C 2+ -dependent enhncement of relese y suthreshold somtic depolriztion Json M Christie 1, Deli N Chiu 1,2 & Crig E Jhr 1 In mny neurons, suthreshold somtic depolriztion cn spred electrotoniclly into the xon nd modulte susequent spike-evoked trnsmission. Although relese proility is regulted y intrcellulr C 2+, the C 2+ dependence of this modultory mechnism hs een deted. Using pired recordings from synpticlly connected moleculr lyer interneurons (MLIs) of the rt cereellum, we oserved C 2+ -medited strengthening of relese following rief suthreshold depolriztion of the som. Two-photon microscopy reveled tht, t the xon, somtic depolriztion evoked C 2+ influx through voltgesensitive C 2+ chnnels nd fcilitted spike-evoked C 2+ entry. Exogenous C 2+ uffering diminished these C 2+ trnsients nd eliminted the strengthening of relese. Axonl C 2+ entry elicited y suthreshold somtic depolriztion lso triggered synchronous trnsmission tht my deplete vesicle vilility nd therey temper relese strengthening. In this cereellr circuit, ctivity-dependent presynptic plsticity depends on C 2+ elevtions resulting from oth su- nd suprthreshold electricl ctivity initited t the som. Short-term ltertion in the strength of neurotrnsmission cn result from the electrotonic spred of suthreshold depolriztion from the som to presynptic sites of relese on the xon 1 3. Mechnisms tht trigger or modulte neurotrnsmission re often medited through control of voltge-sensitive C 2+ chnnels (VSCCs) ecuse the likelihood of vesicle fusion is lrgely determined y the intrcellulr [C 2+ ]. Although high-concentrtion C 2+ elevtions re required to rpidly trigger exocytosis 4,5, low-concentrtion elevtions cn ugment susequent relese vi vesicle recruitment, priming nd sensitiztion 6. Becuse of their lrge mplitude, ction potentils re efficient triggers for VSCC ctivtion 7 1 nd relese. However, direct recordings from clycel nerve terminls revel tht even slight depolriztion cn open VSCCs, leit t low proility, nd strengthen susequent spike-evoked neurotrnsmission 11,12. In contrst, in dentte grnule cells of the hippocmpus, shortterm strengthening of relese elicited y modest somtic depolriztion my e C 2+ independent 1,13. Whether somtic depolriztion enhnces ction potentil evoked relese y ctivting xonl VSCCs in inhiitory neurons hs not een ddressed, lthough synchronous relese is ugmented in C 2+ -dependent mnner in young nimls 14. MLIs of the cereellum, including stellte nd sket cells, mke inhiitory GABAergic synpses onto Purkinje cells s well s onto other MLIs 15. Depolrizing potentils originting in the somtodendritic comprtment of MLIs pssively propgte into their xonl ror 16,17 nd open VSCCs 18. However, ction potentils re required to rpidly coordinte relese from presynptic sites, therey limiting the voltge rnge for VSCC ctivtion to suthreshold depolriztion. Whether xonl C 2+ entry evoked y suthreshold somtic depolriztion is sufficient to lter spikeevoked neurotrnsmission hs not een directly determined in MLIs. We investigted the mechnisms tht medite somtic voltge control of xonl trnsmitter relese etween MLIs using pired electricl recording nd two-photon lser-scnning microscopy (2PLSM). Suthreshold somtic depolriztion ws sufficient to ctivte xonl VSCCs, elicit C 2+ influx, nd strengthen oth ction potentil evoked nd synchronous trnsmitter relese. Enhncement of relese ws diminished or eliminted y chelting intrcellulr C 2+ with EGTA or y locking VSCCs, indicting tht there is direct connection etween somtic voltge control of neurotrnsmission nd C 2+ entry t the site of relese. This suggests tht relese plsticity elicited y suthreshold somtic depolriztion depends on presynptic VSCCs. RESULTS Suthreshold depolriztion enhnces spike-evoked relese GABA A receptor (GABA A R)-medited synptic trnsmission ws exmined etween pirs of MLIs in rt cereellr slices using simultneous whole-cell recording. Action potentils elicited in presynptic cells y somtic current injection ( 6 pa, 3 5 ms) evoked timelocked inwrd currents in voltge-clmped postsynptic cells (E Cl mv) tht were locked y picrotoxin ( µm, 3.8 ± 1.8% of control, n = 5). Irreversile rundown of evoked trnsmission, commonly oserved in pired MLI recordings 19, ws eliminted y presynptic perforted ptch recording 2 (see Online Methods). To determine whether somtic depolriztion ws sufficient to lter the strength of neurotrnsmission, we riefly depolrized presynptic neurons from rest ( 73.3 ±.3 mv, n = 4) to potentil just suthreshold for spiking ( 56.1 ±.9 mv, 3 ms). This depolriztion ws followed y ction potentil stimultion (1-ms dely). Inhiitory postsynptic currents (IPSCs) evoked y ction potentils preceded y suthreshold depolriztion were lrger thn interleved control IPSCs evoked y ction potentils lone (121.1 ± 4.8% of control mplitude, n = 4, P <.1; Fig. 1). Furthermore, the pired-pulse rtio (PPR = IPSC 2 /IPSC 1 ) 1 Vollum Institute, Oregon Helth & Science University, Portlnd, Oregon, USA. 2 Neuroscience Grdute Progrm, Oregon Helth & Science University, Portlnd, Oregon, USA. Correspondence should e ddressed to J.M.C. (json.christie@mxplnckflorid.org). Received 1 Octoer; ccepted 9 Novemer; pulished online 19 Decemer 21; doi:1.138/nn.2718 62 VOLUME 14 NUMBER 1 JANUARY 211 nture NEUROSCIENCE
