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Polychlorinated biphenyl (PCB) levels and the factors affecting PCB accumulation in subadult and maturing Chinook salmon Oncorhynchus tshawytscha from Puget Sound were characterized. Specifically, we (1) determined PCB levels in Chinook salmon from Puget Sound and compared them with levels in Chinook salmon from other West Coast populations, (2) determined whether PCB accumulation mainly occurred in the freshwater or marine habitats, and (3) quantified the relative importance of fish age, fish size (fork length), lipid content, and saltwater age (the number of winters spent in saltwater) on PCB concentration. The average PCB concentration measured in skinless muscle tissue samples of subadult and maturing Chinook salmon collected from Puget Sound was 53 ng/g (wet weight), which was 3-5 times higher than those measured in six other populations of Chinook salmon on the West Coast of North America. Concentrations in the Puget Sound samples varied from 10 to 220 ng/g. A comparison of PCB body burdens...

Cerebral hypoxia has been proposed as a mechanism by which prenatal ethanol exposure causes fetal alcohol spectrum disorder (FASD) in children, but no study had tested this hypothesis using a chronic exposure model that mimicks a common human exposure pattern. Pregnant sheep were exposed to ethanol, 0.75 or 1.75 g kg(-1) (to create blood ethanol concentrations of 85 and 185 mg dl(-1), respectively), or saline 3 days per week in succession (a 'binge drinking' model) from gestational day (GD) 109 until GD 132. Fetuses were instrumented on GD 119-120 and studied on GD 132. The 1.75 g kg(-1) dose resulted in a significant increase in fetal biventricular output (measured by radiolabelled microsphere technique) and heart rate, and a reduction of mean arterial pressure and total peripheral resistance at 1 h, the end of ethanol infusion. The arterial partial pressure of CO(2) was increased, arterial pH was decreased and arterial partial pressure of O(2) did not change. Fetal whole-brain blood flow increased by 37% compared with the control group at 1 h, resulting in increased cerebral oxygen delivery. The elevation in brain blood flow was region specific, occurring preferentially in the ethanol-sensitive cerebellum, increasing by 44% compared with the control group at 1 h. There were no changes in the lower dose group. Assessment of regional differences in the teratogenic effects of ethanol by stereological cell-counting technique showed a reduced number of cerebellar Purkinje cells in response to the 1.75 g kg(-1) dose compared with the control brains. However, no such differences in neuronal numbers were observed in the hippocampus or the olfactory bulb. We conclude that repeated exposure to moderate doses of ethanol during the third trimester alters fetal cerebral vascular function and increases blood flow in brain regions that are vulnerable to ethanol in the presence of acidaemia and hypercapnia, and in the absence of hypoxia.

Controllable shifting of threshold voltage and modulation of current in organic field-effect transistors (OFETs) is demonstrated, resulting in the formation of unipolar inverters by making use of space-charge electrets. Prior to the deposition of the organic semiconductor (OSC), negative corona charges are injected and trapped in the bulk of the organosilsesquioxane glass resin gate dielectrics. The effective surface potential is controlled by the corona-charging and subsequent annealing process. It is found that the shift of the transfer characteristics is governed by the electrostatic induction effects of the charged gate electrets, and this observed shift can be related to the surface potential of the layer next to the transistor channel. The process control, efficiency, and long-term stability of charge storage in spin-on organosilsesquioxane glass resins are sufficient to enable the construction of simple unipolar inverters and to allow for circuit tuning. New OFET unipolar inverters with an enhancement-mode driver and a depletion-mode load are presented, composed of only two simple OFETs with the same channel dimensions and the same p-type OSC on charged electrets. This design allows the implementation of full-swing organic logic circuits and illustrates a potential process simplification for organic electronics.

Wecompute a sequential equilibrium for the U.S.Army'sTargeted Selective Reenlistment Bonus Program in whichtheArmy offers a bonus, soldiers with a low cost of serving in an undesirable location accept the bonus and are sent to an undesirable location, and soldiers with a high cost of serving in an undesirable location reenlist but decline the bonus.Wefind that this program benefits both theArmy and soldiers, increases retention, increases the number of soldierswho serve in an undesirable location, and better matches soldiers to assignments.We discuss implications of our model on human resource management practices for theArmy in its administration of the bonus program and its application to other large organizations that set wages through a rules-based mechanism. © 2007 Wiley Periodicals, Inc.

Background: In spite of the fact that drinking and smoking often occur together, little is known about the pharmacokinetic interaction between alcohol and nicotine. Previous research in neonatal rats demonstrated that nicotine reduces blood alcohol concentrations (BACs) if alcohol and nicotine are administered simultaneously. However, it is unclear whether such a phenomenon can be observed in adult subjects, given the fact that there is an ontogenetic difference in alcohol metabolism. Methods: A range of nicotine doses (0, 0.25, 0.5, 1, 2, 4, and 6 mg/kg) were administered individually with an alcohol dose (4 g/kg) via intragastric (IG) intubation to adult female rats, and the resultant BACs were measured at various time points following drug administration. Furthermore, the hypothesis that nicotine's role in reducing BACs is mediated through factors related to gastric function was examined by comparing the resultant BACs after an IG intubation or intraperitoneal (IP) injection of alcohol. Results: The results from this study showed significant nicotine dose–related decreases in BACs with 0.5, 1, 2, 4, and 6 mg/kg doses of nicotine at the various time points assessed. This effect, however, occurred only when alcohol was administered via IG intubation, but not after an IP injection of alcohol. Conclusions: These results suggest that the nicotine-induced decrease in BAC may be related to gastric function. One possible explanation was related to nicotine's action in delaying gastric emptying. The longer the alcohol was retained in the stomach, the more likely that the alcohol would be metabolized by gastric alcohol dehydrogenase before its absorption into the bloodstream by the small intestine (the major site of alcohol absorption).

