Developing Rat Brain Research Articles

DNA methylation markers in the postnatal developing rat brain

In spite of intense interest in how altered epigenetic processes including DNA methylation may contribute to psychiatric and neurodevelopmental disorders, there is a limited understanding of how methylation processes change during early postnatal brain development. The present study used in situ hybridization to assess mRNA expression for the three major DNA methyltranserases (DNMTs) – DNMT1, DNMT3a and DNMT3b – in the developing rat brain at seven developmental timepoints: postnatal days (P) 1, 4, 7, 10, 14, 21, and 75. We also assessed 5-methylcytosine levels (an indicator of global DNA methylation) in selected brain regions during the first three postnatal weeks. DNMT1, DNMT3a and DNMT3b mRNAs are widely expressed throughout the adult and postnatal developing rat brain. Overall, DNMT mRNA levels reached their highest point in the first week of life and gradually decreased over the first three postnatal weeks within the hippocampus, amygdala, striatum, cingulate and lateral septum. Global DNA methylation levels did not follow this developmental pattern; methylation levels gradually increased over the first three postnatal weeks in the hippocampus, and remained stable in the developing amygdala and prefrontal cortex. Our results contribute to a growing understanding of how DNA methylation markers unfold in the developing brain, and highlight how these developmental processes may differ within distinct brain regions.

Phospholipase A2, Oxidative Stress, and Neurodegeneration in Binge Ethanol-Treated Organotypic Slice Cultures of Developing Rat Brain

Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration. HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined. 3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration. In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.

Reversal effect of monoisoamyl dimercaptosuccinic acid (MiADMSA) for arsenic and lead induced perturbations in apoptosis and antioxidant enzymes in developing rat brain

Oxidative stress (OS) has been implicated in the pathophysiology of many neurodegenerative disorders. Several studies have shown that exposure to arsenic (As) and lead (Pb) produces oxidative stress, one of the most noted molecular mechanisms for the neurotoxicity of these metals. In the present study, we examined the effect of combined exposure to these metals (As and Pb) on the activity levels of antioxidant enzymes and apoptotic marker enzymes in brain regions (cerebral cortex, hippocampus and cerebellum) of rats at postnatal day (PND) 21, 28 and 3 months age and compared the toxicity levels with individual metals (As or Pb). Further, we also evaluated the therapeutic efficacy of a chelating agent, monoisoamyl dimercaptosuccinic acid (MiADMSA) against arsenic and lead induced developmental neurotoxicity. Pregnant rats were exposed to sodium meta-arsenite (50ppm) and lead acetate (0.2%) individually, and in combination (As=25ppm+Pb=0.1%) via drinking water throughout perinatal period (GD 6 to PND 21). MiADMSA (50mg/kg, orally through gavage) was given for three consecutive days to the PND 18 pups (i.e., PND 18 to PND 20). Exposure to metal mixture resulted in a significant decrease in the activity levels of antioxidant enzymes such as manganese-superoxide dismutase (Mn-SOD), Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) while the malondialdehyde (MDA) levels and mRNA expression levels of caspase-3 and caspase-9 were significantly increased in all the three brain regions. The observed alterations were greater with exposure to metal mixture than individual metals (As or Pb) and the changes were more prominent at PND 28 and greater in cerebral cortex than hippocampus and cerebellum. Interestingly, chelation therapy with MiADMSA showed significant recovery in antioxidant enzymes, lipid peroxidation and gene expression levels of caspase-3 and caspase-9. From these findings, it can be concluded that combined exposure to As and Pb showed an additive effect on antioxidant enzymes than individual metal exposure and chelation therapy with MiADMSA significantly reversed the As and Pb induced apoptosis and oxidative stress, a major contributing factor to neurotoxicity.

