Search

The endocannabinoid (eCB) system and the cannabinoid CB1 receptor (CB1R) play key roles in the modulation of brain functions. Although actions of eCBs and CB1Rs are well described at the synaptic level, little is known of their modulation of neural activity at the network level. Using microelectrode arrays, we have examined the role of CB1R activation in the modulation of the electrical activity of rat and mice cortical neural networks in vitro. We find that exogenous activation of CB1Rs expressed on glutamatergic neurons decreases the spontaneous activity of cortical neural networks. Moreover, we observe that the net effect of the CB1R antagonist AM251 inversely correlates with the initial level of activity in the network: blocking CB1Rs increases network activity when basal network activity is low, whereas it depresses spontaneous activity when its initial level is high. Our results reveal a complex role of CB1Rs in shaping spontaneous network activity, and suggest that the outcome of endogenous neuromodulation on network function might be state dependent.

Neurons in many brain regions release endocannabinoids from their dendrites that act as retrograde signals to transiently suppress neurotransmitter release from presynaptic terminals. Little is known, however, about the physiological mechanisms of short-term endocannabinoid-mediated plasticity under physiological conditions. Here we investigate calcium-dependent endocannabinoid release from cartwheel cells (CWCs) of the mouse dorsal cochlear nucleus (DCN) in the auditory brainstem that provide feedforward inhibition onto DCN principal neurons. We report that sustained action potential firing by CWCs evokes endocannabinoid release in response to submicromolar elevation of dendritic calcium that transiently suppresses their parallel fiber (PF) inputs by >70%. Basal spontaneous CWC firing rates are insufficient to evoke tonic suppression of PF synapses. However, elevating CWC firing rates by stimulating PFs triggers the release of endocannabinoids and heterosynaptic suppression of PF inputs. Spike-evoked suppression by endocannabinoids selectively suppresses excitatory synapses, but glycinergic/GABAergic inputs onto CWCs are not affected. Our findings demonstrate a mechanism of transient plasticity mediated by endocannabinoids that heterosynaptically suppresses subsets of excitatory presynaptic inputs to CWCs that regulates feedforward inhibition of DCN principal neurons and may influence the output of the DCN.

Endocannabinoid (eCB) signaling is tightly regulated by eCB biosynthetic and degradative enzymes. The eCB 2-arachidonoylglycerol (2-AG) is hydrolyzed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB signaling, synaptic function, and learning behavior were altered in MAGL knock-out mice. We report that MAGL⁻/⁻ mice exhibited prolonged depolarization-induced suppression of inhibition (DSI) in hippocampal CA1 pyramidal neurons, providing genetic evidence that the inactivation of 2-AG by MAGL determines the time course of the eCB-mediated retrograde synaptic depression. CB₁ receptor antagonists enhanced basal IPSCs in CA1 pyramidal neurons in MAGL⁻/⁻ mice, while the magnitude of DSI or CB₁ receptor agonist-induced depression of IPSCs was decreased in MAGL⁻/⁻ mice. These results suggest that 2-AG elevations in MAGL⁻/⁻ mice cause tonic activation and partial desensitization of CB₁ receptors. Genetic deletion of MAGL selectively enhanced theta burst stimulation (TBS)-induced long-term potentiation (LTP) in the CA1 region of hippocampal slices but had no significant effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The enhancement of TBS-LTP in MAGL⁻/⁻ mice appears to be mediated by 2-AG-induced suppression of GABA(A) receptor-mediated inhibition. MAGL⁻/⁻ mice exhibited enhanced learning as shown by improved performance in novel object recognition and Morris water maze. These results indicate that genetic deletion of MAGL causes profound changes in eCB signaling, long-term synaptic plasticity, and learning behavior.

Endocannabinoids (eCBs) are feedback messengers in the nervous system that act at the presynaptic nerve terminal to inhibit transmitter release. Here we report that in brain slices from rat, eCBs are released from vasopressin (VP) neurons in the paraventricular nucleus of the hypothalamus following coincident bursts of presynaptic and postsynaptic spiking. eCBs transiently depress glutamate release from excitatory terminals and, in doing so, prevent the synapses from undergoing long-term depression (LTD). Specifically, we show that blockade of CB1 receptors unmasks LTD following coincident presynaptic and postsynaptic activity. This LTD is presynaptic in nature, but requires the release of the opioid peptide dynorphin from the postsynaptic neuron. Dynorphin release and subsequent LTD require the activation of postsynaptic metabotropic glutamate receptors (mGluRs). Our findings indicate that eCBs, by transiently depressing glutamate release, limit mGluR activation and indirectly gate release of dynorphin from the postsynaptic neuron. We propose that eCBs, in addition to their well described role in the rapid modulation of transmitter release from the nerve terminal, also regulate the release of other retrograde transmitters and thus encode for multiple temporal windows of synaptic plasticity.

Retrograde synaptic signaling by endocannabinoids (eCBs) is a widespread mechanism for activity-dependent inhibition of synaptic strength in the brain. Although prevalent, the conditions for eliciting eCB-mediated synaptic depression vary among brain circuits. As yet, relatively little is known about the molecular mechanisms underlying this variation, although the initial signaling events are likely dictated by postsynaptic proteins. SAP90/PSD-95-associated proteins (SAPAPs) are a family of postsynaptic proteins unique to excitatory synapses. Using Sapap3 knock-out (KO) mice, we find that, in the absence of SAPAP3, striatal medium spiny neuron (MSN) excitatory synapses exhibit eCB-mediated synaptic depression under conditions that do not normally activate this process. The anomalous synaptic plasticity requires type 5 metabotropic glutamate receptors (mGluR5s), which we find are dysregulated in Sapap3 KO MSNs. Both surface expression and activity of mGluR5s are increased in Sapap3 KO MSNs, suggesting that enhanced mGluR5 activity may drive the anomalous synaptic plasticity. In direct support of this possibility, we find that, in wild-type (WT) MSNs, pharmacological enhancement of mGluR5 by a positive allosteric modulator is sufficient to reproduce the increased synaptic depression seen in Sapap3 KO MSNs. The same pharmacologic treatment, however, fails to elicit further depression in KO MSNs. Under conditions that are sufficient to engage eCB-mediated synaptic depression in WT MSNs, Sapap3 deletion does not alter the magnitude of the response. These results identify a role for SAPAP3 in the regulation of postsynaptic mGluRs and eCB-mediated synaptic plasticity. SAPAPs, through their effect on mGluR activity, may serve as regulatory molecules gating the threshold for inducing eCB-mediated synaptic plasticity.

Inhibitory neurons in the thalamic reticular nucleus (TRN) play a critical role in controlling information transfer between thalamus and neocortex. GABAergic synapses formed by TRN neurons contact both thalamic relay cells and neurons within TRN. These two types of synapses are thought to have distinct roles for the generation of thalamic network activity, but their selective regulation is poorly understood. In many areas throughout the brain, retrograde signaling mediated by endocannabinoids acts to dynamically regulate synaptic strength over both short and long time scales. However, retrograde signaling has never been demonstrated in the thalamus. Here, we show that depolarization-induced suppression of inhibition (DSI) is prominent at inhibitory synapses interconnecting TRN neurons. DSI is completely abolished in the presence of a cannabinoid receptor 1 (CB1R) antagonist and in mice lacking CB1Rs. DSI is prevented by DAG lipase inhibitors and prolonged by blocking the 2-arachidonoylglycerol (2-AG) degradation enzyme monoacylglycerol lipase, indicating that it is mediated by the release of 2-AG from TRN neurons. By contrast, DSI is not observed at TRN synapses targeting thalamic relay neurons. A combination of pharmacological and immunohistochemical data indicate that the differences in endocannabinoid signaling at the two synapses are mediated by a synapse-specific targeting of CB1Rs, as well as differences in endocannabinoid release between the two target neurons. Together, our results show that endocannabinoids control transmitter release at specific thalamic synapses, and could dynamically regulate sensory information processing and thalamus-mediated synchronous oscillations.