The current U.S. military pay structure offers inequitable and inefficient wages across locations. Military personnel are paid less competitive wages in high-cost and/or low-amenity locations compared to low-cost and/or high-amenity locations. This pay system results in unequal reenlistment rates across locations, which leads to production inefficiencies caused by short-term manning shortages in highturnover locations. Wages set according to local civilian compensating wage differentials would result in a more stabilized force (across locations) by equalizing the opportunity cost of staying in the military at each location. Additionally, more personnel would volunteer to serve in the high-cost and/or low-amenity locations because wages would be more commensurate with local costs and amenities. This would result in fewer non-volunteer assignments to undesirable locations and a minimized opportunity cost for personnel serving at each location. Reenlistment simulations on first-term Air Force personnel show that the proposed wage structure would better equalize reenlistment rates across locations. This proposal could be implemented at no cost to the government by cutting wages in low-cost and/or high-amenity locations. A gradual implementation in which such wages are frozen or increased slowly may be more politically palatable. © 2005 by the Association for Public Policy Analysis and Management

A new method for quantifying “sparkle” uses a simple measurement which includes a pixelated source, a test sample, and an eye simulator. The degree of sparkle is calculated from the standard deviation of the pixel powers across a portion of the display. Measurements show excellent correlation to human response studies.

NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N(G)-nitro-L-arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono) bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.

Background: Abnormal thyroid hormone system function in the mother or fetus during pregnancy can result in brain defects, some of which resemble those found in children with fetal alcohol syndrome. It has been hypothesized that ethanol may act to mediate alcohol-related birth defects in part by altering thyroid hormone system function. We investigated whether a binge pattern of maternal ethanol consumption over the last trimester equivalent of gestation in sheep results in an alteration in fetal or maternal thyroid function. Methods: Pregnant ewes received saline or ethanol beginning on day 109 of gestation (term, 145 days) for three 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. Fetal and maternal blood samples were collected, and plasma tri-iodothyronine (T3), thyroxine (T4), and free T4 concentrations were measured. Results: Fetal T3 and T4 on day 118, fetal T3 on day 132, and maternal T3 on day 132 were lower in response to ethanol. Fetal and maternal free T4 and maternal T4 did not change in response to ethanol. Fetal thymus and adrenal weights were reduced in response to ethanol. Conclusions: We conclude that, in sheep, maternal ethanol exposure during the third trimester equivalent resulting in blood ethanol concentrations that are commonly achieved by ethanol abusers decreases circulating thyroid hormone concentrations in the mother and fetus and fetal thyroid and thymus mass.

Background : Although the mechanisms that underlie fetal alcohol-induced neuronal loss have not been determined, hypoxia/hypoxemia has been considered a leading candidate. This study was designed to test the hypothesis that neuronal loss could occur in the developing brain in the absence of fetal hypoxemia. Methods: Three groups of pregnant sheep were used: a control group, a binge-drinking group, and a pair-fed group. The alcohol and pair-fed animals were anesthetized on day 113 of pregnancy, and the mothers and fetuses were instrumented with arterial and venous catheters. All animals were killed on day 133. Stereological cell counting techniques were used to estimate the total number of Purkinje cells in the fetal cerebellum. Results: Peak maternal and fetal blood alcohol concentrations did not produce fetal hypoxemia. Nevertheless, there was a 25% loss of Purkinje cells of the cerebellum in the alcohol-exposed fetuses compared with that in the pair-fed controls. The loss of neurons was not accompanied by microencephaly or a concomitant decrease in either cerebellar weight or volume of the fetal cerebellum. Conclusions: Neuronal loss can be observed after alcohol exposure during the third trimester equivalent in fetal sheep in the absence of alcohol-induced hypoxemia. Furthermore, cell loss in the absence of deficits in gross brain weight or regional brain volume indicates that the lack of gross brain volume deficits from magnetic resonance imaging techniques is not a reliable indication that the brain is unaffected by the alcohol exposure.

Background : We tested the hypothesis that in utero ethanol exposure results in changes in fetal and maternal adrenocorticotropin (ACTH) and cortisol during the third trimester equivalent, by using a chronically instrumented fetal sheep model. Methods: Pregnant ewes received saline or ethanol intravenously 3 consecutive days per week from day 109 to day 132 of gestation. Fetal and maternal blood samples were collected on days 118 and 132. Results: Maternal and fetal ACTH and cortisol values increased on days 118 and 132 of gestation in response to ethanol infusions that created blood ethanol concentrations (BECs) that are easily achievable by human drinkers. Peak ACTH and cortisol values were detected 30 to 60 min after peak BECs were achieved. Conclusions: Chronic ethanol exposure during the third trimester equivalent in sheep resulted in repeated activation of the hypothalamus-pituitary-adrenal axis in both the mother and fetus. Temporally, the patterns of maternal and fetal responses to ethanol infusion were similar. We conclude that ovine maternal ethanol exposure during the third trimester equivalent increases fetal ACTH and cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects.

