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Two experiments examined the effect of the noncompetitive NMDA receptor antagonist, dizocilpine maleate (MK-801), on spatial working memory during development. Rats were trained on spatial delayed alternation (SDA) in a T-maze after ip administration of 0.06 mg/kg MK-801, 0.1 mg/kg MK-801, or saline on postnatal days (P) P23 and P33 (Experiment 1), or following bilateral intrahippocampal administration of 2.5 or 5.0 [micro]g per side MK-801 or saline on P26 (Experiment 2). In Experiment 1, MK-801 dose-dependently impaired SDA learning at both ages. Because the same doses of systemic MK-801 have no effect on T-maze position discrimination learning, impairment of SDA by MK-801 likely reflects disruption of spatial working memory. Both doses of MK-801 abolished acquisition of SDA performance in Experiment 2. Disruption of hippocampal plasticity may account for the effects produced by systemic MK-801 administration. These results confirm and extend earlier lesion studies by implicating plasticity of hippocampal neurons in the ontogeny of spatial delayed alternation. Keywords: development, working memory, MK-801, rat, spatial learning

Classical conditioning of eyeblink responses has been one of the most important models for studying the neurobiology of learning, with many comparative, ontogenetic, and clinical applications. The current study reports the development of procedures to conduct eyeblink conditioning in preweanling lambs and demonstrates successful conditioning using these procedures. These methods will permit application of eyeblink conditioning procedures in the analysis of functional correlates of cerebellar damage in a sheep model of fetal alcohol spectrum disorders, which has significant advantages over more common laboratory rodent models. Because sheep have been widely used for studies of pathogenesis and mechanisms of injury with many different prenatal or perinatal physiological insults, eyeblink conditioning can provide a well-studied method to assess postnatal behavioral outcomes, which heretofore have not typically been pursued with ovine models of developmental insults. Keywords: delay conditioning, associative learning in lamb, ovine model, cerebellum

Four experiments examined the effect of dizocilpine maleate (MK-801), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, on reversal learning during development. On postnatal days (PND) 21, 26, or 30, rats were trained on spatial discrimination and reversal in a T-maze. When MK-801 was administered (intraperitoneally) before both acquisition and reversal, 0.18 mg/kg generally impaired performance, whereas doses of 0.06 mg/kg and 0.10 mg/kg, but not 0.03 mg/kg, selectively impaired reversal learning (Experiments 1 and 3). The selective effect on reversal was not a result of sensitization to the second dose of MK-801 (Experiment 2) and was observed when the drug was administered only during reversal in an experiment addressing state-dependent learning (Experiment 4). Spatial reversal learning is more sensitive to NMDA-receptor antagonism than is acquisition. No age differences in sensitivity to MK-801 were found between PND 21 and 30. Keywords: spatial discrimination, reversal, MK-801, development

Discrimination of the eyeblink conditioned response (CR) between conditioned stimuli (CSs) of different durations and modalities was examined across development in rats. Interstimulus interval (ISI) discrimination was evident at Postnatal Days 23-34 in Experiment 1, and earlier CR peak latencies and enhanced CR amplitudes were seen to the long CS in the ISI discrimination group relative to a control group receiving the short CS without reinforcement. Experiment 2 showed that early CR peak latencies and enhanced CR amplitudes to the long CS in the ISI discrimination group were due to associative pairing of the short CS and unconditioned stimulus. Experiment 3 demonstrated ISI discrimination in adults that was improved relative to younger subjects, but with no enhancement of CR amplitude to the long CS in the ISI discrimination group. Cerebellar cortical maturation may influence the ontogeny of CR timing. Keywords: eyeblink conditioning, cerebellum, conditioned response timing, ISI discrimination, development

The ontogeny of associative learning in delay (750-ms conditional stimulus [CS], 650-ms interstimulus interval [ISI]), long-delay (l,350-ms CS, 1,250-ms ISI), and trace (750-ms CS, 500-ms trace interval, 1,250-ms ISI) eyeblink conditioning was examined in 5-month-old human infants and adults. Infants and adults showed different acquisition rates but reached equivalent asymptotes of conditional responses (CRs) in standard delay conditioning. In long-delay and trace conditions, infants exhibited less robust conditioning than adults and minimal ability to appropriately time CRs. During infancy, the ISI, rather than the conditioning procedure, predicted rate and effectiveness of CRs. These findings suggest that higher order cognitive abilities begin emerging early in development. Across ontogeny, however, there are changes in the limits and parameters that support associative learning.

