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Knowing the site of drug action is important to optimize effectiveness and address any side effects. We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.
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BACKGROUND AND HYPOTHESIS: Type-1 trace amine-associated receptors (TAAR1) modulate dopaminergic and glutamatergic neurotransmission and are targeted by novel antipsychotic drugs. We hypothesized that schizophrenia (SCZ) causes adaptive changes in TAAR1 expression in the prefrontal cortex. STUDY DESIGN: We measured TAAR1 mRNA and protein levels by quantitative PCR and immunoblotting in post-mortem prefrontal cortical samples obtained from 23 individuals affected by SCZ and 23 non-schizophrenic controls (CTRL). Data were correlated with a number of variables in both groups. STUDY RESULTS: TAAR1 mRNA levels were largely increased in the SCZ prefrontal cortex, and did not correlate with age, age at onset and duration of SCZ, or duration of antipsychotic treatment. For the analysis of TAAR1 protein levels, CTRL and SCZ were divided into 2 subgroups, distinguished by the extent of neuropathological burden. CTRL with low neuropathological burden (LNB) had lower TAAR1 protein levels than CTRL with high neuropathological burden (HNB), whereas no changes were found between LNB and HNB in the SCZ group. TAAR1 protein levels were lower in CTRL with LNB with respect to all SCZ samples or to SCZ samples with LNB. In the SCZ group, levels showed an inverse correlation with the duration of antipsychotic treatment and were higher in individuals treated with second-generation antipsychotics as compared with those treated with first-generation antipsychotics. CONCLUSIONS: The up-regulation of TAAR1 observed in the SCZ prefrontal cortex supports the development of TAAR1 agonists as new promising drugs in the treatment of SCZ.
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Antipsicóticos , Esquizofrenia , Humanos , Esquizofrenia/genética , Antipsicóticos/uso terapéutico , Regulación hacia Arriba , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Corteza Prefrontal/metabolismo , Dopamina/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Previous studies suggest that different metabotropic glutamate (mGlu) receptor subtypes are potential drug targets for treating absence epilepsy. However, no information is available on mGlu3 receptors. OBJECTIVE: To examine whether (i) changes of mGlu3 receptor expression/signaling are found in the somatosensory cortex and thalamus of WAG/Rij rats developing spontaneous absence seizures; (ii) selective activation of mGlu3 receptors with LY2794193 affects the number and duration of spikewave discharges (SWDs) in WAG/Rij rats; and (iii) a genetic variant of GRM3 (encoding the mGlu3 receptor) is associated with absence epilepsy. METHODS: Animals: immunoblot analysis of mGlu3 receptors, GAT-1, GLAST, and GLT-1; realtime PCR analysis of mGlu3 mRNA levels; assessment of mGlu3 receptor signaling; EEG analysis of SWDs; assessment of depressive-like behavior. Humans: search for GRM3 and GRM5 missense variants in 196 patients with absence epilepsy or other Idiopathic Generalized Epilepsy (IGE)/ Genetic Generalized Epilepsy (GGE) and 125,748 controls. RESULTS: mGlu3 protein levels and mGlu3-mediated inhibition of cAMP formation were reduced in the thalamus and somatosensory cortex of pre-symptomatic (25-27 days old) and symptomatic (6-7 months old) WAG/Rij rats compared to age-matched controls. Treatment with LY2794193 (1 or 10 mg/kg, i.p.) reduced absence seizures and depressive-like behavior in WAG/Rij rats. LY2794193 also enhanced GAT1, GLAST, and GLT-1 protein levels in the thalamus and somatosensory cortex. GRM3 and GRM5 gene variants did not differ between epileptic patients and controls. CONCLUSION: We suggest that mGlu3 receptors modulate the activity of the cortico-thalamo-cortical circuit underlying SWDs and that selective mGlu3 receptor agonists are promising candidate drugs for absence epilepsy treatment.
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Epilepsia Tipo Ausencia , Receptores de Glutamato Metabotrópico , Ratas , Humanos , Animales , Lactante , Epilepsia Tipo Ausencia/tratamiento farmacológico , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Electroencefalografía , Convulsiones , Genética Humana , Modelos Animales de EnfermedadRESUMEN
Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.
