1 Analysis of parvalbumin-positive interneuron density in auditory and prefrontal cortex in a mouse model of schizophrenia Sandra Saraswati Wenas MSc Neuroscience 2013-2014 Student Number: 13088199 Supervisor: Dr. Jennifer F. Linden UCL Ear Institute Number of Pages: 39 Number of Figures: 11 TOTAL Word Count: 6095 Abstract: 246 Introduction: 747 Methods and Results: 3413 Limitation of Methods: 275 Discussion: 1414 Declaration of ownership:I, Sandra Saraswati Wenas confirm that the work presented in this thesis is my own.Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Perfusion was performed by Dr. Jennifer F. Linden.MatLab code was written by Dr. Jennifer F. Linden and repeated by Sandra S Wenas.Many thanks to Dr. Jennifer F. Linden for her support and guidance during the completion of this project.Many thanks to Dr. Lucy Anderson and Dr. Jennifer Bizley for their advices on immunohistochemistry. 2 Abstract The22q11deletionsyndrome(22q11DS)isaninterstitialchromosomal microdeletion of chromosome 22.Patients with 22q11DS have 25-30% risk factors for schizophrenia.22q11DS is also a risk factor for the development of otitis media (OM)andstudiesin22q11DShaveshownthatamajorityofpatientswiththis syndrome have a history of chronic or recurrent OM.OM is a middle ear disease thatcancauseconductivehearingloss.Changesintheauditorycortexduring developmenthasbeenshowntodisruptexcitation/inhibitionbalance.Recent researchonschizophreniahassuggestedthatabnormalitiesindensityand connectivityofparvalbumin-positive(PV+)interneuronsinthecortexmay contribute to the cognitive deficits that remain the most difficult-to-treat aspect of the disease. Reduction in PV+ interneuron density has also been correlated with hearing loss in a mouse model.We used Df1-knockout mouse (Df1/+) to examine therelationshipbetweenOMandreductionofPV+interneurondensityinthe auditorycortexandprefrontalcortex.TheDf1/+mouseisthegenetically engineered mouse model for human 22q11DS.We found significant reduction in PV+ interneuron density specifically in cortical layers III to VI of the primary auditory cortexofDf1/+mice.Ourdataalsosuggestedacorrelationbetweenboth genotypeandOMwiththereductioninPV+interneurondensityintheauditory cortex of these mice.No significance was shown for data of the prefrontal cortex.However,giventhelimitedprefrontalcortexsectionsthatcouldbeanalyzedwe judged our data to be inconclusive. 3 Introduction 22q11 deletion syndrome (22q11DS) is a relatively common (1:4000 live births) cytogeneticabnormalitythatarisesfrominterstitialchromosomalmicrodeletion on chromosome 22 encompassing at least 30 genes.The syndrome is associated withcardiac,craniofacialandcognitiveabnormalities(Paylor andLindsay,2006).Infact,22q11DSislinkedwithhighfrequencies(80-100%)ofneurocognitive disabilities (Gerdes et al., 1999) and patients with 22q11DS have 25-30% risk factor for schizophrenia, making it one of the most significant cytogenetic risk factors for schizophrenia(PaylorandLindsay,2006).Moreover,withsuchhighlevelofrisk factor,22q11DSisthesecondhighestriskfactorfordevelopingschizophrenia whereonlyhavingamonozygotictwinortwoparentswithschizophreniacan increase the probability (Bassett and Chow, 1999). 22q11DS is also a risk factor for the development of otitis media (OM), a middle ear disease that can cause conductive hearing loss (Reyes et al., 1999).A 30-40% incidence rate of hearing loss has also been shown in 22q11DS (Botto et al., 2003).Studies in 22q11DS have shown that a majority of patients have a history of chronic or recurrent OM (Dyce et al., 2002).In OM with effusion, the Eustachian tube is blocked and fluid cannot be drained out from the middle ear, which disrupts the pressure balance across the tympanic membrane (Yost, 2007). ThehemizygousDf1-knockoutmouse(Df1/+)isthegeneticallyengineered mousemodelforhuman22q11DS.Mousechromosome16isorthologousto chromosome 22 in human.Cre/loxP system has allowed the Df1/+ deletion which encompasses 18 of the 30 genes postulated to be deleted in the syndrome, one of which is Tbx1 (Paylor et al., 2001; Funke et al., 2007).Moreover, cognitive deficits 4 andbehavioralabnormalitiestypicalofschizophreniaandhuman22q11DShave been shown in Df1/+ mice.It has also been shown that approximately half of Df1/+ mice are susceptible to otitis media and conductive hearing loss (Fuchs et al., 2013) Recent research on schizophrenia has suggested that abnormalities in density andconnectivityofparvalbumin-positive(PV+)interneuronsinthecortexmay contribute to the cognitive deficits that remain the most difficult-to-treat aspect of thedisease (Marin,2012).ReductioninPV+interneurondensityhasalsobeen correlated with hearing loss in a mouse model (Martin del Campo et al., 2012). Inhibitoryinterneuronsinthecerebralcortexareinvolvedinlocalneural assembliesbygivinginhibitoryinputsandshapingsynchronizeoscillations (Klausberger and Somogyi, 2008).Parvalbumin-expressing (PV+) interneurons for example