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.

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.

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 exposure to low levels of corticosterone during lactation increases social play behavior in rat adolescent offspring.

Although costly in energy and time, social play is present and evolutionarily conserved in nearly all young mammals. Ontogenetic factors responsible for this particular form of supposed rewarding behavior are incompletely understood. Here, we have focused our attention on maternal glucocorticoid hormone. We used a model in which neonate rats are fed by mothers in which drinking water has been supplemented with 0.2 mg/ml corticosterone. The control groups were lactated by water-drinking mothers. Both male and female adolescent offspring of corticosterone (CORT) supplemented dams (CORT-nursed) showed an increase in social play behavior (i.e., pinning, pouncing, wrestling/boxing and social exploration) when compared to controls. No differences were observed between CORT-nursed progeny of both sexes and controls in the exploration of the arena during the social encounter. Finally, no differences were found in CORT plasma levels in basal conditions and following a social play session in both male and female CORT-nursed rats. These results indicate that variations in the maternal glucocorticoid status are able, directly or indirectly, to influence social play behavior in the offspring, although there is no direct relationship between the level of social play behavior and the intensity of adrenocortical activation.

Effects of cholinergic enhancing drugs on cholinergic transporters in the brain and peripheral blood lymphocytes of spontaneously hypertensive rats.

Cholinergic hypofunction is a trait of Alzheimer's disease and vascular dementia and countering it is one of the main therapeutic strategies available for these disorders. Cholinergic transporters control cellular mechanisms of acetylcholine (ACh) synthesis and release at presynaptic terminals. This study has assessed the influence of 4 week treatment with two different cholinergic enhancing drugs, the cholinergic precursor choline alphoscerate (alpha-glyceryl-phosphorylcholine) or the acetylcholinesterase (AChE) inhibitor galantamine on high affinity choline uptake transporter (CHT) and vesicular ACh transporter (VAChT) expression in the brain of spontaneously hypertensive rats (SHR). SHR represent an animal model of cerebrovascular injury characterized by cholinergic hypofunction. Analysis was performed by immunochemistry, ELISA and immunohistochemistry on frontal cortex, striatum and hippocampus. Immunochemical and ELISA analysis was extended to peripheral blood lymphocytes (PBL), used as a peripheral reference of changes of brain cholinergic markers. An increased expression of VAChT and CHT was observed in brain areas investigated and in PBL of SHR. The similar trend for cholinergic transporters observed in brain and PBL suggests these cells may represent a marker of brain cholinergic transporters. Treatment with choline alphoscerate increased CHT and to a greater extent VAChT expression. Treatment with galantamine countered the increase of CHT and VAChT. The different activity of the cholinergic precursor and of the AChE inhibitor on parameters investigated is likely related to their mechanism of action. Choline alphoscerate increases ACh synthesis and release. This requires an augmentation of systems regulating neurotransmitter uptake and storage. The effect of choline alphoscerate on CHT and VAChT observed in this study suggests an improved synaptic efficiency elicited by the compound. The AChE inhibitor slows-down ACh degradation in the synaptic cleft. A greater availability of neurotransmitter elicited by galantamine counters the enhanced activity of cholinergic transporters compensating cholinergic deficits. Differences in the activity of the cholinergic precursor and AChE inhibitor investigated on CHT and VAChT suggests that association between choline alphoscerate and AChE/cholinesterase inhibitors may represent a strategy for potentiating deficient cholinergic neurotransmission worthwhile of being investigated in clinical trials.

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.

Long-term effects of developmental exposure to low doses of PCB 126 and methylmercury.

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are food contaminants often found in fish. Experimental and epidemiological studies indicate that both PCBs and MeHg are developmental neurotoxicants, and some reports suggest that they may cause additive and/or synergistic neurotoxicity. We had previously investigated the effects of exposure to low doses of MeHg (0.5 mg/kg/day in drinking water) and PCB 126 (100 ng/kg/day in food) alone or in combination, from gestational day 7 to post-partum day 21, on neurobehavioral development in Wistar rats. The main finding was hyperactivity in male rats exposed to PCB 126, and in female animals exposed to PCB 126+MeHg at 4 months of age (Vitalone et al., 2008). Since effects caused by developmental exposure may be exacerbated as the animal ages, aim of the present study was to investigate behavioral effects of the same developmental exposure to PCB 126 and/or MeHg up to the age of 20 months. Results indicate that aging did not enhance the behavioral effects of early exposures; however, behavioral alterations found in the first months of life in male rats exposed to PCB 126, or in female rats exposed to PCB 126+MeHg, were persistent. Furthermore, an additional effect (increased body weight) was unmasked in adulthood in male rats exposed to PCB 126. These results indicate that developmental exposure to a low, environmentally relevant dose of PCB 126 causes long-lasting hyperactivity in male rats, and a significant increase in body weight.

Interaction between Ephrins and mGlu5 metabotropic glutamate receptors in the induction of long-term synaptic depression in the hippocampus.

