Ontogeny of contextual fear conditioning: behavioral, neuropharmacological and molecular mechanisms

Date
2018
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
This dissertation characterizes learning-related molecular changes in developing rodents using a variant of contextual fear conditioning called the context preexposure facilitation effect (CPFE). The CPFE is a variant of contextual fear conditioning in which acquisition of the contextual representation (preexposure phase) and association of the retrieved contextual memory with an immediate foot-shock (training phase) are separated by 24 hrs. When tested 24 hr later, animals exhibit significant fear responding to the training context relative to control animals preexposed to an alternate context (Fanselow, 1986, 1990). During the CPFE, expression patterns of the inducible transcription factor early growth response-1 gene (Egr-1) vary based on training phase and brain sub-region in adult and adolescent rats (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013; Chakraborty, Asok, Stanton, & Rosen, 2016; Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014). Importantly, regions in medial prefrontal cortex (mPFC) show a learning-related increase in Egr-1 expression on the training day, whereby immediate shock increases expression in context-preexposed rats that learn fear, relative to control groups that do not. Behavioral expression of the CPFE emerges between the infant [postnatal day (PD) 17] and juvenile (PD 24) period in the rat (Schiffino et al., 2012; Robinson-Drummer & Stanton, 2015), but it is not known whether Egr-1 expression correlates with this effect. Experiment 1 of this dissertation addressed this question by examining behavior and Egr-1 expression in infant (PD 17) and juvenile (PD 24) rats during the CPFE using preexposure protocols involving a single-exposure or multiple-exposures to the training context. Following a single, 5 min preexposure to the training context, Egr-1 expression in the mPFC, dorsal hippocampus (dHPC) and lateral nucleus of the amygdala (LA) was increased in PD 24 rats but not PD 17 rats. In contrast, increased Egr-1 expression following an immediate foot-shock (1 s, 1.5 mA) did not differ between PD 17 and PD 24 rats, and was not learning-related. Interestingly, increasing the number of exposures to the training chamber on the preexposure day (i.e. multiple-exposure protocol) altered training-day expression such that a learning-related increase in expression was observed in the mPFC in PD 31 and 24 but not PD 17 rats. However, across all experiments, behavioral expression of the CPFE was related to age-differences in Egr-1 expression on the preexposure day but not the training day. ☐ The second experiment in this dissertation used a rodent model of fetal alcohol spectrum disorder (FASD) to examine the effect of neonatal alcohol exposure on Egr-1 mRNA expression following the training day of the CPFE. Prenatal alcohol exposure in humans can adversely affect postnatal cognitive, behavioral and neural development (Del Campo & Jones, 2017; Senturias & Asamoah, 2014). Extensive human and animal research on FASD has been invaluable in elucidating the neurobiological substrates of the disorder with key regions like the hippocampus and frontal cortex being particularly susceptible to the teratogenic effects of early alcohol exposure (Fryer et al., 2007; Lebel, Roussotte, & Sowell, 2011; Norman, Crocker, Mattson, & Riley, 2009). The CPFE is a hippocampus-dependent task (G. F. Hamilton et al., 2011; Matus-Amat, Higgins, & Rudy, 2004; Matus-Amat, Higgins, Sprunger, Wright-Hardesty, & Rudy, 2007; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016) that is also disrupted by neonatal alcohol exposure in adolescent rats (Dokovna, Jablonski, & Stanton, 2013; Jablonski & Stanton, 2014; Murawski & Stanton, 2010, 2011). Experiment 2 characterized Egr-1 expression following the training day in animals preexposed to the training context (Group Pre) and non-associative control animals preexposed to an alternate context (Group Alt-pre). Rat pups were exposed to a 5.25 g/kg/day single binge-like dose of alcohol (Group EtOH) or were sham intubated (Group SI) over postnatal days PD 7-9. Animals exposed to alcohol failed to show the CPFE replicating our previous findings (Jablonski & Stanton, 2014; Murawski & Stanton, 2011). Furthermore, alcohol exposure reduced cortical Egr-1 expression following training such that the learning–related increase in Egr-1 observed in the mPFC of SI animals was abolished in Group EtOH. However, decreases were not observed in the lateral nucleus of the amygdala or the hippocampus relative to Group SI. ☐ The previous results suggest that regional molecular activity in the mPFC is a better indicator of CPFE performance than the hippocampus or amygdala. The mPFC plays a critical role in contextual fear conditioning (Corcoran & Quirk, 2007; Heroux, Robinson-Drummer, Sanders, Rosen, & Stanton, 2017; Zelikowsky et al., 2013) and develops abnormally following neonatal alcohol exposure (G. F. Hamilton, Whitcher, & Klintsova, 2010c; Whitcher & Klintsova, 2008). Additionally, impaired cholinergic function contributes to the learning impairment observed in the CPFE following alcohol exposure (Dokovna et al., 2013). The final experiment examined the specific contribution of the mPFC cholinergic system to each phase of the CPFE in normal adolescent rats (PD 31). Adolescent rats received bilateral infusions of scopolamine (35μg/side) or phosphate buffered saline (PBS) 10 min before all three phases of the CPFE or only prior to a single phase. Intra-mPFC administration of scopolamine prior to all three phases significantly impaired fear conditioning suggesting that mPFC cholinergic function is necessary for successful CPFE performance. Analyses of the individual infusion days revealed a significant impairment of the CPFE when infusions occurred prior to preexposure or training (i.e. immediate foot-shock) but not prior to testing. ☐ These studies demonstrate a clear maturation of Egr-1 expression between PD 17 and PD 24 that is both age- and experience-dependent. The data suggest that regional activity and plasticity within the mPFC on the preexposure day may contribute to impaired context representation processes in infant rats. In addition, the diminished mPFC Egr-1 expression observed following neonatal alcohol exposure on the training day likely reflects impaired memory of the context-shock association. Finally, cholinergic function in the mPFC is critical for successful performance of the CPFE and suggests a potential mechanism by which neonatal alcohol exposure may impair learning or memory in this task. Taken as a whole, this dissertation reveals the versatility of the CPFE in identifying the neurological correlates and mechanisms of contextual learning in both normal and abnormal development.
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Keywords
Biological sciences, Context fear, CPFE, FASD, Learning, Ontogeny
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