Asami Tanimura

The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase α Mediates Retrograde Suppression of Synaptic Transmission

Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

Molecular and Morphological Configuration for 2-Arachidonoylglycerol-Mediated Retrograde Signaling at Mossy Cell–Granule Cell Synapses in the Dentate Gyrus

2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)–granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase α (DGLα) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC–GC synapses, as they were abolished in DGLα-knock-out mice. Immunohistochemistry revealed that DGLα was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB1 receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC–GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC–GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC–GC synapses by escaping from enzymatic degradation. This molecular–anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.

Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression‐related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression‐related behaviour. The NR2A subunit of the NMDA receptor is tyrosine‐phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr‐1325‐Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock‐in mice show antidepressant‐like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock‐in mice, DARPP‐32 phosphorylation at Thr 34, which is important for the regulation of depression‐related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src‐induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor‐mediated downstream signalling to modulate depression‐related behaviour.

Endocannabinoids and Retrograde Modulation of Synaptic Transmission

Since the first reports of endocannabinoid-mediated retrograde signaling in 2001, great advances have been made toward understanding the molecular basis and functions of the endocannabinoid system. Electrophysiological studies have revealed that the endocannabinoid system is functional at various types of synapses throughout the brain. Basic mechanisms have been clarified as to how endocannabinoids are produced and released from postsynaptic neurons and regulate neurotransmitter release through activating presynaptic cannabinoid CB1 receptors, although there remain unsolved questions and some discrepancies. In addition to this major function, recent studies suggest diverse functions of endocannabinoids, including control of other endocannabinoid-independent forms of synaptic plasticity, regulation of neuronal excitability, stimulation of glia-neuron interaction, and induction of CB1R-independent plasticity. Using recently developed pharmacological and genetic tools, behavioral studies have elucidated the roles of the endocannabinoid system in various aspects of neural functions. In this review, we make a brief overview of molecular mechanisms underlying the endocannabinoid-mediated synaptic modulation and also summarize recent findings, which shed new light on a diversity of functional roles of endocannabinoids.

Complementary synaptic distribution of enzymes responsible for synthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in the human hippocampus

Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AGs physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.

Astroglial Glutamate Transporter Deficiency Increases Synaptic Excitability and Leads to Pathological Repetitive Behaviors in Mice

An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette’s syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLASTCreERT2/+/GLT1flox/flox, iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG–mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG–mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol

•  2‐Arachidonoylglycerol (2‐AG), one of the best‐characterized retrograde messengers at central synapses, has been thought to be produced ‘on demand’ through a diacylglycerol lipase α (DGLα)‐dependent pathway upon activation of postsynaptic neurons (on‐demand synthesis hypothesis). •  However, recent studies propose an alternative hypothesis that 2‐AG is pre‐synthesized by DGLα, stored in neurons, and released from such ‘pre‐formed pools’ without the participation of DGLα (pre‐formed pool hypothesis). •  To test these hypotheses, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM‐188, on retrograde 2‐AG signalling. •  We found that 2‐AG‐mediated retrograde signalling was blocked after 1 h treatment with OMDM‐188 in acute slices from the hippocampus, striatum and cerebellum, and was blocked several minutes after OMDM‐188 application in cultured hippocampal neurons. •  These results fit well with the on‐demand synthesis hypothesis, rather than the pre‐formed pool hypothesis.

Not glutamate but endocannabinoids mediate retrograde suppression of cerebellar parallel fiber to Purkinje cell synaptic transmission in young adult rodents.

In the cerebellum of juvenile mice or rats, endocannabinoids are shown to mediate depolarization-induced suppression of excitation (DSE) and retrograde suppression induced by activation of type 1 metabotropic glutamate receptor (mGluR1) at parallel fiber (PF) to Purkinje cell (PC) synapses. However, recent studies showed that glutamate also mediated retrograde signaling through presynaptic kainate receptors in the cerebellum of young adult mice and rats. We reexamined this possibility in C57BL/6 mice at postnatal day 20-35 (P20-P35) and in Sprague-Dawley rats at P18-P24. We found that DSE at PF-PC synapses was abolished by AM251, a cannabinoid receptor antagonist, and by tetrahydrolipstatin (THL), a blocker of diacylglycerol lipase (DGL) that produces an endocannabinoid, 2-arachidonoylglycerol (2-AG). AM251 and THL did not affect depolarization-induced Ca(2+) transients in PCs, and THL did not suppress cannabinoid sensitivity of PFs. Moreover, DSE at PF-PC synapses was absent in CB(1) knockout mice. AM251 also eliminated transient suppression of PF-PC synaptic transmission following a brief burst of PF stimulation, a phenomenon known to be mediated by mGluR1. These results suggest that DSE and mGluR1-mediated suppression in young adult PCs are mediated by endocannabinoids, and that glutamate, if any, has little contribution.

Retrograde BDNF to TrkB signaling promotes synapse elimination in the developing cerebellum

Elimination of early-formed redundant synapses during postnatal development is essential for functional neural circuit formation. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs). A single CF is strengthened whereas the other CFs are eliminated in each PC dependent on postsynaptic activity in PC, but the underlying mechanisms are largely unknown. Here, we report that brain-derived neurotrophic factor (BDNF) from PC facilitates CF synapse elimination. By PC-specific deletion of BDNF combined with knockdown of BDNF receptors in CF, we show that BDNF acts retrogradely on TrkB in CFs, and facilitates elimination of CF synapses from PC somata during the third postnatal week. We also show that BDNF shares signaling pathway with metabotropic glutamate receptor 1, a key molecule that triggers a canonical pathway for CF synapse elimination. These results indicate that unlike other synapses, BDNF mediates punishment signal for synapse elimination in the developing cerebellum.During development, synapses are selectively strengthened or eliminated by activity-dependent competition. Here, the authors show that BDNF-TrkB retrograde signaling is a “punishment” signal that leads to elimination of climbing fiber-onto-Purkinje cell synapses in the developing cerebellum.