We applied the group-I metabotropic glutamate (mGlu) receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), to neonatal or adult rat hippocampal slices at concentrations (10 microM) that induced a short-term depression (STD) of excitatory synaptic transmission at the Schaffer collateral/CA1 synapses. DHPG-induced STD was entirely mediated by the activation of mGlu5 receptors because it was abrogated by the mGlu5 receptor antagonist, MPEP [2-methyl-6-(phenylethynyl)pyridine], but not by the mGlu1 receptor antagonist, CPCCOEt [7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester]. Knowing that ephrin-Bs functionally interact with group-I mGlu receptors (Calò et al., 2005), we examined whether pharmacological activation of ephrin-Bs could affect DHPG-induced STD. We activated ephrin-Bs using their cognate receptor, EphB1, under the form of a preclustered EphB1/Fc chimera. Addition of clustered EphB1/Fc alone to the slices induced a small but nondecremental depression of excitatory synaptic transmission, which differed from the depression induced by 10 microM DHPG. Surprisingly, EphB1/Fc-induced synaptic depression was abolished by MPEP (but not by CPCCOEt) suggesting that it required the endogenous activation of mGlu5 receptors. In addition, coapplication of DHPG and EphB1/Fc, resulted in a large and nondecremental long-term depression. The effect of clustered EphB1/Fc was specific because it was not mimicked by unclustered EphB1/Fc or clustered EphA1/Fc. These findings raise the intriguing possibility that changes in synaptic efficacy mediated by mGlu5 receptors are under the control of the ephrin/Eph receptor system, and that the neuronal actions of ephrins can be targeted by drugs that attenuate mGlu5 receptor signaling.

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.

Enhanced tau phosphorylation in the hippocampus of mice treated with 3,4-methylenedioxymethamphetamine ("Ecstasy").

3,4-Methylenedioxymethamphetamine (MDMA) ("Ecstasy") produces neurotoxic effects, which result into an impairment of learning and memory and other neurological dysfunctions. We examined whether MDMA induces increases in tau protein phosphorylation, which are typically associated with Alzheimer's disease and other chronic neurodegenerative disorders. We injected mice with MDMA at cumulative doses of 10-50 mg/kg intraperitoneally, which are approximately equivalent to doses generally consumed by humans. MDMA enhanced the formation of reactive oxygen species and induced reactive gliosis in the hippocampus, without histological evidence of neuronal loss. An acute or 6 d treatment with MDMA increased tau protein phosphorylation in the hippocampus, revealed by both anti-phospho(Ser(404))-tau and paired helical filament-1 antibodies. This increase was restricted to the CA2/CA3 subfields and lasted 1 and 7 d after acute and repeated MDMA treatment, respectively. Tau protein was phosphorylated as a result of two nonredundant mechanisms: (1) inhibition of the canonical Wnt (wingless-type MMTV integration site family) pathway, with ensuing activation of glycogen synthase kinase-3beta; and (2) activation of type-5 cyclin-dependent kinase (Cdk5). MDMA induced the expression of the Wnt antagonist, Dickkopf-1, and the expression of the Cdk5-activating protein, p25. In addition, the increase in tau phosphorylation was attenuated by strategies that rescued the Wnt pathway or inhibited Cdk5. Finally, an impairment in hippocampus-dependent spatial learning was induced by doses of MDMA that increased tau phosphorylation, although the impairment outlasted this biochemical event. We conclude that tau hyperphosphorylation in the hippocampus may contribute to the impairment of learning and memory associated with MDMA abuse.

Neurobehavioral assessment of rats exposed to low doses of PCB126 and methyl mercury during development.

Epidemiological and laboratory studies have suggested that polychlorinated biphenyls (PCBs) and methyl mercury (MeHg) may have additive or synergistic effects on CNS function. Aim of this study was to characterize the effects of exposure to low levels of MeHg (0.5mg/kgday in drinking water) and PCB126 (100ng/kgday in food), alone and in combination, on neurobehavioral development in Wistar rats. Dams were treated from gestational day 7 to post-natal day (PND) 21. Animals were tested for developmental landmarks and reflexes (PND1-21), attention deficits (PND40), locomotor activity (PND30, 110), spatial learning (PND75), coordination and balance (PND90), object discrimination (PND80), anxiety (PND100), and conditioned learning (PND110). Parameters related to pregnancy, sex ratio at birth, and physical development (at weaning) did not differ among groups, though PCB126 decreased number of pups at birth. A slight delay in negative geotaxis was found in female rats in all treatment groups. No significant effects were seen in attention, coordination and balance, object discrimination, and spatial and conditioned learning. Increased motor activity was present in PCB126-treated male and in MeHg+PCB-treated female rats in the elevated plus maze test, and in PCB126-treated male rats in the open field test (PND110). The results do not support the hypothesis that co-exposure to MeHg and PCB126 results in additive or synergistic effects. This finding is in agreement with more recent in vitro and in vivo studies.

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.

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration.

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.

Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus.

The developmental pattern of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes was investigated in the hippocampus (subfields CA1, CA3 and CA4) and in the dentate gyrus of male and female rats aged 11, 16, 30, 90 and 150 days by immunohistochemistry associated with image analysis. Analysis was centred on stratum radiatum, a hippocampal area rich in GFAP-immunoreactive astrocytes. The volume of different portions of hippocampus, the number and the size of astrocytes, the intensity of cell body GFAP immunostaining as well as the extension of astrocyte were assessed. A maturation pattern consisting in higher cellular expression of GFAP, an increase in overall cell size and expanding arborisation from the 11th to the 30th postnatal day, followed by stabilisation of these parameters until the 90th day of life, and a subsequent decrease in the oldest age group studied was found. A sex-related different temporal pattern of astrocytes maturation in size and GFAP content was observed in the CA1 subfield only. The increase of GFAP content during pre-weaning ages was less pronounced in females than in males as well as the decrease between the 90th and the 150th day of age. Moreover, the size of astrocytes was larger in females than in males at the 11th and 150th days of life. These findings suggest that hippocampal astrocytes undergo rapid maturation in the 1st month of postnatal life, followed by a slow consolidation of this process until the 3rd month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner probably as a response to the increased volume of hippocampus noticeable at this age.