Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB₁ receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D₃ receptor null (D3R^(−/−)) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R^(−/−) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R^(−/−) mice also exhibited different endocannabinoid and TRPV1, but not CB₁, levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB₁ and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R^(−/−) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.     

Anatomical characterization of the cannabinoid CB1 receptor in cell‐type–specific mutant mouse rescue models

Type 1 cannabinoid (CB₁) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB₁ receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell‐type–specific rescue of CB₁ receptors have emerged as excellent tools for investigating CB₁ receptors’ cell‐type–specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB₁ receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB₁ receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu‐CB₁‐RS) or GABAergic neurons (GABA‐CB₁‐RS) was studied by immunoelectron microscopy. Results were compared with conditional CB₁ receptor knockout lines. As expected, CB₁ immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB₁ receptor‐immunopositive excitatory and inhibitory synapses were Glu‐CB₁‐RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA‐CB₁‐RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB₁ receptor‐immunopositive excitatory and inhibitory synapses in the inner one‐third of the dentate molecular layer was Glu‐CB₁‐RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA‐CB₁‐RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu‐CB₁‐RS and GABA‐CB₁‐RS mice show the usual CB₁ receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in‐depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302–318, 2017. © 2016 Wiley Periodicals, Inc.     

Differential effects of the antidepressants tranylcypromine and fluoxetine on limbic cannabinoid receptor binding and endocannabinoid contents

The goal of this study was to determine whether the endocannabinoid system is altered by chronic antidepressant treatment. The effects of 3-week administration of the monoamine oxidase inhibitor, tranylcypromine (10 mg/kg) and the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg) on cannabinoid CB₁ receptor densities and endocannabinoid contents were determined in limbic brain regions of the rat. Tranylcypromine significantly reduced tissue content of the endocannabinoid N-arachidonylethanolamine (anandamide) in the prefrontal cortex, hippocampus and hypothalamus and increased 2-arachidonoylglycerol content in the prefrontal cortex. Tranylcypromine treatment significantly increased CB₁ receptor binding density in the prefrontal cortex and hippocampus, but not in the hypothalamus. Treatment with fluoxetine increased CB₁ receptor density in the prefrontal cortex, but had no effect on endocannabinoid contents in any brain region examined. These data suggest that monoaminergic neurotransmission can regulate the endocannabinoid system and further indicates a role of the endocannabinoid system in affective illness and its treatment. An erratum to this article can be found at     

Modulation of the endocannabinoid system: Neuroprotection or neurotoxicity?

There is now a large volume of data indicating that compounds activating cannabinoid CB₁ receptors, either directly or indirectly by preventing the breakdown of endogenous cannabinoids, can protect against neuronal damage produced by a variety of neuronal “insults”. Given that such neurodegenerative stimuli result in increased endocannabinoid levels and that animals with genetic deletions of CB₁ receptors are more susceptible to the deleterious effects of such stimuli, a case can be made for an endogenous neuroprotective role of endocannabinoids. However, this is an oversimplification of the current literature, since (a) compounds released together with the endocannabinoids can contribute to the neuroprotective effect; (b) other proteins, such as TASK-1 and PPARα, are involved; (c) the CB₁ receptor antagonist/inverse agonist rimonabant has also been reported to have neuroprotective properties in a number of animal models of neurodegenerative disorders. Furthermore, the CB₂ receptor located on peripheral immune cells and activated microglia are potential targets for novel therapies. In terms of the clinical usefulness of targeting the endocannabinoid system for the treatment of neurodegenerative disorders, data are emerging, but important factors to be considered are windows of opportunity (for acute situations such as trauma and ischemia) and the functionality of the target receptors (for chronic neurodegenerative disorders such as Alzheimer's disease).     

