~Stcs' USA 50 , Pp: 616s July Suntmony Transsynaptic neuronal loss induced in bippocamgial licubuniures bon herpes simplex virus vector expressing the GluR6 subunit 6y by a of the kainate receptor J#c FEDEROFFY AND HowARD ZENG Xiu-Liu" BERCOLD't , PATRIZIA CASACCIA-BONNEFILI_ PETER Brooklyo  Ncv Yon . NY Yot-Hle_lth Science Ccntcr Biclczx . State Unitcrsity ol Nc Lnllomy Lic ce Hroeram iUrTT_cna "Epn Albcnt Eunalcin School & Mcdeict BienI Arutoxckt Hedicine "DhirLikhl, 1}; i0J 19931 199} (received for rtvich Junkan Dominick P. Furpura Apa $ Cominiuniceted by of thc GluR6 cncoded channcl: This channel may &e morc epilepsy lemporal lobr Patlents #lth cercrt prescnce Ihan to Cat duc I0 lht pcrmeable lo Na ABSTRACT tbe hippo: bsk acurons withln {he CAJ and hilar regions al residuc 690 (16) . This argininc residue i$ als0 argininc hilar ncurons was also Inductd by Ol an Loss d CAJ similar and non NMDA reccplors displaying othcr cAmpuI nith found slice cunute {rensduclng errezivol erohlppmplepalrUS (HSVHreactor &: not encoded ionic pcmeability (17). The arginine residuc mplicatlon-delective herpes sinibler elutaminc codon by Ihc gene for GluR6; inslcad rtceplor pressing thc GluR6 kainate sublype of Uhe Elulamate argininc codon by RNA convered GluR6 mRNA transduccd @ibroblasts, HSVGluR6 expresscd (HSVGIuR6) editing (16) . #ith anti-CluR6 seruni After prolcIn that rcacted vecior, 4 'M,[IS,00 Wc have conslructed a herpes simplex vinus (HSV) keinale-depcndcnt HSVGluR6, rbrohfasts directs thc exprcssion of a cDNA encoding HSVGluR6, aposure Gells Ghat otiere sPreuousox ,ceombinane loxiclly appeartd the GluR6 kainate Iecepior subunit undcr thc edited fonm kalnale  Microappllcalion oC nanoliter amounts the control of Ihc HSV immediate carly (IE) 4/5 promoler alicr Sulhursile of organotyplc hlpporampa microapplication mclhod stocks This viral veclor #as applied by HSV loczlized transduclion and gcpe Iranster Ihat penmnits localized infeclions of organolypic hipporampal suled mjcroapplicatoa. Microapplicallon of !00 HSVGluR6 Yirtons HSV veclors lransduce Eenes with very high large Ioss o CA} slice cullures. into Crj stralum Pyramidale Induccd necdcd [0 small numbcrs ol vinons elicicncy. Ihat despitc the smell numutt ctlks and hilar neurons 19). The microapplicalion biological elTect produce pimmidal ICQ virions iransduced neurons. Thls cllect #as nol scen whco permits Iransler of genes inlo identificd hippocampal CAI stralum pyremldek mcthod of KSVGluRG mlcroapplicd slicc cultures rclain much of Ihe Iaminar Organi Hcnt N-melhyi-D-Aspartale  reccplor antagonists Joci , since Tetrodoloxin synaptic conncclions o zation, ccllular heterogcneily, and loss of CAJ and hllar ncurons, suegcsting Lnhibitcd Ine Wnge transverse hippocampal slices (20-22). Microapplication ~transduced cclls Induced cluster of HSVGluR6 CAJ stralum pyramidale; bul not CAL siralum snall G that (hc HSVGluR6 loss o non- ~espartate-dependent trantsynaptic Iranssynaplic ncuronal Joss Aemicihi: epraaidhtougcoultedeicA 'Tanasyano denauconusEceions resulled transduced Ocurons_ spread The cell loss scen in the KSVGluR6 of the slice culturc Inthe hippocampus of patients with lemporal-lobe epilepsy; similar t0 Ihc hippocampa transduced slice cullures damaged (1-4) most likely vivo or in palients often CAJ and hilar ncurons arc rcsulting (rom kainate injeclion _ dathajemporal Tobkopilcsy. excessive synaplic release of elutamale (5 , 6). Seizurc experimenlally aclivity and cell can b induccd elutamale 2conis sicreotaxic Kainale_ #Pplication MATERIALS AND METHODS observed both at the sitc of kainale injecction Ncuronal loss sugresling that kainale dcpolarization of Organotypkc Slice Cullures. NIH ZT} mousc and a1 distant silc $ Crll Llnes and crcio Eaglc Lranssynaplic Uc injection silc induccs modilicd Dulbecco ncurons fbroblasts were grown similar excitotoxic mcchanism may be respon- Ercolomycin and toxicity (8). plus 10Z calf serum AmPlulamint Knn paticnts with (emporal-lobx sible for CAJ ncuronal loss (100 units/ml) and penicillin (10 units/ml) (all