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. 2012 Mar 12;590(Pt 10):2305–2315. doi: 10.1113/jphysiol.2011.223511

Long-lasting potentiation of hippocampal synaptic transmission by direct cortical input is mediated via endocannabinoids

Jian-Yi Xu 1, Jian Zhang 1, Chu Chen 1
PMCID: PMC3424754  PMID: 22411015

Abstract

Hippocampal CA1 pyramidal neurons receive sensory inputs from the entorhinal cortex directly through the perforant path (PP) and indirectly through Schaffer collaterals (SC). Direct cortical inputs to CA1 pyramidal neurons through the PP provide instructive signals for hippocampal long-term synaptic plasticity. However, the molecules conveying synaptic signalling in this new form of heterosynaptic plasticity remain unclear. Endocannabinoids, important endogenous signalling mediators, modulate synaptic efficacy primarily through inhibition of GABAergic or glutamatergic synaptic transmission via presynaptically expressed CB1 receptors. Here, we report that pairing of direct and indirect cortical inputs to CA1 pyramidal neurons resulted in a long-lasting potentiation of synaptic responses at SC synapses, but not at the PP. The pairing-potentiated synaptic transmission at the SC was accompanied by a reduced ratio of paired-pulse facilitation (PPR). Enhanced synaptic response at the SC by pairing of PP–SC stimuli is Ca2+ dependent and requires the presence of functional GABAergic and glutamatergic synaptic transmissions and activation of group I metabotropic glutamate receptors. Pharmacological inhibition or genetic deletion of the CB1 receptor eliminated the pairing-induced long-term synaptic plasticity and decreased PPR at the SC. The potentiation induced by pairing of PP–SC stimuli primarily is the glutamatergic synaptic transmission. While the pairing-induced long-lasting potentiation of synaptic response was blocked by inhibitors for diacylglycerol lipase (DGL), which biosynthesizes 2-AG, inhibition of monoacylglycerol lipase (MAGL), which metabolizes 2-AG, facilitated the potentiation at SC synapses by pairing of weak PP–SC stimuli. Our results suggest that 2-AG functions as a signalling mediator tuning synaptic efficacy at the proximal synapses of hippocampal CA1 pyramidal neurons while direct and indirect cortical inputs to the same neurons are spatiotemporally primed.

Key points

  • Hippocampal CA1 pyramidal neurons receive dual sensory inputs from the cortex directly through the perforant path (PP) and indirectly through the Schaffer collaterals (SC).

  • Direct cortical inputs to CA1 pyramidal neurons through the PP are important for synaptic plasticity and memory formation.

  • In this study, we show that long-lasting potentiation of glutamatergic synaptic transmission at SC synapses by pairing of PP–SC inputs was suppressed by pharmacological and genetic inhibition of CB1 receptors.

  • Inhibition of the enzyme synthesizing the endocannabinoid 2-arachidonoylglycerol (2-AG) prevented the pairing-induced potentiation, while inhibition of the enzyme hydrolysing 2-AG facilitated the potentiation.

  • Our results indicate that 2-AG functions as a signalling mediator tuning synaptic efficacy at the proximal synapses of hippocampal CA1 pyramidal neurons while direct and indirect cortical inputs to the same neurons are spatiotemporally primed, suggesting that endocannabinoids are involved in the information processing and storage in the hippocampus.

Introduction

Information processed in hippocampal CA1 pyramidal neurons from the cortex is through the perforant path (PP) pathway, which makes synapses at the distal dendrites of pyramidal neurons in the stratum lacunosum-moleculare (SLM), and the Schaffer collateral (SC) pathway, which makes synapses at more proximal dendrites in the stratum radiatum (SR). While neurons in layer III of the entorhinal cortex directly project (the PP) to the CA1 region, neurons in layer II of the entorhinal cortex send sensory inputs to CA1 neurons indirectly through the trisynaptic path, which consists of the PP dentate granule neuron synapses, mossy fibre CA3 neuron synapses and SC CA1 neuron synapses. The dual sensory inputs the CA1 pyramidal neurons receive are essential for information processing, consolidation, storage and retrieval in the hippocampus (Eichebaum, 2001; Remondes & Schuman, 2002, 2004; Nolan et al. 2004). For instance, direct cortical input to the CA1 through the PP pathway is required for long-term synaptic plasticity and memory formation (Remondes & Schuman, 2004; Nolan et al. 2004; Brun et al. 2008). In addition, LTP- or LTD-inducing stimulation at the PP modulates synaptic plasticity at the SC (Colbert & Levy, 1993; Remondes & Schuman, 2002; Izumi & Zorumski, 2008). Recent studies demonstrate that pairing of PP and SC inputs induces an input-timing-dependent plasticity at the SC in CA1 pyramidal neurons, revealing a new form of heterosynaptic plasticity at the SC synapses when the neurons receive instructive signals from direct cortical inputs in the same CA1 pyramidal neurons (Dudman et al. 2007). However, the biochemical messengers conveying synaptic signalling in this new form of hippocampal long-term synaptic plasticity remain unclear.