appear to drive oscillations in the gamma frequency range (30 80 Hz) as compared to somatostatin-expressing interneurons which seem to be involved in producing the beta-frequency oscillations (15 30 Hz) (Buzsaki and Draguhn, 2004). Ithasbeensuggestedthatexactmodulationandbalanceofexcitatoryand inhibitorysynapticinputsinindividualcellsisessentialforsignalpropagationin processinginformation(VogelsandAbbott,2005).Possibly,themostprofound changestoinhibitorysynapsefunctionareduetodevelopmentalhearingloss.Changestoauditorycortexprocessingduringnormaldevelopmentorafter environmental modifications, including hearing loss are believed to contribute to the modification of intrinsic inhibitory networks (Takesian et al., 2009, Razak et al., 2008).Therefore,itisimportanttounderstandhowinhibitoryneuronsand synapsesarealteredbyhearinglossandhowthesechangesimpacttheneural circuits(Takesianetal.,2009).Moreover,ithasbeensuggestedthatPV+ 5 interneurons in particular are affected in schizophrenia, indicated by a reduction of GAD67inthedorsolateralprefrontalcortexofpatientswithschizophrenia (Hashimoto et al., 2003). Therefore,ourexperimentsaimedtodeterminewhetherthedensityofPV+ interneurons is reduced in the auditory and/or frontal cortex of a mouse model of 22q11DS and schizophrenia, the Df1/+ hemizygous mouse.Here we showed that PV+interneurondensityintheauditorycortexofDf1/+micewassignificantly reduced relative to WT animals.We also showed that this reduction was significant across cortical layers III to VI.To see whether OM can account for the reduction of PV+ interneurons, we examined the correlation between OM and PV+ interneuron densityintheauditoryandprefrontalcortexonthecontralateralandipsilateral brainhemispheres.Ourresultsindicatedthatbothgenotypeandcontralateral hearing loss were associated with reduced PV+ interneuron density in the auditory cortex.Asimilaranalysisintheprefrontalcortexdidnotshowasignificant difference in PV+ interneuron density between Df1/+ mice and WT mice; however less data could be collected in prefrontal than in auditory cortex and therefore only the auditory cortex results could be considered conclusive. 6 Materials and Methods Animals 17 hemizygous Df1/+ mice (10 females, 7 males) were used in this project.TheDf1/+mouselinehadbeenmaintainedonaC57BL/6backgroundfora minimum of 10 generations prior to the analyses.The Df1 deletion was engineered on a 129S5 SvEvBrd genetic background (Lindsay et al., 1999). Perfusion Mice were overdosed with 0.1 ml injection of 200 mg/ml sodium pentobarbital and perfused trancardially using a peristaltic pump with 10 ml of 0.1M autoclaved PBS followed by 30 ml of autoclaved 4% paraformaldehyde (0.1M, pH 7.4).Due to delaysintheissuingofaHomeOfficepersonallicenseforthestudent,all perfusions were performed by the supervisor, Dr. Jennifer Linden. Tympanic Membrane Inspection Tympanic membrane inspection was used to assess OM and conductive hearing loss.Auditory brainstem response was initially considered as the method to assess hearing loss.However, a study done on Df1/+ mice by Fuchs et al., 2013 showed thatseverityofotitismediacorrelatesdirectlywiththeelevatedthresholdof auditory brainstem response.Therefore OM could account for click ABR deficits in Df1/+ mice.Given the time constraints of this MSc research project, we chose to use post-mortem tympanic membrane inspection to assess OM rather than making more time-consuming in-vivo auditory brainstem response measurements. 7 Aftertheanimalwasperfusedwithfixative,theheadwasremovedandthe outerearofbothrightandleftearswerecutaway,exposingthemiddleear.A small pocket microscope was used to examine the tympanic membrane.A scoring systemwasusedwhereobservationofanair-filledvoidbehindthetympanic membrane was scored 0 (no OM), observation of air bubbles behind the tympanic membrane was scored 1 (low-severity OM), and observation of a pus-like substance behind the tympanic membrane was scored 2 (severe OM).Tympanic membrane scoresweredeterminedbothbylookingthroughthetympanicmembraneto detect bubbles or evidence for gray pus rather than a uniformly air-filled cavity, and by puncturing the tympanic membrane with a small pair of scissors to confirmthe presence of air versus fluid in the middle ear.Histology Followingtympanicmembraneinspection,thebrainwasremovedfromthe braincaseandpost-fixedin4%paraformaldehydebeforethenstoredina4C refrigerator. Brainswereprocessedinpairs,eithergender-matched,age-matchedor genotype-matched.Priortocutting,brainswererinsedwith0.1MPB.The olfactory bulb and cerebellum were cut off to better adhere brains to the mounting block.A needle was passed through the right hemisphere from caudal to rostral in ordertodifferentiatetherightandlefthemispheres.Theneedlewaspassed throughthehippocampusandthecortex.Coronalsections,50minthickness werecutusingavibratome.Thebrainwassectionedthoroughlyfromrostralto caudal as we aimed to get the prefrontal cortex and the auditory