ABHD6 Inhibition Rescues a Sex-Dependent Deficit in Motor Coordination in The HdhQ200/200 Mouse Model of Huntington's Disease.

Huntington's Disease is associated with motor behavior deficits that are lessened by few therapeutic options. This preliminary study tested if pharmacological inhibition of α/ß-hydrolase domain containing 6 (ABHD6), a multifunctional enzyme expressed in the striatum, rescues behavioral deficits in HdhQ200/200 mice. Previous work has shown that this model exhibits a reduction in spontaneous locomotion and motor coordination at 8 and 10 months of age, with a more severe phenotype in female mice. Semi-quantitative immunohistochemistry analysis indicated no change in striatal ABHD6 expression at 8 months of age, but a 40% reduction by 10 months in female HdhQ200/200 mice compared to female wild-type (WT) littermates. At 8 months of age, acute ABHD6 inhibition rescued motor coordination deficits in female HdhQ200/200 mice without affecting WT performance. ABHD6 inhibition did not impact spontaneous locomotion, grip strength, or overall weight in either group, showing that effects were specific to motor coordination. At 10 months of age, semi-chronic ABHD6 inhibition by osmotic pump delivery also rescued motor coordination deficits in female HdhQ200/200 mice without affecting female WT littermates. Our preliminary study suggests that ABHD6 inhibition improves motor performance in female HdhQ200/200 mice.

Noradrenergic depletion causes sex specific alterations in the endocannabinoid system in the Murine prefrontal cortex.

Brain endocannabinoids (eCB), acting primarily via the cannabinoid type 1 receptor (CB1r), are involved in the regulation of many physiological processes, including behavioral responses to stress. A significant neural target of eCB action is the stress-responsive norepinephrine (NE) system, whose dysregulation is implicated in myriad psychiatric and neurodegenerative disorders. Using Western blot analysis, the protein expression levels of a key enzyme in the biosynthesis of the eCB 2-arachidonoylglycerol (2-AG), diacylglycerol lipase-α (DGL-α), and two eCB degrading enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) were examined in a mouse model that lacks the NE-synthesizing enzyme, dopamine ß-hydroxylase (DßH-knockout, KO) and in rats treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4). In the prefrontal cortex (PFC), DGL-α protein expression was significantly increased in male and female DßH-KO mice (P < 0.05) compared to wild-type (WT) mice. DßH-KO male mice showed significant decreases in FAAH protein expression compared to WT male mice. Consistent with the DßH-KO results, DGL-α protein expression was significantly increased in male DSP-4-treated rats (P < 0.05) when compared to saline-treated controls. MGL and FAAH protein expression levels were significantly increased in male DSP-4 treated rats compared to male saline controls. Finally, we investigated the anatomical distribution of MGL and FAAH in the NE containing axon terminals of the PFC using immunoelectron microscopy. MGL was predominantly within presynaptic terminals while FAAH was localized to postsynaptic sites. These results suggest that the eCB system may be more responsive in males than females under conditions of NE perturbation, thus having potential implications for sex-specific treatment strategies of stress-related psychiatric disorders.

A novel GPR55-mediated satiety signal in the oval Bed Nucleus of the Stria Terminalis.

Nestled within feeding circuits, the oval (ov) region of the Bed Nucleus of the Stria Terminalis (BNST) may be critical for monitoring energy balance through changes in synaptic strength. Here we report that bidirectional plasticity at ovBNST GABA synapses was tightly linked to the caloric state of male rats, seesawing between long-term potentiation (iLTP, fed) and depression (iLTD, food restricted). L-α-lysophosphatidylinositol (LPI) acting on GPR55 receptors and 2-arachidonoylglycerol (2-AG) through CB1R were respectively responsible for fed (iLTP) and food restricted (iLTD) states. Thus, we have characterized a potential gating mechanism within the ovBNST that may signal metabolic state within the rat brain feeding circuitry.

Monoacylglycerol lipase inhibitors produce pro- or antidepressant responses via hippocampal CA1 GABAergic synapses.

