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GABAB Receptor Agonist R-Baclofen Reverses Social Deficits and ReducesRepetitive Behavior in Two Mouse Models of Autism

J L Silverman1,*,M C Pride1,J E Hayes1,K R Puhger1,H M Butler-Struben1,S Baker1,J N Crawley1
1MIND Institute, Department of Psychiatryand Behavioral Sciences, University of California Davis School of Medicine,Sacramento, CA, USA
*

MIND Institute, Department of Psychiatry and BehavioralSciences, University of California Davis School of Medicine,Sacramento, CA95817, USA, Tel: +1 916 734 8531, Fax: +1 916 7345089, E-mail:jill.silverman@ucdmc.ucdavis.edu

Received 2014 Jul 31; Revised 2015 Feb 28; Accepted 2015 Mar 3; Prepublished 2015 Mar 10; Issue date 2015 Aug.

Copyright © 2015 American College of Neuropsychopharmacology
PMCID: PMC4613612  PMID:25754761

Abstract

Autism spectrum disorder (ASD) is diagnosed by two core behavioral criteria, unusualreciprocal social interactions and communication, and stereotyped, repetitivebehaviors with restricted interests. Excitatory/inhibitory imbalance is aprominent hypothesis for the etiology of autism. The selective GABABreceptor agonist R-baclofen previously reversed social deficits and reducedrepetitive behaviors in a mouse model of Fragile X syndrome, and Arbaclofen improvedsome clinical symptoms in some Fragile X and ASD patients. To evaluate R-baclofen ina broader range of mouse models of ASD, we tested both the R-baclofen enantiomer andthe less potent S-baclofen enantiomer in two inbred strains of mice that display lowsociability and/or high repetitive or stereotyped behaviors. R-baclofen treatmentreversed social approach deficits in BTBR T+ Itpr3tf/J (BTBR), reducedrepetitive self-grooming and high marble burying scores in BTBR, and reducedstereotyped jumping in C58/J (C58), at nonsedating doses. S-baclofen producedminimal effects at the same doses. These findings encourage investigations ofR-baclofen in other preclinical model systems. Additional clinical studies may bewarranted to further evaluate the hypothesis that the GABAB receptorrepresents a promising pharmacological target for treating appropriately stratifiedsubsets of individuals with ASD.

INTRODUCTION

Autism is a neurodevelopmental disorder diagnosed by deficits in two core behavioraldomains: (1) unusual reciprocal social interactions and impaired socialcommunication, and (2) stereotyped, repetitive behaviors with restricted interests.One prominent hypothesis, consistent with comorbid seizures and anxiety in autismspectrum disorder (ASD), is an imbalance of excitatory/inhibitoryneurotransmission (Bourgeron, 2009;Geschwind and Levitt, 2007;Gogollaet al, 2009;LeBlanc and Fagiolini,2011;Rubenstein and Merzenich, 2003).Reduced GABAergic neurotransmission and fewer GABAergic interneurons in mouse modelswith targeted mutations in risk genes for ASD,in vivo spectroscopy, andelectrophysiological biomarkers of lower GABA activity in affected individualssupport the hypothesis that elevating GABAergic activity may offer a therapeutictarget for treating some components of ASD (Blatt and Fatemi,2011;Eaglesonet al, 2010;Gaetzet al, 2014;Hanet al, 2012,2014;Haradaet al, 2011;Moriet al, 2012;Oberman, 2012;Sgadoet al, 2013). One therapeuticstrategy targeting the GABAB receptor subtype evaluated STX209(Arbaclofen), the selective GABAB enantiomer, in clinical trials forFragile X syndrome, Fragile X with an autism diagnosis, and ASD. A phase 2 clinicaltrial detected improvements on Aberrant Behavior Checklist (ABC)-Social Avoidancescores (Berry-Kraviset al, 2012) in Fragile Xpatients. An open-label trial of STX209 in patients with ASD not associated withFragile X showed beneficial effects on ABC-irritability social withdrawal scale, andon the social responsiveness scale (Ericksonet al,2014).

Baclofen (β-p-chlorophenyl-GABA) is a GABA analog that acts at theGABAB receptor and reduces glutamate release (Bowery, 1993;Boweryet al,1983;Hendersonet al, 2012;Kanget al, 2012). STX209 and racemicbaclofen administered toFmr1 knockout mice restored protein synthesis,corrected their increased dendritic spine density, and reduced audiogenic seizures(Hendersonet al, 2012).

BTBR T+ Itpr3tf/J (BTBR) is an inbred strain of mice that exhibits robust,well-replicated impairments in social interactions, minimal vocalizations in socialsettings, high levels of repetitive self-grooming and digging, and cognitive deficits(Amodeoet al, 2012;Bolivaret al, 2007;Chadman,2011;Gouldet al, 2011;McFarlaneet al, 2008;McTigheet al, 2013;Pearsonet al, 2011;Pobbeet al,2010;Scattoniet al, 2008;Silvermanet al, 2013a,2013b;Yanget al,2007b). Reduced spontaneous GABAergic neurotransmission in BTBR wasrecently reported (Gogollaet al, 2014;Hanet al, 2014). C58/J (C58) is anindependent inbred strain of mice that displays robust, well-replicated stereotypedvertical jumping and responds to mGluR5 antagonist treatment (Silvermanet al, 2012), although endogenous GABA systems havenot yet been explored in this strain.

