Norepinephrine and posttraining memory consolidation in neonatal rats

By: D. A. Wilson
Developmental Psychobiology Laboratory, Department of Zoology, University of Oklahoma;
Thu-Cuc Pham
Developmental Psychobiology Laboratory, Department of Zoology, University of Oklahoma
R. M. Sullivan
Developmental Psychobiology Laboratory, Department of Zoology, University of Oklahoma

Acknowledgement: This research was supported by National Science Foundation Grants BNS-9110506 to R. M. Sullivan and IBN-9209929 to D. A. Wilson and National Institutes of Health Grant DC01674 to D. A. Wilson.

Newborn rat pups are capable of learning and remembering olfactory associations early in perinatal development. This early olfactory associative learning is correlated with specific neural changes in the olfactory bulb. For example, spatial-temporal single-unit response patterns of mitral/tufted cells (the primary output neurons of the bulb) to conditioned odors are modified (Wilson, Sullivan, & Leon, 1987; Wilson & Leon, 1988). Furthermore, focal glomerular layer 2-deoxglucose (2-DG) uptake to conditioned odors is enhanced (Coopersmith & Leon, 1984; Sullivan & Leon, 1986). These changes are odor specific, long-lasting, independent of changes in respiration, and can be extinguished by unreinforced presentations of an odor (Wilson & Sullivan, 1994). Additional olfactory bulb changes following long-term training include an increase in focal populations of glomerular layer neurons (Woo & Leon, 1991).

This early olfactory learning is heavily influenced by two systems known to be important memory modulators in the adult—central catecholamines and the limbic system. For example, early olfactory learning involves and requires activation of the noradrenergic projection from the locus coeruleus to the olfactory bulb. Acquisition of olfactory memory can be disrupted in pups by either systemic injections of β-receptor antagonists (Sullivan, Wilson, & Leon, 1989), localized 6-OHDA lesions of the locus coeruleus (Sullivan, Wilson, Lemon, & Gerhardt, 1994) or localized infusion of the β-receptor antagonist propranolol into the olfactory bulb during training (Sullivan, Zyzak, Skierkowski, & Wilson, 1992). Furthermore, association of an odor with the β-receptor agonist isoproterenol, without any other standard reinforcer, is sufficient to induce a learned odor preference in neonates (Sullivan, McGaugh, & Leon, 1991). Norepinephrine is not required, however, for expression of odor memories in neonates once that odor memory has been acquired (Sullivan & Wilson, 1991).

Despite the relative general immaturity of the limbic system during the 1st postnatal week in the rat, the amygdala appears to modulate early learning. Bilateral lesions of the amygdala (primarily including the cortical nucleus of the amygdala) impair acquisition of a learned odor preference in pups on Postnatal Day 6 (Sullivan & Wilson, 1993). However, overtraining can compensate for these lesions. These results have been interpreted as suggesting that the amygdala facilitates this form of learning in neonates, rather than serving a storage role.

In the adult of several species, central and peripheral catecholamines (McGaugh, Liang, Bennett, & Sternberg, 1984) as well as the amygdala and limbic system (Liang, McGaugh, & Yao, 1990; Zola-Morgan, & Squire, 1990) are important mediators of posttraining memory consolidation. For example, posttraining infusions of norepinephrine receptor antagonists into the amygdala impair subsequent memory performance in a passive avoidance task (Gallagher, Kapp, Musty, & Driscoll, 1977). Conversely, posttraining infusion of norepinephrine or norepinephrine agonists into the amygdala can facilitate memory (Liang, McGaugh, & Yao, 1990).