The probability of suffering the mood disorder depression is up to 30% in women and 15% in men during their life span. Pharmacological options for depression are limited: conventional antidepressants have low efficacy and a delayed onset of action (several weeks). Here we investigate the antidepressant actions of inhibitors of monoacylglycerol lipase (MAGL), the major degradative enzyme of the endocannabinoid 2-arachidonoylglycerol. A low-dose of MAGL inhibitors produces antidepressant effects on acute stress-exposed mice, through glutamatergic synaptic long-term depression (LTD), without significant effects on chronic corticosterone-exposed mice. In contrast, a high-dose of MAGL inhibitors produces pro- or antidepressant effects on acute stress- or chronic corticosterone-exposed mice, respectively, through GABAergic synaptic disinhibition. In the hippocampus, in vivo inhibition of MAGL induces a CB1 cannabinoid receptor (CB1R)-dependent suppression of inhibitory GABAergic synapses and an in vivo LTD of excitatory glutamatergic synapses. LTD induction requires CB1R in astroglial cells (but not in GABAergic or glutamatergic neurons) and postsynaptic glutamate receptors. The conventional antidepressant fluoxetine produces rapid or delayed antidepressant effects in acute stress- or chronic corticosterone-exposed mice, respectively. We propose that depression-like behavior of animals in response to acute stress is the normal behavioral response, and thus, MAGL inhibitors, which produce antidepressant effects in chronic corticosterone-exposed animals through GABAergic synaptic disinhibition, represent a new class of rapidly-acting and long-lasting antidepressants.

Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons.

Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-ß-hydroxylase (DßH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DßH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DßH. Triple label immunofluorescence revealed that CB1r and DßH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery.

Inhibiting endocannabinoid biosynthesis: a novel approach to the treatment of constipation.

BACKGROUND AND PURPOSE: Endocannabinoids are a family of lipid mediators involved in the regulation of gastrointestinal (GI) motility. The expression, localization and function of their biosynthetic enzymes in the GI tract are not well understood. Here, we examined the expression, localization and function of the enzyme diacylglycerol lipase-α (DAGLα), which is involved in biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). EXPERIMENTAL APPROACH: Cannabinoid CB1 receptor-deficient, wild-type control and C3H/HeJ mice, a genetically constipated strain, were used. The distribution of DAGLα in the enteric nervous system was examined by immunohistochemistry. Effects of the DAGL inhibitors, orlistat and OMDM-188 on pharmacologically induced GI hypomotility were assessed by measuring intestinal contractility in vitro and whole gut transit or faecal output in vivo. Endocannabinoid levels were measured by mass spectrometry. KEY RESULTS: DAGLα was expressed throughout the GI tract. In the intestine, unlike DAGLß, DAGLα immunoreactivity was prominently expressed in the enteric nervous system. In the myenteric plexus, it was colocalized with the vesicular acetylcholine transporter in cholinergic nerves. In normal mice, inhibiting DAGL reversed both pharmacologically reduced intestinal contractility and pharmacologically prolonged whole gut transit. Moreover, inhibiting DAGL normalized faecal output in constipated C3H/HeJ mice. In colons incubated with scopolamine, 2-AG was elevated while inhibiting DAGL normalized 2-AG levels. CONCLUSIONS AND IMPLICATIONS: DAGLα was expressed in the enteric nervous system of mice and its inhibition reversed slowed GI motility, intestinal contractility and constipation through 2-AG and CB1 receptor-mediated mechanisms. Our data suggest that DAGLα inhibitors may be promising candidates for the treatment of constipation.

AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.

