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Protein synthesis by rat hippocampal slices maintained in vitro
Author(s) -
Phillips L. L.,
Steward O.
Publication year - 1988
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490210103
Subject(s) - paraformaldehyde , hippocampal formation , leucine , amino acid , in vivo , in vitro , incubation , chemistry , fixative , biophysics , biology , biochemistry , microbiology and biotechnology , anatomy , endocrinology , cytoplasm , organic chemistry
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% O 2 /5% CO 2 ) balanced salt medium containing 5 nM 3 H‐leucine. The time course of 3 H‐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 3 H‐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 μm 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 μm thickness there was complete penetration of 3 H‐leucine with no indication of a gradient in the extent of incorporation throughout the slice. The pattern of grain density within 300–600 μm slices matches that previously reported after in vivo injections of radio‐labeled 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 μm thickness showed irregular labeling. Slices of 200 μm were filled with pyknotic nuclei and vacuoles and exhibited patchy labeling. In 800 μm slices there were isolated areas of good preservation within the slice core, but these areas exhibited little incorporation. Relative to the 300–600 μm slices, there was a higher number of pyknotic nuclei and a much deeper layer of necrosis along the cut edges. Slices of 1,000 μm 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.