Target cell-specific plasticity rules of NMDA receptor-mediated synaptic transmission in the hippocampus

ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 1 TYPE O iginal Resea ch PUBLISHED 05 Ap il 2023 DOI 10.3389/ ncel.2023.1068472 OPEN ACCESS EDITED BY Dominique Debanne, INSERM U1072 Neu obiologie des Canaux Ioniques e de la Synapse, F ance REVIEWED BY Diasynou Fio a an e, Uni e si y o Cali o nia, Da is, Uni ed S a es Hélène Ma ie, Cen e Na ional de la Reche che Scien i ique (CNRS), F ance *CORRESPONDENCE Pablo E. Cas illo [email p o ec ed] †PRESENT ADDRESS Ka ina Al iña, Depa men o Neu oscience, Uni e si y o Flo ida, Gaines ille, FL, Uni ed S a es ‡These au ho s ha e con ibu ed equally o his wo k SPECIALTY SECTION This a icle was submi ed o Cellula Neu ophysiology, a sec ion o he jou nal F on ie s in Cellula Neu oscience RECEIVED 13 Oc obe 2022 ACCEPTED 20 Ma ch 2023 PUBLISHED 05 Ap il 2023 CITATION Lu zu S, Al iña K, Puen e N, G andes P and Cas illo PE (2023) Ta ge cell-speci ic plas ici y ules o NMDA ecep o -media ed synap ic ansmission in he hippocampus. F on . Cell. Neu osci. 17:1068472. doi: 10.3389/ ncel.2023.1068472 COPYRIGHT © 2023 Lu zu, Al iña, Puen e, G andes and Cas illo. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. Ta ge cell-speci ic plas ici y ules o NMDA ecep o -media ed synap ic ansmission in he hippocampus S e ano Lu zu1‡, Ka ina Al iña1†‡, Nago e Puen e2,3, Ped o G andes2,3 and Pablo E. Cas illo1,4* 1Dominick P. Pu pu a Depa men o Neu oscience, Albe Eins ein College o Medicine, B onx, NY, Uni ed S a es, 2Depa men o Neu osciences, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y UPV/EHU, Leioa, Spain, 3Achuca o Basque Cen e o Neu oscience, Science Pa k o he Uni e si y o he Basque Coun y UPV/EHU, Leioa, Spain, 4Depa men o Psychia y and Beha io al Sciences, Albe Eins ein College o Medicine, B onx, NY, Uni ed S a es Long- e m po en ia ion and dep ession o NMDA ecep o -media ed synap ic ansmission (NMDAR LTP/LTD) can signi ican ly impac synapse unc ion and in o ma ion ans e in se e al b ain a eas. Howe e , he mechanisms ha de e mine he di ec ion o NMDAR plas ici y a e poo ly unde s ood. He e, using physiologically ele an pa e ns o p esynap ic and pos synap ic bu s ac i i ies, whole-cell pa ch clamp eco dings, 2-pho on lase calcium imaging in acu e a hippocampal slices and immunoelec on mic oscopy, we es ed whe he dis inc calcium dynamics and g oup I me abo opic glu ama e ecep o (I-mGluR) sub ypes con ol he sign o NMDAR plas ici y. We ound ha pos synap ic calcium ansien s (CaTs) in esponse o hippocampal MF s imula ion we e signi ican ly la ge du ing he induc ion o NMDAR-LTP compa ed o NMDAR- LTD a he MF- o-CA3 py amidal cell (MF-CA3) synapse. This di e ence was abolished by pha macological blockade o mGluR5 and was signi ican ly educed by deple ion o in acellula calcium s o es, whe eas blocking mGluR1 had no e ec on hese CaTs. In addi ion, we disco e ed ha MF o hila mossy cell (MF- MC) synapses, which sha e se e al s uc u al and unc ional commonali ies wi h MF-CA3 synapses, also unde goes NMDAR plas ici y. To ou su p ise, howe e , we ound ha he pos synap ic dis ibu ion o I-mGluR sub ypes a hese wo synapses di e , and he same induc ion p o ocol ha induces NMDAR-LTD a MF-CA3 synapses, only igge ed NMDAR-LTP a MF-MC synapses, despi e a compa able calcium dynamics. Thus, pos synap ic calcium dynamics alone canno p edic he sign o NMDAR plas ici y, indica ing ha bo h pos synap ic calcium ise and he ela i e con ibu ion o I-mGluR sub ypes likely de e mine he lea ning ules o NMDAR plas ici y. KEYWORDS CA3, den a e gy us, mossy cell, synap ic plas ici y, calcium signal, wo-pho on lase scanning mic oscopy F on ie s in Cellula Neu oscience 01 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 2 Lu zu e al. 10.3389/ ncel.2023.1068472 In oduc ion Long- e m changes in synap ic s eng h a e widely ega ded as cellula co ela es o mos o ms o lea ning and memo y (Malenka and Bea ,2004;May o d e al.,2012). The N-me hyl- D-aspa a e ecep o (NMDAR) is a c i ical igge o long- e m po en ia ion and dep ession (LTP and LTD) o he as componen o glu ama e gic ansmission, which is mainly media ed by α-amino-3-hyd oxy-5-me hyl-4-isoxazolep opionic acid ecep o s (AMPARs) (Lusche and Malenka,2012). Like AMPARs, NMDARs a e also dynamically egula ed and unde go ac i i y-dependen bidi ec ional long- e m plas ici y (i.e., NMDAR-LTP and LTD) a many cen al synapses (Rebola e al.,2010;Hun and Cas illo,2012). NMDAR plas ici y can s ongly impac synap ic ansmission by changing he h eshold o AMPAR plas ici y (Rebola e al.,2011;Hun e al.,2013) and by modula ing synap ic in eg a ion and neu onal ou pu (Hun e al.,2013;Banks e al., 2015). Howe e , he molecula mechanisms ha con ol NMDAR plas ici y and de e mine i s di ec ion emain unclea . A be e unde s anding o hese mechanisms is impo an since NMDAR dys egula ion has been implica ed in se e al b ain diso de s (Lau and Zukin,2007), including schizoph enia (Ja i ,2010), au ism spec um diso de (Won e al.,2012), addic ion (Bo gland e al., 2006) and neu odegene a i e diseases such as Alzheime ’s (Liu e al.,2019) and Pa kinson’s disease (Ga doni e al.,2010). The induc ion o NMDAR plas ici y elies on pos synap ic calcium ise, which ypically a ises om he ac i a ion o NMDARs hemsel es and he ec ui men o o he calcium sou ces such as in e nal calcium s o es and calcium in lux ia ol age-ga ed calcium channels (Kwon and Cas illo,2008a;Rebola e al.,2008; Ha