Yihui Cui

Vincent Paille

Hao Xu

Stéphane Genet

Bruno Delord

Elodie Fino

Hugues Berry
email: hugues.berry@inria.fr

Laurent Venance
email: laurent.venance@college-de-france.fr

Endocannabinoids mediate bidirectional striatal spike-timing dependent plasticity

Keywords: endocannabinoids, synaptic plasticity, basal ganglia 2-arachidonoylglycerol AP: action potential AM251: N-(piperidin-1-yl)- spike-timing-dependent long-term potentiation MCPG: (S)-α-Methyl-4-carboxyphenylglycine mGluR: metabotropic glutamate receptors STDP: spike-timing dependent plasticity

Synaptic plasticity, a main substrate for learning and memory, is commonly assessed with prolonged stimulations. Since learning can arise from few or even a single trial, synaptic strength is expected to adapt rapidly. However, it remained elusive whether synaptic plasticity occurs in response to limited event occurrences. To answer this question, we investigated if a low number of paired stimulations can induce plasticity in a major synaptic learning rule, the spike-timing dependent plasticity (STDP). It is known that 100 pairings induce bidirectional STDP, i.e. spike-timing-dependent potentiation (tLTP) and depression (tLTD) at most central synapses. In rodent striatum, we found that tLTD progressively disappears when decreasing the number of paired stimulations (below 50 pairings) whereas tLTP displays a biphasic profile: tLTP is observed for 75-100 pairings, absent for 25-50 pairings and reemerges for 5-10 pairings. This tLTP, induced by very few pairings (~5-10), depends on the endocannabinoid (eCB) system. This eCB-tLTP involves postsynaptic endocannabinoid synthesis, is homosynaptic and depends on type-1 cannabinoid receptor (CB1R) and transient receptor potential vanilloid type-1 (TRPV1) activation. eCB-tLTP occurs in both striatopallidal and striatonigral MSNs and is dopamine-dependent. Lastly, we show that eCB-LTP and eCB-LTD can be induced sequentially in the same neuron, depending on the cellular conditioning paradigm. Thus, while usually considered as simply depressing synaptic function, endocannabinoids constitute a versatile system underlying bidirectional plasticity. Our results reveal a novel form of synaptic plasticity, eCB-tLTP, which may underlie rapid learning capabilities characterizing behavioral flexibility.

KEY POINTS SUMMARY

 Although learning can arise from few or even a single trial, synaptic plasticity is commonly assessed under prolonged activation. Here, we explored the existence of rapid responsiveness of synaptic plasticity at corticostriatal synapses in a major synaptic learning rule, the spike-timing-dependent plasticity.

 We found that spike-timing-dependent depression (tLTD) progressively disappears when decreasing the number of paired stimulations (below 50 pairings) whereas spike-timingdependent potentiation (tLTP) displays a biphasic profile: tLTP is observed for 75-100 pairings, is absent for 25-50 pairings and reemerges for 5-10 pairings.

 This tLTP induced by low numbers (5-10) of pairings depends on endocannabinoid system, type-1 cannabinoid receptor and transient receptor potential vanilloid type-1 activation.

 Endocannabinoid-tLTP may represent a physiological mechanism operating in the rapid learning of new associative memories and behavioral rules characterizing the flexible behavior of mammals or during the initial stages of habit learning.

INTRODUCTION

Cardinal cognitive abilities can display rapid learning dynamics. Forming new associative memories and behavioral rules can be learned within a few (5-10) or even a single trial [START_REF] Schultz | Changes in behavior-related neuronal activity in the striatum during learning[END_REF][START_REF] Pasupathy | Different time courses of learning-related activity in the prefrontal cortex and striatum[END_REF][START_REF] Tse | Schemas and memory consolidation[END_REF][START_REF] Quilodran | Behavioral shifts and action valuation in the anterior cingulate cortex[END_REF][START_REF] Ito | Validation of decision-making models and analysis of decision variables in the rat basal ganglia[END_REF]. In cortex and striatum, neurons that respond to behaviorally relevant events (cues, actions or rewards) fire very few spikes (one to a dozen) upon each trial (i.e. they typically discharge at a frequency of 5-25Hz during a 0.1-0.5s period) [START_REF] Schultz | Changes in behavior-related neuronal activity in the striatum during learning[END_REF][START_REF] Pasupathy | Different time courses of learning-related activity in the prefrontal cortex and striatum[END_REF][START_REF] Quilodran | Behavioral shifts and action valuation in the anterior cingulate cortex[END_REF]. This evidence suggests that the discharge of a small number (2-50) of spikes should be sufficient to induce synaptic plasticity, a substrate for learning and memory [START_REF] Martin | New life in an old idea: the synaptic plasticity and memory hypothesis revisited[END_REF]. However, typical cell conditioning experimental protocols for initiating long-term plasticity, such as high-or lowfrequency stimulations, rely on the repetition of hundreds of pre-or postsynaptic spikes.

Noticeable exceptions are reports showing the existence of single-shock LTD in visual cortex or single-burst LTP in hippocampus [START_REF] Holthoff | Single-shock LTD by local dendritic spikes in pyramidal neurons of mouse visual cortex[END_REF][START_REF] Remy | Dendritic spikes induce single-burst long-term potentiation[END_REF].

Besides these reports introducing the possibility of bidirectional plasticity induced by limited stimulation, the possible existence of rapid responsiveness of synaptic plasticity still needs to be extended to other synapses and cell conditioning protocol.

Here, we tested the hypothesis that a low number of spikes could lead to long-term synaptic plasticity. For this purpose, we chose spike-timing dependent plasticity (STDP) as a synaptic learning paradigm. Indeed, STDP (tLTP and tLTD) depends on the relative timing between pre-and postsynaptic spikes, and relies on much fewer events (around 100 paired stimulations) than the high-or low-frequency stimulation protocols (hundreds of stimulations) [START_REF] Caporale | Spike timing-dependent plasticity: a Hebbian learning rule[END_REF][START_REF] Sjöström | Dendritic excitability and synaptic plasticity[END_REF][START_REF] Feldman | The spike-timing dependence of plasticity[END_REF]. We first investigated if limited occurrences of paired stimulations, from 2 to100 pre-post or post-pre pairings, could induce STDP at corticostriatal synapses. In the striatum, bidirectional STDP with NMDAR-mediated tLTP and endocannabinoid(eCB)-mediated tLTD, has been previously reported and was induced with 100 paired stimulations [START_REF] Fino | Bidirectional activity-dependent plasticity at corticostriatal synapses[END_REF][START_REF] Shen | Dichotomous dopaminergic control of striatal synaptic plasticity[END_REF][START_REF] Pawlak | Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity[END_REF]Fino et al., 2010;Fino and Venance, 2010;[START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. In the present study we report that these forms of plasticity disappear when the number of paired stimulations is decreased. However, a reliable and robust tLTP re-emerges for a low number (∼5-10) of paired stimulations. We show that this tLTP is not NMDAR-dependent but eCBmediated. This eCB-tLTP depends on the activation of type-1 cannabinoid receptor (CB1R) and transient receptor potential vanilloid type-1 (TRPV1). eCB-tLTP can be induced in both striatopallidal and striatonigral MSNs and is dopamine-dependent. Finally, we observe that eCB-tLTP and eCB-tLTD can be sequentially induced at the same synapse, thus demonstrating that eCBs serve as a generic signaling system able to encode for bidirectional plasticity. eCBs have emerged as a major signaling system in learning and memory because of their powerful influence on synaptic plasticity that has been associated with the depression of neuronal communication on short or long timescales [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Castillo | Endocannabinoid signaling and synaptic function[END_REF][START_REF] Mathur | Endocannabinoid-dopamine interactions in striatal synaptic plasticity[END_REF][START_REF] Melis | Interplay between synaptic endocannabinoid signaling and metaplasticity in neuronal circuit function and dysfunction[END_REF]. eCB-tLTP reported here shows that eCBs in fact support bidirectional plasticity. This new form of plasticity may underlie the quick reactivity necessary for adapting the response of the synaptic weight during rapid learning.

