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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Neurobiol Learn Mem. 2011 Apr 14;96(2):166–172. doi: 10.1016/j.nlm.2011.04.003

Norepinephrine Mediates Contextual Fear Learning and Hippocampal pCREB in Juvenile Rats Exposed to Predator Odor

Patricia A Kabitzke 1, Lindsay Silva 2, Christoph Wiedenmayer 3
PMCID: PMC3148269  NIHMSID: NIHMS289528  PMID: 21513808

Abstract

Predator odors induce unconditioned fear in the young animal and provide the opportunity to study the mechanisms underlying unlearned and learned fear. In the current study, cat odor produced unlearned, innate fear in infant (postnatal age 14; PN14) and juvenile (PN26) rats, but contextual fear learning occurred only in juveniles. It was hypothesized that contextual fear learning in juveniles is mediated by norepinephrine. Consistent with this hypothesis, pretraining injection of the β-adrenergic antagonist propranolol reduced the unlearned fear response while posttraining injection inhibited contextual fear learning in juvenile rats exposed to cat odor. We suggest that NE mediates the formation of contextual fear memories by activation of the transcription factor CREB in the hippocampus in juveniles but not in infants. Levels of phosphorylated CREB (pCREB) were increased in the dorsal and ventral hippocampi of juvenile rats exposed to cat odor. These levels were not increased in infants or juveniles exposed to a control odor. Further, propranolol blocked these increases in pCREB. In conclusion, although innate fear occurs within the neonatal period, contextual fear learning is a relatively late-occurring event, is hippocampal dependent, and mediated by norepinephrine.

1. Introduction

Contextual fear conditioning is adaptive under natural conditions. Animals that remember specific locations in which predator odors were encountered could increase their survival and subsequent fitness by treating those areas with caution in the future. Indeed, live cat and natural cat fur or skin odors have been found to be strong unconditioned stimuli for contextual fear conditioning in adult rats – altering their behaviors in the conditioned context for long periods of time (Blanchard and Blanchard, 1989; Blanchard et al., 2001). However, fear conditioning in early life differs from fear learning in adulthood as animals fail to form shock-induced contextual fear memories until postnatal day 23 (PN23) (Rudy and Morledge, 1994). It is not clear at what age young animals form contextual fear memories of predator odor. In their life history, young rats become more mobile after weaning and begin to venture from the natal burrow (Barnett, 1958; Bolles and Woods, 1964). Contextual learning ability may be critical to their survival since the location where a threat was encountered is a reliable predictor for future threat. In contrast, the infant rat is typically confined to the nest with minimal locomotor activity (Nowak, 1999). Therefore, the infant is under little selection pressure to learn about the context. Consequently, brain circuits may not be mature at this age. In one of the few experiments using young rats (PN18, 26, and 38), a cat-rubbed or odorless cloth-covered block was placed in the experimental chamber during training and animals were returned to the context containing the odorless cloth-covered block during testing (Hubbard et al., 2004). This experiment showed that rats as young as PN26 could learn from predator odor cues but it remained unclear if this learning was contextual or cued as the block likely was a cue.

