Multimodal antidepressant vortioxetine causes analgesia in a mouse model of chronic neuropathic pain.

Vortioxetine is a multimodal antidepressant that potently antagonizes 5-HT3 serotonin receptors, inhibits the high-affinity serotonin transporter, activates 5-HT1A and 5-HT1B receptors, and antagonizes 5-HT1D and 5-HT7 receptors. 5-HT3 receptors largely mediate the hyperalgesic activity of serotonin that occurs in response to nerve injury. Activation of 5-HT3 receptors contributes to explain why selective serotonin reuptake inhibitors, such as fluoxetine, are not indicated in the treatment of neuropathic pain. Here, we studied the analgesic action of vortioxetine in the chronic constriction injury model of neuropathic pain in mice. Vortioxetine was injected once a day for 27 days at doses (10 mg/kg, intraperitoneally) that determine >90% 5-HT3 receptor occupancy in the central nervous system. The action of vortioxetine was compared to the action of equal doses of the serotonin-noradrenaline reuptake inhibitor, venlafaxine (one of the gold standard drugs in the treatment of neuropathic pain), and fluoxetine. Vortioxetine caused a robust analgesia in chronic constriction injury mice, and its effect was identical to that produced by venlafaxine. In contrast, fluoxetine was inactive in chronic constriction injury mice. Vortioxetine enhanced mechanical pain thresholds in chronic constriction injury mice without changing motor activity, as assessed by the open-field and horizontal bar tests. None of the three antidepressants caused analgesia in the complete Freund's adjuvant model of chronic inflammatory pain. These findings raise the attractive possibility that vortioxetine can be effective in the treatment of neuropathic pain, particularly in patients with comorbid depression and cognitive dysfunction.

Changes in mGlu5 Receptor Signaling Are Associated with Associative Learning and Memory Extinction in Mice.

Using an in vivo method for the assessment of polyphosphoinositide (PI) hydrolysis, we examine whether spatial learning and memory extinction cause changes in mGlu5 metabotropic glutamate receptor signaling in the hippocampus and prefrontal cortex. We use the following five groups of mice: (i) naive mice; (ii) control mice exposed to the same environment as learner mice; (iii) leaner mice, trained for four days in a water maze; (iv) mice in which memory extinction was induced by six trials without the platform; (v) mice that spontaneously lost memory. The mGlu5 receptor-mediated PI hydrolysis was significantly reduced in the dorsal hippocampus of learner mice as compared to naive and control mice. The mGlu5 receptor signaling was also reduced in the ventral hippocampus and prefrontal cortex of learner mice, but only with respect to naive mice. Memory extinction was associated with a large up-regulation of mGlu5 receptor-mediated PI hydrolysis in the three brain regions and with increases in mGlu5 receptor and phospholipase-Cß protein levels in the ventral and dorsal hippocampus, respectively. These findings support a role for mGlu5 receptors in mechanisms underlying spatial learning and suggest that mGlu5 receptors are candidate drug targets for disorders in which cognitive functions are impaired or aversive memories are inappropriately retained.

Selective reduction in the expression of type-1 metabotropic glutamate receptors in the hippocampus of adult rats born by caesarean section.

Perinatal hypoxia causes long-term neurobiological consequences, including alterations in mechanisms of activity-dependent synaptic plasticity and cognitive dysfunction. Changes in neurotransmitter receptors have been associated with these alterations, but little is known on how early hypoxia influences the expression and function of metabotropic glutamate (mGlu) receptors in adult life. This is an important issue because mGlu receptors are implicated in mechanisms of synaptic plasticity. Here, we examined the expression of mGlu1, mGlu5, and mGlu2/3 receptor subtypes in the hippocampus, nucleus accumbens, prefrontal cortex, and dorsal striatum in 6-month old Wistar rats (a) born by vaginal delivery; (b) born by caesarean section; and (c) born by caesarean section followed by 20 min of asphyxia. Unexpectedly, we found a large reduction of mGlu1α protein levels in the hippocampus of rats born by caesarean section regardless of the presence of asphyxia. No changes in mGlu1α receptor protein levels were found in the other brain regions. Levels of mGlu5 and mGlu2/3 receptors and levels of GluA2/3 and GluN1 subunits of AMPA and NMDA receptors did not differ among the three groups of rats in any brain region. These results are consistent with previous findings showing that changes in mGlu1 receptors occur within the epigenetic programming caused by early-life events.

