Effects of lifelong ethanol consumption on rat sympathetic neurons.

In this experiment we studied the effects of aging and lifelong ethanol consumption on rat peripheral sympathetic neurons. The aim was to find out the possible differences in the vulnerability to ethanol-induced neuronal degeneration between rats of both genders, or between the alcohol-avoiding (ANA) and the alcohol-preferring (AA) lines of rat. The superior cervical ganglia (SCG) of 40 male and 41 female AA and ANA rats were analyzed. The ethanol-exposed groups had 12% ethanol as the only available fluid from 3 to 24 months of age. The young (3 months) and old (24 months) control groups had water instead. SCG neuronal density, volume, and total neuron number were measured by unbiased morphometric methods. No gender difference was seen in either the volume of the SCG or in the SCG neuron number. The volume of the ganglion was significantly increased with age, but the total neuron number did not change. Neuronal density was significantly decreased with age, but lifelong ethanol consumption induced no further decrease. SCG neuron number in the ethanol-exposed groups did not differ from the age-matched or young control groups, but a significant negative correlation (r = -0.70, p<0.01) was seen between individual ethanol consumption and the number of SCG neurons in the female rats. The amount of lipopigment in the SCG was increased in the ethanol-exposed male rats. These results suggest that the peripheral sympathetic neurons are rather resistant to ethanol-induced degeneration, and that no major gender or line differences exist in this respect.     

Lead/ethanol interactions II: Pharmacokinetics

Adult male rats were exposed ad libitum to water containing either 500 ppm lead acetate (group-lead) or an equivalent amount of sodium acetate (group-control) for 60 days prior to receiving ip injections of either 1.0, 2.0, or 3.0 g/kg ethanol (20% v/v). Blood alcohol concentrations (BACs) were recorded over a 6-h time period postinjection, and the groups were compared at each dose for differences in the pattern of ethanol pharmacokinetics. While there was a dose-related effect obtained with increasing ethanol dose producing increasing BAC values, at no dose was there any evidence of group separation at any point during the 6-h postinjection period. These data are instructive with respect to understanding the nature of previously demonstrated lead/ethanol interactions, and rule out the possibility that lead-induced disturbances in the catalysis of ethanol, or some other pharmacokinetic operation, is the basis for the effects of lead on ethanol intake and ethanol administration. Alternative possible accounts of this curious interaction between a xenobiotic contaminant and alcohol are discussed.     

Effects of acute ethanol on the Ca2+ response to AMPA in cultured rat cortical GABAergic nonpyramidal neurons

AIMS AND METHODS: Immunocytochemical studies revealed that the vast majority of neurons in our primary cultures of rat cortical cells are GABA-positive and represent nonpyramidal interneuron-like cells. The influence of ethanol on (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-induced Ca2+ influx was investigated in multipolar, medium-sized neurons by using single-cell fura-2 microfluorimetry. RESULTS: In a first series of experiments, the results showed a small but significant decrease of 17-22% by ethanol (100 mm) of the intracellular Ca2+ signals induced by slowly superfused AMPA (10, 30, 100 microm). This finding is comparable with the inhibitory activity of ethanol on N-methyl-D-aspartic acid (NMDA)-induced Ca2+ signals in these cells. Further studies with a fast pressure-application of AMPA (30 microm) showed a similar degree of inhibition by ethanol (100 mm). Superfusion with tetrodotoxin/bicuculline, to rule out possible effects of spontaneously released GABA and synaptic spike activity, did not significantly influence the AMPA-induced Ca2+ response nor the inhibitory effect of ethanol. CONCLUSIONS: The present findings indicate that ethanol at high concentrations inhibits Ca2+ signaling via both AMPA and NMDA glutamate receptors in cortical interneuron-like cells. These effects may contribute to the central depressant action of this drug.     

Brain adenosine modulation of behavioral interactions between ethanol and carbamazepine in mice

The effect of the anticonvulsive drug carbamazepine on ethanol-induced motor incoordination and loss-of-righting reflex was investigated in male CD-1 mice. The results of the investigation showed that carbamazepine significantly potentiated the motor incoordinating effect of ethanol in a dose-dependent fashion. Although carbamazepine did not alter the onset time, it significantly prolonged the duration of ethanol-induced loss-of-righting reflex. Pretreatment with theophylline significantly attenuated the carbamazepine-induced potentiation of ethanol-induced motor incoordination and loss-of-righting reflex. Results from a blood ethanol study indicated no effect of carbamazepine on the clearance of ethanol. The data suggest the involvement of nonadenosinergic mechanism in carbamazepine-ethanol behavioral interactions which is responsible for the accentuating effects of carbamazepine on ethanol-induced motor incoordination and duration of loss-of-righting reflex.     