CA1 pyramidal neurons show different expression and function of ion channels and receptors at synapses between distal SLM and proximal SR regions (Otmakhova et al. 2002; Arrigoni & Greene, 2004; Nolan et al. 2004; Nicholson et al. 2006; Ahmed & Siegelbaum, 2009). Correspondingly, the neurons display distinct synaptic responses to neurotransmitters between PP and SC synapses (Hasselmo & Schnell, 1994; Otmakhova & Lisman, 1998, 2000; Otmakhova et al. 2005; Xu et al. 2010), suggesting that synaptic efficacy at distal and proximal dendrites is differentially modulated by neurotransmitters or modulators.

Endocannabinoids are endogenous signalling mediators involved in a variety of physiological and pathological processes. Modulation of synaptic efficacy by endocannabinoids occurs primarily through their actions on presynaptically expressed CB1 receptors, both in GABAergic and glutamatergic synapses in the brain (Chevaleyre et al. 2006; Lovinger, 2008; Alger, 2009; Kano et al. 2009). Here we report that pharmacological inhibition or genetic deletion of the CB1 receptor eliminated long-lasting potentiation of synaptic response at SC synapses by pairing of distal and proximal synaptic inputs to the same CA1 pyramidal neurons. The pairing-induced long-term synaptic plasticity was blocked by inhibition of diacylglycerol lipase (DGL), an enzyme that biosynthesizes 2-AG. In contrast, strengthening 2-AG by inhibition of monoacylglycerol lipase (MAGL), a key enzyme that hydrolyses brain 2-AG, facilitated the potentiation by pairing of weak PP–SC stimuli. In addition, we provided evidence that the glutamatergic synaptic transmission was potentiated by pairing of PP–SC inputs. Our results suggest that 2-AG functions as a signalling molecule tuning synaptic efficacy at the proximal synapses of CA1 pyramidal neurons while direct and indirect cortical inputs to the same neurons occur with precision priming.

Methods

Hippocampal slice preparation

Hippocampal slices were prepared from Sprague–Dawley rats (Charles River, Wilmington, MA, USA) and CB1 receptor knockout (KO) mice (cnr1−/−, NIMH transgenic core, NIH, Bethesda, MD, USA) as described previously (Chen et al. 2002; Chen & Bazan, 2005; Sang et al. 2005; Xu et al. 2010). CB1R KO mice were backcrossed for more than 10 generations onto the C57BL/6 background strain. Breeding of heterozygous mice produced homozygous CB1R wild-type (WT) and KO mice. Age-matched littermates (either sex) were used in all the studies. Both rats and mice from 4 to 8 weeks were used for the experiments. The care and use of the animals reported in this study were approved by the Institutional Animal Care and Use Committee of the Louisiana State University Health Sciences Center and conform to the principles of UK regulations, as described in Drummond (2009). Briefly, animals were rapidly decapitated after anaesthesia with isoflurane (3% in a mixture of nitrous oxide and oxygen) using a small animal guillotine. Then, brains were rapidly removed and placed in cold oxygenated (95% O2–5% CO2) low-Ca2+/high-Mg2+ slicing solution composed of (in mm): 2.5 KCl, 7.0 MgCl2, 28.0 NaHCO3, 1.25 NaH2PO4, 0.5 CaCl2, 7.0 glucose, 3.0 pyruvic acid, 1.0 ascorbic acid and 234 sucrose. Slices were cut at a thickness of 400 μm and transferred to a holding chamber in an incubator containing oxygenated artificial cerebrospinal fluid (ACSF) composed of (in mm): 125.0 NaCl, 2.5 KCl, 1.0 MgCl2, 25.0 NaHCO3, 1.25 NaH2PO4, 2.0 CaCl2, 25.0 glucose, 3 pyruvic acid and 1 ascorbic acid at 36°C for 0.5–1 h, and maintained in an incubator containing oxygenated ACSF at room temperature (∼22–24°C) for at least 1.5 h before recordings. Slices were then transferred to a recording chamber where they were continuously perfused with the 95% O2–5% CO2-saturated standard ACSF at ∼32–34°C. Individual CA1 pyramidal neurons were viewed with a Zeiss Axioskop microscope and fitted with a 60× water-immersion objective and differential interference contrast (DIC) optics.