Background : Our previous findings suggested that the intragastric coadministration of alcohol and nicotine to neonatal rats resulted in a significant decrease from the predicted peak blood alcohol concentration (BAC). We hypothesized that the coadministration of alcohol and nicotine would produce a nicotine dose–related decrease in peak BAC and a change in the BAC time curve profile. Methods: Sprague-Dawley rat pups were given alcohol and nicotine simultaneously via intragastric infusion. Two sets of nicotine doses were used in two independent studies. The low doses of nicotine were examined after the study of high doses of nicotine administration because of the possible ceiling effects from these nicotine doses on lowering BACs. Results: The results not only confirmed that the peak BAC was lowered by nicotine, but also generated new findings showing that the profile of BAC time curve was affected by these doses of nicotine. Concerns about possible ceiling effects led us to conduct another experiment to examine the effects of lower doses of nicotine on BACs. Those results showed a significant decline in BACs after cotreatment with 0.5 or 1 mg/kg nicotine and less robust changes on the BAC curve profiles. Although the nicotine dose at 0.25 mg/kg/day did not affect significantly the overall BAC profile, it did lower the peak BAC. Conclusions: Nicotine is capable of lowering the peak BAC among neonatal rat pups. Furthermore, the pattern of the BAC time curve seems to be more affected by high doses of nicotine.

Background: Prenatal alcohol exposure disrupts motor performance in affected offspring. The ventrolateral nucleus (VLN) of the thalamus functions to relay information between the cerebellum and motor cortex. Reductions in the size of the thalamus have been found in children with fetal alcohol syndrome, and therefore a rat model system was used to determine whether VLN size and neuronal number were altered by alcohol exposure during development. Methods: Rat pups were exposed to alcohol in utero during the first 10 days of gestation (first trimester equivalent), the second 10 days of gestation (second trimester equivalent), or the first two trimesters equivalent combined. Some pups were exposed to alcohol in utero during the time of VLN neurogenesis. In addition, offspring from some of the dams treated during the first two trimesters equivalent were reared artificially from postnatal day (P) 4 through P9 (part of the third trimester equivalent) and received binge-like alcohol during this time, resulting in offspring exposed to alcohol during all three trimesters equivalent. Other offspring from untreated dams were reared in the same manner but received alcohol only during the third trimester equivalent. Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. Results: A unique effect of alcohol treatment was not found for the VLN volume or the number of neural cells within the VLN. However, the period of VLN neurogenesis was found to be sensitive to both alcohol and nutritional control treatments, resulting in significant decreases in the VLN volume and neural cell number. Conclusions: Motor deficits seen in offspring exposed prenatally to alcohol do not seem to result from direct effects on the structure of the VLN of the thalamus.

Background: Alcohol-induced zinc deficiency is one of the mechanisms proposed as a cause of developmental brain damage associated with fetal alcohol syndrome. It is known that alcohol exposure during the brain growth spurt period leads to cerebellar Purkinje cell loss. Therefore, this study examined whether zinc supplementation was capable of preventing alcohol-induced Purkinje cell loss in the cerebellar vermis in a neonatal rat model system. Methods: Sprague-Dawley rat pups were given alcohol (EtOH; 4.5 g/kg/day), zinc (Zn; 0.54 mg/ml diet; [10 times the regular diet Zn concentration]), or both from postnatal days (PD) 4 through 9 using the artificial-rearing paradigm. A gastrostomy control (GC) and a suckle control group (SC) also were included. All pups were killed on PD 10. Following perfusion, the cerebellar vermis was dissected and processed for stereological cell counting. The total number of Purkinje cells and the volume of the cerebellar vermis were determined. Results: Alcohol produced a significant loss of Purkinje cells compared with that in the GC group (no EtOH and no Zn supplement). The zinc supplementation had no effect in attenuating alcohol-induced Purkinje cell loss in the cerebellar vermis. In fact, the serum zinc concentration data indicated higher zinc concentrations following either EtOH or Zn treatment. Interestingly, the GC group showed a significantly lower zinc concentration compared with the SC group, even though no significant difference in Purkinje cell numbers was observed between these two control groups. Conclusion: These findings indicate that alcohol exposure during the third trimester equivalent did not result in zinc deficiency in this neonatal rat model system, nor did zinc supplementation rescue the alcohol-induced Purkinje cell loss in the cerebellar vermis. These findings showed clearly that the serum zinc concentration was not correlated with Purkinje cell loss, suggesting that alcohol-induced loss of cerebellar Purkinje cells in this neonatal rat model system is independent of the availability of serum zinc.

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 (P a O 2 ); 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 P a O 2 decreased whereas fetal P a O 2 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.

