Mutations in SLITRK1 are found in patients with Tourette's syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an α2-adrenergic agonist that is frequently used to treat patients with Tourette's syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourette's syndrome and trichotillomania.
The Slitrk family of proteins comprises neuronal transmembrane proteins that control neurite outgrowth.1, 2 Structurally, Slitrk proteins share leucine-rich repeat (LRR) domains located N terminus to the transmembrane domain. LRR domains are present in many proteins and mediate protein–protein interactions.3 The LRR domains in Slitrk family proteins are highly similar to those in Slit family proteins, which control axon guidance and branching.4 Another structural feature of Slitrk proteins is C-terminally located tyrosine residues, which are flanked by amino acid sequences similar to the C-terminal domain of the Ntrk neurotrophin receptor.5 The C-terminal domain is conserved in all Slitrk proteins except Slitrk1.1, 2
Several type I transmembrane proteins with LRR domains have been identified till date, and most are produced predominantly in the nervous system.6 In addition to LRR domains, many type I transmembrane proteins have motifs typical of cell adhesion molecules, such as immunoglobulin-like and fibronectin type III domains. Functionally, they modulate neurite outgrowth, at least in vitro, but the precise function of each family member has not been clarified yet.6
Mutations in SLITRK1 are found in patients with Tourette's syndrome (TS) and trichotillomania (TTM).7, 8 TS is a developmental neuropsychiatric disorder that is characterized by persistent motor and vocal tics and often is accompanied by obsessive-compulsive disorder (OCD), attention-deficit hyperactivity disorder (ADHD), anxiety or depression.9, 10 TTM is a chronic behavioral disorder characterized by the irresistible urge to pull out one's hair, resulting in noticeable hair loss.11 TS belongs to the OCD spectrum of diseases, as may TTM, in light of phenomenologic and neurobiologic evidence.
To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior and a depression-like phenotype. Neurochemically, concentrations of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were increased in the brains of Slitrk1-knockout mice. In addition, administration of clonidine, an α2-adrenergic agonist, abrogated the anxiety-like behavior of Slitrk1-knockout mice. These results indicate that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice.
Materials and methods
The mice were maintained by the Laboratory Animal Facility, RIKEN Brain Science Institute. All animal experiments were carried out according to the guidelines for animal experimentation at RIKEN. The mice were housed on a 12-h light–dark cycle, with the dark cycle occurring from 2000 to 0800 hours. All mice used were littermates from mated heterozygotes. All the analyses we present here were performed with adult male mice. The behavioral tests were started when the mice were 8 weeks old and completed before the mice reached the age of 22 weeks.
Generation of Slitrk1-null mutant mice
Slitrk1-null mutant mice were generated as described previously.12 Briefly, to construct the Slitrk1-targeting vector, overlapping Slitrk1 genomic clones were isolated from a phage library derived from mice of the 129SV strain (Stratagene, La Jolla, CA, USA). The targeting construct contained the 3.6-kb 5′ and 5.2-kb 3′ homology regions, and the 2.5-kb fragment containing the open-reading frame of Slitrk1 was replaced with the phosphoglycerol kinase (PGK)–neo expression cassette flanked by a loxP sequence. E14 embryonic stem (ES) cells were electroporated with the targeting construct and selected with G418. Drug-resistant clones were analyzed by Southern blotting. BamHI- and ScaI-digested genomic DNA were hybridized with a 1.5-kb 3′ genomic fragment that corresponded to the genomic sequence outside of the targeting vector and a 0.6-kb PstI PGK–neo probe, respectively. Chimeric mice were generated by the injection of targeted ES cells into C57BL/6J blastocysts. To excise the PGK–neo cassette, mice with germline transmission were first mated with mice transgenic for Cre recombinase under the control of the cytomegalovirus immediate early enhancer-chicken β-actin hybrid (CAG) promotor.13 Correct excision of the PGK–neo cassette was confirmed by Southern blot. Mice carrying the mutated Slitrk1 allele were backcrossed to C57BL/6J for more than six generations before analysis. Genotyping of progenies was performed by Southern blot or PCR analysis of DNA isolated from tail samples; the PCR primers used were Slitrk1S (5′-IndexTermTACTACGCTGCAAACCTGCTTG-3′), Slitrk1WTAS (5′-IndexTermAATAGCCCAGACGCCAGTCA-3′) and Slitrk1KOAS (5′-IndexTermCAATACATTCATGCCTTCGTGCAAC-3′).
