Attenuation of social interaction-associated ultrasonic vocalizations and

Attenuation of social interaction-associated ultrasonic vocalizations and spatial working memory performance in rats exposed to chronic unpredictable stressMuhammad S.Riaz a ,1,Martin O.Bohlen a ,1,Barak W.Gunter a ,Quentin Henry b ,Craig A.Stockmeier b ,Ian A.Paul b ,⁎a Graduate Program in Neuroscience,University of Mississippi Medical Center,2500North State Street,Jackson,MS 39216,USAbDepartment of Psychiatry &Human Behavior,University of Mississippi Medical Center,2500North State Street,Jackson,MS 39216,USAH I G H L I G H T S •CUS exposure did not alter sucrose preference in adult male Sprague Dawley rats.•CUS exposure attenuated social interaction-associated ultrasonic vocalizations.•CUS exposure attenuated cognitive performance on a spatial working memory task.•CUS exposure resulted in attenuation of weight gain in CUS-treated rats.•CUSexposureresulted in increased latency to feed in a novel environment.a b s t r a c ta r t i c l e i n f o Article history:Received 28April 2015Received in revised form 25August 2015Accepted 3September 2015Available online 11September 2015Keywords:Major depressionUnpredictable chronic mild stress (CUS)Ultrasonic vocalizations USVsSprague dawley rats Anhedonia StressWorking memory Spatial navigation Cognitive declineExposure to unpredictable chronic mild stress (CUS)is a commonly used protocol in rats that is reported to evoke antidepressant-reversible behaviors such as loss of preference for a sweetened water solution which is taken as an analog of the anhedonia seen in major depression.However,the induction of anhedonic-like behavior by chronic mild stress,gauged by an animal's preference for sucrose solution,is not fully reproducible and consistent across laboratories.In this study,we compared a widely used behavioral marker of anhedonia —the sucrose pref-erence test,with another phenotypic marker of emotional valence,social interaction-associated ultrasonic vocal-izations as well as a marker of an anxiety-like phenotype,novelty-suppressed feeding,and cognitive performance in the eight arm radial maze task in adult male Sprague –Dawley rats.Chronic four-week exposure to unpredictable mild stressors resulted in 1)attenuation of social interaction-associated ultrasonic vocalizations 2)attenuation of spatial memory performance on the radial arm maze 3)attenuation of body weight gain and 4)increased latency to feed in a novelty-suppressed feeding task.However,chronic exposure to CUS did not re-sult in any signi ficant change in sucrose preference at one-week and three-week intervals.Our results argue for the utility of ultrasonic vocalizations in a social interaction context as a comparable alternative or adjunct to the sucrose preference test in determining the ef ficacy of CUS to generate an anhedonic-like phenotypic state.©2015Elsevier Inc.All rights reserved.1.IntroductionIt has been well established that chronic emotional stress plays a pivotal role in the genesis of many psychiatric disorders with induction of both short-lasting and long-lasting alterations in behavior and phys-iological functions [1,2].Such emotional stressors are one of the main sources of stress in human life,especially for those low in the social hierarchy,and play a major role in the pathogenesis of anxiety anddepressive disorders [3,4].In social settings,stress can occur throughout the lifespan,and can range from childhood neglect to peer abuse such as school bullying in adolescence or workplace harassment in adult-hood [5,6].Furthermore,chronic stress may be associated with fear-ful (and life-threatening)events of traumatic nature such as violence,war,injury or assault [7].There are very few animal models of human major depressive disorder (MDD)that can adequately provide the face,construct and predictive validity for bench research to be translated into bedside d Chronic Unpredictable Stress (CUS)is one such para-digm that has been used to model the human symptom of anhedonia (de fined as loss of interest in daily activities that were previouslyPhysiology &Behavior 152(2015)128–134⁎Corresponding author.E-mail address:ipaul@ (I.A.Paul).1These authors contributed equally to thiswork./10.1016/j.physbeh.2015.09.0050031-9384/©2015Elsevier Inc.All rightsreserved.Contents lists available at ScienceDirectPhysiology &Behaviorj o u r na l ho m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p h benjoyable)[8].While a sufficient volume of literature supports the effi-cacy of CUS in altering observable behaviors,sucrose testing is by far the most cited measure of‘anhedonia’[9].The use of sucrose testing(of either consumption or preference)generalizes palatable taste reactivity as an index of hedonic state in laboratory rodents.However,reduction in preference for a sweetened water solution has been widely criticized as being