Endocannabinoid-LTP Mediated by CB1 and TRPV1 Receptors Encodes for Limited Occurrences of Coincident Activity in Neocortex

Synaptic efficacy changes, long-term potentiation (LTP) and depression (LTD), underlie various forms of learning and memory. Synaptic plasticity is generally assessed under prolonged activation, whereas learning can emerge from few or even a single trial. Here, we investigated the existence of rapid responsiveness of synaptic plasticity in response to a few number of spikes, in neocortex in a synaptic Hebbian learning rule, the spike-timing-dependent plasticity (STDP). We investigated the effect of lowering the number of pairings from 100 to 50, and 10 on STDP expression, using whole-cell recordings from pyramidal cells in rodent somatosensory cortical brain slices. We found that a low number of paired stimulations induces LTP at neocortical layer 4–2/3 synapses. Besides the asymmetric Hebbian STDP reported in the neocortex induced by 100 pairings, we observed a symmetric anti-Hebbian LTD for 50 pairings and unveiled a unidirectional Hebbian spike-timing-dependent LTP (tLTP) induced by 10–15 pairings. This tLTP was not mediated by NMDA receptor activation but requires CB1 receptors and transient receptor potential vanilloid type-1 (TRPV1) activated by endocannabinoids (eCBs). eCBs have been widely described as mediating short- and long-term synaptic depression. Here, the eCB-tLTP reported at neocortical synapses could constitute a substrate operating in the online learning of new associative memories or during the initial stages of learning. In addition, these findings should provide useful insight into the mechanisms underlying eCB-plasticity occurring during marijuana intoxication.


INTRODUCTION
In mammals, cardinal cognitive abilities can display very rapid learning dynamics. Forming new associative memories and behavioral rules can be learned within a few or even a single trial (Schultz et al., 2003;Pasupathy and Miller, 2005;Armstrong et al., 2006;Rutishauser et al., 2006;Whitlock et al., 2006;Tse et al., 2007;Quilodran et al., 2008;Cook and Fagot, 2009;Ito and Doya, 2009;Izquierdo et al., 2016). Cortical and striatal neurons that respond to relevant cues, actions or rewards Abbreviations: ∆t STDP , STDP time interval; CB 1 R, type-1 cannabinoid receptor; eCB, endocannabinoid; mGluR5, type-5 metabotropic glutamate receptor; PLC, phospholipase; STDP, spike-timing dependent plasticity; tLTP, spike-timing dependent LTP; TRPV1, transient receptor potential vanilloid type-1; VSCCs, voltage-sensitive calcium channels. fire few action potentials (∼1-12) upon each trial (Schultz et al., 2003;Pasupathy and Miller, 2005;Quilodran et al., 2008). This indicates that the emission of a low number of action potentials should be sufficient to allow synaptic plasticity expression. However, common cellular conditioning protocols, such as high-or low-frequency stimulations, used for the induction of long-term plasticity involve hundreds (or even more than one thousand) of pre-and/or postsynaptic action potentials. Noticeable exceptions are studies reporting that single-shock synaptic stimulation of layer 5 neocortical pyramidal neurons induced NMDAR-dependent long-term depression (LTD) in visual cortex (Holthoff et al., 2004) and that single-burst of strong and synchronous inputs from hippocampal CA1 to CA3 triggered NMDAR-and L-type voltage-sensitive calcium channels (VSCCs) dependent long-term potentiation (LTP; Remy and Spruston, 2007). Moreover, it was shown that low numbers of paired stimulations (∼20) in spike-timingdependent plasticity (STDP) paradigm (Dan and Poo, 2006;Sjöström et al., 2008;Feldman, 2012), were able to induce spiketiming-dependent potentiation (tLTP) in dissociated culture of hippocampal neurons (Zhang et al., 2009), in cortical slices (Froemke et al., 2006) and in corticostriatal slices (Cui et al., 2015(Cui et al., , 2016. These studies revealed that limited occurrences of coincident activity are able to induce bidirectional plasticity, and this needs to be extended to other synapses and cell conditioning paradigms. We previously reported in striatum the existence of an endocannabinoid-mediated spike-timing dependent LTP (eCB-tLTP) induced with a very low number of pairings (from 5 to 15 pairings; Cui et al., 2015Cui et al., , 2016. STDP is a synaptic Hebbian learning rule in which synaptic weight changes depend on the activity on both sides of the synapse (Dan and Poo, 2006;Sjöström et al., 2008;Feldman, 2012). Since its discovery, STDP has attracted considerable interest