Embryonic cerebral cortical progenitors are resistant to apoptosis, but increase expression of suicide receptor DISC-complex genes and suppress autophagy following ethanol exposure

BACKGROUND: In utero exposure to ethanol can result in severe fetal brain defects. Previous studies showed that ethanol induces apoptosis in differentiated cortical neurons. However, we know little about ethanol's effects on proliferating embryonic cortical progenitors. This study investigated the impact of ethanol exposure on the Fas/Apo-1/CD95 suicide receptor pathway, and on the survival of proliferating cortical neuroepithelial progenitors. METHODS: Murine embryonic-derived primary cortical neuroepithelial cells were maintained as neurosphere cultures and exposed to a dose range of ethanol for periods ranging from 1 to 5 days. Programmed cell death was measured by 4 independent means (Annexin-V staining, caspase activation, DNA fragmentation, and autophagic vacuole formation). Surface Fas/Apo-1 suicide receptor expression was measured by flow cytometry. Expression of Fas/Apo-1-associated DISC-complex genes was measured by quantitative polymerase chain reaction. RESULTS: Ethanol exposure did not substantially increase apoptosis, necrosis, or surface Fas/Apo-1 expression. Moreover, ethanol significantly decreased caspase activation and autophagic activity. Finally, ethanol exposure induced mRNA expression of genes that constitute the death receptor complex. CONCLUSIONS: This study provides surprising evidence that ethanol does not induce either programmed cell death or necrosis of immature progenitors during neurogenesis, although ethanol may render neural progenitors susceptible to future apoptotic insults. Furthermore, our novel observation that ethanol suppresses autophagy is consistent with a hypothesis that ethanol promotes premature neural progenitor maturation. Taken together with our previous data regarding the role of the Fas/Apo-1 receptor in neural development, we conclude that ethanol disrupts basic proliferation and differentiation machinery rather than initiating cell death per se.     

MK-801 can exacerbate or attenuate behavioral alterations associated with neonatal alcohol exposure in the rat, depending on the timing of administration

BACKGROUND: We have reported that administration of MK-801, an NMDA receptor antagonist, during ethanol withdrawal in the developing rat attenuates ethanol's adverse effects on behavioral development. In the present study, we altered the timing of MK-801 delivery in relation to the last alcohol dose to determine if its protective effects were specific to the ethanol withdrawal phase. METHODS: Five groups of rats were artificially reared and exposed to alcohol in a binge-like manner on postnatal day (PD) 6, producing peak blood alcohol levels of 335 mg/dl that cleared to 0 mg/dl by 33 hours. Four groups received MK-801 at various times after alcohol treatment (0, 9, 21, or 33 hr post-ethanol). The fifth alcohol-treated group received saline. Two artificially reared control groups were included: one was injected with saline and the other injected with 0.5 mg/kg MK-801. Finally, a normally reared suckle control group was also included. Activity level and performance on a spatial discrimination reversal-learning task were evaluated at PD 18 and PD 40, respectively. RESULTS: Administration of MK-801 at the same time as ethanol treatment (0 hr) produced a high rate of mortality. Ethanol exposure on PD6 increased activity level relative to controls. Administration of MK-801 at 0 hr exacerbated this ethanol-induced overactivity, whereas administration of MK-801 at 21 and 33 hr reduced the severity of ethanol-related overactivity. Similarly, ethanol exposure on PD 6 significantly increased the number of errors committed on a spatial discrimination reversal-learning task. MK-801 injections 9 hrs after ethanol exacerbated this effect, whereas MK-801 treatment 33 hrs after ethanol attenuated this effect. Thus, MK-801 administration at the time of ethanol treatment was highly toxic, whereas during the withdrawal period it was protective. CONCLUSION: These data are consistent with the hypothesis that ethanol exposure in the neonatal rat inhibits the NMDA receptor, producing a subsequent rebound in NMDA receptor activation and possible excitotoxicity during withdrawal. Both the acute inhibitory effects of ethanol and the excitatory effects of withdrawal may contribute to fetal alcohol effects.     