We employed BTBR and C58 to test the hypothesis that R-baclofen could ameliorateautism-relevant behaviors in mouse models of autism. Rescue of social approach inBTBR, and reductions in repetitive behaviors in both BTBR and C58, were detectedafter acute, systemic R-baclofen treatment. In contrast, the less active enantiomerS-baclofen was less potent or inactive in both strains, using dose comparisonsconsistent with the literature (Boweryet al,1983;Drewet al, 1984;Paredes and Agmo, 1989). These preclinical results lendsupport to further investigations of GABAB agonists as a pharmacologicaltarget for treating core diagnostic symptoms of ASD.

MATERIALS AND METHODS

Mice

Breeding pairs of C57BL/6J (B6), BTBR T+ Itpr3tf/J (BTBR), C58/J(C58), and 129/SvImJ mice were purchased from The Jackson Laboratory (BarHarbor, ME) and bred as harem trios in a conventional mouse vivarium at theUniversity of California Davis School of Medicine in Sacramento. After weaning,juveniles were housed by sex and strain in Tecniplast cages in groups of two tofour per cage. Cages were housed in ventilated racks in a temperature-controlled(68–72 °F) and humidity-controlled (~25%) colony room, ona 12-h circadian cycle, lights on from 0700 to 1900 h. Standard rodent chowand tap water were availablead libitum. In addition to standard bedding,a Nestlet square, shredded brown paper, and a cardboard tube (JonesvilleCorporation, Jonesville, MI) were provided in each cage. Previous studies in ourlaboratory documented no sex differences in either BTBR or B6 on sociability orself-grooming assays (McFarlaneet al,2008;Silvermanet al, 2010a;Yanget al, 2007a,2007b). Therefore, male and female mice were used in all studies inapproximately equal proportions. Behavioral testing arenas were cleaned with70% ethanol between test subjects. At least 5 min between cleaningand the start of the next session was allowed for ethanol evaporation and odordissipation. All procedures were conducted in compliance with the NIH Guidelinesfor the Care and Use of Laboratory Animals and approved by UC Davis InstituteAnimal Care and Use Committee (Protocol no. 16839).

Drug Administration

R- and S-baclofen hydrochloride (Sigma Aldrich, St Louis, MO) were acutelyadministered intraperitoneally in a 10 ml/kg injection volume of0.9% physiological saline vehicle in the first cohort of B6 and BTBR mice.In accordance with the literature on the relativein vivo potencies ofthe two enantiomers (Hendersonet al,2012;Paredes and Agmo, 1989), R-baclofenwas tested at doses of 1.0, 3.0, and 5.0 mg/kg, whereas S-baclofen wastested at doses of 0.1, 1.0, 3.0, and 10.0 mg/kg. In accordance withtmax values and preliminary findings in open fieldlocomotion (Supplementary Figure S6), compoundswere administered 60 min before the start of each behavioral assay(Hendersonet al, 2012). Experimentaldesign was between-subjects, with a 1-week washout period. Each cohort 1 mousereceived a randomized single acute dose of R-baclofen hydrochloride (1.0, 3.0, and5.0 mg/kg) or vehicle and was tested on one task per week. An identicaldesign was employed for the R-baclofen replication cohort 2. A third cohort of B6and BTBR received a single acute dose of S-baclofen hydrochloride (0.1, 1.0, 3.0,and 10.0 mg/kg) or vehicle before testing on social approach. Cohort 3was subsequently treated with only the two highest doses of S-baclofen or vehiclebefore testing on self-grooming and marble burying.

Behavioral Testing

Behavioral assessment of BTBR and of the control strain B6 that displays normalsociability and low repetitive behaviors was conducted at ages 8–12 weeks,body weights 25–35 g, during the light phase of the circadian cycle.Order of testing was open field locomotion (week 1), social approach (week 2),self-groom (week 3), and marble burying (week 4). A second cohort was used toconduct replication. A third cohort of BTBR and B6 mice was used for behavioraltesting following low dose S-baclofen hydrochloride or saline. C58 mice weresimilarly tested for responses to both R- and S-baclofen. The behavioral taskorder for C58 was open field locomotion (week 1), observations of spontaneousvertical jumping and self-grooming (week 2), and social approach (week 3). Thisorder of testing was designed to focus on the main phenotype of C58, highstereotyped jumping, based on our previous unpublished findings of normalsociability in C58. A second cohort of C58 mice was used to conduct a replicationof repetitive jumping after R-baclofen treatment. A third cohort of C58 mice wasused for evaluating effects of the two highest doses of S-baclofen. Drug doses anddigital videotapes of the behavioral sessions were coded by an independentinvestigator to ensure that the raters were blind to the treatment condition.