Figure 1 Suthreshold somtic depolriztion enhnces spike-evoked relese in MLIs. () In MLI pired recording, ction potentils in the presynptic neuron evoked GABA A R-medited IPSCs in the postsynptic neuron. Suthreshold somtic depolriztion (3 ms) elicited efore spiking enhnced relese. Control ction potentil responses re in red nd test responses preceded y suthreshold depolriztion re in lck. An mplified view of the first IPSC is shown in the inset. () Pired-pulse IPSCs from the sme neuron normlized to the pek of the first response. For clrity, spontneous IPSCs were lnked efore verging in (inset) nd. (c) Summry dt showing tht the PPR decresed with somtic depolriztion of the presynptic neuron. P <.5, pired t test. Dt re men vlues ± s.e.m. (d) Liner regression nlysis reveled tht relese enhncement elicited y suthreshold depolriztion ws correlted with the sl PPR. (e,f) Differences in relese enhncement for fcilitting nd depressing responses mesured in 1.5 nd 3. mm extrcellulr C 2+. P <.5, ANOVA nlysis with post hoc Tukey s multiple comprison tests indicted significnce. Dt re men vlues plotted ± s.e.m. of IPSCs evoked y two ction potentils (25-ms intervl, PPR = 1. ±.64, n = 37; Fig. 1,) following suthreshold depolriztion ws significntly reduced compred with nondepolrized control trils (P =.2; Fig. 1,c). This indictes tht suthreshold somtic depolriztion cuses short-term ltertion in presynptic relese resulting from the electrotonic spred of the somtic depolriztion into the xon, s oserved in other neurons 1 3,13. Control recordings included oth fcilitting nd depressing responses (Fig. 1c), indicting tht synpses etween MLIs with different relese proilities coexist in cereellum. Synptic strengthening cused y suthreshold depolriztion depended on sl relese proility, s the mount of enhncement of IPSC 1 ws correlted with the PPR mesured in control trils (Fig. 1d). Depressing synpses, on verge, were not ltered y suthreshold depolriztion (P =.17). However, there ws considerle vriility in this group, with some pirs showing relese enhncement, wheres others were wekened (Fig. 1e). In 3 mm extrcellulr C 2+, relese enhncement cused y suthreshold depolriztion ws less thn in 1.5 mm C 2+ Pre Post Stim 1 c d e f PPR 2 1 n = 37 Control 1 ms Stim 1 4 ms 25 25 3 Percentge of control IPSC 1 2 15 Control r 2 =.54 P <.1 1 Control PPR 2 3 1 mv Percentge of control IPSC 1 (Fig. 1e), ut the increse ws still significnt (P =.3). High concentrtions of extrcellulr C 2+ incresed relese proility, s reflected y lrger control IPSCs (pek mplitude ws 36. ± 67. nd 172.7 ± 22.5 pa for 3. nd 1.5 mm C 2+, respectively; n = 13 nd 4, P =.2) nd pired-pulse depression in ll recordings (n = 11; Fig. 1f). Thus, strengthening of relese y suthreshold depolriztion is similr to other ctivity-dependent forms of relese enhncement, such s spikeevoked fcilittion nd ugmenttion, in tht these processes occur more redily in low relese proility synpses. Suthreshold depolriztion nd presynptic C 2+ entry Intrcellulr C 2+ is known to govern mny spects of trnsmitter relese 6. We proed for presynptic C 2+ elevtion evoked y suthreshold somtic depolriztion using 2PLSM. MLIs were filled through the whole-cell ptch pipette with Alex Fluor 594 to visulize cell morphology nd the C 2+ indictor Fluo-5F to detect intrcellulr C 2+ trnsients (Fig. 2). Axon vricosities were trgeted for opticl recording. These vricosities re known to e hotspots of ction 1.5 mm (fcilit) 1.5 mm (depress) 3. mm Control PPR 2 n = 15 2 n = 15 n = 22 15 n = 11 n = 22 1 n = 11 5 Stim 2 Stim 1 Stim 2 Normlized 1 ms 1.5 mm (fcilit) 1.5 mm (depress) 3. mm Externl [C 2+ ] Externl [C 2+ ] 15 µm c Su C 2+ ( G/R).2.1 4 µm.5.1 AP C 2+ ( G/R) r 2 =.4 P =.26 25 mv 5 ms Som.4 G/R Vricosity 5 ms Integrl.2 G/R 25 ms Su C 2+ AP C 2+ Stim.5 ( G/R) s 1 Figure 2 Suthreshold somtic depolriztion evokes nd enhnces xonl C 2+ entry. () Two-photon fluorescence imge of representtive MLI filled vi ptch pipette with Alex 594 (13 µm) nd Fluo-5F (2 µm). Right, higher mgnifiction view shows xon vricosities. () Somtic current injection elicited control ction potentil evoked responses (red) nd test spikes pired with 3-ms suthreshold depolriztions (lck). Shown directly elow re the resulting C 2+ trnsients recorded in n xon vricosity. Integrtion of the C 2+ signls ccentutes the difference etween control nd depolrized responses efore spike firing. Bottom left, mplified view of C 2+ trnsients with dshed lines (-ms epoch verge) demrcting the mplitude of the C 2+ response evoked y suthreshold depolriztion. Bottom right, ction potentil evoked C 2+ trnsients selined efore spike firing (lck r) isolted depolriztiondependent fcilittion of the spike-evoked C 2+ trnsient. Exponentil fits to the spike-elicited trnsients were used to determine pek mplitudes. (c) Comprison of depolriztion-evoked C 2+ elevtion nd fcilittion of ction potentil elicited C 2+ entry. Line fit of the liner regression is indicted y the dshed line. nture NEUROSCIENCE VOLUME 14 NUMBER 1 JANUARY 211 63
