Memory impairment and hippocampus specific protein oxidation induced by ethanol intake and 3, 4-methylenedioxymethamphetamine (MDMA) in mice.

Ethanol and 3, 4-Methylenedioxymethamphetamine (MDMA) are popular recreational drugs widely abused by adolescents that may induce neurotoxic processes associated with behavioural alterations. Adolescent CD1 mice were subjected to ethanol intake using the drinking in the dark (DID) procedure, acute MDMA or a combination. Considering that both drugs of abuse cause oxidative stress in the brain, protein oxidative damage in different brain areas was analysed 72 h after treatment using a proteomic approach. Damage to specific proteins in treated animals was significant in the hippocampus but not in the prefrontal cortex. The damaged hippocampus proteins were then identified by mass spectrometry, revealing their involvement in energy metabolism, structural function, axonal outgrowth and stability, and neurotransmitter release. Mice treated with MDMA displayed higher oxidative damage than ethanol-treated mice. To determine whether this oxidative damage was affecting hippocampus activity, declarative memory was evaluated at 72 h after treatment using the object recognition assay and the radial arm maze. Although acquisition in the radial arm maze was not impaired by ethanol intake, MDMA treatment impaired long-term memory in both tests. Therefore, oxidative damage to specific proteins observed under MDMA treatment affects important cellular function on the hippocampus that may contribute to declarative memory deficits.

Influence of chronic caffeine on MDMA-induced behavioral and neuroinflammatory response in mice.

RATIONALE: Previous research suggests that chronic daily caffeine administration protects against brain injury in different animal models of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, ischemic and traumatic brain injury, and allergic encephalitis. However, little is known about the effects of chronic caffeine administration on 3,4-methylenedioxymethamphetamine (MDMA)-induced neuroinflammation. OBJECTIVE: The present study examines whether chronic caffeine (10, 20, or 30 mg/kg, i.p, for 21 consecutive days) protects against MDMA-induced astrocytic and microglial activation in mice striatum, impairing its neuroinflammatory effects. Additionally, locomotor activity, sensoriomotor reflexes, body temperature, and anxiety were evaluated after caffeine injection on days 0 (basal), 7, 14, and 21 of the chronic treatment in order to assess possible behavioral alterations due to caffeine administration. METHODS: On day 22, mice pretreated with caffeine or saline received a neurotoxic regimen of MDMA (3 × 20 mg/kg, i.p., 2-h interval) or saline, and changes in body temperature were evaluated. Forty-eight hours after last MDMA or saline injection (day 24), the aforementioned behavioral parameters were investigated and microglia and astroglia activation to MDMA treatment was examined in the mouse striatum. RESULTS: Caffeine (10 mg/kg) chronically administered completely prevented MDMA-induced glial activation without inducing physiological or behavioral alterations in any of the assays performed. CONCLUSION: Chronic caffeine consumption at low doses exerts anti-inflammatory effects and prevents MDMA-induced neuroinflammation.

The orphan receptor GPR3 modulates the early phases of cocaine reinforcement.

BACKGROUND AND PURPOSE: The modulatory activity of the orphan receptor GPR3 in the brain has been related to the control of emotional behaviours. Limbic structures that express GPR3 have been associated with the effects of drug abuse. EXPERIMENTAL APPROACH: The role of GPR3 in different cocaine-elicited behaviours including locomotor activity, behavioural sensitization, conditioned place preference (CPP) and intravenous self-administration was evaluated in Gpr3-/- mice and their Gpr3+/+ littermates. Cocaine-induced dopamine release in the nucleus accumbens was also evaluated to elucidate the effect of Gpr3 deletion on extracellular levels of dopamine. KEY RESULTS: Gpr3-/- mice exhibited higher rewarding responses in the CPP paradigm. Gpr3-/- mice self-administered more cocaine, especially during the first days of training. No differences were found between genotypes regarding behavioural sensitization and the maximal effort required to obtain a cocaine infusion. Non-contingent priming injections of cocaine before operant training eliminated enhanced cocaine self-administration in Gpr3-/- mice. Extracellular levels of dopamine in the nucleus accumbens induced by cocaine did not differ between genotypes. CONCLUSIONS AND IMPLICATIONS: The increased responsiveness of Gpr3-/- mice to the acute locomotor effects of cocaine and the inconsistency to further increase this effect reflected an 'already maximally sensitized' basal state. Enhanced responsiveness of Gpr3-/- mice to cocaine reward and to early phases of reinforcement suggests that an initial alteration increased vulnerability to this type of drug abuse. Overall, altered signalling pathways of GPR3 could contribute to the neurobiological substrate involved in developing addiction to cocaine.

Transgenic over expression of nicotinic receptor alpha 5, alpha 3, and beta 4 subunit genes reduces ethanol intake in mice.

Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol's as well as nicotine's effects.

Alteration of neuropathic and visceral pain in female C57BL/6J mice lacking the PPAR-α gene.