The endocannabinoid system and the regulation of neural development: potential implications in psychiatric disorders

During brain development, functional neurogenesis is achieved by the concerted action of various steps that include the expansion of progenitor cells, neuronal specification, and establishment of appropriate synapses. Brain patterning and regionalization is regulated by a variety of extracellular signals and morphogens that, together with neuronal activity, orchestrate and regulate progenitor proliferation, differentiation, and neuronal maturation. In the adult brain, CB₁ cannabinoid receptors are expressed at very high levels in selective areas and are engaged by endocannabinoids, which act as retrograde messengers controlling neuronal function and preventing excessive synaptic activity. In addition, the endocannabinoid system is present at early developmental stages of nervous system formation. Recent studies have provided novel information on the role of this endogenous neuromodulatory system in the control of neuronal specification and maturation. Thus, cannabinoid receptors and locally produced endocannabinoids regulate neural progenitor proliferation and pyramidal specification of projecting neurons. CB₁ receptors also control axonal navigation, migration, and positioning of interneurons and excitatory neurons. Loss of function studies by genetic ablation or pharmacological blockade of CB₁ receptors interferes with long-range subcortical projections and, likewise, prenatal cannabinoid exposure induces different functional alterations in the adult brain. Potential implications of these new findings, such as the participation of the endocannabinoid system in the pathogenesis of neurodevelopmental disorders (e.g., schizophrenia) and the regulation of neurogenesis in brain depression, are discussed herein.     

Loss of cannabinoid CB1 receptor expression in the 6-hydroxydopamine-induced nigrostriatal terminal lesion model of Parkinson's disease in the rat

The endocannabinoid system is emerging as a potential alternative to the dopaminergic system for the treatment of Parkinson's disease. Like all emerging targets, validation of this system's potential for treating human Parkinsonism necessitates testing in animal models of the condition. However, if components of the endocannabinoid system are altered by the induction of a Parkinsonian state in animal models, this could have an impact on the interpretation of such preclinical experiments. This study sought to determine if expression of the CB₁ subtype of cannabinoid receptor is altered in the two most commonly used rat models of Parkinson's disease. Parkinsonian lesions were induced by stereotaxic injection of 6-hydroxydopamine into the axons (medial forebrain bundle) or terminals (striatum) of the nigrostriatal pathway. On days 1, 3, 7, 14 and 28 post-lesion, rats were sacrificed and brains were processed for tyrosine hydroxylase and CB₁ receptor immunohistochemistry. The CB₁ receptor was expressed strongly in the substantia nigra pars reticulata, minimally overlapping with tyrosine hydroxylase immunoreactivity in the pars compacta. Interestingly, while there was little change in CB₁ receptor expression following axonal lesion, expression of the receptor was significantly reduced following terminal lesion. Loss of CB₁ receptor expression in the pars reticulata correlated significantly with the loss of striatal and nigral volume after terminal lesion indicating this may have been due to 6-hydroxydopamine-induced non-specific damage of striatonigral neurons which are known to express CB₁ receptors. Thus, this result has implications for the choice of model and interpretation of studies used to investigate potential cannabinoid-based therapies for Parkinson's disease as well as striatonigral diseases such as Huntington's disease and Multiple Systems Atrophy.     

The Endocannabinoid System and its Modulation by Phytocannabinoids

The endocannabinoid system is currently defined as the ensemble of the two 7-transmembrane-domain and G protein-coupled receptors for Δ⁹-tetrahydrocannabinol (but not for most other plant cannabinoids or phytocannabinoids)—cannabinoid receptor type-1 (CB₁R) and cannabinoid receptor type-2 (CB₂R); their two most studied endogenous ligands, the “endocannabinoids” N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG); and the enzymes responsible for endocannabinoid metabolism. However, anandamide and 2-AG, and also the phytocannabinoids, have more molecular targets than just CB₁R and CB₂R. Furthermore, the endocannabinoids, like most other lipid mediators, have more than just one set of biosynthetic and degrading pathways and enzymes, which they often share with “endocannabinoid-like” mediators that may or may not interact with the same proteins as Δ⁹-tetrahydrocannabinol and other phytocannabinoids. In some cases, these degrading pathways and enzymes lead to molecules that are not inactive and instead interact with other receptors. Finally, some of the metabolic enzymes may also participate in the chemical modification of molecules that have very little to do with endocannabinoid and cannabinoid targets. Here, we review the whole world of ligands, receptors, and enzymes, a true “endocannabinoidome”, discovered after the cloning of CB₁R and CB₂R and the identification of anandamide and 2-AG, and its interactions with phytocannabinoids.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-015-0374-6) contains supplementary material, which is available to authorized users.     

