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Institut des Sciences Cognitives, Université Claude Bernard Lyon 1, FranceNeurosciences Sensorielles, Comportement, Cognition Centre National de la Recharche Scientifique, Université Claude Bernard Lyon 1, France
Institut des Sciences Cognitives, Université Claude Bernard Lyon 1, FranceNeurosciences Sensorielles, Comportement, Cognition Centre National de la Recharche Scientifique, Université Claude Bernard Lyon 1, France
Institut des Sciences Cognitives, Université Claude Bernard Lyon 1, FranceNeurosciences Sensorielles, Comportement, Cognition Centre National de la Recharche Scientifique, Université Claude Bernard Lyon 1, France
Early life adverse experience alters adult emotional and cognitive development. Here we assess early life learning about adverse experience and its consequences on adult fear conditioning and amygdala activity.
Methods
Neonatal rats were conditioned daily from 8-12 days-old with paired odor (conditioned stimulus, CS) .5mA shock, unpaired, odor-only, or naive (no infant conditioning). In adulthood, each infant training group was divided into three adult training groups: paired, unpaired or odor-only, using either the same infant CS odor, or a novel adult CS odor without or with the infant CS present as context. Adults were cue tested for freezing (odor in novel environment), with amygdala 14C 2-DG autoradiography and electrophysiology assessment.
Results
Infant paired odor-shock conditioning attenuated adult fear conditioning, but only if the same infant CS odor was used. The 14C 2-DG activity correlated with infant paired odor-shock conditioning produced attenuated amygdala but heightened olfactory bulb activity. Electrophysiological amygdala assessment further suggests early experience causes changes in amygdala processing as revealed by increased paired-pulse facilitation in adulthood.
Conclusions
This suggests some enduring effects of early life adversity (shock) are under CS control and dependent upon learning for their impact on later adult fear learning.
Early life adverse experience alters adult emotionality and cognitive function, as modeled by prolonged preweanling separation from the mother, preweanling isolation from mother/siblings, and painful electric shock (
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.
Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.
The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat.
). The variability in the long-term learning effects of early life manipulations may relate to the specific learning task, age of manipulation or testing, and the possibility of early life stress potentiating/attenuating individual differences (
). Previous work on infant adverse experiences using shock suggests that unpredictable shock produces greater changes in adult emotionality than predictable shock (
). While maternal/sibling deprivation models are useful in assessing the adult impact of infant trauma, shock can be placed within the context of predictable versus unpredictable infant pain to assess potential enduring learning effects from early life that could be under the control of the conditioned stimulus (CS).
Here we assess the effects of infant shock within the context of a learning paradigm that permits shock presentation in a predictable (paired odor-shock) and unpredictable (unpaired) fashion. The effects of infant shock were assessed on later adult cognition as measured by adult fear conditioning. Furthermore, we begin to assess the potential neural circuitry underlying the enduring infant effects of early life shock through assessment of the amygdala, which is critically involved in adult fear conditioning (
). Since the cue predictive of shock is an odor, we also assess the adult olfactory bulb, which has a strong input to the amygdala both directly and indirectly through the piriform (olfactory) cortex (
Subjects were 171 (n = 13 Y-maze, Experiment [Exp] 1 = 89, Exp 2 = 51, Exp 3 = 18) male Long Evans rats born and bred at the University of Oklahoma. Additional midgestation rats were purchased for the Institut des Sciences Cognitives (Lyon, France). Only one male per litter was used in each training/testing condition. After weaning, pups were housed two per cage until adult fear conditioning (4 ± 1.5 months). All procedures were approved by respective Institutional Animal Care and Use Committees with National Institute of Health/European guidelines. To ensure consistency of conditioning/testing of infant/adult animals, training/testing personnel overlapped in France and the United States.