Lead/ethanol interactions I: Rate-depressant effects

Adult male rats were exposed to a diet containing 500 ppm added lead as lead acetate (group lead-diet) or a control diet containing no added chemicals (group control-diet) for 61 days prior to commencing fixed-ratio 32 (FR 32) lever press training for water reinforcement. After steady state responding was achieved, all animals received serial administrations of acute doses of ethanol prior to the daily training session. Specifically, lead-diet and control-diet rats received i.p. injections of .25, .5, .75, 1.0, and 1.25 g/kg ethanol, in ascending order, alternating daily with injections of saline. The results revealed a dose-dependent rate-depressant effect, with higher doses of ethanol producing more behavioral suppression than lower doses for both groups. In addition, at the dose of 1.0 g/kg it was observed that the suppressive effects of ethanol on schedule-controlled responding were reduced among lead-treated animals relative to controls. These data are discussed in terms of lead-induced attenuation of the pharmacologic effects of ethanol.     

Effect of neonatal ethanol exposure on parvalbumin-expressing GABAergic neurons of the rat medial septum and cingulate cortex.

This study was performed to determine the long-term effects of ethanol exposure during the brain growth spurt (postnatal days 4-10) on the number of parvalbumin-immunoreactive (PA+) GABAergic neurons in the adult (P60) rat medial septum and anterior cingulate cortex. Significant loss of neurons within each of these populations has previously been demonstrated following prenatal ethanol exposure. In the present study, no significant differences in the number of PA+ neurons were found in either the medial septum or the cingulate cortex when control and ethanol-exposed animals were compared. The cellular densities and volumetric measures in both brain regions were also similar in the two groups. We speculate that compensatory up-regulative mechanisms may have accounted for the protection of the PA neuronal populations in these two areas following the early neonatal exposure.     

Effects of ethanol on cultured embryonic neurons from the cerebral cortex of the rat

Fetal alcohol syndrome is a serious disorder that causes lifelong learning, memory, and behavioral problems. In the current study, we determined the ethanol concentrations that produced detrimental effects on the development of embryonic cortical neurons because mental capacity seems to be proportional to the level of dendritic arborization. Neurons from fetal rat cortices were grown in culture in close proximity to a glial plane. The cells were treated with concentrations of ethanol ranging from 450 nM to 45 mM, and neurite outgrowth was subsequently quantified. A significant decrease in dendritic branching was observed at ethanol concentrations as low as 45 μM after 6 days of ethanol exposure in vitro, whereas changes in primary neurite outgrowth were observed at an ethanol concentration of 4.5 μM. This finding is of particular interest as it seems to indicate that occasional ethanol exposure is detrimental to cortical development at very low concentrations of ethanol.     

Basal firing rate of rat locus coeruleus neurons affects sensitivity to ethanol

Intracellular recordings were made from spontaneously active rat locus coeruleus (LC) neurons in a totally submerged brain slice preparation. Bath application of ethanol (ETOH) (1-60 mM) inhibited the spontaneous firing of LC neurons. These ETOH concentrations are equal to or below ETOH concentrations found in the brain during mild to moderate intoxication. The basal frequency of spontaneous firing of LC neurons ranged from 0.4-7 Hz. For 9 LC neurons which showed complete block of firing by ETOH, the latency to block was found to be directly related to the logarithm of the firing rate (correlation coefficient 0.94). This relationship was not secondary to a relationship between membrane potential and latency to block since for the same 9 neurons, membrane potential and latency to block were not significantly correlated. We conclude that the basal firing rate of a neuron can affect its sensitivity to the inhibitory effects of ETOH.     

Effect of chronic ethanol administration on disposition of ethanol and its metabolites in rat.