Background: The mechanisms that mediate fetal brain injury which results from maternal alcohol consumption are not well understood. Although fetal hypoxia is a popularly proposed mechanism, it has been difficult to assess brain oxygenation in vivo. We measured intracellular high energy phosphate concentrations and estimated intracellular pH (pH i ) in brains of unanesthetized neonatal rat pups by using in vivo 31 P-NMR spectroscopy. We reasoned that decreases in brain oxygenation sufficient to result in brain injury would also reduce high energy phosphates and pH i . Methods: On postnatal day 4, before alcohol administration, pups were placed into a 20 mm diameter NMR probe, their heads were positioned carefully in the center of the 31 P detection coil, and spectra were collected over 20 min. Animals were then fed diet with or without 4.5 g/kg of ethanol in two (in succession) of 12 daily feedings via artificial rearing methods. A second spectrum was collected at 90 min after the beginning of the second alcohol feeding, at the time that coincided with the peak blood alcohol concentration (BAC). Identical feedings were performed daily until day 9, when pre- and postfeeding spectra were again obtained. Positive control groups were fed control diet and were studied in atmospheres of 5% oxygen, 95% nitrogen or 0% oxygen, 100% nitrogen. Results: Phosphocreatine (PCr), β-adenosine triphosphate (ATP), and pH i decreased and inorganic phosphate (P i ) increased in day 4 animals subjected to 0% oxygen (20 min) compared with pretreatment and all other treatment groups. Day 9 animals did not tolerate these conditions. There were no significant changes in response to 5% oxygen on day 4, but P, increased and β-ATP decreased compared with pretreatment values and compared with alcohol and control groups on day 9. There were no changes in PCr, β-ATP, or pH, in response to alcohol treatment at either age. PCr was significantly increased in the alcohol and 5% oxygen groups and apparently increased in the control group on day 9 compared with day 4, most likely due to increases in cranial muscle mass within the NMR coil. Conclusions: We conclude that acute alcohol exposure that results in peak BACs of 315 mg/dl does not alter brain high energy phosphate concentrations or pH i in neonatal rat pups, although these BACs are known to result in significant brain injury. These findings do not support hypoxia as a mechanism of alcohol-mediated brain injury during the third trimester equivalent in the rat pup model.

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.

Although alcohol has been shown to affect brain development adversely, the underlying mechanism of alcohol's actions are poorly understood. The present study addressed the hypothesis that alcohol affects growth factor availability during critical periods of neural growth by measuring the mRNA expression of brain-derived neurotrophic factor (BDNF), a potent developmental growth factor. Multiple offspring of timed-pregnant rat dams given alcohol (6.0 g/kg per day) or control treatments during gestation were sacrificed at either embryonic (E) day 21 or E33 (usually postnatal day 10) when their olfactory bulbs were processed for molecular analyses or neuron counting. BDNF mRNA levels were measured by reverse-transcription–polymerase chain reaction, and DNA methylation of the BDNF gene was quantified by Southern blot analyses following digestion with methylation-sensitive enzymes. Estimates of total granule cell number were obtained by counting those cells using unbiased stereological techniques. There was a significant decrease in BDNF mRNA levels in the alcohol-exposed offspring of both ages compared with controls. In addition, the number of olfactory bulb granule cells significantly decreased in the E33 but not the E21 rat pups exposed to alcohol compared with their appropriate aged controls. Finally, BDNF DNA of alcohol-exposed animals was less susceptible to digestion with the methylation-sensitive enzyme HpaII compared with controls, suggesting that the DNA of the alcohol exposed pups was hypermethylated. Our results indicate that exposure to alcohol during early brain development in the rat, a period equivalent to the first two trimesters in humans, can have a detrimental effect on normal development of the olfactory bulb by reducing the number of BDNF-synthesizing neurons. Although the exact mechanism for the alcohol-induced neuronal loss is unknown, the inappropriate transcription of the BDNF gene is one mechanism that may account for the complexity of effects observed in offspring exposed to heavy alcohol exposurein utero. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 414–423, 1999

Animal models have clearly established that ethanol exposure can deplete neurons in the developing nervous system. However, the mechanism by which ethanol reduces cell number is unclear. In our study, cultures of pheochromocytoma cells, a neuronal-like cell line, were maintained in media, which supported cell proliferation. Although cell numbers continued to increase in the presence of ethanol, this increase was partially inhibited by ethanol exposure. This inhibitory effect was concentration and duration dependent. Cell proliferation was still partially inhibited after removal of ethanol, but this inhibition was temporary and disappeared after a 24-hr recovery period in ethanol-free conditions. Further study indicated that ethanol partially inhibited the increase in cell numbers by two mechanisms: (1) studies with vital stains indicated that ethanol induced cell death; (2) experiments using synchronized pheochromocytoma cell cultures showed that ethanol can induce cell cycle delay, thereby lengthening the doubling time of the cells. Analysis by flow cytometry indicated that with ethanol exposure, the cells accumulated in the G1 phase of the cell cycle. Our results suggest that in the developing nervous system, ethanol may limit the numbers of proliferating, neuronal precursor cells by two simultaneous mechanisms, cell death and cell cycle delay.

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.

Previous research from our laboratory has shown that [ethanol (EtOH)] exposure during the brain growth spurt is detrimental to olfactory bulb development. This study extends those findings by examining the effects of EtOH, nicotine (NIC), and the combination of these drugs (EtOH/NIC) on olfactory bulb mitral cell numbers, as well as on various major neurotransmitter levels in neonatal rats. An artificial rearing paradigm was used in the present studies. These artificially reared pups were given 4 g/kg/day of EtOH and/or 6 mg/kg/day of NIC on postnatal day (PD) 4 to PD 9, except in the case of the acute neurochemistry study, in which the pups received treatment on PD 9 only. An artificially reared gastrostomy control group (GC) and a suckle control group were included. The mean total numbers of mitral cells in the EtOH and NIC groups were significantly reduced from that of the GC, as well as the volume of the left main olfactory bulb. There was no difference among any of the groups in mitral cell density. As for neurochemistry data, there was no difference in neurotransmitter levels among any of the groups in the repeat exposure regimen. There were, however, changes after the acute exposure (exposure on PD 9 only). Both serotonin and GABA levels were significantly increased only after NIC exposure. However, norepinephrine levels were significantly decreased after acute exposure in all three drug treatment groups, compared with that of the control group. Except for the GC control group, dopamine levels were not detected consistently after acute exposure to EtOH, NIC, or EtOH/NIC. Collectively, these findings demonstrate that exposure to EtOH or NIC individually during the brain growth spurt results in developmental deficits in the olfactory bulb, suggesting that both EtOH and NIC are neuroteratogens. Furthermore, this study demonstrated the capability of NIC to antagonize (protect) EtOH-induced mitral cell loss in the developing olfactory bulb.

