Neurons Of Rat Cerebral Cortex Research Articles

Ubiquitin ligase RFWD2 promotes dendritic spine and synapse formation by activating the ERK/PEA3/c-Jun pathway in rat cerebral cortical neurons

The regulation of protein degradation through the ubiquitin–proteasome system is essential for normal brain development, axon growth, synaptic growth and plasticity. The E3 ubiquitin ligase RFWD2 plays a key role in the onset and development of neurological diseases, including the pathogenesis of Alzheimer's disease (AD), but the mechanisms controlling the homeostasis of neuronal synaptic proteins are still poorly understood. Here, we showed that the expression level of RFWD2 gradually decreased with the age of the rats and was negatively correlated with the development of cerebral cortical neurons and dendrites in vivo. RFWD2 was shown to localize to presynaptic terminals and some postsynaptic sides of both excitatory synapses and inhibitory synapses via colocalization with neuronal synaptic proteins (SYN, PSD95, Vglut1 and GAD67). Overexpression of RFWD2 promoted dendrite development and dendritic spine formation and markedly decreased the expression of synaptophysin and PSD95 by reducing the expression of ETV1, ETV4, ETV5 and c-JUN in vitro. Furthermore, the whole-cell membrane slice clamp results showed that RFWD2 overexpression resulted in greater membrane capacitance in neuronal cells, inadequate cell repolarization, and a longer time course for neurons to emit action potentials with decreased excitability. RFWD2 regulates dendritic development and plasticity, dendritic spine formation and synaptic function in rat cerebral cortex neurons by activating the ERK/PEA3/c-Jun pathway via a posttranslational regulatory mechanism and can be used as an efficient treatment target for neurological diseases.

THE INHIBITION OF AUTOPHAGY AND APOPTOSIS BY INSULIN AS A BASIS OF ITS NEUROPROTECTIVE ACTION ON RAT BRAIN CORTICAL NEURONS UNDER CONDITIONS OF OXIDATIVE STRESS IN VITRO

Insulin is one of the most promising neuroprotectors. A significant gap in understanding the mechanism of its action is the lack of data on whether it is able to prevent autophagic neuronal death. The aim of our work was to evaluate the contribution of autophagy and apoptosis to the death of rat cerebral cortex neurons in culture under oxidative stress and to study the ability of insulin to prevent this death and inhibit autophagy and apoptosis in neurons. The influence of hydrogen peroxide and insulin on the level of two main autophagy markers (LC3B-II and SQSTM1/p62) and apoptosis marker (cleaved сaspase-3) was studied. To assess the viability of neurons, the MTT test was used, and Western blotting was applied to measure the level of marker proteins. It was found that oxidative stress caused the activation of autophagy and apoptosis in neurons. This is manifested in a significant increase of the autophagy marker LC3B-II and apoptosis marker (cleaved сaspase-3) and in a decrease in the SQSTM1/p62 protein level. The content of SQSTM1/p62, which is involved in the formation of autophagosomes, decreases with the activation of autophagy, as this protein is degraded in lysosomes. Hydrogen peroxide causes autophagic and apoptotic death of neurons, as the inhibitors of autophagy (3-methyl adenine) and apoptosis (z-DEVD-FMK) were shown to increase the viability of neurons in conditions of oxidative stress. Insulin, in its turn, prevents the death of neurons and hinders autophagy, causing a decrease of the level of lipidated form LC3B-II and the increase of the SQSTM1/p62 protein level, it hinders apoptosis as well decreasing the level of cleaved caspase-3. The protective effect of insulin is mediated by the activation of specific signaling pathways associated with receptors of insulin and IGF-1, as the inhibitor of these receptors BMS-754807 completely blocks the neuroprotective effect of insulin. Thus, the pronounced activation of autophagy under oxidative stress is one of the causes of neuron death, and the protection of neurons by insulin is associated with the suppression of not only apoptotic, but also autophagic cell death.

