Accepted Manuscript Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model Ryan F. Yoshimura, Minhtam B. Tran, Derk J. Hogenkamp, Narielle L. Ayala, Timothy Johnstone, Andrew J. Dunnigan, Timothy K. Gee, Kelvin W. Gee PII:
S0028-3908(17)30396-9
DOI:
10.1016/j.neuropharm.2017.08.029
Reference:
NP 6834
To appear in:
Neuropharmacology
Received Date: 31 May 2017 Revised Date:
18 August 2017
Accepted Date: 21 August 2017
Please cite this article as: Yoshimura, R.F., Tran, M.B., Hogenkamp, D.J., Ayala, N.L., Johnstone, T., Dunnigan, A.J., Gee, T.K., Gee, K.W., Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2017.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title page Title: Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model
Department of Pharmacology, School of Medicine, University of California Irvine, Irvine, CA, 92697-4625
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Corresponding author: Kelvin W Gee 110C Medical Surge I Department of Pharmacology University of California, Irvine ` Irvine, CA 92697-4625 Phone 949-824-8009 Fax 949-824-4855 Email [email protected]
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Authors: Ryan F Yoshimuraa; Minhtam B Trana; Derk J Hogenkampa; Narielle L Ayalaa; Timothy Johnstonea; Andrew J Dunnigana; Timothy K Geea; Kelvin W Geea
ACCEPTED MANUSCRIPT
Abstract
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Autism spectrum disorder (ASD) is associated with two core symptoms (social communication deficits and stereotyped repetitive behaviors) in addition to a number of comorbidities. There are no FDA-approved drugs for the core symptoms and the changes that underlie these behaviors are not fully understood. One hypothesis is an imbalance of the excitation (E)/inhibition (I) ratio with excessive E and diminished I occurring in specific neuronal circuits. Data suggests that both gamma-aminobutyric acidA (GABAA) and α7 nicotinic acetylcholine receptors (nAChRs) significantly impact E/I. BTBR T+ tf/J (BTBR) mice are a model that display an autism-like phenotype with impaired social interaction and stereotyped behavior. A β2/3-subunit containing GABAA receptor (GABAAR) subtype selective positive allosteric modulator (PAM), 2-261, and an α7 nAChR subtype selective PAM, AVL-3288, were tested in social approach and repetitive self-grooming paradigms. 2-261 was active in the social approach but not the self-grooming paradigm, whereas AVL-3288 was active in both. Neither compound impaired locomotor activity. Modulating α7 nAChRs alone may be sufficient to correct these behavioral and cognitive deficits. GABAergic and nicotinic compounds are already in various stages of clinical testing for treatment of the core symptoms and comorbidities associated with ASD. Our findings and those of others suggest that compounds that have selective activities at GABAAR subtypes and the α7 nAChR may address not only the core symptoms, but many of the associated comorbidities as well and warrant further investigation in other models of ASD.
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Keywords: autism; BTBR; GABA; nicotinic; allosteric modulator
ACCEPTED MANUSCRIPT
1. Introduction The Diagnostic Statistical Manual V defines the two core clinical symptoms of ASD as social communication deficits and repetitive patterns of behavior (American Psychiatric Association, 2013). Individuals with ASD also have an increased risk for comorbidities including anxiety disorders, seizures, obsessive
RI PT
compulsive disorder, attention deficit hyperactivity disorder, sleep disorders and cognitive impairments (Leyfer et al., 2006; Kotagal and Broomall, 2012; Srivastava and Schwartz, 2014; Frey et al., 2016). Although there are treatments available for some of these comorbidities, no drugs are approved for the treatment of the core
SC
symptoms.
