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BEHAVIORAL PHENOTYPING OF V-AKT MURINE THYMOMA VIRAL ONCOGENE HOMOLOG 1-DEFICIENT MICE REVEALS A SEX-SPECIFIC PREPULSE INHIBITION DEFICIT IN FEMALES THAT CAN BE PARTIALLY ALLEVIATED BY GLYCOGEN SYNTHASE KINASE-3 INHIBITORS BUT NOT BY ANTIPSYCHOT

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Schizophrenia is a severe mental illness with a strong genetic predisposition. Accumulating evidence from human genetics and animal studies suggest v-akt murine thymoma viral oncogene homolog 1 (Akt1) might contribute to susceptibility for
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  BEHAVIORAL PHENOTYPING OF V-AKT MURINE THYMOMA VIRALONCOGENE HOMOLOG 1-DEFICIENT MICE REVEALS ASEX-SPECIFIC PREPULSE INHIBITION DEFICIT IN FEMALES THATCAN BE PARTIALLY ALLEVIATED BY GLYCOGEN SYNTHASEKINASE-3 INHIBITORS BUT NOT BY ANTIPSYCHOTICS  Y. W. CHEN a AND W. S. LAI a,b * a Department of Psychology, National Taiwan University, Taipei 10617,Taiwan b Neurobiology and Cognitive Science Center, National Taiwan Univer-sity, Taipei 10617, Taiwan Abstract—Schizophrenia is a severe mental illness with astrong genetic predisposition. Accumulating evidence fromhuman genetics and animal studies suggest  v-akt murinethymoma viral oncogene homolog 1 (Akt1)  might contributeto susceptibility for schizophrenia. In contrast to inconclu-sive findings in human genetic studies, a mutant mousemodelisasimplifiedandalternativeapproachtodeterminingthe biological functions of AKT1 and its possible role in thepathogenesis of schizophrenia. In study 1, a comprehensivebattery of behavioral tests was performed on both male andfemale mice. The results of behavioral phenotyping did notreveal significant differences between genotypes or sexes,except increased time of immobility in the tail suspensiontest and acoustic prepulse inhibition (PPI) deficits in  Akt1 -knockoutfemales.OnthebasisoftheobservedPPIdeficit,instudy 2a, neuromorphological alterations were examinedwith morphometric analysis of green fluorescent protein(GFP)-labeled pyramidal neurons in the auditory cortex of female mice. The results indicated abnormalities in the archi-tecture and complexity of the neurons of mutant femalescompared with those of the controls. In study 2b, potentiallyeffective pharmacological treatments were explored to miti-gate the observed PPI deficits in females. Antipsychotics(either raclopride (3 mg/kg) or clozapine (3 mg/kg)) did notalleviate observed PPI deficits in  Akt1 -knockout females butit was partially normalized by 8-hydroxy-N,N-dipropyl-2-ami-notetralin (8-OH-DPAT, 5 mg/kg) and SB216763 (2.5 mg/kg).These findings imply the importance of AKT1 in some behav-ioral phenotypes and dendritic morphology in the auditorycortex of female mice, and they also suggest that subjectswith  Akt1  deficiency are insensitive to antipsychotic drugs,whereas glycogen synthase kinase-3 (GSK3) inhibitors couldhave therapeutic potential for the treatment of acoustic PPIdeficits. © 2011 IBRO. Published by Elsevier Ltd. All rightsreserved.Key words:  Akt1  knockout mice, behavioral phenotyping,prepulse inhibition, neuromorphology of layer V pyramidalneurons, antipsychotics, schizophrenia. Schizophrenia is a multifactorial disorder with a stronggenetic predisposition. Accumulating evidence from hu-man genetic studies suggest functional candidate genesthat contribute to a susceptibility to schizophrenia, includ-ing v-aktmurinethymomaviraloncogenehomolog1 (  Akt1 ;Norton et al., 2006; Schwab and Wildenauer, 2009). Anassociation between schizophrenia and  Akt1  genetic vari-ants was first reported in a Caucasian family of Europeandescent by Emamian et al. in a seminal paper (Emamian etal., 2004). Since this first report, several positive/negativegenetic association studies have been reported in other ethnic groups. For example evidence for   Akt1  as a sus-ceptibility