Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice

Abstract

Although nutrients, including amino acids and their metabolites such as serotonin (5-HT), are strong modulators of anxiety-related behavior, the metabolic pathway(s) responsible for this physiological modulation is not fully understood. Regarding tryptophan (Trp), the initial rate-limiting enzymes for the kynurenine pathway of tryptophan metabolism are tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Here, we generated mice deficient for tdo (Tdo(-/-)). Compared with wild-type littermates, Tdo(-/-) mice showed increased plasma levels of Trp and its metabolites 5-hydroxyindoleacetic acid (5-HIAA) and kynurenine, as well as increased levels of Trp, 5-HT and 5-HIAA in the hippocampus and midbrain. These mice also showed anxiolytic modulation in the elevated plus maze and open field tests, and increased adult neurogenesis, as evidenced by double staining of BrdU and neural progenitor/neuronal markers. These findings demonstrate a direct molecular link between Trp metabolism and neurogenesis and anxiety-related behavior under physiological conditions.

Figure 1

Generation of tdo-deficient (Tdo^-/-) mice. (A) Schema of the Trp metabolic pathways. (a) the Kyn pathway. Over 95% of the dietary Trp is metabolized along this pathway. (b) the serotonin pathway. (c) the transamination pathway. (B) A targeting strategy for tdo gene disruption. Exons are represented as numbered boxes (coding regions; black boxes). The probe for Southern blot analysis is indicated by a solid bar. ApaI, A; PvuII, P; HindIII, H; EcoRI, E; XbaI, X; Neo, PGK-neomycin resistant cassette; DT-A, diphtheria toxin-A. (C) Southern blot analysis of representative progeny. Tail genomic DNA was digested with PvuII for hybridization with a specific probe against the intron sequence between exons 3 and 4 of tdo. The expected sizes of the hybridized DNA fragment for Tdo^+/+ (+/+) and Tdo^-/- (-/-) mice are 6.1 kb and 4.9 kb, respectively. (D) Quantitative real-time RT-PCR for tdo mRNA expression in adult liver. Mouse tdo/gapdh of adult liver in Tdo^+/+ mice was arbitrarily given a value of 100%. Values are means ± S.D. (E) Western blot analysis. Total liver homogenates were immunoblotted with TDO-specific antiserum. (F) Assay for TDO enzyme activity, determined in total liver lysates from 10-week-old animals of each genotype. Values represent means ± S.D.

Figure 2

Effect of tdo deletion on systemic Trp metabolites. (A-H) Plasma amino acid composition and Trp metabolites in 18- to 20-week-old Tdo^+/+ (+/+) and Tdo^-/- (-/-) mice. Plasma Trp concentration (A), other essential amino acid (EAA-Trp) concentrations (B), and the ratio of Trp to large neutral amino acids (LNAA, H) were determined using an amino acid analyzer (means ± S.E.). **, p < 0.0001. Plasma levels of 5-HIAA (C), IAA (D), ILA (E), kynurenine (Kyn: F), kynurenic acid (KYNA: G) were determined using HPLC-FD and HPLC-UV systems. Values represent means ± S.E. *, p < 0.05.

Figure 3

Modulation of brain Trp and serotonin metabolism by tdo disruption. (A-F) Trp and serotonin (5-HT) metabolites of the hippocampus (A-C) and midbrain (D-F) in 18- to 20-week-old Tdo^+/+ (+/+) and Tdo^-/- (-/-) mice. Trp (A, D), 5-HT (B, E), and 5-HIAA (C, F) contents in the hippocampus (upper) and midbrain (middle) were determined using an HPLC-FD system (means ± S.E.). *, p < 0.01. **, p < 0.0001. (G) tryptophan hydroxylase (tph2) mRNA levels and enzymatic activity in the adult midbrain of Tdo^+/+ (+/+) and Tdo^-/- (-/-) mice. (left) tph2 mRNA expression in the midbrain. TaqMan RT-PCR analyses were performed using total RNA extracted from the midbrain of 14- to 16-week-old mice. After normalization to gapdh, data were expressed as % fluorescent units relative to those in Tdo^+/+ mice. Data represent means ± S.E. (right) Assay for TPH enzymatic activity. TPH enzyme activity was measured using midbrain homogenates obtained from 15-week-old mice of each genotype. Values represent means ± S.E. N.S., not significant.