Chemokines in Alzheimer's Disease: New Insights Into Prokineticins, Chemokine-Like Proteins.

Alzheimer's disease is the most common neurodegenerative disorder characterized by the presence of ß-amyloid aggregates deposited as senile plaques and by the presence of neurofibrillary tangles of tau protein. To date, there is a broad consensus on the idea that neuroinflammation is one of the most important component in Alzheimer's disease pathogenesis. Chemokines and their receptors, beside the well-known role in the immune system, are widely expressed in the nervous system, where they play a significant role in the neuroinflammatory processes. Prokineticins are a new family of chemokine-like molecules involved in numerous physiological and pathological processes including immunity, pain, inflammation, and neuroinflammation. Prokineticin 2 (PROK2) and its receptors PKR1 and PKR2 are widely expressed in the central nervous system in both neuronal and glial cells. In Alzheimer's disease, PROK2 sustains the neuroinflammatory condition and contributes to neurotoxicity, since its expression is strongly upregulated by amyloid-ß peptide and reversed by the PKR antagonist PC1. This review aims to summarize the current knowledge on the neurotoxic and/or neuroprotective function of chemokines in Alzheimer's disease, focusing on the prokineticin system: it represents a new field of investigation that can stimulate the research of innovative pharmacotherapeutic strategies.

Maternal exposure to low levels of corticosterone during lactation protects the adult offspring against ischemic brain damage.

A growing body of evidence underscores the importance of early life events as predictors of health in adulthood. Abnormalities in maternal care or other forms of early postnatal stress induce long-term changes in behavior and influence the vulnerability to illnesses throughout life. Some of these changes may be produced by the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is invariably associated with stress. We used a model in which neonate rats are fed by mothers drinking water supplemented with 0.2 mg/ml corticosterone, the main glucocorticoid hormone in rodents. Plasma corticosterone levels increased in the dams to an extent similar to that induced by a mild stress. Corticosterone-treated dams also showed an increase in maternal care. Remarkably, adult rats that had been nursed by corticosterone-treated mothers were protected against neuronal damage and cognitive impairment produced by transient global brain ischemia. Neuroprotection was associated with a reduced HPA response to ischemia and was primarily decreased when corticosterone was injected at a dose that eliminated any difference in endogenous corticosterone levels between rats raised by mothers supplemented with corticosterone and their matched controls. These data suggest that an increased maternal care protects the offspring against ischemic neuronal damage and that at least a component of neuroprotection is mediated by a reduced response of the HPA axis to ischemia.

Faecal corticosterone metabolite assessment in socially housed male and female Wistar rats.

Knowledge of animals' hormonal status is important for conservation studies in wild or semi-free-ranging conditions as well as for behavioural and clinical experiments conducted in laboratory research, mostly performed on rats and mice. Faecal sampling is a useful non-invasive method to obtain steroid hormone assessments. Nevertheless, in laboratory studies, unlike other contexts, faecal sampling is less utilised. One of the issues raised is the necessity to collect samples belonging to different animals, separately. Usually, researchers using faecal sampling solve this problem through the isolation of animals or taking the cage rather than single animal as unit of study. These solutions though, could lead to unreliable measurements, and cannot be applied in many studies. Our aim was to show the biological reliability of individual faecal corticosterone metabolite (FCM) assessments in socially housed male and female Wistar rats. We analytically validated the enzyme immunoassay kit used for FCM assessments. Then, we exposed the animals to two different stress stimuli that are known to activate the hypothalamus-pituitary-adrenal axis and the following release of corticosterone to biologically validate the EIA kit: environmental enrichment and predator odour. Individual faecal sampling from social animals was collected through short-time handling. The results demonstrated that both the stimuli increased FCM levels in male and female rats showing the reliability of EIA kit assessment and the applicability of our sampling method. We also found a diurnal rhythm in FCM levels. These results could help to increase the use of faecal hormone metabolite determinations in studies conducted on rats.

