Neuroprotocols最新文献

The adrenergic receptors belong to a gene superfamily whose members share a great deal of sequence homology. To date, over 60 members of this superfamily have been cloned and sequenced. Ready access to this wealth of sequence information, together with the software tools to analyze it, can facilitate experimental design and interpretation as well as expedite the realization of experimental goals. Many investigators have one or more computers connected to the Internet, often for no purpose other than to exchange electronic mail (e-mail) or to utilize the file-serving capabilities of a Novell network. These investigators are, to a large extent, unaware of the extensive resources that the Internet offers. The aim of this article is to facilitate utilization of the Internet, particularly as a tool to aid in the study of adrenergic receptors, and to guide the readers exploration of this resource. Although the Internet may at first appear to be an unfathomable morass of information, the simple command "help" can often be used to guide one′s path. Wherever possible, the reader is directed to retrieve the latest documentation on each topic directly from the Internet.

Adrenergic receptors have been studied extensively for more than 30 years, first by physiological means, later with pharmacologic and biochemical approaches, and within the past several years by molecular biology. This extensive body of work provided the basis for subdividing the adrenergic receptors into β-, α₁-, and α₂-adrenergic receptor types and, subsequently, into β₁-, β₂-, α_1A-, α_1B-, α_2A-, and α_2B-adrenergic receptor subtypes. Although the pharmacologic approach indicated that there exist multiple subtypes of each type of adrenergic receptor, it was the molecular cloning of adrenergic receptor cDNAs/genes that demonstrated the existence of three genes encoding each adrenergic receptor type in humans and rats (and therefore probably in all mammals). The nine adrenergic receptor proteins expressed in cultured cells faithfully mimic the basic pharmacologic and biochemical properties ascribed to these receptors. In this article, we review the molecular cloning and characterization of the adrenergic receptors with special emphasis on the α₂-adrenergic receptors and we discuss a classification scheme based on the hypothetical molecular evolution of the adrenergic receptors.

The distribution of α- and β-adrenergic receptors in slices of anatomically complex tissues can be delineated autoradiographically with great precision and resolution. This brief review highlights methodological aspects of adrenergic receptor autoradiography and in situ hybridization at the light microscopic level of resolution. It focuses on technical differences between autoradiographic analysis in tissue sections and conventional radioligand binding assays in membranes prepared from tissue homogenates. it emphasizes strategies for characterizing quantitatively the distribution of specific receptor subtypes and classes using autoradiography and ways of detecting naturally occurring low-abundance adrenergic receptor mRNAs using in situ hybridization.

The study of motor neurons is a technically difficult area in neurobiology because of limitations in isolation and culture. Among the many approaches used for isolating and culturing primary motor neurons, few have produced enough cells for biochemical and molecular studies. To overcome this problem, we have used somatic cell hybridization methods to generate hybrid neural cells that have traits typical of motor neurons. Isolated embryonic mouse spinal cord motor neurons were fused to an aminopterin-sensitive and neomycin-resistant mouse neuroblastoma cell line to produce several hybrid neuron cell lines. Those cell lines expressing high levels of choline acetyltransferase activity were selected and cloned. The hybrid nature of the cloned cells was confirmed by analysis of glucose phosphate isomerase allozymes and karyotyping. Availability of these embryonic clonal hybrid cells will make possible molecular, physiologic, and biochemical studies to define the biological properties of spinal motor neurons.

We have isolated several immortal cell lines from Xenopus neuroepithelium and retinal pigment epithelium. These cell lines were initially isolated from primary cultures by serial passaging of proliferating cells, followed by subcloning with limiting dilution techniques. Several morphologically distinct cell lines have been isolated using these procedures. On the basis of immunocytochemical characterization using specific antibodies, we have established that three of these cell lines, the XR1, XRpe1, and XRpe2 cell lines, are glial-like in nature. These cell lines were extensively labeled by antibodies against glial fibrillary acidic protein and vimentin, markers used to identify glial cells. Mono-layers of these cell lines served as useful substrates for axon outgrowth from developing retinal ganglion cells. In addition, analysis of cell-free substrates, prepared by treatment of cell line monolayers with Triton X- 100, revealed that the XR1, XRpe1, and XRpe2 cell lines produce an extracellular matrix (ECM) with potent neurite outgrowth-promoting activity. In contrast, other established retinal and nonretinal Xenopus cell lines were relatively ineffective and did not support axon outgrowth. We propose that neurite outgrowth-promoting activity produced by these cell lines is associated with their ECM and may be glial cell specific. In addition, to further characterize these cell lines, we have recently imaged live cells, using the atomic force microscope (AFM). The use of AFM on living, cultured cells provides a new, high-resolution method for examining dynamic cytoskeletal and morphological events.