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Dolor Crónico , Corteza Somatosensorial , Animales , Dolor Crónico/inducido químicamente , Dolor Crónico/metabolismo , Matriz Extracelular/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Parvalbúminas/metabolismo , Corteza Somatosensorial/metabolismoRESUMEN
Pilots and crew of domestic flights are exposed to transient periods of mild reductions of partial pressure of inspired oxygen each day, and this might have functional consequence on their performance in the long range. Here, we exposed mice to mild reductions of oxygen exposure (ROE) four times per day for 21 days by lowering oxygen partial pressure to levels corresponding to an altitude of about 2300 m, which is the quote of pressurization of the air cabin. Four groups of mice were studied: unstressed or stressed mice exposed to ROE or normoxic conditions. Mice were exposed to chronic unpredictable stress (CUS) for 28 days, and ROE was delivered in the last 21 days of CUS. In normoxic mice, CUS caused anhedonia in the sucrose preference test, anxiety-like behaviour in the open field test, learning impairment in the Morris water maze, reduced hippocampal neurogenesis, increased serum corticosterone levels and increased expression of depression-related genes (Pclo, Mthfr and Grm5) in the hippocampus. All these changes were reversed by ROE, which had little or no effect in unstressed mice. These findings suggest that ROE simulating air cabin conditions of domestic flights may enhance resilience to stress improving mood, anxiety and learning ability.
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Hipocampo , Oxígeno , Resiliencia Psicológica , Estrés Psicológico/psicología , Aeronaves , Animales , Ansiedad , Depresión , Ratones , Presión ParcialRESUMEN
mGlu5 metabotropic glutamate receptors are highly expressed and functional in the early postnatal life, and are known to positively modulate NMDA receptor function. Here, we examined the expression of NMDA receptor subunits and interneuron-related genes in the prefrontal cortex and hippocampus of mGlu5-/- mice and wild-type littermates at three developmental time points (PND9, - 21, and - 75). We were surprised to find that expression of all NMDA receptor subunits was greatly enhanced in mGlu5-/- mice at PND21. In contrast, at PND9, expression of the GluN2B subunit was enhanced, whereas expression of GluN2A and GluN2D subunits was reduced in both regions. These modifications were transient and disappeared in the adult life (PND75). Changes in the transcripts of interneuron-related genes (encoding parvalbumin, somatostatin, vasoactive intestinal peptide, reelin, and the two isoforms of glutamate decarboxylase) were also observed in mGlu5-/- mice across postnatal development. For example, the transcript encoding parvalbumin was up-regulated in the prefrontal cortex of mGlu5-/- mice at PND9 and PND21, whereas it was significantly reduced at PND75. These findings suggest that in mGlu5-/- mice a transient overexpression of NMDA receptor subunits may compensate for the lack of the NMDA receptor partner, mGlu5. Interestingly, in mGlu5-/- mice the behavioral response to the NMDA channel blocker, MK-801, was significantly increased at PND21, and largely reduced at PND75. The impact of adaptive changes in the expression of NMDA receptor subunits should be taken into account when mGlu5-/- mice are used for developmental studies.
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Hipocampo/metabolismo , Corteza Prefrontal/metabolismo , Receptor del Glutamato Metabotropico 5/deficiencia , Receptores de N-Metil-D-Aspartato/metabolismo , Regulación hacia Arriba , Animales , Animales Recién Nacidos , Maleato de Dizocilpina/farmacología , Eliminación de Gen , Interneuronas/metabolismo , Ratones Noqueados , Subunidades de Proteína/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismoRESUMEN
mGlu5 metabotropic glutamate receptors are highly functional in the early postnatal life, and regulate developmental plasticity of parvalbumin-positive (PV+) interneurons in the cerebral cortex. PV+ cells are enwrapped by perineuronal nets (PNNs) at the closure of critical windows of cortical plasticity. Changes in PNNs have been associated with neurodevelopmental disorders. We found that the number of Wisteria Fluoribunda Agglutinin (WFA)+ PNNs and the density of WFA+/PV+ cells were largely increased in the somatosensory cortex of mGlu5-/- mice at PND16. An increased WFA+ PNN density was also observed after pharmacological blockade of mGlu5 receptors in the first two postnatal weeks. The number of WFA+ PNNs in mGlu5-/- mice was close to a plateau at PND16, whereas continued to increase in wild-type mice, and there was no difference between the two genotypes at PND21 and PND60. mGlu5-/- mice at PND16 showed increases in the transcripts of genes involved in PNN formation and a reduced expression and activity of type-9 matrix metalloproteinase in the somatosensory cortex suggesting that mGlu5 receptors control both PNN formation and degradation. Finally, unilateral whisker stimulation from PND9 to PND16 enhanced WFA+ PNN density in the contralateral somatosensory cortex only in mGlu5+/+ mice, whereas whisker trimming from PND9 to PND16 reduced WFA+ PNN density exclusively in mGlu5-/- mice, suggesting that mGlu5 receptors shape the PNN response to sensory experience. These findings disclose a novel undescribed mechanism of PNN regulation, and lay the groundwork for the study of mGlu5 receptors and PNNs in neurodevelopmental disorders.