In Vivo Non-radioactive Assessment of mGlu5 Receptor-Activated Polyphosphoinositide Hydrolysis in Response to Systemic Administration of a Positive Allosteric Modulator.

mGlu5 receptor-mediated polyphosphoinositide (PI) hydrolysis is classically measured by determining the amount of radioactivity incorporated in inositolmonophosphate (InsP) after labeling of membrane phospholipids with radioactive inositol. Although this method is historically linked to the study of mGlu receptors, it is inappropriate for the assessment of mGlu5 receptor signaling in vivo. Using a new ELISA kit we showed that systemic treatment with the selective positive allosteric modulator (PAM) of mGlu5 receptors VU0360172 enhanced InsP formation in different brain regions of CD1 or C57Black mice. The action of VU0360172 was sensitive to the mGlu5 receptor, negative allosteric modulator (NAM), MTEP, and was abolished in mice lacking mGlu5 receptors. In addition, we could demonstrate that endogenous activation of mGlu5 receptors largely accounted for the basal PI hydrolysis particularly in the prefrontal cortex. This method offers opportunity for investigation of mGlu5 receptor signaling in physiology and pathology, and could be used for the functional screening of mGlu5 receptor PAMs in living animals.

Prenatal stress alters the negative correlation between neuronal activation in limbic regions and behavioral responses in rats exposed to high and low anxiogenic environments.

Behavioral adaptation to an anxiogenic environment involves the activity of various interconnected limbic regions, such as the amygdala, hippocampus and prefrontal cortex. Prenatal stress (PS) in rats affects the ability to cope with environmental challenges and alters brain plasticity, leading to long-lasting behavioral and neurobiological alterations. We examined in PS and control animals whether behavioral reactivity was correlated to neuronal activation by assessing Fos protein expression in limbic regions of rats exposed to a low or high anxiogenic environment (the closed and open arms of an elevated plus maze, respectively). A negative correlation was found between behavioral and neuronal activation, with a lower behavioral reactivity and a higher neuronal response observed in rats exposed to the more anxiogenic environment (the open arm) with respect to the less anxiogenic environment (the closed arm). Interestingly, the variation in the neurobehavioral response between the two arms of the maze was less pronounced in rats that had been subjected to PS. This study provides a remarkable example of how long-lasting changes in brain plasticity induced by PS affect the ability of limbic neurons to cope with anxiogenic stimuli of different strength.

Maternal exposure to low levels of corticosterone during lactation protects adult rat progeny against TNBS-induced colitis: A study on GR-mediated anti-inflammatory effect and prokineticin system.

The early phase of life represents a critical period for the development of an organism. Interestingly, early life experiences are able to influence the development of the gastrointestinal tract and the reactivity to colonic inflammatory stress. We recently demonstrated that adult male rats exposed to low doses of corticosterone during lactation (CORT-nursed rats) are protected against experimental colitis induced by the intracolonic infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Based on these interesting results, we wanted to better investigate which cellular actors could be involved in the protection of CORT-nursed rats from TNBS-induced experimental colitis. Therefore, in the present work, we focused our attention on different factors implicated in GR-mediated anti-inflammatory effect. To address this issue, colonic tissues, collected from control and CORT-nursed healthy animals and from control and CORT-nursed colitic rats, were processed and the following inflammatory factors were evaluated: the expression of (i) glucocorticoid receptors (GR), (ii) glucocorticoid-induced leucine zipper (GILZ), (iii) phospho-p65NF-κB, (iv) the pro-inflammatory cytokines IL-1ß and TNF-α, (v) the prokineticins PK2 and PK2L and (vi) their receptors PKR1 and PKR2. We found that adult CORT-nursed rats, in comparison to controls, showed increased expression of colonic GR and reduced expression of pro-inflammatory molecules (IL-1ß, TNF-α, PK2 and PK2L) in response to inflammatory colitis. The observed changes were associated with an increase in GILZ colonic expression and with a reduction in phospo-p65NF-κB colonic expression.

Relocation stress induces short-term fecal cortisol increase in Tonkean macaques (Macaca tonkeana).

