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Gating the Polarity of Endocannabinoid-Mediated Synaptic Plasticity by Nitric Oxide in the Spinal Locomotor Network

Gating the Polarity of Endocannabinoid-Mediated Synaptic Plasticity by Nitric Oxide in the Spinal Locomotor Network

Jianren Song, Alexandros Kyriakatos and Abdeljabbar El Manira

Abstract

The final motor output underlying behavior arises from an appropriate balance between excitation and inhibition within neural networks. Retrograde signaling by endocannabinoids adapts synaptic strengths and the global activity of neural networks. In the spinal cord, endocannabinoids are mobilized postsynaptically from network neurons and act retrogradely on presynaptic cannabinoid receptors to potentiate the locomotor frequency. However, it is still unclear whether mechanisms exist within the locomotor networks that determine the sign of the modulation by cannabinoid receptors to differentially regulate excitation and inhibition. In this study, using the lamprey spinal cord in vitro, we first report that 2-AG (2-arachidonyl glycerol) is mobilized by network neurons and underlies a form of modulation that is embedded within the locomotor networks. We then show that the polarity of the endocannabinoid modulation is gated by nitric oxide to enable simultaneously potentiation of excitation and depression of inhibition within the spinal locomotor networks. Our results suggest that endocannabinoid and nitric oxide systems interact to mediate inversion of the polarity of synaptic plasticity within the locomotor networks. Thus, endocannabinoid and nitric oxide shift in the excitation–inhibition balance to set the excitability of the spinal locomotor network.

Introduction

Neural networks underlying behavior undergo continuous plasticity that adapts their output contingent on previous activity. In networks with relatively fixed connectivity, modulatory systems produce adaptive changes by adjusting neuronal properties and synaptic strengths (Nusbaum et al., 2001; Abbott and Regehr, 2004; Destexhe and Marder, 2004; Kristan et al., 2005; LeBeau et al., 2005; Toledo-Rodriguez et al., 2005; Harris-Warrick, 2011). The resulting shift in the net excitability of the network mostly involves activation of distinct transmitter receptors that differentially affect excitatory and inhibitory synaptic transmission (e.g., dopamine) (Albin et al., 1989; Kreitzer and Malenka, 2007; Surmeier et al., 2007). However, it is still unclear whether mechanisms exist that reverse the polarity of the modulation by a single receptor to differentially regulate excitation and inhibition.

The spinal cord contains networks of neurons [central pattern generator (CPG)] sufficient and necessary to produce coordinated locomotor movements (Grillner, 1975; Kiehn, 2006; Goulding, 2009; Grillner and Jessell, 2009; Fetcho and McLean, 2010; Roberts et al., 2010; Büschges et al., 2011). The level of excitability of the spinal locomotor networks has to be tightly controlled to allow for movements of variable speed and intensity. This is often achieved by involving modulatory systems (El Manira et al., 2008; Sillar et al., 2008; El Manira and Kyriakatos, 2010; Harris-Warrick, 2011; Jordan and Slawinska, 2011; Miles and Sillar, 2011). In the lamprey spinal cord, endocannabinoids are released from CPG neurons and contribute to setting the baseline locomotor frequency (Kettunen et al., 2005; Kyriakatos and El Manira, 2007). Two main endocannabinoids have been identified, 2-arachidonyl glycerol (2-AG) and anandamide, which are synthesized and degraded by separate pathways (Sugiura et al., 2002; Piomelli, 2003; Di Marzo, 2008). They are released from postsynaptic neurons and act retrogradely on presynaptic type 1 cannabinoid receptors (CB1Rs) to match transmitter release to the level of postsynaptic excitability (Kim et al., 2002; Freund et al., 2003; Chevaleyre et al., 2006; Mackie and Stella, 2006; Katona and Freund, 2008; Kano et al., 2009; El Manira and Kyriakatos, 2010).

In many CNS regions, CB1Rs activation by 2-AG has been shown to exclusively induce synaptic depression and in no case has synaptic potentiation been directly linked to direct activation of this receptor (Alger, 2002; Freund et al., 2003; Chevaleyre et al., 2006; Katona and Freund, 2008; Lovinger, 2008; Heifets and Castillo, 2009; Kano et al., 2009) (but see Cachope et al., 2007). In this study, we first show that 2-AG is released in the locomotor network. Blocking its synthesis or degradation affected the locomotor rhythm in a manner consistent with a role in potentiating the frequency by shifting the balance between excitation and inhibition. We then show that the inversion of the polarity of the modulation of excitatory and inhibitory synaptic transmission by the endocannabinoid 2-AG is gated by nitric oxide (NO). Scavenging NO or inhibiting its synthesis blocked the potentiation of excitation, but not the depression of inhibition. Our results thus indicate that NO gates the sign of CB1Rs modulation of synaptic transmission by 2-AG within the locomotor CPG to potentiate excitation and the locomotor output.

Materials and Methods

All experiments were performed on in vitro preparations of the isolated, intact spinal cord from adult lampreys (Lampetra fluviatilis) of either sex. All protocols were approved by the Animal Research Ethical Committee, Stockholm. Lampreys were anesthetized with MS-222 (100 mg/L; Sigma-Aldrich) and eviscerated, and the lateral muscle walls were removed. The spinal cord and notochord were dissected and pinned in a cooled (8–12°C) Sylgard-lined experimental chamber continuously perfused with physiological solution. The control solution was composed of the following (in mm): 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, and 2 HEPES, bubbled with O2 and pH adjusted to 7.4. Fictive swimming activity was induced by adding 50–100 μm NMDA to the physiological solution. Alternating locomotor burst activity was recorded by en passant glass suction electrodes placed on two opposing ventral roots at their exits from the spinal cord. After perfusion of NMDA, the locomotor burst frequency increased gradually over time and reached a stable level after 3–4 h. Pharmacological reagents were normally added only after the burst frequency had been stable for at least 1 h. Two minute recordings of ventral root activity were sampled every 5 min. The cycle duration was measured and averaged from 100 to 200 consecutive cycles. The cycle duration was defined as the time interval between the onsets of two consecutive bursts, and the burst frequency was calculated as the inverse of the averaged cycle duration. To examine the changes in midcycle reciprocal inhibition during locomotion, intracellular recordings were made from motoneurons (MNs) and rhythmically active unidentified interneurons with 3 m potassium acetate-filled thin-walled glass microelectrodes with a resistance of 15–30 MΩ. MNs were identified by recording their axonal action potentials in a one-to-one manner from the corresponding ventral root through the extracellular suction electrode. All MNs recorded during fictive locomotion received phasic excitation alternating with phasic inhibition. The concurrent recordings of the locomotor frequency and of the activity of MNs allowed us to correlate changes in the synaptic amplitude with changes in locomotor frequency. Two minute recordings of intracellular and extracellular activity were sampled every 5 min. The spikes were removed using a digital low-pass filter (30 Hz), and the synaptic input was averaged from 100 to 200 cycles; their duration was normalized and aligned according to the membrane potential of the recorded neurons. The peak-to-trough amplitude of the locomotor-related synaptic input was measured and monitored in control and in the presence of different pharmacological agents.

To examine the changes in ipsilateral excitatory or reciprocal inhibitory synaptic transmission, the recording chamber was divided into two pools by an agar barrier (Dale, 1986). Locomotor activity was induced in the rostral pool by NMDA while the spinal cord in the caudal pool was perfused with physiological solution containing strychnine to block inhibitory synaptic transmission or kynurenic acid to block ionotropic glutamate receptors. After 30 min in strychnine or kynurenic acid, intracellular recordings were made from MNs two to three segments away from the agar barrier. In the presence of strychnine, excitatory synaptic input occurred in phase with the activity of the ipsilateral ventral root in the rostral pool. Inhibitory synaptic input was recorded in the presence of kynurenic acid and occurred in phase with the contralateral ventral root. Two minute recordings of intracellular and extracellular activities were sampled every 5 min, and the amplitude of the excitatory or inhibitory drive and the frequency of the locomotor bursts were monitored in control and in the presence of different pharmacological agents. In experiments in which the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) was tested, it was first applied for 1 h before application of 2-AG either to the entire spinal cord or in the caudal pool in experiments with split-bath configuration. The effects of 2-AG in preparations with preincubation with carboxy-PTIO were compared with those obtained in separate control preparations without carboxy-PTIO preincubation.

Intracellular current-clamp recordings were made in bridge mode with an Axoclamp 2B amplifier (Molecular Devices). pClamp software (Molecular Devices) was used for data acquisition and analysis on a personal computer equipped with an analog/digital interface (Digidata 1300). Recordings were also acquired and stored digitally using pClamp. Data analysis of burst frequency and synaptic transmission was done off-line using the program Spike2 (Cambridge Electronic Design). The values reported correspond to mean ± SEM, and n represents the number of experiments. The statistical significance was assessed with paired or unpaired Student's t tests, and differences were considered significant if p < 0.05.

The following drugs were used in this study: NMDA (100 μm; Tocris), strychnine (5 μm; Sigma-Aldrich), kynurenic acid (2 mm; Sigma-Aldrich), 2-AG (1–2 μm; Tocris), the NO scavenger carboxy-PTIO (150 μm; Tocris), tetrahydrolipstatin (THL) (10–20 μm; Tocris), and 4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL184) (1–2 μm; Tocris). The concentrations of THL and JZL184 used in this study were determined based on previously published work in mammals. All agonists and antagonists were dissolved as stock solutions in water except for 2-AG and JZL184, which were dissolved in DMSO. The same concentration of the solvent DMSO had no effect on the locomotor frequency and was always added to control solutions throughout the experiments.

Results

Identity of the endocannabinoid in the spinal locomotor circuit

Endocannabinoids are released within the spinal locomotor circuit in lamprey and contribute to setting the baseline of locomotor frequency (Kettunen et al., 2005; Kyriakatos and El Manira, 2007). To determine the identity of the endocannabinoid responsible for this embedded modulation, we tested the effects of 2-AG and specific inhibitors of the enzymes involved in its synthesis and degradation. In our experiments, locomotor activity was induced by NMDA (100 μm) and was monitored by recording the activity from two opposing ventral roots while membrane potential oscillations were recorded intracellularly from MNs (Fig. 1A). Application of 2-AG (1–2 μm) increased the locomotor burst frequency by 17.9 ± 4.1% (p < 0.001; n = 6; Fig. 1A,B), which was accompanied by a concurrent decrease in the mean amplitude of the membrane potential oscillation to 83.7 ± 4.7% comparing with control (p < 0.01; n = 5; Fig. 1C,D) and an increase in the number of action potentials during the on-cycle depolarization from 1.1 ± 0.1 spike per cycle in control to 3.2 ± 0.1 in 2-AG (p < 0.001; n = 5; Fig. 1B). These results, which are in accord with previous findings (Kettunen et al., 2005; Kyriakatos and El Manira, 2007), show that exogenous application of 2-AG potentiates burst frequency and decreases the amplitude of the membrane potential oscillation during locomotion.

Increase in the locomotor burst frequency by 2-AG. A, Locomotor rhythm was induced by NMDA and recorded in opposing ventral roots. Intracellular recording from a MN that received on-cycle excitation in phase with the ipsilateral ventral root (vr-i) activity that alternated with midcycle inhibition occurring in phase with the contralateral ventral root (vr-c) burst. B, Application of the endocannabinoid 2-AG (2 μm) increased the burst frequency, decreased the oscillation amplitude, and increased spiking activity. C, Averaged synaptic inputs received by the motoneuron showing a decrease in the amplitude of the oscillation by 2-AG. In these recordings, the action potentials were filtered out using a low-pass filter and normalized. D, Averaged data from all the experiments showing the time course of decrease of the oscillation amplitude (red), which is paralleled with an increase in the locomotor burst frequency (blue) induced by 2-AG.

