Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.
Although organotypic hippocampal slice cultures (OHSCs) are used to study function within the hippocampus, the effect of maintenance in vitro upon protein expression is not fully understood. Therefore, we examined developmental changes in cultures prepared from P8 rats and maintained on porous membranes between medium and atmosphere. Between 7 and 28 days following explantation, altered hippocampal morphology could not be detected despite a significant decrease in both MAP-2c and a mid-range tau isoform by 21 DIV. During the same period, lower GFAP expression was observed, and GFAP labeling suggested a migration of astrocytes to the slice-atmosphere interface. In contrast, levels of the synaptic proteins synaptophysin and PSD-95 were significantly increased, but GAP-43 was not. The preservation of myelinated axons and synapses, along with glial and endothelial cells, was confirmed by ultrastructural analysis. Furthermore, intranuclear inclusion bodies, which are associated with normal aging in vivo, were detected in the CA1 pyramidal layer in cultures older than 14 DIV. When OHSCs were maintained for approximately 3, 4, and 10 weeks, a rise and then fall in the expression of synaptophysin and, especially, PSD-95 were found, and the biphasic trend paralleled by significant changes in Schaffer collateral-evoked excitatory post-synaptic potentials from CA1 neurons. Our data not only describe changes in cytoskeletal, synaptic, and nuclear proteins related to the maintenance of interface OHSCs, but also emphasize the potential of the model for the study of age-related phenomena within the hippocampus.
Mielke
John G
JG
Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Rd., Building M-54, Ottawa, Ontario, Canada K1A 0R6. john.mielke@nrc.ca
Comas
Tanya
T
Woulfe
John
J
Monette
Robert
R
Chakravarthy
Balu
B
Mealing
Geoffrey A R
GA
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D003599:Cytoskeleton / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D019922:GAP-43 Protein / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006624:Hippocampus / Q000254:growth & development* / Q000502:physiology* / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D016708:Synaptophysin / Q000378:metabolism; D013997:Time Factors; D016875:tau Proteins / Q000378:metabolism
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D019922:GAP-43 Protein; D005904:Glial Fibrillary Acidic Protein; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; C489787:MAP2 protein, rat; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D016708:Synaptophysin; D016875:tau Proteins; C007293:DAPI