RESUMEN
ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.
Asunto(s)
Ansiedad , Memoria , Receptores Purinérgicos P2X4 , Sinapsis , Animales , Ansiedad/genética , Técnicas de Sustitución del Gen , Hipocampo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plasticidad Neuronal , Neuronas , Receptores Purinérgicos P2X4/genéticaRESUMEN
The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous and central player in Ca2+ signaling that is best known for its functions in the brain. In particular, the α isoform of CaMKII has been the subject of intense research and it has been established as a central regulator of neuronal plasticity. In contrast, little attention has been paid to CaMKIIß, the other predominant brain isoform that interacts directly with the actin cytoskeleton, and the functions of CaMKIIß in this organ remain largely unexplored. However, recently, the perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric and neurodevelopmental diseases, highlighting CAMK2B as a gene of interest. Herein, after highlighting the main structural and expression differences between the α and ß isoforms, we will review the specific functions of CaMKIIß, as described so far, in neuronal development and plasticity, as well as its potential implication in brain diseases.
Asunto(s)
Encéfalo/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Trastornos Mentales/genética , Trastornos del Neurodesarrollo/genética , Plasticidad Neuronal/fisiología , Neuronas/enzimología , Animales , Encéfalo/fisiopatología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Movimiento Celular , Regulación de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Memoria/fisiología , Trastornos Mentales/enzimología , Trastornos Mentales/fisiopatología , Mutación , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/fisiopatología , Neuronas/ultraestructura , Transducción de Señal , Sinapsis/enzimología , Sinapsis/ultraestructuraRESUMEN
Perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIß as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIß in the brain remain poorly explored. Here, we reveal a novel function for this CaMKII isoform in vivo during neuronal development. By using in utero electroporation, we show that CaMKIIß is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIß causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIß inhibits migration, demonstrating that precise regulation of CaMKIIß expression is required for correct neuronal migration. More precisely, CaMKIIß controls the multipolar-bipolar transition in the intermediate zone and locomotion in the cortical plate through its actin-binding and -bundling activities. In addition, our data indicate that a fine-tuned balance between CaMKIIß and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIß, demonstrating that CaMKIIß has a major biological function in the developing brain.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/fisiología , Neurogénesis/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Corteza Cerebral/metabolismo , Embrión de Mamíferos/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Proteínas de Microfilamentos/genética , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Neuronas/metabolismo , Cultivo Primario de Células , Isoformas de Proteínas/metabolismo , Células Piramidales/metabolismoRESUMEN
Initially hippocampal dependent, memory representations rely on a broadly distributed cortical network as they mature over time. How these cortical engrams acquire stability during systems-level memory consolidation without compromising their dynamic nature remains unclear. We identified a highly responsive "consolidation switch" in the synaptic composition of N-methyl-d-aspartate receptors (NMDARs), which dictates the progressive embedding and persistence of enduring memories in the rat cortex. Cortical GluN2B subunit-containing NMDARs were preferentially recruited upon encoding of associative olfactory memory to support neuronal allocation of memory engrams. As consolidation proceeds, a learning-induced redistribution of GluN2B subunit-containing NMDARs outward synapses increased synaptic GluN2A subunit contribution and enabled stabilization of remote memories. In contrast, synaptic reincorporation of GluN2B subunits occurred during subsequent forgetting. By manipulating the surface distribution of GluN2A and GluN2B subunit-containing NMDARs at cortical synapses, we uncovered that the rearrangement of GluN2B-containing NMDARs constitutes an essential tuning mechanism that determines the fate of cortical memory engrams and controls their malleability.
Asunto(s)
Corteza Cerebral , Memoria , Receptores de N-Metil-D-Aspartato , Sinapsis , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animales , Sinapsis/metabolismo , Sinapsis/fisiología , Ratas , Memoria/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiología , MasculinoRESUMEN
Activity-dependent modulations of N-methyl-D-aspartate glutamate receptor (NMDAR) trapping at synapses regulate excitatory neurotransmission and shape cognitive functions. Although NMDAR synaptic destabilization has been associated with severe neurological and psychiatric conditions, tuning NMDAR synaptic trapping to assess its clinical relevance for the treatment of brain conditions remains a challenge. Here, we report that ketamine (KET) and other clinically relevant NMDAR open channel blockers (OCBs) promote interactions between NMDAR and PDZ-domain-containing scaffolding proteins and enhance NMDAR trapping at synapses. We further show that KET-elicited trapping enhancement compensates for depletion in synaptic receptors triggered by autoantibodies from patients with anti-NMDAR encephalitis. Preventing synaptic depletion mitigates impairments in NMDAR-mediated CaMKII signaling and alleviates anxiety- and sensorimotor-gating-related behavioral deficits provoked by autoantibodies. Altogether, these findings reveal an unexpected dimension of OCB action and stress the potential of targeting receptor anchoring in NMDAR-related synaptopathies.