BACKGROUND AND PURPOSE: Cannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies. EXPERIMENTAL APPROACH: The covalent nature of AM841 was determined in vitro using electrophysiological and receptor internalization studies on isolated cultured hippocampal neurons. Mouse models were used for behavioural analysis of analgesia, hypothermia and hypolocomotion. The motility of the small and large intestine was assessed in vivo under normal conditions and after acute stress. The brain penetration of AM841 was also determined. KEY RESULTS: AM841 behaved as an irreversible CB1 receptor agonist in vitro. AM841 potently reduced GI motility through an action on CB1 receptors in the small and large intestine under physiological conditions. AM841 was even more potent under conditions of acute stress and was shown to normalize accelerated GI motility under these conditions. This compound behaved as a peripherally restricted ligand, showing very little brain penetration and no characteristic centrally mediated CB1 receptor-mediated effects (analgesia, hypothermia or hypolocomotion). CONCLUSIONS AND IMPLICATIONS: AM841, a novel peripherally restricted covalent CB1 receptor ligand that was shown to be remarkably potent, represents a new class of potential therapeutic agents for the treatment of functional GI disorders.

Cover crops influence soil microorganisms and phytoextraction of copper from a moderately contaminated vineyard.

We investigated the ability of summer (Avena sativa [oat], Trifolium incarnatum [crimson clover], Chenopodium [goosefoot]) and winter (Vicia villosa [hairy vetch], Secale Cereale L. [Rye], Brassica napus L. partim [rape]) cover crops, including a mixed species treatment, to extract copper from an organic vineyard soil in situ and the microbial communities that may support it. Clover had the highest copper content (14.3mgCukg(-1) DM). However, it was the amount of total biomass production that determined which species was most effective at overall copper removal per hectare. The winter crop rye produced significantly higher amounts of biomass (3532kgDMha(-1)) and, therefore, removed significantly higher amounts of copper (14,920mgCuha(-1)), despite less accumulation of copper in plant shoots. The maximum annual removal rate, a summation of best performing summer and winter crops, would be 0.033kgCuha(-1)y(-1). Due to this low annual extraction efficiency, which is less than the 6kgCuha(-1)y(-1) permitted for application, phytoextraction cannot be recommended as a general method of copper extraction from vineyards. Copper concentration did not influence aboveground or belowground properties, as indicated by sampling at two distances from the grapevine row with different soil copper concentrations. Soil microorganisms may have become tolerant to the copper levels at this site. Microbial biomass and soil enzyme activities (arylsulfatase and phosphatase) were instead driven by seasonal fluxes of resource pools. Gram+ bacteria were associated with high soil moisture, while fungi seemed to be driven by extractable carbon, which was linked to high plant biomass. There was no microbial group associated with the increased phytoextraction of copper. Moreover, treatment did not influence the abundance, activity or community structure of soil microorganisms.

Parsing the players: 2-arachidonoylglycerol synthesis and degradation in the CNS.

UNLABELLED: The endogenous cannabinoid signalling system, composed of endogenous cannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade the endogenous cannabinoids, is much more complex than initially conceptualized. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid and plays a major role in CNS development and synaptic plasticity. Over the past decade, many key players in 2-AG synthesis and degradation have been identified and characterized. Most 2-AG is synthesized from membrane phospholipids via sequential activation of a phospholipase Cß and a diacylglycerol lipase, although other pathways may contribute in specialized settings. 2-AG breakdown is more complicated with at least eight different enzymes participating. These enzymes can either degrade 2-AG into its components, arachidonic acid and glycerol, or transform 2-AG into highly bioactive signal molecules. The implications of the precise temporal and spatial control of the expression and function of these pleiotropic metabolizing enzymes have only recently come to be appreciated. In this review, we will focus on the primary organization of the synthetic and degradative pathways of 2-AG and then discuss more recent findings and their implications, with an eye towards the biological and therapeutic implications of manipulating 2-AG synthesis and metabolism. LINKED ARTICLES: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

High fat diet and body weight have different effects on cannabinoid CB(1) receptor expression in rat nodose ganglia.