Generation of an anti-Slitrk1 antibody
A polyclonal anti-Slitrk1 antibody was raised in a rabbit against peptides corresponding to the cytoplasmic region of mouse Slitrk1 (SSYWHNGPYNADGSHRVYDC). Peptides were synthesized and conjugated to keyhole limpet hemocyanin through a cysteine residue. After immunization by conventional methods, antisera were obtained, and the antibody was purified by affinity chromatography with the immunized peptide. Specificity of the antibody was confirmed by the absence of immunopositive bands in western blotting analyses of brain lysate from knockout mice, or after preabsorption of the antibody with competing peptide (Supplementary Figure S1).
Specimens were homogenized in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). Approximately 10 μg of extract was loaded onto a 7.5% SDS–PAGE gel, electrophoresed and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Signals were visualized by using an ECL kit (GE Healthcare, Buckinghamshire, UK).
The open-field test was performed as previously described.14 Each mouse was placed in the center of an open-field apparatus (50 × 50 × 40 (H) cm) illuminated by light-emitting diodes (70 lux at the center of the field) and then allowed to move freely for 15 min. Distance traveled (cm) and duration (%) in the center area of the field (30% of the field) were adopted as the indices, and the relevant data were collected every 1 min. Data were collected and analyzed using Image J OF4 (O'Hara, Tokyo, Japan).
Elevated plus-maze test
The elevated plus-maze test was conducted as previously described with slight modification.15 Mice were tested for anxiety-like behavior on a standard plus-maze apparatus (closed arms, 25 × 5 × 15 (H) cm; open arms 25 × 5 × 0.3 (H) cm) arranged orthogonally 60 cm above the floor. Illuminance was 70 lux at the center platform of the maze (5 × 5 cm). Each mouse was placed on the center platform facing an open arm and then was allowed to move freely in the maze for 5 min. Total distance traveled, percentage of time in the open arms and percentage of open arm entries were measured as indices. Data were collected and analyzed using Image J EPM (O'Hara).
Classic fear-conditioning test
The classic fear-conditioning test was conducted as previously described with slight modification.16 Fear conditioning was performed in a rectangular, clear plastic chamber equipped with a stainless-steel grid floor (34 × 26 × 30 (H) cm). White noise (65 dB) was supplied from a loudspeaker as an auditory cue (conditioned stimulus, CS). The conditioning trial consisted of a 2-min exploration period followed by two CS–US (unconditioned stimulus) pairings separated by 1 min. A US (foot shock: 0.5 mA, 2 s) was administered at the end of the 30-s CS period.
At 24 h after the conditioning trial, a context test was performed in the same conditioning chamber for 3 min in the absence of white noise. Further, a cued test was performed in an alternative context with distinct cues: the test chamber was different from the conditioning chamber in brightness (0–1 lux), color (white), floor structure (no grid) and shape (triangular). The cued test was conducted 24 h after completion of the context test and consisted of a 2-min exploration period (no CS) to evaluate nonspecific contextual fear followed by a 2-min CS period (no foot shock) to evaluate the acquired cued fear. Rate of freezing response (immobility excluding respiration and heartbeat) of mice was measured as an index of fear memory. Data were collected and analyzed using Image J FZ2 (O'Hara).