Several neurotransmitter systems have been implicated in fear learning (Davies et al., 2004) (for review, see Benarroch, 2009). In rats, the central release of norepinephrine (NE) and its subsequent effect on β-adrenergic receptors within the brain plays a critical role in unlearned and conditioned fear (Do Monte et al., 2008) and selectively modulates contextual memories (Murchison et al., 2004; Wilson et al., 2004). Two areas, the amygdala and the hippocampus, are necessary for contextual fear conditioning in adult animals (LeDoux, 2000). When an animal is faced with a severe threat, like a predator, NE is released from the locus coeruleus (LC) and increases general neuronal excitability (Joels et al., 2007). As the animal focuses attention on gathering sensory information about the stimulus and context (Berridge and Waterhouse, 2003; Takahashi et al., 2008), NE may be involved in contextual fear acquisition. Memory consolidation, occurring within a few minutes to a few hours after training, requires protein synthesis to support new or reconfigured synaptic connections (Dudai, 2004). NE is one factor that promotes protein synthesis through binding to the amygdala (McGaugh and Roozendaal, 2002; Kogan and Richter-Levin, 2008). However, activation of the β-adrenergic receptors in the hippocampus could also be critical to long-term contextual fear memory. Indeed, blocking these receptors in CA1 disrupts contextual fear memory consolidation (Ji et al., 2003). It has been hypothesized that young animals do not show contextual fear-conditioning to shock because the neural substrates that underlie this ability are late to mature (Rudy, 1993; Rudy and Morledge, 1994). The amygdala is functional in early life as infant rats demonstrate amygdala-dependent cued conditioning by PN10 (Sullivan et al., 2000a). It is likely the hippocampus that is immature in infant rats but whether it mediates contextual learning early in life has not been tested.

If blocking the effects of NE prevents protein synthesis in the hippocampus then this would be evidence for the role of the hippocampus in NE-mediated contextual fear memory. In its active state, phosphorylated cAMP response element binding protein (pCREB) binds to DNA through cAMP response elements (CRE), turns on the transcription of genes leading to the expression of new proteins (Thonberg et al., 2002). These proteins are important for the cellular changes associated with long-term memory (LTM) (Rodrigues et al., 2004; Carlezon et al., 2005; Josselyn and Nguyen, 2005). pCREB is elevated in hippocampal CA1 cells after contextual fear conditioning to shock in adult mice (Sindreu et al., 2007). pCREB is also important for learning in young animals. For example, in rat pups (PN6), pCREB mediates cued odor learning in the olfactory bulb when an odor is paired with stroking (McLean et al., 1999). pCREB levels have been successfully reduced by blocking the actions of NE with the β-adrenergic antagonist propranolol. Although propranolol blocks contextual fear memory when injected systemically or into the CA1 region of the hippocampus in both shock and predator odor paradigms (Ji et al., 2003; Do Monte et al., 2008), the consequences of propranolol administration on hippocampal pCREB remain unknown. Furthermore, how hippocampal CREB is related to long-term contextual fear learning across early development has not yet been investigated.

The goal of the present study was to examine the mechanisms underlying predator-induced contextual fear learning in early life. To evaluate these mechanisms, the role of NE in the unlearned and learned fear response and the effects of NE blockade on hippocampal pCREB were assessed.

2. General methods

All procedures were approved by the New York State Psychiatric Institute Institutional Animal Care and Use Committee (Protocol #470).

2.1. Animals

All experiments used wild-type Long-Evans hooded rats bred in the animal care facility of Columbia University and housed under standard laboratory conditions. Cages were monitored daily in the morning and evening for the presence of newborn pups and the date of birth was considered as day 0. Rats were weaned on PN23 and littermates were kept together in the same cage. The infant animals were trained on PN14 and the juvenile animals PN26. For all behavioral experiments both female and male animals were used since previous work found no sex differences in threat-induced freezing in infant and juvenile rats (Wiedenmayer and Barr, 1998).

2.2. Apparatus

In order to test odor stimuli for different ages of rats, an acrylic box with a hinged top lid and internal width and height of 15.25 × 20.25 cm was used. The box contained a movable divider with a small opening that allowed access to the hide box. The total length of the apparatus was 18 cm for infants and 36 cm for juveniles, 3 times the approximate lengths of animals at these ages. The hide box accounted for the furthest third of the total distance from the odor port, at the opposite end of the apparatus. The wall of the hide box facing the camera was clear to allow for observation when the animal was inside. Since volatility is not a feature of cat fur odor (Dielenberg and McGregor, 1999), the odor port was at the opposite end of the chamber to create a gradient of odor intensity across the apparatus. The size of the hide box was 6 × 4 × 4 cm for infants and 12 × 4 × 4 cm for juveniles. The chamber and hide box were cleaned with alcohol before each use.