The purpose of this study was to investigate the spontaneous activity of mature rat cerebellar neurons that had been exposed to ethanol (EtOH) during postnatal days 4 to 10, which corresponds to the third trimester in humans. Newborn Sprague-Dawley rats were implanted with gastric feeding tubes and were artificially reared from postnatal days 4 to 10 with two different diets. The experimental group received 4.5 g/kg/day of EtOH delivered in a milk solution. Controls received similar feeding with an isocaloric supplement replacing the EtOH. Electrophysiological evaluations were performed after an EtOH-free rearing period. Although lobules IX and X of the cerebellar vermis appeared morphologically smaller in the animals neonatally exposed to EtOH, compared with controls, extracellular recordings from both Purkinje cells and Golgi interneurons in adult rats showed no differences in spontaneous activity or firing pattern between the control and EtOH-exposed animals. Similarly, excitations and inhibitions of Purkinje neuron activity evoked by parallel pathway stimulation appeared unaffected by the developmental EtOH exposure. However, we did observe a significant decrease in the proportion of Purkinje neurons generating complex spike bursts in the group exposed to EtOH neonatally. These data suggest that, although fewer Purkinje neurons may survive the brain growth spurt if exposed to EtOH during this critical period of development, those that do survive appear to function normally. The observed abnormality in complex spike production may result from EtOH effects on developing neurons in the inferior olive that give rise to the climbing fibers that cause this bursting pattern in Purkinje neurons.

Ethanol exposure during development leads to alterations in neuronal differentiation and profound neuronal loss in multiple regions of the developing brain. Although differentiating Purkinje cells of the cerebellum are particularly vulnerable to ethanol exposure, the mechanisms that ameliorate ethanol-induced Purkinje cell loss have not been well defined. Previous research indicates that glial-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β family, promotes the survival of several neuronal populations, including cerebellar Purkinje cells. Therefore, we examined whether GDNF could attenuate ethanol-induced Purkinje cell loss in an in vitro model system using calbindin-D28k-immunoreactivity as a specific marker for Purkinje cells. We found that ethanol led to a significant dose-related decline in calbindin-D28k-immunoreactive cells in explant cultures of the developing cerebellum. However, concurrent administration of GDNF led to a significant rescue of calbindin-D28k-immunoreactive cells. Therefore, our results suggest that GDNF prevents ethanol-associated Purkinje cell loss. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 835–847, 1997

Neuronal cell loss is one of the most debilitating effects of fetal ethanol exposure. Cultures of cerebellar granule cells are a useful model to investigate ethanol neurotoxicity, because ethanol depletes cell numbers in these cultures, which also occurs in vivo. The primary goal of the present study was to identify and characterize agents that can ameliorate the ethanol-induced cell death that occurs in this culture system. Growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) can prevent neuronal degeneration after toxic insult in various experimental paradigms. These growth factors were investigated in the current study to determine whether or not they can mitigate ethanol-induced death of cerebellar granule cells in culture. Results indicate that NGF and bFGF significantly reduced the ethanol-induced cell loss. Both the NGF- and bFGF-mediated neuroprotection required protein and RNA synthesis, because actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) blocked their neuroprotective effects. In addition to its neuroprotective effect, bFGF also had a neurotrophic effect and could enhance cell survival in the absence of ethanol exposure. NGF did not have a neurotrophic effect. Neither EGF nor IGF-I was neuroprotective, although the latter did have a substantial neurotrophic effect. In conclusion, bFGF and NGF have long been recognized for their role in enhancing neuronal cell survival and differentiation. This study suggests that these growth factors can also provide neuroprotection against ethanol-induced cell death.

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms underlying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.

Alcohol was infused intravenously into chronically cannulated adult female sheep as a 40% solution (w/v) at doses of 0.5, 1.0, 1.5, or 2.0 g/kg over 1 hr. Saline infusions, equal in volume to the highest dose, served as a control. Dose-dependent peak blood alcohol concentrations (BACs) were attained 60 min after the beginning of alcohol infusion for all doses (86.5 +/- 3.7, 213.2 +/- 11.0, 373.2 +/- 14.3, and 494.1 +/- 34.5 mg/dl +/- SE, respectively). Plasma cortisol concentrations increased in response to the 0.5 g/kg infusions (BACs less than 100 mg/dl), whereas both ACTH and cortisol concentrations increased in the 1.0 and 2.0 g/kg dose groups. Mean arterial pressure, heart rate, and Paco2 increased, whereas Pao2 decreased in response to the 1.5 and 2.0 g/kg infusions. Arterial pH declined in the highest dose group. Respiratory rate was lower in all groups receiving alcohol compared with that of the control group. Hematocrit did not change. We conclude that BACs in adult female sheep below 100 mg/dl (levels easily achieved by social drinkers) result in activation of the hypothalamus-pituitary-adrenal axis. At high BACs (> 350 mg/dl), pituitary adrenal responses are accompanied by increases in heart rate, blood pressure, and Paco2 and decreases in Pao2 and arterial pH. These findings support the hypothesis that alcohol acts directly on the brain to mediate pituitary adrenal responses and that the additional responses to high BACs (the blood gas and hemodynamic responses), might be mediated by direct actions of alcohol on the brain, by cerebral ischemia, or by alcohol-mediated suppression of ventilatory drive and hypoxemia.