Forced swimming test
We used a slightly modified procedure compared with that reported by Porsolt et al.17 Each mouse was placed for 7 min in a glass cylinder (30 cm high, 10 cm in diameter) containing 10 cm of water maintained at 22–25 °C. The duration of immobility was recorded during the last 3 min of the test period.
Tail suspension test
The tail suspension test was conducted as previously described.18 Mice were attached to a wire by using adhesive tape placed approximately 1.5 cm from the tip of the tail and were suspended 30 cm above the floor. The duration of immobility was recorded for 5 min.
Samples were taken from the prefrontal cortex, striatum and nucleus accumbens (Supplementary Figure S2). All specimens were homogenized in 0.1 M perchloric acid and then centrifuged for 15 min at 20 000 g at 4 °C; the resulting supernatant was assayed for neurotransmitters by high-performance liquid chromatography (HPLC) on C18 columns coupled to electrochemical detection (Eicom, Kyoto, Japan). For analysis of monoamines and their metabolites, the mobile phase consisted of 41.4 mM citrate, 39.2 mM sodium acetate, 17% methanol, 190 mg/l sodium 1-octanesulfonate and 5 mg/l EDTA; the solution was adjusted to pH 3.7 by using glacial acetic acid and the flow rate was 0.5 ml/min. For analysis of acetylcholine and choline, the mobile phase consisted of 100 mM Na2HPO4, 65 mg/ml tetramethylammonium chloride and 200 mg/l sodium 1-decanesulfonate; the solution was adjusted to pH 8.0 with phosphoric acid and was used at a flow rate of 1.0 ml/min. HPLC data were collected automatically and analyzed by Ezchrom Elite (Scientific Software, Tokyo, Japan). Protein was measured by DC Protein Assay kit (BioRad, Hercules, CA, USA).
To evaluate whether clonidine would modify the anxiety-like behavior in Slitrk1-deficient mice, mice were given 10 μg/kg clonidine (Wako Pure Chemical, Osaka, Japan) or an equal volume of saline (10 ml/kg) intraperitonially 30 min before the elevated plus-maze test. Administration of 5–10 μg/kg of clonidine can abrogate the anxiety-like behavior of sleep-deprived mice in the elevated plus-maze test.19
Statistical analyses were conducted by using the SPSS statistical package. Parametric data were analyzed by using Student's t-test, and nonparametric data were analyzed by using Mann–Whitney's U-test. Effects of factors were analyzed using analyses of variance (ANOVAs) (uni-ANOVA, one-way ANOVA with post hoc tests and general linear model (GLM)). Differences were defined as statistically significant when P<0.05.
Slitrk1 protein expression
First, we examined the expression pattern of Slitrk1 protein in the brains of adult male mice by immunoblot analysis using an anti-Slitrk1 antibody. Slitrk1 was ubiquitously detected in the nervous system and was abundant in the olfactory bulb, frontal cortex, hippocampus and amygdala (Figure 1). In Northern blot analyses, Slitrk1 mRNA was detected only in brain among adult mouse organs,1 and human SLITRK1 mRNA was enhanced in frontal cerebral cortex,2 consistent with the mouse expression profile.
Generation of Slitrk1-null mutant mice
To clarify the role of Slitrk1, we generated Slitrk1-deficient mouse lines. A targeting vector was used to replace the open-reading frame of Slitrk1 with a floxed PGK–neo cassette (Figure 2a). We isolated four independent ES clones with homologous recombination, and three ES clones yielded chimeric mice capable of transmitting the disrupted allele (+neo) through the germline. Subsequently, the PGK–neo cassette was removed by crossing the heterozygous mice with CAG–Cre transgenic partners, which express Cre recombinase in their zygotes (Δneo; Figure 2b). Ablation of Slitrk1 protein was confirmed by western blotting (Figure 2c).