2.3. Odor stimuli

The odor stimulus was delivered via an odor box that was affixed over the odor port on the outside of the apparatus. The stimulus consisted of either the cat odor or approximately 0.1mL of pure essential lavender oil (Imperial Drug & Spice Corp., West New York, NJ) presented on kimwipe. The unlearned response of infant and juvenile rats to scatological cat odor cues (soiled cat litter) have been described (Wiedenmayer et al., 2005) but in the present experiment, a mixed cat fur odor stimulus was used that may more closely mimic the actual presence of a cat (Blanchard et al., 2003). Pilot work revealed that both infant and juvenile rats would freeze to a cat odor cue of polyester Berber fleece that had been used as a bed for cats in the colony for approximately 12 months and contained a mix of cat fur, skin oils, saliva, and waste materials. The fleece was cut into squares of approximately 5 × 7.5 cm and stored at −80 degrees Celsius. Lavender oil was used as the control since pilot studies showed that rats do not display fear behaviors to this odor and to control for nonspecific effects in response to a novel odor.

2.4. Behavioral measures

In response to cat odor, rats’ commonly observed pattern of behavior is to terminate exploratory and grooming behaviors, retreat to a location of lower danger (i.e., a hide box) and then engage in freezing and ‘head out’ behavior, which decreases conspicuousness and allows supervision of the threat, respectively (Blanchard and Blanchard, 1989; Dielenberg and McGregor, 2001). Such an escape may be more analogous to natural environmental conditions since rodents are thigmotaxic and tend to operate from a defensible position (Dielenberg et al., 1999; Whishaw et al., 2006). In all experiments a hide box was used and freezing behavior was measured. Freezing, or complete immobility, is a common fear response of adult rats encountering a cat or cat odor in the laboratory (Blanchard and Blanchard, 1971; Hubbard et al., 2004). Although predator exposure induces other behaviors (i.e. avoidance, risk-assessment) freezing behavior is the most commonly used measure of fear.

Behavior was recorded by a Panasonic PV-GS300 digital video camcorder mounted next to the chamber. Videos were transferred to a computer hard-drive then analyzed with The Observer XT (Noldus Information Technology) and screened by an observer unaware of the experimental conditions. Whenever the animal was completely immobile, including from within the hide box, a key was manually pressed on the Noldus keyboard. The total duration of freezing during odor exposure was quantified by the Noldus Observer XT software.

2.5. Procedures

2.5.1. Experiment 1: the unlearned fear response and contextual fear learning to natural predator odor in early ontogeny

The experiment consisted of 3 sessions, each separated by 24hrs: preexposure to context, odor conditioning, and contextual testing. On each of the 3 days, the lights in the behavioral testing room were dimmed in order to reduce light-enhanced aversion. During the first session, a single infant (PN14) or juvenile (PN26) rat was removed from the home cage and placed in the center of the apparatus for five minutes. Conditioned freezing is enhanced when animals have been previously preexposed to the context (Pugh and Rudy, 1996). After preexposure, the tail was marked with permanent ink and the animal was placed in a cage containing home bedding and a littermate. Animals were separated from the home cage until all animals for the session had been run. In order to conserve body heat, infant animals were placed on a heating pad prior to returning to the home cage.

During odor conditioning the next day, each animal was placed in the apparatus and allowed to habituate for two minutes with no odor stimulus present. Cat odor (material that had been used as a cat bed) or the control odor (lavender oil) was then presented via the odor port to the animal in the chamber for five minutes.

Twenty-four hours after training, animals were put back in the chamber for five minutes of contextual testing. To rule out the possibility of between-context generalized fear (as discussed by (Fanselow, 1980) and as a control to test for associative contextual learning, half of the juveniles were tested in a novel context that differed from the original context in color, size, and floor substrate. The order of stimulus conditions and the sex of animals were counterbalanced across litters.