The level of glucocorticoids, especially if obtained from noninvasive sampling, can be used as an index of animal well-being, allowing evaluation of the animal's response to environmental modifications. Despite evidence that these hormones play a relevant role in energy metabolism regulation in perceived or real stress events, little is known regarding the factors that could modify the capability of animals to cope with relocation events. The aim of this research was to assess fecal cortisol metabolite concentrations before, during and after acute stress (transfer and relocation event) in two well-established social groups of Tonkean macaques (Macaca tonkeana). The results showed that the fecal levels of cortisol increased in individuals of both groups in response to the stress event, with a similar trend in males and females. Hormone levels were back to baseline values in both groups a few days after transfer and relocation. The presence of known social partners could be one of the factors that possibly facilitated the adaptation process.

Maternal exposure to environmental enrichment before and during gestation influences behaviour of rat offspring in a sex-specific manner.

The beneficial effects of Environmental Enrichment (EE) applied immediately after weaning or even in adulthood have been widely demonstrated. Less is known about the possible changes in behaviour and brain development of the progeny following the exposure of dams to EE. In order to further investigate this matter, female rats were reared in EE for 12weeks, from weaning until delivery. After having confirmed the presence of relevant behavioural effects of EE, both control and EE females underwent mating. Maternal behaviour was observed and male and female offspring were then administered a battery of behavioural test at different ages. EE mothers showed a decreased frequency of total nursing and, during the first 2days of lactation, an increase in licking/grooming behaviour. Maternal exposure to EE affected offspring behaviour in a sex-specific manner: social play behaviour and anxiety-like behaviour were increased in males but not in females and learning ability was improved only in females. As a general trend, maternal EE had a marked influence on motility in male and female offspring in both locomotor activity and swimming speed. Overall, this study highlights the importance of environmental stimulation, not only in the animals directly experiencing EE, but for their progeny too, opening the way to new hypothesis on the heritability mechanisms of behavioural traits.

Pharmacological blockade of group II metabotropic glutamate receptors reduces the growth of glioma cells in vivo.

U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.

Maternal exposure to low levels of corticosterone during lactation protects against experimental inflammatory colitis-induced damage in adult rat offspring.

Opposing emotional events (negative/trauma or positive/maternal care) during the postnatal period may differentially influence vulnerability to the effects of stress later in life. The development and course of intestinal disorders such as inflammatory bowel disease are negatively affected by persistent stress, but to date the role of positive life events on these pathologies has been entirely unknown. In the present study, the effect of early life beneficial experiences in the development of intestinal dysfunctions, where inflammation and stress stimuli play a primary role, was investigated. As a "positive" experimental model we used adult male rat progeny nursed by mothers whose drinking water was supplemented with moderate doses of corticosterone (CORT) (0.2 mg/ml) during the lactation period. Such animals have been generally shown to cope better with different environmental situations during life. The susceptibility to inflammatory experimental colitis induced by intracolonic infusion of TNBS (2,4,6-trinitrobenzenesulphonic acid) was investigated in CORT-nursed rats in comparison with control rats. This mild increase in maternal corticosterone during lactation induced, in CORT-nursed rats, a long lasting protective effect on TNBS-colitis, characterized by improvements in some indices of the disease (increased colonic myeloperoxidase activity, loss of body weight and food intake) and by the involvement of endogenous peripheral pathways known to participate in intestinal disorder development (lower plasma corticosterone levels and colonic mast cell degranulation, alterations in the colonic expression of both corticotrophin releasing factor/CRF and its receptor/CRH-1R). All these findings contribute to suggesting that the reduced vulnerability to TNBS-colitis in CORT-nursed rats is due to recovery from the colonic mucosal barrier dysfunction. Such long lasting changes induced by mild hormonal manipulation during lactation, making the adult also better adapted to colonic inflammatory stress, constitute a useful experimental model to investigate the etiopathogenetic mechanisms and therapeutic treatments of some gastrointestinal diseases.

Transplacental exposure to AZT induces adverse neurochemical and behavioral effects in a mouse model: protection by L-acetylcarnitine.

Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-effects of antiretroviral therapy in fetus/newborn.

Maternal corticosterone effects on hypothalamus-pituitary-adrenal axis regulation and behavior of the offspring in rodents.

