Reproducibility via coordinated standardization:A multi-center study in a Shank2 genetic rat model for Autism Spectrum Disorders

Inconsistent findings between laboratories are hampering scientific progress and are of increasing public concern. Differences in laboratory environment is a known factor contributing to poor reproducibility of findings between research sites, and well-controlled multisite efforts are an important next step to identify the relevant factors needed to reduce variation in study outcome between laboratories. Through harmonization of apparatus, test protocol, and aligned and non-aligned environmental variables, the present study shows that behavioral pharmacological responses in Shank2 knockout (KO) rats, a model of synaptic dysfunction relevant to autism spectrum disorders, were highly replicable across three research centers. All three sites reliably observed a hyperactive and repetitive behavioral phenotype in KO rats compared to their wild-type littermates as well as a dose-dependent phenotype attenuation following acute injections of a selective mGluR1 antagonist. These results show that reproducibility in preclinical studies can be obtained and emphasizes the need for high quality and rigorous methodologies in scientific research. Considering the observed external validity, the present study also suggests mGluR1 as potential target for the treatment of autism spectrum disorders

Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism

Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD

Reproducibility via coordinated standardization : a multi-center study in a S hank2 genetic rat model for Autism Spectrum Disorders

Inconsistent findings between laboratories are hampering scientific progress and are of increasing public concern. Differences in laboratory environment is a known factor contributing to poor reproducibility of findings between research sites, and well-controlled multisite efforts are an important next step to identify the relevant factors needed to reduce variation in study outcome between laboratories. Through harmonization of apparatus, test protocol, and aligned and non-aligned environmental variables, the present study shows that behavioral pharmacological responses in Shank2 knockout (KO) rats, a model of synaptic dysfunction relevant to autism spectrum disorders, were highly replicable across three research centers. All three sites reliably observed a hyperactive and repetitive behavioral phenotype in KO rats compared to their wild-type littermates as well as a dose-dependent phenotype attenuation following acute injections of a selective mGluR1 antagonist. These results show that reproducibility in preclinical studies can be obtained and emphasizes the need for high quality and rigorous methodologies in scientific research. Considering the observed external validity, the present study also suggests mGluR1 as potential target for the treatment of autism spectrum disorders

Image_2_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.TIF

<p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

Table_1_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.xlsx

<p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

Tetrahydrocarbazole-Based Serotonin Reuptake Inhibitor/Dopamine D2 Partial Agonists for the Potential Treatment of Schizophrenia

A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays

WS-50030 [7-{4-[3-(1H-Inden-3-Yl)Propyl]Piperazin-1-Yl}-1,3-Benzoxazol- 2(3H)-One]a Novel Dopamine D2 Receptor Partial Agonist/Serotonin Reuptake Inhibitor with Preclinical Antipsychotic-Like and Antidepressant-Like Activity

The preclinical characterization of WS-50030 [7-{4-[3-(1Hinden-3-yl)propyl] piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D2 receptor (D2L Ki, 4.0 nM) and serotonin transporter (Ki, 7.1 nM), potent D2 partial agonist activity (EC50, 0.38 nM; Emax, 30%), and complete block of the serotonin transporter (IC50, 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID50, 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D2 partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazole\u27s reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D2 receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants

Evolution Education Questionnaire on Acceptance and Knowledge (EEQ) : standardised and ready-to-use protocols to measure acceptance of evolution and knowledge about evolution in an international context

The lack of standardised assessment of evolutionary knowledge and acceptance of evolution across Europe makes comparisons between studies difficult. The Evolution Education Questionnaire on Acceptance and Knowledge (EEQ) was constructed to measure attitudes and understanding across Europe and beyond. We aimed to compile a brief instrument to allow for easy application in school and university. The target group of the EEQ was freshman university students who had just finished their secondary education. However, several components of the questionnaire were developed and validated for additional target groups. Therefore, this questionnaire may, in addition, be suitable for students in secondary school, in-service teachers as well as the general public.This method report describes the contents and application of the EEQ and provides information on survey conduction, data preparation, analyses and interpretation of results to serve as a standardised and ready-to-use protocol to measure the acceptance of and knowledge about evolution in a local, national or international context. To allow for sampling in different European countries, we present the EEQ in 23 European languages.The lack of standardised assessment of evolutionary knowledge and acceptance of evolution across Europe makes comparisons between studies difficult. The Evolution Education Questionnaire on Acceptance and Knowledge (EEQ) was constructed to measure attitudes and understanding across Europe and beyond. We aimed to compile a brief instrument to allow for easy application in school and university. The target group of the EEQ was freshman university students who had just finished their secondary education. However, several components of the questionnaire were developed and validated for additional target groups. Therefore, this questionnaire may, in addition, be suitable for students in secondary school, in-service teachers as well as the general public. This method report describes the contents and application of the EEQ and provides information on survey conduction, data preparation, analyses and interpretation of results to serve as a standardised and ready-to-use protocol to measure the acceptance of and knowledge about evolution in a local, national or international context. To allow for sampling in different European countries, we present the EEQ in 23 European languages