Electrophysiological recordings

Whole-cell patch-clamp recordings were made using an Axopatch-200A patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) under voltage clamp. Pipettes (2–3 MΩ) were pulled from borosilicate glass with a micropipette puller. The internal pipette solution contained (in mm): 90.0 CsCH3SO3, 40.0 CsCl, 10.0 Hepes, 5.0 CaCl2, 4.0 Mg2ATP, 0.3 Na2GTP and 5.0 QX-314, or 130 KCH3SO4, 10 KCl, 4 NaCl 10 Hepes, 0.1 EGTA, 4 Mg2ATP, 0.3 Na2GTP and 5 QX-314. The membrane potential was held at −70 mV. Whole-cell postsynaptic currents (referred to as synaptic response) were recorded in the soma in response to stimuli of Schaffer collateral synapses (SC) in the stratum radiatum (SR) and perforant path (PP) synapses in the stratum lacunosum-moleculare (SLM) independently (500 to 800 ms apart) at a frequency of 0.05 Hz via bipolar tungsten electrodes. The SLM was identified by PP axons. To ensure that the PP and SC are independently activated by electrical stimulation with electrodes placed at the SR and SLM, we used paired-pulses or tri-pulses to deliver stimuli at SC–PP or PP–SC, as described previously (Xu et al. 2010). If two stimuli delivered from the two stimulation electrodes act on the same pathway, then the second stimulation will induce facilitation (paired-pulse facilitation). In the absence of GABA or glutamate receptor antagonists in the external solution, recorded total whole-cell postsynaptic currents contained both GABA and glutamate receptor-gated currents. To determine the components of GABA and glutamate receptor-gated currents, we applied 20 μm bicuculline. Bicuculline application resulted in reduction of whole-cell currents by 25.4 ± 8.7% (n = 6) based on the external and internal solutions used in recordings. The protocol for pairing of PP and SC synaptic inputs was adopted from Dudman et al. (2007), and consisted of pairs of proximal and distal stimuli (1 Hz for 90 s) in which the distal stimulus preceded the proximal stimulus by 20 ms. Paired-pulse facilitation was induced by delivering two pulses with an inter-pulse interval of 80–100 ms (Chen et al. 2002). The paired-pulse ratio (PPR) was calculated as P2/P1 (P1, the amplitude of the first EPSC; P2, the amplitude of the second EPSC). Bicuculline (20 μm), SR95531 (gabazine, 1 μm) plus CGP55845 (2 μm), DNQX (10 μm) or NBQX (10 μm) plus AP5 (50 μm) were added to the external solution to block GABAergic synaptic transmission, or glutamatergic synaptic transmission in some experiments. In another set of experiments, field excitatory postsynaptic potentials (fEPSPs) at SC synapses were recorded as described previously (Fan et al. 2010). The recording pipettes were filled with ACSF.

Data were presented as mean ± SEM. Unless stated otherwise, Student's t test and analysis of variance (ANOVA) with Student–Newman–Keuls test were used for statistical comparison when appropriate. Differences were considered significant when P < 0.05.

Results

Pairing of PP and SC inputs induces long-lasting potentiation of synaptic transmission at the SC

As shown in Fig. 1A, pairing of distal and proximal stimuli with low frequency (1 Hz for 90 s) resulted in long-lasting potentiation of synaptic response at the SC (187.4 ± 17.6% of base, n = 14), but not at the PP in rat hippocampal slices (112.1 ± 11.6% of base, n = 10). This is consistent with the reports by Dudman et al. (2007). To determine pre- or post-synaptic-mediated mechanisms, we measured PPR. Pairing of PP and SC stimuli resulted in a significant decrease in PPR at the SC (P < 0.01, n = 8, Fig. 1B). However, pairing of SC and SC stimuli did not induce a long-lasting potentiation of the synaptic response or alter the PPR at the SC (Fig. 1C). This means that the pairing-potentiated synaptic response at the SC results from direct cortical input through the PP (Dudman et al. 2007).

Figure 1. Pairing of distal and proximal synaptic inputs induces long-lasting potentiation of synaptic response at the proximal Schaffer collateral (SC) synapses.