In the adult brain, neural stem cells proliferate within the subventricular zone before differentiating into migratory neuroblasts that travel along the rostral migratory stream (RMS) to populate the olfactory bulb with new neurons. Because neuroblasts have been shown to migrate to areas of brain injury, understanding the cues regulating this migration could be important for brain repair. Recent studies have highlighted an important role for endocannabinoid (eCB) signaling in the proliferation of the stem cell population, but it remained to be determined whether this pathway also played a role in cell migration. We now show that mouse migratory neuroblasts express cannabinoid receptors, diacylglycerol lipase α (DAGLα), the enzyme that synthesizes the endocannabinoid 2-arachidonoylglycerol (2-AG), and monoacylglycerol lipase, the enzyme responsible for its degradation. Using a scratch wound assay for a neural stem cell line and RMS explant cultures, we show that inhibition of DAGL activity or CB(1)/CB(2) receptors substantially decreases migration. In contrast, direct activation of cannabinoid receptors or preventing the breakdown of 2-AG increases migration. Detailed analysis of primary neuroblast migration by time-lapse imaging reveals that nucleokinesis, as well as the length and branching of the migratory processes are under dynamic control of the eCB system. Finally, similar effects are observed in vivo by analyzing the morphology of green fluorescent protein-labeled neuroblasts in brain slices from mice treated with CB(1) or CB(2) antagonists. These results describe a novel role for the endocannabinoid system in neuroblast migration in vivo, highlighting its importance in regulating an additional essential step in adult neurogenesis.

Activation of both presynaptic metabotropic cannabinoid type 1 receptors (CB(1)s) and ionotropic kainate receptors (KARs) can efficiently modulate GABA release at many synapses of the CNS. The inhibitory effect of kainic acid (KA) has been ascribed to metabotropic actions, and KAR-induced release of secondary neuromodulatory agents may partly mediate these actions. Here, we investigated the involvement of the endocannabinoid system in the modulation of GABAergic synaptic transmission by pharmacological activation of KARs with KA in CA1 pyramidal neurons of the mouse hippocampus. We show that the depression of GABAergic synaptic transmission induced by KA (3 μm) is strongly inhibited by the simultaneous blockade of CB(1) and GABA(B) receptors with SR141716A (5 μm) and CGP55845 (5 μm), respectively. KA induces a calcium-dependent mobilization of the endocannabinoid anandamide (AEA) by activation of GluK2-containing KARs in postsynaptic pyramidal neurons. Consistently, the effect of KA is prolonged by the inhibitor of AEA degradation URB597 (1 μm) in a CB(1)-dependent manner, but it is not altered by blockade of degradation or synthesis of the other main endocannabinoid 2-arachidonoylglycerol (2AG). Hence, our work reveals that the pharmacological activation of KARs leads to the stimulation of secondary metabotropic signaling systems. In addition, these data further underline the profound mechanistic differences between exogenous and endogenous activation of KARs in the hippocampus.

Endocannabinoids (eCBs) are retrograde neurotransmitters that modulate the function of many types of synapses. The presence of eCBs, their CB1 receptor (CB1R), and metabolizing enzymes at embryonic and early postnatal periods have been linked to developmental processes such as neuronal proliferation, differentiation, and migration, axon guidance, and synaptogenesis. Here, we demonstrate the presence of a functional eCB system in the developing visual system and the role of CB1R during axon growth and retinothalamic development. Pharmacological treatment of retinal explants and primary cortical neuron cultures with ACEA, a selective CB1R agonist, induced a collapse of the growth cone (GC). Furthermore the application of AM251, a CB1R inverse agonist, to the neuronal cultures increased the surface area of GC. In vivo, intraocular injection of ACEA diminished retinal projection growth, while AM251 promoted growth and caused aberrant projections. In addition, compared with their wild-type littermates, CB1R-deficient adult mice revealed a lower level of eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus. Finally, we found that pharmacological modulation of CB1R affected the trafficking of Deleted in colorectal cancer (DCC) receptor to the plasma membrane in a PKA-dependent manner. Moreover, pharmacological inhibition or genetic inactivation of DCC abolished the CB1R-induced reorganization of the GC. Overall, these findings establish a mechanism by which the CB1R influences GC behavior and nervous system development in concerted action with DCC.

Endocannabinoid signaling is distributed throughout the brain, regulating synaptic release of both excitatory and inhibitory neurotransmitters. The presence of endocannabinoid signaling within stress-sensitive nuclei of the hypothalamus, as well as upstream limbic structures such as the amygdala, suggests it may play an important role in regulating the neuroendocrine and behavioral effects of stress. The evidence reviewed here demonstrates that endocannabinoid signaling is involved in both activating and terminating the hypothalamic-pituitary-adrenal axis response to both acute and repeated stress. In addition to neuroendocrine function, however, endocannabinoid signaling is also recruited by stress and glucocorticoid hormones to modulate cognitive and emotional processes such as memory consolidation and extinction. Collectively, these data demonstrate the importance of endocannabinoid signaling at multiple levels as both a regulator and an effector of the stress response.

Endocannabinoids (eCBs) are ubiquitous retrograde signaling molecules in the nervous system that are recruited in response to robust neuronal activity or the activation of postsynaptic G-protein-coupled receptors. Physiologically, eCBs have been implicated as important mediators of the stress axis and they may contribute to the rapid feedback inhibition of the hypothalamic-pituitary-adrenal axis (HPA) by circulating corticosteroids (CORTs). Understanding the relationship between stress and eCBs, however, is complicated by observations that eCB signaling is itself sensitive to stress. The mechanisms that link stress to changes in synaptic eCB signaling and the impact of these changes on CORT-mediated negative feedback have not been resolved. Here, we show that repetitive immobilization stress, in juvenile male rats, causes a functional downregulation of CB(1) receptors in the paraventricular nucleus of the hypothalamus (PVN). This loss of CB(1) receptor signaling, which requires the activation of genomic glucocorticoid receptors, impairs both activity and receptor-dependent eCB signaling at GABA and glutamate synapses on parvocellular neuroendocrine cells in PVN. Our results provide a plausible mechanism for how stress can lead to alterations in CORT-mediated negative feedback and may contribute to the development of plasticity of HPA responses.

Cannabinoids modulate the activity of many neuronal cells, among them sensory neurons in the olfactory epithelium. Here we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) is synthesized in both olfactory receptor neurons and glia-like sustentacular cells in larval Xenopus laevis. Its production in the latter depends on the hunger state of the animal. The essential effect of 2-AG in olfactory receptor neurons is the control of odorant detection thresholds via cannabinoid CB(1) receptor activation. Hunger renders olfactory neurons more sensitive. Endocannabinoid modulation in the nose may therefore substantially influence food-seeking behavior.