Energy balance is regulated, in part, by the orexigenic signaling pathways of the vagus nerve. Fasting-induced modifications in the expression of orexigenic signaling systems have been observed in vagal afferents of lean animals. Altered basal cannabinoid (CB1) receptor expression in the nodose ganglia in obesity has been reported. Whether altered body weight or a high fat diet modifies independent or additive changes in CB1 expression is unknown. We investigated the expression of CB1 and orexin 1 receptor (OX-1R) in the nodose ganglia of rats fed ad libitum or food deprived (24h), maintained on low or high fat diets (HFD), with differing body weights. Male Wistar rats were fed chow or HFD (diet-induced obese: DIO or diet-resistant: DR) or were body weight matched to the DR group but fed chow (wmDR). CB1 and OX-1R immunoreactivity were investigated and CB1 mRNA density was determined using in situ hybridization. CB1 immunoreactivity was measured in fasted rats after sulfated cholecystokinin octapeptide (CCK8s) administration. In chow rats, fasting did not modify the level of CB1 mRNA. More CB1 immunoreactive cells were measured in fed DIO, DR and wmDR rats than chow rats; levels increased after fasting in chow and wmDR rats but not in DIO or DR rats. In HFD fasted rats CCK8s did not reduce CB1 immunoreactivity. OX-1R immunoreactivity was modified by fasting only in DR rats. These data suggest that body weight contributes to the proportion of neurons expressing CB1 immunoreactivity in the nodose ganglion, while HFD blunts fasting-induced increases, and CCK-induced suppression of, CB1-immunoreactivity.

Dopamine D2 and acetylcholine α7 nicotinic receptors have subcellular distributions favoring mediation of convergent signaling in the mouse ventral tegmental area.

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) mediate nicotine-induced burst-firing of dopamine neurons in the ventral tegmental area (VTA), a limbic brain region critically involved in reward and in dopamine D2 receptor (D2R)-related cortical dysfunctions associated with psychosis. The known presence of α7nAChRs and Gi-coupled D2Rs in dopamine neurons of the VTA suggests that these receptors are targeted to at least some of the same neurons in this brain region. To test this hypothesis, we used electron microscopic immunolabeling of antisera against peptide sequences of α7nACh and D2 receptors in the mouse VTA. Dual D2R and α7nAChR labeling was seen in many of the same somata (co-localization over 97%) and dendrites (co-localization over 49%), where immunoreactivity for each of the receptors was localized to endomembranes as well as to non-synaptic or synaptic plasma membranes often near excitatory-type synapses. In comparison with somata and dendrites, many more small axons and axon terminals were separately labeled for each of the receptors. Thus, single-labeled axon terminals were predominant for both α7nAChR (57.9%) and D2R (89.0%). The majority of the immunolabeled axonal profiles contained D2R-immunoreactivity (81.6%) and formed either symmetric or asymmetric synapses consistent with involvement in the release of both inhibitory and excitatory transmitters. Of 160 D2R-labeled terminals, 81.2% were presynaptic to dendrites that expressed α7nAChR alone or together with the D2R. Numerous glial processes inclusive of those enveloping either excitatory- or inhibitory-type synapses also contained single labeling for D2R (n=152) and α7nAChR (n=561). These results suggest that classic antipsychotic drugs, all of which block the D2R, may facilitate α7nAChR-mediated burst-firing by elimination of D2R-dependent inhibition in neurons expressing both receptors as well as by indirect pre-synaptic and glial mechanisms.

The impact of adolescent social isolation on dopamine D2 and cannabinoid CB1 receptors in the adult rat prefrontal cortex.

Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior.

Brain CB1 receptor expression following lipopolysaccharide-induced inflammation.