medium thc recument such palicnts components #ere from GIBCO) Vero monkey kidney ccll cpilepsy. Dunng scizures clusler of cxcitalory synapses and inlrinsic burst firing of exccpt that 10  (ctal idenlical medium Zio4n Wctc excitotoxic cell Iloss Ihroughout Both ccll lincs were mainiaincd CA} ncurons May rcsult in was UScd_ bovine scrum (S) . Burst firing or CA} ncurons Organotypic slice cultures the CA} ad hilar 37*C in S7 COx almosphcrc rezions cnhanccd expression of non-N-melhyl-D; hippocampal sliccs (rom Spra (rom cstablished From WtuM rcsult Wee (non-NMDA) reccplors in thc hippocampus of aller birth (23) . These cultuTes days guc-Dawley rt pups 10 asnanaic #ccks epicptic palients (9 . 10) . wcTc maintaincd at JS C ina S% COzatmosphcere for GluR?, KA-]; and Recenlly livc cDNAs (GluRS, GluR6 All rcagents wcrc oblaincd [rom Sigma unless pnor family of kainatc KA-2) havc bzen cloncd which dcline olherwise spccilied. rcccpior subunits (11-1S). Introduclion and cxpression o GluR6 cDNA Titer o( HSVGluR6. Consiruction 293 embryonic containing the complete coding scqucnce of GluR6 ( Gift % GluR6 cDNA into Xenopus oocyles numun kidncy cclls induces & rapidly desensitizing, kainale-E Heincman , Salk institule, La Jolia, CA) was digested with Faled activaled by kainate, but not by channel (14). This channcl the piutamaic agonists =)a-amino-} hydroxy 5 mcthylisoor Abbreviations: NMDA N-mcthyl-D-aspanale; AMPA;(#haaminz. ~#-propionale (AMPA) and NMDA (41) . Coapplication CNQX, 6 cyano 7: 370l6 - ) hxdroxy-$-mcthylisoxezolc-4 Propionale rcduces the dcscnsitization CPP; = [(R} Kith thc Ieclin Con Vkainalc J} dione; 7 CK nitroquinoralinc T-hlorokynutcna 3-carbox} Piecrazinc + yllpropyl ! phosphonale: HS Mpes S-bromo 4-chloro } indolyl plcx viNus; IE, immedialc carly; X Gul , paee chute ol thisarick Nnb Tbc putlication dclnyed CTo talororeprinosicq cot MytcrL; This arikk must uhcrclore b hcreby marked "advertiscmcnf rcprinl rcqucsts should Ex addrcsscd . Indkale Ihis facl; 417M solcly accudurit #U 6165 40009979
~Stcs' USA 50 , Pp: 616s July Suntmony Transsynaptic neuronal loss induced in bippocamgial licubuniures herpes simplex virus vector expressing the GluR6 subunit by a of the kainate receptor ZENG Xiu-Liu" AND HowARD FEDEROFFY BERCOLD't , PATRIZIA CASACCIA-BONNEFILI_ PETER Biclczx . State Unitcrsity ol Nc  Yot-Hle_lth Science Ccntcr Brooklyo  Ncv Yon . NY iUrTT_cna Hroeram Lnllomy Lic ce "Epn Hedicine Arutoxckt Albcnt Eunalcin School & Mcdeict BienI 1}; i0J "DhirLikhl, Dominick P. Furpura Apa $ 199} (received for rtvich Junkan 19931 Cominiuniceted by cercrt lemporal lobr epilepsy of thc GluR6 cncoded channcl: This channel may &e morc ABSTRACT Patlents #lth pcrmeable lo Na Ihan to Cat duc I0 lht prescnce bsk acurons withln {he CAJ and hilar regions tbe hippo: hilar ncurons was also Inductd by Ol an argininc al residuc 690 (16) . This argininc residue i$ als0 cAmpuI Loss d CAJ and cunute nith found othcr non NMDA reccplors displaying similar slice {rensduclng errezivol erohlppmplepalrUS (HSVHreactor &: ionic pcmeability (17). The arginine residuc not encoded mplicatlon-delective herpes sinibler Elulamate rtceplor by Ihc gene for GluR6; inslcad elutaminc codon pressing thc GluR6 kainate sublype of Uhe GluR6 mRNA convered argininc codon by RNA transduccd @ibroblasts, HSVGluR6 expresscd (HSVGIuR6) prolcIn that rcacted #ith anti-CluR6 seruni After editing (16) . 'M,[IS,00 Wc have conslructed a herpes simplex vinus (HSV)  vecior, rbrohfasts HSVGluR6, keinale-depcndcnt aposure HSVGluR6, directs thc exprcssion of a cDNA encoding loxiclly appeartd Gells Ghat otiere sPreuousox ,ceombinane thc edited fonm the GluR6 kainate Iecepior subunit undcr kalnale  Microappllcalion oC nanoliter amounts the control of Ihc HSV immediate carly (IE) 4/5 promoler HSV stocks organotyplc hlpporampa alicr Sulhursile of This viral veclor #as applied by microapplication mclhod suled  loczlized transduclion and gcpe Iranster Ihat penmnits localized infeclions of organolypic hipporampal mjcroapplicatoa. Microapplicallon of !00 HSVGluR6 