METHODS

Animals

All experiments were performed in accordance with local animal welfare committee (Center for Interdisciplinary Research in Biology and EU guidelines (directive 2010/63/EU). Every precaution was taken to minimize stress and the number of animals used in each series of experiments. Sprague-Dawley rats (Charles River, L'Arbresle, France) and C57BL/6 mice CB 1 R -/- (ledent et al., 1999) and D 1 -eGFP mice were used for brain slice electrophysiology.

Animals were housed in standard 12 hours light/dark cycles and food and water were available ad libitum.

Brain slice preparation

Horizontal brain slices containing the somatosensory cortex and the corresponding corticostriatal projection field were prepared according to the methods previously published [START_REF] Fino | Bidirectional activity-dependent plasticity at corticostriatal synapses[END_REF][START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. Corticostriatal connections (between somatosensory cortex layer 5 and dorsal striatum) are preserved in a horizontal plane. Briefly, horizontal brain slices with a thickness of 330 or 300µm were prepared, respectively, from rats (males and females, P (17-25) ) or mice (males and females P (17-25) and P (60-90) ) using a vibrating blade microtome (VT1200S, Leica Micosystems, Nussloch, Germany). Brains were sliced in a 95% CO2/5% O2-bubbled, ice-cold cutting solution containing (in mM) 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2 , 1 pyruvic acid, and then transferred into the same solution at 34°C for one hour and then moved to room temperature.

Electrophysiology recordings

Patch-clamp recordings were performed as previously described (Fino et al., 2010;[START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. Briefly, borosilicate glass pipettes of 4-6MΩ resistance contained for whole-cell recordings (in mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 0.3 EGTA (adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2 , 10µM pyruvic acid bubbled with 95% O 2 and 5% CO 2 . Signals were amplified using EPC10-2 amplifiers (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at 34°C using a temperature control system (Bath-controller V, Luigs&Neumann, Ratingen, Germany) and slices were continuously superfused at 2-3 ml/min with the extracellular solution. Slices were visualized on an Olympus BX51WI microscope (Olympus, Rungis, France) using a 4x/0.13 objective for the placement of the stimulating electrode and a 40x/0.80 water-immersion objective for localizing cells for whole-cell recordings. Medium-sized spiny neurons (MSNs), the striatal output neurons, were distinguished from interneurons based on passive and active membrane properties [START_REF] Fino | Cell-specific spike-timing-dependent plasticity in GABAergic and cholinergic interneurons in corticostriatal rat brain slices[END_REF]. Series resistance was not compensated. Current-clamp recordings were filtered at 2.5 kHz and sampled at 5 kHz and voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz using the Patchmaster v2x32 program (HEKA Elektronik). Note that none of the bath-applied drugs had a significant effect on basal EPSC amplitudes (Table 1).

Chemicals

Spike-timing-dependent plasticity induction protocols

Electrical stimulations were performed with a bipolar electrode (Phymep, Paris, France) placed in the layer 5 of the somatosensory cortex [START_REF] Fino | Bidirectional activity-dependent plasticity at corticostriatal synapses[END_REF]Fino et al., 2010).

Electrical stimulations were monophasic at constant current (ISO-Flex stimulator, AMPI, Jerusalem, Israel). Currents were adjusted to evoke 50-200pA EPSCs. Repetitive control stimuli were applied at 0.1Hz. STDP protocols consisted in pairings of pre-and postsynaptic stimulations (at 1Hz) with the two events separated by a specific temporal interval (Δt STDP ).

Presynaptic stimulations corresponded to cortical stimulations and the postsynaptic stimulation of an action potential evoked by a depolarizing current step (30ms duration) in MSNs. We chose to evoke a 30ms suprathreshold depolarization to mimic corticostriatal summation of EPSPs induced by cortical activity as observed in in vivo studies [START_REF] Mahon | Distinct patterns of striatal medium spiny neuron activity during the natural sleep-wake cycle[END_REF]. MSNs were maintained all along the STDP experiments at a constant holding membrane potential which corresponds to their initial resting membrane potential (-75±1 mV, n=103). Thus, EPSCs during baseline or after STDP protocol were mesured at the same membrane potential (in voltage-clamp mode); note that the STDP pairings (performed in current-clamp mode) were conducted also at this very same holding membrane potential.

Neurons were recorded for 10 min during baseline and for at least 60 min after STDP protocol; long-term synaptic efficacy changes were measured from 50 min. 30 successive EPSCs were individually measured and then averaged. Variation of series resistance above 20% led to the rejection of the experiment. After recording of 10 min control baseline, drugs

were applied in the bath. A new baseline with drugs was recorded after a time lapse of 10 min (to allow the drug to be fully perfused) for 10 min before STDP protocol (see effects of the bath-applied drugs on baseline in Table 1). Drugs were present until the end of the recording (except when specified). In a subset of experiments (for U73122, THL, BAPTA and GDP-ß-S) drugs were applied intracellularly through the patch-clamp pipette. Once the cell patched, drugs were allowed to diffuse into the cell during at least 15 minutes before starting the baseline recording. Local applications of 2-AG were performed through a patch-clamp pipette placed at the vicinity (50µm) of the recorded neuron and linked to a Picospritzer II system (Parker, USA), which supplies repeatable pressure pulses.

It should be noted that STDP protocol consisting in 5-10 post-pre pairings (with a single postsynaptic spike) were sufficient to induce potent tLTP in rat while in C57BL/6 mice 15 pairings (with 2-3 postsynaptic spikes) were necessary to trigger tLTP.