Expression of CB<Subscript>1</Subscript> cannabinoid receptor in the anterior cingulate cortex in schizophrenia,bipolar disorder,and major depression

Summary The human endogenous cannabinoid system is an appealing target in the investigation of psychiatric disorders. In schizophrenia, endocannabinoids and their receptors are involved in the pathology of the disease. Previous studies reported an increased radioligand binding to cannabinoid receptors 1 (CB₁) in schizophrenia, both in the dorsolateral prefrontal cortex and in the anterior cingulate cortex (ACC). We analyzed the expression of the CB₁ receptors in the ACC at the protein level using immunohistochemistry. In a quantitative postmortem study, 60 patients suffering from schizophrenia, bipolar disorder, major depression and controls were included. Numerical densities of neurons and glial cells immunopositive for CB₁ receptors were evaluated. No evidence of an increased or decreased density of CB₁ receptor immunopositive cells in schizophrenia or bipolar disorder was found. In major depression, CB₁ receptor immunopositive glial cells in the grey matter were decreased. Furthermore, our data show that different medications have an impact on the expression of CB₁ receptors in the ACC.     

Molecular architecture of endocannabinoid signaling at nociceptive synapses mediating analgesia

Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid‐mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2‐arachidonoylglycerol (2‐AG) is mobilized in the lumbar spinal cord in temporal correlation with stress‐induced antinociception. We therefore characterized the precise molecular architecture of 2‐AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase‐alpha (DGL‐α), the synthesizing enzyme of 2‐AG. Peroxidase‐based immunocytochemistry demonstrated high levels of DGL‐α protein and CB₁ cannabinoid receptor, a receptor for 2‐AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High‐resolution electron microscopy uncovered postsynaptic localization of DGL‐α at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB₁ on excitatory axon terminals. Furthermore, DGL‐α in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR₅), whose activation induces 2‐AG biosynthesis. Finally, intrathecal activation of mGluR₅ at the lumbar level evoked endocannabinoid‐mediated stress‐induced analgesia through the DGL–2‐AG–CB₁ pathway. Taken together, these findings suggest a key role for 2‐AG‐mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2‐AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2‐AG levels may be an efficacious way to regulate pain sensation.     

Depolarizing GABAergic synaptic input triggers endocannabinoid‐mediated retrograde synaptic signaling

Endocannabinoids released by postsynaptic neurons inhibit neurotransmitter release from presynaptic axon terminals. One typical stimulus of endocannabinoid production is an increase of calcium concentration in postsynaptic neurons. The aim of the present study was to clarify whether depolarizing GABAergic synaptic input, by increasing calcium concentration in postsynaptic neurons, can trigger endocannabinoid production. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in Purkinje cells in mouse cerebellar slices with patch‐clamp pipettes containing 151 mM chloride (a usual recording mode). sIPSCs were depolarizing inward currents under this condition. Combined electrophysiological and fluorometric calcium imaging experiments indicated that sIPSCs frequently triggered calcium spikes. After the calcium spikes, a short‐term suppression of sIPSCs occurred. This suppression was prevented by the CB₁ cannabinoid receptor antagonist rimonabant and the diacylglycerol lipase inhibitor orlistat, but not changed by URB597, an inhibitor of anandamide degradation. It is, therefore, likely that CB₁ receptors and 2‐arachidonoylglycerol were involved. For testing the physiological significance of the above observation, we carried out experiments on brains of 3‐ to 5‐day‐old mice. The gramicidin‐induced perforated patch‐clamp mode was used for preserving the physiological intracellular chloride concentration of the neurons. Depolarizing GABAergic sIPSCs occurred under this condition, but at a very low rate. Rimonabant did not change the frequency of these sIPSCs, arguing against the persistence of an endocannabinoid tone. The results point to a new kind of trigger of endocannabinoid production: depolarizing GABAergic synaptic input can elicit endocannabinoid production in postsynaptic neurons by activating calcium channels. The produced endocannabinoid suppresses GABA release from presynaptic axon terminals. Synapse 63:643–652, 2009. © 2009 Wiley‐Liss, Inc.     