Third trimester binge ethanol exposure results in fetal hypercapnea and acidemia but not hypoxemia in pregnant sheep

BACKGROUND: The mechanisms by which maternal ethanol abuse during pregnancy causes neurodevelopmental injury in the fetus are not well understood. The purpose of this study was to use a chronically instrumented fetal sheep model system to determine if a binge pattern of ethanol exposure administered throughout the third trimester reduced fetal arterial partial pressure of oxygen (PaO2); a positive finding would support the hypothesis that fetal hypoxemia may play a role in mediating ethanol-related birth defects. METHODS: Pregnant ewes received saline or 0.75, 1.25, 1.5, or 1.75 g/kg of ethanol intravenously over 1 hr beginning on day 109 of gestation (term = 145 days) for 3 consecutive days per week followed by 4 days without exposure. The fetuses were surgically instrumented on day 113, and experiments were performed on days 118 or 132, the 6th and the 12th ethanol exposure, respectively. RESULTS: Ethanol infusions resulted in peak blood ethanol concentrations of 80.8 +/- 6.5, 182.5 +/- 13.5, 224.4 +/- 13.9, and 260.6 +/- 20.0 mg/dl +/- SEM (maternal) and 70.0 +/- 5.9, 149.7 +/- 9.0, 216.9 +/- 14.0, and 233.3 +/- 19.8 mg/dl +/- SEM (fetal) in response to the 0.75, 1.25, 1.5, and 1.75 g/kg doses, respectively. Maternal and fetal heart rate and maternal blood pressure increased whereas fetal blood pressure decreased in a dose-dependent manner in response to ethanol infusions. Maternal and fetal arterial pH decreased and arterial partial pressures of carbon dioxide increased in response to ethanol infusions. Maternal PaO2 decreased whereas fetal PaO2 did not change in response to ethanol infusions. CONCLUSIONS: A binge ethanol exposure paradigm, three consecutive days per week throughout the third trimester at ethanol doses that created blood ethanol concentrations commonly achieved by human ethanol abusers, resulted in changes in maternal and fetal heart rate, changes in blood pressure, hypercapnea, acidemia, and maternal, but not fetal, hypoxemia. We conclude that in an ovine model system, ethanol doses that create blood ethanol concentrations as high as 260 mg/dl do not result in fetal hypoxemia. Remaining issues to address with this model system are whether neurodevelopmental injuries that are associated with maternal ethanol abuse are mediated by a reduction in fetal cerebral blood flow, fetal hypercapnea, or acidemia.     

Evaluation of the Alcopatch, a Transdermal Dosimeter for Monitoring Alcohol Consumption

Background : The Alcopatch is an improved transdermal dosimeter for the measurement of alcohol consumption, by detection of ethanol in fluid excreted from the skin. The device is worn as a band around the ankle and provides a visual signal in the event of tampering. Methods : Fourteen volunteers wore duplicate Alcopatches for a period of 7 or 8 days, while keeping a written record of their beverage alcohol consumption. Ethanol concentration in the Alcopatch was measured by gas chromatography and correlated with self-reported consumption. Results : All alcohol consumption in excess of 0.25 g/kg/day resulted in measurable levels of ethanol in the Alcopatch. A positive correlation was observed between the reported consumption of ethanol (in g/kg/day) and the concentration of ethanol in the Alcopatch (square root, in mg/dl) ( y = 0.91x + 0.28, r = 0.61) in 12 of 14 subjects. Conclusions : The Alcopatch detected the consumption of beverage alcohol with high sensitivity and specificity over a period of 7 to 8 days and may be useful for the study of target populations.     

