Development of Threat Expression Following Infant Maltreatment: Infant and Adult Enhancement but Adolescent Attenuation

Abstract

Early life maltreatment by the caregiver constitutes a major risk factor for the development of later-life psychopathologies, including fear-related pathologies. Here, we used an animal model of early life maltreatment induced by the Scarcity-Adversity Model of low bedding (LB) where the mother is given insufficient bedding for nest building while rat pups were postnatal days (PN) 8-12. To assess effects of maltreatment on the expression of threat-elicited defensive behaviors, animals underwent odor-shock threat conditioning at three developmental stages: late infancy (PN18), adolescence (PN45) or adulthood (>PN75) and tested the next day with odor only presentations (cue test). Results showed that in typically developing rats, the response to threat increases with maturation, although experience with maltreatment in early infancy produced enhanced responding to threat in infancy and adulthood, but a decrease in maltreated adolescents. To better understand the unique features of this decreased threat responding in adolescence, c-Fos expression was assessed within the amygdala and ventromedial prefrontal cortex (vmPFC) associated with the cued expression of threat learning. Fos counts across amygdala subregions were lower in LB rats compared to controls, while enhanced c-Fos expression was observed in the vmPFC prelimbic cortex (PL). Correlational analysis between freezing behavior and Fos revealed freezing levels were correlated with CeA in controls, although more global correlations were detected in LB-reared rats, including the BA, LA, and CeA. Functional connectivity analysis between brain regions showed that LB reared rats exhibited more diffuse interconnectivity across amygdala subnuclei, compared the more heterogeneous patterns observed in controls. In addition, functional connectivity between the IL and LA switched from positive to negative in abused adolescents. Overall, these results suggest that in adolescence, the unique developmental decrease in fear expression following trauma is associated with distinct changes in regional function and long-range connectivity, reminiscent of pathological brain function. These results suggest that early life maltreatment from the caregiver perturbs the developmental trajectory of threat-elicited behavior. Indeed, it is possible that this form of trauma, where the infant's safety signal or "safe haven" (the caregiver) is actually the source of the threat, produces distinct outcomes across development.

Keywords: amygdala; expression threat; infant; learned fear; maltreatment; medial prefrontal cortex; trauma.

FIGURE 1

Schematic of methodology and experimental timeline. From PN8-12, pups were exposed to either Scarcity-Adversity Model of low bedding (LB) rearing or control rearing from the mother. For LB rearing, the mother was provided with insufficient bedding for nest building, which resulted in maltreatment of pups but growth similar to controls. Pups are odor-shock conditioned at one age, either during infancy (PN18) adolescence (PN45) or adulthood (>PN75). All animals were tested in a cue test the next day; in adolescents, neural responses following cue testing were assessed with c-Fos.

FIGURE 2

Low bedding induces maternal maltreatment of pups. (A) Pups were exposed to either Scarcity-Adversity Model of low bedding (LB) rearing or control rearing from postnatal days (PN)8-12. LB rearing involved providing the mother with insufficient bedding for nest building, which produces maltreatment of pups and increased pup vocalizations (B) but growth indistinguishable from controls. Maltreatment was verified by repeated observations of maternal behaviors categorized as rough handling, such as stepping on pups, transporting pups by a body area other than the nape of the neck (i.e., leg), or roughly moving pups within the nest.

FIGURE 3

Acquisition and expression of learned threat across development. During conditioning, responses to the CS increased over consecutive CS-US pairings, a pattern indicative of learning. No statistical difference was seen between LB and control rearing as suggested by rate of learning for infants and adults (A,C), although maltreated adolescents showed some retardation of learning (B). Cue testing 24 h later in a novel environment was analyzed for percent time spent freezing to the CS at the three ages. All paired animals (control and LB) were significantly higher than unpaired and odor-only controls, although freezing to cues following maltreatment increased in infancy (D), decreased in adolescence (E) and increased in adults (F), relative to control-reared animals. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Amygdala and prefrontal cortex activation following memory retrieval at 24 h in adolescent animals. (A) Amygdala subnuclei activation following fear retrieval at 24 h in both control (black) and abused (red) animals; LA: lateral nucleus, B: basal nucleus, CeA: central nucleus, CoA: cortical nucleus, and MeA: medial nucleus. (B) Prefrontal cortex activation following fear memory retrieval at 24 h in both normal (black) and abused (red) animals; PL: prelimbic cortex, IL: infralimibic cortex. (C,D) Bivariate correlation matrices were contructed across regions assessed for c-Fos levels after cue test during adolescents following control or low bedding rearing. White squares denote example nodules (groups of regions) that show dramatic changes in connectivity across condition. Color bar shows Pearson’s r values with positive correlations in red and negative correlations in blue. For statistical comparisons, r values were converted to z scores via Fisher transform and compared using ANOVA. Asterisks indicate pairwise correlations that significantly differed between LB and control. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^#p = 0.05.

FIGURE 5

Correlations between cued threat response and ROI activation during adolescence. Correlations were computed between time spent freezing to CS and c-Fos levels for control-reared and maltreated (LB) pups. Pearson’s r values statistically different from 0 are. ^∗p < 0.05, ^∗∗p < 0.01.