Molecular neuroscienceEndogenous insulin signaling protects cultured neurons from oxygen–glucose deprivation-induced cell death
Section snippets
Primary neuronal cultures
Primary cultured cortical and hippocampal neurons were prepared from Wistar rats at approximately embryonic day 18 in accordance with guidelines for the use of experimental animals established by the Canadian Council on Animal Care. All procedures were approved by the institutional animal care committee, and were designed to minimize both the number of animals employed and their suffering. Briefly, isolated cortices or hippocampi were pooled, dissociated via mechanical trituration, and
IRs are constitutively present at the plasma membrane and are synaptically localized in cultured neurons
Although earlier studies reported that IRs were present in cultured rat neurons (Raizada et al 1982, Boyd and Raizada 1983, Abbott et al 1999), the intracellular versus plasmalemmal expression profile of the receptor had not been examined. To determine the proportion of IRs expressed at the plasma membrane in cultured hippocampal neurons, cell culture ELISAs were performed under both low-permeant (plasmalemmal) and permeant (total) conditions with an antibody recognizing an epitope in the
Discussion
In agreement with earlier studies (Weyhenmeyer et al 1985, Abbott et al 1999), the current immunochemical findings illustrate that the IR is abundant in cultured neurons and may be found throughout both somata and neurites. For the first time, the proportion of IRs present upon the surface of hippocampal neurons was examined, and the receptor was shown to be nearly equally distributed between plasmalemmal and intracellular locations, which is in stark contrast to the pattern observed with
Acknowledgments
The authors would like to thank Edmund Lo for technical contributions, and Dr. Joseph Tauskela for a review of the manuscript and constructive discussion.
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