Delay conditioning was used since it is most effective for contextual fear conditioning (Burman and Gewirtz, 2004). A one-trial learning paradigm was used because, from an ethological standpoint, even a single experience with a potent predator stimulus would represent enough of a threat to promote rapid, long-lasting changes in behavior (Wiedenmayer, 2004).

2.5.2. Experiment 2: effects of systemic injection of propranolol on contextual fear memory acquisition and consolidation

Procedures for Experiment 2 were identical to Experiment 1 except that either a propranolol solution (20 mg/kg) or 0.9% saline was intraperitoneally (i.p., 30-guage needles) injected into juveniles 30 minutes before exposure or immediately following exposure. This dose of propranolol has been shown to be effective in young animals (Sullivan et al., 1989; Sullivan et al., 2000b; Weber et al., 2003; Harley et al., 2006) and pilot work done in our lab confirmed that this was an effective dose in our experimental paradigm. We included pre-training injections since this schedule been used in other studies (e.g. Sullivan et al., 1989; Sullivan et al., 1991; Adamec et al., 2007) to examine the effects of the drug on conditioned tasks. The post-training injection schedule was used since there exists a period for effective propranolol administration, soon after training, when β-adrenergic activity is at its highest (Ji et al., 2003). Wilson (1994) found that i.p. injections of propranolol were effective to block odor preference in pups when administered immediately and 60 minutes, but not at 4 hours, after training. In order to rule out peripheral effects of NE on the contextual learning, juvenile rats were systemically injected with the β-adrenergic receptor antagonist nadolol, which does not cross the blood-brain barrier. When the nadolol solution (10 mg/kg) was used, it was injected immediately following odor exposure since this dose and timing was effective in studies with adult rats (Colussi-Mas et al., 2005; Do Monte et al., 2008). The experimental schedule otherwise remained the same but the only stimulus used was cat odor was used as the comparison of interest was between nadolol and saline.

2.5.3. Experiment 3: effects of predator odor cues and propranolol on hippocampal pCREB

Unlike the behavioral experiments, in Experiment 3, only male rats were used to control for potential neonatal sex differences in CREB phosphorylation (Auger et al., 2001). The first aim of the experiment compared infant and juvenile rats and followed training procedures outlined in Experiment 1. The second aim of the experiment compared juveniles i.p.-injected with either propranolol solution (20 mg/kg) or 0.9% saline and followed training procedures outlined in Experiment 2.

Animals were injected intraperitoneally with an overdose of nembutal (pentobarbital sodium, Abbott Laboratories, North Chicago, IL) and sacrificed by decapitation 60 minutes after odor exposure. It has previously been reported that peak CREB phosphorylation occurs 10–360 minutes after odor-shock pairing (Zhang et al., 2003). DH and VH sections were rapidly collected on cold plate (−16°C) and immediately frozen. Although β-adrenergic receptors are found in CA1 and CA3 and both of these regions are present in the DH and VH, they were processed separately since the region of the hippocampus that is involved in contextual conditioning remains under investigation. All tissue samples are stored at −80° C until tissue homogenization. For homogenization, tissue was lysed in cold buffer containing 0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 2 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5mM PMSF, 100X protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 100X phosphotase I and II inhibitor cocktails (Sigma-Aldrich) until a uniform solution was achieved. The homogenates were centrifuged for 15 min at 14000 rpm at 4°C, pellets were discarded, and protein concentrations of each sample were measured by Bradford Assay (BioRad, Hercules, CA).

