Neonatal Alcohol Exposure Research Articles

Long-term effects of neonatal alcohol exposure on photic reentrainment and phase-shifting responses of the activity rhythm in adult rats

In rats, neonatal alcohol (EtOH) exposure coinciding with the period of rapid brain growth produces structural damage in some brain regions that often persists into adulthood and thus may have long-term consequences in the neural regulation of behavior. Because recent findings indicate that the circadian clock located in the rat suprachiasmatic nucleus is vulnerable to alcohol-induced insults during development, the present study examined the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior in adult rats. Rat pups were exposed to alcohol (3.0, 4.5, or 6.0 g·kg −1·day −1) or isocaloric milk formula on postnatal days 4–9 using artificial-rearing methods. At 2 months of age, animals were housed individually and circadian wheel-running behavior was continuously analyzed to determine the effects of neonatal alcohol treatment on the rate of reentrainment to a 6-h advance in the 12-h light:12-h dark photoperiod and the phase-shifting properties of free-running rhythms in response to discrete light pulses on a background of constant darkness. For all doses, neonatal alcohol exposure had a significant effect in reducing the time for reentrainment such that EtOH-treated rats required four to five fewer days than control animals for stable realignment of the activity rhythm to the shifted light–dark cycle. Coupled with the accelerated rate of reentrainment, the amplitude of light-evoked phase delays at circadian time 14 and advances at circadian time 22 in the 4.5 and 6.0 g·kg −1·day −1 EtOH groups was almost twofold greater than that observed in control animals. The present observations indicate that the mechanisms by which photic signals regulate circadian behavior are permanently altered following alcohol exposure during the period of rapid brain development. These long-term alterations in the photic regulation of circadian rhythms may account, at least partially, for some neurobehavioral consequences of prenatal alcohol exposure in humans such as depression.

Neonatal Alcohol Exposure Permanently Disrupts the Circadian Properties and Photic Entrainment of the Activity Rhythm in Adult Rats

Alcohol exposure during the period of rapid brain development produces structural damage in different brain regions, including the suprachiasmatic nucleus (SCN), that may have permanent neurobehavioral consequences. Thus, this study examined the long-term effects of neonatal alcohol exposure on circadian behavioral activity in adult rats. Artificially reared Sprague-Dawley rat pups were exposed to alcohol (EtOH; 4.5 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4-9. At 2 months of age, rats from the EtOH, GC, and suckle control (SC) groups were housed individually, and properties of the circadian rhythm in wheel-running behavior were continuously analyzed during exposure to a 12-hr light:12-hr dark photoperiod (LD 12:12) or constant darkness (DD). Neonatal alcohol exposure had distinctive effects on the rhythmic properties and quantitative parameters of adult wheel-running behavior. EtOH-treated animals were distinguished by unstable and altered entrainment to LD 12:12 such that their daily onsets of activity were highly variable and occurred at earlier times relative to control animals. In DD, circadian regulation of wheel-running behavior was altered by neonatal alcohol exposure such that the free-running period of the activity rhythm was shorter in EtOH-exposed rats than in control animals. Total amount of daily wheel-running activity in EtOH-treated rats was greater than that observed in the SC group. In addition, the circadian activity patterns of EtOH-exposed rats were fragmented such that the duration of the active phase and the number of activity bouts per day were increased. These data indicate that neonatal alcohol exposure produces permanent changes in the circadian regulation of the rat activity rhythm and its entrainment to LD cycles. These long-term alterations in circadian behavior, along with the developmental alcohol-induced changes in SCN endogenous rhythmicity, may have important implications in clinical sleep-wake disturbances observed in neonates, children, and adults exposed to alcohol in utero.