In the rat and rabbit neonate, memory consolidation processes appear to emerge early in ontogeny. For example, newborn rabbits exposed to a novel odor while suckling on Postnatal Day 2 will subsequently (24 hr later) show conditioned nipple search behavior to that odor. However, disruption of brain activity by whole body cooling (body temperature reduced to 7 °C) immediately after the suckling episode blocks subsequent conditioned responses to the odor (Kindermann, Gervais, & Hudson, 1991). Body cooling 4 hr after training has no effect on memory. Weldon et al. (1982) and Weldon, Travis, and Kennedy (1991) have begun to explore the pharmacology of consolidation in newborn rats (Postnatal Days 5 and 6) using an associative olfactory learning task with odor as the conditioned stimulus (CS) and tactile stimulation as the unconditioned stimulus (US). They found that posttraining systemic injection of dopamine D1 receptor antagonists (Weldon et al., 1991) or glutamate NMDA (N-methyl-D-aspartate) receptor antagonists (Weldon & Fedorcik, 1993) block subsequent memory of the CS. Together, these results suggest that posttraining consolidation mechanisms are present and functional during the 1st postnatal week in rats and rabbits.

The present study was an examination of the role of norepinephrine in memory consolidation in neonates. Given the early development of the noradrenergic system in rats and the role of norepinephrine in consolidation in adults, it seems likely that, in addition to noradrenergic effects on primary sensory system function during training (Wilson & Sullivan, 1991; Wilson & Sullivan, 1992), norepinephrine may modulate posttraining consolidation of early olfactory memories.

Experiment 1

To address whether posttraining norepinephrine is involved in consolidation of early olfactory memories, rat pups were trained on Postnatal Day 5 in an associative conditioning task with citral odor and intraoral infusions of milk. Immediately after training, pups were systemically injected with one of several doses of the β-receptor antagonist propranolol. Memory for a learned relative odor preference was tested 24 hr later.

Method

Subjects

Subjects were male and female Wistar rat pups between 5 and 7 days old. Litters were born to females obtained from Hilltop Lab Animals (Scottdale, PA), maintained with ad-lib food and water on a 12-hr light-dark cycle. Dams and their litters were housed in polypropylene cages lined with wood chips. Pups were randomly assigned to groups with no more than 1 male and 1 female from a single litter assigned to each treatment group.

Procedure

On Postnatal Days 5 and 6, pups were separated from the dam for 6 hr prior to training. At least 3 hr prior to training, pups were anesthetized with ether and implanted with an intraoral cheek cannula (PE-10) implanted so as to allow milk infusion during training. Following the 6-hr deprivation period, pups were voided by stimulation of the perianal area and placed in individual glass breakers for a 10-min habituation period. Cannulas were connected to syringe pumps (Harvard Apparatus, South Natick, MA) with PE-50 tubing. Syringes in the pumps were filled with commercial Half-and-Half. Training chambers were kept at 30 °C (±1 °C) throughout the habituation and training session.

After the 10-min habituation period, pups were trained in one of three conditioning groups. Pups in the paired condition (n = 37) received overlapping presentations of odor and intraoral milk infusions. The odor stimulus consisted of 2.5 μl of citral (Sigma Chemical, St. Louis, MO) applied to a 2-cm square piece of Kimwipe tissue. The tissue was placed near the top of the training beaker and was present throughout the training session. Intraoral milk infusions consisted of a 10-s delivery (0.05 ml/min) of milk through the cheek cannula. Eleven infusions were delivered over the 30-min training session with an intertrial interval of 3 min. Pups in the odor-only condition (n = 38) received only odor presentations, whereas pups in the milk-only condition (n = 40) received only milk presentations. Milk-only pups were trained in a different room from the paired and odor-only pups in order to avoid odor contamination.

Immediately after training, all pups were gently taken to an odor-free room and injected with the β-receptor antagonist propranolol (0 mg/kg, 10 mg/kg, or 20 mg/kg ip) with at least 11 pups per training group per drug dose. Following the injection, cheek cannulas were removed, and pups were returned to the dam until testing.