in experimental and in computational neuroscience because it relies on spike correlation and has emerged as a candidate mechanism for activity-dependent changes in neural circuits, including map plasticity (Abbott and Nelson, 2000;Dan and Poo, 2006;Morrison et al., 2008;Sjöström et al., 2008;Feldman, 2012;Froemke, 2015;Korte and Schmitz, 2016). Here, we tested at neocortical layer 2/3 synapses the hypothesis that a low number of spikes (∼10-15) could lead to long-term synaptic plasticity accounting for fast and flexible learning of new behavioral responses. At neocortical layer 4-2/3 synapses, a hundred of pairings induced Hebbian STDP, i.e., tLTP and tLTD being triggered by causal pre-before-postsynaptic pairings and anti-causal post-before-presynaptic pairings, respectively (Feldman, 2000;Froemke et al., 2006;Nevian and Sakmann, 2006;Banerjee et al., 2009;Itami and Kimura, 2012;Rodríguez-Moreno et al., 2013;Banerjee et al., 2014). STDP polarity was developmentally controlled (Banerjee et al., 2009;Kimura, 2012, 2016). In the present study, we observed that STDP polarity changed depending on the number of pairings: from asymmetric Hebbian STDP for 100 pairings, to symmetric anti-Hebbian tLTD for 50 pairings and to unidirectional tLTP for 10-15 pairings. Notably, a low number of paired stimulations (∼10-15) were sufficient to trigger tLTP. We found that this tLTP displays an unidirectional Hebbian polarity. This tLTP was not NMDARdependent but eCB-mediated and required the activation of CB 1 receptors and transient receptor potential vanilloid type-1 (TRPV1). Our study evidences, together with recent reports (Cui et al., 2015(Cui et al., , 2016Wang et al., 2016Wang et al., , 2018Maglio et al., 2018) that eCB system not only promotes LTD but also LTP. Therefore, endocannabinoids (eCBs) can underlie bidirectional plasticity, depending on the regime of activity pattern on both sides of the synapse.

Animal Models
All experiments were performed in accordance with the guidelines of the local animal welfare committee (Center for Interdisciplinary Research in Biology Ethics Committee) and the EU (directive 2010/63/EU). Every precaution was taken to minimize stress and the number of animals used in each series of experiments. Animals were housed in standard 12 h light/dark cycles and food and water were available ad libitum. Sprague-Dawley rats (Charles River, L'Arbresle, France) and C57BL/6 mice type-1 cannabinoid receptor knockout, (CB 1 R −/− ), and wild-type littermates (CB 1 R +/+ ) mice (Ledent et al., 1999), were used for ex vivo electrophysiology.

Spike-Timing-Dependent Plasticity Induction Protocols
Electrical stimulation was performed with a bipolar electrode (Phymep, Paris, France) placed in the layer 4 of the somatosensory cortex. Electrical stimulation was monophasic at constant current (ISO-Flex stimulator, AMPI, Jerusalem, Israel). Currents were adjusted to evoke 50-250 pA excitatory postsynaptic currents (EPSCs). Repetitive control stimuli were applied at 0.1 Hz. STDP protocols consisted of pairings of preand postsynaptic stimulations (100, 50 or 10 at 1 Hz) separated by a specific time interval (∆t STDP ); ∆t STDP was estimated as the time interval between the stimulation artifact recorded in the postsynaptic cell and the neighboring postsynaptic action potential. Presynaptic stimulation corresponded to cortical layer 4 stimulation and the postsynaptic stimulation to an action potential evoked by a depolarizing current step for 30 ms duration (injected currents were 370 ± 35 pA for the 10 pre-post pairing experiments, n = 14) in one layer 2/3 pyramidal cell. ∆t STDP < 0 ms for post-pre (post-before-pre) pairings, and ∆t STDP > 0 ms for pre-post (pre-before-post) pairings. Pyramidal cells were maintained during the whole duration of the experiments at a constant holding membrane potential which corresponds to their initial resting membrane potential (−66 ± 1 mV, n = 40). Thus, EPSCs during baseline or after STDP protocol were measured at the same membrane potential (in voltage-clamp mode); STDP pairings (performed in current-clamp mode) were conducted also at this same holding membrane potential. A single STDP protocol was applied per cell, and only one cell was recorded per brain slice. Neuronal recordings were made over a period of 10 min at baseline, and for 60 min after the STDP protocols; long-term changes in synaptic efficacy were measured from 45 min to 55 min. We individually measured and averaged 60 successive EPSCs, comparing the last 10 min of the recording with the 10-min baseline recording.