Exploring the molecular mechanisms underlie the endoplasmic reticulum stress-mediated methylmercury-induced neuronal developmental damage

Methylmercury (MeHg) is a ubiquitous environmental pollutant, which can cross the placenta and blood brain barrier, thus affecting fetal growth and development. Although previous studies have demonstrated that MeHg induces endoplasmic reticulum (ER) stress in rat cerebral cortex and primary neurons, the role of ER stress in MeHg-induced neurodevelopmental toxicity remains unclear. Here, we used ICR pregnant mice and hippocampal neurons cells (HT22 cells) to investigate the molecular mechanism by which MeHg exposure during pregnancy affects neurodevelopment. We found that prenatal MeHg exposure caused developmental delay in offspring, accompanied with ER stress, cell apoptosis, cell cycle arrest and abnormal DNA methylation. Then, we used ER stress specific inhibitor 4-PBA and CHOP siRNA to investigate the role of ER stress on HT22 cells damage caused by MeHg. The results showed that 4-PBA pretreatment restored MeHg-induced axonal shortening and alleviated apoptosis, cell cycle arrest and DNA methylation. At the same time, the activation of CHOP/c-Jun/GADD45A signaling pathway was inhibited, and the interaction between CHOP and c-Jun was weakened. In addition, CHOP siRNA reduced the expression of c-Jun and GADD45A, and relieved DNA methylation levels to some extent. In summary, our study suggested that ER stress induced by MeHg mediated cell apoptosis and cell cycle arrest, and may affect DNA methylation through activation of CHOP/c-Jun/GADD45A signaling pathway, thus leading to neuronal damage.

The Study of Neuroprotective Mechanisms of the Ubiquinol Action in Experimental Focal Ischemia

Ischemic stroke is one of the most socially important diseases characterized by impaired cerebral circulation with focal damage of the brain tissue and decreased functionality. Despite the achievements of modern pharmacology, possibilities of pharmacotherapy of stroke remain limited, and the research for new drugs with neuroprotective effects that can prevent brain cell death still attracts much attention. In this study we have investigated the neuroprotective activity of ubiquinol used in a new innovative form for intravenous administration form on a rat model of permanent 24 h cerebral ischemia with evaluation of the mechanisms of its neuroprotective effect. Ubiquinol (30 mg/kg), administered intravenously in the acute period of permanent 24 h focal cerebral ischemia, had a direct neuroprotective effect, characterized by a decrease in the volume of brain tissue necrosis. The protective effect of ubiquinol was determined by its ability to inhibit the development of oxidative stress due to its direct anti-radical action, preventing the increase in the lipid hydroperoxide content in the brain tissue adjacent to the focus of necrosis, decreasing lipid oxidation rate in plasma under conditions of increased total antioxidant activity in the brain and blood of experimental animals. In vitro experiments have shown the ability of ubiquinol to prevent cell death in primary culture of rat cerebral cortex neurons under 4 h oxygen/glucose deprivation followed by 20 h reoxygenation.

Circular RNA circ_0000950 promotes neuron apoptosis, suppresses neurite outgrowth and elevates inflammatory cytokines levels via directly sponging miR-103 in Alzheimer’s disease

ABSTRACT This study aimed to investigate the effect of circ_0000950/miR-103 network on regulating neuron apoptosis, neurite outgrowth and inflammation in Alzheimer’s disease (AD). Cellular AD model of rat pheochromocytoma cell line PC12 cells and cellular AD model of rat cerebral cortex neurons were constructed, and the effect of circ_0000950 on apoptosis, neurite outgrowth and inflammation in both cellular AD models was determined through upregulation and knockdown of circ_0000950 expression by transfection. Compensation experiments and luciferase assay were further performed to validate the sponging effect of circ_0000950 on miR-103 as well as the mechanisms of circ_0000950/miR-103 on regulating apoptosis, neurite outgrowth and inflammation in both cellular AD models. Circ_0000950 reduced miR-103 expression and increased prostaglandin-endoperoxide synthase 2 (PTGS2) expression in both two cellular AD models. And circ_0000950 overexpression promoted neuron apoptosis, suppressed neurite outgrowth and elevated IL-1β, IL-6 and TNF-α levels compared with overexpression control, whereas circ_0000950 knockdown inhibited neuron apoptosis, enhanced neurite outgrowth and lowered IL-1β, IL-6 and TNF-α levels compared with shRNA control in both two cellular AD models. Compensation experiments along with luciferase reporter assay validated that circ_0000950 promoted cell apoptosis, suppressed neurite outgrowth and elevated inflammatory cytokines levels via directly sponging miR-103. In conclusion, circ_0000950 promotes neuron apoptosis, suppresses neurite outgrowth and elevates inflammatory cytokines levels through directly sponging miR-103 in AD.

