Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement

Abstract : Background: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure. Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24–96 hr. Using two‐electrode voltage clamp, membrane currents were recorded after the application of L‐glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (μC). Results: Ethanol (10–100 mM) reduced EAAT3 activity in a time‐dependent and reversible manner. After a 96 hr‐incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr‐exposure to 50 mM ethanol significantly decreased Vmax (3.6 ± 0.3 for control versus 2.6 ± 0.3 μC for ethanol, n = 20, p < 0.05) but had no effect on Km (57.6 ± 12.8 for control versus 51.8 ± 16.3 μM for ethanol, n = 20, p > 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)‐treated oocytes were incubated with phorbol‐12‐myrisate‐13‐acetate (50 or 100 nM for 10 min), ethanol‐induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 μM chelerythrine significantly decreased EAAT3 activity (1.00 ± 0.08 for control versus 0.51 ± 0.09 μC for chelerythrine group, n = 18–20, p < 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 μM for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups. Conclusions: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C‐dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.     

ENT1 Regulates Ethanol‐Sensitive EAAT2 Expression and Function in Astrocytes

Background: Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum. Methods: We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real‐time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol‐induced EAAT2 expression. Results: An ENT1‐specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up‐regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol‐induced EAAT2 up‐regulation. Conclusions: Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.     

The Mammalian Circadian Clock Exhibits Acute Tolerance to Ethanol

Background:  Tolerance to ethanol is observed over a variety of time courses, from minutes to days. Acute tolerance, which develops over 5 to 60 minutes, has been observed for both behavioral and neurophysiological variables and may involve changes in signaling through NMDA, GABA, or other receptors. Previous work has shown that both acute and chronic ethanol treatments modulate photic and nonphotic phase resetting of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Although not specifically tested, the data thus far do not point to the development of chronic tolerance to the modulatory effects of ethanol. Here we investigated whether acute tolerance the ethanol occurs with respect to in vitro phase modulation of the SCN clock.
Methods:  Mouse brain slices containing the SCN were pretreated with ethanol for varying lengths of time, followed by treatment concurrent with either glutamate or the serotonin agonist, 8-hydroxy-DPAT (DPAT). The phase of the SCN circadian clock was assessed the following day through extracellular recordings of SCN neuronal activity. SCN neuronal activity normally peaks during mid-day, and this rhythm can be shifted by treatment with either glutamate or DPAT.
Results:  While concurrent treatment of SCN-containing brain slices with ethanol and glutamate blocks glutamate-induced phase delays of the SCN clock, pretreating the slices with ethanol for ≥15 minutes prevents this inhibition. Likewise, while concurrent treatment with ethanol and DPAT enhances DPAT-induced phase advances of the SCN clock, pretreating the slices with ethanol for ≥30 minutes prevents this enhancement.
Conclusions:  Both the inhibiting and enhancing effects of ethanol on in vitro SCN clock phase resetting show acute tolerance. Additional experiments are needed to determine whether more slowly developing forms of tolerance also occur with respect to the SCN circadian clock.     

Ethanol Elevates Fetal Serum Glutamate Levels in the Rat

Glutamate is an important excitatory neurotransmitter. However, a sustained elevation of glutamate in the extracellular space may be toxic to neurons. Because the blood-brain barrier is incomplete in the developing fetus, an elevation of fetal serum glutamate could expose the immature, growing brain to potentially toxic levels of extracellular glutamate. Chronic ethanol consumption during pregnancy is associated with an increased risk for a complex array of congenital anomalies, including alterations in the CNS, a hallmark of the fetal alcohol syndrome. Some central nervous system changes appear to involve the glutamate receptor, including reduced number and altered function. One mechanism for receptor downregulation may be a sustained elevation in extracellular glutamate. We hypothesize that chronic ethanol exposure during pregnancy leads to an elevation in fetal serum glutamate. When rats were fed ethanol-containing liquid diet throughout pregnancy, growth retardation of fetuses was observed at sacrifice (gestation day 20). Within each group, ethanol-fed, pair-fed, and ad libitum chow-fed, serum glutamate levels were generally higher in the fetus than in the dam. Ethanol treatment had no effect on fetal or maternal serum glutamine, a reciprocal metabolite of glutamate. In contrast, ethanol treatment increased serum glutamate levels in the fetal serum by nearly 50%, compared with either of the control groups. Maternal serum glutamate was not affected. The finding of ethanol-induced elevation of fetal serum glutamate suggests that the developing brain might be concurrently exposed to elevated levels of extracellular glutamate. Chronic exposure to elevated glutamate during critical periods of brain development may contribute to the pathogenesis of the fetal alcohol syndrome.     