A fundamental property of neuronal networks in Ammon's horn is that each area comprises a single glutamatergic cell population and various types of GABAergic neurons. Here we describe an exception to this rule, in the form of granule cells that reside within the CA3 area and function as glutamatergic nonprincipal cells with distinct properties. CA3 granule cells in normal, healthy rats, similarly to dentate gyrus granule cells, coexpressed calbindin and the homeobox protein Prox1. However, CA3 granule cells were located outside of the dentate gyrus, often hundreds of micrometers from the hilar border, in the lucidum and radiatum layers. CA3 granule cells were present in numbers that were comparable to the rarer GABAergic neuronal subtypes, and their somato-dendritic morphology, intrinsic properties, and perforant path inputs were similar to those of dentate gyrus granule cells. CA3 granule cell axons displayed giant mossy fiber terminals with filopodial extensions, demonstrating that not all mossy fibers originate from the dentate gyrus. Somatic paired recordings revealed that CA3 granule cells innervated CA3 pyramidal and GABAergic cells similarly to conventional mossy fiber synapses. However, CA3 granule cells were distinct in the specific organization of their GABAergic inputs. They received GABAergic synapses from cholecystokinin-expressing mossy fiber-associated cells that did not innervate the dentate granule cell layer, and these synapses demonstrated unusually strong activity-dependent endocannabinoid-mediated inhibition of GABA release. These results indicate that granule cells in the CA3 constitute a glutamatergic, nonprincipal neuronal subtype that is integrated into the CA3 synaptic network.

The endocannabinoid (eCB) system plays central roles in the regulation of food intake and energy expenditure. Its alteration in activity contributes to the development and maintenance of obesity. Stimulation of the cannabinoid receptor type 1 (CB(1) receptor) increases feeding, enhances reward aspects of eating, and promotes lipogenesis, whereas its blockade decreases appetite, sustains weight loss, increases insulin sensitivity, and alleviates dysregulation of lipid metabolism. The hypothesis has been put forward that the eCB system is overactive in obesity. Hippocampal circuits are not directly involved in the neuronal control of food intake and appetite, but they play important roles in hedonic aspects of eating. We investigated the possibility whether or not diet-induced obesity (DIO) alters the functioning of the hippocampal eCB system. We found that levels of the two eCBs, 2-arachidonoyl glycerol (2-AG) and anandamide, were increased in the hippocampus from DIO mice, with a concomitant increase of the 2-AG synthesizing enzyme diacylglycerol lipase-alpha and increased CB(1) receptor immunoreactivity in CA1 and CA3 regions, whereas CB(1) receptor agonist-induced [(35)S]GTPgammaS binding was unchanged. eCB-mediated synaptic plasticity was changed in the CA1 region, as depolarization-induced suppression of inhibition and long-term depression of inhibitory synapses were enhanced. Functionality of CB(1) receptors in GABAergic neurons was furthermore revealed, as mice specifically lacking CB(1) receptors on this neuronal population were partly resistant to DIO. Our results show that DIO-induced changes in the eCB system affect not only tissues directly involved in the metabolic regulation but also brain regions mediating hedonic aspects of eating and influencing cognitive processes.

At hippocampal excitatory synapses, endocannabinoids (eCBs) mediate two forms of retrograde synaptic inhibition that are induced by postsynaptic depolarization or activation of metabotropic glutamate receptors (mGluRs). The homer family of molecular scaffolds provides spatial organization to regulate postsynaptic signaling cascades, including those activated by mGluRs. Expression of the homer 1a (H1a) immediate-early gene produces a short homer protein that lacks the domain required for homer oligomerization, enabling it to uncouple homer assemblies. Here, we report that H1a differentially modulates two forms of eCB-mediated synaptic plasticity, depolarization-induced suppression of excitation (DSE) and metabotropic suppression of excitation (MSE). EPSCs were recorded from cultured hippocampal neurons and DSE evoked by a 15 s depolarization to 0 mV and MSE evoked by a type I mGluR agonist. Expression of H1a enhanced DSE and inhibited MSE at the same synapse. Many physiologically important stimuli initiate H1a expression including brain-derived neurotrophic factor (BDNF). Treating hippocampal cultures with BDNF increased transcription of H1a and uncoupled homer 1c-GFP (green fluorescent protein) clusters. BDNF treatment blocked MSE and enhanced DSE. Thus, physiological changes in H1a expression gate the induction pathway for eCB-mediated synaptic plasticity by uncoupling mGluR from eCB production.

Adenosine A(2A) receptor antagonists are psychomotor stimulants that also hold therapeutic promise for movement disorders. However, the molecular mechanisms underlying their stimulant properties are not well understood. Here, we show that the robust increase in locomotor activity induced by an A(2A) antagonist in vivo is greatly attenuated by antagonizing cannabinoid CB(1) receptor signaling or by administration to CB(1)(-/-) mice. To determine the locus of increased endocannabinoid signaling, we measured the amount of anandamide [AEA (N-arachidonoylethanolamine)] and 2-arachidonoylglycerol (2-AG) in brain tissue from striatum and cortex. We find that 2-AG is selectively increased in striatum after acute blockade of A(2A) receptors, which are highly expressed by striatal indirect-pathway medium spiny neurons (MSNs). Using targeted whole-cell recordings from direct- and indirect-pathway MSNs, we demonstrate that A(2A) receptor antagonists potentiate 2-AG release and induction of long-term depression at indirect-pathway MSNs, but not direct-pathway MSNs. Together, these data outline a molecular mechanism by which A(2A) antagonists reduce excitatory synaptic drive on the indirect pathway through CB(1) receptor signaling, thus leading to increased psychomotor activation.

There remains debate regarding the impact of cannabis on neuropsychiatric disorders. Here, we examined the effects of cannabidiol (CBD), a nonpsychoactive constituent of cannabis, on heroin self-administration and drug-seeking behavior using an experimental rat model. CBD (5-20 mg/kg) did not alter stable intake of heroin self-administration, extinction behavior, or drug seeking induced by a heroin prime injection. Instead, it specifically attenuated heroin-seeking behavior reinstated by exposure to a conditioned stimulus cue. CBD had a protracted effect with significance evident after 24 h and even 2 weeks after administration. The behavioral effects were paralleled by neurobiological alterations in the glutamatergic and endocannabinoid systems. Discrete disturbances of AMPA GluR1 and cannabinoid type-1 receptor expression observed in the nucleus accumbens associated with stimulus cue-induced heroin seeking were normalized by CBD treatment. The findings highlight the unique contributions of distinct cannabis constituents to addiction vulnerability and suggest that CBD may be a potential treatment for heroin craving and relapse.