Figure 3 Suthreshold depolriztion dependent C 2+ signling is EGTA sensitive. () Somtic recording of n ction potentil (red) nd n ction potentil preceded y 3-ms suthreshold depolriztion (lck). The resulting C 2+ trnsients in n xon vricosity re shown elow in control nd following EGTA-AM ppliction (1 2 µm, >1 min). Integrls of the C 2+ responses re provided for ech condition. The inset shows comprison of ction potentil evoked responses, elicited without preceding suthreshold depolriztion, in control nd with EGTA. Dshed lines indicte the response mplitude efore spiking (-ms epoch) nd the exponentil fits of ction potentil evoked C 2+ trnsients used to determine pek mplitudes. () The effect of EGTA on C 2+ entry evoked y suthreshold depolriztion. (c) EGTA inhiits fcilittion of spikeevoked C 2+ entry cused y suthreshold depolriztion. Dt re men ± s.e.m., P <.5, pired t test. potentil evoked C 2+ entry nd re presumed to e en pssnt sites of relese 15,18,21,22, lthough it remins possile tht these speciliztions my include nonsynptic sites. In current-clmped MLIs, suthreshold somtic depolriztion (3 ms) often evoked smll C 2+ trnsients in xon vricosities ( Su C 2+ ; Fig. 2). The mplitude of these C 2+ trnsients vried cross recording sites, including mny vricosities without ny pprent C 2+ elevtion (Fig. 2c). However, on verge, C 2+ responses were significntly lrger thn corresponding mesurements otined from interleved control trils without suthreshold depolriztion ( G/R =.1 ±.4 nd.26 ±.1 for control nd depolrized, respectively; P <.1, n = 35). In ddition, we oserved tht C 2+ trnsients evoked y ction potentils following suthreshold depolriztion were lrger thn those without prior depolriztion (Fig. 2). Sutrction of the C 2+ elevtion evoked during the suthreshold depolriztion reveled tht the fcilittion of spike-evoked C 2+ (24.5 ± 4.5% increse in pek mplitude, P <.1) ws not merely the result of shift in seline fluorescence, ut ws insted true enhncement of spike-induced C 2+ influx ( AP C 2+ ; Fig. 2). The mount of spike-evoked C 2+ fcilittion ws not correlted with the c Control Pre Post 2 pa 1 ms Stim 1 2 mv 2 pa 3 ms 2 pa 3 ms Difference in % chnge ( /) Percentge of control IPSC 1 175 15 125 75 5 6 4 2 2 Stim 1 Stim 2 2 mv 2 pa 3 ms Fcilitting n = 5 d PPR 1.5 1..5 Depress n = 5 n = 4 r 2 =.82 P <.1.5 1. 1.5 Control PPR () Fcilitting 25 mv 5 ms Control.5 G/R 5 ms Integrl.5 G/R 5 ms Integrl.5 G/R 5 ms mplitude of C 2+ influx during suthreshold depolriztion (Fig. 2c). Whether this indictes tht the two components contriuting to C 2+ elevtion re not cuslly relted is uncler ecuse the extremely smll mplitude of the suthreshold response interferes with our ility to ccurtely mesure it. However, in 3 mm extrcellulr C 2+, the C 2+ trnsient resulting from suthreshold depolriztion ws lrger thn tht in control conditions (pek G/R =.36 ±.8 nd.61 ±.3 for 1.5 mm nd 3. mm extrcellulr C 2+, respectively; n = 35 nd 74, P =.4) nd ws correlted with fcilittion of the spike-induced C 2+ influx (r 2 =.19, P =.2), suggesting tht the C 2+ elevtion during suthreshold depolriztion cused the spike-induced fcilittion. Inhiition of VSCCs y comintion of ntgonists (1 µm ω-conotoxin MVIIC,.3 µm SNX, 2 µm nimodipine nd 1 µm miefrdil) confirmed tht suthreshold depolriztion evoked C 2+ entry occurs through VSCCs (Supplementry Fig. 1,), s ws previously reported 18. The unlocked component presumly reflects toxin-resistnt VSCCs 22 24, s spike-evoked C 2+ trnsients were locked to similr extent (Supplementry Fig. 1). Suthreshold depolriztion induced fcilittion of spike-evoked C 2+ entry ws lso diminished y VSCC inhiition (8.7 ± 5.% of control, n = 9, P =.2), gin suggesting tht this fcilittion ws dependent on n increse in intrcellulr C 2+ elicited y the preceding suthreshold depolriztion. Stim 1. ( G/R) s 1 1. ( G/R) s 1 c Su C 2+ ( G/R) AP C 2+ ( G/R).15.1.5.3.2.1 n = 9 n = 9 Figure 4 EGTA inhiits enhncement of relese elicited y suthreshold depolriztion. () Action potentil evoked IPSCs (red) recorded from pirs of connected MLIs in control nd following EGTA-AM ppliction. In lternting trils, ction potentils were preceded y 3-ms suthreshold depolriztion (lck). () Top, EGTA eliminted relese strengthening cused y suthreshold depolriztion in fcilitting cell pirs. Relese strengthening ws not pprent in cell pirs tht depressed in sl conditions. Bottom, liner regression nlysis plot of the difference etween the percentge chnge in EGTA nd in control reltive to the sl PPR. (c) The effect of EGTA on pired-pulse responses from trils without preceding suthreshold depolriztion. Inset, mplified view of the first IPSC. (d) EGTA ltered pired-pulse responses in cell pirs tht fcilitted in sl conditions. Dt re men ± s.e.m., P <.5, pired t test. Spontneous IPSCs were lnked efore verging in nd c. 64 VOLUME 14 NUMBER 1 JANUARY 211 nture neuroscience
