nn.2866 synaptogenesis ltp and spatial memory

8/4/2019 Nn.2866 Synaptogenesis LTP and Spatial Memory

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nature neurOSCIenCe VOLUME 14 | NUMBER 8 | AUGUST 2011 1 0 0 1

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The genetic an epigenetic mechanisms un er ying the eve opment,maintenance an function of the mamma ian nervous system havebeen extensive y stu ie over the years. To un erstan what func-tiona ro e DNA methy ation epen ent epigenetic mechanisms may have in any of these bio ogica processes, it is important to stu y how DNA methy ation an the recognition of DNA methy ation by proteins containing the methy DNAbin ing omain (MBDs) may be ynamica y regu ate in neurona nuc ei in response to extra-ce u ar signa s. A though there is pharmaco ogica 1,2 an genetic 3

evi ence that ynamic DNA methy ation is require for earning anmemory, re ative y itt e is known about how bin ing to methy ateDNA by the MBDs may be ynamica y regu ate to support normabrain functions.

As the foun ing member of the MBD gene fami y, Mecp2enco esa protein that specifica y bin s to methy ate DNA an repressestranscription 4,5. Ear ier stu ies have shown that MeCP2 interactswith the transcriptiona repressor mSin3A an histone eacety ases 6,suggesting that it may function as a mo ecu ar inker between DNAmethy ation, chromatin remo e ing an subsequent gene si encing 7.However, recent stu ies have foun that MeCP2 may activate the tran-scription of some genes whi e repressing transcription of others 8,9.Mutations in the X- inke human MECP2 gene ea to Rett syn rome(RTT) 1013, an autism-spectrum neuro eve opmenta isor er thatpre ominant y affects fema es 14,15. At an estimate preva ence of 1in 10,00015,000 gir s, RTT is the secon most common X- inkementa retar ation in fema es.

To better un erstan the functions of MeCP2 an to eterminethe mo ecu ar mechanism of RTT, it is important to stu y both howMeCP2 ynamica y regu ates neurona gene transcription in responseto iverse extrace u ar stimu i an the physio ogica importance of such regu ation. The most common extrace u ar stimu us in the brain

is neurona activity. Two ear ier in vitro stu ies in rat neurons founthat NAIP at serine 421 (S421) prece es the re ease of MeCP2 fromthe neurona specific promoter of the Bdnf gene an the subsequentexpression of BDNF16,17. Recent y, a more etai e ana ysis of themouse brain by mass spectrometry i entifie NAIP at two serines,S421 an S424, of the MeCP2 protein 18. Moreover, both sensory inputs an psychostimu ants have been shown to in uce phosphor-y ation of MeCP2 at S421 in the intact mouse brain 17,19. Co ective y,these fin ings raise the possibi ity that NAIP of MeCP2 may serve

as a mo ecu ar switch on the chromatin for ynamica y mo u atingneurona functions.To etermine what in vivo function requires NAIP of MeCP2, we

generate knock-in mice that carry point mutations in the en ogenous Mecp2gene ocus that abo ish phosphory ation at both S421 an S424( Mecp2S421A;S424A/ymice). We foun that phosphory ation at S421cou be in uce by epo arization, high-frequency e ectrica stimu-ation an behaviora training in the hippocampus of wi -type mice,an that the Mecp2S421A;S424A/ymice acke phosphory ation at S421 inthe brain at both the base ine eve an after seizure. Mecp2S421A;S424A/y mice performe better in two hippocampus- epen ent earning anmemory tests than their wi -type ittermates, presente enhanceong-term potentiation (LTP) at both the Schaffer co atera CA1 syn-apses an the mossy fiberCA3 synapses, ha increase excitatory synaptogenesis an showe correspon ing gene expression changesthat are consistent with these phenotypes. Fina y, to better quantify the promoter occupancy of the phospho-mutant MeCP2 protein, wegenerate two Mecp2 knock-in mice: Flag-Mecp2S421A;S424A/ymiceexpressing F ag-tagge mutant MeCP2 from the en ogenous Mecp2 ocus an Flag-Mecp2+/y mice expressing F ag-tagge wi -typeMeCP2 protein from the en ogenous Mecp2 ocus. We foun thatthe F ag-tagge mutant MeCP2 boun more tight y to known MeCP2

1 Genetics Training Program, University o Wisconsin-Madison, Madison, Wisconsin, USA. 2 Waisman Center, University o Wisconsin-Madison, Madison, Wisconsin,USA. 3 Department o Genetics and Neurology, University o Wisconsin-Madison, Madison, Wisconsin, USA. Correspondence should be addressed toQ.C. ([email protected]).

Received 29 April; accepted 31 May; published online 17 July 2011; doi:10.1038/nn.2866

Loss of activity-induced phosphorylation of MeCP2enhances synaptogenesis, LTP and spatial memoryHongda Li 1,2, Xiaofen Zhong 2, Kevin Fongching Chau 2, Emily Cunningham Williams 1,2 & Qiang Chang 13

DNA methylationdependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2is highly expressed in neurons and functions as a molecular linker between DNA methylation, chromatin remodeling andtranscription regulation. Previous in vitro studies have shown that neuronal activityinduced phosphorylation (NAIP) of methylCpGbinding protein 2 (MeCP2) precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, thein vivo function of this phosphorylation event remains elusive. We generated knock-in mice that lack NAIP of MeCP2 and foundthat they performed better in hippocampus-dependent memory tests, presented enhanced long-term potentiation at two synapses

in the hippocampus and showed increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 proteinbound more tightly to several MeCP2 target gene promoters and altered the expression of these genes. Our results suggest thatNAIP of MeCP2 is required for modulating dynamic functions of the adult mouse brain.
http://www.nature.com/doifinder/10.1038/nn.2866http://www.nature.com/doifinder/10.1038/nn.2866


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target gene promoters than F ag-tagge wi -type MeCP2, in icatingthat the phospho- ea form of MeCP2 may function as a hypermorph.Our fin ings provi e, to the best of our know e ge, the first geneticevi ence that NAIP of MeCP2 is require for the eve opment anfunction of the a u t mouse brain. These resu ts may be re evant forun erstan ing RTT isease mechanisms. Moreover, our resu ts ato the emerging picture of epigenetic regu ation of nervous systemfunction, which inc u es not on y changes in DNA methy ation itse f,but a so a critica ro e for stimu us- epen ent phosphory ation of amethy DNAbin ing protein.

