NR3A-containing NMDA receptors promote neurotransmitter release and spike timing-dependent plasticity
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1 NR3A-ontaining NMDA rptors promot nurotransmittr rlas an spik timing-pnnt plastiity Rylan S. Larsn, Rkah J. Corlw, Mail A. Hnson, Aam C. Rorts, Masayoshi Mishina, Masahiko Watana, Stuart A. Lipton, Nouki Nakanishi, Isal Pérz-Otaño, Rihar J. Winrg, & Bnjamin D. Philpot SUPPLEMENTARY FIGURES Natur Nurosin: oi:.3/nn.7
2 a MW (kd) Antioy NRA (#-9) 7 7 NRA (#AB) NRB NRD (#3) NRD (#s-7) NR3A NR Protin lvls (% rainstm) Protin lvls (% WT) BS VC BS VC NR3A NRD WT / WT / WT / WT / WT / WT / NR NR3A NRD 37 β-atin Supplmntary Figur : NRD lvls ar omparativly low in visual ortx (VC), an ths lvls ar unhang in NR3A / mi. (a) Largr rprsntativ lots show antioy spifiity for th targt antigns with molular wight markrs. () Rprsntativ lots an quantifi protin lvls of NR3A an NRD in P P V (n = ) an rainstm (BS) (n = ). NR3A lvls wr omparal in rainstm an visual ortx, ut NRD lvls wr minimal in visual ortx ompar to rainstm. Protin lvls wr stanariz to an atin loaing ontrol an normaliz to rainstm valus. () Rprsntativ lots an quantifiation of NR (n = pr gnotyp), NR3A (n = 3 pr gnotyp), an NRD (n = 3 pr gnotyp) protin lvls in visual ortx. NR an NRD lvls wr unhang in P NR3A / mi ompar to WT ontrols (P =. an P =., rsptivly) whil NR3A was not tt in NR3A / mi (P <<.). Protin lvls in NR3A / an WT animals wr stanariz to a β-atin loaing ontrol an xprss as a prntag of WT lvls. Error ars rprsnt s..m. P <.. Natur Nurosin: oi:.3/nn.7
3 a P3 WT NRA / pa ms pa ms WT NRA / WT f NRA / 3 3 Supplmntary Figur : Th NRA suunit os not signifiantly ontriut to th aility of prnmdars to nhan spontanous nurotransmittr rlas onto L/3 pyramial nurons in juvnil V. (a,) Sampl voltag-lamp rorings of AMPAR-miat mepscs from L/3 pyramial lls uring as an D- appliation prios in (a) WT an () NRA / mi. mepscs ar iniat y. (, ) Amplitu an (, ) intr-vnt intrval umulativ proaility histograms from th sam lls shown in a an uring as an D- ( µm) appliation rval a hang in mepsc frquny, ut not amplitu, for oth ( ) WT ontrol an ( ) NRA / mi. (,f) Sattr plots of amplitu an frquny, for an aftr D- appliation, in () WT an (f) NRA / mi. L/3 pyramial lls xhiit a signifiant rution in mepsc frquny y D- in oth WT (n = 7, P <.3) an NRA / mi (n = 7, P <.), ut no hangs in amplitu. Th D- fft on mepsc frquny was similar twn WT an NRA / mi. P <.. Natur Nurosin: oi:.3/nn.7
4 P3 a UBP pa ms pa ms UBP f EPSC amp. (%as) mepsc (% as) UBP Amplitu 3 UBP UBP 3 3 UBP Frquny g EPSC amp. (%as) UBP 7 pa ms UBP / UBP 7 pa ms UBP Supplmntary Figur 3: Th NMDAR antagonist UBP affts th amplitu of AMPAR-miat urrnts whn all NMDARs ar lok, suggsting that this rug laks spifiity. (a,) Sampl voltag-lamp rorings of AMPAR-miat mepscs from L/3 pyramial lls from P3 P WT mi uring (a) vhil (ACSF) ontrol or () UBP (3 µm) appliation. mepsc vnts ar iniat y. (,) Sattr plots of mepsc amplitu an frquny for an aftr () vhil ontrol or () UBP appliation. UBP signifiantly ras oth amplitu (n =, P <.3) an frquny (P <.) of AMPAR-miat mepscs, whil thr wr no hangs in amplitu an frquny in vhil ontrol xprimnts (n = ). Baus postsynapti NMDARs wr lok, this ras in amplitu suggsts that UBP might afft AMPAR-miat urrnts y an NMDAR-inpnnt pathway. () mepsc amplitu aftr vhil ACSF vhil ontrol or UBP appliation, avrag an normaliz to as ontrols (ar graph of ata prsnt in an ). (f) UBP appliation ras th amplitu of vok AMPAR urrnts in L /3 synapss ror in voltag-lamp from P P WT mi (n = ), whil thr wr no hangs with vhil ontrol (n = ). All NMDARs wr lok uring this xprimnt y D,L- ( µm) in th xtrnal roring solution. (g) Quantifiation an sampl tras from ata takn from th last fiv minuts of rug tratmnt in f, monstrating that UBP signifiantly ru AMPAR EPSC amplitus ompar to vhil ontrols (P <.). Baus UBP afft our postsynapti raout of glutamat rlas (AMPAR urrnts), w wr unal to us this rug to assss th rol of UBP-targt prnmdar suunits on nurotransmittr rlas. Natur Nurosin: Error ars rprsnt oi:.3/nn.7 s..m. P <., P <., P <..
