Elsevier

Behavioural Brain Research

Volume 212, Issue 2, 15 October 2010, Pages 133-142
Behavioural Brain Research

Research report
Variants of contextual fear conditioning are differentially impaired in the juvenile rat by binge ethanol exposure on postnatal days 4–9

https://doi.org/10.1016/j.bbr.2010.04.003Get rights and content

Abstract

Neonatal ethanol exposure in the rat is known to partially damage the hippocampus, but such exposure causes only modest or inconsistent deficits on hippocampus-dependent behavioral tasks. This may reflect variable sensitivity of these tasks or residual function following partial hippocampal injury. The context preexposure facilitation effect (CPFE) is a variant of context conditioning in which context exposure and immediate shock occur on successive occasions. During testing, preexposed rats freeze more than non-preexposed controls. The CPFE is more sensitive to anterograde hippocampal injury than standard contextual fear conditioning (e.g., Rudy JW, O’Reilly RC. Conjunctive representations, the hippocampus, and contextual fear conditioning. Cogn Affect Behav Neurosci 2001;1:66–82). We report that rats exposed to a high binge dose of ethanol (5.25 g/kg/day) over postnatal days [PD] 4–9 failed to demonstrate the CPFE when preexposed to the conditioning context on PD31, relative to sham-intubated and undisturbed controls (Experiment 1). Neonatal alcohol disrupted conditioned freezing to a much lesser extent relative to controls when context preexposure was followed by a standard context conditioning trial rather than immediate shock (Experiment 2). Fear conditioning to a discrete auditory cue (tone) was unaffected by neonatal alcohol exposure ruling out possible performance effects (Experiment 3). These findings suggest that the CPFE is an especially sensitive task for detecting hippocampal injury produced by neonatal alcohol. Mixed results with other tasks may reflect residual hippocampal function and/or the use of alternate neurobehavioral systems or “strategies” following alcohol-induced brain damage.

Introduction

Fetal alcohol spectrum disorders (FASD) represent a range of adverse neurocognitive, neurobehavioral, and structural effects caused by developmental alcohol exposure [69], [41]. Children with FASD demonstrate lower IQs [45], impairments on executive function tasks (e.g., problem solving, abstract thinking, cognitive flexibility), and significant learning deficits [46]. These deficits may reflect the consequences of neuroanatomical abnormalities commonly seen in human cases of FASD, including aberrations of the corpus callosum, the basal ganglia, the cerebellum, and the hippocampus [60], [61], [70], [81], which are largely a product of the timing, pattern, and dosage of ethanol consumption during pregnancy [39], [40]. Maternal and genetic factors also contribute to the effects of alcohol on the developing fetus [61]. FASD is considered a major public health problem [61] and constitutes one of the leading preventable causes of mental retardation [1]. Although public awareness programs have attempted to educate the public about the dangers of consuming alcohol during pregnancy, FASD persist [19], with an estimated prevalence of around 10 per 1000 births in the United States [49].

The use of animal models of FASD has contributed greatly to our understanding of the effects of developmental alcohol exposure. For example, the craniofacial abnormalities seen in Fetal Alcohol Syndrome are the result of alcohol exposure during the 1st trimester of development [61], [71], [72]. In contrast, the cerebellum, prefrontal cortex, and hippocampus appear to be most sensitive to the damaging effects of alcohol during the brain growth spurt [27], [80], [38], which occurs during the 3rd trimester in humans, and during the first postnatal week in the rodent [17]. The effect of alcohol on the developing hippocampus has been of particular interest because of the known role of this structure in many forms of learning and memory. Alcohol exposure limited to the 3rd trimester equivalent in the rodent causes reductions in hippocampal pyramidal cell number, specifically within the CA1 subregion [8], [9], [38], [43], [77]. Disruptions in hippocampal physiology and adult neurogenesis in the dentate gyrus (DG) are also affected by developmental alcohol exposure [7], [34], [35], [58], [73].

Rats exposed to high binge-like doses of alcohol during the brain growth spurt exhibit deficits on tasks that are impaired by hippocampal damage. For example, neonatal ethanol exposure resulting in a high blood alcohol concentration (BAC; >300 mg/dl) produces deficits during spatial conditional alteration and reversal learning in the T-maze [76], [54], as well as spatial navigation impairments in the water maze [24], [23], [21], [31], [43]. Generally, the water maze impairments induced by neonatal ethanol affects the rate of acquisition, with some studies showing no differences during “free swim” probe sessions [43], [75], the most stringent measure of spatial cognition tested after asymptotic learning. Water maze acquisition is prevented by hippocampal lesions [52], and the milder spatial learning deficits in this task observed in alcohol-exposed rats may or may not indicate hippocampal dysfunction [55], [75]. Importantly, spatial learning in the water maze is also impaired by lesions of the cerebral cortex, striatum, basal forebrain, and cerebellum [16], and the effects of alcohol on these structures cannot be completely discounted. For example, rats administered 5.25 g/kg of alcohol over PD7-9 showed CA1 pyramidal cell reductions and water maze impairments [43]. Vitamin E co-administered with alcohol over PD7-9 prevented CA1 pyramidal cell loss, but did not rescue the water maze deficits [43], suggesting that water maze deficits in these rats was caused by damage to other structures or by an action on hippocampus (e.g., plasticity) that is not related substantially to CA1 cell counts.