cortex.Sections werealternatelyplacedeitheronslidesforNisslstainingorintowellplates 8 containing0.1MPBSforParvalbuminstaining.SlidesforNisslstainingwereair-driedovernightonahotplate,whereasthewellplateswerestoredinthe4C refrigerator.Nissl substance was revealed using cresyl violet. Immunohistochemistrywasdonewithfree-floatingsectionsandalwayswith agitation on a shaker, either in the refrigerator or at room temperature.Sections remained in the same well plates throughout all steps.This was done to minimize the risk of tearing the sections through lifting and handling.Not lifting the sections also kept them in their initial orientations thus minimizing the risk of flipping the right and the left hemispheres.At this point it is worth mentioning that in the effort to wash sections properly, the sections of the first two animals used in this study weremovedfromonewellplatestoanotherwhichresultedinsomesections breaking and became unusable.It was then decided that sections would stay in the samewellplatesfromthebeginningtotheend.Washingstepsweredone frequentlyandthoroughlywiththehelpofamicropipettetoensurethatthere werenoleftoversolutionbeforeaddinganewsolutioncontainingeitherthe primary antibody, secondary antibody or 0.1M PBS. Sections were pretreated with 500 l of Triton in 0.1M PBS for 20 minutes, at room temperature.Sections were then blocked with a block mix made up of 6 ml of 0.1M PBS, 3 ml 0.5% Triton and 1 ml goat serum.These were incubated for 40 minutes,atroomtemperature.Sectionswerethenincubatedovernightina solution containing mouse anti-PV (1:8000, Sigma-Aldrich) and block mix, in a 4C refrigerator.Following overnight incubation, sections were rinsed in 0.1M PBS for 5 x 5 minutes.Sections were incubated in a solution containing Goat-Anti Mouse IgG (1:200, Sigma Aldrich) and block mixfor 1 hour at room temperature, at this 9 point the well plates were covered with Styrofoam lids to avoid exposure to light. Sections were rinsed in 0.1M PBS for 5 x 5 minutes, also covered with Styrofoam lids. Prior to mounting, sections were rinsed with dH2O before then transferred to slides, mounted with FLuoromount-G (Southernbiotech) and cover slipped.Slides werestoredatroomtemperatureandcoveredwithaluminumfoiltoavoid exposure to light. Image Processing Individualsectionswerestudiedatmagnificationsrangingfromx5tox10on Zeiss AxioPlan 2 Imaging microscope.Images were taken at x5 for the purpose of identifying the locations of auditory and prefrontal cortex whereas images taken at x10 were used for cell counting.Images for cell counting were taken at x10 so that all six cortical layers between pia and the white matter would be captured.Images from both the right and left hemispheres were taken. Nissl-stainedsectionswereusedto identifyauditoryandprefrontalcortexon eachcoronalsectionusingthemouseatlasofFranklinandPaxinos(1996), particularly by examining the structure of the hippocampus in the coronal sections.Primaryauditorycortex(A1)onthisatlaslieswithinBregma-2.18to-3.64mm anterior-posterior (i.e., 2.18 to 3.64 mm posterior to Bregma).For consistency and to avoid inclusion of region of the primary sensory cortex (S1), we took into account sections identified as Bregma -2.54 mm.We aimed to take the region of auditory cortex that was more posterior to ensure enough distance from the barrel cortex of S1.Primary auditory cortex was identified by an increase in cortical thickness, changes in cell density most prominently on cortical layer IV and position relative tothehippocampus(Figure1A).ThelocationofauditorycortexonthePV+ 10 sectionswereidentifiedbycomparinghippocampallandmarkstotheadjacent Nissl-stainedsections(Figure1B).Inthechosenexamples,theangleonwhich sectionsweremountedweredifferentbetweenPV+sectionsandNissl-stained sections.The long horizontal line extends from the hippocampus to the center of the auditory cortex thus was used as an approximation to adjust the position and orientation of the rectangle which marks the auditory cortex.PrefrontalcortexwasidentifiedbythepositionsofCingulatecortex,area1 (Cg1),Infralimbiccortex(IL)andPrelimbiccortex(PrL)(Figure2A).Accordingto the mouse atlas by Franklin and Paxinos (1996) this lies within Bregma 1.42 to 2.10 mm.Sections that we took into account for cell counting were identified as Bregma 1.70 mm. ImageJ Software was used to enhance the contrast of the image in order to view thecellsmoreclearlyformanualcounting.Thewidthofcountedareaforthe auditorycortexwasapproximately600mwhereasthewholeprefrontalcortex was taken into account in the counting. Manual counting was performed blind to genotype status of the animals.To analyze the location of cells within the cortical height from pia to the white matter, distance was measured from the pial surface using the straight line selection tool and the built-in routine for measuring distance within ImageJ, for both auditory and prefrontal cortex.Distances were normalized by the total pia-to-white-matter distance for each section.Data from ImageJ was then transferred to Microsoft Excel. 