Abstract
Aim
Tryptophan hydroxylase 2 (Tph2) is a rate‐limiting enzyme for the biosynthesis of 5‐hydroxytryptamine (5‐HT, serotonin). Previous studies have reported that C1473G polymorphism of the murine Tph2 gene leads to decreased 5‐HT levels in the brain and abnormal behavioral phenotypes, such as impaired anxiety‐ and depression‐like behaviors. In this study, to confirm the effect of the C1473G polymorphism on mouse phenotypes, we conducted a comprehensive battery of behavioral tests and measured the amounts of brain free amino acids involved in the production of 5‐HT.
Methods
We obtained C57BL/6J congenic mice that were homozygous for the 1473G allele of Tph2 (1473G) and subjected them and their wild‐type littermates (1473C) to a battery of behavioral tests. Using reverse‐phase high‐performance liquid chromatography (HPLC), we measured the amounts of free amino acids in the 5‐HT and epinephrine synthetic/metabolic pathways in the frontal cortex, hippocampus, striatum, and midbrain.
Results
We failed to detect significant differences between genotypes in depression‐like behaviors, anxiety‐like behaviors, social behaviors, sensorimotor gaiting, or learning and memory, while 1473G mice exhibited a nominally significant impairment in gait analysis, which failed to reach study‐wide significance. In the HPLC analysis, there were no significant differences in the amounts of 5‐HT, dopamine, norepinephrine, and epinephrine in the frontal cortex, hippocampus, striatum, and midbrain.
Conclusion
Our findings do not support the idea that congenic C57BL/6J mice carrying the 1473G allele may represent an animal model of mood disorder under normal conditions without stress.
Keywords: 5‐HT, comprehensive behavioral test battery, depression‐like behavior, tryptophan hydroxylase 2
We assessed the behavioral and biochemical phenotypes of congenic C57BL/6J mice carrying the 1473G allele and failed to identify significant differences between the 1473G allele‐carrying mice and their wild‐type littermates. Thus, our findings do not support the use of 1473G allele‐carrying C57BL/6J mice as an animal model of mood disorder under normal conditions without stress.
MAIN TEXT
Tryptophan hydroxylase (Tph) is a rate‐limiting enzyme in 5‐hydroxytryptamine (5‐HT, serotonin) biosynthesis,1 and to date, two isoforms of Tph have been identified in mammals. Tph1 is mainly expressed in the periphery, and Tph2 is preferentially expressed in the brain.2 Zhang et al. reported that C1473G polymorphism exists in the mouse Tph2 gene, and the mutant mice show decreased synthesis of 5‐HT in the brain.3 Previous studies demonstrated that mice homozygous for the 1473G allele of Tph2 (1473G) exhibit abnormal behavioral phenotypes, such as impaired anxiety‐ and depression‐like behaviors.4, 5 In contrast, other groups have failed to detect these abnormal behaviors in 1473G mice.6, 7, 8, 9, 10 The biological significance of C1473G polymorphism remains controversial. C1473G polymorphism is reported to lead to a proline to arginine substitution and disturbance of 5‐HT synthesis.3 This sequence alteration and the amount of 5‐HT differ depending on the mouse line.3, 4, 8, 11 Some mouse lines, including C57BL/6 and 129X1/SvJ, are homozygous for the 1473C allele (1473C), but other lines, such as BALB/c and DBA/2J, are homozygous for the 1473G allele (Table S1), causing decreased 5‐HT synthesis compared to 1473C mice.3, 12 The objective of the present study was to further investigate the functional significance of C1473G polymorphism in mice.