RATIONALE: Peroxisome proliferator-activated receptors (PPARs) participate in the control of chronic neuropathic and inflammatory pain, and these receptors could play a role on acute pain. OBJECTIVES: We used null (PPAR-α -/-) and wild-type female mice and the PPAR-α blocker GW6471 to evaluate (1) the role of PPAR-α on neuropathic pain, (2) the involvement of PPAR-α on visceral and acute thermal nociception, and (3) tissue levels of pro-inflammatory factors. METHODS: Neuropathic pain was induced by sciatic nerve ligature. Acute thermal nociception was evaluated through hot-plate, tail-immersion, and writhing tests. The pro-inflammatory factors nitric oxide, TNF-α, and interleukins-1ß and -3 were measured. RESULTS: Regarding neuropathic pain, higher sensitivity to thermal and mechanical non-noxious and noxious stimuli was observed in mice lacking PPAR-α. Cold and mechanical allodynia and heat hyperalgesia were augmented in null mice. With respect to visceral nociception, writhes after acetic acid were enhanced in mutant mice. Although basal thermal sensitivity was enhanced in PPAR-α -/- mice, cutaneous thermal nociception did not differ between genotypes. Blockade of PPAR-α was devoid of effects on acute thermal and writhing tests. Finally, nerve ligature enhanced pro-inflammatory factors in plantar tissue, levels being higher in null mice. No changes in pro-inflammatory factors were observed in the hot-plate test. CONCLUSIONS: Genetic ablation of PPAR-α is involved in neuropathic and visceral nociception. Lack of PPAR-α is not involved in acute thermal pain, but it is involved in basal thermal reaction. Changes are biological adaptations to receptor deletion because blockade of PPAR-α does not affect inflammatory pain or thermal reactions.

Behavioural and neuroinflammatory effects of the combination of binge ethanol and MDMA in mice.

RATIONALE: Binge drinking is a common pattern of alcohol consumption among young people. Binge drinkers are especially susceptible to brain damage when other substances are co-administered, in particular, 3,4-methylendioxymethamphetamine (MDMA). OBJECTIVE: To evaluate the behavioural consequences of voluntary binge ethanol consumption, alone and in combination to MDMA. Also, to elucidate the effects of the combined consumption of these two drugs on neuroinflammation. MATERIALS AND METHODS: Adolescent mice received MDMA (MDMA-treated mice), ethanol (ethanol-treated mice group) or both (ethanol plus MDMA-treated mice). Drinking in the dark (DID) procedure was used as a model of binge. Body temperature, locomotor activity, motor coordination, anxiety-like and despair behaviour in adolescent mice were evaluated 48 h, 72 h, and 7 days after the treatments. Also, neuroinflammatory response to these treatments was measured in the striatum. RESULTS: The hyperthermia observed in MDMA-treated mice was abolished by pre-exposition to ethanol. Ethanol plus MDMA-treated mice showed lower locomotor activity. Ethanol-treated mice showed motor coordination impairment and increased despair behaviour. Anxiety-like behaviour was only seen in animals that were treated with both drugs. Contrarily, neuroinflammation was mostly seen in animals treated only with MDMA. CONCLUSIONS: Ethanol and MDMA co-administration increases the neurobehavioural changes induced by the consumption of each one of these drugs. However, as ethanol consumption did not increase neuroinflammatory responses induced by MDMA, other mechanisms, mediated by ethanol, are likely to account for this effect and need to be evaluated.

GPR3 orphan receptor is involved in neuropathic pain after peripheral nerve injury and regulates morphine-induced antinociception.

GPR3 is an orphan G-protein coupled receptor broadly expressed in brain structures controlling emotional-like behaviors and pain. GPR3 receptor up-regulates cAMP and promotes neurite outgrowth in mammalian neurons, being a good candidate to participate in the pathophysiology of neurodegenerative diseases as well as brain and spinal cord injuries. In this study, we evaluated the role of GPR3 receptor in the development and expression of neuropathic pain after sciatic nerve ligature, and the inflammatory reaction in the dorsal horn of the spinal cord in both Gpr3-/- and Gpr3+/+ mice. Hyperalgesia to noxious thermal stimulus and allodynia to cold and mechanical stimuli were evaluated using the plantar test, the cold-plate test and the Von Frey filament model, respectively. Additionally, we evaluated the involvement of GPR3 receptors in morphine-induced antinociception using the tail immersion test. After nerve injury, Gpr3-/- mice showed a higher sensitivity to thermal non-noxious and noxious stimuli than Gpr3+/+ mice, whereas no differences were observed between genotypes in mechanical allodynia. In addition, no differences in microglia and astrocytes activation were found when compared the ipsilateral dorsal horn of Gpr3-/- and Gpr3+/+ mice exposed to nerve ligature. On the other hand, the genetic deletion of GPR3 receptors reduced morphine antinociception in the tail immersion test in mice without any changes in basal thermal threshold. Taken together, our results demonstrate, for the first time, the involvement of the orphan GPR3 receptor in the expression and development of neuropathic pain and in the analgesia induced by morphine. The lack of GPR3 receptors produced hypersensitivity to thermal non-noxious and noxious stimuli without affecting the spinal inflammatory response associated to sciatic nerve injury and reduced morphine antinociception in the tail immersion test. Our findings propose GPR3 receptors as a new molecular target in neuropathic pain therapy as well as a new component of a pro-opioid receptor system.