unreliable due to variable responsiveness of rodents to CUS [10]and having possible relationships to body weight and nutritional status as well as caloric intake which may confound the results[11].In fact,these observed effects may be limited to the gustatory circuitry and metabolic demand.A large body of evidence has accumulated over the last two decades that highlights the difficulties associated with sucrose testing as a con-sistent measure of hedonic drive[12].For example,chronic stress has no effect on sucrose consumption under a progressive ratio schedule as would be expected if hedonic drive were reduced[13].Similarly, sucrose consumption was more dependent on food deprivation and weight changes seen with chronic stress paradigm,and independent of other elements of stress protocol[14].While CUS reproduces charac-teristic behavioral responses,there is a need to validate objective alter-natives to sucrose testing as a measure of a rodent's affective state.The goal of the present study was to compare the effects of CUS on sucrose preference to other behavioral markers relevant to major depressive disorder in order to increase the reproducibility of the paradigm.Therefore,in Experiment A,we examined multiple measures to seek more reliable behavioral markers for the effects of CUS,starting with the sucrose preference test.Based on previously published studies,it was expected that the stressed rats would show a reduced preference for the sucrose solution[15].However,given that sucrose preference has also been shown to paradoxically increase or remain unchanged with exposure to chronic stress,we also tested other behavioral markers of chronic stress in addition to sucrose preference test[16].These markers included tests of body weight gain,separation/anticipatory ultrasonic vocalization and novelty-suppressed feeding.A total of20rats were used for this experiment(10per treatment group).Experiment A was followed by Experiment B with an additional test of a behavioral marker relevant to MDD,namely,spatial working memory in an8-arm radial maze[17].Spatial memory is a type of hippocampal-dependent learning and memory,and evidence suggests frequent impairment of hippocampal-dependent learning and memory in MDD[18].A total of30rats were used for this experiment(14control and16CUS).In total,50rats were used in Experiments A and B.2.Materials and methods2.1.Materials and methods—experiment AThe goal of thefirst experiment was to identify reliable behavioral markers for the effects of mild chronic unpredictable stress(CUS).The markers included assessment of gain in body weight,sucrose prefer-ence,novelty-suppressed feeding,and anticipatory ultrasonic vocaliza-tion after brief separation from cage-mate.2.1.1.Animals and housingFor both Experiments A and B,adult male Sprague–Dawley rats (Charles River,Wilmington,Mass.,USA)weighing200–250g at the start of experiment were housed two per cage(25×48×20cm)in a temperature and humidity-controlled colony room(~21°C,40–50%) at the University of Mississippi Medical Center Laboratory Animal Facility.The rats were maintained on a reverse12:12light/dark cycle, with lights off at0700h.All behavioral testing took place during the dark phase of the cycle.Food and water were available ad libitum, except during testing.Before the beginning of experiments,all animals were handled for approximately15min,daily for3days.All proce-dures were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee and conformed to the guidelines of the National Institutes of Health.2.1.2.Sucrose preferencePrior to beginning of testing,rats were habituated to the presence of the two drinking bottles for5days(4h each day)in their home cages. One of the bottles contained sucrose in increasing concentrations each day(0.1,0.3,1.0,3.0,10%).This allowed us to determine that a3% sucrose solution consistently(S.E.M.b10%of mean)elicited a3-fold preference over tap water while the1%solution did not reliably main-tain a strong preference and the10%solution elicited a larger,but much more variable preference(S.E.M.~20%of the mean).Therefore, for all subsequent studies,we compared the preference for a3%sucrose solution to tap water.Following this acclimation,rats had the free choice of either drinking the3%sucrose solution or tap water for a period of 3consecutive days(4h each day).Sucrose preference was calculated as a percentage of the volume of sucrose intake over the total volume offluid intake[11]and analyzed over the testing period of3days via two-factor ANOVA followed by planned comparisons using uncor-rected univariate F-tests for between-cell comparisons.There was a significant difference in sucrose preference between the three days[F(2,38)=4.316,p b0.05].Post hoc comparisons using the Tukey HSD test indicated that the mean(M)%preference score for the Day-1sucrose preference(M=80.63,SD=11.59)was significantly