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Receptores de Glutamato Metabotrópico , Corteza Somatosensorial , Animales , Matriz Extracelular/metabolismo , Interneuronas/metabolismo , Ratones , Parvalbúminas/metabolismoRESUMEN
Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABAA receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 h and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models.
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Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Niacinamida/análogos & derivados , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/metabolismo , Tálamo/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Relación Dosis-Respuesta a Droga , Masculino , Niacinamida/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Ratas Wistar , Receptores de GABA-A/metabolismo , Tálamo/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.
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Corteza Cerebral/anomalías , Embrión de Mamíferos/anomalías , Neuronas GABAérgicas/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Biomarcadores , Corteza Cerebral/metabolismo , Femenino , Regulación de la Expresión Génica , Genes Homeobox , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , ARN Mensajero , Receptores de Glutamato Metabotrópico/genéticaRESUMEN
Metabotropic glutamate (mGlu) receptors are considered as candidate drug targets for the treatment of schizophrenia. These receptors form a family of eight subtypes (mGlu1 to -8), of which mGlu1 and -5 are coupled to Gq/11, and all other subtypes are coupled to Gi/o. Here, we discuss the possibility that selective ligands of individual mGlu receptor subtypes may be effective in controlling the core symptoms of schizophrenia, and, in some cases, may impact mechanisms underlying the progression of the disorder. Recent evidence indicates that activation of mGlu1 receptors inhibits dopamine release in the meso-striatal system. Hence, selective positive allosteric modulators (PAMs) of mGlu1 receptors hold promise for the treatment of positive symptoms of schizophrenia. mGlu5 receptors are widely expressed in the CNS and regulate the activity of cells that are involved in the pathophysiology of schizophrenia, such as cortical GABAergic interneurons and microglial cells. mGlu5 receptor PAMs are under development for the treatment of schizophrenia and cater the potential to act as disease modifiers by restraining neuroinflammation. mGlu2 receptors have attracted considerable interest because they negatively modulate 5-HT2A serotonin receptor signaling in the cerebral cortex. Both mGlu2 receptor PAMs and orthosteric mGlu2/3 receptor agonists display antipsychotic-like activity in animal models, and the latter drugs are inactive in mice lacking mGlu2 receptors. So far, mGlu3 receptors have been left apart as drug targets for schizophrenia. However, activation of mGlu3 receptors boosts mGlu5 receptor signaling, supports neuronal survival, and drives microglial cells toward an antiinflammatory phenotype. This strongly encourages research of mGlu3 receptors in schizophrenia. Finally, preclical studies suggest that mGlu4 receptors might be targeted by novel antipsychotic drugs, whereas studies of mGlu7 and mGlu8 receptors in animal models of psychosis are still at their infancy.
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Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1-/- and Asic2-/- mice and observed a significant reduction of clinical score in Asic1-/- mice and a significant reduction in the clinical score in Asic2-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.