We studied the effects of chronic alcohol intake on the disposition of alcohol and its metabolites in the rat. We used male Wistar rats for all of the experiments in this study. Using a pair-feeding process, rats were fed a liquid diet containing alcohol or without alcohol for 6 weeks. Ethanol solutions (0.5, 1.0, 1.5, and 2.0 g/kg body weight [BW]) were administered as a bolus, intravenously. We then measured blood ethanol and acetate concentrations. Simultaneous multiline fitting was performed using mean blood alcohol concentration (BAC)-time curves fitted to the one-compartment open model with parallel first-order and Michaelis-Menten elimination kinetics. At low doses (0.5, 1.0, and 1.5 g/kgBW), no differences were observed between the alcohol group and the control group with respect to ethanol elimination rate, area under the curve of ethanol (AUC(EtOH)), and mean residence time of ethanol (MRT(EtOH)). At higher doses (2.0 g/kgBW), ethanol elimination rate in the alcohol group was significantly higher than in the control group (P<.5%). These findings were also substantiated by corresponding changes in AUC(EtOH) and MRT(EtOH). At low doses, no differences were observed between the alcohol group and the control group with respect to plateau concentration of acetate (AcT) (concentration of steady state=C(ss)AcT), area under the curve of AcT (AUC(AcT)), and mean residence time of AcT (MRT(AcT)). However, at higher doses, although there were no differences in C(ss)AcT, both AUC(AcT) and MRT(AcT) were significantly lower in the alcohol group when compared to the control group (P<.5%). Chronic alcohol consumption increases ethanol oxidation and AcT metabolism in rats, as observed at high blood alcohol concentrations (BACs). These effects were observed at BACs of 3.5-4.5 mg/ml, and were not observed at lower doses. Thus, with general alcohol consumption, interindividual differences and intra-individual changes in alcohol metabolism may not take into account increased or accelerated metabolism due to alcohol tolerance.     

Actions of ethanol on hypothalamic and pituitary hormones in prepubertal female rats

To determine the effects of prepubertal ethanol (ETOH) exposure on hypothalamic and pituitary hormones known to be involved in the onset of female puberty, we have chronically exposed female rats to either a liquid-diet containing ETOH or an isocaloric control liquid-diet. An additional set of controls consisted of animals maintained on Lab Chow, and water provided ad lib. Our results indicate that the feeding regimen employed produced no differences with regard to body and reproductive organ weights, as well as any of the hormones measured between the two control groups. Conversely, ETOH-treated animals showed significantly lower body and reproductive organ weights than the control animals and although no differences were detected between ETOH-treated and control animals with regard to the hypothalamic content of somatostatin (SRIF), there was a significant increase in the hypothalamic content of growth hormone releasing hormone (GHRH), with a concomitant and significant decrease in the serum concentration of growth hormone (GH). Furthermore, the ETOH-treated animals showed a significant increase in the hypothalamic content of luteinizing hormone releasing hormone (LHRH) with a significant decrease in the serum concentration of luteinizing hormone (LH), but not follicle stimulating hormone (FSH). These results demonstrate for the first time that chronic, prepubertal ETOH administration alters the concentrations of specific hypothalamic and pituitary hormones which are known to be involved in the female pubertal process.     

铝致大鼠皮层神经元凋亡及应激活化蛋白激酶活性的变化

目的：探讨氯化铝（AlCl3）对大鼠皮层神经元凋亡的诱导作用及其与应激活化蛋白激酶，又称c-jun氨基末端激酶（SAPK／JNK）信号转导通路的关系。方法：原代无血清培养至第7天，用不同剂量AlCl3(0、10、100、1000μmol/L）处理大鼠皮层神经元48h，然后用琼脂糖凝胶电泳及AnnexinⅤ联合PI染色法检测细胞凋亡；用免疫印迹法(Western blot)检测SAPK／JNK磷酸化水平的时间变化规律。结果：各染铝组均出现DNA ladder这一典型的凋亡特征，且随剂量增加渐趋明显，各剂量组细胞凋亡率明显升高，与对照组相比差异有显著性（P＜0．05），呈明显的剂量－效应关系。6h时SAPK／JNK磷酸化程度达到最高，以后逐渐降低，呈时间－效应关系（P＜0．05）。结论：铝诱导大鼠皮层神经元凋亡可能与SAPK／JNK信号转导通路有关。     

900MHz微波电磁辐射对原代培养的大鼠脑皮质神经元的谷氨酸AMPA受体2及胞内钙离子浓度的影响

目的　研究非“致热效应”强度的微波 (90 0MHz)电磁辐射对分离式原代培养的大鼠脑皮质神经元谷氨酸受体 2 (GluR2 )及胞内钙离子浓度的影响。方法　将大鼠脑皮质神经元暴露于 90 0MHz的连续性微波电磁辐射 (SAR =3 .2 2W Kkg、2 .2 3W kg、1. 15W kg) ,进行每天 2小时、连续 4天 (或 6天 )及一次性 12小时暴露(SAR =3. 2 2W kg) ,观察其对上述指标的影响。结果　免疫组织化学及激光扫描共聚焦显微镜研究显示 ,SAR =3. 2 2W kg、2 . 2 3W kg、1. 15W kg时 ,微波使暴露组神经元的GluR2表达明显下调 (P <0 . 0 1) ,同时使胞内钙离子浓度明显上调 (P <0 . 0 1)。结论　 90 0MHz微波电磁辐射 (SAR =3 .2 2W kg、2. 2 3W kg、1. 15W kg)对神经元GluR2表达及胞内钙离子浓度的影响具有蓄积效应 ,且具有一定的剂量反应关系 ,一次性微波暴露的影响在一定程度上是可逆的 ,所选微波对神经元的影响属于电磁辐射的非“致热效应”。     