Infant Odor-Shock Conditioning
Postnatal day (PN) 7-8 pups were conditioned daily for 5 days, producing an odor preference with no amygdala participation (
). Conditioning groups were: Paired—11 pairings of 1 sec hindlimb .5mA shocks overlapping with the last second of the 30 sec CS (McCormick Peppermint; 2L/min, 1:10 peppermint vapor:air) delivered via an olfactometer with 4 min intertrial interval (ITI), Unpaired—shocked 1.5-2 min after each CS odor presentation, Odor—11 CS presentations, and Naïve (
The 45 min conditioning was similar to infant conditioning: 11 pairings of 30 sec CS odor (same concentration) and shock (.8 mA, 1 sec foot shock from grid floor) delivered during the odors’ last sec in a Lafayette chamber under red light. Twelve different behavioral groups were used with each of the 4 infant training groups (Paired, Unpaired, Odor, Naive) split into 3 adult groups (Paired, Unpaired, Odor) for behavior, with select groups used for neural and odor specificity assessment.
While the peppermint odor was always used as the CS in infancy, two different CS’s were used in adulthood: peppermint odor (same odor as infant training; Experiment 1) and a novel citral odor (to assess if attenuated fear conditioning in Experiment 1 was specific to infant odor; Experiment 2A). In the last study, the peppermint odor was present as context (Experiment 2B). This context peppermint odor was also delivered by a flow dilution olfactometer at the same concentration used in infancy but at a lower delivery rate (1 L/min) and present continuously during adult conditioning (
Pavlovian Conditioning of Emotional Responses to Olfactory and Contextual Stimuli: A Potential Model for the Development and Expression of Chemical Intolerance.
CS (peppermint in Experiment 1A, citral in Experiment 2A, 2B) conditioned fear was assessed 24 hours after adult conditioning (novel Plexiglas aquarium; 25.4 × 50.8 × 30.5 cm) to prevent context conditioning influences. After 10 min, 30 sec CS odor (peppermint or citral) was presented (3 times, 4 min ITI) and freezing measured 30 sec before and during each odor presentation. Most testing sessions were filmed.
Autoradiography
Adult rats were injected with 14C 2-deoxyglucose (2-DG; 40μCi sc) 5 min before conditioning. After conditioning, brains were removed, frozen in 2-methylbutane (−45°C) and stored in a −70°C freezer. For analysis, brains were sectioned (20 μm) in a −20°C cryostat, and every other section was placed on a cover slip and exposed for 5 days along with 14C methylmethacrylate standards (
) and analyzed blind to the experimental conditions with the NIH computer-based digital image system for quantitative optical densitometry. An increase in autoradiographic density indicates increased neural activity but does not discriminate between inhibition and excitation.
Autoradiography Amygdala Analysis
Amygdala nuclei (basolateral, lateral, medial, central, Figure 1) were identified by counterstaining sections with cresyl violet to construct a template to overlay the autoradiographs (
). At least three sections were analyzed, all within stereotaxic coordinates for electrophysiology. The 2-DG uptake was expressed relative to corpus callosum uptake (did not vary with conditioning group) to control for differences in section thickness and exposure (
Figure 1(Left) Dark-field image of the cresyl violet stained section alone, showing the clear contrast of individual amygdala nuclei. (Right) Pseudocolor autoradiograph of C14 2-DG uptake in an adult during odor-shock training overlayed on a dark-field image of the same section stained with cresyl violet. The individual nuclei are outlined and labeled in the right image to indicate where optical densitometric measurements were made in this study. BMA, basomedial nucleus; ceA, central nucleus; coA, cortical nucleus; LA/BLA, basolateral complex; PCX, piriform cortex.
), were analyzed in select groups. The odor-specific pattern was identified in at least three sections and five readings were taken from each odor-specific loci along with five readings within the periventricular core to control for differences in section thickness and exposure (
In select conditioning groups of Experiment 1, adult rats were tested for amygdala electrophysiological responses. Two days after the cue-testing, animals were anesthetized with Equithesin (mixture of chloral hydrate and sodium pentobarbital; 3 ml/kg, intraperitoneally [IP]). A bipolar stimulating electrode was lowered into the left olfactory bulb mitral cell layer (A/P −6 mm relative to the nasofrontal suture, L/M 1.3 mm relative to Bregma). A monopolar recording electrode was implanted ipsilaterally in the basolateral amygdala (A/P –2.8 mm, L/M 4.9 mm relative to Bregma). Accurate positioning of recording electrode depth was achieved using field potential profiles evoked by olfactory bulb stimulation.
