Calcium threshold shift enables frequency-independentcontrol of plasticity by an instructive signalClaire Piochona,1, Heather K. Titleya,1, Dana H. Simmonsa, Giorgio Grassellia, Ype Elgersmab, and Christian Hansela,2

aDepartment of Neurobiology, University of Chicago, Chicago, IL 60637; and bDepartment of Neuroscience, Erasmus Medical Center, 3000 CA Rotterdam,The Netherlands

Edited by Masao Ito, RIKEN Brain Science Institute, Wako, Japan, and approved September 23, 2016 (received for review August 19, 2016)

At glutamatergic synapses, both long-term potentiation (LTP) andlong-term depression (LTD) can be induced at the same synapticactivation frequency. Instructive signals determine whether LTP orLTD is induced, by modulating local calcium transients. Synapsesmaintain the ability to potentiate or depress over a wide fre-quency range, but it remains unknown how calcium-controlledplasticity operates when frequency variations alone cause differ-ences in calcium amplitudes. We addressed this problem at cerebel-lar parallel fiber-Purkinje cell synapses, which can undergo LTD orLTP in response to 1-Hz and 100-Hz stimulation. We observed thathigh-frequency activation elicits larger spine calcium transients thanlow-frequency stimulation under all stimulus conditions, but,regardless of activation frequency, climbing fiber (CF) coactiva-tion provides an instructive signal that further enhances calciumtransients and promotes LTD. At both frequencies, bufferingcalcium prevents LTD induction and LTP results instead, identify-ing the enhanced calcium signal amplitude as the critical param-eter contributed by the instructive CF signal. These observationsshow that it is not absolute calcium amplitudes that determinewhether LTD or LTP is evoked but, instead, the LTD threshold slides,thus preserving the requirement for relatively larger calciumtransients for LTD than for LTP induction at any given stimulusfrequency. Cerebellar LTD depends on the activation of calcium/calmodulin-dependent kinase II (CaMKII). Using genetically modified(TT305/6VA and T305D) mice, we identified α-CaMKII inhibitionupon autophosphorylation at Thr305/306 as a molecular event un-derlying the threshold shift. This mechanism enables frequency-independent plasticity control by the instructive CF signal basedon relative, not absolute, calcium thresholds.

calcium/calmodulin-dependent kinase II | cerebellum | long-termdepression | long-term potentiation | Purkinje cell

Synaptic activation frequency is an important factor in theinduction of long-term potentiation (LTP) and long-term

depression (LTD). For example, it has been shown at Schaffercollateral-CA1 pyramidal cell synapses that application of 900pulses at 1–3 Hz causes LTD, whereas the same number ofpulses applied at 50 Hz causes LTP (1). However, LTP and LTDcan also be induced at the same stimulus frequency. This phe-nomenon has been demonstrated at hippocampal, neocortical,and cerebellar synapses, where potentiation and depressionmechanisms operate over a wide range of activation frequencies(2–7). In the neocortex and hippocampus, the level of post-synaptic depolarization determines whether LTP or LTD resultsfrom stimulation at a given frequency (2, 5). These voltage-de-pendent thresholds for LTP and LTD induction reflect thresh-olds in calcium signal amplitudes (3, 4, 8–11) that, whenmaintained for sufficiently long time periods (12), control syn-aptic plasticity in concert with distinct calcium sensors that arerestricted to local microenvironments (13, 14).At cerebellar parallel fiber (PF)-Purkinje cell synapses, both

LTP and LTD can be induced using 1-Hz and 100-Hz PF stim-ulation protocols, and at both frequencies, climbing fiber (CF)coactivation promotes LTD, whereas LTP results from PFstimulation alone (6, 7, 15, 16). CF coactivity leads to supralinear

spine calcium signaling (17), which helps to reach the calciumthreshold for LTD, which is higher than the threshold for LTP atthese synapses (6). It seems that for central synapses, there isa computational advantage to be able to undergo potentiationor depression regardless of activation frequency but under thecontrol of instructive signals, such as CF coactivity in cerebellarplasticity (18–20). Here, we address a fundamental problem thatarises from LTP and LTD induction under the control of in-structive signals over a wide frequency range: How is it possible tomaintain the essential role of calcium thresholds, and the role ofan instructive signal that helps to reach the higher threshold, whenthe calcium levels resulting from high-frequency stimulation arelikely in a higher range than the calcium levels resulting from low-frequency stimulation? The relevance of this problem is illustratedby the observation that at PF synapses, LTP-inducing 100-Hz PFbursts evoke larger calcium transients than paired single-pulse CFactivation, which promotes LTD (21). We thus investigated spinecalcium signaling at PF synapses under low- and high-frequencyLTD- and LTP-inducing conditions, as well as mechanisms thatmay shift the calcium sensitivity of the LTD pathway.

ResultsTo monitor spine calcium transients, we used confocal imagingin slices obtained from P21-75 mice. For the comparison ofcalcium levels that are reached under low- and high-frequencyLTD- and LTP-inducing conditions, we applied defined patternsof PF and CF activation, respectively, that were derived from theplasticity protocols described below. LTD results from PF + CFactivation at 1 Hz for 5 min, whereas LTP is evoked when thesame PF activation pattern is applied in isolation (6, 16). LTD

Significance

Instructive signals play an important role in synaptic plastic-ity and learning. For example, at cerebellar parallel fiber (PF)-Purkinje cell synapses, climbing fiber (CF) coactivation providesan instructive signal that promotes long-term depression (LTD)by amplifying spine calcium transients above a threshold levelthat, at these synapses, is higher than for LTP induction. Here,we show that the CF instructive signal maintains its controlover PF plasticity regardless of the PF synaptic activation fre-quency, which, on its own, alters spine calcium signaling. Wedemonstrate that high-frequency stimulation reduces the calciumsensitivity of LTD, resulting from inhibitory calcium/calmodulin-dependent kinase II autophosphorylation at Thr305/306. Wepropose that this regulatory mechanism causes a horizontalshift of the long-term potentiation/LTD cross-over point, mak-ing plasticity independent from absolute calcium amplitudes.