One hypothesis for ASD is that the clinical symptoms arise from an imbalance of E/I in neural systems (Rubenstein and Merzenich, 2003). Most cases of ASD are idiopathic, supporting a potential interaction of
M AN U
multiple genes with the environment. Evidence from preclinical and human postmortem studies showing deficits in GABAAR function and GABA-mediated inhibition support a role for the GABAergic system. ASD patients demonstrate a selective downregulation of GABAARs which may be associated with abnormalities in chromosomal loci 15q11-q13 which contain genes encoding the α5, β3 and γ3 GABAAR subunits (Shao et al., 2003; Fatemi et al., 2009). Changes in expression of some of these subunits have been observed in brains of ASD patients and autism related genes have been found to be mainly expressed in GABAergic interneurons (Xu
TE D
et al., 2014). Selectively enhancing GABA-mediated inhibition via modulation of GABAAR subtypes may be therapeutically beneficial in correcting the E/I imbalance. The 15q13 chromosome also encompasses the CHRNA7 gene that encodes the α7 nAChR which has a diversity of functions that are highly relevant to ASD (Albuquerque et al., 2009). There are changes in nAChR
EP
binding in the cerebral cortex and cerebellum (Martin-Ruiz et al., 2004) along with a reduction in neuronal α7 nAChR immunoreactivity in the thalamus of ASD patients (Ray et al., 2005). Microdeletions in chromosome
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15q13.3 and reduced expression of CHRNA7 associated with ASD along with the autism-related phenotypes associated with the 15q13.3 homozygous knockout mouse provide strong support for selectively targeting α7 nAChRs (Lowther et al., 2015; Forsingdal et al., 2016). The BTBR T+ tf/J (BTBR) mouse is a strain that has been characterized with ASD-related behaviors. Similar to the core symptoms of ASD, BTBR mice demonstrate low sociability and increased repetitive behaviors. Enhancing GABA mediated inhibition with the GABAAR-active neurosteroid ganaxolone improved social approach behavior, but not repetitive self-grooming behavior in BTBR mice (Kazdoba et al., 2016). Clonazepam, the benzodiazepine (BZ) GABAAR PAM, also improved social approach behavior in BTBR mice (Han et al., 2014).
ACCEPTED MANUSCRIPT
BTBR mice also have lower basal levels of acetylcholine in the medial prefrontal cortex and treatment with an acetylcholinesterase inhibitor reduced social deficiency (Karvat and Kimchi, 2014). Nicotinic receptors have been implicated in the expression of both social interaction and repetitive behaviors in BTBR mice (Wang et al., 2015). Thus the BTBR model may respond to the modulation of GABAergic and/or nicotinic acetylcholinergic
RI PT
systems, providing clues to a potential therapeutic approach for treatment of the core symptoms of ASD. We have previously characterized two PAMs, AVL-3288 and 2-261, that are selective for the α7 nAChR and β2/3-subunit-containing GABAARs (β2/3 GABAAR, 2-261 is equally active at either receptor subtype) respectively (Ng et al., 2007; Gee et al., 2010). These targets were chosen because both receptor subtypes show
SC
potential loss of function in ASD (Lowther et al., 2015; Forsingdal et al., 2016; Shao et al., 2003; Fatemi et al., 2009). PAMs are important tools since unlike direct receptor agonists, they potentially preserve the spatiotemporal integrity of neurotransmission and reduce the influence of various factors associated with direct
M AN U
agonism as confounds in the interpretation of the pharmacological effects (Uteshev, 2014). Additionally, AVL3288 is a type I PAM that is in clinical trials for the treatment of cognitive impairment associated with schizophrenia (CIAS; Gee et al., 2017). Unlike the clinically used BZs, 2-261 is a GABAAR PAM that has reduced side effects and abuse liability in animal models which are important considerations for an ASD drug (Gee et al., 2010; Yoshimura et al., 2014). Using these and other PAMs from our compound library as tools, we examine the effect of selectively modulating the GABAAR subtypes that contain β2/β3 subunits and the α7 nAChR on
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sociability and stereotypic behavior in the BTBR mouse model.
ACCEPTED MANUSCRIPT
2. Materials and Methods 2.1. Chemicals
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GRN-529, a negative allosteric modulator of the metabotropic glutamate receptor 5 (mGluR5), was used as a positive control since previous studies have reported that the compound is active in BTBR mice where similar behavioral outcomes were measured (Silverman et al., 2012). GRN-529 was prepared as described in the literature from commercially available methyl 4-hydroxy-3-iodobenzoate (Aldrich; St. Louis, MO) and 6,7dihydro-5H-pyrrolo[3,4-b]pyridine dihydrochloride (Combi-Blocks) (Li et al., 2010; Sperry et al., 2012). The enaminone amides (2-261, 2-301, 2-313 and 4-327) were prepared as described in the literature (Hogenkamp et al., 2007). AVL-3288 (also known as compound 6, XY-4083) was prepared as described in US patent 7,820,663. Gabazine and MLA were purchased from Sigma-Aldrich (St. Louis, MO).