RESULTSLoss of NAIP of MeCP2 in Mecp2 S421A;S424A/y miceTo compare the eve of S421 phosphory ation in the si ent hippo-campus with that in the active hippocampus, we treate fresh y prepare hippocampa s ices with either tetra otoxin (TTX, whichsuppresses neurona activity) or KC (which epo arizes neurons)an staine s ices with both phospho-S421 (p-S421)-specific anti-bo y an an antibo y to NeuN (a neurona marker). The intensity of p-S421 signa was marke y higher in the KC -treate s ices thanin the TTX-treate s ices ( Fi . 1 ), in icating that S421 phosphory a-tion was in uce by epo arization. As a contro , the intensity of theNeuN signa was the same in KC - an TTX-treate s ices ( Fi . 1a ).We next teste whether phosphory ation of S421 cou be in uceby more physio ogica y re evant stimu i. High-frequency e ectricastimu ation (HFS) is common y use to in uce LTP in hippocampas ices. We use the same p-S421specific antibo y an foun thatthe intensity of the p-S421 signa after HFS in acute hippocampas ices was increase by more than 100% as compare with its eve ins ices un er base ine e ectrica stimu ation ( Fi . 1b , ). Again, NeuNimmunoreactivity was simi ar in s ices receiving either base ine stimu-ation or HFS ( Fi . 1b ). Fina y, we teste whether phosphory ation of S421 cou be in uce by behaviora training, such as contextua fearcon itioning an Morris water maze. In the contextua fear trainingexperiment, we compare p-S421 immunoreactivity in hippocampasections from mice that receive shock imme iate y after being putinto the box (imme iate shock contro ) an mice that receive shock 2 min after being put into the box (context traine ), an foun thatthe p-S421 signa in the context traine mice was increase by 59%

over the imme iate shock contro ( Fi . 1 , ). In the Morris watermaze training experiment, we compare p-S421 immunoreactivity in hippocampa sections from mice that receive the hi en p at-form version of Morris water maze training (water-maze traine )an mice that swam the same amount of time in the same tank withno p atform (yoke /swimming contro ), an foun that p-S421 signain the hippocampus of water mazetraine mice was increaseby 88% over the yoke /swimming contro ( Fi . 1 , ). In a ition toco-staining with NeuN ( Fi . 1 , ), we staine for tota MeCP2 onthe a jacent sections an etecte no ifference in the tota MeCP2expression between the context traine group an the imme iateshock contro an between the water mazetraine group an the

yoke /swimming contro ( Supp ary Fi . 1 ). Taken together,these ata suggest that increase neurona activity eve , in uce by either artificia epo arization, physio ogica y re evant e ectricastimu ation or behaviora training, can ea to phosphory ation of S421 on the MeCP2 protein in the mouse hippocampus.

Recent y, a etai e mass spectrometry ana ysis 18 i entifie twoserines in MeCP2 (S421 an S424) whose phosphory ation is in uceby neurona activity in the mouse brain. However, the same stu y foun NAIP of MeCP2 on y at S421, an not at S424, in the rat brain.To comp ete y abo ish NAIP of MeCP2, we generate mice with pointmutations in the en ogenous Mecp2gene18 ( Mecp2S421A;S424A/ymice).These mice showe none of the RTT phenotypes observe in the Mecp2/ mice (that is, sma brain or hin imb c asping), bre nor-ma y an ha a norma ifespan 18 ( ata not shown). On brain sectionsprepare from the Mecp2S421A;S424A/ymice, the tempora an spatiaexpression patterns of the mutant MeCP2 protein were in istinguish-ab e from those of the wi -type MeCP2 protein in wi -type mice( ata not shown). For examp e, mutant MeCP2 immunoreactivity (Fi . 2a ) in the pyrami a neurons (co-staine by NeuN; Fi . 2b ) of the CA1 region ( Fi . 2a h) in the a u t hippocampus were in istin-guishab e from MeCP2 immunoreactivity in the wi type ( Fi . 2 , ).Moreover, the subce u ar oca ization of the mutant MeCP2 proteinin cortica neurons ( Supp ary Fi . 2a ) was a so i entica tothat of the wi -type MeCP2 protein ( Supp ary Fi . 2 h).In brain ysates prepare from the Mecp2S421A;S424A/ymice, mutantMeCP2 was expresse at the same eve as that of wi -type MeCP2in wi -type mice un er norma con itions an after seizure ( Fi . 2i ).

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Figure 1 Phosphorylation at serine 421 induced by depolarization, HFS and behavioral training. ( ad ) Representative con ocal microscope images oco-staining o DAPI (labeling all cell nuclei), NeuN (labeling all neuronal nuclei) and p-S421 in the CA1 region o hippocampal slices treated with 1 MTTX or 50 mM KCl or 1 h ( a ), receiving baseline stimulation or HFS (100 Hz, 1 s, twice with a 20-s interval; b ), rom mice that received shockimmediately a ter being put into the box (immediate shock control) and mice that received shock 2 min a ter being put into the box (context trained; c ),or rom mice that received the hidden plat orm version o Morris water-maze training and mice that swam the same amount o time in the sametank with no plat orm (yoked/swimming control; d ). Scale bars represent 20 m. ( e ) Quanti ication o relative p-S421 intensity in the depolarizationexperiment ( n = 4 or each group), HFS experiment ( n = 4 or each group), the contextual ear-training experiment ( n = 5 or each group) and the Morriswater-maze training experiment ( n = 3 or each group). Data are presented as mean s.e.m. * P < 0.05, ** P < 0.01.