Human (Homo sapiens) volunteers (N = 72) received saline, a low dose of oral scopolamine (0.6 mg), a high dose of oral scopolamine (1.2 mg), or a peripheral analogue (glycopyrrolate). They then underwent classical conditioning of the eyeblink response to a tone conditioned stimulus (CS) and a corneal airpuff unconditioned stimulus (UCS) in a delay conditioning paradigm. There was a dose-related decline in acquisition of the conditioned response. These drug-induced conditioning deficits were similar to those previously reported in rabbit eyeblink conditioning and could not be attributed to such nonassociative factors as changes in auditory thresholds to the tone CS, magnitude of reflexive blinks to the airpuff UCS, or to changes in spontaneous blink rates.

In Experiment 1, Long-Evans rat pups received medial prefrontal cortex (PFC) aspirations or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform one of two T-maze tasks: discrete-trials delayed alternation (DA) or simple position discrimination. Early PFC damage produced a selective failure to learn the DA task. In Experiment 2, pups given the same lesion or sham surgery were trained on DA on PND19, PND27, or PND33. In relation to sham-operated controls, pups with PFC damage were impaired on PND19, somewhat impaired on PND27, and entirely unimpaired when tested on PND33. In Experiment 3, pups given larger lesions of the frontal cortex on PND10 were impaired on DA when tested on PND23 but not when tested on PND33. These findings indicate that early PFC lesions result in a memory deficit around the time of weaning, which then recovers over the next 10-14 days of development. Moreover, the early deficit is selective for a late developing cognitive process (or processes) that is involved in acquisition of DA.

Long-Evans rat pups, 17 - 18 or 24 days of age, were trained with an eyeblink conditioning (EBC) procedure that was used previously with adult rats (Skelton, 1988). Pups received 3 sessions of delay conditioning in a single day at about 4-hr intervals (100 trials/session). Trials involved pairings of an auditory conditioned stimulus (2.8-kHz, 82-dB tone) and a periocular-shock unconditioned stimulus (US; 100 ms, 2 mA), which were presented 280 ms apart. EBC was observed at both ages, but older pups learned much more rapidly. Subsequent experiments established that this effect is associative (Experiment 2), that age differences in EBC cannot be attributed to differences in ability to respond or in sensitivity to the US (Experiment 3), and that EBC rate can be modulated by motivational state (Experiment 4). This preparation may help elucidate the relation between neural development and the ontogeny of learning.

In Experiment 1, Long-Evas rat pups received fimbria-fornix transections or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform either a discrete-trials delayed alternation (DA) or a simple position discrimination (PD) task in a T maze. Rat pups in both surgical conditions learned the PD task within five 12-trial blocks of training. However, only sham-operated pups learned the DA task. In Experiment 2, performance of DA emerged between PND19 and PND27 in sham-operated pups but failed entirely to develop in pups with early lesions. In Experiment 3, fornix-transected pups that were given extended DA training (132 trials) on PND23-PND24 showed some improvement in performance but remained impaired in relation to sham-operated controls. These findings implicate the limbic system in the postnatal development of DA but not PD and suggest that dual-process theories of memory may be relevant to the psychobiology of cognitive development.