Experiments were excluded if input resistance (Ri) varied by more than 20%. 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 the STDP protocol. Drugs were present until the end of the recording; except for picrotoxin, which was bath-applied 40 min after pairing protocol. In a subset of experiments, THL were applied intracellularly via the patch-clamp pipette (i-THL). Once the cell patched, drugs were allowed to diffuse into the cell during at least 10 min before starting recording of the baseline. STDP protocols consisting of 10 pre-post pairings (with 2-3 postsynaptic spikes) were sufficient to induce potent tLTP in rats whereas in C57BL/6 mice 15 pairings (still with 2-3 postsynaptic spikes per postsynaptic discharge) were required to trigger tLTP. Note that 2-3 action potentials per pairing were required for 10 pairings to induce tLTP since single backpropagating action potentials paired with presynaptic stimulation, did not induce plasticity (105 ± 5%, p = 0.4029, n = 4; Figure 1H).
Post-pre and pre-post ∆t STDP were comprised between 5 ms and 20 ms (absolute values) which is within the temporal domain of expression of STDP (Feldman, 2012). We ensured that ∆t STDP (absolute) values did not display significant variations among experimental groups for post-pre or pre-post pairing protocol (one-way ANOVA: F = 1.782; p = 0.0968, Dunnett's multiple comparisons test; with absolute values of ∆t STDP ; Supplementary Table S1).

Patch-Clamp 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 (San Diego, CA, USA). ''n'' refers to an experiment on a single cell from a single brain slice (the number of animals for each experimental group are indicated in the legends of the figures). Experimenters were blind to the genotype of CB 1 R −/− and CB 1 R +/+ littermate mice. All results are expressed as mean ± SEM in the text and as mean ± SD for visualization purposes in the figures, and statistical significance was assessed using two-sided Student's t test or the one sample t test as appropriate using the indicated significance threshold (p). We analyzed all datasets (Prism 6.0 software) and all of them fitted Gaussian distribution with equal variance.
Frontiers in Cellular Neuroscience | www.frontiersin.org presynaptic) loci of plasticity. The case where an absence of variation of normalized CV −2 is associated with a variation of normalized EPSC amplitude reflects mainly postsynaptic modifications (Clements and Silver, 2000).

Polarity of STDP Varies Upon Number of Pairings at Neocortical Layer 4-2/3 Synapses
To examine the effect of a few number of pairings on long-term synaptic efficacy changes, we made whole-cell recordings from pyramidal cells of the somatosensory cortex in horizontal brain slices ( Figure 1A). In cortical slices, layer 2/3 pyramidal cells receive monosynaptic inputs from the layer 4 as illustrated by EPSC latency standard deviation (0.47 ± 0.04 ms, n = 26), which is inferior to 1 ms ( Figure 1B). We investigated the effect of lowering the number of pairings from 100 to 50 and 10 on STDP (Figure 1). Baseline EPSCs were recorded for 10 min followed by pairing a single presynaptic stimulation with a postsynaptic brief depolarization of the recorded pyramidal cell which induces 2-3 spikes. The STDP protocol consisted in pairing pre-and postsynaptic stimulations with a fixed timing interval, ∆t STDP (∆t STDP < 0 indicates that postsynaptic stimulation preceded presynaptic stimulation and ∆t STDP > 0 indicates that presynaptic stimulation preceded postsynaptic stimulation), repeated n times at 1 Hz ( Figure 1C). Post-pre and pre-post ∆t STDP stood between 5 ms and 20 ms (absolute values) i.e., within the temporal domain of expression of neocortical STDP (see in ''Materials and Methods'' section and Figure Legends for detailed values of ∆t STDP for each experimental condition; Dan and Poo, 2006;Sjöström et al., 2008;Feldman, 2012). After the STDP pairings, EPSCs were monitored for 1 h.
