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The perception of pain is a multidimensional sensory and emotional/affective experience arising from distributed brain activity. However, the involved brain regions are not specific for pain. Thus, how the cortex distinguishes nociception from other aversive and salient sensory stimuli remains elusive. Additionally, the resulting consequences of chronic neuropathic pain on sensory processing have not been characterized. Using in vivo miniscope calcium imaging with cellular resolution in freely moving mice, we elucidated the principles of nociceptive and sensory coding in the anterior cingulate cortex, a region essential for pain processing. We found that population activity, not single-cell responses, allowed discriminating noxious from other sensory stimuli, ruling out the existence of nociception-specific neurons. Additionally, single-cell stimulus selectivity was highly dynamic over time, but stimulus representation at the population level remained stable. Peripheral nerve injury-induced chronic neuropathic pain led to dysfunctional encoding of sensory events by exacerbation of responses to innocuous stimuli and impairment of pattern separation and stimulus classification, which were restored by analgesic treatment. These findings provide a novel interpretation for altered cortical sensory processing in chronic neuropathic pain and give insights into the effects of systemic analgesic treatment in the cortex.
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Giro del Cíngulo , Neuralgia , Humanos , Ratones , Animales , Giro del Cíngulo/diagnóstico por imagen , Nocicepción/fisiología , Encéfalo , NociceptoresRESUMEN
GABAergic neurons and GABAA receptors (GABAARs) are critical elements of almost all neuronal circuits. Most GABAARs of the CNS are heteropentameric ion channels composed of two α, two ß and one γ subunit. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABAARs. Most GABAAR classifications rely on the heterogeneity of the α subunit (α1 - α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1 - γ3), which mediate synaptic clustering of GABAARs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABAARs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord specific loss of γ2 subunits was well tolerated. GABAAR clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a non-sedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABAARs in this site. They further suggest that γ1 containing GABAARs contribute to the spinal control of nociceptive information flow.Significance statement Our results identify for the first time a CNS area (the spinal dorsal horn) in which atypical GABAA receptors containing the γ1 subunit serve a physiological role in the synaptic clustering of GABAA receptors. They also show that pharmacological modulation of γ1 GABAA receptors by a non-sedative GABAA receptor modulator alleviates chronic pain in neuropathic mice.
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BACKGROUND: Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood. METHODS: To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling. RESULTS: The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings. CONCLUSIONS: Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Nav1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.
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Epilepsia Benigna Neonatal/genética , Síndromes Epilépticos/genética , Discapacidad Intelectual/genética , Canal de Sodio Activado por Voltaje NAV1.2/fisiología , Adolescente , Niño , Epilepsia Benigna Neonatal/fisiopatología , Síndromes Epilépticos/fisiopatología , Estudios de Asociación Genética , Células HEK293 , Humanos , Discapacidad Intelectual/fisiopatología , Fenotipo , Adulto JovenRESUMEN
Glycinergic neurotransmission has long been known for its role in spinal motor control. During the last two decades, additional functions have become increasingly recognized-among them is a critical contribution to spinal pain processing. Studies in rodent pain models provide proof-of-concept evidence that enhancing inhibitory glycinergic neurotransmission reduces chronic pain symptoms. Apparent strategies for pharmacological intervention include positive allosteric modulators of glycine receptors and modulators or inhibitors of the glial and neuronal glycine transporters GlyT1 and GlyT2. These prospects have led to drug discovery efforts in academia and in industry aiming at compounds that target glycinergic neurotransmission with high specificity. Available data show promising analgesic efficacy. Less is currently known about potential unwanted effects but the presence of glycinergic innervation in CNS areas outside the nociceptive system prompts for a careful evaluation not only of motor function, but also of potential respiratory impairment and addictive properties.