These results are consistent with the idea that 2-AG is released within the spinal circuit. To test this hypothesis, we interfered with 2-AG biosynthesis and degradation pathways by using specific inhibitors. 2-AG is liberated from diacylglycerol (DAG) by DAG lipase (DGL) and is metabolized principally by monoglyceride lipase (MGL) in the nervous system. If 2-AG is released in the lamprey spinal cord, inhibition of DGL would impact the locomotor activity by decreasing the basal levels of 2-AG. Application of the specific DGL inhibitor THL (10–20 μm) (Bisogno et al., 2003; Hashimotodani et al., 2008) decreased the locomotor burst frequency by 8.1 ± 2.7% (p < 0.01; n = 6) and increased the amplitude of the membrane potential oscillation by 13.8 ± 3.8% (p < 0.01; n = 5; Fig. 2A,B). Conversely, inhibition of 2-AG degradation should increase the basal levels of 2-AG and enhance its modulatory actions. Indeed, inhibition of MGL with the specific blocker JZL184 (1–2 μm) (Pan et al., 2009) increased the locomotor burst frequency by 17.5 ± 3.5% and decreased the amplitude of the membrane potential oscillation by 17.7 ± 3.6% (p < 0.001; n = 6; Fig. 2C,D).

Identification of 2-AG as an endocannabinoid released within the locomotor network. A, Averaged membrane potential oscillation displayed by a MN during locomotor activity. Inhibition of the 2-AG synthesizing enzyme DAG lipase by THL (10–20 μm) increased the amplitude of the oscillation received by this MN during locomotor activity induced by NMDA. B, Graph showing averaged data points over time in control and in the presence of THL. The increase in the oscillation amplitude (red) was accompanied with a decrease in the locomotor burst frequency (blue). C, Inhibition of MGL, the degradation enzyme of 2-AG, by JZL184 (1–2 μm) decreased the amplitude of the oscillation displayed by the recorded MN during locomotor activity induced by NMDA. D, Graph showing averaged data points in control and in JZL184, which increased the locomotor burst frequency (blue) and decreased the oscillation amplitude (red).

By interfering with the synthesis and degradation machinery, our results support the conclusion that 2-AG is released within the locomotor circuit. Furthermore, the data suggest that 2-AG is continuously synthesized to help in setting the appropriate balance between inhibition and excitation from network neurons and thus regulates the baseline locomotor burst frequency.

Endocannabinoids change the balance between excitation and inhibition

The change in the synaptic drive received by network neurons during locomotion could be due to a shift in the balance between excitation and inhibition. We therefore tested how 2-AG and its endogenous mobilization affect synaptic transmission between network neurons during locomotion. In these experiments, the recording chamber was divided into two pools by an agar barrier, and locomotor activity was induced in the rostral pool with NMDA (100 μm). The caudal pool was perfused with normal saline (without NMDA), and strychnine (5 μm) was added to isolate the excitatory component of the rhythmic locomotor synaptic drive arising from interneurons whose axons project ipsilaterally across the agar barrier (Fig. 3A). In this configuration, the effect of endocannabinoids on the excitatory drive to motoneurons relevant for the generation of locomotor activity can be monitored (Fig. 3B). Application of 2-AG in the caudal pool increased the amplitude of the excitatory synaptic drive in the recorded motoneurons to 113.9 ± 3.3% of control values (p < 0.01; n = 6; Fig. 3C–E).

Figure 3.

Potentiation of excitatory synaptic transmission by the endocannabinoid 2-AG. A, The recording chamber was divided into two pools with an agar barrier, and locomotor activity was induced by NMDA in the rostral part while locomotor-driven excitation was recorded in a MN in the caudal pool, which was perfused with normal saline solution containing strychnine to block inhibition. B, The recorded MN received excitatory inputs occurring in phase with the ipsilateral ventral root burst. C, Application of 2-AG to the caudal pool increased the amplitude of excitatory synaptic input received by the MN. D, Averaged excitatory synaptic input received by the MN showing an increase in amplitude induced by 2-AG. E, Plot showing average data from different experiments showing the time course of the change in the amplitude of excitation in the caudal pool (red) and the locomotor burst frequency in the rostral pool (blue) before and during 2-AG application. F, Average excitatory synaptic input showing a decrease in amplitude induced by THL. G, Plot of averaged data from different experiments showing the time course of the change in the amplitude of the excitation in the caudal pool (red) and the locomotor burst frequency in the rostral pool (blue) in control and in THL. H, Application of JZL184 in the caudal pool increased the amplitude of the excitatory input received by the MN. I, Plot of averaged data from different experiments showing the change in the amplitude of excitation in the caudal pool (red) and the locomotor burst frequency in the rostral pool (blue) in control and in JZL184.

The above results show that activation of cannabinoid receptors by exogenous application of 2-AG potentiates excitatory synaptic transmission within the locomotor network. The question that arises is whether there is a tonic modulation of excitatory synaptic transmission by endocannabinoids released within the spinal cord that contributes to the regulation of the burst frequency during locomotion. To answer this question, inhibitors of the enzymes responsible for 2-AG biosynthesis and degradation were used. Inhibition of the DAG lipase with THL (10–20 μm) decreased the amplitude of the locomotor-related excitatory synaptic drive to motoneurons by 16.3 ± 2.2% (p < 0.001; n = 7; Fig. 3F,G). However, inhibition of the 2-AG degradation enzyme MGL with JZL184 (1–2 μm) increased the amplitude of the excitatory drive received by 13.1 ± 3.3% on motoneurons during locomotion (p < 0.001; n = 6; Fig. 3H,I). In these experiments, 2-AG, THL, and JZL184 had no effect on the locomotor burst frequency induced by NMDA in the rostral pool (Fig. 3E,G,I).

The potentiation of the excitatory drive could explain the increase in the firing of MNs and burst frequency during locomotion. However, it does not account for the overall decrease in the membrane potential oscillation amplitude (Fig. 1), which could involve a concomitant effect on inhibitory synaptic transmission. To test this possibility, experiments using the split-bath configuration with the caudal pool being perfused with normal saline containing kynurenic acid (2 mm) were performed to block excitatory synaptic transmission (Fig. 4A). Midcycle inhibitory synaptic transmission originating from active commissural interneurons in the rostral pool was monitored in motoneurons recorded in the caudal pool (Fig. 4B). Application of 2-AG in the caudal pool depressed the amplitude of the midcycle inhibition in motoneurons by 26.8 ± 2.9% (p < 0.001; n = 7; Fig. 4C–E). Conversely, blockade of the synthesizing enzyme DAG lipase with THL (10–20 μm) increased the amplitude of the midcycle inhibition by 10.4 ± 2.5% (p < 0.01; n = 5; Fig. 4F,G), indicating that newly synthesized endocannabinoids are responsible for a tonic decrease of the midcycle inhibition during locomotion. If there is a tonic mobilization of the endocannabinoid 2-AG, the modulation of midcycle inhibition should be further enhanced by blockade of its degradation enzyme MGL. Indeed, application of the MGL inhibitor JZL184 decreased the amplitude of the midcycle inhibition by 15.8 ± 2.6% (p < 0.001; n = 6; Fig. 4H,I). In these experiments, 2-AG, THL, and JZL184 had no effect on the locomotor burst frequency induced by NMDA in the rostral pool (Fig. 4E,G,I).


Depression of inhibitory synaptic transmission by the endocannabinoid 2-AG. A, The recoding chamber was divided into two pools with a agar barrier, and locomotor activity was induced by NMDA in the rostral part while locomotor-driven excitation was recorded in a MN in the caudal pool, which was perfused with normal saline solution containing kynurenic acid (KynA) to block excitation. B, The recorded MN received inhibitory inputs occurring in phase with the contralateral ventral root burst. C, Application of 2-AG to the caudal pool decreased the amplitude of inhibitory synaptic input received by the MN. D, Averaged inhibitory synaptic input received by the MN showing a decrease in amplitude induced by 2-AG. E, Plot showing average data from different experiments showing the time course of the change in the amplitude of the inhibition in the caudal pool (red) and the locomotor burs frequency in the rostral pool (blue) before and during 2-AG application. F, Average inhibitory synaptic input in control and in THL. G, Plot of averaged data from different experiments showing the time course of the change in the amplitude of the inhibition in the caudal pool (red) and the locomotor burst frequency in the rostral pool (blue) in control and in THL. H, Application of JZL184 in the caudal pool decreased the amplitude of the inhibitory input received by the MN. I, Plot of averaged data from different experiments showing the change in the amplitude of the inhibition in the caudal pool (red) and the locomotor burst frequency in the rostral pool (blue) in control and in JZL184.

These results indicate that there is a tonic modulation by endocannabinoids of excitatory and inhibitory synaptic transmission from network neurons during locomotion. 2-AG is the endocannabinoid synthesized and mobilized within the locomotor network that tips the excitation–inhibition balance within the locomotor network in favor of excitation by differentially potentiating excitatory and depressing inhibitory synaptic transmission and thereby synergistically increasing the locomotor burst frequency.

Inversion of the polarity of endocannabinoid modulation by nitric oxide

The endocannabinoid 2-AG appears to modulate excitatory and inhibitory synaptic transmission within the locomotor network in opposite directions. While the depression of synaptic transmission has been reported previously, potentiation of excitation is novel. What mechanism is responsible for the inversion of the polarity of the endocannabinoid modulation of excitation and inhibition? We previously showed that endocannabinoids and NO are recruited by metabotropic glutamate receptor 1 (mGluR1) activation and act synergistically to modulate the locomotor activity and synaptic transmission (Kyriakatos and El Manira, 2007; Kyriakatos et al., 2009). We therefore tested whether NO is involved in reversing the polarity of endocannabinoid modulation. For this, we performed experiments using the split-bath chamber to monitor the modulation of excitatory (with strychnine in the caudal pool) and inhibitory (with kynurenic acid in the caudal pool) synaptic transmission by 2-AG during locomotion in preparations preincubated with carboxy-PTIO (150 μm; 1 h in the caudal pool), which is preferentially an extracellular NO scavenger (Griffiths et al., 2003). Preincubation with carboxy-PTIO completely blocked 2-AG-induced increase in excitatory synaptic transmission (p < 0.01; n = 7; Fig. 5A–D; in Fig. 5D: carboxy-PTIO, black, and control, gray), indicating that the potentiation of the excitatory drive during locomotion by endocannabinoids is entirely dependent on NO signaling. In contrast, the 2-AG-induced depression of inhibitory synaptic transmission arising from commissural interneurons was not affected by carboxy-PTIO (Fig. 6A,B). The decrease in the amplitude of the inhibitory synaptic potentials by 2-AG in preparations preincubated with carboxy-PTIO (black) was not significantly different from that obtained in control (gray) experiments (p > 0.05; n = 6; Fig. 6C,D). These results show that NO acts to switch the sign of cannabinoid-induced modulation of synaptic transmission, hence resulting in a more pronounced shift in the balance between excitation and inhibition within the locomotor circuit


Nitric oxide is required for the potentiation of the excitation by the endocannabinoid 2-AG. A, Excitatory synaptic input received by a motoneuron was recorded in a split-bath chamber with strychnine in the caudal pool (Fig. 3). The caudal pool was preincubated with the NO scavenger carboxy-PTIO for 1 h. B, Application of 2-AG in the caudal pool in the presence of carboxy-PTIO failed to potentiate the amplitude of the excitatory input received by the MN. C, Average excitatory synaptic input recorded in a MN preincubated with carboxy-PTIO before and during 2-AG application. D, Plot of averaged data from different experiments showing the time course of the change in the amplitude of the excitation in the presence of carboxy-PTIO before and during 2-AG application (black data points). The gray data points show the change in the excitation induced by 2-AG in the absence of carboxy-PTIO (same data as in Fig. 3E).