Antioxidant Pretreatment Does Not Ameliorate Alcohol‐Induced Purkinje Cell Loss in the Developing Rat Cerebellum

Recent research has suggested that oxidative stress is a potential mechanism for alcohol-induced injury and that supplementation with antioxidants can ameliorate alcohol-induced damage. In this study, two known antioxidants, melatonin and U83836E, were assessed for their effectiveness in blocking the expected alcohol-induced cerebellar Purkinje cell loss in neonatal rat pups. Sprague-Dawley rat pups were artificially reared from postnatal days (PDs) 4-9 and were exposed to either alcohol or antioxidants (melatonin or U83836E) individually or in combination. A normal control group (raised by rat dams) was included in this study. On PD 9, the brain from each pup was removed and weighed, and the cerebellar vermis was processed for stereological cell counting. Alcohol exposure during the brain growth spurt produced microencephaly, in addition to significant decreases in the number and density of Purkinje cells in lobule I and the volume of lobule I. The antioxidants did not reduce any of the adverse effects observed from alcohol exposure, and they did not decrease the Purkinje cell number when administered alone. Furthermore, antioxidants did not change the only blood alcohol concentration measured on PD 6. The results confirmed alcohol-induced microencephaly and cerebellar Purkinje cell loss from neonatal alcohol exposure, and they showed that neither antioxidant could attenuate these adverse effects on the developing brain. The inability of antioxidants to reduce Purkinje cell loss from neonatal alcohol exposure suggests the existence of alternative mechanisms for developmental alcohol-induced Purkinje cell loss.

Fetal Alcohol Effects: Potential Treatments From Basic Science

This article represents the proceedings of a symposium presented at the 2004 annual meeting of the International Society for Biomedical Research on Alcoholism, held in Mannheim, Germany. The presentations were as follows: 1) “Antioxidants Prevent Ethanol-Induced Cell Death in Developing Brain and in Cultured Neural Cells” by M. Pascual, M. C. García-Minguillán, and Consuelo Guerri; 2) “Rational Development of Ethanol Antagonists” by Michael E. Charness and Michael F. Wilkemeyer; 3) “Choline Supplementation as a Treatment for Fetal Alcohol Effects” by Jennifer D. Thomas and Hector D. Dominguez; 4) “Cerebellar and Cortical Plasticity After Neonatal Alcohol Exposure: Model of Intervention” by Anna Y. Klintsova, Charles R. Goodlett, and William T. Greenough; and 5) “Circadian Rhythms in Prenatally Ethanol-Exposed Rats” by Hiromi Sakata-Haga.

Effects of neonatal alcohol and/or cocaine exposure on stress in juvenile and adult female rats

The effects of neonatal exposure to alcohol and/or cocaine on two measures of stress were studied in juvenile and young adult female rats. After implantation with an intragastric cannula, subjects were artificially reared from postnatal days 4–10. This “brain growth spurt” period is roughly equivalent to CNS development during the third trimester of human pregnancy. There were five treatment groups: alcohol (6 g/kg/day), cocaine (60 mg/kg/day), alcohol/cocaine (6 g/kg/day alcohol and 60 mg/kg/day cocaine), stock (an artificially reared control), and sham (a suckled control). Subjects were tested in open field and forced swim tests beginning at 21 or 60–70 days of age, respectively. Compared to controls, alcohol-exposed females displayed longer latencies to become immobile in the forced swim test as juveniles and cocaine-exposed females showed increased immobility as adults. Increased immobility can be interpreted as hyporesponsiveness to stress. In contrast, very few differences were observed in the open field. Furthermore, the group exposed to alcohol and cocaine in combination did not differ from controls in either paradigm. These findings suggest that the forced swim test may be more sensitive to neonatal drug effects than open field, although these effects may not be consistent across age.