Behavioral Scoring

Self-grooming in BTBR and stereotyped jumping in C58

Mice were scored for spontaneous self-grooming and jumping as previouslydescribed (Silvermanet al, 2010a,2012). Each mouse was individually placedinto a standard mouse cage, illuminated at ~40 lux. Cages were empty toeliminate digging in the bedding, a potentially competing behavior. After a10-min habituation period in the test cage, each mouse was scored by a trainedobserver uninformed of the drug treatment using Noldus Observer event recording(Noldus Observer 8.0XT, Leesburg, VA), using parameters of cumulative timespent grooming all body regions, or cumulative bouts of jumping, during a10-min session.

Marble burying assay

Repetitive marble burying was measured as previously described (Hendersonet al, 2012;Thomaset al, 2009,2012). Twenty black glass marbles (15 mm in diameter) werearranged in a symmetrical 4 × 5-cm grid on top of 2–3 cmdeep bedding in clean, standard mouse cages (27 × 16.5 ×12.5 cm) with a filter top lid. Each mouse was placed in the center ofthe cage for a 30-min exploration period, after which the number of marblesburied was tallied by the investigator. ‘Buried’ was defined as>50% covered by bedding (Thomaset al,2009). Testing was performed under dim light (~15 lux).

Open field locomotion

General exploratory locomotion in a novel open field environment was assayed aspreviously described (Silvermanet al,2010b;Flanneryet al,2014). Open field activity was considered an essential control fordirect drug effects on physical activity, for example, sedation orhyperactivity (Silvermanet al, 2010a,2012,2013a),that could confound the interpretation of results from the self-grooming,marble burying, and social approach tasks. The testing room was illuminated at~40 lux.

Sociability

Social approach was tested in an automated three-chambered apparatus usingmethods similar to those previously described (McFarlaneet al, 2008;Silvermanetal, 2010b,2011,2012,2013a;Yanget al, 2011). Newly automated EthovisionXT videotracking software (Version 9.0, Noldus Information Technologies,Leesburg, VA) and modified nonreflective materials for the chambers wereemployed to maximize throughput. The updated apparatus (40 cm ×60 cm × 23 cm) was a rectangular, three-chambered box madefrom matte white finished acrylic (P95 White, Tap Plastics, Sacramento, CA).Opaque retractable doors (12 cm × 33 cm) were designed tocreate optimal entryways between chambers (5 cm × 10 cm)while providing maximal manual division of compartments. Three zones, definedusing the EthoVision XT software, detected time in each chamber for each phaseof the assay. Zones were defined as the annulus extending 2 cm from eachnovel object or novel mouse enclosure (inverted wire cup, Galaxy Cup, KitchenPlus,http://www.kitchenplus.com). Direction of the head, facing towardthe cup enclosure, defined sniff time. A top-mounted infrared sensitive camera(Ikegami ICD-49, B&H Photo, New York, NY) was positioned directly aboveevery two 3-chambered units. Infrared lighting (Nightvisionexperts.com) provideduniform, low-level illumination. The subject mouse was first contained in thecenter chamber for 10 min, then explored all three empty chambers duringa 10 min habituation session, and then explored the three chamberscontaining a novel object in one side chamber and a novel mouse in the otherside chamber. Lack of innate side preference was confirmed during the initial10 min of habituation to the entire arena. Novel stimulus mice were129Sv/ImJ, a relatively inactive strain, aged 10–14 weeks, andmatched to the subject mice by sex. Number of entries into the side chambersserved as a within-task control for levels of general exploratory locomotion.In addition to the automated Ethovision videotracking scoring method, a trainedrater scored the same videos using Noldus Observer XT event coding software(Noldus Information Technologies). Direct comparison of scores obtained withautomated videotrackingversus human observation of videos ispresented inSupplementary Figure S7.

Statistical Analysis

Repeated Measures ANOVA (~ pairedt-test) was used to analyzethree-chambered social approach data. Comparisons of time spent in the chamberwith the novel mouseversus time spent in the chamber with the novelobject were conducted within each drug treatment group and within each strain.Similarly, time sniffing the novel mouseversus time sniffing the novelobject were compared within each drug treatment group and within each strain. Thisstatistical approach is consistent with our original development and validation ofthe three-chambered social approach task over 10 years ago (Nadleret al, 2004) and with data obtained in testinghundreds of cohorts of mice using this assay (Brielmaieret al, 2014;Chadmanet al,2008;Crawleyet al, 2007;Eyet al, 2012;Moyet al, 2007;Silvermanetal, 2012;Wohret al,2013;Yanget al, 2012). Theabsolute number of seconds spent with the novel mouse is highly variable acrosscohorts of the same genotype or strain, and is therefore not a sufficiently stableparameter for biologically meaningful comparisons across genotypes or acrosstreatment groups. This assay primarily provides a yes-or-no comparison of meangroup time with the novel mouseversus mean group time with the novelobject. If this comparison is significant, then the group displays sociability. Ifit is not, the group does not display sociability. As described in our previouspublications (Brielmaieret al, 2014;Chadmanet al, 2008;Eyet al, 2012;Silvermanet al, 2010b,2012;Wohret al, 2013;Yanget al, 2011,2012) andothers (Clipperton-Allen and Page, 2014;Kerret al, 2013;Tsaiet al, 2012), this within-group comparison is validwithin a genotype, or within a drug treatment dose, but not across genotypes noracross drug treatment groups. Center chamber times are shown in the graphs only tovisually display the absence of treatment effects on time spent in the centralstarting area that could indicate sedation or hyperactivity. For number of chamberentries during social approach, drug effects were compared within each strain by aseparate between-groups drug × entries ANOVA. In cases where the overallANOVA for drug was significant on number of entries, the treatment factor for eachstrain was further analyzed with Dunnett’spost hoc test to compareeach drug dose group with its vehicle control group.

