Fetal Cortical Neurons Research Articles

Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol‐induced apoptosis

Ethanol ingestion during pregnancy elicits damage to the developing brain, some of which appears to result from enhanced apoptotic death of neurons. A consistent characteristic of this phenomenon is a highly differing sensitivity to ethanol within specific neuron populations. One possible explanation for this "selective vulnerability" could be cellular variations in glutathione (GSH) homeostasis. Prior studies have illustrated that ethanol elicits apoptotic death of neurons in the developing brain, that oxidative stress may be an underlying mechanism, and that GSH can be neuroprotective. In the present study, both multiphoton microscopy and flow cytometry demonstrate a striking heterogeneity in GSH content within cortical neuron populations. Ethanol differentially elicits apoptotic death and oxidative stress in these neurons. When neuron GSH content is reduced by treatment with butathione sulfoxamine, the ethanol-mediated enhancement of reactive oxygen species is exacerbated. Sorting of cells into high- and low-GSH populations further exemplifies ethanol-mediated oxidative stress whereby apoptotic indices are preferentially elevated in the low-GSH population. Western blot analysis of the low-GSH subpopulations shows higher ethanol-mediated expression of active caspase 3 and 24-kDa PARP-1 fragments compared with the high-GSH subpopulation. In addition, neuronal content of 4-hydroxynonenal adducts is higher in low-GSH neurons in response to ethanol. These studies suggest that GSH content is an important predictor of neuronal sensitivity to ethanol-mediated oxidative stress and subsequent cell death. The data support the proposition that the differences in proapoptotic responses to ethanol within specific neuron populations reflect a heterogeneity of neuron GSH content.

Bioactive multilayer thin films of charged N, N-disubstituted hydrazine phosphorus dendrimers fabricated by layer-by-layer self-assembly

Charged N, N-disubstituted hydrazine phosphorus-containing dendrimers are deposited either as alternate all-dendrimers multilayers or alternating with linear polymers on 3-mercaptopropionic acid or 3-aminopropyldimethylethoxysilane coated surfaces via electrostatic layer-by-layer self-assembly. The behavior of the film formation is investigated by surface plasmon resonance spectroscopy and ellipsometry. Fetal cortical rat neurons were cultured on the dendrimer films in order to investigate the influence of the surface charge of the outermost layer on their adhesion and maturation. It was found that neurons attached preferentially and matured slightly faster on film surfaces terminated with positively charged dendrimers than on negatively charged surfaces.

Caspase-dependent Apoptosis Induced by Thapsigargin was Prevented by Glycogen Synthase Kinase-3 Inhibitors in Cultured Rat Cortical Neurons

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca(2+)-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9-10). Blockade of N-methyl-D-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3beta activation in rat cortical neurons.

Membrane vesicles shed by oligodendroglioma cells induce neuronal apoptosis

In order to investigate the mechanism by which oligodendrogliomas cause neuronal damage, media conditioned by G26/24 oligodendroglioma cells, were fractionated into shed vesicles and vesicle-free supernatants, and added to primary cultures of rat fetal cortical neurons. After one night treatment with vesicles, a reproducible, dose-dependent, inhibitory effect on neurite outgrowth was already induced and, after 48-72 h of incubation, neuronal apoptosis was evident. Vesicle-free supernatants and vesicles shed by NIH-3T3 cells had no inhibitory effects on neurons. Western blot analyses showed that treated neurons expressed a decreased amount of neurofilament (NF), growth-associated protein (GAP-43) and microtubule-associated protein (MAP-2). Moreover procaspase-3 and -8 were activated while Bcl-2 expression was reduced. Vesicles were found positive for the proapoptotic molecule, Fas-ligand (Fas-L), and for the B isoform of Nogo protein, a myelin component with inhibitory effects on neurons. Nogo B involvement in the vesicle effects was analyzed both by testing the neutralizing capability of anti-Nogo antibodies and by removing the Nogo receptor from neurons by phospholipase C digestion. These treatments did not revert the vesicle effects. To test the role of Fas-L, vesicles were treated with functional anti-Fas-L monoclonals. Vesicle inhibitory and proapoptotic effects were reduced. Vesicles shed by ovarian carcinoma cells (OvCa), which are known to vehicle biologically active Fas-L, had similar effects on neurons to those of oligodendroglioma vesicles, and their inhibitory effects were also reduced by anti Fas-L antibodies. We therefore conclude that vesicles shed by G26/24 cells induce neuronal apoptosis at least partially by a Fas-L mediated mechanism.

