Palmitoylethanolamide induces microglia changes associated with increased migration and phagocytic activity: involvement of the CB2 receptor.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABAA receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABAA receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, ß1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, ß1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/ß1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/ß1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABAA receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

The A1 adenosine receptor as a new player in microglia physiology.

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

New horizons on the role of cannabinoid CB1 receptors in palatable food intake, obesity and related dysmetabolism.

Excessive consumption of high-energy, palatable food contributes to obesity, which results in the metabolic syndrome, heart disease, type-2 diabetes and death. Current knowledge on the function of the hypothalamus as the brain 'feeding centre' recognizes this region as the main regulator of body weight in the central nervous system. Because of their intrinsically fast and adaptive activities, feeding-controlling neural circuitries are endowed with synaptic plasticity modulated by neurotransmitters and hormones that act at different hierarchical levels of integration. In the hypothalamus, among the chemical mediators involved in this integration, endocannabinoids (eCBs) are ideal candidates for the fast (that is, non-genomic), stress-related fine-tuning of neuronal functions. In this article, we overview the role of the eCB system (ECS) in the control of energy intake, and particularly in the consumption of high-energy, palatable food, and discuss how such a role is affected in the brain by changes in the levels of feeding-regulated hormones, such as the adipose tissue-derived anorexigenic mediator leptin, as well as by high-fat diets. The understanding of the molecular mechanisms underlying the neuronal control of feeding behaviours by eCBs offers many potential opportunities for novel therapeutic approaches against obesity. Highlights of the latest advances in the development of strategies that minimize central ECS overactivity in 'western diet'-driven obesity are discussed.

Protective role of palmitoylethanolamide in contact allergic dermatitis.

BACKGROUND: Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD. OBJECTIVE: We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro. METHODS: Eight- to ten-week-old female C57BL/6J wild-type and CB(1)/CB(2) double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-alpha receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors. RESULTS: 2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-alpha and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 microM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-alpha. PEA (5-10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism. CONCLUSIONS: We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

The endocannabinoid system as a link between homoeostatic and hedonic pathways involved in energy balance regulation.

The endocannabinoid system (ECS) and, in particular, cannabinoid CB(1) receptors, their endogenous agonists (the endocannabinoids anandamide and 2-arachidonoylglycerol) and enzymes for the biosynthesis and degradation of the latter mediators are emerging as key players in the control of all aspects of food intake and energy balance. The ECS is involved in stimulating both the homoeostatic (that is, the sensing of deficient energy balance and gastrointestinal load) and the hedonic (that is, the sensing of the salience and the incentive/motivational value of nutrients) aspects of food intake. The orexigenic effects of endocannabinoids are exerted in the brain by CB(1)-mediated stimulatory and inhibitory effects on hypothalamic orexigenic and anorectic neuropeptides, respectively; by facilitatory actions on dopamine release in the nucleus accumbens shell; and by regulating the activity of sensory and vagal fibres in brainstem-duodenum neural connections. In turn, the levels of anandamide and 2-arachidonoylglycerol and/or CB(1) receptors in the brain are under the control of leptin, ghrelin and glucocorticoids in the hypothalamus, under that of dopamine in the limbic forebrain and under that of cholecystokinin and ghrelin in the brainstem. These bi-directional communications between the ECS and other key players in energy balance ensure local mediators such as the endocannabinoids to act in a way coordinated in both 'space' and 'time' to enhance food intake, particularly after a few hours of food deprivation. Alterations of such communications are, however, also among the underlying causes of overactivity of the ECS in hyperphagia and obesity, a phenomenon that provided the rationale for the development of anti-obesity drugs from CB(1) receptor antagonists.

Endocannabinoids: some like it fat (and sweet too).

There is growing interest in the commercialisation of the CB(1) receptor antagonist Rimonabant in Europe for the treatment of obesity and the metabolic syndrome. Clinical trials have shown that CB(1) receptor blockers are able to reduce not only food intake but also abdominal adiposity and its metabolic sequelae. Accordingly, CB(1) receptors, and tissue concentrations of endocannabinoids sufficient to activate them, are present in all brain and peripheral organs involved in the control of energy balance, including the hypothalamus, nucleus accumbens, pancreas, adipose tissue, skeletal muscle and liver. At the central level, the endocannabinoid system seems to play a dual role in the regulation of food intake by hedonic and homeostatic energy regulation. At the peripheral level, the endocannabinoid system seems to behave as a system that reduces energy expenditure and directs energy balance towards energy storage into fat. The emerging role of the endocannabinoid system in energy balance at both central and peripheral levels will be discussed in this review.

Immunohistochemical localization of anabolic and catabolic enzymes for anandamide and other putative endovanilloids in the hippocampus and cerebellar cortex of the mouse brain.