Twenty-four hours after training, pups were tested for a relative odor preference for citral in a two-odor choice test. The test apparatus consisted of a Plexiglas arena (24 cm long × 14 cm wide) with a wire mesh floor. The floor was divided in half by a 2-cm wide midline. On one side of the midline under the floor were clean wood shavings and on the other side was a Kimwipe scented with 2.5 μl of citral. Testing consisted of placing the pup on the midline and monitoring the amount of time the pup spent over each odor. The test trial lasted 60 s, and each pup received three trials. The direction the pup was placed on the midline was counterbalanced across trials. The floor was wiped clean with water between trials. Total time over the citral odor was analyzed across training per drug groups with analyses of variance (ANOVA) and post hoc Fisher tests.

Results

As shown in Figure 1, saline-injected pups trained with paired presentations of citral and milk showed a relative preference for citral 24 hr after training compared with milk-only and odor-only pups. This acquired preference was blocked by posttraining injections of either 10 mg/kg or 20 mg/kg propranolol; 3 × 3 ANOVA, Training Group × Dose interaction, F(4, 106) = 3.14, p < .05. Post hoc Fisher tests revealed paired-saline pups spent significantly more time over the CS odor than any other group (p < .05).

Experiment 2

The results of Experiment 1 suggest that posttraining propranolol blocks consolidation of early olfactory memories. Previous work has shown that propranolol injected just prior to testing (24-hr posttraining) does not interfere with expression of olfactory memories in pups (Sullivan & Wilson, 1991); thus, this posttraining effect must be limited in duration. Experiment 2 investigated the time course of the norepinephrine-mediated consolidation period. Rats were trained as described above and injected with propranolol either 0 or 60 min after training.

Method

Pups were trained as described above in the paired (n = 37), odor-only (n = 38), and milk-only conditions (n = 36). Following training, half of the pups in each group were immediately injected with either saline or 20 mg/kg propranolol, as described above. All pups were then returned to the dam. One hour later, the noninjected pups were removed from the litter and injected with saline or 20 mg/kg propranolol and then returned to the dam. All groups had at least 8 pups, with no more than 1 male and 1 female from a single litter in any group. Twenty-four hours after training, pups were tested in the two-odor choice test.

Results

As shown in Figure 2, propranolol injected at either 0 or 60 min posttraining blocked olfactory memory; Training Group × Drug interaction, F(6, 97) = 3.04, p < .01. Post hoc analysis revealed that paired pups injected with saline at 0 min showed a significant odor preference compared with propranolol-injected paired pups (Fisher, p < .05). However, the odor preference expressed by paired-saline pups injected at 60 min did not significantly differ from paired-saline pups injected at 0 min, whereas the preference magnitude of the 60 min injected group was reduced. The mean time spent over the CS odor was not significantly different between paired-saline at 60 min and paired-propranolol at 60 min (see Figure 2).

Experiment 3

The results of Experiment 2 suggest that the duration of norepinephrine mediated consolidation in pups is greater than 60 min. Furthermore, there is the potential that even saline injections at 60 min posttraining influence memory consolidation. Experiment 3 further explored the time course of propranolol effects on consolidation and reassessed the effects of saline injections.

Method

On Postnatal Days 5 and 6, pups were trained in the paired condition as described above (n = 46). Following training, pups were injected either immediately (saline, n = 7; 20 mg/kg propranolol, n = 8), 1 hr (saline, n = 8; 20 mg/kg propranolol, n = 7), or 4 hr later (saline, n = 8; 20 mg/kg propranolol, n = 8). Pups not injected immediately were returned to the dam during the delay period. Twenty-four hours later, pups were tested in the two-odor choice test.

Results

As shown in Figure 3, the effects of propranolol on olfactory memory in pups dissipate by 4 hr posttraining. Propranolol injected 0 or 60 min posttraining impaired memory performance, but propranolol injected 240 min posttraining had no significant effect; Drug × Time interaction, F(2, 40) = 15.27, p < .01. Post hoc analyses revealed that pups injected with saline at 0 or 240 min posttraining and pups injected with propranolol 240 min posttraining showed a significant odor preference compared with pups injected with propranolol at 0 or 60 min. Interestingly, similar to Experiment 2 results, pups injected with saline 60 min after training also failed to show a learned odor preference. The saline effect at 60 min cannot account for the effects of propranolol in general, however, because saline injections at 0 min had no effect on memory, whereas propranolol injected at that time did.

