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Exp Neurobiol 2018; 27(6): 539-549
Published online December 28, 2018
© The Korean Society for Brain and Neural Sciences
Yunjin Lee1,†, Hannah Kim1,†, and Pyung-Lim Han1,2*
1Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul 03760, Korea.
2Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Korea.
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-3277-4130, FAX: 82-2-3277-3419
†These authors contributed equally to this work.
Autism spectrum disorder (ASD) is a heterogeneous group of neurobehavioral disorders characterized by the two core domains of behavioral deficits, including sociability deficits and stereotyped repetitive behaviors. It is not clear whether the core symptoms of ASD are produced by dysfunction of the overall neural network of the brain or that of a limited brain region. Recent studies reported that excessive glutamatergic or dopaminergic inputs in the dorsal striatum induced sociability deficits and repetitive behaviors. These findings suggest that the dorsal striatum plays a crucial role in autistic-like behaviors. The present study addresses whether functional deficits of well-known ASD-related genes in the dorsal striatum also produce ASD core symptoms. This study also examines whether these behavioral changes can be modulated by rebalancing glutamate and/or dopamine receptor activity in the dorsal striatum. First, we found that the siRNA-mediated inhibition of
Keywords: Autism spectrum disorder, dorsal striatum, MeCP2, TSC1
Autism spectrum disorder (ASD) is a heterogeneous group of neurobehavioral disorders that is characterized by sociability deficits and restricted stereotypies . Recent studies have identified a number of genes associated with ASD. Statistical modeling based on published results predicts that 1,000~1,500 genes may be associated with ASD [2,3]. The complexity of the functional profiles of known ASD genes makes it difficult to define shared mechanisms in ASD. It is also unclear whether ASD core symptoms are produced by dysfunction of the brain's overall neural network or by that of a specific brain region.
A growing body of evidence indicates that functional changes in the dorsal striatum promote sociability deficits and repetitive behaviors [4,5,6]. The dorsal striatum receives excitatory glutamatergic inputs from cortical and subcortical regions including the prefrontal cortex and thalamus , and dopaminergic inputs from the substantia nigra . Adenylyl cyclase 5 (AC5) is preferentially expressed in the dorsal striatum, where it functions as an essential mediator of D1 and D2 dopamine receptors . Recently, we reported that AC5 KO or D2 KO mice exhibit typical autistic-like behaviors. Similarly, siRNA-mediated inhibition of AC5 or D2 dopamine receptors locally in the dorsal striatum also produces the autistic-like phenotypes displayed by AC5 KO or D2 KO mice [5,6,10]. Furthermore, siRNA-mediated inhibition of
Of the well-characterized ASD-related genes in previous studies, SHANK (SH3 and multiple ankyrin repeat domains), NLGN (neuroligin), and NRXN (neurexin) [11,12,13,14] are key components in synapse formation, MeCP2 (methyl CpG binding protein 2)  and FMR1 (fragile X mental retardation 1) play a role in transcription/post-transcriptional processes, and TSC1 and TSC2 (tuberous sclerosis proteins 1 and 2)  are cytosolic factors that regulate the mTOR signaling pathway.
In this study, we investigated whether striatal inhibition of well-known ASD-related genes could produce autistic-like behaviors. We found that siRNA-mediated suppression of
Seven-week-old C57BL/6 male mice were purchased from Daehan BioLink (Eumsung, Chungbuk, Republic of Korea). Upon arrival, mice were housed in pairs in standard plastic cages and were fed lab chow and water ad libitum. The animal room was maintained at a temperature of 22~23℃ with 50~60% humidity and with a normal light-dark cycle (light on at 7:00 a.m.). It was a specific-pathogen-free environment. All animals were handled in accordance with the Guidelines of Animal Care at Ewha Womans University through permission of EWU-IACUC (No. 16-020).
