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 sec 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
], control rats (Groups SI & UD) preexposed to the testing context demonstrated the CPFE, with elevated levels of contextual freezing to an immediate shock compared to control groups preexposed to the alternate context, which displayed the immediate shock deficit (ISD;[18
]). In contrast, rats neonatally exposed to ethanol (Group EtOH) demonstrated the ISD regardless of preexposure experience. This is the first report of a CPFE deficit in this rodent model of third trimester ethanol exposure. Experiment 2 replicated and extended this finding by including a standard contextual fear conditioning procedure. Preexposure to the testing context facilitated conditioning during sCFC for all groups. In addition, Group EtOH rats displayed a significant reduction in contextual freezing compared to control groups. Comparisons between the two variants of contextual fear conditioning reveal that the CPFE is more sensitive to developmental alcohol exposure than is sCFC. Additionally, Experiment 3 ruled out various performance effects that may have been caused by developmental alcohol exposure by showing intact post-shock and cued (tone) freezing in alcohol-exposed rats.
Relatively few experiments have examined fear conditioning in rodents exposed to ethanol during development. Wagner & Hunt [78
] demonstrated trace fear conditioning deficits in juvenile rats exposed to 5.25 g/kg/day of ethanol over PD4-9 compared to control rats. However, ethanol-exposed rats and controls did not differ during delay fear conditioning (consistent with our results in Exp. 3). Lesions of the dorsal hippocampus (DH) have been show to disrupt trace fear conditioning, while leaving delay conditioning intact [14
]. Wagner & Hunt [78
] conclude that trace conditioning deficits in ethanol exposed rodents are likely to result from ethanol-induced disruptions in the hippocampus and/or basal forebrain. Two additional studies examined sCFC in adult rodents prenatally
exposed to ethanol [79
]. In these studies, conditioning involved pairing a tone CS with an aversive footshock US over a number of trials. Ethanol-exposed rodents showed reduced levels of freezing when tested in the training context 24h later compared to controls, but showed normal levels of freezing during tone presentation, a task dissociation that has been seen in rodents with hippocampal lesions [56
]. The hippocampus has also been implicated in contextual fear conditioning (see below), and deficits in sCFC in rodents prenatally exposed to ethanol may result from hippocampal dysfunction. Our results are in general agreement with these findings. Rats exposed to ethanol over PD4-9 showed disruptions in contextual freezing in two separate variants of the task whereas these rats showed conditioned freezing to a tone CS that is comparable to control groups.
Contextual fear conditioning requires that the shock become associated with the context in which the shock occurs and is mediated by interactions between the hippocampus and the amygdala [56
]. The standard contextual fear circuit involves reciprocal connections between the CA1/subiculum border regions and the basaolateral complex (BLA), which includes the basal and accessory basal nuclei of the amygdala [36
]. Although posttraining lesions of the hippocampus have been shown to disrupt contextual fear conditioning [32
], pretraining lesions of the hippocampus have inconsistently affected contextual fear conditioning [42
], suggesting that an extrahippocampal system is capable of mediating contextual conditioning when the hippocampus is “offline” [66
]. The extrahippocampal system is likely to involve reciprocal connections between the entorhinal cortex and the BLA [57
for review]. The hippocampus actively competes with and dominates the extrahippocampal system at the level of the BLA during contextual conditioning [42
]. However, when the competition from the hippocampus is disrupted (e.g., through lesions, inactivations, etc.), the extrahippocampal system is capable of mediating contextual learning [5
]. In Experiment 2, ethanol-exposed rats showed a more modest but significant impairment of standard contextual conditioning (sCFC) relative to control groups, a reduction similar to those induced by pretraining excitotoxic lesions of the dorsal hippocampus [82
]. The more modest impairment of sCFC relative to the CPFE (Experiment 2) may occur because sCFC may be mediated by an extrahippocampal system following neonatal alcohol exposure.