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We next examine the phosphory ation status of S421 in the Mecp2S421A;S424A/yan contro mice. Consistent with our expectation,a though phosphory ation at S421 was present at base ine eve s(Fi . 2 ,i) an great y in uce by seizure in the wi -type brain(Fi . 2i ), it was comp ete y abo ishe in the Mecp2S421A;S424A/ymiceboth at base ine ( Fi . 2 ,i) an after seizure ( Fi . 2i ). Using reverse-transcription PCRbase sequencing of the expresse Mecp2S421A;S424A RNA, we previous y foun that S424 ha been change to A424 (ref. 18).Given that the mutation to a anine is a proven way to abo ishserine phosphory ation, it can be assume that A424 cannot bephosphory ate un er any con itions in the Mecp2S421A;S424A/ymice.Taken together, these resu ts suggest that the mutations of the serinesat positions 421 an 424 to a anine specifica y abo ishe NAIP of MeCP2, without changing the expression eve , expression patternan intrace u ar oca ization of the mutant protein.

Enhanced hippocampal memory in Mecp2 S421A;S424A/y miceTo revea the physio ogica re evance of NAIP of MeCP2, we car-rie out a series of behaviora ana yses on the Mecp2S421A;S424A/y mice. To e iminate the potentia inf uence of ifferent genetic back-groun s on anima behavior, we backcrosse the Mecp2S421A;S424A mice with C57BL/6 mice for a minimum of ten generations. As aninitia screen for phenotypes covering anxiety, exp oratory activ-ity, motor coor ination, an earning an memory, we comparethe Mecp2S421A;S424A/ymice with their wi -type ittermates in theopen fie test, the rotaro test an the fear-con itioning test. In the

open fie test, the Mecp2S421A;S424A/ymice showe a simi ar eve of anxiety, as measure by the percentages of istance trave e in themi e an the corners of the fie , as their wi -type ittermates(Supp ary Fi . 3a ,b), an showe the same amount of exp ora-tory activity, as measure by the tota istance trave e , as their wi -type ittermates ( Supp ary Fi . 3 ). To confirm these resu ts, weperforme another wi e y use anxiety test, the e evate p us maze,with another cohort of Mecp2S421A;S424A/ymice an their wi -typeittermates, an observe simi ar eve s of exp oratory activity ananxiety in the Mecp2S421A;S424A/ymice an their wi -type ittermates(Supp ary Fi . 3 f ). In a test for motor coor ination, the Mecp2S421A;S424A/ymice fe off the rotating ro s ight y quicker thantheir wi -type ittermates ( Supp ary Fi . 3 ). However, theifference was not statistica y significant ( P = 0.3).

After the first two screening tests yie e no ifference, we observea strong phenotype in the ast test of the screening, the fear-con ition-ing test. In this c assic Pav ovian con itioning preparation aime atstu ying fear earning an memory ( Fi . 3a ), the Mecp2S421A;S424A/y mice froze more than their wi -type ittermates ( Fi . 3 b) in the contexttest, whereas they froze the same as the wi -type ittermates ( Fi . 3 b)in the cue test. It is wi e y be ieve that performance in the contexttest is epen ent on the function of both the hippocampus an theamyg a a, whereas performance in the cue test is on y epen ent

on the function of the amyg a a. Thus, the resu ts of both the con-text test an the cue test strong y suggest that there is a ifferencein hippocampus function between Mecp2S421A;S424A/yan wi -typemice. To confirm the context test resu ts, we performe context-on y training (no tone p aye uring training; Fi . 3 ) an testing witha fresh cohort of Mecp2S421A;S424A/ymice an their wi -type itter-mates. The Mecp2S421A;S424A/ymice again froze more than the witypes (Fi . 3 ) in the context test. In both training proce ures, nosignificant ifference in base ine freezing (pre-shock uring trainingsessions, an pre-tone in the cue test sessions, P > 0.3) was observebetween the Mecp2S421A;S424A/ymice an their wi -type ittermates(Fi . 3b , ). To further confirm the invo vement of the hippocampusin an in epen ent assay, we performe the Morris water maze test(Fi . 4a ), a we -estab ishe hippocampus- epen ent spatia earningan memory test, on yet another cohort of Mecp2S421A;S424A/ymice antheir wi -type ittermates. The hi en p atform version of the watermaze test was use in this experiment. Both the Mecp2S421A;S424A/y mice an their wi -type ittermates swam at the same spee uringtraining an testing ( Fi . 4b ). Throughout the training perio , bothgenotypes earne the ocation of the hi en p atform equa y we ,as in icate by the persistent ecrease in the time nee e to fin anc imb onto the p atform ( Fi . 4 ). In the probe tria s, a though boththe Mecp2S421A;S424A/yan the wi -type mice remembere where thehi en p atform was p ace uring training by spen ing significant y more time in the target qua rant than the other qua rants (in icatingthat both genotypes remembere the ocation of the p atform, P < 0.01),the Mecp2S421A;S424A/ymice spent significant y more time than the

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Figure 2 Loss o NAIP at S421 in the Mecp2 S421A;S424A/y mice.(ah ) Representative images o the CA1 region o the hippocampus rom theMecp2 S421A;S424A/y mice ( a ,b ,e ,f) and their wild-type littermates ( c ,d ,g,h)stained with MeCP2 ( a ,c ) and NeuN ( b ,d ) or p-S421 ( e ,g) and NeuN ( f,h ).Images on the le t correspond with the adjacent image on the right (redand green channels, respectively). Arrowheads indicate the pyramidalcell layer in CA1. Scale bar represents 100 m. ( i) Western blot analysiswas per ormed to compare the expression levels o p-S421 (top), MeCP2(middle) and actin (bottom) in the brains o the wild-type (le t two lanes)and Mecp2 S421A;S424A/y mice (right two lanes) under normal condition anda ter seizure. Full-length blots are shown in Supplementary Figure 8 .


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20 min of recor ings were significant y if-ferent between the two genotypes ( P = 0.03;Fi . 5 ). These resu ts are consistent withthe enhance hippocampus- epen ent earning an memory inthese mice that was observe in the fear-con itioning test an theMorris water maze.