We prepared congenic C57BL/6J (B6J) mice using a backcrossing breeding strategy.4, 7 In brief, heterozygous mice were created from hybrids between Balb/c AJc1 and B6J strains, which are homozygous for the 1473G and 1473C allele, respectively. After six successive backcrossings of heterozygous mice with the B6J strain, the heterozygous backcrosses were intercrossed to generate congenic B6J mice homozygous for the 1473G and 1473C allele. We subjected those 1473G and 1473C mice to a comprehensive behavioral test battery that included the wire hang, grip strength, rotarod, hot plate, gait analysis, tail suspension, Porsolt forced swim, open field, light/dark transition, elevated plus maze, social interaction, sociability and social novelty preference, startle response/prepulse inhibition (PPI), and fear conditioning tests, as previously described.13, 14, 15, 16 All behavioral tests were carried out with male mice that were at least 19 weeks old at the start of testing (Table S3). The behavioral results are summarized in Table 1. There were no significant differences between the genotypes in physical characteristics or on the wire hang, grip strength, rotarod, and hot plate tests. In the gait analysis, 1473G mice exhibited nominally significant impairments in the stance width of the hind paws and the step angles of the front paws, but these results failed to reach study‐wide significance. None of the indices of the tail suspension and Porsolt forced swim tests (Figure S1) showed significant differences between the genotypes. No genotype‐specific differences were observed in the open field (Figure S2), light/dark transition, and elevated plus‐maze tests. There were no significant genotype effects in the social interaction, sociability, and social novelty preference tests. In the startle response/PPI tests, 1473G mice displayed normal acoustic startle responses and sensorimotor gating. No obvious differences between the genotypes were detected in the fear conditioning tests (Figure S3).
Table 1.
Comprehensive behavioral battery in 1473G and 1473C mice
| Test | 1473G (n = 7) | 1473C (n = 7) | Genotype effect | |
|---|---|---|---|---|
| F‐value | P‐value | |||
| Physical characteristics | ||||
| Weight (g) | 31.743 (±0.566) | 31.786 (±0.635) | F 1,12 = 0.003 | 0.9606 |
| Body temperature (°C) | 37.100 (±0.298) | 37.299 (±0.252) | F 1,12 = 0.109 | 0.7474 |
| Neurological screen and neuromuscular strength test | ||||
| Grip strength (N) | 0.893 (±0.037) | 0.929 (±0.054) | F 1,12 = 0.295 | 0.5968 |
| Wire hang (% falling within 60 s) | 48.143 (±5.230) | 52.000 (±6.904) | F 1,12 = 0.198 | 0.664 |
| Rotarod test | ||||
| Latency to fall (s) | 148.143 (±17.358) | 179.452 (±11.009) | F 1,12 = 2.32 | 0.1536 |
| Hot plate test | ||||
| Latency (s) | 3.014 (±0.122) | 3.457 (±0.473) | F 1,12 = 0.822 | 0.3826 |
| Gait analysis | ||||
| Stance width (cm) | ||||
| Front | 1.421 (±0.041) | 1.357 (±0.048) | F 1,12 = 1.043 | 0.3273 |
| Hind | 2.029 (±0.036) | 1.900 (±0.038) | F 1,12 = 6.075 | 0.0298 |
| Step angles (°) | ||||
| Front | 68.914 (±2.171) | 59.900 (±3.393) | F 1,12 = 5.008 | 0.045 |
| Hind | 49.850 (±3.301) | 60.343 (±5.215) | F 1,12 = 2.891 | 0.1148 |
| Tail suspension test | ||||
| Immobility (%) | 28.901 (±7.352) | 14.549 (±2.925) | F 1,12 = 3.29 | 0.0948 |
| Porsolt forced swim test | ||||
| Immobility (%) | ||||
| Day 1 | 57.28 (±3.447) | 56.667 (±2.559) | F 1,12 = 0.02 | 0.8889 |
| Day 2 | 62.839 (±5.179) | 58.45 (±5.755) | F 1,12 = 0.321 | 0.5813 |
| Distance traveled (cm) | ||||
| Day 1 | 83.629 (±3.431) | 87.823 (±3.525) | F 1,12 = 0.727 | 0.4106 |
