Dcx reexpression reduces subcortical band heterotopia and seizure threshold in an animal model of neuronal migration disorder

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1 Dx reexpression reues suortil n heterotopi n seizure threshol in n niml moel of neuronl migrtion isorer Jen-Bernr Mnent, Yu Wng, YoonJeung Chng, Murugn Prmsivm & Joseph J LoTuro 29 Nture Ameri, In. All rights reserve. Disorers of neuronl migrtion n le to mlformtions of the ererl neoortex tht gretly inrese the risk of seizures. It remins unteste whether mlformtions use y isorers in neuronl migrtion n e reue y retivting ellulr migrtion n whether suh repir n erese seizure risk. Here we show, in rt moel of suortil n heterotopi (SBH) generte y in utero RNA interferene of the Dx gene, tht errntly positione neurons n e stimulte to migrte y reexpressing Dx fter irth. Restrting migrtion in this wy oth reues neoortil mlformtions n restores neuronl ptterning. We further fin tht the pity to reue SBH ontinues into erly postntl evelopment. Moreover, intervention fter irth reues the onvulsnt-inue seizure threshol to level similr to tht in mlformtion-free ontrols. These results suggest tht isorers of neuronl migrtion my e eventully tretle y reengging evelopmentl progrms oth to reue the size of ortil mlformtions n to reue seizure risk. A usl onnetion etween isruptions in neuronl migrtion uring fetl evelopment n ltere neoortil exitility is well estlishe. Foi of normlly migrte neurons re prtiulrly prevlent in iniviuls with phrmologilly intrtle epilepsies, n surgil resetion of mlforme ortex n often effetively tret suh rug-resistnt epilepsy 6,7. Mny ses, however, remin untretle y surgery euse of the lotion n/or wiespre istriution of mlformtion(s). One suh mlformtion ours in oule ortex synrome, in whih n of heterotopi gry mtter ompose of normlly migrte neurons is lote etween the ventriulr wll n the ortil mntle n is seprte from oth y n of white mtter 8,9. Fol resetion of epileptogeni tissue in oule ortex synrome shows poor linil outome. Doule ortex synrome or SBH is lso ssoite with mil to moerte mentl retrtion n with intrtle epilepsy in out 6% of ffete iniviuls 2, n it is most often use in femles y muttions in the X-linke gene DCX, mirotuule-ining protein essentil to neuronl migrtion 3 6. DCX muttions in mles usully use nterior lissenephly, ut SBH ssoite with DCX muttions hve lso een esrie in mles 7. Stuies in niml moels hve revele tht severl types of migrtion isruptions n mlformtions inrese neuronl exitility n seizure risk. For exmple, spontneous seizures re oserve in the tish (telenephli internl struture heterotopi) mutnt rt 8, n signifintly reue threshols to onvulsnt gents re oserve in rts with ortil migrtion nomlies use y prentl exposure to tertogens suh s methylzoxymethnol 9,2, oine 2 or irrition 22. Similrly, in freeze-lesion moel of mirogyri, epileptiform ishrges re relily evoke in rin slies ontining mlformtions, n the threshol ose of onvulsnts to inue seizures is reue 23,24. A reent stuy lso reporte tht spontneous onvulsive seizures n osionlly e oserve in suset of Dxknokout mie showing isrete hippompl mlformtions ut no ortil normlities 2. Together, results from niml moels n stuies on surgilly remove humn tissue inite tht mlforme neoortex is ssoite with reorgnize neuronl networks n ltere ellulr physiologies tht rete hyperexitle tissue. It is urrently unknown whether there is time in evelopment tht interventions to reverse or reue forme or forming mlformtions woul lso prevent neuronl hyperexitility n seizure risk. We previously evelope rt moel of SBH y eresing Dx expression with in utero RNA interferene (RNAi) 26. This moel reproues ntomil fetures of the mlformtions present in the humn oule ortex synrome, n we hve reently shown tht