Acute Alcohol Effects on Opiomelanocortinergic Regulation

To assess acute effects of alcohol on forebrain and pituitary opiomelanocortinergic regulation, a model was developed in which “experienced” (previously introduced to ethanol administration, so the subjective response was not a novel stimulus) male Sprague-Dawley rats received pulsatile intragastric ethanol infusions during the dark (active) photophase to produce and sustain (for 3 hr) behaviorally relevant (0, 40 to 70, 80 to 110, or 120 to 150 mg/dl) plasma ethanol levels. The effects of alcohol on hypothalamo-pituitary-adrenal (H-P-A) axis function were biphasic with respect to dosage (inhibition with low dosage and stimulation with higher dosages) and time (initial stimulation with higher dosages was followed by rapid return to control levels even though elevated plasma ethanol levels were maintained). The effects of alcohol on H-P-A activation were also inconsistent; some of the animals did not appear to respond even though elevated (i.e., > 100 mg/dl) plasma ethanol levels were produced. Induction of moderate (80 to 110 mg/dl) plasma ethanol levels acutely (within 30 min) increased immunoreactive (i) β-endorphin concentrations in the ventral tegmental area of the brain; higher (120 to 150 mg/dl) plasma ethanol levels increased iβ-endorphin concentrations in both the ventral tegmental area and the nucleus accumbens, whereas iβ-endorphin concentrations were not significantly altered in other brain areas. High (120 to 150 mg/dl) plasma ethanol levels also increased mediobasohypothalamic pro-opiomelanocortin (biosynthetic precursor of forebrain β-endorphin) mRNA concentrations at 3 and 6 hr after initiation of ethanol infusions. Results demonstrate that atraumatic induction of physiologically meaningful plasma alcohol levels by gastric ethanol infusion activates the forebrain opiomelanocortinergic opioid system and exerts complex effects on the interrelated H-P-A system, consistent with evidence that these systems may interact to mediate or modulate some responses to alcohol ingestion.     

Increased Cell Death and Reduced Neural Crest Cell Numbers in Ethanol-Exposed Embryos: Partial Basis for the Fetal Alcohol Syndrome Phenotype

Fetal alcohol syndrome (FAS) is characterized by growth retardation, craniofacial malformations, and heart and neural defects; the cellular and molecular mechanism(s) responsible for ethanol's teratogenicity remains unknown. Although the phenotype suggests that prenatal ethanol exposure perturbs neural crest cell development, direct proof that these cells are an in utero target is still lacking. Previous research suggested that cranial neural crest cells are eliminated by ethanol-induced apoptosis. We tested this hypothesis using a chick embryo model of FAS. A single dose of ethanol, chosen to achieve a concentration of 35–42 mg/dl, was injected in ovo at gastrulation and resulted in growth retardation, craniofacial foreshortening, and disrupted hindbrain segmentation. Ethanol exposure enhanced cell death within areas populated by cranial neural crest cells, particularly in the hindbrain and craniofacial mesenchyme. In contrast, control embryos had limited cell death within these regions. Subsequent immunolabeling with neural crest cell-specific antibody revealed that ethanol treatment resulted in fewer neural crest cell numbers, whereas neural crest migration patterns were unaffected by ethanol. These results suggest that prenatal ethanol exposure leads to loss of cranial neural crest cells. Such a loss could result, in part, in the phenotype characteristic of FAS.     

Brain Neuronal Degeneration Caused by Episodic Alcohol Intoxication in Rats: Effects of Nimodipine, 6,7-Dinitro-quinoxaline-2,3-dione, and MK-801

Rats repeatedly intoxicated with alcohol (ethanol, three times daily) over a 4-day period display neuronal degeneration in the dentate gyrus; entorhinal, piriform, insular, orbital, and perirhinal cortices; and in the olfactory nerve fibers and terminals in the olfactory bulb. Postulating a role for excitotoxicity, we have attempted to prevent the degeneration using antagonists that are neuroprotective in this type of brain damage. In an initial study, continuous subcutaneous infusion of a high dose of the glutamate/NMDA receptor antagonist MK-801 (2 mg/kg/day) by itself caused extensive neuronal degeneration in several brain regions and severe behavioral intoxication that precluded survival if combined with high blood alcohol levels (～300 mg/dl). Moreover, the lower, nonneurotoxic blood alcohol levels (～150 mg/dl) that were compatible with survival worsened the MK-801-induced brain damage. In a subsequent experiment, daily intraperitoneal injections of a lower dose of MK-801 (1 mg/kg/day) resulted in no MK-801 toxicity and, when combined with neurotoxic levels of alcohol, no reduction in alcohol-induced neurotoxicity. Nimodipine, a voltage-gated Ca²⁺ channel blocker, reduced the neuronal damage in the dentate gyrus, but greatly increased it in the piriform cortex when administered intragastrically at 600 mg/kg/day; it provided no protection from alcohol-dependent degeneration when given intragastrically at 100 mg/kg/day. Continuous intracere-broventricular delivery of 0.24 to 0.29 mg/day of 6,7-dinitro-quinoxa-line-2,3-dione, a glutamate/α-amino-3-hydroxy-5-methyl-4-isoxazole receptor antagonist, failed to diminish alcohol-dependent neuronal damage in any brain region. We conclude that brain damage from episodic “binge” alcohol intoxication is not primarily mediated by excitotoxic mechanisms, implying that other, nonexcrtotoxic pathophysiological mechanisms, are involved. Furthermore, MK-801, far from protecting from the alcohol-induced damage, at high doses causes widespread neuropathology that is significantly potentiated by alcohol.     

