Laura Orio

3,4‐Methylenedioxymethamphetamine increases interleukin‐1β levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5‐HT depletion

3,4‐Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5‐HT release. Interleukin‐1β (IL‐1β) is a pro‐inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL‐1β concentration and glial activation and their relationship with acute hyperthermia and 5‐HT depletion. MDMA, given to rats housed at 22°C, increased IL‐1β levels in hypothalamus and cortex from 1 to 6 h and [3H]‐(1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)3‐isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX‐42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL‐1 receptor antagonist did not modify MDMA‐induced hyperthermia indicating that IL‐1β release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4°C, hyperthermia was abolished and the IL‐1β increase significantly reduced. The MDMA‐induced acute 5‐HT depletion was prevented by fluoxetine coadministration but the IL‐1β increase and hyperthermia were unaffected. Therefore, the rise in IL‐1β is not related to the acute 5‐HT release but is linked to the hyperthermia. Contrary to IL‐1β levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.

Elevation of Ambient Room Temperature has Differential Effects on MDMA-Induced 5-HT and Dopamine Release in Striatum and Nucleus Accumbens of Rats

3,4-Methylenedioxymethamphetamine (MDMA) produces acute dopamine and 5-HT release in rat brain and a hyperthermic response, which is dependent on the ambient room temperature in which the animal is housed. We examined the effect of ambient room temperature (20 and 30°C) on MDMA-induced dopamine and 5-HT efflux in the striatum and shell of nucleus accumbens (NAc) of freely moving rats by using microdialysis. Locomotor activity and rectal temperature were also evaluated. In the NAc, MDMA (2.5 or 5 mg/kg, i.p.) produced a substantial increase in extracellular dopamine, which was more marked at 30°C. 5-HT release was also increased by MDMA given at 30°C. In contrast, MDMA-induced extracellular dopamine and 5-HT increases in the striatum were unaffected by ambient temperature. At 20°C room temperature, MDMA did not modify the rectal temperature but at 30°C it produced a rapid and sustained hyperthermia. MDMA at 20°C room temperature produced a two-fold increase in activity compared with saline-treated controls. The MDMA-induced increase in locomotor activity was more marked at 30°C due to a decrease in the activity of the saline-treated controls at this high ambient temperature. These results show that high ambient temperature enhances MDMA-induced locomotor activity and monoamine release in the shell of NAc, a region involved in the incentive motivational properties of drugs of abuse, and suggest that the rewarding effects of MDMA may be more pronounced at high ambient temperature.

A comparative study on the acute and long‐term effects of MDMA and 3,4‐dihydroxymethamphetamine (HHMA) on brain monoamine levels after i.p. or striatal administration in mice

1 This study investigated whether the immediate and long‐term effects of 3,4‐methylenedioxymethamphetamine (MDMA) on monoamines in mouse brain are due to the parent compound and the possible contribution of a major reactive metabolite, 3,4‐dihydroxymethamphetamine (HHMA), to these changes. The acute effect of each compound on rectal temperature was also determined. 2 MDMA given i.p. (30 mg kg−1, three times at 3‐h intervals), but not into the striatum (1, 10 and 100 μg, three times at 3‐h intervals), produced a reduction in striatal dopamine content and modest 5‐HT reduction 1 h after the last dose. MDMA does not therefore appear to be responsible for the acute monoamine release that follows its peripheral injection. 3 HHMA does not contribute to the acute MDMA‐induced dopamine depletion as the acute central effects of MDMA and HHMA differed following i.p. injection. Both compounds induced hyperthermia, confirming that the acute dopamine depletion is not responsible for the temperature changes. 4 Peripheral administration of MDMA produced dopamine depletion 7 days later. Intrastriatal MDMA administration only produced a long‐term loss of dopamine at much higher concentrations than those reached after the i.p. dose and therefore bears little relevance to the neurotoxicity. This indicates that the long‐term effect is not attributable to the parent compound. HHMA also appeared not to be responsible as i.p. administration failed to alter the striatal dopamine concentration 7 days later. 5 HHMA was detected in plasma, but not in brain, following MDMA (i.p.), but it can cross the blood–brain barrier as it was detected in the brain following its peripheral injection. 6 The fact that the acute changes induced by i.p. or intrastriatal HHMA administration differed indicates that HHMA is metabolised to other compounds which are responsible for changes observed after i.p. administration.