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2.2. Oocyte electrophysiology
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cDNA clones of human receptor subunits were synthesized by GENEWIZ (South Plainfield, NJ) and the subunit mRNA was prepared by TriLink Biotechnologies (San Diego, CA). 2-electrode voltage clamp oocyte electrophysiology was performed as previously described (Ng et al., 2007). Briefly, all compounds were tested with a 30 second pretreatment prior to co-application with EC10 agonist (concentration of agonist that evokes 10% of the maximum response). Recorded currents in the presence of test compound were calculated as % modulation relative to the control currents ((Imodulated / Icontrol * 100%) – 100%). Concentration-response curves were fit to non-linear regression analysis on Prism 4.0 (GraphPad, San Diego, CA) for % maximal stimulation, EC50 values and their 95% confidence limits.
2.3. Animals
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Adult, male BTBR (Center for Autism Research and Translation[CART], UCI, Irvine, CA) and C57BL/6J or DBA/2J mice (The Jackson Laboratory, Bar Harbor, ME) weighing 20-30 g were group housed and allowed access to food and water ad libitum. Animals were bred at their originating facilities (CART for BTBR and The Jackson Laboratory for C57BL/6J and DBA/2J). Animals were tested in the behavioral experiments between 10 and 16 weeks of age. Some animals were reused once in both a single social approach and a single self-grooming experiment. Animals that were reused were allowed a two week washout period. All animals in the locomotor activity experiment were naïve. Animals were group housed under a 12-hour light:dark cycle and tested according to U.C. Irvine Institutional Animal Care and Use Committee approved protocols.
2.4. Social approach task Testing used a Plexiglas box split into three equal chambers with removable doors to allow passage between each chamber. The chamber floor was dimly lit at ~8 lux. The subject mice (DBA/2J) were placed inside a cup
ACCEPTED MANUSCRIPT
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and habituated to the cup and chamber in two 15 minute sessions prior to testing. A different subject mouse was used for each mouse tested. Prior to testing, each BTBR mouse was habituated to an empty apparatus in the center chamber only for 10 minutes, and then the doors were opened and the mouse was habituated to the entire empty apparatus for another 10 minutes. Following habituation, the test mouse was removed and two identical cups were placed in the side chambers, one empty and the other containing the subject mouse. The test mouse was then placed in the center chamber and allowed to explore the entire apparatus for 10 minutes while under video surveillance. The amount of time spent interacting with the novel object or subject/stranger mouse, the amount of time spent in each chamber and the number of passages between chambers was recorded by a blinded observer. The chamber was cleaned with 70% ethanol between subjects.
2.5. Self-grooming (repetitive behavior)
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Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol and 80-90% saline), the positive control (3 mg/kg GRN-529) and 0.1 - 10 mg/kg of test compounds. All test compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. In the antagonist study, 3 mg/kg gabazine or 3 mg/kg MLA were dissolved in saline and administered as an intraperitoneal injection 15 minutes prior to test compounds. Drug administration was ordered with a counter-balanced design. Statistics were determined by unpaired t-tests.
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Self-grooming was monitored in an empty home cage using video recording under normal lighting conditions. The BTBR mouse was placed in the empty home cage (without bedding) and allowed to habituate for 10 minutes. Following habituation, the mouse was then recorded for a 10 minute test session. The videos were scored for the amount of time the animals spent grooming by a blinded observer. The cages were cleaned with 70% ethanol between subjects.
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Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol and 80-90% saline), the positive control (3 mg/kg GRN-529) and 0.1 - 10 mg/kg of test compounds. All test compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. Drug administration was ordered with a counter-balanced design. Statistics were determined by oneway ANOVA with post hoc analyses using Dunnett’s multiple comparison test, with treatment dose as the independent variable and self-grooming time as the dependent variable.