RESUMEN
Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca(2+) concentration. However the mechanism by which a short-lasting rise in Ca(2+) concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca(2+) elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that αCaMKII, but not ßCaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and αCaMKII. We then show that interfering with GluN2B/αCaMKII interaction prevents synaptic activity from inducing ERK-dependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and αCaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 4-Aminopiridina/farmacología , Análisis de Varianza , Animales , Bicuculina/farmacología , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Células Cultivadas , Corteza Cerebral/citología , Espinas Dendríticas/metabolismo , Homólogo 4 de la Proteína Discs Large , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Guanilato-Quinasas/metabolismo , Inmunoprecipitación , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fotoblanqueo , Bloqueadores de los Canales de Potasio/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/genética , TransfecciónRESUMEN
GluN2B-containing NMDA receptors are involved in many important physiological functions and play a pivotal role in mediating pain as well as in several neurodegenerative disorders. We aimed to develop fluorescent probes to target the GluN2B subunit selectively in order to allow better understanding of the relationships between receptor localisation and physiological importance. Ifenprodil, known as the GluNR2B antagonist of reference, was chosen as the template for the elaboration of probes. We had previously reported a fluorescein conjugate that was shown (by confocal microscopy imaging of DS-red-labelled cortical neurons) to bind specifically to GluN2B. To elaborate this probe, we explored the influence of both the nature and the attachment point of the spacer between the fluorophore and the parent compound, ifenprodil. We performed chemical modifications of ifenprodil at the benzylic position and on the phenol ring by introducing secondary amine or amide functions and evaluated alkyl chains from two to 20 bonds either including or not including secondary amide functions as spacers. The previously developed probe was found to display the greatest activity in the inhibition of NMDA-induced Ca(2+) influx by calcium imaging experiments on HEK293 cells transfected with the cDNA encoding for GluN1-1A and GluN2B. Further investigations revealed that this probe had a neuroprotective effect equivalent to that of ifenprodil in a standard test for neurotoxicity. Despite effects of lesser amplitude with these probes relative to ifenprodil, we demonstrated that they displaced [(3) H]ifenprodil in mouse brain slices in a similar manner.
Asunto(s)
Fluoresceína/química , Fármacos Neuroprotectores/química , Piperidinas/química , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Sitios de Unión , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Calcio/metabolismo , Células Cultivadas , Fluoresceína/metabolismo , Fluoresceína/farmacología , Células HEK293 , Humanos , Masculino , Ratones , Modelos Moleculares , N-Metilaspartato/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Piperidinas/metabolismo , Piperidinas/farmacología , Radiografía , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
N-Methyl-D-aspartate ionotropic glutamate receptors (NMDARs) play key roles in synaptogenesis, synaptic maturation, long-term plasticity, neuronal network activity, and cognition. Mirroring this wide range of instrumental functions, abnormalities in NMDAR-mediated signaling have been associated with numerous neurological and psychiatric disorders. Thus, identifying the molecular mechanisms underpinning the physiological and pathological contributions of NMDAR has been a major area of investigation. Over the past decades, a large body of literature has flourished, revealing that the physiology of ionotropic glutamate receptors cannot be restricted to fluxing ions, and involves additional facets controlling synaptic transmissions in health and disease. Here, we review newly discovered dimensions of postsynaptic NMDAR signaling supporting neural plasticity and cognition, such as the nanoscale organization of NMDAR complexes, their activity-dependent redistributions, and non-ionotropic signaling capacities. We also discuss how dysregulations of these processes may directly contribute to NMDAR-dysfunction-related brain diseases.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Transducción de Señal , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica , Plasticidad Neuronal/fisiología , Neuronas/metabolismoRESUMEN
We describe the synthesis and pharmacological characterization of a first generation of ifenprodil conjugates 4-7 as fluorescent probes for the confocal microscopy imaging of the NR2B-containing NMDA receptor. The fluorescein conjugate 6 displayed a moderate affinity for NMDAR but a high selectivity for the NR2B subunit and its NTD. Fluorescence imaging of DS-red labeled cortical neurons showed an exact colocalization of the probe 6 with small protrusions along the dendrites related to a specific binding on NR2B-containing NMDARs.
