Takashi Maejima

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.

Presynaptic Inhibition Caused by Retrograde Signal from Metabotropic Glutamate to Cannabinoid Receptors

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

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.

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.

Endogenous cannabinoid as a retrograde messenger from depolarized postsynaptic neurons to presynaptic terminals.

Cannabinoid receptors are the molecular targets for the active component Delta(9)-tetrahydrocannabinol of marijuana and hashish, and constitute a major family of G protein-coupled seven-transmembrane-domain receptors. They consist of type 1 (CB1) and type 2 (CB2) receptors of which the CB1 is rich in various regions of the CNS. Accumulated evidence suggests that endogenous cannabinoids function as diffusible and short-lived intercellular messengers that modulate synaptic transmission. Recent studies have provided strong experimental evidence that endogenous cannabinoids mediate signals retrogradely from depolarized postsynaptic neurons to presynaptic terminals to suppress subsequent neurotransmitter release, driving the synapse into an altered state. In hippocampal neurons, depolarization of postsynaptic neurons and resultant elevation of [Ca(2+)](i) lead to transient suppression of inhibitory transmitter release (depolarization-induced suppression of inhibition, DSI). In cerebellar Purkinje cells, on the other hand, depolarization-induced elevation of [Ca(2+)](i) causes transient suppression of excitatory transmitter release (depolarization-induced suppression of excitation, DSE). DSI and DSE appear to share the same properties and may be a general and important mechanism by which the postsynaptic neuronal activity can influence the amount of transmitter release.

Pet-1 is required across different stages of life to regulate serotonergic function.

Transcriptional cascades are required for the specification of serotonin (5-HT) neurons and behaviors modulated by 5-HT. Several cascade factors are expressed throughout the lifespan, which suggests that their control of behavior might not be temporally restricted to programming normal numbers of 5-HT neurons. We used new mouse conditional targeting approaches to investigate the ongoing requirements for Pet-1 (also called Fev), a cascade factor that is required for the initiation of 5-HT synthesis, but whose expression persists into adulthood. We found that Pet-1 was required after the generation of 5-HT neurons for multiple steps in 5-HT neuron maturation, including axonal innervation of the somatosensory cortex, expression of appropriate firing properties, and the expression of the Htr1a and Htr1b autoreceptors. Pet-1 was still required in adult 5-HT neurons to preserve normal anxiety-related behaviors through direct autoregulated control of serotonergic gene expression. These findings indicate that Pet-1 is required across the lifespan of the mouse and that behavioral pathogenesis can result from both developmental and adult-onset alterations in serotonergic transcription.

Substitution of 5-HT1A receptor signaling by a light-activated G protein-coupled receptor.

Understanding serotonergic (5-HT) signaling is critical for understanding human physiology, behavior, and neuropsychiatric disease. 5-HT mediates its actions via ionotropic and metabotropic 5-HT receptors. The 5-HT1A receptor is a metabotropic G protein-coupled receptor linked to the Gi/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT1A signaling, we created a light-activated G protein-coupled receptor that targets into 5-HT1A receptor domains and substitutes for endogenous 5-HT1A receptors. To induce 5-HT1A-like targeting, vertebrate rhodopsin was tagged with the C-terminal domain (CT) of 5-HT1A (Rh-CT5-HT1A). Rh-CT5-HT1A activates G protein-coupled inward rectifying K+ channels in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT1A receptor. The intracellular distribution of Rh-CT5-HT1A resembles that of the 5-HT1A receptor; Rh-CT5-HT1A localizes to somatodendritic sites and is efficiently trafficked to distal dendritic processes. Additionally, neuronal expression of Rh-CT5-HT1A, but not Rh, decreases 5-HT1A agonist sensitivity, suggesting that Rh-CT5-HT1A and 5-HT1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT5-HT1A is able to rescue 5-HT1A signaling of 5-HT1A KO mice in cultured neurons and in slices of the dorsal raphe showing that Rh-CT5-HT1A is able to functionally compensate for native 5-HT1A. Thus, as an optogenetic tool, Rh-CT5-HT1A has the potential to directly correlate in vivo 5-HT1A signaling with 5-HT neuron activity and behavior in both normal animals and animal models of neuropsychiatric disease.

Pharmacological evidence for the involvement of diacylglycerol lipase in depolarization-induced endocanabinoid release

Depolarization-induced suppression of inhibition (DSI) or excitation (DSE) is a well-known form of endocannabinoid-mediated short-term plasticity that is induced by postsynaptic depolarization. It is generally accepted that DSI/DSE is triggered by Ca(2+) influx through voltage-gated Ca(2+) channels. It is also demonstrated that DSI/DSE is mediated by 2-arachidonoylglycerol (2-AG). However, how Ca(2+) induces 2-AG production is still unclear. In the present study, we investigated molecular mechanisms underlying the Ca(2+)-driven 2-AG production. Using cannabinoid-sensitive inhibitory synapses of cultured hippocampal neurons, we tested several inhibitors for enzymes that are supposed to be involved in 2-AG metabolism. The chemicals we tested include inhibitors for phospholipase C (U73122 and ET-18), diacylglycerol kinase (DGK inhibitor 1), phosphatidic acid phosphohydrolase (propranolol), and diacylglycerol lipase (DGL; RHC-80267 and tetrahydrolipstatin (THL)). However, unfavorable side effects were observed with these inhibitors, except for THL. Furthermore, we found that RHC-80267 hardly inhibited the endocannabinoid release driven by G(q/11)-coupled receptors, which is thought to be DGL-dependent. By contrast, THL exhibited no side effects as long as we tested, and was confirmed to inhibit the DGL-dependent process. Using THL as a DGL inhibitor, we demonstrated that DGL is involved in both hippocampal DSI and cerebellar DSE. To test a possible involvement of PLCdelta in DSI, we examined hippocampal DSI in PLCdelta1, delta3 and delta4-knockout mice. However, there was no significant difference in the DSI magnitude between these knockout mice and wild-type mice. The present study clearly shows that DGL is a prerequisite for DSI/DSE. The enzymes yielding DG remain to be determined.

Cooperative Interaction between Hepatocyte Nuclear Factor 4α and GATA Transcription Factors Regulates ATP-Binding Cassette Sterol Transporters ABCG5 and ABCG8

ABSTRACT Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4α (HNF4α) in the ABCG5/ABCG8 intergenic promoter, through which HNF4α strongly activated the expression of a reporter gene in both directions. The HNF4α-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4α. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4α leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4α and GATA were essential for maximal synergism. We also show that HNF4α, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4α and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4α acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.