MDMA-induced neurotoxicity: long-term effects on 5-HT biosynthesis and the influence of ambient temperature

1 3,4‐Methylenedioxymethamphetamine (MDMA or ‘ecstasy’) decreases the 5‐HT concentration, [3H]‐paroxetine binding and tryptophan hydroxylase activity in rat forebrain, which has been interpreted as indicating 5‐HT neurodegeneration. This has been questioned, particularly the 5‐HT loss, as MDMA can also inhibit tryptophan hydroxylase. We have now evaluated the validity of these parameters as a reflection of neurotoxicity. 2 Male DA rats were administered MDMA (12.5 mg kg−1, i.p.) and killed up to 32 weeks later. 5‐HT content and [3H]‐paroxetine binding were measured in the cortex, hippocampus and striatum. Parallel groups of treated animals were administered NSD‐1015 for determination of in vivo tryptophan hydroxylase activity and 5‐HT turnover rate constant. 3 Tissue 5‐HT content and [3H]‐paroxetine binding were reduced in the cortex (26–53%) and hippocampus (25–74%) at all time points (1, 2, 4, 8 and 32 weeks). Hydroxylase activity was similarly reduced up to 8 weeks, but had recovered at 32 weeks. The striatal 5‐HT concentration and [3H]‐paroxetine binding recovered by week 4 and hydroxylase activity after week 1. In all regions, the reduction in 5‐HT concentration did not result in an altered 5‐HT synthesis rate constant. 4 Administering MDMA to animals when housed at 4°C prevented the reduction in [3H]‐paroxetine binding and hydroxylase activity observed in rats housed at 22°C, but not the reduction in 5‐HT concentration. 5 These data indicate that MDMA produces long‐term damage to serotoninergic neurones, but this does not produce a compensatory increase in 5‐HT synthesis in remaining terminals. It also highlights the fact that measurement of tissue 5‐HT concentration may overestimate neurotoxic damage.

3,4-Methylenedioxymethamphetamine increases pro-interleukin-1β production and caspase-1 protease activity in frontal cortex, but not in hypothalamus, of Dark Agouti rats: Role of interleukin-1β in neurotoxicity

3,4-Methylenedioxymethamphetamine (ecstasy) increases mature interleukin-1beta production in rat brain shortly after injection. This effect is a consequence of the 3,4-methylenedioxymethamphetamine-induced hyperthermia and is reduced when rats are maintained at low ambient room temperature. Since interleukin-1beta is generated as an inactive 31-kDa precursor protein and processed into mature form by caspase-1, we have now examined the effect of 3,4-methylenedioxymethamphetamine on pro-interleukin-1beta production and caspase-1-like protease activity in the hypothalamus and frontal cortex of Dark Agouti rats. 3,4-Methylenedioxymethamphetamine increased the immunoreactivity of pro-interleukin-1beta in frontal cortex, not in hypothalamus, 3 h and 6 h after administration. Caspase-1-like protease activity was increased in frontal cortex 3 h after 3,4-methylenedioxymethamphetamine injection compared with saline-treated animals. 3,4-Methylenedioxymethamphetamine did not modify the expression of pro-caspase-1 but increased the immunoreactivity for the caspase-1 active cleavage product (p20) in frontal cortex 3 h after dosing. No change on caspase-1-like protease activity was observed in hypothalamus. The basal immunoreactivity of pro-interleukin-1beta and caspase-1-like protease activity was higher in the hypothalamus than in frontal cortex of control (saline-treated) animals. These data indicate that 3,4-methylenedioxymethamphetamine alters, in a region-specific manner, the mechanisms which regulate interleukin-1beta production in the brain of Dark Agouti rats and suggest that the release of interleukin-1beta in hypothalamus may be regulated independently of caspase-1 activation. Administration (i.c.v.) of interleukin-1beta enhanced the 3,4-methylenedioxymethamphetamine-induced long-term loss of brain 5-HT parameters and immediate hyperthermia. Neither of these effects was observed when interleukin-1beta was given into hippocampus. These results indicate that exogenous interleukin-1beta potentiates 3,4-methylenedioxymethamphetamine neurotoxicity as a consequence of its effect on body temperature and suggest that the 3,4-methylenedioxymethamphetamine-induced rise in interleukin-1beta levels could in turn contribute to the maintenance of 3,4-methylenedioxymethamphetamine-induced hyperthermia and subsequent neurotoxicity.