CREB, pCREB, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH – a housekeeping gene) in DH and VH were measured by western blot. Samples containing 50 μg of protein per lane were loaded on 10% SDS-PAGE gels and transferred by electroblotting to Immobilon-P (polyvinylidene difluoride) transfer membranes (Millipore, Bedford, MA). To probe for pCREB, membranes were pretreated with PBS 3% milk buffer and incubated overnight at 4°C in anti-phosphorylated CREB (1:1000, Milipore). Membranes were washed, treated with a secondary HRP-labeled goat anti-rabbit antibody (1:4000, Milipore) and HRPlinked anti-biotin (1:2000, Cell Signaling) for 90 min, and washed again. Membranes were then developed with ECL detection reagents (GE Healthcare) and exposed to ECL Hyperfilm (GE Healthcare). Membranes were then stripped in buffer containing 10% SDS, 0.5 M Tris-CL (pH 6.8), and 14.3 M β-ME and re-probed for CREB. To probe for CREB, membranes were pretreated with TBS-T (0.05%) 3% milk buffer and incubated overnight at 4°C in anti-CREB (1:1000, Cell Signaling). Membranes were washed, treated with a secondary HRP-labeled goat anti-rabbit antibody (1:4000, Milipore) for 60 min, and washed again. After ECL detection, membranes were again blocked and reprobed for GAPDH. To probe for GAPDH, membranes were pretreated with TBS-T (0.1%) 5% milk buffer and incubated overnight at 4°C in anti-GAPDH (1:2000, Cell Signaling). Membranes were washed, treated with a secondary HRP-labeled goat anti-rabbit antibody (1:4000, Milipore) for 60 min, and washed again. Membranes were then developed and exposed as previously described. Membranes were stripped between probing since the molecular weights of pCREB (43 kDa), CREB (43 kDa), and GAPDH (37 kDa) are within too narrow a range to be run simultaneously.

2.6. Data Analysis

When a significant main effect was found without a significant interaction in two-way factorial analyses of variance (ANOVA), the Tukey HSD procedure was used to control for Type 1 error across the pairwise comparisons. When a significant interaction was found, follow-up tests were conducted to evaluate the two pairwise differences among the means with alpha set at .0125 (.025/2 = .0125) to control for Type I error. An independent samples t-test was used in the nadolol experiment.

For western blot films, quantitative densitometric analysis was performed using ImageJ sofware. Normalized pCREB was calculated by normalizing optical densities of pCREB and CREB to GAPDH, then dividing pCREB by CREB. This value was then divided by the average density of the control condition. All statistical tests were performed using SPSS (v. 16 for Windows, Chicago, IL) and all graphs were generated using GraphPad Prism (v. 5 for Windows, San Diego, CA).

3. Results

3.1 Experiment 1

3.1.1. Cat odor is a highly-effective fear-inducing stimulus across early ontogeny

Both infants and juveniles froze significantly more when exposed to cat odor compared to the control odor (significant main effect for stimulus: F(1, 20) = 28.42, p < .001) (Fig. 1A). There was no effect of age (F(1, 20) = 0.02, p > .05) as well as no interaction between the stimulus and age on freezing (F(1, 20) = 0.00, p > .05) (Fig. 1A).

Figure 1.

Figure 1

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Cat odor is a highly-effective fear-inducing stimulus across early ontogeny and supports contextual fear conditioning in juveniles. (A) Mean and SEM percent time freezing during training in Experiment 1. (B) Juvenile, but not infant, rats exhibit fear memory when returned to the context 24 hours after exposure to the cat odor. When juveniles were tested in a new context 24 hours after exposure to the cat odor, they did not display an increase in freezing. Mean and SEM percent time freezing during contextual test in Experiment 1 (*** p < .001, significantly different from every other group; N = 6 per group).

3.1.2. Juvenile, but not infant, rats exhibit fear memory when returned to the same context 24 hours after exposure to the cat odor

Upon return to the context the following day, juveniles previously exposed to the cat odor froze significantly more than any other group (significant 2 × 2 interaction: F(1, 20) = 6.51, p < .05). Juvenile animals that had been exposed to cat odor froze significantly more than juveniles that had been exposed to the control odor (p < .001) and more than infants that had been exposed to cat odor (p < .001) (Fig. 1B). Importantly, juveniles that had been exposed to cat odor froze more when tested in the same context than juveniles that had been exposed to cat odor and tested in a new context (p < .001) (Fig. 1B). There were no significant differences in freezing in infant animals previously exposed to either cat or the control odor when they were returned to the context the following day nor did infants differ from juveniles that had previously been exposed to the control odor. There were additionally no significant differences in freezing in juvenile animals previously exposed to either cat or the control odor when they were placed in a new context the following day nor were there differences between the two contexts when they had previously been exposed to the control odor. The role of NE in this ability at this age was addressed in the next experiment.