Olfactory bulb electrical stimulation, which was delivered through a Master-8 stimulator (AMPI, Jerusalem, Israel), was a single monophasic square pulse, .1 msec duration, 300-500 μA amplitude, .1Hz frequency. Stimulation test intensity was set to induce a response amplitude of approximately 70% of maximum. The basolateral signal was amplified (Grass Model 12, Astro-Med, Warwick, Rhode Island), filtered (1-300 Hz) and digitized (sampling frequency: 5 kHz) using a data acquisition system (Wavebook 512, Iotech, Cleveland, Ohio).
Olfactory bulb paired-pulse stimulation was used to assess the time course of short-term basolateral inhibition and facilitation. The effect of the conditioning (first) pulse was assessed by measuring changes in the response to the test (second) pulse. Paired pulses were delivered at interpulse intervals of 20 and 30 msec. Twelve responses were recorded for averaging at each interpulse interval.
Off-line, individual evoked field potentials were averaged (n = 12 sweeps) and analyzed using the data acquisition software Dasylab (Iotech). Peak amplitudes of both conditioning and test pulses were measured. Test signal amplitude was expressed as a percentage of conditioning signal amplitude and obtained ratios compared between the groups.
Statistics
For statistical analysis we performed analysis of variance (ANOVA) followed by post hoc Fisher tests, always between individual groups at p < .05, at least.
Results
Infant Odor-Shock Training Produces An Odor Preference
PN13 pups were Y-maze tested, which verified odor preference acquisition (Supplement 1; F(2,10) = 5.311, p < .05); post hoc tests revealed the Infant Paired group differed significantly from unpaired and odor controls (
Experiment 1: Long-Term Effects of Early Learning on Adult Fear Conditioning
Experiment 1A: Behavior
In adulthood, the effects of infant odor-shock conditioning on adult fear conditioning were assessed using the same CS odor in infancy and adulthood. The day after adult fear conditioning, animals were given a cue CS test in a novel environment (Figure 2A). Animals that received Infant Paired peppermint-shock conditioning and reconditioning as Adult Paired peppermint-shock showed decreased freezing during subsequent CS odor cue test. Infant conditioning in control groups (Unpaired, Odor, Naive) had no effect on Adult Paired conditioning (Figure 2A). The freezing behavior ANOVA analysis revealed a main effect of condition (F(11,50) = 22.018, p < .001). Post hoc tests revealed that the Adult Paired groups each differed significantly from control and the Infant Paired/Adult Paired group exhibited significantly lower freezing level than Infant Naive/Adult Paired, Infant Unpaired/Adult Paired or Infant Odor only/Adult Paired groups.
Figure 2Following infant odor-shock conditioning, adult rats were reconditioned using the same conditioned stimulus (CS). The three adult conditioning groups are on the X axis as a function of the four infant conditioning groups. (A) Freezing (±SEM) during the peppermint CS cue test in a novel environment, (B) Mean relative 2-DG uptake (±SEM) in the olfactory bulb during adult fear conditioning, (C) Mean relative 2-DG uptake (±SEM) in amygdala nuclei during adult fear conditioning. One asterisk represents significant differences from each of the control groups and two asterisks represent significant differences from other Adult Paired conditioning groups (at least, p < .05).
The reduced freezing seen in animals with Infant Paired/Adult Paired odor-shock conditioning (same CS) was associated with increased olfactory bulb activity during conditioning (F(5,18) = 3.441, p < .05; Figure 2B). Post hoc tests revealed Infant Paired/Adult Paired group differed significantly from other groups.
Experiment 1C: Amygdala Autoradiography
The reduced freezing seen in animals with Infant Paired/Adult Paired odor-shock conditioning (same CS) was associated with reduced amygdala activity during conditioning (Figure 2C). Specifically, the ANOVA revealed a main effect for condition of the basolateral (F(5,21) = 10.990, p < .001) and lateral (F(5,21) = 9.855, p < .001) amygdala nuclei. Post hoc tests revealed the Infant Naive/Adult Paired, Infant Unpaired/Adult Paired and Infant Odor/Adult Paired differed from each of the other groups for the basolateral and lateral nuclei. Infant Paired/Adult Paired amygdala nuclei did not differ from controls. No statistical differences were found for the medial or central amygdala.