Methamphetamine differentially affects BDNF and cell death factors in anatomically defined regions of the hippocampus

Methamphetamine exposure reduces hippocampal long-term potentiation (LTP) and neurogenesis and these alterations partially contribute to hippocampal maladaptive plasticity. The potential mechanisms underlying methamphetamine-induced maladaptive plasticity were identified in the present study. Expression of brain-derived neurotrophic factor (BDNF; a regulator of LTP and neurogenesis), and its receptor tropomyosin-related kinase B (TrkB) were studied in the dorsal and ventral hippocampal tissue lysates in rats that intravenously self-administered methamphetamine in a limited access (1h/day) or extended access (6h/day) paradigm for 17days post baseline sessions. Extended access methamphetamine enhanced expression of BDNF with significant effects observed in the dorsal and ventral hippocampus. Methamphetamine-induced enhancements in BDNF expression were not associated with TrkB receptor activation as indicated by phospho (p)-TrkB-706 levels. Conversely, methamphetamine produced hypophosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit 2B (GluN2B) at Tyr-1472 in the ventral hippocampus, indicating reduced receptor activation. In addition, methamphetamine enhanced expression of anti-apoptotic protein Bcl-2 and reduced pro-apoptotic protein Bax levels in the ventral hippocampus, suggesting a mechanism for reducing cell death. Analysis of Akt, a pro-survival kinase that suppresses apoptotic pathways and pAkt at Ser-473 demonstrated that extended access methamphetamine reduces Akt expression in the ventral hippocampus. These data reveal that alterations in Bcl-2 and Bax levels by methamphetamine were not associated with enhanced Akt expression. Given that hippocampal function and neurogenesis vary in a subregion-specific fashion, where dorsal hippocampus regulates spatial processing and has higher levels of neurogenesis, whereas ventral hippocampus regulates anxiety-related behaviors, these data suggest that methamphetamine self-administration initiates distinct allostatic changes in hippocampal subregions that may contribute to the altered synaptic activity in the hippocampus, which may underlie enhanced negative affective symptoms and perpetuation of the addiction cycle.

Systemic Administration of Oleoylethanolamide Protects from Neuroinflammation and Anhedonia Induced by LPS in Rats

Background: The acylethanolamides oleoylethanolamide and palmitoylethanolamide are endogenous lipid mediators with proposed neuroprotectant properties in central nervous system (CNS) pathologies. The precise mechanisms remain partly unknown, but growing evidence suggests an antiinflammatory/antioxidant profile. Methods: We tested whether oleoylethanolamide/palmitoylethanolamide (10mg/kg, i.p.) attenuate neuroinflammation and acute phase responses (hypothalamus-pituitary-adrenal (HPA) stress axis stress axis activation, thermoregulation, and anhedonia) induced by lipopolysaccharide (0.5mg/kg, i.p.) in rats. Results: Lipopolysaccharide increased mRNA levels of the proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6, nuclear transcription factor-κB activity, and the expression of its inhibitory protein IκBα in cytoplasm, the inducible isoforms of nitric oxide synthase and cyclooxygenase-2, microsomal prostaglandin E2 synthase mRNA, and proinflammatory prostaglandin E2 content in frontal cortex 150 minutes after administration. As a result, the markers of nitrosative/oxidative stress nitrites (NO2 -) and malondialdehyde were increased. Pretreatment with oleoylethanolamide/ palmitoylethanolamide reduced plasma tumor necrosis factor-α levels after lipopolysaccharide, but only oleoylethanolamide significantly reduced brain tumor necrosis factor-α mRNA. Oleoylethanolamide and palmitoylethanolamide prevented lipopolysaccharide-induced nuclear transcription factor-κB (NF-κB)/IκBα upregulation in nuclear and cytosolic extracts, respectively, the expression of inducible isoforms of nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E2 synthase and the levels of prostaglandin E2. Additionally, both acylethanolamides reduced lipopolysaccharide-induced oxidative/nitrosative stress. Neither oleoylethanolamide nor palmitoylethanolamide modified plasma corticosterone levels after lipopolysaccharide, but both acylethanolamides reduced the expression of hypothalamic markers of thermoregulation interleukin-1β, cyclooxygenase-2, and prostaglandin E2, and potentiated the hypothermic response after lipopolysaccharide. Interestingly, only oleoylethanolamide disrupted lipopolysaccharide-induced anhedonia in a saccharine preference test. Conclusions: Results indicate that oleoylethanolamide and palmitoylethanolamide have antiinflammatory/neuroprotective properties and suggest a role for these acylethanolamides as modulators of CNS pathologies with a neuroinflammatory component.