We next lowered the number of pairings down to 10 and observed an unidirectional Hebbian tLTP ( Figure 1F). Indeed, as exemplified in the Supplementary Figures S1C1,C2, 10 post-pre pairings did not induce synaptic efficacy changes, whereas 10 pre-post pairings induced tLTP. To summarize, 10 post-pre pairings failed to induce significant long-term plasticity (95.5 ± 7% of the baseline, p = 0.5884, n = 7; 6/7 cells displayed an absence of significant plasticity; Figure 1F1), whereas 10 pre-post pairings were able to induce a potent tLTP (168.2 ± 11.1%, p < 0.0001, n = 15; 15/15 cells displayed tLTP; Figures 1F2,3). We analyzed the relationship between the synaptic efficacy changes after 10 pre-post pairings and the EPSC amplitude during the baseline. There was no significant correlation between plasticity and EPSC amplitude (50-260 pA) during baseline (10 pre-post pairings, R 2 = 0.1012, p = 0.2478, n = 15; Supplementary Figure S1D). Interestingly, 2-3 postsynaptic action potentials (per pairing) were necessary to induce tLTP with 10 pre-post pairings, whereas a single action potential was sufficient to induce tLTD and tLTP with post-pre and pre-post pairings, respectively ( Figure 1H). Indeed, when a postsynaptic single action potential was evoked (per pairing), no plasticity was observed ( Figure 1H). Accordingly, we have tested the occurrence of plasticity for 10 post-pre pairings in similar conditions than the ones used for pre-post pairings, i.e., with 2-3 postsynaptic action potentials for every pairings.
10 Pairings-tLTP Is NMDAR-Independent but Relies on mGluR5 and L-Type VSCC Activation We next questioned the mechanism of tLTP induced by N pairings = 10 and first tested whether this tLTP would be NMDAR-mediated. Indeed, neocortical tLTP induced by pre-post pairings (using various frequencies and numbers of pairings) has been reported to be NMDAR-mediated (Sjöström et al., 2003;Nevian and Sakmann, 2006;Corlew et al., 2007;Rodríguez-Moreno and Paulsen, 2008;Banerjee et al., 2014;reviewed in Sjöström et al., 2008;Feldman, 2012). For this purpose, we bath-applied the selective NMDAR blocker D-AP5 (50 µM), which had no effect on synaptic transmission (normalized EPSC (baseline−drugs/baseline−control) amplitude: 96.1 ± 1.7%, p = 0.0703, n = 6). Here, tLTP induced by N pairings = 10 was not NMDAR-activation dependent. Indeed, an example of tLTP induced by 10 pre-post pairings in presence of D-AP5 is shown in the Supplementary Figure S2A. To summarize, tLTP induced with 10 pre-post pairings was not significantly affected by D-AP5 (132.3 ± 14.5% of the baseline, p = 0.0437, n = 8; 5/8 displayed tLTP; Figure 2), questioning the identity of the signaling pathways underlying this tLTP.
