Visceral Fat Dysfunctions in the Rat Social Isolation Model of Psychosis.

Medication with neuroleptics has been associated with adipose tissue dysfunctions and, in particular, with increased visceral fat amount. However, several studies suggested that antipsychotic treatment might not be the main responsible of fat mass accumulation, as this has been also described in not treated psychotic patients. One of the most used "drug-free" rodent models of psychosis is the social isolation rearing of young adult rats, which provides a non-pharmacologic method of inducing long-term alterations reminiscent of symptoms seen in psychotic patients. Recent data highlighted a crucial role of redox imbalance in adipose tissue dysfunctions, in terms of decreased antioxidant defense and increased reactive oxygen species (ROS). Here, we investigated possible oxidative stress-related biomolecular alterations associated with visceral fat increase in 7 week isolated rats. To this purpose, we quantified total and visceral fat amount by using dual-energy X-ray (DEXA) absorptiometry. On visceral fat, we analyzed the expression of specific ROS-producer genes (Nox1, Nox4, Hmox-1), antioxidant enzymes (Prdx1 and Ucp-1) and oxidative stress-induced damage markers (Cidea, Slc2a4, and Acacb). The impact of oxidative stress on beta3-adrenergic receptors (Adrb3), at both mRNA and protein level, was also assessed. We found that 7 weeks of social isolation induced an increase in total and visceral fat, associated with a decrease in Prdx1 (mRNA and protein) as well as Ucp-1 mRNA levels and an enhanced expression of Nox1 (mRNA and protein) and Hmox-1 mRNA. No differences were detected in Nox4 mRNA levels between grouped and isolated animals. Elevations in Cidea, Slc2a4, and Acacb expression in visceral fat of isolated animals accounted for oxidative stress-related damage in this tissue, further associated with a significant increase in Adrb3 mRNA and protein. Our results provide a novel understanding of the pathological link existing among psychosocial stress-induced psychosis, adipose tissue dysfunctions and redox imbalance, opening new therapeutic perspectives for the treatment of alterations in peripheral tissues associated with this mental disorder.

Toll-like receptor 4-dependent glial cell activation mediates the impairment in memory establishment induced by ß-amyloid oligomers in an acute mouse model of Alzheimer's disease.

BACKGROUND: Amyloid-ß oligomers (AßO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AßO-mediated mouse model of AD, we explored whether the memory alteration AßOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved. METHODS: C57 naïve mice were given a single intracerebroventricular injection of synthetic AßO-containing solution (1µM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1ß receptor antagonist) to prevent impairment in the novel object recognition task, and compared AßO's effects in TLR4 knockout mice. RESULTS: A single AßO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AßO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AßO's action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation. CONCLUSIONS: These data provide new information on AßO's mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.

Soluble beta amyloid evokes alteration in brain norepinephrine levels: role of nitric oxide and interleukin-1.

Strong evidence showed neurotoxic properties of beta amyloid (Aß) and its pivotal role in the Alzheimer's disease (AD) pathogenesis. Beside, experimental data suggest that Aß may have physiological roles considering that such soluble peptide is produced and secreted during normal cellular activity. There is now suggestive evidence that neurodegenerative conditions, like AD, involve nitric oxide (NO) in their pathogenesis. Nitric oxide also possess potent neuromodulatory actions in brain regions, such as prefrontal cortex (PFC), hippocampus (HIPP), and nucleus accumbens (NAC). In the present study, we evaluated the effect of acute Aß injection on norepinephrine (NE) content before and after pharmacological manipulations of nitrergic system in above mentioned areas. Moreover, effects of the peptide on NOS activity were evaluated. Our data showed that 2 h after i.c.v. soluble Aß administration, NE concentrations were significantly increased in the considered areas along with increased iNOS activity. Pre-treatment with NOS inhibitors, 7-Nitroindazole (7-NI), and N6-(1-iminoethyl)-L-lysine-dihydrochloride (L-NIL), reversed Aß-induced changes. Ultimately, pharmacological block of interleukin1 (IL-1) receptors prevented NE increase in all brain regions. Taken together our findings suggest that NO and IL-1 are critically involved in regional noradrenergic alterations induced by soluble Aß injection.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.

Memantine prevents memory consolidation failure induced by soluble beta amyloid in rats.