Ethanol inhibits the sensory responses of cerebellar granule cells in anesthetized cats

BACKGROUND: Granule cells occupy a strategic position in the transmission of afferent information to the cerebellar cortex. They are also the most abundant type of neurons in the cerebellum. The functions of the cerebellum are thought to be sensitive to acute alcohol intoxication. The effects of acute alcohol intoxication on the in vivo physiology of cerebellar granule cells are, however, not completely known. METHODS: We studied chloralose-anesthetized cats at ethanol doses relevant to human drinking (0.3-1.2 g/kg). We recorded the electrophysiological responses of granule cell clusters to auditory and visual stimulation, and simultaneously monitored the concentration of ethanol in the cerebrospinal fluid (CSF). RESULTS: At an intravenous ethanol dose of 0.3 g/kg, CSF ethanol concentration peaked in 10 minutes at 17 mM, equivalent to a blood alcohol concentration (BAC) of about 0.08 g/dL. Ethanol quickly and almost completely abolished both auditory and visual responses from granule cells. Complete or near-complete inhibition lasted 15 to 20 minutes; approximately 50% recovery required an additional 15 minutes, and a full recovery yet another 15 minutes. A higher ethanol dose at 1.2 g/kg resulted in a more severe inhibition and required longer time for recovery. The relationship between ethanol dose, CSF ethanol concentration, and granule cell responses was dynamic and nonlinear, critically depending upon the elapsed time. CONCLUSIONS: Cerebellar granule cell sensory responses are highly sensitive to ethanol inhibition. A rapid development of acute tolerance appears to be a major factor contributing to the dynamic and nonlinear relationship among ethanol dosage, CSF ethanol concentration, and granule cell responses. It is likely that a generalized de-afferentation of the cerebellum from its mossy fiber afferents, followed by the subsequent development of acute tolerance may play major roles by which alcohol intoxication affects cerebellar functions.     

Human Neutrophil Functions Are Inhibited In Vitro by Clinically Relevant Ethanol Concentrations

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs—chemotaxis and production of reactive oxygen species—and on changes in cytosolic-free calcium ([Ca²⁺]_i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b), fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca²⁺]_i, as assessed by videomicroscopy using the Ca²⁺-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca²⁺]_i, levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca²⁺ influx or in [Ca²⁺]_i.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x_L and Bcl-x_S, which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma.   In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Significant Increase of Blood Alcohol by Cimetidine after Repetitive Drinking of Small Alcohol Doses

