Protein synthesis by rat hippocampal slices maintained in vitro.
The present study evaluates protein synthesis in rat hippocampal slices maintained in vitro. Transverse slices of hippocampus were prepared from both adult rats and rat pups during postnatal development and incubated in a gassed (95% O2/5% CO2) balanced salt medium containing 5 nM 3H-leucine. The time course of 3H-leucine incorporation into TCA-precipitable protein was determined using slices removed from the media after 5, 10, 20, 30, 40, 60, and 120 min of incubation. The pattern of 3H-amino acid incorporation was evaluated by fixing slices with paraformaldehyde, embedding the slices in plastic, and sectioning the slices end on and en face for autoradiographic analysis. Biochemical analysis of 300 and 400 micron slices revealed that incorporation of leucine into protein proceeds at a constant rate. The autoradiographic analysis revealed that in adult hippocampal slices of 300-600 micron thickness there was complete penetration of 3H-leucine with no indication of a gradient in the extent of incorporation throughout the slice. The pattern of grain density within 300-600 micron slices matches that previously reported after in vivo injections of radiolabeled amino acid, where grain density is highest over neuronal cell bodies and lower over the laminae that contain dendritic processes and axons (Phillips et al: Mol Brain Res 2:251-261, 1987). Hippocampal slices of 200, 800, and 1,000 micron thickness showed irregular labeling. Slices of 200 micron were filled with pyknotic nuclei and vacuoles and exhibited patchy labeling. In 800 micron slices there were isolated areas of good preservation within the slice core, but these areas exhibited little incorporation. Relative to the 300-600 micron slices, there was a higher number of pyknotic nuclei and a much deeper layer of necrosis along the cut edges. Slices of 1,000 micron thickness showed poor preservation throughout and low levels of incorporation. Biochemical studies revealed a much higher rate of incorporation in the slices prepared from postnatal animals. Autoradiography of the slices from developing rats revealed that penetration was excellent and incorporation appeared to be greater as judged by an overall higher grain density. We believe that rat hippocampal slices provide a good in vitro model of protein metabolism that will be useful for studies of protein synthesis in isolated cell body and dendritic laminae and for the evaluation of whether protein synthesis in particular laminae is regulated by synaptic activity.
Phillips
L L
LL
Department of Neuroscience, University of Virginia School of Medicine, Charlottesville 22908.
Steward
O
O
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D006624:Hippocampus / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D000596:Amino Acids; D009419:Nerve Tissue Proteins