Dose-Dependent Effects of Acute In Vivo Ethanol Exposure on Extracellular Glutamate Concentration in the Cerebral Cortex of the Near-Term Fetal Sheep

The cerebral cortex is a target site of ethanol teratogenesis. l -Glutamate is a major excitatory neurotransmitter that plays an important neurotrophic role in brain development. It has been proposed that optimal function of the glutamate neuronal system is required for normal brain development; overactivation could lead to excitotoxic-induced neuronal injury, whereas underactivation could delay/restrict brain development. The objective of this study was to test the hypothesis that acute in vivo ethanol exposure alters basal glutamate release in the fetal cerebral cortex. The experimental approach involved measuring fetal cortical extracellular glutamate concentration using the technique of in vivo microdialysis. Near-term fetal sheep were chronically instrumented with a microdialysis probe placed in the parasagittal cortex. At 124 ± 3 days of gestation, the effects of maternal intravenous infusion of 2 g or 4 g ethanol/kg maternal body weight or an equivalent volume of saline, given as four equally divided doses over 5 hr, on fetal cerebral cortical extracellular glutamate concentration were determined. None of the three treatment regimens produced fetal or maternal demise during the time course of the study. There was an ethanol dose-dependent increase, p = 0.005, in extracellular glutamate concentration in the fetal cerebral cortex. This increase was paroxysmal in nature and was not directly related to the fetal blood ethanol concentration. In view of the proposed role for glutamate in neuronal development, this apparent ethanol-induced increase in glutamate release may be important in the pathogenesis of ethanol teratogenesis involving the cerebral cortex.     

新生大鼠缺血缺氧后胶质性谷氨酸转运体的表达及GM1的干预研究

目的探讨新生大鼠缺血缺氧性脑损伤后胶质性谷氨酸转运体的表达及神经节苷脂（GM1）的干预作用。方法通过建立新生大鼠缺血缺氧性脑损伤动物模型，应用免疫组化方法，观察缺血缺氧后不同时期大脑皮质胶质性谷氨酸转运体EAATI、EAAT2的动态表达度GM1对其表达的影响。结果缺血缺氧后6hEAAT1的表达开始上升、第2d达高峰，第3d恢复到假手术组水平；EAAT2的表达在缺血缺氧后12h开始上升，第3d达高峰，第5d恢复到假手术组水平；GM1干预组脑组织损伤明显减轻，EAAT1和EAAT2的表达较单纯缺血缺氧组显著增加（P〈0．01），持续时间延长。结论缺血缺氧诱导胶质性谷氨酸转运体的表达，GM1提高胶质性谷氨酸转运体的表达可能是GM1脑保护作用的重要机制之一。     

Inhibition of the activity of excitatory amino acid transporter 4 expressed in Xenopus oocytes after chronic exposure to ethanol

Background: The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in these effects. Methods: EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol‐containing solution for 24 to 96 hours. Membrane currents induced by l ‐aspartate were recorded using 2‐electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (μC). Results: Ethanol dose‐ and time‐dependently reduced EAAT4 activity. EAAT4 activity after a 96‐hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 μC for control vs. 1.6 ± 0.2 μC for ethanol, n = 18, p < 0.05) of EAAT4 for l ‐aspartate. Preincubation of ethanol‐treated (50 mM for 96 hours) oocytes with phorbol‐12‐myrisate‐13‐acetate (100 nM for 10 minutes) abolished the ethanol‐induced decrease in EAAT4 activity. While staurosporine (2 μM for 1 hour) or chelerythrine (100 μM for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 μM for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions: The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri‐Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion.     