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Analgésicos/uso terapéutico , Descubrimiento de Drogas , Proteínas de Transporte de Glicina en la Membrana Plasmática/fisiología , Terapia Molecular Dirigida/métodos , Receptores de Glicina/fisiología , Analgésicos/aislamiento & purificación , Animales , Drogas en Investigación , HumanosRESUMEN
Cerebral ischemia frequently leads to long-term disability and death. Excitotoxicity is believed to be the main cause for ischemia-induced neuronal death. Although a role of glutamate receptors in this process has been firmly established, the contribution of metabotropic GABAB receptors, which control excitatory neurotransmission, is less clear. A prominent characteristic of ischemic insults is endoplasmic reticulum (ER) stress associated with the up-regulation of the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). After inducing ER stress in cultured cortical neurons by sustained Ca(2+) release from intracellular stores or by a brief episode of oxygen and glucose deprivation (in vitro model of cerebral ischemia), we observed an increased expression of CHOP accompanied by a strong reduction of cell surface GABAB receptors. Our results indicate that down-regulation of cell surface GABAB receptors is caused by the interaction of the receptors with CHOP in the ER. Binding of CHOP prevented heterodimerization of the receptor subunits GABAB1 and GABAB2 and subsequent forward trafficking of the receptors to the cell surface. The reduced level of cell surface receptors diminished GABAB receptor signaling and, thus, neuronal inhibition. These findings indicate that ischemia-mediated up-regulation of CHOP down-regulates cell surface GABAB receptors by preventing their trafficking from the ER to the plasma membrane. This mechanism leads to diminished neuronal inhibition and may contribute to excitotoxicity in cerebral ischemia.
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Estrés del Retículo Endoplásmico , Neuronas/metabolismo , Receptores de GABA-B/metabolismo , Factor de Transcripción CHOP/metabolismo , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Femenino , Expresión Génica , Glucosa/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Neuronas/citología , Oxígeno/metabolismo , Unión Proteica , Ratas , Ratas Wistar , Receptores de GABA-B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción CHOP/genéticaRESUMEN
Metabotropic GABAB receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABAB receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABAB receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABAB receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABAB receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys(48)-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABAB2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABAB receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABAB receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABAB receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.
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Degradación Asociada con el Retículo Endoplásmico/genética , Neuronas/metabolismo , Receptores de GABA-B/metabolismo , Ubiquitina/metabolismo , Animales , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ratas , Ratas Wistar , Receptores de GABA-B/genética , Ubiquitina/genética , Ubiquitinación/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
ABSTRACT: The anterior cingulate cortex (ACC) plays a crucial role in the perception of pain. It is consistently activated by noxious stimuli and its hyperactivity in chronic pain indicates plasticity in the local neuronal network. However, the way persistent pain effects and modifies different neuronal cell types in the ACC and how this contributes to sensory sensitization is not completely understood. This study confirms the existence of 2 primary subtypes of pyramidal neurons in layer 5 of the rostral, agranular ACC, which we could classify as intratelencephalic (IT) and cortico-subcortical (SC) projecting neurons, similar to other cortical brain areas. Through retrograde labeling, whole-cell patch-clamp recording, and morphological analysis, we thoroughly characterized their different electrophysiological and morphological properties. When examining the effects of peripheral inflammatory pain on these neuronal subtypes, we observed time-dependent plastic changes in excitability. During the acute phase, both subtypes exhibited reduced excitability, which normalized to pre-inflammatory levels after day 7. Daily conditioning with nociceptive stimuli during this period induced an increase in excitability specifically in SC neurons, which was correlated with a decrease in mechanical sensitization. Subsequent inhibition of the activity of SC neurons projecting to the periaqueductal gray with in vivo chemogenetics, resulted in reinstatement of the hypersensitivity. Accordingly, it was sufficient to enhance the excitability of these neurons chemogenetically in the inflammatory pain condition to induce hypoalgesia. These findings suggest a cell type-specific effect on the descending control of nociception and a cellular mechanism for pain-induced analgesia. Furthermore, increased excitability in this neuronal population is hypoalgesic rather than hyperalgesic.
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Persistent pain is a prevalent medical concern correlating with a hyperexcitable anterior cingulate cortex (ACC). Its activity is modulated by inputs from several brain regions, but the maladjustments that these afferent circuits undergo during the transition from acute to chronic pain still require clarification. We focus on ACC-projecting claustrum (CLAACC) neurons and their responses to sensory and aversive stimuli in a mouse model of inflammatory pain. Using chemogenetics, in vivo calcium imaging, and ex vivo electrophysiological approaches, we reveal that suppression of CLAACC activity acutely attenuates allodynia and that the claustrum preferentially transmits aversive information to the ACC. With prolonged pain, a claustro-cingulate functional impairment develops, which is mediated by a weakened excitatory drive onto ACC pyramidal neurons, resulting in a diminished claustral influence on the ACC. These findings support an instrumental role of the claustrum in the processing of nociceptive information and its susceptibility to persistent pain states.