Depression of inhibition by the endocannabinoid 2-AG is not dependent on nitric oxide. A, Inhibitory synaptic input received by a motoneuron was recorded in a split-bath chamber with kynurenic acid in the caudal pool (Fig. 4). The caudal pool was preincubated with the NO scavenger carboxy-PTIO for 1 h. B, Application of 2-AG in the caudal pool in the presence of carboxy-PTIO was still able to depress the amplitude of the inhibitory input received by the MN. C, Average inhibitory synaptic input recorded in a MN preincubated with carboxy-PTIO before and during 2-AG application. D, Plot of averaged data from different experiments showing the time course of the change in the amplitude of the inhibition in the presence of carboxy-PTIO before and during 2-AG application (black data points). The gray data points show the change in the inhibition induced by 2-AG in the absence of carboxy-PTIO (same data as in Fig. 4E).

The question that stems from the above results is whether the potentiation of the burst frequency induced by endocannabinoids in the whole locomotor circuit is a direct consequence of the differential modulation of excitatory and inhibitory synaptic transmission. To test for this, the effect of 2-AG on the locomotor burst frequency and membrane potential oscillation was tested in the presence of carboxy-PTIO (150 μm). Application of 2-AG in preparations preincubated with carboxy-PTIO could still induce a potentiation of the locomotor burst frequency; however, the increase was significantly smaller than that in control [carboxy-PTIO (blue), 108.4 ± 2.5%; n = 6; control (gray), 117.9 ± 3.9%; p < 0.01; n = 6; Fig. 7A,B]. This is despite the fact that the decrease in the amplitude of the membrane potential oscillations was significantly more pronounced in carboxy-PTIO by 23.4 ± 3.7% (red) than that without carboxy-PTIO by 16.3 ± 4.7% (gray) (p < 0.01; n = 6; Fig. 7C,D). These results thus indicate that the interplay between cannabinoid and NO signaling underlies a concomitant enhancement of the excitatory drive and a decrease of midcycle inhibition, which produces a more pronounced shift in favor of increased excitability in the spinal locomotor circuit and hence a potentiation of the burst frequency.

Nitric oxide contributes to the increase in the locomotor burst frequency by the endocannabinoid 2-AG. A, Locomotor rhythm was induced by NMDA in the presence of the NO scavenger carboxy-PTIO and was recorded in opposing ventral roots. Intracellular recording from a MN that received on-cycle excitation in phase with the ipsilateral ventral root (vr-i) activity that alternated with midcycle inhibition occurring in phase with the contralateral ventral root (vr-c) burst. B, Application of the endocannabinoid 2-AG (2 μm) in the presence of carboxy-PTIO was still able to increase the burst frequency that was associated with a decrease in the oscillation amplitude. C, Averaged synaptic inputs received by the MN showing a decrease in the amplitude of the oscillation by 2-AG. In these recordings, the action potentials were filtered out using a low-pass filter, normalized, and aligned in relation to the membrane potential of the MN. D, Averaged data from all the experiments showing the time course of the effect of 2-AG on synaptic transmission and the locomotor burst frequency in the presence of carboxy-PTIO. The decrease of the oscillation amplitude was more pronounced (red), while the potentiation of the locomotor burst frequency was attenuated (blue) in preparations preincubated with carboxy-PTIO compared with controls (gray; same data as in Fig. 1D).

Discussion

Embedded endocannabinoid modulation and its gating by nitric oxide

In this study, we first show that 2-AG is responsible for the endocannabinoid-embedded modulation of the locomotor frequency and synaptic transmission by interfering with its synthesis and degradation. Blocking the synthesis or degradation of 2-AG produced opposite effects on the locomotor frequency as well as on the underlying on-cycle excitation and midcycle inhibition. In addition, we show that NO switches the sign of endocannabinoid modulation of synaptic transmission within the locomotor CPG. The NO gating enables endocannabinoids to simultaneously potentiate excitation and depress inhibition (Fig. 8). Our results indicate that the endocannabinoid 2-AG is mobilized from network neurons during locomotor activity and plays a significant role, in synergy with NO, in setting the baseline burst frequency by adjusting the excitability balance within the locomotor CPG. Thus, although the final locomotor output is generated by activation of ionotropic receptors, it is also heavily dependent on the embedded modulation by endocannabinoids, which plays a buffering role to set the baseline operation of the locomotor CPGs.

Figure 8.

Summary of the interplay between the endocannabinoid 2-AG and nitric oxide. The intimate interplay between endocannabinoid and MN induces a switch in the balance between excitation and inhibition within the locomotor circuit and thus mediates an increase in swimming frequency.

Identification of the endocannabinoid mobilized in the spinal locomotor CPGs

The two main endocannabinoids are 2-AG and anandamide, and they are synthesized and metabolized by separate pathways (Sugiura et al., 2002, 2006; Piomelli, 2003; Di Marzo, 2008). However, the identity of the endocannabinoid regulating the locomotor activity has until now been unclear. In the dorsal horn, anatomical evidence using electron microscopy showed postsynaptic localization of DGL-α at nociceptive synapses formed by primary afferents, and a presynaptic positioning of CB1Rs on excitatory axon terminals (Nyilas et al., 2009; Pernía-Andrade et al., 2009). DGL-α in postsynaptic elements receiving nociceptive input was colocalized with mGluR5, whose activation induces 2-AG biosynthesis (Nyilas et al., 2009). Blocking of the DGL, which hydrolyzes DAG into 2-AG, decreased the frequency of the locomotor rhythm by depressing the excitation and enhancing the inhibition. This indicates that there is a continuous synthesis of 2-AG within the locomotor networks that helps to maintain the baseline level of activity. In addition, blocking MGL, the enzyme that degrades 2-AG, increased the locomotor burst frequency by potentiating the excitation and depressing the inhibition. Indeed, by affecting the synthesizing (DGL) and degrading (MGL) enzymes, we produce opposite effects on the locomotor rhythm and the underlying on-cycle excitation and midcycle inhibition. These results argue that 2-AG is released in the spinal locomotor CPG that is continuously synthesized and helps modulating the baseline excitability underlying the locomotor output. Our results do not exclude the possibility that, in addition to the on-demand release of endocannabinoids, there is also a constitutive synthesis and mobilization of endocannabinoids independent of the activity of the spinal locomotor network.

In keeping with these results, we have previously shown that activation of mGluR1 induces short- and long-term potentiation of the locomotor activity (Krieger et al., 2000; El Manira et al., 2002, 2010; Kyriakatos and El Manira, 2007). This receptor is coupled to the G-protein Gq/11, which activates phospholipase C (Kettunen et al., 2002, 2003; Nanou et al., 2009; Nanou and El Manira, 2010); this signaling pathway yields to accumulation of DAG that in turn is hydrolyzed by DGL into 2-AG (Safo and Regehr, 2005; Chevaleyre et al., 2006; Heifets and Castillo, 2009; Kano et al., 2009; Tanimura et al., 2010). The long-term potentiation of the locomotor burst frequency was completely dependent on activation of CB1Rs because it was blocked by the antagonist AM251 (Kyriakatos and El Manira, 2007). Evidence exists to suggest that 2-AG or anandamide represents the endocannabinoid released in many regions in the CNS. A direct activation of CB1R by endocannabinoids, be it 2-AG or anandamide, has been shown to produce exclusively depression of both excitatory and inhibitory synaptic transmission in many regions of the CNS (Alger, 2002; Wilson and Nicoll, 2002; Freund et al., 2003; Chevaleyre et al., 2006; Katona and Freund, 2008; Kreitzer and Malenka, 2008; Lovinger, 2008; Heifets and Castillo, 2009; Kano et al., 2009). In hippocampus, cerebellum, and hypothalamus, the depression of synaptic transmission has been suggested to involve an interaction between NO and endocannabinoid signaling (Safo and Regehr, 2005; Makara et al., 2007; Crosby et al., 2011). In goldfish, however, it has been shown that endocannabinoids indirectly potentiate excitatory synaptic transmission via release of dopamine (Cachope et al., 2007).

Nitric oxide and endocannabinoids in the spinal locomotor networks

Several classes of neurons are known to be involved in motor control and sensorimotor integration in the lamprey spinal cord. These include gray matter neurons (motoneurons and interneurons), proprioceptive edge cells, which express NO synthase (NOS) and are therefore potential intrinsic sources of NO (Kyriakatos et al., 2009). We previously showed that the NOS inhibitor l-NAME and the NO scavenger carboxy-PTIO decrease the locomotor frequency, and the latter also blocked NO donor-induced modulation. These results indicate that NO mediates a positive-feedback control of the network during swimming to maintain the rhythm at a high frequency (Kyriakatos et al., 2009). In Xenopus tadpoles, NO exerts mainly inhibitory actions by selectively enhancing GABAergic descending inhibition from mid-hindbrain reticulospinal interneurons and by enhancing glycinergic inhibition during swimming (McLean and Sillar, 2002). This latter effect involves metamodulation of the effects of noradrenaline (McLean and Sillar, 2004). Endocannabinoids are mobilized within the lamprey locomotor network and modulate inhibitory synaptic transmission and locomotor frequency (Kettunen et al., 2005; El Manira et al., 2008; El Manira and Kyriakatos, 2010). Endocannabinoid and NO signaling were thought to act only in parallel to modulate the activity of the spinal locomotor network. Our results now show that endocannabinoids and NO signaling are in addition intimately coupled to mediate selective modulation of excitatory and inhibitory synaptic transmission and regulate the locomotor network output.

Nitric oxide switches the sign of endocannabinoid modulation of synaptic transmission

An important, previously unknown interaction between endocannabinoids and NO is in the selective potentiation of excitatory synaptic transmission. This potentiation, combined with a direct depression of the inhibition by activation of presynaptic CB1R, adjusts the excitability balance in favor of increased excitatory transmission and hence a potentiation of the locomotor burst frequency (Fig. 8). The interaction between NO and endocannabinoids is restricted to excitatory synaptic transmission and was blocked by the membrane-impermeable scavenger carboxy-PTIO (Griffiths et al., 2003), indicating that NO diffuses outside the neurons where it is produced. This leads to a decrease of inhibitory synaptic transmission by activation of presynaptic CB1Rs. One prevalent mechanism of NO synthesis is via Ca2+ influx through NMDA receptors, leading to activation of NOS, which is commonly tethered to NMDA receptors (Brenman and Bredt, 1997; Prast and Philippu, 2001; Garthwaite, 2008). We previously showed that inhibition of NOS by l-NAME affects the locomotor frequency in the lamprey spinal cord (Kyriakatos et al., 2009) and blocked the endocannabinoid-dependent potentiation of the burst frequency induced by mGluR1 (Kyriakatos and El Manira, 2007). The NO-generated and the mobilized endocannabinoids interact to produce potentiation of excitatory synaptic transmission. It thus seems that NO acts as a metamodulator to determine the polarity of the endocannabinoid-mediated plasticity of excitation. Ultimately, the balance between excitatory and inhibitory synaptic transmission determines the speed and force of the locomotor movements produced by the spinal locomotor CPG. The gating of the sign of endocannabinoid-mediated synaptic plasticity by NO represents a novel mechanism by which the excitability balance is appropriately set within the locomotor CPG to allow for a more flexible execution of motor behavior.


New perspectives in neuroprotection and anti-inflammatory therapy of this Parkinson's neurodegenerative disease

 

ABSTRACT

Cannabinoid type-1 receptors (CB1Rs) modulate synaptic neurotransmission by participating in retrograde signaling in the adult brain. Increasing evidence suggests that cannabinoids through CB1Rs play an important role in the regulation of motor activities in the striatum. In the present study, we used human brain samples to examine the relationship between CB1R and dopamine receptor density in case of Parkinson’s disease (PD).