Author contributions: C.P., H.K.T., D.H.S., G.G., Y.E., and C.H. designed research; C.P., H.K.T.,D.H.S., and G.G. performed research; Y.E. contributed new reagents/analytic tools; C.P.,H.K.T., D.H.S., and G.G. analyzed data; and Y.E. and C.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1C.P. and H.K.T. contributed equally to this work.2To whom correspondence should be addressed. Email: chansel@bsd.uchicago.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613897113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613897113 PNAS | November 15, 2016 | vol. 113 | no. 46 | 13221–13226

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020

can also be induced with a train of eight PF stimuli (100 Hz)followed 120 ms after stimulus onset by single-pulse CF stimu-lation. This activation pattern is applied at 1 Hz for 5 min. Withthis protocol, too, omission of CF activation causes LTP instead(7). All stimulus protocols cause postsynaptically expressedforms of LTD/LTP (6, 7). In the following, we will use the terms“low-frequency protocol” and “high-frequency protocol” to referto the highest lead frequency within the PF stimulus patterns.Thus, 100-Hz PF burst stimulation is referred to as the high-frequency protocol, whereas single-pulse PF stimulation at 1 Hzis referred to as the low-frequency protocol. For the compari-son of calcium levels that are reached under the four activationconditions, we chose to break down the protocols into thestimulus patterns that are repeated at 1 Hz for 5 min. For thehigh-frequency protocol, this stimulus pattern is a 100-Hz PFburst (eight pulses), and for the low-frequency protocol, it is asingle PF pulse, which are paired/not paired with CF activation.Calcium transients evoked in spines on secondary or higher or-der branches of Purkinje cell dendrites were calculated as ΔG/R =(G(t) − G0)/R, where G is the calcium-sensitive fluorescence ofOregon Green 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA)-2 (OGB-2; 200 μM), G0 is the baselinesignal, and R is the calcium-insensitive fluorescence of Alexa 633(30 μM) (22). Calcium transients were monitored under all fourstimulation conditions (applied two to three times in randomizedorder, with 30-s intervals between stimuli) from the same spinethat was defined as the region of interest (ROI). The ROI wasselected based on the strength of calcium responses to synapticactivation (maximally responding spine). To obtain a calciumtransient measure, ΔG/R values were averaged over a period of200 ms after stimulus onset (17). Single-pulse PF stimulationresulted in small calcium signals (0.02 ± 0.02 ΔG/R; n = 11; Fig. 1A–D). Paired PF + CF activation resulted in significantly largercalcium transients (0.06 ± 0.01 ΔG/R; P = 0.001). Application ofthe 100-Hz PF burst evoked calcium signals that were aboutthreefold as large as the calcium signals obtained with paired PF +CF single-pulse stimulation (0.18 ± 0.02 ΔG/R; P = 0.00005).When this 100-Hz PF burst was followed after 120 ms by a singleCF pulse, the resulting calcium transient was further enhanced(0.24 ± 0.04 ΔG/R; P = 0.041). OGB-2 is a high-affinity calciumindicator (Kd = 485 nM), allowing for accurate measurement ofcalcium in the low-amplitude range. To obtain a better dynamicrange at high calcium amplitudes, we repeated these measurements

using the low-affinity (Kd = 1.8 μM) indicator Fluo-5F (300 μM).Single-pulse PF stimulation resulted in ΔG/R values in the noiserange (−0.01 ± 0.01 ΔG/R; n = 11; Fig. 1 E–H). However, with CFcoactivation, the signal was significantly enhanced (0.04 ± 0.01ΔG/R; P = 0.035). A 100-Hz PF burst stimulation resulted incalcium transients that were about 12-fold larger than the calciumtransients obtained with single-pulse PF + CF activation (0.46 ±0.15 ΔG/R; P = 0.013). When this 100-Hz burst was followed after120 ms by a single CF pulse, the calcium signal amplitude wasfurther enhanced (0.59 ± 0.19 ΔG/R; P = 0.039). With both OGB-2and Fluo-5F, calcium transients monitored in adjacent shaft areasshowed the same amplitude relationships as calcium transientsmeasured in the spines (Fig. S1). The results obtained with bothindicators show that 100-Hz PF bursts evoke larger calciumtransients than single pulses, and that CF coactivation furtherenhances calcium signals at both frequencies. To compare spinecalcium transients evoked by 100-Hz PF bursts with and withoutCF coactivation under conditions that are as remote as possiblefrom dye saturation, we went a step further and used the ultralowcalcium affinity (Kd = 22.0 μM) indicator OGB-5N (300 μM).These recordings confirmed our previous observations: paired100-Hz PF burst + CF activation caused spine calcium transients(0.32 ± 0.09 ΔG/R) that were significantly larger than spine cal-cium transients observed with 100-Hz PF burst stimulation alone(0.21 ± 0.05 ΔG/R; n = 9; P = 0.04338; Fig. S2).To examine whether larger calcium transients are required for

LTD than for LTP induction in the low-frequency range as wellas the high-frequency range, we applied both LTD protocolswhen the calcium chelator BAPTA was added to the pipettesaline. Under control conditions, paired PF + CF activation at1 Hz for 5 min caused PF-LTD (65.7 ± 6.3%; t = 31–35 min; n = 6;P = 0.0029; Fig. 2). In line with previous observations (6) appli-cation of the 1-Hz LTD protocol instead resulted in LTP (149.1 ±14.5%; n = 6; P = 0.0198; Fig. 2) when BAPTA (20 mM) waspresent in the pipette saline. Excitatory postsynaptic current(EPSC) amplitude changes observed in these groups differedsignificantly (P = 0.0051). Application of the 100-Hz PF burst-LTD protocol resulted under control conditions in PF-LTD(77.9 ± 3.8%; n = 6; P = 0.0008; Fig. 2). For these high-frequencystimulus experiments, we selected a lower BAPTA concen-tration (5 mM) than the concentrations that are typically usedto block 1-Hz LTD (10–40 mM) (6, 23, 24). The exact BAPTAconcentration was arbitrarily chosen, but the underlying idea

0.60.40.2

s

PF burst + CF PF burst PF + CF PF

80

60

40

20

0

G/R

(% o

f max

imum

)

2.01.51.00.50.0Time (s)

PF burst + CF PF burst PF+ CF PF

80

60

40

20

0

G/R

(% o

f max

imum

)

2.01.51.00.50.0Time (s)

