RESUMEN
The neuropathic pain phenotype is the consequence of functional and morphological reorganization of the PNS and CNS. This reorganization includes DRGs and the spinal cord, and extends to multiple supraspinal areas including the limbic and reward systems. Several recent papers show that acute manipulation of cortical and subcortical brain areas causally correlates with the cognitive, emotional and sensory components of neuropathic pain, yet mechanisms responsible for pain chronification remain largely unknown. Here we show that nucleus accumbens expression of ΔFos-B, a transcription factor that plays a critical role in addiction and in the brain response to stress, is reduced long term following peripheral neuropathic injury. Conversely, boosting ΔFos-B expression in the nucleus accumbens by viral transfection causes a significant and long-lasting improvement of the neuropathic allodynia. We suggest that ΔFos-B in the nucleus accumbens is a key modulator of long term gene expression leading to pain chronification.
Asunto(s)
Dolor Crónico/metabolismo , Neuralgia/metabolismo , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Dolor Crónico/fisiopatología , Dolor Crónico/psicología , Frío , Dependovirus/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Neuralgia/fisiopatología , Neuralgia/psicología , Fenotipo , Estimulación Física , Proteínas Proto-Oncogénicas c-fos/genética , Ratas Sprague-Dawley , TactoRESUMEN
In chronic pain, the medial prefrontal cortex (mPFC) is deactivated and mPFC-dependent tasks such as attention and working memory are impaired. We investigated the mechanisms of mPFC deactivation in the rat spared nerve injury (SNI) model of neuropathic pain. Patch-clamp recordings in acute slices showed that, 1 week after the nerve injury, cholinergic modulation of layer 5 (L5) pyramidal neurons was severely impaired. In cells from sham-operated animals, focal application of acetylcholine induced a left shift of the input/output curve and persistent firing. Both of these effects were almost completely abolished in cells from SNI-operated rats. The cause of this impairment was an â¼60% reduction of an M1-coupled, pirenzepine-sensitive depolarizing current, which appeared to be, at least in part, the consequence of M1 receptor internalization. Although no changes were detected in total M1 protein or transcript, both the fraction of the M1 receptor in the synaptic plasma membrane and the biotinylated M1 protein associated with the total plasma membrane were decreased in L5 mPFC of SNI rats. The loss of excitatory cholinergic modulation may play a critical role in mPFC deactivation in neuropathic pain and underlie the mPFC-specific cognitive deficits that are comorbid with neuropathic pain.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) undergoes major reorganization in chronic pain. Deactivation of mPFC output is causally correlated with both the cognitive and the sensory component of neuropathic pain. Here, we show that cholinergic excitation of commissural layer 5 mPFC pyramidal neurons is abolished in neuropathic pain rats due to a severe reduction of a muscarinic depolarizing current and M1 receptor internalization. Therefore, in neuropathic pain rats, the acetylcholine (ACh)-dependent increase in neuronal excitability is reduced dramatically and the ACh-induced persisting firing, which is critical for working memory, is abolished. We propose that the blunted cholinergic excitability contributes to the functional mPFC deactivation that is causal for the pain phenotype and represents a cellular mechanism for the attention and memory impairments comorbid with chronic pain.
Asunto(s)
Acetilcolina/metabolismo , Umbral del Dolor/fisiología , Corteza Prefrontal/metabolismo , Receptor Muscarínico M1/metabolismo , Ciática/patología , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/fisiopatología , Masculino , Picrotoxina/farmacología , Corteza Prefrontal/patología , Corteza Prefrontal/ultraestructura , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1/genética , Ciática/fisiopatología , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/patología , Transmisión Sináptica/efectos de los fármacos , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Neuropathic pain is associated with hyperexcitability and intrinsic firing of dorsal root ganglia (DRG) neurons. These phenotypical changes can be long lasting, potentially spanning the entire life of animal models, and depend on altered expression of numerous proteins, including many ion channels. Yet, how DRGs maintain long-term changes in protein expression in neuropathic conditions remains unclear. DNA methylation is a well-known mechanism of epigenetic control of gene expression and is achieved by the action of three enzymes: DNA methyltransferase (DNMT) 1, 3a, and 3b, which have been studied primarily during development. We first performed immunohistochemical analysis to assess whether these enzymes are expressed in adult rat DRGs (L4-5) and found that DNMT1 is expressed in both glia and neurons, DNMT3a is preferentially expressed in glia and DNMT3b is preferentially expressed in neurons. A rat model of neuropathic pain was then used to determine whether nerve injury may induce epigenetic changes in DRGs at multiple time points after pain onset. Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats. Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week. We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.