gene for schizophrenia has also been reported inEuropean sib-pair families (Schwab et al., 2005), Irish families (Thiselton et al., 2008), the Iranian population (Bajestan et al., 2006), the Japanese population (Ikeda et al., 2004, 2006), and the Chinese population (Xu et al., 2007), and weakly in a UK case–control sample (Norton et al., 2007), but not in a family sample from Taiwan (Liu et al., 2006), other Japanese samples (Ohtsuki et al., 2004), or the Korean population (Lee et al., 2010). The biological functions of AKT1 and the mechanism by which it contrib-utes to a susceptibility to schizophrenia remain unclear. AKT1 (PKB  ), a serine/threonine kinase of the AKTfamily, is involved in multiple biological processes anddiverse signal transduction pathways (Franke, 2008). The activation of AKT1 and the phosphatidylinositol 3-kinase(PI3K)-AKT–glycogen synthase kinase-3 (GSK3) cascadehas been implicated in many neural functions, such as N  -methyl- D -aspartate receptor signaling, dendritic spinedevelopment, the expression of long-term potentiation,and neurite outgrowth (Scheid and Woodgett, 2001; Readand Gorman, 2009). Together with genetic studies, accu-mulating evidence from human brain studies also suggestthat AKT1 signaling plays a role in the pathogenesis of schizophrenia. Particularly, there was a reduction in AKT1and phosphorylated GSK3   protein levels in the lympho-cytes and postmortem brains of individuals with schizo-phrenia (Emamian et al., 2004). A follow-up study also exhibited a functional reduction in the insulin receptor con-tent and insulin-dependent AKT signal transduction in the *Correspondence to: W. S. Lai, Department of Psychology, NationalTaiwan University, 1 Roosevelt Road, Section 4, Taipei 10617, Tai-wan. Tel:  886-2-3366-3112; fax:  886-2-3362-9909.E-mail address: wslai@ntu.edu.tw (W. S. Lai).  Abbreviations:  Akt1, v-akt murine thymoma viral oncogene homolog 1; ANOVA, analysis of variance; DAD2R, dopamine D2 receptor; GFP,green fluorescent protein; GSK3, glycogen synthase kinase-3; PI3K,phosphatidylinositol 3-kinase; PLSD, protected least significant differ-ence; PP2A, protein phosphatase 2A; PPI, prepulse inhibition; SEM,standard error of the mean; WT, wild type. Neuroscience  174 (2011) 178–189 0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2010.09.056 178  dorsolateral prefrontal cortex of medicated schizophrenicpatients (Zhao et al., 2006). It was also evident that there is an epistatic effect of genetic variations in the dopami-nergic system and  Akt1  on the functional neuroimaging inschizophrenic patients (Tan et al., 2008). Nevertheless, an association between human genetic and neuroimagingstudies does not establish causation, and the observedmutation usually has no proven functional consequences. A simplified and alternative approach to examining thisassociation is to use mutant mouse models.Despite some obvious limitations in the use of mousemodels to study human disorders, emerging studies usingmutant and wild-type mice have shown great sensitivity tothe sensorimotor-gating-disruptive effect of amphetaminein  Akt1 -knockout mice (Emamian et al., 2004), andchanges in the dopamine-related prefrontal cognitive func-tions and medial prefrontal dendritic structure in  Akt1 -knockout mice (Lai et al., 2006), as well as pharmacolog- ical evidence that the drugs used in the management of psychosis, such as lithium, haloperidol, and clozapine, canenhance AKT signaling  in vivo  (Beaulieu et al., 2004;Emamian et al., 2004) and  in vitro  (Chalecka-Franaszekand Chuang, 1999). In the search for AKT1 functions or itspossible involvement in the schizophrenia-like symptoms,however, most animal studies either did not specify thedifferent isoforms of the AKT family or only concentratedon male or mixed subjects with limited behavioral tasks.Given the findings from epidemiological surveys and meta-analyses that numerous sex