Electrophysiological data analysis

Off-line analysis was performed using Fitmaster (Heka Elektronik) and Igor-Pro 6.0.3 (Wavemetrics, Lake Oswego, OR, USA). Statistical analysis was performed using Prism 5.0 software (San Diego, CA, USA). In all cases "n" refers to a single cell experiment from single slice. Experimenters were blind to the genotype of CB 1 R -/-and CB 1 R

RESULTS

A low number of paired stimulations induces spike-timing dependent potentiation

We investigated if a low number of post-and presynaptic paired stimulations can induce plasticity in a major synaptic learning rule such as STDP. As previously described [START_REF] Fino | Bidirectional activity-dependent plasticity at corticostriatal synapses[END_REF][START_REF] Shen | Dichotomous dopaminergic control of striatal synaptic plasticity[END_REF][START_REF] Pawlak | Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity[END_REF]reviewed in Kreitzer and Malenka, 2008;Fino and Venance, 2010;[START_REF] Feldman | The spike-timing dependence of plasticity[END_REF], 100 pairings induced bidirectional STDP in MSNs:

post-pre pairings (-30<Δt STDP <0 ms) induced tLTP (mean value of the EPSC amplitude recorded 60 min after STDP protocol: 142±16%, p=0.0262, n=10), while pre-post pairings (0<Δt STDP <+30 ms) induced tLTD (66±10%, p=0.0124, n=7) (Fig. 1A-C and 1I). We observed a similar bidirectional STDP with 75 pairings: post-pre and pre-post pairings induced tLTP (167±26%, p=0.0378, n=8) and tLTD (64±5%, p=0.0010, n=6), respectively (Fig. 1D and1I). We then decreased the number of pairings to 50 and observed contrasting effects on synaptic plasticity: while a potent tLTD persisted with pre-post pairings (66±6%, p=0.0013, n=7), the tLTP usually associated with post-pre pairings disappeared (103±12%, p=0.7902, n=7) (Fig. 1E and1I). In turn tLTD disappeared for 25 pre-post pairings (94±3%, p=0.0801, n=8) (Fig. 1F and1I). On the post-pre side there was still no significant plasticity (132±24%, p=0.1985, n=11) following 25 pairings although half of the cells displayed tLTP and the other half no plasticity (Fig. 1F and1I). Unexpectedly, decreasing the number of paired stimulations further to 10-5 pairings unveiled another trend: whereas 10 pre-post pairings failed to induce significant plasticity (99±10%, p=0.9267, n=9), 10 post-pre pairings were sufficient to induce a potent tLTP (165±11%, p<0.0001, n=27) (Fig. 1G and1I). A similar picture was obtained even with 5 pairings: post-pre and pre-post pairings induced tLTP (139±13%, p=0.0087, n=16) and no significant plasticity (93±6%, p=0.2417, n=6), respectively (Fig. 1H-I). No significant plasticity was detected with 2 post-pre pairings (108±9%, p=0.4013, n=6) (Fig. 1I). In conclusion, tLTD disappears with decreasing the number of paired stimulations whereas tLTP displays a biphasic profile since tLTP is observed for 75-100 pairings and 5-10 pairings (with similar amplitudes) and absent for 25-50 pairings.

pairings-tLTP is NMDAR-independent

We then questioned the mechanism of tLTD and tLTP induced by these different numbers of pairings. We observed that the corticostriatal 100 pairings-tLTD was CB 1 R-dependent, as previously demonstrated [START_REF] Shen | Dichotomous dopaminergic control of striatal synaptic plasticity[END_REF][START_REF] Pawlak | Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity[END_REF]Fino et al., 2010;[START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. Indeed, pharmacological inhibition of CB 1 R with AM251 (3µM) prevented the expression of 100 pairings-induced tLTD (106±6%, p=0.4153, n=5) (Fig. 2A). Note that AM251 alone (without electrical stimulation) had no effect on basal synaptic transmission (99±5%, p=0.7988, n=8) (Table 1), indicating that CB 1 R had no constitutive activity at corticostriatal synapses. Similarly to the 100 pairings-tLTD, the 50 pairings-tLTD was prevented with AM251 (3µM) (114±17%, p=0.4541, n=7) (Fig. 2B). Thus, the pre-post corticostriatal tLTD was CB 1 R-mediated.

Concerning the post-pre tLTP, we confirmed that the 100 pairings-tLTP was NMDARmediated since prevented with the selective NMDAR blocker D-AP5 (50µM) (104±5%, p=0.4310, n=5) (Fig. 2C), as previously reported [START_REF] Shen | Dichotomous dopaminergic control of striatal synaptic plasticity[END_REF][START_REF] Pawlak | Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity[END_REF]Fino et al., 2010;[START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. We then explored the mechanism of the tLTP induced by 10 post-pre pairings and found that it does not rely on the same signaling pathway (i.e NMDAR). Indeed, the tLTP induced by 10 pairings was not significantly affected by D-AP5 (169±22%, p=0.0098, n=13) (Fig. 2C), questioning the identity of the signaling pathways underlying this new form of tLTP.

pairings-tLTP involves postsynaptic 2-AG signaling

The corticostriatal synapse is glutamatergic and we first tested whether glutamatergic Gprotein coupled receptors were required for the expression of tLTP induced by 10 pairings.

MSNs express group-I metabotropic glutamate receptors (mGluRs) [START_REF] Testa | Metabotropic glutamate receptor mRNA expression in the basal ganglia of the rat[END_REF] belonging to the class of Gq/11-coupled receptors. 10 pairings-tLTP was prevented by the inhibition of group-I mGluR with MCPG (500µM) (100±8%, p=0.9636, n=5) (Fig. 3A-B).

More specifically, among group-I mGluRs, MSNs express prominently the mGluR5 isoform [START_REF] Uchigashima | Subcellular arrangement of molecules for 2-arachidonoyl-glycerol-mediated retrograde signaling and its physiological contribution to synaptic modulation in the striatum[END_REF]. MPEP (10µM), a blocker of mGluR5, prevented the induction of 10 pairings-tLTP and a slight depression was observed (68±8%, p=0.0108, n=6) (Fig. 3B).

Besides glutamate, acetylcholine is also released following a cortical activation of the corticostriatal synapses: indirectly from striatal cholinergic interneurons (tonically active and directly contacted by cortical pyramidal cells). Interestingly, MSNs express the M1 muscarinic receptors that are also another class of Gq/11-coupled receptors [START_REF] Hersch | Distribution of m1-m4 muscarinic receptor proteins in the rat striatum: light and electron microscopic immunocytochemistry using subtype-specific antibodies[END_REF][START_REF] Yamasaki | Preferential localization of muscarinic M1 receptor on dendritic shaft and spine of cortical pyramidal cells and its anatomical evidence for volume transmission[END_REF]. We thus tested if these receptors could be involved in the 10 pairings-tLTP. We found that the inhibition of M1 muscarinic receptors with pirenzepine (1µM) prevented tLTP (95±20%, p=0.8037, n=6) (Fig. 3B). Altogether, these results show that the tLTP triggered by 10 pairings requires the concomitant activation of mGluR5 and M1 muscarinic receptors. We investigated the postsynaptic localization of these receptors with the application in the recorded postsynaptic neuron of a non-hydrolysable nucleotide GDPβS that prevents G-protein activation (2mM i-GDPβS applied intracellularly through the patch-clamp pipette). i-GDPβS precluded tLTP (90±10%, p=0.3249, n=10) indicating that mGluR5 and M1 receptors were postsynaptically located (Fig. 3B). Group-I mGluRs and M1 receptors are Gq/11-coupled receptors and thus activate phospholipase Cβ (PLCβ) [START_REF] Rebecchi | Structure, function, and control of phosphoinositidespecific phospholipase C[END_REF]. We then tested if PLCβ activation was involved in the 10 pairings-tLTP. In the presence of a PLCβ inhibitor applied intracellularly through the patch-clamp pipette (i-U73122, 5µM) 10 post-pre pairings failed to induce any significant plasticity (94±7%, p=0.4255, n=5) (Fig. 3B), confirming the implication of postsynaptic PLCβ.