Real-time PCR analysis was performed as described previously [5,6]. Total RNA was purified from striatal tissues using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), and treated with DAase I to avoid genomic contamination. Real-time PCR was performed with iQTM SYBR Green Supermix (Bio-Rad Laboratories, Foster City, CA, USA) using the CFX 96 Real-Time PCR System Detector (Bio-Rad Laboratories; Foster City, CA, USA). The following primer sets were used: 5′- ACGAAGTGCCTGCGTCTGGAC-3′ and 5′-CTCTTGCCAACCATTCTCATCAGTG-3′ for Shank3; 5′- TGGAATGGGGACCAGGATGC-3′ and 5′-CAGTACTGTTGAACCCTGCGGC-3′ for Nlgn3; 5′-GCGTAGATGGGCTGCAAC-3′ and 5′-CCTGTGCCATCTTGCCTACT-3′ for Fmr1; 5′ -TGCTTTCAGTCATTTGGCTATA-3′ and 5′ TGTCGTATTCACCTTCAGTT-3′ for Mecp2; 5′-GAGGTAGAGTCACTGGAGGC-3′ and 5′-GAACTGGGAAGTGAGCCAAC-3′ for Tsc1; 5′-GCTGCCATCTGTTTTACGG-3′ and 5′-TGACTGGTGCCTGATGAACT-3′ for Gapdh; and 5′-GCTGCCATCTGTTTTACGG-3′ and 5′-TGACTGGTGCCTGATGAACT-3′ for L32.
Stereotaxic injection of siRNA was performed as described previously [5,6]. Briefly, mice were anesthetized using an injection of a mixture of ketamine hydrochloride and xylazine hydrochloride (the ratio of 2.5: 1) at a dose of 2.5 µl/g of body weight. The siRNA was injected at a volume of 1.5 µL (18 ng of siRNA) into each side of the dorsal striatum (AP, +1.0; ML, ±1.5; DV, −3.6 mm) using a stereotaxic apparatus (Stoelting Company, Wood Dale, IL, USA). Behavioral tests were examined 48 h after siRNA injection based on the known knockdown profile of target transcripts and behavioral modification effects from previous studies [17,18]. The following siRNAs were used: control-siRNA (SN-1012), Shank3-siRNA (1424750, NM_021423.2), Nlgn3-siRNA (1392209, NM_172932.1), Fmr1-siRNA (1358762, NM_008031.2), Mecp2-siRNA (1385135, NM_010788.2), and Tsc1-siRNA (1441941, NM_022887.2). These siRNAs were purchased from Bioneer Co. (Deajun, Korea). The FAM-labeled RISK-independent siRNA transfection control siGLO Green (D-001630-01-05) was purchased from Dharmacon Inc. (Chicago, IL, USA).
SCH23390, ecopipam, and D-cycloserine (DSC) were administered intraperitoneally (i.p.) 30 min prior to the behavioral tests. Fenobam was administered 1 h prior to the behavioral tests. The drug doses used were as follows: SCH23390 (0.02 mg/kg), ecopipam (0.02 mg/kg), DCS (20 mg/kg), and fenobam (30 mg/kg). These doses were chosen based on the dose tests in this study and in previous studies [5,6].
Behavioral assessments were carried out using a computerized video tracking system (Panlab SMART, Harvard Apparatus, Barcelona, Spain) as described previously [5,6]. The behavior testing room was illuminated by 20 lux in the three-chamber assays, U-field assays, open field test, and repetitive behavior test. In contrast, it was illuminated by 250 lux in the marble burying test. White noise (65 dB) was used to mask background noise in the behavior testing room. Test equipment was frequently cleaned using 70% ethanol.
The sociability test and social novelty preference test were carried out as described previously [5,6,10]. The U-field two-choice field was also used to test for social preference as described previously [5,6,19]. For the social novelty preference test, a subject mouse was allowed to freely explore the U-shaped two-choice field (45×45 cm2) for 10 min (habituation). While the subject mouse was returned to its home cage for two min, a familiar mouse (a cage mate) and an unfamiliar mouse (a new social target) were separately loaded into wire cages (12 cm in diameter) and placed at the corner of each closed square of the U-shaped arena. The subject mouse was placed in the center of the U-shaped field and allowed to freely explore the two-choice field for 10 min. During this time, the time spent and trajectory in each field were recorded.
Cage mates and strangers were prepared as described previously [5,6]. Mice were randomly housed in pairs two to three days prior to behavioral tests. The cage mate conditions were maintained until the behavioral tests were started. One cage mate was used as the subject mouse, while the other was used as the familiar target.
