Glutamate-induced transient modification of the postsynaptic density - PubMed (original) (raw)

Glutamate-induced transient modification of the postsynaptic density

A Dosemeci et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5-3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca(2+) from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity.

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Figures

Figure 1

Figure 1

Measurement of the thickness and the amount of gold label associated with PSDs. A cytoplasmic outline of the PSD was traced by hand. This area was then enclosed by tracing the postsynaptic membrane separately. The average thickness of the PSD was calculated by dividing the outlined area by the length of the postsynaptic membrane. The intensity of the gold label was estimated as the number of silver-enhanced gold particles within the outline around the PSD divided by the length of the corresponding postsynaptic membrane.

Figure 2

Figure 2

Brief depolarization with high K+ induces thickening of PSDs in hippocampal cultures (A and B) and hippocampal slices (C and D). Hippocampal cultures were incubated for 3 min either in normal incubation medium (A) or in medium containing 90 mM KCl (B) before fixation. After recovery hippocampal slices were perfused for 90 s in either normal ACSF (C) or in ACSF containing 90 mM KCl (D) and fixed immediately. In media containing 90 mM KCl, osmolarity was adjusted by an equivalent reduction in [NaCl]. (Bar = 100 nm.)

Figure 3

Figure 3

Reversible thickening of PSDs induced by high K+ is accompanied by a reversible increase in CaMKII labeling. Cultures were incubated in either normal incubation medium (A) or medium containing 90 mM KCl (B and C) and fixed immediately. One group of cultures was allowed to recover for 30 min after exposure to high K+ (D). After fixation, samples were immunogold labeled with an antibody against CaMKII. Silver-enhanced gold particles appear as black grains of variable size. (Bar = 100 nm.) (Bottom) Scatter plots show measurements of the thickness (Bottom Left) and CaMKII label intensity (Bottom Right) of PSDs in parallel experiments. Each point corresponds to a measurement from an individual PSD. Between 22 and 24 PSDs were analyzed per group.

Figure 4

Figure 4

Glutamate/glycine application mimics the effects of high-K+ depolarization. Cultures were incubated in either normal incubation medium (A) or the same medium containing 100 mM glutamate/10 mM glycine for 90 s (B and C). After exposure to glutamate, one group of samples was washed and incubated for an additional 5 min in Ca2+-free incubation medium (D). A, B and C, D represent pairs from two separate experiments. (Bar = 100 nm.) (Bottom) Measurements of the thickness of the PSDs (Bottom Left) and intensity of CaMKII gold label (Bottom Right) are presented as scatter plots. Each point corresponds to a measurement from an individual PSD. Between 15 and 20 PSDs were analyzed per group.

Figure 5

Figure 5

Immunogold labeling pattern for PSD-95 is distinct from that for CaMKII. Cultures were labeled with an antibody against PSD-95 (A and B) or with an antibody against CaMKII (C and D) after 3-min exposure to either normal incubation medium (A and C) or medium containing 100 μM glutamate/10 μM glycine (B and D). (Bar = 100 nm.)

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