We recently demonstrated the acceptability and feasibility of a randomized, double‐blind choline supplementation intervention for heavy drinking women during pregnancy. In this study, we report our results relating to the efficacy of this intervention in mitigating adverse effects of prenatal alcohol exposure (PAE) on infant growth and cognitive function. Sixty‐nine Cape Coloured (mixed ancestry) heavy drinkers in Cape Town, South Africa, recruited in mid‐pregnancy, were randomly assigned to receive a daily oral dose of either 2 g of choline or placebo from time of enrollment until delivery. Each dose consisted of an individually wrapped packet of powder that, when mixed with water, produced a sweet tasting grape‐flavored drink. The primary outcome, eyeblink conditioning (EBC), was assessed at 6.5 months. Somatic growth was measured at birth, 6.5, and 12 months, recognition memory and processing speed on the Fagan Test of Infant Intelligence, at 6.5 and 12 months. Infants born to choline‐treated mothers were more likely to meet criterion for conditioning on EBC than the placebo group. Moreover, within the choline arm, degree of maternal adherence to the supplementation protocol strongly predicted EBC performance. Both groups were small at birth, but choline‐treated infants showed considerable catch‐up growth in weight and head circumference at 6.5 and 12 months. At 12 months, the infants in the choline treatment arm had higher novelty preference scores, indicating better visual recognition memory. This exploratory study is the first to provide evidence that a high dose of choline administered early in pregnancy can mitigate adverse effects of heavy PAE on EBC, postnatal growth, and cognition in human infants. These findings are consistent with studies of alcohol‐exposed animals that have demonstrated beneficial effects of choline supplementation on classical conditioning, learning, and memory. Choline supplementation has been shown to mitigate adverse effects of prenatal alcohol exposure on neurobehavior in animals, including eyeblink conditioning (EBC). In a randomized, double‐blind trial, human infants born to choline‐supplemented mothers showed greater increases in conditioned responses, compared with those whose mothers received placebo. Within the choline arm, degree of maternal adherence strongly predicted EBC performance. Choline‐treated infants also showed considerable catch‐up growth in weight and head circumference and better visual recognition memory compared to the placebo group.

The context preexposure facilitation effect (CPFE) is a contextual fear conditioning paradigm in which learning about the context, acquiring the context-shock association, and retrieving/expressing contextual fear are temporally dissociated into three distinct phases. In contrast, learning about the context and the context-shock association happens concurrently in standard contextual fear conditioning (sCFC). By infusing the GABAAreceptor agonist muscimol into medial prefrontal cortex (mPFC) in adolescent Long-Evans rats, the current set of experiments examined the functional role of the mPFC in each phase of the CPFE and sCFC. In the CPFE, the mPFC is necessary for the following: acquisition and/or consolidation of context memory (Experiment 1), reconsolidation of a context memory to include shock (Experiment 2), and expression of contextual fear memory during a retention test (Experiment 3). In contrast to the CPFE, inactivation of the mPFC prior to conditioning in sCFC has no effect on acquisition, consolidation, or retention of a contextual fear memory (Experiment 4). Interestingly, the mPFC is not required for acquiring a context-shock association (measured by post-shock freezing) in the CPFE or sCFC (Experiment 2b and 4). Taken together, these results indicate that the mPFC is differentially recruited across stages of learning and variants of contextual fear conditioning (CPFE versus sCFC). More specifically, separating out learning about the context and the context-shock association necessitates activation of the medial prefrontal cortex during early learning and/or consolidation.

In two experiments we examined the ability of 15-, 21-, and 27-day-old rats to perform two spatial working memory problems (delayed alternation and discrete-trials delayed alternation) and a reference memory problem (position habit) in a T-maze. In the delayed alternation problem, each animal was presented with a series of free-choice trials and was rewarded for regularly alternating responses to the left and right arms of the T-maze. In the discrete-trials delayed alternation problem, each animal was forced to one maze arm and rewarded (forced run) and was then placed back into the start box and given a choice of arms (choice run). The direction of forced runs followed an irregular, counterbalanced series, and animals were rewarded for choosing the alternate maze arm on choice runs. In the position habit problem, animals were rewarded for consistently choosing one of the two arms of the T-maze. Performance on these problems was assessed relative to control conditions in which reward was not contingent on choice behavior. At all ages, rat pups learned to perform the delayed alternation and position habit problems (Experiment 1). However, only 21- and 27-day-old rats were able to learn the discrete-trials delayed alternation problem. The 15-day-olds were unable to learn this task (Experiment 2). The results of these experiments show that reference memory capacity is present by at least 15 days of age in the rat and does not develop further at later ages. However, working memory capacity develops between 15 and 21 days of age on tasks involving trial-specific memory (Experiment 2) but not on recent-memory tasks involving a series of regularly alternating trials (Experiment 1).