Protective effects of pentoxifylline on lipopolysaccharide-induced white matter injury in a rat model of periventricular leukomalasia

Objective: To investigate the potential neuroprotective effect of maternal pentoxifylline (PNTX) treatment in endotoxin-induced periventricular leukomalasia (PVL) in the developing rat brain.Method: Intraperitoneal injection of lipopolysaccharide was administered on two of three Wistar pregnant rats to establish PVL. To obtain PNTX-treated group, one of the two dams were injected with PNTX. The control group was treated with saline. Rat pups were grouped as control, maternal LPS-treated group and PNTX + LPS-treated group. At 7th postnatal days, apoptosis and hypomyelination were evaluated. Apoptosis was evaluated by caspase-3 and terminal deoxynucleotidyl transferase [TdT] dUTP nick endlabelling reaction (TUNEL) immunostaining. To assess hypomyelination, myelin basic protein (MBP) staining, as a marker of myelination, was evaluated.Results: MBP staining was significantly less and weaker in the brains of the LPS-treated group as compared with the PNTX-treated group. PNTX treatment significantly reduced the number of apoptotic cells in the periventricular WM shown on Tunel and caspase-3.Conclusions: Presented study is first indicated that PNTX may provide protection against an LPS-induced inflammatory response and WMI in the developing rat brain. Our results also suggest that PNTX treatment in pregnant women with maternal or placental infection may minimize the risk of PVL and cerebral palsy.

Neonatal treatment with monosodium glutamate lastingly facilitates spreading depression in the rat cortex

AimsMonosodium glutamate (MSG) is a neuroexcitatory amino acid used in human food to enhance flavor. MSG can affect the morphological and electrophysiological organization of the brain. This effect is more severe during brain development. Here, we investigated the electrophysiological and morphological effects of MSG in the developing rat brain by characterizing changes in the excitability-related phenomenon of cortical spreading depression (CSD) and microglial reaction. Main methodsFrom postnatal days 1–14, Wistar rat pups received 2 or 4g/kg MSG (groups MSG-2 and MSG-4, respectively; n=9 in each group), saline (n=10) or no treatment (naïve group; n=5) every other day. At 45–60days, CSD was recorded on two cortical points for 4h. The CSD parameters velocity, and amplitude and duration of the negative potential change were calculated. Fixative-perfused brain sections were immunolabeled with anti-IBA-1 antibodies to identify and quantify cortical microglia. Key findingsMSG-4 rats presented significantly higher velocities (4.59±0.34mm/min) than the controls (saline, 3.84±0.20mm/min; naïve, 3.71±0.8mm/min) and MSG-2 group (3.75±0.10mm/min). The amplitude (8.8±2.2 to 11.2±1.9mV) and duration (58.2±7.1 to 73.6±6.0s) of the negative slow potential shift was similar in all groups. MSG-treatment dose-dependently increased the microglial immunolabeling. SignificanceThe results demonstrate a novel, dose-dependent action of MSG in the developing brain, characterized by acceleration of CSD and significant microglial reaction in the cerebral cortex. The CSD effect indicates that MSG can influence cortical excitability, during brain development, as evaluated by CSD acceleration. Data suggest caution when consuming MSG, especially in developing organisms.

Dexamethasone induces apoptosis of progenitor cells in the subventricular zone and dentate gyrus of developing rat brain

The use of dexamethasone in premature infants to prevent and/or treat bronchopulmonary dysplasia adversely affects neurocognitive development and is associated with cerebral palsy. The underlying mechanisms of these effects are multifactorial and likely include apoptosis. The objective of this study was to confirm whether dexamethasone causes apoptosis in different regions of the developing rat brain. On postnatal day 2, pups in each litter were randomly divided into the dexamethasone-treated (n = 91) or vehicle-treated (n = 92) groups. Rat pups in the dexamethasone group received tapering doses of dexamethasone on postnatal days 3-6 (0.5, 0.25, 0.125, and 0.06 mg/kg/day, respectively). Dexamethasone treatment significantly decreased the gain of body and brain weight and increased brain caspase-3 activity, DNA fragments, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and cleaved caspse-3-positive cells at 24 hr after treatment. Dexamethasone increased cleaved caspse-3-positive cells in the cortex, thalamus, hippocampus, cerebellum, dentate gyrus, and subventricular zone. Double-immunofluorescence studies show that progenitor cells in the subventricular zone and dentate gyrus preferentially undergo apoptosis following dexamethasone exposure. These results indicate that dexamethasone-induced apoptosis in immature cells in developing brain is one of the mechanisms of its neurodegenerative effects in newborn rats.

Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina

Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2′-, 3′-cyclic-nucleotide-3′-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developing rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology.