11 A. B. Figure 1: Identifying the auditory cortex. (A) Nissl-stained coronal section. The long horizontal line extends from the hippocampus to the center of the auditory cortex sampled for counting.Scale bar: 500 m. (B) Coronal section stained for PV located adjacent to the Nissl-stained section in (A).Scale bar: 500 m. 12 A. B. Figure2: Identifying the prefrontal cortex.(A) Nissl-stained coronal section. Prefrontal cortex areaisidentified as the locations of Cg1, PrL and IL.Scale bar: 300 m. (B) Coronal section stained for PV located adjacent to the Nissl- stained section in (A).Scale bar: 300 m. 13 Data Analysis AlldatawasreadfromMicrosoftExcelspreadsheetsintoMatLab,whichwas used to plot and analyze the data.The Wilcoxon rank-sum test was used to test for statistical significance.P-values are stated in full in the text and figures, and were considered significant if below 0.05. Limitation of Methods Animals Although we managed to yield preliminary data with statistical significance for the auditory cortex, it would have been more ideal to use a bigger sample size.We aim to process more animals in order to yield enough data for publication. Tympanic Membrane Inspection IthasbeensuggestedthatOMcanaccountfortheelevatedclickevokedABR thresholdinDf1/+mice(Fuchsetal.,2013).Ourdatawouldhavebeenmore comprehensiveifweweretocombineboththeABRandtympanicmembrane inspection.AnothermethodthatcouldhavebeenconsideredwasPreyerreflex whichisaflickofthepinnaeevokedbyatransientloudsound,asthismethod wouldtakeshortertimethanABR.However,althougheffectiveforidentifying profound sensorineural hearing loss, Preyer reflex might not be sensitive enough to detect less severe auditory dysfunction. 14 Sectioning the Prefrontal Cortex Cryostatwouldperhapsbeabettermethodinsectioningespeciallyforthe prefrontal cortex.As the tissue can be snap-freeze within the mounting medium thus giving it something to adhere to.We imagine that we would be able to yield better sections with prefrontal cortex with this method. Cell Counting Cell counting was done manually thus may not be free from human error.However the counting was performed blind to the genotype of the animal in each sample, andthereforeanyerrorsshouldnothaveaffectedtheexperimentalresultsfor comparisonofDf1/+andWTmice.Forthepublication,weaimtoanalyzedata from the remaining samples with automated counting, as an additional check.This will be done through collaboration with Gatsby Computational Neuroscience Unit, University College London. 15 Results Tympanic Membrane Inspection 17animalsunderwenttympanicmembraneinspectiondonebytwo investigators for cross confirmation (Table 1).We compared the appearance of the tympanic membrane with photos from a previous study as shown in Figure 3A C (courtesy of Jennifer Fuchs and Abigail Tucker, Kings College London). A.B.C.Figure3:Photosshowingthetympanicmembrane.(A)Normalmiddleearwithair-filledspacebehindthe tympanic membrane, scored: 0. (B) Middle ear with air bubbles behind the tympanic membrane, scored: 1. (C) Middle ear with effusion or pus-like substance behind the tympanic membrane, scored: 2. Photos courtesy of Jennifer Fuchs and Abigail Tucker, Kings College London. 16 Animal IDGenderAge (Weeks) GenotypeRight Ear Score Left Ear Score M10038M8WT00 M10039F12HET10 M10040F12WT00 M10041F12HET21 M10042F12HET00 M10043M16WT00 M10044M16WT00 M10045M16HET02 M10046M16HET20 M10047M16WT00 M10048M12WT00 M10051F8WT01 M10052F8HET00 M10053F8WT00 M10054F8HET00 M10055F8WT00 M10056F8WT00 Table 1: Tympanic membrane scores on both ears of Df1/+ mice and their WT littermates. Out of 10 wild type mice, 9 mice showed no signs of otitis media whereas 1 mouse (animal ID M10051) clearly had air bubbles in the left ear.Out of 7 HET mice, 3 showed no signs of otitis media, 3 had otitis media on one ear and 1 had otitis media on both ears with different levels of severity. Histology Brains from the first 11 mice (M10038 M10048) were further processed for this project while brains from the rest of the mice were stored.We aimed to obtainpreliminarydatafromthefirst11micewhichwehopedwouldbe 17 informative enough to justify further study and to set the right questions to be asked; however due to time constraints on the project only data from the first 11 mice could ultimately be analyzed for this report. ThefirsttwoWTbrainsthatwereprocessed(M10038andM10043)were unusableasthePV+sectionswerenotrinsedwithdH2Opriortomounting therefore did not adhere and were either lost or stacked on top of the other.The remaining few sections that adhered were not usable for counting but were enough to show that there was a high background staining.Therefore, with the remaining9brains,brainfromanimalIDM10048wasusedtooptimizethe stainingthroughatrialofdifferentprimaryandsecondaryantibodydilution.Forconsistency,sectionsthatweretakenintoaccountforcountingofanimal M10048 were only those sections processed with the antibody dilution chosen for further experiments (1:8000 for primary antibody and 1:200 for secondary antibody). Image Processing Auditory Cortex Fluorescence microscopy on auditory cortex comparing WT, HET without OM andHETwithOM(ononeear)mice(Figures4A-C)showedanapparent reduction of PV+ interneuron cells in HET animals with OM.Observation by eye couldnotdistinguishanyapparentdifferencebetweenWTandHETwithout OM,althoughthereseemstobefewercellsonthelater.Whetherthis difference was significant would be further analyzed. 