5-HT transporter sites and 5-HT1A and 5-HT3 receptors in Fawn-Hooded rats: a quantitative autoradiography study

BACKGROUND: The neuroanatomical profiles of 5-HT1A receptors, 5-HT3 receptors, and 5-HT transporters (5-HTT) in the brain of the Fawn-Hooded (FH) rat, particularly mesocorticolimbic regions, are not fully elucidated. METHODS: By means of in vitro quantitative autoradiography, we used [3H]citalopram, [3H]8-OH-DPAT, and [3H]GR65630 to label 5-HTT, 5-HT1A receptors, and 5-HT3 receptors in the brain of alcohol-na?ve FH rats, Wistar-Kyoto (WKY) rats, and FH rats given free access to 5% ethanol and/or after 24 to 48 hr withdrawal. RESULTS AND CONCLUSIONS: In alcohol-na?ve rats, FH rats displayed significantly higher (p < 0.05) densities of [3H]citalopram binding in the nucleus accumbens (+30%), lateral septum (+37%), ventral pallidum (+21%), and ventral tegmental area (+24%), as well as an increased binding of [3H]8-OH-DPAT to 5-HT1A receptors in the frontal and parietal cortex (+33%), occipital and temporal cortex (+25%), and hippocampal CA3 region (+31%), compared with WKY rats, whereas both strains exhibited comparable [3H]GR65630 binding to 5-HT3 receptors. Compared with control FH (naive) rats, chronic ethanol consumption significantly decreased (p < 0.(15)[3H]8-OH-DPAT binding in the frontal and parietal cortex (-15%) but significantly increased binding (p < 0.05) in the entorhinal cortex (+25%), retrosplenial granular cortex (+20%), and hippocampal CA1 (+14%) and CA3 regions (+18%). Moreover, ethanol withdrawal induced the same extent of increased [3H]8-OH-DPAT binding in the entorhinal and retrosplenial cortex as seen in FH (chronic) rats. In contrast, [3H]8-OH-DPAT binding in the hippocampal CA1 and CA3 regions was decreased by -9% and -20% from the level of chronic ethanol-treated FH rat (p < 0.05) and returned to the control level seen in FH (na?ve) rats. SIGNIFICANCE: The elevated 5-HT transporters and 5-HT1A receptors in the mesocorticolimbic areas in FH rats may reflect a potential innate altered transmission at serotonergic synapses, which possibly may affect the high intake of alcohol in FH rats. The region-specific alterations of 5-HT1A receptors in FH rat brain after ethanol challenges suggest that 5-HT1A receptors are sensitive to ethanol challenges, whereas 5-HTT are apparently insensitive.     

Regional brain metabolism during alcohol intoxication

BACKGROUND: Ethanol has a broad range of actions on many neurotransmitter systems. The depressant actions of ethanol in the brain are related in part to facilitation of gamma-aminobutyric acid (GABA) neurotransmission via its interaction with the benzodiazepine/GABA receptor complex. The purpose of this study was to evaluate the effects of ethanol on regional brain metabolism in 10 healthy right-handed men. The results were compared with those we previously published in a different group of 16 normal male subjects who received intravenous lorazepam, a benzodiazepine drug that also enhances GABA neurotransmission. METHODS: The subjects were scanned with positron emission tomography and [F-18] fluorodeoxyglucose twice: 40 min after the end of placebo (diet soda) or ethanol (0.75 g/kg) oral administration. Image data sets were analyzed by using both the region of interest and the statistical parametric mapping (SPM) approach. SPM was used to generate a difference image between baseline and ethanol, which we compared to the difference image between baseline and lorazepam (30 microg/kg). RESULTS: Ethanol significantly increased self-reports of "high" (p < or = 0.0001), dizziness (p < or = 0.004), and intoxication (p < or = 0.0001). Ethanol significantly decreased whole brain (-25 +/- 6%, p < or = 0.0001) and regional metabolism. Normalization of the regional measures by whole brain metabolism (relative measures) showed that ethanol decreased relative metabolic activity in occipital cortex (-4.9 +/- 4.1%, p < or = 0.006), whereas it increased relative metabolic act in left temporal cortex (+3.5 +/- 2.9%, p < or = 0.006) and left basal ganglia (+9 +/- 6.3%, p < or = 0.0009). SPM analyses revealed the same pattern of responses as the relative measures, showing decreases in occipital cortex and increases in left temporal cortex. Comparison of the relative measures and the SPM analyses obtained with lorazepam data revealed a similar pattern of effects, with relative decreases in occipital cortex (-7.8 +/- 4.8%) and relative increases in left temporal cortex (+3.8 +/- 5.7%). Lorazepam, but not ethanol, also decreased thalamic metabolism (-11.2 +/- 7.2%). CONCLUSIONS: These results support similar though not identical mechanisms for the effects of alcohol and benzodiazepines on brain glucose metabolism. The fact that lorazepam, but not alcohol, reduced thalamic metabolism, an effect associated with sleepiness, could explain the higher sedative effects of lorazepam than of alcohol.     