Ghrelin-Induced Orexigenic Effect in Rats Depends on the Metabolic Status and Is Counteracted by Peripheral CB1 Receptor Antagonism

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions.

Role of the satiety factor oleoylethanolamide in alcoholism

Oleoylethanolamide (OEA) is a satiety factor that controls motivational responses to dietary fat. Here we show that alcohol administration causes the release of OEA in rodents, which in turn reduces alcohol consumption by engaging peroxisome proliferator‐activated receptor‐alpha (PPAR‐α). This effect appears to rely on peripheral signaling mechanisms as alcohol self‐administration is unaltered by intracerebral PPAR‐α agonist administration, and the lesion of sensory afferent fibers (by capsaicin) abrogates the effect of systemically administered OEA on alcohol intake. Additionally, OEA is shown to block cue‐induced reinstatement of alcohol‐seeking behavior (an animal model of relapse) and reduce the severity of somatic withdrawal symptoms in alcohol‐dependent animals. Collectively, these findings demonstrate a homeostatic role for OEA signaling in the behavioral effects of alcohol exposure and highlight OEA as a novel therapeutic target for alcohol use disorders and alcoholism.

Exposure to a Highly Caloric Palatable Diet During Pregestational and Gestational Periods Affects Hypothalamic and Hippocampal Endocannabinoid Levels at Birth and Induces Adiposity and Anxiety-Like Behaviors in Male Rat Offspring

Exposure to unbalanced diets during pre-gestational and gestational periods may result in long-term alterations in metabolism and behavior. The contribution of the endocannabinoid system to these long-term adaptive responses is unknown. In the present study, we investigated the impact of female rat exposure to a hypercaloric-hypoproteic palatable diet during pre-gestational, gestational and lactational periods on the development of male offspring. In addition, the hypothalamic and hippocampal endocannabinoid contents at birth and the behavioral performance in adulthood were investigated. Exposure to a palatable diet resulted in low weight offspring who exhibited low hypothalamic contents of arachidonic acid and the two major endocannabinoids (anandamide and 2-arachidonoylglycerol) at birth. Palmitoylethanolamide, but not oleoylethanolamide, also decreased. Additionally, pups from palatable diet-fed dams displayed lower levels of anandamide and palmitoylethanolamide in the hippocampus. The low-weight male offspring, born from palatable diet exposed mothers, gained less weight during lactation and although they recovered weight during the post-weaning period, they developed abdominal adiposity in adulthood. These animals exhibited anxiety-like behavior in the elevated plus-maze and open field test and a low preference for a chocolate diet in a food preference test, indicating that maternal exposure to a hypercaloric diet induces long-term behavioral alterations in male offspring. These results suggest that maternal diet alterations in the function of the endogenous cannabinoid system can mediate the observed phenotype of the offspring, since both hypothalamic and hippocampal endocannabinoids regulate feeding, metabolic adaptions to caloric diets, learning, memory, and emotions.