Postnatal ethanol exposure disrupts signal detection in adult rats

Neurotoxicology and Teratology 27 (2005) 815 – 823 www.elsevier.com/locate/neutera

Postnatal ethanol exposure disrupts signal detection in adult rats Kevin M. Woolfrey, Pamela S. Hunt, Joshua A. Burk * Department of Psychology, College of William & Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, USA Received 21 January 2005; accepted 21 June 2005 Available online 22 August 2005

Abstract Human prenatal ethanol exposure that occurs during a period of increased synaptogenesis known as the ‘‘brain growth spurt’’ has been associated with significant impairments in attention, learning, and memory. The present experiment assessed whether administration of ethanol during the brain growth spurt in the rat, which occurs shortly after birth, disrupts attentional performance. Rats were administered 5.25 g/kg/day ethanol via intragastric intubation from postnatal days (PD) 4 – 9, sham-intubation, or no intubation (naı¨ve). Beginning at PD 90, animals were trained to asymptotic performance in a two-lever attention task that required discrimination of brief visual signals from trials with no signal presentation. Finally, manipulations of background noise and inter-trial interval duration were conducted. Early postnatal ethanol administration did not differentially affect acquisition of the attention task. However, after rats were trained to asymptotic performance levels, those previously exposed to ethanol demonstrated a deficit in detection of signals but not of non-signals compared to sham-intubated and naı¨ve rats. The signal detection deficit persisted whenever these animals were re-trained in the standard task, but further task manipulations failed to interact with ethanol pretreatment. The present data support the hypothesis that early postnatal ethanol administration disrupts aspects of attentional processing in the rat. D 2005 Elsevier Inc. All rights reserved. Keywords: Alcohol; Vigilance; Neonatal; Rat

1. Introduction High levels of ethanol consumption during pregnancy in humans can have serious cognitive consequences that are part of the symptoms associated with fetal alcohol syndrome (FAS) or, in milder cases, fetal alcohol effects (FAE) [23,49,53]. In particular, alterations of attention are characteristic and persistent in these cases [3,9,10,27,52]. For example, Streissguth and colleagues have consistently noted impaired performance on tests of sustained attention and vigilance in children and adolescents with a history of prenatal alcohol exposure [42,50 – 52]. Attentional processing is hypothesized to mediate the efficacy and capacity of later stages of information processing, including working memory [1,12,30]. The deficits in attention may contribute to other cognitive impairments in cases of FAS or FAE, which include aspects * Corresponding author. Tel.: +1 757 221 3882; fax: +1 757 221 3896. E-mail address: [email protected] (J.A. Burk). 0892-0362/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2005.07.002

of working memory and executive functioning [11,24, 25,32,57]. An understanding of the neural systems that contribute to the attentional deficits following prenatal exposure to high levels of ethanol in humans is likely to be beneficial in the development of treatments for the associated cognitive deficits. Animal models of attentional deficits following early exposure to ethanol are useful for clarifying the relevant neural substrates associated with cognitive impairments. However, there has been relatively limited progress in the development of animal models of attentional deficits in fetal alcohol syndrome or fetal alcohol effects. A growing body of evidence suggests that high levels of ethanol consumption throughout pregnancy in humans results in more severe cognitive deficiencies than exposure restricted to the first half of gestation [3,8,48]. While the total amount of ethanol exposure is confounded in such analyses, the results suggest that third trimester exposure may be more detrimental to brain development and cognitive capabilities than first and second trimester