We next examine whether there were any changes at the ce u areve that may un er ie the a tere synaptic physio ogy an behavioraoutput of the hippocampus in the Mecp2S421A;S424A/ymice. Primary hippo-campa an cortica neurons were iso ate from postnata ay 01 Mecp2S421A;S424A/yan wi -type ittermate pups an cu ture sep-arate y for 21 in vitro (DIV). We staine neurons for VGLUT1(excitatory presynaptic marker), PSD95 (postsynaptic marker) anMAP2 ( en ritic marker) to examine the eve opment of excita-tory synapses (Fi . 7 ). Quantification of the ensities of VGLUT1

puncta, PSD95 puncta, an co oca ize VGLUT1 an PSD95puncta was performe a ong MAP2- abe e neurites ( Fi . 7b , ).In the hippocampa cu ture, our ana ysis revea e a 45% increase inthe number of VGLUT1 puncta ( P = 0.00003), a 28% increase in thenumber of PSD95 puncta ( P = 0.005), an a 152% increase in thenumber of co oca ize VGLUT1 an PSD95 puncta ( P = 0.000002)in Mecp2S421A;S424A/yneurons compare with those in the wi -typeneurons ( Fi . 7 ). In the cortica cu ture, a simi ar ana ysis revea ea 99% increase in the number of VGLUT1 puncta ( P = 0.00002),

a 37% increase in the number of PSD95 puncta ( P = 0.002), an a 84%increase in the number of co oca ize VGLUT1 an PSD95 puncta(P = 0.04) in Mecp2S421A;S424A/yneurons compare with those in wi -type neurons ( Fi . 7 ). Given that co oca ize VGLUT1 an PSD95puncta ike y represent functiona excitatory synapses, our resu tsstrong y suggest that there is an increase in excitatory synaptogenesisin both the hippocampa an cortica neurons of the Mecp2S421A;S424A/y mice. These resu ts are consistent with the enhance hippocampaLTP an the enhance earning an memory phenotypes that weobserve in these mice.

Altered gene expression in Mecp2 S421A;S424A/y hippocampus

Given the we -estab ishe ro e of MeCP2 in mo u ating gene tran-scription5,8, we suspecte that potentia gene transcription changesmay un er ine the ce u ar, synaptic an behaviora phenotypes thatwe observe in the Mecp2S421A;S424A/y mice. To etermine whichtranscription changes are cause by the oss of NAIP of MeCP2 inthese mice, we first examine known MeCP2 target genes with estab-ishe neurona functions, such as Bdnf 16,20. We micro issecte thehippocampus from the Mecp2S421A;S424A/ymice (n = 14) an theirwi -type ittermates ( n = 14), extracte RNA, performe rea -time

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Figure 5 Enhanced Scha er collateralCA1 LTPin Mecp2 S421A;S424A/y mice. ( a ,b) Representativerecording o ield excitatory postsynaptic potential( EPSP) rise slope in wild-type ( a) andMecp2 S421A;S424A/y (b) slices. Sample traces ineach genotype represent be ore (1) and 160 mina ter (2) the tetanic stimulation (100 Hz, 1 s,twice with a 20-s interval). ( c) Average normalized(against baseline) EPSP rise slope in the wild-type (closed circle) and the Mecp2 S421A;S424A/y (open circle) slices. Error bars represent s.e.m.Statistical analysis was carried out using two-wayANOVA with repeated measure ( F 1,30 = 4.67,P = 0.04). ( d) Average normalized (againstbaseline) EPSP rise slope 160180 min a ter theLTP induction in wild-type and Mecp2 S421A;S424A/y slices. The bar graph shows the mean s.e.m.16 slices rom 14 Mecp2 S421A;S424A/y mice and16 slices rom 15 wild-type littermates wereincluded in c and d . *P < 0.05.

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Figure 6 Enhanced mossy iberCA3 LTP in Mecp2 S421A;S424A/y mice. ( a ,b ) Representative recording o EPSP amplitudes in wild-type ( a ) andMecp2 S421A;S424A/y (b ) slices. Sample traces in each genotype represent be ore (1) and 40 min a ter (2) the tetanic stimulation (100 Hz, 1 s, twicewith a 20-s interval), and a ter the application o DCG-IV (3). ( c ) Average normalized (against baseline) amplitudes in wild-type (closed circle)and Mecp2 S421A;S424A/y (open circle) slices. Error bars represent s.e.m. Statistical analysis was carried out using two-way ANOVA with repeatedmeasures ( F 1 ,32 = 11.203, P = 0.002). ( d ) Average normalized (against baseline) amplitudes 4060 min a ter the LTP induction in wild-type andMecp2 S421A;S424A/y slices. The bar graph shows the mean s.e.m. 18 slices rom 12 Mecp2 S421A;S424A/y mice and 16 slices rom 11 wild-typelittermates were included in c and d . * P < 0.05, ** P < 0.01.


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PCR an etecte an increase in Bdnf transcript covering its pro-tein-co ing region (B nf CDS) in the Mecp2S421A;S424A/yhippocam-pus (Fi . 8a ). In a ition, we foun an increase in Bdnf transcript

covering its neurona -specific promoter (exon IV, B nf E4) in the Mecp2S421A;S424A/yhippocampus ( Fi . 8a ). The increase in Bdnf tran-scription is consistent with the observe phenotypes of enhancehippocampa LTP an hippocampus- epen ent earning an memory in the Mecp2S421A;S424A/ymice.

Recognizing that the observe phenotypes of the Mecp2S421A;S424A/y mice, better contextua fear memory, enhance hippocampa LTP,increase excitatory synaptogenesis an the increase in Bdnf tran-scription, are simi ar to those observe in mice overexpressing Mecp2 ( Mecp2Tg )8,21,22, we examine the expression of three a itiona geneswhose eve s were a tere in Mecp2Tg mice8: myocyte enhancer factor 2( Mef2c), bone morphogenetic protein 4 ( Bmp4) an metabotropic

g utamate receptor 1 ( Grm1), a of which areimportant for the eve opment an functionof the nervous system. Moreover, MeCP2 hasbeen shown to irect y bin to the promoterof Mef2c an repress its transcription 8. Wefoun increase expression of Bmp4 anecrease expression of Mef2c an Grm1 (Fi . 8a ) in the Mecp2S421A;S424A/y hippo-campus. The expression change of each of the three genes in the Mecp2S421A;S424A/ymicewas very simi ar to that in the Mecp2Tg mice.However, it is not imme iate y c ear how

expression changes in Bmp4, Mef2c an Grm1 may he p to exp ainthe observe phenotypes in the Mecp2S421A;S424A/ymice.