A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol

Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?

Abstract: NMDA receptors not only play a pivotal role in normal physiological processes in the central nervous system (CNS), but have been identified as an important target of ethanol. Chronic exposure to ethanol induces a number of adaptive processes in the CNS, including an upregulation of NMDA receptor number and function. The increase in NMDA receptor number in response to chronic ethanol exposure both in vivo and in vitro is accompanied by an increase in NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that these adaptive changes play an important role in the development of alcohol dependence and withdrawal syndrome. At the molecular level, chronic ethanol exposure of fetal cortical neurons selectively increases expression of NMDAR1 splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetal cortical neurons also increases NMDAR1 mRNA half‐life in these neurons. However, when new protein synthesis is inhibited, the half‐life of NR1 mRNA in these neurons returns to control values, strongly suggesting that ethanol induces the synthesis of protein(s) that may regulate the decay of NR1 mRNA. In recent years, it has become apparent that regulation of mRNA stability is an important aspect of regulation of gene expression. Changes in mRNA stability can be accomplished by interaction between cis‐acting sequences in the 3′ untranslated region (3′UTR) of mRNAs and trans‐acting proteins expressed in cells. Such interactions may protect RNAs from degradation by ribonucleases, thereby increasing the half‐life of mRNAs.

An Old Story with a New Twist: Do NMDAR1 mRNA Binding Proteins Regulate Expression of the NMDAR1 Receptor in the Presence of Alcohol?

NMDA receptors not only play a pivotal role in normal physiological processes in the central nervous system (CNS), but have been identified as an important target of ethanol. Chronic exposure to ethanol induces a number of adaptive processes in the CNS, including an upregulation of NMDA receptor number and function. The increase in NMDA receptor number in response to chronic ethanol exposure both in vivo and in vitro is accompanied by an increase in NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that these adaptive changes play an important role in the development of alcohol dependence and withdrawal syndrome. At the molecular level, chronic ethanol exposure of fetal cortical neurons selectively increases expression of NMDAR1 splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetal cortical neurons also increases NMDAR1 mRNA half-life in these neurons. However, when new protein synthesis is inhibited, the half-life of NR1 mRNA in these neurons returns to control values, strongly suggesting that ethanol induces the synthesis of protein(s) that may regulate the decay of NR1 mRNA. In recent years, it has become apparent that regulation of mRNA stability is an important aspect of regulation of gene expression. Changes in mRNA stability can be accomplished by interaction between cis-acting sequences in the 3' untranslated region (3'UTR) of mRNAs and trans-acting proteins expressed in cells. Such interactions may protect RNAs from degradation by ribonucleases, thereby increasing the half-life of mRNAs.

Astrocytes protect neurons from ethanol‐induced oxidative stress and apoptotic death

Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol-mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-, 12-, and 24-hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol-mediated apoptotic death and that this may be related to maintenance of neuron GSH.

Potential Role of cAMP Response Element-Binding Protein in Ethanol-InducedN-Methyl-d-aspartate Receptor 2B Subunit Gene Transcription in Fetal Mouse Cortical Cells

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as (32)P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is probably involved in mediating ethanol-induced up-regulation of NR2B gene.

Neuron-Restrictive Silencer Factor Regulates theN-Methyl-d-aspartate Receptor 2B Subunit Gene in Basal and Ethanol-Induced Gene Expression in Fetal Cortical Neurons