Together, the results of Experiment 1 indicate the Infant Paired/Adult Paired group (same CS) had attenuated freezing, enhanced olfactory bulb activity and attenuated amygdala (basolateral and lateral nuclei) activity compared to all other Adult Paired groups. Since our two-DG method of assessing neural activity cannot measure subtle differences, a lack of significant difference between groups should not be interpreted as the same neural activity.
Experiment 2: Are the Long-Term Effects of Infant Conditioning Odor Specific?
We assessed whether attenuation of adult odor conditioning was specific to the odor used in infancy by adult fear conditioning with a novel CS (citral odor). This novel CS was presented without (Exp 2A; Supplement 2) or with (Exp 2B; Figure 3) the infant CS peppermint odor present as context (continuous peppermint presentation during conditioning to novel odor).
Figure 3Following infant odor-shock conditioning with peppermint odor, adult rats were reconditioned using a novel citral conditioned stimulus (CS) odor, with peppermint odor continuously present as context. During testing, only the citral CS used in adult conditioning was present (peppermint odor was not present during testing). (A) Freezing (±SEM) during the citral CS cue test in a novel environment as a function of infant and adult training. One asterisk indicates a significant difference from the Infant Naive/Adult paired group and two asterisks indicate a significant difference from all groups except the Infant Unpaired/Adult Paired group (at least, p < .05). Thus, the Infant Unpaired/Adult Paired group is intermediate between the other Adult Paired groups. (B) Mean relative 2-DG uptake (±SEM) in the olfactory bulb during adult fear conditioning. One asterisk indicates a significant difference from the controls and two asterisks represent a significant difference from the other Adult Paired groups (at least, p < .05). (C) Mean relative 2-DG uptake (±SEM) in amygdala nuclei during adult fear conditioning. One asterisk indicates a significant difference from the controls and two asterisks represent a significant difference from the other Adult Paired groups (at least, p < .05).
Experiment 2A: Behavior - Novel Citral CS in Adult Conditioning
Attenuated learned freezing is specific to the odor used in infant training. The ANOVA analysis revealed a main effect of group (F(2,11) = 28.147, p < .0001); post hoc Fisher tests further showed the Infant Paired Peppermint/Adult Paired Citral and Infant Naive/Adult Paired Citral groups, which did not differ from one another, demonstrated significantly higher levels of freezing compared to the control group (Infant Naive/Adult Unpaired Citral). As outlined below, the effect of infant odor-shock conditioning on fear learning (Exp 2B, Figure 3A) and amygdala autoradiography (Exp 2C, Figure 3B) appear to be specific to the odor experienced in infancy.
Experiment 2B: Behavior - Novel Citral CS in Adult Conditioning with Infant Peppermint CS as Context
Figure 3A shows adult freezing performance following conditioning to a novel citral CS with the infant peppermint CS used as context during acquisition. Only the novel CS citral odor was present during adult cue testing. Only Adult Paired animals learned to freeze to the citral odor CS, although differences were found in this conditioning group dependent upon infant experience. Specifically, Infant Paired Peppermint/Adult Paired Citral with peppermint context demonstrated lower levels of freezing than Infant Naive/Adult Paired Citral with peppermint context. There was also an effect for infant unpaired presentations. Specifically, Infant Unpaired Peppermint/Adult Paired Citral with peppermint context showed significantly less freezing behavior, however not as attenuated as the Infant Paired Peppermint/Adult Paired Citral with peppermint context. The freezing behavior ANOVA analysis revealed a main effect of condition (F(4,13) = 16.197, p < .0001); post hoc Fisher tests revealed that the Infant Unpaired Peppermint/Adult Paired Citral with peppermint context and Infant Naive/Adult Paired Citral with peppermint context groups differed significantly from one another, all the control groups and Infant Paired Peppermint/Adult Paired Citral with peppermint context at the p < .05 level. Infant Paired Peppermint/Adult Paired Citral with peppermint context did not significantly differ from control groups. Therefore, the presence of the infant odor as context during acquisition in adulthood attenuates fear learning to a novel odor in both Infant Paired and Unpaired odor-shock animals.