ne e al.,2009;Fe nandez de Se illa and Buno,2010;Jo e al., 2010;Hun e al.,2013). The impo ance o calcium signaling in he bidi ec ionali y o NMDAR plas ici y is highligh ed by he ac ha changing he in acellula calcium bu e capaci y wi h calcium chela o s can ei he block o change he sign o NMDAR plas ici y (Ha ney e al.,2006). In addi ion, se e al G p o ein-coupled ecep o s (GPCRs) ha e been implica ed in he induc ion o NMDAR plas ici y (MacDonald e al.,2007;Lu zu and Cas illo,2020). Chie among hem is he g oup I me abo opic glu ama e ecep o s (I-mGluRs), which play an essen ial ole in he induc ion o NMDAR plas ici y a se e al cen al synapses (Yang e al.,2014;Lu zu and Cas illo,2020). I-mGluRs a e Gq- coupled p o ein ecep o s whose ac i a ion induces calcium elease om in e nal s o es (Niswende and Conn,2010), indica ing ha mGluRs signi ican ly con ibu e o he pos synap ic calcium ise ha igge s NMDAR plas ici y. A he hippocampal mossy ibe - o-CA3 py amidal cells (MF-CA3) synapse, bidi ec ional NMDAR plas ici y can be elici ed by pai ing physiologically ele an pa e ns o p esynap ic and pos synap ic bu s i ing (i.e., bu s iming-dependen plas ici y), whe e he o de in which p e- and pos synap ic bu s s a e p esen ed is c ucial o es ablish he di ec ion o NMDAR plas ici y (Hun e al.,2013). Speci ically, NMDAR-LTP is igge ed i p esynap ic bu s i ing p ecedes pos synap ic i ing, while simply e e sing he o de o he bu s s induces NMDAR- LTD. Bo h NMDAR-LTP and LTD ely on pos synap ic calcium ise bu display di e en molecula equi emen s. Whe eas NMDAR- LTP equi es NMDAR/mGluR5 co-ac i a ion and calcium elease om in acellula s o es, NMDAR-LTD equi es NMDAR/mGluR1 co-ac i a ion bu no calcium elease om in e nal s o es (Hun e al.,2013), sugges ing ha highe le els o pos synap ic calcium de e mine he di ec ion o NMDAR plas ici y, a possibili y ne e di ec ly es ed. Mo eo e , wha addi ional ac o s con ibu e o inducing LTP o LTD o NMDAR-media ed ansmission emain unclea . In he p esen s udy, we combined whole-cell pa ch clamp eco dings, 2-pho on (2P) calcium imaging and glu ama e uncaging in acu e a hippocampal slices, and ana omical analysis ia immunoelec on mic oscopy, o de e mine he ole o pos synap ic calcium ise and I-mGluRs in shaping bidi ec ional NMDAR plas ici y. In addi ion o he MF-CA3 synapse, we also examined he synapse be ween MF and hila mossy cells (MF-MC synapse), which sha es se e al ana omical and unc ional p ope ies wi h MF-CA3 synapses (Scha man,2016). Ou da a indica e ha NMDAR plas ici y is a ge -cell speci ic, and ha exp ession o I-mGluRs sub ypes combined wi h pos synap ic calcium dynamics de e mine he exp ession and di ec ion o NMDAR plas ici y a MF synapses. Ma e ials and me hods All expe imen al p ocedu es desc ibed we e pe o med in acco dance wi h he Ins i u ional Animal Ca e and Use Commi ee guidelines o he Albe Eins ein College o Medicine and he Uni e si y o he Basque Coun y and adhe ed o he guidelines o he USA Na ional Ins i u es o Heal h (NIH). Acu e ans e se slices we e p epa ed om young adul Sp ague Dawley a s o bo h sexes (P17-27). Hippocampi we e dissec ed om he b ain and 400 µm hick slices we e cu using a ib a ome (Leica VT1200S) in ice-cold suc ose solu ion con aining (in mM): 215 suc ose, 20 glucose, 26 NaHCO3, 4 MgCl2, 4 MgSO4, 1.6 NaH2PO4, 2.5 KCl, and 1 CaCl2. Slices we e hen allowed o eco e o ∼20 min a 34◦C in 50% suc ose solu ion and 50% a i icial ce eb al spinal luid (ACSF) eco ding solu ion con aining (in mM): 124 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, and 1.3 MgSO4. A e his eco e y pe iod, slices we e incuba ed in ACSF a oom empe a u e (∼25◦C) o a leas one hou . Fo elec ophysiology and calcium imaging expe imen s slices we e pe used wi h ACSF bubbled wi h 95% O2and 5% CO2. Unless o he wise s a ed, du ing calcium imaging expe imen s ACSF was supplemen ed wi h he GABAA ecep o an agonis pic o oxin (30 µM) o block as synap ic inhibi ion, and 0.5–1 µM LY303070, a non-compe i i e selec i e AMPAR an agonis , o minimize he ac i a ion o he CA3 ecu en ne wo k (Kwon and Cas illo, 2008b). Elec ophysiology Slices we e ans e ed o a eco ding chambe pe used wi h ACSF bubbled wi h 95% O2and 5% CO2and kep a oom empe a u e (∼25◦C). The low a e was adjus ed o 2 ml/min. Visualized whole-cell pa ch clamp eco dings om CA3 py amidal neu ons and MCs we e pe o med using bo osilica e glass pipe es (3–6 M ip esis ance) illed wi h a po assium-based in e nal F on ie s in Cellula Neu oscience 02 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 3 Lu zu e al. 10.3389/ ncel.2023.1068472 solu ion con aining: 135 mM KMeSO3, 10 mM HEPES, 4 mM MgCl2, 4 mM Na2ATP, 0.4 mM NaGTP and 10 mM Na+c ea ine phospha e; 290 mOsm; pH 7.3 co ec ed wi h KOH. Fo calcium imaging, he low-a ini y calcium indica o Fluo5 (300 µM) and he mo phological luo escen indica o Alexa 594 (20 µM) we e added o he in e nal solu ion. Da a we e collec ed using a Mul iClamp 700B ampli ie (Molecula De ices, San Jose, CA). Signals we e il e ed a 2.4 kHz and acqui ed a 5 kHz wi h cus om so wa e w i en in Igo P o. CA3 py amidal cells and MCs we e ini ially ol age clamped a ∼–65 mV and he in e nal solu ion was allowed o equilib a e o a leas 30 min. Du ing his ime window, we checked o he p esence and s abili y o MF-de i ed AMPAR exci a o y pos synap ic cu en s (EPSCs) using an ex acellula bipola he a glass s imula o which was placed in he s a um lucidum o MF-CA3 eco dings, o in he subg anula zone o he den a e gy us o MF-MC eco