different from the Day-2sucrose preference(Mean=71.99,SD= 7.84).However,the Day-3sucrose preference(Mean=76.74,SD= 11.64)did not significantly differ from either Day-1or Day-2sucrose preference.Since the sucrose preference differed significantly between thefirst and second day only,a two-day protocol of sucrose preference testing was employed during the CUS treatment.2.1.d chronic unpredictable stress(CUS)Rats were assigned to one of two groups(n=10each per group) and were either exposed to mild chronic unpredictable stress(CUS)or handled to serve as no stress controls.Rats in each group were matched as closely as possible for body weight,baseline sucrose preference and totalfluid intake.Rats assigned to the CUS group were exposed to the CUS protocol shown in Table1.This10-day protocol was systematically repeated to maintain the element of unpredictability throughout the experiment and for a total of35days(5weeks of CUS treatment). During this period,control animals were regularly handled,weighed and housed separately without any exposure to the CUS paradigm.Body weight was measured before CUS(Day0),2days after the be-ginning of CUS(Day2)and every4days thereafter.Sucrose preference testing was conducted after1and3weeks exposure to Vs wereTable1CUS treatment paradigm—2stressors/day repeated daily:This10-day protocol was systematically repeated to maintain the element of unpredictability throughout the exper-iment and for a total of35days(5weeks of CUS treatment)for Experiment-A,and28days (4weeks of CUS treatment)for Experiment-B.During both experiments,control animals were regularly handled,weighed and housed separately without any exposure to the CUS paradigm.Day Stressor1Stressor2Day150min cold room60min cage rotationDay24h wet bedding12h lights on during dark cycleDay360min restraint stress3h lights off during light cycleDay450min cage rotation15h food and water deprivation duringthe dark cycleDay515min cold room isolation17h isolation housing during light cyclein clean cageDay64h wet bedding3h lights on during dark cycleDay730min cage rotation1h lights on during dark cycleDay85min swimming exposureduring dark cycle60min restraint stressDay94h wet bedding12h food deprivation during dark cycle Day1045min cold room isolation6h lights on during dark cycle129M.S.Riaz et al./Physiology&Behavior152(2015)128–134tested1to5days after the conclusion of CUS and novelty-suppressed feeding24h after the last USV recording.2.1.4.Sucrose preference testingSucrose preference testing was carried out in a quiet dimly lit testing area.The animals were transported from the colony,separated into two clean cages and allowed to acclimate for2h during week-1and week-3 of CUS treatment.After two hours acclimation,rats were presented with a choice of2bottles during the2consecutive test days(4h each day)in both week-1and week-3.One bottle contained tap water,and the sec-ond contained a3%sucrose solution.Water and sucrose solution intake was measured daily,and the position of the two bottles was randomly switched to reduce any confound produced by a side bias.Sucrose pref-erence was calculated as outlined above and analyzed over the testing period of2days via single factor(CUS exposure),repeated measures ANOVA followed by planned comparisons using uncorrected univariate F-tests for between-cell comparisons.2.1.5.Body weight analysisDuring the course of CUS treatment(35days),animals from both the control group and CUS-treated group were weighed every third day(at09:00h)to monitor their overall health.The difference in weight gain between the two groups was analyzed by single factor(CUS expo-sure)repeated measures ANOVA,and followed by planned comparisons using uncorrected univariate F-tests for between-cell comparisons.2.1.6.Anticipatory USV response testV recording apparatus.The USV recording apparatus consisted of four Plexiglas experimental chambers constructed of2.5cm thick black Plexiglas.After preliminary studies identified a sound echo,a layer of sound absorbing foam approximately5mm thick was applied to the chamber walls which reduced echo to low levels that permitted manual evaluation of the sonogram.Each chamber was equipped with a ventilation fan which also emitted b20dB white noise with no detect-able ultrasonic noise.In addition,each chamber was equipped with a6Wfluorescent light which was masked to produce red light.High-sensitivity ultrasonic microphones(Sonotrack,Metris B.V.,KA Hoofd-dorp,Netherlands)with a frequency response range of15to125kHz were securely placed via small sealed holes in the top panels of each chamber.The main Sonotrack unit was connected to a Dell Computer workstation running the Sonotrack software in a Microsoft Windows environment.The entire apparatus was placed in a dedicated sound-attenuated room.Ultrasonic vocalizations were measured using Sonotrack ultrasonic microphones,bandpassfiltered