It has been well documented that ß-amyloid (Aß) peptide accumulation and aggregation in the brain plays a crucial role in the pathophysiology of Alzheimer's disease (AD). However, a new orientation of the amyloid cascade hypothesis has evidenced that soluble forms of the peptide (sAß) are involved in Aß-induced cognitive impairment and cause rapid disruption of the synaptic mechanisms underlying memory. The primary aim of this study was to elucidate the effects of sAß, acutely injected intracerebrally (i.c.v., 4 µM), on the short term and long term memory of young adult male rats, by using the novel object recognition task. Glutamatergic receptors have been proposed as mediating the effect of Aß on synaptic plasticity and memory. Thus, we also investigated the effects of sAß on prefrontal cortex (PFC) glutamate release and the specific contribution of N-methyl-D-aspartate (NMDA) receptor modulation to the effects of sAß administration on the cognitive parameters evaluated. We found that a single i.c.v. injection of sAß 2 h before testing did not alter the ability of rats to differentiate between a familiar and a novel object, in a short term memory test, while it was able to negatively affect consolidation/retrieval of long term memory. Moreover, a significant increase of glutamate levels was found in PFC of rats treated with the peptide 2 h earlier. Interestingly, memory deficit induced by sAß was reversed by a NMDA-receptor antagonist, memantine (5 mg/kg i.p), administered immediately after the familiarization trial (T1). On the contrary, memantine administered 30 min before T1 trial, was not able to rescue long term memory impairment. Taken together, our results suggest that an acute i.c.v. injection of sAß peptide interferes with the consolidation/retrieval of long term memory. Moreover, such sAß-induced effect indicates the involvement of glutamatergic system, proposing that NMDA receptor inhibition might prevent or lead to the recovery of early cognitive impairment.

Chronic nandrolone administration induces dysfunction of the reward pathway in rats.

Data in animal models and surveys in humans have revealed psychiatric complications of long-term anabolic androgenic steroid abuse. However, the neurobiochemical mechanisms behind the observed behavioral changes are poorly understood. The aim of the present study was to investigate the effects of nandrolone decanoate on emotional behavior and neurochemical brain alterations in gonadally intact male rats. The behavioral reactivity to the elevated plus maze and the social interaction test was used to assess anxiety-related symptoms, and the sucrose preference test was used to evaluate anhedonia. Dopaminergic, serotonergic and noradrenergic transmissions were also evaluated in selected brain areas. The chronic administration of nandrolone, at 5 mg kg(-1) injected daily for 4 weeks, induced the loss of sweet taste preference, a sign of anhedonia and dysfunction of the reward pathway. The behavioral outcomes were accompanied by reductions in the dopamine, serotonin and noradrenaline contents in the nucleus accumbens. Alterations in the time spent in the open arms and in the social interaction test were not found, suggesting that nandrolone did not induce an anxiogenic profile. No differences were revealed between the experimental groups in the amygdala in terms of the neurotransmitters measured. Our data suggest that nandrolone-treated rats have a depressive, but not anxiogenic-like, profile, accompanied by brain region-dependent changes in dopaminergic, serotonergic and noradrenergic neurotransmission. As anabolic androgenic steroid dependence is plausibly the major form of worldwide substance dependence that remains largely unexplored, it should be highlighted that our data could contribute to a better understanding of the altered rewards induced by nandrolone treatment and to the development of appropriate treatments.

Modulatory activity of soluble beta amyloid on HPA axis function in rats.

Despite the consolidation of the amyloid hypothesis, the main component of senile plaques in Alzheimer's disease (AD), recent findings have led to a conceptual shift opening new questions about the potential physiological role of this peptide. In addition, soluble beta amyloid (sBA), in transgenic AD model, resulted to be increased after chronic and acute stress and alterations in cortisol levels have been reported in AD. Impaired hypothalamic pituitary adrenal (HPA) axis has been linked to depressive state and, consistently, we have previously demonstrated that BA is able to provoke depressive-like profile in rats. Here we further analysed the effect of the peptide in behavioural paradigms used to study emotional and cognitive response, by using the passive avoidance task, for cognitive parameters, and the sucrose preference test (SPT), to evaluate anhedonia. Moreover, in order to correlate behavioural with neurochemical and neuroendocrinal data, we investigated the effects of the peptide on noradrenergic system in amygdala (AMY), prefrontal cortex (PFC) and hippocampus (HIPP) along with plasmatic corticosterone and hypothalamic corticotrophin releasing factor (CRF). We found that BA-treated animals showed an impaired memory consolidation of inhibitory avoidance training, while no effect was evident in SPT. These results lead us to hypothesize a different response to stress coping behaviour in BA treated rats. Moreover, BA caused a significant increase in noradrenaline (NA) in PFC and HIPP, while in AMY was decreased. Consistently, we found a significant decrease in plasma corticosterone concentrations in BA-treated rats. Taken together, our data suggest that BA exerts an inhibitory effect on HPA axis activation.