The 92 kDa matrix metalloproteinase (gelatinase B, MMP-9) plays a major role in the facilitation of tumor metastasis and in inflammatory disorders characterized by excessive matrix protein destruction. MMP-9 is transcriptionally induced in multiple cell types by exposure to the inflammatory mediators bacterial endotoxin, interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-alpha). CT-2519, (1-(5-isothiocyanatohexyl)-3,7-dimethylxanthine), a synthetic small molecule from an anti-inflammatory compound library, was evaluated for its effect on endotoxin and cytokine-induced MMP-9 synthesis by a monocytic leukemic cell line, THP-1, and a monocyte/macrophage line, RAW 264.7. CT-2519 dose-dependently inhibited endotoxin and cytokine-induced synthesis of MMP-9 by these cells. Furthermore, both MMP-9 secretion and matrix invasion by cells of a human fibrosarcoma cell line, HT-1080, were inhibited by CT-2519 in a dose-dependent manner. Northern blot analyses and studies utilizing MMP-9 promoter constructs indicated that the inhibitory action of CT-2519 occurs at the level of transcriptional suppression. Given the observation that cellular activation by endotoxin, IL-1 and TNF-alpha may be mediated, at least in part, through induction of certain species of phosphatidic acid (PA), the effect of CT-2519 on lipid levels was analyzed. CT-2519 effectively reduced endotoxin-mediated increases in particular cellular lipid levels. Pharmacologic modulation of cytokine-dependent gene products, such as MMP-9, may offer an important therapeutic approach to the treatment of neoplastic and inflammatory disorders.

Behavioral and morphological studies suggest that exposure to alcohol during development may cause damage in the neocortex. In this study, rat pups were exposed to alcohol during the brain growth spurt and examined at adulthood to ascertain the long-term effect of alcohol exposure on the neocortex. Four-day-old rat pups were surgically implanted with an intragastric cannula while under ether anesthesia and artificially reared from postnatal day (PN) 4 through PN11. Two of the consecutive 12 daily feeds contained either alcohol (4.5 g/kg; alcohol-exposed) or an isocaloric maltose/dextrin solution (gastrostomy control) from PN4 through PN9. On PN115, animals were perfused intracardially and the brains removed. Unbiased stereological methods were used to determine the neocortical volume, the total number of neurons and glial cells in the entire neocortex and in layer V, and the mean cell volume of neurons or mean nuclear volume of glial cells in layer V. No effect of alcohol was seen in the neuronal population on either cell number or mean cell volume, nor was there any difference in the total number or mean nuclear volume of glial cells in layer V. These findings suggest that neither the entire neocortex nor layer V alone are vulnerable to permanent alcohol-induced cell death.

The most serious features of fetal alcohol syndrome (FAS) are mental retardation and other behavioral problems resulting from alcohol-induced damage to the developing central nervous system (CNS). The mechanism by which alcohol induces its neuroteratogenic effects is unknown. One hypothesis is that gestational alcohol exposure results in a reduction in neuronal number. This study demonstrates that gestational exposure to ethanol in a non-human primate species induces permanent dose-related deficits in the number of cerebellar Purkinje cells. Ethanol was administered via nasogastric tube once per week to 15 gravid pigtailed macaques (Macaca nemistrina) in one of the following doses : 0.0 (intubated controls), 1.2, 1.8, 2.5, 3.3, and 4.1 g/kg/dose. Offspring were reared with parental surrogates and were sacrificed at 6 months of age ; 8-μm-thick parasagittal sections were cut through the paraffin-embedded cerebellar vermis. Purkinje cells were quantified, the length of the Purkinje cell line was determined stereologically, and Purkinje cell linear frequency was calculated. The number of Purkinje cells and their linear frequencies were significantly reduced in the alcohol-treated subjects, and the deficits were dose-dependent. The groups receiving 2.5 g/kg/dose and above were most severely affected and had an average deficit in Purkinje cell number of 11.8%, relative to controls. Alcohol had no effect on the length of the Purkinje cell line. The findings suggest that alcohol-induced reduction in neuronal number may be an important factor underlying the CNS dysfunction in FAS.

Previous studies in our laboratory indicated that cocaine exposure during the brain growth spurt period, a developmental stage vulnerable to various teratogens, did not produce microencephaly (gross brain weight measures). However, neonatal cocaine exposure has been shown to affect motor coordination and balance, which are both sensitive to cerebellar damage. The purpose of this study was to investigate whether cocaine exposure during the brain growth spurt period could result in the loss of cerebellar Purkinje cells, a neuronal population known to be vulnerable to other teratogenic insults. Sprague-Dawley rat pups were randomly assigned to either cocaine-treated groups (40, 80 mg/kg s.c.) or a gastrostomy control group, and were reared using an artificial-rearing method from postnatal days (PDs) 4 through 9. On PD 10, these animals were perfused and the cerebella were extracted and processed for cell counts. Estimates of Purkinje cell numbers were obtained using a 3-dimensional optical dissector method. The results using this stereological method demonstrated no significant Purkinje cell loss in response to cocaine treatment, even at a dose which has been shown to result in high mortality. The failure of cocaine to produce significant Purkinje cell loss (present finding) or microencephaly (previous finding) odds to the evidence indicating that cocaine is not a potent neuroteratogen.