dings. In bo h cases, he s imula ing pipe e was placed ∼50–100 µm om he pa ched cell. MF- e oked esponses we e elici ed wi h a sho (80 µs) cu en squa e pulse. S imula ion in ensi y anged om 20 o 50 µA. Fo bo h ypes o eco dings, MF-de i ed esponses we e iden i ied acco ding o he high p esynap ic acili a ion (P2/P1 >2), as ise ime (<2 ms) and high sensi i i y o he mGluR2/3 agonis DCG-IV (1 µM) (>80% educ ion o he synap ic esponse). A e he ini ial ∼30 min in ol age clamp, eco dings we e swi ched o cu en clamp mode while keeping he memb ane es ing po en ial a −70 mV. The es ing po en ial was adjus ed o mo e hype pola ized po en ials (∼–75 mV) i synap ically- d i en spikes we e e oked du ing MF bu s s imula ion. The induc ion p o ocols o NMDAR-LTP and LTD we e designed as p e iously desc ibed (Hun e al.,2013). B ie ly, NMDAR-LTP and LTD induc ion p o ocols consis ed o pai ings o p esynap ic MF bu s s (PRE) (5 pulses a 50 Hz) wi h pos synap ic bu s s o CA3 py amidal cells (POST) (3 ac ion po en ials a 100 Hz) which we e e oked wi h b ie (2–3 ms) injec ions o cu en (1–1.5 nA) in he pos synap ic cell. The NMDAR-LTP p o ocol consis ed o 100 PRE-POST pai ings (i.e., PRE leading POST s imula ion) wi h a 10 ms delay in be ween, deli e ed a a 2 Hz in e -pai ing in e al. NMDAR-LTD was induced by e e sing he bu s o de (i.e., POST-PRE) wi h he same 10 ms delay. All calcium measu emen s we e pe o med using single pai ings excep hose in Figu es 1G–I, whe e he ull induc ion p o ocol wi h one hund ed pai ings was used, and in Figu e 3 whe e only PRE s imula ion pa e ns we e used. Reco dings showing access esis ance >25 Mo changes exceeding 15% we e disca ded. To measu e DHPG-induced cu en s we pa ched CA1 py amidal cells in ol age-clamp mode a −60 mV in p esence o 10 µM NBQX, 100 µM pic o oxin, and 3 µM CGP and 25 µM D-APV ( o block AMPA/Kaina e, GABAA, GABAB, and NMDA ecep o s, espec i ely). The DHPG-media ed change in holding cu en was measu ed by sub ac ing he a e age alue o he holding cu en du ing he las 3 min o a 10-min DHPG applica ion o he a e age alue o he baseline pe iod (1I Holding). Fo he induc ion o NMDAR plas ici y in MCs, isualized whole-cell pa ch clamp eco dings om MCs we e pe o med using a K+-based in e nal solu ion con aining: 135 mM KMeSO4, 5 mM KCl, 1 mM CaCl2, 5 mM NaOH, 10 mM HEPES, 5 mM MgATP, 0.4 mM Na3GTP, 5 mM EGTA, 10 mM D-glucose, pH 7.2 (280–290 mOsm). MCs we e iden i ied using p e iously es ablished c i e ia (La ime and S owb idge,2008): ele a ed spon aneous synap ic ac i i y, li le o no a e hype pola iza ion and non-bu s i ing pa e ns. These c i e ia we e alida ed by pa ch-loading cells wi h Alexa 594, which allowed us o image he cell and con i m he p esence o TEs on MCs (Scha man,2016). To enhance esponse de ec abili y, MF-de i ed NMDAR EPSCs we e elici ed wi h 2 pulses a 200 Hz in he p esence o 10 µM NBQX, 100 µM pic o oxin, and 3 µM CGP ( o block AMPA/Kaina e, GABAA, and GABAB ecep o s espec i ely). 2-pho on calcium imaging and glu ama e uncaging Calcium imaging and glu ama e uncaging measu emen s we e pe o med using an Ul ima 2-pho on lase scanning mic oscope (B uke Co po a ion, Bille ica, MA) equipped wi h bo h an Insigh Ti:Sapphi e lase and a MaiTai lase (Spec a Physics, MKS Ins umen s, Inc., Ando e , MA). The exci a ion wa eleng h o imaging and uncaging we e se a 830 and 720 nm, espec i ely. Fo calcium imaging, luo escence ac oss he egion o in e es was de ec ed in line scan mode a high equency (500 Hz) using P ai ie View 5.4 so wa e (B uke Co po a ion). Pos synap ic CaTs we e es ima ed by calcula ing he 1G/R a io, which ep esen s he s imula ion-induced change in luo escence om he baseline o he g een channel (Fluo-5 signal), no malized by he luo escence o he Red channel (Alexa-594 signal). PRE, POST, PRE-POST, and POST-PRE CaTs we e quan i ied as he a e age peak 1G/R o a leas 5 ials. All ials we e acqui ed wi h a leas 1 min delay. To assess he e ec o d ug applica ion on PRE CaTs, he peak 1G/R a e d ugs wash-in (a e age o a leas 5 aces acqui ed du ing a 5–10 min long baseline) was no malized o he a e age o he CaTs acqui ed du ing he baseline pe iod (a e age o a leas 5 aces acqui ed du ing he las 15 min o 30 min d ug wash-in). Only ials in which synap ic MF s imula ion did no induce spikes we e analyzed. To a oid po en ial p iming e ec s due o he PRE-POST, o POST-PRE s imula ion, we deli e ed hese p o ocols in an in e lea ed ashion in di e en cells. Fo glu ama e uncaging expe imen s 2.5 mM MNI-caged- L-glu ama e was added o he ACSF and used o pe use he slice. The uncaging lase was pa ked ∼1µm away om he head o he a ge dend i ic spine and 5 uncaging pulses (0.5 ms du a ion, a 50 Hz) we e used o mimic glu ama e elease om MFs wi h he same equency used o elec ical s imula ion o MF. The uncaging lase powe was adjus ed o de ec a measu able calcium signal and a oid pos synap ic spikes du ing he sho bu s . To compa e he di e ence be ween 100 PRE- POST and POST-PRE pai ings, measu emen s we e pe o med in he same cell. To a oid any po en ial con ounding e ec due o changes in NMDAR unc ion, he wo p o ocols we e elici ed wi hin 5 min om each o he , be o e he onse o he exp ession o NMDAR plas ici y (Hun e al.,2013). Mo eo e , he wo p o ocols we e elici ed by al e na ing he o de in di e en cells o accoun o po en ial p iming e ec s be ween p o ocols. F on ie s in Cellula Neu oscience 03 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 4 Lu zu e al. 10.3389/ ncel.2023.1068472 