An increasing body of evidence indicates that: 1) the endocannabinoid anandamide (AEA) and other unsaturated N-acylethanolamines (NAEs), 2) 12-(S)-lipoxygenase (12-LOX) products of arachidonic acid, and 3) unsaturated N-acyldopamines (NADAs), act as endogenous ligands of transient receptor potential vanilloid type 1 (TRPV1) channels at intracellular binding sites. AEA is synthesized and released "on demand" in neurons from its membrane precursor, N-arachidonoyl-phosphatidylethanolamine, by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), and is inactivated by intracellular hydrolysis by fatty acid amide hydrolase (FAAH), whereas catechol-O-methyl-transferase (COMT) was suggested to inactivate NADAs. However, it is not known whether these enzymes or 12-LOX co-localize to any extent with TRPV1 receptors in the brain. In this study we used immunohistochemical techniques (single peroxidase and double immunofluorescence staining), and analyzed the localization of the TRPV1 channel in mouse hippocampal and cerebellar neurons with respect to NAPE-PLD, FAAH, 12-LOX and COMT. Cycloxygenase-2 (COX-2), another putative AEA-degrading enzyme, was also studied. Co-localization between TRPV1 and either NAPE-PLD or FAAH, COX-2, 12-LOX and COMT was found in Ammon's horn (CA3) hippocampal pyramidal neurons and (with the exception of 12-LOX) in some Purkinje cells. At the cellular level, both anabolic and catabolic enzymes appeared as fine grains with immunoperoxidase labeling and were observed in the somatodendritic compartment of CA3 pyramidal cells as well as (with the exception of 12-LOX) in the cytoplasm of Purkinje neurons, in which FAAH and COX-2 immunoreactivities were, however, preferentially localized in the large extension of the dendritic arbor. Our data agree with the hypothesis that, in potential "endovanillergic" neurons, endogenous TRPV1 agonists, and AEA in particular, act as intracellular mediators by being produced from and/or degraded by the same mouse brain cells that express TRPV1 receptors.

Arvanil, anandamide and N-arachidonoyl-dopamine (NADA) inhibit emesis through cannabinoid CB1 and vanilloid TRPV1 receptors in the ferret.

Cannabinoid (CB) agonists suppress nausea and vomiting (emesis). Similarly, transient receptor potential vanilloid-1 (TRPV1) receptor agonists are anti-emetic. Arvanil, N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide, is a synthetic 'hybrid' agonist of CB1 and TRPV1 receptors. Anandamide and N-arachidonoyl-dopamine (NADA) are endogenous agonists at both these receptors. We investigated if arvanil, NADA and anandamide were anti-emetic in the ferret and their mechanism of action. All compounds reduced the episodes of emesis in response to morphine 6 glucuronide. These effects were attenuated by AM251, a CB1 antagonist that was pro-emetic per se, and TRPV1 antagonists iodoresiniferatoxin and AMG 9810, which were without pro-emetic effects. Similar sensitivity to arvanil and NADA was found for prodromal signs of emesis. We analysed the distribution of TRPV1 receptors in the ferret brainstem and, for comparison, the co-localization of CB1 and TRPV1 receptors in the mouse brainstem. TRPV1 immunoreactivity was largely restricted to the nucleus of the solitary tract of the ferret, with faint labeling in the dorsal motor nucleus of the vagus and sparse distribution in the area postrema. A similar distribution of TRPV1, and its extensive co-localization with CB1, was observed in the mouse. Our findings suggest that CB1 and TRPV1 receptors in the brainstem play a major role in the control of emesis by agonists of these two receptors. While there appears to be an endogenous 'tone' of CB1 receptors inhibiting emesis, this does not seem to be the case for TRPV1 receptors, indicating that endogenously released endocannabinoids/endovanilloids inhibit emesis preferentially via CB1 receptors.

Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain.

Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 channels have been proposed to act as metabotropic and ionotropic receptors, respectively, for two classes of endogenous polyunsaturated fatty acid amides, the acylethanolamides and the acyldopamides. Furthermore, we and others have shown that functional crosstalk occurs between these two receptors when they are expressed in the same cell. Although demonstrated in sensory neurons of the dorsal root ganglia, spinal cord and myenteric neurons, co-expression of cannabinoid type 1 and transient receptor potential vanilloid type 1 has not yet been studied in the brain. In the present study, we addressed this issue by using commercially available specific antibodies whose specificity was confirmed by data obtained with brains from cannabinoid type 1(-/-) and transient receptor potential vanilloid type 1(-/-) mice. Double cannabinoid type 1/transient receptor potential vanilloid type 1 immunofluorescence and single cannabinoid type 1 or transient receptor potential vanilloid type 1 avidin-biotin complex immunohistochemistry techniques were performed and both methods used point to the same results. Cannabinoid type 1/transient receptor potential vanilloid type 1 expression was observed in the hippocampus, basal ganglia, thalamus, hypothalamus, cerebral peduncle, pontine nuclei, periaqueductal gray matter, cerebellar cortex and dentate cerebellar nucleus. In particular, in the hippocampus, cannabinoid type 1/transient receptor potential vanilloid type 1 expression was detected on cell bodies of many pyramidal neurons throughout the CA1-CA3 subfields and in the molecular layer of dentate gyrus. In the cerebellar cortex, expression of cannabinoid type 1/transient receptor potential vanilloid type 1 receptors was found surrounding soma and axons of the vast majority of Purkinje cell bodies, whose cytoplasm was found unstained for both receptors. Cannabinoid type 1 and transient receptor potential vanilloid type 1 immunoreactivity was also detected in: a) the globus pallidus and substantia nigra, in which some intensely transient receptor potential vanilloid type 1 immunopositive cell bodies were found in dense and fine cannabinoid type 1/transient receptor potential vanilloid type 1 positive and cannabinoid type 1 positive nerve fiber meshworks, respectively; b) the cytoplasm of thalamic and hypothalamic neurons; and c) some neurons of the ventral periaqueductal gray. These data support the hypothesis of a functional relationship between the two receptor types in the CNS.