Homeostatic regulation of synaptic strength in response to persistent changes of neuronal activity plays an important role in maintaining the overall level of circuit activity within a normal range. Absence of miniature EPSCs (mEPSCs) for a few hours is known to cause upregulation of excitatory synaptic strength, suggesting that mEPSCs contribute to the maintenance of excitatory synaptic functions. In the present study, we found that the absence of mEPSCs for 1-3 h also resulted in homeostatic suppression of presynaptic functions of inhibitory synapses in acute cortical slices from juvenile rats, as suggested by the reduced frequency (but not amplitude) of miniature IPSCs (mIPSCs) as well as the reduced amplitude of IPSCs. This homeostatic regulation depended on endocannabinoid (eCB) signaling, because blockade of either the activation of cannabinoid type-1 receptors (CB1Rs) or the synthesis of its endogenous ligand 2-arachidonoylglycerol (2-AG) abolished the suppression of inhibitory synapses caused by the absence of mEPSCs. Blockade of group I metabotropic glutamate receptors (mGluR-I) also abolished the suppression of inhibitory synapses, consistent with the mGluR-I requirement for eCB synthesis and release in cortical synapses. Furthermore, this homeostatic regulation also required eukaryotic elongation factor-2 (eEF2)-dependent protein synthesis, but not gene transcription. Activation of eEF2 alone was sufficient to suppress the mIPSC frequency, an effect abolished by inhibiting CB1Rs. Thus, mEPSCs contribute to the maintenance of inhibitory synaptic function and the absence of mEPSCs results in presynaptic suppression of inhibitory synapses via protein synthesis-dependent elevation of eCB signaling.

Substance P is thought to play an essential role in several forms of supraspinally mediated analgesia. The actions of substance P on synaptic transmission within descending analgesic pathways, however, are largely unknown. Here, we used whole-cell recordings from rat midbrain slices to examine the effects of substance P on GABAergic and glutamatergic transmission within the periaqueductal gray (PAG), a key component of a descending analgesic pathway that projects via the rostral ventromedial medulla (RVM) to the spinal cord dorsal horn. We found that substance P reversibly decreased the amplitude and increased the paired-pulse ratio of evoked IPSCs recorded from identified PAG-RVM projection neurons and from unidentified PAG neurons. Substance P had no effect on miniature IPSCs, implying an indirect mode of action. The effects of substance P were abolished by metabotropic glutamate type 5 and cannabinoid CB1 receptor antagonists, but unaltered by NMDA, GABA(B), mu,delta-opioid, adenosine A(1), and 5HT(1A) receptor antagonists. Consistent with a role for endogenous glutamate in this process, substance P increased the frequency of action potential-dependent spontaneous EPSCs. Moreover, the effect of substance P on evoked IPSCs was mimicked and occluded by a glutamate transport inhibitor. Finally, these effects were dependent on postsynaptic G-protein activation and diacylglycerol lipase activity, suggesting the requirement for retrograde signaling by the endocannabinoid 2-arachidonoylglycerol. Thus, substance P may facilitate descending analgesia in part by enhancing glutamate-mediated excitation and endocannabinoid-mediated disinhibition of PAG-RVM projection neurons.

Fatty acid amide hydrolase (FAAH) is a degradative enzyme for a group of endogenous signaling lipids that includes anandamide (AEA). AEA acts as an endocannabinoid and an endovanilloid by activating cannabinoid and vanilloid type 1 transient receptor potential (TRPV1) receptors, respectively, on dorsal root ganglion (DRG) sensory neurons. Inhibition of FAAH activity increases AEA concentrations in nervous tissue and reduces sensory hypersensitivity in animal pain models. Using immunohistochemistry, Western blotting, and reverse transcription-PCR, we demonstrate the location of the FAAH in adult rat DRG, sciatic nerve, and spinal cord. In naive rats, FAAH immunoreactivity localized to the soma of 32.7 +/- 0.8% of neurons in L4 and L5 DRG. These were small-sized (mean soma area, 395.96 +/- 5.6 mum(2)) and predominantly colabeled with peripherin and isolectin B4 markers of unmyelinated C-fiber neurons; 68% colabeled with antibodies to TRPV1 (marker of nociceptive DRG neurons), and <2% colabeled with NF200 (marker of large myelinated neurons). FAAH-IR was also present in small, NF200-negative cultured rat DRG neurons. Incubation of these cultures with the FAAH inhibitor URB597 increased AEA-evoked cobalt uptake in a capsazepine-sensitive manner. After sciatic nerve axotomy, there was a rightward shift in the cell-size distribution of FAAH-immunoreactive (IR) DRG neurons ipsilateral to injury: FAAH immunoreactivity was detected in larger-sized cells that colabeled with NF200. An ipsilateral versus contralateral increase in both the size and proportion of FAAH-IR DRG occurred after spinal nerve transection injury but not after chronic inflammation of the rat hindpaw 2 d after injection of complete Freund's adjuvant. This study reveals the location of FAAH in neural tissue involved in peripheral nociceptive transmission.

Glucocorticoids exert an opposing rapid regulation of glutamate and GABA synaptic inputs to hypothalamic magnocellular neurons via the activation of postsynaptic membrane-associated receptors and the release of retrograde messengers. Glucocorticoids suppress synaptic glutamate release via the retrograde release of endocannabinoids and facilitate synaptic GABA release via an unknown retrograde messenger. Here, we show that the glucocorticoid facilitation of GABA inputs is due to the retrograde release of neuronal nitric oxide and that glucocorticoid-induced endocannabinoid synthesis and nitric oxide synthesis are mediated by divergent G-protein signaling mechanisms. While the glucocorticoid-induced, endocannabinoid-mediated suppression of glutamate release is dependent on activation of the G(alpha)s G-protein subunit and cAMP-cAMP-dependent protein kinase activation, the nitric oxide facilitation of GABA release is mediated by G(beta)gamma signaling that leads to activation of neuronal nitric oxide synthase. Our findings indicate, therefore, that glucocorticoids exert opposing rapid actions on glutamate and GABA release by activating divergent G-protein signaling pathways that trigger the synthesis of, and glutamate and GABA synapse-specific retrograde actions of, endocannabinoids and nitric oxide, respectively. The simultaneous rapid stimulation of nitric oxide and endocannabinoid synthesis by glucocorticoids has important implications for the impact of stress on the brain as well as on neural-immune interactions in the hypothalamus.

5-HT(2)-type serotonin receptors (5-HT(2)Rs) are widely expressed throughout the brain and mediate many of the modulatory effects of serotonin. It has been thought that postsynaptic 5-HT(2)Rs act primarily by depolarizing neurons and thereby increasing their excitability. However, it is also known that 5-HT(2)Rs are coupled to G(q/11)-type G-proteins and that some other types of G(q/11)-coupled receptors can regulate synapses by evoking endocannabinoid release and activating presynaptic cannabinoid-type 1 receptors (CB(1)Rs). Here, we examine whether activation of 5-HT(2)Rs can regulate synapses through such a mechanism by studying excitatory synapses onto cells in the inferior olive (IO). These cells express 5-HT(2)Rs on their soma and dendrites, and the IO receives extensive serotonergic input. We find that the excitatory synaptic inputs onto IO cells are strongly suppressed by serotonin receptor agonists as well as release of endogenous serotonin. Both 5-HT(2)Rs and 5-HT(1B)Rs contribute to this modulation by decreasing the probability of glutamate release from presynaptic boutons. The suppression by 5-HT(2)Rs is of particular interest because it is prevented by CB(1)R antagonists, and 5-HT(2)Rs are thought to be located only postsynaptically on IO cells. Our results indicate that serotonin activates 5-HT(2)Rs on IO neurons, thereby releasing endocannabinoids that act retrogradely to suppress glutamate release by activating presynaptic CB(1)Rs. These findings establish a link between serotonin signaling and endocannabinoid signaling. Based on the extensive distribution of 5-HT(2)Rs and CB(1)Rs, it seems likely that this mechanism could mediate many of the actions of 5-HT(2)Rs throughout the brain.

Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control (postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB(1) cannabinoid receptor mRNA was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-alpha, an enzyme responsible for 2-arachidonoylglycerol synthesis, was also reduced by approximately 60%, whereas its related beta isoform levels were unchanged. Expression level of N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and 2-arachidonoylglycerol's degrading enzyme monoacylglycerol lipase did not change. The density of CB(1) immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal changes in the ratio of CB(1)-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB(1)-positive glutamatergic axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human hippocampus and imply that downregulation of CB(1) receptors and related molecular components of the endocannabinoid system may facilitate the deleterious effects of increased network excitability.

Endocannabinoids are regarded as retrograde signaling molecules at various types of synapses throughout the CNS. The lipid derivatives anandamide and 2-arachidonoylglycerol (2-AG) are generally thought to be the key molecular players in this process. Previous anatomical and electrophysiological studies provided compelling evidence that the biosynthetic enzyme of 2-AG is indeed localized in the postsynaptic plasma membrane, whereas its target, the CB1 cannabinoid receptor, and the enzyme responsible for its inactivation are both found presynaptically. This molecular architecture of 2-AG signaling is a conserved feature of most synapses and supports the retrograde signaling role of 2-AG. Conversely, the molecular and neuroanatomical organization of synaptic anandamide signaling remains largely unknown. In contrast to its predicted role in retrograde signaling, here we show that N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of anandamide and its related bioactive congeners, the N-acylethanolamines (NAEs), is concentrated presynaptically in several types of hippocampal excitatory axon terminals. Furthermore, high-resolution quantitative immunogold labeling demonstrates that this calcium-sensitive enzyme is localized predominantly on the intracellular membrane cisternae of axonal calcium stores. Finally, the highest density of NAPE-PLD is found in mossy terminals of granule cells, which do not express CB1 receptors. Together, these findings suggest that anandamide and related NAEs are also present at glutamatergic synapses, but the sites of their synthesis and action are remarkably different from 2-AG, indicating distinct physiological roles for given endocannabinoids in the regulation of synaptic neurotransmission and plasticity.

Glutamate spillover regulates GABAergic synaptic transmission at several CNS synapses via presynaptic ionotropic and metabotropic glutamate receptors (mGluRs). We have previously demonstrated that activation of group I-III mGluRs inhibits GABAergic transmission in the midbrain periaqueductal gray (PAG), a region involved in organizing behavioral responses to threat, stress, and pain. Here, we examined the role of glutamate spillover in the modulation of GABAergic transmission in the PAG. Using whole-cell recordings from rat PAG slices, we found that evoked IPSCs were reduced by the nonspecific glutamate transport blockers DL-threo-beta-benzyloxyaspartic acid (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylic acid, but not by the glial GLT1-specific blocker dihydrokainate. In contrast, TBOA had no effect on evoked IPSCs when glutamate uptake into the postsynaptic neuron was selectively impaired. TBOA increased the paired-pulse ratio of evoked IPSCs and reduced the rate but not the amplitude of spontaneous miniature IPSCs. The effect of TBOA on evoked IPSCs was abolished by the broad-spectrum mGluR antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (100 microM), reduced by the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and mimicked by the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Furthermore, the effects of both TBOA and DHPG were reduced by the cannabinoid CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251). Finally, although MPEP and AM251 had no effect on single evoked IPSCs, they increased evoked IPSCs during repetitive stimulation. These results indicate that neuronal glutamate transporters limit mGluR5 activation and endocannabinoid signaling, but may be overwhelmed during conditions of elevated glutamate release. Thus, neuronal glutamate transporters play a key role in regulating endocannabinoid-mediated cross talk between glutamatergic and GABAergic synapses within the PAG.

Retrograde signaling by endocannabinoids is known to induce short- and long-term synaptic plasticity, but the significance of this modulation for the activity of neural networks underlying motor behavior is largely unclear. Here, we used the isolated lamprey spinal cord to show that endocannabinoids released by activation of metabotropic glutamate receptor 1 (mGluR1) induce long-term synaptic plasticity during an ongoing locomotor rhythm and how this is translated onto the integrated activity of the spinal circuitry. A brief activation of mGluR1 induces a long-term increase in the locomotor frequency that is mediated by a concomitant long-term depression of midcycle reciprocal inhibition and long-term potentiation of ipsilateral synaptic excitation arising from locomotor circuit interneurons. Blockade of cannabinoid receptors with AM251 prevented the mGluR1-mediated long-term plasticity of both inhibitory and excitatory synaptic transmission, as well as that of the locomotor activity. Similarly, inhibition of nitric oxide signaling blocked the mGluR1-mediated long-term plasticity. These results show that the locomotor circuitry is endowed with a "memory" capacity mediated by a long-term shift in the balance between synaptic inhibition and excitation. This is triggered by activation of mGluR1 and requires subsequent endocannabinoid and nitric oxide signaling.

In the cerebellum, the process of retrograde signaling via presynaptic receptors is important for the induction of short- and long-term changes in inhibitory synaptic transmission at interneuron-Purkinje cell (PC) synapses. Endocannabinoids, by activating presynaptic CB1 receptors, mediate a short-term decrease in inhibitory synaptic efficacy, whereas glutamate, acting on presynaptic NMDA receptors, induces a longer-latency sustained increase in GABA release. We now demonstrate that either low-frequency climbing fiber stimulation or direct somatic depolarization of Purkinje cells results in SNARE-dependent vesicular release of glutamate from the soma and dendrites of PCs. The activity-dependent release of glutamate caused the activation of postsynaptic metabotropic glutamate receptor 1 (mGluR1) on PC somatodendritic membranes, resulting in the cooperative release of endocannabinoids and an mGluR1-mediated slow membrane conductance. The activity of excitatory amino acid transporters regulated the spatial spread of glutamate and thus the extent of PC mGluR1 activation. We propose that activity-dependent somatodendritic glutamate release and autocrine activation of mGluR1 on PCs provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission in the cerebellar cortex.

Endocannabinoid (eCB)-mediated forms of long-term synaptic plasticity occur in several brain regions, but much remains unknown about their basic properties and underlying mechanisms. Here, we present evidence that eCB-mediated long-term depression (eCB-LTD) at excitatory synapses on medium spiny neurons in the striatum requires presynaptic activity coincident with CB1 receptor activation. This dual requirement for CB1 activation and presynaptic activity is a mechanism by which eCB-LTD may be made synapse specific.