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exposure. This hypothesis is strengthened by numerous findings from rodent studies that ethanol exposure during the third-trimester equivalent period, shortly following birth in rats [15], results in greater impairments in working and spatial memory tasks as well as greater cell loss in some brain regions, than exposure restricted to earlier stages of development (e.g., [18,31,41]). Thus, early postnatal ethanol exposure in the rat is most likely to model many of the cognitive deficits reported in humans following prenatal ethanol exposure, but does not reproduce the range of symptoms associated with ethanol administration throughout human pregnancy [53]. A taxonomy concerning the features of a task that impact sustained attention performance has been described [14,43]. Based on this taxonomy, a two-lever attention task has been developed for use in the rat [6,33]. This task requires discrimination of sequentially and variably presented visual signals and non-signals. As would be predicted by this taxonomy [14,43], decreasing the inter-trial interval (ITI) or increasing the background noise by flashing a houselight has been shown to decrease accuracy in this task [33], thus supporting that it is a valid measure of attention in the rat. Furthermore, this task is thought to assess similar aspects of attention in rats and humans [7]. The present experiment tested the effects of early postnatal ethanol exposure on acquisition of this two-lever attention task. Attentional demands were further manipulated by flashing the houselight and varying the duration of the inter-trial interval to assess whether ethanol exposed animals were differentially affected by these manipulations. The experiment was guided by the hypothesis that early postnatal ethanol administration would not affect acquisition of more basic aspects of operant behavior, but would impair performance when attentional demands were increased. This ethanol exposure regimen and dose have been shown by our laboratory [21,22] and others (e.g., [17,29,31,37,54]) to impair aspects of learning and memory and to produce region-specific neuronal cell loss.

2. Methods 2.1. Subjects Subjects were Long-Evans rats from six litters housed and bred in the College of William and Mary vivarium. Litters were culled to 10 animals (6 males and 4 females) on postnatal day (PD) 2. Only the males were used in this experiment. Ethanol administration began on PD 4 (or when the animals’ weight had reached a 12 g minimum). Two male siblings from each litter were randomly assigned to one of three treatment groups: ethanol, sham or naı¨ve. The litter remained intact until rats were weaned on PD 21 and housed in separate cages with same-sex littermates. The animals were housed singly in hanging wire cages on PD 60. The vivarium ran on a 14:10 h light / dark cycle (lights

on 0600– 2000). Food and water were available ad libitum until the beginning of behavioral testing. Subjects were treated in accordance with the guidelines of the Animal Care and Use Committee at the College of William and Mary. 2.2. Apparatus 2.2.1. Ethanol exposure An 11.9% v/v ethanol solution was created daily by dissolving 95% ethanol into Similac\ baby formula with iron. Ethanol intubations were accomplished using Intramedic PE-10 polyethylene tubing (Clay Adams, Sparks, MD) lubricated with corn oil and 1 ml syringes with 30 gauge needles. While separated from the dam, pups were kept in bins that were warmed with heating pads (34 -C). 2.2.2. Experimental chambers Behavioral testing was conducted in eight chambers (Med Associates, Inc., Georgia, VT). The front of each chamber contained two retractable levers, a central panel light, and a water dispenser equipped with a pair of photocells to detect head entries. The water dispenser was located between the two levers and under the central panel light. A houselight was located in the back of each chamber. Measures of illuminance for these chambers have been reported previously [4]. A Gateway PC running Med-PC software (v. IV) was used to control and record stimuli presentation, lever operation, and reward delivery. 2.3. Procedure 2.3.1. Design and intubation procedure Male rats were assigned randomly to one of three groups: ethanol-intubated, sham-intubated, or non-intubated (naı¨ve). Each litter was removed from the dam as a group and housed in a holding cage that was placed on a heating pad for the duration of the intubation procedure. Intubations for ethanol and sham animals occurred two hours apart, three times each day, beginning at 09:00 daily. The litters were separated from the dam for approximately 20 min during each intubation session. All intubations were delivered via corn oil-lubricated polyethylene tubing through the animal’s mouth, esophagus and into the stomach. The ethanolexposed group was administered the alcohol and formula solution during the first two daily administrations (2.625 g/ kg/feeding for a total ethanol dose of 5.25 g/kg/day) from PD 4 through PD 9, followed by a third formula—only administration for nutritive purposes. This ethanol dose and delivery schedule closely paralleled procedures used by other laboratories [17,44,47], with blood – ethanol concentrations reported in the range of 260– 270 mg/dl. Shamintubated animals received polyethylene intubations, but were not administered formula. Previous work in our lab indicated that intubations with the Similac\ formula resulted in abnormal weight gain in sham controls (see also [17]). Intubations without infusions of formula have been