5 P3 WT a NRD / pa ms NRD / WT pa ms NRD / f s AP s AP 3 WT 3 s AP s AP Supplmntary Figur : Th NRD suunit is not rquir for prnmdars to nhan spontanous nurotransmittr rlas onto L/3 pyramial nurons in juvnil V. (a,) Sampl voltag-lamp rorings of AMPAR-miat mepscs from L/3 pyramial lls uring as an D- appliation prios in (a) WT an () NRD / mi. mepscs ar iniat y. (,) Amplitu an (,) intr-vnt intrval umulativ proaility histograms from th sam lls shown in a an uring as an D- appliation rval a hang in mepsc frquny ut not amplitu for oth () WT ontrol an () NRD / mi. (,f) Sattr plots of amplitu an frquny, for an aftr D- appliation, in () WT an (f) NRD / mi. L/3 pyramial lls xhiit a signifiant rution in mepsc frquny y D- in oth WT (n =, P =.) an NRD / mi (n =, P <.7), ut no hangs in amplitu. Th fft on mepsc frquny was similar twn WT an NRD / mi. P <.. Natur Nurosin: oi:.3/nn.7
6 P3 a Ro -9 Ro -9 pa ms pa ms Ro Ro f Ro -9 s C on tro l s C on tro l 3 3 Ro -9 3 B R as o - 9 B R as o l in - 9 Supplmntary Figur : NRB-ontaining prnmdars nhan spontanous nurotransmittr rlas onto L/3 pyramial nurons in juvnil V. (a-) Sampl voltag-lamp rorings of AMPAR-miat mepscs from WT L/3 pyramial lls in (a) ontrol xprimnts with vhil (ACSF) appliation or () with. µm Ro -9 appliation. mepscs ar iniat y. (,) Amplitu an (,) intr-vnt intrval umulativ proaility histograms from th sam lls shown in a an uring as an () vhil ontrol or () Ro -9 appliation. Thr was no hang in ithr mepsc amplitu or frquny in th ontrol xprimnt with vhil appliation, whil Ro -9 appliation ras mepsc frquny without hanging amplitu. (,f) Sattr plots of amplitu an frquny for an aftr () vhil (ACSF) ontrol or (f) Ro -9 appliation. Thr wr no signifiant hangs in amplitu or frquny in ontrol xprimnts (n = ). Ro -9 aus a signifiant rution in mepsc frquny (n = 7, P <.7), ut i not altr mepsc amplitu. P <.. Natur Nurosin: oi:.3/nn.7
7 P3 a Ifnproil pa ms pa ms Ifnproil.. Ifnproil Ifnproil Ifnproil.. 3 f 3 Ifnproil Ifnproil Ifnproil Supplmntary Figur : Bloka of NRB-ontaining prnmdars y ifnproil rus spontanous nurotransmittr rlas onto L/3 pyramial nurons. (a-) Sampl voltag-lamp rorings of AMPAR-miat mepscs from WT L/3 pyramial lls in (a) ontrol xprimnts with vhil (ACSF) appliation or () with 3 µm ifnproil appliation. mepscs ar iniat y. (, ) Amplitu an (, ) intr-vnt intrval umulativ proaility histograms from th sam lls shown in a an uring as an () vhil ontrol or () ifnproil appliation. (,f) Sattr plots of amplitu an frquny for an aftr () vhil (ACSF) ontrol or (f) ifnproil appliation. Thr wr no signifiant hangs in amplitu or frquny in ontrol xprimnts (n = 7). Ifnproil aus a signifiant rution in mepsc frquny (n =, P <.), ut i not altr mepsc amplitu. P <.. Natur Nurosin: oi:.3/nn.7
8 a pa ms WT NR3A / P3 pa ms WT NR3A / Prntag Prntag Prntag.. Prntag.. 3 WT f NR3A / Supplmntary Figur 7: Th NR3A suunit is rquir for th aility of prnmdars to nhan spontanous nurotransmittr rlas in juvnil V. (a,) Sampl voltag-lamp rorings of AMPAR-miat mepscs from L/3 pyramial lls uring as an D- appliation prios in (a) WT an () NR3A / mi. mepscs ar iniat y. (, ) Amplitu an (, ) intr-vnt intrval umulativ proaility histograms from th sam lls shown in a an. D- ras mepsc frquny ut not amplitu in L/3 nurons in WT mi, whil thr was no hang in ithr amplitu or frquny in NR3A / mi. (,f) Sattr plots of amplitu an frquny for an aftr D- appliation in () WT an (f) NR3A / mi. mepsc frquny (n =, P <.7), ut not amplitu, was signifiantly ru in L/3 pyramial nurons from WT mi. Thr wr no hangs in mepsc frquny an amplitu osrv in L/3 pyramial nurons from NR3A / mi (n = ). P <.. Natur Nurosin: oi:.3/nn.7