After activation by Gq/11-coupled receptors, PLCβ triggers large elevations in the concentration of calcium ions [START_REF] Rebecchi | Structure, function, and control of phosphoinositidespecific phospholipase C[END_REF]. We therefore tested the involvement of calcium in the expression of 10 pairing-induced tLTP. We first showed that postsynaptic calcium elevation, in the recorded striatal neuron, was mandatory for tLTP induction. Indeed, specific loading of the recorded postsynaptic neuron with the fast calcium buffer BAPTA (10mM i-BAPTA applied intracellularly) prevented tLTP induction (108±10%, p=0.4774, n=5) (Fig. 3B). We next demonstrated that calcium entry via L-type voltage-sensitive calcium channels (VSCCs), the main type of activated VSCCs in MSNs [START_REF] Carter | State-dependent calcium signaling in dendritic spines of striatal spiny neurons[END_REF], is responsible since their blockade (1µM nimodipine, 95±9%, p=0.6092, n=5) precluded tLTP (Fig. 3B). Further downstream in the signaling pathway, these concomitant activations are expected to promote diacylglycerol lipase-α (DAGLα) activity and therefore 2-arachidonoylglycerol (2-AG) synthesis [START_REF] Hashimotodani | Phospholipase Cβ serves as a coincidence detector through its Ca2+ dependency for triggering retrograde endocannabinoid signal[END_REF][START_REF] Piomelli | A neuroscientist's guide to lipidomics[END_REF][START_REF] Marzo | Endocannabinoids: synthesis and degradation[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Tanimura | The Endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase α mediates retrograde suppression of synaptic transmission[END_REF][START_REF] Alger | Supply and demand for endocannabinoids[END_REF]. 2-AG is produced from the PLCβ product diacylglycerol by calcium-activated DAGLα and is the principal eCB involved in modulating synaptic strength by selectively activating CB 1 R [START_REF] Piomelli | A neuroscientist's guide to lipidomics[END_REF]. We found that DAGLα inhibitor, tetrahydrolipstatin (10µM i-THL applied intracellularly) prevented tLTP (91±7%, p=0.2607, n=5) (Fig. 3A-B).

Importantly, since i-THL application was restricted to the recorded neuron, this result indicates that the production of 2-AG needed to activate CB 1 R arises from the postsynaptic striatal neuron engaged in the paired stimulations. In summary, tLTP induced with 10 pairings involved the 2-AG synthesis pathway.

To further demonstrate the key role of 2-AG in bidirectional STDP (tLTD and tLTP with 100 pre-post pairings and 10 post-pre pairings, respectively), we applied local puffs of 2-AG of different duration in the vicinity (50-100µm) of the recorded striatal neuron. We first applied 100 brief (300ms) puffs of 2-AG (100µM) at 1Hz, i.e the same total duration as a 100 pairings STDP protocol at 1Hz. In these conditions, we observed that without STDP protocol, 2-AG local application was able to induce a significant LTD (68±10%, p=0.0156, n=8) (Fig. 3C)

with magnitude similar to the tLTD induced by 100 pre-post pairings (p=0.8624). This LTD involved CB 1 R activation since 2-AG puffs did not induce plasticity anymore when AM251 (3µM) was bath-applied (96±5%, p=0.5000, n=6) (Fig. 3C).

We then aimed at mimicking the 10 pairing-induced LTP, by applying brief puffs of 2-AG (100µM) 10 times at 1Hz, thus with the same total duration as a 10 pairings STDP protocol at 1Hz. We observed that even in the absence of STDP protocol, 2-AG local application was able to induce a significant LTP (139±24%, p=0.0391, n=8) (Fig. 3D) with magnitude similar to the tLTP induced by 10 post-pre pairings (p=0.3705). This LTP involved CB 1 R activation since 2-AG puff did not induce plasticity anymore in presence of AM251 (3µM) (92±4%, p=0.1542, n=5) (Fig. 3D).

pairings-tLTP is CB 1 R activation mediated

Since 2-AG is a specific ligand of CB 1 Rs [START_REF] Piomelli | A neuroscientist's guide to lipidomics[END_REF][START_REF] Marzo | Endocannabinoids: synthesis and degradation[END_REF][START_REF] Alger | Supply and demand for endocannabinoids[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF], we then asked whether tLTP induced by 10 pairings was indeed CB 1 R-mediated. Pharmacological inhibition of CB 1 R with AM251 (3µM) prevented the expression of 10 pairings-induced tLTP (80±11%, p=0.1424, n=6) (Fig. 4A).

This pharmacological result was further confirmed by experiments with CB 1 R-knockout (CB 1 R -/-) mice [START_REF] Ledent | Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice[END_REF], where no significant plasticity was observed following 15 pairings (93±4%, p=0.0882, n=16) whereas tLTP could be induced in the wild-type CB 1 R +/+ mice (135±5%, p=0.0001, n=10) (Fig. 4B-C). Note that in C57BL/6 mice 15 pairings with 2-3 APs were required to efficiently induced tLTP while 5-10 pairings with a single postsynaptic AP per pairing were sufficient to trigger tLTP in rats. This CB 1 R-mediated tLTP was expressed up to adulthood since we observed a reliable tLTP in P (60-90) CB 1 R +/+ mice (137±8%, p=0.0109, n=5), which was absent in P (60-90) CB 1 R -/-mice (93±6%, p=0.3048, n=5) (Fig. 4C). Pharmacological and genetic evidence demonstrated that tLTP induced by 10 pairings is eCB-mediated. We thus refer to this new form of LTP as eCB-tLTP.