| Day 2 | 68.351 (±4.33) | 83.61 (±6.752) | F 1,12 = 3.619 | 0.0814 |
| Open field test | ||||
| Distance traveled (cm) | 571.798 (±83.344) | 623.577 (±55.978) | F 1,12 = 0.266 | 0.6154 |
| Number of vertical activities | 61.905 (±7.868) | 72.232 (±5.937) | F 1,12 = 1.098 | 0.3154 |
| Center time (s) | 52.904 (±10.598) | 41.015 (±5.684) | F 1,12 = 0.977 | 0.3424 |
| Stereotypic counts | 653.768 (±48.016) | 621.137 (±66.817) | F 1,12 = 0.157 | 0.6986 |
| Light/dark transition test | ||||
| Stay time in light compartment (s) | 193.929 (±18.927) | 190.143 (±14.931) | F 1,12 = 0.025 | 0.8778 |
| Number of transitions | 20.571 (±3.108) | 21.429 (±1.837) | F 1,12 = 0.056 | 0.8163 |
| Elevated plus‐maze test | ||||
| Open arms entries per total entries (%) | 30.292 (±3.696) | 31.19 (±4.417) | F 1,12 = 0.024 | 0.8787 |
| Stay time ratio on open arms (%) | 13.667 (±3.321) | 13.69 (±2.641) | F 1,12 < 0.0001 | 0.9956 |
| Social interaction test | ||||
| Total duration of contact (s) | 75.333 (±5.487) | 69.333 (±8.098) | F1,4 = 0.376 | 0.5728 |
| Number of contacts | 55.667 (±8.511) | 56.333 (±2.028) | F1,4 = 0.006 | 0.9429 |
| Total duration of active contacts (s) | 17.567 (±2.969) | 17.567 (±0.696) | F1,4 = 0 | 1 |
| Mean duration per contacts | 1.433 (±0.145) | 1.233 (±0.133) | F1,4 = 1.029 | 0.3679 |
| Distance traveled (cm) | 3847.5 (±268.099) 430.667 (±317.643) | F1,4 = 1.968 | 0.2333 | |
| Sociability and social novelty preference test | ||||
| Sociability test | ||||
| Empty side | 97.571 (±13.811) | 95.0 (±10.207) | F 1,12 = 0.022 | 0.8835 |
| Time spent around cage (s) | ||||
| Stranger side | 143.143 (±20.036) | 123.286 (±13.852) | F 1,12 = 0.665 | 0.4308 |
| Social novelty preference test | ||||
| Familiar side | 127.714 (±20.757) | 121.429 (±20.903) | F 1,12 = 0.046 | 0.8346 |
| Time spent around cage (s) | ||||
| Stranger side | 152.571 (±20.752) | 135.143 (±17.07) | F 1,12 = 0.421 | 0.5288 |
| Startle response/prepulse inhibition test | ||||
| Startle amplitude | ||||
| 110 dB | 0.614 (±0.077) | 0.844 (±0.203) | F 1,12 = 0.842 | 0.377 |
| 120 dB | 1.219 (±0.215) | 1.397 (±0.158) | ||
| Prepulse inhibition (%) (Prepulse sound level/startle) | ||||
| 74/110 dB | 28.393 (±14.088) | 28.81 (±11.223) | F 1,12 = 0.099 | 0.7579 |
| 78/110 dB | 48.843 (±10.047) | 35.283 (±28.902) | ||
| 74/120 dB | 21.15 (±8.938) | 10.787 (±10.207) | F 1,12 = 0.805 | 0.3873 |
| 78/120 dB | 47.225 (±9.853) | 34.074 (±13.286) | ||
| Fear conditioning test | ||||
| Freezing (%) | ||||
| Conditioning | 27.229 (±3.602) | 26.452 (±4.429) | F 1,12 = 0.019 | 0.894 |
| Context testing | 26.514 (±8.652) | 34.697 (±7.528) | F 1,12 = 0.509 | 0.4892 |
| Cued testing with altered context | 58.331 (±4.568) | 54.807 (±3.57) | F 1,12 = 0.369 | 0.5546 |
We next quantified the amount of free amino acids that are involved in the 5‐HT metabolic pathway (eg, 5‐HT and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA)) in the prefrontal cortex, hippocampus, striatum, and midbrain using reverse‐phase high‐performance liquid chromatography (HPLC), as previously described.17 We also measured the amounts of free amino acids in the epinephrine (Epi) synthetic/metabolic pathway (eg, dopamine (DA), 3‐methoxytyramine (3‐MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), normetanephrine (NM), 4‐hydroxy‐3‐methoxyphenylglycol (MHPG), and Epi). The quantitative results are summarized in Table S2. The amounts of free amino acids in the hippocampus, striatum, and midbrain did not significantly differ between the genotypes, while there was a non‐significant tendency toward decreased 5‐HT in the prefrontal cortex in 1473G mice.
