Carlo Cinque

Prenatal stress and long-term consequences: implications of glucocorticoid hormones.

We have shown that prenatal restraint stress (PNRS) induces higher levels of anxiety, greater vulnerability to drugs, a phase advance in the circadian rhythm of locomotor activity and an increase in the paradoxical sleep in adult rats. These behavioral effects result from permanent modifications to the functioning of the brain, particularly in the feedback mechanisms of the hypothalamic-pituitary-adrenal (HPA) axis: the secretion of corticosterone is prolonged after stress and the number of the central glucocorticoid receptors is reduced. These abnormalities are associated with modifications in the synthesis and/or release of certain neurotransmitters. Dysfunction of the HPA axis is due, in part, to stress-induced maternal increase of glucocorticoids, which influences fetal brain development. Some biological abnormalities in depression can be related to those found in PNRS rats reinforcing the idea of the usefulness of PNRS rats as an appropriate animal model to study new pharmacological approaches.

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.

Maternal corticosterone influences behavior, stress response and corticosteroid receptors in the female rat

In infancy, glucocorticoids have been shown to affect hypothalamus-pituitary-adrenal (HPA) axis activity and behavior. Both the activity of the HPA axis and many aspects of behavior exhibit important gender-dependent differences physiologically. In our previous studies, male offspring of hypercorticosteronemic mothers show long-lasting changes of learning as well as adrenocortical activity. In the light of these findings, this study aims to determine the long-term effects of glucocorticoids in the early stages of life in female rats. Corticosterone (200 microg/ml) was added to the drinking water of the dams. Female offspring exhibited lower adrenocortical secretory response to stress, improvement in learning (water maze at 21, 30 and 90 days; active avoidance at 15 months) and reduced fearfulness in anxiogenic situations (dark-light test at 1 and 15 months; conditioned suppression of drinking at 3 months; plus maze at 15 months) after weaning, from 21 days up to 15 months of age, but not before. No difference in hippocampal adrenocorticoid receptors was observed. These results, together with previous data on male offspring, show that the outcomes of maternal hypercorticosteronemia on hormonal stress response and behavior are similar in males and females, but the effects on some aspects of the HPA axis activity are gender-dependent. Possible explanations for these differences are discussed.

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 offsprings 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.

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 α‐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.

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.

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 Alzheimers 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(Ser404)-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-3β; 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.

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 μm) 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 μm 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.