Endoplasmic reticulum rather than mitochondria plays a major role in the neuronal apoptosis induced by polybrominated diphenyl ether-153

Polybrominated diphenyl ether-153 (BDE-153) has been demonstrated to induce neuronal apoptosis in rat cerebral cortex and primary neurons, however, the roles of mitochondria and endoplasmic reticulum (ER) remain unclear in the BDE-153-induced neuronal apoptosis. To this purpose, we observed the mitochondria and ER ultrastructure changes in the neuronal apoptosis in rats following BDE-153 treatment, detected the mitochondrial membrane potential (MMP), Ca2+-Mg2+-ATP enzyme activity, and the changes of mitochondria and ER apoptosis related molecules in rat cerebral cortex and in primary neurons following BDE-153 treatment. Results showed that compared to the control group, neuronal apoptosis was significantly increased in a dose-dependent manner in rat cerebral cortex and in primary neurons following BDE-153 treatment. In comparison with control, BDE-153 treatment induced remarkable ultrastructural changes in ER rather than in mitochondria, and the severity of ER damage was worse with the increasing BDE-153 dose. Meanwhile, ER apoptosis related molecules including caspase-12 (at mRNA level), cleaved caspase-12 (at protein level), and Tmem132a (at mRNA and protein levels) were significantly increased in the cerebral cortex in rats following BDE-153 treatment, while procaspase-12 protein was significantly decreased, comparing with control. In contrast, mitochondria apoptosis related molecules (MMP, Ca2+-Mg2+-ATP enzyme activity, cyt-C protein, caspase-3, 8, 9 mRNA, caspase-8, 9 enzyme activities) did not significantly changed in the cerebral cortex of rats or in primary neurons following BDE-153 treatment, except for the elevated caspase-3 mRNA and enzyme activity. Therefore, we conclude that BDE-153 induced neuronal apoptosis was dependent on p53, and mediated more by ER than mitochondria in the cerebral cortex of rats and in primary neurons. The findings suggest that ER is a potential sensitive target of BDE-153 neurotoxicity, providing a scientific evidence for the mechanism and intervention study on PBDE’s neurotoxicity.

Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques.

With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluorescence protocols, allowing for isolation of multiple target proteins in one tissue sample. Combining multiple immunofluorescence labeling with stereological data collection can provide a powerful tool to maximize explanatory power and efficiency, while minimizing tissue use. Multiple cell classes, subtypes of larger populations, or different cell states can be quantified in one case and even in one sampling run. Here, we present a protocol integrating stereological data collection and multiple immunofluorescence using commonly employed widefield epifluorescence filter sets, optimized for blue (DAPI), green (FITC), and far red (CY5) channels. Our stereological protocol has been designed to accommodate the challenges of fluorescence imaging to overcome limitations like fixed filter sets, photobleaching, and uneven immunolabeling. To enhance fluorescence signal for stereological sampling, our immunolabeling protocol utilizes both high temperature antigen retrieval to improve primary antibody binding and secondary antibodies conjugated to optimally stable fluorophores. To illustrate the utility of this approach, we estimated the number of Ctip2 immunoreactive subcerebral projection neurons and NeuN immunoreactive neurons in rat cerebral cortex at postnatal day 10. We used DAPI (blue) to define the neocortex, anti-NeuN (far red) to identify neurons, and co-labeling of anti-Ctip2 (green) and anti-NeuN (far red) to isolate only subcerebral projection neurons. Our protocol resulted in estimates with low sampling error (CE < 0.05) and high intrarater reliability (ICC > 0.98) that fall within the range of published values, attesting to its efficacy. We show our immunofluorescence techniques can be used to reliably identify other cell types, e.g., different glial cell classes, to highlight the broader applications of our approach. The flexibility of the technique, increasingly reduced costs of fluorescence technologies, and savings in experimental time and tissue use make this approach valuable for neuroscientists interested in incorporating stereology to ask precise neurophysiological and neuroanatomical questions.

Insulin Increases Viability of Neurons in Rat Cerebral Cortex and Normalizes Bax/Bcl-2 Ratio under Conditions of Oxidative Stress.