2.6. Open field locomotor activity Locomotor activity was assessed in a 26 cm x 26 cm open field chamber (Coulbourn Instruments; Holliston, MA) under normal lighting conditions (~350 lux). The BTBR mouse was placed in the center of the open field at the start of the 30 minute test session. Total distance, number of movements and time spent moving were recorded by infrared tracking beams using TruScan software (Coulbourn Instruments). The chamber was cleaned with 70% ethanol between subjects.
ACCEPTED MANUSCRIPT
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Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol, 80-90% saline), the positive control (3 mg/kg GRN-529) and 3 mg/kg 2-261 or 3 mg/kg AVL-3288. All compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. Drug administration was done with a counter-balanced design. Statistics were determined by two-way ANOVA with post hoc analyses using Bonferroni’s post test, with treatment and time as the independent variables and distance traveled as the dependent variable.
ACCEPTED MANUSCRIPT
3. Results 3.1. In vitro activity of AVL-3288, 2-261 and various test compounds at human α7 nAChR and GABAAR subtypes
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PAMs from our compound library were chosen based on their activity at human α7 nAChR and β2-containing GABAAR subtypes. The structures of these compounds and their activity on specific human α7 nAChR, β1- or β2containing GABAAR and α4β3δ GABAAR subtypes expressed in Xenopus oocytes are summarized in Table 1. AVL3288 has activity at α7, but has limited activity at the GABAAR subtypes. 2-261 has activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2 as previously reported (Gee et al., 2010), but inactive at α7 nAChRs. 2-301 has limited activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2, but inactive at α7 nAChRs. 2-313 has activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2, but inactive at α7 nAChRs. 4-327 has activity at α7, has selectivity for α1β2γ2 over α1β1γ2, but inactive at α4β3δ.
3.2. Effect of treatments on social approach behavior
In the phenotype control experiment (Supplementary Figure S1), BTBR mice (t=2.345, df=42, P
S0028-3908(17)30396-9
DOI:
10.1016/j.neuropharm.2017.08.029
Reference:
NP 6834
To appear in:
Neuropharmacology
Received Date: 31 May 2017 Revised Date:
18 August 2017
Accepted Date: 21 August 2017
Please cite this article as: Yoshimura, R.F., Tran, M.B., Hogenkamp, D.J., Ayala, N.L., Johnstone, T., Dunnigan, A.J., Gee, T.K., Gee, K.W., Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2017.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title page Title: Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model
Department of Pharmacology, School of Medicine, University of California Irvine, Irvine, CA, 92697-4625
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Corresponding author: Kelvin W Gee 110C Medical Surge I Department of Pharmacology University of California, Irvine ` Irvine, CA 92697-4625 Phone 949-824-8009 Fax 949-824-4855 Email [email protected]
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Authors: Ryan F Yoshimuraa; Minhtam B Trana; Derk J Hogenkampa; Narielle L Ayalaa; Timothy Johnstonea; Andrew J Dunnigana; Timothy K Geea; Kelvin W Geea
ACCEPTED MANUSCRIPT
Abstract
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Autism spectrum disorder (ASD) is associated with two core symptoms (social communication deficits and stereotyped repetitive behaviors) in addition to a number of comorbidities. There are no FDA-approved drugs for the core symptoms and the changes that underlie these behaviors are not fully understood. One hypothesis is an imbalance of the excitation (E)/inhibition (I) ratio with excessive E and diminished I occurring in specific neuronal circuits. Data suggests that both gamma-aminobutyric acidA (GABAA) and α7 nicotinic acetylcholine receptors (nAChRs) significantly impact E/I. BTBR T+ tf/J (BTBR) mice are a model that display an autism-like phenotype with impaired social interaction and stereotyped behavior. A β2/3-subunit containing GABAA receptor (GABAAR) subtype selective positive allosteric modulator (PAM), 2-261, and an α7 nAChR subtype selective PAM, AVL-3288, were tested in social approach and repetitive self-grooming paradigms. 2-261 was active in the social approach but not the self-grooming paradigm, whereas AVL-3288 was active in both. Neither compound impaired locomotor activity. Modulating α7 nAChRs alone may be sufficient to correct these behavioral and cognitive deficits. GABAergic and nicotinic compounds are already in various stages of clinical testing for treatment of the core symptoms and comorbidities associated with ASD. Our findings and those of others suggest that compounds that have selective activities at GABAAR subtypes and the α7 nAChR may address not only the core symptoms, but many of the associated comorbidities as well and warrant further investigation in other models of ASD.