Background: Prenatal alcohol exposure is related to a wide range of neurocognitive effects. Eyeblink conditioning (EBC), which involves temporal pairing of a conditioned with an unconditioned stimulus, has been shown to be a potential biomarker of fetal alcohol exposure. A growing body of evidence suggests that white matter may be a specific target of alcohol teratogenesis, and the neural circuitry underlying EBC is known to involve the cerebellar peduncles. Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that has proven useful for assessing central nervous system white matter integrity. This study used DTI to examine the degree to which the fetal alcohol‐related deficit in EBC may be mediated by structural impairment in the cerebellar peduncles.

Background: Alcohol exposure in the rat on postnatal days (PD) 4 to 9 is known to partially damage the hippocampus and to impair hippocampus‐dependent behavioral tasks. We previously reported that PD4 to 9 alcohol exposure eliminated the context preexposure facilitation effect (CPFE) in juvenile rats, a hippocampus‐dependent variant of contextual fear conditioning. In the CPFE, context exposure and immediate shock occur on successive occasions and this produces conditioned freezing relative to a control group that is not preexposed to the training context. Here, we extend our earlier findings by examining the effects of neonatal alcohol administered at multiple doses or over different neonatal exposure periods.

Background: Classical eyeblink conditioning (EBC) involves contingent temporal pairing of a conditioned stimulus (e.g., tone) with an unconditioned stimulus (e.g., air puff). Impairment of EBC has been demonstrated in studies of alcohol‐exposed animals and in children exposed prenatally at heavy levels.

The striatum plays a major role in both motor control and learning and memory, including executive function and "behavioral flexibility." Lesion, temporary inactivation, and infusion of an N-methyl-D-aspartate (NMDA)-receptor antagonist into the dorsomedial striatum (dmSTR) impair reversal learning in adult rats. Systemic administration of MK-801 disrupts reversal learning in developing rats, as reported in an earlier work by Chadman et al., but it is not known whether NMDA-receptor function within the dmSTR plays a role in this effect. In Experiment 1, reversal learning was dose-dependently impaired following bilateral dmSTR administration of MK-801 (either 2.5 or 5.0 µg) only during the reversal phase relative to saline in postnatal day (P) 26 rats. In Experiment 2, separate groups of P26 rats were trained on the same reversal learning task, but were administered bilateral dmSTR infusions during acquisition only (MK–SAL), reversal only (SAL–MK), both phases (MK–MK), or neither phase (SAL–SAL). The MK-801 effect was specific to the reversal training phase. The drug did not alter acquisition of the initial discrimination. Analysis of the pattern of errors indicates that dmSTR MK-801 treatment increased perseveration of the choice response trained in acquisition. NMDA receptors in the dmSTR play a role in reversal learning in the weanling rat.

Background: Eyeblink conditioning (EBC) is a Pavlovian paradigm that involves contingent temporal pairing of a conditioned stimulus (e.g., tone) with an unconditioned stimulus (e.g., air puff). Animal studies have shown that binge consumption of alcohol during pregnancy impairs EBC and that this impairment is likely mediated by a loss of neurons in the inferior olive and the cerebellar cortex and deep nuclei, as well as by a reduction in neural plasticity in the cerebellar deep nuclei.

Background: Neonatal alcohol consumption in rats is widely used to model cerebellar injury arising from 3rd‐trimester human fetal alcohol exposure. Binge alcohol exposure of 5 g/kg/day or more over postnatal days (PD) 4 to 9 in rats damages the cerebellum and consequently impairs classical eyeblink conditioning (EBC). The present study sought to identify deficits in EBC using doses lower than those that have been reported previously following alcohol exposure limited to PD4‐9. Complex conditioned response (CR) timing tasks utilizing 2 interstimulus intervals (ISIs) were used to test the hypothesis that 3 g/kg/day of alcohol would produce early onset and early peaked CRs, whereas 4 and 5 g/kg/day would impair CR acquisition.