Carvacrol: from ancient flavoring to neuromodulatory agent.

Oregano and thyme essential oils are used for therapeutic, aromatic and gastronomic purposes due to their richness in active substances, like carvacrol; however, the effects of the latter on the central nervous system have been poorly investigated. The aim of our study was to define the effects of carvacrol on brain neurochemistry and behavioural outcome in rats. Biogenic amine content in the prefrontal cortex and hippocampus after chronic or acute oral carvacrol administration was measured. Animals were assessed by a forced swimming test. Carvacrol, administered for seven consecutive days (12.5 mg/kg p.o.), was able to increase dopamine and serotonin levels in the prefrontal cortex and hippocampus. When single doses were used (150 and 450 mg/kg p.o.), dopamine content was increased in the prefrontal cortex at both dose levels. On the contrary, a significant dopamine reduction in hippocampus of animals treated with 450 mg/kg of carvacrol was found. Acute carvacrol administration only significantly reduced serotonin content in either the prefrontal cortex or in the hippocampus at the highest dose. Moreover, acute carvacrol was ineffective in producing changes in the forced swimming test. Our data suggest that carvacrol is a brain-active molecule that clearly influences neuronal activity through modulation of neurotransmitters. If regularly ingested in low concentrations, it might determine feelings of well-being and could possibly have positive reinforcer effects.

Neuroendocrine profile in a rat model of psychosocial stress: relation to oxidative stress.

AIMS: Psychosocial stress alters the hypothalamic-pituitary-adrenal axis (HPA-axis). Increasing evidence shows a link between these alterations and oxidant elevation. Oxidative stress is implicated in the stress response and in the pathogenesis of neurologic and psychiatric diseases. NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the central nervous system. Here, we investigated the contributory role of NOX2-derived ROS to the development of neuroendocrine alterations in a rat model of chronic psychosocial stress, the social isolation. RESULTS: Significant elevations in the hypothalamic levels of corticotropin-releasing factor and plasmatic adrenocorticotropic hormone were observed from 4 weeks of social isolation. Increased levels of peripheral markers of the HPA-axis (plasmatic and salivary corticosterone) were observed at a later time point of social isolation (7 weeks). Alteration in the exploratory activity of isolated rats followed the same time course. Increased expression of markers of oxidative stress (8-hydroxy-2-deoxyguanosine [8OhdG] and nitrotyrosine) and NOX2 mRNA was early detectable in the hypothalamus of isolated rats (after 2 weeks), but later (after 7 weeks) in the adrenal gland. A 3-week treatment with the antioxidant/NOX inhibitor apocynin stopped the progression of isolation-induced alterations of the HPA-axis. Rats with a loss-of-function mutation in the NOX2 subunit p47(phox) were totally protected from the alterations of the neuroendocrine profile, behavior, and increased NOX2 mRNA expression induced by social isolation. INNOVATION: We demonstrate that psychosocial stress induces early elevation of NOX2-derived oxidative stress in the hypothalamus and consequent alterations of the HPA-axis, leading ultimately to an altered behavior. CONCLUSION: Pharmacological targeting of NOX2 might be of crucial importance for the treatment of psychosocial stress-induced psychosis.

Neurochemical consequence of steroid abuse: stanozolol-induced monoaminergic changes.