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

The probability of neurotransmitter release at the nerve terminal is an important determinant of synaptic efficacy. At some central synapses, the postsynaptic, or target, neuron determines neurotransmitter release probability (P(r)) at the presynaptic terminal. The mechanisms responsible for this target-cell dependent control of P(r) have not been elucidated. Using whole-cell patch-clamp recordings from magnocellular neurosecretory cells in the paraventricular and supraoptic nuclei of the hypothalamus, we demonstrate that inhibitory, GABA synapses specifically onto oxytocin (OT)-producing neurosecretory cells exhibit a low P(r) that is relatively uniform at multiple synapses onto the same cell. This low P(r) results from a two-step process that requires the tonic release of OT from the postsynaptic cell. The ambient extracellular levels of neuropeptide are sufficient to activate postsynaptic OT receptors and trigger the Ca2+-dependent production of endocannabinoids, which act in a retrograde manner at presynaptic cannabinoid CB1 receptors to decrease GABA release. The functional consequence of this tonic inhibition of GABA release is that all inhibitory inputs facilitate uniformly when activated at high rates of activity. This causes inhibition in the postsynaptic cell that is sufficiently powerful to disrupt firing. Blockade of CB1 receptors increases P(r) at these synapses, resulting in a rapid depression of IPSCs at high rates of activity, thereby eliminating the ability of afferent inputs to inhibit postsynaptic firing. By playing a deterministic role in GABA release at the afferent nerve terminal, the postsynaptic OT neuron effectively filters synaptic signals and thereby modulates its own activity patterns.

Transient surges of dopamine in the nucleus accumbens are associated with drug seeking. Using a voltammetric sensor with high temporal and spatial resolution, we demonstrate differences in the temporal profile of dopamine concentration transients caused by acute doses of nicotine, ethanol, and cocaine in the nucleus accumbens shell of freely moving rats. Despite differential release dynamics, all drug effects are uniformly inhibited by administration of rimonabant, a cannabinoid receptor (CB1) antagonist, suggesting that an increase in endocannabinoid tone facilitates the effects of commonly abused drugs on subsecond dopamine release. These time-resolved chemical measurements provide unique insight into the neurobiological effectiveness of rimonabant in treating addictive disorders.

Long-term depression (LTD) at the corticostriatal synapse is postsynaptically induced but presynaptically expressed, the depression being a result of retrograde endocannabinoid signaling that activates presynaptic cannabinoid CB1 receptors and reduces the probability of glutamate release. To study the role of protein synthesis in striatal LTD, we used a striatum-only preparation in which the presynaptic cell body is cut off, leaving intact only its axons, whose terminals synapse on medium spiny neurons. LTD (duration >150 min) was induced in this preparation, thus providing evidence that transcription in the presynaptic cell nucleus is not necessary for this form of plasticity. The maintenance of striatal LTD, however, was blocked by bath application of protein translation inhibitors but not by the same inhibitors loaded into the postsynaptic cell. These results suggest that local translation is critical for the expression of striatal LTD, distinguishing this form of mammalian synaptic plasticity from other forms that require postsynaptic protein synthesis. Possible roles of axonal or glial translation in striatal LTD are considered.

Neurons release endocannabinoids from their dendrites to trigger changes in the probability of transmitter release. Although such retrograde signaling has been described for principal neurons, such as hippocampal pyramidal cells and cerebellar Purkinje cells (PCs), it has not been demonstrated for local interneurons. Here we tested whether inhibitory interneurons in the cerebellum, stellate cells (SCs) and basket cells, regulate the strength of parallel fiber (PF) synapses by releasing endocannabinoids. We found that depolarization-induced suppression of excitation (DSE) is present in both SCs and basket cells. The properties of retrograde inhibition were examined more thoroughly for SCs. Both DSE and synaptically evoked suppression of excitation (SSE) triggered with brief PF bursts require elevations of postsynaptic calcium, are blocked by a type 1 cannabinoid receptor (CB1R) antagonist, and are absent in mice lacking the CB1R. SSE for SCs is similar to that described previously for PCs in that it is prevented by BAPTA and DAG lipase inhibitors in the recording pipette; however, unlike in PCs, NMDA receptors (NMDARs) play an important role in SSE for SCs. Although SCs express CB1Rs postsynaptically, neither high-frequency firing of SCs nor PF bursts lead to autocrine suppression of subsequent SC activity. Instead, PF bursts decrease the amplitude of disynaptic inhibition in PCs by evoking endocannabinoid release that transiently reduces the ability of PF synapses to trigger spikes in SCs. Thus, local interneurons within the cerebellum can release endocannabinoids through metabotropic glutamate receptor- and NMDAR-dependent mechanisms and contribute to use-dependent modulation of circuit properties.

Cannabinoids exert their psychomotor actions through the CB1 cannabinoid receptor in the brain. Genetic deletion of CB1 in mice causes various symptoms, including changes in locomotor activity, increased ring catalepsy, supraspinal hypoalgesia, and impaired memory extinction. Although the cerebellar cortex contains the highest level of CB1, severe cerebellum-related functional deficits have not been reported in CB1 knock-out mice. To clarify the roles of CB1 in cerebellar function, we subjected CB1 knock-out mice to a delay version of classical eyeblink conditioning. This paradigm is a test for cerebellum-dependent discrete motor learning, in which conditioned stimulus (CS) (352 ms tone) and unconditioned stimulus (US) (100 ms periorbital electrical shock) are coterminated. We found that delay eyeblink conditioning performance was severely impaired in CB1 knock-out mice. In contrast, they exhibited normal performance in a trace version of eyeblink conditioning with 500 ms stimulus-free interval intervened between the CS offset and the US onset. This paradigm is a test for hippocampus-dependent associative learning. Sensitivity of CB1 knock-out mice to CS or US was normal, suggesting that impaired delay eyeblink conditioning is attributable to defects in association of responses to CS and US. We also found that intraperitoneal injection of the CB1 antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide] to wild-type mice caused severe impairment in acquisition but not extinction of delay eyeblink conditioning. SR141716A treatment had no effect on trace eyeblink conditioning with a 500 or 750 ms trace interval. These results indicate that endogenous cannabinoid signaling through CB1 is essential for cerebellum-dependent discrete motor learning, especially for its acquisition.

Endocannabinoid signaling has been demonstrated to mediate depolarization-induced suppression of excitation at climbing fiber (CF) and parallel fiber (PF) synapses onto cerebellar Purkinje cells. Here, we show that CF-evoked release of cannabinoids (CBs) additionally suppresses a presynaptic form of long-term potentiation (LTP) at PF synapses. PF-LTP can be induced by 8 Hz PF tetanization but is blocked when the PF tetanization is paired with 4 or 1 Hz CF coactivation. CF activity can be substituted for by bath application of the CB receptor agonist WIN55,212-2 [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone]. In the presence of the CB1 receptor antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], CF activity no longer suppresses PF-LTP. Presynaptic potentiation can also be obtained by the adenylyl cyclase activator forskolin. WIN55,212-2 blocked this forskolin-mediated enhancement, showing that CB1 receptor activation interferes with the adenylyl cyclase-protein kinase A cascade, which participates in LTP induction. CF activity has been described to promote the induction of postsynaptic PF-long-term depression (LTD) and to impair postsynaptic PF-LTP. Our observation that CF activity blocks the induction of presynaptic LTP suggests that the CF input controls all forms of presynaptic and postsynaptic PF plasticity and that CF activity provides a "safety lock" to prevent an enhancement of transmitter release while postsynaptic AMPA receptor function is downregulated during LTD.