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Keywords: autism; BTBR; GABA; nicotinic; allosteric modulator
ACCEPTED MANUSCRIPT
1. Introduction The Diagnostic Statistical Manual V defines the two core clinical symptoms of ASD as social communication deficits and repetitive patterns of behavior (American Psychiatric Association, 2013). Individuals with ASD also have an increased risk for comorbidities including anxiety disorders, seizures, obsessive
RI PT
compulsive disorder, attention deficit hyperactivity disorder, sleep disorders and cognitive impairments (Leyfer et al., 2006; Kotagal and Broomall, 2012; Srivastava and Schwartz, 2014; Frey et al., 2016). Although there are treatments available for some of these comorbidities, no drugs are approved for the treatment of the core
SC
symptoms.
One hypothesis for ASD is that the clinical symptoms arise from an imbalance of E/I in neural systems (Rubenstein and Merzenich, 2003). Most cases of ASD are idiopathic, supporting a potential interaction of
M AN U
multiple genes with the environment. Evidence from preclinical and human postmortem studies showing deficits in GABAAR function and GABA-mediated inhibition support a role for the GABAergic system. ASD patients demonstrate a selective downregulation of GABAARs which may be associated with abnormalities in chromosomal loci 15q11-q13 which contain genes encoding the α5, β3 and γ3 GABAAR subunits (Shao et al., 2003; Fatemi et al., 2009). Changes in expression of some of these subunits have been observed in brains of ASD patients and autism related genes have been found to be mainly expressed in GABAergic interneurons (Xu
TE D
et al., 2014). Selectively enhancing GABA-mediated inhibition via modulation of GABAAR subtypes may be therapeutically beneficial in correcting the E/I imbalance. The 15q13 chromosome also encompasses the CHRNA7 gene that encodes the α7 nAChR which has a diversity of functions that are highly relevant to ASD (Albuquerque et al., 2009). There are changes in nAChR
EP
binding in the cerebral cortex and cerebellum (Martin-Ruiz et al., 2004) along with a reduction in neuronal α7 nAChR immunoreactivity in the thalamus of ASD patients (Ray et al., 2005). Microdeletions in chromosome
AC C
15q13.3 and reduced expression of CHRNA7 associated with ASD along with the autism-related phenotypes associated with the 15q13.3 homozygous knockout mouse provide strong support for selectively targeting α7 nAChRs (Lowther et al., 2015; Forsingdal et al., 2016). The BTBR T+ tf/J (BTBR) mouse is a strain that has been characterized with ASD-related behaviors. Similar to the core symptoms of ASD, BTBR mice demonstrate low sociability and increased repetitive behaviors. Enhancing GABA mediated inhibition with the GABAAR-active neurosteroid ganaxolone improved social approach behavior, but not repetitive self-grooming behavior in BTBR mice (Kazdoba et al., 2016). Clonazepam, the benzodiazepine (BZ) GABAAR PAM, also improved social approach behavior in BTBR mice (Han et al., 2014).
