Summary of the experimental procedure.

<p>CS: conditioned stimulus; CTR: control; ELS: early-life stress; IMO: immobilization; PND: postnatal day; US: unconditioned stimulus.</p

Sign-tracking and goal-tracking behavior in day 1.

<p>Time (s) spent in behavior towards the lever (left panels) and time (s) spent inside the magazine during lever presentation (right panels) for day 1. Results are shown for control (CTR) and early-life stress (ELS) rats of both sexes, exposed to immobilization (IMO) stress or not when adults. && p < 0.01 versus non-IMO. The same scale as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190044#pone.0190044.g005" target="_blank">Fig 5</a> is maintained to allow comparisons between days 1 and 5. In the insets, the same results with different scale are provided. Means and SEM are shown.</p

Types of maternal behavior.

<p>Distribution of different types of maternal behavior in control (CTR) and early-life stress (ELS) dams during PND 2 to 8.</p

Expression of dopaminergic markers.

<p>Tyrosine hydroxylase (TH) expression in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNpc) and D1 dopamine receptors in the nucleus accumbens shell (AcbS) in arbitrary units (AU). The results are shown for control (CTR) and early-life stress (ELS) rats of both sexes, exposed to immobilization (IMO) stress or not when adults. * p < 0.05 versus corresponding CTR; + p < 0.05 versus corresponding males; & p < 0.05, && p < 0.01 versus corresponding non-IMO. Means and SEM are shown.</p

Magazine entries.

<p>Magazine entries during the ITI (left panels) and magazine entries during pellet delivery (right panels) for day 5. Results are shown for control (CTR) and early-life stress (ELS) rats of both sexes, exposed to immobilization (IMO) stress or not when adults. * p < 0.05 versus corresponding CTR. Means and SEM are shown.</p

Sex-dependent impact of early-life stress and adult immobilization in the attribution of incentive salience in rats

<div><p>Early life stress (ELS) induces long-term effects in later functioning and interacts with further exposure to other stressors in adulthood to shape our responsiveness to reward-related cues. The attribution of incentive salience to food-related cues may be modulated by previous and current exposures to stressors in a sex-dependent manner. We hypothesized from human data that exposure to a traumatic (severe) adult stressor will decrease the attribution of incentive salience to reward-associated cues, especially in females, because these effects are modulated by previous ELS. To study these factors in Long-Evans rats, we used as an ELS model of restriction of nesting material and concurrently evaluated maternal care. In adulthood, the offspring of both sexes were exposed to acute immobilization (IMO), and several days after, a Pavlovian conditioning procedure was used to assess the incentive salience of food-related cues. Some rats developed more attraction to the cue predictive of reward (sign-tracking) and others were attracted to the location of the reward itself, the food-magazine (goal-tracking). Several dopaminergic markers were evaluated by in situ hybridization. The results showed that ELS increased maternal care and decreased body weight gain (only in females). Regarding incentive salience, in absolute control animals, females presented slightly greater sign-tracking behavior than males. Non-ELS male rats exposed to IMO showed a bias towards goal-tracking, whereas in females, IMO produced a bias towards sign-tracking. Animals of both sexes not exposed to IMO displayed an intermediate phenotype. ELS in IMO-treated females was able to reduce sign-tracking and decrease tyrosine hydroxylase expression in the ventral tegmental area and dopamine D1 receptor expression in the accumbens shell. Although the predicted greater decrease in females in sign-tracking after IMO exposure was not corroborated by the data, the results highlight the idea that sex is an important factor in the study of the long-term impact of early and adult stressors.</p></div

Maternal behavior measures.

<p>Left panels: Number of episodes of arched-back (A), licking-grooming (B) and “off” nest (C) behaviors during postnatal days (PND) 2 to 8, and on PND 13 and PND 18 (sum of all the times of the day analyzed). Right panels: Number of episodes of licking-grooming (D), arched-back (E) and off-nest (F) behaviors at 4 different times of day (sum of PND 2 to 8). The results are shown for control (CTR) and early-life stress (ELS) dams. ** p < 0.01 and *** p < 0.001 versus CTR. Means and SEM are shown.</p

Motor activity (cm) inside the chamber for day 1 and 5.

<p>Results are shown for control (CTR) and early-life stress (ELS) rats of both sexes, exposed to immobilization (IMO) stress or not when adults. ** p < 0.01 versus corresponding CTR; + p < 0.05, ++ p < 0.01, +++ p < 0.001 versus corresponding males; && p < 0.01 versus corresponding non-IMO. Means and SEM are shown.</p

Sign-tracking (ST)–Goal-tracking (GT) score for day 5.

<p>A positive score indicates that ST behavior predominates over GT, whereas a negative score indicates that GT behavior predominates over ST. The results are shown for control (CTR) and early-life stress (ELS) rats of both sexes, exposed to immobilization (IMO) stress or not when adults. ** p < 0.01 versus corresponding CTR; +++ p < 0.001 versus corresponding males. When compared against a “zero” value, the score was only statistically significant for IMO males and females not exposed to ELS ($ p < 0.05; &&& p < 0.001). Means and SEM are shown.</p

Functionality of dopamine D₄ receptors in pineal gland and pinealocytes.

<p>Pineal glands extracted at 9:00 h were treated for 10 min with increasing amounts of dopamine or with 1 µM of RO 10-5824 (RO). The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A) and Akt (Ser⁴⁷³) phosphorylation (B), of two separate experiments performed in duplicate were quantified and values represent the mean ± S.D. of the fold increase relative to basal levels found in untreated cells. Significant differences with respect to basal levels were determined by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001). A representative Western blot is shown at the top (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). (C) Pinealocytes were isolated from pineal glands extracted at 9:00 h and were treated with medium (Control), 1 µM of RO 10-5824 (RO), 1 µM phenylephrine (Phenyl), or 1 µM isoproterenol (Iso) for 10 min before labeling with anti-S-arrestin (green) and anti-phospho-ERK1/2 (red), as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cell nuclei were stained with DAPI (blue). Scale bar, 5 µm.</p