There are inconsistencies in the biochemical phenotype of the Tph2 1473G allele‐carrying mice among the present and previous studies. The present and a previous study failed to detect significant changes in the free amino acid (5‐HT, 5‐HTP, and 5‐HIAA) level in the frontal cortex, hippocampus, stratum, or midbrain,6 but other studies have reported significant genotype effects in some of these areas 3, 4, 8, 9, 10, 11 (Table S1). No major changes were detected in the behavioral phenotype of the C57BL/6J mice carrying the 1473G allele in the present and some previous studies,6, 7, 8, 9, 10 while a few previous studies have reported significant changes in depression‐like and/or anxiety‐like behavior 4, 5 (Table S1). These inconsistencies may be due to differences in factors such as genetic background, flanking genes, age, exposure of the animals to stress, and/or experimental environments/conditions.
In conclusion, we failed to detect major differences in depression‐ and anxiety‐like behaviors or levels of brain free amino acids in 1473G mice on a C57BL/6J genetic background, while 1473G mice exhibited nominally significant impairments in the gait analysis, which failed to reach study‐wide significance. Under conditions without stress or drug administration, C57BL/6J mice homozygous for the Tph2 1473G allele displayed no significant behavioral or physiological phenotype, indicating that these congenic mice may not represent an animal model of mood disorder.
CONFLICT OF INTERESTS
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interests.
AUTHORS’ CONTRIBUTIONS
TM was responsible for the original conception and overall design of the research. KTa and KTo established the congenic mice and performed the comprehensive behavioral test battery. TI and SF conducted the quantification of the amino acids. HK, NH, KTa, KTo, TI, SF, and TM analyzed the data. HK, NH, and TM wrote the manuscript. All authors read and approved the final manuscript.
DATA REPOSITORY
Raw data on the behavioral tests and the information about each mouse are accessible on the public database “Mouse Phenotype Database” (http://www.mouse-phenotype.org/).
ANIMAL STUDIES
All behavioral testing procedures were approved by the Institutional Animal Care and Use Committee of Graduate School of Medicine of Kyoto University and Fujita Health University.
Supporting information
ACKNOWLEDGMENTS
This research was supported by a Grant‐in‐Aid for Scientific Research on Priority Areas (200163013), a Grant‐in‐Aid for Scientific Research (B) (21300121), a Grant‐in‐Aid for Scientific Research (C) (25430077), a Grant‐in‐Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, a grant from the Institute for Bioinformatics Research and Development (BIRD) of the Japan Science and Technology Agency (JST).
Koshimizu H, Hirata N, Takao K, et al. Comprehensive behavioral analysis and quantification of brain free amino acids of C57BL/6J congenic mice carrying the 1473G allele in tryptophan hydroxylase‐2. Neuropsychopharmacol Rep. 2019;39:56–60. 10.1002/npr2.12041
Koshimizu and Hirata contributed equally to this work.