10 Pairings-tLTP Involves Postsynaptic 2-AG Signaling and Is CB 1 R-Mediated With a Presynaptic Induction Locus According to our previous results obtained in striatum (Cui et al., 2015(Cui et al., , 2016 and because mGluR5 and VSCCs are involved in eCB synthesis (Piomelli et al., 2007;Di Marzo, 2008;Kano et al., 2009;Alger and Kim, 2011), we then asked whether tLTP induced by 10 pre-post pairings was endocannabinoid-and CB 1 R-mediated. Concomitant activations of mGluR5 (belonging to Gq/11coupled receptors, whose stimulation results in PLCβ activation) and VSCC promote diacylglycerol lipase-α activity and therefore 2-arachidonoylglycerol (2-AG) synthesis (Piomelli et al., 2007;Di Marzo, 2008;Kano et al., 2009;Alger and Kim, 2011). 2-AG is produced from the PLCβ product diacylglycerol by calciumactivated diacylglycerol lipase-α and is the principal eCB involved in modulating synaptic weight via CB 1 R activation (Piomelli et al., 2007). We applied intracellularly via the patch-clamp pipette a diacylglycerol lipase-α inhibitor, tetrahydrolipstatin (10 µM, i-THL) and we observed that i-THL prevented tLTP as illustrated in the example in Supplementary Figure S2D and in the summary graph (58.5 ± 6.5%, p = 0.0004, n = 8; 0/8 cells displayed tLTP; Figure 3C). Because the i-THL application was confined to the recorded neuron, this indicates that the production of 2-AG needed to activate CB 1 R arises from the postsynaptic pyramidal cell subjected to the paired stimulations.
CB 1 Rs are mainly located on presynaptic terminals (Katona and Freund, 2012), the locus of eCB-tLTP is thus expected to be presynaptic. To test this, we used the mean variance analysis of EPSCs (Clements and Silver, 2000). The coefficients of variation (CV) of EPSC amplitude were estimated during baseline and after plasticity induction (40 min after pairing protocol). The normalized CV −2 (CV −2 after plasticity induction /CV −2 during baseline) is plotted with the normalized EPSC amplitude; see ''Materials and Methods'' section). We obtained a CV −2 value of 3.5 ± 1.2 (p = 0.0048, n = 17) associated with a change in normalized EPSC amplitude of 1.6 ± 0.2 (p = 0.0063). Normalized CV −2 ≥ normalized EPSC amplitude, indicated a presynaptic locus for eCB-tLTP ( Figure 4C). This was confirmed by applying paired pulses with 50 ms interpulse interval (which induced a significant EPSC paired-pulse facilitation, PPF) before and after STDP protocol (Figure 4D). EPSC facilitation was 149.6 ± 37.1% (p = 0.0210) and 130.6 ± 24.8% (p = 0.0699) before and after STDP pairings (n = 7), respectively. We observed a significant decrease of PPF (PPF plasticity/baseline = 0.923 ± 0.046, p = 0.040, n = 7) indicating a presynaptic locus of the plasticity downstream of CB 1 Rs.
The magnitude of neocortical eCB-tLTP could be affected by a decrease of the GABA release, via an activation of CB 1 Rs located on GABAergic terminals. Indeed, eCB-induced depression of GABAergic transmission leading to a facilitation of LTP magnitude has been observed in the hippocampus (Carlson et al., 2002;Chevaleyre and Castillo, 2004;Zhu and Lovinger, 2007;Lin et al., 2011;Xu et al., 2012). To this aim, we blocked the GABA A receptors with bath-applied picrotoxin (50 µM) after STDP induction. First, we tested whether picrotoxin affects EPSC transmission during baseline and found no significant variation of EPSC amplitude after picrotoxin application (normalized EPSC (baseline−drugs/baseline−control) amplitude: 105.3 ± 7.0, p = 0.6358, n = 8). Forty minutes after pairings; with 10 pre-post pairings, tLTP was still observed as shown with the example in the Supplementary Figure S3C: the mean baseline EPSC amplitude was 136 ± 32 pA before pairings, was increased by 93% to 263 ± 37 pA 40 min after pairings and was further increased by 116% (compared with baseline) to 294 ± 32 pA after picrotoxin application (1 h after pairings). In summary, picrotoxin did not impair tLTP (153.1 ± 13.9%, p = 0.0089, n = 7, before picrotoxin and 198.3 ± 13.8%, p = 0.0004, n = 7; 7/7 cells displayed tLTP) but induced an increase in the tLTP magnitude (p = 0.0350; Figure 5), illustrating that GABAergic microcircuits exert an inhibitory brake on eCB-tLTP.
Altogether, our results demonstrate that 10 pairings-tLTP is mediated by both CB 1 R and TRPV1 activation.