Relese strengthening cused y direct presynptic suthreshold depolriztion t the clyx of Held is inhiited y C 2+ cheltion 11,12. In MLI xonl vricosities, depolriztion-evoked C 2+ entry ws Post Pre not significntly ltered y the cell-permele C 2+ uffer EGTA-AM (1 2 µm, >1 min, P =.8; Fig. 3,). In mny cses, however, suthreshold depolriztion did not result in mesurle C 2+ trnsient (Fig. 2c) nd n effect of cheltion overll could therefore only e determined indirectly, using pired recording (see elow). To test for Averge direct effect of cheltion on C 2+ elevtion induced y suthreshold depolriztion, we focused our nlysis on lrge-mplitude responses ( G/R >.5, n = 4; Fig. 3). In this group, EGTA significntly reduced the C 2+ trnsients ( G/R =.9 ±.19 nd.43 ±.27 for control nd EGTA, respectively; n = 4, P =.3). Fcilittion of spike-evoked C 2+ entry following suthreshold depolriztion ws gretly diminished y EGTA cross ll cells (Fig. 3c), indicting tht, even in cses in which depolriztion did not produce mesurle increse in C 2+, fcilittion of the spike-evoked C 2+ trnsient ws C 2+ dependent 25,26. EGTA lso reduced C 2+ trnsients evoked y single ction potentils lone (7.1 ± 3.3% of control, n = 9, P =.8; Fig. 3), suggesting tht there is competition etween Fluo-5F nd EGTA for C 2+ inding 27, tht sl C 2+ levels re sufficient to prtilly fcilitte VSCC opening or tht there is sustntil rundown of VSCC opening. Enhncement of relese is C 2+ dependent In pired MLI recordings, the enhncement of IPSC 1 y suthreshold depolriztion ws locked y EGTA-AM (2 µm, >1 min, 122.2 ± 7.4 nd 97.5 ± 6.9% of control mplitude for nd, respectively; n = 9, P =.9; Fig. 4,). This indictes tht the increse in spike-evoked relese cused y suthreshold depolriztion depends on the EGTA-sensitive C 2+ elevtion oserved in xon vricosities. The lock of relese-strengthening y EGTA occurred minly in recordings showing pired-pulse fcilittion in control conditions. On verge, IPSCs c +VSCC lock Pre V Mem 7 mv Pre V Mem 7 mv Pre V Mem 7 mv 5 mv 4 mv 4 mv 7 mv 7 mv 7 mv 1 mv 2 pa 4 ms 4 pa 4 ms Post-verge integrl (n = 34 cells) 1 pa s 1 Figure 5 Suthreshold depolriztion evokes synchronous relese. () Pired recording of MLIs showing spontneous IPSCs in unstimulted control responses (red) nd recruitment of synchronous events with 3-ms presynptic suthreshold depolriztion (lck). Three individul sweeps from the postsynptic neuron re shown superimposed for ech condition with the verge response (27 nd 28 sweeps for control nd depolrized, respectively) t the ottom. () The verge chrge integrtion of the postsynptic response cross ll cell recordings (±.s.e.m.). P <.5, pired t test. in depressing cell pirs were unltered y suthreshold depolriztion nd remined unchnged with the ddition of EGTA (Figs. 1e nd 4). It follows tht the mgnitude of the EGTA effect ws correlted with the control PPR (Fig. 4). This reinforces the conclusion tht suthreshold depolriztion differentilly ffects relese t MLI synpses in relese proility dependent mnner. EGTA did not ffect the mplitude of IPSC 1 evoked without preceding suthreshold depolriztion (IPSC mplitudes = 97.5 ± 4.8 nd 82.8 ± 11.3% of control, n = 5 nd 4, P =.47 nd.16, for fcilitting, nd depressing responses, respectively; Fig. 4c). This implies tht the C 2+ domin responsile for triggering exocytosis is tightly ssocited with relese-competent vesicles 28,29. However, EGTA ltered the PPR, chnging fcilittion to depression, though it hd no cler effect on depressing cell pirs (PPR for depressing cells =.67 ±.9 nd.97 ±.9 for control nd EGTA, respectively; n = 4, P =.13; Fig. 4c,d). This suggests tht, in sl conditions, residul C 2+ following n initil spike fcilittes susequent relese 27 nd my msk depression 3,31. EGTA did not induce significnt chnge in the PPR in these responses when suthreshold depolriztion preceded spiking (PPR =.79 ±.5 nd 1.1 ±.11 for nd, respectively; n = 5, P =.18), suggesting little dditionl or underlying modifiction of relese in this condition. Suthreshold depolriztion nd synchronous relese At some synpses, synchronous relese cn result from the low concentrtion of C 2+ remining fter spike firing 32,33. In our pired recordings, there ws often smll increse in the frequency of synchronous IPSCs during suthreshold somtic depolriztion (Fig. 5), likely cused y the elevtion of C 2+ y the depolrizing stimulus. Although spikeevoked IPSCs from unclmped surrounding MLIs likely overwhelmed d Percentge frequency increse from 7 mv 1, 1 n.s. n = 8 1 6 5 4 Pre V Mem (mv) e Percentge control frequency ( 4 mv) n = 5 1,5 5 15 5 +VSCC lock 4 7 4 Pre V Mem (mv) pa ms Figure 6 Depolriztion-dependent synchronous relese is triggered y VSCCs. (,) Pired recording from MLIs in tetrodotoxin with representtive sweeps from the postsynptic neuron showing recruitment of synchronous IPSCs with presynptic depolriztion. The presynptic holding potentil is indicted in gry ove ech sweep. Responses were recorded in 3 mm extrcellulr C 2+ to enhnce relese. (c) In the sme cell, IPSC frequency ws unltered y presynptic depolriztion fter VSCC lock ( µm Cd 2+ nd Ni 2+ ). (d) Asynchronous IPSC frequency depended on the presynptic holding potentil. Dt re men ± s.e.m. P <.5, ANOVA nlysis (Tukey s multiple comprison test). (e) Block of VSCCs eliminted the depolriztion-dependent increse in synchronous trnsmission. Dt re men ± s.e.m., P <.5, ANOVA nlysis. nture NEUROSCIENCE VOLUME 14 NUMBER 1 JANUARY 211 65