The behavioral and physiological traits of an individual are strongly influenced by early life events. One of the major systems implicated in the responses to environmental manipulations and stress is the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoid hormones (cortisol in humans and corticosterone in rodents) represent the final step in the activation of the HPA system and play an important role in the effects induced by the perinatal environment. We demonstrated, in rats with some differences between males and females, that mothers whose drinking water was supplemented with moderate doses of corticosterone throughout the lactation period, give birth to offspring better able to meet the demands of the environment. The progeny of these mothers, as adults, show improved learning capabilities, reduced fearfulness in anxiogenic situations, lower metabotropic glutamate receptors and higher glucocorticoid receptors in the hippocampus with a persistent hyporeactivity of the HPA axis leading to a resistance to ischemic neuronal damage. Other studies performed in mice showed that low doses of corticosterone in the maternal drinking water, which, as in our rat model, may reflect a form of mild environmental stimulation, enhanced the offspring's ability to cope with different situations, while elevated doses, comparable to those elicited by strong stressors, caused developmental disruption. Significantly, adult rats and mice that had been nursed by mothers with a mild hypercorticosteronemia provide an example of how a moderate corticosterone increase mediates the salutary effects of some events occurring early in life. Both maternal and infantile plasma levels of the hormone may play a role in these effects, the first influencing maternal behavior, the second acting directly on the central nervous system of the developing rat.

Prenatal restraint stress generates two distinct behavioral and neurochemical profiles in male and female rats.

Prenatal Restraint Stress (PRS) in rats is a validated model of early stress resulting in permanent behavioral and neurobiological outcomes. Although sexual dimorphism in the effects of PRS has been hypothesized for more than 30 years, few studies in this long period have directly addressed the issue. Our group has uncovered a pronounced gender difference in the effects of PRS (stress delivered to the mothers 3 times per day during the last 10 days of pregnancy) on anxiety, spatial learning, and a series of neurobiological parameters classically associated with hippocampus-dependent behaviors. Adult male rats subjected to PRS ("PRS rats") showed increased anxiety-like behavior in the elevated plus maze (EPM), a reduction in the survival of newborn cells in the dentate gyrus, a reduction in the activity of mGlu1/5 metabotropic glutamate receptors in the ventral hippocampus, and an increase in the levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF in the hippocampus. In contrast, female PRS rats displayed reduced anxiety in the EPM, improved learning in the Morris water maze, an increase in the activity of mGlu1/5 receptors in the ventral and dorsal hippocampus, and no changes in hippocampal neurogenesis or BDNF levels. The direction of the changes in neurogenesis, BDNF levels and mGlu receptor function in PRS animals was not consistent with the behavioral changes, suggesting that PRS perturbs the interdependency of these particular parameters and their relation to hippocampus-dependent behavior. Our data suggest that the epigenetic changes in hippocampal neuroplasticity induced by early environmental challenges are critically sex-dependent and that the behavioral outcome may diverge in males and females.

Inhibition of COX-2 reduces the age-dependent increase of hippocampal inflammatory markers, corticosterone secretion, and behavioral impairments in the rat.

Brain aging as well as brain degenerative processes with accompanying cognitive impairments are generally associated with hyperactivity of the hypothalamus-pituitary-adrenal axis, the end product of which, the glucocorticoid hormone, has been warranted the role of cell damage primum movens ("cascade hypothesis"). However, chronic inflammatory activity occurs in the hippocampus of aged rats as well as in the brain of Alzheimer's disease patients. The concomitant increase in the secretion of the glucocorticoid hormone, the endogenous anti-inflammatory and pro-inflammatory markers, has prompted us to investigate the two phenomena in the aging rat, and to work out its meaning. This study shows that: (I) interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and prostaglandin E(2) (PGE(2)) increase with age in the rats hippocampus, and (II) chronic oral treatment with celecoxib, a selective cycloxygenase-2 (COX-2) inhibitor, is able to contrast the age-dependent increase in hippocampal levels of pro-inflammatory markers and circulating anti-inflammatory corticosterone, provided that it is started at an early stage of aging. Under these conditions, age-related impairments in cognitive ability may be ameliorated. Taken together, these results indicate that there is a natural tendency to offset the age-dependent increase in brain inflammatory processes via the homeostatic increase of the circulating glucocorticoid hormone.