ACCEPTED MANUSCRIPT
BTBR mice also have lower basal levels of acetylcholine in the medial prefrontal cortex and treatment with an acetylcholinesterase inhibitor reduced social deficiency (Karvat and Kimchi, 2014). Nicotinic receptors have been implicated in the expression of both social interaction and repetitive behaviors in BTBR mice (Wang et al., 2015). Thus the BTBR model may respond to the modulation of GABAergic and/or nicotinic acetylcholinergic
RI PT
systems, providing clues to a potential therapeutic approach for treatment of the core symptoms of ASD. We have previously characterized two PAMs, AVL-3288 and 2-261, that are selective for the α7 nAChR and β2/3-subunit-containing GABAARs (β2/3 GABAAR, 2-261 is equally active at either receptor subtype) respectively (Ng et al., 2007; Gee et al., 2010). These targets were chosen because both receptor subtypes show
SC
potential loss of function in ASD (Lowther et al., 2015; Forsingdal et al., 2016; Shao et al., 2003; Fatemi et al., 2009). PAMs are important tools since unlike direct receptor agonists, they potentially preserve the spatiotemporal integrity of neurotransmission and reduce the influence of various factors associated with direct
M AN U
agonism as confounds in the interpretation of the pharmacological effects (Uteshev, 2014). Additionally, AVL3288 is a type I PAM that is in clinical trials for the treatment of cognitive impairment associated with schizophrenia (CIAS; Gee et al., 2017). Unlike the clinically used BZs, 2-261 is a GABAAR PAM that has reduced side effects and abuse liability in animal models which are important considerations for an ASD drug (Gee et al., 2010; Yoshimura et al., 2014). Using these and other PAMs from our compound library as tools, we examine the effect of selectively modulating the GABAAR subtypes that contain β2/β3 subunits and the α7 nAChR on
AC C
EP
TE D
sociability and stereotypic behavior in the BTBR mouse model.
ACCEPTED MANUSCRIPT
2. Materials and Methods 2.1. Chemicals
SC
RI PT
GRN-529, a negative allosteric modulator of the metabotropic glutamate receptor 5 (mGluR5), was used as a positive control since previous studies have reported that the compound is active in BTBR mice where similar behavioral outcomes were measured (Silverman et al., 2012). GRN-529 was prepared as described in the literature from commercially available methyl 4-hydroxy-3-iodobenzoate (Aldrich; St. Louis, MO) and 6,7dihydro-5H-pyrrolo[3,4-b]pyridine dihydrochloride (Combi-Blocks) (Li et al., 2010; Sperry et al., 2012). The enaminone amides (2-261, 2-301, 2-313 and 4-327) were prepared as described in the literature (Hogenkamp et al., 2007). AVL-3288 (also known as compound 6, XY-4083) was prepared as described in US patent 7,820,663. Gabazine and MLA were purchased from Sigma-Aldrich (St. Louis, MO).
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2.2. Oocyte electrophysiology
TE D
cDNA clones of human receptor subunits were synthesized by GENEWIZ (South Plainfield, NJ) and the subunit mRNA was prepared by TriLink Biotechnologies (San Diego, CA). 2-electrode voltage clamp oocyte electrophysiology was performed as previously described (Ng et al., 2007). Briefly, all compounds were tested with a 30 second pretreatment prior to co-application with EC10 agonist (concentration of agonist that evokes 10% of the maximum response). Recorded currents in the presence of test compound were calculated as % modulation relative to the control currents ((Imodulated / Icontrol * 100%) – 100%). Concentration-response curves were fit to non-linear regression analysis on Prism 4.0 (GraphPad, San Diego, CA) for % maximal stimulation, EC50 values and their 95% confidence limits.
2.3. Animals
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Adult, male BTBR (Center for Autism Research and Translation[CART], UCI, Irvine, CA) and C57BL/6J or DBA/2J mice (The Jackson Laboratory, Bar Harbor, ME) weighing 20-30 g were group housed and allowed access to food and water ad libitum. Animals were bred at their originating facilities (CART for BTBR and The Jackson Laboratory for C57BL/6J and DBA/2J). Animals were tested in the behavioral experiments between 10 and 16 weeks of age. Some animals were reused once in both a single social approach and a single self-grooming experiment. Animals that were reused were allowed a two week washout period. All animals in the locomotor activity experiment were naïve. Animals were group housed under a 12-hour light:dark cycle and tested according to U.C. Irvine Institutional Animal Care and Use Committee approved protocols.