Experiment 4

In adults, the relationship between posttraining norepinephrine and memory consolidation is an inverted U-shaped function, with moderate levels improving memory and low or high levels impairing memory (McGaugh, 1983). In neonates, the results of Experiments 1 through 3 demonstrate that low levels of posttraining norepinephrine (through blockade of noradrenergic β-receptors) impair early olfactory memories. Experiment 4 examined whether memory consolidation could be enhanced by raising posttraining noradrenergic activity through systemic injection of the β-receptor agonist isoproterenol.

Method

On Postnatal Days 5 and 6, pups were trained as described above in the paired (n = 56), odor-only (n = 57), and milk-only (n = 54) conditions. Immediately following training, pups were injected with isoproterenol (1 mg/kg, 2 mg/kg, or 4 mg/kg) or saline vehicle. The pups were then returned to the dam until testing 24 hr later. Learned relative odor preferences were tested in the two-odor choice test described above.

Results

As shown in Figure 4, isoproterenol injected immediately after training impaired memory for a learned odor preference. There were no significant differences between different doses of isoproterenol, so the data were collapsed across dose and an ANOVA performed comparing saline- and isoproterenol-injected rats. Paired-saline pups had a significant preference for the CS odor compared with paired-isoproterenol pups and all pups in the milk- and odor-only conditions; Training Group × Drug interaction, F(2, 161) = 4.45, p < .05; post hoc Fisher tests revealed that paired-saline was significantly different from all other groups, p < .05.

Experiment 5

All doses of isoproterenol used in Experiment 4 produced an impairment in odor preference memory. In adults, low doses of norepinephrine posttraining facilitate memory (Liang et al., 1990), and high doses of noradrenergic agonists posttraining impair memory (Ellis & Kesner, 1983). Thus, the failure to find isoproterenol-induced memory enhancement in pups in Experiment 4 may be due to the doses used. Therefore, Experiment 5 repeated the posttraining isoproterenol paradigm with substantially lower doses. In addition, in order to test the generalizability of these effects, a different US was used—tactile stimulation.

Method

On Postnatal Days 5 and 6, pups were trained similarly to those in the previous experiments, with the exception that tactile stimulation (stroking) was used as the US. Tactile stimulation consisted of stroking all areas of the pup's body vigorously with a sable hairbrush for 30 s. Stroking has been shown to have similar reinforcing properties to milk in pups (Sullivan & Hall, 1988). Pups were placed in glass beaker for a 10-min habituation period and then trained in either paired or odor-only conditions. Immediately following training, pups were injected with isoproterenol (0.25 mg/kg, 0.5 mg/kg, or 1.0 mg/kg) or saline vehicle (ns = 8 per conditioning group per dose). The pups were then returned to the dam until testing 24 hr later. Learned relative odor preferences were tested in the two-odor choice test described above, except that timing was done automatically with a videotracking system (Videomex V; Columbus Instrument, Columbus, OH).

Results

As shown in Figure 5, paired saline-injected pups (odor and tactile stimulation) showed a significant relative odor preference for citral compared with odor-only, saline-injected pups. All three doses (0.25, 0.5, and 1.0 mg/kg) of isoproterenol injected immediately after paired training blocked this relative odor preference, F(4, 35) = 4.33, p < .01. Post hoc analysis revealed that paired-saline pups spent significantly more time over citral than all other groups (Fisher, p < .05), which did not differ significantly from each other.

General Discussion

The present series of experiments demonstrates that posttraining norepinephrine modulates consolidation of early olfactory memories. The norepinephrine dependent consolidation period lasts less that 4 hr. Norepinephrine levels for optimal consolidation appear to have a very limited range, with either increases or decreases from baseline activity producing an impairment in memory.