DISCUSSION
In rodent neocortex, we report here the existence of a Hebbian coincidence-activity dependent LTP induced by a low number of pairings (∼10), which involved the eCB system. eCB-tLTP induction relies on activation of CB 1 R and TRPV1 triggered by coupled rises of calcium mediated by mGluR5 (via PLCß, diacylglycerol lipase-α activation and calcium released from internal stores) and VSCCs. Most of the steps of eCB synthesis and release (mainly 2-AG and anandamide) tightly depend on postsynaptic calcium levels (and time course; Piomelli et al., 2007;Di Marzo, 2008;Kano et al., 2009;Alger and Kim, 2011). Due to their on-demand intercellular signaling (Piomelli et al., 2007;Alger and Kim, 2011), eCB action is expected to be controlled by precisely timed stimuli. Here we show that STDP, a Hebbian synaptic learning rule (Dan and Poo, 2006;Sjöström et al., 2008;Feldman, 2012;Froemke, 2015), efficiently triggers eCB signaling, even for a low number of pairings, and can promote the expression of eCB-tLTP. Therefore, in addition, to the widespread eCB-LTD, eCBs can aslo mediate potentiation. Bidirectionality of eCB-plasticity is a crucial property of eCB system because it allows eCB-LTP and -LTD to reverse each other at a single synapse. We have previously proposed a mechanism accounting for corticostriatal eCB-tLTP using a combination of patch-clamp recordings and a mathematical model (Cui et al., 2016). In our model, low to moderate peak levels of eCB would lead to tLTD whereas high eCB levels would yield tLTP. It is thus expected that the first 10 pairings produce large peak levels of eCB synthesis, thus inducing tLTP. If the amplitude of the 2-AG peaks decreases for subsequent pairings, this initial tLTP would be de-potentiated; its expression would be thus restricted for few coincident activity and eCB would have no significant impact on tLTP induced with 100 pairings. Supporting this, it has been reported that in the somatosensory cortex NMDAR-tLTP expression (induced with 100 pre-post pairings) was not modified after the inhibition of CB 1 Rs Nevian and Sakmann, 2006).
We observed eCB-tLTP in both somatosensory cortex and in dorsolateral striatum, demonstrating that this form of plasticity is not restricted to a single type of synapses, but could serve as a general system in various brain structures to allow the engram of salient events from few spikes. Neocortical eCB-tLTP is similar to those we recently described in the dorsal striatum (Cui et al., 2015(Cui et al., , 2016 with the noticeable difference that this new form of plasticity displays, a distinct polarity depending on its synaptic site of expression: Hebbian in neocortex and anti-Hebbian in striatum. Interestingly, eCB-tLTP only requires a single postsynaptic spike per paired stimulation in the dorsal striatum but 2-3 in the neocortex, possibly consistent with the general observation that learning dynamics are usually faster in sub-cortical structures than in the neocortex (Pasupathy and Miller, 2005). It remains to be investigated whether neuromodulators control eCB-tLTP expression and/or polarity, as reported for dopamine for the control of the NMDAR-tLTP in prefrontal or visual cortex (Seol et al., 2007;Xu and Yao, 2010). Furthermore, it has been reported that in a STDP paradigm noradrenaline and serotonin transform eligibility traces into plasticity in the visual cortex (He et al., 2015). In the same line, it is important to explore whether neuromodulators could promote the emergence of eCB-tLTP for lower number of pairings or with a single backpropagating action potential (instead of 2-3 as it is the case in the present study).