To assess effects of repetitive alcohol drinking and pre-existing first-pass metabolism on the cimetidine induced increase in blood alcohol concentrations, 20 healthy men (aged 20 to 40) of varied ethnicity and consuming less than 60 g alcohol per week underwent baseline quantitation of first-pass metabolism of alcohol. This was followed by oral administration of 0.6 g/kg ethanol given postprandially in 3 to 4 drinks spread over 135 min, before and after cimetidine (400 mg twice a day for 7 days). Blood alcohol concentrations were determined by breath analysis. First-pass metabolism was quantified by applying Michaelis-Menten kinetics to blood alcohol curves after intravenous or oral administration of equal alcohol doses. At baseline, 15 subjects had a substantial first-pass metabolism (over one sixth of the dose); their alcohol levels increased with repeated doses with a mean peak of 27 ± 3 mg/dl before and 39 ± 5 after cimetidine ( P < 0.01), an effect much greater and longer than after a single alcohol dose. Three subjects exceeded 50 mg/dl, the legal limit for driving in several countries. By contrast, in the five subjects with minimal first-pass metabolism, cimetidine did not increase alcohol levels. Thus, under conditions mimicking social drinking, cimetidine increased blood alcohol to concentrations known to impair psychomotor skills and they persisted at those levels over prolonged periods of time. In a minority of subjects, no such interaction was found, but their first-pass metabolism at baseline was absent or minimal and thus no inhibition by the drug was to be expected.     

Intensity and Duration of Chronic Ethanol Exposure Is Critical for Subsequent Escalation of Voluntary Ethanol Drinking in Mice

Background: Excessive alcohol drinking continues to be an important health problem. Recent studies from our laboratory and others have demonstrated that animal models of ethanol dependence and relapse can contribute to understanding factors that contribute to excessive drinking. In this study, we tested the hypothesis that the amount and duration of ethanol exposure is critical for promoting the escalation in drinking by mice given access to ethanol in a limited access paradigm. Methods: We used several methods of chronic intermittent ethanol exposure in male C57BL/6J mice that would vary in the amount and duration of exposure to ethanol as indicated by blood ethanol concentrations (BEC). After establishing baseline drinking in the mice using a 2 hours, 2 bottle choice drinking paradigm, each study involved alternating between periods of ethanol exposure and periods of limited access to ethanol (1 cycle) for a total of 3 cycles. In Study 1, mice were allowed extended access (16 hours) to ethanol for oral consumption or remained in the home cage. In Study 2, the ethanol exposure consisted of intragastric gavage of increasing doses of ethanol or isocaloric sucrose as the control. Study 3 compared intragastric gavage combined with pyrazole, an alcohol dehydrogenase inhibitor, with vapor inhalation of ethanol using procedures known to lead to increased drinking in mice. Finally, Study 4 was a retrospective review of several studies conducted in our laboratory using inhalation procedures. The retrospective review encompassed a range of postvapor chamber BEC values and ethanol intakes that would allow a relationship between increased drinking and BEC to be examined. Results: Allowing mice to drink for longer periods of time did not cause increased drinking in subsequent limited access sessions. Likewise, gastric intubation of ethanol which produced high BEC (>300 mg/dl) with or without pyrazole did not increase drinking. Only the vapor inhalation procedure, which was associated with sustained BEC above 175 mg/dl for the entire exposure period resulted in increased drinking. The retrospective study provided further evidence that sustained BEC levels above 175 mg/dl was critical to the escalation in drinking. Conclusions: We found that the intensity (amount) and duration of ethanol exposure, indexed by BEC, is critical to produce increased drinking in mice. Specifically, BEC must regularly exceed 175 mg/dl for the escalation in drinking to occur. Future studies will examine neurobiological adaptations that may underlie the increased drinking behavior caused by chronic intermittent ethanol exposure.     

Behavioral Effects and Pharmacokinetics of Low-Dose Intravenous Alcohol in Humans

Alcohol has physiological effects on the human central nervous system at blood alcohol concentrations (BACs) as low as 9 mg/dl. It is unknown, however, if humans can perceive the effects of such low doses of alcohol. Furthermore, low BACs can be difficult to measure. The purpose of this experiment was to: (1) assess the ability of humans to perceive subjective effects of low BACs; (2) measure behavioral effects of low BACs on a psychomotor performance task; and (3) test the sensitivity and accuracy of the transdermal alcohol sensor (TAS) for measuring low BACs from skin. Five men and seven women were administered single-blind intravenous infusions of ethyl alcohol in 5% dextrose/water to achieve peak BACs of 0,10,20, and 40 mg/dl. Subjective intoxication scales and a computer administered continuous performance task (CPT) were used to assess alcohol effects. BACs were estimated from skin, blood, and breath. The only alcohol-induced sensation significantly increased during the alcohol infusions was anesthesia measured by the Alcohol Sensation Scale on the descending limb of the BAC curve. The subjective positive-reinforcing stimulant and mood effects of alcohol were not reported until subjects were administered the 40 mg/dl alcohol infusion. Other measures of subjective intoxication and sedation, and the CPT were unaffected by the alcohol infusions. The TAS provided a noninvasive method for estimating BACs that was comparable with estimates obtained from blood and breath, although delayed in time.     