3.2 Experiment 2

3.2.1. Pretraining injection of the b-adrenergic antagonist propranolol blocks the unlearned fear response and contextual fear learning to cat odor in juveniles

Juveniles injected with propranolol before exposure to cat odor froze significantly less than vehicle-injected animals during that odor training (significant 2 × 2 interaction: F(1, 20) = 9.81, p < .01) and during testing the next day (significant 2 × 2 interaction: F(1, 20) = 6.82, p < .05) (Fig. 2A). There were no significant differences in freezing between animals injected with propranolol before exposure to either the cat or the control odor during training and testing. Animals injected with the vehicle before exposure to cat odor froze significantly more than animals injected with the vehicle before exposure to the control odor during training (p < .001) and testing (p < .01) (Fig. 2A). There were no significant differences in freezing in animals injected with either propranolol or the vehicle before exposure to the control odor during training and testing.

Figure 2.

Figure 2

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The b-adrenergic antagonist propranolol effectively blocks juveniles’ unlearned and learned fear response to cat odor. (A) Pretraining injection of propranolol attenuated freezing to cat odor during training and freezing to the context during testing in Experiment 2. Rats were injected with 20 mg/kg of propranolol or the vehicle 30 min prior to exposure (*** p < .001, significantly different from every other group; N = 6 per group). (B) Posttraining injection of propranolol blocks contextually conditioned fear in juveniles. The peripheral b-adrenergic antagonist nadolol did not affect contextual fear conditioning. Mean and SEM percent time freezing during training and contextual test in Experiment 2. Rats were injected with 20 mg/kg of propranolol, 10 mg/kg nadolol or vehicle immediately following training and tested 24 h later (*** p < .001, significantly different from every other group; N = 6 per group).

3.2.2. The b-adrenergic antagonist propranolol effectively blocks the consolidation of long-term fear memory in juveniles

Animals injected with propranolol after exposure to cat odor froze significantly less than animals injected with the vehicle after exposure to cat odor (significant 2 × 2 interaction: F(1, 20) = 28.88, p < .001) (Fig. 2B). There were no significant differences in freezing in animals injected with propranolol after exposure to either the cat or the control odor. Animals injected with the vehicle after exposure to cat odor froze significantly more than animals injected with the vehicle after exposure to the control odor (p < .001) (Fig. 2B). There were no significant differences in freezing between animals injected with either propranolol or the vehicle after exposure to the control odor.

3.2.3. The peripheral β-adrenergic receptor antagonist nadolol does not block the expression of long-term fear memory in juveniles

Juvenile rats were systemically injected after cat odor exposure with the peripheral β-adrenergic receptor antagonist nadolol, which does not cross the blood-brain barrier (Colussi-Mas et al., 2005; Do Monte et al., 2008). During contextual testing, the nadolol-injected animals froze at the same rates as cat odor-exposed, saline-injected animals (nadolol: M = 38.52, SEM = 4.21; vehicle: M = 37.98, SEM = 8.26; t(10) = −0.06, p > .05) (Fig. 2B). These levels of freezing were similar to the other groups that had exhibited contextual fear learning.

3.3. Experiment 3

3.3.1. Juveniles exhibited a significant increase in CREB phosphorylation in both the dorsal (DH) and ventral hippocampus (VH) after they were exposed to cat odor than when they were exposed to the control odor

A 2 × 2 interaction (DH: F(1, 20) = 9.70, p < .01; VH: F(1, 20) = 5.92, p < .05) revealed that juveniles exhibited significantly higher pCREB levels in both the DH (p < .001) and VH (p < .01) after they were exposed to cat odor than after exposure to the control odor and compared to infants exposed to cat odor (DH: p < .001; VH: p < .01) (Fig. 3A). There were no significant differences in pCREB levels in infants after exposure to either the cat or the control odor or between juveniles and infants exposed to the control odor.