REFERENCES
- 1. Grenett HE, Ledley FD, Reed LL, Woo SL. Full‐length cDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases. Proc Natl Acad Sci USA. 1987;84(16):5530–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Walther DJ, Peter J‐U, Bashammakh S, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science. 2003;299(5603):76. [DOI] [PubMed] [Google Scholar]
- 3. Zhang X, Beaulieu J‐M, Sotnikova TD, Gainetdinov RR, Caron MG. Tryptophan hydroxylase‐2 controls brain serotonin synthesis. Science. 2004;305(5681):217. [DOI] [PubMed] [Google Scholar]
- 4. Berger SM, Weber T, Perreau‐Lenz S, et al. A functional Tph2 C1473G polymorphism causes an anxiety phenotype via compensatory changes in the serotonergic system. Neuropsychopharmacology. 2012;37(9):1986–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Jiao J, Nitzke AM, Doukas DG, Seiglie MP, Dulawa SC. Antidepressant response to chronic citalopram treatment in eight inbred mouse strains. Psychopharmacology. 2011;213(2–3):509–20. [DOI] [PubMed] [Google Scholar]
- 6. Tenner K, Qadri F, Bert B, Voigt J‐P, Bader M. The mTPH2 C1473G single nucleotide polymorphism is not responsible for behavioural differences between mouse strains. Neurosci Lett. 2008;431(1):21–5. [DOI] [PubMed] [Google Scholar]
- 7. Osipova DV, Kulikov AV, Popova NK. C1473G polymorphism in mouse tph2 gene is linked to tryptophan hydroxylase‐2 activity in the brain, intermale aggression, and depressive‐like behavior in the forced swim test. J Neurosci Res. 2009;87(5):1168–74. [DOI] [PubMed] [Google Scholar]
- 8. Siesser WB, Zhang X, Jacobsen JPR, Sotnikova TD, Gainetdinov RR, Caron MG. Tryptophan hydroxylase 2 genotype determines brain serotonin synthesis but not tissue content in C57Bl/6 and BALB/c congenic mice. Neurosci Lett. 2010;481(1):6–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Bazhenova EY, Bazovkina DV, Kulikova EA, et al. C1473G polymorphism in mouse tryptophan hydroxylase‐2 gene in the regulation of the reaction to emotional stress. Neurosci Lett. 2017;640:105–10. [DOI] [PubMed] [Google Scholar]
- 10. Mosienko V, Matthes S, Hirth N, et al. Adaptive changes in serotonin metabolism preserve normal behavior in mice with reduced TPH2 activity. Neuropharmacology. 2014;85:73–80. [DOI] [PubMed] [Google Scholar]
- 11. Isles AR, Hathway GJ, Humby T, de la Riva C, Wilkinson LS. An mTph2 SNP gives rise to alterations in extracellular 5‐HT levels, but not in performance on a delayed‐reinforcement task. Eur J Neurosci. 2005;22(4):997–1000. [DOI] [PubMed] [Google Scholar]
- 12. Osipova DV, Kulikov AV, Mekada K, et al. Distribution of the C1473G polymorphism in tryptophan hydroxylase 2 gene in laboratory and wild mice. Genes Brain Behav. 2010;9(5):537–43. [DOI] [PubMed] [Google Scholar]
- 13. Shoji H, Irino Y, Yoshida M, Miyakawa T. Behavioral effects of long‐term oral administration of aluminum ammonium sulfate in male and female C57BL/6J mice. Neuropsychopharmacol Rep. 2018;38(1):18–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hattori S, Takao K, Funakoshi H, Miyakawa T. Comprehensive behavioral analysis of tryptophan 2,3‐dioxygenase (Tdo2) knockout mice. Neuropsychopharmacol Rep. 2018;38(2):52–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Maeta K, Hattori S, Ikutomo J, et al. Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap‐associating domain. Mol Brain. 2018;11:27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Hirata N, Hattori S, Shoji H, Funakoshi H, Miyakawa T. Comprehensive behavioral analysis of indoleamine 2,3‐dioxygenase knockout mice. Neuropsychopharmacol Rep. 2018;38(3):133–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Esaki K, Takashi K, Ohmori T, Tsukino M, Ohshima T, Furuya S. Soy peptide ingestion increases neuroactive amino acids in the adult brain of wild‐type and genetically engineered serine‐deficient mice. J Nutr Food Sci. 2011;1(4):1–6. [Google Scholar]
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