Long-term behavioral consequences of brief, repeated neonatal isolation

Abstract

Rats subjected to stressful stimuli during the stress hyporesponsive period exhibit varied neuroendocrine and behavioral changes as neonates, adolescents and adults. The current work examined the effects of neonatal isolation stress, using a within-litter design, on adult anxiety-related behavior and endocrine stress reactivity. Neonatal rats were isolated daily for 1 h from postnatal day (P) 4 to 9, a manipulation previously shown to induce hypothalamic-pituitary-adrenal (HPA) responses on P9 (Knuth, E.D., Etgen, A.M. (2005) Corticosterone secretion induced by chronic isolation in neonatal rats is sexually dimorphic and accompanied by elevated ACTH. Horm Behav 47:65-75.). Control animals were either handled briefly or left undisturbed (with-dam). Adult rats were tested for anxiety-related behavior using the elevated plus maze and open field, and for endocrine responses following restraint stress. Neonatal isolation decreased center exploration of the open field following 1 h restraint, including decreased time in the center compared to with-dam or handled controls and decreased center entries and distance traveled in the center compared to with-dam controls. It also decreased time in and entries into the open arms of the elevated plus maze compared to handled controls, suggesting enhanced anxiety-related behavior. Neonatal isolation had no effect on basal or restraint-induced levels of ACTH or corticosterone. These findings indicate that neonatal isolation may enhance anxiety-related behaviors, especially in response to stress, without altering HPA function.

Figure 1

Behavior in the EPM. Behavior in the EPM was recorded for 5 min in adult rats that were isolated, handled, or with-dam on P4-9. Neonatal isolation significantly decreased the fraction of time spent in open arms (open/open+closed) (a), as well as open arm entries (b) compared to handled rats. Handled rats also make a higher proportion of open arm entries than with-dam rats (b). Data were collapsed across sex because there were no significant differences between males and females. ^* significantly different from handled controls (p<0.05); ^** significantly different from with-dam controls (p<0.05). Values represent mean ± SEM. n=16-19 per group.

Figure 2

General motor activity in the open field following stressful challenge (1 hr restraint) in adult rats that were isolated, handled, or with-dam on P4-9. Adult rats were challenged with 1 hr restraint stress prior to being placed in the open field for 15 min. Neonatal stress condition had no influence on total distance traveled (a) or average velocity (b) in the open field after stressful challenge. —^*— denotes a main effect of sex (p<0.0001). Values represent mean ± SEM for the 15-min test period. n=8-10 per group.

Figure 3

Anxiety-related behavior in the open field following a stressful challenge. Activity in the center of an open field and rearing were recorded following 1 hr restraint in adult rats that were isolated, handled, or with-dam on P4-9. Neonatal isolation significantly decreased time in the center compared to handled and with-dam rats (a), grid crosses to the center compared to with-dam rats (b), and distance traveled in the center compared to with-dam rats (c). Isolated females exhibited less exploratory activity than with-dam females (d). ^* significantly different from handled controls (p<0.05); ^** significantly different from with-dam controls (p<0.05). —^*— denotes a significant effect of sex (p<0.05). Values represent mean ± SEM for the 15-min test period. n=8-10 per group.

Figure 4

Neonatal isolation did not influence basal or stress-induced plasma corticosterone. Basal and stress-induced levels of plasma corticosterone were measured in adult rats that were isolated (black circles), handled (gray squares), or with-dam (open triangles) on P4-9. Restraint stress (20 min) increased corticosterone in females (a) and males (b) immediately after restraint (0 min), and levels remained high for 30 min after release from restraint (^*p<0.0001 vs. basal). Females had more corticosterone than males at 0 min (p<0.0001). Females handled as neonates had higher integrated corticosterone secretion than with-dam females (a, inset, ^**p<0.05), an effect not detected in males (b, inset). Values represent mean ± SEM. n=5-7 per group. AUC values expressed as ng/ml•min.

Figure 5

Neonatal isolation did not influence baseline or stress-induced plasma ACTH. Basal and stress-induced levels of plasma ACTH were measured in adult rats that were isolated (black circles), handled (gray squares), or with-dam (open triangles) on P4-9. Restraint stress (20 min) increased ACTH in females (a) and males (b) immediately after restraint (0 min), and levels remained high for 30 min after release from restraint (^*p<0.001 vs. basal). Females and males displayed equivalent plasma ACTH at all time points, as well as equivalent integrated ACTH release across neonatal stress groups (insets). Values represent mean ± SEM. n=5-7 per group. AUC values expressed as pg/ml•min.