Cannabinoid 1 receptors (CB(1)) are highly expressed on presynaptic terminals in the brain where they are importantly involved in the control of neurotransmitter release. Alteration of CB(1) expression is associated with a variety of neurological and psychiatric disorders. There is now compelling evidence that peripheral inflammatory disorders are associated with depression and cognitive impairments. These can be modeled in rodents with peripheral administration of lipopolysaccharide (LPS), but central effects of this treatment remain to be fully elucidated. As a reduction in endocannabinoid tone is thought to contribute to depression, we asked whether the expression of CB(1) in the CNS is altered following LPS treatment. CD1 mice received LPS (0.1-1mg/kg, ip) and 6h later activated microglial cells were observed only in circumventricular organs and only at the higher dose. At 24h, activated microglial cells were identified in other brain regions, including the hippocampus, a structure implicated in some mood disorders. Immunohistochemistry and real-time polymerase chain reaction (PCR) were utilized to evaluate the change of CB(1) expression 24h after inflammation. LPS induced an increase of CB(1) mRNA in the hippocampus and brainstem. Subsequent immunohistochemical analysis revealed reduced CB(1) in the hippocampus, especially in CA3 pyramidal layer. Analysis of co-localization with markers of excitatory and inhibitory terminals indicated that the decrease in CB(1) expression was restricted to glutamatergic terminals. Despite widespread microglial activation, these results suggest that peripheral LPS treatment leads to limited changes in CB(1) expression in the brain.

Cannabinoid-1 receptors in the mouse ventral pallidum are targeted to axonal profiles expressing functionally opposed opioid peptides and contacting N-acylphosphatidylethanolamine-hydrolyzing phospholipase D terminals.

The ventral pallidum (VP) is a major recipient of inhibitory projections from nucleus accumbens (Acb) neurons that differentially express the reward (enkephalin) and aversion (dynorphin)-associated opioid peptides. The cannabinoid-1 receptor (CB1R) is present in Acb neurons expressing each of these peptides, but its location in the VP is not known. To address this question, we used electron microscopic dual immunolabeling of the CB1R and either dynorphin 1-8 (Dyn) or Met(5)-enkephalin (ME) in the VP of C57BL/6J mice, a species in which CB1R gene deletion produces a reward deficit. We also used similar methods to determine the relationship between the CB1R and N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), an anandamide-synthesizing enzyme located presynaptically in other limbic brain regions. CB1R-immunogold was principally localized to cytoplasmic endomembranes and synaptic or extrasynaptic plasma membranes of axonal profiles, but was also affiliated with postsynaptic membrane specializations in dendrites. The axonal profiles included many single CB1R-labeled axon terminals as well as terminals containing CB1R-immunogold and either Dyn or ME immunoreactivity. Dually labeled terminals comprised 26% of all Dyn- and 17% of all ME-labeled axon terminals. Both single- and dual-labeled terminals formed mainly inhibitory-type synapses, but almost 16% of these terminals formed excitatory synapses. Approximately 60% of the CB1R-labeled axonal profiles opposed or converged with axon terminals containing NAPE-PLD immunoreactivity. We conclude that CB1Rs in the mouse VP have subcellular distributions consistent with on demand activation by endocannabinoids that can regulate the release of functionally opposed opioid peptides and also modulate inhibitory and excitatory transmission.

The CB2-preferring agonist JWH015 also potently and efficaciously activates CB1 in autaptic hippocampal neurons.

The G protein coupled receptors CB(1) and CB(2) are targets for the psychoactive constituents of cannabis, chief among them Δ(9)-THC. They are also key components of the multifunctional endogenous cannabinoid signaling system. CB(1) and CB(2) receptors modulate a wide variety of physiological systems including analgesia, memory, mood, reward, appetite and immunity. Identification and characterization of selective CB(1) and CB(2) receptor agonists and antagonists will facilitate understanding the precise physiological and pathophysiological roles of cannabinoid receptors in these systems. This is particularly necessary in the case of CB(2) because these receptors are sparsely expressed and problematic to detect using traditional immunocytochemical approaches. 1-Propyl-2-methyl-3-(1-naphthoyl)indole (JWH015) is an aminoalkylindole that has been employed as a "CB(2)-selective" agonist in more than 40 published papers. However, we have found that JWH015 potently and efficaciously activates CB(1) receptors in neurons. Using murine autaptic hippocampal neurons, which express CB(1), but not CB(2) receptors, we find that JWH015 inhibits excitatory postsynaptic currents with an EC50 of 216nM. JWH015 inhibition is absent in neurons from CB(1)(-/-) cultures and is reversed by the CB(1) antagonist, SR141716 [200nM]. Furthermore, JWH015 partially occludes CB(1)-mediated DSE (∼35% remaining), an action reversed by the CB(2) antagonist, AM630 [1 and 3µM], suggesting that high concentrations of AM630 also antagonize CB(1) receptors. We conclude that while JWH015 is a CB(2)-preferring agonist, it also activates CB(1) receptors at experimentally encountered concentrations. Thus, CB(1) agonism of JWH015 needs to be considered in the design and interpretation of experiments that use JWH015 to probe CB(2)-signaling.