FIGURE 1 NMDAR LTP was associa ed wi h a la ge pos synap ic calcium ise han NMDAR LTD a he MF-CA3 synapse. (A) Expe imen al eco ding con igu a ion ( op). Simul aneous exci a ion o Alexa 594 and Fluo-5 loaded ia a pa ch pipe e was pe o med wi h a 2P lase uned a 830 nm. Rep esen a i e CA3 py amidal cell loaded wi h Alexa 594 (middle) and inse showing magni ica ion o he imaged pos synap ic TE (yellow box). High magni ica ion image showing he scanning line (yellow dashed line) on he TE (bo om). s.l.: s a um lucidum; θs im: he a glass s imulus pipe e; ec: eco ding pipe e. (B) Elec ophysiological eco dings and calcium esponses ob ained om a CA3 py amidal neu on. S imula ion pa e ns employed ( op): PRE (MF elec ical s imula ion – 5 pulses a 50 Hz); POST (CA3 py amidal cell ac ion po en ials – 3 spikes a 100 Hz); PRE-POST (delay 10 ms be ween bu s s); and POST-PRE (delay 10 ms be ween bu s s). Fluo escence p o ile (middle) o he g een signal (Fluo5 ) supe imposed wi h he ed signal (mo phological dye Alexa 594). S: spine; d: dend i e. Fluo escence quan i ica ion epo ed as 1G/R o e ime (bo om). (C) Summa y o mean peak 1G/R ac oss all pa e ns o s imula ion. He e and in all igu es, ci cles linked by a line ep esen indi idual expe imen s pe o med in he same cell. (D) Expe imen al con igu a ion used o pe o m elec ophysiology, calcium imaging and glu ama e uncaging ( op). Exci a ion wa eleng hs employed we e 830 nm ( o imaging) and 720 nm ( o glu ama e uncaging). High magni ica ion image showing he posi ion o he uncaging lase beam (yellow ci cle) he scanning line (yellow dashed line) on he TE (bo om). (E,F) Same as panels (B,C) bu employing glu ama e uncaging ins ead o MF s imula ion. (G) Single elec ophysiological esponse (le ) and luo escence p o ile du ing ull induc ion p o ocol o NMDAR LTP and LTD ( igh ). (H) Calcium esponses o he expe imen showed in panel (G) exp essed as 1G/R o e ime. Boxed a ea shows a magni ica ion o 1G/R du ing he i s 4 pai ings. (I) Summa y o 1G/R change du ing he ull induc ion p o ocols exp essed as in eg a ed a ea unde he cu e. Da a a e p esen ed as mean ±SEM. **p<0.01, ***p<0.001. Double p e-embedding immunoelec on mic oscopy Long E ans a s (n= 3) we e deeply anes he ized by in ape i oneal injec ion o ke amine/xylazine (80/10 mg/kg body weigh ) and ansca dially pe used a oom empe a u e (RT, 20– 25◦C) wi h phospha e bu e ed saline (0.1 M PBS, pH 7.4) o 20 s, ollowed by he ixa i e solu ion (4% o maldehyde eshly depolyme ized om pa a o maldehyde, 0.2% pic ic acid and 0.1% glu a aldehyde) in PBS (0.1 M, pH 7.4) o 10–15 min. B ains we e emo ed om he skull and pos - ixed in he ixa i e solu ion o abou 1 week a 4◦C and s o ed a 4◦C in 1:10 dilu ed ixa i e solu ion un il use. The p ocedu e has been p e iously desc ibed (Puen e e al., 2019). B ie ly, co onal hippocampal ib osec ions we e cu a 50 µm and collec ed in phospha e bu e (0.1 M PB, pH 7.4) F on ie s in Cellula Neu oscience 04 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 5 Lu zu e al. 10.3389/ ncel.2023.1068472 FIGURE 2 mGluR5 bu no mGluR1 ac i a ion was c i ical o he di e ence in pos synap ic calcium ise du ing PRE-POST and POST-PRE a MF-CA3 synapses. (A) Rep esen a i e aces o PRE-POST and POST-PRE luo escence p o iles in con ol condi ions ( op) and summa y plo s showing he a e age 1G/R measu ed du ing PRE-POST and POST-PRE pa e ns (same da ase om Figu e 1C) (bo om). (B,C) Same expe imen al design as in A bu in p esence o he mGluR5 an agonis MPEP (4 µM) (B) and he mGluR1 an agonis YM (1 µM) (C).(D) Rep esen a i e inwa d cu en media ed by ba h applica ion o he g oup I mGluR agonis DHPG (30 µM) in con ol and du ing YM co-applica ion ( op). Time-cou se summa y plo o he DHPG-media ed change in holding cu en (1I Holding) eco ded om ol age-clamped CA3 py amidal cells unde con ol condi ions and in he p esence o YM. He e and in all igu es: a, numbe o animals and c, numbe o cells. Da a a e p esen ed as mean ±SEM). **p<0.01, ***p<0.001, n.s. no signi ican . wi h 0.1% sodium azide a RT. They we e ans e ed and p e- incuba ed in a blocking solu ion o 10% bo ine se um albumin (BSA), 0.1% sodium azide and 0.02% saponin p epa ed in T is- hyd ogen chlo ide bu e ed saline 13 (TBS), pH 7.4 o 30 min a RT. Then, he hippocampal sec ions we e incuba ed wi h he p ima y abbi polyclonal an i-mGluR1b ecep o an ibody (2 mg/ml, mGluR1b–Rb–A 250, F on ie Ins i u e Co. L d, Ishika i, Hokkaido, Japan, RRID:AB_2616586) o guinea pig polyclonal an i-mGluR5 an ibody (2 mg/ml, mGluR5b–GP–A 270–1, F on ie Ins i u e Co. L d, Ishika i, Hokkaido, Japan, RRID:AB_2571804) dilu ed in 10% BSA/TBS con aining 0.1% sodium azide and 0.004% saponin on a shake o 2 days a 4◦C. Bo h an ibodies we e ex ensi ely cha ac e ized p e iously, and he cha ac e is ic labeling in wild ype issue disappea ed in he b ains o mGluR1 and mGluR5 knock–ou mice (Fe agu i e al.,1998;Ma eos e al., 1998;Tanaka e al.,2000;Uchigashima e al.,2007;Oh ani e al., 2014). Tissue was incuba ed a e se e al washes in 1% BSA/TBS wi h he bio inyla ed seconda y an ibody (1:200 goa bio inyla ed an i- abbi , Ca # BA-1000, Vec o Labs, Bu lingame, CA, USA; RRID:AB_2313606 o 1:200 donkey bio inyla ed an i-guinea pig Ca #, 706-065-148, Jackson ImmunoResea ch Labs, Wes G o e, PA, USA; RRID:AB_2340451) in 1% BSA/TBS wi h 0.004% saponin o 3 h a RT. The sec ions we e hen washed in 1% BSA/TBS o e nigh on a shake a 4◦C and incuba ed wi h ei he 1.4 nm gold-labeled goa an i- abbi IgG an ibody (Fab’ agmen , 1:100, Ca # #2004, Nanop obes Inc., Yaphank, NY, USA) o 1.4 nm gold-labeled goa an i-guinea pig IgG an ibody (Fab’ agmen , 1:100, Ca # #2055, Nanop obes Inc., Yaphank, NY, USA) in 1% BSA/TBS wi h 0.004% saponin on a shake o 3 h a RT, o mGluR1b o mGluR5 labeling, espec i ely. Nex , he sec ions we e washed in 1% BSA/TBS and subsequen ly incuba ed in he a idin- bio in complex (1:50; PK-7100, Vec o Labs, Bu lingame, CA, USA; RRID:AB_2336827) dilu ed in he wash solu ion o 1.5 h. A e washing in 1% BSA/TBS o e nigh a 4◦C, issue was pos - ixed wi h 1% glu a aldehyde in TBS o 10 min and washed in double- dis illed wa e . A e wa d, he gold pa icles we e sil e in ensi ied wi h an HQ Sil e ki (Ca #2012; Nanop obes Inc., Yaphank, NY, USA) o abou 12 min in he da k, washed in 0.1 M PB (pH 7.4) and subsequen ly incuba ed in 0.05% DAB (Millipo eSigma, Ca #D5637; RRID:AB_2336819) and 0.01% hyd ogen pe oxide p epa ed in 0.1 M PB o 3 min. Finally, he sec ions we e osmica ed (1% osmium e oxide, Elec on Mic oscopy Sciences, Ca #19150) in 0.1 M PB pH 7.4 o 20 min, washed in 0.1 M PB (pH 7.4), dehyd a ed in g aded alcohols (50%–100%) o p opylene oxide and plas ic-embedded in Epon esin 812. 50–60 nm-ul a hin sec ions we e cu wi h a diamond kni e (Dia ome USA), collec ed on nickel mesh g ids, s ained wi h 2.5% lead ci a e, and examined wi h a JEOL JEM 1400 Plus elec on mic oscope. Tissue samples we e imaged using a digi al came a (sCMOS). The double immunocy ochemical me hod was epea ed h ee imes on he sec ions ob ained om each o he h ee b ains s udied. Immunope oxidase and immunogold-labeling we e isualized on he hippocampal sec ions wi h a ligh mic oscope and po ions o he CA3 s a um lucidum and he den a e hilus wi h good and consis en mGluR1b and mGluR5 ecep o s immunolabeling we e iden i ied and immed down o ul a hin sec ioning. Th ee o ou semi- hin sec ions (1 µm- hick) we e hen cu wi h a his o-diamond kni e (Dia ome USA) and s ained wi h 1% oluidine blue. To u he s anda dize he condi ions, only he i s 20 ul a hin sec ions (50–60 nm hick) we e cu , collec ed on o he g ids and pho og aphed. The elec on mic og aphs we e aken a 8,000×wi h a Digi al Mo ada Came a om Olympus (Hambu g, Ge many). Sampling was always ca e ully and accu a ely ca ied ou in he same way o he b ains s udied and i was blinded o expe imen e s du ing mGluRs quan i ica ion. Da a analysis and s a is ics Elec ophysiology and calcium imaging. G aphs and s a is ical analysis we e pe o med using O iginP o (O igin Labo a o y, No hamp on, MA). The no mali y o da a dis ibu ion was assessed wi h he Shapi o–Wilk es . Da a wi h a no mal F on ie s in Cellula Neu oscience 05 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 6 Lu zu e al. 10.3389/ ncel.2023.1068472 FIGURE 3 NMDAR and mGluR5 bu no mGluR1 con ibu ed o pos synap ic calcium ise e oked du ing p esynap ic MF bu s ac i i y. (A) Calcium signals induced by p esynap ic s imula ion (PRE) be o e and a e ba h applica ion o he NMDAR an agonis D-APV (25 µM). Rep esen a i e aces ( op) and summa y plo o he a e aged peak 1G/R be o e (no malized o baseline) and a e d ug applica ion. (B–D) Same expe imen al design as in panel (A) bu ollowing applica ion o he mGluR5 an agonis MPEP (4 µM) (B), he mGluR1 an agonis YM (1 µM) and in naï e condi ions (i.e., calcium signals moni o ed du ing 15–30 min in he absence o d ug applica ions) o e alua e s abili y. Da a a e p esen ed as mean ±SEM. ***p<0.001, n.s. no signi ican . dis ibu ion we e analyzed using a pa ame ic es (One-Sample Tes o unpai ed da a and Pai ed Tes o pai ed da a), whe eas da a ha did no display a no mal dis ibu ion we e analyzed wi h a non-pa ame ic es (Mann–Whi ney). Fo mul iple compa isons, we employed a One-Way ANOVA wi h a Bon e oni pos hoc analysis o mean compa ison. S a is ical signi icance was se a P<0.05. Immunoelec on mic oscopy. The double immunocy ochemical me hod was epea ed h ee imes on he sec ions ob ained om each o he h ee b ains ha we e analyzed. In pa icula , a o al o 181 dend i ic ho ny exc escences (TEs) o py amidal neu ons con ac ed by MF e minals in CA3 s a um lucidum (a ea s udied: 1406.1 µm2) and 153 mossy cell TEs wi h MF synapses in den a e hilus (a ea s udied: 1406.1 µm2) we e analyzed. Posi i e mGluR1b and/o mGluR5 TEs we e iden i ied by he p esence o DAB immunodeposi s and/o by he p esence o a leas one gold pa icle wi hin 30 nm o he pos synap ic memb ane. Collec ing da a o mGluR1b/mGluR5 posi i e TEs ob ained o each an ibody in CA3 and in he den a e hilus we e pooled. The pe cen age o mGluR1b and/o mGluR5 posi i e TEs was analyzed and displayed using a s a is ical so wa e package (G aphPad P ism 8, G aphPad So wa e Inc, San Diego, CA, USA; RRID:SCR_002798). Da a a e p esen ed as mean ±SEM. The da a we e pooled since h ee analyzed samples did no di e in mGluRs labeling (Kolmogo o Smi no es , P>0.19). Then, he a io o he o al mGluR5 e sus o al mGluR1b in TEs o CA3 and den a e hilus was compa ed. The no mali y es (Kolmogo o –Smi no no mali y es ) was applied be o e s a is ical es s and subsequen ly, da a we e analyzed using a pa ame ic unpai ed Tes . D ugs and chemicals All chemicals and d ugs used we e pu chased om Millipo eSigma (S . Louis, MO, USA) excep D-APV, NBQX, CGP-55845, DCG-IV, MNI-caged-L-glu ama e and CPA which we e ob ained om Toc is Bioscience (Minneapolis, MN, USA), LY 303070 which was ob ained om ABX ad anced biochemical compounds (Radebe g, Ge many), and Fluo5 and Alexa which we e pu chased om In i ogen (Ca