PFburst + CF PFburst PF + CF PF

PF burst + CF PF burst PF + CF PFB

C G

D H

Fluo-5FOGB-2

0.8

0.6

0.4

0.2

0.0

G/R

PF

burs

t + C

FP

F bu

rst

PF

+ C

FP

F

**

*

*

0.3

0.2

0.1

0.0

G/R

PF

burs

t + C

FP

F bu

rst

PF

+ C

FP

F

***

**

**

F

EAFig. 1. Spine calcium transients evoked by LTP- andLTD-inducing PF and CF activation patterns. (A–D)OGB-2 measurements. (A, Left) Purkinje cell filledwith Alexa 633. (Scale bar: 20 μm.) (A, Center) En-larged view. The circle outlines the ROI. (Scale bar:1 μm.) (A, Right) Green fluorescence of OGB-2. Thecircle outlines the ROI. (Scale bar: 1 μm.) (B, Top)Electrophysiological responses to the followingstimuli: 100-Hz PF burst + CF, PF burst alone, single-pulse PF + CF, and PF pulse alone. (Scale bars: verti-cal, 20 mV; horizontal, 100 ms.) (B, Bottom)Simultaneously recorded calcium transients. (Scalebars: vertical, 0.1 δG/R; horizontal, 500 ms.) (C) Cal-cium transients averaged from all Purkinje cellrecordings (n = 11). Calcium signals are expressed asthe percentage of the peak amplitude in each re-cording. (D) Bar graph summarizing calcium signalamplitudes (ΔG/R; average over a 200-ms periodstarting at stimulus onset, n = 11). (E–H) Fluo-5Fmeasurements. (E, Left) Purkinje cell filled withAlexa 633. (Scale bar: 20 μm.) (E, Center) Enlargedview. The circle outlines the ROI. (Scale bar: 1 μm.)(E, Right) Green fluorescence of Fluo-5F. The circleoutlines the ROI. (Scale bar: 1 μm.) (F) Electrophysi-ological responses and calcium transients arrangedas in B. (Scale bars: Top, vertical, 20 mV; Top, horizontal, 100 ms; Bottom, vertical, 0.5 δG/R; Bottom, horizontal, 500 ms.) (G) Averaged calcium transients (n =11). (H) Bar graph summarizing signal amplitudes (n = 11). Error bars indicate SEM. **P < 0.01; *P < 0.05.

13222 | www.pnas.org/cgi/doi/10.1073/pnas.1613897113 Piochon et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020

of selecting a lower concentration was that moderate manipu-lation of the higher calcium signals observed with the high-frequency LTD protocol (Fig. 1) might be sufficient to evoke aswitch from LTD toward LTP, allowing for a qualitative as-sessment of the relative calcium dependencies of LTP andLTD. We indeed observed that LTP was induced instead ofLTD when the 100-Hz burst protocol was applied in the pres-ence of BAPTA (134.3 ± 12.2%; n = 7; P = 0.031; Fig. 2). EPSCchanges observed under these two conditions differed signifi-cantly (P = 0.0043).Calcium transients evoked by 100-Hz PF burst + CF activation

in the presence of BAPTA (5 mM) were significantly lowerthan in the absence of BAPTA (+BAPTA: 0.16 ± 0.06 ΔG/R,n = 8; −BAPTA: 0.59 ± 0.19 ΔG/R, n = 11; P = 0.0349; Fig. S3),while they did not differ statistically from calcium transientsevoked by LTP-inducing PF burst stimulation (−BAPTA: 0.46 ±0.15 ΔG/R; n = 11; P = 0.1471). Moreover, these transients weresignificantly higher than calcium signals evoked by the LTD-inducing single-pulse PF + CF activation (−BAPTA: 0.04 ±0.01 ΔG/R; n = 11; P = 0.0093; Fig. S3). Similarly, in thepresence of 20 mM BAPTA, single-pulse PF + CF activa-tion caused significantly lower calcium transients than withoutBAPTA (+BAPTA: 0.01 ± 0.002 ΔG/R, n = 8; −BAPTA: 0.04 ±0.01 ΔG/R, n = 11; P = 0.0349; Fig. S3). These calcium transientswere significantly higher than the calcium transients reached withLTP-inducing single-pulse PF stimulation (−BAPTA; −0.01 ± 0.01ΔG/R; n = 11; P = 0.0147), but it should be noted that as a resultof the high Kd of Fluo-5F, these latter stimulus conditions do notreliably evoke transients above noise levels (traces in Fig. S3). Toobtain an approximation of calcium amplitudes reached in spinesduring these stimulus conditions, we calculated [Ca2+]i valuesbased on the Fluo-5F G/R measures. The calcium signals reachedpeak values of about 0.4 μM for single-pulse PF + CF stimulation,2.8 μM for 100-Hz PF burst stimulation, and 7.1 μM for 100-HzPF burst + CF activation (consistent measurements of calciumlevels; see ref. 17). In the presence of BAPTA (5 mM), peak cal-cium levels evoked by 100-Hz PF burst + CF activation were re-duced to about 2.2 μM. No attempt was made to calculate [Ca2+]i

for single-pulse PF stimulation or single-pulse PF + CF stimu-lation in the presence of BAPTA because these signals were tooclose to noise levels in the Fluo-5F recordings.Together, these observations suggest that regardless of synaptic

activation frequency, additional calcium influx contributed by theinstructive CF signal is required to induce LTD. Hence, while theabsolute calcium levels reached in the high-frequency range aresignificantly higher than in the low-frequency range, the relativecalcium thresholds are preserved at both stimulus frequencies.The transition from absolute to relative calcium thresholds re-quires that high-frequency stimulation lower the calcium sensi-tivity of the LTD induction process, in effect shifting the LTDthreshold toward a higher value.To identify the mechanism behind this threshold shift, we