Figure 1

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Inset, recording setup. One stimulating electrode was placed in the site of the distal perforant path (PP) in the SLM, and another stimulating electrode was placed in the site of SC synapses in the SR. Somatic recordings under the whole-cell voltage clamp mode were made in CA1 pyramidal neurons. Aa, representative traces of whole-cell postsynaptic currents in response to independent PP and SC stimuli recorded from the same rat hippocampal CA1 pyramidal neurons before and after pairing of PP–SC stimuli. Ab, time courses of PP–SC pairing-induced changes in postsynaptic response at the PP and SC. Ac, mean values of synaptic response averaged from 36 to 40 min following pairing of PP–SC stimuli. **P < 0.01, compared with SC. Ba, representative traces of whole-cell postsynaptic currents recorded at SC before and after PP–SC pairing stimuli. Bb, time courses of normalized PPR before and after PP–SC pairing. PPR was normalized to the baseline. Bc, mean values of PPR before and after PP–SC pairing. **P < 0.01 compared with baseline. Ca, representative traces of whole-cell postsynaptic currents recorded at SC before and after pairing of SC–SC stimuli. Inset, recording setup. One stimulating electrode was placed in one site of the SC, and another stimulating electrode was placed in the other site of SC. Cb, time courses of whole-cell postsynaptic currents before and after SC–SC pairing. Cc, mean values of normalized PPR at the SC before and after SC–SC pairing.

Facilitation of synaptic transmission at the SC by pairing is Ca2+ dependent and requires activation of group I metabotropic glutamate receptors

As demonstrated before (Dudman et al. 2007), potentiation of synaptic response at the SC by pairing of PP and SC inputs depends on a rising intracellular Ca2+ and activation group I metabotropic glutamate receptors (mGluRs). To confirm these observations in our system, we first used MPEP (20 μm) and LY367385 (100 μm) to block group I mGluRs. As shown in Fig. 2A, blockade of group I mGluRs eliminated the pairing-potentiated synaptic transmission at the SC. We also included BAPTA (20 mm), a Ca2+ chelater, in recording patch pipettes. Decrease of intracellular Ca2+ also diminished the potentiation (Fig. 2B). These results confirm that long-lasting potentiation of synaptic response at the SC by pairing of PP and SC stimuli is Ca2+- and group I mGluR-dependent (Dudman et al. 2007). As mentioned in the Methods section, recorded total whole-cell currents contained both the GABA and glutamate receptor-gated currents. To determine whether glutamate receptor-gated currents were potentiated by pairing of PP and SC stimuli, we applied bicuculline to isolate the glutamate receptor-gated currents. As seen in Fig. 2C, PP–SC pairing-induced potentiation was absent in the external solution containing bicuculline (20 μm), indicating that potentiation of synaptic transmission at the SC by pairing of PP and SC inputs requires the presence of functional GABAergic synaptic transmissions (but see Dudman et al. 2007). To confirm this speculation, we used SR95531 (gabazine) plus CGP55845, which were used in the report by Dudman et al. (2007). However, PP–SC pairing stimulation still failed to potentiate EPSCs in the presence of gabazine (GABAA antagonist) and CGP55845 (GABAB receptor antagonist). This is consistent with the data from the experiment where bicuculline was used. It has been shown that a priming stimulation that induces long-term depression at inhibitory synapses (I-LTD) also potentiates LTP at the SC (Chevaleyre & Castillo, 2004; Zhu & Lovinger, 2007). To determine whether pairing of PP–SC stimuli results in I-LTD at the SC, we isolated IPSCs by application of DNQX (10 μm) plus AP5 (50 μm) in the external solution. As shown in Fig. 2D, PP–SC pairing did not induce I-LTD.

Figure 2. Potentiation of synaptic response at the SC by pairing of PP–SC inputs is Ca2+ dependent and requires activation of group I metabotropic glutamate receptors (mGluRs) and presence of functional GABAergic synaptic transmission.

