Mei Qiang

Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons.

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-d-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.

Chronic Intermittent Ethanol Exposure and Its Removal Induce a Different miRNA Expression Pattern in Primary Cortical Neuronal Cultures

BACKGROUND Increasing evidence indicates that repeated exposure to and withdrawal from alcohol can result in persistent molecular and cellular adaptations. One molecular adaptation that occurs is the regulation of gene expression, which is thought to lead to the functional alterations that characterize addiction: tolerance, dependence, withdrawal, craving, and relapse. MicroRNAs (miRNAs) have been recently identified as master regulators of gene expression through post-transcriptional regulation. However, the role of miRNAs in the neuroadaptations after alcohol removal has not yet been directly addressed. METHODS We employed a chronic intermittent ethanol (CIE) model in primary cortical neuronal cultures to examine the global extent of differential miRNA expression using a TaqMan real-time PCR miRNA array. RESULTS Sixty-two miRNAs were differentially expressed after 10 days of CIE (CIE10) treatment (n = 42 with false discovery rate [FDR] < 0.05 and fold change > 2) and 5 days post-CIE (P5) treatment (n = 26) compared with untreated control values. Compared to CIE10, ethanol (EtOH) removal experience in P5 induced a distinct expression pattern, including 20 differentially expressed miRNAs, which did not exhibit a significant change at CIE10. The predicted target molecules of EtOH removal-induced miRNAs function mainly in the regulation of gene transcription, but also function in neuron differentiation, embryonic development, protein phosphorylation, and synaptic plasticity. Interestingly, some of the miRNAs differentially expressed 5 days after CIE treatment were found to cluster on chromosomes near CpG islands, suggesting that they share functional similarity by targeting alcohol-related genes. CONCLUSIONS Taken together, these results suggest a potential role of differentially expressed miRNAs in mediating EtOH removal-related phenotypes.

The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.

Background The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5′-regulatory area following CIE treatment. Methods Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5′-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. Results Analysis of individual CpG methylation sites within the NR2B 5′regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors. Conclusions These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.

Second trimester prenatal alcohol exposure alters development of rat corpus callosum

Prenatal alcohol exposure produces many developmental defects of the central nervous system (CNS), such as in the corpus callosum (CC). This study was designed to observe the effect of prenatal alcohol exposure during the second trimester equivalent on the development of dendritic arbors of CC projection neurons (CCpn) in rat visual cortex. In addition, the effect of second trimester equivalent prenatal alcohol exposure on brain weight was determined. Pregnant dams received 1.2-6.0 g/kg ethanol (EtOH) during gestational day (G) 11-20. Controls consisted of normal and nutritionally matched pairfed (PF) dams. Pups were sacrificed on the day of birth, G26, G29 and G33. DiI crystals were placed in the midsagittal CC bundle to retrogradely label CCpn. Images of visual cortex were obtained from tissue slices using a confocal laser scanning microscope. The number and length of apical and basilar dendrite branches were determined. The results show that prenatal alcohol exposure restricted to the second trimester equivalent alters the development of the CCpn dendritic arbor and the brain weight in a blood alcohol concentration (BAC)-dependent manner. The alteration in the EtOH CCpn is manifested as an increase in the number and length of CCpn apical and basilar dendrite branches, while brain weight is reduced compared with Controls.

Role of AP‐1 in ethanol‐induced N‐methyl‐d‐aspartate receptor 2B subunit gene up‐regulation in mouse cortical neurons

Activator protein 1 (AP‐1) has been reported to regulate the gene expression in a wide variety of cellular processes in response to stimuli. In this study, we investigated the DNA‐protein binding activities and promoter activity in the N‐methyl‐d‐aspartate R2B (NR2B) gene AP‐1 site in normal and ethanol‐treated cultured neurons. The identity of the AP‐1 site as the functional binding factor is suggested by the specific binding of nuclear extract derived from cultured cortical neurons to the labeled probes and the specific antibody‐induced supershift. Mutations in the core sequence resulted in a significantly reduced promoter activity and the ability to compete for the binding. Moreover, treatment of the cultured neuron with 75 mm ethanol for 5 days caused a significant increase in the AP‐1 binding activity and promoter activity. The AP‐1 DNA‐binding complex in control and ethanol‐treated nuclear extract was composed of c‐Fos, FosB, c‐Jun, JunD, and phosphorylated CREB (p‐CREB). Western blot analysis showed that p‐CREB and FosB significantly increased, whereas c‐Jun decreased. The DNA affinity precipitation assay indicated that FosB, p‐CREB, and c‐Jun increased in the AP‐1 complex following ethanol treatment. These results suggest that AP‐1 is an active regulator of the NR2B transcription and ethanol‐induced changes may result at multiple levels in the regulation including AP‐1 proteins expression, CREB phosphorylation and perhaps reorganization of dimmers.

Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.

Epigenetic Mechanisms Are Involved in the Regulation of Ethanol Consumption in Mice

Background: Repeated alcohol exposure is known to increase subsequent ethanol consumption in mice. However, the underlying mechanisms have not been fully elucidated. One postulated mechanism involves epigenetic modifications, including histone modifications and DNA methylation of relevant genes such as NR2B or BDNF. Methods: To investigate the role of epigenetic mechanisms in the development of alcohol drinking behavior, an established chronic intermittent ethanol exposure reinforced ethanol drinking mouse model with vapor inhalation over two 9-day treatment regimens was used. The DNA methyltransferase inhibitor, 5-azacytidine or the histone deacetylase inhibitor, Trichostatin A was administered (intraperitoneally) to C57BL/6 mice 30min before daily exposure to chronic intermittent ethanol. Changes in ethanol consumption were measured using the 2-bottle choice test. Results: The results indicated that systemic administration of Trichostatin A (2.5 µg/g) facilitated chronic intermittent ethanol-induced ethanol drinking, but systemic administration of 5-azacytidine (2 µg/g) did not cause the same effect. However, when 5-azacytidine was administered by intracerebroventricular injection, it facilitated chronic intermittent ethanol-induced ethanol drinking. Furthermore, the increased drinking caused by chronic intermittent ethanol was prevented by injection of a methyl donor, S-adenosyl-L-methionine. To provide evidence that chronic intermittent ethanol- or Trichostatin A-induced DNA demethylation and histone modifications of the NR2B promoter may underlie the altered ethanol consumption, we examined epigenetic modifications and NR2B expression in the prefrontal cortex of these mice. Chronic intermittent ethanol or Trichostatin A decreased DNA methylation and increased histone acetylation in the NR2B gene promoter, as well as mRNA levels of NR2B in these mice. Conclusions: Taken together, these results indicate that epigenetic modifications are involved in regulating ethanol drinking behavior, partially through altering NR2B expression.

Effects of the Analgesic Acetaminophen (Paracetamol) and its para‐Aminophenol Metabolite on Viability of Mouse‐Cultured Cortical Neurons

Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

Effect of nitric oxide synthesis inhibition on c-Fos expression in hippocampus and cerebral cortex following two forms of learning in rats: an immunohistochemistry study.

This study investigated the effect of NO on c-Fos expression using immunocytochemical techniques, following training in the one-trial passive avoidance response and Morris water maze tasks. Fos-like immunoreactive (FLI) neurons were examined in the hippocampus and cerebral cortex of rats in which intracerebroventricular (i.c.v.) injection of Nomega-nitro-L-arginine (NAME) was given 30 min before training in the experimental groups. The results showed that: (1) when compared with untreated rats or rats receiving i.c.v. saline injection, fos-like immunoreactive neurons were significantly increased in both brain regions following training in either task, and (2) preceding i.c.v. injections of NAME blocked both the formation of memory and the increased expression of c-Fos in both brain regions subjected to training on the two models. These results suggest that NO production may be a critical factor for the formation of memory and the enhanced c-Fos expression, and so might contribute to the long-term plasticity of synaptic organization in the two brain regions.

The distribution of β-tubulin isotypes in cultured neurons from embryonic, newborn, and adult mouse brains.

Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α- and β-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of β-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the βI, βII, and βIII isotypes, but apparently not βIV or βV. βI, βII, and βIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although βI and βIII are present in these neurons in both cell bodies and neurites and βIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, βV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the βV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3days, and almost disappeared after 5days. Second, the distribution of βII is different from other isotypes. After newborn mouse neurons were cultured for 3days, βII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that βIIs major function may involve the neurites and not the cell body. They also raise the possibility that βV has a unique role in the neurons of newborn mice.