Repeated three-hour maternal deprivation as a model of early-life stress alters maternal behavior, olfactory learning and neural development.

Early life stress such as physical abuse, trauma or neglect during a critical period of development can elicit negative long-lasting effects on health. Neonatal maternal deprivation (MD) is a stressful event capable of triggering structural and neurobiological changes in Central Nervous System (CNS) development during proliferative and migratory cell differentiation. In this study, we investigated the maternal behavior of lactating rats submitted to protocol of chronic neonatal maternal deprivation (MD) during postnatal day (PND) 1 until 10. We analyzed the effects of the MD in the olfactory memory and cellular proliferation and differentiation in the hippocampus and olfactory bulb in Wistar rat pups on 7, 11 and 21 days postpartum. Analysis in active neurons, cellular differentiation and proliferation, were marked and evaluated by flow cytometry in tissue samples of hippocampi and olfactory bulb. Our results demonstrated an increase in maternal behavior immediately after dam's return to the home-cage in MD group compared to the non-deprived group. In addition, MD pups spent more time (higher latency) to identify the nest odor in comparison to the non-deprived rat pups in the olfactory learning task and showed a significant delay in the neural differentiation and proliferation in the hippocampus and olfactory bulb. These results reveal that disruptions in the mother-infant bonding by the MD induce changes in maternal behavior and interaction with the offspring that could be leading to delayed CNS development and significant impairment in offspring's olfactory learning.

Effect of co-culture of mesenchymal stem/stromal cells with pancreatic islets on viability and function outcomes: a systematic review and meta-analysis.

The maintenance of viable and functional pancreatic islets is crucial for successful islet transplantation from brain-dead donors. To overcome islet quality loss during culture, some studies have co-cultured islets with mesenchymal stem/stromal cells (MSC). However, it is still uncertain if MSC-secreted factors are enough to improve islet quality or if a physical contact between MSCs and islets is needed. Therefore, we performed a systematic review and meta-analysis to clarify the effect of different culture contact systems of islets with MSCs on viability and insulin secretion outcomes. Pubmed and Embase were searched. Twenty studies fulfilled the eligibility criteria and were included in the qualitative synthesis and/or meta-analysis. For both outcomes, pooled weighted mean differences (WMD) between islet cultured alone (control group) and the co-culture condition were calculated. Viability mean was higher in islets co-cultured with MSCs compared with islet cultured alone [WMD = 18.08 (95% CI 12.59-23.57)]. The improvement in viability was higher in islets co-cultured in indirect or mixed contact with MSCs than in direct physical contact (P <0.001). Moreover, the mean of insulin stimulation index (ISI) was higher in islets from co-culture condition compared with islet cultured alone [WMD = 0.83 (95% CI 0.54-1.13)], independently of contact system. Results from the studies that were analyzed only qualitatively are in accordance with meta-analysis data. Co-culture of islets with MSCs has the potential for protecting islets from injury during culture period. Moreover, culture time appears to influence the beneficial effect of different methods of co-culture on viability and function of islets.

High postnatal susceptibility of hippocampal cytoskeleton in response to ethanol exposure during pregnancy and lactation.

Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity.

Methylglyoxal-induced cytotoxicity in neonatal rat brain: a role for oxidative stress and MAP kinases.

Carbonyl compounds such as methylglyoxal (MGO) seem to play an important role in complications resulting from diabetes mellitus, in aging and neurodegenerative disorders. In this study, we are showing, that MGO is able to suppress cell viability and induce apoptosis in the cerebral cortex and hippocampus of neonatal rats ex-vivo. These effects are partially related with ROS production, evaluated by DCFH-DA assay. Coincubation of MGO and reduced glutathione (GSH) or Trolox (vitamin E) totally prevented ROS production but only partially prevented the MGO-induced decreased cell viability in the two brain structures, as evaluated by the MTT assay. Otherwise, L-NAME, a nitric oxide (NO) inhibitor, partially prevented ROS production in the two structures but partially prevented cytotoxicity in the hippocampus. Pharmacological inhibition of Erk, has totally attenuated MGO-induced ROS production and cytotoxicity, suggesting that MEK/Erk pathway could be upstream of ROS generation and cell survival. Otherwise, p38MAPK and JNK failed to prevent ROS generation but induced decreased cell survival consistent with ROS-independent mechanisms. We can propose that Erk, p38MAPK and JNK are involved in the cytotoxicity induced by MGO through different signaling pathways. While Erk could be an upstream effector of ROS generation, p38MAPK and JNK seem to be associated with ROS-independent cytotoxicity in neonatal rat brain. The cytotoxic damage progressed to apoptotic cell death at MGO concentration higher than those described for adult brain, suggesting that the neonatal brain is resistant to MGO-induced cell death. The consequences of MGO-induced brain damage early in life, remains to be clarified. However, it is feasible that high MGO levels during cortical and hippocampal development could be, at least in part, responsible for the impairment of cognitive functions in adulthood.

Exposure of young rats to diphenyl ditelluride during lactation affects the homeostasis of the cytoskeleton in neural cells from striatum and cerebellum.

