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Ubiquitin-specific peptidase 46 (Usp46) regulates mouse immobile behavior in the tail suspension test through the GABAergic system.

Imai S, Mamiya T, Tsukada A, Sakai Y, Mouri A, Nabeshima T, Ebihara S - PLoS ONE (2012)

Bottom Line: This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system.However, it is not known precisely how the immobile behavior is regulated by the GABAergic system.These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABA(A) receptor might participate in the regulation of TST immobility time.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.

ABSTRACT
The tail suspension test (TST) is widely recognized as a useful experimental paradigm for assessing antidepressant activity and depression-like behavior. We have previously identified ubiquitin-specific peptidase 46 (Usp46) as a quantitative trait gene responsible for decreasing immobility time in the TST in mice. This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system. However, it is not known precisely how the immobile behavior is regulated by the GABAergic system. Therefore, in the present study, we examined whether the immobility time is influenced by drugs affecting the action mediated by GABA(A) receptor using both 3-bp deleted (the Usp46 mutant) and Usp46 (Usp46 KO) mice. Nitrazepam, an agonist at the benzodiazepine-binding site of the GABA(A) receptor, which potentiates the action of GABA, produced a dose-dependent increase in TST immobility time in the Usp46 mutant mice without affecting general behaviors. The Usp46 KO mice exhibited short immobility times comparable to the Usp46 mutant mice, which was also increased by nitrazepam administration. The effects of nitrazepam in the Usp46 mutant and KO mice were antagonized by flumazenil. These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABA(A) receptor might participate in the regulation of TST immobility time.

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Generation of the Usp46 KO mice.(A) Integration site of the pU-17 trap vector. The trap vector is inserted approximately 16 kbp downstream from exon 1. The trap vector contains a splice acceptor (SA), the β-galactosidase/neomycin-resistance fusion (β-geo) gene, a polyadeylation signal (pA) and pSP73 vector sequences [10]. The white arrows (FP3, RP1, and SA6AS) indicate the primers used for genotyping. (B) Genotyping by the polymerase chain reaction. DNA fragments of 772 bp from the wild type allele and 1.4 kbp from the inserted allele were amplified by the primer pairs FP3-RP1 and FP3-SA6AS, respectively. (C) Immobility time in wild-type (WT) and Usp46 KO mice (KO) in the tail suspension test (TST). The KO mice showed a significantly shorter immobility time than the WT mice in the TST. Data represent the mean + S.E.M. for 5–6 mice in each group. **P<0.01 (Student’s t-test).
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pone-0039084-g001: Generation of the Usp46 KO mice.(A) Integration site of the pU-17 trap vector. The trap vector is inserted approximately 16 kbp downstream from exon 1. The trap vector contains a splice acceptor (SA), the β-galactosidase/neomycin-resistance fusion (β-geo) gene, a polyadeylation signal (pA) and pSP73 vector sequences [10]. The white arrows (FP3, RP1, and SA6AS) indicate the primers used for genotyping. (B) Genotyping by the polymerase chain reaction. DNA fragments of 772 bp from the wild type allele and 1.4 kbp from the inserted allele were amplified by the primer pairs FP3-RP1 and FP3-SA6AS, respectively. (C) Immobility time in wild-type (WT) and Usp46 KO mice (KO) in the tail suspension test (TST). The KO mice showed a significantly shorter immobility time than the WT mice in the TST. Data represent the mean + S.E.M. for 5–6 mice in each group. **P<0.01 (Student’s t-test).

Mentions: The Usp46 knockout (KO) mice (17-13906 Usp46 gene trapped mice, TG Resource Bank #6072) were descendants of the mouse strain generated by Trans Genic Inc. (Kumamoto, Japan) using the gene trap technique (Fig. 1) [10]. Because the original Usp46 KO mice were generated from B6 and CBA mice, these mice were backcrossed to B6 a minimum of 9 times in order to remove any possible phenotypic variations caused by a different genomic background. To determine their genotypes, ear biopsies were performed at 4 weeks of age for DNA detection using PCR. F1 heterozygous and B6 alleles were amplified using a neo primer pair: 5′-CTGAATGAACTGCAGGACGAG-3′ and 5′-GTCCAGATCATCCTGATCGAC-3′, and lox-SA primer pair: 5′-AGGTCGAGGGACCTATACCG-3′ and 5′-GAGGCCGCTTGTCCTCTTTG-3′. The F1 heterozygous mice were crossed between themselves to obtain the F2 generation: wild-type, heterozygous, and homozygous. To determine their genotypes, FP3 primer 5′-ATAGACTCTGCTGTTTCTCCTATGCTCC-3′, RP1 primer 5′-AATGTTGAGGCAAAGCTGCCAAGCTCAC-3′, and SA6AS primer 5′-CCGGCTAAAACTTGAGACCT-3′ were used.