Endocannabinoids can act as retrograde messengers that allow postsynaptic cells to regulate the strength of their synaptic inputs. In the cerebellum, Purkinje cells (PCs) release endocannabinoids through two mechanisms. Synaptic activation evokes local endocannabinoid release that relies on a pathway that involves the metabotropic glutamate receptor mGluR1 and phospholipase-C (PLC). In contrast, depolarization evokes endocannabinoid release from the entire dendritic arbor. This leads to depolarization-induced suppression of inhibitory (DSI) and excitatory (DSE) synapses by a mechanism that does not involve mGluR1 or PLC. This latter mechanism of endocannabinoid release has only been observed under artificial conditions that transiently elevate postsynaptic calcium to >5 microm. Here, we tested the possibility that this mechanism could lead to retrograde inhibition in response to more realistic calcium signals. At both climbing fiber and inhibitory synapses onto PCs, we found that prolonging the elevation of calcium significantly lowered the peak calcium required to evoke PLC-independent endocannabinoid release. This suggests that the mechanism of endocannabinoid release involved in DSI and DSE is likely to evoke endocannabinoid release in response to physiologically relevant levels of calcium. When dendritic calcium was elevated to 0.4-1 microm for 15 s or more, endocannabinoid release from PCs selectively suppressed inhibitory synapses. This suggests that inhibitory synapses are more sensitive to prolonged calcium increases. Thus, in contrast to localized retrograde inhibition evoked by synaptic activation, modest but sustained calcium elevation could globally suppress inhibitory synapses onto PCs.

The hypothalamic paraventricular nucleus (PVN) integrates preautonomic and neuroendocrine control of energy homeostasis, fluid balance, and the stress response. We recently demonstrated that glucocorticoids act via a membrane receptor to rapidly cause endocannabinoid-mediated suppression of synaptic excitation in PVN neurosecretory neurons. Leptin, a major signal of nutritional state, suppresses CB(1) cannabinoid receptor-dependent hyperphagia (increased appetite) in fasting animals by reducing hypothalamic levels of endocannabinoids. Here we show that glucocorticoids stimulate endocannabinoid biosynthesis and release via a Galpha(s)-cAMP-protein kinase A-dependent mechanism and that leptin blocks glucocorticoid-induced endocannabinoid biosynthesis and suppression of excitation in the PVN via a phosphodiesterase-3B-mediated reduction in intracellular cAMP levels. We demonstrate this rapid hormonal interaction in both PVN magnocellular and parvocellular neurosecretory cells. Leptin blockade of the glucocorticoid-induced, endocannabinoid-mediated suppression of excitation was absent in leptin receptor-deficient obese Zucker rats. Our findings reveal a novel hormonal crosstalk that rapidly modulates synaptic excitation via endocannabinoid release in the hypothalamus and that provides a nutritional state-sensitive mechanism to integrate the neuroendocrine regulation of energy homeostasis, fluid balance, and the stress response.

Understanding the relationship between dendritic excitability and synaptic plasticity is vital for determining how dendrites regulate the input-output function of the neuron. Dendritic calcium spikes have been associated with the induction of long-term changes in synaptic efficacy. Here we use direct recordings from cerebellar Purkinje cell dendrites to show that synaptically activated local dendritic calcium spikes are potent triggers of cannabinoid release, producing a profound and short-term reduction in synaptic efficacy at parallel fiber synapses. Enhancing dendritic excitability by modulating dendritic large-conductance calcium-activated potassium (BK) channels improves the spread of dendritic calcium spikes and enhances cannabinoid release at the expense of spatial specificity. Our findings reveal that dendritic calcium spikes provide a local and tunable coincidence detection mechanism that readjusts synaptic gain when synchronous activity reaches a threshold, and they reveal a tight link between the regulation of dendritic excitability and the induction of synaptic plasticity.

2-arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha (DAGLalpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLalpha was predominantly expressed in Purkinje cells. DAGLalpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLalpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLalpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLalpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLalpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLalpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.

The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.

Endocannabinoids are important mediators of short- and long-term synaptic plasticity, but the mechanisms of endocannabinoid release have not been studied extensively outside the hippocampus and cerebellum. Here, we examined the mechanisms of endocannabinoid-mediated long-term depression (eCB-LTD) in the dorsal striatum, a brain region critical for motor control and reinforcement learning. Unlike other cell types, strong depolarization of medium spiny neurons was not sufficient to yield detectable endocannabinoid release. However, when paired with postsynaptic depolarization sufficient to activate L-type calcium channels, activation of postsynaptic metabotropic glutamate receptors (mGluRs), either by high-frequency tetanic stimulation or an agonist, induced eCB-LTD. Pairing bursts of afferent stimulation with brief subthreshold membrane depolarizations that mimicked down-state to up-state transitions also induced eCB-LTD, which not only required activation of mGluRs and L-type calcium channels but also was bidirectionally modulated by dopamine D2 receptors. Consistent with network models, these results demonstrate that dopamine regulates the induction of a Hebbian form of long-term synaptic plasticity in the striatum. However, this gating of plasticity by dopamine is accomplished via an unexpected mechanism involving the regulation of mGluR-dependent endocannabinoid release.

Endocannabinoid release from a single neuron has been shown to cause presynaptic inhibition of transmitter release at many different sites. Here, we demonstrate that hypothalamic proopiomelanocortin (POMC) neurons release endocannabinoids continuously under basal conditions, unlike other release sites at which endocannabinoid production must be stimulated. The basal endocannabinoid release selectively inhibited GABA release onto POMC neurons, although exogenous administration of cannabinoid agonists also inhibited glutamate release. The CB1 cannabinoid receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked endocannabinoid-mediated inhibition of GABA release without affecting excitatory synaptic currents, whereas the CB1 receptor agonist WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate] inhibited both inhibitory and excitatory synaptic currents in POMC neurons. These data demonstrate that endogenously released cannabinoids and exogenously applied CB1 receptor agonists can have markedly different effects on synaptic inputs. Furthermore, the data suggest a novel form of endocannabinoid-mediated retrograde inhibition, whereby the regulation of a subset of inputs requires either the removal of tonic presynaptic inhibition caused by endocannabinoids or the engagement of a mechanism that actively inhibits endocannabinoid production.

Intercellular signaling dynamics critically influence the functional roles that the signals can play. Small lipids are synthesized and released from neurons, acting as intercellular signals in regulating neurotransmitter release, modulating ion channels on target cells, and modifying synaptic plasticity. The repertoire of biological effects of lipids such as endocannabinoids (eCBs) is rapidly expanding, yet lipid signaling dynamics have not been studied. The eCB system constitutes a powerful tool for bioassaying the dynamics of lipid signaling. The eCBs are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The dramatic effects of these lipid signals are detected electrophysiologically as CB1-dependent alterations in conventional synaptic transmission, which therefore serve as a sensitive reporter of eCB actions. We used electrophysiological recording, photolytic release of caged glutamate and a newly developed caged AEA (anandamide), together with rapid [Ca2+]i measurements, to investigate the dynamics of retrograde eCB signaling between CA1 pyramidal cells and GABAergic synapses in rat hippocampus in vitro. We show that, at 22 degrees C, eCB synthesis and release must occur within 75-190 ms after the initiating stimulus, almost an order of magnitude faster than previously thought. At 37 degrees C, the time could be < 50 ms. Activation of CB1 and downstream processes constitute a significant fraction of the total delay and are identified as major rate-limiting steps in retrograde signaling. Our findings imply that lipid messenger dynamics are comparable with those of metabotropic neurotransmitters and can modulate neuronal interactions on a similarly fast time scale.