eCB system is deeply involved in learning and memory (Mechoulam and Parker, 2013) because its major role in synaptic plasticity expression (Augustin and Lovinger, 2018). Indeed, eCBs (2-AG and anandamide) have been extensively reported to mediate short-or long-term depression, via the activation of CB 1 R (Kano et al., 2009;Castillo et al., 2012;Katona and Freund, 2012;Melis et al., 2014) or TRPV1 (Gibson et al., 2008;Maione et al., 2009;Chávez et al., 2010;Grueter et al., 2010;Puente et al., 2011). Nevertheless, it exists now as a growing body of evidence showing an indirect role of eCBs in promoting short-or LTP (reviewed in: Araque et al., 2017;Augustin and Lovinger, 2018): at mixed synapses of the goldfish Mauthner cell via intermediary dopaminergic neurons (Cachope et al., 2007) or at CA1 synapses in hippocampus via a GABA A receptormediated mechanism (Lin et al., 2011;Xu et al., 2012). It has also been reported in the hippocampus that eCB-induced presynaptic depression of GABAergic transmission facilitates LTP (Carlson et al., 2002;Chevaleyre and Castillo, 2004;Zhu and Lovinger, 2007), or that eCBs mediate heterosynaptic short-term potentiation via intermediary astrocytes (Navarrete and Araque, 2010). More recently, it has been reported a direct role of eCBs in promoting LTP at cortical inputs to the granule cells of the dentate gyrus (Wang et al., 2016(Wang et al., , 2018, to the mediumsized spiny neurons of the dorsolateral striatum (Cui et al., 2015(Cui et al., , 2016 or to the basal dendrites of layer 5 pyramidal cells (in this later case the eCB-LTP is also BDNF-and NMDARmediated; Maglio et al., 2018). At hippocampal CA1 synapses, eCB-mediated LTP induced with high-frequency (Lin et al., 2011), low-frequency (Zhu and Lovinger, 2007) or paired (Xu et al., 2012) stimulations were prevented by inhibition of CB 1 R and GABA A receptors. Here, we show at neocortical synapses that GABA, which does not affect EPSC amplitude during baseline, controls the plasticity magnitude by exerting a brake on eCB-tLTP. In line with previous studies showing the existence of eCB-LTP at striatal (Cui et al., 2015(Cui et al., , 2016, hippocampal (Wang et al., 2016(Wang et al., , 2018 and cortical (Maglio et al., 2018) synapses, the present neocortical eCB-tLTP constitutes an example of a paired-activity eCB-LTP with a direct implication of eCBs (see i-THL experiments) in the induction of tLTP of the stimulated synapse itself. It remains to be determined whether eCB-tLTP expression could be extended to other brain structures and synapses.
Evidence for TRPV1 activation by physiological neuronal activity remains unclear. In rodent neocortex, it is fair to say that the expression of TRPV1 in pyramidal cells was debated. Indeed, although an immunoelectronmicroscopy study reported TRPV1 protein in neocortex at the postsynaptic dendritic spines of pyramidal cells (Tóth et al., 2005), a multi-approach investigation (in situ hybridization, calcium imaging) detects TRPV1 only at the level of the arteriolar smooth muscle cells (Cavanaugh et al., 2011). Recent studies using real-time PCR and western blot (Huang et al., 2014) and patch-clamp recordings (Pezzoli et al., 2014) showed expression of TRPV1 by pyramidal cells of the neocortex. Here, EPSC recordings, before and after TRPV1 agonist (capsaicin) application, show a marked decrease of EPSC amplitude in presence of capsaicin, in line with previous observations (Pezzoli et al., 2014). In addition, we found that two competitive TRPV1 antagonists prevent eCB-tLTP expression. As previously described in striatum (Cui et al., 2015), our study confirms that STDP efficiently triggers eCB signaling and is able to recruit the TRPV1 signaling pathway. TRPV1 is a cationic channel highly permeable to calcium (Starowicz et al., 2007;Di Marzo, 2008) and may contribute to eCB-tLTP induction by boosting the calcium transients. Here, TRPV1 is most likely activated by anandamide arising from the stimulated postsynaptic cell. In the dorsolateral striatum, anandamide appears necessary but not sufficient for tLTP induction (Cui et al., 2016). In the neocortex, it remains to be determined whether anandamide alone could trigger eCB-tLTP induction following limited occurrences of coincident activity. As described for eCB-mediated LTD (Puente et al., 2011), our results illustrate the bidirectionality of eCBs as a system exhibiting polymodal activation through CB 1 R and TRPV1, to induce LTD and LTP.