differences occur in schizo-phrenia ( Aleman et al., 2003; McGrath et al., 2004), further  examinations of the behavioral phenotypes and relatedalterations in both male and female mice are warranted. Itis of great interest to investigate the biological functions of  AKT1 and its involvement in the pathogenesis of schizo-phrenia using genetically modified mice or transgenic micewith/without  Akt1  gene as a model.Taking advantage of   Akt1 -knockout mice, there arethree objectives in the present study. In study 1, we wouldlike to perform a battery of behavioral tasks to characterizesome basic and important behavioral phenotypes of bothmale and female  Akt1 -knockout mice. On the basis of thegenotypic prepulse inhibition (PPI) deficits observed in thestudy 1, two exploratory studies to probe for neuromorpho-logical alterations and pharmacological treatments wereconducted. In study 2a, given the importance of AKT1 inseveral aspects of neurite outgrowth (Read and Gorman,2009) and the involvement of auditory cortex in the regu-lation of acoutstic startle and PPI (Swerdlow et al., 2001;Bowen et al., 2003; Floody et al., 2010), the dendriticarchitecture of output neurons in the auditory cortex wereexamined to reveal conceivable clues for interpreting theobserved PPI deficits in females. In study 2b, since the AKT-GSK3 signaling cascade was proposed to be relevantto the actions of dopamine and psychotropic drugs (Beau-lieu et al., 2007; Del’Guidice and Beaulieu, 2010), poten-tially effective pharmacological treatments were also ex-plored to normalize the observed PPI deficits in females. EXPERIMENTAL PROCEDURES Animals  All  Akt1  /  mice and their wild-type littermates used in this studywere generated from  Akt1 -heterozygous breeding pairs in aC57BL/6 genetic background (backcrossed over 10 generations)and genotyped using PCR analysis of mouse-tail DNA, as de-scribed previously (Cho et al., 2001; Lai et al., 2006). As described before (Cho et al., 2001), loss of expression of AKT1 resulted in partial lethality occurring some time between mid-embryonic de-velopment and the time of weaning. Fewer than expected  Akt1  /  mice (  1/12) were available and they were tested repeatedly insome experiments to meet the reduction of the 3R’s principle inanimal use. After weaning, animals were housed with food andwater available  ad libitum  in polysulfone individually ventilatedcages (Alternative Design Manufacturing & Supply, Arkansas, AR,USA) within the animal rooms of the Psychology Department,National Taiwan University. All animals were 2–3 month-old at thebeginning of experiments and preliminary observations in their home cages revealed normal physical conditions, except bothmale and female mutant mice exhibited a reduction of body weightcompared with controls as reported previously (Cho et al., 2001).  Animals were handled and weighed daily at least 1 week beforethe behavioral experiments. All animal procedures were per-formed according to protocols approved by the appropriate AnimalCare and Use Committees established by the National TaiwanUniversity. Every effort was made to limit the number of animalsused and minimize their suffering. Behavioral phenotyping procedure In study 1, both male and female adult  Akt1  /  mice and their wild-type littermates ( n  8 each) were housed individually for atleast 1 week before behavioral testing in a room maintained on a12-h light/dark cycle. All behavioral studies were performed duringthe dark cycles. For behavioral phenotyping, a series of sevenbehavioral tests (run from the first to the seventh week), whichincluded an open-field locomotor assay, a dark/light transition test,an elevated plus maze, tail suspension test, PPI, auditory tracefear conditioning, and a Morris water maze, were performed insequence, with a 1 week interval between tests to evaluate thebasic motor function, anxiety, anxiety-like behaviors, depressive-like behavior, sensorimotor gating function, auditory associativelearning and memory, and the spatial learning and memory func-tion of the mice, respectively. The general principle of the arrange-ment is to avoid a more stressful task before a less stressful oneand to minimize carryover effects. The details of each of the seventasks were described briefly as follows. Open-field locomotor assay (the 1st week).  