Post mortem putamen, nucleus caudatus and medial frontal gyrus samples obtained from PD patients were used for CB1R and dopamine D2/D3 receptor autoradiography. [125I]SD7015, a novel selective CB1R inverse agonist, developed by a number of the present co-authors, and [3H]raclopride, a dopamine D2/D3antagonist, were used as radioligands. Our results demonstrate unchanged CB1R density in the putamen and nucleus caudatus of deceased PD patients, treated with levodopa (l-DOPA). At the same time dopamine D2/D3 receptors displayed significantly decreased density levels in case of PD putamen (control: 47.97 ± 10.00 fmol/g, PD: 3.73 ± 0.07 fmol/g (mean ± SEM), p < 0.05) and nucleus caudatus (control: 30.26 ± 2.48 fmol/g, PD: 12.84 ± 5.49 fmol/g, p < 0.0005) samples. In contrast to the putamen and the nucleus caudatus, in the medial frontal gyrus neither receptor densities were affected.

Our data suggest the presence of an unaltered CB1R population even in late stages of levodopa treated PD. This further supports the presence of an intact CB1R population which, in line with the conclusion of earlier publications, may be utilized as a pharmacological target in the treatment of PD. Furthermore we found discrepancy between a maintained CB1R population and a decreased dopamine D2/D3 receptor population in PD striatum. The precise explanation of this conundrum requires further studies with simultaneous examination of the central cannabinoid and dopaminergic systems in PD using higher sample size.

Keywords: Parkinson’s disease, Endocannabinoid CB1 receptor, Dopamine D2/D3 receptor, Molecular imaging biomarker, Human brain autoradiography, Striatum

1. Introduction

The endocannabinoid (EC) system is commonly described as a neuromodulatory system that interacts with and regulates the functions of many neurotransmitter systems, including cholinergic (Ach), dopaminergic (DA), serotoninergic, adrenergic, opiate, glutamatergic and GABAergic systems [,,]. The main contribution of ECs to the control of synaptic neurotransmission is to act as retrograde messengers through type 1 cannabinoid receptors (CB1R) [,]. Presynaptic CB1Rs are abundant in the adult mammalian brain []. CB1Rs are coupled to Gi/o proteins and, under specific conditions to Gs proteins [,,]. CB1Rs regulate the activity of various plasma membrane proteins and signal transduction pathways, including ion channels, context-dependent recruitment of second messengers (Erks, STATs, etc.) and various kinases. In addition, CB1Rs activate G protein-independent pathways, as well [,,]. Among various other functions, endocannabinoids have neuromodulatory functions, as well, [] and play an important role in long term potentiation (e.g. []).

Multiple levels of evidence suggest that ECs have a potential to protect neurons under chronic degenerative conditions via CB1R-dependent and -independent mechanisms [,,,,]. An increasing number of studies have demonstrated that CB1R density and binding is altered in the extrapyramidal system of humans in e.g., Huntington disease and PD [,,,,,,,].

However, the observed alterations in CB1R’s in various neurodegenerative diseases, such as HD or PD, may be of diverse origins. The GABAergic spiny neurons (MSNs) are the most populous neuronal cell type of the striatum (90–95% in rats and over 85% in humans), along with several small populations of interneurons [,]. CB1Rs are primarily expressed my MSNs. The HD brain is characterized by loss of the MSNs of the striatum, which results in robust down-regulation of CB1Rs. A severe loss of CB1R’s in the striatum has, consequently, been described as a landmark of HD [,]. On the other hand, the alteration of striatal CB1R population in PD is full of controversies and the most important striatal cells expressing the CB1R are affected in a lesser extent compared to HD and the EC systems shows a strong tendency for reorganization [,].

It is well known that a progressive degeneration of the dopaminergic system, especially the dopaminergic neurons of the substantia nigra pars compacta (SNc) [,], underlies the pathogenesis and clinical manifestations of PD. The decrease in striatal dopamine (DA) alters the regulation of synaptic dopamine levels, and dopamine receptor density and functional state [,,,,,,,,,]. Alterations in basal ganglia CB1R density or EC levels have been described in rat models of PD on the basis of which a strong functional connection between the striatal dopamine and endocannabinoid systems has been hypothesized [,]. However, experimental models in small animals are inconclusive regarding the direction of changes of CB1R density in Parkinson models: whereas there is evidence for the decrease of CB1Rs in the striatum, as a consequence of 6-hydroxydopamine-induced nigrostriatal terminal lesion in rats []; studies using postmortem human PD brain samples, 6-hydroxydopamine (6-OHDA) or reserpine-treated rat models of PD, MPTP-lesioned marmoset and mouse mutant models of PD indicate an up-regulation [,,,,,], no change [,,,] or down-regulation [,] of CB1Rs in Parkinson’s disease.

In order to investigate changes in CB1R: D2/D3 balance in PD in the human basal ganglia, we explore correlative alterations in dopamine D2/D3 receptors, key players in the disease process [,,], and the alteration in CB1R using selective radioligands – [3H]raclopride [,], [125I]SD7015 [] – in brain tissues obtained from PD and age-matched control subjects.

Discussion

We investigated the relationship of CB1R and D2/D3 receptor densities in PD human brains by means of receptor autoradiography. [125I]SD7015, a novel CB1R agonist, [] and [3H]raclopride, a dopamine D2/D3 receptor antagonist [], were applied as radioligands.

CB1R densities in putamen, nucleus caudatus and frontal cortex samples seem to be unchanged in PD while in contrast, dopamine D2/D3 receptor density in PD putamen and nucleus caudatus decreases. The latter is in line with previous findings, namely this decrease of D2/D3 receptor density in PD putamen and nucleus caudatus could be the consequence of longterm antiparkinsonian treatment. It is generally agreed that dopaminergic denervation leads to striatal D2 dopamine receptor up-regulation as postsynaptic compensatory mechanism in response to deficiencies in synaptic dopamine signaling [,,,,]. Treatment of PD patients with dopaminergic drugs returns the striatal dopamine D2 receptor expression to near normal levels [,,]. Frontal cortex samples presented no difference between the subject cohorts.

The CB system in experimental PD models and PD patients has been extensively studied, yet with contradictory conclusions. In rat Parkinson models (reserpine treatment or 6-OHDA-lesion models) an increase in endogenous endocannabinoid levels was observed in the striatum [,,,] as it was also observed in the CNS of 16 untreated PD patients []. Other authors found significantly altered CB1R mRNA expression in animal models of PD or in postmortem human PD brain specimens. [,,,]. Finally, changes in CB1R binding sites [,,] and activation of GTP-binding proteins in the basal ganglia of PD patients and of MPTP-treated marmosets were also reported []. On the other hand, using the 6-OHDA rat model Romero et al. [] did not find significant changes in CB1 receptor binding, measured by [3H]WIN-55,212,2 autoradiography, or in the activation of signal transduction mechanisms, measured by WIN-55,212,2-stimulated [35S]GTPgammaS binding autoradiography, between the lesioned and non-lesioned sides at the level of the lateral caudate-putamen, globus pallidus and substantia nigra.

In the present study we did not find changes in CB1R densities in the striatum and frontal cortex of PD subjects. One of the explanations for the unaltered CB1R density found by us could be the unchanged density of high affinity CB1Rs in the investigated PD brain regions [,]. This may be possible due to the presence of the large reserve of CB1Rs and their likely inter-conversion between low and high affinity states [,]. Due to low sample size results are only suggestive in the aspect of an intact CB1R density. This statement requires further justification in the future by studies using more specimens and performing the detailed investigation of this problem.

On the other hand, functional relationship have been reported between CB1Rs and both dopamine D1 and D2 receptors [,,,,,,,,, ]. For instance, Giuffrida et al. [] proved that striatal administration of D2 agonist results in release of endocannabinoids. Furthermore, activation of CB1 or dopamine D2 receptors alone resulted in inhibition of cAMP accumulation whereas simultaneous activation of both receptors increased cAMP levels []. Kreitzer and Malenka [] reported in animal models of Parkinson’s disease that DA depletion blocked the generation of endocannabinoid-mediated long-term depression (eCB-LTD) in indirect striatal pathway but administration of dopamine D2 receptor agonist together with inhibitors of endocannabinoid degradation rescued indirect-pathway eCB-LTDs and in vivo reduced parkinsonian motor deficits. Dopamine receptor antagonists inhibited cannabinoid induced striatal mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activation [].

Administration of CB1R agonists increased dopamine turnover and release [,,,], excited dopaminergic neurons in the ventral tegmental area and substantia nigra [], decreased the tremor associated with overactivity of the subthalamic nucleus and improved motor impairment [,,], at the same time a CB1 antagonist (rimonabant) alleviated hypokinesia in animal models of Parkinson’s disease [,], probably effecting on the lateral globus pallidus. It has been demonstrated that glutamatergic and GABAergic terminals strongly express CB1Rs and CB1R agonists significantly inhibited glutamate and GABA release, however, cannabinoids in vitro, directly did not affect the release of dopamine [,,]. Yin et al. [] observed that presynaptic reduction in glutamate release was the consequence of a retrograde signal through eCBs; this eCB synthesis and release from the postsynaptic cell results from cooperating, convergent glutamate and dopamine inputs. A potential indirect dopamine–CB1R interaction through the cannabinoid induced regulation of the upper mentioned neurotransmitters (GABA, glutamate) of striatal neuronal pathways could be the basis of cannabinoid effect on motor activity. However, the findings of [] that dopaminergic cells also express CB1R as well as observations about functional interactions between CB1Rs and both dopamine D1 and D2 receptors [,,,,,,,,,]. could contribute to the upper mentioned effect as well. Due to the complexity of this cannabinoid–dopamine receptor conundrum further researches are required with well designed study protocols. Although our results base on relatively small sample size, they refer to the presence of an apparently intact CB1 receptor population may be usable in PD therapy, even in advanced PD.

On the other hand, it is accepted that classical neuroinflammatory diseases such as multiple sclerosis present aspects of neurodegeneration, while classical degenerative disorders such as Alzheimer’s disease, Parkinson’s disease are demonstrably affected by inflammation []. In CNS CB1 receptors exist in all types of neural cells, in astrocytes [,], microglia [,], and oligodendrocytes [] whereas CB2receptors are expressed on cells of immune system and microglia []. Studies report neuroprotective [,,] and anti-inflammatory effects through CB receptors [,,,,,]; CBs protected against dopaminergic cell death, as well []. Thus CB1 and CB2 receptors could provide substrate for neuroprotective and anti-inflammatory actions of cannabinoids in neurodegenerative diseases, however, the effects through CB1Rs are more relevant to neuroprotection, whereas CB2Rs modulate the immune response primarily, although a potential overlap as well as non CB1/CB2-mediated mechanisms may exist [].

In this study we used PD putamen, nucleus caudatus, medial frontal gyrus samples in order to correlate CB1 receptor density with dopamine D2/D3 receptor density. Our results refer to an unchanged CB1R and decreased dopamine D2/D3 receptor density in nucleus caudatus and putamen of PD patients whereas medial frontal gyrus sections did not show any alteration. Our data suggest that in case of long-term l-DOPA treatment and long disease progression CB1R density does not fall under control levels, although, dopamine D2/D3 receptor density is significantly decreased. Various explanations could exist: (1) neurodegeneration induced affinity or sensitivity increase of ‘reserve’ CB1Rs could compensate CB1R density changes, (2) reactive changes of CB system could go along with PD progression, until more effective compensatory mechanisms come into action, (3) despite the decreased density, dopamine D2receptor signal transduction seems functionally intact []) and possible physiological interactions with CB1 receptors could be maintained even in later stages of PD, which could result in unchanged CB1R density; however, functional receptor crosstalks between these receptor types are, yet, unequivocally unproven. Nevertheless the combination up to various degree of the aforementioned or other, yet, unknown mechanisms is the most probable phenomenon.

We concluded that an intact CB1R population could represent alternative target for treatment of PD; additionally it could open new perspectives in neuroprotection and anti-inflammatory therapy of this neurodegenerative disease. Better understanding and further exploration of central cannabinoid system and related questions need further in vivo and in vitro detailed designed studies, emphasizing study population/sample group homogeneity regarding to data about PD neuropathological stage, disease duration and l-DOPA substitution/dopamine agonist therapy. This is suggested since one of the limitation of this study is the low sample number and consequently the questionable reliability of statistical calculations on these data. By the parallel investigation of CB1 and DA D2/3 receptors in PD striatum we wished to base and pioneer future researches aiming this field of neuroscience.