Vitamin E protects against alcohol-induced cell loss and oxidative stress in the neonatal rat hippocampus

Oxidative stress has been proposed as a possible mechanism underlying nervous system deficits associated with Fetal Alcohol Syndrome (FAS). Current research suggests that antioxidant therapy may afford some level of protection against the teratogenic effects of alcohol. This study examined the effectiveness of antioxidant treatment in alleviating biochemical, neuroanatomical, and behavioral effects of neonatal alcohol exposure. Neonatal rats were administered alcohol (5.25g/kg) by intragastric intubation on postnatal days 7, 8, and 9. A subset of alcohol-exposed pups were co-administered a high dose of Vitamin E (2g/kg, or 71.9IU/g). Controls consisted of a non-treated group, a group given the administration procedure only, and a group given the administration procedure plus the Vitamin E dose. Ethanol-exposed animals showed impaired spatial navigation in the Morris water maze, a decreased number of hippocampal CA1 pyramidal cells, and higher protein carbonyl formation in the hippocampus than controls. Vitamin E treatment alleviated the increase in protein carbonyls and the reduction in CA1 pyramidal cells seen in the ethanol-exposed group. However, the treatment did not improve spatial learning in the ethanol-exposed animals. These results suggest that while oxidative stress-related neurodegeneration may be a contributing factor in FAS, the antioxidant protection against alcohol-induced oxidative stress and neuronal cell loss in the rat hippocampus does not appear to be sufficient to prevent the behavioral impairments associated with FAS. Our findings underscore the complexity of the pathogenesis of behavioral deficits in FAS and suggest that additional mechanisms beyond oxidative damage of hippocampal neurons also contribute to the disorder.

Developmental alcohol exposure disrupts circadian regulation of BDNF in the rat suprachiasmatic nucleus

In rats, damage to neuronal populations in some brain regions occurs in response to neonatal alcohol exposure coinciding with the period of rapid brain growth. These alcohol-induced defects in brain development may persist into adulthood and thus have long-term implications for the functional characteristics of damaged neuronal populations. The present study examined the effects of neonatal alcohol exposure on endogenous rhythmicity of the circadian clock located in the rat suprachiasmatic nucleus (SCN). Specifically, experiments were conducted to determine whether neonatal alcohol exposure alters the circadian rhythm of brain-derived neurotrophic factor (BDNF) content in the rat SCN because this neurotrophin is an important rhythmic output from the SCN clock. Male rat pups were exposed to alcohol (4.5 g/kg/day) or isocaloric milk formula on postnatal days 4–9 using artificial rearing methods. At 5–6 months of age, SCN and hippocampal tissue was harvested and subsequently examined for content of BDNF protein. Time-dependent fluctuations in BDNF protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). In alcohol-treated rats, SCN levels of BDNF were significantly decreased and were characterized by a loss of circadian rhythmicity relative to those observed in control animals. In comparison, hippocampal levels of BDNF displayed no evidence of circadian regulation in all three treatment groups, but were slightly lower in alcohol-treated animals than in control groups. Importantly, these observations suggest that alcohol exposure during the period of rapid brain development may cause permanent changes in the SCN circadian clock.

Perinatal choline supplementation does not mitigate motor coordination deficits associated with neonatal alcohol exposure in rats

Prenatal alcohol exposure can disrupt brain development, leading to a variety of behavioral alterations including learning deficits, hyperactivity, and motor dysfunction. We have been investigating the possibility that perinatal choline supplementation may effectively reduce the severity of alcohol's adverse effects on behavioral development. We previously reported that perinatal choline supplementation can ameliorate alcohol-induced learning deficits and hyperactivity in rats exposed to alcohol during development. The present study examined whether perinatal choline supplementation could also reduce the severity of motor deficits induced by alcohol exposure during the third trimester equivalent brain growth spurt. Male neonatal rats were assigned to one of three treatment groups. One group was exposed to alcohol (6.6 g/kg/day) from postnatal days (PD) 4 to 9 via an artificial rearing procedure. Artificially and normally reared control groups were included. One half of subjects from each treatment received daily subcutaneous injections of a choline chloride solution from PD 4 to 30, whereas the other half received saline vehicle injections. On PD 35–37, subjects were tested on a parallel bar motor task, which requires both balance and fine motor coordination. Ethanol-exposed subjects exhibited significant motor impairments compared to both control groups whose performance did not differ significantly from one another. Perinatal choline treatment did not affect motor performance in either ethanol or control subjects. These data indicate that the beneficial effects of perinatal choline supplementation in ethanol-treated subjects are task specific and suggest that choline is more effective in mitigating cognitive deficits compared to motor deficits associated with developmental alcohol exposure.