Diffusion tensor magnetic resonance histology reveals microstructural changes in the developing rat brain

The postnatal period is a remarkably dynamic phase of brain growth and development characterized by large-scale macrostructural changes, as well as dramatic microstructural changes, including myelination and cortical layering. This crucial period of neurodevelopment is uniquely susceptible to a wide variety of insults that may lead to neurologic disease. MRI is an important tool for studying both normal and abnormal neurodevelopmental changes, and quantitative imaging strategies like diffusion tensor imaging (DTI) allow visualization of many of the complex microstructural changes that occur during postnatal life. Diffusion tensor magnetic resonance histology (DT-MRH) provides particularly unique insight into cytoarchitectural changes in the developing brain. In this study, we used DT-MRH to track microstructural changes in the rat brain throughout normal postnatal neurodevelopment. We provide examples of diffusion tensor parameter changes in both white matter and gray matter structures, and correlate these changes with changes in cytoarchitecture. Finally, we provide a comprehensive database of image sets as a foundation for future studies using DT-MRH to characterize abnormal neurodevelopment in rodent models of neurodevelopmental disease.

Analysis of EphA5 receptor in the developing rat brain: an in vivo study in congenital hypothyroidism model

The EphA5 receptor has recently been known to play an important role in the initiation of the early phase of synaptogenesis, during which irreparable harm would be done to the developing brain in the absence of sufficient thyroid hormone (TH). In the present article, we aimed to analyze the characteristics of EphA5 receptor expression in the brain of congenital hypothyroid rats. The results showed that the levels of the EphA5 receptor were downregulated by TH deficiency in the developing rat brain with remarkable spatial and temporal characteristics. In the hypothyroid rats, the mRNA and protein levels of EphA5 receptor decreased significantly in the hippocampus (27.92-53.26%), cerebral cortex (12.52-47.16%), and cerebellum (8.72-31.69%) compared with those in the normal rats from postnatal day 0 (P0) to P21 (p < 0.01). The expression of EphA5 receptor was highest and declined most as much as 53% in the hippocampus with TH deficiency. At P7, the EphA5 receptor decreased most prominently during all the observed time point. The EphA5 receptor plays actively in the brain development in congenital hypothyroid rats. Our study highlights the high expression of EphA5 receptor protein in hippocampus and dramatic changes at P7 in condition of TH deficiency, which may provide important basis for further investigations in manipulating congenital hypothyroidism.

Early Exposure to General Anesthesia Disturbs Mitochondrial Fission and Fusion in the Developing Rat Brain

General anesthetics induce apoptotic neurodegeneration in the developing mammalian brain. General anesthesia (GA) also causes significant disturbances in mitochondrial morphogenesis during intense synaptogenesis. Mitochondria are dynamic organelles that undergo remodeling via fusion and fission. The fine balance between these two opposing processes determines mitochondrial morphometric properties, allowing for their regeneration and enabling normal functioning. As mitochondria are exquisitely sensitive to anesthesia-induced damage, we examined how GA affects mitochondrial fusion/fission. Seven-day-old rat pups received anesthesia containing a sedative dose of midazolam followed by a combined nitrous oxide and isoflurane anesthesia for 6 h. GA causes 30% upregulation of reactive oxygen species (n = 3-5 pups/group), accompanied by a 2-fold downregulation of an important scavenging enzyme, superoxide dismutase (n = 6 pups/group). Reactive oxygen species upregulation is associated with impaired mitochondrial fission/fusion balance, leading to excessive mitochondrial fission. The imbalance between fission and fusion is due to acute sequestration of the main fission protein, dynamin-related protein 1, from the cytoplasm to mitochondria, and its oligomerization on the outer mitochondrial membrane. These are necessary steps in the formation of the ring-like structures that are required for mitochondrial fission. The fission is further promoted by GA-induced 40% downregulation of cytosolic mitofusin-2, a protein necessary for maintaining the opposing process, mitochondrial fusion (n = 6 pups/group). Early exposure to GA causes acute reactive oxygen species upregulation and disturbs the fine balance between mitochondrial fission and fusion, leading to excessive fission and disturbed mitochondrial morphogenesis. These effects may play a causal role in GA-induced developmental neuroapoptosis.

Activation of UTP-Sensitive P2Y2Receptor Induces the Expression of Cholinergic Genes in Cultured Cortical Neurons: A Signaling Cascade Triggered by Ca2+Mobilization and Extracellular Regulated Kinase Phosphorylation

ATP functions as an extracellular signaling molecule that is costored and coreleased with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP upregulates the expression of synaptic genes in muscle-including the genes for nicotine acetylcholine receptor (α-, δ-, and ε-subunits) and acetylcholinesterase (AChE)-via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it colocalized with the postsynaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE), and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca(2+) mobilization and extracellular regulated kinases 1/2 activation. The importance of P2Y2R action was further shown by the receptor's synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca(2+) influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrated the influence of costimulation of P2Y1R and P2Y2R in neurons.