In the present report we examined the effect of maternal exposure to diphenyl ditelluride (PhTe)(2) (0.01 mg/kg body weight) during the first 14 days of lactational period on the activity of some protein kinases targeting the cytoskeleton of striatum and cerebellum of their offspring. We analyzed the phosphorylating system associated with glial fibrillary acidic protein (GFAP), and neurofilament of low, medium and high molecular weight (NF-L, NF-M and NF-H, respectively) of pups on PND 15, 21, 30 and 45. We found that (PhTe)(2) induced hyperphosphorylation of all the proteins studied on PND 15 and 21, recovering control values on PND 30 and 45. The immunocontent of GFAP, NF-L, NF-M and NF-H in the cerebellum of 15-day-old pups was increased. Western blot assays showed activation/phosphorylation of Erk1/2 on PND 21 and activation/phosphorylation of JNK on PND 15. Otherwise, p38MAPK was not activated in the striatum of (PhTe)(2) exposed pups. On the other hand, the cerebellum of pups exposed to (PhTe)(2) presented activated/phosphorylated Erk1/2 on PND 15 and 21 as well as activated/phosphorylated p38MAPK on PND 21, while JNK was not activated. Western blot assays showed that both in the striatum and in the cerebellum of (PhTe)(2) exposed pups, the immunocontent of the catalytic subunit of PKA (PKAcα) was increased on PND 15. Western blot showed that the phosphorylation level of NF-L Ser55 and NF-M/NF-H KSP repeats was increased in the striatum and cerebellum of both 15- and 21-day-old pups exposed to (PhTe)(2). Diphenyl diselenide (PhSe)(2), the selenium analog of (PhTe)(2), prevented (PhTe)(2)-induced hyperphosphorylation of striatal intermediate filament (IF) proteins but it failed to prevent the action of (PhTe)(2) in cerebellum. Western blot assay showed that the (PhSe)(2) prevented activation/phosphorylation of Erk1/2, JNK and PKAcα but did not prevent the stimulatory effect of (PhTe)(2) on p38MAPK in cerebellum at PND 21. In conclusion, this study demonstrated that dam exposure to low doses of (PhTe)(2) can alter cellular signaling targeting the cytoskeleton of striatum and cerebellum in the offspring in a spatiotemporal manner, which can be related to the neurotoxic effects of (PhTe)(2).

Signaling mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neurons and astrocytes.

The studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein - GFAP) and neurons (low, medium and high molecular weight neurofilament subunits - NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100 µM QUIN was mainly due to increased Ca(2+) influx through NMDA and L-type voltage-dependent Ca(2+) channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca(2+) through NMDA receptors and L-VDCC, as well as Ca(2+) release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP(3) contributed to Ca(2+) release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca(2+) contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons.

Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats.

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 µM DL-AP5 and 50 µM MK801, 100 µM CNQX and 100 µM DNQX. Also, 10 µM verapamil and 10 µM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 µM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 µM) totally prevented this effect. Results obtained with 0.2 µM calyculin A (PP1 and PP2A inhibitor), 1 µM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 µM FK-506 or 100 µM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.

Cross-talk among intracellular signaling pathways mediates the diphenyl ditelluride actions on the hippocampal cytoskeleton of young rats.

In the present report, we showed that diphenyl ditelluride (PhTe)(2) induced in vitro hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) subunits in hippocampus of 21 day-old rats. Hyperphosphorylation was dependent on L-voltage dependent Ca(2+) channels (L-VDCC), N-methyl-d-aspartate (NMDA) and metabotropic glutamate receptors, as demonstrated by the specific inhibitors verapamil, DL-AP5 and MCPG, respectively. Also, dantrolene, a ryanodine channel blocker, EGTA and Bapta-AM, extra and intracellular Ca(2+) chelators respectively, totally prevented this effect. Activation of metabotropic glutamate receptors by (PhTe)(2) upregulates phospholipase C (PLC), producing inositol 1, 4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). Therefore, high Ca(2+) levels and DAG directly activate Ca(2+)/calmodulin-dependent protein kinase (PKCaMII) and protein kinase C (PCK), resulting in the hyperphosphorylation of Ser-57 in the carboxyl-terminal tail domain of the low molecular weight NF subunit (NF-L). Also, the activation of Erk1/2, and p38MAPK resulted in hyperphosphorylation of KSP repeats of the medium molecular weight NF subunit (NF-M). It is noteworthy that PKCaMII and PKC inhibitors prevented (PhTe)(2)-induced Erk1/2MAPK and p38MAPK activation as well as hyperphosphorylation of KSP repeats on NF-M, suggesting that PKCaMII and PKC could be upstream of this activation. Taken together, our results highlight the role of Ca(2+) as a mediator of the (PhTe)(2)-elicited signaling targeting specific phosphorylation sites on IF proteins of neural cells of rat hippocampus. Interestingly, this action shows a significant cross-talk among signaling pathways elicited by (PhTe)(2), connecting glutamate metabotropic cascade with activation of Ca(2+) channels. The extensively phosphorylated amino- and carboxyl- terminal sites could explain, at least in part, the neural dysfunction associated with (PhTe)(2) exposure.