Altered promoter association by the mutant MeCP2As a first step to un erstan how the oss of phosphory ation may a ter gene transcription, we examine the abi ity of the mutantMeCP2 to bin to gene promoters. To better quantify the promoteroccupancy of the phospho-mutant MeCP2 protein, we generate two Mecp2knock-in mice: Flag-Mecp2S421A;S424A/ymice, which expressF ag-tagge mutant MeCP2 from the en ogenous Mecp2 ocus, anFlag-Mecp2+/y mice, which express F ag-tagge wi -type MeCP2from the en ogenous Mecp2 ocus. These unique mouse ines a oweus to carry out chromatin immunoprecipitation (ChIP) using a very re iab e antibo y to F ag. Western b ot ana ysis confirme that bothF ag-tagge mutant an wi -type MeCP2 were expresse at the same

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Figure 7 Increased excitatory synaptogenesisin Mecp2 S421A;S424A/y mice. ( a ) Representativecon ocal microscope images o culturedhippocampal neurons (21 DIV) rom eitherthe Mecp2 S421A;S424A/y mice or their wild-typelittermates stained with antibodies to VGLUT1,PSD95 and MAP2. ( b ,d ) Representative imageso neurites rom both genotypes that wereanalyzed in the hippocampal culture ( b ) and thecortical culture ( d ). Scale bars represent 10 m.(c ,e ) Quanti ication o the densities (numbero puncta per m) o VGLUT1 puncta, PSD95puncta, and colocalized VGLUT1 and PSD95puncta in the hippocampal ( n = 10 in eachgenotype; c ) and the cortical cultures(n = 12 in each genotype; e ). Numbers rom theMecp2 S421A;S424A/y neurons were normalizedagainst those rom the wild-type neurons.*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 8 Altered expression o MeCP2

target genes in the hippocampus o theMecp2 S421A;S424A/y mice and increasedpromoter binding by the Flag-tagged mutantMeCP2. ( a ) Relative expression levels oBdnf CDS, Bdnf E4, Bmp4 , Mef2c and Grm1 in the adult hippocampus o wild-type andMecp2 S421A;S424A/y mice ( n = 14 in eachgenotype or Bdnf CDS, Bdnf E4; n = 8 in eachgenotype or Bmp4 , Mef2c and Grm1 ; P = 0.02or Bdnf CDS, P = 0.04 or Bdnf E4, P = 0.01or Bmp4 , P = 0.05 or Mef2c , P = 0.02 orGrm1 ). ( b ) Relative binding o Flag-tagged wild-type and mutant MeCP2 to the promoters oBdnf (exon IV), Bmp4 , Mef2c and Grm1 in the adult hippocampus o the Flag-Mecp2 +/y and Flag-Mecp2 S421A;S424A/y mice, respectively ( n = 6 in eachgenotype, P = 0.03 or Bdnf , P = 0.01 or Bmp4 , P = 0.05 or Mef2c , P = 0.04 or Grm1 ). * P < 0.05, ** P < 0.01. Error bars represent s.e.m.

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eve as wi -type MeCP2 acking F ag ( ata not shown). Behavioratesting confirme that the Flag-Mecp2S421A;S424A/ymice ha enhancehippocampus- epen ent earning an memory ( Supp ary Fi . 3i ), simi ar to that observe in the Mecp2S421A;S424A/ymice, whereasthe Flag-Mecp2+/y mice performe simi ar to the Mecp2+/y mice ( atanot shown). These ata suggest that the Flag-Mecp2S421A;S424A/ymiceare functiona y in istinguishab e from Mecp2S421A;S424A/ymice

an that Flag - Mecp2+/y

mice are functiona y in istinguishab e Mecp2+/y mice.Because the transcriptiona changes that we etecte were from

the in vivo hippocampus ( Fi . 8a ), we reasone that it wou be themost appropriate to use hippocampi from the Flag-Mecp2S421A;S424A/y an the Flag-Mecp2+/y mice for ChIP ana ysis to examine promoteroccupancy of the mutant form an the wi -type form of MeCP2.Bdnf , Bmp4, Mef2c an Grm1 promoter occupancy by F ag-taggemutant MeCP2 was increase compare with that by F ag-taggewi -type MeCP2 ( Fi . 8b ). Thus, the oss of phosphory ation appearsto enhance the bin ing of MeCP2 to its target gene promoters. BecauseMeCP2 has been shown to be capab e of simu taneous y activatingthe transcription of some genes an repressing the transcription of others 8,9, it is possib e that the increase promoter occupancy of themutant MeCP2 may ea to opposite transcriptiona outcomes in themutant hippocampus. More etai e stu ies in the future wi he p toetermine the mechanisms of such regu ations.

DISCUSSIONIt has been suggeste that NAIP of MeCP2 represents a mechanismfor ynamica y regu ating bin ing of MeCP2 to methy ate DNAan therefore regu ating MeCP2s abi ity to coor inate neuronagene transcription 1618. However, it is not c ear what, i f any, functionrequires such phosphory ation in the in vivo brain. NAIP of MeCP2was abo ishe our Mecp2S421A;S424A/ymice, an our resu ts provi e,to the best of our know e ge, the first genetic evi ence that NAIP of MeCP2 is invo ve in regu ating severa important functions in the

in vivobrain, inc u ing promoter occupancy, gene transcription, exci-tatory synaptogenesis, LTP, an earning an memory.The range of phenotypes that we observe in the Mecp2S421A;S424A/y

mice spans the mo ecu ar, ce u ar, synaptic an anima behavioraeve s. A though it is not easy to estab ish causa inks across a eve s,the phenotypes observe in Mecp2S421A;S424A/y mice, inc u ingincrease Bdnf expression, increase excitatory synaptogenesis,enhance hippocampa LTP, an enhance hippocampus- epen entearning an memory, are very consistent with each other. Moreover,the Mecp2S421A;S424A/ymice an the Mecp2Tg mice8,21,22 showe nota-b e phenotypic simi arities across the eve s of promoter occupancy,gene expression, excitatory synaptogenesis, synaptic physio ogy ananima behavior, raising the possibi ity that these two mouse mo e smay share some common un er ying mechanisms. A thoughthese two mouse mo e s appear to converge on the same mo ecu areventincrease promoter occupancy, they achieve it through if-ferent means. In Mecp2S421A;S424A/ymice, the expression eve of themutant protein was not a tere . However, the oss of NAIP cause it tobin more tight y to gene promoters. In Mecp2Tg mice, the increase inMeCP2 eve resu te in enhance bin ing of MeCP2 at the promotersof its target genes. Together, these fin ings high ight the nee to pre-cise y regu ate the abi ity of MeCP2 to bin to gene promoters.

