Sarah A. Jablonski

Role of age, post-training consolidation, and conjunctive associations in the ontogeny of the context preexposure facilitation effect

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which context learning and context-shock associations occur on separate occasions. The CPFE with an immediate shock emerges between Postnatal Day (PND) 17 and 24 in the rat and depends on hippocampal NMDA-receptor function in PND 24 rats (Schiffino et al. [2011] Neurobiology of Learning and Memory 95(2):190-198). This study investigated this ontogenetic effect further and reports three findings: First, the CPFE is absent on PND 19 but emerges modestly in rats given exposure on PND 21. Second, the absence of the CPFE on PND 17 does not reflect inability to consolidate the context-shock association established on the training day. Lastly, the CPFE on PND 24 requires exposure to the combined features of the context. These results are the first to show that the early development of contextual fear conditioning depends on conjunctive representations and that processes underlying the CPFE begin to emerge around PND 21 in the rat.

Exercise and environment as an intervention for neonatal alcohol effects on hippocampal adult neurogenesis and learning

Neonatal alcohol exposure impairs cognition and learning in adulthood and permanently damages the hippocampus. Wheel running (WR) improves hippocampus-associated learning and memory and increases the genesis and survival of newly generated neurons in the hippocampal dentate gyrus. WR significantly increases proliferation of newly generated dentate granule cells in alcohol-exposed (AE) and control rats on Postnatal Day (PD) 42 but only control rats show an increased number of surviving cells thirty days after WR (Helfer et al., 2009b). The present studies examined whether proliferation-promoting WR followed by survival-enhancing environmental complexity (EC) during adolescence could increase survival of new neurons in AE rats. On PD 4-9, pups were intubated with alcohol in a binge-like manner (5.25g/kg/day, AE), were sham-intubated (SI), or were reared normally (suckle control, SC). On PD 30 animals were assigned to WR (PD 30-42) followed by EC (PD 42-72; WR/EC) or were socially housed (SH/SH) for the duration of the experiment. All animals were injected with 200mg/kg bromodeoxyuridine (BrdU) on PD 41. In Experiment 1, survival of newly generated cells was significantly enhanced in the AE-WR/EC group in comparison with AE-SH/SH group. Experiment 2A examined trace eyeblink conditioning. In the SH/SH condition, AE impaired trace eyeblink conditioning relative to SI and SC controls. In the WR/EC condition, AE rats performed as well as controls. In Experiment 2B, the same intervention was examined using the context preexposure facilitation effect (CPFE); a hippocampus-dependent variant of contextual fear conditioning. Again, the WR/EC intervention reversed the deficit in conditioned fear to the context that was evident in the SH/SH condition. Post-weaning environmental manipulations promote cell survival and reverse learning deficits in rats that were exposed to alcohol during development. These manipulations may provide a basis for developing interventions that ameliorate learning impairments associated with human fetal alcohol spectrum disorders.

DETERMINANTS OF NOVEL OBJECT AND LOCATION RECOGNITION DURING DEVELOPMENT

In the novel object recognition (OR) paradigm, rats are placed in an arena where they encounter two sample objects during a familiarization phase. A few minutes later, they are returned to the same arena and are presented with a familiar object and a novel object. The object location recognition (OL) variant involves the same familiarization procedure but during testing one of the familiar objects is placed in a novel location. Normal adult rats are able to perform both the OR and OL tasks, as indicated by enhanced exploration of the novel vs. the familiar test item. Rats with hippocampal lesions perform the OR but not OL task indicating a role of spatial memory in OL. Recently, these tasks have been used to study the ontogeny of spatial memory but the literature has yielded conflicting results. The current experiments add to this literature by: (1) behaviorally characterizing these paradigms in postnatal day (PD) 21, 26 and 31-day-old rats; (2) examining the role of NMDA systems in OR vs. OL; and (3) investigating the effects of neonatal alcohol exposure on both tasks. Results indicate that normal-developing rats are able to perform OR and OL by PD21, with greater novelty exploration in the OR task at each age. Second, memory acquisition in the OL but not OR task requires NMDA receptor function in juvenile rats [corrected]. Lastly, neonatal alcohol exposure does not disrupt performance in either task. Implications for the ontogeny of incidental spatial learning and its disruption by developmental alcohol exposure are discussed.