To assessspontaneous locomotor activity, each subject was placed into thecenter of an open-field apparatus (25.40  25.40  40.64 cm 3 ,Coulbourn Instruments, Whitehall, PA, USA) under dim lightingcondition (60 lx). Motor activity parameters (including total traveldistance, number of rearing, and number of center entries) weremonitored and recorded over a 15 min period by using TruScan2.01 photobeam activity system (Coulbourn Instruments, White-hall, PA, USA). Dark/light transition test (the 2nd week).  To assess brightlight-induced anxiety, the open-field apparatus was also used for the light/dark transition test. A dark insert containing a smallopening equally divided the open-field arena into two chambers.One chamber was brightly illuminated (900 lx), whereas the other chamber was dark. Each mouse was placed into the lit compart-ment with facing away from the door of the dark chamber andallowed to move freely between the two chambers for 10 min. Thelatency until the first transition, the number of transitions betweenY. W. Chen and W. S. Lai / Neuroscience 174 (2011) 178–189 179  the two compartments, the time spent in each chamber, and thetotal travel distance were recorded. Elevated plus maze (the 3rd week).  An elevated plus mazewas used to measure anxiety-like behaviors. The maze wasshaped like a plus sign in white plastics, with two un-walled (open)arms (50  10 cm 2 ) and two walled (closed) arms (50  10  50cm 3 ). The apparatus was elevated 50 cm from the floor. Eachanimal was placed in the center of the plus maze (10  10 cm 2 )facing an open arm and allowed to explore the maze for 5 min.Time spent and traveled distance in the open arms were recordedon line by using EthoVision tracking system (Noldus InformationTechnology, Wageningen, the Netherlands). The ratio of timespent in the open arm divided by the total time was used as anindex of anxiety in the maze. Tail suspension test and stress-induced locomotor activity (the 4th week).  The tail suspension test and the open-fieldapparatus were used to assess depressive-like behaviors andstress-induced locomotor activity. Each mouse was first placed inthe center of an open-field apparatus (Coulbourn Instruments,Whitehall, PA, USA) and allowed to explore freely for 5 min. After a 5 min exploration, each mouse was suspended for 6 min byclipping the animal’s tail in a constant position, two-thirds of thedistance from the base of the tail. The behavior of each animalwas recorded continuously with a digital video camera. After tailsuspension for 6 min, each mouse was placed back in the open-field apparatus for another exploration for 5 min. Travel distance inthe open field was recorded using the TruScan 2.01 photobeamactivity system (Coulbourn Instruments, Whitehall, PA, USA). Thetime of immobility during the 6 min tail suspension period wasscored by a video tracking system (TopScan, Cleaver SystemIncorporated, Reston, VA, USA). Prepulse inhibition (the 5th week).  