RESUMEN
NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Muerte Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/toxicidad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/toxicidad , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo I/fisiología , Péptidos/toxicidad , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: Anatomical studies have shown that the paraventricular nucleus of the thalamus (PVT) innervates areas of the forebrain involved in the expression and regulation of emotional behaviors including fear and anxiety. In addition, the PVT is densely innervated by fibers containing orexin-A (OXA) and orexin-B (OXB), peptides that are well-known for their arousal effects on behavior. OBJECTIVES: In this study, we investigate whether microinjections of orexin receptor agonists and antagonists in the PVT region alter expression of anxiety-like behaviors in the rat as measured in the elevated plus maze. RESULTS: We report that microinjections of OXA and OXB in the PVT region elicited anxiety-like response as indicated by a reduction in open arm time and entries. In addition, OXA and OXB produced changes in ethological measures indicative of an anxiety state. Central administrations of antagonists for corticotropin releasing factor (CRF) or the opioid kappa receptors attenuated the anxiogenic effects produced by microinjections of OXA in the PVT region. We also provide evidence that endogenously released orexins act at the PVT to produce anxiety by showing that microinjections of TCSOX229, an orexin-2 receptor antagonist, in the PVT region attenuated the anxiogenic effects produced by a previous exposure to footshock stress. CONCLUSIONS: This study indicates that endogenously released orexins act on the PVT to regulate anxiety levels through mechanisms involving the brain kappa and CRF receptors.
Asunto(s)
Ansiedad/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Núcleos Talámicos de la Línea Media/metabolismo , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Ansiedad/inducido químicamente , Conducta Animal/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/toxicidad , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Microinyecciones , Neuropéptidos/toxicidad , Receptores de Orexina , Orexinas , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Neuropéptido/agonistas , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores Opioides kappa/metabolismoRESUMEN
The aim of this study was to test the role of the perifornical hypothalamus and adjacent areas in the behavioral and cardiovascular responses to two forms of stress, conditioned fear to context and restraint. Of particular interest was the role of the hypocretin (orexin) containing neurons in these responses. Rats implanted with radio-telemetric probes and fear conditioned to a context received bilateral injections of the neurotoxin hypocretin-2-saporin centered on the perifornical area. One week later, the animals were tested for conditioned fear to context and restraint while recording freezing, 22 kHz ultrasonic vocalizations, activity, mean arterial pressure and heart rate. Histological verification revealed that the lesions were not specific since virtually all the neurons within the injection area were lost. Nevertheless, these lesions, which were centered on the perifornical area, markedly reduced all recorded components of the contextual fear response (by 70%) but had no effect on the response to restraint. The lesions also caused a reduction in body weight and reduced the circadian rhythm of the recorded parameters. The results show (i) that hypocretin-2-saporin was not specific enough to produce lesions restricted to the hypocretin system, (ii) that neurons of the perifornical area are necessary for the expression of both the cardiovascular and behavioral components of conditioned fear to context, and (iii) that the same neurons are not necessary for the cardiovascular response to restraint. Thus, the perifornical hypothalamus is critical for some forms of stress but not others. We propose that it is a necessary relay for emotional responses in which the psychological component is stronger than the sensory component.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Miedo , Hipotálamo/lesiones , Neurotoxinas/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Reacción Cataléptica de Congelación , Frecuencia Cardíaca/efectos de los fármacos , Hipotálamo/fisiopatología , Inmunohistoquímica/métodos , Péptidos y Proteínas de Señalización Intracelular/toxicidad , Masculino , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/toxicidad , Orexinas , Ratas , Ratas Wistar , Saponinas/toxicidad , Factores de Tiempo , Vocalización Animal/efectos de los fármacosRESUMEN
Orexins (hypocretins) are neuropeptides expressed specifically in neurons in the lateral hypothalamic area and are known to be involved in the regulation of vigilance and feeding behavior. However, the relationship between orexin and emotional behaviors like anxiety is still poorly understood. Therefore, in this report we evaluated the effect of intracerebroventricular injection of orexin-A in two major anxiety tests, the light-dark exploration test (mouse) and the elevated plus-maze test (mouse, rat). Orexin increased time spent in the dark compartment in the light-dark test and time spent on the closed arms in the elevated plus-maze test. These results were not caused by a hypothetical sedative or activity-inducing effect of orexin-A because spontaneous locomotor activity did not alter upon orexin-A application under novel conditions. We therefore suggest an anxiogenic effect of orexin-A. To our knowledge, this is the first report about a relationship between orexin-A and anxiety.
Asunto(s)
Ansiedad/etiología , Conducta Animal/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/toxicidad , Neuropéptidos/toxicidad , Análisis de Varianza , Animales , Ansiedad/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Inyecciones Intraventriculares/métodos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Orexinas , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The medial septum and diagonal band of Broca (MSDB) provide a major input to the hippocampus and are important for spatial learning and memory. Although electrolytic MSDB lesions have prominent memory impairing effects, selective lesions of either cholinergic or GABAergic MSDB neurons do not or only mildly impair spatial memory. MSDB neurons are targets of orexin-containing neurons from the hypothalamus. At present, the functional significance of orexin afferents to MSDB is unclear, and the present study investigated a possible involvement of orexin innervation of the MSDB in spatial memory. Orexin-saporin, a toxin that damages neurons containing the hypocretin-2 receptor, was administered into the MSDB of rats. Rats were subsequently tested on a water maze to assess spatial reference memory and a plus maze to assess spatial working memory. At 100 ng/microl, orexin-saporin destroyed primarily GABAergic septohippocampal neurons, sparing the majority of cholinergic neurons. At 200 ng/microl, orexin-saporin almost totally eliminated GABAergic septohippocampal neurons and destroyed many cholinergic neurons. Spatial reference memory was impaired at both concentrations of orexin-saporin with a dramatic impairment observed for 24-h retention. Short-term reference memory was also impaired at both concentrations. Rats treated with 200 ng/microl, but not 100 ng/microl, of orexin-saporin were also impaired on a spontaneous alternation task, showing a deficit in spatial working memory. Our results, together with previous studies, suggest that orexin innervation of the MSDB may modulate spatial memory by acting on both GABAergic and cholinergic septohippocampal neurons.