Perinatal choline supplementation attenuates behavioral alterations associated with neonatal alcohol exposure in rats

Children exposed to alcohol prenatally suffer from a variety of behavioral alterations, including hyperactivity and learning deficits. Given that women continue to drink alcohol during pregnancy, it is critical that effective interventions and treatments be identified. Previously, we reported that early postnatal choline supplementation can reduce the severity of learning deficits in rats exposed to alcohol prenatally. The present study examined whether choline supplementation can reduce the severity of behavioral alterations associated with alcohol exposure during the third trimester equivalent brain growth spurt. Male neonatal rats were assigned to one of three treatment groups. One group was exposed to alcohol (6.6 g/kg/day) from postnatal days (PD) 4–9 via an artificial rearing procedure. Artificially reared and normally reared control groups were included. One half of subjects from each treatment received daily subcutaneous injections of a choline chloride solution from PD 4–30, whereas the other half received saline vehicle injections. On PD 31–34, after choline treatment was complete, activity level was monitored and, on PD 40–42, subjects were tested on a serial spatial discrimination reversal learning task. Subjects exposed to alcohol were significantly hyperactive compared to controls. The severity of ethanol-induced hyperactivity was attenuated with choline treatment. In addition, subjects exposed to ethanol during the neonatal period committed a significantly greater number of perseverative-type errors on the reversal learning task compared to controls. Exposure to choline significantly reduced the number of ethanol-related errors. Importantly, these behavioral changes were not due to the acute effects of choline, but were related to long-lasting organizational effects of early choline supplementation. These data suggest that early dietary interventions may reduce the severity of fetal alcohol effects.

Neonatal Alcohol Exposure Induces Long-Lasting Impairment of Visual Cortical Plasticity in Ferrets

Fetal alcohol syndrome is a major cause of learning and sensory deficits. These disabilities may result from disruption of neocortex development and plasticity. Alcohol exposure during the third trimester equivalent of human gestation may have especially severe and long-lasting consequences on learning and sensory processing, because this is when the functional properties and connectivity of neocortical neurons start to develop. To address this issue, we used the monocular deprivation model of neural plasticity, which shares many common mechanisms with learning. Ferrets were exposed to ethanol (3.5 mg/kg, i.p.) on alternate days for 3 weeks starting on postnatal day (P) 10. Animals were then monocularly deprived at the peak of ocular dominance plasticity after a prolonged alcohol-free period (15-20 d). Quantitative single-unit electrophysiology revealed that alcohol exposure disrupted ocular dominance plasticity while preserving robust visual responses. Moreover, optical imaging of intrinsic signals revealed that the reduction in visual cortex area driven by the deprived eye was much less pronounced in ethanol-treated than in control animals. Alcohol exposure starting at a later age (P20) did not disrupt ocular dominance plasticity, indicating that timing of exposure is crucial for the effects on visual plasticity. In conclusion, alcohol exposure during a brief period of development impairs ocular dominance plasticity at a later age. This model provides a novel approach to investigate the consequences of fetal alcohol exposure and should contribute to elucidate how alcohol disrupts neural plasticity.