To assess the sensori-motor gating function, each mouse was tested with the SR-LABstartle apparatus (San Diego Instruments, San Diego, CA, USA).The background noise was 72 dB during testing. Each sessionwas initiated with a 5 min acclimatization period followed by 64trials, consisting of pulse-alone (P-alone) trials, prepulse  pulse(pp  P) trials, and no stimulation (nostim) trials. A P-alone trialwas a 120 dB white noise burst of 40 msec. In the pp  P trials, the120 dB pulse was preceded (by 100 msec) by a 20 msec whitenoise prepulse burst of 78 dB (PP6), 82 dB (PP10), or 90 dB(PP18). The nostim trials consisted of background noise only. Thesession began and ended with a block of six presentations of theP-alone trial. Between these two blocks, the rest 52 trials wereperformed pseudorandomly and separated by intertrial intervals of 15 sec on average (varying between 10 and 20 sec). PPI wascalculated as a percentage of the startle response using theformula: %PPI  100   [(P-alone score)—(pp  P score)]/(pulse-alone score), where the pulse-alone score was the average of thepulse-alone values from the in-between block of 52 trials.  Auditory trace fear conditioning (the 6th week).  In order tomeasure auditory learning and associative learning, auditory tracefear conditioning assay was conducted by using a commerciallyavailable fear-conditioning system that is controlled automaticallyby using a PC with TruScan 2.01 system (Coulbourn Instruments,Whitehall, PA, USA). On the day of conditioning, each mouse wasindividually placed into the conditioning chamber. After one 3 minexploratory period, each subject was exposed to six tone-footshock pairings (tone (CS), 16 sec; foot shock, 2 sec, 0.5 mA, 18sec after the termination of the tone; separated by 3 min intertrialinterval). Three min after the last foot shock, mice were returnedto their home cage. Twenty-four hours after the foot shock, eachmouse was tested for auditory (tone) fear conditioning in a novelchamber. Different environmental cues (e.g. different visual cues,50 ml of 10% vanilla scent, and gray plastic floor) were providedin the novel chamber. Each mouse was tested there for a 3 minbaseline period (pre-CS) and three 30 sec periods (CS) duringwhich the tone was presented consistently for 30 sec, separatedby 1 min intertone interval. All behaviors were videotaped by avideo camera in front of the testing chamber and total freezingtime was measured as an index of fear memory. Freezing behav-ior is defined as a complete lack of movement excluding respira-tion. Freezing behavior was scored by using a stopwatch, andconverted to freezing percentage (freezing percentage  (totalfreezing time/total testing time)  100%). Morris water maze (the 7th week).  Spatial learning andmemory abilities were examined in a modified version of hidden-platform acquisition and 1-day retention tasks in a standard Morriswater maze. A circular pool (diameter, 100 cm) that was filled withwater, clouded with nontoxic white paint, and kept at 22  1 °C wasin the center of a testing room surrounding with several visualcues. A platform (12  12 cm 2 ) was hidden 1 cm beneath thesurface of the water. Swimming paths of each subject was re-corded using EthoVision tracking system (Noldus InformationTechnology, Wageningen, the Netherlands). To access acquisi-tion and 1-day retention of spatial memory, 15 swimming trials(with 1 min inter-trial interval) started pseudo-randomly from eachof the other three quadrants except the target one where theplatform was placed. Mice that failed to find the platform within 1min were gently guided to the platform, where they remained therefor 30 sec before being returned to their cages. Escape latency(sec) to find the hidden platform and path length (cm) were re-corded. Twenty-four hours later, each subject was placed back tothe pool without platform for 2 min for a 1-day retention test. Thetime spent and swimming distance in each of the quadrant wererecorded and used as an index of reference memory ability. Dendritic architecture and neuromorphologicalanalysis of pyramidal neurons in the auditory cortex In study 2a, as a follow up to the observed PPI impairment infemale mice, auditory cortex of females were examined to revealany neuromorphological alterations in mutant mice that could inpart account for the PPI deficits. Because pyramidal neurons of layer V in the neocortex are the primary output cells of the cortex,the specific transgenic