RESUMEN
Sensory whiskers are mapped to rodent layer 4 somatosensory cortex as discrete units termed barrels, which can be visualized at high resolution in living brain slices. Both anatomical and physiological properties of the layer 4 neuronal network can thus be investigated in the context of the functional boundaries of this sensory map. Large-scale confinement of neuronal arbors to single barrels was suggested by restricted lateral diffusion of DiI across septa between barrels. Morphological analysis of dendritic and axonal arborizations of individual excitatory neurons showed that neuronal processes remain within the barrel of origin through polarization toward the center of the barrel. Functionally, the large-scale properties of the neuronal network were investigated through mapping the spatial extent of field EPSPs, which were found to attenuate at barrel borders. This ensemble property of a layer 4 barrel was further investigated by analyzing the connectivity of pairs of excitatory neurons with respect to the locations of the somata. Approximately one-third of the excitatory neurons within the same barrel were synaptically coupled. At the septum between adjacent barrels the connectivity dropped rapidly, and very few connections were found between neurons located in adjacent barrels. Each layer 4 barrel is thus composed of an excitatory neuronal network, which to a first order approximation, acts independently of its neighbors.
Asunto(s)
Red Nerviosa/fisiología , Corteza Somatosensorial/fisiología , Animales , Axones/fisiología , Axones/ultraestructura , Mapeo Encefálico , Dendritas/fisiología , Dendritas/ultraestructura , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes , Técnicas In Vitro , Lisina/análogos & derivados , Red Nerviosa/citología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Neuronas/clasificación , Neuronas/fisiología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Corteza Somatosensorial/citología , Transmisión Sináptica/fisiología , Tálamo/citología , Tálamo/fisiología , Vibrisas/inervaciónRESUMEN
The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.
Asunto(s)
Encéfalo/fisiología , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/genética , Glutamatos/farmacología , Ácido Glutámico , Glicina/farmacología , Sustancias Macromoleculares , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Familia de Multigenes , N-Metilaspartato/farmacología , Sondas de Oligonucleótidos , Especificidad de Órganos , Péptidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.