Ubiquitin-specific peptidase 46 (Usp46) regulates mouse immobile behavior in the tail suspension test through the GABAergic system.

Imai S, Mamiya T, Tsukada A, Sakai Y, Mouri A, Nabeshima T, Ebihara S - PLoS ONE (2012)

Generation of the Usp46 KO mice.(A) Integration site of the pU-17 trap vector. The trap vector is inserted approximately 16 kbp downstream from exon 1. The trap vector contains a splice acceptor (SA), the β-galactosidase/neomycin-resistance fusion (β-geo) gene, a polyadeylation signal (pA) and pSP73 vector sequences [10]. The white arrows (FP3, RP1, and SA6AS) indicate the primers used for genotyping. (B) Genotyping by the polymerase chain reaction. DNA fragments of 772 bp from the wild type allele and 1.4 kbp from the inserted allele were amplified by the primer pairs FP3-RP1 and FP3-SA6AS, respectively. (C) Immobility time in wild-type (WT) and Usp46 KO mice (KO) in the tail suspension test (TST). The KO mice showed a significantly shorter immobility time than the WT mice in the TST. Data represent the mean + S.E.M. for 5–6 mice in each group. **P<0.01 (Student’s t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375232&req=5

pone-0039084-g001: Generation of the Usp46 KO mice.(A) Integration site of the pU-17 trap vector. The trap vector is inserted approximately 16 kbp downstream from exon 1. The trap vector contains a splice acceptor (SA), the β-galactosidase/neomycin-resistance fusion (β-geo) gene, a polyadeylation signal (pA) and pSP73 vector sequences [10]. The white arrows (FP3, RP1, and SA6AS) indicate the primers used for genotyping. (B) Genotyping by the polymerase chain reaction. DNA fragments of 772 bp from the wild type allele and 1.4 kbp from the inserted allele were amplified by the primer pairs FP3-RP1 and FP3-SA6AS, respectively. (C) Immobility time in wild-type (WT) and Usp46 KO mice (KO) in the tail suspension test (TST). The KO mice showed a significantly shorter immobility time than the WT mice in the TST. Data represent the mean + S.E.M. for 5–6 mice in each group. **P<0.01 (Student’s t-test).
Mentions: The Usp46 knockout (KO) mice (17-13906 Usp46 gene trapped mice, TG Resource Bank #6072) were descendants of the mouse strain generated by Trans Genic Inc. (Kumamoto, Japan) using the gene trap technique (Fig. 1) [10]. Because the original Usp46 KO mice were generated from B6 and CBA mice, these mice were backcrossed to B6 a minimum of 9 times in order to remove any possible phenotypic variations caused by a different genomic background. To determine their genotypes, ear biopsies were performed at 4 weeks of age for DNA detection using PCR. F1 heterozygous and B6 alleles were amplified using a neo primer pair: 5′-CTGAATGAACTGCAGGACGAG-3′ and 5′-GTCCAGATCATCCTGATCGAC-3′, and lox-SA primer pair: 5′-AGGTCGAGGGACCTATACCG-3′ and 5′-GAGGCCGCTTGTCCTCTTTG-3′. The F1 heterozygous mice were crossed between themselves to obtain the F2 generation: wild-type, heterozygous, and homozygous. To determine their genotypes, FP3 primer 5′-ATAGACTCTGCTGTTTCTCCTATGCTCC-3′, RP1 primer 5′-AATGTTGAGGCAAAGCTGCCAAGCTCAC-3′, and SA6AS primer 5′-CCGGCTAAAACTTGAGACCT-3′ were used.

Bottom Line: This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system.However, it is not known precisely how the immobile behavior is regulated by the GABAergic system.These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABA(A) receptor might participate in the regulation of TST immobility time.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.

ABSTRACT
The tail suspension test (TST) is widely recognized as a useful experimental paradigm for assessing antidepressant activity and depression-like behavior. We have previously identified ubiquitin-specific peptidase 46 (Usp46) as a quantitative trait gene responsible for decreasing immobility time in the TST in mice. This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system. However, it is not known precisely how the immobile behavior is regulated by the GABAergic system. Therefore, in the present study, we examined whether the immobility time is influenced by drugs affecting the action mediated by GABA(A) receptor using both 3-bp deleted (the Usp46 mutant) and Usp46 (Usp46 KO) mice. Nitrazepam, an agonist at the benzodiazepine-binding site of the GABA(A) receptor, which potentiates the action of GABA, produced a dose-dependent increase in TST immobility time in the Usp46 mutant mice without affecting general behaviors. The Usp46 KO mice exhibited short immobility times comparable to the Usp46 mutant mice, which was also increased by nitrazepam administration. The effects of nitrazepam in the Usp46 mutant and KO mice were antagonized by flumazenil. These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABA(A) receptor might participate in the regulation of TST immobility time.

Show MeSH
Related in: MedlinePlus