The sociability test using three chambers has been previously described [5,6]. Briefly, a subject mouse was allowed to freely explore the three chambers (22×32 cm2 each) for 10 min. After this habituation, a circular wire cage (12 cm in diameter) containing a social target (stranger; a naïve C57BL/6, same sex, same age) was placed in one side chamber, and while an empty wire cage was placed in the other side chamber. The subject mouse was allowed to freely explore both chambers for 10 min. Similarly, the time spent and the trajectory between chambers were recorded during this test. This test step was regarded as the sociability test.
The novel object preference test was performed as described previously [5,6]. Mice were presented with two identical objects (100-ml-glass-flasks containing cage bedding at a 3-cm-depth) placed 20 cm apart in the open field (30 cm×45 cm) and were familiarized with the objects for 10 min (habituation session). While the subject mouse was returned to its home cage for 2 min, one familiar object was exchanged for a novel object (a wooden block; 3.5 cm×3.5 cm×7 cm). After 2 min, the mice were placed in the center of the open-field arena, and allowed to freely explore the open-field. The time spent investigating an object by directing its nose within 2 cm of the object and/or sniffing was recorded for 5 min. The familiar object and novel object were used randomly and counterbalanced.
The open field test was conducted as described previously . In brief, mice were allowed to freely explore the open field (45×45 cm2) for 1 hour, and the moving distance was recorded.
Repetitive behavior assessments were carried out as described previously [5,6]. In brief, mice were placed individually in a home cage with new bedding. The times spent performing repetitive behaviors including grooming and digging were measured from video recordings of the 10 minute test session.
The marble burying test was carried out as described previously [5,6]. Mice were placed individually in a home cage that was filled with moderately fine wood chip bedding (JRS 3-4, Rosenberg, Germany). The bedding was layered up to 5 cm from the cage floor. Twelve marbles (diameter: 1.5 cm) were placed uniformly throughout the cage. Mice were allowed to freely explore the cage for 30 min. The number of buried marbles was recorded every five minutes. A marble was considered “burying” when <25% of it was visible.
Two-sample comparisons were performed using Student's t-test. Multiple comparisons were performed by one-way and two-way ANOVA or two-way repeated measures ANOVA followed using a post hoc test using Graphpad Prism 6 (San Diego, CA, USA). All data are presented as mean±SEM or box plot diagrams. Statistical significance was defined by the 5% level.
To determine whether functional deficits of well-known ASD-related genes in the dorsal striatum would produce autistic-like behaviors, Shank3-siRNA, Nlgn3-siRNA, Fmr1-siRNA, Mecp2-siRNA, or Tsc1-siRNA was injected bilaterally in the dorsal striatum of normal mice. Two days later, when the siRNA-mediated knockdown effects are fully effective [10,21,22] (Fig. 1B, I), standard behavioral tests were used to assess the mice (Fig. 1A).
Mice injected with Nlgn3-siRNA, Mecp2-siRNA, or Tsc1-siRNA showed increased grooming behavior compared to those that received control-siRNA injection. Mice injected with Nlgn3-siRNA or Shank3-siRNA exhibited reduced digging behavior or only a tendency of reduction in digging compared to those with control-siRNA injection. However, mice injected with Fmr1-siRNA injection displayed no significant change in grooming or digging behavior (Fig. 1C, D). We recently demonstrated that the sociability test using a U-shaped two-choice field is useful to determine whether an animal is sociable with a novel social target in the presence of a familiar cage-mate [5,6]. This test involves presenting the subject mouse with a social novelty (a non-mate) and a socially familiar target (a cage-mate) in the U-shaped two-choice field. In this U-shaped two-choice field test, mice injected with Fmr1-siRNA, Mecp2-siRNA, or Tsc1-siRNA in the dorsal striatum exhibited decreased time interacting with an unfamiliar target over a cage-mate compared to that of mice injected with the control-siRNA. In contrast, mice injected with Shank3-siRNA or Nlgn3-siRNA did not show significant changes in this test (Fig. 1E, F). In the novel object preference test, mice injected with Shank3-siRNA, Nlgn3-siRNA, Fmr1-siRNA, Mecp2-siRNA, or Tsc1-siRNA spent less time sniffing a novel object over a familiar one compared to that of mice injected with the control-siRNA (Fig. 1G, H).