the mlformtions re resue or prevente y onurrent emryoni expression of Dx 27. Here we use onitionl vrition of this resue pproh to etermine whether elye Dx expression, fter SBH hs forme, n reue heterotopi n restore neuronl ptterning. We show tht oth lminr isplement of neurons n SBH size re reue upon elye expression of Dx uring erly postntl perios. We show further tht rts with SBH re more suseptile to seizures inue y the onvulsnt pentylenetetrzol n tht reution of SBH restores seizure threshols to levels similr to those of unffete ontrols. Deprtment of Physiology n Neuroiology, 7 North Egleville Ro U-36, University of Connetiut, Storrs, Connetiut 6269, USA. Corresponene shoul e resse to J.J.L. (joseph.loturo@uonn.eu). Reeive My; epte 6 Novemer; pulishe online 2 Deemer 28; oi:.38/nm VOLUME [ NUMBER [ JANUARY 29 NATURE MEDICINE

2 29 Nture Ameri, In. All rights reserve. I V wm Dx off? + 4-OHT I V wm Dx on Mlformtion free 3UTRm3hp CALNL- CAG-mRFP 3UTRhp CALNL- CAG-mRFP P, P or P resue 3UTRhp CALNL- CAG-mRFP CAG promoter RESULTS Conitionl reexpression of Dx The purpose of the present stuy ws to investigte whether neoortil lmintion efiits n SBH mlformtions n e reue y reexpression of Dx fter irth. Our pproh ws to initite SBH formtion n lminr isplement y in utero RNAi of Dx n then reexpress Dx in mispositione neurons (Fig. ). To omplish this, we pte previously evelope in vivo onitionl trnsgene expression system 28 to onitionl RNAi resue pproh. Beuse enogenous Dx expression ereses with neuronl evelopment, we oul not hieve onitionl reexpression y turning off Dx RNAi. Inste we rete system in whih version of Dx tht is insensitive to Dx RNAi ws turne on in ells in whih enogenous Dx ws knoke own y RNAi. We onstrute onitionl DCX expression vetor (CALNL-) tht ontins stop oon flnke y two loxp sites ownstrem from the CAG promoter whih uiquitously rives expression n upstrem from the DCX sequene (Fig. ). The Dx sequene in this plsmi vetor is missing the 3 untrnslte region (UTR) of Dx, n the vetor enoing short hirpin RNA (shrna) ginst Dx (3UTRhp) tht we previously evelope 26 trgets this region. Thus, unlike enogenous Dx, expresse from CALNL- is not trgete y Dx RNAi. Another requirement of this strtegy is tht e expresse only fter the ition of 4-hyroxytmoxifen (4-OHT). To test for suh ontrolle reexpression in vivo, we trnsfete neoortil neuronl progenitors in utero t emryoni y 4 (E4) with the plsmi DNA vetors CALNL-, n 3UTRhp (see Methos). At irth, rt pups were given single intrperitonel injetions of either 4-OHT or vehile? e CAG promoter + 4-OHT Cre Mlformtion free CAG promoter In utero eletroportion P resue P resue P resue ER loxp Cre neo Dx knokown ER pa loxp DCX DCX polya 4-OHT injetion 4-OHT inue expression E4 P P P polya polya CALNL- Merge DCX ntioies CALNL- CAG-mRFP + 4-OHT Vehile Figure Temporl ontrol of Dx expression in mispositione neurons y using Cre-epenent expression vetors n 4-OHT tivtle Cre reominse. () A shemti igrm of the experimentl pproh use to test the hypothesis tht reexpression of Dx n retivte migrtion n regress mlformtion. () A 4-OHT tivtle Cre reominse ompose of two estrogen reeptor (ER) ining omins is expresse uner the ontrol of the CAG promoter ( ). The Cre-epenent Dx expression vetor ontins neomyin resistne gene (neo) with stop oon flnke y loxp sites (CALNL-). In the presene of 4-OHT, reomintion ours n is expresse. pa n polya inite the polyenosine sequene in the plsmis. () Confol imges showing trnsfete neurons in neoortex of P rts tht hve reeive 4-OHT or vehile injetion t irth. Rts were eletroporte t E4 with four plsmis: CAG-mRFP,, CALNL- n 3UTRhp. In 4-OHT trete rts (left), DCX-GFP is expresse n Dx is etetle with ntioies in trnsfete mispositione neurons. In vehile-trete rts (right), no signl is