18 A. B. C. Figure 4. PV+ cells in the auditory cortex. (A) WT mouse with no OM. (B) Df1/+ mouse with no OM. (C) Df1/+ mouse with OM scored 2. Scale bar: 300 m 19 Prefrontal Cortex Outofbrainsfrom9micethatwentthroughthewholehistologyprocess, only 7 could be imaged for prefrontal cortex.The size of the section that lies withinBregma1.70mmlevelisrelativelysmall,thereforesectioningwitha vibratome was not an ideal method.At this level of Bregma, sections tend to not hold together and separated during cutting thus making it difficult to mount with the right orientation on slides.Some sections were also damaged by the vibratome blade mostly at the top middle part were the prefrontal cortex lies.A different approach such as snap-freezing the brain on a mounting block and cuttingwithcryostatwouldbemoreideal.Thisapproachwouldallowsmall sectionofthebraintoatleastadheretothefrozenmountingblockduring cutting. We could not obtain good sections for prefrontal cortex from animals M10041 and M10044.Animal M10044 in particular was not well perfused. Fluorescence microscopy onprefrontal cortex comparing WT, HET without OMandHETwithOM(ononeear)didnotseemtoshowanyapparent difference between WT and HET either with or without OM (Figure 5A and B). Figure B showed the prefrontal cortex taken from a HET mouse with OM on 1 ear and none on the other, observation by eye suggested no difference in PV+ interneuron density between the two hemispheres.This observation however would be further analyzed with MatLab. 20 A. B. Figure 5. PV+ cells in the prefrontal cortex (A) WT with no OM. (B) Df1/+ with OM on the right ear scored 1 and no OM on the left ear.Scale bar: 300 m 21 Data Analysis Auditory Cortex Sectionsfrom9brainswereusedforcellcounting.Dataoncellcounts consistedofthenumberofPV+interneuroncellsfromeachhemisphereand their location in respect to the cortical depth from pia to white matter, for each animal.A box plot (Figure 6) comparing the total cell counts of PV+ interneuron showedsignificantreductioninDf1/+mice(Wilcoxonrank-sumtestfor differenceinmediancellcount,p=0.00297),confirmingwhatisevidentin example sections in Figure 4. We were interested to see how much of PV+ interneuron reduction can be accountedbyhearingloss.Cumulativecellcounts(Figure7A)wereusedto checkforcorrelationbetweentympanicmembrane(TM)scores(ourindexof OM) and PV+ interneuron cell density in the auditory cortex. Severity of OM was differentiatedbythedifferentweightofthelinesusedforthecumulative distribution, with the thinnest line indicating HET or WT with no OM (TM score =0forbothears),themediumlineindicatingHETwithOMseverityof1 (maximum TM score = 1) and the thickest line indicating HET with OM severity of 2 (maximum TM score = 2).Note that there were no WT mice in this data subset with TM scores above 0.The graph suggested that the PV+ interneuron density in the WT tend to be higher than that of HET in agreement to what was shown by the box plot; however there was no obvious impact of OM severity when only maximum TM score across both ears was taken into account. 22 Figure 6:Boxplot comparing the total cell counts in the auditory cortex between WT (in blue) and HET (in red) TofindoutwhetherPV+interneurondensitydependedspecificallyonthe OMstatusofthecontralateral(oripsilateral)ear,weplottedthecumulative histograms for each hemisphere separately, and used line widths to indicate the TMscoreinthecontralateralearonly(ratherthanthemaximumTMscore across both contralateral and ipsilateral ears, as in Figure 7A). We were interested to see if the genotype and OM status observed in HET could be explained by the PV+ interneuron density of the contralateral auditory cortex.Figure 7B suggested a correlation between OM in the middle ear of HET mice to the auditory cortex in the contralateral brain hemisphere.Comparison of HET mice with different severity of OM on the right ears for example, showed that PV+ interneuron cell density on the auditory cortex in the left hemisphere 23 was higher for HET mice with OM severity of 1 than 2.The same pattern was evidentinthegraphshowingthecellcountsfromtherighthemisphere.Therefore,althoughtherewasnoapparentdependenceofcumulativecell counts on maximum TM score across ears (Figure 7A), the cumulative cell counts ineachhemisphereappearedtobelowerwhentheTMscoreinthe contralateral ear was higher (Figure 7B). To check the specificity for the contralateral ear, we also replotted the data with line widths reflecting TM score in the ipsilateral ear (Figure 7C).Our data suggestednocorrelationbetweenPV+interneurondensityintheauditory cortex and TM score / OM severity in the ipsilateral ear (Figure 7C). A. 24 B. C. Figure 7: Cumulative cell counts showing PV+ interneuron cell counts in the auditory cortex and TM score / OM status plotted on normalized distance from pia to white matter (WM).WT is shown in blue whereas HET is shown in red. (A) Total cumulative cell counts showing total cell counts of both right and left hemispheres.Line width indicates maximum TMscore(i.e.,mostsevereOMstatus)foreitherear.