2.4. Social approach task Testing used a Plexiglas box split into three equal chambers with removable doors to allow passage between each chamber. The chamber floor was dimly lit at ~8 lux. The subject mice (DBA/2J) were placed inside a cup
ACCEPTED MANUSCRIPT
RI PT
and habituated to the cup and chamber in two 15 minute sessions prior to testing. A different subject mouse was used for each mouse tested. Prior to testing, each BTBR mouse was habituated to an empty apparatus in the center chamber only for 10 minutes, and then the doors were opened and the mouse was habituated to the entire empty apparatus for another 10 minutes. Following habituation, the test mouse was removed and two identical cups were placed in the side chambers, one empty and the other containing the subject mouse. The test mouse was then placed in the center chamber and allowed to explore the entire apparatus for 10 minutes while under video surveillance. The amount of time spent interacting with the novel object or subject/stranger mouse, the amount of time spent in each chamber and the number of passages between chambers was recorded by a blinded observer. The chamber was cleaned with 70% ethanol between subjects.
2.5. Self-grooming (repetitive behavior)
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Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol and 80-90% saline), the positive control (3 mg/kg GRN-529) and 0.1 - 10 mg/kg of test compounds. All test compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. In the antagonist study, 3 mg/kg gabazine or 3 mg/kg MLA were dissolved in saline and administered as an intraperitoneal injection 15 minutes prior to test compounds. Drug administration was ordered with a counter-balanced design. Statistics were determined by unpaired t-tests.
TE D
Self-grooming was monitored in an empty home cage using video recording under normal lighting conditions. The BTBR mouse was placed in the empty home cage (without bedding) and allowed to habituate for 10 minutes. Following habituation, the mouse was then recorded for a 10 minute test session. The videos were scored for the amount of time the animals spent grooming by a blinded observer. The cages were cleaned with 70% ethanol between subjects.
AC C
EP
Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol and 80-90% saline), the positive control (3 mg/kg GRN-529) and 0.1 - 10 mg/kg of test compounds. All test compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. Drug administration was ordered with a counter-balanced design. Statistics were determined by oneway ANOVA with post hoc analyses using Dunnett’s multiple comparison test, with treatment dose as the independent variable and self-grooming time as the dependent variable.
2.6. Open field locomotor activity Locomotor activity was assessed in a 26 cm x 26 cm open field chamber (Coulbourn Instruments; Holliston, MA) under normal lighting conditions (~350 lux). The BTBR mouse was placed in the center of the open field at the start of the 30 minute test session. Total distance, number of movements and time spent moving were recorded by infrared tracking beams using TruScan software (Coulbourn Instruments). The chamber was cleaned with 70% ethanol between subjects.
ACCEPTED MANUSCRIPT
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SC
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Naïve mice were randomly divided into groups. The treatment groups were the vehicle (5-10% DMSO, 5-10% solutol, 80-90% saline), the positive control (3 mg/kg GRN-529) and 3 mg/kg 2-261 or 3 mg/kg AVL-3288. All compounds were administered as intraperitoneal injections 30 minutes prior to testing at a volume of 1 mL/kg. Drug administration was done with a counter-balanced design. Statistics were determined by two-way ANOVA with post hoc analyses using Bonferroni’s post test, with treatment and time as the independent variables and distance traveled as the dependent variable.
ACCEPTED MANUSCRIPT
3. Results 3.1. In vitro activity of AVL-3288, 2-261 and various test compounds at human α7 nAChR and GABAAR subtypes
M AN U
SC
RI PT
PAMs from our compound library were chosen based on their activity at human α7 nAChR and β2-containing GABAAR subtypes. The structures of these compounds and their activity on specific human α7 nAChR, β1- or β2containing GABAAR and α4β3δ GABAAR subtypes expressed in Xenopus oocytes are summarized in Table 1. AVL3288 has activity at α7, but has limited activity at the GABAAR subtypes. 2-261 has activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2 as previously reported (Gee et al., 2010), but inactive at α7 nAChRs. 2-301 has limited activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2, but inactive at α7 nAChRs. 2-313 has activity at α4β3δ and has selectivity for α1β2γ2 over α1β1γ2, but inactive at α7 nAChRs. 4-327 has activity at α7, has selectivity for α1β2γ2 over α1β1γ2, but inactive at α4β3δ.
3.2. Effect of treatments on social approach behavior
In the phenotype control experiment (Supplementary Figure S1), BTBR mice (t=2.345, df=42, P