Figure 4

Anxiety-related behavior tests of 13- to 15-week-old Tdo^+/+ (+/+) and Tdo^-/- (-/-) mice. (A-C) Elevated plus maze tests. Duration in the open arms (A) and center zone (B), and total distance traveled (C) were scored for 5 min. Data represent the mean ± S.E. (D -F) Open field tests. The ratio between locomotion in the center and total locomotion (D), duration in the center zone (E), and total distance traveled (F) were scored for 30 min. Data represent the mean ± S.E. *, p < 0.05. **, p < 0.01. N.S., not significant.

Figure 5

Increase in neurogenesis in the subgranular zone (SGZ) following tdo deletion. (A) H&E staining of the dentate gyrus (DG) of paraffin-embedded coronal brain sections (5 μm) of 13-week-old Tdo^+/+ and Tdo^-/- mice. Center and right panels, higher magnification views. Right panels, PSA-NCAM-immunostaining. (B-I) Estimation of proliferating neural precursors and neurogenesis in the hippocampus of 13-week-old Tdo^+/+ and Tdo^-/- mice. (B) Total number of nuclei/section in the SGZ and granular cell layer (GCL) is shown as total cells. (C) BrdU/Ki67-double staining in the DG at 24 h after BrdU injection. (D-F) BrdU-, Ki67-, and double-labeled cells are indicated as a percentage of total cells. (G) Double-immunostaining of anti-BrdU and neural markers (Nestin, GFAP, PSA-NCAM, or DCX) in the SGZ of Tdo^-/- mice at 24 h after BrdU injection. (H) BrdU-immunostaining in the DG at 28 days after BrdU injection. Nuclei were stained with TO-PRO-3 (blue). (I) TuJ1 and NeuN were co-labeled with anti-BrdU at 28 days after BrdU injection. Orthogonal images show three dimensional analyses of individual cells marked by intersecting lines in the x, y, and z axes. Bars: 100 μm (C, H) and 30 μm (G, I). Data represent means ± S.E. *, p < 0.05 versus Tdo^+/+ mice.

Figure 6

Reduction in LV size and increased neural progenitors proliferation in the SVZ of Tdo^-/- mice. (A) Paraffin-embedded coronal brain sections (5 μm) were stained with H&E, and the size of each ventricle was measured. The boxed area in the subventricular zone (SVZ) was used for studies in Figures 6C-F. Arrows illustrate the change in the size of the LV in Tdo^-/- mice. (B) Quantitative size of each ventricle in both genotypes. Relative mean size in each ventricle of Tdo^+/+ mice was defined as 100%. Results are expressed as the means ± S.E. and tested for significance with ANOVA and Scheffe's post hoc test (p < 0.05). (C-F) Incorporation of BrdU in neural progenitors of SVZ of Tdo^+/+ and Tdo^-/- mice. Frozen coronal sections (20 μm) of the SVZ of the brain of 13-week-old mice were stained with TO-PRO-3 (nuclei, Ca and Cb), BrdU (Cc-f, Dc-f, E, and F), DCX (E), PSA-NCAM (F), nestin (Da, Db, De and Df) (boxed area in Figure 6A) at 24 h after BrdU injection. Inset, a merged view at high magnification (Df) of BrdU (green) and nestin (red). Ctx, cortex; Str, striatum; Sep, septum; Hip, hippocampus; D3V, dorsal 3rd ventricle; Aq, Aqueduct; and CC, corpus callosum. Bars: 100 μm.