9 a P P P3 GFP-NR3A µm P3 STg NR3A OE pa ms pa ms f STg STg g.. NR3A OE NR3A OE Supplmntary Figur : Ovrxprssion of th NR3A suunit in xitatory nurons nows prnmdars with a gratr aility to nhan spontanous nurotransmittr rlas in L/3 pyramial lls of mor matur V. (a) Dvlopmntal timours of GFP-NR3A ovrxprssion in V monstrating minimal xprssion of th transgn for P. (,) Sampl voltag-lamp rorings of AMPAR-miat mepscs from L/3 pyramial lls uring as an D- appliation prios in () singl-transgni (STg) mi an () oul-transgni mi ovrxprssing (OE) NR3A. mepscs ar iniat y. (, ) Amplitu an (, ) intr-vnt intrval umulativ proaility histograms from th sam lls shown in an uring as an D- appliation. (f,g) Sattr plots of amplitu an frquny for an aftr D- appliation in (f) STg an (g) NR3A OE mi. Thr was no hang in amplitu with D- appliation in ithr STg or NR3A OE mi. Th ras in mepsc frquny with D- appliation was muh largr in OE mi (n =, P <.) ompar to thir STg littrmats (n = 7, P <.3). frquny prior to D- was also highr in OE mi as ompar to STg mi, although this fft i not rah statistial signifian (OE = 7. Hz, STg =.3 Hz, P <.7). P <. an Natur Nurosin: P <.. oi:.3/nn.7
10 P3, Low Mg +,NR3A / a pa ms pa ms f Supplmntary Figur 9: In low Mg + onitions, prnmdars laking NR3A ar al to nhan spontanous nurotransmittr rlas in juvnil V. (a,) Sampl voltag-lamp rorings in low Mg + ACSF from L/3 pyramial lls in NR3A / mi for or aftr (a) vhil (ACSF) or () D- appliation. mepscs ar iniat y. (, ) Amplitu an (, ) intr-vnt intrval umulativ proaility histograms from lls shown in a an uring as an D- appliation prios. Not that D- ru mepsc frquny. (,f) Sattr plots of amplitu an frquny for an aftr () vhil or (f) D- appliation in NR3A / mi. In low Mg + ACSF, mepsc amplitu an frquny wr onstant in ontrol xprimnts (n = 3), ut D- appliation ru mepsc frquny (n =, P <.) without affting amplitu. P <.. Natur Nurosin: oi:.3/nn.7
11 P Low Mg + Normal Mg + f Ro -9 pa ms Low Mg g + 3 f 3 j 3 Low Mg+ h k Low Mg+ h Low Mg + 3 Ro -9 Ro s AP s AP s AP Normal Mg + + s AP.. 3. i g Low Mg pa ms pa ms l Low Mg + 3 R o s - 9 pa ms Low Mg+ Low Mg + R s o - 9 s C on tro l Normal Mg + s C on tro l a Supplmntary Figur : In low Mg+, NRB-ontaining prnmdars nhan nurotransmittr rlas in P WT mi. (a) Sampl voltag-lamp roring in normal Mg+ ( mm) ACSF from L/3 pyramial lls in a P mous uring as an D- appliation prios. (,,) Rorings in low Mg+ ACSF from WT L/3 pyramial lls in P mi uring () vhil (ACSF) ontrol, () D-, or () Ro -9 appliation prios. mepscs ar iniat y. (-h) Amplitu an intr-vnt intrval umulativ proaility histograms from th sam lls shown in a-. Thr is no hang in mepsc amplitu in any onition, ut mepsc frquny ras with D- or Ro -9 appliation in th low Mg+ onition. (i-l) Sattr plots of mepsc amplitu an frquny monstrating rsponss to ontrol or NMDAR antagonist appliation whn rorings wr ma in (i) normal Mg+ (n = 9), or (j,k,l) low Mg+ (n = for ontrol xprimnts, n = 9 for xprimnts, n = 7 for Ro -9 xprimnts). A signifiant rution in mepsc frquny was osrv only in th low magnsium onition with D- appliation (P <.) or with Ro -9 appliation (P <.). P <.. Natur Nurosin: oi:.3/nn.7
12 Slop (% as) P3 AP-EPSP NRD / (n = ) 3 3 Supplmntary Figur : NRD-ontaining NMDARs ar not rquir for spik timing-pnnt long-trm prssion (tltd) in juvnil V. tltd was inu in L/3 V synapss in rspons to ation potntials pair with L-gnrat EPSPs whil postsynapti NMDARs wr lok in P3 P NRD / mi (n =, quantifiation of th last minuts = ± % of as EPSP slop, P <.). Natur Nurosin: oi:.3/nn.7
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