Dendritic spines elongate after stimulation of group 1 metabotropic glutamate receptors in cultured hippocampal neurons - PubMed (original) (raw)

Dendritic spines elongate after stimulation of group 1 metabotropic glutamate receptors in cultured hippocampal neurons

Peter W Vanderklish et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Changes in the morphology of dendritic spines are correlated with synaptic plasticity and may relate mechanistically to its expression and stabilization. Recent work has shown that spine length can be altered by manipulations that affect intracellular calcium, and spine length is abnormal in genetic conditions affecting protein synthesis in neurons. We have investigated how ligands of group 1 metabotropic glutamate receptors (mGluRs) affect spine shape; stimulation of these receptors leads both to calcium release from intracellular stores and to dendritic protein synthesis. Thirty-minute incubation of cultured hippocampal slices and dissociated neurons with the selective group 1 mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced a significant increase in the average length of dendritic spines. This elongation resulted mainly from the growth of existing spines and was also seen even in the presence of antagonists of ionotropic receptors, indicating that activation of these receptors by mGluR-induced glutamate release was not required. Prolonged antagonism of group 1 mGluRs with (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) did not result in shorter average spine length. Elongation of dendritic spines induced by DHPG was blocked by calcium chelation and by preincubation with the protein synthesis inhibitor puromycin. The results suggest that in vivo activation of group 1 mGluRs by synaptically released glutamate affects spine shape in a protein synthesis-dependent manner.

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Figures

Figure 1

Figure 1

Examples of the effect of mGluR stimulation on the length of dendritic spines in two hippocampal culture systems. Slices (A and B) and dissociated neurons (C and D) were fixed after treatment with vehicle control (Left) or 100 μM DHPG (Right). Control and treated cultures prepared from the same animals were processed in parallel. Scale bar = 5 μm.

Figure 2

Figure 2

The effect of DHPG on the average length of dendritic spines of dentate gyrus granule cells in slice culture and dissociated hippocampal neurons. A 30-min incubation with 100 μM DHPG resulted in a significant increase in the average length of dendritic spines (1.35 ± 0.06 μm) relative to controls acutely treated with AP5/CNQX (0.99 ± 0.03 μm; n = 10 experiments, 2–3 granule cells per experiment, ≥50 spines per cell; P < 0.001, two-tailed Mann–Whitney test). Similar effects were obtained in dissociated neuronal culture (control 1.03 ± 0.03, DHPG 1.33 ± 0.05; P < 0.001). Prolonged incubation with MCPG did not alter the average length of dendritic spines (dentate granule cells, 0.99 ± 0.03; dissociated neurons, 1.05 ± 0.05).

Figure 3

Figure 3

Frequency distribution histograms of dendritic spine length in control and DHPG-treated cultures. (Upper) The length of spines on the dendrites of dentate granule cells in slice culture were measured and segregated into bins of 0.3 μm. The composite frequency distribution from 10 experiments shows that the occurrence of longer spines increases whereas that of shorter spines decreases. (Lower) The same effect is evident in composite frequency distributions of spine lengths in dissociated neuronal cultures.

Figure 4

Figure 4

Example of spine types and analysis of shifts in the relative abundance of these types accompanying DHPG treatment. (A) Maximum intensity projection of an image stack taken from a DHPG-treated dentate granule cell. Four spine types are labeled, classified as described in Methods and differentiated with respect to length and shape characteristics. (B) Relative abundance of spine types in control slices and in slices treated acutely with DHPG or chronically for 2–3 days with MCPG (control and DHPG treatments also included AP5 + CNQX). Spines identified as type 3 or 4, characterized by longer, thinner profiles with smaller spine heads, are increased in frequency with DHPG treatment whereas shorter (“nubbin”) spines, type 1, are decreased. A slight shift in the opposite direction with chronic MCPG is seen in this experiment, but this result did not occur reliably enough to influence the average spine length. The proportion of type 2 spines, the classic mushroom-shaped profiles, did not change with DHPG treatment.

Figure 5

Figure 5

(A) The effect of puromycin on DHPG-induced spine lengthening in hippocampal slice cultures. Mean spine lengths in slices treated with puromycin (Puro), DHPG, and DHPG in the presence of puromycin (DHPG + Puro) are graphed with sample images from each treatment above. (B) The effect of simultaneous manipulation of group 1 mGluRs and protein synthesis on spine density. The average number of dendritic spines per 50-μm segment of secondary dendrites of dentate granule cells was not significantly different from controls (70.9 ± 4.1, n = 7) after treatment with DHPG (82.6 ± 5.1, n = 9) or after 2–3 days of incubation with MCPG (66.5 ± 3.9, n = 11). Addition of puromycin simultaneously with either drug also did not significantly change the density of spines, although a trend toward fewer spines was seen with DHPG + puromycin (56.3 ± 1.0, n = 3; P = 0.06, Mann–Whitney U test, two-tailed).

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