Effects of ethanol on the rat glutamate excitatory amino acid transporter type 3 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase

Background: Glutamate is a major excitatory neurotransmitter in the central nervous system. Glutamate transporters play a critical role in maintaining extracellular glutamate concentrations. We investigated the effects of ethanol on a neuronal glutamate transporter, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3 K) in mediating these effects. Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 messenger RNA. By using a two‐electrode voltage clamp, membrane currents were recorded after the application of l‐glutamate. Responses were quantified by integration of the current trace and reported as microcoulombs. Data are mean ± SEM. Results: Ethanol enhanced EAAT3 activity in a concentration‐dependent manner. At 25, 50, 100, and 200 mM of ethanol, the responses were significantly increased compared with control values. Kinetic study demonstrated that ethanol (50 mM) significantly increased V_max (3.48 ± 0.2 μC for control versus 4.16 ± 0.24 μC for ethanol; n= 19; p < 0.05) without a significant change in the K_m (65.6 ± 11.1 μM for control versus 55.8 ± 9.6 μM for ethanol; n= 19; p > 0.05) of EAAT3 for glutamate. Preincubation of the oocytes with phorbol‐12‐myristate‐13‐acetate (PMA) significantly increased EAAT3 activity (0.98 ± 0.08 μC for control versus 1.28 ± 0.09 μC for ethanol; n= 19; p < 0.05). However, there was no statistical difference among the responses of EAAT3 to PMA, ethanol, or PMA plus ethanol. Although the PKC inhibitors chelerythrine and staurosporine did not decrease the basal EAAT3 activity, they abolished the enhancement of EAAT3 activity by ethanol. Pretreatment with wortmannin, a PI3 K inhibitor, also abolished the ethanol‐enhanced EAAT3 activity. Conclusions: These results suggest that acute ethanol exposure increases EAAT3 activity at clinically relevant concentrations and that PKC and PI3 K may be involved in mediating these ethanol effects.     

Long-Term Behavioral Changes in Response to Early Developmental Exposure to Ethanol in Zebrafish

Background:  Zebrafish is becoming an important research tool for the analysis of brain function and behavior. It has been proposed to model human alcoholism as well as fetal alcohol syndrome. Previous studies investigating the consequences of exposure to ethanol during early development of zebrafish employed robust dosing regimens (high ethanol concentration and long exposure) that may model a rare situation in the human clinic. These studies found major structural abnormalities developing in the exposed fish.
Methods:  Here we hope to avoid such gross changes and administer only low doses of ethanol (0.00, 0.25, 0.50, 0.75, 1.00 vol/vol %) at 24-hour postfertilization and for only a short period of time (for 2 hours). We analyze the behavior of exposed fish at adult stage using computerized stimulus presentation and automated videotracking response quantification.
Results:  Despite the short ethanol exposure period and the modest concentrations, significant behavioral alterations were found: fish exposed to higher doses of ethanol swam at an increased distance from a computer-animated zebrafish shoal while their activity levels did not change.
Conclusions:  Although the interpretation of and the mechanisms underlying this finding will require further investigation, the results suggest that zebrafish will be an appropriate model organism for the analysis of the effects of moderate to mild prenatal ethanol exposure.     