We genotyped pups born from heterozygous matings at birth and at weaning. When counted in two different populations, the numbers of (Slitrk1+/+, Slitrk1+/− and Slitrk1−/− mice) were (28, 61 and 31) at birth, and were (162, 326 and 123) at weaning, respectively. The percentage of null homozygotes at weaning (20.1%) was lower than the expected Mendelian ratio (25%; P<0.05, χ2-test). In addition, male Slitrk1−/− mice weighed about 11% less than did their wild-type littermates (at 8 weeks of age: Slitrk1+/+, 24.8±1.4 g (n=15); Slitrk1−/−, 22.0±0.7 g (n=13); P<0.01, Student's t-test), whereas female null mice did not exhibit a reduction in body weight. Slitrk1-deficient mice did not display any external abnormalities except the males' reduced body weight. The cause of death before weaning and the low body weight is unclear at this point. However, none of the mice died during the behavioral testing period. Therefore, specific behavioral abnormalities in these mice are unlikely to reflect general health effects. Anatomic and histologic analyses of adult Slitrk1-deficient brains did not reveal any obvious abnormalities (Supplementary Figure S3). We did not find any unusual behaviors, including stereotypy, tremor, seizure or abnormal repetitive behaviors during the global observation or the timed video recordings of Slitrk1-deficient mice (Supplementary Material 1). We therefore applied the following behavioral tests to reveal their behavioral phenotypes.
Slitrk1-deficient mice displayed elevated anxiety-like behavior
Slitrk1-deficient mice consistently showed a pronounced decrease in locomotor activity. Total distance traveled by Slitrk1-deficient mice was significantly (P<0.01) less than that of controls during open-field (Figure 3a), light–dark transition and elevated plus-maze tests (data not shown). Their locomotor activity in the home cage was significantly (P<0.01) lower than that of the wild-type during the light phase but was not different during the dark phase (Supplementary Figure S4a to c).
In addition, Slitrk1-deficient mice showed behavioral abnormalities that may be related to anxiety, including a slight (albeit nonsignificant) reduction in the percentage of time spent in the center compartment of the open-field test (Figure 3b). In the elevated plus-maze test, Slitrk1-deficient mice exhibited significant (P<0.05) decreases both in the percentage of time spent in open arms and entries into open arms (Figures 3c and d). In the light–dark transition test, Slitrk1-deficient mice displayed relative (albeit nonsignificant) decreases in the percentage of distance traveled and the duration of time spent in the light box (Supplementary Figure S4d and e). Furthermore, we observed significantly (P<0.01) increased freezing in response to the context alone in Slitrk1-deficient mice in the fear-conditioning test (Figure 3e). However, the freezing response did not differ between wild-type and null mice presented with a conditioned tone in an unfamiliar context (Figure 3f).
Slitrk1-deficient mice exhibit depression-like behavior
In the forced swimming test, a well-established paradigm to detect depression-like behavior in rodents, duration of floating immobile on the surface of the water was increased in Slitrk1-deficient mice (P<0.05; Figure 3g). They also displayed increased immobility time in the tail suspension test (P<0.05; Figure 3h). In addition, we performed an auditory startle response test with and without prepulse, Morris's water maze test and marble burying behavior test, but none of the responses differed between wild-type and Slitrk1-deficient mice (Supplementary Figure S4f and data not shown). The results of the behavioral analyses are summarized in Supplementary Table 1.
Neurochemical analysis of Slitrk1-deficient mice
To determine whether neurochemical changes were associated with the behavioral abnormalities of Slitrk1-deficient mice, we measured the levels of monoamines in the prefrontal cortex, striatum, and nucleus accumbens by using HPLC–electrochemical detection (Table 1). Norepinephrine and its metabolite MHPG tended to be higher in all three regions in Slitrk1-knockout mice. Norepinephrine content was significantly higher in prefrontal cortex (P<0.05) and MHPG in nucleus accumbens (P<0.01) compared with the levels in wild-type mice. In addition, compared with wild-type levels, the choline and acetylcholine contents in the striatum were decreased in Slitrk1-knockout mice, and choline content was significantly lower (P<0.05; Table 1).