Egr-1 increases in the prefrontal cortex following training in the context preexposure facilitation effect (CPFE) paradigm

The context pre-exposure facilitation effect (CPFE) is a modified form of standard contextual fear conditioning that dissociates learning about the context during a preexposure phase from learning the context-shock association during an immediate shock training phase conducted on separate days. Fear conditioning in the CPFE is an associative process in which only animals that are preexposed to the same context they are later given an immediate shock in demonstrate freezing when tested for conditioned fear memory. Previous research has shown that the hippocampus and amygdala are necessary for different phases of the CPFE, but whether other brain regions are also involved is unknown. The present study examined expression of the immediate-early gene early growth response gene 1 (Egr-1; also called Zif268, Ngfi-a, Krox-24) in the dorsal hippocampus, lateral nucleus of the amygdala, retrosplenial cortex, and several prefrontal cortex regions (infralimbic and prelimbic medial prefrontal cortex, anterior cingulate, and orbitofrontal cortex) following each phase of the CPFE in juvenile rats. Animals preexposed to the conditioning context displayed fear conditioned freezing during a retention test whereas rats preexposed to an alternate context did not. Following context preexposure, Egr-1 mRNA was elevated in context and alternate context exposed animals compared to home-cage control rats in almost all regions analyzed. Following the context-shock training phase, fear conditioned rats displayed significantly more Egr-1 mRNA expression in the infralimbic, prelimbic, and orbitofrontal cortices compared to the alternate context preexposed control rats. These differences in Egr-1 expression were not found in amygdala between the preexposed context and alternate context rats. No sex differences were observed following preexposure or training in any regions analyzed. The findings suggest that increased expression of Egr-1 within the prefrontal cortex is associated with contextual fear conditioning in the CPFE paradigm.

Neonatal alcohol exposure impairs contextual fear conditioning in juvenile rats by disrupting cholinergic function.

The context preexposure facilitation effect (CPFE) is a variant of context fear conditioning in which context preexposure facilitates conditioning to immediate foot shock. Learning about context (preexposure), associating the context with shock (training), and expression of context fear (testing) occur in successive phases of the protocol. The CPFE develops postnatally, depends on hippocampal NMDA receptor function, and is highly sensitive to neonatal alcohol exposure during the weanling/juvenile period of development [15,16]. The present study examined some behavioral and pharmacological mechanisms through which neonatal alcohol impairs the CPFE in juvenile rats. We found that a 5-min context preexposure plus five 1-min preexposures greatly increases the levels of conditioned freezing compared to a single 5-min exposure or to five 1-min preexposures (Experiment 1). Increasing conditioned freezing with the multiple- exposure CPFE protocol does not alter the neonatal alcohol-induced deficit in the CPFE (Experiment 2). Finally, systemic administration of 0.01 mg/kg physostigmine prior to all three phases of the CPFE reverses this ethanol-induced deficit. These findings show that impairment of the CPFE by neonatal alcohol is not confined to behavioral protocols that produce low levels of conditioned freezing. They also support recent evidence that this impairment reflects a disruption of cholinergic function [18].

Effects of exercise and environmental complexity on deficits in trace and contextual fear conditioning produced by neonatal alcohol exposure in rats

In rodents, voluntary exercise and environmental complexity increases hippocampal neurogenesis and reverses spatial learning and long-term potentiation deficits in animals prenatally exposed to alcohol. The present experiment extended these findings to neonatal alcohol exposure and to delay, trace, and contextual fear conditioning. Rats were administered either 5.25 g/kg/day alcohol via gastric intubation or received sham-intubations (SI) between Postnatal Day (PD) 4 and 9 followed by either free access to a running wheel on PD 30-41 and housing in a complex environment on PD 42-72 (wheel-running plus environmental complexity; WREC) or conventional social housing (SHSH) from PD 30 to 72. Adult rats (PD 80 ± 5) received 5 trials/day of a 10-s flashing-light conditioned stimulus (CS) paired with .8 mA footshock either immediately (delay conditioning) or after a 10-s trace interval (trace conditioning) for 2 days. Neonatal alcohol exposure impaired context and trace conditioning, but not short-delay conditioning. The WREC intervention did not reverse these deficits, despite increasing context-related freezing in ethanol-exposed and SI animals.