Self-grooming and marble burying were analyzed using one-way ANOVA within strainfor drug dose, using Statistica 10.0 software (Statsoft,www.statsoft.com).Repetitive vertical jumping was analyzed using a one-way ANOVA for drug dose. Incases where the overall ANOVA for the drug was significant,post hocanalysis was performed with Dunnett’spost hoc test to compare eachdrug dose with its vehicle control.

Open field data were analyzed with a repeated measures ANOVA using abetween-groups factor of drug within strain, and a within-group factor of timecourse, for the parameters of total distance, horizontal activity, or center time.Comparisons with a significant ANOVA were followed by Dunnett’sposthoc analysis, using SigmaPlot version 12.0 (Systat, San Jose, CA) toidentify treatment group differences.

RESULTS

R-Baclofen Increased Sociability in BTBR in the Three-Chambered SocialApproach Task

Figure 1 illustrates the sociability scores from theautomated three-chambered social approach task following a single dose ofR-baclofen or saline vehicle (i.p.) in B6 and BTBR mice. Significant sociabilitywas detected in B6, but not BTBR, in the saline vehicle groups, consistent withprevious reports (Figure 1a, B6 saline: F(1,11) =11.11,p<0.05;Figure1b, BTBR saline: F(1, 11) =1.44, NS). B6 continuedto exhibit significantly more time in the chamber with the novel mouse than timein the chamber with the novel object for all doses of R-baclofen (Figure 1a, B6 1 mg/kg: F(1, 11)=31.17,p<0.001; B6 3 mg/kg: F(1, 11)=16.56,p <0.01), providing evidence for no deleterious actionsof R-baclofen at these doses on normal sociability. R-baclofen, at doses of 1 and3 mg/kg, reversed the sociability deficit in BTBR on the chamber timeparameter (Figure 1b, BTBR 1 mg/kg:F(1, 11)=6.32,p<0.05; BTBR 3 mg/kg:F(1, 11) = 8.12,p<0.02).

Figure 1.

Figure 1

R-baclofen rescued social approach deficits in the BTBR mouse model of autism.R-baclofen or saline vehicle was administered acutely, 60 min before thethree-chambered social approach test session. (a) B6 mice displayed normalsociability on the chamber time parameter, spending more time in the side chamberwith the novel mouse as compared with the side chamber with the novel object,after treatment with vehicle and at each dose of R-baclofen. (b) BTBR miceexhibited characteristic lack of sociability on the chamber time parameter aftervehicle treatment, spending approximately equal time in the side chamber with thenovel mouse and the side chamber with the novel object. R-baclofen at doses of 1and 3 mg/kg reversed the sociability deficits in BTBR. (c) Automatedsniffing was measured by Noldus Ethovision 9.0XT software using settings thatincluded (1) multiple body point dynamic subtraction detection, (2) thesubject’s nose in a discrete zone that surrounded the novel mouse or object,and (3) the subject’s head oriented toward the novel mouse or object. B6mice treated with vehicle or R-baclofen exhibited characteristic sociability onthe directed sniffing parameter. (d) BTBR exhibited its characteristic lack ofsociability on the directed sniffing parameter, that is, did not spend more timesniffing the novel mouseversus the novel object, after vehicletreatment. R-baclofen at doses of 1 and 3 mg/kg reversed the socialsniffing deficits in BTBR. Number of entries into the side chambers was unaffectedby R-baclofen treatment in (e) B6 and (f) BTBR, indicating the absence ofconfounding hyper- or hypo-exploratory locomotion during the social approach task.*P<0.05, novel mouseversus novel object. SeeSupplementary Figure S1 for the replication ofR-baclofen social approach in B6 and BTBR in cohort 2.

Social sniffing was defined as time spent within 2 cm of the wire cup, withthe head facing the wire cup containing the stimulus mouse, as compared with thetime spent sniffing the novel object, using the same body point detectionsettings. B6 subject mice displayed significant sociability on social sniffing inall saline and R-baclofen groups (Figure 1c, B6saline: F(1, 11) = 40.41,p<0.001; B61 mg/kg: F(1, 11) = 7.36,p<0.05; B63 mg/kg: F(1, 11) = 10.35,p<0.01). BTBRfailed to display significant sociability on social sniffing time (Figure 1d, BTBR saline: F(1, 11) = 3.81,NS), as previously reported for BTBR at baseline and with various vehicles(Chadman, 2011;Pobbeet al, 2011;Silvermanetal, 2010a,2012,2013b). R-baclofen reversed the low social sniffing inBTBR (Figure 1d, BTBR 1 mg/kg: F(1,11) = 26.75,p<0.01; BTBR 3 mg/kg:F(1, 11) = 8.66,p<0.02). Replication with asecond independent cohort of B6 and BTBR yielded similar findings (Supplementary Figure S1).