Burst Pre Post 4 ms Stim 2 mv 1 ms Stim ( depol) 1 ms +Burst Burst Figure 7 Burst firing reduces the cpcity for relese enhncement cused y depolriztion. () In recording from connected MLIs, high-frequency ursts of ction potentils (ten spikes, 6 Hz) preceded 3-ms suthreshold stimultion nd test spikes in interleved trils. Amplified views on the right show relese enhncement cused y suthreshold depolriztion nd wekening of trnsmission when suthreshold excittion ws pired with urst firing. () The effect of urst firing on the first IPSC in control trils without suthreshold stimultion. For mplified views (,), spontneous events were lnked efore verging. (c) Burst firing eliminted relese strengthening induced y suthreshold depolriztion. Dt re men ± s.e.m., P <.5, pired t test. +Burst 4 ms this smll increse in synchronous trnsmission in mny recordings, chrge integrtion of verged postsynptic currents reveled difference etween control nd depolrized trils cross cells (Fig. 5). The increse of synchronous IPSCs y presynptic somtic depolriztion ws much clerer when ckground ction potentil driven relese from spontneously firing surrounding cells ws locked with tetrodotoxin (1 µm) nd the stimulus durtion ws prolonged (15 s, 3 mm extrcellulr C 2+ ; Fig. 6). The frequency of synchronous relese ws steeply dependent on the holding potentil of the presynptic neuron (Fig. 6,,d). Block of VSCCs y Cd 2+ nd Ni 2+ ( µm) eliminted depolriztion-evoked synchronous relese without ffecting sl spontneous trnsmission (Fig. 6c,e), confirming the connection etween somtic depolriztion, xonl VSCC ctivtion nd synchronous relese. Asynchronous relese elicited y repetitive firing wekens susequent ction potentil evoked phsic trnsmission y depleting the pool of redily-relesle vesicles 33,34. Vesicle vilility my, in prt, regulte relese strengthening induced y suthreshold depolriztion. To explore this ide, we reduced the relesle pool of vesicles with trin of presynptic ction potentils (ten spikes, 6 Hz) nd then tested for suthreshold depolriztion induced enhncement of relese (Fig. 7). The mplitude of spike-evoked test IPSCs following urst firing ws reduced compred with interleved trils without the urst (77.3 ± 7.% of control, n = 1, P =.5; Fig. 7), result consistent with depression induced y vesicle depletion. In ddition, urst firing eliminted relese-strengthening elicited y suthreshold depolriztion (Fig. 7,c). This indictes tht in conditions tht diminish the relesle pool of vesicles, suthreshold depolriztion is less effective in ltering relese proility. By extension, synchronous relese elicited y suthreshold depolriztion likely limits the strengthening of spike-evoked trnsmission y depleting the redily-relesle pool of vesicles. This my explin why in some cell pirs (four of ten) the dditive effect of suthreshold depolriztion nd ursting resulted in wekening of trnsmission rther thn enhncement (Fig. 7,c). DISCUSSION We found tht suthreshold somtic depolriztion pssively spreds into the xon ror of MLIs nd strengthens susequent ction Stim 2 mv 1 ms c Percentge of control IPSC 1 (+depol) 25 2 15 n = 1 Burst +Burst potentil evoked relese of GABA onto neighoring cells. Thus, suthreshold depolriztion enhncement of relese is not limited to excittory neurons, ut lso includes inhiitory neurons. We found tht, for MLIs, this enhncement of relese depends on the opening of VSCCs, which results in C 2+ elevtion nd fcilittion of spike-evoked C 2+ influx. An increse in synchronous relese tht ccompnies the suthreshold depolriztion is C 2+ dependent nd my decrese the cpcity for relese strengthening owing to vesicle depletion. Our results suggest tht suthreshold potentils my modify neurotrnsmission t synpses throughout the nervous system in C 2+ -dependent mnner, s do other forms of short-term fcilittion. Somtic depolriztion nd C 2+ influx t sites of relese Axons re not electrotoniclly isolted from the somtodendritic comprtment, ut insted support the electrotonic spred of suthreshold somtodendritic potentils 1 3,16,18. As result, the open proility of voltge-sensitive ion chnnels in xons cn e modified y somtodendritic depolriztion. Our C 2+ mesurements indicte tht somtic depolriztions electrotoniclly spred into MLI xons nd re sufficient to open VSCCs. Although we did not directly determine them here, xonl length constnts cn exceed 4 µm in some neurons 1 3,35. This prmeter must e convolved with the numer nd composition of VSCCs t sites of relese to determine suthreshold depolriztion-medited C 2+ entry 36. In clycel terminls, P/Qtype VSCCs open t low proility following slight depolriztion 11 (< 6 mv), n ctivtion rnge elow tht previously reported 25,37. Although MLI vricosities express P/Q/N-type