etete in the green hnnel or with Dx ntioies. () Trnsfetion onitions. (e) Shemti igrm of experiments. Sle r, mm. ontrol. In postntl y (P) neoortil setions from 4-OHT injete rts, trnsfete ells were positive for signl n were immunopositive for Dx (Fig. ). In ition, expression ws etete s erly s fter 4-OHT injetion n peke to over 8% of trnsfete ells fter 2 (Supplementry Fig. online). In ontrst, in littermte ontrols reeiving the sme in utero trnsfetions ut injete with vehile only, Dx n expression were not etete (Fig. ). Thus, this system llows for temporlly ontrolle reexpression of Dx t times when expression of enogenous Dx is erese. Reexpression of Dx reues SBH n restores lminr position To etermine when to first reexpress Dx in mispositione neurons, we next estlishe when SBH n lmintion efiits were present fter irth in the Dx RNAi moel 26. Dx knokown use lminr isplement n prominent SBH y the y of irth (P; Fig. 2). Consequently, we onute two types of experiments to inue reexpression of Dx in mispositione neurons on P n nlyze the rts t P2. In the first, we ontrolle for the ition of 4-OHT n for Cre reominse tivity n vrie whether or DCX ws onitionlly expresse from CALNL- or CALNL-, respetively, in rts eletroporte t E4 with shrna vetors trgeting Dx (Fig.,e). This experiment ontrolle for possile effets of Cre reominse, s oth groups reeive trnsfetion of the plsmi n oth reeive 4-OHT injetions. We foun tht, when ws expresse t P, SBH mlformtions were signifintly reue in size (Fig. 2 e) reltive to when ws expresse. In ition, wheres trnsfete ells in the group umulte in white mtter n in eep lyers, NATURE MEDICINE VOLUME [ NUMBER [ JANUARY 29 8

3 29 Nture Ameri, In. All rights reserve. trnsfete ells in the group were lote preominntly in upper lyers in pttern similr to tht foun in rts tht reeive shrna vetor (3UTRm3hp) ineffetive in eresing Dx expression (Fig. 2). In seon version of the elye resue experiment, we ontrolle for trnsfetion of ll plsmis, ut vrie whether the rts in litter reeive n injetion of 4-OHT or vehile solution (Fig. 3). The results of this experiment were nerly ientil to the first experiment. SBH size, ssesse either y the ensity of neurons within the white mtter tht were positive for the upper lyer neuron mrker CAAT isplement protein, known s CDP/Cux, or y the ross setionl re of the mlformtion efine y umultion of CDP/Cux + neurons, ws signifintly reue in the group tht reeive 4-OHT ompre to the vehile ontrol group (Fig. 3 ). In oth types of reexpression experiments, expression use reistriution of trnsfete ells from errnt positions to upper neoortil lyers without signifint hnge in totl numer of ells (Fig. 3). Together, these results inite tht ells normlly fte for the upper lyers of the neoortex ut stlle t errnt positions were inue y Dx reexpression to migrte to their normlly fte lyer. Resue neurons re morphologilly norml We next ssesse whether the mispositione neurons inue to migrte to upper lyers y Dx reexpression were morphologilly similr to neurons tht migrte normlly to their fte upper lyer positions. We ompre severl morphologil properties Figure 2 Restortion of neoortil lmintion n regression of SBH fter reexpression of Dx t P. () Representtive neoortil setions showing lminr position of trnsfete ells in rts eletroporte t E4 with either ineffetive, ontrol shrna (3UTRm3hp; top row) or effetive DCX-trgeting shrna vetors (3UTRhp; ottom row) together with CAG-mRFP n proesse postntlly from P to P. () Representtive neoortil setions showing restortion of neoortil lmintion t P2 fter reexpression of Dx in mispositione neurons t P. Four plsmis were eletroporte t E4: CAG-mRFP,, CALNL- n 3UTRhp, n 4-OHT ws ministere t irth. () Quntifition of trnsfete ell istriution within the neoortil gry mtter fter inution of