eCB-LTP induction involves TRPV1

Besides 2-AG, the production of another eCB, anandamide, could also be increased upon cellular activity [START_REF] Piomelli | A neuroscientist's guide to lipidomics[END_REF][START_REF] Alger | Supply and demand for endocannabinoids[END_REF]. Whereas 2-AG is a specific ligand of CB 1 R, anandamide activates both CB 1 R (albeit less potently than 2-AG) and TRPV1. TRPV1 is a nonselective cationic channel [START_REF] Ross | Anandamide and vanilloid TRPV1 receptors[END_REF][START_REF] Starowicz | Biochemistry and pharmacology of endovanilloids[END_REF][START_REF] Marzo | Endocannabinoids: synthesis and degradation[END_REF] involved in eCB-mediated short-and long-term depression [START_REF] Gibson | TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons[END_REF][START_REF] Maione | TRPV1 channels control synaptic plasticity in the developing superior colliculus[END_REF][START_REF] Chávez | TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus[END_REF][START_REF] Grueter | Postsynaptic TRPV1 triggers cell typespecific long-term depression in the nucleus accumbens[END_REF][START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF]. We therefore tested whether TRPV1 was implicated in eCB-tLTP. Note that in absence of paired stimulation, application of capsazepine (10µM), a TRPV1 antagonist, had no significant effect on basal EPSC (100±9%, p=0.9778, n=6) (Table 1), indicating that TRPV1 have no constitutive activity at corticostriatal synapses. We then found that the application of capsazepine (10µM) during the STDP stimulation protocol (10 post-pre pairings) blocked eCB-tLTP (83±11%, n=6, p=0.1133) (Fig. 5). To confirm this result, we used AMG9810, another competitive TRPV1 antagonist, structurally distinct from capsazepine, and observed that AMG9810 (1µM) also blocked eCB-tLTP (93±6%, p=0.3046, n=5) (Fig. 5B; Table 1).

Altogether, our results demonstrate that 10 pairings-tLTP is mediated by eCB (2-AG and anandamide), acting on both CB 1 R and TRPV1.

eCB-tLTP occurs in both striatopallidal and striatonigral MSNs and is dopaminedependent

Pyramidal cells from cortex layer 5 contact two MSN subpopulations belonging to the direct (striatonigral) or indirect (striato-pallido-subthalamo-nigral) trans-striatal pathways [START_REF] Gerfen | Modulation of striatal projection systems by dopamine[END_REF][START_REF] Calabresi | Direct and indirect pathways of basal ganglia: a critical reappraisal[END_REF]. We investigated wether eCB-tLTP are similarly induced in both striatopallidal and striatonigral MSNs. The two MNs subtypes express different dopaminergic receptors (D 1 R-like and D 2 R-like for the direct and indirect pathways, respectively) allowing us to identify them with transgenic D 1 -eGFP mice and to investigate eCB-tLTP occurrence in D 1 + and non-D 1 + MSNs (Fig. 6A-B). We observed that 15 post-pre pairings (see Methods) induced tLTP in both D 1 + (165±18%, p=0.0166, n=7) and non-D 1 + (156±20%, p=0.0215, n=9) (Fig. 6A-B). This indicates that tLTP can be induced with few pairings in both striatopallidal and striatonigral MSNs.

Striatum receives excitatory afferents from the cortex as well as a dense innervation from midbrain dopaminergic neurons. Dopamine, a key regulator of action selection and associative learning [START_REF] Yin | The role of the basal ganglia in habit formation[END_REF][START_REF] Redgrave | The short-latency dopamine signal: a role in discovering novel actions?[END_REF][START_REF] Schultz | Multiple dopamine functions at different time courses[END_REF], efficiently modulates corticostriatal synaptic plasticity and particularly the "classical" eCB-LTD [START_REF] Kreitzer | Striatal Plasticity and Basal Ganglia Circuit Function[END_REF][START_REF] Filippo | Short-term and long-term plasticity at corticostriatal synapses: Implications for learning and memory[END_REF][START_REF] Gerfen | Modulation of striatal projection systems by dopamine[END_REF]. We then asked whether dopamine was involved in eCB-tLTP induction. We examined which dopaminergic receptor subtype is involved in eCB-tLTP. For this purpose we first bathapplied a mixture of D 1 R and D 2 R antagonists, SCH23390 (4µM) and sulpiride (10µM), respectively. We found that this cocktail prevented the induction of eCB-tLTP and a depression was observed (75±2%, p<0.0001, n=7) (Fig. 6B). Thus, eCB-tLTP is dopaminedependent. We then selectively inhibited either D 1 R or D 2 R. When we applied a D 1 R antagonist, SCH23390 (4µM), we observed a potent tLTP (149±16%, p=0.0211, n=7) while tLTD could be elicited when a D 2 R antagonist, sulpiride (10µM), was bath-applied (68±10%, p=0.0209, n=7) (Fig. 6C). This indicates that eCB-tLTP is D 2 R-mediated and not dependent on D 1 R.

Corticostriatal eCB-tLTP is homosynaptic

To test the homosynaptic feature of eCB-tLTP we performed paired recordings of two neighboring MSNs (perisomatic distance<50µm) in which one neuron was subjected to 10 post-pre pairings (STDP protocol) while the second received only 10 presynaptic stimulations (n=6 pairs) (Fig. 6A). We observed a potent tLTP (150±13%, p=0.0143, n=6) only in the neuron subjected to post-pre pairings, while the neighboring neuron, that received only the presynaptic stimulation, did not show any significant plasticity (106±3%, p=0.0833, n=6)

indicating that corticostriatal eCB-tLTP is homosynaptic.

In hippocampus, facilitation of LTP via eCB-induced presynaptic depression of GABAergic transmission has been reported [START_REF] Carlson | Endocannabinoids facilitate the induction of LTP in the hippocampus[END_REF][START_REF] Chevaleyre | Endocannabinoid-mediated metaplasticity in the hippocampus[END_REF][START_REF] Zhu | Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity[END_REF]. Here, the observed eCB-tLTP could arguably arise from a decrease of GABA release, through an activation of CB 1 Rs located on GABA terminals, thus decreasing the inhibitory tonus during the pairing paradigm. To test this hypothesis, we blocked the GABA A receptors with picrotoxin (50µM). A significant tLTP was still observed for 10 pairings (135±12, n=6; p=0.0300) (Fig. 7B). The magnitude of 10 pairings-tLTP was not affected by a blockade of GABA A transmission since there was no significant difference with those observed in control condition (p=0.1200). As we recently described for STDP induced by 100 pairings [START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF], GABA controls the polarity of the timing-dependance of STDP:

with a blockade of GABA A transmission, tLTP was induced for 10 pre-post pairings (n=6) while post-pre pairings did not induce plasticity (96±7, p=0.5865, n=7) (Fig. 7B). We confirmed that this tLTP was eCB-mediated: the co-application of picrotoxine (50µM) and AM251 (3µM) prevented the induction of tLTP (103±7%, p=0.7454, n=5) (Fig. 7B). In conclusion, GABAergic microcircuits are not involved in the synaptic efficacy changes themselves induced by 10 pairings but control the polarity of the timing-dependence of the eCB-tLTP.

