Experience-dependent, rapid structural changes in hippocampal pyramidal cell spines

Abstract

Morphological changes in dendritic spines may contribute to the fine tuning of neural network connectivity. The relationship between spine morphology and experience-dependent neuronal activity, however, is largely unknown. In the present study, we combined 2 histological analyses to examine this relationship: 1) Measurement of spines of neurons whose morphology was visualized in brain sections of mice expressing membrane-targeted green fluorescent protein (Thy1-mGFP mice) and 2) Categorization of CA1 neurons by immunohistochemical monitoring of Arc expression as a putative marker of recent neuronal activity. After mice were exposed to a novel, enriched environment for 60 min, neurons that expressed Arc had fewer small spines and more large spines than Arc-negative cells. These differences were not observed when the exploration time was shortened to 15 min. This net-balanced structural change is consistent with both synapse-specific enhancement and suppression. These results provide the first evidence of rapid morphological changes in spines that were preferential to a subset of neurons in association with an animal's experiences.

Figure 1.

Simultaneous imaging of experience-dependent Arc expression and fine neuronal structure. (A) Time course of paradigm for mouse exposure to the novel environment (left) and representative images of experience-dependent Arc expression in hippocampal area CA1 (right). Thy1-mGFP mice were exposed to a novel environment for 15 min (N15) or 60 min (N60), whereas littermate HC controls remained in their HCs. Images of Arc immunohistochemistry (red) are shown with Nissl counterstain (blue). (B) Percentage of Arc(+) cells. n = 9, 6, and 5 mice for HC, N15, and N60 groups, respectively. **P < 0.01 versus HC, Tukey's post hoc test. Error bars indicate the standard error of the mean. (C) The level of Arc expression did not differ between the N15 and N60 groups. Somatic immunoreactivity (i.r.) of Arc(+) neurons was normalized to HC Arc(−) cells; P > 0.05 by Student's t-test. (D) Representative image of an mGFP (green)–expressing pyramidal cell, Arc immunoreactive cells (red), and Nissl stain (blue) in an N60 mouse. The inset shows spines (arrowheads) on a basal dendrite of an mGFP(+) cell. SO, stratum oriens where basal dendrites extend; SP, stratum pyramidale; SR, stratum radiatum. Scale bars, 20 μm in (A) and (D), 1 μm in the inset of (D).

Figure 2.

Effects of exposure to a novel, enriched environment on spine density. Spine density per micron of dendrite length and distributions of spine head sizes in N15 (A, B) and N60 (C, D) cells are shown. Arc(+) cells in the N60 group possessed fewer small spines compared with Arc(−) cells in both the N60 and HC groups, whereas there was not a statistically significant difference in the N15 group. (A, B) HC, n = 19 (1081 spines) from 4 mice; Arc(−), n = 23 cells (1513 spines); Arc(+), n = 10 cells (564 spines) from 4 mice. (C, D) HC, n = 16 cells (851 spines) from 6 mice; Arc(−), n = 19 cells (1283 spines); Arc(+), n = 8 cells (350 spines) from 8 mice. Error bars indicate standard error of the mean. *P < 0.05 by Tukey's post hoc test in (A, C). **P < 0.01/3, *P < 0.05/2 by Bonferroni–Holm test after repeated-measures 2-way ANOVA in (B, D). These significant differences were reproduced in another independent experiment. (E) There was a pronounced decrease in spines smaller than 0.5 μm on Arc(+) cells in the N60 group (red), but not in the N15 (gray) group. (F) Spine density ratio between the Arc (+) and Arc (−) cells for the small (≤0.5 μm) and large (>0.5 μm) spines. Note the distinctly opposite patterns for small and large spines in the N60 group.

Figure 3.

Distributions of spines with large heads. (A–F) Arc(+) cells possessed more large spines than did corresponding control cells in the N60 group, whereas such differences were rarely observed in the N15 group. For each of 2 environment-exposed groups (N15 or N60), pooled datasets of spines from the 3 groups, 1) Arc(−) cells in HC (black), 2) Arc(−) cells in either N15 or N60 (blue), or 3) Arc(+) cells in either N15 or N60 (pink) were analyzed. Head sizes of all spines in the dataset were ranked in descending order, and then we defined large-head spines as spines whose size was among the largest x% (5–25%) of all measured spines in the each of the pooled datasets. Then the ratios of the large-head spines belongs to each group (normalized by analyzed dendrite length of the group) were calculated (see Materials and Methods). n = 3289 spines (59 cells) in the N15 group and 2484 spines (43 cells) in the N60 group. (A, D) Comparisons of the ratio of spines within the top 5% of head size for the N15 or N60 groups, respectively. (B, E) The ratio distributions of spines in the top 25% of head size. Arc(+) groups were compared with their corresponding HC and Arc(−) groups. Dotted line, 33.3%. (C, F) To estimate possible stochastic fluctuations, we created 200 surrogate data points by randomly shuffling the rank order in the pooled dataset. The averages (lines) and standard deviations (gray areas) of the randomdata points are shown. Data from Arc(+) groups are shown as open (P > 0.05) or closed (P < 0.05 vs. the randomized data) pink circles. The significant differences observed for the N60 group were reproduced in another independent experiment. (G–I) Densities of very-large spines (head sizes > 0.8 μm) were plotted against that of small spines (head sizes < 0.5 μm) in HC (G), N15 (H), and N60 (I) groups. Open and closed symbols indicate data from each cell and means ± SEM, respectively. Regression lines were drawn for the data including both Arc(−) and Arc(+) cells. n = 38 (G), 37 (H), and 27 (I). P = 0.11 (G), 0.02 (H), and 0.004 (I). By Pearson's correlation test, correlation coefficients were −0.26 (G), −0.38 (H), and −0.53 (I).