Takako Ohno-Shosaku

Endocannabinoid-Mediated Control of Synaptic Transmission

The discovery of cannabinoid receptors and subsequent identification of their endogenous ligands (endocannabinoids) in early 1990s have greatly accelerated research on cannabinoid actions in the brain. Then, the discovery in 2001 that endocannabinoids mediate retrograde synaptic signaling has opened up a new era for cannabinoid research and also established a new concept how diffusible messengers modulate synaptic efficacy and neural activity. The last 7 years have witnessed remarkable advances in our understanding of the endocannabinoid system. It is now well accepted that endocannabinoids are released from postsynaptic neurons, activate presynaptic cannabinoid CB(1) receptors, and cause transient and long-lasting reduction of neurotransmitter release. In this review, we aim to integrate our current understanding of functions of the endocannabinoid system, especially focusing on the control of synaptic transmission in the brain. We summarize recent electrophysiological studies carried out on synapses of various brain regions and discuss how synaptic transmission is regulated by endocannabinoid signaling. Then we refer to recent anatomical studies on subcellular distribution of the molecules involved in endocannabinoid signaling and discuss how these signaling molecules are arranged around synapses. In addition, we make a brief overview of studies on cannabinoid receptors and their intracellular signaling, biochemical studies on endocannabinoid metabolism, and behavioral studies on the roles of the endocannabinoid system in various aspects of neural functions.

Endogenous Cannabinoids Mediate Retrograde Signals from Depolarized Postsynaptic Neurons to Presynaptic Terminals

Endogenous cannabinoids are considered to function as diffusible and short-lived modulators that may transmit signals retrogradely from postsynaptic to presynaptic neurons. To evaluate this possibility, we have made a paired whole-cell recording from cultured hippocampal neurons with inhibitory synaptic connections. In about 60% of pairs, a cannabinoid agonist greatly reduced the release of the inhibitory neurotransmitter GABA from presynaptic terminals. In most of such pairs but not in those insensitive to the agonist, depolarization of postsynaptic neurons and the resultant elevation of intracellular Ca2+ concentration caused transient suppression of inhibitory synaptic currents, which is mainly due to reduction of GABA release. This depolarization-induced suppression was completely blocked by selective cannabinoid antagonists. Our results reveal that endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals to cause the reduction of transmitter release.

The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum

Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocannabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, “CB3,” and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s) of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specific antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum.

Phospholipase Cβ Serves as a Coincidence Detector through Its Ca2+ Dependency for Triggering Retrograde Endocannabinoid Signal

Endocannabinoids mediate retrograde signal and modulate transmission efficacy at various central synapses. Although endocannabinoid release is induced by either depolarization or activation of G(q/11)-coupled receptors, it is markedly enhanced by the coincidence of depolarization and receptor activation. Here we report that this coincidence is detected by phospholipase Cbeta1 (PLCbeta1) in hippocampal neurons. By measuring cannabinoid-sensitive synaptic currents, we found that the receptor-driven endocannabinoid release was dependent on physiological levels of intracellular Ca(2+) concentration ([Ca(2+)](i)), and markedly enhanced by depolarization-induced [Ca(2+)](i) elevation. Furthermore, we measured PLC activity in intact neurons by using exogenous TRPC6 channel as a biosensor for the PLC product diacylglycerol and found that the receptor-driven PLC activation exhibited similar [Ca(2+)](i) dependence to that of endocannabinoid release. Neither endocannabinoid release nor PLC activation was induced by receptor activation in PLCbeta1 knockout mice. We therefore conclude that PLCbeta1 serves as a coincidence detector through its Ca(2+) dependency for endocannabinoid release in hippocampal neurons.

Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2‐mediated direct suppression and M1/M3‐mediated indirect suppression through endocannabinoid signalling

The cholinergic system in the CNS plays important roles in higher brain functions, primarily through muscarinic acetylcholine receptors. At cellular levels, muscarinic activation produces various effects including modulation of synaptic transmission. Here we report that muscarinic activation suppresses hippocampal inhibitory transmission through two distinct mechanisms, namely a cannabinoid‐dependent and cannabinoid‐independent mechanism. We made paired whole‐cell recordings from cultured hippocampal neurons of rats and mice, and monitored inhibitory postsynaptic currents (IPSCs). When cannabinoid receptor typeu20031 (CB1) was blocked, oxotremorineu2003M (oxo‐M), a muscarinic agonist, suppressed IPSCs in a subset of neuron pairs. This suppression was associated with an increase in paired‐pulse ratio, blocked by the M2‐prefering antagonist gallamine, and was totally absent in neuron pairs from M2‐knockout mice. When CB1 receptors were not blocked, oxo‐M suppressed IPSCs in a gallamine‐resistant manner in cannabinoid‐sensitive pairs. This suppression was associated with an increase in paired‐pulse ratio, blocked by the CB1 antagonist AM281, and was completely eliminated in neuron pairs from M1/M3‐compound‐knockout mice. Our immunohistochemical examination showed that M2 and CB1 receptors were present at inhibitory presynaptic terminals of mostly different origins. These results indicate that two distinct mechanisms mediate the muscarinic suppression. In a subset of synapses, activation of M2 receptors at presynaptic terminals suppresses GABA release directly. In contrast, in a different subset of synapses, activation of M1/M3 receptors causes endocannabinoid production and subsequent suppression of GABA release by activating presynaptic CB1 receptors. Thus, the muscarinic system can influence hippocampal functions by controlling different subsets of inhibitory synapses through the two distinct mechanisms.

Synaptically Driven Endocannabinoid Release Requires Ca2+-Assisted Metabotropic Glutamate Receptor Subtype 1 to Phospholipase C β4 Signaling Cascade in the Cerebellum

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of Gq/11-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) β4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCβ4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCβ4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCβ4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

Cooperative endocannabinoid production by neuronal depolarization and group I metabotropic glutamate receptor activation.

Endocannabinoids are retrograde messengers that are released from central neurons by depolarization‐induced elevation of intracellular Ca2+ concentration [Ca2+]I or by activation of a group I metabotropic glutamate receptor (mGluR). We studied the interaction between these two pathways for endocannabinoid production in rat hippocampal neurons. We made a paired whole‐cell recording from cultured hippocampal neurons with inhibitory synaptic connections. Activation of group I mGluRs, mainly mGluR5, by the specific agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG), suppressed inhibitory postsynaptic currents (IPSCs) in about half of the neuron pairs. A cannabinoid agonist, WIN55,212–2, suppressed IPSCs in all DHPG‐sensitive pairs but not in most of DHPG‐insensitive pairs. The effects of both DHPG and WIN55,212–2 were abolished by the cannabinoid antagonists, AM281 and SR141716A, indicating that activation of group I mGluR releases endocannabinoids and suppress inhibitory neurotransmitter release through activation of presynaptic cannabinoid receptors. Depolarization of the postsynaptic neurons caused a transient suppression of IPSCs, a phemomenon termed depolarization‐induced suppression of inhibition (DSI) that was also abolished by cannabinoid antagonists. Importantly, DSI was enhanced significantly when group I mGluRs were activated simultaneously by DHPG. This enhancement was much more prominent than expected from the simple summation of depolarization‐induced and group I mGluR‐induced endocannabinoid release. DHPG caused no change in depolarization‐induced Ca2+ transients, indicating that the enhanced DSI by DHPG was not due to the augmentation of Ca2+ influx. Enhancement of DSI by DHPG was also observed in hippocampal slices. These results suggest that two pathways work in a cooperative manner to release endocannabinoids via a common intracellular cascade.

Endocannabinoids and Synaptic Function in the CNS

Marijuana affects neural functions through the binding of its active component (Δ9-THC) to cannabinoid receptors in the CNS. Recent studies have elucidated that endogenous ligands for cannabinoid receptors, endocannabinoids, serve as retrograde messengers at central synapses. Endocannabinoids are produced on demand in activity-dependent manners and released from postsynaptic neurons. The released endocannabinoids travel backward across the synapse, activate presynaptic CB1 cannabinoid receptors, and modulate presynaptic functions. Retrograde endocannabinoid signaling is crucial for certain forms of short-term and long-term synaptic plasticity at excitatory or inhibitory synapses in many brain regions, and thereby contributes to various aspects of brain function including learning and memory. Molecular identities of the CB1 receptor and enzymes involved in production and degradation of endocannabinoids have been elucidated. Anatomical studies have demonstrated unique distributions of these molecules around synapses, which provide morphological bases for the roles of endocannabinoids as retrograde messengers. CB1-knockout mice exhibit various behavioral abnormalities and multiple defects in synaptic plasticity, supporting the notion that endocannabinoid signaling is involved in various aspects of neural function. In this review article, the authors describe molecular mechanisms of the endocannabinoid-mediated synaptic modulation and its possible physiological significance. NEUROSCIENTIST 13(2):127—137, 2007.

Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde endocannabinoid signalling in the hippocampus.

The cholinergic system is crucial for higher brain functions including learning and memory. These functions are mediated primarily by muscarinic acetylcholine receptors (mAChRs) that consist of five subtypes (M1–M5). A recent study suggested a novel role of acetylcholine as a potent enhancer of endocannabinoid signalling that acts retrogradely from postsynaptic to presynaptic neurons. In the present study, we further investigated the mechanisms of this cholinergic effect on endocannabinoid signalling. We made paired whole‐cell recordings from cultured hippocampal neurons, and monitored inhibitory postsynaptic currents (IPSCs). The postsynaptic depolarization induced a transient suppression of IPSCs (DSI), a phenomenon known to involve retrograde signalling by endocannabinoids. The cholinergic agonist carbachol (CCh) markedly enhanced DSI at 0.01–0.3u2003µm without changing the presynaptic cannabinoid sensitivity. The facilitating effect of CCh on DSI was mimicked by the muscarinic agonist oxotremorine‐M, whereas it was eliminated by the muscarinic antagonist atropine. It was also blocked by a non‐hydrolizable analogue of GDP (GDP‐β‐S) that was applied intracellularly to postsynaptic neurons. The muscarinic enhancement of DSI persisted to a substantial degree in the neurons prepared from M1‐knockout and M3‐knockout mice, but was virtually eliminated in the neurons from M1/M3‐compound‐knockout mice. CCh still enhanced DSI significantly under the blockade of postsynatpic K+ conductance, and did not significantly influence the depolarization‐induced Ca2+ transients. These results indicate that the activation of postsynaptic M1 and M3 receptors facilitates the depolarization‐induced release of endocannabinoids.

Presynaptic Monoacylglycerol Lipase Activity Determines Basal Endocannabinoid Tone and Terminates Retrograde Endocannabinoid Signaling in the Hippocampus

Endocannabinoids function as retrograde messengers and modulate synaptic transmission through presynaptic cannabinoid CB1 receptors. The magnitude and time course of endocannabinoid signaling are thought to depend on the balance between the production and degradation of endocannabinoids. The major endocannabinoid 2-arachidonoylglycerol (2-AG) is hydrolyzed by monoacylglycerol lipase (MGL), which is shown to be localized at axon terminals. In the present study, we investigated how MGL regulates endocannabinoid signaling and influences synaptic transmission in the hippocampus. We found that MGL inhibitors, methyl arachidonoyl fluorophosphonate and arachidonoyl trifluoromethylketone, caused a gradual suppression of cannabinoid-sensitive IPSCs in cultured hippocampal neurons. This suppression was reversed by blocking CB1 receptors and was attenuated by inhibiting 2-AG synthesis, indicating that MGL scavenges constitutively released 2-AG. We also found that the MGL inhibitors significantly prolonged the suppression of both IPSCs and EPSCs induced by exogenous 2-AG and depolarization-induced suppression of inhibition/excitation, a phenomenon known to be mediated by retrograde endocannabinoid signaling. In contrast, inhibitors of other endocannabinoid hydrolyzing enzymes, fatty acid amide hydrolase and cyclooxygenase-2, had no effect on the 2-AG-induced IPSC suppression. These results strongly suggest that presynaptic MGL not only hydrolyzes 2-AG released from activated postsynaptic neurons but also contributes to degradation of constitutively produced 2-AG and prevention of its accumulation around presynaptic terminals. Thus, the MGL activity determines basal endocannabinoid tone and terminates retrograde endocannabinoid signaling in the hippocampus.