postulated two requirements that a candidate signaling pathwayneeds to fulfill: (i) It requires a calcium sensor to detect thehigher calcium levels resulting from high-frequency activation,and (ii) it needs to tip the balance from a kinase-dominatedLTD pathway toward a phosphatase-activated LTP pathway atPF synapses (25, reviewed in ref. 26). This description profilematches the inhibitory autophosphorylation that has been de-scribed for the alpha subunit of calcium/calmodulin-dependentkinase II (α-CaMKII) at Thr305 and Thr306, which lowers theaffinity of CaMKII for CaM, and thus reduces Ca/CaM-medi-ated activation (27). β-CaMKII is expressed in Purkinje cells aswell (28), but the inhibitory autophosphorylation has only beenexamined in α-CaMKII and has been shown to have a dominantnegative effect on the holoenzyme (27). Both α-CaMKII andβ-CaMKII are essential for proper LTD induction (28, 29).CaMKII promotes LTD by suppressing the activity of proteinphosphatase 2A through negative regulation of phosphodies-terase 1 and subsequent disinhibition of a cGMP/protein ki-nase G pathway (30). Negative regulation of CaMKII by Thr305/306 autophosphorylation requires prior calcium/calmodulin-medi-ated activation of CaMKII and subsequent calmodulin dissociation(31). Moreover, it has been shown that Thr305/306 phosphoryla-tion, triggered by 10-Hz priming, reduces hippocampal LTP (32),whereas genetic manipulation of the Thr305/306 phosphorylationsite lowers the LTP threshold and prevents priming (27, 32). Thus,CaMKII inhibitory autophosphorylation depends on previous cal-cium signaling and directly affects the kinase/phosphatase balance,thus fulfilling the two requirements outlined above. To test whetherThr305/306 phosphorylation is indeed involved in reducing theprobability for LTD induction at high stimulus frequencies,we examined whether LTP is still induced by the 100-Hz PFburst protocol, or whether LTD is restored, in α-CaMKII mutantmice in which Thr305 and Thr306 are substituted by the non-phosphorylatable amino acids valine and alanine, respectively [re-ferred to as TT305/6VA mice (27)]. In wild-type (WT) littermates,application of the 100-Hz LTP protocol potentiated EPSC ampli-tudes (131.8 ± 12.4%; t = 36–40 min; n = 12; P = 0.026; Fig. 3B). Incontrast, application of the same protocol resulted in LTD inTT305/6VA mice (81.5 ± 8.4%; n = 15; P = 0.044; Fig. 3B). Thedifference between these groups was significant (P = 0.0021). Wenext examined whether the same switch toward LTD occurs whenthe 1-Hz LTP protocol is applied. In WT mice, single-pulse PF ac-tivation potentiated EPSC amplitudes (122.3 ± 4.3%; t = 36–40 min;n = 7; P = 0.002; Fig. 3A). In TT305/6VA mice, LTP wasobserved as well (124.9 ± 7.7%; n = 8; P = 0.007; group compari-son: P = 0.42; Fig. 3A). Likewise, LTD was unaffected in TT305/6VA mice, whether it was triggered by the 1-Hz protocol (WT:72.8 ± 4.4%, t = 36–40 min, n = 8, P = 0.0005; TT305/6VA: 67.6 ±3.0%, n = 5, P = 0.0004; group comparison: P = 0.38; Fig. S4A) orthe 100-Hz protocol (WT: 71.5 ± 5.0%, n = 6, P = 0.002; TT305/6VA: 70.2 ± 5.3%, n = 5, P = 0.005; group comparison: P = 0.93;Fig. S4B). We also tested LTD/LTP protocols in T305D mice, inwhich Thr305 is replaced by a negatively charged Asp, which servesas a phosphomimetic resembling persistent Thr305 phosphorylationand preventing Ca/CaM binding (27). In slices prepared from T305Dmice, application of the 1-Hz LTD protocol and the 100-Hz LTDprotocol, respectively, resulted in LTP instead (1 Hz: 127.9 ± 10.2%,

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

3530252015105Time (min)

**

Control (n=6)BAPTA (n=6)

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

3530252015105Time (min)

**

Control (n=6) BAPTA (n=7) -100

-50

0

50

100

% c

hang

e

Con

trol

BA

PTA

-100

-50

0

50

100

% c

hang

e

Con

trol

BA

PTA20 ms

100 pA

Baseline Post-tetanization

1 Hz LTD protocol

100 Hz LTD protocol

Control

BAPTA

Control

BAPTA

A

B

20 ms 100 pA

Fig. 2. In the low- and high-frequency activation range, BAPTA reverses LTDtoward LTP. (A) Application of the 1-Hz LTD protocol results in LTP whenBAPTA (20 mM) is added to the pipette saline. (Left) Time graph showing thatLTD is induced under control conditions (n = 6), but LTP is induced in thepresence of BAPTA (n = 6). (Center) Traces show EPSCs before and after tet-anization. (Right) Individual cell data (t = 31–35 min). (B) Application of the100-Hz LTD protocol results in LTP when BAPTA (5 mM) is added to the pipettesaline. (Left) Time graph showing LTD under control conditions (n = 6) and LTPin the presence of BAPTA (n = 7). (Center) Typical traces. (Right) Individual celldata. Arrows indicate tetanization. Error bars indicate SEM. **P < 0.01.

Piochon et al. PNAS | November 15, 2016 | vol. 113 | no. 46 | 13223

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020

t = 36–40 min, n = 6, P = 0.041; 100 Hz: 138.3 ± 12.4%, n = 7, P =0.021; Fig. 4). These results are significantly different from therespective LTD measures in WT controls (same as reported for theTT305/6VA mice; 1-Hz group: P = 0.0019; 100-Hz group: P =0.0027). LTP induction was not affected in T305Dmice [1-Hz LTP:136.3 ± 12.3%, n = 5, P = 0.018 compared with WT (P = 0.123);100-Hz LTP: 134.0 ± 9.6, n = 6, P = 0.017 compared with WT (P =0.93); Fig. S5]. These results suggest that high-frequency PF activationcauses CaMKII phosphorylation at Thr305/306, which blocks LTDinduction despite calcium levels above the threshold for 1-Hz LTD.CF coactivation prevents inhibitory autophosphorylation andrestores LTD. However, LTD is not induced when CaMKII isinactivated in a phosphomimetic mutant, which demonstrates theexistence of one (rather than multiple) threshold(s) for LTD in-duction that is regulated by the availability of CaMKII for activation.