chnnels, mong others 22, the VSCC sutypes mediting depolriztion-evoked C 2+ influx remin uncler. It is possile tht low-ctivtion threshold VSCCs, such s R-type chnnels, preferentilly medite this C 2+ influx, s predicted for hippocmpl mossy fier outons 36. It is unknown whether C 2+ -induced C 2+ relese from intrcellulr stores 38 contriutes to suthreshold depolriztion evoked C 2+ trnsients. Suthreshold depolriztion not only induces direct influx of C 2+ in MLI xons, ut lso fcilittes ction potentil evoked C 2+ entry. This effect is proly dependent on the preceding influx of C 2+, s oth inhiition of VSCCs nd C 2+ cheltion diminished the fcilittion of spike-evoked C 2+ entry. At some synpses, VSCCmedited currents re enhnced following C 2+ inding y neuronl clcium sensor 1 (NCS-1), which induces shortening of the ctivtion phse of VSCCs 26,39. VSCC fcilittion is elicited y oth supr- nd suthreshold depolriztion evoked C 2+ elevtion nd is mitigted y exogenous C 2+ uffering 12,25,26. In MLIs, enhncement of spikeevoked C 2+ entry following suthreshold depolriztion could reflect comprle C 2+ -dependent fcilittion of VSCC ctivity, s MLIs express NCS-1 t presynptic sites of relese 4,41. It remins possile tht fcilittion of ction potentil evoked C 2+ entry does not depend directly on the C 2+ elevtion evoked y the suthreshold depolriztion. Alterntively, ecuse spike-evoked C 2+ influx in presynptic terminls is, in prt, 66 VOLUME 14 NUMBER 1 JANUARY 211 nture neuroscience
controlled y the mplitude nd durtion of the ction potentil wveform 9,42, fcilittion of spike-evoked C 2+ entry following suthreshold depolriztion my reflect n ltertion of the underlying ction potentil. Prolonged suthreshold somtic depolriztion inctivtes xonl Kv1 potssium chnnels in L5 pyrmidl cells of visul cortex, leding to spike rodening 3, which should in turn enhnce C 2+ entry 42. However, potssium chnnel inctivtion is not expected to e EGTA sensitive or to depend on VSCCs, rguing ginst this possiility. Somtic depolriztion nd enhncement of relese Presynptic receptor medited depolriztion of clycel nerve terminls elicits VSCC C 2+ elevtion nd strengthening of relese 11,43. Our results extend this finding; suthreshold excittory potentils need not only e elicited on or ner terminls to enhnce relese. Depolrizing potentils evoked in the dendrites or som spred through the xon, opening VSCCs, elevting C 2+ t presynptic speciliztions nd therey incresing the likelihood of trnsmission. Smll C 2+ trnsients increse the proility of fusion y incresing microdomin C 2+ (refs. 44,45), recruiting or sensitizing vesicles through C 2+ inding y high-ffinity sensor tht is distinct from the exocytosis trigger 27,46, nd sturting endogenous C 2+ uffers 44,45. If these mechnisms re medited y residul C 2+ following ction potentil firing, then they my lso e triggered y C 2+ influx during suthreshold depolriztion 12. Given tht relese proility is steeply dependent on the intrcellulr [C 2+ ] 4,5, enhncement of spike-evoked C 2+ entry will lso strengthen trnsmission 12,25,26,42 unless the relese process is sturted. If this occurs in MLIs, it my explin why high relese-proility synpses hve diminished cpcity for relese strengthening induced y suthreshold depolriztion. We found tht xonl VSCC-medited C 2+ entry elicited during presynptic depolriztion is sufficient to evoke synchronous relese in MLIs. C 2+ -induced synchronous fusion hs reltively high sensitivity to intrcellulr [C 2+ ], with n elevtion to s little s 1 µm triggering relese 4,5. If the C 2+ sensor for synchronous exocytosis t MLIs hs similr sensitivity, then the intrcellulr [C 2+ ] reched t MLI vricosities my e much higher thn depolriztionevoked C 2+ elevtion in the clyx 11 ( nm). The supr-liner C 2+ coopertivity for exocytosis 4,5,47 might suggest greter mount of spike-evoked relese enhncement following presynptic depolriztion nd C 2+ entry thn we oserved. However, synchronous trnsmission cn deplete the pool of relese-redy vesicles, diminishing vilility for phsic trnsmission 33,34. The resulting short-term depression of relese cn develop concomitntly with fcilittion, one effect msking the other 3,31. In this wy, the cpcity for depolriztion-evoked strengthening of trnsmission in MLIs is limited y the superimposition of C 2+ -dependent depression medited y vesicle pool depletion. Although IPSC mplitudes were unchnged y suthreshold depolriztion in EGTA, it my e tht relese enhncement ws merely lnced y depression rther thn fully locked. EGTA only prtilly diminished depolriztion-induced C 2+ elevtion suggesting tht the C 2+ -dependent effects on relese persisted in this condition. The expression of fcilittion nd depression will depend on the reltive C 2+ sensitivity of ech mechnism. As implied in our urst-firing experiment, the cpcity for relesestrengthening y suthreshold depolriztion my e strongly limited y depletion. Nonhomogenous pools of vesicles my populte presynptic sites t MLIs, s they do t the clyx of Held 48. Suthreshold depolriztion my help recruit more reluctnt