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suggested as a control group that avoids the potential confound of differential increases in body weight following administration of formula alone [17]. Naı¨ve rats accompanied their littermates to the feedings but were not intubated. 2.3.2. Behavioral training On PD 82, water deprivation began, with animals receiving four hours of water for two days, two hours of water for two days, one hour of water for two days, and 30 min of water for two days. Animals were maintained on 30 min of water immediately after testing for the remainder of the experiment. Behavioral training began on PD 90. Animals were initially trained to lever press for water using a fixed-ratio 1 reinforcement schedule. Animals were required to receive 120 rewards (0.1 ml tap water) per day for three consecutive days before proceeding to the next stage of training. The next stage of training involved presentation of visual signals (1 sec illumination of the central panel light) and of non-signals (no illumination of the central panel light). Immediately following a signal or non-signal, both levers would extend into the chamber for three seconds or until a lever press occurred. For half of the animals in each condition, on signal trials, pressing the left lever was considered correct, scored as a hit and reinforced while a press on the right lever was considered incorrect and scored as a miss. For these animals, on non-signal trials, pressing the right lever was considered correct, scored as a correct rejection and rewarded while a press on the left lever was considered incorrect and scored as a false alarm. For the other half of the animals in each condition, the rules were reversed (right lever press correct on signal trials and left lever press correct on nonsignal trials). Failure to press a lever within 3 s after extension was scored as an omission. All incorrect responses were followed by a trial identical to the previous trial. After three consecutive incorrect responses, a forced-choice trial was introduced in which only the correct lever was extended until it was pressed or 90 s elapsed. On signal trials, the central panel light also remained illuminated during the period that the lever was extended. Each session consisted of 162 trials with signal and non-signal events occurring in a pseudo-random order. The houselight was illuminated throughout each session. The ITI was 12 T 3 s. Rats were required to achieve a criterion of > 70% on both hits and correct rejections for three consecutive sessions. After reaching criterion, the task was changed in three ways, (1) the signals were briefer and variable (500, 100, and 25 ms), (2) the ITI was reduced to 9 T 3 s, and (3) correction and forced-choice trials were eliminated. Within each session, there were three blocks of 54 trials (total of 162 trials/session) with 27 signal trials (nine at each signal duration) and 27 non-signal trials within each block. Animals were trained until they reached 70% accuracy on both 500 ms signal duration trials and non-signal trials for

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three consecutive sessions. After reaching criterion, the rats were then trained for at least five additional sessions until asymptotic performance was reached. Asymptotic performance was defined as three consecutive sessions in which accuracy on 500 msec signal duration hits did not vary more than 15% and correct rejection accuracy did not vary more than 10% (see [5] for previous use of similar criteria). Rats required approximately three months of training to reach asymptotic performance. Training then continued for an additional three sessions to establish baseline performance. After baseline performance was established, three task manipulations were conducted. Each task manipulation was preceded by at least three days of training in the standard task to re-establish asymptotic performance. Also, each task manipulation was tested for three consecutive sessions. First, the houselight was flashed (1.0 s on/off) throughout a session. Rats were then returned to the baseline task and then tested with the ITI reduced to 4.5 T 3 s. Animals were then returned to the baseline task until re-establishing asymptotic performance and then tested with the ITI increased to 18 T 3 s. 2.4. Behavioral measures The total number of hits (h), misses (m), correct rejections (cr), and false alarms (fa) were collected for each signal duration (where appropriate) and for each of three blocks of 54 trials within a session. Relative number of hits [h / (h + m)] and correct rejections [cr / (cr + fa)] were calculated. Omissions were not included in measures of accuracy. The latency to press levers after extension was also collected for each trial outcome. Finally, the latency to reward retrieval, defined as the time between a correct lever press and the breaking of the water dispenser photocell, was also recorded. 2.5. Statistical analysis Body weights were analyzed using mixed factor ANOVAs during ethanol exposure (PD 4 – 9) for ethanolintubated and sham-intubated rats (naı¨ve animals were not weighed during this time to minimize handling of this group of animals). Body weights were also compared for animals in all three conditions in adulthood once rats reached asymptotic task performance levels. The reported body weights were from rats that were 6 –8 months old (once they had established asymptotic performance). The total number of sessions to reach criterion in lever pressing, in the attention task with correction trials, and in the final version of the attention task were analyzed. Once animals reached asymptotic performance, further analyses of task performance were conducted. Accuracy was assessed using mixed model analyses of variance (ANOVAs) of relative hits and correct rejections that included condition (naı¨ve, sham, and ethanol), signal duration (for hits), and block. Baseline accuracy was assessed by an overall analysis that included the initial