DiscussionThe observation that CF signaling promotes LTD regardless oflarge variations in calcium signaling at different activation fre-quencies sheds light on the importance of instructive signals inbidirectional plasticity and the way they operate. Our data showthat the LTD threshold slides, enabling the instructive CF signalto operate over a wide frequency range by providing, at a givenfrequency, a spine calcium transient that is higher than in theabsence of this signal (Fig. 5). The observation that the differ-ence in the calcium signal amplitude caused by the switch inactivation frequency is larger than the difference resulting fromCF coactivation (Fig. 1) leads to the question of whether CFactivity indeed promotes LTD by amplifying calcium transients,or whether it contributes other signaling factors that trigger theswitch toward LTD. For example, CF activity results in the re-lease of corticotropin-releasing factor, which promotes LTD atPF and CF synapses by activation of PKC (33, 34). In addition, inthe mature cerebellum, CF stimulation recruits a specific calciumsource that is not available at PF synapses, postsynaptically lo-cated NMDA receptors (7, 35, 36). It is conceivable that theseCF-specific signaling components contribute to LTD. However,the BAPTA experiments demonstrate that a critical factor in theswitch from LTP to LTD is the local calcium signal amplitude,

which is significantly enhanced upon CF coactivation. A criticalprediction of this conclusion is that strong PF activation caninduce LTD in the absence of CF coactivation. This replacementeffect has indeed been demonstrated (37, 38, also ref. 39), sug-gesting that strong calcium influx, regardless of synaptic origin,promotes LTD but that CF activity facilitates the inductionprocess by amplifying calcium transients.The concept of synaptic modification under control of post-

synaptic activity thresholds was introduced in the Bienenstock–Cooper–Munro (BCM) theory, which was originally developedas a model for developmental plasticity in the visual cortex (8). Akey assumption of the BCM theory is that the modificationthreshold θM, which marks the level of postsynaptic activity atwhich the polarity of synaptic plasticity changes from depressionto potentiation, is not fixed but, instead, may slide as a functionof synaptic activation history. Our data show that in the low-frequency stimulus range, the [Ca2+]i threshold for LTD in-duction is ≤0.4 μM, whereas in the high-frequency range, it is>3 μM. These values fall into the range of [Ca2+]i amplitudesthat have been reported as triggering LTD induction in the ab-sence of synaptic activation (40, 41). Thus, these data demon-strate that the shift in the LTD threshold upon high-frequencysynaptic activation is a process that prevents the LTD that wouldotherwise take place at the same [Ca2+]i levels. Previous reportshave assigned a key role for such threshold shifts to CaMKII. Ithas been shown that CaMKII autophosphorylation at Thr286shifts the threshold for hippocampal LTP to the right, thus fa-cilitating LTD at potentiated synapses (42, also ref. 43). More-over, it has been demonstrated that priming-induced Thr305/306phosphorylation prevents subsequent LTP induction (32). Inthese examples, threshold shifts follow prior synaptic activity;thus, they constitute forms of metaplasticity as predicted by theBCM rule. The threshold shift described here does not constitutea classic form of metaplasticity [i.e., “plasticity of synaptic plas-ticity” (44)], because the LTD threshold does not shift as aconsequence of activity that occurred before stimulus protocolapplication and separated by an activity-free interval from it but,instead, results exclusively from sensing enhanced calcium levels

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

40302010Time (min)

**

Wild-type (n=6) T305D (n=7)

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

40302010Time (min)

**

Wild-type (n=8) T305D (n=6)

-100

-50

0

50

100

% c

hang

e

Wild

-type

T305

D

-100

-50

0

50

100

% c

hang

e

Wild

-type

T305

D

Wild-type

T305D

Wild-type

T305D

1 Hz LTD protocol

100 Hz LTD protocol

A

B

20 ms 100 pA

Baseline Post-tetanization

20 ms 100 pA

Fig. 4. LTD is prevented in T305D mice, in which Thr305 replacement by Aspmimics constitutive inhibitory CaMKII autophosphorylation. (A, Left) Timegraph showing that 1-Hz PF + CF activation induces LTD inWTmice (n = 8), butLTP in T305D mice (n = 6). (A, Center) Typical traces show EPSCs before andafter application of the 1-Hz LTD protocol in WT mice (Top) and T305D mice(Bottom). (A, Right) Individual cell data (t = 36–40 min). (B, Left) Time graphshowing that 100-Hz PF burst + CF activation induces LTD in WT mice (n = 6),but LTP in T305D mice (n = 7). (B, Center) Typical traces show EPSCs before andafter application of the 100-Hz LTD protocol in WT mice (Top) and T305D mice(Bottom). (B, Right) Individual cell data. Error bars indicate SEM. **P < 0.01.

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

40302010Time (min)

Wild-type (n=7) TT305/6VA (n=8)

200

150

100

50

0

PF-

EP

SC

(% o

f bas

elin

e)

40302010Time (min)

**

Wild-type (n=12) TT305/6VA (n=15)

-100

-50

0

50

100

% c

hang

e

Wild

-type

TT30

5/6V

A

-100

-50

0

50

100

% c

hang

e

Wild

-type

TT30

5/6V

A

20 ms 100 pA

0.240.20

s

20 ms 100 pA

Baseline Post-tetanization

0.240.20

s

Wild-type

TT305/6VA

Wild-type

TT305/6VA

1 Hz LTP protocol

100 Hz LTP protocol

A

B

Fig. 3. LTP induced by 100-Hz PF burst stimulation, but not 1-Hz PF stim-ulation, is prevented in mice that express CaMKII and cannot undergoThr305/306 phosphorylation. (A, Left) Time graph showing that 1-Hz PFstimulation induces LTP in WT mice (n = 7) and TT305/6VA mice (n = 8). Thearrow indicates time of tetanization. (A, Center) Typical traces show EPSCsbefore and after tetanization in WT mice (Top) and TT305/6VA mice (Bot-tom). (A, Right) Individual cell data (t = 36–40 min). (B, Left) Time graphshowing that 100-Hz PF burst stimulation induces LTP in WT mice (n = 12),but LTD in TT305/6VA mice (n = 15). (B, Center) Typical traces show EPSCsbefore and after tetanization in WT (Top) and TT305/6VA mice (Bottom). (B,Right) Individual cell data. Error bars indicate SEM. **P < 0.01.