An endocannabinoid tone limits excitotoxicity in vitro and in a model of multiple sclerosis

The aim of this study was to evaluate how endocannabinoids interact with excitotoxic processes both in vitro, using primary neural cell cultures, and in vivo, in the TMEV-IDD model of multiple sclerosis. First, we observed that neuronal cells respond to excitotoxic challenges by the production of endocannabinoid molecules which in turn exerted neuroprotective effects against excitotoxicity. The inhibitor of endocannabinoid uptake, UCM707, protected specifically against AMPA-induced excitotoxicity, by activating CB₁ and CB₂ cannabinoid receptors, as well as the nuclear factor, PPARγ. This neuroprotective effect was reverted by blocking the glial glutamate transporter, GLT-1. Mice subjected to the model of multiple sclerosis showed a decrease in the expression of GLT-1. UCM707 reversed this loss of GLT-1 and induced a therapeutic effect. Our data indicate that the enhancement of the endocannabinoid tone leads to neuroprotection against AMPA-induced excitotoxicity and provides therapeutic effects in this model of multiple sclerosis.     

Regulation of subthalamic neuron activity by endocannabinoids

High levels of anandamide are located in the basal ganglia. The subthalamic nucleus (STN) is considered to be an important modulator of basal ganglia output. The present study aims at characterizing the modulation of the electrical activity of STN neurons by exogenous anandamide or endocannabinoids. Single‐unit extracellular recordings in anesthetized rats and patch‐clamp techniques in rat brain slices containing the STN were performed. Immunohistochemical assays were used. In vivo, anandamide administration produced two opposite effects (inhibition or stimulation) on STN neuron firing rates, depending of the precise location of the neuron within the nucleus. These effects were enhanced by prior inhibition of fatty acid amide hydrolase with URB597, but not by the inhibitor of carrier‐mediated anandamide transport AM404. Rimonabant, a specific CB₁ receptor antagonist, also produced inhibition or stimulation of STN neuron activity when administered alone or after anandamide. These effects seem to be mediated by indirect mechanisms since: (1) STN neuron activity is not modified by the cannabinoid agonist Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) in vitro; (2) no depolarization‐induced suppression of inhibition phenomena were observed; and (3) CB₁ receptor immunolabeling was not detected in the STN, but was abundant in areas which project efferents to this nucleus. Moreover, chemical lesion of the globus pallidus abolished the stimulatory effect of anandamide and microinfusion of anandamide into the prefrontal cortex led to inhibition of STN neuron activity. The present results show that endocannabinoids exert a tonic control on STN activity via receptors located outside the nucleus. These findings may contribute to enhance our understanding of the role of the endocannabinoid system in motor control. Synapse 64:682–698, 2010. © 2010 Wiley‐Liss, Inc.     

In utero cannabinoid exposure alters breathing and the response to hypoxia in newborn mice

Cannabis is one of the most commonly used recreational drugs at ages highly correlated with potential pregnancy. Endocannabinoid signalling regulates important stages of neuronal development. When cannabinoid receptors, which are widely distributed through the nervous system, are activated by exogenous cannabinoids, breathing in adult rats is depressed. Here, we show that, in newborn mice, endocannabinoids, through the activation of cannabinoid receptor type 1 (CB₁R), participate in the modulation of respiration and its control. Blocking CB₁Rs at birth suppressed the brake exerted by endocannabinoids on ventilation in basal and in hypoxic conditions. The number of apnoeas and their duration were also minimized by activation of CB₁Rs in normoxic and in hypoxic conditions. However, prenatal cannabis intoxication, caused by a daily injection of WIN55,212‐2, in pregnant mice durably modified respiration of the offspring, as shown by hyperventilation in basal conditions, an altered chemoreflex in response to hypoxia, and longer apnoeas. When CB₁Rs were blocked in WIN55,212‐2 treated newborns, persistent hyperventilation was still observed, which could partly be explained by a perturbation of the central respiratory network. In fact, in vitro medullary preparations from WIN55,212‐2 treated pups, free of peripheral or of supramedullary structures, showed an altered fictive breathing frequency. In conclusion, the endocannabinoid pathway at birth seems to modulate breathing and protect the newborn against apnoeas. However, when exposed prenatally to an excess of cannabinoid, the breathing neuronal network in development seems to be modified, probably rendering the newborn more vulnerable in the face of an unstable environment.     