Multiple Functions of Endocannabinoid Signaling in the Brain

Authors, István Katona and Tamás F. Freund

Abstract

Despite being regarded as a hippie science for decades, cannabinoid research has finally found its well-deserved position in mainstream neuroscience. A series of groundbreaking discoveries revealed that endocannabinoid molecules are as widespread and important as conventional neurotransmitters like glutamate or GABA, yet act in profoundly unconventional ways. We aim to illustrate how uncovering the molecular, anatomical and physiological characteristics of endocannabinoid signaling revealed new mechanistic insights into several fundamental phenomena in synaptic physiology. First, we summarize unexpected advances in the molecular complexity of biogenesis and inactivation of the two endocannabinoids, anandamide and 2-arachidonoylglycerol. Then we show how these new metabolic routes are integrated into well-known intracellular signaling pathways. These endocannabinoid-producing signalosomes operate in phasic and tonic modes thereby differentially governing homeostatic, short-term and long-term synaptic plasticity throughout the brain. Finally, we discuss how cell type- and synapse-specific refinement of endocannabinoid signaling may explain the characteristic behavioral effects of cannabinoids.

Keywords: retrograde signaling, feed-back inhibition, synaptic plasticity, G-protein-coupled receptors, diacylglycerol lipase

The “Grass Route” to the Discovery of the Endocannabinoid System

Predator-prey competition is a major driving force behind evolution. For example, most plants developed a dedicated repertoire of chemical molecules to distract consumption. These allelochemicals often mimic or perturb endogenous signaling pathways in the nervous system, thereby becoming behaviorally effective. It seems that the underlying evolutionary processes were robust enough to invent receptor agonists or antagonists and even allosteric activators or inhibitors of enzymes with excellent potency and affinity. This natural treasure trove served traditional medicine for several thousand years and still remains a major frontier for drug discovery (). Moreover, neuroscience research has also greatly profited from deciphering the mechanisms by which these plant products are behaviorally active, paving the way for the discovery of several endogenous signaling systems primarily active in the brain ().

A prime example is the cannabis plant (Cannabis sativa L.) and the discovery of the endocannabinoid system in animals. Cannabis plants produce a unique mixture of chemical constituents, the most famous products being the C21 terpenophenolic compounds, which are collectively called phytocannabinoids. Detailed chemical analysis have identified about 70 molecular species of phytocannabinoids (), but unquestionably, one molecule and its discovery stands out. Intriguingly, this aromatic terpenoid has been more famous (or infamous, as one wishes) among the public than among neuroscientists until very recently. This compound called (−)-Δ9-tetrahydrocannabinol 9-THC) was isolated from confiscated hashish by Yechiel Gaoni and Raphael Mechoulam (Fig. 1A-B) (), and was shown to account for the psychotropic effects of cannabis preparations in rhesus monkeys (). This seminal discovery transformed cannabinoid research from an anecdote-based practice into an evidence-based modern research field. The use of the chemically defined Δ9-THC molecule made it possible to obtain qualitatively and quantitatively reproducible pharmacological, physiological or behavioral data, which then helped to uncover the neurobiological substrates of psychoactive effects of cannabis.

Figure 1
A tribute to the discoveries unraveling the endocannabinoid system

The second major breakthrough in cannabinoid research provided answer to the conceptual question of why our brain reacts to cannabis. Using [3H]-CP55,940, a potent radioactively-labeled synthetic cannabinoid, Bill Devane, Allyn Howlett and their colleagues obtained the first unequivocal evidence for the presence of a specific cannabinoid receptor, which inhibits adenylate cyclase via Gi-protein signaling in the brain (Fig. 1C-D) (; ). This discovery is also considered as the first direct evidence for existence of the endocannabinoid system. The subsequent qualitative and quantitative radioligand binding studies quickly revealed the distribution of cannabinoid receptors in the brain (Fig 1E) (). First, lesion experiments showed that the vast majority of cannabinoid binding sites in the brain are on neurons, and most likely on their axonal bundles (). Second, the quantitative distribution pattern fitted well with the brain regions underlying the behavioral effects of cannabis. Third, this pattern was remarkably similar across species indicating a conserved physiological function for cannabinoid receptors. Finally, and most importantly, the density of cannabinoid receptors in the brain was comparable to the levels of glutamate, GABA or striatal dopamine receptors (). Thus, these observations collectively predicted in advance that cannabinoid receptors are as ubiquitous components of chemical synapses as conventional neurotransmitter receptors.

This period was the golden age for the cloning of G-protein-coupled receptors, thus, the molecular identification of the first cannabinoid receptor has followed very soon (). The CB1cannabinoid receptor indeed turned out to be a class A G-protein-coupled receptor, and has a notably similar sequence (97-99% amino acid sequence identity) across mammalian species, supporting once again a phylogenetically conserved function for CB1. In situ hybridization confirmed neuronal expression and revealed a heterogeneous distribution pattern largely corresponding to the ligand binding sites (). With the help of significant homology (44% at the amino acid level), a second cannabinoid receptor was also discovered thereafter (). These two receptors originated from a common ancestor, and it is now fairly safe to conclude that a third, phylogenetically closely related third cannabinoid receptor is unlikely to be found ().

Compelling evidence shows that CB1 receptors are the major neurobiological substrates for Δ9-THC effects on the human brain. The acute psychological consequences of marijuana smoking such as the subjective “high” experience was efficiently blocked by pretreatment with the CB1 antagonist, rimonabant in healthy human subjects (). Moreover, the development of novel inverse agonist radioligands for positron emission tomography now allows the monitoring of CB1 receptor availability in the living human brain () (Fig. 1F). The tremendous potential of this new approach is reflected by the emerging data showing robust changes in CB1 receptor availability in patients with Huntington disease, or temporal lobe epilepsy (; ), or by the demonstration of cortex-specific downregulation of CB1 availability in chronic cannabis smokers, which is a long-suspected mechanism of cannabis tolerance ().

While CB1 receptors are considered as primarily neuronal receptors, CB2 receptors are highly expressed in the spleen and regarded as the predominant cannabinoid receptor of the immune system. This somewhat simplified concept has been ideal to provide a framework for the potential therapeutic exploitation of the endocannabinoid system; one particularly exciting example is that the beneficial effects of Δ9-THC in multiple sclerosis is mediated by neuronal CB1 receptors and by CB2 receptors on autoreactive T cells in tandem (). However, accumulating data also support important physiological and pathophysiological functions for peripheral CB1 receptors (), whereas central effects of CB2 receptors are well-documented in emesis regulation or in rewarding effects of cocaine (; ).

The discovery of cannabinoid receptors initiated an immediate quest for their endogenous ligands, the so-called endocannabinoids. Because of the lipophilic nature of phytocannabinoids, Devane, Mechoulam and their colleagues argued that lipid-soluble fractions of the brain should contain the putative endocannabinoid molecule, which finally led them to its chemical identification (Fig. 1G-H) (). This compound, N-arachidonoylethanolamide was termed anandamide based on the Sanskrit word “ananda” meaning “inner bliss”. This name reflects a good foresight, because anandamide plasma levels are significantly reduced in patients with major depression (), and blockade of anandamide hydrolysis exerts robust anti-depressant-like effects (). Anandamide turned out to be a partial agonist of the two cannabinoid receptors, which is unusual for an endogenous natural ligand, and it is found at low concentrations (pmol/g tissue) in the brain (). Because its pharmacological activity did not fully recapitulate the behavioral effects of Δ9-THC (), the existence of a second endocannabinoid was postulated, and soon identified as 2-arachidonoylglycerol(2-AG) (Fig. 1I-J) (; ). 2-AG is a full agonist at both CB1 and CB2 receptors and it is present at much higher levels (nmol/g tissue) in the brain (). With the discovery of anandamide, 2-AG and the cannabinoid receptors, the “grass route” has gloriously ended after 3 decades, and gave the green light to neuroscientists to continue with hypothesis-driven questions on the function of endocannabinoid signaling, which has culminated in a paradigm shift in neuroscience.

The “Retrograde Route” of Endocannabinoids in the Brain

While anterograde synaptic transmission has been extensively studied for decades, much less information was known about retrograde signaling pathways at chemical synapses. Several candidates such as gases (e.g. nitric oxide), peptides (e.g. dynorphin), growth factors (e.g. brain-derived neurotrophic factor) or even conventional amino acid transmitters such as glutamate or GABA were shown to be released by the somatodendritic domain of postsynaptic neurons and then to act on the axon terminals of presynaptic neurons (). However, most of these retrograde messengers operate at specific synapses or restricted to a few cell types, and none of them could fully explain most common forms of retrograde synaptic communication. Based on high anandamide synthase activity in the hippocampus and the then prevailing view that the chemically related lipid molecule arachidonic acid is a retrograde messenger in hippocampal long-term potentiation (), Devane and Axelrod proposed first that anandamide may also play a retrograde messenger role on axonal CB1 receptors (). In accordance, the first immunohistochemical studies visualizing CB1 protein localization reported dense meshwork of fibers throughout the brain at the light microscopic level (; ). These fibers proved to be axons in both the rat and human brain, as revealed by high-resolution immunogold staining and electron microscopy (; ). In fact, the majority of CB1 receptors accumulated presynaptically on axon terminals of GABAergic interneurons in the hippocampus (Fig. 2A). Furthermore, CB1 receptor agonists reduced electrically-evoked GABA release (Fig. 2B), in accordance with a proposed retrograde messenger function of endocannabinoid signaling (). The presynaptic localization of CB1 receptors and its inhibitory effect on neurotransmitter release have proved to be a general feature of most axon terminals in the central (), and peripheral nervous system ().

Figure 2
The retrograde route of endocannabinoid signaling in the brain

The most important support for the retrograde scenario came subsequently from the study of an electrophysiological paradigm, in which selective depolarization of a postsynaptic neuron induces short-term depression of GABA release from axon terminals innervating the same postsynaptic neuron (DSI) (; ). This robust phenomenon premised the existence of a bona fideretrograde messenger, and thus, the demonstration that three independent antagonists of CB1 receptors block DSI at hippocampal GABAergic synapses (Fig. 2C-D) (; ), together with the presynaptic localization of CB1 receptors (), were in agreement with the plausible scenario that endocannabinoids may be the long-awaited retrograde messengers. Importantly, depolarization-induced suppression of excitation (DSE) was also inhibited by a CB1 antagonist at cerebellar excitatory synapses (). Several other forms of synaptic plasticity were subsequently reported to be CB1-dependent including e.g. long-term depression (Fig 2E-F) (; ; ). Collectively, these findings indicated that endocannabinoid signaling plays a conceptually similar role at distinct types of synapses throughout the brain, and represent the turning point, when endocannabinoid research finally became a major focus for mainstream neuroscience.

Although the most parsimonious scenario is the retrograde route for endocannabinoids, the manner of how anandamide and/or 2-AG get through the extracellular space is still an unresolved issue considering that these lipid messengers are fairly hydrophobic with logP values of 5.1 and 5.39 for anandamide and 2-AG, respectively (). Nevertheless, converging evidence from the combination of biochemical, anatomical, pharmacological and physiological experiments fully supports the retrograde scenario. As the first step, molecular identification of enzymes involved in endocannabinoid metabolism helped to define which endocannabinoid molecule is responsible for given forms of endocannabinoid-mediated plasticity. For example, the inactivation of anandamide is carried out by a serine hydrolase called fatty acid amide hydrolase (FAAH) (). Thus, the lack of effect of FAAH inhibitors in a given paradigm indicates that anandamide is not involved in that particular phenomenon as it was stated for example for hippocampal DSI (). The second candidate 2-AG is synthesized by two isoforms of diacylglycerol lipase, α and β, (), whereas a monoacylglycerol lipase (MGL) was found to degrade the majority (85%) of 2-AG in the brain (; ). Genetic inhibition of MGL consistently enhanced short-term synaptic depression (; ), whereas genetic inactivation of DGL-α fully eliminated all forms of endocannabinoid-mediated synaptic plasticity in the prefrontal cortex (), hippocampus (; ), striatum and cerebellum (). Thus, the indispensable involvement of DGL-α and the regulatory role of MGL in short-term synaptic depression clearly supports the view that 2-AG is the enigmatic synaptic endocannabinoid.