Bidirectional eCB-STDP in the same neuron eCB-tLTP and eCB-tLTD could represent functional inverses of each other. This could be demonstrated if both phenomena could be sequentially triggered in the same neuron to modify the synaptic weight and then bring it back to its baseline. We tested this hypothesis by applying successively two protocols leading to unidirectional plasticity, which exclusively imply eCBs: 10 post-pre pairings (eCB-tLTP) (Fig. 4) and 50 pre-post pairings (eCB-tLTD) (Fig. 2B). We found that eCB-tLTP and eCB-tLTD can indeed be induced sequentially in the very same neuron independently of the order of induction protocols (n=5) (Fig. 8). Following eCB-LTP induction by 10 post-pre pairings, the potentiated synaptic weight can be decreased back to its basal level by applying 50 pre-post pairings (Fig. 7A). Symmetrically, the synaptic weight depressed by an eCB-tLTD could be re-increased by eCB-tLTP induction in the same neuron (Fig. 8). These results demonstrate that eCB-tLTP and eCB-tLTD can be induced sequentially in the same neuron.

DISCUSSION

Corticostriatal long-term plasticity provides a fundamental mechanism for the function of the basal ganglia in action selection and in procedural learning [START_REF] Yin | The role of the basal ganglia in habit formation[END_REF][START_REF] Yin | Dynamic reorganization of striatal circuits during the acquisition and consolidation of a skill[END_REF][START_REF] Koralek | Corticostriatal plasticity is necessary for learning intentional neuroprosthetic skills[END_REF]. Thus, characterizing striatal plasticity repertoire in physiological conditions is crucial. The striatum receives a wide range of patterns of cortical activities from isolated trains of few spikes to prolonged bursting events. While corticostriatal plasticity under prolonged activation is well elucidated, its occurrence to few spikes remained unexplored. Here, we uncovered the existence of an eCB-tLTP induced by a low number of pairings in the striatum of both juvenile and adult rodents. Indeed, few (5-10) coincident preand postsynaptic spikes were found to strengthen synaptic efficacy through a signaling pathway that relies on eCB system. eCB-tLTP induction relies on activation of CB 1 R and TRPV1 and on 2-AG elevations triggered by coupled rises of calcium and DAGLα activity (mediated by mGluR5, muscarinic M1 receptors and VSCCs) in MSNs. Both activation of glutamatergic afferents from cerebral cortex and striatal cholinergic interneurons (which are monosynaptically contacted by cortical pyramidal cells [START_REF] Fino | Cell-specific spike-timing-dependent plasticity in GABAergic and cholinergic interneurons in corticostriatal rat brain slices[END_REF]) promote the induction of eCB-tLTP.

We also found that eCB-tLTP is dopamine-dependent. More precisely, eCB-tLTP is D 2 Rmediated and not dependent on D 1 R. We then questioned the localization (pre-or postsynaptic) of the D 2 R involved in eCB-tLTP. The postsynaptic localization at MSNs was a priori less likely. Indeed, due to the segregation of expression of D 1 R and D 2 R among MSNs [START_REF] Kreitzer | Striatal Plasticity and Basal Ganglia Circuit Function[END_REF][START_REF] Gerfen | Modulation of striatal projection systems by dopamine[END_REF][START_REF] Calabresi | Direct and indirect pathways of basal ganglia: a critical reappraisal[END_REF], roughly half of MSNs are expected to be D 2 R-expressing neurons. If eCB-tLTP was supported by the postsynaptic D 2 R MSNs, one would expect to induce eCB-tLTP in ~50% of the (randomly chosen) neurons. In our experiments, eCB-tLTP was successfully induced in 83% of the (randomly chosen) tested MSNs in rats, thus suggesting a presynaptic localization of the D 2 R.

Moreover, this was confirmed with experiments performed in D 1 R-eGFP mice show that eCB-tLTP can be induced in both striatopallidal and striatonigral MSNs. This suggests that D 2 Rs involved in eCB-tLTP are not postsynaptically located. Presynaptic D 2 R are expressed at three different locations: the nigrostriatal dopaminergic afferents [START_REF] Mei | Getting specialized: presynaptic and postsynaptic dopamine D2 receptors[END_REF], the cholinergic interneurons [START_REF] Hersch | Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with motor corticostriatal afferents[END_REF] and the corticostriatal glutamatergic afferents [START_REF] Bamford | Dopamine modulates release from corticostriatal terminals[END_REF]. The precise locus of presynaptic D 2 R involved in eCB-tLTP remains to be determined.

We describe here a homosynaptic tLTP in mammals, wherein eCB signaling directly underlies both the induction and the long-term maintenance of synaptic weight increase. eCB signaling exhibits bidirectional plasticity with eCB-tLTP and eCB-tLTD. Bidirectionality is of paramount functional importance since it allows LTP and LTD to reverse each another at a single synapse.

eCBs have emerged as a major actor in learning and memory because of their powerful influence on synaptic plasticity [START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Castillo | Endocannabinoid signaling and synaptic function[END_REF][START_REF] Melis | Interplay between synaptic endocannabinoid signaling and metaplasticity in neuronal circuit function and dysfunction[END_REF]. The eCB system is mainly composed of biolipids synthesized and released on-demand acting as retrograde neurotransmitters on presynaptic CB 1 R (one of the most abundant G protein-coupled receptors in the brain) and postsynaptic TRPV1. eCBs have been reported to depress synaptic weight, i.e. short-or long-term depression, through the activation of CB 1 R [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Castillo | Endocannabinoid signaling and synaptic function[END_REF][START_REF] Melis | Interplay between synaptic endocannabinoid signaling and metaplasticity in neuronal circuit function and dysfunction[END_REF] or TRPV1 [START_REF] Gibson | TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons[END_REF][START_REF] Maione | TRPV1 channels control synaptic plasticity in the developing superior colliculus[END_REF][START_REF] Chávez | TRPV1 activation by endogenous anandamide triggers postsynaptic long-term depression in dentate gyrus[END_REF][START_REF] Grueter | Postsynaptic TRPV1 triggers cell typespecific long-term depression in the nucleus accumbens[END_REF][START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF]. Noticeable exceptions are reports of an indirect role of eCBs in promoting LTP at mixed (chemical and electrical) synapses of the goldfish Mauthner cell via intermediary dopaminergic neurons [START_REF] Cachope | Potentiation of electrical and chemical synaptic transmission mediated by endocannabinoids[END_REF] or at hippocampal CA1 synapses via a GABA A receptor-mediated mechanism [START_REF] Lin | Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation[END_REF][START_REF] Xu | Long-lasting potentiation of hippocampal synaptic transmission by direct cortical input is mediated via endocannabinoids[END_REF], facilitation of LTP in the hippocampus via eCB-induced presynaptic depression of GABAergic transmission [START_REF] Carlson | Endocannabinoids facilitate the induction of LTP in the hippocampus[END_REF][START_REF] Chevaleyre | Endocannabinoid-mediated metaplasticity in the hippocampus[END_REF][START_REF] Zhu | Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity[END_REF], and mediation of heterosynaptic short-term potentiation via intermediary astrocytes [START_REF] Navarrete | Endocannabinoids potentiate synaptic transmission through stimulation of astrocytes[END_REF]. However, to our knowledge, the present study is the first report of a homosynaptic eCB-dependent LTP in mammals, with direct implication of eCBs in the induction and longterm maintenance of spike-timing-dependent potentiation of the stimulated synapse itself. Just like in hippocampal pyramidal cells [START_REF] Shouval | A unified model of NMDA receptor-dependent bidirectional synaptic plasticity[END_REF][START_REF] Graupner | Calcium-based plasticity model explains sensitivity of synaptic changes to spike pattern, rate, and dendritic location[END_REF], postsynaptic calcium levels (or time course) could be crucial in the induction of eCB-STDP in the striatum. Since many of the steps along the eCB pathways are Ca 2+ -dependent (incl. 2-AG and anandamide synthesis), the "Ca 2+ hypothesis" would translate to the CB 1 R pathway. This would lead to a scenario where low to moderate peak levels of eCB would lead to LTD whereas high eCB levels would yield LTP. According to this scenario, our results could be reconciled if the first 5-20 post-pre pairings produce very large peak levels of 2-AG, thus tLTP. If the amplitude of the 2-AG peaks decreases for subsequent post-pre pairings, this initial tLTP would be de-potentiated by the subsequent pairings, thus restricting the expression of eCB-tLTP to the first 5 to 20 pairings. On the other hand, the LTP observed with 100 post-pre pairings entirely results from an increase of the postsynaptic weight through activation of the CaMKII pathway by NMDARs, with no additional contribution of the eCB-LTP; indeed, when CB 1 R was inhibited, the NMDAR-tLTP induced with 100 post-pre pairings was not significantly affected (Fino et al., 2010). Thus, eCB-LTP would start to be expressed after 5-20 post-pre pairings. Subsequent pairings would then erase this potentiation while, independently, triggering the expression of NMDA-LTP.