(B)Cumulativecellcountsfromeachhemisphereshown separately, with line width indicating OM status for the contralateral ear. 25 To assess the effects of hearing loss versus genotype on total cell counts, we generatedascatterplotcomparingnumberofPV+cellsontheauditorycortex versus the TM score of both ears, contralateral ear or ipsilateral ear.We aimed to see how much hearing loss accounts for the difference in cell counts.The scatter plotinFigure8showedthatthecellcountsarelowestwhenthecontralateral tympanic membrane score is > 0.Rank-sum test also showed significant differences in cell counts between WT with all TM = 0 versus HET contralateral TM = 0, WT with all TM = 0 versus HET contralateral TM > 0, WT with all TM = 0 versus HET ipsilateral TM = 0 and WT with all TM = 0 versus HET ipsilateral TM > 0 (Wilcoxon rank-sum testfordifferenceinmediancellcount,p=0.028,p=0.0109,p=0.0109,and p=0.028).Whereas comparison of WT with all TM = 0 versus HET where all TM=0 didnotshowstatisticalsignificance(p=0.0889).ComparisonbetweenHET contralateral TM = 0 versus HET contralateral TM > 0 showed that there is a trend forgenotypeandcontralateralhearinglosshavinganeffect(p=0.0556).Comparison between HET ipsilateral TM = 0 versus HET ipsilateral TM > 0 as well as HETcontralateralTM>0andHETipsilateralTM>0showednosignificant difference(p=0.333,p=0.246).Thesesuggestedthatthereareeffectsofboth contralateral and ipsilateral hearing loss and possibly also of genotype.However, we have 1 animal where the TM scores for both ears = 0 and the cell counts tend to be lower than that of WT with all TM = 0.Therefore we cannot say that there is noeffectofgenotype.Although,sincethereisonlyoneHETwithTM=0inboth ipsilateral and contralateral ears, we could not draw a conclusion.26 Figure 8: Scatter plot comparing TM scores to number of PV+ cells. By referring to a study done by Anderson et al., 2009 on laminar distribution of mouse auditory cortex, we were able to determine the location of cells across the6corticallayers.Theboundariesoflayersweredeterminedasthe approximatepercentageofcorticaldepthfromthepiatothewhitematter; layer I= 0 8.5%, layer II= 8.5 20%, layer III= 20 37.5%, layer IV= 37.5 50%, layer V= 50 70% and layer VI= 70 100% (Anderson et al., 2009). As shown in the box plot of Figure 9, there were almost no PV+ interneuron cellsinlayerIofauditorycortexandveryfewinlayerII.Mostcellswerein layers III-VI.Figure 8 also shows that there was a clear difference of cell counts between WT and HET in the layers with the highest PV+ interneuron density in WT animals.Cell counts of WT were higher than HET across layers and these 27 differences were significant from layer III to VI (rank-sum test for difference in medians:p=0.0139,p=0.00233,p=0.00932,p=0.00302,forlayerIII-VI respectively).In agreement with the observation from Figure 7A, the difference incellcountsbetweenWTandHETwassignificantstartingfromlayerIII onwards. Figure 9: Box plot showing number of PV+ cells from layer I-VI of the auditory cortex.Significant difference in median cell counts seen in layer III-VI. Prefrontal Cortex A comparison of the total counts of PV+ interneurons in the prefrontal cortex showedthattherewasnosignificantreductionintheDf1/+mice(rank-sum test on medians, p=0.396) (Figure 10).Cumulative cell counts (Figure 11A) was 28 performedtocheckforcorrelationbetweenOMandPV+interneuroncell density in the prefrontal cortex with the same approach that was done for the auditory cortex.The difference between WT and HET was not as apparent as thatoftheauditorycortex.Therewasanoverlapincumulativecellcount between WT and HET with OM severity of 1 and2, whereas HET with noOM showed the lowest cell count. Correlation between genotype, OM status and PV+ interneuron cell density in the prefrontal cortex of the contralateral brain hemisphere was also analyzed (Figure.11B)Asopposedtodataobservedfromtheauditorycortex,there seemstobenocorrelationineitherhemispherebetweenPV+interneuron density and either genotype or TM score / OM status of the contralateral ear.Data on the left hemisphere for example showed cell counts of HET mice with TM score / OM severity of 2 on the right ear that were almost as high as that of WT mice.Whereas cell counts from some WT mice were as low as those of HET miceeitherwithorwithoutOM.Similarly,therewasnoclearcorrelation between PV+ interneuron density and either genotype or OM status observed in data of the right hemisphere. To check if there was a possible ipsilateral correlation, we ran a second test comparing genotype and OM status to the PV+ interneuron cell density of the prefrontal cortex on the ipsilateral brain hemisphere; the result of this analysis was also negative (Figure 11C). 