Experiment 2C: Olfactory Bulb Autoradiography - Novel Citral CS in Adult Conditioning with the Infant Peppermint CS Used as Context
Figure 3B illustrates olfactory bulb responses during adult conditioning with a novel citral odor along with the infant peppermint odor used as context. Infant Paired Peppermint/Adult Paired Citral and Infant Unpaired Peppermint/Adult Paired Citral with peppermint context showed significant increases in 2-DG uptake relative to controls (F(4, 13) = 8.037, p < .0017: post hoc p < .05).
Experiment 2D: Amygdala Autoradiography - Novel Citral CS in Adult Conditioning with Infant Peppermint CS as Context
Figure 3C illustrates amygdala nuclei responses during adult conditioning with a novel odor (citral) and the infant peppermint odor used as context. The Infant Naive/Adult Paired Citral group with peppermint context had significantly more amygdala activity (basolateral and lateral) during acquisition compared to Infant Paired Peppermint/Adult Paired Citral with peppermint context. The Infant Unpaired Peppermint/Adult Paired Citral with peppermint context had intermediate amygdala activation (basolateral, lateral) compared to these two groups (Infant Naive/Adult Paired Citral group with peppermint context and Infant Paired Peppermint/Adult Paired Citral with peppermint context). ANOVA analysis revealed statistical differences for the basolateral (F(4,14) = 14.749, p < .0001), lateral (F(4,14) = 14.138, p < .0001), medial (F(4,14) = 3.192, p < .0464), central (F(4,14) = 3.114, p < .0499) amygdala nuclei. Post hoc Fisher tests revealed that Infant Naive/Adult Paired animals were significantly different from control groups (Adult Unpaired, as well as the Adult Odor only groups), as well as the Infant Paired/Adult Paired groups. Significant differences also emerged for the Infant Unpaired/Adult Paired group versus the Infant Paired/Adult Paired group for basolateral and lateral nuclei, suggesting some attenuation of neural activity for the Infant Unpaired group later conditioned in the Adult Paired group. Infant Paired/Adult Paired amygdala nuclei did not differ significantly from controls.
Experiment 3: Electrophysiological Adult Amygdala Assessment Following Infant Odor-Shock Conditioning
Due to the robust effects required to produce detectable 2-DG autoradiography differences, we used electrophysiological amygdala assessment to detect more subtle differences in synaptic efficacy.
Animals were conditioned as infants and again in adulthood. The electrophysiology was performed 2 days after adult conditioning on anesthetized rats from three groups: Infant Naive/Adult Paired, Infant Paired/Adult Paired and Infant Unpaired/Adult Paired (examples in Figure 4A). Olfactory bulb mitral cell layer was electrically stimulated with recording in the basolateral amygdala (
). A decrease in size of the response to the second pulse suggests inhibition and reflected in a score below 1 in Figure 4B. The paired-pulse ratio ANOVA revealed a main effect of group (F(2,17) = 6.29, p = .009) and interpulse (20 vs. 30 msec) interval (F(1,17) = 67.81, p < .001). Post hoc Fisher tests showed that for the 20 msec interval, the paired-pulse ratio obtained in Infant Paired/Adult Paired animals was significantly different from Infant Naive/Adult Paired animals (p = .02) but not from that in Infant Unpaired/Adult Paired rats. For the 30 msec interval, Infant Paired/Adult Paired group differed significantly from both Infant Naive/Adult Paired and Infant Unpaired/Adult Paired groups. These data suggest that the normal inhibition found in the second pulse (Infant Naive/Adult Paired) was significantly attenuated in the Infant Paired/Adult Paired animals with the 20 msec interval and switched to excitation at the 30 msec interval. There was a trend towards inhibition attenuation in the Infant Unpaired/Adult Paired animals at the 20 msec interval. These data suggest the basolateral amygdala’s synaptic interactions are altered following early life experiences with predicable shock, with a trend towards an effect following unpredictable shock.
Figure 4Basolateral amygdala response to olfactory bulb paired pulse stimulation was assessed following infant and adult odor-shock conditioning. (A) Representative averaged (n = 12) field potentials evoked in the recording site by conditioning (dotted line) and test (plain line) pulses delivered to the olfactory bulb at either 20 msec or 30 msec intervals in the different experimental groups. (B) Mean test pulse amplitude was expressed as percent variation (+SEM) from the conditioning pulse amplitude, for the two interpulse intervals (IPI). Asterisk represents a significant between groups difference (at least p < .05).
