supplementary material nmda receptor … (stdp) was induced by pairing each presynaptic stimulation...

1

SUPPLEMENTARY MATERIAL

NMDA receptor-dependent metaplasticity at hippocampal mossy fiber synapses

Nelson Rebola, Christophe Blanchet, Mario Carta, Frederic Lanore, Christophe Mulle.

Supplementary Figure 1 – NMDA/AMPA ratio is smaller at Mf-synapses than at A/C synapses. (A,B) Average values and representative traces of EPSCs recorded at

negative (–70mV) and positive potentials (+30 mV, in the presence of NBQX)

illustrating the marked difference in NMDA/AMPA ratios at A/C and Mf synapses. Error

bars represent s.e.m.

Nature Neuroscience: doi:10.1038/nn.2809

2

Supplementary Figure 2 – LTP of NMDARs affects EPSP time course and

facilitates spike transfer at hippocampal Mf-CA3 synapses. (A) A train of Mf-

stimulation induces LTP of NMDA-EPSPs recorded in the current-clamp mode with 1.3

mM extracellular Mg2+. A train of 5 stimuli at 25 Hz was used to record NMDA-EPSPs

in the presence of 20 µM NBQX. (B) Representative traces of NMDA-EPSPs and full

Mf-EPSPs (no glutamate receptor antagonists) before and after bursts of stimulation

used to induce LTP of NMDARs at Mf-CA3 synapses. (C) Bursts of stimulation used to

induce LTP of NMDARs at Mf-CA3 synapses do not durably potentiate Mf-EPSPs. (D) D-AP5 does not alter the time course of Mf-EPSPs (represented by the area) in control


3

conditions at both 0.1 and 1 Hz. (E) Representative traces of Mf-EPSPs recorded at

1Hz before and after induction of LTP of NMDARs in the absence or presence of D-

AP5. (F) D-AP5 alters the time course of Mf-EPSPs recorded at 1 Hz after LTP of

NMDARs. (G) Discharge probability of CA3 pyramidal cells in response to a train of

five Mf stimuli at 25 Hz before and after induction of LTP of NMDARs. (H)

Representative traces illustrating the increase in spike transfer at Mf-CA3 synapses

after LTP of NMDARs. (I) If induction of LTP NMDARs is blocked by D-AP5 no

increase in spike probability of CA3 pyramidal cells is observed. Error bars represent

s.e.m.


4

Supplementary Figure 3 – Time course and amplitude of Mf-EPSPs before and

after LTP of NMDARs. (A-B) Normalized area values of Mf-EPSPs before and after

D-AP5 (50 µM) in control conditions (A) and after inducing LTP of NMDARs (B). (C-D) Amplitude values of Mf-EPSPs before and after D-AP5 (50 µM) in control conditions (C) and after inducing LTP of NMDARs (D). Error bars represent s.e.m.


5

Supplementary Figure 4 – Induction of LTP of NMDARs does not change CA3 pyramidal cell intrinsic excitability. (A,B) Average values and representative traces

illustrating the lack of effect of LTP of NMDARs on CA3 pyramidal cell firing in

response to step current injections. (C) Induction of LTP of NMDARs did not alter

action potential threshold of CA3 pyramidal cells. Error bars represent s.e.m.


6

Supplementary Figure 5 – Metaplasticity is prevented by the mGluR5 antagonist MPEP (A,B) Time course and representative traces illustrating that blocking induction

of LTP of NMDARs with the mGluR5 antagonist (MPEP, 10 µM) prevents the depo-

pairing protocol to induce LTP at Mf-synapses. Error bars represent s.e.m.


7

Supplementary Figure 6 – Metaplasticity can also be induced by high frequency stimulation (HFS) protocols known to trigger presynaptic LTP at Mf-synapses.

(A, B, C) Time course, representative traces and average values illustrating that HFS

stimulation can also trigger metaplasticity at Mf-synapses. HFS alone induces a clear

LTP at Mf-synapses. A depo-pairing protocol applied 20 min after HFS induced

additional potentiation. Error bars represent s.e.m.


8

Supplementary Figure 7 – D-serine potentiates NMDARs at Mf-synapses. (A-B) Time course and representative Mf-NMDA-EPSCs showing the potentiation by D-

serine (100 µM) of Mf-NMDARs. Error bars represent s.e.m.


9

Supplementary Figure 8 – D-serine mimics the effects of LTP of NMDARs at Mf- synapses. (A) In the presence of D-serine depo-pairing protocol induced LTP at Mf-

synapses. (B) The LTP triggered by the depo-pairing protocol at Mf-synapses in the

presence of D-serine is blocked by D-AP5. (C) Representative traces of Mf-EPSCs

before and after applying a depo-pairing protocol in the presence of D-serine and in D-

serine + D-AP5. (D) Average values of depo-LTP induced at Mf-synapses in D-serine

and D-serine + D-AP5. Error bars represent s.e.m.


10

Supplementary Figure 9 – STDP-LTP at Mf-synapses is blocked by intracellular inclusion of MK-801. (A,B) Inclusion of 1 mM MK-801 in the patch pipette prevented

STDP-LTP at Mf-synapses induced in the presence of D-serine. Error bars represent

s.e.m.


11

Supplementary Figure 10 – Potentiation of NMDARs with the adenosine A2A

receptor CGS 21680 renders Mf-synapses responsive to the STDP protocol. (A,B) Time course and representative traces of Mf-EPSPs illustrating the STDP-LTP

induced in the presence of the adenosine A2A receptor CGS 21680 (30 nM). Error bars

represent s.e.m.