This study demonstrates that exposure to an alcohol regimen that resulted in low, uniform blood alcohol concentrations during a period of rapid brain growth can lead to a permanent deficit in the number of Purkinje cells and granule cells in the floccular-parafloccular region of the cerebellum. Sprague-Dawley rat pups were artificially reared and were administered alcohol over postnatal days 4 through 9, a period of brain development similar to that of the human third trimester. Two groups received a daily alcohol dose of 4.5 g/kg, administered either as a 10.2% solution in two of the 12 daily feedings (10.2% group) or as a 5.1% solution in four of the 12 feedings (5.1% group). A third group received a daily dose of 6.6 g/kg administered as a 2.5% solution in every feeding (2.5% group). The condensed patterns of alcohol administration resulted in high peak blood alcohol concentrations with near total clearance while the higher daily dose (6.6 g/kg), administered continuously, resulted in low but continuous blood alcohol concentrations. Pups were allowed to grow to adulthood and killed on postnatal day 115. The total number of Purkinje cells and granule cells in the floccular-parafioccular region of the cerebellum was estimated using unbiased stereological methods. Exposure to alcohol resulted in significant deficits in the number of both Purkinje cells and granule cells at 115 days of age in all three treatment groups. Most importantly a significant deficit of Purkinje cells and granule cells was found following continuous exposure to low blood alcohol concentrations, i.e., in the 2.5% group. The total number of Purkinje cells in the 2.5% group was 2.33 ± 0.31 x 104 compared with 3.18 ± 0.30 x 104 in the artificially reared controls. The total number of granule cells in the 2.5% group and the controls was 1.24 ± 0.10 x 107 and 1.64 ± 0.19 x 107 respectively. These results support the hypothesis that exposure to a continuous, low blood alcohol concentration can result in the death of developing neurons and lead to permanent neuronal deficits. The degree of neuronal loss does not correlate with the magnitude of the peaks of blood alcohol concentration © Wiley-Liss, Inc.

The purpose of this study was to examine whether exposure of rat pups to alcohol postnatally over a period of brain development similar to that of the human 3rd trimester results in a permanent loss of cells in the inferior olivary nucleus. It was hypothesized that a deficit of neurons in the inferior olive, the sole source of climbing fibers, may contribute to the cerebellar dysfunction observed following exposure to alcohol during development. Sprague-Dawley rat pups were artificially reared and administered alcohol over postnatal days 4–9. One artificially reared group received a daily alcohol dose of 4.5 g/kg, administered as a 10.2% solution in 2 of 12 daily feedings (10.2% group). This pattern of alcohol administration resulted in high peak blood alcohol concentrations with near total clearance. The other artificially reared group was fed a diet made isocaloric to the alcohol-containing diet (gastrostomy control group). Pups were allowed to grow to adulthood and killed on postnatal day 115. The total number of neurons in the inferior olivary nucleus was estimated using unbiased stereological methods. Exposure to alcohol resulted in a significant deficit in the number of neurons in the inferior olive at 115 days of age. The total number of neurons in the alcohol-exposed group was 40.12 ± 8.7 ± 103, compared with 53.37 ± 3.7 ± 103 in the artificially reared controls. These results indicate that there is a permanent deficit of neurons in the inferior olive after postnatal exposure to alcohol. This supports the hypothesis that cerebellar dysfunction after exposure to alcohol during the brain growth spurt may be the result of more extensive neuronal deficits than previously thought.

The prevalence of concomitant use of alcohol and cocaine among drug abusers has raised concern about the possible increased risk of fetal damage. The aim of this study was to assess the interactive effects of alcohol and cocaine on lethality, somatic growth, and brain growth using an animal model system. Sprague-Dawley rat pups were used as subjects. They were randomly assigned to 1 of the 9 artificially reared groups which varied with respect to the combination treatments of cocaine (0, 40, or 60 mg/kg) and alcohol (0, 3.3, or 4.5 g/kg). All artificially reared pups were given daily cocaine and alcohol treatments during a major part of the brain growth spurt period (postnatal days 4–9). An additional group of suckled control animals raised by their natural dams was included to control for artificial rearing. The results are summarized as follows: (1) Drug-induced lethality was higher in cocaine-treated groups when compared with non-cocaine-treated groups, and the concurrent administration of high doses of alcohol and cocaine significantly increased the mortality rate. (2) Somatic growth, in terms of body weight, was not affected by alcohol, cocaine, or the combination of both drugs using the artificial rearing technique. (3) Alcohol exposure during this brain growth spurt period significantly reduced whole brain weight, as well as forebrain, cerebellum, and brain stem weights. (4) In contrast to alcohol, cocaine failed to exert a detrimental effect on brain weight measures during this early postnatal period. (5) There were no antagonistic or synergistic effects in inducing brain growth restrictions by concurrent exposure to alcohol and cocaine during the brain growth spurt. The potential involvement of cocaethylene in mediating the observed lethality is discussed. © 1994 Wiley-Liss, Inc.