and digitized by Sonotrack A/D conversion card and stored as.dat-files(internal Sonotrack format for sound data storage)and.wav-files(external stor-age format for media player usage)for later analysis.Sound analysis was performed using Metris Sonotrack software(Sonotrack,Metris B.V.,KA Hoofddorp,Netherlands).Although Sonotrack is equipped for automatic detection of USVs,we found that significant numbers of USVs were missed in a background noise of undetermined origin and therefore decided to complete all analyses manually.Soundfiles were converted to spectrograms for manual visual counts of calls.A sound was considered to be a50kHz USV if it fell between35and70kHz with duration of30–80ms.Calls were considered in the22kHz category if they fell between18and32kHz with duration of300–3000ms.All soundfiles were manually scored by trained experimenters without knowledge of test group assignment.2.1.6.2.Anticipatory USV response testing.Anticipatory Ultrasonic Vocali-zation(USV)response testing was carried out for4days following the 28-day CUS exposure paradigm,and involved both the control and the stressed animal groups.On the day prior to test onset,CUS-treated and control animals were placed individually in the test chamber for30min to habituate them to the testing situation.On test days,experimental subjects(CUS-treated or control)and cage-mate partners were individually isolated in holding cages for4h prior to testing to assess social USVs.On each of the4test days,cage-mates were taken from their holding cages and each animal was placed alone into one of the recording chambers for a5-min period.One control pair and one CUS-treated pair were tested at a time and the assignment of testing chambers was randomized.After5min of recording individual animals alone,the cage-mate partners were reunited in one of their test cham-bers,and the dyad was allowed to interact Vs were record-ed during the anticipatory(5min)period on test days1,2,3,and5and the(5min)social interaction period on test days1and5for this exper-iment and the digitized recordings were saved for later analysis.Day4 of testing is omitted due to construction near the testing area which pre-cluded recording.Unless otherwise stated,all animals were returned to their home-cage immediately after testing on each of the4test days. After examining automatic counting in low,middle and high frequency bands,it became clear that rats were emitting a significant number of calls of low intensity that were not picked up from the background noise and therefore were not automatically counted.We therefore decided to manually count all soundfiles.Initially,individual calls were counted above and below45kHz to represent high frequency (~55kHz)and low frequency(~22kHz)vocalizations.However,close examination of the resulted data set indicated that few if any vocaliza-tions were emitted below40kHz and all were of short duration (b100msec)indicating that they did not have the characteristics of the low frequency vocalizations often attributed to distress.Therefore all vocalizations were counted as high frequency vocalizations.After all soundfiles had been manually scored as described above,a single factor ANOVA was used for statistical analysis followed by planned comparisons using uncorrected univariate F-tests for between cell comparisons.The number of daily vocalizations,particularly among the control groups were fairly variable and so were transformed as log (raw value+1).Aggregate call data over all four days was analyzed with a single factor(CUS)repeated measures ANOVA(trials)followed by comparison between the two groups using a two-tailed Fisher's Exact test.2.1.7.Novelty suppressed feedingThe Novelty Suppressed Feeding(NSF)test was carried out at the end of last andfinal week5of this experiment,following the4days of anticipatory USV response testing.Prior to the NSF test,the rats were food deprived overnight(~16h).On the day of testing,4h after lights off,the animals were presented with a single food pellet(chow)placed in the middle of a novel environment,which was a circular enclosure of 100cm diameter with clean bedding.Each of the rats was placed near the enclosure wall and allowed to explore freely for10min,and the latency to start feeding(during the10min assay)was recorded.The enclosure was thoroughly wiped down between subjects with a10% ethanol/water solution to eliminate scents and olfactory traces.The difference in latency to feed between the two groups was analyzed via Student's t-test,with statistical significance set atαof0.05.2.2.Materials and methods—experiment BThe goal of the second experiment was to establish the effects of CUS on hippocampal-dependent spatial memory performance.2.2.1.Spatial(hippocampal-dependent)memory performance on radial arm maze2.2.1.1.Radial arm maze apparatus.The Plexiglas radial maze was elevat-ed30cm off the ground,and