13224 | www.pnas.org/cgi/doi/10.1073/pnas.1613897113 Piochon et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020

during stimulus application. Our finding that application of thehigh-frequency LTP protocol in TT305/6VA mice induces LTDinstead, whereas the same polarity switch is not observed whenusing the low-frequency LTP protocol, suggests that inhibitoryautophosphorylation of CaMKII selectively occurs upon high-frequency PF stimulation but is overcome by CF coactivation. Notmuch is known about the biochemical requirements for Thr305/306 autophosphorylation or its prevention. It has been shownthat Thr305/306 autophosphorylation requires Ca/CaM binding toCaMKII and subsequent dissociation (31). A possible, but notexperimentally tested, scenario is that repeated 100-Hz PF burstsprovide the calcium influx required for Ca/CaM binding to CaMKII,but that during the pauses between the bursts, which last almost 1 s,CaM dissociates from CaMKII, allowing inhibitory phosphorylationto take place. Single PF pulses (1-Hz LTP protocol) do not seem toprovide sufficient calcium to trigger Ca/CaM binding to CaMKII.Conversely, the larger calcium transient that results from paired100-Hz PF burst + CF activation might provide sufficient calciumto prevent CaM dissociation, thus allowing CaMKII to remain inthe activated state.The frequency-dependent calcium threshold shift described here

might also explain why it has been difficult to confirm the existenceof distinct calcium thresholds for LTD and LTP induction usingphotolysis of caged calcium compounds (40, 45). In the absence ofsynaptic activity, the thresholds might assume different, possiblyoverlapping, values, and additional parameters, such as the in-volvement of specific calcium sources/sensors, might gain impor-tance. The latter possibility has been demonstrated in corticalpyramidal cells, in which larger calcium transients are needed forLTP than for LTD induction, but activation of metabotropic glu-tamate receptors provides a switch for LTD. Blockade of this switchallows for LTP induction if the calcium signals reach the higherLTP threshold (14). An important aspect of calcium amplitudethresholds is that they can vary with the duration of calcium ex-posure. Using calcium uncaging experiments, for example, it hasbeen shown that lower calcium amplitudes are sufficient for cere-bellar LTD induction if they are presented for longer periods oftime (40). Note that our data do not contradict this “leaky in-tegrator” model. Rather, we kept the overall duration of the teta-nization period constant (all stimulus patterns were applied at1 Hz for 5 min) to study the shift in LTP/LTD induction proba-bilities that specifically results from a variation in the synaptic

activation frequency alone. A consequence of calcium thresholdsthat shift depending on stimulus conditions, such as variations induration or frequency, is that threshold values do not generalize,but only apply to very specific activation conditions. For this reason,we only report peak [Ca2+]i values for protocol-typical individualstimuli (Fig. 5), but otherwise describe relations between thresholdamplitudes. Our findings have important implications for the con-trol of activity-dependent synaptic plasticity by dendritic calciumtransients. The study identifies a fundamental problem that syn-apses have to solve when enabling LTD and LTP under control ofan instructive signal, but independent of the induction frequency:Spine calcium transients reach different amplitudes at differentactivation frequencies. At PF-Purkinje cell synapses, this problem issolved by a shift of the LTD threshold upon Thr305/306 phos-phorylation of CaMKII, allowing for LTD/LTP induction inde-pendent of absolute calcium levels, but under control of relativecalcium thresholds.

Materials and MethodsAnimals. All procedures were performed in accordance with the guidelines ofthe University of Chicago’s Animal Care and Use Committee. Experimentswere performed using P21-75 mice (C57BL/6). In some experiments, we usedP21-75 TT305/6VA or T305D mutant mice and WT littermate controls, whichare also in a congenic C57BL/6 background (27).

Slice Preparation. Animals were anesthetized with isoflurane and de-capitated. The cerebellar vermis was removed and cooled in artificial cere-brospinal fluid (ACSF) containing 124mMNaCl, 5 mMKCl, 1.25 mMNa2HPO4,2 mM CaCl2, 2 mM MgSO4, 26 mM NaHCO3, and 10 mM D-glucose, bubbledwith 95% (vol%) O2 and 5% (vol%) CO2. Parasagittal slices of the cerebellarvermis (200 μm) were prepared using a Leica VT-1000S vibratome, and weresubsequently kept for at least 1 h at room temperature in oxygenated ACSF.Throughout recording, slices were perfused with ACSF that was supple-mented with picrotoxin (100 μM) to block GABAA receptors.

Whole-Cell Patch-Clamp Recordings. Patch-clamp recordings from the Purkinjecell soma were performed at room temperature using an EPC-10 amplifier(HEKA Electronics). Currents were filtered at 3 kHz, digitized at 25 kHz, andacquired using Patchmaster software (HEKA Electronics). Patch pipettes (2–5 MΩ)were filled with a solution containing 9 mM KCl, 10 mM KOH, 120 mMK-gluconate, 3.48 mM MgCl2, 10 mM Hepes, 4 mM NaCl, 4 mM Na2ATP,0.4 mM Na3GTP, and 17.5 mM sucrose (osmolarity: 295–305 mmol/kg,pH 7.25). In two groups of experiments, 5 and 20 mM BAPTA (tetrapotas-sium salt) was added to the internal saline. In the BAPTA experiments, CaCl2was added (2.5 and 10 mM) to maintain the resting calcium concentration(46). To evoke synaptic responses, PFs and CFs were activated using glass elec-trodes filled with ACSF. In the LTD and LTP experiments (Figs. 2–4 and Figs.S3–S5), test responses were recorded in voltage-clamp mode before andafter application of the induction protocol at a frequency of 0.05 Hz. Tet-anization was applied in current-clamp mode. Series and input resistanceswere monitored by applying hyperpolarizing voltage steps (−10 mV) at theend of each sweep. Recordings were excluded if series or input resistancesvaried by >15% over the course of the experiments.

Confocal Calcium Imaging. Calcium transients were monitored using a ZeissLSM 5 Exciter confocal microscope equipped with a 63× Apochromat ob-jective (Carl Zeiss MicroImaging). Calcium signals were calculated as ΔG/R =(G(t) − G0)/R (22), where G is the calcium-sensitive fluorescence (G0 = base-line signal) of either OGB-2 (200 μM), Fluo-5F (300 μM), or OGB-5N (300 μM)and R is the calcium-insensitive fluorescence of Alexa 633 (30 μM). UsingG/R values from the Fluo-5F measurements, peak [Ca2+]i was estimated bycalculating (22)

�Ca2+

�i =KdðG=R−G=RminÞ=ðG=Rmax −G=RÞ,

which is analogous to the equation (47)

�Ca2+

�i =KdðR−RminÞ=ðRmax −RÞ,

where R is the ratio of the fluorescence intensities F1 and F2 of a dual-wavelength indicator (e.g., fura-2) at excitation wavelengths λ1 and λ2, re-spectively. For Fluo-5F, G/Rmax (1.57) was measured in dye-filled Purkinjecells. To obtain maximal calcium signal amplitudes, the CF input was teta-nized at 100 Hz in ACSF containing 4 mM CaCl2. G/Rmin (0.0026) was