A previous stu y 17 use a entivirus-base approach to simu -taneous y knock own en ogenous MeCP2 an express exogenousMeCP2S421A in cu ture neurons, an foun that a oss of phosphor-y ation at S421 e to re uce Bdnf transcription. Severa factors may have contribute to the apparent iscrepancy between these fin ings

an our fin ings. First, we abo ishe potentia phosphory ation atboth S421 an S424 by mutating both serines to a anines, whereasthe previous stu y 17 on y mutate S421. Secon , our resu ts camefrom the a u t hippocampus of iving mice, whereas the resu ts of the previous stu y 17 came from cu ture neurons that were iso atefrom embryonic rat brain. Thus, not on y is our in vivo system if-ferent from the previous y use in vitro system17, but the age an

species of the anima s use in the two stu ies are substantia y iffer-ent. Thir , it is a so possib e that the efficiencies of knocking ownen ogenous wi -type MeCP2 an vira infection may not have been100% in the previous stu y 17, which may have resu te in eitherthe wi -type MeCP2 an the mutant MeCP2 S421A being presentin the same neuron, or a mixture of wi -type an mutant neuronsin the same cu ture. Nonethe ess, our fin ing of the e evate Bdnf eve in vivo is consistent with the enhance LTP an better spatiamemory in the Mecp2S421A;S424A/ymice, which were a so observein the Mecp2Tg mice.

Severa important questions remain unanswere an await moreextensive stu ies in the future. First, the precise synaptic mechanismsinking MeCP2 phosphory ation with hippocampa LTP remaine usive. As it is wi e y be ieve that presynaptic changes un er ieCA3 LTP an postsynaptic changes un er ie CA1 LTP, the oss of MeCP2 phosphory ation appears to cause both pre- an postsynapticchanges. This is possib e because activity-in uce MeCP2 phosphor-y ation cou happen in both pre- an postsynaptic neurons. Secon ,a though our fin ing of enhance CA1 LTP in the Mecp2S421A;S424A/y mice at ate time points (160180 min after LTP in uction) are con-sistent with a change in the transcription- epen ent phase of LTP( ate LTP), no unequivoca conc usion can be rawn because no tran-scription inhibitors were inc u e in our experiment. We a so notethat, a though CA1 LTP in the Mecp2S421A;S424A/ymice appeare tobe enhance at ear y time points after LTP in uction, it is not c earhow MeCP2 phosphory ation may be invo ve in transcription- in epen ent regu ation of synaptic p asticity. Fina y, because we

focuse our attention on the functiona re evance of activity-in uceMeCP2 phosphory ation in vivo, the gene transcription an promoteroccupancy ana ysis inc u e here was imite to on y a few can i ategenes. However, given that MeCP2 is capab e of bin ing to a sing emethy ate CpG pair 4 an is wi e y istribute across the genome 23,it is high y ike y that a tere occupancy of the mutant MeCP2 may occur at other genomic oci an that a itiona gene expressionchanges may exist an a so un er ie the ce u ar, synaptic an behav-iora phenotypes observe in the Mecp2S421A;S424A/ymice. To fu y un erstan the mo ecu ar mechanisms un er ying these phenotypes,it is important to etermine where the mutant MeCP2 protein bin sacross the genome, whether an how the oss of phosphory ation may affect its association with other chromatin remo e ing comp exes,an u timate y how neurona gene transcription is a tere by the ossof phosphory ation.

METHODSMetho s an any associate references are avai ab e in the on ine version of the paper at http://www.nature.com/natureneuroscience/.

Note: Supplementary information is available on the Nature Neuroscience website.

AcknowledgmentSWe thank A. Messing, J. Svaren an J. Yin for critica y rea ing the manuscript,S. Marwaha for assistance with mouse work, an M. An rzejewski for assistancewith statistica ana ysis. H.L. was supporte by a pre- octora fe owshipfrom the Stem Ce an Regenerative Me icine Center at the University of Wisconsin, Ma ison. E.C.W. was supporte by a pre octora training grant(5T32GM07133) an a gra uate stu ent fe owship from the Frien s of the
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Waisman Center. Q.C. was supporte by a Young Investigator Awar from theNationa A iance for Research on Schizophrenia an Depression. This work was partia y supporte by grants from the Nationa Institute of Chi Hea than Human Deve opment (R01 HD064743 an R21 HD066560 to Q.C., an P30HD03352 to the Waisman Center).

AUtHoR contRIBUtIonSQ.C. irecte the stu ies. H.L., X.Z. an Q.C. conceive an esigne theexperiments. H.L. performe the western b ot, mouse behaviora tests, ChIPexperiments, gene expression stu ies, imaging an image ana ysis. K.F.C.performe mouse behaviora tests. X.Z. performe a of the e ectrophysio ogy experiments, neurona cu ture an hippocampa s ice experiments, animmunostaining. E.C.W. performe immunostaining. The paper was written by Q.C., H.L. an X.Z. an commente on by a authors.

comPetIng FInAncIAl InteReStSThe authors ec are no competing financia interests.

Published online at http://www.nature.com/natureneuroscience/.Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html .

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2. Miller, C.A. et al. Cortical DNA methylation maintains remote memory. Nat. Neurosci. 13 ,664666 (2010).

3. Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synapticunction in adult orebrain neurons. Nat. Neurosci. 13 , 423430 (2010).4. Lewis, J.D. et al. Purifcation, sequence, and cellular localization o a novel

chromosomal protein that binds to methylated DNA. Cell 69 , 905914 (1992).5. Nan, X., Campoy, F.J. & Bird, A. MeCP2 is a transcriptional repressor with abundant

binding sites in genomic chromatin. Cell 88 , 471481 (1997).6. Jones, P.L. et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress

transcription. Nat. Genet. 19 , 187191 (1998).