Overall, these results indicate that injection of Shank3-siRNA, Nlgn3-siRNA, Fmr1-siRNA, Mecp2-siRNA, or Tsc1-siRNA in the dorsal striatum produced mild to severe repetitive behavior, and deficits in sociability or novel object preference. Of these, the knockdown of
Next, we examined the behavioral features of mice with striatal knockdown of
Collectively, these results suggest that local inhibition of
Recent pharmacological studies combined with siRNA-mediated functional analyses [5,6] demonstrated that the autistic-like phenotypes of AC5 KO mice or D2 KO mice were modulated by D-cycloserine (an NMDA agonist), fenobam (an mGluR5 antagonist), SCH23390 (a D1 antagonist) and/or ecopipam (a D1/5 antagonist). We examined whether the autistic-like phenotypes induced by inhibition of MeCP2 or TSC1 in the dorsal striatum could be modulated by pharmacologically targeting the dorsal striatum.
Mice with siRNA-mediated inhibition of
Overall, these results suggest that autistic-like phenotypes induced by siRNA-mediated inhibition
In this study, we investigated the neuroanatomical correlates of ASD core symptoms. Our results and evidence from the literatures support the hypothesis that striatal dysfunction can induce impairments in the two core domains of ASD behavioral symptoms, including sociability deficits and repetitive behaviors. First, as demonstrated here, local inhibition of MeCP2 and TSC1 in the dorsal striatum, and although to a much lesser extent, that of SHANK3, NLGN3, and FMR1, produced ASD-related behaviors. These findings are based on the results of the typical behavioral tests used in preclinical studies. Second, optogenetic stimulation of cortico-striatal glutamatergic neurons in wildtype mice produced sociability deficits (Fig. 3A; ref ). Thus, increased glutamatergic inputs to the dorsal striatum, which might induce the activation of various types of glutamatergic receptors, were pro-autistic . Partly consistent with this finding, AC5 KO mice , D2 KO mice , or
As demonstrated here, siRNA-mediated inhibition of
In this study, we demonstrated that siRNA-mediated inhibition of
Mice with Mecp2-siRNA or Tsc1-siRNA injection in the dorsal striatum exhibited increased grooming, but decreased marble burying behavior (Fig. 2). Thus, mice with Mecp2-siRNA or Tsc1-siRNA injection exhibited complex phenotypes in the view of the repetitive behaviors. The marble burying test is commonly used to measure not only repetitive behavior, but also anxiety in rodents. In addition, rodents display burying behavior in response to an aversive stimulus, and this behavior is referred to “defensive burying” . Thus, marble burying behavior could present multiple behavioral aspects. Furthermore,
We recently demonstrated that autistic-like phenotypes displayed by AC5 KO mice or D2 KO mice were rescued by pharmacological inhibition of mGluR5 with fenobam and of D1 receptors with SCH23390/ecopipam (SCH39166) or pharmacological activation of GluN with D-cycloserine [5,6]. Although functional inhibition of MeCP2 or TSC1 is not directly related to the genetic deletion of AC5 or D2 receptor, autistic-like behaviors induced by striatal inhibition of MeCP2 or TSC1 (Fig. 2) were also rescued by treatment with D-cycloserine, fenobam, SCH23390 and/or ecopipam (Fig. 2). These results raise the possibility that autistic-like phenotypes induced by functional deficits in the dorsal striatum could be reversed by modulation of the glutamate or dopamine receptor systems. It will be worth to examine whether similar mechanisms work for other cases of ASD animal models.
Concerning the pharmacological effects of SCH23390 and ecopipam, the impairment in novel object preference in mice with MeCP2 inhibition was rescued by ecopipam, but not by SCH23390 (Fig. 2G, H). Thus, there seems to be a subtle difference in pharmacological effects of SCH23390 and ecopipam. SCH23390 is a halobenzazepine that acts as a selective antagonist of D1 and D5 (Kis=0.2 and 0.3 nM, respectively) , and also show high affinity at the serotonin receptor subtype 5-HT2C (Ki, 9.3 nM) , and 5-HT1C. However, the doses required to induce a similar response in vivo are greater than 10-fold higher than those required to induce a D1-mediated response . Ecopipam (SCH 39166) is a synthetic benzazepine derivative that acts as a selective antagonist of D1 and D5. Ecopipam has reduced affinity at 5-HT2 to compared that of SCH23390 . Therefore, it might be possible that the subtle difference in pharmacological profiles of SCH23390 and ecopipam produces slightly differential behavioral responses.