or expression in rts eletroporte t E4 with either effetive (3UTRhp) or noneffetive (3UTRm3hp) Dxtrgeting shrna vetor together with CAG-mRFP, n either CALNL- or CALNL- ( 2 setions from two or three rts per onition)., not signifint. () Size n position of SBH t three rostroul levels fter inution of (top) or (ottom) expression in rts eletroporte t E4 with CAG-mRFP,, 3UTRhp n either CALNL (top) or CALNL- (ottom). (e) Quntifition of SBH surfe fter inution of or in the sme experimentl onitions (eight or nine setions from two or three rts per onition; re units in squre pixels, px 2 ¼ mm 2 ). P o., P o.. Sle rs, mm. 3UTRhp 3UTRm3hp E4 : P CAG-mRFP n 3UTRhp of pil enrites in two onitions: mlformtion-free onition, in whih neurons were trete with n ineffetive Dx RNAi n were inste inue to express strting t P, n P resue group, in whih neurons were initilly rreste in SBH y Dx RNAi n then stimulte to migrte y inue expression of (Fig. 4 ). Lele neurons in oth groups were uniformly positive for the upper lyer mrker CDP/Cux (Fig. 4 ). Aitionlly, there were no signifint ifferenes etween the morphologies of lele neurons in the two groups, s ssesse y the numer of enriti proesses (Fig. 4e), the verge length of the proesses (Fig. 4f) n the totl proess length (Fig. 4g). In ition to hving norml enriti evelopment, the resue neurons extene xons ross the orpus llosum, n these xons projete to pproprite lminr territories in the ontrlterl hemisphere (Supplementry Fig. 2 online). We lso foun no signifint ifferene in the numer of interneurons in neoortex ontining SBH, in resue neoortex or in mlformtion-free ontrlterl hemispheres (Supplementry Figs. 3 n 4 online). These results inite tht resue neurons stimulte to migrte wy from errnt positions settle in the pproprite lyers, ontinue to express lminr-speifi mrkers n showe pproprite enriti n xonl ifferentition ptterns. E4 : P E4 : P E4 : P CALNL- e Reltive position of trnsfete ells in the grey mtter (in numer) Mlformtion free P resue Perentge of ells P resue Merge (+ Blue Nissl) Men surfe of heterotopi ( 3 px 2 ) 2 P resue 86 VOLUME [ NUMBER [ JANUARY 29 NATURE MEDICINE

4 29 Nture Ameri, In. All rights reserve. Merge CDP/Cux ntioies CALNL- CAG-mRFP Vehile-trete 4-OHT trete Vehile SBH Dx knokown E4 + 4-OHT SBH Inue expression Heterotopi reution eomes less effetive with ge To ientify the ltest evelopmentl time point t whih inue Dx reexpression oul still resue neoortil lmintion n use SBH to regress, we ministere 4-OHT t P or P n ompre the results to those in pups tht reeive 4-OHT t P. The results of these three resue tretments were lso ompre to those in rts tht reeive only inution of t P n thus evelope SBH. The totl numer of ells, the positions of the trnsfete ells n the size of SBH were nlyze t P2 (Fig. ). There ws no signifint ifferene in the totl numer of trnsfete ells per setion etween ny of the onitions, initing tht there ws no ell eth inue y either or expression t P, P or P (Fig. e). In ontrst, in oth the P n the P resue groups there ws signifint shift of neurons towr upper lyer positions reltive to P No Men surfe of CDP/Cux + ells ( 3 px 2 ) Men ensity of CDP/Cux + ells (ells per 3 px 2 ) Doule ortex P resue Doule P ortex resue Figure 3 Migrtion of fte upper lyer neurons from SBH. () A shemti igrm of experiments. Four plsmis were eletroporte t E4: CAG-mRFP,, CALNL- n 3UTRhp. 