RESUMEN
The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily and is strongly expressed in the nervous system. NCAM is found in three major forms, of which two--NCAM-140 and NCAM-180--are transmembrane proteins, while the third--NCAM-120--is attached to the membrane via a glycosylphosphatidyl inositol anchor. In addition, soluble NCAM forms exist in brain, cerebrospinal fluid, and plasma. NCAM mediates cell adhesion through homophilic as well as through heterophilic interactions. Following NCAM binding, transmembrane signalling is believed to be activated, resulting in increased intracellular calcium. By mediating cell adhesion to other cells and to the extracellular matrix and by activating intracellular signaling pathways, NCAM influences cell migration, neurite extension, and fasciculation, and possibly formation of synapses in the brain. From studies on NCAM knock-out mice, NCAM have been shown to be crucial for the formation of the olfactory bulb and the mossy fiber system in the hippocampus. In addition, NCAM is important for neuronal plasticity in the adult brain associated with learning and regeneration.
Asunto(s)
Envejecimiento/metabolismo , Feto/fisiología , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Feto/metabolismo , Sistema Nervioso/metabolismoRESUMEN
The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(pid/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.
Asunto(s)
Recuento de Células/métodos , Técnicas de Cultivo de Célula/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Sistema Nervioso/citología , Sistema Nervioso/embriología , Neuritas/ultraestructura , Animales , Recuento de Células/instrumentación , Recuento de Células/normas , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/normas , Diferenciación Celular/fisiología , Tamaño de la Célula/fisiología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Sistema Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Células PC12/citología , Células PC12/metabolismo , RatasRESUMEN
Neuronal Kv7 channels are recognized as potential drug targets for treating hyperexcitability disorders such as pain, epilepsy, and mania. Hyperactivity of the amygdala has been described in clinical and preclinical studies of anxiety, and therefore, neuronal Kv7 channels may be a relevant target for this indication. In patch-clamp electrophysiology on cell lines expressing Kv7 channel subtypes, Maxipost (BMS-204352) exerted positive modulation of all neuronal Kv7 channels, whereas its R-enantiomer was a negative modulator. By contrast, at the Kv7.1 and the large conductance Ca2+-activated potassium channels, the two enantiomers showed the same effect, namely, negative and positive modulation at the two channels, respectively. At GABA(A) receptors (alpha1beta2gamma2s and alpha2beta2gamma2s) expressed in Xenopus oocytes, BMS-204352 was a negative modulator, and the R-enantiomer was a positive modulator. The observation that the S- and R-forms exhibited opposing effects on neuronal Kv7 channel subtypes allowed us to assess the potential role of Kv7 channels in anxiety. In vivo, BMS-204352 (3-30 mg/kg) was anxiolytic in the mouse zero maze and marble burying models of anxiety, with the effect in the burying model antagonized by the R-enantiomer (3 mg/kg). Likewise, the positive Kv7 channel modulator retigabine was anxiolytic in both models, and its effect in the burying model was blocked by the Kv7 channel inhibitor 10,10-bis-pyridin-4-ylmethyl-10H-anthracen-9-one (XE-991) (1 mg/kg). Doses at which BMS-204352 and retigabine induce anxiolysis could be dissociated from effects on sedation or memory impairment. In conclusion, these in vitro and in vivo studies provide compelling evidence that neuronal Kv7 channels are a target for developing novel anxiolytics.
Asunto(s)
Ansiolíticos/farmacología , Carbamatos/farmacología , Indoles/farmacología , Neuronas/efectos de los fármacos , Fenilendiaminas/farmacología , Canales de Potasio con Entrada de Voltaje/agonistas , Algoritmos , Animales , Ansiolíticos/química , Ansiedad/fisiopatología , Ansiedad/psicología , Ataxia/inducido químicamente , Conducta Animal/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Emociones/efectos de los fármacos , Conducta Exploratoria/efectos de los fármacos , Femenino , Fuerza de la Mano , Indoles/química , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Masculino , Potenciales de la Membrana , Ratones , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , ARN Complementario/biosíntesis , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/genética , Estereoisomerismo , Xenopus laevisRESUMEN
The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.