A B

Fig. 5. Sliding plasticity thresholds and role of CaMKII inhibitory auto-phosphorylation. (A) Schematic presents a model of the relationship be-tween calcium amplitudes and LTD/LTP as assessed in this study (the dashedlines indicate that possible LTP thresholds were not investigated). Thenumbers below show approximations of peak [Ca2+]i values, which werecalculated from ΔG/R measures recorded at each stimulus condition. Notethat these peak values were determined from individual protocol-typicalstimuli, and not from complete stimulus trains (Fig. S6). [Ca2+]i values are notpresented for the low-amplitude signals (N.D.), because no reliable measuresabove noise could be obtained with the low-affinity indicator Fluo-5F.(B) Diagram showing the role of CaMKII in LTD induction. CaMKII indirectlypromotes LTD (dashed arrow) by negative regulation of phosphodiesterase1 (PDE1), and the resulting facilitation of a cGMP/protein kinase G (PKG)cascade, which ultimately removes a blockade of LTD induction pathways byprotein phosphatase 2A (PP2A) (also ref. 30). Inhibitory autophosphorylationof CaMKII at Thr305/306 may disable this negative regulation of PP2A.

Piochon et al. PNAS | November 15, 2016 | vol. 113 | no. 46 | 13225

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020

determined in cuvettes using a solution containing 0 mM [Ca2+], supplementedwith 10 mM K2EGTA. The Kd values for all dyes were determined using cuvettemeasurements of solutions prepared from a calcium calibration buffer kit(Molecular Probes). The green fluorescence G resulted from excitation at 488 nmusing an argon laser. The red fluorescence R resulted from excitation at 633 nmusing a HeNe laser (both from Lasos Lasertechnik). Purkinje cells were loadedwith the dyes through diffusion from the patch pipette. The experiments wereperformed at room temperature andwere initiated after the fluorescence at theselected dendritic ROI reached a steady-state level, which typically required≥30 min. In each recording, the ROI was the spine on a secondary (or higherorder) dendritic branch in the field of view close to the stimulus electrode thatresponded maximally to synaptic activation. The maximally responding spinewas selected to ensure comparability between the calcium measures in differentneurons. The strategy to focus on protocol-typical stimulus units, rather thanmeasuring calcium levels during the entire tetanization period (5 min), waschosen to be able to test all four stimulus conditions in the same Purkinje cell.This strategy would not be possible with the application of complete protocols,which trigger either LTD or LTP, and would thus affect subsequent measures.This approach is validated in control experiments (Fluo-5F) that show there isno calcium build-up between stimuli over the course of 5-min periods of

tetanization, not even when the high-frequency LTD protocol is applied (Fig. S6).Image acquisition was restricted to the first 2 s of each beginning minute ofongoing stimulation to reduce the amount of phototoxicity. This strategyallowed us to monitor baseline and peak calcium levels during tetanizationwhile minimizing transient calcium build-up that may result from prolongedlight exposure [compare with studies that focused on capturing the completecalcium profile during ongoing tetanization (48, 49)]. Our recordings show thatunder these imaging conditions, baseline calcium levels do not plateau.

Data Analysis. Data were analyzed using Patchmaster software (HEKA Elec-tronics) and Igor Pro software (Wavemetrics). Imaging data were analyzedusing ZEN software (Carl Zeiss MicroImaging). Statistical significance wasdetermined by using the paired Student’s t test (within-group comparison ofpaired events) and the Mann–Whitney U test (between-group comparison),when appropriate. All data are shown as mean ± SEM.

ACKNOWLEDGMENTS. We thank N. Brunel, S. Dieudonné, and J. MacLeanfor helpful discussions and P. Vezina and Q. Wang for technical support andadvice. This work was supported by National Institute of Neurological Dis-orders and Stroke Grant NS062771 (to C.H.).

1. Dudek SM, Bear MF (1992) Homosynaptic long-term depression in area CA1 of hip-pocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad SciUSA 89(10):4363–4367.

2. Artola A, Bröcher S, Singer W (1990) Different voltage-dependent thresholds for in-ducing long-term depression and long-term potentiation in slices of rat visual cortex.Nature 347(6288):69–72.

3. Mulkey RM, Malenka RC (1992) Mechanisms underlying induction of homosynapticlong-term depression in area CA1 of the hippocampus. Neuron 9(5):967–975.

4. Cummings JA, Mulkey RM, Nicoll RA, Malenka RC (1996) Ca2+ signaling requirementsfor long-term depression in the hippocampus. Neuron 16(4):825–833.

5. Ngezahayo A, Schachner M, Artola A (2000) Synaptic activity modulates the inductionof bidirectional synaptic changes in adult mouse hippocampus. J Neurosci 20(7):2451–2458.

6. Coesmans M, Weber JT, De Zeeuw CI, Hansel C (2004) Bidirectional parallel fiberplasticity in the cerebellum under climbing fiber control. Neuron 44(4):691–700.

7. Piochon C, Levenes C, Ohtsuki G, Hansel C (2010) Purkinje cell NMDA receptors assumea key role in synaptic gain control in the mature cerebellum. J Neurosci 30(45):15330–15335.

8. Bienenstock EL, Cooper LN, Munro PW (1982) Theory for the development of neuronselectivity: Orientation specificity and binocular interaction in visual cortex. J Neurosci2(1):32–48.

9. Bear MF, Cooper LN, Ebner FF (1987) A physiological basis for a theory of synapsemodification. Science 237(4810):42–48.

10. Hansel C, Artola A, Singer W (1997) Relation between dendritic Ca2+ levels andthe polarity of synaptic long-term modifications in rat visual cortex neurons. Eur JNeurosci 9(11):2309–2322.

11. Graupner M, Brunel N (2012) Calcium-based plasticity model explains sensitivity ofsynaptic changes to spike pattern, rate, and dendritic location. Proc Natl Acad Sci USA109(10):3991–3996.

12. Sabatini BL, Oertner TG, Svoboda K (2002) The life cycle of Ca(2+) ions in dendriticspines. Neuron 33(3):439–452.

13. Franks KM, Sejnowski TJ (2002) Complexity of calcium signaling in synaptic spines.BioEssays 24(12):1130–1144.

14. Nevian T, Sakmann B (2006) Spine Ca2+ signaling in spike-timing-dependent plas-ticity. J Neurosci 26(43):11001–11013.

15. Lev-Ram V, Wong ST, Storm DR, Tsien RY (2002) A new form of cerebellar long-termpotentiation is postsynaptic and depends on nitric oxide but not cAMP. Proc NatlAcad Sci USA 99(12):8389–8393.