RESUMEN
Neuropathic pain is associated with hyperexcitability of DRG neurons. Despite the importance of leakage potassium channels for neuronal excitability, little is known about their cell-specific expression in DRGs and possible modulation in neuropathic pain. Multiple leakage channels are expressed in DRG neurons, including TASK1, TASK3, TRESK, TRAAK, TWIK1, TREK1 and TREK2 but little is known about their distribution among different cell types. Our immunohistochemical studies show robust TWIK1 expression in large and medium size neurons, without overlap with TRPV1 or IB4 staining. TASK1 and TASK3, on the contrary, are selectively expressed in small cells; TASK1 expression closely overlaps TRPV1-positive cells, while TASK3 is expressed in TRPV1- and IB4-negative cells. We also studied mRNA expression of these channels in L4-L5 DRGs in control conditions and up to 4 weeks after spared nerve injury lesion. We found that TWIK1 expression is much higher than TASK1 and TASK3 and is strongly decreased 1, 2 and 4 weeks after neuropathic injury. TASK3 expression, on the other hand, decreases 1 week after surgery but reverts to baseline by 2weeks; TASK1 shows no significant change at any time point. These data suggest an involvement of TWIK1 in the maintenance of the pain condition.
Asunto(s)
Regulación hacia Abajo , Ganglios Espinales/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Ganglios Espinales/citología , Masculino , Neuronas/clasificación , Especificidad de Órganos , Canales de Potasio de Dominio Poro en Tándem/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Febrile seizures are associated with increased brain temperature and are often resistant to treatments with antiepileptic drugs, such as carbamazepine and phenytoin, which are sodium channel blockers. Although they are clearly correlated with the hyperthermic condition, the precise cellular mechanisms of febrile seizures remain unclear. We performed patch-clamp recordings from pyramidal cells in acute rat brain slices at temperatures up to 40°C and found that, at ≥37°C, L-type calcium channels are active at unexpectedly hyperpolarized potentials and drive intrinsic firing, which is also supported by a temperature-dependent, gadolinium-sensitive sodium conductance. Pharmacological data, RT-PCR, and the current persistence in Cav1.3 knock-out mice suggested a critical contribution of Cav1.2 subunits to the temperature-dependent intrinsic firing, which was blocked by nimodipine. Because intrinsic firing may play a critical role in febrile seizures, we tested the effect of nimodipine in an in vivo model of febrile seizures and found that this drug dramatically reduces both the incidence and duration of febrile seizures in rat pups, suggesting new possibilities of intervention for this important pathological condition.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Hipocampo/patología , Células Piramidales/fisiología , Convulsiones Febriles/patología , Temperatura , Anilidas/farmacología , Animales , Animales Recién Nacidos , Cloruro de Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/genética , Cinamatos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Electroencefalografía , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Nimodipina/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Long-Evans , Convulsiones Febriles/genética , Convulsiones Febriles/prevención & controlRESUMEN
Cellular mechanisms of central pH chemosensitivity remain largely unknown. The nucleus of the solitary tract (NTS) integrates peripheral afferents with central pathways controlling breathing; NTS neurons function as central chemosensors, but only limited information exists concerning the ionic mechanisms involved. Acid-sensing ion channels (ASICs) mediate chemosensitivity in nociceptive terminals, where pH values â¼6.5 are not uncommon in inflammation, but are also abundantly expressed throughout the brain where pHi s tightly regulated and their role is less clear. Here we test the hypothesis that ASICs are expressed in NTS neurons and contribute to intrinsic chemosensitivity and control of breathing. In electrophysiological recordings from acute rat NTS slices, â¼40% of NTS neurons responded to physiological acidification (pH 7.0) with a transient depolarization. This response was also present in dissociated neurons suggesting an intrinsic mechanism. In voltage clamp recordings in slices, a pH drop from 7.4 to 7.0 induced ASIC-like inward currents (blocked by 100 µM amiloride) in â¼40% of NTS neurons, while at pH ≤ 6.5 these currents were detected in all neurons tested; RT-PCR revealed expression of ASIC1 and, less abundantly, ASIC2 in the NTS. Anatomical analysis of dye-filled neurons showed that ASIC-dependent chemosensitive cells (cells responding to pH 7.0) cluster dorsally in the NTS. Using in vivo retrograde labelling from the ventral respiratory column, 90% (9/10) of the labelled neurons showed an ASIC-like response to pH 7.0, suggesting that ASIC currents contribute to control of breathing. Accordingly, amiloride injection into the NTS reduced phrenic nerve activity of anaesthetized rats with an elevated arterial P(CO(2)) .