The endocannabinoid system has been suggested to elicit signals that defend against several disease states including excitotoxic brain damage. Besides direct activation with CB1 receptor agonists, cannabinergic signaling can be modulated through inhibition of endocannabinoid transport and fatty acid amide hydrolase (FAAH), two mechanisms of endocannabinoid inactivation. To test whether the transporter and FAAH can be targeted pharmacologically to modulate survival/repair responses, the transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor palmitylsulfonyl fluoride (AM374) were assessed for protection against excitotoxicity in vitro and in vivo. AM374 and AM404 both enhanced mitogen-activated protein kinase (MAPK) activation in cultured hippocampal slices. Interestingly, combining the distinct inhibitors produced additive effects on CB1 signaling and associated neuroprotection. After an excitotoxic insult in the slices, infusing the AM374/AM404 combination protected against cytoskeletal damage and synaptic decline, and the protection was similar to that produced by the stable CB1 agonist AM356 (R-methanandamide). AM374/AM404 and the agonist also elicited cytoskeletal and synaptic protection in vivo when coinjected with excitotoxin into the dorsal hippocampus. Correspondingly, potentiating endocannabinoid responses with the AM374/AM404 combination prevented behavioral alterations and memory impairment that are characteristic of excitotoxic damage. The protective effects mediated by AM374/AM404 were (1) evident 7 d after insult, (2) correlated with the preservation of CB1-linked MAPK signaling, and (3) were blocked by a selective CB1 antagonist. These results indicate that dual modulation of the endocannabinoid system with AM374/AM404 elicits neuroprotection through the CB1 receptor. The transporter and FAAH are modulatory sites that may be exploited to enhance cannabinergic signaling for therapeutic purposes.

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 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) beta4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCbeta4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCbeta4 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-PLCbeta4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

The wake-promoting neuropeptides orexins (hypocretins) play a crucial role in controlling neuronal excitability and synaptic transmission in the CNS. In this study, using whole-cell patch-clamp recordings in an acute dorsal raphe nucleus (DRN) slice preparation, we report that orexin B (Orx-B) depresses the evoked glutamate-mediated synaptic currents in DRN 5-HT neurons. The Orx-B-induced depression is accompanied by an increase in the paired-pulse ratio and the coefficient of variance, suggesting a presynaptic site of action. Orx-B also reduces the frequency but not the amplitude of miniature EPSCs, indicating that depression of glutamatergic transmission is mediated by a decrease in glutamate release. Surprisingly, the Orx-B-induced inhibition of glutamatergic transmission is abolished by postsynaptic inhibition of G-protein signaling with GDPbetaS, suggesting that this effect is signaled by postsynaptic orexin receptors and expressed presynaptically, presumably through a retrograde messenger. Interestingly, the Orx-B-induced depression of glutamate release is mimicked and occluded by the cannabinoid receptor agonist WIN 55,212-2, and is abolished by the CB1 cannabinoid receptor antagonist AM 251. These results imply that the Orx-B-induced depression of glutamatergic transmission to DRN 5-HT neurons is mediated by retrograde endocannabinoid release. Examination of downstream signaling pathways involved in this response indicates that the effect of Orx-B requires the activation of phospholipase C and DAG lipase enzymatic pathways but not a rise in postsynaptic intracellular calcium. Therefore, our findings reveal a previously unsuspected mechanism by which postsynaptic orexin receptors can modulate glutamatergic synaptic transmission to DRN 5-HT neurons.

Dopamine (DA) neurons in the ventral tegmental area have been implicated in psychiatric disorders and drug abuse. Understanding the mechanisms through which their activity is regulated via the modulation of afferent input is imperative to understanding their roles in these conditions. Here we demonstrate that endocannabinoids liberated from DA neurons activate cannabinoid CB1 receptors located on glutamatergic axons and on GABAergic terminals targeting GABA(B) receptors located on these cells. Endocannabinoid release was initiated by inhibiting either presynaptic type-III metabotropic glutamate receptors or postsynaptic calcium-activated potassium channels, two conditions that also promote enhanced DA neuron excitability and bursting. Thus, activity-dependent release of endocannabinoids may act as a regulatory feedback mechanism to inhibit synaptic inputs in response to DA neuron bursting, thereby regulating firing patterns that may fine-tune DA release from afferent terminals.

Singing by adult male zebra finches is a learned behavior important for courtship, kin recognition, and nest defense (Zann, 1996) and is inhibited by both brief periods of limited food availability and systemic injection of cannabinoids. These similar effects on singing, combined with increasing evidence for endocannabinoid involvement in feeding behavior, led us to evaluate a possible shared mechanism. We found that limited food availability both reduces singing in a cannabinoid antagonist-reversible manner and increases levels of the endocannabinoid 2-arachidonyl glycerol in various brain regions including the caudal telencephalon, an area that contains auditory telencephalon including the L2 subfield of L (L2) and caudal medial nidopallium (NCM). Development and use of an anti-zebra finch cannabinoid receptor type 1 (CB1) antibody demonstrates distinct, dense cannabinoid receptor expression within song regions including Area X, lMAN (lateral magnocellular nucleus of anterior nidopallium), HVC, RA (robust nucleus of arcopallium), and L2. NCM receives L2 projections and is implicated in integration of auditory information. Activity in this area, determined through expression of the transcription factor ZENK, is increased after exposure to unfamiliar song. Because previous work has shown that these novel song-stimulated increases in NCM activity are mitigated by cannabinoid exposure, we tested and found that similar effects on ZENK expression are produced by limiting food. Limited food-related reductions in the activity of NCM neurons were reversed by the cannabinoid antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide), implicating CB1 cannabinoid receptor involvement. Taken together, these experiments indicate a link between feeding state and gene expression related to auditory perception that is mediated by endocannabinoid signaling.

In the nucleus accumbens (NAc), a key structure to the effects of all addictive drugs, presynaptic cannabinoid CB1 receptors (CB1Rs) and postsynaptic metabotropic glutamate 5 receptors (mGluR5s) are the principal effectors of endocannabinoid (eCB)-mediated retrograde long-term depression (LTD) (eCB-LTD) at the prefrontal cortex-NAc synapses. Both CB1R and mGluR5 are involved in cocaine-related behaviors; however, the impact of in vivo cocaine exposure on eCB-mediated retrograde synaptic plasticity remains unknown. Electrophysiological and biochemical approaches were used, and we report that a single in vivo cocaine administration abolishes eCB-LTD. This effect of cocaine was not present in D1 dopamine receptor (D1R) -/- mice and was prevented when cocaine was coadministered with the selective D1R antagonist 8-chloro-2,3,4,5-tetrahydro-3-5-1h-3-benzazepin-7-ol (0.5 mg/kg) or with the NMDA receptor (NMDAR) blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (1 mg/kg), suggesting the involvement of D1R and NMDAR. We found that the cocaine-induced blockade of retrograde signaling was correlated with enhanced expression levels of Homer scaffolding proteins containing the coiled-coil domain and accompanied by a strong reduction of mGluR5 surface expression. The results suggest that cocaine-induced loss of eCB retrograde signaling is caused by a reduction in the ability of mGluR5 to translate anterograde glutamate transmission into retrograde eCB signaling.