Figure 2

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Aa, representative traces of whole-cell postsynaptic currents recorded at PP and SC from the rat hippocampal CA1 pyramidal neurons before and after pairing of PP–SC stimuli in the presence of group 1 mGluR antagonists MPEP (20 μm) and LY367385 (100 μm). Ab, time courses of synaptic response in the presence of group 1 mGluR antagonists. Ac, mean values of synaptic response averaged from 36 to 40 min following PP–SC pairing. Ba, time courses of synaptic response recorded from neurons treated with BAPTA (20 mm). BAPTA was dialysed through the recording patch pipette for at least 10 min before starting recordings. Bb, mean values of synaptic response averaged from 36 to 40 min following PP–SC pairing. C, time courses of EPSCs at the SC in slices treated with bicuculline (20 μm) or SR95531 (1 μm) + CGP55845 (2 μm). D, time courses of IPSCs at the SC in slices treated with DNQX (10 μm) + AP5 (50 μm). Pairing of PP–SC stimuli does not induce I-LTD at the SC synapses.

Glutamatergic synaptic transmission is potentiated by pairing of PP and SC inputs

To determine which component is potentiated by pairing of PP–SC stimuli, we used NBQX plus AP5 10 min after pairing of PP–SC stimuli. For comparison, we also made recordings where pairing of PP–SC stimuli was not delivered before application of NBQX and AP5. As shown in Fig. 3, 90% of the currents were inhibited by NBQX (10 μm) plus AP5 (50 μm) 10 min after application of NBQX–AP5 and there were no significant differences in the remaining currents (GABA currents) in the absence and presence of PP–SC pairing stimuli, indicating that the potentiation induced by PP–SC pairing stimulation primarily is the glutamatergic synaptic transmission.

Figure 3. Glutamatergic synaptic transmission is potentiated by pairing of PP–SC stimuli.

Figure 3

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A, representative traces of whole-cell postsynaptic currents recorded at SC synapses before and after NBQX + AP5 in the absence and presence of pairing of PP–SC synaptic inputs. NBQX (10 μm) + AP5 (50 μm) were applied 10 min after pairing stimulation. Pairing of PP–SC stimuli were not delivered in one group of rat hippocampal slices. B, time courses of synaptic response at SC synapses before and after NBQX + AP5 in the absence and presence of pairing of PP–SC synaptic inputs.

Long-lasting potentiation of synaptic response at the SC by pairing of PP and SC is CB1 receptor dependent

It has been well documented that endocannabinoids function as a retrograde messenger released from postsynaptic sites to modulate synaptic efficacy by acting on presynaptic CB1 receptors. Since endocannabinoid synthesis requires a rise in intracellular Ca2+ and activation of group I mGluRs is one of the important mechanisms underlying endocannabinoid-mediated synaptic modification (Chevaleyre et al. 2006), we wondered whether the endocannabinoid system is involved in the PP–SC pairing-induced potentiation of hippocampal synaptic transmission since the pairing-induced potentiation also depends on intracellular Ca2+ and group I mGluR activation (Dudman et al. 2007). To test this hypothesis, we used mice deficient in CB1 receptors to determine the involvement of the endocannabinoid system in the pairing-induced potentiation. While PP–SC pairing stimuli still induced the long-lasting potentiation and reduced PPR in age-matched wild-type (WT) mice, the pairing failed to induce potentiation and alter PPR in CB1 receptor knockout (KO) mice (Fig. 4A). Then, we used Rimonabant (RIM, SR141716, a selective CB1 receptor antagonist) to further confirm the importance of the CB1 receptor in the pairing-induced potentiation. As shown in Fig. 4Ba, PP–SC pairing-induced potentiation was prevented in rat slices pretreated with RIM (2 μm) and with continued perfusion during recordings. These results suggest that the endocannabinoid system is indeed involved in the direct cortical input-potentiated synaptic transmission at the proximal synapses of CA1 pyramidal neurons. To determine whether the endocannabinoid signalling contributes to induction or maintenance of the potentiation, we applied RIM (2 μm) 10 min after pairing of PP and SC stimuli in fEPSP recordings. As seen in Fig. 4Bb, inhibition of the CB1 receptor did not prevent the potentiation when RIM was washed in 10 min after the pairing stimulation, suggesting that actions of the endocannabinoid signalling on synaptic potentiation occur only during pairing of PP and SC inputs.

Figure 4. Potentiation of synaptic transmission at SC synapses by pairing of PP–SC synaptic inputs is mediated by the CB1 receptor.