29 Figure 10: Boxplot comparing the total cell counts in the prefrontal cortex between wild type (WT) and Df1/+ (HET) A. 30 B.C. Figure 11: Cumulative distributions of PV+ interneuron cell counts in the prefrontal cortex as a function of normalized distance from pia to white matter (WM).WT is shown in blue whereas HET is shown in red. (A) Total cumulative cell counts showing total cell counts in both right and left hemispheres.Line width indicates maximum TM score / OM severity across both ears.(B) Cumulative cell counts from each hemisphere with line width indicating OM status from the contralateral ear.(C) Cumulative cell counts from each hemisphere with line width indicating OM status from the ipsilateral ear. 31 Cell counts on the six cortical layers of the prefrontal cortex of WT and HET mice werealsocompared(Figure12).Thelayerboundariesoftheauditorycortexas explained by Anderson et al., 2009 were applied to the prefrontal cortex; while the layer boundaries may be different in prefrontal cortex we decided to use the same normalizedborderstofacilitatecomparisonbetweenthetwobrainareas.In agreement with Figure 11, there was no significant difference in PV+ interneuron cell density between the WT and HET across all layers. Figure 12: Box plot showing number of PV+ cells from layers I-VI of the prefrontal cortex.No significant differences were observed in any of the six layers. 32 Discussion Numbers of PV-expressing cells are significantly reduced in the auditory cortex of Df1/+ mice Toourknowledge,thisisthefirststudytospecificallylookatPV+ interneuron density inDf1/+ mice. Themain finding of this study is a significant reductioninthenumberofcellsexpressingPVintheauditorycortexofDf1/+ mouse, the mouse model for schizophrenia.We found significant reduction in PV+ interneurondensityspecificallyincorticallayersIIItoVIoftheprimaryauditory cortex,A1.A1istheonlyregionoftheauditorycortexthathasbeenstudiedin terms of PV expression with aging and hearing loss.Such study for example was conducted in a prebyscusis (are-related hearing loss) rat model showing a decline in PV+ neurons in the A1 region (Martin del Campo et al., 2012). We were interested to divide cell counts across the cortical layers as other studies have shown that auditory response selectivity changes with cortical depth, reflectingdifferencesininhibitorycircuitryacrossdepth(Atencioetal.,2009; Trujillo et al., 2011).It has been suggested that temporal response properties of neuronsvarywithcorticaldepthinmouseprimaryauditorycortexandanterior auditoryfield(Christiansonetal.,2011).Inthisstudy,neuralresponsesacross corticaldepthintheprimaryauditorycortexandanteriorauditoryfieldwere simultaneouslyrecorded.Systemicdepthdependenciesinresponsetosecond-and-laternoiseburstsinslow(1-10bursts)trainsofnoiseburstswereevident.Responsestonoiseburstswithinatrainatalldepthsdecreasedwithincreasing train rate, however the rolloff with increasing train rate happened at faster rates in themoresuperficiallayers(Christiansonetal.,2011).Hughesetal.,2010also 33 showed that differences in responses to broadband noise between young and old ratinA1neuronswerelayer-specific,suggestinglayer-specificsusceptibilitiesof inhibitoryneurotransmissionwithage.Moreover,thereseemstobegreater reductionininhibitioninsuperficialthandeepercorticallayers.Layer-specific susceptibilityofPV+cellsintheauditorycortexofagingC57micehasalsobeen shown in a study done by Martin del Campo and colleagues (2012).In this study by Martin del Campo and colleagues (2012), significant reduction in PV+ expressing cells of old mice compared to young mice was seen in layer I-IV with no change in layer V-VI.On the contrary, our data on Df1/+ mice show significant reduction in PV+ expression cells in layer III-VI of the auditory cortex.Differences in cell counts wereinfactwashighlysignificantinlayerV-VI.Therefore,contrarytoprevious findings,ourdatasuggestgreaterreductionininhibitionindeeperratherthan superficialcorticallayers.Thisdiscrepancymightbeduetothedifferentmouse models, Hughes et al., 2010 and Martin del Campo et al., (2012) worked with mouse model for hearing loss due to aging whereas our mouse is a model for hearing loss during development. Otitis Media contributes to reduction in number of PV-expressing cells in the auditory cortex of Df1/+ mice InthestudydonebyMartindelCampoetal.,2012,thelayer-specific differenceinPV-expressingcellsinA1werenotage-dependent,assimilarcell countsacrosslayerswereshownacrossagegroups.Theirdatadidnotallow disambiguation of