Figure 3.

Figure 3

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Juveniles exhibited a significant increase in hippocampal CREB phosphorylation after cat odor exposure and this effect was blocked by propranolol. (A) Juvenile, but not infant, rats had increased levels of pCREB in both the dorsal (DH) and ventral hippocampus (VH) after they were exposed to cat odor compared to the control odor. Mean and SEM normalized pCREB levels in DH and VH in Experiment 3. (B) Propranolol blocked pCREB increases in the DH and VH of juveniles exposed to cat odor. Rats were injected with 20 mg/kg of propranolol or the vehicle immediately following training (** p < .01, *** p < .001, significant differences in pCREB in cat odor-exposed, vehicle-injected animals from all other groups in the DH and from cat odor-exposed, propranolol-injected and control odor-exposed, vehicle-injected animals in the VH; N = 6 per group). A representative western blot film is shown for each age and condition above the corresponding bar.

3.3.2. Propranolol blocked the increase in CREB phosphorylation in both the DH and VH of juveniles after they were exposed to cat odor

There was a 2 × 2 interaction (DH: F(1, 20) = 12.92, p < .01; VH: F(1, 20) = 6.98, p < .05), in that vehicle-injected animals exhibited significantly increased pCREB levels in both the DH and VH after they were exposed to cat odor compared to cat odor-exposed propranolol-injected animals (DH: p < .001; VH: p < .001) or when they were exposed to the control odor (DH: p < .001; VH: p < .01) (Fig. 3B). There were no significant differences in pCREB levels in propranolol-injected animals after exposure to either the cat or the control odor. There were no significant differences in pCREB levels in vehicle- and propranolol-injected animals following exposure to the control odor.

4. Discussion

These experiments examined the role of the noradrenergic system and CREB in hippocampal-dependent contextual fear learning during early ontogeny. Results can be summarized as follows. Natural predator odor elicited freezing, an unlearned fear response, in both infant and juvenile rats. However contextual fear learning, shown by increased freezing 24 hours later, was only observed in juveniles. The juveniles tested in a context different than the training context also did not freeze, demonstrating that freezing was the result of the contextual conditioning to the specific test context and not an artifact of a generalized or sensitized fear response following exposure to cat odor. Pre-training systemic injection of the β-adrenergic antagonist propranolol reduced the unlearned fear response and prevented fear memory acquisition. Additionally, propranolol injections following fear conditioning prevented consolidation in juveniles. Post-training injection of nadolol did not reduce conditioned freezing, indicating that the central, not peripheral, release of NE is critical to contextual fear conditioning. pCREB, a necessary transcription factor for LTM formation, was elevated in the hippocampi of juveniles, but not of infants, exposed to the cat odor in the third experiment. This increase was blocked by systemic injection of propranolol, indicating that NE is required for CREB phosphorylation. Taken together, these results suggest that centrally-released NE facilitates contextual fear conditioning by inducing CREB phosphorylation in juvenile rats. The absence of a NE-pCREB pathway is but one of many possible mechanisms contributing to the impaired learning in infant rats.

Our results are consistent with the hypothesis that β-adrenergic receptor activation contributes to long-term memory consolidation. We did not test short-term memory (STM) for contextual fear. However, Ji et al. (2003) found no evidence that propranolol impaired short-term contextual fear memory when it was injected directly into the hippocampus following conditioning. Thus, β-adrenergic blocking did not affect STM, but it did affect LTM as there were significant decreases in freezing in the propranolol-injected animals when they were re-tested after 24 hours (Ji et al., 2003).