813 Preoperative concurrent chemoradiotherapy in non-small-cell lung cancer. Feasibility, toxicity and long-term results of a phase II study

Neonatal alcohol exposure impairs cognition and learning in adulthood and permanently damages the hippocampus. Wheel running (WR) improves hippocampus-associated learning and memory and increases the genesis and survival of newly generated neurons in the hippocampal dentate gyrus. WR significantly increases proliferation of newly generated dentate granule cells in alcohol-exposed (AE) and control rats on Postnatal Day (PD) 42 but only control rats show an increased number of surviving cells thirty days after WR (Helfer et al., 2009b). The present studies examined whether proliferation-promoting WR followed by survival-enhancing environmental complexity (EC) during adolescence could increase survival of new neurons in AE rats. On PD 4–9, pups were intubated with alcohol in a binge-like manner (5.25 g/kg/day, AE), were sham-intubated (SI), or were reared normally (suckle control, SC). On PD 30 animals were assigned to WR (PD 30–42) followed by EC (PD 42–72; WR/EC) or were socially housed (SH/SH) for the duration of the experiment. All animals were injected with 200 mg/kg bromodeoxyuridine (BrdU) on PD 41. In Experiment 1, survival of newly generated cells was significantly enhanced in the AE-WR/EC group in comparison with AE-SH/SH group. Experiment 2A examined trace eyeblink conditioning. In the SH/SH condition, AE impaired trace eyeblink conditioning relative to SI and SC controls. In the WR/EC condition, AE rats performed as well as controls. In Experiment 2B, the same intervention was examined using the context preexposure facilitation effect (CPFE); a hippocampus-dependent variant of contextual fear conditioning. Again, the WR/EC intervention reversed the deficit in conditioned fear to the context that was evident in the SH/SH condition. Post-weaning environmental manipulations promote cell survival and reverse learning deficits in rats that were exposed to alcohol during development. These manipulations may provide a basis for developing interventions that ameliorate learning impairments associated with human fetal alcohol spectrum disorders.

Differential vulnerability to motor deficits in second replicate HAS and LAS rats following neonatal alcohol exposure

Children exposed prenatally to alcohol suffer from a variety of behavioral alterations. However, variation exists in the pattern and severity of these alcohol-related neurodevelopmental disorders. We examined the influence of alcohol sensitivity in the etiology of fetal alcohol effects by studying rat lines selectively bred for extremes in alcohol-induced sleep time: high-alcohol-sensitive (HAS) and low-alcohol-sensitive (LAS) rats. Using subjects from the first replicate, we previously reported that HAS rats exposed to alcohol during development were more vulnerable to ethanol-induced hyperactivity and motor deficits compared to LAS rats. To determine if these effects were, in fact, related to the trait for which these subjects were selected, the present study examined the consequences of developmental alcohol exposure in second replicate HAS and LAS rats. Second replicate HAS and LAS rats, as well as Sprague–Dawley rats, were exposed to 6.0 g/kg/day ethanol on Postnatal Days (PD) 4–9, a period of brain development equivalent to the third trimester, via an artificial rearing procedure. Artificially and normally reared controls were included. Activity was measured on PD 18–21 and parallel bar motor coordination on PD 30–32. Ethanol exposure produced hyperactivity in all genetic groups, and there were no differences among HAS and LAS rats. In contrast, consistent with findings from the first replicate, ethanol-exposed HAS rats were more impaired on the motor coordination task compared with LAS rats. These data suggest that genetically mediated responses to alcohol may relate to behavioral vulnerability to motor deficits following developmental alcohol exposure. They also provide evidence that genetic factors play a role in fetal alcohol effects and suggest that phenotypic markers may indicate individuals at high risk for some fetal alcohol effects.

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.

MK‐801 Can Exacerbate or Attenuate Behavioral Alterations Associated With Neonatal Alcohol Exposure in the Rat, Depending on the Timing of Administration

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. 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. 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. 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.

