Giovanni Sebastiano Alemà

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.

Effect of increased maternal corticosterone during lactation on hippocampal corticosteroid receptors, stress response and learning in offspring in the early stages of life

The influence of maternal corticosterone during lactation on the development of the hippocampal corticosteroid receptor system, hypothalamus-pituitary-adrenal axis activity and spatial learning/retention performance was investigated in the rat during postnatal days 11 to 30. We increased the plasma levels of corticosterone by adding the hormone (200 microg/ml) to the drinking water of the dams. When compared to controls corticosterone-nursed offspring displayed: i) higher number of hippocampal type I and type II corticosteroid receptors at 30 days of life, but no changes at 11 and 16 days; ii) higher plasma levels of corticosterone in the basal condition and after 15 min of maternal separation at 11 but not at 16 days: iii) lower adrenal weights at 11 and 16 days, but which were no longer present at the age of 30 days; iv) no difference in performance in the place learning version of the Morris water task and T aquatic maze at 16 days. The present results, together with our previous findings showing that 90-day-old corticosterone-nursed rats have lower basal and restraint stress corticosterone levels and improved learning performance, indicate that the effects of maternal treatment appears only after weaning, thereby suggesting that increased corticosteroid receptors may be responsible, at least partially, for the endocrine and learning modifications induced by pre-weaning corticosterone exposure. The role played by maternal circulating corticosterone during the period of lactation in shaping the characteristics of the hypothalamus-pituitary-adrenal axis and brain of the offspring is outlined.

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.

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.

Cannabinoid agonist WIN55,212 in vitro inhibits interleukin‐6 (IL‐6) and monocyte chemo‐attractant protein‐1 (MCP‐1) release by rat pancreatic acini and in vivo induces dual effects on the course of acute pancreatitis

Background  Cannabinoids (CBs) evoke their effects by activating the cannabinoid receptor subtypes CB1‐r and CB2‐r and exert anti‐inflammatory effects altering chemokine and cytokine expression. Various cytokines and chemokines are produced and released by rodent pancreatic acini in acute pancreatitis. Although CB1‐r and CB2‐r expressed in rat exocrine pancreatic acinar cells do not modulate digestive enzyme release, whether they modulate inflammatory mediators remains unclear. We investigated the CB‐r system role on exocrine pancreas in unstimulated conditions and during acute pancreatitis. Methods We evaluated in vitro and in vivo changes induced by WIN55,212 on the inflammatory variables amylasemia, pancreatic edema and morphology, and on acinar release and content of the cytokine interleukin‐6 (IL‐6) and chemokine monocyte chemo‐attractant protein‐1 (MCP‐1) in untreated rats and rats with caerulein (CK)‐induced pancreatitis. Key Results In the in vitro experiments, WIN55,212 (10−6 mol L−1) inhibited IL‐6 and MCP‐1 release from acinar cells of unstimulated rats and after CK‐induced pancreatitis. In vivo, when rats were pretreated with WIN55,212 (2 mg kg−1, intraperitoneally) before experimentally‐induced pancreatitis, serum amylase, pancreatic edema and IL‐6 and MCP‐1 acinar content diminished and pancreatic morphology improved. Conversely, when rats with experimentally‐induced pancreatitis were post‐treated with WIN55,212, pancreatitis worsened. Conclusions & Inferences These findings provide new evidence showing that the pancreatic CB1‐r/CB2‐r system modulates pro‐inflammatory factor levels in rat exocrine pancreatic acinar cells. The dual, time‐dependent WIN55,212‐induced changes in the development and course of acute pancreatitis support the idea that the role of the endogenous CB receptor system differs according to the local inflammatory status.

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 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.

[EXPRESS] The 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.

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.