Effect of sex and HD mutation on saccharin-preference test and total fluid intake.

<p>(A) We found a significant interaction between sex and genotype (F_(1,44) = 5.84, p<0.05) on saccharin preference (expressed as % of total fluid intake). Indeed compared to WT animals, only HD female mice exhibited reduced saccharin preference. (B) Interestingly looking at total fluid intake (expressed in mL), we revealed an overall effect of genotype (F_(1,44) = 12.3, p<0.01) but no significant effect of sex or interactions. Values represent means (± SEM) of n = 8–14 mice per group. WT vs. HD: (*) p<0.05, (**) p<0.01.</p

In vivo assessment of 5-HT_1A and 5-HT₂ post-synaptic receptor function using 8-OH-DPAT-induced change of corticosterone levels and DOI-induced head-twitches.

<p>Compared to paired-saline injected animals, administration of the 5-HT_1A agonist 8-OH-DPAT (0.3 mg/kg, s.c.) significantly increased corticosterone levels in both (A) male and (B) female mice, regardless of the genotype. In the DOI-induced head-twitches experiment (bottom panel), there was a significant effect of genotype in both sexes. Indeed, the number of head-twitches following DOI (1 mg/kg) were decreased in (C) HD male and (D) HD female mice when compared to WT animals. Values represent means (± SEM) of n = 5–10 mice per group. Saline vs. DPAT0.3: (+) p<0.05, (++) p<0.01, (+++) p<0.001 WT vs. HD: (*) p<0.05, (**) p<0.01, (***) p<0.001.</p

Effect of sex and HD mutation on the novelty suppressed feeding test (NSFT).

<p>(A) Analyzing the time (expressed in sec) to complete the task in the NSFT, we found a significant interaction between sex and genotype (F_(1,55) = 4.2, p<0.05). Indeed compared to WT animals, only HD female mice exhibited greater delay to complete the NSFT. (B) Interestingly measuring the amount of food consumed after NSFT, we did not find any effect of genotype (F_(1,55) = 1.7, p = 0.19) or interaction with the sex (F_(1,55) = 0.39, p = 0.54). Values represent means (± SEM) of n = 12–17 mice per group. WT vs. HD: (***) p<0.001.</p

Gene expression of 5-HT transporter (5-HTT), 5-HT_1A receptor (5-HT_1AR) and tryptophan hydroxylase-2 (TPH2) in the raphe.

<p>Measuring mRNA levels of several genes encoding proteins that regulate 5-HT homeostasis, we found significant genotype-sex interactions for both mRNA levels of (A) 5-HTT and (B) TPH2 but not for (C) 5-HT_1AR gene expression. Both 5-HTT and TPH2 mRNA levels were decreased in female WT when compared to male WT. In addition, HD mutation decreased both 5-HTT and TPH2 gene expressions in male only. Finally 5-HT_1AR mRNA levels were not affected by either the sex or the genotype. Values represent means (± SEM) of n = 5–6 mice per group. WT vs. HD: (**) p<0.01. Male vs. female: (##) p<0.01.</p

Concentrations of 5-HT and 5-HIAA in brain tissue.

<p>Using the HPLC system, we measured the tissue levels of serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in several brain areas. Both 5-HT and 5-HIAA concentrations were decreased in the hippocampus (A/B), the cortex (C/D) and the striatum (E/F) in female HD mice when compared to WT animals. Levels of 5-HIAA were also reduced in male HD. Finally, male mice exhibited higher hippocampal tissue levels of both 5-HT and 5-HIAA when compared to female animals. 5-HT and 5-HIAA are expressed in ng/g tissue. Values represent means (± SEM) of n = 6–8 mice per group. WT vs. HD: (*) p<0.05, (**) p<0.01, (***) p<0.001. Male vs. female: (##) p<0.01.</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

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

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

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

Proposed functional properties of CB₁R-5-HT_2AR heteromers.

<p>In (A), agonist binding to CB₁R (blue) or 5-HT_2AR (light green) triggers the conformational changes of TMs 5 and 6, opening the intracellular cavity for Gi and Gq binding, respectively. In (B), the formation of the CB₁R-5-HT_2AR heteromer makes both receptors signal via Gi. In (C), rimonabant binding to CB₁R or MDL 100,907 to 5-HT_2AR stabilizes the closed conformation of the receptor, facilitating heterodimerization via TMs 5 and 6 as in the crystal structure of the μ-opioid receptor. In this assembly, both protomers are locked in the closed conformation since the opening of TMs 5 and 6 for G-protein binding is not feasible (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002194#pbio.1002194.s008" target="_blank">S7 Fig</a>). Bidirectional cross antagonism is due to the fact that antagonist binding to any protomer must, in addition to its common role in a monomeric signaling unit, disrupt this very stable four-helix association. (D) In agreement, bidirectional cross antagonism is abrogated following treatment with TM 5 or TM 6 interference peptides (dark blue), which disrupt the heteromer structure.</p