Spine-type-specific recruitment of newly synthesized AMPA receptors with learning

Abstract

The stabilization of long-term memories requires de novo protein synthesis. How can proteins, synthesized in the soma, act on specific synapses that participate in a given memory? We studied the dynamics of newly synthesized AMPA-type glutamate receptors (AMPARs) induced with learning using transgenic mice expressing the GluR1 subunit fused to green fluorescent protein (GFP-GluR1) under control of the c-fos promoter. We found learning-associated recruitment of newly synthesized GFP-GluR1 selectively to mushroom-type spines in adult hippocampal CA1 neurons 24 hours after fear conditioning. Our results are consistent with a "synaptic tagging" model to allow activated synapses to subsequently capture newly synthesized receptor and also demonstrate a critical functional distinction in the mushroom spines with learning.

Fig. 1

Basic features of GFP-GluR1^c-fos Tg mice. (A) Schematic representation of the transgenic system. The c-fos promoter was used to drive rapid and transient expression of the tetracycline-regulated transactivator (tTA) in activated neurons. The tTA in turn activates transcription of a tetO promoter-linked GFP-GluR1 in a Dox-regulated manner. (B-E) Confocal microscopy images of intrinsic GFP fluorescence (upper two panels) and GFP immunoreactivity (lower 3 color panels) in the hippocampal slices. Nuclei were stained with TO-PRO-3 (blue). Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiatum; Mo, molecular layer; Gr, granule cell layer; DG, dentate gyrus. (F) Proportion of CA1 neurons with cytoplasmic GFP immunoreactive signals. Data in this and all subsequent figures are represented as mean ± s.e.m. ON Dox: n = 5 mice for home and FC24h. OFF Dox: n = 5 for home; n = 4 for FC1h, FC4h, FC24h, CT24h, and UP24h; n = 3 for FC4h and FC6h. (G) Western blot analysis showing the expression of GFP-GluR1 and endogenous GluR1 in the hippocampus. (H) Co-immunoprecipitation of GFP-GluR1 and endogenous GluR2 in the hippocampus.

Fig. 2

Preferential recruitment of newly synthesized GFP-GluR1 to mushroom spines 24 hours after fear-conditioned learning. (A) Experimental design. (B) Confocal image of a hippocampal slice labeled with anti-GFP antibody (green) and DiI (red). (C) Confocal image showing surface GFP-GluR1 localization on apical dendrite after fear conditioning. Some spines were GFP positive (arrow heads) but some were negative, showing an uneven synaptic trafficking of newly synthesized GFP-GluR1. (D) Representative images of spine morphology and surface GFP-GluR1 localization. Scale bar, 1μm. (E) Relative immunoreactivity from western blot analysis (CT24h: n = 4, FC24h: n = 3). (F) The percentage of GFP-GluR1⁺ mushroom spines was significantly higher (apical: F_2,16 = 8.86, p = 0.0026, basal: F_2,16 = 15.43, p = 0.0002) in FC24h group (apical: n = 7 mice, 1927 spines, basal: n = 7 mice, 1295 spines) as compared to the CT24h group (apical: n = 6 mice, 1626 spines, basal: n = 6 mice, 1292 spines) and UP24h group (apical: n = 6 mice, 2240 spines, basal: n = 6 mice, 1974 spines). The proportion was significantly decreased (apical: p = 0.0056, basal: p = 0.0010, t-test) at 72 hours after conditioning (apical: n = 6 mice, 1233 spines, basal: n = 6 mice, 1138 spines). *p < 0.01.

Fig. 3

Distribution of pre-existing GFP-GluR1 following memory extinction training. (A) Experimental design. (B) The percentage of time spent freezing before fear conditioning (basal), after fear conditioning (after FC), and during three trials of extinction training (EXT1∼3). n = 7. (C) The proportion of GFP-GluR1⁺ mushroom spines was significantly higher (apical: p = 0.045, basal: p = 0.033, t-test) in the extinction trained group (apical: n = 7 mice, 1870 spines, basal: n = 6 mice, 1845 spines) compared to the control group (FC72h, the same data as in Fig. 2F). (D) Relative immunoreactivity from western blot analysis (n = 3 for each condition).

Fig. 4

Spine remodeling following behavioral trainings. (A) No significant changes were observed in spine density (apical: F_4,27 = 0.72, p = 0.58, basal: F_4,27 = 0.88, p = 0.49). n = 6 mice for UP24h, CT24h, and FC72h. n = 7 for FC24h and FC72h+EXT. (B) No significant changes were observed in spine-type distribution. n = 6 mice for UP24h, CT24h, and FC72h. n = 7 for FC24h and FC72h+EXT.