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Table 1 Body weights (means T SEMs in g) during early postnatal intubation PD

4

5

6

7

8

9

Sham11.8 T 0.3 14.2 T 0.4 16.3 T 0.5 18.7 T 0.5 21.1 T 0.6 23.4 T 0.8 intubated: Ethanol12.4 T 0.4 13.1 T 0.5 14.1 T 0.6 15.8 T 0.7 17.8 T 0.8 19.6 T 0.8 intubated:

baseline period and the baselines re-established prior to the short ITI and long ITI conditions. Subsequent analyses of the effects of the distracter, short ITI, and long ITI involved comparisons with the average of the three baseline sessions immediately prior to the task manipulation. The appropriate level of analysis, the individual rat or the litter, for early postnatal exposure has not been established. The present study uses the individual rat as the level of analysis as litter effects are thought to be minimized in experiments with postnatal exposure that include small numbers of animals from each litter and that test the animals at time points much later than the drug exposure [13,46]. All p-values for within-subjects main effects and interactions were adjusted using the Huynh –Feldt correction. Post hoc tests were conducted using Tukey’s honestly significant differences test. An a level of 0.05 was adopted. All data analyses were conducted using SPSS 11.5 software (SPSS, Chicago, IL).

3. Results

intubations. One sham-intubated and two naı¨ve rats chosen at random were not trained in the attention task in order to maintain an equal number of animals in each intubation condition. Thus, the data reported below are from 10 animals in each condition (ethanol-intubation, sham-intubation, naı¨ve). One sham animal became ill and failed to achieve criterion after the distracter sessions. The data from this animal, where available, are included in the analyses below. Finally, the data from one ethanol-exposed animal were not collected during the third session with the short ITI due to technical malfunction. 3.1. Body weights A 2 (condition: sham-intubated and ethanol-intubated)  6 (day) ANOVA for body weights during PD 4 – 9 exposure yielded significant main effects of condition ( F(1,18) = 7.30, p = 0.015) and day ( F(5,90) = 471.8, p < 0.001) and a significant condition X day interaction ( F(5,90) = 24.6, p < 0.001). Post hoc t tests for each day indicated that body 1.0

0.8

Relative Hits

818

Two animals assigned to the ethanol-intubated group and one animal in the sham-intubated group died during

0.6

0.4 Naive Sham EtOH

0.2

0.0 3

25

2

1

Log Signal Duration (msec) Naive Sham EtOH

15

10

5

0 Shaping

Attn Corr

Attn Test

Training Conditions

1.0

Relative Correct Rejections

Sessions to Criterion

20

0.9

0.8

0.7

0.6

0.5 Naive

Fig. 1. The mean sessions to criterion (ordinate) for each training condition (abscissa). Rats were trained to press levers for water (shaping), to respond based on the rules of the task with correction and forced trials that followed incorrect response (Attn Corr; see methods for details), and to criterion performance in the final version of the attention task (Attn Test; see methods for details). There were no significant differences between naı¨ve, sham-intubated (sham), or ethanol-intubated (EtOH) animals in the number of sessions to reach criterion for any of these stages of training. Errors bars represent SEMs.

Sham

EtOH

Fig. 2. In the top figure, the ordinate represents the relative mean hits for the three treatment groups (naı¨ve, sham-intubated [sham], and ethanolintubated [EtOH]) for each signal duration (abscissa). The bottom figure represents the mean relative correct rejections (ordinate) for the three treatment groups (abscissa). Ethanol-intubated animals demonstrated a selective deficit in detection of signals (hits) relative to sham-intubated and naı¨ve animals ( p < 0.05). These groups did not differ from each other in detection of non-signals (correct rejections). The error bars represent SEMs.