Application of BrdU in studying postnatal neurogenesis

PURPOSES: We applied the thymidine analog, 5-bromo-2'-deoxyuridine (BrdU) that labels DNA to study neurogenic events in many brain regions of the postnatal mammalian brain and to evaluate its paradigms, pitfalls and limitations. MATERIAL AND METHODS: Different BrdU paradigms and various time points between postnatal day P0 and P51 were applied to Wistar rats. Paraffin sections for single or double BrdU immunohistochemistry combined with neuronal markers nestin, Dcx, MAP(2) and NeuN by peroxidase or fluorescence labeling were examined. Sections were analyzed using standard or confocal microscopy and stereology. RESULTS: Peak neurogenesis was found out in the dentate gyrus during the first postnatal week of life with a progressive decline after P9. Areas with substantial neurogenesis such as subventricular zone of the lateral ventricles, subgranular zone of the hippocampal dentate gyrus, and cerebellum were observed. By P14 to P18, cell proliferation was reduced in most brain areas with exception of the dentate gyrus. Numerical densities of BrdU(+) cells in all evaluated brain regions were higher in the younger animals and decreased with age. Most newly-generated cells became nonneuronal cells (endothelial and glial cells). BrdU colocalization with neuronal markers was rarely found out mainly within the dentate gyrus and cerebellum. CONCLUSION: In the developing rat brain, BrdU labeling showed regional distribution. It was dose-, age- and survival time-dependent. Our findings provided useful information on BrdU application in the study of postnatal neurogenesis. Scripta Scientifica Medica 2013; 45(1): 24-28.

Effects of Perinatal Lipopolysaccharide (LPS) Exposure on the Developing Rat Brain; Modeling the Effect of Maternal Infection on the Developing Human CNS

In the present study, we examined the effect of perinatal Escherichia coli lipopolysaccharide (LPS) exposure on the developing rat cerebellum and tested the hypothesis that maternal infections impact brain structure and function by mechanisms involving increase in oxidative stress and changes in brain type 2 iodothyronine deiodinase (D2)- and thyroid hormone (TH)-responsive genes. Spontaneously hypertensive rat (SHR) and Sprague-Dawley (SD) rat dams were challenged with LPS (200 μg/kg body weight) exposure during pregnancy (G10-G15) and lactation (P5-P10), the time periods corresponding, respectively, to the first/second and the third trimesters of human pregnancy. LPS exposure resulted in a significantly decreased motor learning in SD male (29.8 %) and in female (55.0 %) pups (p < 0.05); changes in rollover and startle response showed only a trend. The LPS challenge also resulted in a trend (p = 0.09) toward increased cerebellar levels of the oxidative stress marker 3-nitrotyrosine (3-NT) in SD male (16.2 %) and female (21.2 %) neonates, while 3-NT levels were significantly decreased (p < 0.05) in SHR female pups. D2 activity, responsible for local intra-brain conversion of thyroxine (T4) to the active hormone, 3',3,5-triiodothyronine (T3), was significantly (p < 0.05) decreased in LPS-challenged SHR male (40.3 %) and SD female (47.4 %) pups. Several genes were affected by LPS. Notably, D2 (DIO2) and brain-derived neurotrophic factor (BDNF) were significantly elevated in SHR females, while transthyretin (TTR) expression was decreased in both SD males and females (P < 0.05). In vitro chronic exposure of cerebellar cultures to LPS resulted in decreased arborization of Purkinje cells while D2 was only increased transiently. Our data demonstrate that perinatal LPS exposure impacts the developing cerebellum in strain- and sex-dependent manner via complex mechanisms that involve changes in oxidative stress, enzymes involved in maintaining local TH homeostasis, and downstream gene expression.