Fear conditioning, including variants such as delay and trace conditioning that depend on different neural systems, is widely used to behaviorally characterize genetically altered mice. We present data from three strains of mice, C57/BL6 (C57), 129/SvlmJ (129), and a hybrid strain of the two (F(1) hybrids), trained on various versions of a trace fear-conditioning protocol. The initial version was taken from the literature but included unpaired control groups to assess nonassociative effects on test performance. We observed high levels of nonassociative freezing in both contextual and cued test conditions. In particular, nonassociative freezing in unpaired control groups was equivalent to freezing shown by paired groups in the tests for trace conditioning. A number of pilot studies resulted in a new protocol that yielded strong context conditioning and low levels of nonassociative freezing in all mouse strains. During the trace-CS test in this protocol, freezing in unpaired controls remained low in all strains, and both the C57s and F(1) hybrids showed reliable associative trace fear conditioning. Trace conditioning, however, was not obtained in the 129 mice. Our findings indicate that caution is warranted in interpreting mouse fear-conditioning studies that lack control conditions to address nonassociative effects. They also reveal a final set of parameters that are important for minimizing such nonassociative effects and demonstrate strain differences across performance in mouse contextual and trace fear conditioning.

The effects of bilateral hippocampal aspiration lesions on later acquisition of eyeblink conditioning were examined in developing Long-Evans rat pups. Lesions on postnatal day (PND) 10 were followed by evaluation of trace eyeblink conditioning (Experiment 1) and delay eyeblink conditioning (Experiment 2) on PND 25. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US, 100 ms) were presented in one of three conditioning paradigms: trace (380 ms CS, 500 ms trace interval, 880 ms interstimulus interval [ISI]), standard delay (380 ms CS, 280 ms ISI), or long delay (980 ms CS, 880 ms ISI). The results of two experiments indicated that hippocampal lesions impaired trace eyeblink conditioning more than either type of delay conditioning. In light of our previous work on the ontogeny of trace, delay, and long-delay eyeblink conditioning (Ivkovich, Paczkowski, & Stanton, 2000) showing that trace and long-delay eyeblink conditioning had similar ontogenetic profiles, the current data suggest that during ontogeny hippocampal maturation may be more important for the short-term memory component than for the long-ISI component of trace eyeblink conditioning. The late development of conditioning over long ISIs may depend on a separate process such as protracted development of cerebellar cortex.

Eyeblink conditioning depends on an identified brainstem‐cerebellar circuit and may be useful in functional studies of early cerebellar damage produced by neurotoxicants. The present study asked whether binge‐like neonatal ethanol exposure that damages the cerebellum would also result in eyeblink conditioning deficits. On postnatal day (PND) 23 to PND24, three groups of Long‐Evans rat pups were tested for eyeblink conditioning: (1) ETOH, a group that received intragastric administration of 5.25 g/kg/day of ethanol on PND4 through PND9 via artificial rearing; (2) GC, a gastrostomy control group that received calorically matched milk formula on those days; and (3) SC, suckle controls that were reared normally with their dams. Eyeblink conditioning was severely impaired in the ethanol‐treated group relative to the GC and SC groups, which did not differ. This impairment did not reflect sensory, motor, or motivational effects of ethanol treatment, because startle responses to the auditory conditioned stimulus and reflexive eyeblink responses to the unconditioned stimulus did not differ across the three treatment groups. These results suggest that neonatal binge ethanol exposure disrupted brain development in a manner that selectively impaired associative processes involved in eyeblink conditioning, consistent with alcohol‐induced damage to the brainstem‐cerebellar circuit necessary for this form of learning.

Eyeblink conditioning depends critically on an identified brainstem-cerebellar circuit and is modulated under some circumstances by the hippocampus, amygdala, and other forebrain regions. Developmental studies of eyeblink conditioning could help elucidate questions concerning the behavioral expression of plasticity within these brain circuits and regions, and of their functional interactions, as they unfold during ontogeny. Recently, this laboratory has shown that conditioning of the eyeblink reflex develops dramatically between Postnatal Days (PND) 17 and PND 24 in the rat. The present study asked whether the developmental emergence of the eyeblink conditioned response (CR) occurs gradually or abruptly over this age range, and whether it reflects developmental changes in acquisition or expression of the learned eyeblink reflex. In Experiment 1, rat pups received two consecutive days of training beginning on PND 17, 20, or 24. Conditioned responses occurred at low levels on PND 17–18, intermediate levels on PND 20–21, and high levels on PND 24–25. In Experiment 2, 17-day-old rats received 2 days of training, 72 h apart, so that effects of training on PND 17 could be examined at an age, PND 20, when expression of the eyeblink CR was clearly possible. On PND 20, rat pups that had received paired training on PND 17 showed significantly faster conditioning than controls that had received unpaired training or no training on PND 17. These findings suggest that neural plasticity underlying associative learning developmentally precedes its overt expression in behavior. Hypotheses concerning the nature and locus of this learning are discussed.