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weights for ethanol-intubated and sham-intubated rats did not differ on PD 4 or PD 5, but did differ from PD 6– PD 9 (all p < 0.013; Table 1). Body weights during adulthood did not differ between any treatment conditions (mean T SEMs; naı¨ve: 593.7 T 31.3 g; sham-intubated: 640.4 T 25.2 g; ethanol-intubated: 636.0 T 20.6 g). 3.2. Attention test training Early postnatal ethanol exposure did not differentially affect sessions to criterion for any of the stages of training (Fig. 1). 3.3. Baseline attention task performance After reaching asymptotic performance, rats were trained for three additional sessions to establish baseline performance. Immediately preceding each task manipulation (flashing houselight, varying the duration of the ITI), asymptotic performance over three consecutive sessions was reestablished. Comparisons among these periods were made to test whether any changes in performance persisted each time baseline was re-established. The hit rate was averaged across each three session period (original baseline, pre-short ITI, and pre-long ITI) and assessed using a 3 (condition)  3 (period)  3 (session)  3 (signal duration) ANOVA. The effect of condition was significant ( F(2,26) = 5.58, p = 0.01) with post hoc analysis showing poorer performance by ethanol-intubated animals relative to sham-intubated and naı¨ve animals (Fig. 2). Importantly, condition did not interact with period, indicating that the effects of ethanol 0.8 Naive Sham EtOH

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Table 2 Additional measures of performance averaged across all baseline sessions (Means T SEMs) Task parameters

Omissions/ Session

Lever press response latencies (ms)

Photocell latencies (ms)

Naı¨ve: Sham-intubated: Ethanol-intubated:

10.3 T 2.8 12.2 T 5.3 11.4 T 3.5

491.0 T 22.4 497.0 T 25.2 496.6 T 35.2

654.5 T 60.3 597.0 T 61.1 514.5 T 71.5

treatment on the hit rate persisted with subsequent training (Fig. 3). Neonatal ethanol exposure did not differentially affect the correct rejection rate (Fig. 2), the omission rate, lever press latency, or reward retrieval latencies (Table 2). 3.4. Flashing houselight The effect of the flashing houselight (distracter) on signal detection accuracy was assessed using a 3 (condition)  4 (session)  3 (block)  3 (signal duration) ANOVA. An average of the three baseline sessions immediately prior to the flashing houselight session and three consecutive sessions of the distracter task comprised the four levels of ‘‘session’’ in the analysis. For hits, the effect of condition remained significant ( F(2,27) = 4.21, p = 0.026), but did not interact with any other factors (Fig. 4). The lack of an interaction with session indicates that ethanol-exposed animals were not differentially affected by flashing the houselight. Compared to the average of the three baseline sessions, the distracter decreased the relative number of hits ( F(3,81) = 19.13, p < 0.001, Fig. 4). Similarly, the correct rejection rate ( F(3,81) = 12.65, p < 0.001; mean relative correct rejections T SEMs of three baseline sessions: 0.882 T 0.009; mean relative correct rejections T SEMs of three Naive Sham EtOH

0.7

0.7

Relative Hits

Relative Hits

0.8

0.6

0.6 0.5 0.4

0.5 1

2

3

0.3

Baseline 0.2

Fig. 3. The relative mean number of hits (ordinate) for the average of the three baseline sessions each time baseline performance was established (abscissa) for each of the treatment groups. Thus, baseline 1 is immediately before the flashing houselight sessions, baseline 2 is immediately before the short ITI sessions, and baseline 3 is immediately before the long ITI sessions. An ANOVA across each time baseline yielded a main effect of condition, with ethanol-intubated animals (EtOH) demonstrating a decreased hit rate relative to sham-intubated (Sham) or naı¨ve animals. The lack of a condition X session effect indicates that the decrease in the hit rate following early postnatal ethanol administration persisted each time baseline was established. The error bars represent SEMs.

Base 1

Distract Base 2 Short ITI Base 3 Long ITI

Task manipulations Fig. 4. The mean relative hits (ordinate) during each task manipulation: flashing houselight (Distract), a 4.5 T 3 s ITI (Short ITI), and an 18 T 3 s ITI (Long ITI), along with average of the three baseline sessions immediately before each task manipulation (Base 1, Base 2, Base 3). The distracter decreased hits for naı¨ve, sham-intubated (Sham), and ethanol-intubated (EtOH) animals, but did not differentially affect the groups. For both the short ITI and long ITI manipulations, the effect of condition was not significant. The error bars represent SEMs.