Remediation of copper in vineyards--a mini review.

Viticulturists use copper fungicide to combat Downy Mildew. Copper, a non-degradable heavy metal, can accumulate in soil or leach into water sources. Its accumulation in topsoil has impacted micro and macro organisms, spurring scientists to research in situ copper removal methods. Recent publications suggest that microorganism assisted phytoextraction, using plants and bacteria to actively extract copper, is most promising. As vineyards represent moderately polluted sites this technique has great potential. Active plant extraction and chelate assisted remediation extract too little copper or risk leaching, respectively. However, despite interesting pot experiment results using microorganism assisted phytoextraction, it remains a challenge to find plants that primarily accumulate copper in their shoots, a necessity in vineyards where whole plant removal would be time consuming and financially cumbersome. Vineyard remediation requires a holistic approach including sustainable soil management, proper plant selection, increasing biodiversity and microorganisms.

Plasticity of mouse enteric synapses mediated through endocannabinoid and purinergic signaling.

BACKGROUND: The enteric nervous system (ENS) possesses extensive synaptic connections which integrate information and provide appropriate outputs to coordinate the activity of the gastrointestinal tract. The regulation of enteric synapses is not well understood. Cannabinoid (CB)(1) receptors inhibit the release of acetylcholine (ACh) in the ENS, but their role in the synapse is not understood. We tested the hypothesis that enteric CB(1) receptors provide inhibitory control of excitatory neurotransmission in the ENS. METHODS: Intracellular microelectrode recordings were obtained from mouse myenteric plexus neurons. Interganglionic fibers were stimulated with a concentric stimulating electrode to elicit synaptic events on to the recorded neuron. Differences between spontaneous and evoked fast synaptic transmission was examined within preparations from CB(1) deficient mice (CB(1)(-/-)) and wild-type (WT) littermate controls. KEY RESULTS: Cannabinoid receptors were colocalized on terminals expressing the vesicular ACh transporter and the synaptic protein synaptotagmin. A greater proportion of CB(1)(-/-) neurons received spontaneous fast excitatory postsynaptic potentials than neurons from WT preparations. The CB(1) agonist WIN55,212 depressed WT synapses without any effect on CB(1)(-/-) synapses. Synaptic activity in response to depolarization was markedly enhanced at CB(1)(-/-) synapses and after treatment with a CB(1) antagonist in WT preparations. Activity-dependent liberation of a retrograde purine messenger was demonstrated to facilitate synaptic transmission in CB(1)(-/-) mice. CONCLUSIONS & INFERENCES: Cannabinoid receptors inhibit transmitter release at enteric synapses and depress synaptic strength basally and in an activity-dependent manner. These actions help explain accelerated intestinal transit observed in the absence of CB(1) receptors.

Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice.

BACKGROUND AND PURPOSE: Gastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility. EXPERIMENTAL APPROACH: We examined the effect of AM3506 on electrically-evoked contractility in vitro and GI transit and colonic faecal output in vivo, in normal and FAAH-deficient mice treated with saline or LPS (100 µg·kg(-1), i.p.), in the presence and absence of cannabinoid (CB) receptor antagonists. mRNA expression was measured by quantitative real time-PCR, EC levels by liquid chromatography-MS and FAAH activity by the conversion of [(3)H]-AEA to [(3)H]-ethanolamine in intestinal extracts. FAAH expression was examined by immunohistochemistry. KEY RESULTS: FAAH was dominantly expressed in the enteric nervous system; its mRNA levels were higher in the ileum than the colon. LPS enhanced ileal contractility in the absence of overt inflammation. AM3506 reversed the enhanced electrically-evoked contractions of the ileum through CB(1) and CB(2) receptors. LPS increased the rate of upper GI transit and faecal output. AM3506 normalized the enhanced GI transit through CB(1) and CB(2) receptors and faecal output through CB(1) receptors. LPS did not increase GI transit in FAAH-deficient mice. CONCLUSIONS AND IMPLICATIONS: Inhibiting FAAH normalizes various parameters of GI dysmotility in intestinal pathophysiology. Inhibition of FAAH represents a new approach to the treatment of disordered intestinal motility.