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.
The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat.
The role of corticotropin-releasing factor-norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress.
Persistent changes in corticotropin-releasing factor systems due to early life stress: relationship to the pathophysiology of major depression and posttraumatic stress disorder.
). The present study also brings new information concerning the importance of learning and the predictable versus unpredictable nature of early life adverse experiences on later adult learning at both the behavioral and neurobiological levels.
Behavioral Assessment
In Experiment 1, where the same olfactory CS was used in infancy and adulthood, we found attenuated fear conditioning in adulthood only following predictable infant shock (paired odor-shock). This effect was specific to the CS odor used in early life conditioning since adult conditioning to a novel odor was not disrupted. In Experiment 2, using the infant CS as contextual cue during conditioning to a novel odor (i.e. present continuously during adult acquisition to the novel odor but not present during testing) revealed additional long-term effects of infant odor-shock conditioning in unpaired odor-shock pups. The unpaired pups showed no learning in infancy and were conditioned at an age before the emergence of context conditioning (
). It should be noted that odor-malaise (LiCl or > 1.0 mA shock) conditioning in pups produces an odor aversion, even in fetal rats, but does not incorporate the amygdala until just before weaning (
While we did not detect differences in emotionality in our adult rats within the context of fear conditioning, our infant odor-shock paradigm does produce heightened adult anxiety in the Infant Unpaired animals as measured by latency to leave a dark box and enter a lighted alley (
). It is possible that emotional differences between our groups altered adult performance since early life stress, as well as differences in maternal behavior, have been found to modulate emotionality and learning (
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.
The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat.
The role of corticotropin-releasing factor-norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress.
Persistent changes in corticotropin-releasing factor systems due to early life stress: relationship to the pathophysiology of major depression and posttraumatic stress disorder.
). This literature also suggests significant long-term effects of infant odor-shock conditioning may be age specific, although this needs to be assessed in future studies.
Additional literature, which has modeled early life adversity using infant shock, suggests the predictable versus unpredictable manner of early shock influences emotional outcome and learning. This is consistent with the divergence in outcome from predictable versus unpredictable shock seen in both infancy and in adulthood (
The ability of the infant CS odor to attenuate adult fear conditioning suggests this effect is under the control of learning, at least in part, since the suppression was specific to the odor present in early life. The present results are in agreement with those of Kosten et al. (
). In similar adult fear conditioning studies, which assessed effects of preweanling maternal separation and preweanling isolation, no effect of early life stress was found for cue testing, although the cue used in adulthood was not present during the early life separation (
The differences in adult fear conditioning following infant shock experience cannot be explained by the present series of experiments, although interpretations are suggested based on the existing literature. First, the attenuated learning seen in adults in response to the infant odor presentation is consistent with reward devaluation literature where both the learned response and amygdala activity are decreased (
). Specifically, the infant odor-shock conditioning, which produced a positive odor, may have simply devalued the adult fear conditioning in a manner similar to devaluation of fear conditioning if natural (i.e. food) or learned preferred odors (i.e. odors previously paired with food) were presented. Second, our paradigm assessing the role of the infant learned odor as context is consistent with experimental design for blocking, where the first odor paired with reward blocks conditioning for a subsequent session of novel odor–reward conditioning (
). Indeed, blocking could account for the results of Experiment 2, which found attenuated adult fear conditioning to a novel citral odor in the presence of peppermint odor experienced in infancy. However, blocking cannot account for the attenuated learning of Exp 1 or the attenuated learning in the Unpaired Infant/Adult Paired group, which did not exhibit detectable learning in infancy. Additionally, these behavioral data are not readily interpretable based on latent inhibition or novelty (
) since infant exposure to odor (Odor group) or a novel odor does not produce a consistent effect across groups. Together, this suggests that well-documented behavioral paradigms that attenuate learning may contribute to the effects seen in our adult animals following infant conditioning, although other mechanisms contribute as well and are discussed below.