4-OHT or its vehile solution ws ministere t irth. expression is inue in the 4-OHT trete group only. () Immunohistohemistry of P2 neoortil setions showing E4 trnsfete n nontrnsfete ells immunopositive for the upper lyers neurons mrker CDP/Cux within SBH t P2 in the sene of Dx reexpression (left) or fter Dx reexpression (right). Some trnsfete ells exten out of the SBH into neoortil lyer. (,) Quntifitions of surfe re oupie y SBH () n ensity () of CDP/Cux + ells within SBH in the sme experimentl onitions (six setions from two rts per onition). P o., P o.. Sle r, mm. neurons in rts tht reeive no resue (Fig. f,g). Although neurons in the P n P resue groups migrte further into upper lyers thn neurons in the unresue group, neurons resue t P or P i not migrte s fr into upper lyers s i neurons tht were resue t P (Fig. f,g). However, the P resue group, similr to the P resue group, h signifintly smller SBH mlformtions thn i the P resue group or the group tht reeive no resue (Fig. h,i). These results inite tht reexpression of Dx t P or P n inue prtil reovery of neuronl position, tht reexpression t P n use SBH to regress n tht y P reexpression is no longer effetive t reuing the size of SBH mlformtions. Reution of SBH reues seizure suseptiility n severity SBH mlformtions in humns re well known risk ftor for epilepsy,2. Therefore, we investigte whether the suseptiility to pentylenetetrzol-inue seizures ws hnge in rts with regresse SBH reltive to unresue rts. A series of suonvulsive oses of pentylenetetrzol (2 mg kg ) were ministrte intrperitonelly every min to rts in three ifferent groups (esrie in Fig.,e): Mlformtion free P resue Figure 4 Morphology of resue neurons. (,) Immunohistohemistry for the upper lyer neuron mrker CDP/Cux on P2 frontl neoortil setions. Rts were eletroporte t E4 with either noneffetive (3UTRm3hp; ) or effetive (3UTRhp; ) Dx-trgeting shrna vetors together with CAG-mRFP, n either CALNL- () or CALNL- () n injete with 4-OHT t irth. Both initilly orretly positione trnsfete neurons () n initilly mispositione trnsfete neurons inue to migrte to pproprite positions fter Dx reexpression () (oth green n re) re lote within the CDP/Cux + n of upper lyer neurons (in lue). Trnsfete ells were lote within neoortil lyers II/III n. (,) Reonstrute ortil neurons in n showing enriti roriztion ptterns from the sme experimentl onitions s in n, respetively. (e g) Quntifition of enriti roriztion: men numer of pil proesses per neuron (e), their men length (f) n totl length (g) (36 or 37 reonstrute neurons from three or four rts per onition). Sle r, 2 mm. e 7 f,2 g 2, 6, Mlformtion free 2, 8 P resue 4, 6 3, Men numer of proesses Seonry Tertiry Quternry Men length of proesses Primry Seonry Tertiry Quternry Totl length NATURE MEDICINE VOLUME [ NUMBER [ JANUARY 29 87

5 29 Nture Ameri, In. All rights reserve. Mlformtion free P resue P resue e f Reltive position of trnsfete ells in the grey mtter (in numer) P resue P resue 2 P resue Perentge of ells one tht h reeive 4-OHT to inue expression t P (P resue), one tht reeive 4-OHT t P to inue expression (oule ortex, no resue) n thir group tht reeive ineffetive shrnas n so ws free of mlformtions (mlformtion free). Injetions of pentylenetetrzol were ontinue until eh rt h generlize seizure. Seizure suseptiility n severity ws quntifie with the nonil Rine s sle of seizure severity, inluing ose n lteny to onset of the first miniml motor seizure n the first generlize toni-loni seizure (Fig. 6). Generlize seizures in the oule ortex group ourre t signifintly lower oses of pentylenetetrzol (Fig. 6) n with signifintly shorter ltenies (Fig. 6) thn they i in either mlformtion-free or P resue rts. Notly, mlformtion-free n P resue groups showe similr seizure threshols y ll mesures (Fig. 6 ). These results suggest tht Dx reexpression t P restore levels of seizure risk k to levels seen in rts tht h no neoortil mlformtion. DISCUSSION To our knowlege, this is the first stuy to emonstrte tht moleulr intervention n reue the size n funtionl effets of g Reltive position of trnsfete ells in the grey mtter (in numer) i Men surfe of heterotopi ( 3 px 2 ) P resue P resue h P resue Perentge of ells 2 P resue P resue P resue Figure Cruil evelopmentl perio for regressing mlformtion. ( e) Representtive reonstrute neoortil setions showing the lminr position of neurons t P2 fter inution of (,) or expression t P ( ), P () np(e). Blk ots represent positions of trnsfete ells. Four plsmis were eletroporte t E4: CAG-mRFP ( e), ( e), either CALNL (,) or CALNL- ( e) n either 3UTRhp ( e) or3utrm3hp(). 