Previous research suggested that time spent sniffing the novel mouse is a moredirect and sensitive measure of sociability than the chamber time parameter(Fairlesset al, 2011;Yanget al, 2011). To be sure that socialsniffing using the new automated scoring method was consistent with manualobserver scoring methods previously employed, we compared scores from theautomated directed sniffing software, including proximity and directionalcomponents, and manual scoring of digital videos by a trained observer uninformedof drug treatment. The same direction of drug effects, along with the expectedvariability in absolute number of seconds, were obtained for social sniffing withboth methods (Supplementary Figure S2).

Number of entries into the side chambers was not affected by R-baclofen in B6(Figure 1e, F(2, 33) = 2.57, NS)or BTBR (Figure 1f, F(2, 33) = 2.58,NS), indicating that R-baclofen administration had no effect on generalexploratory activity throughout the three-chambered apparatus during the socialapproach assay. No innate side preference was present in B6 (F(2, 33)= 0.54, NS) or BTBR (F(2, 33) = 0.36, NS) during thetask.

Normal sociability was seen in C58, consistent with our recent unpublishedfindings with other cohorts of C58, but in contrast to previous findings inanother laboratory environment (Ryanet al,2010). No deleterious effects of R-baclofen on a cohort of C58subject mice were detected in the three-chambered social approach task (Supplementary Figure S3).

Additional data related to sociability using a single dose of R-baclofen in themale–female social interaction assay that simultaneously collects maleultrasonic vocalization emissions are presented inSupplementary Figure S4.

Additional control data that revealed no alteration in olfactory abilities byR-baclofen on a cohort of B6 and BTBR subject mice are presented inSupplementary Figure S5.

R-Baclofen Reduced Repetitive Behavior in BTBR and Stereotyped Behavior inC58

Figure 2a–d illustrates self-grooming and marbleburying for B6 and BTBR. BTBR mice treated with saline displayed higherself-grooming times (F(1, 23) =7.84,p<0.02) andburied a greater number of marbles (F(1, 22) = 16.91,p<0.001) in observational assays compared with control B6 treated withsaline, consistent with earlier findings from our group and others (Silvermanet al, 2010a,2012;Amodeoet al, 2012.R-baclofen had no significant effect on self-grooming scores in B6 (Figure 2a, F(2, 33) = 1.25, NS) or onmarble burying (Figure 2c, F(2, 33)= 0.12, NS). A significant reduction on self-grooming scores in BTBRtreated with R-baclofen was detected (Figure 2b,F(2, 33) = 3.53,p<0.05, vehicleversusdrug post hoc comparisonp=0.02). R-baclofen similarly reduced thenumber of marbles buried in BTBR (Figure 2d, F(2,33) = 18.73,p<0.001, vehicleversus drugpost hoc comparisonp=0.001).

Figure 2.

Figure 2

R-baclofen reduced repetitive behaviors in BTBR and stereotyped jumping inC58/J. Cumulative time spent engaged in repetitive behaviors, includingself-grooming behavior, number of marbles buried, and number of vertical jumpsduring a session was scored by investigators blind to drug treatment. (a) B6 micedisplayed their normally low levels of self-grooming after administration ofsaline or R-baclofen. (b) High levels of repetitive self-grooming in BTBR werereduced by R-baclofen at the highest dose, 3 mg/kg.*P<0.05, by one-way ANOVA followed by Dunnett’sposthoc, R-baclofen compared with saline. (c) B6 mice displayed their normallylow levels of marble burying after treatment with saline or R-baclofen. (d) Highlevels of marble burying in BTBR mice were reduced by R-baclofen at the3 mg/kg dose. *P<0.05, by one-way ANOVA followed byDunnett’spost hoc, R-baclofen compared with saline. (e)Stereotyped vertical jumping in C58/J mice was significantly reduced byR-baclofen treatment at doses of 1 and 3 mg/kg.*P<0.05, by one-way ANOVA followed by Dunnett’sposthoc, R-baclofen compared with saline. SeeSupplementary Figure S2 for the R-baclofen repetitive behaviorassays in cohort 2 of B6, BTBR, and C58/J.

Figure 2e illustrates stereotyped jumping in C58 micetreated with saline or R-baclofen. R-baclofen significantly reduced jumping(F(3, 35) = 3.47,p<0.03) at the 1 and3 mg/kg doses, vehicleversus drug post hoc comparison withsaline: 1 mg/kgp=0.043, 3 mg/kgp=0.019).

Replication in a second cohort is shown inSupplementaryFigure S6.