Clonidine treatment attenuated the anxiety-like behavior of Slitrk1-deficient mice
Previous studies have indicated that noradrenergic neurotransmission is altered in some TS patients, and treatment with clonidine, an α2-adrenergic agonist, is sometimes successful.9, 20, 21 Our current neurochemical analysis revealed increased norepinephrine and MHPG levels in Slitrk1-deficient brains. Because clonidine acts on presynaptic α2-adrenergic receptors to inhibit norepinephrine activity,22 we evaluated the effects of clonidine treatment on the behavioral abnormalities of Slitrk1-deficient mice. Administration of clonidine attenuated the anxiety-like behavior of Slitrk1-knockout mice in the elevated plus-maze test, whereas the drug had little effect on wild-type mice (Figures 4a and b). In contrast, clonidine treatment did not alter the locomotor activity of either wild-type or Slitrk1-deficient mice (Figure 4c).
In the present study, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in elevated plus-maze test and increased immobility time in forced swimming and tail suspension tests. The brains of Slitrk1-deficient mice had increased levels of norepinephrine and MHPG, and administration of clonidine, an α2-adrenergic agonist, attenuated the anxiety-like behavior of Slitrk1-knockout mice.
The results of the present study strongly suggest the involvement of norepinephrine mechanisms in the elevated anxiety-like behavior of Slitrk1-knockout mice. Recently, Hu et al. revealed that norepinephrine is involved in the formation of emotional memory by regulating AMPA-receptor trafficking, and injection of epinephrine enhanced the formation of fear-conditioned contextual memory.23 The result also suggests that the increased norepinephrine content of Slitrk1-deficient mice contributes to the increased freezing in the contextual fear-conditioning experiment (Figure 3e). Slitrk1 protein is not abundant in the pons and medulla oblongata, which contain noradrenergic neuronal cell bodies, including those in the locus coeruleus.24 Indeed, Slitrk1-deficient mice lacked overt abnormalities in noradrenergic neurons of the locus coeruleus (Supplementary Figure S5). However, Slitrk1 is produced abundantly in brain areas receiving projections of noradrenergic neurons, such as cerebral cortex, hippocampus, amygdala, thalamus and hypothalamus (Figure 1).24 Although the molecular properties of Slitrk1 have not been clarified yet, our present results indicate that it contributes to noradrenergic neurotransmission.
In this study, Slitrk1-deficient mice displayed not only elevated anxiety-like behavior but also depression-like behavioral abnormality, characterized by increased immobility time in forced swimming and tail suspension tests (Figures 3g and h). The monoamine-deficiency hypothesis postulates that depression is caused by the deficiency in serotonin and/or norepinephrine neurotransmission,25 and compounds that can inhibit reuptake of norepinephrine or serotonin are widely used as antidepressants. Increased levels of norepinephrine and MHPG were observed in Slitrk1-deficient brain, suggesting an altered status of the noradrenergic neurotransmission. Although further analyses are needed to clarify the causes and effects of the transmitter content abnormality, preclinical and clinical evidence suggests a relationship between increased noradrenergic neurotransmission and symptoms associated with stress and anxiety.24, 26 Considering the anxiety-like behavior in the Slitrk1-deficient mice, the altered noradrenergic transmission may be involved in the appearance of depression-like behavior.
As the noradrenergic system, the cholinergic system is involved in fear and anxiety. Systemic injection of mice with muscarinic antagonists increases anxiety, whereas administration of nicotinic agonists decreases anxiety, in the elevated plus-maze test.27, 28 Single injections of nicotine also decrease the freezing response of rats in the contextual fear-conditioning test.29 Furthermore, clinical evidence from patients with Alzheimer's disease also supports a relationship between the cholinergic system and anxiety. Alzheimer's disease is associated with decreased cholinergic levels, and roughly 33% of patients with Alzheimer's syndrome also suffer from anxiety disorders. More importantly, treatment with acetylcholinesterase inhibitors has decreased anxiety in these patients.30 Therefore, the decreased choline and acetylcholine levels of Slitrk1-deficient mice may be important in their increased anxiety-like behavior.
