Article Figures & Data
Figures
- Figure 1.
RNA extracted from sensitization-trained donor animals induces long-term enhancement of the SWR in recipient Aplysia. A, Experimental protocol for inducing LTS in the donor animals. B, Mean posttest duration of the SWR in the untrained control (1.2 ± 0.1 s, n = 31) and trained (56.4 ± 2.0 s, n = 34) groups. The trained group exhibited significant sensitization, as indicated by the comparison with control group (Mann–Whitney test, U = 496, p < 0.001). C, Experimental protocol for the RNA injection experiments. The first pretest occurred 2–3 h after the posttest for the behavioral training (A). D, Mean duration of the SWR measured at ∼24 h after the injection of RNA for the control RNA (5.4 ± 3.9 s, n = 7) and trained RNA (38.0 ± 4.6 s, n = 7) groups. The two groups differed significantly (U = 30, p < 0.003). Furthermore, Wilcoxon tests indicated that the difference between the pretest and posttest for the trained RNA group was significant (W = 28, p < 0.02), whereas it was not significant for the control RNA group (p > 0.2). The bar graphs in this and the following figures display means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, n.s., nonsignificant.
- Figure 2.
DNA methylation is required for RNA-induced enhancement of the SWR. A, Experimental protocol for inducing sensitization in the second donor group. B, Mean posttest duration of the SWR (n = 38). The training produced sensitization (mean posttest SWR = 56.4 ± 1.4 s, and mean pretest SWR = 1.1 ± 0.1 s; W = 741, p < 0.001). C, Experimental protocol for testing the effect of DNMT inhibition on RNA-induced enhancement of the SWR. RG-108/vehicle was injected into animals 5–10 min after the RNA injection. D, Mean postinjection duration of the SWR in the RNA-Veh (n = 3) and RNA-RG (n = 7) groups. The mean duration of the SWR in the RNA-Veh group (35.7 ± 7.7 s) was significantly longer than that in the RNA-RG group (1.4 ± 0.3 s; U = 27, p < 0.02). Moreover, the posttest SWR was sensitized compared to the pretest reflex in the RNA-Veh group (paired t test, p < 0.05), but not in the RNA-RG group (p > 0.4).
- Figure 3.
Treatment with RNA from trained animals increases excitability in dissociated sensory neurons but not in dissociated motor neurons. A, Sample electrophysiological traces from excitability tests on sensory neurons. Scale bars: 20 mV, 0.25 s. B, Changes in the excitability of the sensory neurons induced by RNA/vehicle treatment. The mean change in evoked APs in each group was: vehicle = –17.29 ± 12.86% (n = 19); control RNA = –35.76 ± 19.88% (n = 16); and trained RNA = 56.66 ± 22.07% (n = 19). The group differences were significant (Kruskal–Wallis; H = 11.81, p < 0.04). Dunn’s post hoc tests indicated that the increased firing in the trained RNA group was greater than that in the vehicle group (q = 2.44, p < 0.05) and control RNA group (q = 3.25, p < 0.004), respectively. The difference between vehicle and control RNA groups was not significant (p > 0.9). C, Sample traces from tests of motor neuron excitability. Scale bars: 25 mV, 0.25 s. D, Summary of posttreatment changes in the excitability of motor neurons. The mean changes were: vehicle group = –29.28 ± 19.16% (n = 15); control RNA group = 5.278 ± 34.36% (n = 12); and trained RNA group = –1.136 ± 34.01% (n = 14). The group differences in excitability were insignificant (p > 0.7).
- Figure 4.
Exposure of in vitro sensorimotor synaptic connections to RNA from trained animals enhanced the strength of a subpopulation of synapses. A, Representative records of EPSPs evoked in motor neurons by a single presynaptic AP before and 24 h after the RNA/vehicle treatments. Scale bars: 5 mV, 0.1s. B, Box and whiskers plots showing the distribution of posttreatment changes in EPSP amplitude in the three experimental groups. The boxes delineate the second and third quartiles, the horizontal lines in the boxes represent the medians, and the vertical bars (whiskers) show the extent of the data spread. The crosses indicate the means, whereas individual data points are represented by circles. Mean posttreatment changes in EPSP amplitudes were: vehicle group = –23.38 ± 10.59% (n = 23); control RNA group = –21.32 ± 10.23% (n = 34); and trained RNA group = 22.71 ± 26.70% (n = 32). A Kruskal–Wallis test revealed no significant differences among the groups with respect to the mean changes in EPSP amplitude (p > 0.8). Note, however, that five of the 32 synapses treated with RNA from trained animals showed an increase of >150%, whereas none of the synapses treated with vehicle or RNA from control animals showed an increase of this magnitude. A Levene’s test confirmed that the three groups displayed significantly unequal variances (F(2,86) = 5.883, p < 0.005).
Tables
Data structure Type of test Power (α = 0.05) a (Fig. 1B) Non-normally distributed Mann–Whitney test Not applicable b (Fig. 1D) Non-normally distributed Mann–Whitney test Not applicable c (Fig. 1D) Non-normally distributed Wilcoxon test Not applicable d (Fig. 1D) Non-normally distributed Wilcoxon test Not applicable e (Fig. 2B) Non-normally distributed Wilcoxon test Not applicable f (Fig. 2D) Non-normally distributed Mann–Whitney test Not applicable g (Fig. 2D) Normally distributed Paired t test 0.647 h (Fig. 2D) Non-normally distributed Wilcoxon test Not applicable i (Fig. 3B) Non-normally distributed Kruskal–Wallis test followed by Dunn’s test Not applicable j (Fig. 3D) Non-normally distributed Kruskal–Wallis test Not applicable k (Fig. 4B) Non-normally distributed Levene’s test Not applicable l (Fig. 4B) Non-normally distributed Kruskal–Wallis test Not applicable
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