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Dolor Crónico , Giro del Cíngulo , Ratones , Animales , Giro del Cíngulo/fisiología , Neuronas/fisiología , HiperalgesiaRESUMEN
Depression is a common comorbidity of chronic pain with many patients being affected. However, efficient pharmacological treatment strategies are still lacking. Therefore, it is desirable to find additional alternative approaches. Environmental enrichment has been suggested as a method to alleviate pain-induced depression. However, the neuronal mechanisms of its beneficial effects are still elusive. The anterior cingulate cortex (ACC) plays a central role in processing pain-related negative affect and chronic pain-induced plasticity in this region correlates with depressive symptoms. We studied the consequences of different durations of environmental enrichment on pain sensitivity and chronic pain-induced depression-like behaviors in a mouse model of neuropathic pain. Furthermore, we correlated the behavioral outcomes to the activity levels of pyramidal neurons in the ACC by analyzing their electrophysiological properties ex vivo. We found that early exposure to an enriched environment alone was not sufficient to cause resilience against pain-induced depression-like symptoms. However, extending the enrichment after the injury prevented the development of depression and reduced mechanical hypersensitivity. On the cellular level, increased neuronal excitability was associated with the depressive phenotype that was reversed by the enrichment. Therefore, neuronal excitability in the ACC was inversely correlated to the extended enrichment-induced resilience to depression. These results suggest that the improvement of environmental factors enhanced the resilience to developing chronic pain-related depression. Additionally, we confirmed the association between increased neuronal excitability in the ACC and depression-like states. Therefore, this non-pharmacological intervention could serve as a potential treatment strategy for comorbid symptoms of chronic pain.
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Chronic pain is a complex experience with multifaceted behavioral manifestations, often leading to pain avoidance at the expense of reward approach. How pain facilitates avoidance in situations with mixed outcomes is unknown. The anterior cingulate cortex (ACC) plays a key role in pain processing and in value-based decision-making. Distinct ACC inputs inform about the sensory and emotional quality of pain. However, whether specific ACC circuits underlie pathological conflict assessment in pain remains underexplored. Here, we demonstrate that mice with chronic pain favor cold avoidance rather than reward approach in a conflicting task. This occurs along with selective strengthening of basolateral amygdala inputs onto ACC layer 2/3 pyramidal neurons. The amygdala-cingulate projection is necessary and sufficient for the conflicting cold avoidance. Further, low-frequency stimulation of this pathway restores AMPA receptor function and reduces avoidance in pain mice. Our findings provide insights into the circuits and mechanisms underlying cognitive aspects of pain and offer potential targets for treatment.
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Complejo Nuclear Basolateral , Dolor Crónico , Ratones , Animales , Giro del Cíngulo/metabolismo , Amígdala del Cerebelo/fisiología , EmocionesRESUMEN
Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABAA receptors (α2GABAAR) constitute the most abundant GABAAR subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2-/- mice that lack α2GABAAR from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2-/- mice. Our results confirm that hoxb8-α2-/- mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABAAR modulator HZ-166 is reduced in hoxb8-α2-/- mice and that hoxb8-α2-/- mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did not show significant changes in hoxb8-α2-/- mice rendering a compensatory up-regulation of other GABAAR subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2-/- mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABAAR to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2-/- mice from hyperalgesia.
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Neuronas GABAérgicas/metabolismo , Hiperalgesia/metabolismo , Fenotipo , Receptores de GABA-A/deficiencia , Médula Espinal/metabolismo , Transmisión Sináptica/fisiología , Animales , Femenino , Células HEK293 , Humanos , Hiperalgesia/genética , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Receptores de GABA-A/genéticaRESUMEN
Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABAA receptors (α1, α2, α3, and α5GABAARs), are highly effective against anxiety disorders, but also cause severe side effects greatly limiting their clinical application. Both, preclinical studies in genetically engineered mice, and preclinical and clinical trials with subtype-selective compounds indicate that undesired effects can in principle be avoided by targeting specific GABAAR subtypes. While there is general consensus that activity at α1GABAARs should be avoided, controversy exists as to whether α2 or α3GABAARs need to be targeted for anxiolysis. While previous experiments in GABAAR point-mutated mice demonstrated a critical role of α2GABAARs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABAARs. As most α1GABAAR-sparing BDZ site agonists discriminate little between α2 and α3GABAARs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABAAR modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABAAR triple point-mutated mice, in which only either α1, α2, or α3GABAARs were left BDZ sensitive. These experiments revealed that TP003 behaves as a partial, rather non-selective BDZ site agonist in vitro that acts in vivo through α1, α2, and α3GABAARs (α5GABAAR-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABAARs, but not of α3GABAARs. TP003 should therefore not be considered an α3GABAAR selective agent. Previously published studies using TP003 should be interpreted with caution.