However, these pharmacological and genetic perturbations would not exclude a scenario that 2-AG plays an autocrine role on axon terminals, where CB1 receptors are located. The additional support derives from anatomical observations, which revealed that the subcellular segregation of endocannabinoid-metabolizing enzymes paves a retrograde way for 2-AG throughout the brain. DGL-α was found postsynaptically at several synapse types in the spinal cord (), cerebellum (), ventral tegmental area (), striatum (), basolateral amygdala (), hippocampus (; ), prefrontal cortex () and even in the human hippocampus (). Conversely, MGL was observed presynaptically in axon terminals throughout the brain (; ;  ). Taken together, these anatomical and physiological experiments outlined that the most parsimonious scenario for retrograde synaptic signaling involves 2-AG, which is synthesized and released postsynaptically and then acts on CB1 receptors located on nearby presynaptic axon terminals.

Common Principles of Anterograde Amino Acid Transmission and Retrograde Endocannabinoid Transmission

In their influential review, Sudhof and Malenka recently argued that one of the most important advances in our understanding of synaptic transmission in the last 20 years was the discovery that endocannabinoids are the principal mediators of retrograde synaptic communication (). Since the breakthrough discoveries ten years ago, several hundred studies have dealt with the role of endocannabinoids in synaptic transmission and the retrograde scenario has become widely accepted. It is vital in most biological signaling systems that information flow is precisely controlled by feed-back mechanisms. Thus, it is not surprising that synaptic transmission in chemical synapses requires a similar feed-back mechanism, although it was clearly unforeseen that the consensus molecule for this function would be an endocannabinoid. Also unexpected was that the basic operational principles of retrograde endocannabinoid signaling have so many features in common with conventional anterograde synaptic transmission. In the following sections, we aim to highlight the striking conceptual similarities between classical amino acid transmitter-mediated neurotransmission and retrograde endocannabinoid signaling arguing that the endocannabinoid system is a component of chemical synapses as basic as the conventional neurotransmitter systems (Table 1). It is impossible in a single review to detail these biological phenomena, hence, we picked a few notable examples to summarize key features of endocannabinoid signaling together with some physiological and pathophysiological implications focusing on burning questions in endocannabinoid research.

Table 1
Analogies can be observed in many respects between anterograde transmission mediated by classical amino acid transmitters and endocannabinoid-mediated retrograde signaling. See main list for abbreviations.

Molecular Complexity of Endocannabinoid Mobilization and Degradation

Why the brain needs - at least - two endocannabinoid molecules? Neurons exploit several messengers to operate anterograde synaptic transmission in the brain, predominantly the classical amino acids glutamate and GABA, but glycine and the aromatic amino acid derivative monoamines such as dopamine, serotonin or noradrenaline have also important functions. As retrograde messenger, 2-AG is the key player in most forms of homo- and heterosynaptic short-term depression and in some forms of long-term depression (; ). It is clear however that anandamide also acts through CB1receptors in several neurobiological paradigms (; ; ), where it may function as a retrograde synaptic messenger and mediate certain forms of synaptic homeostasis and plasticity in presynaptic CB1 receptor-dependent manner (, ). Alternatively, anandamide’s synaptic function can also be the activation of postsynaptic TRPV1 receptors (; ). The division of labor between anandamide and 2-AG may be reflected in spatial segregation, if anandamide and 2-AG serve as messengers at different synapses, in distinct microcircuits, or in separate brain regions. This is supported by observations that specific FAAH and MGL inhibitors recapitulate only subsets of behavioral components of cannabinoid effects in vivo (). These behaviors are regulated by CB1 receptors located in distinct cell types and brain circuits (), and spatial isolation also occurs at the subcellular level, because MGL and FAAH are segregated into the presynaptic and postsynaptic domains of neurons, respectively (). The division of labor may also happen at different time scales. Phasic endocannabinoid signaling, such as DSI is mediated by 2-AG (, ), whereas tonic endocannabinoid signaling involves the mobilization of both 2-AG () and anandamide () at hippocampal GABAergic synapses, though likely under different physiological conditions. Finally, the two signaling systems may even interplay in certain behavioral processes. Neither FAAH, nor MAGL inhibitors alone could recapitulate catalepsy or drug discrimination, typical CB1 agonist-evoked behavioral effects. However, a dual FAAH/MGL inhibitor produces catalepsy and also Δ9-THC-like drug discrimination response (). Thus, the combined action of 2-AG and anandamide may be required to fully engage CB1 receptors at specific synapses, and if the mobilization of two messenger molecules require different physiological signals, then presynaptic CB1 receptors may operate as coincidence detectors to underlie synaptic plasticity analogously as postsynaptic NMDA receptors.

The level of complexity in the operational principles and functional significance of retrograde endocannabinoid signaling is further increased by the emerging concept that, in contrast to the primarily amino acid-based anterograde transmission, a multifaceted lipid signaling system evolved to fulfill the complex physiological tasks reliant on retrograde signaling. For example, both anandamide and 2-AG are oxygenated by cyclooxygenase-2 (COX-2) at postsynaptic sites (; ; ; ). The resulting prostanoids, e.g. prostaglandin E2 glycerol ester (PGE2-G) increase neurotransmitter release from axon terminals (), which is the opposite effect to that of 2-AG. Thus, COX-2, which is transported to synapses in an activity-dependent manner may be an important molecular switch to change the direction of synaptic plasticity. Therapeutically important in vivo manifestation of this phenomenon occurs in supraspinal and spinal pain circuitries, where 2-AG has an overall anti-nociceptive effect (; ), whereas 2-AG-derived PGE2-G causes hyperalgesia (). Remarkably, the highly potent analgesic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) like paracetamol (acetaminophen), a selective COX-2 inhibitor (), or ibuprofen, a potent inhibitor of 2-AG oxidation by COX-2 () require the activation of CB1 receptors (; ). Thus, these NSAIDs may partially exhibit their analgesic effects via vetoing the metabolism from anti-nociceptive 2-AG to hyperalgesic prostaglandin caused by hyperalgesia-induced elevations in COX-2 activity.

The discovery of 2-epoxyeicosatrienoylglycerols (2-EGs) also revealed a link to lipoxygenase and cytochrome P450 pathways. These novel lipids are present in the brain (especially 2-11,12-EG) and surprisingly, they are potent endogenous ligands of both cannabinoid receptors in vivo (). Anandamide can be transformed to 5′,6′-epoxyeicosatrienoic acid by CP450 epoxygenases, and activates TRPV4 channels at submicromolar concentrations (), whereas the related eicosanoid 12-(S)-hydroperoxyeicosatetraenoic acid is the retrograde mediator of TRPV1-dependent long-term depression at hippocampal Schaffer collateral-interneuron synapses (). Although the puzzling question, why lipids are utilized so extensively as retrograde messengers in contrast to the predominantly amino acid-based anterograde transmitters may not be easy to answer, this perplexing diversity of lipid signaling pathways should definitely be in focus for neuroscience.

A striking example of parsimony in biology is the way in which neurons exploit amino acids (like glutamate or glycine) as neurotransmitters, which are otherwise basic building blocks of proteins. These amino acids can even be metabolized to each other to produce additional messengers, as GABA is synthesized from glutamate by decarboxylation. This parsimony is also shared by the endocannabinoid system. Glycerol, ethanolamine and arachidonic acid are at the crossroads of several metabolic pathways, included in several major components of biological membranes or provide energy for cellular functions. Arachidonic acid, the common constituent of 2-AG and anandamide is a conditionally essential n-6 polyunsaturated fatty acid (n-6 PUFA) and makes up a significant fraction of brain lipids (). It is present in the diet together with its precursor linoleic acid, and consequently, when their dietary concentrations are increased, anandamide and 2-AG levels also increase (). Remarkably, this has an impact on synaptic endocannabinoid signaling, because changing the dietary n-6/n-3 PUFA ratio abolishes endocannabinoid-mediated LTD in the prefrontal cortex and nucleus accumbens (). Although the underlying mechanistic process is not fully understood, chronic elevation of 2-AG levels also disrupts CB1-mediated signaling highlighting the importance of precise metabolic regulation of 2-AG levels in the brain (; ). Anandamide and 2-AG can also be metabolized to each other like amino acid transmitters and this metabolic pathway can even be region- and subcellular domain-specific. First, anandamide levels are decreased in the hippocampus, but not in the prefrontal cortex of DGL-α knockout animals (, , ). Second, free arachidonic acid can be released from 2-AG by at least three serine hydrolases (), and one of these, α/β-hydrolase-6 (ABHD6) regulates synaptic endocannabinoid signaling in long-term depression (). Moreover, ABHD6 colocalizes postsynaptically in dendrites with FAAH, which conjugates ethanolamine with arachidonic acid to form anandamide ().

The biogenesis of endocannabinoids is also highly complex. Anandamide and related N-acylethanolamines (NAEs) were postulated to be synthesized by at least 5 metabolic pathways. Using N-acyl-phosphatidylethanolamines (NAPEs) as precursors, the PLA route involves phospholipase A2 and lysophospholipase D (), the PLB route includes α/β-hydrolase 4 and glycerophosphodiesterase 1 (; ), the PLC route is mediated by a phospholipase C and protein tyrosine phosphatase type-22 (), whereas the PLD route exploits NAPE-hydrolyzing phospholipase D (NAPE-PLD) (). In addition, anandamide can be formed by conjugation of arachidonic acid and ethanolamine in brain synaptosomes (). A most exciting question for endocannabinoid research is to understand the functional significance of this metabolic diversity. Spatial segregation of distinct anandamide- and NAE-synthesizing pathways may underlie division of labor. Indeed, while FAAH is distributed in the somatodendritic domain of neurons (, ), NAPE-PLD was found inside glutamatergic axon terminals (). Another indication of functional diversity is the different kinetics of anandamide synthesis, which suggests that the PLC route may operate at a different time scale than the PLB route ().

The biosynthesis of 2-AG may seem to be more simple, but this can be misleading. While the role of DGL-α in synaptic plasticity is unequivocal (), the precursor DAG can be synthesized in several ways. The canonical pathway includes phospholipase C-βs, which hydrolyze phophatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and DAG (). Most receptor-driven 2-AG synthesis may go through this route. However, PLCβ1 is not required for depolarization-induced forms of endocannabinoid-mediated synaptic plasticity such as hippocampal DSI (). DAG-independent 2-AG biosynthesis may occur via PLA1 and lyso-PI-specific PLC activity, the latter of which is found in brain synaptosome preparations (; ). Finally, brain homogenates can also synthesize 2-AG from 2-arachidonoyl-lysophosphatidic acid by an unidentified phosphatase (). Thus, another important task for endocannabinoid research is to dissect which biochemical pathways are responsible for 2-AG mobilization at given locations in neuronal networks and to identify which physiological or pathophysiological stimuli activate these pathways.

A spatial segregation indicating functional division of labor has already been reported for the two isoforms of DGL (). While DGL-α is found in the plasma membrane (, ; ), DGL-β is restricted to intracellular membrane segments, including peri-nuclear lipid droplets (). This spatial segregation suggests that DGL-α may play a role in intercellular 2-AG signaling, whereas DGL-β has an intracellular function. In parallel, basal 2-AG levels in the brain of DGL-α knockout mice were dropped by 80%, but remained unaffected or only partially reduced in DGL-β knockout mice (, ). In contrast to DGL-α knockout mice, synaptic endocannabinoid signaling was not impaired by genetic inactivation of DGL-β (; ). It is interesting to note that neurite outgrowth triggered by overexpression of DGL-α could be blocked by a CB1 receptor antagonist, whereas neuritogenesis induced by overexpression of DGL-β was CB1 receptor-independent (). Similarly, adult neurogenesis is impaired in DGL-α, but not in DGL-β knockout mice ().