We studied the protective effect of insulin in various concentrations and its effect on the Bax/ Bcl-2 ratio in neurons of rat cerebral cortex under conditions of oxidative stress. The protective effect of insulin was dose-dependent within the nanomolar range (1 nM<10 nM<100 nM). Preincubation with insulin in concentrations of 100 nM and 1 μM significantly increased Bcl-2 content in neurons in 5, 30, and 45 min and 1, 2, and 4 h after the start of cell exposure to H2O2. This prooxidant increased the Bax/Bcl-2 ratio in neurons to 141-164% in comparison with the control (100%); preincubation of neurons with insulin returned this ratio to normal.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

Neurotrophins and cholinergic enzyme regulated by calpain-2: New insights into neuronal apoptosis induced by polybrominated diphenyl ether-153

Polybrominated diphenyl ether-153 (BDE-153) has been demonstrated to induce neuronal apoptosis in rat cerebral cortex and primary neurons. Neurotrophins and cholinergic enzymes play critical roles in the neuronal survival, maintenance, synaptic plasticity and learning memory, however, their roles in neuronal apoptosis following the BDE-153 treatment remain unclear. In this study, we firstly explored the possible predominant pathway underlying the neuronal apoptotic induced by the BDE-153 treatment in rat cerebral cortex, by measuring expression levels (mRNA and protein) of p53, caspase-3, 8, 9, calpain-1, and calpain-2, detected the levels (protein contents and mRNA) of neurotrophins including brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), and measured acetylcholinesterase (AchE) and choline acetyltransferase (ChaT) activities in rat cerebral cortex and primary neurons following BDE-153 treatment with or without pretreatment with inhibitors. Results showed that the neuronal apoptosis induced by BDE-153 was dependent on p53, and dependent on more calpain-2 than caspase-3 in the cerebral cortex of rats. Following the BDE-153 treatment, the protein contents and mRNA levels of BDNF, GDNF, NGF, NT-3, and NT-4, as well as the AchE and ChaT activities were significantly decreased in the cerebral cortex and primary neurons when compared to the untreated group. When pretreated primary neurons with calpain inhibitor PD150606 or cyclin-dependent kinase (cdk5, the downstream complex of calpain) inhibitor Roscovitine, the neurotrophins contents and activities of ChaT and AchE were reverted, along with the improvement of neuron survival compared with BDE-153 treatment alone. We conclude that neurotrophins and cholinergic enzymes were regulated by the calpain-2 activation and its downstream cdk5 pathway, and which was involved in the neuronal apoptosis induced by the BDE-153 treatment.

Exogenous Brain-Derived Neurotrophic Factor at a 50 ng/mL Concentration has a Significant Protective Effect on Bilirubin-Induced Cerebral Cortex Neuronal Injury.

Bilirubin encephalopathy is a common disabling disease in neonates. Brain-derived neurotrophic factor (BDNF) plays an important role in the repair and regeneration of nerves following injury, as well as the maintenance of neuronal growth and differentiation. This study aimed to investigate the effect of exogenous BDNF on the apoptosis and survival of in vitro cultured neurons injured by bilirubin as well as determine the optimal BDNF concentration. Rat cerebral cortex neurons cultured for 8 days were randomly divided into the control, bilirubin, and BDNF groups. A bilirubin injury model was established. BDNF was added to the BDNF group at a final concentration of 5, 50, and 100 ng/mL 4 hours prior to the addition of bilirubin. The cell morphology was observed via phase contrast microscopy, and the cell viability and apoptotic rate were detected using the methyl thiazolyl tetrazolium method and flow cytometry. The neuronal viability in the bilirubin group was significantly decreased and the apoptotic rate was significantly increased compared with the control group. BDNF significantly improved the impact of bilirubin on neuronal activity; the apoptotic rate was significantly decreased (p < 0.05); however, within 24 - 48 hours, BDNF had no significant effect on the nerve cell viability (p > 0.05). Moreover, the protective effect of different BDNF concentrations also varied (p < 0.05), with the most substantial protective effect at a final concentration of 50 ng/mL. Bilirubin may damage rat cerebral cortex neurons and induce their apoptosis, whereas BDNF has a protective effect on bilirubin-induced cerebral cortex neuronal injury.