age versus hearing loss as the factor underlying reduction in the number of PV-expressing cells.Data in the Fischer 344 rat A1, a strain susceptible to hearing loss, show a decline in cortical PV expression with age.There was no age-related decline in the Long-Evans rat strain in which accelerated hearing loss is 34 notseen(Oudaetal.,2008).Totheextentthatthegeneticsofdifferentstrains was not a factor, this indicateshearing loss dependent changes in activity levels, and not age, was the driving factor in reduced PV expression. Our data suggested that there was a correlation between OM and reduction in PV+ interneuron density, another important finding in this study.Therefore, in agreement to previous suggestions, reduction in PV+ interneuron in the auditory cortexisnotonlydrivenbygenotypebutseemstobeexperience-dependent,in this case associated with hearing loss due to middle ear infection. Click-evokedABRthresholdswassignificantlyelevatedin40%ofDf1/+ animals, often in only one ear (Fuchs et al., 2013).The deficit was monolateral in most cases.This finding is suggestive of a conductive hearing loss because most causesofsensorineuralhearinglosswouldbeexpectedtoaffectbothears. Consistent with this observation, Fuchs et al. 2013 showed a correlation between click ABR threshold deficits and severity of OM across Df1/+ mouse ears, suggesting thathearinglossinDf1/+micearisesfromfrequentlymonolateralOM.In agreement to this suggestion, all but 1 of the Df1/+ mice assessed in our tympanic membrane inspection showed evidence for OM in only 1 ear. NodifferenceinnumberPV-expressingcellsintheprefrontalcortex between Df1/+ and WT mice It has been suggested that gamma frequency oscillations are abnormal in the prefrontalcortexofpatientswithschizophreniawhoareperformingworking memorytasks(UhlhaasandSinger,2010).Slightdifferencesinthetimingof excitation and inhibition can significantly hamper signal gating and lead to gating defects related to schizophrenia and autism (Vogels and Abbott, 2009). 35 ReductioninPV+interneuronactivityinschizophrenia,mightalsodisturb inhibitory transmission from interneurons to pyramidal cells.A reduced excitatory drivetoPV+interneuronsconsistentlycauseareductioninGAD67mRNAlevels, whichmightinturncontributetoreducedactivityofthispopulationof interneurons(Carlenetal.,2011;Belforteetal.,2010).Moreover,ithasbeen suggested that PV+ interneurons in the dorsolateral prefrontal cortex of adults with schizophreniashowadecreaseintheexpressionofGAD67(Hashimotoetal., 2003).With these previous suggestions in mind, we expected to see a reduction of PV+ interneuron numbers in the prefrontal cortex of Df1/+ mice.However this was not what our data suggested.Given the small sample size and the fact that we have limitedwell-sectionedprefrontalcortexsamplestoimageandcount,wejudged our data to be inconclusive. No correlation between Otitis Media and reduction in numbers of PV-expressing cells in the prefrontal cortex of Df1/+ mice Developmental hearing loss although in general originates in the periphery, may lead to central deficits that persist even after peripheral function is restored.One class of deficits is due to changes in central inhibitory synapses.Inhibitory synapse dysfunction may lead to psychiatric disorders, such as schizophrenia.(Sanes et al., 2009).Moreover,thetemporallobe,whichislocateddirectlyabovethemiddle ear, has been implicated in the neuropathology of schizophrenia.It was suggested that middle ear disease might predispose to schizophrenia through inflammatory damage to the overlying temporal lobe (Mason et al., 2008). 36 Studyingtheratesofbilateral,rightandleft-sidedmiddleeardiseasepre-datingtheonsetofschizophreniainthecaseandcontrolgroupsrevealsa particularlystrikingincreaseintheoddsratio(Masonetal.,2008).Moreover, auditoryhallucinationsaresignificantlyassociatedwithmiddleeardiseasepre-dating schizophrenia, and this is particularly striking for middle ear disease on the side of cerebral dominance, adding to the speculation that damage to the overlying temporallobemaybeofetiologicalsignificance.Itisthenreasonableto hypothesizethatseveremiddle-eardiseasemayincreasetheriskofaperson developing schizophrenia later in life, or that middle-ear disease and schizophrenia may share some etiological factors (Mason et al., 2008). Aspreviouslymentioned,weexpectedsignificantreductioninPV+ interneuron numbers in the prefrontal cortex of our Df1/+ mice, however our data couldnotproveastatisticallysignificantreduction.Inthecaseofcorrelation between OM and reduction in PV-expressing cell numbers in the prefrontal cortex, we expected to see at least a trend which did not have to be necessarily significant aswedidnotexpecttoseeasmuchcorrelationasbetweenOMandPV+cell numbers in auditory cortex. However, our data suggested no trend or correlation between OM and PV+ interneuron reduction in the prefrontal cortex of Df1/+ mice. 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