Developmental Neurotoxicity: Evaluation of Testing Procedures with Methylazoxymethanol and Methylmercury. Goldey, E. S., O'Callaghan, J. P., Stanton, M. E., Barone, S., Jr., and Crofton, K. M. (1994). Fundam. Appl. Toxicol.23, 447-464.

This study examined the effects of intrahippocampal injections of scopolamine (a muscarinic antagonist drug) on performance of a working-memory task (contingently) reinforced T-maze alternation) and a reference-memory task (visual discrimination) by the same rats in the same maze. Rats in the first shipment were trained in delayed alternation, received bilateral implantation of cannulae aimed at the CA3 field of the dorsal hippocampus, and were tested for retention with 1 µl microinjections of scopolamine (35 µg) and saline on alternate days. These rats were then trained on visual discrimination and tested alternately under scopolamine or saline as described above. It was found that scopolamine impaired performance of delayed alternation to a greater extent than performance of visual discrimination. Data from rats in the second shipment replicated this finding, with the order of the tasks reversed, and, additionally, showed that delayed alternation, but not visual discrimination, was impaired at a dose of 12 µg/µl. A dose of 4 µg/µl had no effect on either task. It is concluded that performance of a workingmemory task is significantly more sensitive to disruption of cholinergic mechanisms in the hippocampus than performance of a reference-memory task.

Assessment of Offspring Development and Behavior Following Gestational Exposure to Inhaled Methanol in the Rat. Stanton, M. E., Crofton, K. M., Gray, L. E., Gordon, C. J., Boyes, W. K., Mole, M. L., Peele, D. B, and Bushnell, P. J. (1995). Fundam. Appl. Toxicol. 28, 100-110. The prospect of widespread human exposure associated with its use as an alternative fuel has sparked concern about the toxic potential of inhaled methanol (MeOH). Previous studies have revealed congenital malformations in rats following inhaled MeOH (Nelson et al. (1985). Fundam. Appl. Toxicol. 5, 727-736) but these studies did not include postnatal behavioral assessment. In the present study, pregnant Long-Evans rats were placed in exposure chambers containing 15,000 ppm MeOH or air for 7 hr/day on Gestational Days (GD) 7-19. The total alveolar dose of methanol was estimated at about 6.1 g/kg/day, for a total dose of about 42.7 g/kg for the entire study. Maternal body weights were recorded daily and blood methanol concentrations were determined at the end of exposure on GD 7, 10, 14, and 18. Following birth (Postnatal Day 0 [PND 0]), a number of tests were performed at various points in development, including: offspring mortality and body wt (PND 1, 3), motor activity (PND 13-21, 30, 60), olfactory learning (PND 18), behavioral thermoregulation (PND 20-21), T-maze learning (PND 23-24), acoustic startle response (PND 24, 60), reflex modification audiometry (PND 60), pubertal landmarks (PND 31-56), passive avoidance (PND 72), and visual-evoked potentials (PND 160). Maternal blood MeOH levels, measured from samples taken within 15 min after removal from the exposure chamber, declined from about 3.8 mg/ml on the first day of exposure to 3.1 mg/ml on the 12th day of exposure. MeOH transiently reduced maternal body wt (4-7%) on GD 8-10, and offspring BW (5%) on PND 1. No. other test revealed significant effects of MeOH. Prenatal exposure to high levels of inhaled MeOH appears to have little effect on this broad battery of tests beyond PND 1 in the rat. Copyright 1995, 1999 Academic Press

The pituitary-adrenal response of mother and infant squirrel monkeys was assessed under basal conditions and after 5, 10, or 20 min of separation followed by 30 min of reunion. All separation conditions produced equal increments of cortisol (approximately 50%) in both mothers and infants. These increments were lower than would have occurred in the absence of reunion. These results extend previous reports that reunion ameliorates the psychoendocrine effects of separation and indicate that mother-infant attachment can be studied with shorter periods of separation than have been used customarily.