Neuron-restrictive silencer factor (NRSF) is a transcriptional repressor of multiple neuronal genes. This study addressed the role of NRSF in N-methyl-D-aspartate (NMDA) receptor NR2B promoter activity and the molecular mechanisms of ethanol-induced NR2B up-regulation in fetal cortical neurons. The 5'-flanking region of the NR2B gene contains five NRSE-like elements. Functional analysis of the upstream regions of the NR2B gene by transient transfection of neurons revealed that neuron-restrictive silencer element (NRSE) motifs located between base pair -1407 and -2741 represses transcription of the gene. Analysis by electrophoretic mobility shift assay and reporter gene assay identified NRSE2 and 3 as responsible for repressing NR2B gene transcription. The identity of NRSF as the functional binding factor is suggested by the specific binding of in vitro synthesized NRSF or cell lysate to the labeled probes and the specific antibody-induced supershift. Furthermore, whereas mutations of NRSE2 and 3 motifs increased the promoter activity, overexpression of NRSF reduced it significantly. The pattern of NRSF expression during development was investigated and demonstrated that the highest expression is on embryonic day 14 with moderate expression on postnatal day 0, reflecting a possible role of NRSF as a regulator during development. Treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the NRSF mRNA and protein levels, NRSF/NRSE binding activity, and an increase in the promoter activity. Therefore, our studies suggest that NRSF is a negative regulator of NR2B expression and may contribute to the ethanol-induced up-regulation of the NR2B gene in fetal cortical neurons.

Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain

We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227–5567), (5647–6003), (6091–6445), (6424–7024) of adult mouse cortex. Our results indicate that methylation of NR2B gene in the mouse brain is age-dependent phenomenon.

Role of CpG islands in the up-regulation of NMDA receptor NR2B gene expression following chronic ethanol treatment of cultured cortical neurons of mice

Our earlier studies have indicated that chronic ethanol treatment produces an increase in the rate of NR2B gene expression but acute ethanol treatment doesn’t cause any change in the expression in adult cortex and cultured fetal cortical neurons. To determine the molecular mechanism involved in the up-regulation of NR2B gene expression, we studied methylation in CpG islands in the regions (5843–6276) and (6477–6763) in mouse fetal cortical cultured neurons. However, acute administration of ethanol (3.5 g/kg, i.p.) as a 25% solution diluted in 0.9% saline to the adult mice did not cause any change in the methylation status of the NR2B gene. Therefore, these observations indicate that chronic ethanol causes demethylation in CpG islands in the regions (5843–6276) and (6477–6763), thereby probably resulting in increased expression of the NR2B gene. In contrast, acute ethanol treatment did not cause any change in the NR2B gene expression or its demethylation. We also investigated the changes in hypersensitive sites in CpG islands and transcription initiation sites in the cortical cultured neurons following chronic ethanol treatment. These experiments revealed that chronic ethanol treatment does not alter the pattern of hypersensitive sites in adult cortex and cultured cortical neurons.

Fetal cortical allografts project massively through the adult cortex

Allogeneic embryonic CNS tissue grafts placed in the mature brain are classically considered to lack significant long-range efferents. This problem was reexamined using ‘green’ cells from mice expressing ubiquitously an ‘enhanced’ green fluorescent protein as an alternative to classical tract tracing methods. The present study shows that fetal cortical neurons (E15; occipital origin) grafted in the occipitoparietal region of the adult cortex project massively throughout ipsilateral telencephalic structures. Two out of the nine grafted subjects had additional but sparse efferents in the visual thalamus, superior colliculus and pons.

Calcium calmodulin dependent phosphorylation of proteins: fetal cortical neurons and adult cortex.

In the present investigation, changes in the calcium calmodulin-dependent phosphorylation of proteins have been examined in murine fetal cortical neurons and adult cortex. An approximately 80-kD protein in the fetal neurons was not phosphorylated/dephosphorylated in a calmodulin-dependent manner. However, this protein was phosphorylated by PMA both in the presence and absence of calcium. These data suggest that calmodulin inhibits the phosphorylation of a approximately 80-kD protein by inhibiting PKC in murine fetal cortical neurons but not in the adult cortex. More importantly, we demonstrate that the calmodulin-mediated inhibition of phosphorylation was restored by preincubating the cortical neurons with KN-62, a CaM kinase inhibitor.

Changes in methylation pattern of NMDA receptor NR2B gene in cortical neurons after chronic ethanol treatment in mice

Chronic ethanol treatment produced an increase in the rate of NR2B gene expression in adult cortex and cultured fetal cortical neurons. To determine the possible molecular mechanism involved in the upregulation of NR2B gene expression, we studied methylation in CpG island in mouse fetal cortical neurons. Our results demonstrate that chronic ethanol administration causes demethylation of cytosine residues in CpG island in cortical neurons which may be responsible for the changes in the expression of NR2B gene.