lsbad, CA, USA). Resul s The magni ude o pos synap ic calcium ise du ing NMDAR-LTP and LTD induc ion di e s To assess he pos synap ic calcium signals elici ed by dis inc pa e ns o ac i i y ha induce NMDAR-LTP and NMDAR- LTD a MF-CA3 synapses, we combined whole-cell pa ch clamp eco dings and 2P calcium imaging in acu e a hippocampal slices. We pa ch-loaded CA3 py amidal cells wi h a po assium-based in e nal solu ion con aining he low-a ini y calcium indica o Fluo5 (300 µM), and Alexa 594 (20 µM) which was used as a mo phological indica o . We pe o med eco dings in cu en - clamp con igu a ion in he p esence o pic o oxin (30 µM) o block GABAA-media ed ansmission, and a low concen a ion o LY303070 (0.5–1 µM) o minimize AMPAR-media ed synap ic po en ials a ising om he ecu en associa ional-commissu al ne wo k (Kwon and Cas illo,2008b). Unde hese eco ding condi ions, p esynap ic MF s imula ion (e.g., 5 s imuli, 50 Hz) e oked sub h eshold EPSPs ha we e associa ed wi h calcium ansien s (CaTs) in he a ge pos synap ic CA3 spines ho ny exc escences (TEs) (Figu es 1A–C;1G/R PRE = 0.09 ±0.01) and, as expec ed, di ec ac i a ion o CA3 py amidal neu ons (3 ac ion po en ials, 100 Hz) also induced CaTs in TEs (Figu es 1A– C; POST = 0.12 ±0.01). As p e iously epo ed, ac i a ion o pos synap ic glu ama e ecep o s, calcium elease om he in e nal F on ie s in Cellula Neu oscience 06 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 7 Lu zu e al. 10.3389/ ncel.2023.1068472 s o es, and ac i a ion o ol age-ga ed calcium channels likely accoun s o hese CaTs (Kapu e al.,1998,2001;Reid e al., 2001). We nex analyzed CaTs elici ed by single PRE-POST and POST-PRE pai ings which a e known o igge bu s iming- dependen NMDAR-LTP and LTD, espec i ely (Hun e al., 2013). Single pai ings p o ide an es ima ion o he pos synap ic calcium changes occu ing du ing he ull induc ion p o ocol o plas ici y (Koes e and Sakmann,1998;Ne ian and Sakmann, 2004;Mish a e al.,2016). We ound ha PRE-POST pai ings elici ed la ge CaTs han POST-PRE pai ings (Figu es 1B, C,1G/R PRE-POST = 0.25 ±0.02; POST-PRE = 0.16 ±0.01; n= 13; PRE-POST s POST-PRE: pai ed Tes p= 0.00026), indica ing ha he induc ion o NMDAR-LTP is likely associa ed wi h la ge pos synap ic calcium ise han NMDAR-LTD. Ex acellula MF s imula ion could ec ui ex insic neu omodula o y ibe s and igge he elease o modula o s om he MF bou ons, bo h o which could po en ially a ec pos synap ic CaTs. To di ec ly add ess his issue, we employed 2P glu ama e uncaging and es ed whe he glu ama e alone could mimic he di e en CaTs ollowing PRE-POST and POST-PRE pai ing p o ocols wi h MF ex acellula elec ical s imula ion. We ound ha 2P glu ama e uncaging ∼1µm om TEs (Figu e 1D) elici ed ema kably simila PRE-POST and POST-PRE CaTs as obse ed wi h elec ical s imula ion (Figu es 1E,F,1G/R uncaging PRE = 0.09 ±0.03; POST = 0.05 ±0.01; PRE-POST = 0.15 ±0.02; POST-PRE: 0.10 ±0.02; PRE-POST s POST-PRE: pai ed Tes p= 0.0099). These esul s indica e ha he di e en calcium signals elici ed by PRE-POST and POST-PRE pai ing p o ocols a e due o he di ec ac i a ion o pos synap ic glu ama e ecep o s. To de e mine whe he he di e ence in calcium signals obse ed wi h single pai ings was p ese ed du ing he ull induc ion p o ocol, which comp ises 100 PRE-POST (NMDAR- LTP) o POST-PRE (NMDAR-LTD) pai ings deli e ed a 2 Hz espec i ely (Hun e al.,2013), we measu ed CaTs while deli e ing hese p o ocols in he same cell (Figu e 1G). An equal numbe o cells ecei ed a single LTP o LTD p o ocol i s , and he second p o ocol was deli e ed 5 min a e he i s one (see Me hods). We ound ha he NMDAR-LTP induc ion p o ocol elici ed la ge CaTs compa ed o he NMDAR-LTD induc ion p o ocol (Figu es 1H,I). To quan i y his di e ence, we calcula ed he in eg a ed a ea unde he cu e in a bi a y uni s (a.u.) (Figu es 1H, I, PRE-POST = 18,414.30 ±2,833.90 a.u.; POST- PRE = 12,229.05 ±1,964.84 a.u.; PRE-POST s POST-PRE: n= 8, pai ed Tes , p= 0.00529). Al oge he , ou esul s indica e ha NMDAR-LTP induc ion is associa ed wi h a la ge calcium ise han NMDAR-LTD induc ion and ha pos synap ic glu ama e ecep o s mos likely accoun o his di e ence. mGluR1 and mGluR5 con ibu e di e en ly o pos synap ic calcium ise e oked by PRE-POST and POST-PRE pa e ns o ac i i y A he MF-CA3 synapse, mGluR5 and mGluR1 a e equi ed o bu s iming-induced NMDAR-LTP and LTD, espec i ely (Hun e al.,2013). Because Gq-coupled ecep o s ac i a e he phosphoinosi ide pa hway ha media es calcium elease om calcium s o es (Niswende and Conn,2010), we hypo hesized ha co-ac i a ion o NMDAR and mGluR1 o mGluR5 de e mines he magni ude o he pos synap ic calcium ha igge s MF-CA3 NMDAR-LTP and LTD. To es his hypo hesis, we deli e ed he PRE-POST and POST-PRE s imula ion pai ings in he p esence o mGluR1 o mGluR5 selec i e an agonis s. As p e iously epo ed, mGluR1 and mGluR5 an agonism has no signi ican e ec on basal MF-CA3 synap ic ansmission (Hun e al.,2013). In compa ison o con ol condi ions (Figu e 2A, Da a eplo ed om Figu e 1C o compa ison easons), ba h applica ion o he non-compe i i e mGluR5 an agonis MPEP (4 µM) abolished he di e ence be ween PRE-POST and POST-PRE CaTs (Figu e 2B, MPEP 1G/R PRE- POST = 0.20 ±0.03; POST-PRE = 0.18 ±0.03; n= 12, pai ed Tes p= 0.347). Con e sely, blockade o mGluR1 wi h he non-compe i i e an agonis YM298198 (1 µM) did no al e he di e ence be ween PRE-POST and POST-PRE obse ed in con ol condi ions (Figu e 2C, YM 1G/R PRE-POST = 0.25 ±0.04; POST- PRE = 0.16 ±0.04; n= 12, pai