Neuroprotocols最新文献

We describe methods for grafting cultured cells into the newborn and the adult rat using as an example the immortalized hippocampal cell line HiB5. We discuss cell labeling strategies and animal preparation prior to as well as animal care after surgery. The principles of stereotaxic surgery are presented with a standard protocol for transplantation into the adult. A grafting technique that allows micro-volumes of cell suspension to be precisely placed in very small target sites with minimum trauma is described. Also described is a detailed protocol for stereotaxic placement in the newborn that utilizes a new instrument that allows safe, prolonged anesthesia and accurate, reproducible placement of cell suspensions.

Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in the catecholamine biosynthetic pathway, converting norepinephrine to epinephrine. In transgenic mice, 2 kb of the human PNMT promoter directs the expression of the simian virus 40 (SV4O) early region in three classes of retinal neurons and in chromaffin cells of the adrenal medulla. These transgenic animals develop retinal and adrenal medullary tumors between 3 and 4 months of age. We have adapted these tumor cells to cell culture and have derived immortalized retinal neuronal and adrenal chromaffin cell lines. These cell lines express a number of cell- and tissue-specific markers indicative of the differentiated cells of origin.

Avian embryos, especially those of chick and quail, offer powerful systems for studying very early steps of both central and peripheral neurogenesis, but the lack of permanent neural avian cell lines constitutes an obstacle to analyzing differentiating events at the molecular level. In this paper, we present a strategy whereby new avian cell lines can be generated by infecting, in vitro, cells arising from different parts of the nervous system with natural occurring retroviruses. In particular, the cells from neural crest, neuroretina, peripheral ganglia, and neural tube were induced to proliferate highly by either MC29 or RSV infection. We also discuss some investigations performed with retroviral-infected cells aimed at testing factors or searching for genes that are implicated in the cellular cycle or in differentiating events. Finally, we include a discussion concerning the potential uses and limitations of such a system.

The c-myc and the N-myc proto-oncogenes were employed to immortalize neural progenitor cells. Infection of neural precursors isolated from the mouse at the 10th day of embryonic development (E10) with myc-containing retroviruses resulted in immortalized cell lines representing bipotential E10 neuroepithelial cells. These cell lines have the capacity to differentiate into both glial and neuronal cells either spontaneously in the case of the Zen(myc) cell lines or after addition of fibroblast growth factor to the Dol(myc) cell lines. Infection of migrating neural crest cells with the myc retroviruses gave rise to three different types of immortalized cell lines: (i) cell lines resembling freshly isolated neural crest cells; (ii) cell lines that can differentiate into cells expressing Schwann cell markers when grown at high cell concentrations; and (iii) cell lines that have the ability to differentiate in culture to process-bearing cells which expressed neuronal markers or have the characteristics of Schwann cells. Olfactory epithelial cell lines were generated by infection with Zen retrovirus bearing the N-myc proto-oncogene. Some of the cell lines resemble basal cells and others grow as bipolar cells resembling neurons and expressing the neuronal marker neurofilaments.

The use of lipophilic compounds in the study of signal transduction and other cellular processes is often complicated by the need to deliver compounds that are minimally soluble in water to cells in culture. A typical approach is to dissolve the lipophilic compound at high concentration in organic solvent and then to dilute the solvent in aqueous medium. However, such an approach usually results in precipitation of the compound in the aqueous medium and may minimize delivery of the compound to cells. Three techniques to keep the lipophilic compound In a nonprecipitated and stable form that will be readily available to the cells are described. They involve the use of protein carriers, liposomes, and emulsions. Particular attention is given to the use of emulsions, since this technique combines the advantages of a high capacity for lipophilic compounds, ease of assembly, and minimum contact between the lipophilic compound and water. Although the techniques differ with respect to the mechanics of combining the compound and the carrier, they all consist of a two-phase system dependent on partitioning between the carrier and the cells. Due to the need to take partitioning into account, all of these techniques differ from homogeneous solution-phase delivery. Therefore, in addition to descriptions of the techniques, criteria for both experimental design and analysis of data generated using two-phase systems are presented. In combination, the use of methods appropriate for delivery of lipophilic compounds to cells and the application of relevant calculations and analytical procedures provide the means necessary for design and Interpretation of experiments using lipophilic compounds.