Fetal cortical allografts project massively through the adult cortex

Allogeneic embryonic CNS tissue grafts placed in the mature brain are classically considered to lack significant long-range efferents. This problem was reexamined using ‘green’ cells from mice expressing ubiquitously an ‘enhanced’ green fluorescent protein as an alternative to classical tract tracing methods. The present study shows that fetal cortical neurons (E15; occipital origin) grafted in the occipitoparietal region of the adult cortex project massively throughout ipsilateral telencephalic structures. Two out of the nine grafted subjects had additional but sparse efferents in the visual thalamus, superior colliculus and pons.

Ethanol-induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons.

In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P <.05) and by 82% within 2 hr (P <.05), accompanied by increases of mitochondrial 4-hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P <.05) at the 6- to 24-hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P <.05), concomitant with activation of caspase 3 (52% at 12 hr, P <.05). Pretreatment with N-acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol-mediated apoptotic death of fetal cortical neurons.

Novel Role of Vitamin K in Preventing Oxidative Injury to Developing Oligodendrocytes and Neurons

Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Glutamate, cystine deprivation, homocysteic acid, and the glutathione synthesis inhibitor buthionine sulfoximine all cause oxidative injury to immature neurons and oligodendrocytes by depleting intracellular glutathione. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Neither oligodendrocytes nor neurons possess significant vitamin K-dependent carboxylase or epoxidase activity. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Effect of ethanol on lipid-mediated transfection of primary cortical neurons.

Successful introduction of nucleic acids into mammalian neurons has revolutionized the analyses of gene regulation and cellular function. Various methods, including viral infection, have been developed to introduce plasmid DNA into primary neuronal cultures. However, transfection of primary cultures of neurons using the calcium phosphate precipitation method and electroporation have been comparatively inefficient. In this paper, we describe a method to successfully transfect cultured fetal cortical neurons using a cationic lipid reagent, lipofectamine. Cells were cultured in the absence and presence of 50 mM ethanol. To monitor transfection of neurons, we employed three mammalian expression vectors containing Renilla luciferase and/or firefly luciferase, or the beta-galactosidase reporter gene. Fetal cortical neurons were isolated and cultured in the absence or presence of 50 mM ethanol, for two days. On day 3, neurons were washed, fed with serum-free medium, and transfected with the DNA-lipofectamine complex. After two hours, cells were washed, fed complete medium lacking or containing 50 mM ethanol and cultured for two additional days with a change of medium after 24 h. Cultures were terminated 48 h after transfection. Cells were either stained for beta-galactosidase activity using X-gal or lysed to prepare cell extracts to assay for luciferase activity using a luminometer. When neurons were cotransfected, Renilla luciferase was used as an internal control to normalize the expression of the firefly luciferase reporter gene. Analysis of results showed that expression of the reporter gene, firefly luciferase, was approximately 2.5 times greater in ethanol treated neuronal cultures than for neurons cultured in the absence of ethanol. An increased number of neurons expressing beta-galactosidase was also observed in ethanol-treated neurons. These data suggest that perhaps ethanol treatment of fetal cortical neurons improved the DNA uptake and/or increased the expression of the reporter genes.

The molecular effects of alcohol: clues to the enigmatic action of alcohol.

Chronic ethanol treatment (50 mM, five days) induces stabilization of NR1 receptor subunit mRNA in cultured fetal cortical neurons. In this paper, we investigate the mechanism(s) by which ethanol mediates its effects on NR1 mRNA. Specifically, we have determined if cellular localization of NR1 mRNA in cortical neurons and/or de novo protein synthesis is essential for ethanol-mediated stabilization of NR1 mRNA. Subcellular fractionation studies show that all detectable NR1 mRNA is associated with rough endoplasmic reticulum, indicating that subcellular distribution of NR1 mRNA in fetal cortical neurons does not play a role in ethanol-mediated NR1 mRNA stabilization. However, inhibition of protein synthesis by cycloheximide abolished the mRNA stabilizing effect of ethanol on NR1 mRNA, thus suggesting de novo protein synthesis is crucial for the action of ethanol on NR1 mRNA stabilization.