Contextual fear conditioning emerges around post-natal day (PD) 23 in the rat. This is thought to reflect hippocampus-dependent conjunctive learning, which binds the individual features of the context into a unified representation (Rudy, 1993). However, context conditioning can also be supported by hippocampus-independent, feature-based simple associations (Rudy, 2009) and these may operate at PD23–24 (Pugh & Rudy, 1996). To address this issue, we studied the ontogeny of a variant of contextual fear conditioning, termed the context-preexposure-facilitation-effect (CPFE), in which exposure to context and (immediate) foot shock occur on successive occasions. This variant requires conjunctive as opposed to feature-based simple associations (Rudy, 2009). We tested PD17, 24, and 31 rats on the CPFE vs. conventional context conditioning (Exp. 1) and on the CPFE with stronger reinforcement (Exp. 2). The CPFE emerged on PD24 regardless of reinforcer strength and in parallel with context conditioning. Infusions of the NMDA antagonist, MK-801, into the dorsal hippocampus just before preexposure on PD24 eliminated the CPFE, whereas infusions occurring after preexposure had no effect (Exp. 3). These findings demonstrate a role of hippocampal NMDA receptors in the CPFE as early as PD24 and implicate conjunctive learning mechanisms in the ontogeny of contextual fear conditioning.

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 & O’Reilly, 2001, Cogn Affect Behav Neurosci, 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 (Exp. 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 (Exp. 2). Fear conditioning to a discrete auditory cue (tone) was unaffected by neonatal alcohol exposure ruling out possible performance effects (Exp. 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.

Background-Alcohol exposure in the rat on Postnatal Days [PD] 4-9 is known to partially damage the hippocampus and to impair hippocampus dependent behavioral tasks. We previously reported that PD4-9 alcohol exposure eliminated the context preexposure facilitation effect (CPFE) in juvenile rats, a hippocampal-dependant variant of contextual fear conditioning. In the CPFE, context exposure and immediate shock occur on successive occasions and this produces conditioned freezing relative to a control group that is not preexposed to the training context. Here we extend our earlier findings by examining the effects of neonatal alcohol administered at multiple doses or over different neonatal exposure periods.

Highlights d Acute activation of dorsal and ventral HPC engrams in mice drives reward and aversion d The ventral DG is preferentially reactivated in emotionally salient contexts d Chronic activation of HPC engrams decreases or increases context-specific freezing d Memory enhancement is disrupted when BLA cells processing fear are silenced

Developmental alcohol exposure can permanently alter brain structures and produce functional impairments in many aspects of behavior, including learning and memory. This study evaluates the effect of neonatal alcohol exposure on adult neurogenesis in the dentate gyrus of the hippocampus and the implications of such exposure for hippocampus-dependent contextual fear conditioning. Alcohol-exposed rats (AE) received 5.25 g/kg/day of alcohol on postnatal days (PD) 4-9 (third trimester in humans), in a binge-like manner. Two control groups were included: sham-intubated (SI) and suckle-control (SC). Animals were housed in social cages (3/cage) after weaning. On PD80, animals were injected with 200 mg/kg BrdU. Half of the animals were sacrificed two hours later. The remainder were sacrificed on PD114 to evaluate cell survival; separate AE, SI, and SC rats not injected with BrdU were tested for the context preexposure facilitation effect (CPFE; ∼PD117). There was no difference in the number of BrdU+ cells in AE, SI and SC groups on PD80. On PD114, cell survival was significantly decreased in AE rats, demonstrating that developmental alcohol exposure damages new cells' ability to incorporate into the network and survive. Behaviorally tested SC and SI groups preexposed to the training context 24h prior to receiving a 1.5mA 2s footshock froze significantly more during the context test than their counterparts preexposed to an alternate context. AE rats failed to show the CPFE. The current study shows the detrimental, long-lasting effects of developmental alcohol exposure on hippocampal adult neurogenesis and contextual fear conditioning.

Rats exposed to a high binge-like dose of alcohol over postnatal days (PD) 4-9 show reductions in CA1 pyramidal cells and impairments on behavioral tasks that depend on the hippocampus. We first examined hippocampal c-Fos expression as a marker of neuronal activity in normally developing rats following different phases of the context preexposure facilitation effect (CPFE) paradigm (Exp. 1). During the CPFE, preexposure to the training context facilitates contextual conditioning to an immediate shock given on a subsequent occasion. We then examined the relationship between CPFE impairment, hippocampal cell loss and c-Fos expression in rats exposed to alcohol over PD 4-9 (Exp. 2). Normally developing (Exp. 1), sham-intubated control (SI), and PD 4-9 alcohol-exposed (4.00g and 5.25g/kg/day; Exp. 2) juvenile male rats were trained on the CPFE. The CPFE occurs over three phases separated by 24h. Starting on PD 31, rats were preexposed to Context A or Context B for five minutes. 24h later, all rats received an immediate, 1.5 mA foot shock in Context A. Finally, rats were tested for contextual conditioning in Context A on PD 33. Normally developing and SI rats preexposed to Context A showed enhanced contextual fear compared to those preexposed to Context B (Exp. 1) or alcohol-exposed rats preexposed to Context A (Exp. 2). Rats were sacrificed 2h following different phases of the CPFE and processed for c-Fos immunohistochemistry (Exp. 1 and 2) and CA1 pyramidal cell quantification (Exp. 2). In Exp. 1, c-Fos+ cells in the DG were consistently high among rats preexposed to Context A (Pre), Context B (No Pre), or sacrificed directly from their home cage (Home) and did not differ across CPFE phases. CA3 and CA1 c-Fos+ cells were highest during preexposure and decreased across training phases, with Group No Pre showing greater numbers of c-Fos+ cells during training than Group Pre and Controls. In Exp. 2, SI rats had greater numbers of CA1 c-Fos+ cells compared alcohol-exposed rats, differing significantly from rats exposed to the high alcohol dose (5.25g) over PD4-9. Exp. 2 also revealed a linear decline in CA1 pyramidal cells across treatment groups, again with rats from the high alcohol dose group showing significantly fewer CA1 pyramidal cells compared to SI. Our results reveal that context novelty may be a significant contributor to differential hippocampal c-Fos expression following different phases of the CPFE. In addition, lower levels of c-Fos+ cells in alcohol-exposed rats following preexposure may be related to general reductions in the number of CA1 pyramidal cells in these rats. The significant CPFE impairments in rats exposed to the lower alcohol dose (4.00g), who show a 15% reduction in CA1 pyramidal cells compared to SI rats, highlights the sensitivity of the CPFE to hippocampal insult.

Although contextual fear conditioning emerges later in development than explicit-cue fear conditioning, little is known about the stimulus parameters and biological substrates required at early ages. The current experiments adapted methods for investigating hippocampus function in adult rodents to identify determinants of contextual fear conditioning in developing rats. Experiment 1 examined the duration of exposure required by weanling rats at postnatal day (PND) 23 to demonstrate contextual fear conditioning. This experiment demonstrated that 30 s of context exposure is sufficient to support conditioning. Furthermore, preexposure enhanced conditioning to an immediate footshock, the context preexposure facilitation effect (CPFE), but had no effect on contextual conditioning to a delayed shock. Experiment 2 demonstrated that NMDA receptor inactivation during preexposure impairs contextual learning at PND 23. Thus, the conjuctive representations underlying the CPFE are NMDA-dependent as early as PND23 in the rat.

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