The A2a adenosine receptor modulates the reinforcement efficacy and neurotoxicity of MDMA.

Adenosine is an endogenous purine nucleoside that plays a neuromodulatory role in the central nervous system. A2a adenosine receptors have been involved in reward-related processes, inflammatory phenomena and neurotoxicity reactions. In the present study, we investigated the role of A2a adenosine receptors on the acute pharmacological effects, reinforcement and neuroinflammation induced by MDMA administration. First, the acute effects of MDMA on body temperature, locomotor activity and anxiety-like responses were measured in A2a knockout mice and wild-type littermates. Second, MDMA reinforcing properties were evaluated using the intravenous self-administration paradigm. Finally, we assessed striatal astrogliosis and microgliosis as markers of MDMA neurotoxicity. Our results showed that acute MDMA produced a biphasic effect on body temperature and increased locomotor activity and anxiogenic-like responses in both genotypes. However, MDMA reinforcing properties were dramatically affected by the lack of A2a adenosine receptors. Thus, wild-type mice maintained MDMA self-administration under a fixed ratio 1 reinforcement schedule, whereas the operant response appeared completely abolished in A2a knockout mice. In addition, the MDMA neurotoxic regime produced an enhanced inflammatory response in striatum of wild-type mice, revealed by a significant increase in glial expression, whereas such activation was attenuated in mutant mice. This is the first report indicating that A2a adenosine receptors play a key role in reinforcement and neuroinflammation induced by the widely used psychostimulant.

CRF2 mediates the increased noradrenergic activity in the hypothalamic paraventricular nucleus and the negative state of morphine withdrawal in rats.

BACKGROUND AND PURPOSE: Recent evidence suggests that corticotropin-releasing factor (CRF) receptor signalling is involved in modulating the negative symptoms of opiate withdrawal. In this study, a series of experiments were performed to further characterize the role of CRF-type 2 receptor (CRF2) signalling in opiate withdrawal-induced physical signs of dependence, hypothalamus-pituitary-adrenal (HPA) axis activation, enhanced noradrenaline (NA) turnover in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) phosphorylation (activation), as well as CRF2 expression in the nucleus of the solitary tract-A2 noradrenergic cell group (NTS-A2). EXPERIMENTAL APPROACH: The contribution of CRF2 signalling in opiate withdrawal was assessed by i.c.v. infusion of the selective CRF2 antagonist, antisauvagine-30 (AS-30). Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with AS-30 or saline 10 min before naloxone and the physical signs of abstinence, the HPA axis activity, NA turnover, TH activation and CRF2 expression were measured using immunoblotting, RIA, HPLC and immunohistochemistry. KEY RESULTS: Rats pretreated with AS-30 showed decreased levels of somatic signs of naloxone-induced opiate withdrawal, but the corticosterone response was not modified. AS-30 attenuated the increased production of the NA metabolite, 3-methoxy-4-hydroxyphenylglycol, as well as the enhanced NA turnover observed in morphine-withdrawn rats. Finally, AS-30 antagonized the TH phosphorylation at Serine40 induced by morphine withdrawal. CONCLUSIONS AND IMPLICATIONS: These results suggest that physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by CRF2 signalling. Furthermore, they indicate a marginal role for the HPA axis in CRF2-mediation of opiate withdrawal.

Intracranial self-stimulation improves memory consolidation in rats with little training.

Post-training intracranial electrical self-stimulation can improve learning and memory consolidation in rats. However, the molecular mechanisms involved are not known yet. Since previous paradigms of this kind of facilitation are relatively unsuitable to try a molecular approach, here we develop a single and short model of learning and memory facilitation by post-training self-stimulation that could make easier the research of its neural and molecular basis. Thus, three consecutive experiments were carried out to ascertain whether post-training self-stimulation is able to facilitate memory when learning consists of only a brief (5 trials) two-way active avoidance conditioning session. The results of Experiment 1 showed that it is actually possible, and that 48 h after the acquisition session is a very good time to observe the memory improvement. As a way to probe the retroactive effect of self-stimulation, in Experiment 2 we observed that the same self-stimulation treatment given to the subjects not post-training but 48 h before a single two-way active avoidance session does not improve the acquisition of conditioning. In Experiment 3, we showed that the SS facilitative effect observed 48 h after the acquisition session in Experiment 1 was still maintained one week later. We concluded that post-training intracranial self-stimulation can consistently improve memory consolidation even when little acquisition training is given to the animals in a single training session.