Altered reaction of facial motoneurons to axonal damage in the presymptomatic phase of a murine model of familial amyotrophic lateral sclerosis.

In transgenic mice carrying the G93A human mutation of Cu/Zn superoxide dismutase (SOD1), which provide a model of familial amyotrophic lateral sclerosis, we investigated, before the onset of symptoms, two parameters of the response of facial motoneurons to nerve transection, i.e. nitric oxide synthase induction and motoneuron loss. Axotomy elicited after 2 and 3 weeks high nitric oxide synthase expression in facial motoneurons of wild-type mice, whereas the induction was very weak or absent in transgenic mice. At 1 month post-axotomy, loss of facial motoneurons was significantly higher in mutant mice than in wild-type littermates. Thus, SOD1 mutation interferes with the oxidative cascade elicited by axonal injury in cranial motoneurons. The results also indicate that the adverse gain of function of the mutant SOD1 enhances the vulnerability of motoneurons to peripheral stressful conditions.

Co-induction of nitric oxide synthase, bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail.

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15days post-caudotomy. These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko. II. The response of dorsal root ganglion cells to tail amputation and regeneration.

The lizard tail regenerates after amputation, which severs the spinal cord and spinal nerves. Dorsal root ganglia (DRGs) do not regenerate in the regrowing tail, which is innervated by DRGs rostral to the amputation. With Nissl staining, NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we investigated NOS expression and its relationship with structural changes in DRG neurons of caudotomized lizards. First, by horseradish peroxidase retrograde tracing we here provided evidence that the sensory innervation of the regenerated tail derives only from the three pairs of DRGs rostral to the amputation plane. These ganglia were then analyzed in control animals with original intact tail, at 5, 15 and 30 days after caudotomy, and at 8 months in lizards with mature regenerates. Caudotomy elicited in DRG neurons marked hypertrophy that persisted after tail regeneration. In control ganglia, most neurons were lightly NADPH-diaphorase-positive, a few were unstained or intensely stained. Tail transection elicited marked staining up-regulation, and an increase in the proportion of intensely positive neurons. The staining intensity peaked in DRG neurons at 15 days and was still significantly increased in respect to controls several months after complete tail regeneration. NOS immunoreactivity in DRGs matched the histochemical findings. NADPH-diaphorase positivity was also enhanced in the dorsal horn superficial laminae of the corresponding spinal segments. We demonstrate that transection of the lizard spinal nerves, provoked by tail loss, elicits in the axotomized primary sensory neurons marked NOS enhancement, which accompanies axon elongation in the regrowing tail and persists after the end of this process.

Plastic changes and nitric oxide synthase induction in neurons that innervate the regenerated tail of the lizard Gekko gecko: I. Response of spinal motoneurons to tail amputation and regeneration.

The lizard tail regenerates after autotomy or amputation. After horseradish peroxidase injections in the regenerate, motoneurons were retrogradely labeled only in the three spinal segments rostral to the amputation, whose spinal nerves are severed by tail loss. The changes in these motoneurons, compared to those of lizards with original intact tails, were investigated 5, 15, and 30 days after caudotomy and at 8 months in lizards with mature regenerates. Morphometric analysis of Nissl-stained motoneurons rostral to the amputation revealed marked hypertrophy, peaking at 15 days, when chromatolysis and nuclear eccentricity were also evident; motoneuron perikarya remained significantly larger than in controls after tail regeneration. The dUTP nick-end labeling (TUNEL) stain for apoptotic neurons did not reveal labeled cells in the spinal cord 5 and 15 days after caudotomy. Nitric oxide synthase (NOS) expression was studied with nicotinamide adenine-dinucleotide phosphate (NADPH)-diaphorase histochemistry and evaluated quantitatively with densitometry. A few caudal spinal motoneurons were lightly stained in lizards with intact tails. Induction of NADPH-diaphorase positivity was evident in the vast majority of these cells 5 days after caudotomy and was very marked at 15 and 30 days, during tail regrowth. These data were confirmed by neuronal NOS immunohistochemistry. After tail regeneration, histochemical positivity was markedly down-regulated in the tail spinal motoneurons but persisted in the majority of these cells. The findings show that in the lizard caudotomy elicits in axotomized caudal spinal motoneurons NOS induction associated with plasticity phenomena and in particular with vigorous regeneration of axons that innervate the regrowing tail.