mouse line, C57BL/6- Tg  (GFPm) driven by Thy1  promoter, was selected and used to facilitate analyses of theneuromorphology of green fluorescent protein (GFP)-labeled py-ramidal neurons in the auditory cortex (Feng et al., 2000). Based on the observed PPI deficits in female mice, additional femalesubjects generated from  Akt1 -heterozygous breeding pairs in aC57BL/6- Tg  (GFPm) background were used in this experiment. Adult mice were euthanized and transcardially perfused with PBS,followed by 4% paraformaldehyde in PBS. Fixed brains weresectioned coronally using a vibratome (Microm, Thermo Fisher Scientific Walldorf, Germany). A series of 150   m coronal sec-tions were collected and mounted on slides for scanning. Confocalmicroscopy stack images of GFP-labeled neurons were obtainedat intervals of 0.4   m using 20  , 40  -oil and 63  -oil objectivesby Leica SP5 confocal microsystem (Leica Microsystems, Wet-zlar, Germany). The Neurolucida software (Microbrightfield Incor-porated, Williston, VT, USA) were used to trace and reconstruct3-dimensional neurons.Layer V pyramidal neurons were almost exclusively labeledwith GFP in the auditory cortex of the mutant mice, throughout thecell body and dendritic tree. A morphometric analysis of GFP-labeled pyramidal neurons in the layer V of auditory cortex (be-tween bregma   2.30 and   2.80 mm) was performed on adultfemale  Akt1  /  and wild-type mice ( n  5 each) to reveal neuro-morphological abnormalities between genotypes. The classic py-ramidal neurons of layer V have distinguishing larger cell bodieswith a relatively thick apical dendrite (i.e. a thicker dendritic pro-cess that clearly emanates from the apical part of the soma andextends toward the upper cortical layers). All protrusions initiatingY. W. Chen and W. S. Lai / Neuroscience 174 (2011) 178–189180  from the cell soma longer than 5   m were defined as primarydendrites. For each complete and available neuron in the auditorycortex, a total of 14 morphological variables which were modifiedand chosen based on a previous study (Lai et al., 2006) were examined in this experiment, including (1) soma size (the cellsoma size was obtained by outlining the cell soma, followed byautomatic calculation of pixel area in   m 2 ); (2) distance to apicalbifurcation (base of the apical tuft) measured from the cell body tothe major branch point of the apical dendrite; (3) number of branches of apical branches; (4) number of apical tips; (5) totallength of the apical tuft, which is the sum of the lengths of theapical stem and the branches that form the tuft; (6) apical dendriticfield area (ADFA), which measures the area of the dendritic fieldof a neuron calculated as the area enclosed by a polygon that joins the most distal points of dendritic processes (convex area);(7) branch angle of primary apical dendrites (from the distal end of apical bifurcation); (8) number of primary basal dendrites (notincluding apical dendrite and axon); (9) the total length of primarybasal dendrites; (10) number of branches of basal branches; (11)number of basal tips; (12) the total length of basal dendrites; (13)basal dendritic field area (BDFA), which measures the area of thedendritic field of a neuron calculated as the area enclosed by apolygon that joins the most distal points of dendritic processes(convex area); and (14) Sholl analysis of basal dendritic complex-ity. Exploration of pharmacological treatments Probable pharmacological interventions for the observed PPIdeficits in female mice were explored in study 2b. To reduceanimal use, two batches of   Akt1  /  and wild-type females wereused repeatedly to test the effects of two antipsychotic drugsand two potential drugs on the mitigation of PPI impairment.The testing procedure for PPI was the same as describedpreviously in the PPI procedure. The four drugs (all purchasedfrom Sigma-Aldrich, St Louis, MO, USA) were chosen to miti-gate