This study examined the effects of alcohol exposure on the viability of cerebellar granule cells in culture. Continuous alcohol exposure, starting 1 day after the cultures were established, significantly reduced granule cell numbers, even with a single day of exposure to an alcohol concentration as low as 100 mg/dl. The depletion of cerebellar granule cells by alcohol was concentration-dependent (greater loss of cells at higher alcohol concentrations) and duration-dependent (greater loss of cells at longer exposure durations). The loss of granule cells also depended on the number of days the granule cells were in culture before alcohol exposure. Alcohol was significantly more effective in reducing the cell numbers of newly established granule cell cultures (1 day in vitro) compared with older cultures (4 or 7 days in vitro). Cell cycle analysis established that the cerebellar granule cells did not proliferate in culture, indicating that alcohol exposure did not reduce cell numbers by interfering with cell proliferation in this system. Instead, alcohol-induced killing of the granule cells was the most likely mechanism to account for the depletion of granule cells in vitro. Granule cell cultures are a useful in vitro model system to study the cellular and molecular aspects of neuronal cell depletion associated with fetal alcohol exposure. The potential role of the JV-methyl-D-aspartate receptor in this alcohol-induced neuronal cell death is discussed.

The purpose of this study was to determine whether develop- mental alcohol exposure could induce permanent neuronal deficits, whether the peak blood alcohol concentration (BAC) influences the severity of the effects, and whether the effects are gender related. Rat pups were reared artifically over postnatal days (PD) 4 through 11 (a period of rapid brain growth, comparable to part of the human third trimester). Alcohol treatments were administered on PD 4 through 9. Patterns of alcohol exposure that produce different peak BACs have been shown to affect differentially the amount of brain weight deficits and neuron loss shortly after the exposure period, so this study investigated whether the pattern of alcohol exposure was also effective in producing permanent def- icits. Two groups received a daily alcohol dose of 4.5 g/kg, condensed into either four or two feedings. A third group received a higher daily alcohol dose of 6.6 g/kg administered in 12 uniformly spaced daily feedings. Pups were fostered back to dams on PD 11 and perfused on PD 90. Brain weights were measured, and Purkinje cells and granule cells were counted in each of the 10 lobules of the cerebellar vermis. In the hippocampal formation, cell counts were made of the pyramidal cells of fields CA1 and CA2/3, the multiple cell types of CA4 and the granule cells of the dentate gyrus. The groups receiving the lower daily dose (4.5 g/kg) condensed into either four or two feedings were exposed to higher peak BACs and suffered significant permanent brain weight deficits and neuronal losses, relative to controls. The group receiving the higher daily dose (6.6 g/kg) in continuous fractions had no significant brain weight reductions or neuronal loss. Vulnerability to alcohol-induced neuronal loss varied among regions and cell populations and as a function of peak BAC. In the hippocampus, only the CA1 pyramidal cells were significantly reduced in number and only in group receiving the most condensed alcohol treatment. In the cerebellum, the severity of Purkinje cell and granule cell losses varied among lobules, and Purkinje cell vulnerability appeared to depend on the maturational state of the neuron at the time of the alcohol exposure, with the more mature Purkinje cells being the more vulnerable.

Neuronal death is one of the most serious consequences of alcohol exposure during development. Studies described in this paper used a neonatal rat model to address factors affecting neuronal death following alcohol exposure during the period of rapid brain growth, and relate them to possible mechanisms of damage. The profile of blood alcohol concentrations (BACs) is an important variable influencing both brain growth deficits and neuronal death--a smaller daily dose of alcohol can be more damaging than a larger daily dose, if it is consumed in a binge-like pattern that produces relatively higher BACs. Alcohol exposure for a single day also can be damaging, producing both brain growth deficits and neuron loss, if high BACs are obtained. Various brain regions and different neuronal populations within a given brain area exhibit different degrees of vulnerability. Some neuronal loss clearly is a function of cell death due to direct effects of alcohol, while other deficits may be due to either primary or secondary effects of the alcohol insult. In the cerebellum, a maturational or metabolic factor also appears to be involved with alcohol-induced neuronal death. Immunocytochemical studies using a monoclonal antibody against microtubule-associated protein 2 (MAP2) indicated that cerebellar lobules containing Purkinje cells that are in the process of extending dendrites are ones that are more vulnerable to alcohol than lobules containing Purkinje cells that mature later. Alcohol exposure during brain development may be producing neuron attrition in multiple ways, including disruption of membrane integrity, inhibition of protein synthesis or other alterations such as lipid solubility, or by disruption of cytoskeletal elements.

As the summary presentation of a symposium on prenatal alcohol-induced brain damage and long-term postnatal consequences, this paper proposes the establishment of two main research priorities--to begin to correlate long-term behavioral effects with alterations in underlying neural substrates, and to explore the mechanisms of neuroteratogenicity. To reach these goals, three objectives are described. First, animal and human research must become more interrelated. Second, experimental observations should be integrated into formal models that incorporate both neural structure and function. Third, researchers should choose well-defined dependent measures that are derived from models of brain function based on modern concepts of cognitive neuroscience. Examples of neuropsychological tests that may serve as the bases for structure/function relationships are presented. Incorporating these objectives into future research will facilitate understanding of the fundamental issues concerning prenatal alcohol exposure and will begin to provide the bases for rational intervention or treatment.