Choroid plexus dysfunction: the initial event in the pathogenesis of Wernicke's encephalopathy and ethanol intoxication

Background:  In both acute ethanol intoxication and in thiamin deficient glucose metabolism, previous studies have detected blood-brain barrier (BBB) and/or blood-CSF-barrier (BCSFB) impairment but were unable to assess their significance in relation to other changes in the brain.
Methods:  Contrast-enhanced, magnetic resonance imaging (MRI) was used to detect and time any impairment of the BBB or BCSFB in rats given an acute ethanol load or in rats made thiamin deficient to the point of mild ataxia and then given an acute glucose load.
Results:  The BCSFB at the choroid plexus (CP) was impaired within 10 minutes by either (i) a single i.p. dose of glucose in thiamin-deficiency, an effect that was attenuated by prior MK801 and preceded the published onset of exacerbation of motor incoordination and elevation of brain glutamate derivatives; or (ii) a single i.p. dose of ethanol in thiamin-sufficiency, an effect that was proportional to the blood alcohol concentration and preceded the published onset of signs of intoxication. In contrast to the BCSFB, the BBB remained intact throughout the 90 minutes period of these experiments.
Conclusions:  In both ethanol intoxication and thiamin-deficient glucose metabolism, BCSFB impairment exposes the CSF and hence the brain extracellular fluid to neuroactive substances from the blood. CP impairment is the earliest detected event in both these animal models; and explains the paraventricular location of WE neuropathology and why WE is associated with, but not dependent on, alcoholism.     

The role of excitatory amino acid transporter 2 (EAAT2) in epilepsy and other neurological disorders

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) have important roles in the uptake of glutamate and termination of glutamatergic transmission. Up to now, five EAAT isoforms (EAAT1-5) have been identified in mammals. The main focus of this review is EAAT2. This protein has an important role in the pathoetiology of epilepsy. De novo dominant mutations, as well as inherited recessive mutation in this gene, have been associated with epilepsy. Moreover, dysregulation of this protein is implicated in a range of neurological diseases, namely amyotrophic lateral sclerosis, alzheimer’s disease, parkinson’s disease, schizophrenia, epilepsy, and autism. In this review, we summarize the role of EAAT2 in epilepsy and other neurological disorders, then provide an overview of the therapeutic modulation of this protein.

     

Ethanol increases the activity of rat excitatory amino acid transporter type 4 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase

Background: Glutamate is the major excitatory neurotransmitter in the central nervous system and is critical for essentially all physiological processes, such as learning, memory, central pain transduction, and control of motor function. Excitatory amino acid transporters (EAATs) play a key role in regulating glutamate neurotransmission by uptake of glutamate into cells. EAAT4 is the major EAAT in the cerebellar Purkinje cells. The authors investigated the effects of ethanol on EAAT4 and the mediatory effects of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in this context. Methods: Excitatory amino acid transporter 4 was expressed in Xenopus oocytes by injecting EAAT4 mRNA. l ‐aspartate‐induced membrane currents were measured using a two‐electrode voltage clamp. Responses were quantified by integrating current traces and are represented in microCoulombs (μC). Results: Ethanol increased EAAT4 activity in a dose‐dependent manner. At ethanol concentrations of 25, 50, 100, and 200 mM, the responses were significantly higher than untreated control values. Ethanol (25 mM) significantly increased the V_max (1.5 ± 0.1 μC for control vs. 2.0 ± 0.1 μC for ethanol, p < 0.05), but did not affect K_m (2.3 ± 0.6 μM for control vs. 1.7 ± 0.7 μM for ethanol, p > 0.05) of EAAT4 for l ‐aspartate. Preincubation of oocytes with phorbol‐12‐myristate‐13‐acetate (PMA, a PKC activator) significantly increased EAAT4 activity. However, combinations of PMA and ethanol versus PMA or ethanol alone did not increase responses further. Two PKC inhibitors, chelerythrine and staurosporine did not reduce basal EAAT4 activity but abolished ethanol‐enhanced EAAT4 activity. Pretreatment with wortmannin (a PI3K inhibitor) also abolished ethanol‐enhanced EAAT4 activity. Conclusions: These results demonstrate that acute ethanol exposure increases EAAT4 activity at clinically relevant concentrations and that PKC and PI3K may mediate this. The effects of ethanol on EAAT4 may play a role in the cerebellar dysfunction caused by ethanol intoxication.     