Effect of ethanol, acetaldehyde and acetate on the antioxidant-sensitive respiration in the perfused rat liver: influence of fasting and diethylmaleate treatment

The infusion of 45 mM ethanol or 50 microM acetaldehyde into the perfused rat liver produced comparable and significant antioxidant-sensitive respiratory rates, while marginal responses were obtained with 10 mM acetate. Ethanol-induced antioxidant-sensitive respiration was markedly increased in perfused livers from fasted rats or animals given diethylmaleate which exhibit low hepatic glutathione levels, compared to fed rats. These data point out the significant role of acetaldehyde in ethanol-induced liver oxidative stress, which can be exacerbated by glutathione depletion.     

Adolescent and adult rat cortical protein kinase A display divergent responses to acute ethanol exposure

Adolescent rats display reduced sensitivity to many dysphoria-related effects of alcohol (ethanol) including motor ataxia and sedative hypnosis, but the underlying neurobiological factors that contribute to these differences remain unknown. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway, particularly the type II regulatory subunit (RII), has been implicated in ethanol-induced molecular and behavioral responses in adults. Therefore, the current study examined cerebral cortical PKA in adolescent and adult ethanol responses. With the exception of early adolescence, PKA RIIα and RIIβ subunit levels largely did not differ from adult levels in either whole cell lysate or P2 synaptosomal expression. However, following acute ethanol exposure, PKA RIIβ P2 synaptosomal expression and activity were increased in adults, but not in adolescents. Behaviorally, intracerebroventricular administration of the PKA activator Sp-cAMP and inhibitor Rp-cAMP prior to ethanol administration increased adolescent sensitivity to the sedative-hypnotic effects of ethanol compared to controls. Sp-cAMP was ineffective in adults whereas Rp-cAMP suggestively reduced loss of righting reflex (LORR) with paralleled increases in blood ethanol concentrations. Overall, these data suggest that PKA activity modulates the sedative/hypnotic effects of ethanol and may potentially play a wider role in the differential ethanol responses observed between adolescents and adults.     

Effects of nicotine and ethanol on rat atrial membrane potentials.

The purpose of this research was to study the effects of nicotine and ethanol, alone and in combination, on cardiac membrane potentials (MP). Rat atrial preparations driven at 5 Hz were superfused with Tyrode's solution (37 degrees C) while recording MP with intracellular microelectrodes. Nicotine concentrations below and including 6.2 x 10(-5) M did not affect MP. Within 15 s, nicotine 3.1 x 10(-3) M shortened the action potential duration (APD) and depressed the overshoot of the action potential (OS). This action was blocked by atropine. After 3 min, nicotine prolonged the APD and depressed Vmax of phase O, OS and the amplitude of the action potential (AAP), without affecting the resting membrane potential (RMP). Nifedipine blocked the depression of the OS while tetraethylammonium chloride blocked the prolongation of the APD. Acute exposure to ethanol depressed OS and AAP and shortened APD, but it did not affect RMP or Vmax of phase O. When nicotine and ethanol were administered simultaneously, the APD-prolonging effects of nicotine prevailed. The influence of chronic ethanol ingestion on the acute action of nicotine and/or ethanol was studied in rats pair-fed a liquid diet with (ER) or without (NR) ethanol (35% of total caloric intake) for 24 weeks. Chronic ethanol ingestion accentuated the depressant effect of nicotine 3.1 x 10(-3) M on OS and AAP, but it did not modify the APD-prolonging action of nicotine. The same results were observed when ER and NR were exposed to nicotine and ethanol simultaneously.     

900MHz微波电磁辐射对原代培养的大鼠脑皮质神经元能量代谢的影响

目的　研究 90 0MHz微波电磁辐射对原代培养的大鼠脑皮质神经元能量代谢的影响。方法　将大鼠脑皮质神经元暴露于 90 0MHz的连续性微波电磁辐射 (SAR =3 2 2mW g、PD =9mW cm2 ) ,每天暴露 2h ,连续 4d或 5d ,及一次性 1 2h暴露 ,以细胞色素氧化酶为观察指标 ,研究微波对神经元能量代谢的影响。结果　微波电磁辐射可使神经元细胞色素氧化酶活性降低。结论　神经元细胞色素氧化酶活性的改变并非“致热效应”所致 ;微波电磁辐射对神经元细胞色素氧化酶活性影响有蓄积毒性作用 ,其影响在一定程度上是可恢复的 ,并且与神经元接受微波辐射时细胞培养年龄关系不密切。     