Acute Hypocalcemic Effect of Ethanol in Dogs

Ethanol has been shown to reduce serum calcium in multiple animal studies. However, in human studies done using lower doses of alcohol, only inconclusive results have been obtained. This study was undertaken to evaluate the effects of varying doses of oral ethanol on total serum calcium. Fifteen adult mongrel dogs (17-25 kg) were divided into three groups which differed in the dosage of ethanol given. Group I animals received 0.5 g/kg of ethanol; Group II, 1.0 g/kg ethanol; and Group III, 2.0 g/kg of ethanol. Venous blood was sampled for estimation of concentrations of total serum calcium and ethanol. In the animals in Group I, serum calcium levels were unchanged by the ethanol. In both Groups II and III, significant reductions in serum calcium were demonstrated, which occurred within 5 min of intoxication. The mean decrease in serum calcium in Group III animals was significantly greater than that in either Group I and II. We conclude that the rapid hypocalcemic effect requires a threshold amount of ethanol before it becomes chemically evident. This critical value in dogs approximated 1 g/kg which results in a mean peak serum alcohol concentration of 117 +/- 6 mg/dl.     

A three-stage alcohol clamp procedure in human subjects

BACKGROUND: Multiple factors can influence blood alcohol levels (BALs) after oral ethanol intake. To minimize the variability in BALs, an alcohol "clamping" method in which alcohol is administered intravenously has been previously reported. We have modified this method and developed a multistage alcohol clamp procedure in humans. METHODS: Infusion of alcohol to achieve sequentially increasing steady-state BALs at 50, 100, and 150 mg/dl (e.g., an alcohol clamp procedure) was performed in five subjects. The method used intravenous infusion of 6% alcohol in saline and followed a protocol generated by a physiologically based pharmacokinetic model for alcohol infusion. Real-time measurement of BALs was used to base decisions for necessary adjustments in the infusion rates on a negative feedback principle to achieve and maintain the desired BALs. Additionally, in three of these subjects, breath alcohol levels were determined simultaneously with BAL measurements. RESULTS: A three-stage alcohol clamp procedure was used to achieve BALs of 50, 100, and 150 mg/dl. The intra-assay and interassay variations for measurement of BALs were 1.6 and 2.1%, respectively. The coefficients of variation for the 50, 100, and 150 mg/dl alcohol clamps were 9.8, 5.6, and 4.6%, respectively. The deviation from target BALs for the three clamps were less than 5%. Breath alcohol levels were lower than BALs across all time points, with the mean ratio between the two being 0.84. Correlation analysis showed a very strong correlation between the two alcohol measurement methods ( r= 0.96; p < 0.001). CONCLUSIONS: A three-stage alcohol clamp procedure (50, 100, and 150 mg/dl) has been developed by following a protocol generated with a physiologically based pharmacokinetic model for alcohol infusion in human subjects. This is a suitable procedure to examine the responses on cognitive measures and physiologic parameters under steady states of desired BALs.     