the PPI deficits based on previous studies (Dulawa et al.,2000; Brody et al., 2004; Duncan et al., 2006; Gogos et al.,2008; Miller et al., 2009). A maximal effective dose for eachdrug was chosen based on the following criteria: (1) This dosehas been previously reported and confirmed to effectively mit-igate PPI or related behavioral deficits, especially in mice. (2)This dose has less or relatively minimal motor side effect. Allfemales in the first batch ( n  8 per group) were i.p. adminis-tered one saline and two antipsychotic treatments in sequence,with at least a 1 week washout interval between treatments tominimize carryover effects. The three treatments consisted of a0.9% saline injection 15 min before the first PPI test, a 3 mg/kgraclopride (dissolved in 0.9% saline) injection 15 min before thesecond PPI test, and a 3 mg/kg clozapine (dissolved with 20%acetic acid in 0.9% saline) injection 30 min before the last PPItest. All females in the second batch ( n  8 per group) wererepeatedly administered one saline and two drugs treatments insequence, with at least a 1 week washout interval betweentreatments. The three treatments consisted of a 0.9% salineinjection (i.p.) 10 min before the first PPI test, a 5 mg/kg8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT, dissol-ved in 0.9% saline) injection (s.c.) 10 min before the secondPPI test, and a 2.5 mg/kg SB216763 (dissolved in DMSO andbrought up to volume in a mixed solution of propylene glycoland distilled water [10:60:30]) injection (i.p.) 10 min before thelast PPI test. Statistics and data analyses  All Data for the behavioral phenotyping except PPI were ana-lyzed by two-way analysis of variance (ANOVA). A significantinteraction effect is further analyzed as the simple main effectsof genotype differences within each sex and sex differenceswithin each genotype. Data for PPI and pharmacological treat-ments of PPI were analyzed using a repeated measure three-way ANOVA or further analyzed by two-way ANOVA to revealgenotypic difference under each pharmacological treatmentwhere appropriate.  F  -values reaching significant difference( P   0.05) were evaluated further by post hoc analysis using theFisher’s protected least significant difference (PLSD) test. Theresults of each morphological parameter were analyzed bytwo-tailed Student’s  t  -test or ANOVA. Statistic analysis wasdone by StatView 5.0.1 (SAS Institute Incorporated, Cary, NC,USA).  P   values of    0.05 were considered statistically signifi-cant. RESULTS Results of study 1: behavioral phenotyping of   Akt1 -deficient mice revealed sex-specific alterations Compared with the wild-type mice,  Akt1 -knockout micedisplayed normal behavioral profiles in a series of behav-ioral tasks, including a spontaneous locomotor activity as-say (during baseline and after stress), a dark/light transi-tion test, an elevated plus maze task, auditory trace fear conditioning, and the learning and memory of Morris water maze.AssummarizedinTable1,nosignificantdifferences were found between the genotypes or sexes (all  P   0.05),suggesting some basic functions (e.g. motor, auditorylearning, and visual functions) appear to be normal in  Akt1 -knockout mice. In contrast, significant differenceswere observed in the tail suspension test and acoustic PPIin female mice but not in male mice. In the tail suspensiontest, genotype ( F  (1,28)  8.299,  P   0.01), sex ( F  (1,28)  9.177,  P   0.01), and the genotype    sex interaction( F  (1,28)  6.982,  P   0.05) had a significant main effect onthe time of immobility. As shown in Table 1, statistical analysis further showed significant differences in the sim-ple main effects of genotype in females ( P   0.05), and of sex difference in  Akt1 -knockout mice ( P   0.05) and inwild-type mice ( P   0.05). Fisher’s PLSD  post hoc   analysisshowed that female  Akt1 -knockout mice displayed a sig-nificantly increased period of