Morphine-enhanced apoptosis in selective brain regions of neonatal rats

Prolonged neonatal opioid exposure has been associated with: antinociceptive tolerance, long-term neurodevelopmental delay, cognitive, and motor impairment. Morphine has also been shown to induce apoptotic cell death in vitro studies, but its in vivo effect in developing rat brain is unknown. Thus, we hypothesized that prolongued morphine administration in neonatal rats in a model of antinociceptive tolerance and dependence is associated with increased neuroapoptosis. We analyzed neonatal rats from the following groups (1) naïve group (n=6); (2) control group (normal saline (NS), n=5), and (3) morphine group (n=8). Morphine sulfate or equal volume of NS was injected subcutaneously twice daily for 6½ days starting on postnatal day (PD) 1. Development of antinociceptive tolerance was previously confirmed by Hot Plate test on the 7th day. Evidence of neuronal and glial apoptosis was determined by cleaved caspase-3 immunofluorescence combined with specific markers. At PD7, morphine administration after 6½ days significantly increased the density of apoptotic cells in the cortex and amygdala, but not in the hippocampus, hypothalamus, or periaqueductal gray. Apoptotic cells exhibited morphology analogous to neurons. Irrespective of the treatment, only a very few individual microglia but not astrocytes were caspase-3 positive. In summary, repeated morphine administration in neonatal rats (PD1–7) is associated with increased supraspinal apoptosis in distinct anatomical regions known to be important for sensory (cortex) and emotional memory processing (amygdala). Brain regions important for learning (hippocampus), and autonomic and nociceptive processing (hypothalamus and periaqueductal gray) were not affected. Lack of widespread glial apoptosis or robust glial activation following repeated morphine administration suggests that glia might not be affected by chronic morphine at this early age. Future studies should investigate long-term behavioral sequelae of demonstrated enhanced apoptosis associated with prolonged morphine administration in a neonatal rat model.

Dual Effects of Isoflurane on Proliferation, Differentiation, and Survival in Human Neuroprogenitor Cells

Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. Using the ReNcell CX (Millipore, Billerica, MA) human neural progenitor cell line, the authors investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, the authors assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of inositol-1,4,5-trisphosphate or ryanodine receptor activation to these processes. Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate and promoted glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with inositol-1,4,5-trisphosphate or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane-preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation, and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized inositol-1,4,5-trisphosphate and/or ryanodine receptors.

Oxygen Toxicity Is Reduced by Acetylcholinesterase Inhibition in the Developing Rat Brain

The cholinergic anti-inflammatory pathway is a neural mechanism that suppresses the innate inflammatory response and controls inflammation employing acetylcholine as the key endogenous mediator. In this study, we investigated the effects of the cholinergic agonists, physostigmine and donepezil, on neurodegeneration, inflammation and oxidative stress during oxygen toxicity in the developing rat brain. The aim of this study was to investigate the level of neurodegeneration, expression of proinflammatory cytokines, glutathione and lipid peroxidation after hyperoxia and treatment with the acetylcholinesterase (AChE) inhibitors, physostigmine and donepezil in the brain of neonatal rats. Six-day-old Wistar rats were exposed to 80% oxygen for 12-24 h and received 100 μg/kg physostigmine or 200 μg/kg donepezil intraperitoneally. Sex-matched littermates kept in room air and injected with normal saline, physostigmine or donepezil served as controls. Treatment with both inhibitors significantly reduced hyperoxia-triggered activity of AChE, neural cell death and the upregulation of the proinflammatory cytokines IL-1β and TNF-α in the immature rat brain on the mRNA and protein level. In parallel, hyperoxia-induced oxidative stress was reduced by concomitant physostigmine and donepezil administration, as shown by an increased reduced/oxidized glutathione ratio and attenuated malondialdehyde levels, as a sign of lipid peroxidation. Our results suggest that a single treatment with AChE inhibitors at the beginning of hyperoxia attenuated the detrimental effects of oxygen toxicity in the developing brain and may pave the way for AChE inhibitors, which are currently used for the treatment of Alzheimer's disease, as potential candidates for adjunctive neuroprotective therapies to the immature brain.