Ethanol induces Fas/Apo [apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex

INTRODUCTION: Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry. METHODS: Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide. RESULTS: A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol. CONCLUSIONS: Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.     

Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS)

Transgenic overexpression of Cu(+2)/Zn(+2) superoxide dismutase 1 (SOD1) harboring an amyotrophic lateral sclerosis (ALS)-linked familial genetic mutation (SOD1(G93A)) in a Sprague-Dawley rat results in ALS-like motor neuron disease. Motor neuron disease in these rats depended on high levels of mutant SOD1 expression, increasing from 8-fold over endogenous SOD1 in the spinal cord of young presymptomatic rats to 16-fold in end-stage animals. Disease onset in these rats was early, approximately 115 days, and disease progression was very rapid thereafter with affected rats reaching end stage on average within 11 days. Pathological abnormalities included vacuoles initially in the lumbar spinal cord and subsequently in more cervical areas, along with inclusion bodies that stained for SOD1, Hsp70, neurofilaments, and ubiquitin. Vacuolization and gliosis were evident before clinical onset of disease and before motor neuron death in the spinal cord and brainstem. Focal loss of the EAAT2 glutamate transporter in the ventral horn of the spinal cord coincided with gliosis, but appeared before motor neuron/axon degeneration. At end-stage disease, gliosis increased and EAAT2 loss in the ventral horn exceeded 90%, suggesting a role for this protein in the events leading to cell death in ALS. These transgenic rats provide a valuable resource to pursue experimentation and therapeutic development, currently difficult or impossible to perform with existing ALS transgenic mice.     

Fatty Acid Ethyl Esters in Meconium: Are They Biomarkers of Fetal Alcohol Exposure and Effect?

BACKGROUND: Biomarkers of fetal exposure to alcohol are important to establish so that early detection and intervention can be made on these infants to prevent undesirable outcomes. The aim of this study was to analyze long-chain fatty acid ethyl esters (FAEEs) in meconium as potential biomarkers of fetal alcohol exposure and effect. METHODS: Fatty acid ethyl esters were analyzed in the meconium of 124 singleton infants by positive chemical ionization gas chromatography/mass spectrometry (GC/MS) and correlated to maternal ethanol use. RESULTS: A total of 124 mother/infant dyads were enrolled in the study: 31 were in the control group and 93 were in the alcohol-exposed group. The incidence (28% vs 9.7%, p = 0.037) of ethyl linoleate detected in meconium was significantly higher in the alcohol-exposed groups than the control groups. Similarly, when the concentrations of ethyl linoleate in meconium were grouped (trichotomized), there was a significant linear by linear association between alcohol exposure and group concentrations of ethyl linoleate (p = 0.013). Furthermore, only alcohol-exposed infants were found in the group with the highest ethyl linoleate concentration. The sensitivity of ethyl linoleate in detecting prenatal alcohol exposure was only 26.9%, and its specificity and positive predictive value were 96.8 and 96.2%, respectively. There was no significant correlation between the concentration of ethyl linoleate in meconium and absolute alcohol consumed (oz) per drinking day across pregnancy, although a trend toward a positive correlation is seen at lower amounts of alcohol consumed. Among the polyunsaturated, long-chain FAEEs, there was weak evidence that the incidence (21.5% vs 6.5%, p = 0.057) and concentration (p = 0.064) of ethyl arachidonate (AA) were significantly higher in the alcohol-exposed groups than the control groups. Ethyl linolenate and ethyl docosahexanoate (DHA) in meconium were found only in the alcohol group, although not at statistically significant levels. Highly significant correlations were found among the concentrations of ethyl linoleate, ethyl linolenate, ethyl AA, and ethyl DHA in meconium (correlations ranged between rs = 0.203, p = 0.024; and rs = 0.594, p < 0.001). CONCLUSION: We conclude that FAEEs in meconium, particularly ethyl linoleate and ethyl AA, are biomarkers of high specificity for prenatal exposure to alcohol in newborn infants. We also propose that ethyl AA and DHA could be potential biomarkers of fetal alcohol effects on the developing fetal brain and should be investigated further.