Prefrontal mechanisms of impaired cognition in a rat model of FASD

Date
2020
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Fetal Alcohol Spectrum Disorders (FASDs) represent a significant medical and societal problem in the United States and abroad, representing a leading preventable cause of severe intellectual and developmental disability. Neurodevelopmental damage resulting from fetal alcohol exposure causes a severe disruption in prefrontal and hippocampal neuroanatomy and function, and thus causes pervasive cognitive impairments in learning and memory dependent upon these structures. While alcohol-induced disruptions in hippocampal anatomy and function have been extensively explored in both rodent models and humans, rodent work examining prefrontal mechanisms of impaired cognition is sparse. Accordingly, this dissertation characterizes the neurobiological mechanisms underlying context and contextual fear learning and memory and their impairment by third-trimester equivalent alcohol exposure in a rat model of FASD. This dissertation uses a variant of contextual fear conditioning (CFC) called the Context Preexposure Facilitation Effect (CPFE). In the CPFE, learning about the context, acquiring a context-shock association, and retrieving/expressing this association is temporally dissociated across three phases (context preexposure, immediate-shock training, and retention). Third-trimester equivalent exposure from postnatal day (PD) 4-9 abolishes retention test freezing in the CPFE in adolescent and adult rats (G. F. Hamilton et al., 2011; Murawski, Klintsova, & Stanton, 2012; Murawski & Stanton, 2010). Despite this, these previous studies are unable to dissociate PD4-9 alcohol effects on preexposure or training day processes because only retention test freezing was measured. In addition, while this deficit was previously attributed to impaired hippocampal function, our lab has recently discovered that the medial prefrontal cortex (mPFC) is required during all three phases of the CPFE in adolescent rats (Heroux et al., 2017; Robinson-Drummer et al., 2017). This discovery largely motivated the experiments in this dissertation. ☐ The first aim of this dissertation characterizes the effects of prefrontal (Experiment 6.1) or ventral hippocampal (Experiment 6.2) inactivation via local muscimol infusion during context exposure on expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 in the mPFC, dorsal hippocampal (dHPC), ventral hippocampus (vHPC), and ventral midline thalamus (VMT; consisting of reunions [NR] and rhomboid nuclei) in normally-developing adolescent rats. In Experiment 6.1 and 6.2, prefrontal or ventral hippocampal inactivation via muscimol infusion during context exposure abolished subsequent post-shock and retention test freezing in behaviorally-tested littermates of the sacrificed groups. In Experiment 6.1, we found that prefrontal inactivation impaired IEG expression in the mPFC, VMT, and vHPC but not dHPC during context preexposure. In Experiment 6.2, we found that ventral hippocampal inactivation during context preexposure disrupted IEG expression in the vHPC, mPFC, and dHPC but not VMT during context preexposure. ☐ The second aim of this dissertation uses a rat model of FASD to characterize the effects of neonatal alcohol exposure from PD4-9 on regional neural activity and contextual learning and memory in the CPFE in adolescent rats. In Experiments 7.1 and 7.3, rat pups received oral intubation of alcohol (EtOH; 5.25 g/kg/day, split into two doses) or underwent sham-intubation (SI) from PD4-9 were tested on the CPFE from PD31-33. In Experiment 7.1, PD4-9 alcohol-exposed rats showed abolished post-shock and retention test freezing in the CPFE. In Experiment 7.2, alcohol-exposed rats were unimpaired in standard contextual fear conditioning, in which context and context-shock learning occurs within the same trial. This task is “prefrontal-independent” but “hippocampal-dependent,” defined by effects of loss-of-function manipulations in these structures. These data suggest that abolished post-shock freezing in the CPFE likely reflects disrupted prefrontal function supporting consolidation of the context representation. Accordingly, in Experiment 7.3, EtOH and SI rats were sacrificed 30 min after context preexposure and IEG expression in the mPFC and dHPC was analyzed via qPCR. Alcohol exposure impaired expression of the IEGs c-Fos, Arc, Egr-1, and Npas4 in the mPFC but not dHPC during context exposure. ☐ The third aim of this dissertation attempts to reverse these alcohol-induced neural and behavioral deficits by acute, pharmacological enhancement of cholinergic signaling during the CPFE. Our lab has shown that systemic administration of the acetylcholinesterase inhibitor physostigmine (PHY) prior to each phase rescues retention test freezing in PD7-9 alcohol-exposed rats (Dokovna, Jablonski, & Stanton, 2013). Whether this is also true of PD4-9 exposure and the exact phase of the CPFE that mediates this rescue and underlying neural mechanisms is unclear. Rats received oral intubation of alcohol (5.25g/kg/day) or SI from PD4-9, and then received a systemic injection of saline (SAL) or PHY (0.01mg/kg) prior to all three phases (Experiment 8.1.1) or just context exposure (Experiment 8.1.2) during the CPFE protocol from PD31-33. Administration of PHY prior to all three phases or just context preexposure rescued both post-shock and retention test freezing in EtOH rats without altering performance in SI rats. In Experiment 8.2, a subset of rats were sacrificed 30 min after context preexposure to assay changes in IEG expression in the mPFC, dHPC, and vHPC. ETOH-SAL rats had significantly reduced mPFC but not dHPC expression of c-Fos, Arc, Egr-1, and Npas4. ETOH-PHY treatment rescued mPFC expression of c-Fos in alcohol-exposed rats and increased Arc and Npas4 regardless of dosing condition. While there was no effect of PHY on dHPC or vHPC expression of Arc, Egr-1, or Npas4, this treatment significantly boosted hippocampal expression of c-Fos regardless of alcohol treatment. ☐ The results of this dissertation have broad implications for basic behavioral neuroscience and FASD research. First, these studies were the first to suggest a role of mPFC-vHPC circuitry in incidental context learning and memory during the CPFE (Heroux, Horgan, Pinizzotto, Rosen, & Stanton, 2019). These findings shed further light on prefrontal involvement in contextual processes of Pavlovian contextual fear conditioning in normally- and abnormally-developing (e.g., alcohol-exposed) rodents. Second, these studies capture prefrontal dysfunction in a rat model of FASD, and highlight the importance of re-examining cognitive deficits resulting from developmental alcohol exposure that historically has been attributed solely to hippocampal dysfunction. Finally, these studies demonstrate the efficacy of acute treatment with drugs that enhance cholinergic signaling in reversing neural and cognitive impairments seen in a rat model of FASD. These studies provide a foundation for future work examining the efficacy of similar treatments in other models of FASD and in humans.
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Keywords
Fetal Alcohol Spectrum Disorders, Hippocampus, Learning, Memory, Prefrontal cortex
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