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Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/fisiología , Encéfalo/metabolismo , Esclerosis Múltiple/fisiopatología , Canales Iónicos Sensibles al Ácido/genética , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/fisiopatología , Humanos , Hiperalgesia/complicaciones , Hiperalgesia/genética , Hiperalgesia/fisiopatología , Masculino , Ratones Noqueados , MicroARNs/metabolismo , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/genética , Mielitis/complicaciones , Mielitis/genética , Mielitis/fisiopatología , Umbral del Dolor , Polimorfismo de Nucleótido SimpleRESUMEN
In cerebellar Purkinje cells (PCs) type-1 metabotropic glutamate (mGlu1) receptors play a key role in motor learning and drive the refinement of synaptic innervation during postnatal development. The cognate mGlu5 receptor is absent in mature PCs and shows low expression levels in the adult cerebellar cortex. Here we found that mGlu5 receptors were heavily expressed by PCs in the early postnatal life, when mGlu1α receptors were barely detectable. The developmental decline of mGlu5 receptors coincided with the appearance of mGlu1α receptors in PCs, and both processes were associated with specular changes in CpG methylation in the corresponding gene promoters. It was the mGlu1 receptor that drove the elimination of mGlu5 receptors from PCs, as shown by data obtained with conditional mGlu1α receptor knockout mice and with targeted pharmacological treatments during critical developmental time windows. The suppressing activity of mGlu1 receptors on mGlu5 receptor was maintained in mature PCs, suggesting that expression of mGlu1α and mGlu5 receptors is mutually exclusive in PCs. These findings add complexity to the the finely tuned mechanisms that regulate PC biology during development and in the adult life and lay the groundwork for an in-depth analysis of the role played by mGlu5 receptors in PC maturation.
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Regulación hacia Abajo , Epigénesis Genética , Células de Purkinje/metabolismo , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/biosíntesis , Sinapsis/metabolismo , Animales , Islas de CpG , Metilación de ADN , Masculino , Ratones , Ratones Noqueados , Células de Purkinje/citología , Receptores AMPA/genética , Receptores de Ácido Kaínico/genética , Sinapsis/genéticaRESUMEN
mGlu5 receptors are involved in mechanisms of activity-dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor-mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor-mediated somatic Ca2+ mobilization, and mGlu3 receptor-mediated long-term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field.
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Sistema Nervioso Central/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Sistema Nervioso Central/citología , Embrión de Mamíferos , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Hidrólisis/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ratas , Receptor del Glutamato Metabotropico 5/genética , Receptores de Glutamato Metabotrópico/genéticaRESUMEN
OBJECTIVES: Thrombospondins, which are known to interact with the α2 δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). METHODS: We measured the transcripts of thrombospondin-1 and α2 δ subunit, and protein levels of α2 δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. RESULTS: Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. SIGNIFICANCE: These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs.
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Canales de Calcio/genética , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/genética , Epilepsia Generalizada/genética , Trombospondina 1/genética , Animales , Canales de Calcio/biosíntesis , Estudios de Cohortes , Epilepsia Tipo Ausencia/metabolismo , Epilepsia Generalizada/metabolismo , Humanos , Masculino , Ratas , Ratas Wistar , Trombospondina 1/biosíntesisRESUMEN
We have recently shown that pharmacological blockade of mGlu2 metabotropic glutamate receptors protects vulnerable neurons in the 4-vessel occlusion model of transient global ischemia, whereas receptor activation amplifies neuronal death. This raised the possibility that endogenous activation of mGlu2 receptors contributes to the pathophysiology of ischemic neuronal damage. Here, we examined this possibility using two models of transient focal ischemia: (i) the monofilament model of middle cerebral artery occlusion (MCAO) in mice, and (ii) the model based on intracerebral infusion of endothelin-1 (Et-1) in rats. Following transient MCAO, mGlu2 receptor knockout mice showed a significant reduction in infarct volume and an improved short-term behavioural outcome, as assessed by a neurological disability scale and the "grip test". Following Et-1 infusion, Grm2 gene mutated Hannover Wistar rats lacking mGlu2 receptors did not show changes in the overall infarct volume as compared to their wild-type counterparts, although they showed a reduced infarct area in the agranular insular cortex. Interestingly, however, mGlu2 receptor-deficient rats performed better than wild-type rats in the adhesive tape test, in which these rats did not show the laterality preference typically observed after focal ischemia. These findings support the hypothesis that activation of mGlu2 receptors is detrimental in the post-ischemic phase, and support the use of mGlu2 receptor antagonists in the experimental treatment of brain ischemia.