茶多酚对甲基汞致大鼠大脑皮质神经元氧化损伤的防护作用

目的探讨茶多酚(tea polypheonols,TP)对甲基汞(methylmercury,MeHg)所致大鼠大脑皮质神经元氧化损伤的防护作用及机制。方法进行大鼠大脑皮质神经元原代培养,细胞成熟后给予0.01、0.1、1、2μmol/L MeHg分别处理0.5、1、3、6、12 h,通过测定培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活力来进行MeHg细胞毒性分析;根据测定结果选择最具代表性的1μmol/L MeHg暴露6 h作为MeHg染毒组。应用同样方法进行TP预处理组选定,向培养液中分别加入终浓度为5、10、20及40μmol/L TP,分别预处理0.5、1、3及6 h后,再加入终浓度为1μmol/L MeHg,继续培养6 h后测定培养液LDH漏出,根据实验结果选定5、10、20μmol/L预处理3 h作为TP预处理剂量及时间;细胞经各剂量TP预处理后,再暴露于1μmol/L MeHg 6 h,测定神经元细胞凋亡率、非蛋白巯基(non-protein sulfhydryl,NPSH)含量、活性氧簇(reactive oxygen species,ROS)水平及Na+-K+-ATPase和Ca2+-ATPase活力。结果与对照组比较,随着染MeHg剂量的升高,培养液中LDH活力逐渐升高,呈现剂量和时间依赖性的效应关系。TP预处理后,LDH活力逐渐降低,在10、20μmol/L TP预处理组显著降低(P0.05或P0.01);1μmol/L MeHg导致神经元凋亡率显著升高,NPSH含量显著降低,ROS水平显著升高,Na+-K+-ATPase和Ca2+-ATPase活力显著降低,差异均有统计学意义(P0.01),TP预处理对上述指标的拮抗作用呈现剂量-效应关系,差异均有统计学意义(P0.05或P0.01)。结论 TP对MeHg所致大鼠大脑皮质神经元氧化损伤具有一定的防护作用。     

Interaction of flufenamic acid on ethanol metabolism in rat.

Flufenamic acid (FA) is a widely used non-steroidal anti-inflammatory drug. It is also known to be an uncoupling agent of oxidative phosphorylation in mitochondria. The interaction of FA and ethanol has been of concern in the occupational health field, since alcohol consumption is a common habit among members of the working population. Thus, we investigated the effects of FA on ethanol metabolism in the rat. In the first experiment, FA and ethanol were administered intragastrically to male Wistar rats. Ethanol and acetaldehyde were measured in blood samples collected from the tail vein by head-space gas chromatography. In the second experiment, the interaction of FA and ethanol was observed in the perfused rat liver. The following items were monitored in perfused livers from both fed and fastd rats: uptake rate of ethanol, production rate of acetaldehyde, level of reduced pyridine nucleotides, and oxygen consumption rate. In the first experiment, the rats with FA showed significantly higher concentrations of both ethanol and acetaldehyde in blood after ethanol intake than the rats without FA. In the perfused liver, FA suppressed ethanol uptake, and increased acetaldehyde concentration in the effluent. FA decreased the level of intracellular reduced pyridine nucleotides which had been elevated by ethanol. FA caused an increase in the oxygen consumption rate, which was not altered by the coexistence of ethanol. It was concluded that FA suppressed ethanol metabolism due to suppression of acetaldehyde oxidation in the liver, despite its uncoupling effect on oxidative phosphorylation in mitochondria.     

Fura-2 measurement of cytosolic free calcium in rat brain cortical synaptosomes and the influence of ethanol

The effects of ethanol on resting and KCl-depolarized cytosolic calcium concentrations ([Ca2+]i) were measured in purified rat cortical synaptosomes using the calcium indicator fura-2. Ethanol (500-700 mM) significantly elevated the resting [Ca2+]i by 13-25% in the presence of 100 microM external calcium. In the absence of external Ca2+, ethanol (50-200 mM) significantly increased [Ca2+]i by 17-23%. Ethanol (200-700 mM) also significantly decreased KCl-induced rise in [Ca2+]i (delta K) by 40-82% in fura-2 loaded preparations incubated in the absence or presence of external calcium. Ethanol did not produce a significant change in delta K at 50 and 100 mM concentrations. These results suggest that ethanol may cause an elevation in [Ca2+]i via mobilization of intracellular calcium stores which may be linked to a calcium-dependent inactivation of voltage-sensitive calcium channels.