine disinhibition makes tactile stimuli able to activate astrocytes, which may provide d-serine to enable NMDA receptor activation and thus allodynia.
Assuntos
Citratos/uso terapêutico , Glicina/metabolismo , Hiperalgesia/tratamento farmacológico , Serina/metabolismo , Análise de Variância , Animais , Astrócitos/química , Astrócitos/efeitos dos fármacos , Antígeno CD11b/metabolismo , Citratos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Proteínas Oncogênicas v-fos/metabolismo , Medição da Dor , Ratos , Ratos Sprague-Dawley , Raízes Nervosas Espinhais/citologia , Estricnina/uso terapêutico , Fatores de TempoRESUMO
Windup is a progressive, frequency-dependent increase in the excitability of trigeminal and spinal dorsal horn wide dynamic range (WDR) nociceptive neurons to repetitive stimulation of primary afferent nociceptive C-fibers. Superficial dorsal horn neurokinin 1 receptor (NK1R)-expressing neurons were recently shown to regulate sensitization of WDR nociceptive neurons through activation of a defined spino-bulbo-spinal loop. However, the windup of WDR nociceptive neurons was not regulated through this loop. In the present study, we sought to identify the alternative circuit activated by dorsal horn NK1Rs that mediates WDR neuron windup. As a model we used the rat spinal trigeminal nucleus, in which the subnucleus oralis (Sp5O) contains a pool of WDR neurons that receive their nociceptive C-input indirectly via interneurons located in the medullary dorsal horn (MDH). First, we found that intravenous injection of NK1R antagonists (SR140333 and RP67580) produced a reversible inhibition of Sp5O WDR neuron windup. Second, we anatomically identified in the MDH lamina III a subpopulation of NK1R-expressing local interneurons that relay nociceptive information from the MDH to downstream Sp5O neurons. Third, using microinjections of NK1R antagonists during in vivo electrophysiological recordings from Sp5O WDR neurons, we showed that WDR neuron windup depends on activation of NK1Rs located in the MDH laminae I-III. We conclude that, in contrast to central sensitization that is controlled by a spino-bulbo-spinal loop, Sp5O WDR neuron windup is regulated through a local circuit activated by MDH lamina III NK1Rs.
Assuntos
Vias Aferentes/metabolismo , Nociceptores/metabolismo , Dor/metabolismo , Células do Corno Posterior/metabolismo , Receptores da Neurocinina-1/metabolismo , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Vias Aferentes/citologia , Animais , Masculino , Fibras Nervosas Amielínicas/metabolismo , Fibras Nervosas Amielínicas/ultraestrutura , Vias Neurais/citologia , Vias Neurais/metabolismo , Antagonistas dos Receptores de Neurocinina-1 , Neurônios Aferentes/citologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Nociceptores/citologia , Dor/fisiopatologia , Piperidinas/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Quinuclidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Substância P/metabolismo , Transmissão Sináptica/fisiologia , Núcleo Inferior Caudal do Nervo Trigêmeo/citologia , Nervo Trigêmeo/citologia , Nervo Trigêmeo/metabolismo , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/metabolismoRESUMO
Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. During tactile allodynia, activation of the sensory fibers which normally detect touch elicits pain. Here we provide a new behavioral investigation into the dynamic component of tactile allodynia that developed in rats after segmental removal of glycine inhibition. Using in vivo electrophysiological recordings, we show that in this condition innocuous mechanical stimuli could activate superficial dorsal horn nociceptive specific neurons. These neurons do not normally respond to touch. We anatomically show that the activation was mediated through a local circuit involving neurons expressing the gamma isoform of protein kinase C (PKCgamma). Selective inhibition of PKCgamma as well as selective blockade of glutamate NMDA receptors in the superficial dorsal horn prevented both activation of the circuit and allodynia. Thus, our data demonstrates that a normally inactive circuit in the dorsal horn can be recruited to convert touch into pain. It also provides evidence that glycine inhibitory dysfunction gates tactile input to nociceptive specific neurons through PKCgamma-dependent activation of a local, excitatory, NMDA receptor-dependent, circuit. As a consequence of these findings, we suggest that pharmacological inhibition of PKCgamma might provide a new tool for alleviating allodynia in the clinical setting.