Expressions of cannabinoid receptor 1 and L1 cell adhesion molecule in brain of rats with intrauterine growth retardation

Objective To study the mechanism of brain development delay in rats with intrauterine growth retardation(IUGR) through establishing IUGR animal model by low protein diet during pregnancy and examining the expressions of cannabinoid receptor1(CB1) and L1 cell adhesion molecule (NCAM-L1)in IUGR and normal rats. Methods Thirty-two pregnant rats were randomly fed with normal diet or lower protein diet during pregnancy (16 rats in each group). The offspring rats were divided into IUGR group and control group by birth weight, and sacrificed on day 0, 7, 14, 21 after birth, brain weight was recorded.The expressions of CB1 and NCAM-L1 in the brain were examined by immunohistochemistry staining.Image-Pro Plus 5.1 image processing software was used for semi-quantitative analysis.The integrated optical density of the CB1 and NCAM-L1 of the positive cells was calculated. Results The expressions of CB1 and NCAM-L1 in offspring rats brain were basically in the same area, the changes in both was in accord with the brain weight change.The expression of CB1 in the brain of the pup rats in the IUGR group was significantly lower than that in the control group 0, 7, 14, 21 days after birth, by contrast, the expression of NCAM-L1 was significantly higher(all P<0.05), and the expressions of CB1 and NCAM-L1 were negatively correlated between both groups(P=0.032, 0.010). Conclusions CB1 and NCAM-L1 were involved in the process of rat brain development.The effect of CB1 on rat brain development may be achieved by NCAM-L1. Key words: Intrauterine growth retardation; Cannabinoid receptor 1; L1 cell adhesion molecule; Rat

Ontogeny repeats the phylogenetic recruitment of the cargo exporter cornichon into AMPA receptor signaling complexes

Besides mediating most of the fast excitatory neurotransmission in the mammalian CNS, ionotropic glutamate receptors of the AMPA subtype (AMPARs) serve highly diverse functions in brain development controlling neuronal migration, synaptic growth, and synaptic maturation. Pioneering proteomic studies suggest that this functional diversity is met by a great molecular complexity in native AMPAR composition. Here, we have investigated the expression patterns of two recently identified AMPAR constituents, the cornichon homologues CNIH-2 and CNIH-3, and their assembly with the AMPAR core subunits GluA1-4 in developing rat brain. Unlike GluA1-4 expression, which is up-regulated during postnatal brain development, the two cornichon homologues show maximum mRNA and protein expression early after birth, which then decline towards adulthood. Despite rather reciprocal expression profiles, the overall ratio of CNIH-2/3 complexed with GluAs remains constant throughout development. Our data reveal an excess amount of AMPAR-free CNIH-2/3 early in development, which might serve the evolutionarily conserved role of cornichon as a cargo exporter. With progressing development, however, the amount of AMPAR-free CNIH-2/3 subsides, whereas the one being integrated into AMPAR complexes increases. Hence, the cornichon homologues CNIH-2/3 gain importance in their role as auxiliary subunits of native AMPARs during ontogeny, which reflects their functional evolution in phylogeny.

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

BackgroundNeural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.Methodology/Principal FindingsIn this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.ConclusionsThis study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.

Decreased Zinc Availability Affects Glutathione Metabolism in Neuronal Cells and in the Developing Brain

A deficit in zinc (Zn) availability can increase cell oxidant production, affect the antioxidant defense system, and trigger oxidant-sensitive signals in neuronal cells. This work tested the hypothesis that a decreased Zn availability can affect glutathione (GSH) metabolism in the developing rat brain and in neuronal cells in culture, as well as the capacity of human neuroblastoma IMR-32 cells to upregulate GSH when challenged with dopamine (DA). GSH levels were low in the brain of gestation day 19 (GD19) fetuses from dams fed marginal Zn diets throughout gestation and in Zn-deficient IMR-32 cells. γ-Glutamylcysteine synthetase (GCL), the first enzyme in the GSH synthetic pathway, was altered by Zn deficiency (ZD). The protein and mRNA levels of the GCL modifier (GCLM) and catalytic (GCLC) subunits were lower in the Zn-deficient GD19 fetal brain and in IMR-32 cells compared with controls. The nuclear translocation of transcription factor nuclear factor (erythroid-derived 2)-like 2, which controls GCL transcription, was impaired by ZD. Posttranslationally, the caspase-3-dependent GCLC cleavage was high in Zn-deficient IMR-32 cells. Cells challenged with DA showed an increase in GCLM and GCLC protein and mRNA levels and a consequent increase in GSH concentration. Although Zn-deficient cells partially upregulated GCL subunits after exposure to DA, GSH content remained low. In summary, results show that a low Zn availability affects the GSH synthetic pathway in neuronal cells and fetal brain both at transcriptional and posttranslational levels. This can in part underlie the GSH depletion associated with ZD and the high sensitivity of Zn-deficient neurons to pro-oxidative stressors.