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

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

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

Histological characterization of serotonergic neurons.

<p>Detection of serotonergic-specific markers was performed on coronal brain sections of adult <i>wt</i> control (left panel) and <i>Tph2</i>−/− mice (right panel). Protein labeling was obtained by light immunohistochemistry (<i>a-c</i>) and immunohistofluorescence (<i>e-g</i>). (<i>a</i>) Labeling of Tph2 demonstrated its complete absence in the raphe of <i>Tph2</i>−/− mice. (<i>b</i>) The serotonin transporter (Sert) could be detected in both <i>wt</i> and <i>Tph2</i>−/− mice, in the raphe as well as along fibers in projection areas, e.g. in the frontal cortex (FC) as shown in (<i>c</i>). (<i>d</i>) Detection of the serotonergic-specific transcription factor <i>Pet1</i> in the raphe by <i>in situ</i> hybridization occurred similarly in <i>wt</i> and <i>Tph2</i>−/− mice. (<i>e</i>) Detection of serotonin (5-HT) in the raphe showed the absence of specific 5-HT immunoreactivity in <i>Tph2</i>−/− mice. Cell nuclei were also labeled by DAPI staining. (<i>f</i>) The vesicular monoamine transporter-2 (Vmat2) could be detected similarly in the raphe of both <i>wt</i> and <i>Tph2</i>−/− mice. (<i>g</i>) Merged images from (<i>e-f</i>) showed the colocalization of 5-HT and Vmat2 in the serotonergic neurons of <i>wt</i> (yellow in <i>g</i>) while <i>Tph2</i>−/− neurons were only labeled with Vmat2 (red in <i>g</i>). Taken together these results demonstrate that despite 5-HT synthesis deficiency, serotonergic neurons of <i>Tph2</i>−/− mice can develop and be maintained. Moreover, except Tph2 and 5-HT, they possess all known 5-HT-specific markers showing that their serotonergic specification took place. Bars represent 100 µm in (<i>c</i>) and 200 µm in (<i>a</i>), (<i>b</i>), (<i>d</i>), (<i>e-g</i>).</p

Quantitative autoradiography of 5-HT_1A and 5-HT_1B receptors in various brain regions.

<p>(<b><i>A</i></b>) Representative photomicrographs of autoradiograms following the binding of [³H]WAY100635 to 5-HT_1A receptors on whole coronal sections. The signal was visibly increased in e.g. the dorsal raphe, CA1 of hippocampus, frontal cortex and septum of <i>Tph2</i>−/− mice. (<b><i>B</i></b>) Binding density of 5-HT_1A receptors labeled by the radioligand [³H]WAY100635 was up-regulated in most of the brain regions of <i>Tph2</i>−/− mice. (<b><i>C</i></b>) 5-HT_1A receptor-mediated increase in [³⁵S]GTP-γ-S binding after stimulation revealed enhanced 5-HT_1A coupling in the frontal cortex and septum of <i>Tph2</i>−/− mice. (<b><i>D</i></b>) Binding density of 5-HT_1B receptors labeled by the radioligand [¹²⁵I]ICYP was also increased in some brain regions of <i>Tph2</i>−/− mice. For (<b><i>B, C, D</i></b>) results are expressed as optical density (OD = specific OD – nonspecific OD) and presented as means ± sem (n = 5). * indicates ANOVA significant output for genotype effect with *p<0.05, **p<0.01, ***p<0.001. For detailed statistical results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043157#pone.0043157.s001" target="_blank">Table S1</a>. c: cortex, Ss: somatosensory, Retrospl.: retrosplenial, CA1: cornu ammonis area 1 of hippocampus, g: gyrus, D: dorsal, Enth.: enthorinal, Caud. put.: caudate putamen, V pal.: ventral pallidus, Globus pal.: globus pallidus, Hypo. lat.: lateral hypothalamus, D subic.: dorsal subicullum, Subst. nigra: substantia nigra.</p

Neurotransmitter concentrations and TH positive cells counting in different brain regions.

<p>(<b><i>A</i></b>) HPLC analysis of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in four different brain regions showed a drastic reduction of 5-HT and 5-HIAA concentrations in the brain of <i>Tph2</i>−/− compared to <i>wt</i> and +/− mice (n = 8). This reduction was further increased by an improved perfusion protocol (see corresponding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043157#s2" target="_blank">results</a> section) with e.g. 98.9% reduction in rostral raphe. A general reduction of NE concentration was also observed across brain regions of <i>Tph2</i>−/− mice compared to <i>wt</i> and +/− and a reduction of DA concentration in the hippocampus compared to <i>wt</i> mice. Kruskall-Wallis followed by Mann-Whitney-U-test: *p<0.05, **p<0.01, ***p<0.001. (<b><i>B</i></b>) Top: density of tyrosine hydroxylase (TH) positive cells in the locus coeruleus (LC, main noradrenergic cell cluster in the brain) showing a reduction of cell number in the anterior LC (aLC) of <i>Tph2</i>−/− mice (n = 8). Botom: density of TH positive cells in the main dopaminergic cell clusters: subtantia nigra (SN), ventral tegmental area (VTA) and retrorubal field A8 (n = 8). cLC: central LC, pLC: posterior LC. ANOVA followed by Tuckey-HSD: *p<0.05. Data are presented as means ± sem.</p

Electrophysiological characteristics of serotonergic raphe neurons.

<p><i>Tph2</i>−/− mice displayed electrophysiological characteristics similar to +/− and <i>wt</i> mice comprising (<b><i>A</i></b>) mean firing rate of the recorded neurons measured over a 3 min interval; (<b><i>B</i></b>) Up-to-Downstroke Interval (UDI) measured as shown in (<b><i>C</i></b>); <i>wt</i>: n = 19; <i>Tph2</i>+/−: n = 25; <i>Tph2</i>−/−: n = 21. (<b><i>D</i></b>) Representative time-course of the effect of tryptophan (Trp 30 and 100 µM) and R-8-OH-DPAT (DPAT; 30 nM) application on the firing rate of serotonergic neurons in slices taken from <i>wt</i> (upper panel), <i>Tph2</i>+/− (middle panel) and <i>Tph2</i>−/− (lower panel) mice. Lower panel also illustrates the response of <i>Tph2</i>−/− mice to the application of 30 µM L-5-hydroxytryptophan (5-HTP). 5-HTP stopped the firing of serotonergic neurons in all three genotypes. (<b><i>E</i></b>) Bar graph summarizes the responses to Trp application shown in (<b><i>D</i></b>). Both concentrations of Trp (30 and 100 µM) did not change firing of serotonergic neurons in <i>Tph2</i>−/−, but significantly inhibited serotonergic neuron firing in <i>wt</i> and <i>Tph2+/−</i> mice (p<0.05, Wilcoxon Signed Rank Test). When compared across genotypes, the responses of <i>Tph2</i>−/− serotonergic neurons were statistically different from those of +/− and <i>wt</i> mice both for Trp 30 µM (H₍₂₎ = 16.28, p<0.0003, <i>wt</i> n = 7; <i>Tph2</i>+/− n = 10; <i>Tph2</i>−/− n = 12) and 100 µM (H₍₂₎ = 10.43, p = 0.0054, <i>wt</i> n = 7; <i>Tph2</i>+/− n = 8; <i>Tph2</i>−/− n = 8; Kruskal-Wallis, followed by Dunn’s multiple comparison test). Diagram bars represent means ± SD.</p