In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex.

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABAA receptors (GABAA Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CBSH3- or CBSH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2SH3- or CB2SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CBSH3- or CBSH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291-1311, 2017. © 2016 Wiley Periodicals, Inc.

Upregulation of PRDM5 Is Associated with Astrocyte Proliferation and Neuronal Apoptosis Caused by Lipopolysaccharide.

PRDM5 (PR domain containing 5) belongs to PRDM family which consists of transcriptional regulators that modulate cellular processes such as cell growth, differentiation and apoptosis. However, the function of PRDM5 in central nervous system (CNS) inflammatory response is unknown. In recent study, an adult rat neuroinflammation model via lipopolysaccharide (LPS) lateral ventricle injection was constructed. PRDM5 expression was increased in activated astrocytes and apoptotic neurons of the adult rat cerebral cortex after LPS injection. In vitro studies showed that the remarkable upregulation of PRDM5 might be involved in rat primary astrocyte proliferation and rat primary neuronal apoptosis in the cerebral cortex following LPS administration. In addition, using PRDM5 RNA interference both in rat primary asrtocytes and neurons, further indicated that PRDM5 was required for astrocyte proliferation and neuronal apoptosis induced by LPS. Our findings on the cellular signaling pathway may provide a new therapeutic strategy against neuroinflammation in the CNS.

Modulation of GABA- and Kainate-Mediated Ion Currents in Isolated Rat Cerebral Cortex Neurons by Metabotropic Receptors

Patch clamp experiments with fixation of the membrane potential were performed on isolated rat prefrontal cortex neurons to study the modulation of ion currents induced by applications of GABA and kainate by GABAB receptors and group I metabotropic glutamate receptors. Blockade of GABAB receptors with the selective antagonist CGP-55845 (5 μM) increased the peak amplitude of the ion current induced by application of GABA (40 μM) by 26 ± 13% (n = 6). The amplitude of the ion current plateau at 14 sec of application of GABA was the same in controls as with blockade of GABAB receptors. The long-term effects of activation of GABAB receptors were studied in terms of the responses to application of GABA and kainate in the presence of baclofen (50 μM), a selective GABAB receptor agonist. Prolonged prior application of baclofen increased the amplitude of responses to application of GABA by 9 ± 2% (n = 8) as compared with controls. Responses to application of kainate did not change in the presence of baclofen. Analogous experiments using trans-ACPD, a selective agonist of groups I and II metabotropic glutamate receptors, did not reveal any changes in responses to application of GABA or kainate. These data point to modulation of GABAA receptors by postsynaptic metabotropic GABAB receptors in rat cerebral cortex neurons

Summation of GABA- and Glutamate-Mediated Ion Currents in Isolated Rat Cerebral Cortex Neurons

Patch clamp experiments in the whole cell configuration were performed on isolated rat prefrontal cortex neurons to study the summation of ion currents evoked by application of glutamate and GABA. Ion currents were recorded using two different pipette solutions, based on cesium chloride and fluoride. In recordings made using the cesium chloride-based solution, the peak amplitude of the current evoked by simultaneous application of GABA and glutamate (each at 200 μM) coincided with the peak amplitude of the current evoked by application of GABA alone, and was significantly smaller than the arithmetic sum of the responses to application of the two neurotransmitters individually. When the pipette solution based on cesium fluoride was used, the response to simultaneous application of glutamate and GABA was essentially the same as the arithmetic sum of the individual responses. On exposure to these neurotransmitters at saturating concentrations (5 mM), the response recorded to simultaneous application was significantly smaller than the response to application of GABA alone. These results suggest that there is a mechanism of interaction between GABAA and ionotropic glutamate receptors (AMPA and kainate). On simultaneous application of glutamate and GABA, activation of GABAA receptors evidently has a greater influence on glutamate receptors than activation of glutamate receptors of GABAA receptors.

