Enriched early life experiences reduce adult anxiety-like behavior in rats: a role for insulin-like growth factor 1

Abstract

Early life experiences can affect brain development, contributing to shape interindividual differences in stress vulnerability and anxiety-like behavior. In rodents, high levels of maternal care have long-lasting positive effects on the behavior of the offspring and stress response; post-weaning rearing in an enriched environment (EE) or massage counteract the negative effects of maternal separation or prenatal stressors. We recently found that insulin-like growth factor 1 (IGF-1) is a key mediator of early EE or massage on brain development. Whether early enrichment of experience can induce long-lasting effects on anxiety-like behavior and whether IGF-1 is involved in these effects is not known. We assessed anxiety-like behavior by means of the elevated plus maze in control adult rats and in adult rats subjected to early EE or to massage. We found that both EE and massage reduced adult anxiety-like behavior. Early IGF-1 systemic injections in rat pups reared in standard condition mimic the effects of EE and massage, reducing anxiety-like behavior in the adult; blocking early IGF-1 action in massaged and EE animals prevents massage and EE effects. In EE and IGF-1-treated animals, we assessed the hippocampal expression of glucocorticoid receptors (GRs) at postnatal day 12 (P12) and P60, finding a significantly higher GR expression at P60 for both treatments. These results suggest that IGF-1 could be involved in mediating the long-lasting effects of early life experiences on vulnerability/resilience to stress in adults.

Figure 1.

Early postnatal enriched experience is sufficient to affect adult anxiety-like behavior. A, Experimental protocols. B, Examples of movement tracking in the EPM for one EE up to P12 animal (EE), one MSG animal, and one CNT animal. C, D, Mean percentage of time spent in the open arms (C) and mean percentage of entries in the open arms (D) assessed at P60 for CNT, MSG, EE–P12, EE–P45, and EE–P60 rats. All treated groups significantly differ from the CNT group (asterisks, p < 0.01), but they do not differ one from each other (one-way ANOVA). E, F, Vertical bars are SEM. Mean total arm entries (E) and mean total distance moved (F) for all experimental groups. No difference was found between groups (one-way ANOVA). Vertical bars are SEM.

Figure 2.

Early postnatal IGF-1 subcutaneous administration decreases adult anxiety-like behavior. A, Experimental protocol. B, C, Mean percentage of time spent in the open arms (B) and mean percentage of entries in the open arms (C) at P60 for IGF-1-treated, Veh-treated, and CNT rats. CNT and Veh groups differ from IGF-1-treated group (asterisks, p < 0.05); the former two groups do not differ (one-way ANOVA). Vertical bars are SEM. D1, D2, Mean total arm entries (D1) and mean distance moved (D2) during the test for all experimental groups. No difference was found between groups (one-way ANOVA). Vertical bars are SEM.

Figure 3.

Molecular mechanisms underlying EE and IGF-1 action on anxiety-like behavior: Western blot analysis of hippocampal IGF-1R, GR, and BDNF levels at P12 and P60. In all panels, the inset is a representative immunoblotting showing the corresponding protein levels in the hippocampus of animals in the different experimental groups [animals reared in standard conditions (CNT), systemically injected with saline (Veh), reared in enriched conditions up to P45 (EE), and reared in standard conditions but subcutaneously treated with IGF-1 (IGF-1)]. α-Tubulin (α-TUB) is the internal standard. A–F, Quantification of protein levels; data are mean ± SEM. Asterisks denote significant differences (p < 0.05). A, Quantification of IGF-1R levels at P12. In animals reared in EE conditions and in those treated with IGF-1, the amount of IGF-1R is significantly higher than in CNT animals, whereas saline injections have no effects (one-way ANOVA). B, Quantification of GR levels at P12. At this developmental stage, there is no difference among experimental groups (one-way ANOVA). C, Quantification of BDNF levels at P12. No difference among experimental groups is detectable (one-way ANOVA). D, Quantification of IGF-1R levels at P60. In animals reared in EE conditions, the hippocampal expression of IGF-1R is significantly higher than in CNT animals (one-way ANOVA). E, Quantification of GR levels at P60. In animals reared in EE conditions and in those treated with IGF-1, the hippocampal expression of GR is significantly higher than in CNT animals, whereas saline injections do not affect the amount of GR protein (one-way ANOVA). F, Quantification of BDNF levels at P60. In animals reared in EE conditions, the hippocampal expression of BDNF is significantly higher than in CNT animals (one-way ANOVA).

Figure 4.

Early IGF-1 blockade prevents the effects of early postnatal massage and EE on adult anxiety-like behavior. A, Experimental protocols. B, C, Mean percentage of time spent in the open arms (B) and mean percentage of entries in the open arms (C) at P60 for MSG and EE rats treated with JB1, for MSG and EE rats treated with Veh, and for CNT rats. CNT or JB1-treated rats differ from Veh-treated EE or MSG rats (asterisks, p < 0.001; §p = 0.01); CNT and JB1-treated groups do not differ (one-way ANOVA). Vertical bars are SEM. D1, D2, Mean total arm entries (D1) and mean distance moved (D2) for all experimental groups during the test session. No difference was found between groups (one-way ANOVA). Vertical bars are SEM.