There is a large diversity of the STDP rules at play in the brain and even within the same structure, variety seems to be the rule [START_REF] Feldman | The spike-timing dependence of plasticity[END_REF]. Indeed, in striatum the main neuronal population, the MSNs, express NMDAR-tLTP and eCB-tLTD [START_REF] Shen | Dichotomous dopaminergic control of striatal synaptic plasticity[END_REF][START_REF] Pawlak | Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity[END_REF]Fino et al., 2010;[START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF] and eCB-tLTP (the present report). Whereas neighbouring striatal fast-spiking GABAergic interneurons express solely NMDARdependent STDP, both for LTP and LTD, for 100 pairings [START_REF] Fino | Cell-specific spike-timing-dependent plasticity in GABAergic and cholinergic interneurons in corticostriatal rat brain slices[END_REF] but lack plasticity at low numbers of pairings (data not shown). In addition, our results evidence that eCB-tLTP is anti-Hebbian at corticostriatal synapses and tightly controlled by GABAergic interneurons similarly to the bidirectional corticostriatal STDP (i.e. NMDAR-tLTP and eCB-tLTD; [START_REF] Paillé | GABAergic circuits control spike-timing-dependent plasticity[END_REF]. It has been reported that the endocannabinoid-mediated LTP at hippocampal CA1 synapses induced with high-frequency [START_REF] Lin | Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation[END_REF], low-frequency [START_REF] Zhu | Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity[END_REF] or paired stimulations [START_REF] Xu | Long-lasting potentiation of hippocampal synaptic transmission by direct cortical input is mediated via endocannabinoids[END_REF] were prevented not only by inhibition of CB 1 R but also by inhibition of GABA A receptors. Here, we show that GABA is not involved in eCB-tLTP induction or magnitude at corticostriatal synapses but controls the polarity of eCB-tLTP.

Due to their on-demand intercellular signaling modus operandi [START_REF] Alger | Supply and demand for endocannabinoids[END_REF] eCB biosynthesis and release are evoked by precisely timed and positioned physiological stimuli [START_REF] Katona | Endocannabinoid signaling as a synaptic circuit breaker in neurological disease[END_REF]. As previously described, our study confirms that STDP indeed efficiently triggers eCB signaling. Evidence for TRPV1 activation by physiological neuronal activity patterns was lacking. We demonstrate here that STDP is able to engage the TRPV1 signaling pathway. TRPV1 being a cationic channel highly permeable to calcium [START_REF] Ross | Anandamide and vanilloid TRPV1 receptors[END_REF][START_REF] Starowicz | Biochemistry and pharmacology of endovanilloids[END_REF][START_REF] Marzo | Endocannabinoids: synthesis and degradation[END_REF] it may contribute to eCB-tLTP induction by boosting the calcium transients in the postsynaptic element. As recently described for short-and longterm depression [START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF], our results illustrate the versatility of eCB signaling as a system displaying polymodal activation through CB 1 R and TRPV1, to trigger LTP.

eCB-LTP is promoted by very low numbers of pairings (∼5-10), therefore providing a mechanism whereby synapses react to the very first occurrences of incoming activity. This ability contrasts strongly with NMDAR-dependent LTP that requires the iteration of at least 75-100 paired stimulations to be expressed in the classical (1Hz) STDP context. In mammals, associative memories and behavioral rules can be learned within few (5-10) trials or even sometimes within a single trial [START_REF] Pasupathy | Different time courses of learning-related activity in the prefrontal cortex and striatum[END_REF][START_REF] Tse | Schemas and memory consolidation[END_REF][START_REF] Schultz | Changes in behavior-related neuronal activity in the striatum during learning[END_REF][START_REF] Quilodran | Behavioral shifts and action valuation in the anterior cingulate cortex[END_REF][START_REF] Ito | Validation of decision-making models and analysis of decision variables in the rat basal ganglia[END_REF]. In cortex or striatum, neurons with behavior-related activities fire a few spikes upon behaviorally relevant events during each trial (i.e. at frequency 5-25Hz and during 0.1-0.5s, typically <10 spikes) [START_REF] Pasupathy | Different time courses of learning-related activity in the prefrontal cortex and striatum[END_REF][START_REF] Schultz | Changes in behavior-related neuronal activity in the striatum during learning[END_REF][START_REF] Quilodran | Behavioral shifts and action valuation in the anterior cingulate cortex[END_REF], suggesting that a few trials should be sufficient to induce synaptic plasticity. eCB-tLTP may be used for learning and memorizing salient events from a few spikes. Hence, eCB-LTP may represent a molecular substrate operating in rapid learnings of new arbitrary associative memories and behavioral rules characterizing the flexible behavior of mammals or during initial stages of slower habit learnings [START_REF] Barnes | Advance cueing produces enhanced action-boundary patterns of spike activity in the sensorimotor striatum[END_REF]. Moreover, marijuana intoxication leads to impairment of working memory. This impairment was hitherto interpreted solely as the effect of cannabinoids on the promotion of synaptic depression. Our results together with recent reports [START_REF] Lin | Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation[END_REF][START_REF] Xu | Long-lasting potentiation of hippocampal synaptic transmission by direct cortical input is mediated via endocannabinoids[END_REF] open new perspectives since they suggest that synaptic potentiation may as well be implied in the effects of marijuana. *: None of the bath-applied drugs had a significant effect on baseline. In any cases, only baselines with the bath-applied drugs were compared to EPSC measured after 60 minutes recordings. (B) Inhibition of the GABA A transmission with picrotoxin did not affect eCB-tLTP magnitude but controls the time-dependence of eCB-tLTP. With bath-applied picrotoxin (50µM), a potent tLTP was induced by 10 pre-post pairings (n=6; blue) while no significant plasticity was observed with 10 post-pairings (n=7; black). The occurrence and magnitude of tLTP were not affected by a blockade of GABA A transmission since induced plasticities were not significantly different from the ones observed in control conditions. This tLTP induced with 10 pre-post pairings under GABA A receptors blockade is eCB-mediated since prevented with bath-application of AM251 (3µM). GABAergic microcircuits are not involved in the synaptic efficacy changes themselves but control the time-dependence of the eCB-tLTP.