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Eliminación de Gen , Ataque Isquémico Transitorio/genética , Receptores de Glutamato Metabotrópico/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conducta Animal , Corteza Cerebral/patología , Infarto Cerebral/patología , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Ratas Wistar , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/deficienciaRESUMEN
Background L-acetylcarnitine, a drug marketed for the treatment of chronic pain, causes analgesia by epigenetically up-regulating type-2 metabotropic glutamate (mGlu2) receptors in the spinal cord. Because the epigenetic mechanisms are typically long-lasting, we hypothesized that analgesia could outlast the duration of L-acetylcarnitine treatment in models of inflammatory and neuropathic pain. Results A seven-day treatment with L-acetylcarnitine (100 mg/kg, once a day, i.p.) produced an antiallodynic effect in the complete Freund adjuvant mouse model of chronic inflammatory pain. L-Acetylcarnitine-induced analgesia persisted for at least 14 days after drug withdrawal. In contrast, the analgesic effect of pregabalin, amitryptiline, ceftriaxone, and N-acetylcysteine disappeared seven days after drug withdrawal. L-acetylcarnitine treatment enhanced mGlu2/3 receptor protein levels in the dorsal region of the spinal cord. This effect also persisted for two weeks after drug withdrawal and was associated with increased levels of acetylated histone H3 bound to the Grm2 gene promoter in the dorsal root ganglia. A long-lasting analgesic effect of L-acetylcarnitine was also observed in mice subjected to chronic constriction injury of the sciatic nerve. In these animals, a 14-day treatment with pregabalin, amitryptiline, tramadol, or L-acetylcarnitine produced a significant antiallodynic effect, with pregabalin displaying the greatest efficacy. In mice treated with pregabalin, tramadol or L-acetylcarnitine the analgesic effect was still visible 15 days after the end of drug treatment. However, only in mice treated with L-acetylcarnitine analgesia persisted 37 days after drug withdrawal. This effect was associated with an increase in mGlu2/3 receptor protein levels in the dorsal horns of the spinal cord. Conclusions Our findings suggest that L-acetylcarnitine has the unique property to cause a long-lasting analgesic effect that might reduce relapses in patients suffering from chronic pain.
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Acetilcarnitina/farmacología , Acetilcarnitina/uso terapéutico , Epigénesis Genética/efectos de los fármacos , Inflamación/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Amitriptilina/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Adyuvante de Freund/efectos adversos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Manejo del Dolor , Pregabalina/uso terapéutico , Receptores de Glutamato Metabotrópico/metabolismo , Factores de Tiempo , Tramadol/uso terapéuticoRESUMEN
Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-ß and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs.
RESUMEN
BACKGROUND: To examine whether metabotropic glutamate (mGlu) receptors have any role in mechanisms that shape neuronal vulnerability to ischemic damage, we used the 4-vessel occlusion (4-VO) model of transient global ischemia in rats. 4-VO in rats causes a selective death of pyramidal neurons in the hippocampal CA1 region, leaving neurons of the CA3 region relatively spared. We wondered whether changes in the expression of individual mGlu receptor subtypes selectively occur in the vulnerable CA1 region during the development of ischemic damage, and whether post-ischemic treatment with drugs targeting the selected receptor(s) affords neuroprotection. RESULTS: We found that 4-VO caused significantly reduction in the transcript of mGlu2 receptors in the CA1 region at times that preceded the anatomical evidence of neuronal death. Down-regulation of mGlu2 receptors was associated with reduced H3 histone acetylation at the Grm2 promoter. The transcripts of other mGlu receptor subtypes were unchanged in the CA1 region of 4-VO rats. Ischemia did not cause changes in mGlu2 receptor mRNA levels in the resistant CA3 region, which, interestingly, were lower than in the CA1 region. Targeting the mGlu2 receptors with selective pharmacologic ligands had profound effects on ishemic neuronal damage. Post-ischemic oral treatment with the selective mGlu2 receptor NAM (negative allosteric modulator), ADX92639 (30 mg/kg), was highly protective against ischemic neuronal death. In contrast, s.c. administration of the mGlu2 receptor enhancer, LY487379 (30 mg/kg), amplified neuronal damage in the CA1 region and extended the damage to the CA3 region. CONCLUSION: These findings suggest that the mGlu2 receptor is an important player in mechanisms regulating neuronal vulnerability to ischemic damage, and that mGlu2 receptor NAMs are potential candidates in the experimental treatments of disorders characterized by brain hypoperfusion, such as hypovolemic shock and cardiac arrest.