A hypothetical mechanism for interactions between neuromodulators during paradoxical sleep

A mechanism for interdependent changes in the concentrations of neuromodulators during paradoxical sleep is proposed. According to this mechanism, the release of dopamine in the striatum and modulation of corticostriatal inputs promote disinhibitions via the basal ganglia of cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei, thus initiating paradoxical sleep. Simultaneously, dopamine activates postsynaptic G_i/0-protein-coupled D3 receptors on serotonergic and noradrenergic neurons, thereby promoting depression of their excitation and suppression of their activity. An increase in the concentration of acetylcholine, in turn, leads to an increase in the level of dopamine, serotonin, and noradrenaline, due to activation of nicotinic α-receptors and depolarization of neurons that release these neuromodulators. Serotonergic and noradrenergic neurons enable reciprocal excitation due to activation of G_q/11-protein-coupled 5-HT2 and α1 adrenergic receptors. However, an increase in the activity of serotonergic and noradrenergic cells suppresses paradoxical sleep, first, due to depression of excitation of cholinergic neurons via G_i/0-protein-coupled 5-HT1 and α2 adrenergic receptors. Second, serotonin and noradrenaline suppress the activity of dopaminergic cells by affecting 5-HT2 and α1 adrenergic receptors and by potentiating excitation of GABAergic interneurons projecting onto dopaminergic cells. Owing to the specified changes in the efficiency of inputs to neuromodulatory neurons, opposite changes in concentrations of acetylcholine and dopamine, on the one hand, and serotonin and noradrenaline, on the other hand, lead to alternation of various phases of sleep and wakefulness.     

Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G-protein activation in the 3D reconstructed epileptic rat brain

Purpose: The endocannabinoid system is known to modulate seizure activity in several in vivo and in vitro models, and CB₁‐receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy (AE). In these epileptic rats, a unique redistribution of the CB₁ receptor occurs within the hippocampus; however, an anatomically inclusive analysis of the effect of status epilepticus (SE)–induced AE on CB₁ receptors has not been thoroughly evaluated. Therefore, statistical parametric mapping (SPM), a whole‐brain unbiased approach, was used to study the long‐term effect of pilocarpine‐induced SE on CB₁‐receptor binding and G‐protein activation in rats with AE. Methods: Serial coronal sections from control and epileptic rats were cut at equal intervals throughout the neuraxis and processed for [³H]WIN55,212‐2 (WIN) autoradiography, WIN‐stimulated [³⁵S]GTPγS autoradiography, and CB₁‐receptor immunohistochemistry (IHC). The autoradiographic techniques were evaluated with both region of interest (ROI) and SPM analyses. Key Findings: In rats with AE, regionally specific increases in CB₁‐receptor binding and activity were detected in cortex, discrete thalamic nuclei, and other regions including caudate‐putamen and septum, and confirmed by IHC. However, CB₁ receptors were unaltered in several brain regions, including substantia nigra and cerebellum, and did not exhibit regional decreases in rats with AE. Significance: This study provides the first comprehensive evaluation of the regional distribution of changes in CB₁‐receptor expression, binding, and G‐protein activation in the rat pilocarpine model of AE. These regions may ultimately serve as targets for cannabinomimetic compounds or manipulation of the endocannabinoid system in epileptic brain.     

Changes in endocannabinoid transmission in the basal ganglia in a rat model of Huntington's disease.

Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the basal ganglia in Huntington's disease (HD), but there are no data on endocannabinoid levels in this disease. In the present study, we have addressed this question by using rats with bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that, through the selective damage of striatal GABAergic efferent neurons, produces a useful model of HD. Twelve days after the lesion, 3-NP-lesioned rats exhibited motor disturbances, characterized by an ambulatory hyperactivity accompanied by a loss of guided activities. Analysis of GABA contents in the basal ganglia showed a trend towards a reduction compatible with motor hyperactivity. In addition, CB1 receptor binding and, to a greater extent, CB1 receptor activation of GTP-binding proteins, were also reduced in the basal ganglia. These changes were paralleled by a decrease of the contents of the two endocannabinoids, anandamide and 2-arachidonoylglycerol, in the striatum, and by an increase, particularly of anandamide, in the ventral mesencephalon where the substantia nigra is located. Both CB1 receptors and endocannabinoid levels were not altered in the cerebral cortex, an area not affected by the lesion. In summary, behavioral and biochemical changes observed in rats intrastriatally lesioned with 3-NP were similar to those occurring in the brain of HD patients. As expected, a loss of CB1 receptor function was evident in the basal ganglia of these rats and this was accompanied by different changes in endocannabinoid levels.     