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Ansiolíticos/farmacología , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Agonistas de Receptores de GABA-A/farmacología , Imidazoles/farmacología , Piridinas/farmacología , Animales , Ansiolíticos/química , Sitios de Unión , Agonistas de Receptores de GABA-A/química , Células HEK293 , Humanos , Hipnóticos y Sedantes/química , Hipnóticos y Sedantes/farmacología , Imidazoles/química , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones de la Cepa 129 , Ratones Transgénicos , Estructura Molecular , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Relajantes Musculares Centrales/química , Relajantes Musculares Centrales/farmacología , Piridinas/química , Ratas , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABAA receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABAA receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABAA receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABAA receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.
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Prurito/tratamiento farmacológico , Receptores de GABA-A/metabolismo , Médula Espinal/patología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Perros , Péptido Liberador de Gastrina/metabolismo , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Hidrocarburos Fluorados/farmacología , Hidrocarburos Fluorados/uso terapéutico , Hipersensibilidad/complicaciones , Hipersensibilidad/tratamiento farmacológico , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones Endogámicos C57BL , Inhibición Neural/efectos de los fármacos , Mutación Puntual/genética , Prurito/complicacionesRESUMEN
Determining the functional significance of post-translational modifications advances our understanding of many broadly-expressed proteins, and particularly ion channels. The enzymes that catalyze these modifications are often expressed in a cell-type specific manner, resulting in considerable structural diversity among post-translationally modified proteins that are expressed across a variety of cell types. TRP channels exhibit notably variable behavior between cell types in vitro and in vivo , and they are frequently modified with N-glycans that contribute to protein function. TRPA1 possesses two putative N-linked glycosylation sites at N747 and N753 that have not yet been studied in detail. Here, we show that both of these sites can be modified with an N-glycan and that the glycan at position N747 modulates agonist-sensitivity of TRPA1 in vitro Additionally, we found that N-glycosylation also modulates cooperative effects of temperature and the agonist cinnamaldehyde on TRPA1 channel activation. Collectively, these findings suggest a dynamic role played by the N-glycosylation of human TRPA1. They also provide further evidence of the versatility of N-glycans and will assist in efforts to fully understand the complex regulation of TRPA1 activity.
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Data from genetically modified mice suggest that benzodiazepine (BDZ)-site agonists with improved selectivity for α2-subtype GABAA receptors (α2GABAAR) are potentially useful for the treatment of neuropathic pain. Subtype-selective compounds available for preclinical tests in rodents support this concept but have not been approved for human use, hindering proof-of-concept studies in patients. We recently proposed that N-desmethyl clobazam (NDMC), the main metabolite of the licensed BDZ clobazam (CBZ), is responsible for most of the antihyperalgesia observed in mice after CBZ administration. In order to assess a potentially favorable pharmacological profile of NDMC, we analyzed differences in the GABAAR subtype specificity of CBZ, NDMC and diazepam (DZP) in recombinant receptors. DZP and CBZ potentiated sedating α1GABAARs and antihyperalgesic α2GABAARs with similar efficacies, whereas NDMC preferred α2GABAARs over α1GABAARs across a wide concentration range. In vivo, DZP and NDMC reduced neuropathic pain at doses between 3 and 30 mg/kg. At these doses, DZP had strong locomotor sedating effects while NDMC caused no or only weak sedation. Sedative effects of NDMC became apparent when the action of NDMC was restricted to α1GABAARs. However, when GABAAR point-mutated mice were studied that allow the analysis of antihyperalgesia and sedation in isolation, we found that, compared to DZP, NDMC had a significantly improved therapeutic window, consistent with its more favorable α2/α1 in vitro activity ratio. Given that NDMC should share the safety profile of its parent compound CBZ, it should be well-suited for proof-of-concept studies in human volunteers or patients.
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Analgésicos no Narcóticos/metabolismo , Benzodiazepinas/metabolismo , Hiperalgesia/metabolismo , Receptores de GABA-A/metabolismo , Analgésicos no Narcóticos/uso terapéutico , Animales , Benzodiazepinas/uso terapéutico , Clobazam , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Hiperalgesia/tratamiento farmacológico , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Unión Proteica/fisiologíaRESUMEN
Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αßGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αß heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-ß subunit interface and suggests that in α3ßGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.