ed Tes : p= 0.0025). As posi i e con ol ha YM was e icien ly blocking mGluR1, in a sepa a e se o expe imen s we ound ha he inwa d cu en media ed by he I-mGluR agonis DHPG (30 µM) in CA1 py amidal neu ons was signi ican ly educed by YM co-applica ion, as p e iously epo ed (Mannaioni e al.,2001;Rae and I ing,2004) (Figu e 2D, Con ol: −19.49 ±1.28 pA, n= 3; YM: −4.46 ±2.63 pA, n= 3; wo sample - es p= 0.0068). These esul s indica e ha mGluR1 and mGluR5 con ibu e di e en ly o pos synap ic calcium ise a MF- CA3 synapses, consis en wi h hei dis inc ole in NMDAR-LTP and LTD. Glu ama e elease du ing MF bu s ac i i y engages di e se pos synap ic calcium sou ces P e ious s udies a he MF-CA3 synapse epo ed ha glu ama e igge s pos synap ic calcium ise ia ac i a ion o iono opic ecep o s and/o ia mGluR-media ed calcium elease om in acellula s o es (Ja e and B own,1997;Kapu e al., 2001;Reid e al.,2001). O no e, calcium signals in hese s udies we e elici ed by non-physiological e anic s imula ion (e.g., 100 Hz, 1 s) (Ja e and B own,1997), single s imula ion in o gano ypic slice cul u es (Reid e al.,2001), o we e analyzed using a low- spa ial esolu ion imaging app oach (Kapu e al.,2001). We he e o e es ed he con ibu ion o iono opic glu ama e ecep o s and mGluRs in gene a ing pos synap ic calcium signals elici ed by physiologically ele an MF bu s s imula ion (PRE only) and moni o ed using 2P calcium imaging in acu e a hippocampal slices. A e ob aining baseline CaTs in esponse o a sho bu s o MF s imula ion (5 pulses, 50 Hz), we blocked NMDARs wi h he compe i i e an agonis D-APV (25 µM). This manipula ion nea ly abolished pos synap ic CaTs (Figu e 3A, PRE + D- APV = 0.10 ±0.02 1G/R no malized o con ol; n= 16, pai ed Tes : p= 0.000005), indica ing ha NMDARs a e obus ly engaged du ing MF bu s ac i i y and media e mos o he pos synap ic calcium ise despi e being exp essed a a lowe le el han AMPARs (Takumi e al.,1999). In sepa a e expe imen s, he mGluR5 an agonis MPEP (4 µM) signi ican ly educed pos synap ic CaTs (Figu e 3B, F on ie s in Cellula Neu oscience 07 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 8 Lu zu e al. 10.3389/ ncel.2023.1068472 FIGURE 4 Calcium deple ion om in e nal s o es signi ican ly educed he di e ence be ween PRE-POST and POST-PRE CaTs a MF-CA3 synapses. (A) Rep esen a i e CaTs induced by PRE-POST and POST-PRE s imula ion in he p esence o he SERCA pump blocke cyclopiazonic acid (CPA) ( op). B ain slices we e incuba ed o 30 min in 30 µM CPA which was also con inuously pe used. Summa y plo showing he e ec o CPA on peak 1G/R measu ed du ing PRE-POST and POST-PRE s imula ion (bo om). (B) PRE-POST and POST-PRE CaT a io unde di e en expe imen al condi ions: C l (no d ugs), 4 µM MPEP, 30 µM CPA and 1 µM YM. One Way ANOVA; F= 5.86458; p= 0.00194; DF = 3; C l s MPEP, p= 0.0036; C l s CPA, p= 0.0198; C l s YM, p= 0.136; Bon e oni pos hoc analysis. (C) Non-linea summa ion o pos synap ic calcium signals unde di e en expe imen al condi ions. Non-linea i y was measu ed as he a io be ween he measu ed PRE-POST o POST-PRE CaTs and he a i hme ical sum o he PRE and POST componen s alone. Analysis was pe o med on da a shown in Figu es 2B–D,4A. All da a poin s we e compa ed agains a non-linea i y ac o o 1 using One Sample Tes . Da a a e p esen ed as mean ±SEM. **p<0.01, *p<0.05, n.s. no signi ican . PRE + MPEP = 0.49 ±0.04 1G/R no malized o con ol, n= 11, pai ed Tes : p= 0.000041), sugges ing ha MF bu s ac i i y inc eases pos synap ic calcium ia mGluR5 and NMDAR co- ac i a ion. In con as , mGluR1 an agonism wi h YM (1 µM) had no signi ican e ec on CaTs (Figu e 3C, PRE + YM = 0.84 ±0.08 1G/R no malized o con ol, n= 12. pai ed Tes : p= 0.139). Likewise, no signi ican changes we e obse ed in naï e slices du ing 15–30 min, i.e., a e no d ug applica ion (Figu e 3D, PRE – 15–30 min = 1.02 ±0.13 no malized 1G/R, n= 11, Wilcoxon signed anks es : p= 0.83). O e all, ou esul s s ongly sugges ha mGluR5 and mGluR1 engage di e en in acellula cascades implica ed in MF-CA3 pos synap ic calcium ise. In acellula calcium s o es de e mine he calcium ise magni ude elici ed by NMDAR-LTP and LTD induc ion The pos synap ic calcium ise media ed by mGluRs is likely due o hei posi i e coupling o calcium elease om in e nal s o es, such as he endoplasmic e iculum (ER) (Kapu e al., 2001;Reid e al.,2001). The e o e, we p edic ed ha deple ion o calcium om he ER should educe he di e ence be ween single PRE-POST and POST-PRE pai ings. Indeed, his di e ence was signi ican ly educed in hippocampal slices incuba ed o a leas 30 minu es wi h he SERCA pump blocke cyclopiazonic acid (CPA, 30 µM) which deple es he calcium con en om he ER (Figu e 4A,1G/R PRE-POST = 0.26 ±0.04; 1G/R POST- PRE = 0.22 ±0.03; PRE-POST s POST-PRE: n= 9, Pai ed Tes p= 0.01116; see Figu e 2A). To be e quan i y he e ec o CPA, we calcula ed he PRE-POST/POST-PRE a io o CaTs, which allows he compa ison ac oss di e en eco ding condi ions. This analysis e ealed ha mGluR5 an agonism and/o CPA- media ed deple ion o calcium s o es, bu no mGluR1 an agonism, abolished he di e ence be ween PRE-POST and POST-PRE CaTs (Figu e 4B, PRE-POST/POST-PRE; C l = 1.62 ±0.13, n= 13; MPEP = 1.15 ±0.07 n= 12; CPA = 1.20 ±0.06, n= 9; YM = 1.45 ±0.07, n= 12; One Way ANOVA, F= 5.865; p= 0.00194; DF = 3.; C l s MPEP p= 0.0036; C l s CPA p= 0.0198; C l s YM p= 0.136; Bon e oni pos hoc es ). These esul s indica e ha he PRE-POST, bu no he POST-PRE p o ocol engages mGluR5 signaling and calcium elease