Endocannabinoid Signalosomes

The convincing demonstration that DGL-α is indispensable for various forms of retrograde synaptic signaling () calls for investigations of how this important enzyme is integrated into neuronal operations. At hippocampal glutamatergic synapses, DGL-α accumulates around the postsynaptic density at the edge of synapses (; ). Group I mGlu receptors are located within the same perisynaptic annulus () and their agonists evoke 2-AG release through the canonical Gq/11 and PLC-β pathway (). Because both mGlu receptors and DGL-α contain binding motifs for the synaptic scaffold protein Homer (), we proposed that they form a macromolecular complex around the postsynaptic density, the so-called perisynaptic signaling machinery (PSM), which evolved to translate excess presynaptic activity - glutamate spillover in this case - into a negative feed-back signal (). Since then, new findings confirmed and extended the PSM concept. PLC-β1, another molecular constituent of this pathway was also found in the perisynaptic annulus (). Moreover, the long-isoform Homer2b turned out to be necessary for mGlu-triggered 2-AG release (, ), whereas the activity-dependent short isoform (Homer1a) dismantled the PSM and ablated this process ().

Importantly, DGL-α may not only function as a 2-AG-synthesizing enzyme, but could play another role in regulating DAG levels. This function may even be phylogenetically more ancient, because insects lack cannabinoid receptors, but express a DGL-α ortholog encoded by the inaE gene, which is necessary for the opening of TRP channels by DAG (). In mammals, TRP channels like TRPC1 or TRPC3 are also known to be anchored by Homer, concentrated perisynaptically, and stimulated by DAG (, ). In addition, activation of TRPC channels accounts for group I mGlu receptor-triggered feed-forward enhancement of excitability of postsynaptic neurons (; ). Taken together, some molecular players within the PSM are primarily responsible for feed-forward excitation. However this signal is controlled by DGL-α, which may act as a molecular switch by transforming a feed-forward excitatory DAG signal into a negative feed-back signal via 2-AG (Fig. 3). Perisynaptic metabotropic glutamate receptors cannot be activated by single synaptic volleys as intrasynaptic ionotropic glutamate receptors, but require elevated, usually bursting presynaptic population activity (; ). Therefore, we propose that the essential physiological function of the PSM domain of excitatory synapses is to monitor the magnitude of presynaptic activity. Increased presynaptic activity may need to be transformed to a feed-forward excitatory response to increase the excitability of the postsynaptic neuron and to support synaptic potentiation. However, excess presynaptic activity can also become pathological, hence efficient negative feed-back mechanisms should kick in. We suggest that DGL-α may be involved in this mechanism in a strikingly parsimonious manner by terminating feed-forward excitation and initating feed-back inhibition, in other words, by eliminating the cause and the consequence of the excess presynaptic activity at the same time (Fig. 3A-B).

Figure 3
DGL-α as a molecular switch between GPCR-mediated feed-forward excitation and feed-back inhibition

We recently termed the second, 2-AG leg of the pathway a “synaptic circuit-breaker” to indicate that this process may not only happen at single synapses in a homosynaptic manner, but may be a general network mechanism that has a pivotal role in regulating the overall level of network excitability under pathophysiological conditions with an excess glutamatergic tone (). Neuronal insults e.g. convulsions or closed-head injury evoke 2-AG release (; ), whereas perturbations of the synaptic circuit-breaker leads to reduced seizure thresholds and increased incidence of epileptic seizures. Double Gq/11 and PLC-β1 knockout animals die at a young age as a result of spontaneous seizures (; ), whereas glutamatergic cell-specific overexpression or deletion of CB1 receptors are protective or convulsive, respectively (; ; ). Finally, breakdown of the circuit-breaker also occurs in human patients with chronic, intractable temporal lobe epilepsy, whose hippocampus has reduced levels of DGL-α and CB1 receptors (). Taken together, the significance of the PSM and retrograde 2-AG signaling is also reflected at the pathophysiological level and may be exploited for therapy in the future.

A salient emerging concept is that the PSM at glutamatergic synapses may only be a specialized case of a much more fundamental cell physiological mechanism involving the same macromolecular complex and retrograde endocannabinoid signaling. Following the initial observations that Gq/11-coupled, postsynaptic mGlu1 and mGlu5 receptors can elicit synaptic endocannabinoid signaling in the cerebellum and hippocampus (; ), at least 16 other neurotransmitter molecules were shown to trigger 2-AG release and CB1 receptor activation via Gq/11-coupled receptors, PLC-β and DGL-α (Fig 3). Notably, besides the homosynaptic feed-back processes, upstream activation of this macromolecular complex can often lead to heterosynaptic depression of the release of another neurotransmitter as is the case for serotonin and 5HT2A/5HT2B/5HT2C receptors in the inferior olive and elsewhere (; ). These signalosomes may not always be restricted to the perisynaptic zone of synapses, but their subcellular location reflects the source and chemical nature of the given transmitter. For example acetylcholine, which primarily reaches its receptors by volume transmission can evoke endocannabinoid signaling in several brain regions through M1receptors, which are distributed throughout the somatodendritic surface of postsynaptic neurons (; ; ). Some of these signaling mechanisms can be surprisingly cell type-specific as has been shown for the neuropeptide cholecystokinin and CCK2 receptors in the hippocampus (; ). Some may convey information about the general physiological or metabolic state of the animal like the wake-promoting peptide orexin-B and its Gq/11-coupled receptors OX1 and OX2 (), endothelin-1 and its ETA receptor (), oxytocin and its OT receptor () or ghrelin and its receptor (), and then regulate feed-back hormonal responses via the modification of synaptic weights in subcortical and cortical circuits. Particularly interesting is how these signalosomes may be involved in pathophysiological processes upon CNS injury. Thrombin-induced arachidonic acid release led to the original discovery of a PLC-DGL pathway (), and thrombin regulates GABAergic synaptic currents through PAR-1 receptors and retrograde 2-AG signaling in the hippocampus (). Other functionally related pathways also stimulate endocannabinoid signaling as has been demonstrated for thromboxane A2 via the prostanoid receptor TP (), and for the platelet-activating factor through its receptor PAF (). Some other GPCR-endocannabinoid signalosomes are also expected to be found in the CNS, as they are widely distributed throughout the body such as angiotensin II and its AT1 receptor (), the bombesin’s neuromedin B, gastrin-releasing peptide and its BB1/BB2/BB3 receptors (), bradykinin and its B1 and B2 receptors (), noradrenaline and the adrenoceptors α1A α1B α1D () and vasopressin and its V1A and V1B receptors (). It may be too early to claim that the DGL-α - 2-AG - CB1 (and maybe also CB2) endocannabinoid signaling pathway is a built-in feature of the downstream signaling pathway upon Gq/11-coupled receptor activation, nevertheless, when present, it can efficiently translate a feed-forward signal into a feed-back signal to regulate cellular functions.

Molecular Complexity of Endocannabinoid-targeted Receptors

As a conceptual similarity again with classical amino acid transmitters, which act on several ionotropic (AMPA, NMDA, kainate or GABAA, GABAC and Gly) and metabotropic (mGlu or GABAB) receptors to accomplish their diverse responsibilities, these lipid messengers can also interact with several other molecular targets besides CB1 and CB2 cannabinoid receptors. Among these potential targets are ligand-gated ion channels like 5-HT3, glycine and nicotinic acetylcholine receptors, non-selective cation channels like TRPV1, TRPA1 or TRPM8, voltage-gated ion channels like T-type calcium channels or the TASK potassium channels, and metabotropic receptors like GPR55 (for review see ). An especially exciting research direction is to delineate the neurobiological significance of these interactions, which, despite some promising progress (; ), is largely unknown ().

An additional level of signaling complexity for anterograde transmission comes from the variable subunit compositions of amino acid receptors, some well-known examples of which are the synapse-specific segregation of calcium-permeable AMPA receptors determined by absence of the GluR2 subunit (), or the observation that tonic and phasic modes of GABA signaling are mediated by GABAA receptors with different subunit compositions (). A potential mechanism to increase the complexity of endocannabinoid signaling may be the phenomenon of receptor heteromerization. Heterodimers of CB1 receptors have been observed for example with D2 dopamine receptors (), μ-opioid receptors () or OX1 orexin receptors (). Heteromerization may impact ligand sensitivity, downstream signaling and even compartmentalization of a given receptor, which all contribute to the ultimate physiological role of the receptor complex. Thus, it will be a very important task to exploit the latest available microscopy techniques offering appropriate spatial resolution like super-resolution microscopy to characterize the cell type- and synapse type-specific distributions of given cannabinoid receptor heterodimers in brain circuits.

It is widely accepted that besides their primary ligands, the activity of most receptors is controlled by endogenous allosteric modulators. Some famous examples for glutamate receptors include the role of glycine or D-serine in the regulation of NMDA receptors (; , or the profound impact of neurosteroids on δ-subunit-containing GABAA receptors (). Similar endogenous modulators for cannabinoid receptors are just starting to appear. The lipid virodhamine was the first reported endogenous antagonist of CB1 receptors, which surprisingly acts as an agonist on CB2 receptors (). An unexpected new family of CB1 receptor modulators comprises the hemoglobin-derived nonapeptide hemopressin and its longer congeners, which act as an inverse agonist or agonist, respectively (; ). The potential presence of allosteric binding sites on CB1 receptors and the design of selective agents targeting these sites would be especially advantageous, because full agonists evoke robust internalization of cannabinoid receptors (), which lead to in vivo tolerance (), and renders pharmacological exploitation difficult. On the other hand, internalization of presynaptic CB1 receptors on axon terminals offers a new level of physiological control (), whereby the efficacy of endocannabinoid-mediated synaptic plasticity can be dynamically adjusted in an activity-dependent manner. Besides internalization, lateral movement of CB1 receptors on the surface of axon terminals is also well-suited to regulate CB1 receptor availability and desensitization (). Notably, lateral mobility and internalization of postsynaptic AMPA receptors are also key underlying mechanisms for experience-dependent plasticity of anterograde excitatory synaptic transmission ().

Functional Complexity of Endocannabinoid-mediated Synaptic Plasticity

Probably the most spectacular evidence for the profound neurobiological significance of endocannabinoid signaling is the wide repertoire of synaptic physiological processes which are mediated by endocannabinoids. It is again conceivable to suppose that nature followed the same rule of parsimony just as for glutamatergic and GABAergic neurotransmission by adapting the activity of the same conserved molecular players throughout the central and peripheral nervous system to accomplish so many different synaptic physiological tasks for the proper operation at the brain circuit levels. It is out of the scope of this work to summarize the hundreds of studies describing the specific function of retrograde endocannabinoid signaling from the spinal cord to the neocortex, but we recommend two excellent reviews for further reading (; ). Instead, we aim to illustrate with a few select examples the conceptual similarities in which the brain exploits endocannabinoids for retrograde signaling processes, whereas classical amino acid transmitters for anterograde communication.