Figure 4

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Aa, representative traces of whole-cell postsynaptic currents recorded at the SC synapses before and after pairing of PP–SC synaptic inputs in CA1 pyramidal neurons of CB1 receptor knockouts (KO) and their age-matched wild-type controls (WT). Ab, time courses of changes in synaptic response at the SC synapses in WT and KO mice. Ac, mean values of synaptic response potentiation averaged from 36 to 40 min following PP–SC pairing. **P < 0.01 compared with WT at the SC. Ad, mean value of normalized PPR at the SC before and after PP–SC pairing recorded from WT and KO mice. *P < 0.05 compared with the baseline. Ba, time courses of synaptic response recorded at the PP and SC in rat CA1 pyramidal neurons in the presence of Rimonabant (RIM, 2 μm). RIM was pre-treated and continuously perfused during recordings. Bb, time courses of fEPSPs recorded at the SC in rat CA1 pyramidal neurons. RIM (2 μm) was applied 10 min following PP–SC pairing stimulation. There are no differences in the potentiation between RIM and the control.

2-AG mediates PP–SC pairing-facilitated synaptic transmission

Growing evidence suggests that 2-AG is probably a retrograde messenger in modulation of both GABAergic and glutamatergic synaptic activities (Makara et al. 2005; Chevaleyre et al. 2006; Szabo et al. 2006; Kano et al. 2009; Gao et al. 2010). To determine whether 2-AG is a signalling molecule mediating pairing-induced potentiation, we used RHC-80267 (RHC) and tetrahydrolipstatin (THL), inhibitors of diacylglycerol kinase, which synthesizes 2-AG. As shown in Fig. 5A and B, extracellular application of RHC (100 μm) or intracellular application of THL (5 μm) through the recording pipette blocked the pairing-induced potentiation, suggesting that 2-AG may be a signalling mediator in this postsynaptically induced and presynaptically expressed heterosynaptic plasticity. We then looked to see if strengthening 2-AG signalling by inhibiting its metabolism would augment pairing-induced potentiation. To this end, we used JZL184, a potent and selective inhibitor for MAGL that hydrolyses 2-AG (Long et al. 2009a,b; Pan et al. 2009). As seen in Fig. 6A, pairing of PP–SC inputs potentiated synaptic transmission at the SC in rat slices treated with JZL184 (1 μm), but the potentiation was not further augmented when compared with the control (Control: 187.4 ± 17.6% of base versus JZL184: 200.5 ± 19.0% of base, P > 0.05). It has been reported that JZL184 is more potent in inhibiting MAGL in mouse and human tissues than in rat tissue (Long et al. 2009b; Pan et al. 2009). To determine whether JZL184 augments the potentiation by pairing stimulation, we made recordings in wild-type mouse hippocampal slices. As shown in Fig. 6A, it seems that JZL184 increased the potentiation in mouse slices when compared with that in rats. However, JZL184 essentially did not further enhance the potentiation in mouse slices when compared with the potentiation in control mouse slices. This information suggests that 2-AG released during pairing stimulation may be sufficient to strengthen synaptic efficacy at the SC synapses. Our results also suggest that the magnitude of potentiation in mice was greater than that in rats (271.1 ± 37.3%versus 187.4 ± 17.7%, P < 0.01). This may account for the differences in the magnitude of potentiation between ours and others (Dudman et al. 2007). To further delineate the involvement of 2-AG signalling in the pairing-induced synaptic potentiation, we used a weak or ‘milder’ pairing stimulation protocol by reducing stimulation duration from 90 s to 60 s in mouse hippocampal slices. As shown in Fig. 6B, while pairing of weak PP–SC stimuli induced a small potentiation (128.8 ± 12.2%) in the absence of JZL184, it induced a relatively greater potentiation (169.9 ± 14.5%, P < 0.05) in the presence of JZL184 (1 μm). These results suggest that 2-AG signalling may mediate the initiation process of synaptic potentiation at SC synapses when direct and indirect inputs are spatiotemporally primed.

Figure 5. Inhibition of 2-AG synthesis eliminates the potentiation of synaptic transmission by pairing of PP–SC stimuli.

Figure 5

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Aa, representative traces of whole-cell postsynaptic currents recorded before and after PP–SC pairing at the PP and SC in the presence of RHC-80267 (RHC, 100 μm) in rat hippocampal CA1 pyramidal neurons. Ab, time courses of synaptic response recorded before and after pairing at the PP and SC in the presence of RHC. Ba, representative traces of whole-cell postsynaptic currents recorded before and after PP–SC pairing at the PP and SC in the presence of tetrahydrolipstatin (THL, 5 μm) in rat CA1 pyramidal neurons. Bb, time courses of synaptic response recorded before and after PP–SC pairing at the PP and SC in the presence of THL.

Figure 6. Inhibition of 2-AG hydrolysis facilitates potentiation by pairing of weak PP–SC stimuli.