R-Baclofen Did Not Produce Sedation or Hyperactivity or Anxiolytic-LikeResponses in B6 or BTBR

Figure 3a illustrates the lack of effect of R-baclofenon open field exploratory locomotion in B6 and BTBR, tested 60 min afterdrug or saline administration. In B6 and BTBR, habituation to the novelenvironment was significant for total distance traversed in the novel open field(Figure 3a, B6: F(5, 33) = 65.01,p<0.001;Figure 3b, BTBR: F(5,33) = 86.52,p<0.001). In B6 and BTBR, R-baclofen didnot significantly affect total distance traversed (Figure3a, B6: F(2, 33) =1.12, NS;Figure 3b, BTBR: F, (2, 33) = 1.27, NS) orhorizontal activity (Figure 3d, B6: F(2,33)=1.26, NS;Figure 3e, BTBR:F(2, 33)=1.77, NS). No dose × distance traveledinteractions were detected (B6: F(10, 33)=0.99, NS; BTBR:F(10, 33)=0.52, NS).

Figure 3.

Figure 3

R-baclofen did not induce hyperactivity or sedation in B6 or BTBR or an increaseof time spent in the center of the open field in B6, BTBR, or C58/J.Exploratory locomotion and time spent in the center of the arena were measured bytotal distance traversed, horizontal activity, and center time parameters across a30 min test session in an Accuscan open field in B6, BTBR, and C58/Jfollowing administration of saline vehicle or R-baclofen. Total distance wasunaffected by R-baclofen treatment in (a) B6 and (b) BTBR. Horizontal activity wasunaltered by R-baclofen treatment in (d) B6 and (e) BTBR. R-baclofen reduced timein the center of the open field arena in B6, at the 3 mg/kg dose (g)but had no effect on center time in BTBR (h). R-baclofen treatment reduced totaldistance traversed, horizontal activity, and center time in C58/J at the3 mg/kg dose (c, f, i). Data are shown in 5-min time bins.*P<0.05, drug by repeated measures ANOVA (over time), followedby Dunnett’spost hoc as compared with saline. Based on these openfield results, social and repetitive behavior tests were initiated at60 min after treatment to avoid potential sedative confounds. SeeSupplementary Figure S7 for additional post-treatmentintervals in the open field used to select the treatment regimen.

Figure 3c and f illustrates sedating effects of thehighest dose of R-baclofen on open field exploratory locomotion in C58, tested60 min after drug or saline administration. Similar to B6 and BTBR, in C58,habituation to the novel environment was significant for total distance traversedin the novel open field (Figure 3c, C58: F(5,38)=7.89,p<0.001). Moderate sedation was detected inC58, at the 3 mg/kg dose, as compared with saline (Figure 3c, C58 total distance: F(3, 38)=5.72,p<0.05;Figure 3f, C58 horizontalactivity: F(3, 38)=11.64,p<0.001).

Figure 3g–i illustrates the absence ofanxiolytic-like effects of R-baclofen using center time in an open field in B6,BTBR, and C58, tested 60 minutes after drug or saline administration. R-baclofenat 3 mg/kg reduced time in the center of the open field arena in B6(Figure 3g, B6: F(2, 33)=6.30,p<0.05, vehicleversus drugpost hoc comparisonp=0.006) and in C58/J (Figure3i, C58: F(3, 38)=7.66,p<0.001, vehicleversus drug post hoc comparisonp=0.0001). R-baclofenhad no effect on time in the center of the open field arena in BTBR (Figure 3h, BTBR: F(2, 33)=0.89, NS). Asincreases in center time would indicate anxiolytic actions, and the present datashow only decreases in center time consistent with sedation at the highest dose inB6 and in C58, these center time findings indicate that R-baclofen is notproducing anxiolytic effects, consistent with findings on two specificanxiety-related tasks shown inSupplementary Figures S8 andS9.

No anxiolytic effects of R-baclofen on a cohort of B6 and BTBR subject mice weredetected in the elevated plus-maze or the light↔dark exploration assays(Supplementary Figures S8 and S9), suggestingthat R-baclofen reduces repetitive behaviors through a non-anxiolyticmechanism.

When administered at a shorter time interval before testing, 30 min,R-baclofen produced some sedative effects during open field locomotion in B6 atthe highest dose tested, 5 mg/kg, and at both 3 and 5 mg/kgin BTBR (Supplementary Figure S7). These data incombination with publishedtmax values for Arbaclofen(Hendersonet al, 2012), determined ourchoice of the 60 min pretreatment interval for the sociability, repetitive,and stereotypy assays.