Mutations in SLITRK1 are found in patients with TS or TTM.7, 8, 31 These two syndromes are believed to belong to the OCD spectrum of diseases. However, Slitrk1-deficient mice did not display any abnormalities in the marble-burying behavior test (Supplementary Figure S4f), a paradigm used to detect OCD symptoms as well as anxiety-like behavior in animals.32, 33 Although the behavioral phenotypes of Slitrk1-knockout mice are not fully consistent with those of TS patients, these mice display some of the phenomena of TS patients and may yield insight into the pathogenesis of TS. Tic disorders including TS often are associated with anxiety disorders, mood disorders including major depression and phobias.34, 35 In the present study, Slitrk1-deficient mice displayed elevated anxiety-like behavior that was attenuated by the administration of clonidine, a drug frequently used to treat patients with TS. These results suggest possible association of the elevated anxiety-like behavior induced by the dysfunction of Slitrk1 and the symptoms of TS.
In agreement with the behavioral abnormalities of Slitrk1-deficient mice, SLITRK1 mutation in humans seems to be associated with anxiety- or mood-related disorders. For example, one proband diagnosed with TS and ADHD demonstrated a frameshift mutation in SLITRK1; the patient's mother, who had TTM, had the same mutation.7 In addition, a patient with TTM and SLITRK1 mutation also had mild anxiety and a history of depression; her mother, who carried the mutation, had a history of depression, low self-esteem, and a fear of heights.8 In the other case, a patient with TTM and SLITRK1 mutation had no other clinically significant mood, anxiety or behavior problems; however, her father, who was a mutation carrier, was formally diagnosed with TTM and social phobia and a history of bulimia, and his sister, who was not available for genetic testing, had also been diagnosed with TTM and had a history of anxiety and depressive disorders.8 Combining these findings with the results of present study, we hypothesize that SLITRK1 may be involved in the control of anxiety, fear and mood, which are closely related to TS and TTM.
Human SLITRK1 is located on 13q31.1, a region linked strongly with panic disorder, schizophrenia, bipolar disorder and recurrent depressive disorder.36, 37, 38, 39 Clinical studies suggest a relationship between norepinephrine and behaviors of anxiety and fear, as well as alterations in noradrenergic function in patients with psychiatric disorders related to anxiety and stress, panic disorder and posttraumatic stress disorder.26, 40, 41 Further analysis of Slitrk1-deficient mice will be beneficial for understanding the pathogenesis of TS and other related neuropsychiatric diseases.
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We thank Dr Tadafumi Kato and Dr Takeo Yoshikawa (RIKEN BSI) for their critical comments on the paper, Dr Mika Tanaka and Ms Chieko Nishioka for their assistance in generating Slitrk1-knockout mice, Ms Chihiro Homma for her assistance in behavioral analysis and Mr Masaki Kumai (Support Unit for Animal Experiments, RIKEN BSI) for his help in generating the anti-Slitrk1 antibody. This study was supported by RIKEN BSI funds and the Japan Society for the Promotion of Science.
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Cite this article
Katayama, K., Yamada, K., Ornthanalai, V. et al. Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities. Mol Psychiatry 15, 177–184 (2010). https://doi.org/10.1038/mp.2008.97
- leucine-rich repeat
- Tourette's syndrome
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Relationship between Slitrk1 Gene and Depression Symptoms in Chinese Han Patients with Tourette Syndrome
Advances in Psychology (2019)
NGL-3 in the regulation of brain development, Akt/GSK3b signaling, long-term depression, and locomotive and cognitive behaviors
PLOS Biology (2019)