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distracter sessions: 0.823 T 0.013) was decreased during the three distracter sessions relative to the average of the three baseline sessions. There was no main effect of condition and condition did not interact with any factors for analyses of correct rejections, omissions, lever press latencies or reward retrieval latencies. 3.5. Short ITI Rats were trained for three consecutive sessions with a 4.5 T 3 s ITI. Reducing the average ITI by half resulted in poorer signal detection accuracy relative to baseline performance as a main effect of session was yielded by a 3 (condition)  4 (session)  3 (block)  3 (signal duration) ANOVA ( F(3,75) = 4.20, p = 0.008, Fig. 4). For hits, the main effect of condition was not significant, as shamintubated and naive animals tended to exhibit a greater decrease in the hit rate with a briefer ITI compared with ethanol-intubated animals (Fig. 4). However, condition did not interact with any other factor, indicating that the ethanolexposed rats were not differentially affected by the ITI reduction. No effects of condition were evident for correct rejections, omissions, lever press latencies or reward retrieval latencies. 3.6. Long ITI The final stage of training employed three consecutive sessions with an ITI of 18 T 3 s. For hits, there were no main effects of increasing the inter-trial interval or of condition (Fig. 4). Also, the condition X session interaction was not significant. Accuracy on nonsignal trials was not affected by increasing the ITI. Condition did not interact with any other factor, nor were there main effects of condition for correct rejections, omissions, bar press latencies or reward retrieval latencies.

4. Discussion Early postnatal ethanol exposure disrupted detection of signals in this attention task relative to sham-intubated or naı¨ve animals when all rats were trained to asymptotic performance levels. Moreover, the signal detection deficit was reliable, as it was observed each time the animals were trained on the attention task between task manipulations (flashing the houselight and varying the duration of the inter-trial interval). Ethanol-exposed rats did not demonstrate differences in the number of sessions to reach criterion when training to press levers, to respond based on the rules of the task, or when attentional demands were increased in the final version of the task by presenting a visual distracter or decreasing the inter-trial interval, compared with shamintubated or naı¨ve animals. The fact that ethanol-exposed animals were able to acquire criterion levels of performance at a comparable rate to sham-intubated or naı¨ve animals

indicates that this administration regimen did not result in severe deficits in motivation or sensorimotor functioning. Furthermore, even when trained to asymptotic performance levels, ethanol-exposed rats did not demonstrate deficits in correct rejections, omissions, lever press latencies, or the latency to retrieve water following a correct response. Thus, although ethanol exposure decreased body weights beginning on PD 6 compared to sham-intubated animals, as we have observed in previous experiments [58], it is unlikely that these changes in body weight resulted in motivational or motoric deficits that can account for the relatively selective effects of ethanol exposure on the hit rate. Furthermore, group differences in body weight did not persist into adulthood when the animals reached asymptotic task performance. The use of this intubation procedure in the present study allows the delivery of precise doses of ethanol directly into the stomach and minimizes the maternal deprivation that is inherent in artificial rearing procedures. One limitation of the intubation approach however, is the extensive handling and repeated removal of the pups from the dam that, in theory, could exacerbate ethanol-induced deficits. However, the lack of any differences between naı¨ve and sham-treated animals suggests that the intubation procedure itself is not sufficient to affect attentional performance. Finally, the present conclusions need to be tempered by the lack of a control group receiving formula isocalorically adjusted to match the ethanol-exposed animals. Collectively, these findings support the position that early postnatal ethanol exposure disrupts aspects of attentional processing in rats. The deficit in the hit rate was persistent as it was observed each time baseline performance was re-established prior to a task manipulation. The percent change in ethanol-exposed animals was not extremely large (approximately 10% averaged across all signal durations; Fig. 3) relative to other manipulations previously assessed in this task, including lesions of the prefrontal cortex [38] and of basal forebrain corticopetal cholinergic neurons [34]. Thus, early postnatal ethanol exposure appears to produce a somewhat subtle, but enduring deficit in attentional processing in rats. In a previous report [10] it was observed that deficits in sustained attention performance in adult FAS/ FAE patients were greater when the task involved an auditory as opposed to visual stimulus. It would be interesting to see if the same pattern of greater impairment would be evident in alcohol-exposed animals with the use of an auditory variant of this task. Deficits in attention are considered to be hallmark features of individuals with a history of prenatal alcohol exposure [25]. Several prospective studies have reported significant attentional disturbances even in individuals with modest in utero alcohol exposure (e.g., [26,50,51]). The present data demonstrate that exposure to ethanol during the early postnatal period in the rat, which corresponds to the third trimester in humans in terms of neural development, can lead to long-term disruptions in attentional performance.