It has been suggested (see Ji et al., 2003) that β-adrenergic receptor activation might influence memory consolidation by initiating intracellular signaling cascades. When NE binds to β-adrenergic receptors, a signal transduction cascade triggers the second messenger cyclic adenosine monophosphate pathway (cAMP) that activates protein kinase A (PKA) (McLean and Harley, 2004). PKA enters the cell nucleus and turns on the transcription factor CREB (McLean and Harley, 2004). In infants, some factor between NE binding and CREB phosphorylation may not be functional. Alternatively, the absence of change in hippocampal pCREB levels could be due to decreased NE release or binding or to a lack of presynaptic plasticity (Dumas, 2005). However, NE binding in the hippocampus occurs as early as PN3 in rats (Teicher et al., 1986) and infant rats (PN10) express hippocampal pCREB (Bender et al., 2001). Although amygdalar activation is critical for the formation of long-term contextual fear memory (McGaugh and Roozendaal, 2002; Kogan and Richter-Levin, 2008), activation of the β-adrenergic receptors in the hippocampus could also be critical for this type of learning. Perhaps the most important contribution of our experiments is they support this idea as (a) contextual fear conditioning induced pCREB in juvenile but not infant rats and (b) the systemic propranolol injection reduced both pCREB in the hippocampus and prevented contextual fear.

pCREB activity was increased in both the DH and VH, supporting the hypothesis that contextual conditioning requires both areas (Otto and Poon, 2006). Differences in DH function in contextual conditioning may be US-dependent. DH lesioning does not affect contextual conditioning to predator odor as it does with shock (Pentkowski et al., 2006). Hippocampal field may be more critical than dorsal-ventral subdivision as CA3 is involved in contextual memory acquisition (Lee and Kesner, 2004; Daumas et al., 2005) and CA1 in consolidation (Daumas et al., 2005).

It remains unknown if pCREB levels in the amygdala of young animals during contextual fear memory consolidation are elevated as they are in adults (Hubbard et al., 2007) and responsive to NE manipulation. Our preliminary data suggest that pCREB is elevated in the BLA of infants exposed to cat odor and that this increase is blocked with propranolol. Since both the hippocampus and amygdala are densely populated with β-adrenergic receptors (Do Monte et al., 2008; Qu et al., 2008), NE may promote contextual learning by binding in either site. However, propranolol is effective in reducing fear memory consolidation associated with a context but not with a cue (Ji et al., 2003; Debiec and Ledoux, 2004; Grillon et al., 2004; Qu et al., 2008), which suggests that NE critically affects the hippocampus in this type of learning. It is possible that changes in hippocampal pCREB are mediated not by NE binding to the hippocampus directly, but via the amygdala. Although a limitation of the current study is causality between propranolol administration and the reduction of hippocampal pCREB, other studies have shown that infusion of propranolol into CA1 interferes with contextual fear learning in adult mice and rats (Ji et al., 2003). These findings support the central hypothesis that activation of the noradrenergic system supports contextual fear learning by elevating levels of hippocampal pCREB, although future research should further investigate the role of NE in contextual odor fear learning in the hippocampus and amygdala via local injection. Additionally, elements that are potentially missing in the molecular cascade between the binding of NE and pCREB level in infant hippocampi should be explored.

Acknowledgments

I would like to thank my mentor, Christoph Wiedenmayer, who passed away during the final preparation of this manuscript. His scientific curiosity and generosity were an inspiration to many. Others who contributed to this work were students Benjamin Ragen, Anastasiya Mararenko, and Faye Korich who assisted in scoring. Alexei Shemyakin, Thomas Chann, and Brian Davis provided invaluable support in the lab. Maria Milekic and Michelle Riley spent countless hours with me to develop the western blot protocol. Lastly, thanks to Gordon Barr, Robert Ristuccia, Christoph Kellendonk, Peter Balsam, and Harry Shair who provided thoughtful feedback for this paper.

Footnotes

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Contributor Information

Patricia A. Kabitzke, New York State Psychiatric Institute

Lindsay Silva, Hunter College.

Christoph Wiedenmayer, Columbia University.

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