To better examine the role of the hippocampus during contextual fear conditioning Rudy and colleagues have developed the CPFE paradigm, where learning about the context and associating the context with the shock occur on separate occasions. This paradigm appears to be more sensitive to hippocampal insult than is sCFC [66
]. The CPFE is negatively affected by 1) anterograde damage of the DH [63
]; 2) blocking protein synthesis in the DH after context preexposure [4
]; 3) muscimol infusions into the DH prior to context preexposure, prior to immediate shock, and prior to contextual fear testing [47
]; 4) inactivating NMDA receptors in the DH prior to preexposure [48
]; and 5) inactivation of the VH prior to preexposure or inhibiting protein synthesis in the VH after preexposure [65
]. These studies demonstrate that the hippocampus makes important contributions during each of the three phases of the CPFE paradigm. The CPFE deficits observed in our ethanol-exposed rats may be related to hippocampal dysfunction during one or more of these phases.
There are a number of factors that could contribute to the deficits in contextual fear conditioning seen in rats neonatally exposed to ethanol. First, neonatal ethanol exposure that results in high BACs (>350 mg/dl) causes a significant reduction of CA1 pyramidal neurons [38
]. Selective lesions of both dorsal and ventral CA1 disrupt sCFC [29
]. Though CA1 pyramidal neurons are only reduced by 20% in most studies of neonatal ethanol exposure in rats, these small reductions in CA1 may contribute to the contextual fear deficits exhibited. Further support for the involvement of CA1 in sCFC comes from studies of the activity-regulated cytoskeletal protein (Arc), an immediate-early gene, linked to neural-plasticity mechanisms mediating learning [26
shows a selective increase in mRNA signal in the CA1/subiculum 30 minutes after sCFC [30
expression in the hippocampus is increased during both context exploration and during context-shock association during sCFC, but not after an immediate shock [28
]. Future studies are planned to examine Arc expression during contextual fear conditioning in rats neonatally exposed to ethanol. Together, both lesion and Arc studies suggest an important role of CA1 in sCFC. Examining Arc expression during contextual fear conditioning as a function of EtOH exposure will provide more direct evidence for a link between disruptions in CA1 function and impaired contextual conditioning induced by developmental ethanol exposure.
Rudy & O’Reilly [66
] suggest that during the CPFE paradigm, rats form a conjunctive representation of the context during preexposure. During training to an immediate shock, the rat samples a subset of features of the context and, through the process of pattern completion, the previously acquired conjunctive representation is retrieved and it is this retrieved representation that becomes associated with shock, producing the CPFE. Rats that are not preexposed to the context show the ISD to immediate shock because the individual features of the context sampled prior to immediate shock are insufficient to support conditioning. Area CA3 of the hippocampus has been proposed to support the process of pattern completion due to its abundance of recurrent circuits [44
]. Rats with CA3 and DG lesions, but not rats with CA1 or sham lesions, were impaired during the test phase of a spatial pattern completion task [22
], giving experimental support to this view. In addition, CA3 NMDA receptors are required for the rapid formation of context representation during contextual conditioning at short (between 20 and 40 seconds) placement-to-shock intervals [50
]. In light of these considerations, Livy et al. [38
] report reductions in CA3 density and cell number at PD10 using an artificial rearing method for developmental ethanol exposure over PD4-9, though other studies have failed to show CA3 reductions (e.g., [77
]). If our dosing method produced CA3 reductions at PD31-33, the disruptions in the CPFE demonstrated by our ethanol-exposed rats may represent a disruption in the pattern completion process during the training phase of the CPFE paradigm. A deficit in pattern completion, however, is not necessary to account for deficits observed in sCFC.
An additional brain region contributing to our contextual fear conditioning deficits may be the cerebellum. Neonatal ethanol exposure has been shown to reduce the number of Purkinje cells in rodents [25
] and there is some evidence that sCFC is attenuated by inactivation of the cerebellar vermis [67
]. This issue has been noted in studies using the Morris water maze as a behavioral assay in neonatally exposed rodents, for mutant mice with Purkinje cell dysfunction show acquisition deficits in a spatial Morris water maze [83
]. To address the issue of cerebellar effects future studies could restrict neonatal ethanol exposure to PD7-9, a period that does not result in Purkinje cell reductions [27
The current set of experiments demonstrates reductions in contextual conditioning on two variants of contextual fear conditioning in juvenile rats neonatally exposed to a high binge dose of ethanol during early development. Further research is needed to better characterize the neural basis of these conditioning deficits, including the link between alcohol effects on hippocampus and performance on these tasks.