7. Bird, A.P. & Wol e, A.P. Methylation-induced repression: belts, braces, andchromatin. Cell 99 , 451454 (1999).

8. Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates andrepresses transcription. Science 320 , 12241229 (2008).

9. Yasui, D.H. et al. Integrated epigenomic analyses o neuronal MeCP2 reveal a roleor long-range interaction with active genes. Proc. Natl. Acad. Sci. USA 104 ,1941619421 (2007).

10. Amir, R.E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encodingmethyl-CpG-binding protein 2. Nat. Genet. 23 , 185188 (1999).

11. Van den Veyver, I.B. & Zoghbi, H.Y. Methyl-CpG-binding protein 2 mutations in Rettsyndrome. Curr. Opin. Genet. Dev. 10 , 275279 (2000).

12. Wan, M. et al. Rett syndrome and beyond: recurrent spontaneous and amilialMECP2 mutations at CpG hotspots. Am. J. Hum. Genet. 65 , 15201529 (1999).

13. Xiang, F. et al. Mutation screening in Rett syndrome patients. J. Med. Genet. 37 ,250255 (2000).

14. Hagberg, B. Retts syndrome: prevalence and impact on progressive severe mentalretardation in girls. Acta Paediatr. Scand. 74 , 405408 (1985).

15. Hagberg, B. & Hagberg, G. Rett syndrome: epidemiology and geographical variability.Eur. Child Adolesc. Psychiatry 6 (suppl. 1), 57 (1997).

16. Chen, W.G. et al. Derepression o BDNF transcription involves calcium-dependentphosphorylation o MeCP2. Science 302 , 885889 (2003).

17. Zhou, Z. et al. Brain-specifc phosphorylation o MeCP2 regulates activity-dependentBdn transcription, dendritic growth, and spine maturation. Neuron 52 , 255269(2006).

18. Tao, J. et al. Phosphorylation o MeCP2 at Serine 80 regulates its chromatinassociation and neurological unction. Proc. Natl. Acad. Sci. USA 106 , 48824887(2009).

19. Deng, J.V. et al. MeCP2 in the nucleus accumbens contributes to neural andbehavioral responses to psychostimulants. Nat. Neurosci. 13 , 11281136 (2010).

20. Martinowich, K. et al. DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science 302 , 890893 (2003).21. Collins, A.L. et al. Mild overexpression o MeCP2 causes a progressive neurological

disorder in mice. Hum. Mol. Genet. 13 , 26792689 (2004).22. Chao, H.T., Zoghbi, H.Y. & Rosenmund, C. MeCP2 controls excitatory synaptic

strength by regulating glutamatergic synapse number. Neuron 56 , 5865 (2007).23. Skene, P.J. et al. Neuronal MeCP2 is expressed at near histone-octamer levels and

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ONLINE METHODSm us ypi . Two PCR genotyping assays were eve ope . Primers forthe first assay were 5-TCA CAA GTT AAC AGT GCC AGC TGC-3 (forwar )an 5 -ACC CCC TTG GGA CTG AAG TTA CAG A-3 (reverse). A ~100-bpban in this assay represents the wi -type Mecp2a e e. A ~200-bp ban in thisassay represents the Mecp2S421A;S424Aa e e. Primers for the secon assay were5-CGA GGA GGC GCA CTG GAA GCA-3 (forwar ) an 5 -ACC TGA CTGTGC TTG TCG GTA AG-3 (reverse). A 413-bp ban in this assay represents

the Mecp2S421A;S424A

a e e.

I u s ai i a s r b a a ys s f S421 ph sph ry a i . Weuse antibo ies to MeCP2 (Abcam), p-S421 (custom ma e by Covance),tubu in (Sigma), NeuN (Santa Cruz) an actin (Sigma). For immunostaining,1012-week-o mice were ki e by transcar ia perfusion with norma sa ineso ution fo owe by 4% paraforma ehy e (PFA, wt/vo ). Brains were issecte ,post-fixe in 4% PFA, cryoprotecte in 30% sucrose (wt/vo ), embe e an fro-zen in Tissue Tek. Seria corona sections were cut at 40 m throughout the brain,an staine with appropriate primary an secon ary antibo ies. For western b ot,brains were quick y issecte out from 1012-week-o mice an homogenizein RIPA buffer containing both the protease inhibitor cocktai (Roche) an theprotein phosphatase inhibitor cocktai (Roche). Lysate was run on 10% NuPAGEBis-Tris ge (Invitrogen), an transferre to Protran BA 85 nitroce u ose mem-branes (Whatman). The membrane was incubate with appropriate primary an

infrare ye-conjugate secon ary antibo ies (Thermo Scientific), an scanneby the O yssey infrare imaging system.

m us b havi ra s s. We use 1012-week-o mice in a tests. A protoco swere approve by the Institutiona Anima Care an Use Committee at University of Wisconsin-Ma ison.

op fi ar a. Each mouse was p ace in the center of a transparent p asticchamber (41 41 30 cm) an a owe to exp ore free y for 15 min, whi e anoverhea computer-contro e camera monitore its activity. Automate ataana ysis was one using LimeLight2 (Cou bourn Instruments).

e va p us- az . Each mouse was p ace in the center of the maze, consist-ing four perpen icu ar arms (two enc ose by 20 cm wa s; the other two open)e evate 40 cm above groun , an a owe exp ore free y for 10 min, whi e anoverhea computer-contro e camera monitore its behavior. The percentageof time each mouse spent in the open arms an the c ose arms was measureusing the LimeLight2 software.

R ar s . Mice were p ace onto a horizonta rotating ro , with the rotationspee acce erating from 2 to 20 rpm over 5 min. A test aste from the time themouse was p ace on the ro unti it fe off or unti 5 min ha e apse .