Acute Effects of Ethanol and the First Suckling Episode in the Newborn Rat

In humans, early postnatal experience with alcohol is far from rare and includes exposure to alcohol through breast milk or through the bottle to attain sedative effects (Croce, 1987). Exposure to alcohol though mother's milk alters the infant's suckling behavior. However, acute effects of alcohol on suckling behavior using animal models remain to be investigated. The present study was designed to examine the effects of neonatal alcohol exposure on attachment to a surrogate nipple and ingestion of milk in rat pups, naive both to suckling and to maternal care. Cesarean-delivered rat pups were pre-exposed to four dosages of ethanol (0, 1, 2, and 3 g/kg intragastrically) and tested 30 min after ethanol administration. Suckling behavior was impaired only in pups exposed to a dosage of 3 g/kg of ethanol. Pups in this group demonstrated longer latency to attach followed by less efficient suckling--the pups maintained contact with the nipple but showed decreased ingestion of milk from it. Pups treated with 1 g/kg of ethanol showed the greatest suckling efficiency, ingesting far more milk per minute attached to the surrogate nipple than pups in all other groups. At the same time, nonevoked motor activity was significantly suppressed by all three dosages of ethanol. Blood alcohol levels showed a linear relationship with dose at 30 min postintubation. These findings suggest a dissociation between acute ethanol effects on suckling and overall motor activity, with suckling apparently less sensitive to suppressive and more sensitive to activating effects of ethanol.

Neonatal alcohol exposure produces more severe motor coordination deficits in high alcohol sensitive rats compared to low alcohol sensitive rats

Prenatal exposure to alcohol can produce a number of behavioral alterations, including hyperactivity, learning deficits and motor impairments. However, the severity and nature of behavioral alterations varies markedly among children of women who drink during pregnancy. One important determinant of this variation may be genetic differences in the response to alcohol. Recently, we demonstrated that exposure to alcohol during development produced hyperactivity in rats bred for high alcohol sensitivity (HAS), but not in rats bred for low alcohol sensitivity (LAS). These lines were selectively bred for extremes in alcohol-induced “sleep time.” The present study investigated the effects of ethanol exposure during development on motor coordination later in life in both HAS and LAS rats. Using an artificial rearing procedure, neonatal pups from each line were exposed to a binge-like alcohol treatment on postnatal days (PD) 4–9. Within each line, one group was exposed to ethanol (6.0 g/kg/day), one group served as an artificially reared control, and a third served as a normally reared control group. On PD 30, parallel bar motor performance was evaluated. Exposure to ethanol during development severely impaired motor performance in the HAS rats compared to their controls. In LAS rats, early ethanol exposure produced only mild and nonsignificant effects on motor performance. Thus, HAS rats were more vulnerable to ethanol-induced motor deficits compared to the LAS rats. Importantly, there were no differences in peak blood alcohol level between the lines, indicating that vulnerability to ethanol's teratogenic effects was not due to differences in metabolic rate. These results suggest that genetic differences in response to alcohol may serve as a predictor for susceptibility to ethanol's teratogenic effects.

Therapeutic motor training ameliorates cerebellar effects of postnatal binge alcohol.

We have used training on complex motor tasks to ameliorate effect of neonatal alcohol exposure. On postnatal days 4-9, alcohol-exposed (AE) rats were given 4.5 g/kg/day of alcohol by artificial rearing; gastrostomy control (GC) rats were given an isocaloric mixture of maltose/dextrin; suckling control (SC) rats were suckled normally. At 6 months of age, animals from the three groups underwent either rehabilitation training on a series of complex motor tasks, motor conditioning on a flat runway, or an inactive home cage condition. Subsequently, animals were either tested on three tests of balance and coordination, or were used for cerebellar morphology. After rehabilitation, but not after motor conditioning, male and female AE rats exhibited significant improvement in independent tests of motor skills. Using unbiased stereological morphological techniques, rehabilitated SC and AE animals were found to exhibit significantly more parallel fiber synapses per Purkinje cell in the paramedian lobule.