S-Baclofen Was Less Effective on Autism-Relevant Behaviors in BTBR andC58

Figure 4 illustrates the sociability scores in B6 andBTBR mice treated with the less active enantiomer S-baclofen. Sociability wassignificant in B6 but not BTBR on chamber time (Figure4a, B6 saline: F(1, 9)=36.19,p<0.001;Figure 4b, BTBR saline: F(1,11)=0.83, NS). S-baclofen had no effect on chamber time in B6(Figure 4a, B6 0.1 mg/kg: F(1,11)=6.28,p<0.05; B6 1 mg/kg: F(1,11)=6.34,p<0.05; B6 3 mg/kg: F(1,9)=12.36,p<0.01; B6 10.0 mg/kg: F(1,9)=37.2,p<0.01). S-baclofen did not reverse the lackof sociability on chamber time in BTBR (Figure 4b,BTBR 0.1 mg/kg: F(1, 11)=1.37, NS; BTBR1 mg/kg: F(1, 10)=1.86, NS; BTBR 3 mg/kg:F(1, 11)=2.23, NS; BTBR 10.0 mg/kg: F(1,13)=0.92, NS). Social sniffing was unaffected by S-baclofen in B6(Figure 4c, B6 saline: F(1,9)=244.59,p<0.001; B6 0.1 mg/kg: F(1,11)=6.63,p<0.05; B6 1 mg/kg: F(1,11)=19.16,p<0.005; B6 3 mg/kg: F(1,9)=32.75,p<0.001; B6 10.0 mg/kg: F(1,9)=25.39,p<0.001). S-baclofen increased sniffing timein BTBR at doses of 3 and 10 mg/kg (Figure4d, BTBR 3 mg/kg: F(1, 11)=11.76,p<0.006; BTBR 10.0 mg/kg: F(1,13)=12.73,p<0.005), but not at 0.1 and1 mg/kg or vehicle (Figure 4d, BTBR saline:F(1, 11)=3.50, NS; BTBR 0.1 mg/kg: F(1,11)=2.70, NS; BTBR 1 mg/kg: F(1,10)=1.25, NS), consistent with its reported lower potency as comparedwith R-baclofen.

Figure 4.

Figure 4

The less potent enantiomer S-baclofen was less effective on social approach in theBTBR mouse model of autism. S-baclofen was administered acutely 60 minbefore the test session. Vehicle control subject mice received saline. (a) B6 micedisplayed normal sociability on the chamber time parameter, spending more time inthe side chamber with the novel mouse as compared with the side chamber with thenovel object, after treatment with saline and at each dose of S-baclofen. (b) BTBRmice exhibited its characteristic lack of sociability on the chamber timeparameter after treatment with saline. BTBR did not spend more time in the sidechamber with the novel mouse as compared with the side chamber with the novelobject, after treatment with saline and at each dose of S-baclofen. (c) B6 micetreated with saline or S-baclofen exhibited characteristic sociability on thedirected sniffing parameter, as described in the Materials and Methods andFigure 1. (b) BTBR exhibited its characteristic lack ofsociability and did not spend more time sniffing the novel mouseversusthe novel object, after treatment with saline. Although 1 mg/kg ofR-baclofen improved sociability on the sniffing parameter in BTBR, higher doses ofS-baclofen (3 and 10 mg/kg) were required to reverse the directedsocial sniffing deficits in BTBR. Number of entries into the side chambers wasunaffected by S-baclofen treatment in (e) B6 and (f) BTBR, indicating the absenceof confounding increased hyper- or hypo-exploratory locomotion during the socialapproach task. *P<0.05, novel mouseversus novel object.Absence of effects of S-baclofen on repetitive behavior assays in B6, BTBR, andC58 are illustrated inSupplementary Material FigureS10.Supplementary Material FigureS11 confirmed a lack of confounding sedative or activating effectsat the doses of S-baclofen tested in open field locomotion.

Transitions into the chambers were not altered by S-baclofen in B6 (Figure 4e, F(4, 49) = 1.47, NS) or BTBR(Figure 4f, F(4, 56)=2.39, NS),indicating that the drug administration had no effect on exploratory activityduring the social approach assay.

Repetitive self-grooming and marble burying in BTBR and vertical jumping in C58were unaffected by S-baclofen treatment (Supplementary FigureS10). No sedation was detected in B6 at the two higher doses ofS-baclofen in open field locomotion (Supplementary FigureS11).

DISCUSSION

Autism is a multifaceted neurodevelopmental disorder with high variability in symptompresentation and biomarkers across individuals. Extensive consortium studies of ASDhave revealed copy number variants and single gene mutations in GABA receptor subunitgenes across a relative large percentage of cases of ASD and in comorbid syndromesthat meet diagnostic criteria for ASD (Conantet al,2014;Hogartet al, 2009;Kimet al, 2008;Maet al, 2005;McCauleyet al,2004;Nurmiet al, 2001;Pitonet al, 2013;Schroeret al, 1998;Vincentetal, 2006). Seizures appear in approximately one-third of ASDcases, including comorbid neurodevelopmental disorders such as tuberous sclerosis,Fragile X, Rett, 15q11-13 duplication, 16p11.2 deletion, and Phelan-McDermidsyndromes, in which seizures present as a primary symptom (Chaoet al, 2010;Hagermanet al,2010;Sahin, 2012;Sarasuaet al, 2014;Shinawietal, 2010). GABAergic spectroscopy and postmortem biomarkers havereported lower GABA in certain brain regions in ASD (Blatt andFatemi, 2011;Fatemiet al,2002;Gaetzet al, 2014;Haradaet al, 2011;Moriet al, 2012;Yipet al,2009).

Mouse models with mutations in GABA receptor subunits and/or reduced GABAergic orparvalbumin positive interneurons display social deficits, repetitive behaviors, andother ASD-relevant behavioral phenotypes (Bissonetteetal, 2014;Brielmaieret al,2014;DeLoreyet al, 2008;Karayanniset al, 2014;Penagarikanoet al, 2011;Tripathiet al, 2009). Impaired GABA inhibitory transmissionhas been reported in multiple preclinical models of Fragile X syndrome (Gattoet al, 2014;Martinet al, 2014;Paluszkiewiczetal, 2011).