An extensive literature has documented adverse effects on mental health in anabolic androgenic steroids (AAS) abusers. Depression seems a common adverse reaction in AAS abusers. Recently it has been reported that in a rat model of AAS abuse stanozolol induces behavioural and biochemical changes related to the pathophysiology of major depressive disorder. In the present study, we used the model of AAS abuse to examine possible changes in the monoaminergic system, a neurobiological substrate of depression, in different brain areas of stanozolol-treated animals. Wistar rats received repeated injections of stanozolol (5mg/kg, s.c.), or vehicle (propylene glycol, 1ml/kg) once daily for 4weeks. Twenty-four hours after last injection, changes of dopamine (DA) and relative metabolite levels, homovanilic acid (HVA) and 3,4-dihydroxy phenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite levels, 5-hydroxy indolacetic acid (5-HIAA), and noradrenaline (NA) amount were investigated in prefrontal cortex (PFC), nucleus accumbens (NAC), striatum (STR) and hippocampus (HIPP). The analysis of data showed that after chronic stanozolol, DA levels were increased in the HIPP and decreased in the PFC. No significant changes were observed in the STR or in the NAC. 5-HT and 5-HIAA levels were decreased in all brain areas investigated after stanozolol exposure; however, the 5-HIAA/5-HT ratio was not altered. Taken together, our data indicate that chronic use of stanozolol significantly affects brain monoamines leading to neurochemical modifications possibly involved in depression and stress-related states.

Novel highly potent and selective sigma 1 receptor antagonists related to spipethiane.

Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel potent sigma(1) ligands. sigma(1) affinity and sigma(1/)sigma(2) selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing sigma(1) receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the sigma(1) antagonist profile of this series of compounds. In particular, due to its high sigma(1) affinity (pK(i) = 10.28) and sigma(1)/sigma(2) selectivity ratio (29510), compound 9 might be a novel valuable tool for sigma receptor characterization and a suitable template for the rational design of potential therapeutically useful sigma(1) antagonists.

Involvement of NOX2 in the development of behavioral and pathologic alterations in isolated rats.

BACKGROUND: Social stress leads to oxidative stress in the central nervous system, contributing to the development of mental disorders. Loss of parvalbumin in interneurons is an important feature of these diseases. We studied the role of the superoxide-producing nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) in rats exposed to social isolation. METHODS: Male rats were kept for 7 weeks in group or in social isolation (n = 6-10 per group). Behavioral tests, immunohistochemistry, and analysis of NOX2 expression were performed at the end of social isolation. Apocynin was given in the drinking water (5 mg/kg/day). RESULTS: NOX2 was below detection level in the brains of control animals, whereas it was highly expressed in isolated rats, particularly in nucleus accumbens and prefrontal cortex. Indirect markers of oxidative stress (oxidized nucleic acid 8-hydroxy-2'-deoxyguanosine, redox-sensitive transcription factor c-fos, and hypoxia-inducible factor-1alpha) were increased after social isolation in brain areas with high NOX2 expression. An increase in immunoreactive microglia suggested that oxidative stress could be in part due to NOX2 activation in microglia. In response to social isolation, rats showed increased locomotor activity, decreased discrimination, signs of oxidative stress in neurons, and loss of parvalbumin-immunoreactivity. Treatment of isolated rats with the antioxidant/NOX inhibitor apocynin prevented the behavioral and histopathological alterations induced by social isolation. CONCLUSIONS: Our data suggest that NOX2-derived oxidative stress is involved in loss of parvalbumin immunoreactivity and development of behavioral alterations after social isolation. These results provide a molecular mechanism for the coupling between social stress and brain oxidative stress, as well as potential new therapeutic avenues.

Determination of dimethylarginine levels in rats using HILIC-MS/MS: an in vivo microdialysis study.

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.

Further studies on the effect of lysine at the C-terminus of the Dmt-Tic opioid pharmacophore.

A wide range of activities are induced by Lys when introduced at C-terminus of the delta-opioid Dmt-Tic pharmacophore through the alpha-amine group, including: improved delta-antagonism, mu-agonism and mu-antagonism. Here we report the synthesis of a new series of compounds with the general formula H-Dmt-Tic-NH-(CH(2))(4)-CH(R)-R' (R=-NH(2), -NH-Ac, -NH-Z; R'=CO-NH-Ph, -CO-NH-CH(2)-Ph, -Bid) in which Lys is linked to Dmt-Tic through its side-chain amine group. All new compounds (1-9) displayed potent and selective delta-antagonism (MVD, pA(2)=7.81-8.27), which was independent of the functionalized alpha-amine and carboxylic groups of C-terminal Lys. This behaviour suggests a direct application as a prototype intermediate, such as Boc-Dmt-Tic-epsilon-Lys(Z)-OMe, which could be successfully applied in the synthesis (after Z or methyl ester removal) of unique designed multiple ligands containing the pharmacophore of the quintessential delta-antagonist Dmt-Tic and another opioid or biologically active non-opioid ligand.

Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore.

Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.