vesicles, distinguished y their reltive C 2+ sensitivity nd/or distnce from VSCCs, to prticipte in relese during susequent ction potentil evoked trnsmission. Bulk C 2+ elevtion evoked y repetitive firing my lso recruit these vesicles, depleting their vilility nd therey decresing the cpcity for suthreshold depolriztion induced enhncement of relese. Tht is, multiplictive nonliner processes compete for overlpping vesicle pools to determine the expression of shortterm plsticity. We expect tht ny reduction in the vesicle pool(s), medited y preceding su- or suprthreshold ctivity, would diminish enhncement of relese induced y suthreshold depolriztion. Suthreshold depolriztion-induced regultion of spike-evoked relese is clerly importnt in numer of cell types, lthough the mechnisms underlying the regultion my vry cross rin regions 1 3,13,18,35. In MLIs, the phenomenon ppers to e entirely dependent on xonl C 2+, s oserved for other forms of relese strengthening 6. In contrst, t hippocmpl mossy fier synpses, it my e tht the C 2+ dependence is restricted to proximl portions of the xon, wheres other mechnisms of strengthening, such s potssium chnnel inctivtion 3,42, re involved t more distl sites 13. One cler difference etween these two cell types is xonl length, with mossy fiers eing much longer thn MLI xons. Even in MLIs, the increses in xonl C 2+ re, on verge, lrger in proximl thn in distl portions of the xon, s expected from cle properties 18. However, there ws no correltion etween the depolriztion-induced enhncement of relese nd distnce etween the somt of synpticlly connected pirs (r 2 =.6, P =.23, n = 27), lthough this comprison is likely compromised y the inccurcy of this mesurement in predicting xonl length. Our results suggest tht C 2+ -dependent plsticity induced y somtic depolriztion will ffect the gin of most synpses in short xon cells nd t lest proximl synpses in other cell types. However, it is likely tht depolriztion-induced plsticity does not simply monotoniclly decy with distnce from the som given the mount of vrince tht we oserved in our C 2+ imging nd pired-recording experiments. The control of trnsmitter relese y suthreshold synptic input tht precedes ction potentil initition dds new dimension to synptic signling nd requires considertion when nlyzing informtion trnsfer cross synpses. Methods Methods nd ny ssocited references re ville in the online version of the pper t http://www.nture.com/ntureneuroscience/. Note: Supplementry informtion is ville on the Nture Neuroscience wesite. Acknowledgments We thnk M. Hermn, M. McGinley, B. Nhir nd J. Pugh for their helpful discussions nd comments on the mnuscript. This work ws supported y US Ntionl Institutes of Helth grnt NS6637 (C.E.J.). AUTHOR CONTRIBUTIONS Ech of the uthors contriuted extensively to the design nd implementtion of the experiments, interprettion of the dt nd writing of the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t http://www.nture.com/ntureneuroscience/. Reprints nd permissions informtion is ville online t http://www.nture.com/ reprintsndpermissions/. 1. Alle, H. & Geiger, J.R.P. Comined nlog nd ction potentil coding in hippocmpl mossy fiers. Science 311, 129 1293 (26). 2. Shu, Y., Hsenstu, A., Duque, A., Yu, Y. & McCormick, D.A. Modultion of intrcorticl synptic potentils y presynptic somtic memrne potentil. Nture 441, 761 765 (26). 3. Kole, M.H.P., Letzkus, J.J. & Sturt, G.J. Axon initil segment Kv1 chnnels control ction potentil wveform nd synptic efficcy. Neuron 55, 633 647 (27). 4. Bollmnn, J.H., Skmnn, B. & Borst, J.G.G. Clcium sensitivity of glutmte relese in clyx-type terminl. Science 289, 953 957 (2). nture NEUROSCIENCE VOLUME 14 NUMBER 1 JANUARY 211 67
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Neuron 23, 821 832 (1999). 68 VOLUME 14 NUMBER 1 JANUARY 211 nture neuroscience
ONLINE METHODS Slice preprtion. Prsgittl slices from cereellr vermi were prepred from Sprgue Dwley rts (postntl dy 14 2) in ccordnce with Oregon Helth nd Science University Institutionl Animl Cre nd Use Committee pproved protocols. Following isoflurne nesthesi, rts were decpitted nd the cereellum ws isolted. Slices (25 3 µm) were cut on viroslicer (Leic Instruments) in n ice-cold solution contining 87 mm NCl, 25 mm NHO 3, 2.5 mm KCl, 1.25 mm NH 2 PO 4, 7 mm MgCl 2,.5 mm CCl 2, 1 mm glucose nd 75 mm sucrose. Slices were susequently trnsferred to holding chmer contining 119 mm NCl, 26.2 mm NHO 3, 2.5 mm KCl, 1 mm NH 2 PO 4, 2 mm MgCl 2, 1 mm CCl 2 nd 11 mm glucose nd mintined t 34 C for 3 min nd then t 22 25 C until use. For whole-cell recording, slices were plced in sumersion chmer nd superfused with the sme solution (22 25 C) ltered y elevting CCl 2 to 1.5 mm nd reducing MgCl 2 to 1.5 mm, except where noted. All solutions were oxygented y pre- or continuous equilirtion with crogen gs (95% O 2, 5% CO 2 ). Electrophysiology. Whole-cell recordings were otined from MLIs identified with grdient-contrst infrred video microscopy 49 sed on their loction in the moleculr lyer nd distinct morphology 15. Ptch