The saline-induced impairment observed in Experiments 2 and 3 could also be described in terms of noradrenergic activity. The results of Experiments 4 and 5 suggest that too much norepinephrine posttraining impairs memory consolidation. In mature animals, stress induces norepinephrine release in the amygdala (Tanaka et al., 1991), and high levels of norepinephrine agonists infused into the amygdala posttraining disrupt memory for aversive conditioning (Ellis & Kesner, 1983). Therefore, it is easy to speculate that removing pups from the litter 60 min posttraining, handling, and injecting them may have been sufficient to produce a noradrenergic surge that impaired consolidation. The same handling and injection may have been less stressful if it immediately followed the prolonged training session, and thus, saline injection at 0 min would have no effect on consolidation (as observed). Interestingly, a similar saline effect on neonatal associative olfactory memory at 60 min posttraining has been reported by Weldon and Fedorcik (1993).

Combined with previous work, these results suggest that norepinephrine may have at least two roles in early olfactory learning. First, within the primary sensory pathway, norepinephrine increases excitability to stimuli and increases signal:noise ratios (Foote, Bloom, & Aston-Jones, 1983). These combined effects may be involved in the role of norepinephrine in experience-induced sensory system plasticity in newborns (Bear & Singer, 1986; Kasamatsu & Pettigrew; 1979; Sullivan, Wilson, & Leon, 1989). Specifically, during early olfactory learning, norepinephrine enhances olfactory bulb excitability and reduces mitral/tufted cell single-unit habituation to odors during conditioning (Wilson & Sullivan, 1992). This enhanced and maintained response may promote acquisition to odor associations through mechanisms such as long-term potentiation.

The second role of norepinephrine in early learning appears to occur posttraining through modulation of memory consolidation as described in this article. Because the present experiments involved systemic manipulations, the locus of effects cannot be determined. In the adult, however, one site of noradrenergic action related to consolidation is the amygdala (Liang et al., 1990). Although the limbic system in rats is relatively immature during the 1st postnatal week, the amygdala complex is involved in early learning (Sullivan & Wilson, 1993). Thus, it is possible that norepinephrine influences consolidation of early memories through structures similar to those in the adult. On the other hand, recent work on adult olfactory memory suggests a role of the olfactory bulb in olfactory memory consolidation (Mouly, Kindermann, Gervais, & Holley, 1993). Xylocaine infusions into the olfactory bulb immediately after training in a discrimination task significantly impair long-term retention, whereas infusions 2 hr after training do not (Mouly et al., 1993).

As stated above, norepinephrine is not the sole transmitter system responsible for acquisition and consolidation of early olfactory memories. Dopamine (Weldon et al., 1991), serotonin (McLean, Darby-King, Sullivan, & King, 1993), opiates (Kehoe & Blass, 1986), GABA (Smart, Wilson, & Sullivan, 1993), and glutamate (Weldon & Fedorcik, 1993) have all been implicated as playing a role in memory formation during the early postnatal period. It is assumed that, as in the adult, rich and complex interactions occur among these systems to produce long-lasting, adaptive changes in neonatal behavior based on early postnatal experience.

Finally, these results provide further evidence that mechanisms of consolidation emerge early in the postnatal development of the rat. This early emergence is interesting in light of some explanations of infantile amnesia—the inability of humans and animals to recall early postnatal events. For example, research in rats suggests that, even when initial acquisition levels in neonates are equated to acquisition levels in older pups, neonates display more rapid forgetting (Campbell & Alberts, 1979). One hypothesis regarding the cause of infantile amnesia suggests that early memories decay rapidly because of the late maturation of limbic structures involved in consolidation like the hippocampus, amygdala, and overlying temporal cortex (Bachevalier, 1990; Nadel & Zola-Morgan, 1984). The present findings suggest that in the rat mechanisms of consolidation for simple associations are functional in the 1st postnatal week. Disruption of these posttraining processes interferes with normal memory storage.

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Submitted: December 28, 1993 Revised: May 13, 1994 Accepted: May 13, 1994