16. Schonewille M, et al. (2010) Purkinje cell-specific knockout of the protein phospha-tase PP2B impairs potentiation and cerebellar motor learning. Neuron 67(4):618–628.

17. Wang SS-H, Denk W, Häusser M (2000) Coincidence detection in single dendriticspines mediated by calcium release. Nat Neurosci 3(12):1266–1273.

18. Ito M, Kano M (1982) Long-lasting depression of parallel fiber-Purkinje cell trans-mission induced by conjunctive stimulation of parallel fibers and climbing fibers in thecerebellar cortex. Neurosci Lett 33(3):253–258.

19. Ito M, Sakurai M, Tongroach P (1982) Climbing fibre induced depression of bothmossy fibre responsiveness and glutamate sensitivity of cerebellar Purkinje cells.J Physiol 324:113–134.

20. Piochon C, Kruskal P, Maclean J, Hansel C (2013) Non-Hebbian spike-timing-dependentplasticity in cerebellar circuits. Front Neural Circuits 6:124.

21. Canepari M, Vogt KE (2008) Dendritic spike saturation of endogenous calcium bufferand induction of postsynaptic cerebellar LTP. PLoS One 3(12):e4011.

22. Yasuda R, et al. (2004) Imaging calcium concentration dynamics in small neuronalcompartments. Sci STKE 2004(219):pl5.

23. Konnerth A, Dreessen J, Augustine GJ (1992) Brief dendritic calcium signals initiatelong-lasting synaptic depression in cerebellar Purkinje cells. Proc Natl Acad Sci USA89(15):7051–7055.

24. Safo PK, Regehr WG (2005) Endocannabinoids control the induction of cerebellarLTD. Neuron 48(4):647–659.

25. Belmeguenai A, Hansel C (2005) A role for protein phosphatases 1, 2A, and 2B incerebellar long-term potentiation. J Neurosci 25(46):10768–10772.

26. Jörntell H, Hansel C (2006) Synaptic memories upside down: Bidirectional plasticity atcerebellar parallel fiber-Purkinje cell synapses. Neuron 52(2):227–238.

27. Elgersma Y, et al. (2002) Inhibitory autophosphorylation of CaMKII controls PSD as-sociation, plasticity, and learning. Neuron 36(3):493–505.

28. Hansel C, et al. (2006) alphaCaMKII is essential for cerebellar LTD and motor learning.Neuron 51(6):835–843.

29. van Woerden GM, et al. (2009) betaCaMKII controls the direction of plasticity atparallel fiber-Purkinje cell synapses. Nat Neurosci 12(7):823–825.

30. Kawaguchi SY, Hirano T (2013) Gating of long-term depression by Ca2+/calmodulin-dependent protein kinase II through enhanced cGMP signalling in cerebellar Purkinjecells. J Physiol 591(7):1707–1730.

31. Coultrap SJ, Bayer KU (2012) CaMKII regulation in information processing and stor-age. Trends Neurosci 35(10):607–618.

32. Zhang L, et al. (2005) Hippocampal synaptic metaplasticity requires inhibitoryautophosphorylation of Ca2+/calmodulin-dependent kinase II. J Neurosci 25(33):7697–7707.

33. Miyata M, Okada D, Hashimoto K, Kano M, Ito M (1999) Corticotropin-releasingfactor plays a permissive role in cerebellar long-term depression. Neuron 22(4):763–775.

34. Schmolesky MT, De Ruiter MM, De Zeeuw CI, Hansel C (2007) The neuropeptidecorticotropin-releasing factor regulates excitatory transmission and plasticity at theclimbing fibre-Purkinje cell synapse. Eur J Neurosci 25(5):1460–1466.

35. Piochon C, et al. (2007) NMDA receptor contribution to the climbing fiber response inthe adult mouse Purkinje cell. J Neurosci 27(40):10797–10809.

36. Renzi M, Farrant M, Cull-Candy SG (2007) Climbing-fibre activation of NMDA recep-tors in Purkinje cells of adult mice. J Physiol 585(Pt 1):91–101.

37. Hartell NA (1996) Strong activation of parallel fibers produces localized calciumtransients and a form of LTD that spreads to distant synapses. Neuron 16(3):601–610.

38. Han VZ, Zhang Y, Bell CC, Hansel C (2007) Synaptic plasticity and calcium signalingin Purkinje cells of the central cerebellar lobes of mormyrid fish. J Neurosci 27(49):13499–13512.

39. Ke MC, Guo CC, Raymond JL (2009) Elimination of climbing fiber instructive signalsduring motor learning. Nat Neurosci 12(9):1171–1179.

40. Tanaka K, et al. (2007) Ca2+ requirements for cerebellar long-term synaptic de-pression: Role for a postsynaptic leaky integrator. Neuron 54(5):787–800.

41. Nakamura Y, Hirano T (2016) Intracellular Ca(2+) thresholds for induction of excit-atory long-term depression and inhibitory long-term potentiation in a cerebellarPurkinje neuron. Biochem Biophys Res Commun 469(4):803–808.

42. Mayford M, Wang J, Kandel ER, O’Dell TJ (1995) CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP.Cell 81(6):891–904.

43. Bear MF (1995) Mechanism for a sliding synaptic modification threshold. Neuron15(1):1–4.

44. Abraham WC, Bear MF (1996) Metaplasticity: The plasticity of synaptic plasticity.Trends Neurosci 19(4):126–130.

45. Neveu D, Zucker RS (1996) Postsynaptic levels of [Ca2+]i needed to trigger LTD andLTP. Neuron 16(3):619–629.

46. Dzubay JA, Otis TS (2002) Climbing fiber activation of metabotropic glutamate re-ceptors on cerebellar purkinje neurons. Neuron 36(6):1159–1167.

47. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators withgreatly improved fluorescence properties. J Biol Chem 260(6):3440–3450.

48. Eilers J, Takechi H, Finch EA, Augustine GJ, Konnerth A (1997) Local dendritic Ca2+signaling induces cerebellar long-term depression. Learn Mem 4(1):159–168.

49. Schmidt H, Arendt O, Eilers J (2012) Diffusion and extrusion shape standing calciumgradients during ongoing parallel fiber activity in dendrites of Purkinje neurons.Cerebellum 11(3):694–705.

13226 | www.pnas.org/cgi/doi/10.1073/pnas.1613897113 Piochon et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

3, 2

020