pipettes used for currentclmped MLIs contined solution of 128 mm potssium gluconte, 2 mm KCl, 9 mm HEPES, 4 mm MgCl 2, 4 mm NATP, nd.5 mm NGTP nd 9 mm K 4 -BAPTA. For pired recordings, perforted ccess 5 to the presynptic MLI ws chieved y including mphotericin B (3 µg ml 1, previously liquoted in DMSO). Alex Fluor 488 hydrzide (3 µm, Moleculr Proes) ws lso included in the pipette to fluorescently monitor the integrity of the perforted recording with light microscopy. If the ptch memrne ruptured, resulting in cell fluorescence, recording ws terminted. For C 2+ -imging experiments, K 4 -BAPTA ws replced with 2 µm Fluo-5F nd 13 µm Alex Fluor 594 hydrzide (Moleculr Proes). Current-clmped cells were held ner 7 mv with constnt current injection. Action potentils were stimulted etween.3 nd.1 Hz (~25 6 trils per condition). MLIs were voltge clmped with pipette solution contining 135 mm CsCl, 1 mm HEPES, 4 mm Mg 2 ATP,.3 mm NGTP nd 5 mm EGTA. The holding potentil ws kept t 6 mv giving rise to inwrd GABA A R-medited currents (E Cl mv). All pipettes hd open-tip resistnces of 3 12 MΩ. Electrophysiologicl potentils nd currents were recorded with Multiclmp 7B mplifier (Moleculr Devices). Electrode series resistnce ws compensted y ridge lnce in current-clmped cells nd ws uncompensted in the voltgeclmp configurtion. Anlog signls were filtered t 3 1 khz nd digitized t 2 5 khz with 16-it A/D converter (Instrutech). High ccess resistnce (4 8 MΩ) in the presynptic perforted recording lso likely contriuted to filtering nd ction potentil ttenution, lthough ccess often improved during n experiment s result of continued prtitioning of the ntiiotic. Dt were collected using custom softwre (J.S. Dimond, Ntionl Institute of Neurologicl Disorders nd Stroke) written in IgorPro (Wvemetrics) nd nlyzed using Axogrph (Axogrph Scientific). GABA A R-medited synptic responses were isolted with NBQX (1 µm) nd d-ap5 (5 µm) (Tocris Cookson) to lock fst glutmte receptor medited responses. Picrotoxin ( µm) ws lso included during imging experiments to eliminte GABA A R-medited trnsmission. EGTA-AM (1 2 µm, Moleculr Proes), mde from stock mm solution in DMSO, ws pplied for t lest 1 min, during which dt cquisition cesed. On resumption, EGTA-AM ws continuously pplied until the end of the experiment. In mplified views shown in figures, spontneous IPSCs tht were not time-locked to the presynptic ction potentil were lnked efore verging for illustrtive purposes; events were only included within 1 ms of the ction potentil pek. In our nlysis, however, spontneous events were left unmodified. Two-photon lser-scnning microscopy. Fluorescence ws monitored with l-uilt 2PLSM using n Olympus upright microscope, ojective (6, 1. NA) nd oil-immersion condenser (1.4 NA). A Ti:spphire lser (Coherent) tuned to 81 nm ws used for excittion. Emitted green nd red fluorescence were simultneously collected y photomultiplier modules (H8224, Hmmtsu) in oth epi- nd trnsfluorescence pthwys using 565 nm dichroic nd 525/5 nd 62/6 ndpss filters (Chrom). Imges were cquired using ScnImge softwre (K. Svood, Howrd Hughes Medicl Institute, Jneli Frm Reserch Cmpus). Line scns were otined t 5 Hz. Cells were filled for minimum of 2 min efore cquisition to llow for dye equilirtion (xon vricosities <15 µm from the som). For vricosities >15 µm from the som, longer equilirtion times were used. Imging nlysis ws performed offline using ImgeJ (Ntionl Institutes of Helth) nd Axogrph. Fluorescence chnges were quntified s increses in green fluorescence normlized to red fluorescence ( G/R). Pek mplitude mesurements of suthreshold depolriztion evoked C 2+ trnsients were determined y verging -ms epoch efore ction potentil firing. Alternting trils in which no stimultion occurred were used to sutrct ckground fluorescence levels; the difference reflecting the pek response. The pek of ction potentil evoked C 2+ trnsients were determined from exponentil fits of the fluorescence decy following stimultion (mesured t t = ). Chnges in the pek mplitude of ction potentil evoked responses might e underestimted if the indictor were in the nonliner rnge. The C 2+ trnsients evoked y suthreshold depolriztion were usully very smll. For illustrtive purposes only, we integrted these responses to ccentute the rise in C 2+ from the seline noise during the suthreshold stimulus (Figs. 2 nd 3). This method ws not included in ny quntifiction. Sttisticl nlysis. Reported vlues re men ± s.e.m. Excel (Microsoft) nd InStt (GrphPd Softwre) were used for sttisticl nlysis using pired nd unpired t tests (two tiled). ANOVA with post hoc Tukey s tests were used when mking multiple comprisons. Differences were considered to e sttisticlly significnt with α vlues of P <.5. 49. Dodt, H.U., Eder, M., Schierloh, A. & Zieglgnserger, W. Infrred-guided lser stimultion of neurons in rin slices. Sci. STKE 12, 12 (22). 5. Re, J., Cooper, K., Gtes, P. & Wtsky, M. Low ccess resistnce perforted ptch recordings using mphotericin B. J. Neurosci. Methods 37, 15 26 (1991). doi:1.138/nn.2718 nture NEUROSCIENCE