4-OHT ws ministere t P ( ), P () or P(e). (f,g) Quntifition of trnsfete ell istriution within the neoortil gry mtter fter inution of or expression t P (f) n P (g), ompre to Dx reexpression t P (8 2 setions from two or three rts per onition). (h) Size n position of SBH t three rostro-ul levels fter inution of Dx expression t P (top) n P (ottom). (i) Quntifition of SBH surfe fter inution of Dx expression t P n P (eight setions from two or three rts per onition). P o., P o., P o.. Sle r, mm. preexisting isruption in neuronl migrtion. In generl, our stuy rises the possiility tht, in some ontexts, neuronl migrtion is form of neuronl plstiity tht my e engge to inue neurl repir. Previous exmples of ontinue neuronl migrtion in the rin inlue neuronl migrtion of enogenous neurons to the olftory ul n entte gyrus throughout life 29 s well s migrtion of trnsplnte neuronl progenitors into neoortex n other regions of the entrl nervous sytem 3. Our stuy extens this phenomenon of ontinue migrtion to inlue inue migrtion of mispositione neurons in the neoortex. Our stuy lso suggests tht reversing migrtion isruption in evelopment n reue errnt exitility levels in more mture rin. In this stuy, we show tht there re onsierle evelopmentl onstrints on stimulting migrtion in the postntl neoortex inue y reexpression of Dx. Wheres Dx reexpression t P inue migrtion tht le to mrke regression of heterotopi n restore neoortil lmintion, reexpression t P le to prtil restortion of neuronl position with heterotopi regression, n reexpression t P le to prtil reovery of position without signifint heterotopi reution. These ges in rts orrespon in Figure 6 Derese seizure suseptiility to pentylenetetrzol-inue seizures. Quntifition of oses of pentylenetetrzol (PTZ; ), lteny to inue generlize toni-loni seizures () n intervl etween miniml n generlize seizures () in three groups of rts: mlformtionfree rts (ineffetive shrnas n expression inue t P), oule ortex rts with SBH n lmintion efiits (effetive shrnas n expression inue t P) n P resue rts with restore lmintion n regresse SBH (effetive shrnas n expression inue t P). () Exmple of oule ortex rt experiening rerings, fllings n onvulsions (top) n P resue rt t the sme ose of PTZ n sme time fter injetion (ottom). The P resue rt experiene seizures min lter, fter n itionl injetion of PTZ ws given. P o., P o., P o.. Cumultive ose of PTZ inuing toni-loni seizures (mg kg ) P resue Mlformtion free Doule P Mlformtion Doule ortex resue free ortex Men lteny to inue toni-loni seizures (min) P resue Intervl etween miniml n toni-loni seizures (min) Mlformtion free Doule ortex P resue VOLUME [ NUMBER [ JANUARY 29 NATURE MEDICINE

6 29 Nture Ameri, In. All rights reserve. humns roughly to preterm gesttionl weeks 2 3 (for P) n to full-term infnts (for P). We o not urrently know the reson for the sene of regression t P or the reue migrtion fter P intervention. It is likely, however, tht evelopmentl hnges in neoortil tissue n within ifferentiting neurons limit ontinue neuronl migrtion into the postntl perio. Migrtion of pyrmil neurons uring norml neoortil evelopment is guie y ril glil proesses 3, n it is likely tht evelopmentl loss of ril gli, whih normlly ours y the mile of the seon postntl week in the rt, reues the potentil for ontinue migrtion 32,33. In ition, the evelopmentl winow for migrtion out of SBH my lose euse of