Neonatal Alcohol Exposure Produces Hyperactivity in High-Alcohol-Sensitive But Not in Low-Alcohol-Sensitive Rats

Children of women who consume high amounts of alcohol during their pregnancies vary greatly in physical and behavioral outcomes. Although many factors, such as dose and timing of exposure, undoubtedly contribute to this variation, one important determinant may be genetic differences in the response to alcohol. The present study examined activity levels in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats following neonatal alcohol exposure. These lines were selectively bred for extremes in ethanol-induced “sleep times.” The HAS and LAS offspring were exposed to alcohol via an artificial rearing procedure using the “pup-in-the-cup” technique. Rat pups were exposed to ethanol (6 g/kg/day) from postnatal day (PD) 4 through 7 and faded to a dose of 3 g/kg/day on PD 8 and 9. An artificially reared gastrostomy control group (GC) and a normally reared suckle control group (SC) were also included. Activity level was measured on PD 18 through PD 21 for 30 min daily in automated activity monitors. Neonatal ethanol exposure produced overactivity in HAS rats, relative to their controls, but the same ethanol treatment had no effect on the LAS rats. Importantly, there were no differences in blood alcohol concentrations (around 420 mg/dl) between the two lines during the treatment period. These data suggest that genetic differences in response to alcohol may be a predictor for some of the behavioral teratogenic effects of alcohol.

Neonatal alcohol exposure reduces NMDA induced Ca 2+ signaling in developing cerebellar granule neurons

Glutamatergic neurotransmission through NMDA receptors is critical for both neurogenesis and mature function of the central nervous system (CNS), and is thought to be one target for developmentally-induced damage by alcohol to brain function. In the current study we examined Ca 2+ signaling linked to NMDA receptor activation as a potential site for alcohol's detrimental effects on the developing nervous system. We compared Ca 2+ signals to NMDA in granule neurons cultured from cerebella of rat neonates exposed to alcohol (ethanol) during development with responses to NMDA recorded in separated control groups. Alcohol exposure was by the vapor chamber method on postnatal days 4–7. An intermittent exposure paradigm was used where the pups were exposed to alcohol vapor for 2.5 h/day to produce peak BALs of ∼320 mg%. Control pups were placed in an alcohol-free chamber for a similar time period or remained with their mother. After culture under alcohol-free conditions for up to 9 days, Ca 2+ signaling in response to NMDA was measured using fura-2 Ca 2+ imaging. Results show that the peak amplitude of the Ca 2+ signal to NMDA was significantly smaller in cultured granule neurons obtained from alcohol-treated pups compared to granule neurons from control pups. In contrast, the Ca 2+ signal to K + depolarization was not depressed by the alcohol treatment. Resting Ca 2+ levels were also altered by the alcohol treatment. These results show that intermittent alcohol exposure during development in vivo can induce long-term changes in CNS neurons that affect the Ca 2+ signaling pathway linked to NMDA receptors and resting Ca 2+ levels. Such changes could play an important role in the CNS dysfunction associated with alcohol exposure during CNS development.

Neonatal alcohol and nicotine exposure limits brain growth and depletes cerebellar Purkinje cells

The present study examined the effects of coexposure of alcohol and nicotine during the brain growth spurt period on brain weights and cerebellar Purkinje cell numbers. Sprague-Dawley rat pups were randomly assigned into five groups (four artificially reared groups and one suckle control). Artificially reared pups were given alcohol (0 or 4 g/kg/day) and/or nicotine (0 or 6 mg/kg/day) daily from postnatal days (PDs) 4 to 9, and the suckle controls received no experimental treatments. The results are summarized below. (1) Surprisingly, nicotine reduced the peak blood alcohol concentration from about 300 to 230 mg/dl, but alcohol did not affect urine cotinine levels (≈12,000–13,000 ng/ml). (2) Alcohol significantly reduced the weights of forebrain, cerebellum, and brain stem, but nicotine limited only the growth of the forebrain. (3) Purkinje cell numbers in the cerebellar vermis were significantly reduced in response to alcohol, nicotine, and the combination of both drugs. (4) No statistically significant interactive effect was found following the cotreatment of alcohol and nicotine. Collectively, the present study replicated our previous findings demonstrating alcohol's detrimental effects on brain development; it also presented new evidence documenting nicotine's neuroteratogenic effects on restricting brain growth and depleting cerebellar Purkinje cells during the brain growth spurt.