Genetic disruption of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in models sensitive to antidepressants

Abstract

The neurotransmitter serotonin (5-HT) plays an important role in both the peripheral and central nervous systems. The biosynthesis of serotonin is regulated by two rate-limiting enzymes, tryptophan hydroxylase-1 and -2 (TPH1 and TPH2). We used a gene-targeting approach to generate mice with selective and complete elimination of the two known TPH isoforms. This resulted in dramatically reduced central 5-HT levels in Tph2 knockout (TPH2KO) and Tph1/Tph2 double knockout (DKO) mice; and substantially reduced peripheral 5-HT levels in DKO, but not TPH2KO mice. Therefore, differential expression of the two isoforms of TPH was reflected in corresponding depletion of 5-HT content in the brain and periphery. Surprisingly, despite the prominent and evolutionarily ancient role that 5-HT plays in both vertebrate and invertebrate physiology, none of these mutations resulted in an overt phenotype. TPH2KO and DKO mice were viable and normal in appearance. Behavioral alterations in assays with predictive validity for antidepressants were among the very few phenotypes uncovered. These behavioral changes were subtle in the TPH2KO mice; they were enhanced in the DKO mice. Herein, we confirm findings from prior descriptions of TPH1 knockout mice and present the first reported phenotypic evaluations of Tph2 and Tph1/Tph2 knockout mice. The behavioral effects observed in the TPH2 KO and DKO mice strongly confirm the role of 5-HT and its synthetic enzymes in the etiology and treatment of affective disorders.

Figure 1. Generation and confirmation of TPH1, TPH2 and TPH1/TPH1 deficient animals.

(A) Genomic organization of exon 1 and exon 2 of TPH1 gene (WT), the targeting construct (Vector), and the mutant allele (Null). The MC1 Neo selection cassette replaces exon 2 by homologous recombination. The recombined allele is shown as the null allele with the restriction enzyme of choice Spe1 (s) and the 5′ external probe (black bar) used for southern hybridization. (B) Genomic organization of exon 1 and exon 2 of TPH2 gene, the targeting vector construct, and the mutant allele. The MC1 Neo selection cassette replaces exons 1 and 2 by homologous recombination. The recombined allele is shown as the null allele with the restriction enzyme of choice Bgl1 (b) and the external probe (black bar) used for southern hybridization. (C) TPH1 targeted ESC genomic DNA was restriction digested with Spe1 and hybridized with the 5′ external probe to show the wild type (+/+) 9.2 kb band and the mutant band (+/−) at 5.9 kb in the heterozygous ES cells. (D) Targeted ES cell genomic DNA was restriction digested with Bgl1 (b) and hybridized with the 3′ external probe to show the wild type (+/+) 12.5 kb band and the mutant band (+/−) at 5.9 kb in the heterozygous ES cells. (E) The pups were genotyped by isolating tail genomic DNA and PCR amplifying sequences using specific TPH1 primers that amplified a 654 bp product in the wildtypes (+/+) and hets (+/−) and primers specific to the deletion insertion that produced a 483 bp product in the Hets (+/−) and the Homs (−/−). (F) The TPH2 null pups were genotyped by isolating tail genomic DNA and PCR amplifying sequences using specific primers that amplified a 348 bp product in the wildtypes (+/+) and hets (+/−) and primers specific to the deletion insertion that produced a 235 bp product in the Hets (+/−) and the Homs (−/−). (G) The DKO pups were genotyped with the TPH1 and TPH2 specific primers as in Fig.1E, F. Individual animals (1–5), of each relevant genotype is shown: 1) TPH1^+/+/TPH2^+/+; 2) TPH1^+/−/TPH2^+/−, 3) TPH1^+/−/TPH2^−/−; 4) TPH1^−/−/TPH2^+/−, 5) TPH1^−/−/TPH2^−/−. 100 bp molecular weight markers are also indicated.

Figure 2. Open Field total distance (A) and center time (B) in TPH2KO mice.

M – males, F- females.

Figure 3. Marble burying test. Male and female data combined on the plots since one-way ANOVA did not reveal significant effect of sex or sex×genotype interactions.

(A) Marble burying in TPH2KO mice. Males: 15 WT, 16 KO; females: 19 WT, 15 KO. (B) Marble burying in TPH1/TPH2 DKO mice. Males: 10 WT, 6 DKO; females: 9 WT, 12 DKO. * P<0.05 compared to WT mice, ** - p<0.0001 compared to WT mice.

Figure 4. Immobility and struggling in the forced swim test.

(A) TPH2KO Males. (B) TPH2KO Females. (C) TPH1/TPH2 DKO Males. (D) TPH1/TPH2 DKO Females. *** - p<0.001, ** - P<0.01, * - p<0.05 compared to WT mice (unpaired t-test).

Figure 5. Tail suspension test.

(A) Immobility time in TPH2KO male mice over days. # - p<0.05, compared to Day 1 for the same genotype (Tukey's HSD following repeated measures ANOVA). (B) Immobility time in TPH2KO male mice on day 3 of repeated TS testing. (C) Immobility time in TPH1/TPH2 DKO. Male and female data combined on the plot (one-way ANOVA did not reveal significant effect of sex or sex×genotype interactions). Males: 7 WT, 6 DKO; females: 8 WT, 9 DKO. Mice that climbed tails during testing were excluded from analysis. ** - p<0.01 compared to WT mice.

Figure 6. Latency to hind paw response in the hot plate acute thermal pain test in TPH1/TPH2 DKO mice.

Male and female data combined on the plot (one-way ANOVA did not reveal significant effect of sex or sex×genotype interactions). Males: 10 WT, 6 DKO; females: 9 WT, 12 DKO. * - p<0.05, compared to WT mice.