Characterizing the impact of neonatal alcohol exposure on the hippocampus may benefit from other, more sensitive tests. For example, contextual fear conditioning has been widely used as a test of hippocampal function [56], [33], [28]. A variant of standard contextual fear conditioning is the context preexposure facilitation effect (CPFE), where a rat preexposed to the testing context 24 h prior to an immediate shock shows enhanced contextual conditioning relative to rats not preexposed to the context [18], [66], [64]. In a series of experiments, Rudy and co-workers have demonstrated the enhanced sensitivity of the CPFE to hippocampal insult ([63], [4], [47], [65], see [62], [64] for review). The CPFE as a test of hippocampal dysfunction in ethanol-exposed rats has a number of advantages. First, robust learning can occur with a single context-shock association. Second, learning about the context and associating the context with the shock occur on separate occasions. And finally, learning about the context during preexposure and retrieval of the context memory during training and during testing requires an intact hippocampus (ibid), and therefore slight disruptions of hippocampal function may disrupt the CPFE.

In the current set of experiments we tested rats exposed to a high binge dose of ethanol (5.25 g/kg/day) over PD4-9 on two variants of contextual fear conditioning. In Experiment 1, ethanol-exposed and control rats were trained on the CPFE paradigm starting at PD31, an age where the CPFE is robust in developing rats [68]. We hypothesized that if ethanol exposure over this window disrupts hippocampal function, then ethanol-exposed rats should fail to show the CPFE. In Experiment 2, the findings of Experiment 1 were replicated and extended with the inclusion of a standard contextual fear conditioning (sCFC) paradigm, where rats received an unsignaled shock 2 min after placement in the conditioning chamber. The CPFE and sCFC are differentially affected by pretraining lesions of the hippocampus [64]. If PD4-9 ethanol produces hippocampal dysfunction, we hypothesized that ethanol-exposed rats would show greater deficits in the CPFE than during sCFC. Finally, in Experiment 3 ethanol-exposed rats and controls were trained on PD32 in a tone fear conditioning task that is unaffected by hippocampal lesions [56]. We hypothesized that ethanol-exposed and control rats would not differ in their ability to condition to the tone conditioned stimulus (CS). Portions of this paper have been presented in abstract form [53].

Section snippets

Experiment 1: Neonatal alcohol and the context preexposure facilitation effect

Experiment 1 examined the effects of neonatal binge ethanol exposure on the context preexposure facilitation effect (CPFE). The CPFE is sensitive to hippocampal insult [63], [4], [47], [65]. Similar ethanol dosing methods utilized for this experiment produce reductions in CA1 pyramidal cells [77], [43]. We therefore predicted that juvenile rats exposed to a binge dose of ethanol over PD4-9 would show deficits in the CPFE compared to control rats.

Experiment 2: The effects of neonatal alcohol exposure on two variants of contextual fear conditioning.

Experiment 2 examined the effects of neonatal alcohol exposure on two variants of contextual fear conditioning: the CPFE and standard contextual fear conditioning. In standard contextual fear conditioning (sCFC), the subject receives a footshock some time (e.g., 2 min) after placement into the context. Although many studies show disruptions in sCFC after posttraining hippocampal lesions [56], [32], under certain conditions, rats with pretraining lesions of the hippocampus show normal context

Experiment 3: Neonatal alcohol exposure and discrete CS (tone) fear conditioning

Experiment 3 examined the effects of developmental alcohol exposure on fear conditioning to a discrete CS in juvenile rats. The previous experiments demonstrated deficits in contextual fear conditioning in rodents neonatally exposed to alcohol. These deficits are consistent with hippocampal injury produced by developmental exposure to alcohol. However, they could also reflect more general “performance deficits” such as the inability to freeze because of hyperactivity or to learn fear

General discussion

The current set of experiments examined the performance of rats neonatally exposed to a high binge dose of ethanol over postnatal days (PD) 4–9 on two variants of contextual fear conditioning—the context preexposure facilitation effect (CPFE, Experiments 1 and 2) and standard context fear conditioning (sCFC) involving 120 s of context exposure terminating with a single footshock (Experiment 2), as well as on cued (tone) fear conditioning (Experiment 3). As in previous studies of the CPFE [18],

Acknowledgements

The authors would like to thank Jeff B. Rosen for graciously providing access to the fear conditioning equipment used in these experiments. We would also like to thank Mike A. Burman for initial training on the fear conditioning paradigm, and to Felipe L. Schifino and Henry S. Lange for technical support. This work was supported by the University of Delaware and by NIH grant RO1 AA014288-01.

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