Figure 6

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Aa, representative traces of whole-cell postsynaptic currents recorded at SC synapses before and after PP–SC pairing in the presence of JZL184 (1 μm) in rat and mouse hippocampal CA1 pyramidal neurons. Ab, time courses of synaptic response recorded before and after PP–SC pairing at the SC in the presence of JZL184 in rat and mouse hippocampal CA1 pyramidal neurons. Ac, mean values of the potentiation at SC synapses by pairing of PP–SC inputs in rats and mice in the absence and presence of JZL184. Ba, representative traces of whole-cell postsynaptic currents recorded at SC synapses before and after pairing of weak PP–SC stimuli (60 s) in the absence and presence of JZL184 (1 μm) in mouse hippocampal slices. Bb, Time courses of synaptic response recorded before and after PP-SC pairing at the SC in the absence and presence of JZL184 in mouse hippocampal CA1 pyramidal neurons. A weak stimulus protocol was used for pairing of PP and SC synaptic inputs (1 Hz for 60 sec instead of 90 sec). Bc, mean values of the potentiation induced by pairing of weak PP–SC stimuli. *P < 0.05 when compared with the control.

Discussion

Direct cortical inputs to CA1 pyramidal neurons through the PP provide instructive signals for hippocampal long-term synaptic plasticity (Dudman et al. 2007). However, the biochemical messengers mediating this new form of heterosynaptic plasticity are not clear. We demonstrate in this report that pharmacological inhibition or genetic deletion of the CB1 receptor eliminated long-lasting enhancement of synaptic response at SC synapses by pairing of distal and proximal synaptic inputs to the same CA1 pyramidal neurons. The pairing-induced long-term synaptic plasticity at the SC was blocked by inhibition of diacylglycerol lipase (DGL), an enzyme that biosynthesizes the endocannabinoid 2-AG. In contrast, inhibition of MAGL, an enzyme that hydrolyses 2-AG, facilitates the potentiation by pairing of weak PP–SC stimuli. We provide evidence that synaptic transmission at SC synapses potentiated by pairing of PP–SC inputs is glutamatergic, suggesting that 2-AG signalling may target glutamatergic synapses during pairing, resulting in long-lasting synaptic potentiation. Our results suggest that the endocannabinoid 2-AG serves as a signalling mediator tuning synaptic efficacy at hippocampal CA3–CA1 synapses through a CB1 receptor-dependent mechanism when direct and indirect cortical inputs to the same pyramidal neurons are spatiotemporally primed.

It has been well documented that activation of the endocannabinoid system primarily induces inhibitory effects on both GABAergic and glutamatergic synaptic transmission and plasticity. However, endocannabinoid signalling is also capable of facilitating LTP of excitatory synaptic transmission at SC synapses in the hippocampus by a priming stimulation that induces depolarization-induced suppression of inhibition (DSI) or I-LTD (Carlson et al. 2002; Chevaleyre & Castillo, 2004; Zhu & Lovinger, 2007). The phenomenon of potentiated synaptic plasticity observed in the present study may be similar to that reported previously (Carlson et al. 2002; Chevaleyre & Castillo, 2004; Zhu & Lovinger, 2007). For instance, the facilitation occurs at the proximal SC synapses by a primed stimulation, it is long-lasting, group I mGluR- and CB1 receptor-mediated, and it requires the presence of functional GABAergic synaptic transmission (Chevaleyre & Castillo, 2004). The priming effect requires a weak LTP induction stimulus protocol following the priming stimulation (Carlson et al. 2002; Chevaleyre & Castillo, 2004; Zhu & Lovinger, 2007). Similarly, we observed that strengthening 2-AG signalling by inhibition of MAGL facilitates synaptic response when a weak pairing protocol was used. However, there are several differences in terms of the priming stimulus protocols, LTP induction, locations and pathways stimulated between our and previous studies. In the previous studies, for example, DSI- or I-LTD-priming effects are highly localized and input specific in the stratum radiatum region. In the present study, the long-lasting potentiation of synaptic transmission at the SC is induced by pairing of direct cortical input that activates distal synapses of pyramidal neurons in the stratum lacunosum-moleculare and indirect cortical input that activates proximal synapses in the stratum radiatum. There are a few hundred micrometers between the two stimuli that independently activate the PP and SC (Xu et al. 2010). No LTP-inducing stimulation (e.g. theta-burst stimulation (TBS)) is required for the potentiation and no I-LTD is induced at the SC following the pairing stimulations. In particular, we demonstrate that 2-AG functions as the signalling molecule via a CB1 receptor-dependent mechanism in mediating long-lasting potentiation of synaptic transmission at the SC by pairing of PP and SC stimuli. This is consistent with the notion that 2-AG is a primary candidate in endocannabinoid-mediated synaptic modification (Makara et al. 2005; Chevaleyre et al. 2006; Szabo et al. 2006; Kano et al. 2009; Gao et al. 2010).