Asunto(s)
Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Piperidinas/análisis , Piperidinas/química , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/química , Células Cultivadas , Células HEK293 , Humanos , Microscopía Confocal , Estructura Molecular , EstereoisomerismoRESUMEN
Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of ß-amyloid (Aß), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aß release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aß. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aß production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aß release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aß release, representing a causal risk factor for developing AD.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/biosíntesis , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Although tissue type plasminogen activator (tPA) and brain derived neurotrophic factor (BDNF) have been extensively described to influence brain outcomes in a number of disorders, their roles during physiological aging are poorly investigated. In the present study, we investigated whether maintenance of mice in different environmental conditions could influence age-associated changes in hippocampal tPA expression and BDNF maturation in relation with modifications of their cognitive performances. Our data indicate that maintenance in enriched housing led to a reversal of age-associated decrease in expression of hippocampal tPA. A subsequent increase in the level of mature BDNF and an improvement in emotional and spatial memories were observed. Taken together, these data suggest that the tPA-BDNF axis could play a critical role in the control of cognitive functions influenced both by the age and housing conditions.
Asunto(s)
Envejecimiento/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/fisiología , Ambiente , Neuronas/fisiología , Activador de Tejido Plasminógeno/metabolismo , Animales , Aprendizaje por Asociación/fisiología , Conducta Animal/fisiología , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Femenino , Vivienda para Animales , Memoria/fisiología , RatonesRESUMEN
An operationally simple and concise synthesis of anilinoethanolamines, as NMDA NR2B receptor antagonist ifenprodil analogues, was developed via a copper-catalyzed amination of the corresponding bromoarene. Coupling was achieved with linear primary alkylamines, alpha,omega-diamines, hexanolamine and benzophenone imine, as well as with aqueous ammonia, in good yields using CuI and N,N-diethylsalicylamide, 2,4-pentadione or 2-acetylcyclohexanone as catalytic systems. Amination with ethylene diamine was efficient even in the absence of an additive ligand, whereas no reaction occurred with ethanolamine whatever the conditions used. The anilinoethanolamines were evaluated as NR2B receptor antagonists in a functional inhibition assay. Aminoethylanilines displayed inhibition effects close to that of ifenprodil.
Asunto(s)
Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Etanolamina/química , Etanolamina/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Aminación , Compuestos de Anilina/síntesis química , Compuestos de Bromina/química , Catálisis , Línea Celular , Cobre/química , Etanolamina/síntesis química , Humanos , Piperidinas/químicaRESUMEN
Neurocognitive impairment (NCI) is one of the most relevant clinical manifestations of multiple sclerosis (MS). The profile of NCI and the structural and functional changes in the brain structures relevant for cognition in MS share some similarities to those in Alzheimer's disease (AD), the most common cause of neurocognitive disorders. Additionally, despite clear etiopathological differences between MS and AD, an accumulation of effector/memory CD8+ T cells and CD8+ tissue-resident memory T (Trm) cells in cognitively relevant brain structures of MS/AD patients, and higher frequency of effector/memory CD8+ T cells re-expressing CD45RA (TEMRA) with high capacity to secrete cytotoxic molecules and proinflammatory cytokines in their blood, were found. Thus, an active pathogenetic role of CD8+ T cells in the progression of MS and AD may be assumed. In this mini-review, findings supporting the putative role of CD8+ T cells in the pathogenesis of MS and AD are displayed, and putative mechanisms underlying their pathogenetic action are discussed. A special effort was made to identify the gaps in the current knowledge about the role of CD8+ T cells in the development of NCI to "catalyze" translational research leading to new feasible therapeutic interventions.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Linfocitos T CD8-positivos/inmunología , Disfunción Cognitiva/inmunología , Esclerosis Múltiple/inmunología , Animales , Humanos , SinapsisRESUMEN
Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (K(D)=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.