Role of medial prefrontal NMDA receptors in spatial delayed alternation in 19-, 26-, and 33-day-old rats

Long-Evans rats were trained on spatial delayed alteration (SDA) in a T-maze following medial prefrontal cortical (mPFC) infusions of different doses of the noncompetitive NMDA-receptor antagonist, MK-801 (.125 microl; .25 microl; or .25 microlsaline, bilaterally), on postnatal day (PND) 19, 26, or 33. Pups trained on PND 19 showed almost no learning of SDA, regardless of drug condition (including saline). On PND 26, both doses of MK-801 significantly and equivalently prevented SDA learning, with performance during the final three training blocks remaining near chance levels, in contrast with 85% correct performance in the saline control group. On PND 33, substantial SDA learning was evident regardless of dose, although a modest impairment appeared in mid-training at both doses. These findings confirm previous reports of mPFC involvement in the early postnatal ontogeny of SDA and suggest a developmentally transient role of mPFC NMDA-receptor function in this task.

Egr-1 mRNA expression patterns in the prefrontal cortex, hippocampus, and amygdala during variants of contextual fear conditioning in adolescent rats

We report activation of the immediate-early gene Egr-1 in the lateral amygdala (LA), hippocampus (CA1), and medial prefrontal cortex (mPFC) 30-min following the training phase in the context pre-exposure facilitation effect (CPFE) and standard context fear conditioning (180 s context exposure→shock). On day one of the CPFE paradigm, postnatal day (PD) 31 rats (±1) were pre-exposed to Context A (Pre) or Context B (Alt-Pre) for 5 min followed by five additional 1-min exposures. A day later, Pre and Alt-Pre rats received a 2-s, 1.5 mA footshock immediately upon placement in Context A. Animals included in in situ hybridization were then sacrificed 30 (±3) min later. On day three, the behaviorally-tested Pre rats showed significantly more fear-conditioned freezing in Context A than Alt-Pre rats. Standard context fear conditioning groups showed much greater freezing than the Pre group, as well as no shock and immediate-shock controls. Thirty minutes after immediate shock training, Pre rats showed increased Egr-1 mRNA in the prelimbic mPFC relative to Alt-Pre rats. Standard context conditioning selectively increased Egr-1 in CA1. In the LA and mPFC, Egr-1 increased to a similar extent in no shock, immediate shock, and standard context conditioning relative to homecage controls. The present study demonstrates that Egr-1 mRNA expression has a complex relationship to fear learning in different brain regions and variants of context conditioning.

Impairment of the context preexposure facilitation effect in juvenile rats by neonatal alcohol exposure is associated with decreased Egr-1 mRNA expression in the prefrontal cortex.

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which learning about the context (preexposure) and associating the context with a shock (training) occur on separate occasions. The CPFE is sensitive to a range of neonatal alcohol doses (Murawski & Stanton, 2011). The current study examined the impact of neonatal alcohol on Egr-1 mRNA expression in the infralimbic (IL) and prelimbic (PL) subregions of the mPFC, the CA1 of dorsal hippocampus (dHPC), and the lateral nucleus of the amygdala (LA), following the preexposure and training phases of the CPFE. Rat pups were exposed to a 5.25 g/kg/day single binge-like dose of alcohol (Group EtOH) or were sham intubated (SI; Group SI) over postnatal days (PD) 7–9. In behaviorally tested rats, alcohol administration disrupted freezing. Following context preexposure, Egr-1 mRNA was elevated in both EtOH and SI groups compared with baseline control animals in all regions analyzed. Following both preexposure and training, Group EtOH displayed a significant decrease in mPFC Egr-1 mRNA expression compared with Group SI. However, this decrease was greatest after training. Training day decreases in Egr-1 expression were not found in LA or CA1 in Group EtOH compared with Group SI. A second experiment confirmed that the EtOH-induced training-day deficits in mPFC Egr-1 mRNA expression were specific to groups which learned contextual fear (vs. nonassociative controls). Thus, memory processes that engage the mPFC during the context-shock association may be most susceptible to the teratogenic effects of neonatal alcohol.