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A recent report indicates that prenatal ethanol exposure in rats can disrupt choice serial reaction time performance [19]. A more substantial analysis of the attentional effects of ethanol exposure at different time points during neural development as well as administration throughout brain development (prenatal and postnatal exposure in the rat) would seem to be warranted based on the available literature. The pattern of impairment observed here following early postnatal ethanol exposure, a decrease in the hit rate, has been previously demonstrated following manipulations that alter the integrity or decrease the activity of basal forebrain corticopetal cholinergic neurons. Lesions of basal forebrain corticopetal cholinergic neurons induced by intra-basalis infusions of the selective immunotoxin 192IgG-saporin have been shown to decrease hits in the present task [34] and to decrease signal detection with increased time on the task, an effect exacerbated by decreasing the event rate, in a five-choice serial reaction time task [36]. Intra-basalis infusions of benzodiazepine receptor agonists [20] or nmethyl-d-aspartate receptor antagonists [56] which decrease cortical acetylcholine release [16,39] also decrease signal detection in this task without affecting the correct rejection rate. Importantly, lesions of the dorsal noradrenergic fiber bundle do not affect performance in this task [35], suggesting at least some selectivity in the neural structures that are necessary for normal task performance. The present data are consistent with the hypothesis that early postnatal ethanol exposure alters the integrity, activity, or regulation of basal forebrain corticopetal cholinergic neurons. Interestingly, immunotoxic lesions of basal forebrain cholinergic neurons on PD 7 lead to deficits in markers of cholinergic functioning on PD 20, supporting the idea that insults to the cholinergic system at this age may have long-term functional consequences on aspects of cognitive processing that are dependent upon the normal activity of basal forebrain corticopetal cholinergic neurons [2]. Prenatal and postnatal exposure to ethanol has been shown to decrease cortical acetylcholinesterase-positive fibers [45, but see Ref 28]. Another study found that prenatal exposure to ethanol altered the effects of cholinergic receptor ligands on delayed alternation [40]. Recent reports indicate that neonatal choline supplementation ameliorated memory deficits induced by neonatal alcohol exposure, further indicating that the cholinergic system is negatively impacted by early ethanol insult [55]. Thus, there is some evidence that early exposure to ethanol can alter the integrity and responsivity of the cholinergic system. However, the effects of the specific timing, dose, and method of delivery used in the present experiment on basal forebrain corticopetal neurons have not been explicitly established. Flashing the houselight decreased the hit rate for all animals but did not interact with ethanol exposure. Thus, increasing background noise does not appear to affect differentially ethanol-pretreated animals, at least under the

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present experimental conditions. This finding is in contrast to what was predicted based upon the clinical literature that individuals with a history of prenatal alcohol exposure are more easily distracted and have difficulty remaining on-task when a distracting event is introduced [51]. Furthermore, both increasing and decreasing the inter-trial interval had relatively modest effects on performance. This finding may be due to the fact that total session duration was confounded by changes in the event rate. That is, in the brief ITI condition, total session duration was approximately half that of the baseline condition, while in the long ITI condition, total session duration was approximately twice that of the baseline condition. Future studies that assess more comprehensively the relationship between total number of trials, session duration, and event rate may be more successful in revealing differential effects of ethanol exposure on performance. The present data support the hypothesis that early postnatal ethanol exposure disrupts attentional processing and provides a model of some of the attentional disturbances that are persistent in humans that were exposed to ethanol in utero. Further validation of this model would entail the additional demonstration of conditions that differentially disrupt performance in ethanol-exposed animals. Finally, the present data suggest that the assessment of changes in the basal forebrain cholinergic system may be a fruitful approach to understanding the neural basis underlying attentional deficits following early ethanol exposure.

Acknowledgements The present work was supported by NIAAA grant AA12466 (P.S.H.). J.A.B. was supported by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression. This work partially fulfilled the requirements for a M.A. thesis for Kevin Woolfrey from the College of William & Mary.

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