Serotonin1A receptor agonists induce Fos protein expression in the locus coeruleus of the conscious rat

The azapirones, which are partial agonists of the serotonin (5-HT)_1A receptor, possess anxiolytic activity. These agents may act at the pre- or postsynaptic 5-HT_1A receptors, and involve the noradrenergic system. To determine whether these drugs activate noradrenergic neurons via 5-HT_1A receptors, we have evaluated the expression of the immediate early gene c-fos in the locus coeruleus. Tandospirone and ipsapirone each induced expression of Fos protein in the noradrenergic neurons of the locus coeruleus of conscious rats. This effect was reversed by pretreatment with (+)-WAY100135, a specific 5-HT_1A antagonist. These results clearly demonstrate that azapirones activate noradrenergic neurons via 5-HT_1A receptors.     

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones

Abstract Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB₁ receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI‐related drug development such as in obesity treatment. The aim of the study was to investigate how CB₁ ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea‐pig myenteric neurones were loaded with different fluorescent markers: Fluo‐4 to measure network activity, FM1‐43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB₁ receptors was confirmed by RT‐PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB₁ receptor antagonists (AM‐251 and SR141716), abolished by CB₁ activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA‐5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB₁ agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin‐positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB₁ receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.     

Endocannabinoids in appetite control and the treatment of obesity

Research into the endocannabinoid 'system' has grown exponentially in recent years, with the discovery of cannabinoid receptors and their endogenous ligands, such as anandamide and 2-arachidonoylglycerol (2-AG). Important advances have been made in our understanding of endocannabinoid transduction mechanisms, their metabolic pathways, and of the biological processes in which they are implicated. A decade of endocannabinoid studies has promoted new insights into neural regulation and mammalian physiology that are as revolutionary as those arising from the discovery of the endogenous opioid peptides in the 1970s. Thus, endocannabinoids have been found to act as retrograde signals: released by postsynaptic neurons, they bind to presynaptic heteroceptors to modulate the release of inhibitory and excitatory neurotransmitters through multiple G-protein-coupled receptor (GPCR)-linked effector mechanisms. The metabolic pathways of anandamide and 2-AG have now been been characterised in great detail, and we can anticipate that these pathways -- together with endocannabinoid uptake mechanisms -- will complement cannabinoid receptors as targets for the pharmacological analysis of the physiological functions of these substances. Specific insights into the potential role of endocannabinoid-CB1 receptor systems in central appetite control, peripheral metabolism and body weight regulation herald the clinical application of CB1 receptor antagonists in the management of obesity and its associated disorders.     

Biosynthesis of endocannabinoids and their modes of action in neurodegenerative diseases

Endocannabinoids are thought to function as retrograde messengers, which modulate neurotransmitter release by activating presynaptic cannabinoid receptors. Anandamide and 2-arachidonoylglycerol (2-AG) are the two best studied endogenous lipids which can act as endocannabinoids. Together with the proteins responsible for their biosynthesis, inactivation and the cannabinoid receptors, these lipids constitute the endocannabinoid system. This system is proposed to be involved in various neurodegenerative diseases such as Parkinson's and Huntington's diseases as well as Multiple Sclerosis. It has been demonstrated that the endocannabinoid system can protect neurons against glutamate excitotoxicity and acute neuronal damage in both in vitro and in vivo models. In this paper we review the data concerning the involvement of the endocannabinoid system in neurodegenerative diseases in which neuronal cell death may be elicited by excitotoxicity. We focus on the biosynthesis of endocannabinoids and on their modes of action in animal models of these neurodegenerative diseases.