om in acellula s o es, wo key de e minan s o he dis inc CaTs be ween NMDAR-LTP and LTD. Pai ing o p esynap ic and pos synap ic ac ion po en ials wi h sho delays can elici non-linea summa ion o pos synap ic calcium signals (Yus e and Denk,1995;Koes e and Sakmann, 1998), which is hough o be impo an o bidi ec ional plas ici y o AMPAR-media ed ansmission (Ne ian and Sakmann,2004, 2006). To es o po en ial non-linea summa ion o pos synap ic calcium signals gene a ed by MF-CA3 PRE-POST and POST-PRE pai ing unde di e en condi ions, we pe o med a non-linea i y analysis by calcula ing a non-linea i y ac o (NLF), which is he a io be ween he measu ed PRE + POST summa ion (i.e., PRE- POST CaTs peak) and he p edic ed PRE + POST summa ion (i.e., he a i hme ical sum be ween PRE and POST peak CaTs) (Ne ian and Sakmann,2004). We ound ha unde con ol condi ions, he PRE-POST pai ing is sup alinea (i.e., NLF >1), while he POST-PRE coupling is signi ican ly sublinea (i.e., NLF <1) (Figu e 4C). Consis en wi h a c i ical ole o mGluR5 and hei downs eam a ge s calcium s o es in he gene a ion o di e en CaTs le els be ween PRE-POST and POST-PRE, bo h MPEP and CPA abolished he sup alinea summa ion o PRE and POST CaTs. In con as , he blockade o mGluR1 wi h YM, did no F on ie s in Cellula Neu oscience 08 on ie sin.o g ncel-17-1068472 Ma ch 30, 2023 Time: 15:35 # 9 Lu zu e al. 10.3389/ ncel.2023.1068472 a ec he non-linea i y ac o o he PRE-POST and POST-PRE pai ings, (Figu e 4C, NLF C l PRE-POST = 1.22 ±0.07, n= 13, p= 0.01; C l POST-PRE = 0.78 ±0.05, n= 13, p= 0.003; MPEP PRE-POST = 0.97 ±0.06, n= 12, p= 0.72; MPEP POST- PRE = 0.87 ±0.05, n= 12, p= 0.054; YM PRE-POST = 1.20 ±0.05, n= 12, p= 0.002; YM POST-PRE = 0.84 ±0.03, n= 12, p= 0.001; CPA PRE-POST = 0.90 ±0.07, n= 9, p= 0.16; CPA POST- PRE = 0.75 ±0.05, n= 9, p= 0.001; One Sample Tes , all compa isons we e agains he alue NLF = 1; C l PRE-POST s POST-PRE, p= 0.00001; MPEP PRE-POST s POST-PRE, p= 0.91; CPA PRE-POST s POST-PRE, p= 0.846; YM PRE-POST s POST-PRE, p= 0.0011; One-Way ANOVA, F= 9.19144; DF = 7; Bon e oni pos hoc es ). Taken oge he hese esul s sugges ha mGluR5, bu no mGluR1, ac i a ion and calcium elease om he in e nal s o es a e c i ical o he la ge calcium ise associa ed wi h he induc ion o NMDAR-LTP. NMDAR plas ici y a he MF- o-Hila mossy cell synapse MFs also make synapses wi h hila mossy cells (MCs), and hese synapses sha e s uc u al and unc ional p ope ies wi h MF- CA3 synapses, including he p esence o gian MF bou ons and pos synap ic TEs, a high deg ee o synap ic acili a ion, egula ion o glu ama e elease by mGluR2/3 agonis s and he exp ession o p esynap ic LTP (Lyse skiy e al.,2005;Scha man,2016). Howe e , whe he MF-MC synapses can unde go NMDAR plas ici y was ne e es ed. We he e o e eco ded MCs and deli e ed PRE-POST and POST-PRE pa ings like o MF-CA3 synapses. As p e iously epo ed, MCs in a hippocampal slices we e iden i ied by he high equency o spon aneous synap ic ac i i y, egula ac ion po en ial i ing, and minimal- o-no a e hype pola iza ion (La ime and S owb idge,2008). In addi ion, MCs we e pa ch-loaded wi h Alexa 594 and hei iden i y was con i med pos hoc by he isualiza ion o TEs (Figu e 5A). We ound ha he PRE-POST pai ing p o ocol induced obus NMDAR-LTP a MF-MC (Figu e 5B, MC PRE- POST induc ion = 174 ±14% o baseline; n= 6; p= 0.00075; pai ed Tes ). In con as , he POST-PRE p o ocol did no igge NMDAR-LTD, bu a small, ye signi ican , LTP (Figu e 5B, MC POST-PRE induc ion = 121 ±4% o baseline; n= 7; p= 0.0021; pai ed Tes ). Impo an ly, he PRE-POST p o ocol did no induce LTP o he AMPAR-media ed componen o MF-MC synap ic ansmission (Figu e 5C, AMPAR EPSC = 111 ±9% o baseline, n= 6; C l s AMPAR p= 0.00089, One Way ANOVA). The selec i e s eng hening o he NMDAR- bu no AMPAR-media ed componen suppo s a pos synap ic mechanism o NMDAR-LTP exp ession a MF-MC synapses. To de e mine whe he MF-MC NMDAR-LTP equi ed pos synap ic calcium ise, we i s loaded MCs wi h he calcium chela o BAPTA (20 mM). This manipula ion signi ican ly educed he magni ude o NMDAR-LTP (Figu e 5C, Con ol MC NMDAR LTP = 180 ±7% o baseline, n= 9; NMDAR LTP + BAPTA = 120 ±15% o baseline, n= 6; C l s BAPTA p= 0.00442, One Way ANOVA; F= 10.41736; p= 0.0000161; DF = 4; Bon e oni pos hoc). Gi en he c i ical ole o I-mGluRs o he induc ion o NMDAR-LTP a mos cen al synapses (Hun and Cas illo,2012), including he MF-CA3 synapse FIGURE 5 NMDAR plas ici y a he MF- o-Hila Mossy Cells synapse. (A) Rep esen a i e 2P econs uc ion o a MC pa ch-loaded wi h biocy in (le ). MCs displayed egula ac ion po en ial i ing induced by cu en injec ion and minimal o no a e hype pola iza ion ( igh ). (B) Bo h PRE-POST and POST-PRE pai ing p o ocols induced NMDAR LTP bu no LTD a MF- o-Hila Mossy Cells (MF-MC) synapses. (C) MF-MC NMDAR LTP induced by PRE-POST pai ing unde di e en expe imen al condi ions: con ol, 20 mM in acellula BAPTA, 1 µMYMand4µM MPEP, and 30 µM CPA. The PRE-POST pai ing p o ocol had no e ec on AMPAR-media ed synap ic ansmission. One Way ANOVA; F= 9.19144; p= 0.0000161; DF = 4.; C l s BAPTA p= 0.00442; C l s YM p>0.05; C l s MPEP p= 0.0243; C l s CPA p= 0.04. Bon e oni pos hoc analysis. (D) Rep esen a i e MC TE depic ing he line scan (yellow line) whe e 2P calcium imaging was pe o med ( op le ), PRE-POST s POST-PRE MF-MC calcium signals (bo om le ), and summa y plo ( igh ). Da a a e p esen ed as mean ±SEM. **p<0.01, *p<0.05, n.s. no signi ican . F on ie s in Cellula Neu oscience 09 on ie sin.o g