neuronl mturtion. Neurons within SBH form synpses n sen xonl projetions to suortil trgets 26,27, n suh neuronl ifferentition my prelue inue migrtion y reexpression of Dx. However, it is noteworthy tht even s lte s t P our results show tht there is still pity for plstiity n tht mispositione neurons n e shifte from eeper to more superfiil positions y expression of Dx. In the future it will e useful to etermine whih speifi ellulr hnges re responsile for evelopmentl restrition of inue remigrtion n whether these n e mnipulte to llow for heterotopi regression in even more mture rts. Virtully ll neuronl migrtion isorers, inluing lissenephly, periventriulr noulr heterotopis n suortil n heterotopi, re risk ftors for seizures In ition, fol ortil ysplsis re estimte to e ssoite with 3% of intrtle temporl loe epilepsies 37,38. The speifi mehnisms tht use ysplsti orties to use epileptiform tivity remin unler. For exmple, the point in evelopment t whih the presene of neoortil mlformtion first isposes ortil tissue to overexitility hs remine unknown,3,39 4. Our results suggest tht n unerlying use of seizure suseptiility to reue pentylenetetrzol ose is iretly relte to errnt neuronl positioning n to the mlformtion. We nnot yet istinguish from our results t wht point in evelopment migrtion impirment uses inrese exitility, ut regression initite fter irth is suffiient to restore pentylenetetrzol threshols to ontrol levels. Future experiments n now e irete t etermining the time ourse of hnges in synpti n ellulr physiology tht orrelte with reue seizure suseptiility. The speifi experimentl intervention we use to restore migrtion ptterns, eletroportion of n inuile onstrut, is lerly not pplile to humns. There re t lest two possile iretions inite y our finings for interventions tht my one y e therpeutilly vile. First, Dx expression my e inue y virl trnsution of ells within SBH mlformtions. Our results urrently suggest tht suh gene therpy woul proly nee to e pplie perintlly ut oul e restrite to mispositione neurons within SBH. An lterntive to gene therpy oul e to enhne the funtion of other memers of the DCX superfmily tht my hve reunnt funtion with DCX. For exmple, in mouse knokouts of Dx there re no ortil ysplsis 42, n only when Dx n the relte gene Dlk re oth isrupte o migrtion isorers similr to humn lissenephly pper 43.Thus,Dlk in the mouse seems to e ple of ompensting for Dx funtion. There re now multiple efine memers of the DCX superfmily with similr ellulr n iohemil funtions 44, n perhps phrmologil enhnement of the funtion of one or severl of these my filitte regression of SBH in humns. Finlly, the present stuy serves s proof of onept tht mismigrte ells n e mnipulte to restore norml morphologil pttern n level of neuronl exitility in the ererl ortex. METHODS In utero eletroportion n Dx RNA interferene. We performe in utero eletroportion s esrie efore 26.WeuseshRNAstrgetingthe3 UTR of Dx (3UTRhp) or n ineffetive shrna with three point muttions reting mismthes (3UTRm3hp). We previously showe 26 tht the 3UTRhp onstrut effiiently knoks own Dx expression in vivo, wheres the 3UTRm3hp onstrut is ineffetive in using Dx knokown. In ition, the Dx-speifi RNAi inue phenotype is resue y Dx expression, onfirming the speifiity of shrnas 27. We eletroporte plsmis enoing mrfp (CAGmRFP,. mg ml ) to fluoresently lel trnsfete ells long with plsmis expressing shrnas ginst Dx (3UTRhp or 3UTRm3hp,. mg ml ), 4-OHT tivtle form of Cre reominse (,.mg ml ) n Cre-epenent inuile expression vetors (CALNL-,. mg ml ; gift from T. Mtsu n C. Cepko) n the plsmi CALNL- (. mg ml ), whih ws me for this stuy. We onstrute pcaggs-dcx plsmi on the sis of the previously esrie pcaggs-dcx plsmi 27 y fusing to the roxy terminus. To generte CALNL-, we sulone the fusion frgment from pcaggs- into the EoRI n NotI sites of the CALNL- plsmi. All rt protools were pprove y the University of Connetiut Institutionl Animl Cre n Use Committee. 