immobility compared withthat of the wild-type controls ( P   0.01; Fig. 1 A).In the acoustic PPI task, a sex-specific PPI deficit wasobserved in female mice but not in male mice. Female  Akt1 -knockout mice exhibited a profoundly reduced PPIcompared with that in the wild-type controls. Genotype( F  (1,28)  8.721,  P   0.01) and the genotype  sex interac-tion ( F  (1,28)  8.945,  P   0.01) had significant main effectson PPI. Statistical analysis further revealed significant dif-ferences in the simple main effects of genotype in females( P   0.05), and of sex differences in  Akt1 -knockout mice( P   0.05). Fisher’s PLSD  post hoc   analysis showed thatfemale  Akt1 -knockout mice displayed significantly reducedlevels of PPI across all three prepulse intensities com-pared with those of the wild-type controls (all  P   0.05; Fig.1B). The results also indicated that there was no genotypicdifference in the average startle amplitude in response to120 dB pulses in the first and last blocks (wild-type vs.  Akt1  /  : 230.5  43.6 vs. 196.5  50.4 in the first block;131.0  26.3 vs. 80.3  19.5 in the last block; both  P   0.05). Y. W. Chen and W. S. Lai / Neuroscience 174 (2011) 178–189 181  Table 1.  A summary of statistical analyses and behavioral phenotyping results (mean  SEM) in detailsBehavioral task Genotype Sex Interaction Simple main effect Male FemaleWT  Akt1  /  WT  Akt1  /  Open fieldTravel distance (cm) ns ns ns 3262.275  82.47 3165.887  209.617 3637.325  249.647 3423.65  256.92Rearing (#) ns ns ns 129.25  14.908 133.625  15.545 101.75  16.739 97  10.377Center entries (#) ns ns ns 114.625  7.982 106.75  13.64 130.25  17.298 109.625  8.685Dark light assayLatency to enter the dark box(sec)ns ns ns 124.541  41.154 212.992  61.206 141.952  67.917 113.66  46.004Time in the dark box (sec) ns ns ns 388.5  59.33 274.313  47.619 337.906  65.64 394.844  32.551Dark/light transitions (#) ns ns ns 9.5  1.955 9  1.982 12  3.836 10.625  1.614Total travel distance (cm) ns ns ns 2543.463  141.598 2391.262  182.986 2470.387  332.033 2720.225  146.023Elevated plus mazeTime spent in open arms (%) ns ns ns 19.44  10.623 14.061  5.286 14.908  4.165 15.014  3.775Tail suspension   (WT vs.  Akt1  /  ):  P   0.025Immobility (sec)  P   0.01  P   0.01  P   0.05 WT (  vs.  ):  P   0.025 207.545  28.648 212.804  19.553 82.487  19.487 *  204.271  19.026 * Stress effect on locomotion (cm) ns ns ns  Akt1  /  (  vs.  ):  P   0.025 885.45  88.049 924.05  82.717 1037.9  116 1115.575  119.481Prepulse inhibitionPP6 (78dB, %)  P   0.01 ns  P   0.05   (WT vs.  Akt1  /  ):  P   0.025 62.217  1.948 59.533  3.929 67.638  3.79 *  38.642  7.874 * PP10 (82dB, %)  P   0.01 ns  P   0.01  Akt1  /  (  vs.  ):  P   0.025 66.908  4.064 66.924  3.94 79.082  4.13 *  48.724  5.757 * PP18 (90dB, %) ns ns  P   0.053 75.738  4.827 78.87  4.26 86.457  2.194 *  72.551  4.978 * Trace fear conditioningFreezing to tone (%) ns ns ns 34.537  8.361 40.482  7.178 41.319  5.333 27.832  4.684Freezing during interval (%) ns ns ns 37.253  7.409 40.846  7.781 48.321  7.338 29.717  6.855Morris water mazeTime spent in target quadrant(%)ns ns ns 29.222  3.413 30.28  3.269 27.13  2.308 32.711  3.408Travel distance in targetquadrant (%)ns ns ns 29.978  3.125 28.811  3.133 28.253  1.913 32.186  3.167 A series of seven behavioral tests (left column, from top to bottom), including an open-field locomotor assay, a dark/light anxiety assay, an elevated plus maze, tail suspension, prepulse inhibition,trace fear conditioning, and the Morris water maze, were performed in sequence, with a 1 wk interval between the tests. Genotype:  Akt1  /  or wild-type; sex: male or female; interaction:genotype  sex; ns, not significant;  P  ,  P  -value. *  P   0.05 between genotypes. Y .W. C h  en an d W. S .L  ai    /   N e ur   o s  c i    en c  e1 7 4   (   2  0 1 1   )   1 7  8 –1  8  9 1  8 2 
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