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Fibrinolíticos/toxicidad , Receptores de N-Metil-D-Aspartato/metabolismo , Accidente Cerebrovascular/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Células Cultivadas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Activadores Plasminogénicos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal , Técnicas de Cultivo de Tejidos , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/metabolismoAsunto(s)
Conducta Animal , Mapeo Encefálico , Encéfalo/fisiología , Neuronas/fisiología , Optogenética , Semiconductores , Animales , HumanosRESUMEN
BACKGROUND AND PURPOSE: Whereas the effects of chronic arterial hypertension on the cerebral vasculature have been widely studied, its effects on brain tissue have been studied less so. Here we examined if spontaneously hypertensive rats (SHRs) or the normotensive control Wistar Kyoto rats (WKYs) made hypertensive by renal artery stenosis (R-WKYs) are vulnerable to an excitotoxic brain lesion provoked by an overactivation of glutamate receptors. METHODS: Lesion volumes were quantified by histology in WKYs and SHRs subjected to striatal administration of N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). The expression of AMPA receptors subunits and calcium/calmodulin kinase-II alpha was analyzed by real-time polymerase chain reaction and Western blot. RESULTS: NMDA (50 and 75 nmol) induced similar lesions in both SHRs (10+/-2 mm(3) and 16+/-4 mm(3), respectively) and WKYs (11+/-2 mm(3) and 19+/-7 mm(3), respectively). However, AMPA-induced (2.5 and 5 nmol) lesions were significantly greater in 14-week-old SHRs (14+/-3 mm(3) and 20+/-5 mm(3), respectively) than WKYs (4+/-2 mm(3), P<0.05 and 7+/-4 mm(3), P<0.001, respectively). Furthermore, normotensive 7-week-old SHRs also displayed an aggravated AMPA-induced lesion compared with age-matched WKYs (10+/-3 mm(3) vs 6+/-3 mm(3); P<0.05). Neither NMDA nor AMPA produced increased lesion volumes in R-WKYs (12+/-3 mm(3) and 5+/-4 mm(3), respectively) compared with WKYs. Striatal levels of AMPA receptors subunits, GluR1 and GluR2, were not different between SHRs and WKYs. However, SHRs displayed an increase in phosphorylated form of GluR1 at Ser-831 (P<0.05), as well as in calcium/calmodulin kinase-II alpha (P<0.002). Selective inhibition of this kinase by KN-93 reduced AMPA-induced damage in SHRs (P<0.01 vs vehicle). CONCLUSIONS: These findings show that an increase in phosphorylated GluR1, which increases AMPA receptor conductance, may be involved in the vulnerability of SHRs to AMPA.
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Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/fisiopatología , Isquemia Encefálica/fisiopatología , Ácido Glutámico/metabolismo , Hipertensión/complicaciones , Receptores AMPA/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Daño Encefálico Crónico/genética , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Predisposición Genética a la Enfermedad/genética , Hipertensión/genética , Hipertensión/fisiopatología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neurotoxinas/toxicidad , Fosforilación/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores AMPA/agonistas , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/fisiología , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidadRESUMEN
Rats have the ability to learn about potential food sources by sampling their odors on the breath of conspecifics. Although this ethologically based social behavior has been transposed to the laboratory to probe nonspatial associative olfactory memory, only a few studies have taken full advantage of its unique features to examine the organization of recently and remotely acquired information. We provide a set of standardized procedures and technical refinements that are particularly useful in achieving this goal while minimizing confounding factors. These procedures, built upon a three-stage protocol (odor exposure, social interaction and preference test), are designed to optimize performance across variable retention delays, thus enabling the reliable assessment of recent and remote memory, and underlying processes, including encoding, consolidation, retrieval and forgetting. The different variants of the social transmission of food preference paradigm, which take a few days to several weeks to perform, make it an attractive and versatile tool that can be coupled to many applications in CNS research. The paradigm can be easily implemented in a typical rodent facility by personnel with standard animal behavioral expertise.
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Técnicas de Observación Conductual/métodos , Conducta Alimentaria , Preferencias Alimentarias , Memoria , Olfato , Conducta Social , Animales , RatasRESUMEN
Laser Scanning Confocal Microscopy (LSCM) imaging using an appropriate fluorescent probe enables the visualization of a molecular target with high resolution, and represents a method of choice for studying expression, subcellular location, and trafficking of receptors in living cells. The chemical, physical, and pharmacological properties of the probe remain essential. Here, we describe (1) the preparation of a specific probe for NMDAR GluN2B receptor by conjugation of fluorescein to an ifenprodil-based ligand, (2) an in vitro functional assay by calcium imaging for GluN2B binding and inhibition evaluation of the probe, and (3) the labeling and confocal imaging of GluN2B in DS-red labeled living cortical neurons.
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Calcio/química , Microscopía Confocal/métodos , Receptores de N-Metil-D-Aspartato/química , Colorantes Fluorescentes/química , Piperidinas/químicaRESUMEN
We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.
Asunto(s)
Astrocitos/patología , Lesiones Encefálicas/patología , Gliosis/etiología , Receptor PAR-1/metabolismo , Amidas/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Northern Blotting/métodos , Western Blotting/métodos , Lesiones Encefálicas/fisiopatología , Bromodesoxiuridina/metabolismo , Butadienos/farmacología , Recuento de Células/métodos , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Colforsina/farmacología , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Ratones Noqueados , Microglía/patología , Nitrilos/farmacología , Oligopéptidos/farmacología , Piridinas/farmacología , ARN Mensajero/biosíntesis , Receptor PAR-1/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Trombina/farmacología , Factores de TiempoRESUMEN
Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer's disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Aßos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aßos on SWRs in cognitively-challenged animals, we submitted vehicle- and Aßo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aß mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aß mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aß-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer's disease.