5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised alertness, possibly impeding optimal learning performance in the more stressful WM

Increased number of cells expressing the immediate early genes cFos and Arc in the granule cell layer of the hippocampus of 5-HTT-/- compared to 5-HTT+/+ mice.

<p>In the quantitative immunohistochemistry study septal and temporal hippocampus as well as suprapyramidal and infrapyramidal blade of the GCL were analyzed separately. Significant differences of the number of cFos and Arc-immunoreactive (ir) cells were exclusively revealed between mice of the two 5-HTT genotypes investigated. Different treatments such as the experience of BM, WM or control situation did not significantly impact the expression of these two immediate early genes. (A) Representative image of the dentate gyrus of the hippocampus after cFos immunohistochemistry. Most of the cFos-ir cell nuclei (dark brown) are located in the granule cell layer as indicated by arrows in (a). (C) Quantitative evaluation of cFos-ir cells. (B) Representative image of the dentate gyrus after Arc immunohistochemistry. Most of the Arc-ir cells are located in the GCL. In (b) Arc-ir cells bodies are indicated by black arrows and stained processes by grey arrow heads. (D) Quantitative evaluation of Arc-ir cells. Two-way ANOVA, Bars represent the mean number of ir-cells per section +SEM; #=p<0.1; *=p<0.05; ***=p≤ 0.005. GCL, granule cell layer; SGZ, subgranular zone; Hi, hilus; CONT, control mice; BM, Barnes maze tested mice; WM, Morris water maze tested mice. Scale bar in A represents 200 μm for A and B, Scale bar in a represents 60 μm for a and b. </p

Learning performance in the Morris water maze.

<p>Mice of all three genotypes (5-HTT+/+, 5-HTT+/-, 5-HTT-/-) were tested three times per day for 4 consecutive days (acquisition phase; trials 1 - 12). Trials on day five represent reversal trials (trials 13-15). (A) Learning curve for the latency to find the platform. RM-ANOVA revealed a significant effect of trial (indicating learning performance) and a significant genotype effect. (B) The area under the learning curve (AuC) was calculated for each individual for statistical comparison of learning in the acquisition phase. ANOVA revealed a significant effect of genotype. Post hoc analysis using Bonferroni corrected t-tests revealed significant differences between 5-HTT-/- and both other genotypes. (C) Curve depicting the number of stops for each trial. RM-ANOVA revealed a significant effect of trial and genotype. (D) The AuC for the number of stops revealed a significant effect of genotype. Post hoc analysis using Bonferroni corrected t-tests revealed significant differences between 5-HTT-/- and both other genotypes. Data in all figures represent means + SEM. ***=p<0.001. </p

Increased number of cells expressing the two adult neurogenesis marker Ki67 and NeuroD in the granule cell layer of the hippocampus of 5-HTT-/- compared to 5-HTT+/+ mice and gene by environment interaction of the number of Ki67-positive proliferating cells in both blades of the SGZ of the septal hippocampus.

<p>In the quantitative immunohistochemistry study septal and temporal hippocampus as well as suprapyramidal and infrapyramidal blade of the GCL were analyzed separately. Significant differences of the number of Ki67 and NeuroD-immunoreactive (ir) cells were primarily revealed between mice of the two 5-HTT genotypes (5-HTT-/- and 5-HTT+/+) investigated. Only in both blades of the SGZ the number of Ki67-ir cells was significantly increased in 5-HTT+/+ mice after WM treatment vs. naïve 5-HTT+/+ mice (CONT). Additionally, a trend towards higher amounts of Ki67-ir cells in naïve 5-HTT-/- compared to naïve 5-HTT+/+ was found. (A) Representative image of the dentate gyrus of the hippocampus after Ki67 immunohistochemistry. Most of the Ki67-ir cell nuclei (dark brown) are located in the subgranular zone (SGZ) as inidicated by arrows in (a). (C) Quantitative evaluation of Ki67-ir cells. (B) Representative image of the dentate gyrus after NeuroD immunohistochemistry. Most of the NeuroD-ir cell nuclei are located in the SGZ as indicated by arrows in (b). (D) Quantitative evaluation of NeuroD-ir cells. Two-way ANOVA, data represent arithmetic means of the number of ir-cells per section + SEM; #=p<0.1; *=p<0.05. GCL, granule cell layer; SGZ, subgranular zone; Hi, hilus; CONT, control mice; BM, Barnes maze tested mice; WM, Morris water maze tested mice. Scale bar in A represents 200 μm for A and B, Scale bar in a represent 60 μm for a and b. </p

In 5-HTT-/- mice subjected to the WM a low number of NeuroD positive cells is correlated with a poor performance in the Morris water maze.

<p>Applying the Spearman`s test in the quantitative immunohistochemistry study revealed a linear dependence between the learning performance (decreasing learning performance is expressed as increasing area under the learning curve; AuC) and the number of NeuroD-immunoreactive (ir) cells could be found in the temporal hippocampus of 5-HTT-/- WM mice. A significant negative correlation between AuC and the number of NeuroD-ir (Data represent arithmetic mean per section) cells in both, the suprapyramidal blade of the subgranular zone (SGZ) as well as in both blades of the SGZ. A trend towards a negative correlation was found in the infrapyramidal blade of the SGZ. </p