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Hiperalgesia/metabolismo , Inflamación/metabolismo , Manejo del Dolor/métodos , Receptores de Glicina/metabolismo , Médula Espinal/metabolismo , Sitio Alostérico , Animales , Femenino , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Neuronas , Dolor , Fenoles/química , Fenilalanina/química , Fosforilación , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.
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Proteínas Portadoras/fisiología , Neuronas GABAérgicas/fisiología , Proteínas de la Membrana/fisiología , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Acetilación , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/fisiología , Proteínas Portadoras/metabolismo , Femenino , Flavonas/farmacología , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Neocórtex/citología , Neocórtex/metabolismo , Fosforilación , Cultivo Primario de Células , Ratas , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transducción de Señal/efectos de los fármacos , Sumoilación/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
GABAA receptors (GABA(A)Rs) and glycine receptors are key elements of the spinal control of nociception and pain. Compromised functioning of these two transmitter systems contributes to chronic pain states. Restoring their proper function through positive allosteric modulators should constitute a rational approach to the treatment of chronic pain syndromes involving diminished inhibitory spinal pain control. Although classical benzodiazepines (i.e., full agonists at the benzodiazepine binding site of GABA(A)Rs) potentiate synaptic inhibition in spinal pain controlling circuits, they lack clinically relevant analgesic activity in humans. Recent data obtained from experiments in GABA(A)R point-mutated mice suggests dose-limiting sedative effects of classical nonspecific benzodiazepines as the underlying cause. Experiments in genetically engineered mice resistant to the sedative effects of classical benzodiazepines and studies with novel less sedating benzodiazepines have indeed shown that profound antihyperalgesia can be obtained at least in preclinical pain models. Present evidence suggests that compounds with high intrinsic activity at α2-GABA(A)R and minimal agonistic activity at α1-GABA(A)R should possess relevant antihyperalgesic activity without causing unwanted sedation. On-going preclinical studies in genetically engineered mice and clinical trials with more selective benzodiazepine site agonists should soon provide additional insights into this emerging topic.
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Analgésicos/farmacología , Dolor/tratamiento farmacológico , Receptores de GABA-A/efectos de los fármacos , Animales , Benzodiazepinas/farmacología , Modelos Animales de Enfermedad , Agonistas de Receptores de GABA-A/farmacología , Humanos , Ratones , Dolor/fisiopatología , Mutación Puntual , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.
Asunto(s)
Benzodiazepinas/farmacología , Moduladores del GABA/farmacología , Hiperalgesia/genética , Hipnóticos y Sedantes/farmacología , Dolor/genética , Mutación Puntual , Receptores de GABA-A/genética , Analgesia/métodos , Animales , Arginina/genética , Arginina/metabolismo , Diazepam/farmacología , Expresión Génica , Histidina/genética , Histidina/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Masculino , Ratones , Midazolam/farmacología , Dolor/tratamiento farmacológico , Dolor/metabolismo , Dolor/fisiopatología , Manejo del Dolor/métodos , Receptores de GABA-A/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiopatologíaRESUMEN
Human Down syndrome (DS) is determined by the trisomy of autosome 21 and is expressed by multiple abnormalities, being mental retardation the most striking feature. The condition results in altered electrical membrane properties (EMPs) of fetal neurons, which are qualitatively identical to those of trisomy 16 fetal mice (Ts16), an animal model of the human condition. Ts16 hippocampal cultured neurons reportedly exhibit increased voltage-dependent calcium currents (I (Ca)) amplitude. Since Ts16 animals are unviable, we have established immortalized cell lines from the cerebral cortex of Ts16 (named CTb) and normal littermates (named CNh). Using the whole-cell patch-clamp technique, we have now studied I (Ca) in CTb and CNh cells. Current activation occurs at -40 mV in both cell lines (V (holding) = -80 mV). Trisomic cells exhibited a 2.4 fold increase in the maximal Ca(2+) current density compared to normal cells (CNh = -6.3 ± 0.77 pA/pF, n = 18; CTb = -16.4 ± 2.423 pA/pF; P < 0.01, n = 13). Time dependent kinetics for activation and inactivation did not differ between the two cell types. However, steady state inactivation studies revealed a 15 mV shift toward more depolarized potentials in the trisomic condition, suggesting that altered voltage dependence of inactivation may underlie the increased current density. Further, the total charge movement across the membrane is increased in CTb cells, in agreement with that expected by the potential sensitivity shift. These results indicate that CTb cells present altered Ca(2+) currents, similar to those of Ts16 primary cultured central neurons. The CTb cell line represents a model for studying DS-related impairments of EMPs.