4-hyroxytmoxifen ministrtion. We issolve 4-OHT (Sigm) in 9% ethnol t onentrtion of 2 mg ml n ilute it with nine volumes of orn oil. We ministere ilute 4-OHT (2 mg per g oy weight) to the rts through intrperitonel injetions. We injete vehile-trete rts with the sme solution without 4-OHT. Histologil proeures n mirosopy. We trnsrilly perfuse rts uner eep nesthesi with 4% prformlehye in PBS. We remove their rins n post-fixe them for 48 h in the sme fixtive solution efore frontl setioning with virtome (Lei). We proesse rin setions for immunohistohemistry s floting setions. We use the following primry ntioies: got ntioy to ouleortin ( in, Snt Cruz Biotehnology), rit ntioy to CDP/Cux ( in 2, Snt Cruz Biotehnology), mouse n rit ntioies to GFP ( in 2, n in 3,, respetively, Moleulr Proes), rit ntioy to RFP ( in 3,, Chemion). We use the pproprite seonry ntioies ( in 2) onjugte to Alex 488, Alex 68, Alex 633 (Chemion) or Cy (Jkson ImmunoReserh) n onterstine setions with the fluoresent Nissl stining NeuroTre 43/4 ( in 2, Moleulr Proes). We took photomirogrphs with Nikon Elipse E4 mirosope equippe with igitl Spot Cmer (Dignosti Instruments) or with Lei TCS SP2 onfol mirosope. Seizure inution with pentylenetetrzol. We issolve pentylenetetrzol (Sigm) in.9% sline n ministere it intrperitonelly to rts t P3 t onentrtion of 2 mg per kg of oy weight. There ws no sttistilly signifint ifferene in weight or sex rtio etween groups of rts. Rts were ple in Plexiglss ges, oserve y n experimenter nive to the sttus of the rt n their ehvior ws sore with the Rine s sle 4.Weministere pentylenetetrzol every min until generlize seizures ourre. Quntifitions n sttistil nlyses. We performe quntifitions with the imge nlysis softwre ImgeJ.39e on oronl setions (6 mm) lote from regm 3. to regm. oring to the Rt Brin Atls 46.We quntifie the reltive positions of trnsfete ells in the ortex y ounting mrfp + ells in 2 res of interests normlize in iniviul setions to fit within the whole thikness of the ortil wll, from eep to superfiil lyers n exluing the white mtter. We quntifie the size of SBH mlformtions in squre pixels with ImgeJ mesuring tools. We me morphologil nlyses of enriti roriztion fter neuronl reonstrutions with the ImgeJ plugin NeuronJ.2 (Erik Meijering). We performe sttistil tests with InStt 3. (GrphP). We heke the normlity of the t istriution with the Kolmogorov n Smirnov metho. We use two-smple Stuent s t-test to ompre mens of two inepenent groups. When three groups of t were ompre, we use one-wy nlysis of vrine with the Tukey-Krmer post-test for multiple omprisons. We NATURE MEDICINE VOLUME [ NUMBER [ JANUARY 29 89

7 29 Nture Ameri, In. All rights reserve. onsiere vlues s signifint when P o.. All t re presente s mens ± s.e.m. Note: Supplementry informtion is ville on the Nture Meiine wesite. ACKNOWLEDGMENTS CALNL- n were gifts from T. Mtsu n C. Cepko, Hrvr Meil Shool. ImgeJ softwre ws from W. Rsn, US Ntionl Institutes of Helth. NeuronJ.2 ws from E. Meijering (Ersmus University Meil Center Rotterm). This work ws supporte y the US Ntionl Institutes of Helth (MH624 n 6246 to J.J.L.) n the Jerome Lejeune fountion (reserh fellowship to J.-B.M.). AUTHOR CONTRIBUTIO J.-B.M. n J.J.L. ontriute to ll spets of the projet; J.-B.M. onute histologil proeures, seizure inution experiments, quntifitions n t nlysis; Y.W. performe intruterine surgeries n interneuron nlysis; Y.C. performe onfol mirosopy, interneuron nlysis n ontriute to seizure inution experiments n M.P. onstrute the plsmi vetors n ontriute to onfol mirosopy. 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