Benzodiazepines and GABA agonists reversed behavioral and electrophysiologicalabnormalities inFmr1,Scn1a, and BTBR mouse models (Hanet al, 2014;Hanetal, 2012;Olmos-Serranoet al,2010;Pobbeet al, 2011) andArbaclofen treatment reversed symptoms in Fragile X mice (Hendersonet al, 2012). Given the circumscribed but intriguingreversal of some elements of Fragile X and ASD in the first clinical trials ofArbaclofen (Berry-Kraviset al, 2012;Ericksonet al, 2014), we reasoned that extensivepreclinical evaluation of R-baclofen in mouse models of autism could beinformative.

R-baclofen normalizes multiple aspects of excitatory/inhibitory (E/I) circuitbalance in mouse models of E/I dysfunction (Gandaletal, 2012). The efficacy of R-baclofen could be the result of itsability to dampen hyperexcitability via both pre- and postsynaptic mechanisms.GABAB receptors on the presynaptic neuron inhibit GABA releasepresynaptically and activate postsynaptic, inward-rectifying potassium channels thatcause neuronal hyperpolarization. R-baclofen may be beneficial in BTBR because of thereduced frequency of inhibitory synaptic events, reduced inhibitoryneurotransmission, and increased excitatory neurotransmission reported for BTBR(Hanet al, 2014).

Here we report a strongly significant reduction in stereotyped and repetitivebehaviors in two unrelated inbred strain mouse models of autism. High self-groomingand high marble burying in BTBR mice and high vertical jumping in C58 mice werenormalized by doses of 1 and 3 mg/kg R-baclofen. Furthermore, these lowdoses of R-baclofen reversed sociability deficits in BTBR, an inbred strain thatdisplays low social interactions on multiple social assays (Bolivaret al, 2007;Defensoretal, 2011;Lipina and Roder, 2013;McFarlaneet al, 2008;Pearsonet al, 2012;Pobbeet al, 2011;Silvermanet al,2012,2013a,2013b). These R-baclofen doses produced no sedative effects on openfield locomotion or on number of entries during the three-chambered social approachsession. B6, a control inbred strain with normal sociability and low repetitivebehaviors, was unaffected by R-baclofen on these assays, indicating lack ofdeleterious effects in normal controls. Higher doses of the less potent enantiomerS-baclofen were required to reverse abnormalities in BTBR and C58, supporting the useof the R-enantiomer.

It is important to recognize that the replicated rescues of sociability by R-baclofenin BTBR mice did not generalize to male–female reciprocal social interactions.In our initial investigation with a single dose, 1 mg/kg, R-baclofen didnot improve or impair the normally high reciprocal social interactions in B6 controlmice, confirming no deleterious effects on social interactions. However, this dose ofR-baclofen did not reverse the low male–female interactions in BTBR mice. It ispossible that the optimal doses of R-baclofen will vary across behavioral assays inmice. Given the sedation detected at the higher dose of R-baclofen in many of ourmouse behavior assays, chronic treatment may be useful to provide an opportunity fordesensitization to the sedative effects. It may be necessary to conduct comprehensivedose–response curves, for both acute and chronic treatments, across a range ofpreclinical assays relevant to autism to work out the optimal therapeutic regimen. Itis conceivable that similar dosing issues may have affected results in the previousclinical trial with Arbaclofen (Ericksonet al,2014), and will require attention in future clinical studies with thiscompound and other GABAergic agonists.

There are no FDA-approved pharmacologic compounds to improve deficiencies in the corebehavioral symptom domains of ASD, deficits in social communication, and restricted,repetitive behaviors. Our novel preclinical results using two mouse models of ASD arestrikingly promising. These data support the hypothesis that enhancing inhibitorytransmission improves ASD relevant deficits. We discovered that by increasingGABAergic signaling via the GABAB receptor agonist R-baclofen, lack ofsociability of BTBR was reversed on two parameters of sociability and replicated intwo independent cohorts. Furthermore, we found dose-dependent reductions in highlevels of repetitive self-grooming and marble burying in BTBR and in stereotypedvertical jumping in C58 treated with R-baclofen. Future investigations of chronicR-baclofen treatment, in these and other rodent models, will provide additionalinsights. Our findings support the hypothesis that enhancing inhibitory synaptictransmission offers a therapeutic strategy for improving the diagnostic symptoms ofASD. GABAB agonists represent one potential hypothesis-based therapeuticintervention. Although the first clinical trials with Arbaclofen produced significantimprovement on only a subset of measures, our preclinical findings suggest that thestrategy justifies additional trials with refined outcome measures in abiomarker-stratified patient population.

FUNDING AND DISCLOSURE

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the UC Davis MIND Institute.

Footnotes

Supplementary Information accompanies the paper on theNeuropsychopharmacology website (http://www.nature.com/npp)

Supplementary Material

Supplementary Information
Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure 4
Supplementary Figure 5
Supplementary Figure 6
Supplementary Figure 7
Supplementary Figure 8
Supplementary Figure 9
Supplementary Figure 10
Supplementary Figure 11
Supplementary Figure Legends

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