F ar- i i i s . In training proce ure 1, mice were p ace into a shock chamber an a owe to exp ore for 2 min. Then, a white-noise tone (87 B)soun e for 30 s (con itione stimu us). During the ast 1.5 s of the tone, micereceive a mi foot shock (0.5 mA, uncon itione stimu us). The same tonefootshock (con itione -uncon itione stimu us) combination was e ivere again2 min ater. The mice were returne to their home cages 1 min after the en of the secon con itione -uncon itione stimu us combination. The context testwas performe 22 h after the training. During the test, mice were p ace back into the same training chamber, an monitore by an overhea camera in thechamber for 5 min. The cue test was performe 2 h after the context test: co orep exig ass inserts were p ace into the training chamber to hi e the shock grian to change the context of the chamber. Mice were then p ace in the chamberan monitore by the overhea camera for 6 min, uring which two con itionestimu i (space the same way as in the training session) were given. In trainingproce ure 2, everything staye the same except that no tones were given. On y the context test was a ministere on the mice traine in training proce ure 2. Aevents in the fear con itioning test were programme an ata recor e throughthe FreezeFrame2 an FreezeView software from (Actimetrics Software).

m rris a r az . Mice were traine to ocate a hi en p atform (1 cm be owthe surface of the water) in a circu ar poo (1.20 m in iameter) of opaque water

using ista visua cues. Mice were given two b ocks of four training tria s a ay for 4 consecutive ays. During a training tria , each mouse was re ease into thepoo from 1 of 4 starting positions in a ran om or er. The ocation of the hi enp atform remaine constant throughout training. Time to fin the p atform wasmeasure in each tria . If a mouse i not fin the p atform in 60 s, it was gent y gui e by han onto the p atform an a owe to remain there for 10 s. After 4 ,the probe test was performe with no p atform. Each mouse was a owe tosearch the p atform for 60 s whi e an overhea camera recor e its movement.

Time spent searching in each qua rant by a mouse was use to characterize itssearch behavior.

e r physi y. We anesthetize an ecapitate 812-week-o mice anprepare 400- M transverse hippocampa s ices using a Vibratome TissueChopper. S ices were recovere in oxygenate artificia cerebrospina f ui(ACSF) at 25 C for at east 1 h. The ACSF containe 124 mM NaC , 2.5 mMKC , 1.25 mM NaH 2PO4, 26 mM NaHCO 3, 1.2 mM MgC 2, 2.5 mM CaC 2an 15 mMg ucose. A recor ings were performe at 30 1 C in a submerge chamber(Warner Instruments) continuous y superfuse with oxygenate ACSF. Schafferco atera CA1 synaptic responses an mossy fiberCA3 synaptic responses wererecor e as extrace u ar fEPSPs from the CA1 stratum ra iatum an the CA3stratum uci um area, respective y. Un ess otherwise mentione , test stimu i weree ivere at a frequency of 0.05 Hz. The stimu us intensity was set to pro uce2530% of the maximum response in a the experiments. PPF was performe at

25, 50, 100, 200, 300, 400 an 500-ms interstimu us interva s. Frequency faci ita-tion was investigate by increasing the stimu ation rate from 0.10.5 Hz. LTP wasin uce by two trains of 100-Hz stimu ation asting 1 s with a 20-s intertetanusinterva . No bicucu ine was app ie uring any recor ing. For CA3 LTP in uc-tion, tetanus was e ivere in the presence of 50 M d-AP5 to prevent contami-nation of the NMDA- epen ent pathway converging on CA3 neurons. Mossy fiber synaptic responses were i entifie by arge PPF an frequency faci itation.Experiments were inc u e for ata ana ysis on y if DCG-IV (1 M) cause agreater than 75% re uction in the synaptic response. For Schaffer co atera CA1recor ings, fEPSP rise s ope was ca cu ate as the s ope of the rising phase 2080%of the peak response. For CA3 recor ings, the amp itu e of in ivi ua synapticresponse was measure after subtraction of the response that remaine in thepresence of DCG-IV. Data were acquire an ana yze with pC amp10.2 soft-ware (Axon Instruments). Signa s were fi tere at 2 Hz an samp e at 10 kHzby Digi ata 1440A (Axon Instruments). Traces were average for every 2-mininterva .

A a ysis f x i a ry sy ap sis i u ur hipp a pa a r i aur s. Dissociate hippocampa an cortica neurons were iso ate from post-

nata ay 0 or 1 Mecp2S421A;S424A/yan Mecp2+/y pups, an p ate at a ensity of 1 104 cm2 on po y-l- ysinecoate (Sigma P2636) covers ips. Cu ture me iumwas Neurobasa (Invitrogen 21103-049) containing 2% B27 (vo /vo , Invitrogen12587-010), 1% N2 (vo /vo , Invitrogen 17502-048) an 0.5 mM G utaMax(Invitrogen 35050-061). Cu ture hippocampa an cortica neurons were fixe at21 DIV with 4% PFA an 4% sucrose in phosphate-buffere sa ine (PBS), washewith PBS, permeabi ize with Triton X-100 in PBS, b ocke with 10% normaonkey serum (vo /vo ) in PBS (a at 25 C), an incubate at 4 C overnight withmouse antibo y to MAP2 (Mi ipore 05-346, 1:300), rabbit antibo y to PSD95(Ce Signa ing 2507, 1:200) an goat antibo y to VGLUT1 (Santa Cruz sc-13320,1:200) in PBS containing 10% norma onkey serum. The next ay, after beingwashe in PBS, the covers ips were incubate with secon ary antibo ies, inc u -ing AffiniPure F(ab) 2 fragment onkey IgG(H+L) conjugate with DyLight-488(antibo y to goat), DyLight-549 (antibo y to rabbit) an DyLight-649 (anti-bo y to mouse), respective y (1:200, Jackson immunoResearch Laboratories).Covers ips were then washe in PBS an staine with DAPI before being mounteon g ass s i es with F uoromount G (SouthernBiotech 0100-01).

I a a a ysis. Images in Fi ur s 1 an 7 were capture on a Nikon A1RSI con-foca microscope. For each experimenta series, a of the images were acquirewith i entica settings for aser power, photomu tip ier gain an offset. Z stackswere co apse in a maximum projection an ana yze with ImageJ software(US Nationa Institutes of Hea th). For quantification of MeCP2 p-S421 eve inCA1, a of the images were quantifie using a sing e thresho va ue, which isetermine by the backgroun eve of the contro sections in each experiment.


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