12


13

Supplementary Figure 11 – LTP observed at Mf-synapses cannot be explained

by contamination of synaptic responses by A/C EPSCs. (A, B and C) - Time

course, representative traces of Mf-EPSCs and average values showing that 10-30 nM

TTX does not affect depo-LTP obtained at Mf-synapses in the presence of D-serine.

(D, E and F) or after prior induction of LTP of NMDARs (G, H and I) The L-type

calcium channel blocker nifedipine (20 µM) blocks induction of depo-LTP at Mf-

synapses but not at A/C synapses. Error bars represent s.e.m.


14

MATERIALS AND METHODS

Electrophysiology

Parasagittal hippocampal slices (350 µm thick) were obtained from 18-25 day-old

C57Bl/6 mice. Slices were transferred to a recording chamber in which they were

continuously superfused with an oxygenated extracellular medium (95% O2 and 5%

CO2) containing (mM): 125 NaCl, 2.5 KCl, 2.3 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 26

NaHCO3, 20 glucose, pH 7.4. Whole-cell recordings were made at ∼32ºC from CA3

pyramidal cells under infrared differential interference contrast imaging using

borosilicate glass capillaries which had resistances between 4-8 MΩ. For current

clamp recordings the intracellular solution contained (in mM): 130 KGluconate, 10 KCl,

10 HEPES, 0.2 EGTA, 0.02 CaCl2, 4 MgATP, 0.3 GTP, 15 phosphocreatine, pH

adjusted to 7.3 with KOH. When studying the modulation of NMDA receptor-mediated

EPSCs (NMDA-EPSCs) by D-serine as was well as measuring the AMPA/NMDA ratio

in the voltage-clamp mode, the patch electrodes were filled with a solution containing

(mM): 120 cesium methanesulfonate, 2 MgCl2, 4 NaCl, 5 phospho-creatine, 2 Na2ATP,

20 BAPTA, 10 HEPES, 0.33 GTP, pH 7.3 adjusted with CsOH. For depo-pairing LTP

done in the voltage-clamp configuration the intracellular solution contained (mM): 125

cesium gluconate, 8 NaCl, 15 phospho-creatine, 4 MgATP, 0.2 EGTA, 10 HEPES,

0.33 GTP, 5 TEA-Cl, pH adjusted to 7.3 with CsOH. On average, using a KGluconate

intracellular solution, CA3 pyramidal cells had a resting membrane potential of (-72 ± 6

mV) and only neurons that had a resting membrane potential more negative than -55

mV were used. No liquid-juntion correction was used. Unless otherwise indicated,

bicuculline (10 µM) and CGP 55845 (3 µM) were present in the superfusate of all

experiments. A patch pipette (open tip resistance ~ 5 MΩ (less than 1 µm)) was placed

in the dentate gyrus to stimulate mossy fibers and in the stratum radiatum of the CA3

area to stimulate associative/commissural fibers. Mossy fiber synaptic currents were

first identified in the voltage-clamp mode before switching to current clamp and were

identified according to the following criteria: Robust low frequency facilitation, low

release probability at 0.1 Hz, rapid single rise times (around 1 ms) and decays free of

secondary peaks that may indicate the presence of polysynaptic contamination. At the

end of each experiment DCG-IV (0.1 µM) an mGluR2 agonist was used to verify the

mossy fiber origin of the EPSPs. To record AMPA/NMDA ratios, AMPA-EPSCs were

first recorded in the voltage-clamp mode at -70 mV in the presence of bicuculline (10


15

µM) and CGP 55845 (3µM). To record NMDA-EPSCs the membrane potential was

changed to +30 mV and NBQX (20 µM) was added to the extracellular medium.

Decay time course of Mf-EPSPs was calculated by dividing the area of the EPSP by

its amplitude. In the current-clamp configuration input resistance was monitored with

small current steps (-20 pA for 300 ms) and cells were excluded if changed by >25%.

The access resistance was <20 MΩ, and cells were discarded if it changed by

>20%. No series resistance compensation was used. Recordings were made using an

EPC 9.0 or EPC 8.0 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) and were

filtered at 0.5-1 kHz, digitized at 1-5 kHz, and stored on a personal computer for

additional analysis (IGOR PRO 5.0; Wave- Metrics, Lake Oswego, OR).

Synaptic responses were evoked every 20 sec (0.05 Hz). LTP of NMDARs was

induced by a train consisting of 6 bursts of 6 stimuli at 50 Hz repeated every 140 ms.

Spike-timing-dependent-plasticity (STDP) was induced by pairing each presynaptic

stimulation with 3 postsynaptic action potentials (50 Hz) triggered 10 ms after the

presynaptic stimulations. This pairing was repeated 30 times every 5 sec. This STDP

protocol induced robust long-term potentiation at associative/commissural synapses

into CA3 pyramidal cells that was dependent on NMDAR (data not shown) activation

and did not occur if either the action potentials or presynaptic stimulation was applied

alone (data not shown). For depolarization-pairing LTP 100-150 EPSCs evoked at 2-

3Hz were paired with continuous postsynaptic depolarization to 0 mV.

All drugs were obtained from Tocris Cookson (Bristol, UK), Sigma (St. Louis, MO) or

Ascent Scientific (Bristol, UK.)

Statistical analysis

Values are presented as mean ± SEM of n experiments. For statistical analysis non-

parametric test were used. In supplementary figure 2 and 3 a Wilcoxon matched pairs

test was used. Mann-Whitney test was used for two group’s comparison and Kruskal-

Wallis test followed by a Dunn´s multiple comparison test for comparison between

more than two groups. Statistical differences were considered as significant at P<0.05.


supplementary material nmda receptor … (stdp) was induced by pairing each presynaptic stimulation...

Documents