consisted of8arms each measuring 10×80cm with blackflooring and clear walls.Arms were connected by a round central platform35cm in diameter.Each arm contained a de-pressed circular food cup at its terminal end,which was baited during testing with one45mg bacon-flavored sugar pellet(Purina TestDiet130M.S.Riaz et al./Physiology&Behavior152(2015)128–134#1,813,244,Richmond,IN).The principle behind testing on a radial arm maze apparatus is that an animal quickly learns that there is only one pellet in each arm and thus avoids the already visited and de-baited arm.The animal keeps on exploring all the unexplored arms until all of the arms have been visited.The animal uses spatial cues that surround the maze to remember which arms it has entered,and hence radial arm maze testing is used to test for intact spatial memory.2.2.1.2.Spatial working memory training.Prior to CUS treatment,animals were trained on the radial arm maze task for3weeks with one daily trial during the dark cycle.At the beginning of each trial,the rat was placed in the central platform and was allowed to freely move on the maze until either all eight arms were entered at least once,or until300s had passed;whichever camefirst.An arm entry was recorded when all four of the animal's legs had crossed the demarcated threshold of the arm.Performance was scored for the number of correct(unvisited)arm entries in thefirst eight arm visits.Success rate for both training and testing trials was calculated as the percentage of correct entries infirst eight visits(Previously unvisited arms entered/first eight arm visits ×100).All the animals achieved the acquisition criteria of80%success rate by the18th trial(1trial/day)as shown in Fig.6A.2.2.1.3.CUS treatment and spatial working memory testing.Following the training period,animals were assigned to either CUS-treatment (n=16)or no stress control(n=14)groups.Rats in each group were matched as closely as possible for body weight.Animals from both CUS-treated and control groups were tested for spatial working memory performance on the radial arm maze,once every week during the4weeks of CUS treatment.The spatial memory performance over of the course of testing period was analyzed via single factor(CUS)ANOVA with repeated measures(trials),followed by planned comparisons using uncorrected univariate F-tests for between cell comparisons.2.3.Statistical analysisAll data were analyzed as described in2.1.2.,2.1.4.–2.1.7,and2.2.1.3. using IBM SPSS version22.Significance for all tests was defined asαor p value b0.05.3.Results3.1.Experiment A3.1.1.Body weightThe control animals showed significant weight gain,compared to baseline(Day0)from day15to33,whereas no such weight gain was observed in CUS-exposed animals[F(9162)=12.95,p b0.05,Fig.1].Between-group analyses showed that CUS-exposed animals weighed significantly less compared to control animals through Day-26till Day-33(Week-5)of CUS treatment.In addition,weight of CUS animals at Day-33was similar to before CUS,suggesting that they did not gain weight.In contrast,the control animals showed a significant gain in weight at Day-33compared to before CUS[t(19)=2.09,p b0.05,Fig.1].3.1.2.Sucrose preference testCUS-treated rats did not significantly differ from control group in sucrose preference at the end of week-1of CUS treatment,or week-3 of CUS treatment[F(1,38)=0.02,n.s.,Fig.2A-B].3.1.3.Novelty suppressed feeding testCUS-treated rats showed a significant increase in latency to feed dur-ing the testing period compared to the control group[10min;CUS vs. control,[t(19)=2.09,p b0.05,Fig.3].3.1.4.Anticipatory ultrasonic vocalizationsCUS-treated rats produced fewer total anticipatory USVs than con-trol rats through all4days of testing,with a trend nearing significance for between-subjects group effect[F(1,18)=4.115,p=0.058,Fig.4A]. Pair-wise comparisons between the two groups across all4testing days showed a significant reduction in total anticipatory USVs in CUS-treated rats vs.control rats on Day-1of testing[F(1,18)=7.507, p b0.05,Fig.4A],and a trend towards reduction in total anticipatory USVs on Day-3of testing,however the observed difference between the2groups did not achieve statistical significance[F(1,18)=3.595, p=0.08,Fig.4A].During the4testing days,anticipatory USVs were positively recorded for28out of40testing sessions for control animals, while CUS-treated animals emitted detectable anticipatory USVs in only 15out of40testing sessions[Fig.4B].Comparing the2groups for only the testing sessions with detectable emitted USVs using a two-tailed Fischer's Exact test revealed a significant difference in USVproductionFig.1.Weight change:Posthoc analysis showed that control animals gained significantweight,compared to CUS-treated group during the CUS-treatment period(Day-10toDay-33),[F(9,162)=12.95,p b0.05].