Migration of Cortical Neurons Is Altered by Gestational Exposure to Ethanol

Rats prenatally exposed to ethanol exhibit a variety of structural anomalies in the central nervous system. One of the key features of experimental fetal alcohol syndrome is microencephaly. Cerebral cortex is particularly susceptible to the effects of prenatal exposure to ethanol. Its total mass is reduced, it is thinner, and it contains fewer neurons and glia. Various studies in rats and humans suggest that chronic early exposure to ethanol leads to a massive reorganization of cortex characterized by heterotopic clusters of neurons. A pulse and chase study with [³H]thymldine autoradiography was used to determine if gestational exposure to ethanol leads to defects in neuronal migration. Rats were fed an ethanol-containing diet between gestational day (G) 6 and G21, pair-fed a liquid control diet, or fed chow. Ethanol delayed the migration of early-generated neurons (those born on G13) to deep cortex by 2 days. Moreover, the migration of late-generated neurons (those born on G21) was delayed 4 to 6 days by ethanol, and often these neurons terminated their migration in ectopic locations. Ethanol significantly decreased the rate of migration and the time postmitotic cells remained in the proliferative zones. On the other hand, ethanol did not have a significant effect on the rate of cortical expansion and the fraction of cells that left the proliferating population in order to migrate to cortex. Thus, the migration of young neurons was profoundly altered by prenatal exposure to ethanol. Such delays may lead to a desynchronization of cortical development that makes it impossible for cortical neurons to establish a normal circuitry.     

Intragastric Intubation of Alcohol During Postnatal Development of Rats Results in Selective Cell Loss in the Cerebellum

Postnatal alcohol exposure produces reductions in the number of Purkinje cells in the rat cerebellum. The goal of this study was to determine if the method of postnatal alcohol exposure would influence the degree of vulnerability of the Purkinje cells. Previously reported studies from other laboratories have demonstrated cerebellar Purkinje cell count reductions following postnatal alcohol exposure via artificial rearing and vapor inhalation techniques. This study used gastric intubation to administer alcohol (3.6 g ethanol/kg body weight, bid) to male rat pups from postnatal days 4–10. Peak blood alcohol levels were 203 ± 12.7 mg/dl on postnatal day 6. On postnatal day 10, the animals were perfused, and brain weights were obtained. Body weight was not significantly altered by the postnatal alcohol exposure, yet the wet weights of the cerebral cortex and whole brain were significantly reduced. Although the cerebellar weight was not significantly reduced, the overall number of Purkinje cells measured in the cerebellar vermis was significantly reduced by 24% compared with the isocaloric and normal control groups. The pattern of vulnerability for the individual cerebellar lobules was similar to the previously reported studies, indicating that alcohol's teratogenicity transcends experimental paradigm and is remarkedly consistent, when relatively similar blood alcohol profiles are established.     

Neuroanatomic and Neurochemical Abnormalities in Non-human Primate Infants Exposed to Weekly Doses of Ethanol during Gestation

Ethanol was orally administered once per week to 54 gravid pigtailed macaques ( Macaca nemestrina ) in doses of 0.0, 0.3, 0.6, 1.2, 1.8, 2.5 or 4.1 gm/kg from the 1st week in gestation or in doses of 2.5, 3.3 or 4.1 gm/kg from the 5th week. Mean maternal peak plasma ethanol concentrations (MPPEC's) ranged from 24 ± 6 mg/dl at the 0.3 g/kg dose to 549 ± 71 mg/dl at the 4.1 g/kg dose. Thirty-three live born infants were assessed for abnormalities of physical and behavioral development. Ocular pathology, neuropathologic and neurochemical assessments were done on 31 animals at 6 months postnatal age. Microphthalmia was noted in three of the 26 animals exposed to ethanol. Retinal ganglion cell loss was significantly associated with intra-uterine ethanol exposure. Microphthalmia and retinal ganglion cell loss was observed in both the delayed and full-gestational exposed animals. No structural anomalies were found in the brains via gross inspection or light microscopy. Chemical abnormalities in the striatal nuclei were identified. Striatal dopamine concentrations increased with increasing MPPEC exposure (0–249 mg/dl) among animals exposed weekly to ethanol throughout gestation. Striatal dopamine concentrations decreased with increasing MPPEC exposure (260–540 mg/dl) among animals whose weekly exposure to ethanol was delayed until the 5th week of gestation. The same pattern of association was also noted between MPPEC and ultrastructural alterations in the caudate nucleus. The extent of ultrastructural alterations increased with increasing MPPEC among the fullgestational exposed animals and decreased with increasing MPPEC among the delayed-dose animals.