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 CB1 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 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.

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.

The influence of the antidiabetic sulphonylurea tolbutamide on K+ channels of mouse pancreatic beta-cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K+ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of beta-cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 microM. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K+ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca2+-dependent K+ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K+ channels.

Recent studies have clarified that endogenous cannabinoids (endocannabinoids) are released from depolarized postsynaptic neurons in a Ca(2+)-dependent manner and act retrogradely on presynaptic cannabinoid receptors to suppress inhibitory or excitatory neurotransmitter release. This type of modulation has been found in the hippocampus and cerebellum and was called depolarization-induced suppression of inhibition (DSI) or excitation (DSE). In this study, we quantitatively examined the effects of postsynaptic depolarization and a cannabinoid agonist on excitatory and inhibitory synapses in rat hippocampal slices and cultures. We found that both DSE and DSI can be induced, but DSE was much less prominent than DSI. For the induction of DSE, the necessary duration of depolarization was longer than for DSI. The magnitude of DSE was much smaller than that of DSI. To explore the reasons for these differences, we tested the sensitivity of EPSCs and IPSCs to a cannabinoid agonist, WIN55,212-2, in hippocampal cultures. IPSCs were dichotomized into two distinct populations, one with a high sensitivity to WIN55,212-2 (50% block at 2 nm) and the other with no sensitivity. In contrast, EPSCs were homogeneous and exhibited a low sensitivity to WIN55,212-2 (50% block at 60 nm). We estimated that the 5 sec depolarization elevated the local endocannabinoid concentration to a level equivalent to several nanomoles of WIN55,212-2. Using CB1 knock-out mice, we verified that both DSI and DSE were mediated by the cannabinoid CB1 receptor. These results indicate that presynaptic cannabinoid sensitivity is a major factor that determines the extent of DSI and DSE.

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.

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarizationinduced elevation of intracellular Ca2ϩ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G q/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) ␤4 cascade without detectable Ca 2ϩ elevation. The second mode is Ca 2ϩ elevation to a micromolar range without activation of the mGluR1-PLC␤4 cascade. The third mode is the Ca 2ϩ -assisted mGluR1-PLC␤4 cascade that requires weak mGluR1 activation and Ca 2ϩ 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 Ca 2ϩ -assisted mGluR1-PLC␤4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

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 type 1 (CB1) was blocked, oxotremorine M (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 M(2)-preferring antagonist gallamine, and was totally absent in neuron pairs from M(2)-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 M(1)/M(3)-compound-knockout mice. Our immunohistochemical examination showed that M(2) 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 M(2) receptors at presynaptic terminals suppresses GABA release directly. In contrast, in a different subset of synapses, activation of M(1)/M(3) 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.