Representative traces are the average of 15 EPSCs during baseline (black traces) and 50 min after STDP protocol (grey traces). Error bars represent SD. *: p<0.05. ns: not significant. Representative experiments illustrating in (A) an eCB-tLTP event (induced by 10 post-pre pairings, red vertical line) followed by an eCB-tLTD occurrence (induced by 50 pre-post pairings, blue vertical line) and in (B) the reversed sequence (eCB-tLTD followed by eCB-tLTP). In both cases, the neurons come back to baseline level after the full sequence.

Single EPSC amplitudes (empty grey circles) and averaged data (empty white circles) are represented.

  Chemicals were bath-applied or injected only in the recorded postsynaptic neuron through the patch-clamp pipette. DL-2-amino-5-phosphono-pentanoic acid (D-AP5, 50 µM) (Tocris, Ellisville, MO, USA), 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP hydrochloride, 10 µM) (Tocris), 5,11-Dihydro-11-[(4-methyl-1-piperazinyl)acetyl]-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one dihydrochloride (pirenzepine dihydrochloride, 1 µM) (Sigma) were dissolved directly in the extracellular solution and bath applied. N-(piperidin-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 3 µM) (Tocris), picrotoxin (50 µM) (Sigma), 1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-methyloxyethyl 1-methylethyl ester (nimodipine, 1 µM) (Tocris) and (2E)-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2propenamide (AMG9810, 1µM) (Tocris), R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390, 4µM, Sigma) and (S-)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide (sulpiride, 10µM, Tocris) were dissolved in ethanol and then added in the external solution at a final concentration of ethanol of 0.01-0.1%. BAPTA (1 mM) (Sigma) and GDP-ß-S (2mM) were dissolved directly into the intracellular solution and applied via the patch-clamp pipette. U73122 (5 µM) (Sigma) was dissolved in ethanol and then added in the intracellular solution at a final concentration of ethanol of 0.033%. Tetrahydrolipstatin (THL, 10 µM) (Sigma) was dissolved in DMSO and applied internally via the patch-clamp pipette at a final concentration of DMSO of 0.1%. (S)-α-Methyl-4-carboxyphenylglycine (MCPG, 500 µM) (Tocris) was dissolved in 1.1 eq. NaOH, and then added in the external solution. N-[2-(4-Chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine, 10 µM) (Tocris) and 2-arachidonoylglycerol (2-AG, 100 µM) (Tocris) were dissolved in DMSO and then added in the external solution at a final concentration of DMSO of 0.0025 and 0.1%, respectively.

  mGluR5 and M1R need to be simultaneously activated to elicit eCB-tLTP. Both receptors (mGluR5 and M1) are Gq/11-coupled receptors and positively coupled to PLCβ, thus leading to DAG production, and favoring the synthesis of 2-AG. Whereas cholinergic activation is not sufficient to trigger eCB-LTP, it remains necessary for the eCB-LTP induction. Our hypothesis is that eCB-LTP is induced only when large levels of 2-AG are produced. Our results of M1, mGluR5, VSCC and TRPV1 blocking indicate that it is mandatory to activate all possible cumulative contributions to 2-AG production (PLCβ activation for DAG production, VSCC and TRPV1 to increase calcium surge, thus activating DAGLα) in order to reach large levels of 2-AG, which would promote eCB-tLTP.

Figure

  Figure 1. A low number of paired stimulations induces spike-timing dependent

Figure 2 .

 2 Figure 2. 10 pairings-tLTP is NMDAR-independent.

Figure 3 .

 3 Figure 3. 10 pairings-tLTP induction involves postsynaptic 2-AG signaling.

Figure 4 .

 4 Figure 4. 10 pairings-tLTP is CB 1 R-mediated.

Figure 5 .

 5 Figure 5. eCB-LTP is also TRPV1-mediated

Figure 6 .

 6 Figure 6. eCB-tLTP is induced in both striatopallidal and striatonigral MSNs and is

Figure 7 .

 7 Figure 7. Corticostriatal eCB-tLTP is homosynaptic

Figure 8 .

 8 Figure 8. Bidirectional eCB synaptic plasticity in a single neuron

  Figure1

TABLETable 1 : Effects of bath-applied drugs on corticostriatal EPSC baseline amplitude.

 1

	Bath-applied drugs	Normalized EPSC (baseline-	n, p *
		drugs/Baseline-control)
		amplitude
	D-AP5 (50µM)	98±4%	n=6, p=0.6076
	AM251 (3 µM)	99±5%	n=8, p=0.7988
	MCPG (500µM)	94±6%	n=5, p=0.4029
	MPEP (10µM)	100±4%	n=4, p=0.9243
	Pirenzepine (1µM)	92±7%	n=6, p=0.3739
	Nimodipine (1µM)	96±4%	n=4, p=0.3790
	Capsazepine (10µM)	100±4%	n=5, p=0.9778
	AMG9810 (1µM)	97±8%	n=5, p=0.6800
	Picrotoxine (50µM)	96±3%	n=6, p=0.1493
	SCH23390 (4µM) + sulpiride (10µM)	102±2%	n=7, p=0.4468

Acknowledgments:

We thank O Manzoni, S Hormuzdi, P Faure, for helpful suggestions and critical comments;

We thank C Ledent (ULB, Belgium) and Denis Hervé (Institut du fer a Moulin, France) for kindly providing CB 1 R -/-and D 1 R-eGFP mice, respectively.

Funding:

This work has been supported by grants from INSERM, INRIA, Collège de France, the Agence Nationale pour la Recherche and the Ecole des Neurosciences de Paris.

ADDITIONAL INFORMATION SECTION

Competing interests:

The authors declare that they have no competing financial interest and conflict of interest.