*p b0.05vs.control.Control animals also showed asignificant weight gain on Day-33compared to pre-CUS Day-0[t(19)=2.09,p b0.05,§].Fig.2.A)Week-1of CUS:CUS-treated rats did not differ from control group in sucrosepreference at the end of week-1of CUS treatment[F(1,38)=0.02,n.s.]B)Week-3ofCUS:CUS-treated rats did not differ from control group in sucrose preference at the endof week-3of CUS treatment either.131M.S.Riaz et al./Physiology&Behavior152(2015)128–134between the two groups p b 0.05,Fig.4B.There were no Main or Inter-action effects on average call duration and even though CUS animals emitted consistently shorter USVs than controls,this did not achieve statistical signi ficance [F (1,18)=3.474,p =0.08].When cage mates were reunited for 5min on the first or fifth testing day,CUS pairs consis-tently emitted less than half the number of calls of unstressed controls [One tailed t (8)=2.128,p =0.033]Fig.5.3.2.Experiment B3.2.1.Effect of CUS on memory retrievalCUS-treated rats showed progressively more errors every week in radial arm maze testing during the 4weeks of CUS treatment compared to the pretreatment baseline.In contrast,the performance of the control animals was unchanged over those four trials when compared to base-line.Although the error rate of CUS-treated rats and control rats did not differ at baseline,by the second week of CUS treatment,there was a signi ficant increase in errors in CUS-treated rats compared to controlrats that persisted throughout the rest of testing.Single factor (Treat-ment group)repeated measures (trials)ANOVA revealed a signi ficant main effect of trials [F (4112)=9.789,p b 0.001]and of group [F (1,28)=9.570,p b 0.001]as well as a treatment group by trials interaction [F (4112)=9.732,p b 0.001,Fig.5B].4.DiscussionThis study used the stress exposure paradigm of CUS,and compared multiple behavioral measures for their predictive ef ficacy as markers of chronic stress.The CUS-treated animals showed attenuation of social interaction associated USVs,attenuated weight gain,suppressed feed-ing when exposed to novelty,and attenuation of spatial memory.The stress-induced reduction in weight gain is in accordance with previous-ly published studies,which show a similar effect [19,20].One possible explanation for this decrease in weight gain could be the use of food deprivation as one of the unpredictable mild stressors.However,not only was food deprivation stress an infrequent event during CUS,evidence suggests that decreased hedonic responsiveness following CUS is not secondary to loss of body weight [21].In the first experiment of this study,there was no reduction in sucrose preference between CUS-treated and control animals.Further-more,the duration of stress exposure did not change sucrose preference as CUS-treated animals showed no change in preference at end either the first week or third week of treatment.Our observation supports pre-vious studies that have reported dif ficulty in eliciting a decreased sucrose preference using multiple variants of chronic stress procedures [11,14,22–24].These inconsistencies have been recorded in chronic stress models of both mice and rats,and such equivocal and even con-tradictory findings weaken the reliability of sucrose preference testing as a behavioral marker of hedonic state [1,25–27].Interestingly,how-ever,recent studies by Wiborg and coworkers [10]suggest that these inconsistencies may result from a differential population change in sucrose preference during CUS exposure with some animals displaying profound reductions,while other display either no or very little re-sponse to stress.If this is replicable,it would lend population validity [28,29]to CUS as a model of depression since not all individuals subject-ed to stress develop major depressive disorders.Sucrose preference testing was used for this study,since previous studies have shown that sucrose consumption is a weak and inconsis-tent index of reward responsiveness in chronic stress paradigm [11].In fact,previous studies show that any observed effect of reduced sucrose consumption in CUS-exposed animals is lost,when corrected for stress-induced weight changes [14].Thus,the sucrosepreferenceFig.3.Novelty suppressed feeding (NSF)test:CUS-treated rats showed a signi ficantly increased latency to feed during the testing period [10min;CUS vs.control (CTRL),t (19)=2.09,p b 0.05,*].Fig.4.A)50-kHz USVs:CUS-treated rats produced signi ficantly fewer anticipatory 50kHz USVs (Band-3;44.5–100kHz)through all 4days of testing [F (1,8)=7.20,p b 0.05,*].B)Total USVs:CUS-treated rats emitted signi ficantly fewer total USVs,with all bands combined [F (1,8)=7.20,p b 0.05,*].Fig.5.50-kHz USVs of reunited cage mates.CUS-exposed rats produced signi ficantly fewer 50kHz USVs than controls (CTRL)(Band 3:44.5–100kHz)when reunited with cage mates on days 1and 5of testing [One tailed t (8)=2.128,p =0.033].132M.S.Riaz et al./Physiology &Behavior 152(2015)128–134。