Regarding the two major modes of endocannabinoid signaling, one has to differentiate between tonic and phasic actions. Just as ambient GABA has a crucial role in establishing the excitability of postsynaptic neurons through extrasynaptic GABAA receptors (), the pivotal role of ambient extracellular endocannabinoid concentrations in the determination of neurotransmitter release probability has just begun to unfold. Paired recordings from a special subtype of GABAergic interneuron and postsynaptic CA3 pyramidal neurons in the hippocampus revealed that tonic endocannabinoid signaling can mute GABA release from axon terminals through CB1 receptor activation (). Subsequent research has shown that this phenomenon is not due to the constitutive activity of CB1receptors per se, but depends on the constitutive release of endocannabinoids from the postsynaptic neuron, as postsynaptic BAPTA chelation of intracellular Ca2+ signals abolished the tonic endocannabinoid signaling (; ). Remarkably, tonic endocannabinoid signaling is also cell type-specific. It can depend on the type of the postsynaptic neuron, e.g. proopiomelanocortin (POMC) neurons, but not the neighboring non-POMC neurons, release endocannabinoids constitutively and regulate their incoming GABAergic inputs in the arcuate nucleus of the hypothalamus, although both cell populations could produce endocannabinoids in a stimulation-dependent manner (). Alternatively, the same postsynaptic neuron can also regulate GABAergic inputs in a different manner as has been elegantly demonstrated in the hippocampus, where CA1 pyramidal neurons regulate perisomatic inhibition via endocannabinoids both in a tonic and phasic manner, whereas only phasic endocannabinoid signaling was found at dendritic inhibitory inputs (). This latter observation at unitary connections also confirms that even constitutive endocannabinoid release can act in a homosynaptic and subcellularly restricted manner (; ). Tonic endocannabinoid signaling may involve both anandamide and 2-AG, although probably at different time scales (; ). The presence of tonic endocannabinoid control of neurotransmitter release indicates that physiological signals must exist to override it whenever necessary. On the other hand, this phenomenon can also serve to integrate the efficacy of a dedicated unitary connection into network activity, because high-frequency firing of the presynaptic neuron can eliminate the tonic endocannabinoid blockade (; ).

Salient features of synaptic endocannabinoid signaling ideally support the induction of changes in synaptic strength in a phasic, activity-dependent manner. The governing rules for these retrograde forms of synaptic plasticity also follow similar logic, and these mechanisms are nicely integrated into several well-known forms of anterograde synaptic plasticity mediated by glutamate or GABA. Homosynaptic short-term synaptic depression of excitation and inhibition was the first described form of endocannabinoid-mediated synaptic plasticity (; ; ), and supposed to be mediated predominantly by 2-AG (; ; ; ). Although the extracellular spread of endocannabinoids is limited (), endocannabinoid-mediated short-term depression is also involved indirectly in heterosynaptic forms of plasticity (). Endocannabinoid-mediated forms of long-term depression were first described in the dorsal and ventral striatum and in the amygdala, but are also present at most synapses in the brain (; ; , ; ). A special, potentially homosynaptic form is the so-called spike timing-dependent long-term depression, which was described first in the neocortex and requires presynaptic NMDA or postsynaptic mGlu receptors (; ). Heterosynaptic forms of long-term depression are also known to exploit endocannabinoids ().

Another special form of synaptic plasticity serves to re-adjust synaptic gain in response to persistent changes of neuronal activity. This phenomenon of synaptic homeostasis involves increase in the action-potential independent release probability of glutamate and in the density of postsynaptic glutamate receptors, but also a decrease in the number of postsynaptic GABA receptors to restore circuit activity (). There are likely to be multiple forms of synaptic scaling and correspondingly, both anandamide and 2-AG was reported to contribute to homeostatic regulation of GABAergic synapses in the hippocampus (, ). In addition, just as glutamate and GABA are known to act also in an autocrine manner to regulate their own release, compelling evidence supports that both endocannabinoids may also have autocrine functions. The postsynaptic role of anandamide in long-term depression was postulated in the hippocampus and striatum (; ), whereas 2-AG is the mediator of the autocrine phenomenon of slow-self inhibition in neocortical interneurons (). Although it took some time for neuroscientists to accept that glutamate can also be a gliotransmitter (there are multiple types of glutamate and GABA receptors on glial cells), the idea that molecules can be utilized for multiple physiological functions gained wider recognition. In parallel, exciting new discoveries revealed that endocannabinoid signaling not only depresses, but also potentiates glutamatergic transmission via a novel form of neuron-glia crosstalk ().

Cell Type- and Synapse-specific Differences in Endocannabinoid Signaling underlying Circuit-dependent Behaviors

In the previous section, we aimed to illustrate the perplexing chemical and functional diversity of the endocannabinoid system in the brain and to highlight that the same neurobiological principles may govern anterograde synaptic transmission and retrograde endocannabinoid signaling. Finally, we consider the idea that subtle, but important refinements in the logic of endocannabinoid signaling were evolved to provide the most optimal contribution of retrograde communication to the functional operation of microcircuits. Probably all brain circuits require detailed studies to fully delineate how given endocannabinoid signalosomes in particular subcellular compartments mediate certain forms of synaptic plasticity and thereby regulate network activity and behavioral processes. Here we describe a few striking examples from the hippocampus (Fig. 4).

Figure 4
Quantitatively differential distribution of the molecular players of retrograde 2-AG signaling in cortical areas

In contrast to the qualitative uniformity of synaptic 2-AG signaling, each glutamatergic and GABAergic synapse is quantitatively different regarding the density of the molecular components of the 2-AG pathway. DGL-α has the highest density in the inner molecular layer at mossy cell-granule cell synapses, is abundant at the Schaffer collateral-CA1 pyramidal neuron synapses, and is localized at other glutamatergic synapses throughout the hippocampal formation () (Fig. 4A). The functional consequence of this input-specific pattern is reflected in the distinct thresholds necessary to evoke 2-AG mediated DSE at different glutamatergic synapses (). This complexity is further increased at the ultrastructural level, and may underlie the contribution of 2-AG to different homo- or heterosynaptic forms of synaptic plasticity. DGL-α is concentrated in a perisynaptic annulus in the head of dendritic spines in the CA1 subfield (; ), conversely, it is accumulated around the necks of spines of dentate gyrus granule cells (). In contrast to glutamatergic synapses, only a very small amount of DGL-α is present at hippocampal or neocortical GABAergic synapses (I. Katona pers. comm.), as clearly reflected by the lack of DGL-α immunostaining in the cell body layers (Fig. 4A-B). On the other hand, GABAergic synapses in the basolateral amygdala show an extremely high density of DGL-α (Fig. 4C) ().

CB1 receptors also exhibit a characteristic expression pattern and layer-specific distribution (Fig. 4E-F). In contrast to DGL-α, CB1 receptors have the highest density on GABAergic synapses derived from the CCK-positive class of interneurons in the rodent and human hippocampus (; ). However, even distinct types of CCK-positive interneurons, the perisomatic basket cells and the Schaffer collateral-associated dendritic inhibitory cells, differ in their CB1 content, and this matches the distinct efficacy of endocannabinoid-mediated synaptic plasticity at these synapses (). CB1receptors are also present at glutamatergic synapses, albeit at much lower levels (; ). The distribution of the degrading enzyme MGL displays an even more surprising pattern. While axon terminals of GABAergic interneurons and recurrent collaterals of CA3 pyramidal neurons bear a high density of MGL (Fig. 4I-J), Schaffer collaterals derived from the same CA3 pyramidal cells may contain less MGL (Fig. K-L) (; ). Moreover, MGL density is strikingly low in the inner molecular layer on axon terminals of mossy cells (Fig. 4G-H) (; ). Although the physiological consequences of these quantitative differences are not yet fully understood, it suggests that the contribution of 2-AG signaling to distinct behavioral phenomena and pathophysiological processes may be qualitatively different at specific synapses and microcircuits.

The development of cell type-specific CB1 receptor knockout models represent the key innovation to elucidate how endocannabinoid signaling at specific microcircuit locations contributes to network activity and behavior (; , ). Although GABAergic axon terminals carry much more CB1 receptors than their glutamatergic counterparts, they are not involved in seizure susceptibility (). Instead, these receptors play a pivotal role in Δ9-THC-induced long-term memory deficits (), and protect against age-related cognitive decline (). A similar cell type-specific functional dichotomy was observed in the regulation of feeding and energy balance, where CB1 receptors on striatal GABAergic neurons reduce food intake, whereas those on forebrain glutamatergic axons convey an orexigenic signal (Bellochio et al., 2010). Opposite function of CB1 receptors on different cell types is also exemplified in the pain transmission circuitry, where deletion of CB1 from primary nociceptive neurons in the dorsal root ganglia proved to be pro-nociceptive, whereas removal from GABA/glycinergic terminals protects from central hyperalgesia (; ). Collectively, these findings demonstrate that endocannabinoid signaling at different synapses contributes to distinct behavioral components controlled by certain neuronal circuits.

Closing remarks

The ultimate mission of life sciences in the post-genomic era of biology is to provide a full understanding of the function of all molecular players encoded in our genome together with all small-molecule metabolites comprising our metabolome. In neuroscience, we will even need to integrate these emerging data with the cell type-catalog of the brain and functional connectomics. One admitted expectation is that these enormous datasets generated by large-scale community efforts will support systems biology approaches to uncover conceptually new principles of biology, whereas another major force fuelling these efforts is the tremendous potential for evidence-based novel therapeutics. The unfolding of the molecular, anatomical, physiological and behavioral features of endocannabinoid signaling in the last decade fully justifies this expectation, because it has led to our appreciation of the fundamental role of retrograde communication in the brain. Besides its multiple functions, molecular complexity and selective impairment in distinct brain disorders also offer hopes that the endocannabinoid system may be exploited therapeutically.

Acknowledgements

The authors are grateful M. Herkenham, R. Hargraeves, A. Howlett, M. Kano, D. Lovinger, O. Manzoni, R. Mechoulam, R. Nicoll, Y. Shim, K. Van Laere and M. Watanabe for permission to modify figures from their original work. We thank Balázs Baksa for the artwork, Drs. Chris Henstridge, Ewen Legg, Barna Dudok, Eszter Horváth, Balázs Pintér for help with the preparation of the manuscript, and to members of the Katona and Freund labs for discussions. The authors were supported by grants from the Swiss Contribution (SH7/2/18), the Hungarian Scientific Research Fund (NK77793), the Norwegian Financial Mechanism Joint Program (NNF 78918), the European Research Council (No: 243153), and NIH (MH 54671 and NS30549). I.K. is the recipient of a Wellcome Trust International Senior Research Fellowship.

Abbreviations

2-AG 2-arachidonoylglycerol
2-EG 2-epoxyeicosatrienoylglycerol
Δ9-THC (−)-Δ9-tetrahydrocannabinol
AA arachidonic acid
AAT aspartate aminotransferase
ABHD6 α/β hydrolase 6
AEA anandamide
Ang angiotensin
CNS central nervous system
COX-2 cyclooxygenase-2
DAG diacylglycerol
DGL diacyglycerol lipase
DSE depolarization-induced suppression of excitation
DSI depolarization-induced suppression of inhibition
EA ethanolamine
FAAH fatty acid amide hydrolase
GABA gamma-aminobutyric acid
GAD glutamic acid decarboxylase
Glu glutamate
Gly glycine
GPCR G protein-coupled receptor
IP3 inositol 1,4,5-triphosphate
IUPHAR International Union of Basic and Clinical Pharmacology
LTD long-term depression
lyso-PI lyso-phosphatidylinositol
MGL monoacylglycerol lipase
nAChR nicotinic acetylcholine receptor
NAE N-acylethanolamine
NAPE N-acyl-phosphatidylethanolamine
NAPE-PLD N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D
NSAID nonsteroidal anti-inflammatory drug
PGE2-G prostaglandin E2 glycerol ester
PIP2 phophatidylinositol 4,5-bisphosphate
PLA phospholipase A
PLB phospholipase B
PLC phospholipase C
PLD phospholipase D
POMC proopiomelanocortin
PSM perisynaptic signaling machinery
PUFA polyunsaturated fatty acid

Terms for mini-glossary

Retrograde signaling Retrograde messengers are released from the somatodendritic domain of neurons and then modify release properties of afferent axon terminals or regulate activity in nearby glial processes.
Depolarization-induced suppression of inhibition or excitation (DSI or DSE) Depolarized neurons release endocannabinoids that transiently inhibit GABA or glutamate release, respectively from their afferent synaptic terminals.

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