The neocortex receives a broad range of cortical activity patterns, from isolated trains of few spikes to sustained bursting events. At neocortical layer 4-2/3 synapses, STDP exhibits symmetric Hebbian tLTP during the first two postnatal weeks (Itami and Kimura, 2012), then asymmetric Hebbian STDP (Feldman, 2000;Sjöström et al., 2001Sjöström et al., , 2003Froemke et al., 2006;Nevian and Sakmann, 2006;Banerjee et al., 2014); a lack of tLTD has been reported for older animals (Banerjee et al., 2009 but see Min and Nevian, 2012). It should be noted that depending on the site of stimulation (layer 4 or at within layer 2/3), the temporal window and the locus of NMDAR involved of tLTD are different: for post-pre pairings, stimulation in layer 4 induced a tLTD dependent on presynaptic NMDAR which is expressed in a broad ∆t STDP , whereas stimulation within layer 2/3 triggered a tLTD dependent on postsynaptic NMDAR which is expressed in a more restricted ∆t STDP (Banerjee et al., 2014). Although cortical plasticity under prolonged activation (lowand high-frequency stimulations, theta bursts or 100 pairings STDP) is well elucidated, its expression in response to few spikes remained elusive. Nevertheless, it has been observed that dendritic spike(s) induced by single-shock and single-burst were responsible for, respectively, LTD in visual cortex (Holthoff et al., 2004) and LTP in hippocampus (Remy and Spruston, 2007). These both plasticity was NMDAR-mediated and could thus account for single-trial learning. eCB-LTP is promoted by about 10 of pairings, allowing for the synapses to react to the first occurrences of incoming activity. In the same line, ∼20-25 STDP pairings induced LTP in hippocampal neurons (Zhang et al., 2009) or at layer 2/3 cortical pyramidal cells (Froemke et al., 2006); interestingly, in hippocampal neurons bath-application of dopamine allows the induction of tLTP with a lower number of pairings (10 instead of 20 pairings; Zhang et al., 2009). Associative memories and behavioral rules can be learned with few trials (5-10) or even with one trial (Schultz et al., 2003;Pasupathy and Miller, 2005;Armstrong et al., 2006;Rutishauser et al., 2006;Whitlock et al., 2006;Tse et al., 2007;Quilodran et al., 2008;Cook and Fagot, 2009;Ito and Doya, 2009;Izquierdo et al., 2016). Upon behaviorally pertinent events, neurons with behavior-related activities fire a few spikes during each trial (one to a dozen) upon each trial (i.e., a discharge at frequency 5-10 Hz during 0.1-0.5 s; Schultz et al., 2003;Pasupathy and Miller, 2005;Quilodran et al., 2008). This suggests that a low number (2-50) of spikes should be sufficient for the expression of synaptic plasticity. The present results suggest that eCB-tLTP could be involved for learning salient events from a low number of action potentials and may constitute a neuronal substrate for single-trial or online learning, such as cortical episodic memory. It remains to be investigated if eCB-tLTP occurs in vivo and to evaluate the involvement of eCB-tLTP in the initial phases of online learning, which could be thereafter reinforced by NMDAR-LTP when stimuli are subsequently repeated.
Frequent cannabis use leads to impairment of working memory in the left superior parietal cortex (Jager et al., 2006) as well as long-term memory via activation of CB 1 R (Mechoulam and Parker, 2013;Augustin and Lovinger, 2018). This impairment was mainly interpreted as the effect of cannabinoids on the induction of short-and long-term synaptic depression. Our results, in line with other studies (Lin et al., 2011;Xu et al., 2012;Cui et al., 2015;Wang et al., 2016;Maglio et al., 2018), indicate that potentiation of synaptic transmission may also be involved in the effects of marijuana intoxication.

AUTHOR CONTRIBUTIONS
LV, YC and SP: conception and design of the experiments. YC and SP performed electrophysiological experiments. YC, SP and LV performed data analysis. LV: writing of original draft with contributions from YC.

FUNDING
This work has been supported by grants from INSERM, Collège de France, the Agence Nationale pour la Recherche (ANR-CRCNS; award id: Dopaciumcity), LabEx MemoLife and the Ecole des Neurosciences de Paris.

ACKNOWLEDGMENTS
We thank C. Ledent (ULB, Brussels, Belgium) for kindly providing CB 1 R −/− mice and their CB 1 R +/+ littermates. We thank the LV lab members for helpful suggestions and critical comments.