Reduced numbers of cortical GABA-immunoreactive neurons in the chronic d-galactose treatment model of brain aging

Chronic administration of d-galactose (d-gal) is widely used to mimic the process of brain aging; however, the neural mechanisms are still poorly understood. In this study, we investigated the effect of long-term d-gal treatment on the number of GABA-immunoreactive neurons in rat cerebral cortex and the behavioral correlates. After eight weeks of daily subcutaneous injection of d-gal (100mg/ml/kg), rats showed reduced exploratory activity and lower ambulation in the open field compared to controls. There was no significant reduction in total neurons in the cortex, but there was a marked decrease in the number of GABA-immunoreactive neurons in all cortical layers of d-gal-treated rats. The ratio of GABA-immunoreactive neurons to total neurons was significantly lower in all cortical layers of d-gal-treated rats, with greatest reductions in output layers III (39.9% reduction), V (46.3%), and VI (48.4%). Our study provides the first evidence that chronic d-gal treatment may decrease cortical GABAergic neurotransmission, especially in cerebral output layers. The reduction in GABA-immunoreactive cell number likely disrupts the intracortical excitatory/inhibitory balance and may contribute to the behavioral deficits observed in this aging model.

Injury of cortical neurons is caused by the advanced glycation end products-mediated pathway.

Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

Effect of pulsed electromagnetic field exposure on adenosine receptors in rat brain

Different effects of pulsed electromagnetic field (PEMF) exposure on brain tissue have been described in pre-clinical models and in clinical settings. Nevertheless, the mechanism of action and the possible interaction with membrane receptors such as adenosine receptors (ARs) has not been investigated. The present study focused on the effect of PEMFs on A1 and A2A ARs in the rat cerebral cortex and cortical neurons. Affinity and density of ARs were evaluated by means of saturation binding experiments while mRNA expression was investigated through retro-transcription polymerase chain reaction (RT-PCR). PEMF treatment of the intact rat cerebral cortex or cortical neurons at 1.5 mT mediated a transient and significant increase in A2A ARs after 4 h (2.0-fold increase) and 6 h (1.4- and 1.8-fold increase, respectively) of exposure. In addition, PEMF treatment of the rat cerebral cortex and rat cortical neurons at 3 mT upregulated A2A ARs after 2 h (2.0- and 2.2-fold increase, respectively) and 4 h (1.6- and 1.9-fold increase, respectively). The treatment of rat cortex membranes with PEMFs at 1.5 and 3 mT induced an increase in A2A AR density after 2 h (1.9- and 2.2-fold increase, respectively) and was constant at all incubation times investigated. In rat cortical neurons, mRNA levels of A1 and A2A ARs were not affected by PEMF exposure for the times and intensities used. These results suggest that PEMF treatment has different biological effects in whole organs or cells in comparison with isolated membranes.

Ultrastructural characterization of rat neurons in primary culture

Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal™ medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14-21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1-10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30-50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission.

Effects of the flavonoid casticin from Brazilian Croton betulaster in cerebral cortical progenitors in vitro: Direct and indirect action through astrocytes

Neurodegenerative diseases are a major constraint on the social and economic development of many countries. Evidence has suggested that phytochemicals have an impact on brain pathology; however, both their mechanisms of action and their cell targets are incompletely known. Here, we investigated the effects of the flavonoid casticin, extracted from Croton betulaster, a common plant in the state of Bahia in Brazil, on rat cerebral cortex neurons in vitro. Treatment of neural progenitors with 10 microM casticin increased the neuronal population positive for the neuronal marker beta-tubulin III and the neuronal transcriptional factor Tbr2 by approximately 20%. This event was followed by a 50% decrease in neuronal death. Pools of astrocyte (GFAP and S100beta), neural (nestin), and oligodendrocyte (Olig2 and NG2) progenitors were not affected by casticin. Neither neuronal commitment nor proliferation of progenitors was affected by casticin, suggesting a neuroprotective effect of this compound. Culture of neural progenitors on casticin-treated astrocyte monolayers increased the neuronal population by 40%. This effect was reproduced by conditioned medium derived from casticin-treated astrocytes, suggesting the involvement of a soluble factor. ELISA assays of the conditioned medium revealed a 20% increase in interleukin-6 level in response to casticin. In contrast to the direct effect, neuronal death was unaffected, but a 52% decrease in the death of nestin-positive progenitors was observed. Together our data suggest that casticin influences the neuronal population by two mechanisms: 1) directly, by decreasing neuronal death, and 2) indirectly, via astrocytes, by modulating the pool of neuronal progenitors.