As discussed above, 2-AG is involved in this new form of hereosynaptic plasticity at the SC by direct cortical inputs. We observed that the pairing-induced potentiation was not suppressed when CB1 receptors were inhibited 10 min following pairing of PP–SC stimulation. This indicates that action of 2-AG signalling in mediating potentiation occurs during the pairing stimulation and might play a role in initiation of the potentiation process. We have shown previously that synaptic response to exogenous application of the cannabinoid WIN55212-2 is smaller at the PP than at the SC. Also expression of the CB1 receptor in the SLM is lower than that in the SR (Xu et al. 2010). Therefore, it is unlikely that the released 2-AG at distal dendrites during pairing of PP and SC stimuli contributes to the potentiation at the proximal SC synapses. Previous studies suggest that the priming stimulation induces endocannabinoid-mediated DSI or I-LTD by suppression of inhibitory GABAergic synaptic transmission (disinhibition), which in turn facilitates excitatory synaptic transmission at the proximal synapses in the stratum radiatum (Carlson et al. 2002; Chevaleyre & Castillo, 2004; Zhu & Lovinger, 2007). We speculate that a rising intracellular Ca2+ concentration, through activation of NMDA and mGluR signalling pathways, triggers synthesis and release of 2-AG in the postsynaptic sites, primarily at the proximal synapses. Spatial and temporal summation of EPSCs/EPSPs, resulting from pairing of PP and SC inputs, prolongs membrane depolarization at proximal dendrites and leads to the release of 2-AG, which acts on presynaptic CB1 receptors on both GABAergic and glutamatergic synapses. Based on our results, the glutamatergic synaptic transmission is potentiated by PP–SC pairing stimulation, thus 2-AG signalling may primarily act on the glutamatergic synapses at the SC. However, we cannot exclude the possibility that 2-AG signalling produced inhibitory effects on GABAergic synapses that are greater than on glutamatergic synapses during pairing of PP–SC inputs, resulting in a facilitation of excitatory synaptic transmission at the SC because the density of CB1 receptors at GABAergic terminals is higher than that at glutamatergic (Kano et al. 2009; Pertwee et al. 2010). The precise signal transduction downstream of the CB1 receptor for the 2-AG-mediated long-lasting potentiation of synaptic transmission by pairing of PP and SC inputs remains to be determined.

Direct cortical input to CA1 through the PP pathway is required for hippocampal long-term memory and synaptic plasticity (Remondes & Schuman, 2004; Nolan et al. 2004; Dudman et al. 2007; Brun et al. 2008). Here, we provide evidence that 2-AG is the signalling mediator that strengthens hippocampal synaptic efficacy through a CB1 receptor-dependent mechanism when instructive signals from direct cortical inputs are temporally primed with indirect cortical inputs to pyramidal neurons in the CA1 region. This suggests that the role of the endocannabinoid system in hippocampal long-term synaptic plasticity and memory formation is greater than what was previously thought.

Acknowledgments

The authors thank NIMH transgenic core for providing CB1 receptor knockout mice and NIMH Chemical Synthesis and Drug Supply Program for providing SR141716 and AP5. This work was supported by National Institutes of Health grants NS054886 and AG039669.

Glossary

Abbreviations

2-AG

2-arachidonoylglycerol

DGL

diacylglycerol lipase

(I-LTD), long-term depression at inhibitory synapses; MAGL

monoacylglycerol lipase

PP

perforant path

PPR

ratio of paired-pulse facilitation

RIM

Rimonabant

SC

Schaffer collateral

SLM

stratum lacunosum-moleculare

SR

stratum radiatum

Author contributions

J.-Y.X. and C.C. both contributed to the conception and design of experiments. J.-Y.X., J.Z. and C.C. performed experiments, and analysed and interpreted the data. C.C. drafted the manuscript, with critical comments from J.-Y.X. All authors approved the final version for publication. These experiments were performed in the Neuroscience Centre of Excellence, Louisiana State University Health Sciences Center, New Orleans.

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