Yirtons large Ioss o CA} slice cullures. HSV veclors lransduce Eenes with very high into Crj stralum Pyramidale Induccd numutt elicicncy. Ihat small numbcrs ol vinons necdcd [0 pimmidal ctlks and hilar  neurons despitc the smell ICQ virions produce biological elTect 19). The microapplicalion iransduced neurons. Thls cllect #as nol scen whco CAI stralum pyremldek mcthod permits Iransler of genes inlo identificd hippocampal of KSVGluRG Hcnt mlcroapplicd Joci , since slicc cultures rclain much of Ihe Iaminar Organi Tetrodoloxin N-melhyi-D-Aspartale  reccplor antagonists zation, ccllular heterogcneily, and synaptic conncclions o Lnhibitcd Ine Wnge loss of CAJ and hllar ncurons, suegcsting ~transduced cclls Induced transverse hippocampal slices (20-22). Microapplication that (hc snall G cluster of HSVGluR6 loss o non- HSVGluR6 CAJ stralum pyramidale; bul not CAL siralum Aemicihi: ~espartate-dependent trantsynaptic resulled Iranssynaplic ncuronal Joss transduced Ocurons_ epraaidhtougcoultedeicA 'Tanasyano denauconusEceions spread of the slice culturc The cell loss scen in the KSVGluR6 Inthe hippocampus of patients with lemporal-lobe epilepsy; transduced slice cullures similar t0 Ihc hippocampa CAJ and hilar ncurons arc often damaged (1-4) most likely rcsulting (rom kainate injeclion _ vivo or in palients excessive synaplic release of elutamale (5 , 6). Seizurc dathajemporal Tobkopilcsy. aclivity and cell can b induccd experimenlally sicreotaxic #Pplication Kainale_ elutamale 2conis observed both at the sitc of kainale injecction MATERIALS AND METHODS Ncuronal loss and a1 distant silc $ sugresling that kainale dcpolarization of crcio Crll Llnes and Organotypkc Slice Cullures. NIH ZT} mousc ncurons Uc injection silc induccs Lranssynaplic fbroblasts were grown Dulbecco modilicd Eaglc toxicity (8). similar excitotoxic mcchanism may be respon- Knn plus 10Z calf serum AmPlulamint and Ercolomycin sible for CAJ ncuronal loss paticnts with (emporal-lobx (100 units/ml) and penicillin (10 units/ml) (all medium such palicnts thc recument cpilepsy. Dunng scizures components #ere from GIBCO) Vero monkey kidney ccll cxcitalory synapses and inlrinsic burst firing of clusler of Wctc Zio4n idenlical medium exccpt that 10  (ctal CA} ncurons May rcsult in excitotoxic cell Iloss Ihroughout bovine scrum was UScd_ Both ccll lincs were mainiaincd the CA} ad hilar rezions (S) . Burst firing or CA} ncurons 37*C in S7 COx almosphcrc Organotypic slice cultures rcsult From cnhanccd expression of non-N-melhyl-D; Wee cstablished (rom WtuM hippocampal sliccs (rom Spra asnanaic (non-NMDA) reccplors in thc hippocampus of guc-Dawley rt pups 10 days aller birth (23) . These cultuTes epicptic palients (9 . 10) . wcTc maintaincd at JS C ina S% COzatmosphcere for #ccks Recenlly livc cDNAs (GluRS, GluR6 GluR?, KA-]; and pnor All rcagents wcrc oblaincd [rom Sigma unless KA-2) havc bzen cloncd which dcline family of kainatc olherwise spccilied. rcccpior subunits (11-1S). Introduclion and cxpression o Consiruction Titer o( HSVGluR6. GluR6 cDNA GluR6 cDNA into Xenopus oocyles numun 293 embryonic containing the complete coding scqucnce of GluR6 ( Gift % kidncy cclls induces & rapidly desensitizing, kainale-E Faled Heincman , Salk institule, La Jolia, CA) was digested with channel (14). This channcl activaled by kainate, but not by the piutamaic agonists =)a-amino-} hydroxy 5 mcthylisoor Abbreviations: NMDA N-mcthyl-D-aspanale; AMPA;(#haaminz. 370l6 - ~#-propionale (AMPA) and NMDA (41) . Coapplication ) hxdroxy-$-mcthylisoxezolc-4 Propionale CNQX, 6 cyano 7: Vkainalc Kith thc Ieclin Con rcduces the dcscnsitization nitroquinoralinc J} dione; 7 CK T-hlorokynutcna CPP; = [(R} 3-carbox} Piecrazinc + yllpropyl ! phosphonale: HS Mpes Nnb paee chute plcx viNus; IE, immedialc carly; X Gul , S-bromo 4-chloro } indolyl Tbc putlication cot ol thisarick dclnyed MytcrL; This arikk must uhcrclore b hcreby marked "advertiscmcnf CTo talororeprinosicq Indkale Ihis facl; rcprinl rcqucsts should Ex addrcsscd . accudurit #U 417M solcly 6165 bon 6y J#c 4 40009979