Neural Assessment
The enhanced olfactory bulb activity seen in adult groups in response to odors learned in early life suggests differential neural processing of the odor between groups occurs very early on in the olfactory pathway. These olfactory bulb differences in odor processing have previously been demonstrated in pups following infant experience (
) (Ressler, personal communication). These olfactory bulb data also suggest the bulb is sending altered odor input to the amygdala and may contribute to the amygdala’s response to adult conditioning following infant experience. Indeed, the enhanced 2-DG olfactory bulb response is associated with an increased inhibitory signal from mitral cells (
Robust amygdala (basolateral and lateral nuclei) activation was detected during adult odor fear conditioning using 2-DG. This observation is consistent with previous data suggesting that, similar to its well-documented role in tone fear conditioning, the amygdala is critically involved in odor fear conditioning (
). However, this enhanced 2-DG response was attenuated in groups that showed attenuated fear conditioning following infant odor-shock experience.
The 2-DG technique showed attenuation of amygdala 2-DG uptake in animals that had received infant paired odor shock (predictable) conditioning compared to animals receiving odor-shock conditioning for the first time (Infant Naive/Adult Paired). These data certainly indicate that the amygdala is processing information differently following infant experience. However, the lack of significant difference between Infant Paired/Adult Paired and adult control conditioning groups using autoradiography does not indicate these groups are processing information similarly. Indeed, the 2-DG method of assessing amygdala activity cannot measure subtle differences and requires prolonged and robust differences in neural activity. Furthermore, 2-DG cannot determine whether an increase in uptake reflects increased excitatory or inhibitory neural activity.
We used paired-pulse stimulation of the olfactory bulb to assess short-term inhibition and facilitation (
) and further characterize differences in amygdala activity between conditioning groups. These data show that following adult conditioning, the paired-pulse inhibition normally observed in Infant Naive/Adult Paired animals was strongly attenuated in Infant Paired/Adult Paired animals and even switched to paired-pulse facilitation depending upon the interpulse interval. These electrophysiological data suggest broad infant experience effects on amygdala activity since the modifications were not dependent on the presence of the odor experienced in infancy. Indeed, these data may highlight previously documented altered amygdala development associated with myriad early life manipulations (
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat.
Integration of the autoradiography and physiology data suggests infant experience alters amygdala neural activity, although the mechanism is not known. 2-DG autoradiography does not discriminate between excitatory or inhibitory neural activity, but simply indicates changes in long-term neural activity. Additionally, paired-pulse assessment of the amygdala does not indicate either an increase or decrease in neural activity but does suggest changes associated with modifications of excitatory and inhibitory activity. However, combined, these data suggest that the context (paired vs. unpaired) of infant trauma is critical in defining alterations in brain development trajectory but that additional learning associated effects of infant trauma further defines the long-term outcome. Further work will delineate potential age-specific effects.
Neurobiology of Infant Trauma
Useful paradigms of infant trauma have been the maternal deprivation paradigm (prolonged separation of pups from mother) and shock. These manipulations modify emotional expression, including fear, learning and associated limbic structures (
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
The role of corticotropin-releasing factor-norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress.
Persistent changes in corticotropin-releasing factor systems due to early life stress: relationship to the pathophysiology of major depression and posttraumatic stress disorder.
). Our new infant model aligns with these data but contributes new information: i.e. some aspects of early life trauma are under CS control and dependent upon learning for its impact on later adult fear learning.
This work was supported by National Institute of Child Health and Human Development (NICHD) HD33402, OCAST, University of Oklahoma Presidential International Travel Funds to RMS, Eurodoc grant to YS, and financial support of the Agence Nationale de la Recherche (ANR), The French National Research Agency under the Programme National de Recherche en Alimentation et nutrition humaine project ANR-05-PNRA-1.E7 AROMALIM to AMM and RG.
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders.
Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.
The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat.
Pavlovian Conditioning of Emotional Responses to Olfactory and Contextual Stimuli: A Potential Model for the Development and Expression of Chemical Intolerance.
The role of corticotropin-releasing factor-norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress.
Persistent changes in corticotropin-releasing factor systems due to early life stress: relationship to the pathophysiology of major depression and posttraumatic stress disorder.
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