Fig 1.
D3-treated mice do not spend significantly more time in the target quadrant during the probe trials.
This experiment was replicated two independent times. (A) Probe Trial 1: Vehicle-treated (control, n = 4) mice spent significantly more time in the target quadrant ‘4’ (T) than in the remaining quadrants (p<0.001). Neither the D3 9μg-treated mice (n = 3) nor the D3 15 μg-treated mice (n = 5) spent significantly more time in the target quadrant. (B) Probe Trial 2: One week later, equivalent results were obtained. Controls spent significantly more time in the target quadrant (p<0.001), mice treated with 9 μg had a significant preference for a quadrant, though it was not the target quadrant (p = 0.01), and mice treated with 15 μg of D3 did not spend significantly more time in any specific quadrant.
Fig 2.
Acute D3 increases TrkA phosphorylation and CREB in the hippocampus.
(A) Densitometric quantification of Western blots shows a significant increase in pTrkA in the hippocampus at 5 hours (p<0.05, n = 3) and a trend at 24 hours (p = 0.11, n = 2), compared to vehicle (n = 3). (B) Representative Western blots of samples immunoprecipitated with TrkA antibody and blotted with phospho-tyrosine antibody 4G10. Protein was extracted from the hippocampi of mice sacrificed at the times indicated. (C) Densitometric analysis shows a significant increase in CREB (p<0.005) at the 24-hour time point after treatment with D3 (n = 4) compared to vehicle (n = 3). (D) Representative Western blots of protein extracted from the hippocampi of mice sacrificed at the times indicated.
Fig 3.
A 2-week treatment with D3 increases pErk5 Erk5, pAkt, pCREB and CREB in the hippocampus.
(A) Representative Western Blots of proteins analyzed from hippocampus tissue. Increases in pAkt, total and pErk5, and total and pCREB were observed. Densitometry quantification of the bands shows a significant increase in (B) total Erk5 (p<0.05), pErk5 (p<0.05), (C) pAkt (p<0.01), (D) total CREB (p<0.01) and pCREB (p<0.01) (controls: n = 3, D3: n = 3; two repeats of Western Blots).
Fig 4.
D3 reduces dendritic arborization in the basal CA1 region.
(A) Representative confocal image, 3D filament, and magnified segments from apical and basal CA1 tertiary dendrites of an independent neuron from a control mouse. (B) Representative confocal image, 3D filament and magnified segments from apical and basal CA1 tertiary dendrites of an independent neuron from a 2-week D3-treated mouse. (C) Quantification of branching in the apical and basal region shows a decrease in branching points, significant in the basal dendrites of mice treated with D3 (p<0.05). A trend to a decrease is also observed in the apical region (p = 0.10). The values are shown as a fold decrease from control ± SEM. Total branching points of neurons belonging to D3-treated mice were normalized to the total branching points of the controls (controls n = 8, D3 n = 9, 2 cells measured per mouse, one repeat). (D) Mean Sholl analysis performed in the apical and basal regions yielded a significant decrease in arborization in the basal region (p<0.05). Values are shown as the average ± SEM of the mean number of intersections. (E) Sholl analysis performed in the apical and basal regions did not yield any statistically significant differences, but does show a trend to a decrease in arborization in the basal region (p = 0.1). Values are shown as the average ± SEM of the total number of intersections. (F) Spine density was quantified in the CA1 region and is expressed in spines/10μm, no significant differences were observed with D3 treatment in either of the regions.
Fig 5.
D3 has no effect on neuronal arborization in the CA3 region.
(A) Representative confocal image, 3D filament, and magnified segments from apical and basal CA3 tertiary dendrites of an independent neuron from a control mouse. (B) Representative confocal image, 3D filament, and magnified segments from apical and basal CA3 tertiary dendrites of an independent neuron from a 2-week D3-treated mouse. (C) Quantification of branching in the apical and basal regions shows no difference between the control and the D3-treated mice (p = 0.796; controls n = 8, D3 n = 9). Total branching points of neurons belonging to D3-treated mice were normalized to the total branching points of the controls. (D) Spine density in the CA3 region was quantified and is expressed in spines/10μm; no significant differences were observed with D3 treatment in either of the regions.
Fig 6.
Acute D3 treatment conveys STM impairment that mainly affects hippocampal-dependent spatial memory consolidation.
(A) Mice were trained in the Morris Water Maze (MWM) with three trials per day for 5 consecutive days. Values are shown as mean latency to find the platform (seconds). In the probe trials, values are displayed as the percentage of time the mice spent in the target quadrant. Probe Trial 1 (PT1) was performed two hours after the last trial on day 5 and Probe Trial 2 one week after PT1. Controls (n = 5), D3 9 μg (n = 3) and D3 15 μg (n = 5). Significant decreases in the percentage of time the mice spent in the target quadrant are framed in the dotted-line box. This experiment was performed twice independently, obtaining equal results. (B) Mice were trained as previously described and underwent PT1. They were not manipulated for the following 2 days to allow for memory consolidation. Next, they were randomly assigned to one of two groups: mice treated with vehicle (n = 6) or those treated with 15 μg of D3 (n = 5). Four days later, controls and D3-treated mice were re-tested (PT2) to assess retrieval, and no significant difference was observed, suggesting that D3 treatment impairs memory consolidation.
Fig 7.
D3 impairment in STM consolidation after acute ICV injection is reversible.
Mice were trained and tested through the Morris Water Maze (MWM) as previously described. Controls (n = 5), D3 9 μg (n = 3) and D3 15 μg (n = 5). No learning or memory deficits were observed (A) two months, nor (B) three monts after treatment, implying reversibility of the effects. This experiment was performed twice independently, obtaining equal results.
Table 1.
Summary of data.