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Figure 1. Stability and dynamics of dendritic spines during 5-day time-lapse imaging.(a) Each single granule cell in the odor and control groups was labeled by electroporation with GFP. Overlay images of bright field and fluorescence are shown in a1 and a3, with amplification of fluorescence images in a2 and a4. (a5) High magnification views of the four types of dendritic spines in a single granule cell are shown in a4. f indicates filopodia, t indicates thin spine, s indicates stubby spine, m indicates mushroom spine. (b,c) Serial time-lapse images of a single neuron each in the control and the odor groups at 1-day interval are shown. The red arrow indicates stable spines, yellow arrow indicates newly added spines, and blue arrow indicates eliminated spines. Scale bar corresponds to 5 μm. (d) Percentages of the large and small dendritic spines at 1-day interval in odor stimulation compared with the control group were analyzed. (e) The stability of large and small spines at 1-day intervals was analyzed. (f) The dynamic changes (added or eliminated) of large and small spines at 1-day intervals were analyzed. Error bars indicate the mean ± SEM. N = 6 neurons in 6 tadpoles, n = 18 dendrites in each group. Numbers of spines in control group are 952 at 1d, 962 at 2d, 973 at 3d, 1013 at 4d, and 1035 at 5d. Numbers of spines in odor group are 1164 at 1d, 1223 at 2d, 1357 at 3d, 1390 at 4d, and 1418 at 5d. The significance levels were *p < 0.05, **p < 0.01, and ***p < 0.001. Two-sample unpaired t-tests.
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Figure 2. Long-term imaging of dendritic spines in vivo.(a) Olfactory manipulation process. (b,c) Time-lapse imaging of spines in experimental group (b, normally raising for 1 week, odor treating for 1 week, and odor removing for 1 week), compared with imaging of spines in control group (c, normal raising for 3 weeksâ development), showed sustained number changes in dendritic spines under the two conditions. The red arrow indicates the stable spines, yellow arrow indicates the newly added spines, and blue arrow indicates the eliminated spines. Scale bar corresponds to 2âμm. (d) Quantitative analysis of spine densities showed changed numbers of spines per 10âμm dendrite in the control and experimental groups. Nâ=â6 neurons in 6 tadpoles and nâ=â21 dendrites were used for the 2-day interval observation. Numbers of spines in control group are 994 at 1d, 1123 at 3d, 1135 at 5d, 1182 at 7d, 1207 at 9d, 1214 at 11d, 1245 at 13d, 1271 at 15d, 1292 at 17d, 1267 at 19d, and 1360 at 21d. Numbers of spines in experimental group are 971 at 1d, 1164 at 3d, 1160 at 5d, 1169 at 7d, and 1525 at 9d, 1583 at 11d, 1654 at 13d, 1678 at 15d, 1599 at 17d, 1633 at 19d, and 1700 at 21d. Error bars indicate the meanâ±âSEM. The significance levels were *pâ<â0.05, **pâ<â0.01, and ***pâ<â0.001. Two-sample unpaired t-tests.
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Figure 3. Ratio changes of the four spines influenced by odor-enriched and odor-removed environment.(a) Statistical analysis of filopodia (a1), thin (a2), stubby (a3), and mushroom (a4) spines in the experimental group showed the dynamic changing percentages of spines influenced by normal raising for 1 week, odor enrichment for 1 week, and odor removing for 1 week. (b) Respective percentages of the four types of spines during natural development for 3 weeks in the control group were analyzed, showing differences from the experimental group. (c) In the experimental group, the stability of each type of spine, respectively treated with normal raising, odor stimulation, and odor removal, was analyzed. N = 6 neurons in 6 tadpoles, n = 21 dendrites. Total numbers of spines in control group are 1109 at the 1st week, 1234 at the 2nd week, and 1306 at the 3rd week. Total numbers of spines in experimental group are 1116 at the 1st week, 1610 at the 2nd week, and 1644 at the 3rd week. Error bars indicate mean ± SEM. The significance levels were *p < 0.05, **p < 0.01, and ***p < 0.001. One-way ANOVA Tukey’s multiple comparison tests.
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Figure 4. The morphological transformation of spines in different olfactory environments.(a) The time-lapse imaging of spines transformed into other types in control, odor enrichment and olfactory deprivation groups. In control group, a thin spine transformed into a mushroom at 4âh, then reverted to a thin at 24âh. A mushroom spine transformed into a thin at 4âh, then transformed into a filopodia at 12âh. In the odor group, a stubby spine transformed into a thin at 4âh, then reverted to a stubby at 12âh. A filopodia spine disappeared at 4âh, then a thin spine appeared at 8âh, transformed into a filopodia at 12âh, and reverted to a thin at 24âh. In the olfactory deprivation group, a filopodia spine transformed into a thin at 4âh, then the thin disappeared at 8âh until 24âh. A thin spine transformed into a stubby at 8âh, then the stubby disappeared at 24âh. Scale bar corresponds to 5âμm. Percentages of filopodia (b), thin (c), stubby (d) and mushroom (e) spines that transformed into other types of spines were statistically analyzed. (f) Simplified diagram of spine transformation in response to sensory stimulation. For the purpose of illustration, only a sample of spine transformation events is shown and the proportion of spine transformation does not strictly follow the statistical figures. Spines were first imaged at T0 and the spine transformation events happened at T1. The red spines illustrate that the spine transformation preferences were changed. The black spines illustrate the unchanged preferences. âfâ indicates filopodia, âtâ indicates thin, âsâ indicates stubby, and âmâ indicates mushroom. The solid arrowhead indicates different spines, and the hollow arrowhead indicates disappeared spines. Nâ=â6 neurons in 6 tadpoles and nâ=â36 dendrites were used for each group analysis. The total numbers of all transformed spines are 674 in control, 748 in odor enrichment, and 739 in olfactory deprivation. The bars indicate meanâ±âSEM. Significance was set at *pâ<â0.05, **pâ<â0.01, and ***pâ<â0.001. One-way ANOVA Tukeyâs multiple comparison tests.
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