Maternal deprivation and adolescent cannabinoid exposure impact hippocampal astrocytes, CB1 receptors and brain-derived neurotrophic factor in a sexually dimorphic fashion.

We have recently reported that early maternal deprivation (MD) for 24 h [postnatal day (PND) 9-10] and/or an adolescent chronic treatment with the cannabinoid agonist CP-55,940 (CP) [0.4 mg/kg, PND 28-42] in Wistar rats induced, in adulthood, diverse sex-dependent long-term behavioral and physiological modifications. Here we show the results obtained from investigating the immunohistochemical analysis of CB1 cannabinoid receptors, glial fibrillary acidic protein (GFAP) positive (+) cells and brain-derived neurotrophic factor (BDNF) expression in the hippocampus of the same animals. MD induced, in males, a significant increase in the number of GFAP+ cells in CA1 and CA3 areas and in the polymorphic layer of the dentate gyrus (DG), an effect that was attenuated by CP in the two latter regions. Adolescent cannabinoid exposure induced, in control non-deprived males, a significant increase in the number of GFAP+ cells in the polymorphic layer of the DG. MD induced a decrease in CB1 expression in both sexes, and this effect was reversed in males by the cannabinoid treatment. In turn, the drug "per se" induced, in males, a general decrease in CB1 immunoreactivity, and the opposite effect was observed in females. Cannabinoid exposure tended to reduce BDNF expression in CA1 and CA3 of females, whereas MD counteracted this trend and induced an increase of BDNF in females. As a whole, the present results show sex-dependent long-term effects of both MD and juvenile cannabinoid exposure as well as functional interactions between the two treatments.

COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation.

BACKGROUND AND PURPOSE: Depolarization-induced suppression of inhibition (DSI) and excitation (DSE) are two forms of cannabinoid CB(1) receptor-mediated inhibition of synaptic transmission, whose durations are regulated by endocannabinoid (eCB) degradation. We have recently shown that in cultured hippocampal neurons monoacylglycerol lipase (MGL) controls the duration of DSE, while DSI duration is determined by both MGL and COX-2. This latter result suggests that DSE might be attenuated, and excitatory transmission enhanced, during inflammation and in other settings where COX-2 expression is up-regulated. EXPERIMENTAL APPROACH: To investigate whether it is possible to control the duration of eCB-mediated synaptic plasticity by varied expression of eCB-degrading enzymes, we transfected excitatory autaptic hippocampal neurons with putative 2-AG metabolizing enzymes: COX-2, fatty acid amide hydrolase (FAAH), α/ß hydrolase domain 6 (ABHD6), α/ß hydrolase domain 12 (ABHD12) or MGL. KEY RESULTS: We found that overexpression of either COX-2 or FAAH shortens the duration of DSE while ABHD6 or ABHD12 do not. In contrast, genetic deletion (MGL(-/-)) and overexpression of MGL both radically altered eCB-mediated synaptic plasticity. CONCLUSIONS AND IMPLICATIONS: We conclude that both FAAH and COX-2 can be trafficked to neuronal sites where they are able to degrade eCBs to modulate DSE duration and, by extension, net endocannabinoid signalling at a given synapse. The results for COX-2, which is often up-regulated under pathological conditions, are of particular note in that they offer a mechanism by which up-regulated COX-2 may promote neuronal excitation by suppressing DSE while enhancing conversion of 2-AG to PGE(2) -glycerol ester under pathological conditions.