BEHAVIORAL AND HORMONAL EFFECTS OF CHRONIC RESTRAINT STRESS IN ADOLESCENT AND ADULT RATS Hansen C, Virgolini MB, De Giovanni L, Miranda-Morales RS, Willie-Billie A, Acevedo MB, Pautassi RM. Instituto de Investigación Médica M. y M. Ferreyra (INIMEC – CONICET-UNC), Instituto de Farmacología Experimental de Córdoba (IFEC-CONICET), Córdoba, Argentina; Departamento de Farmacología, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina. Laboratorio de Análisis Clínicos Especializados (LACE), Córdoba, Argentina. INTRODUCTION It is important to assess age-related differences in sensitivity to stress that may facilitate adolescent ethanol drinking. We have previously observed that 5 days of restraint stress enhance alcohol (i.e., ethanol) intake in adolescent but not in adult rats (see Figure 1). It is thus important to analyze age-related differences in response to restraint stress (RS), which may explain the higher predisposition to stress-induced ethanol drinking in adolescents. This experiment measured anxiety response in a light-dark box (LDB) test as well as stress-induced corticosterone (CORT) levels in adolescent and adult Wistar rats that had been exposed to immobilization (restraint) stress (120 min of restraint a day, for 5 days; or non-stressed). Methods: Animals were given daily RS sessions (120 min each, between 9 00 to 11 00 AM) on PD30 to 34, or 70 to 74 (adolescent and adult groups, respectively) or remain undisturbed in their home cage (non-stressed control group). Three days after termination of the last stressor, animals were assessed for anxiety response in a light-dark test (5 min) and then exposed for 5 min to inescapable stress [confinement in the white section of the light-dark box (LDB) with illumination of 1200 lux]. Blood samples were collected 90 minutes before LDB test (i.e., baseline), via tail tip bleeds, and immediately after termination of the inescapable stress (trunk blood samples). These samples were used for CORT levels measurement. Results: Chronic RS affected later behavioral response to acute stress (i.e., immobilization was increased) in adults (t22 = -2.98, p < 0.01) but not in adolescents; whereas adolescent, but not adult, rats exhibit lower baseline levels of two anxiety-like responses in the LDB as a function of prior RS. Stressed adolescents spent more time in the bright compartment [t22 = 2.09, p<0.05] and moved significantly more often between compartments [t22 = 2.31, p<0.05] than control, non-stressed adolescents. These results have been depicted in Figure 2. Corticosterone release (see Figure 3) was significantly enhanced by acute stress exposure (F1,21 = 60.20, p <0.001 and F1,21 = 77.86, p <0.001; for adolescent and adults, respectively). This response was fairly similar in both ages and in rats exposed or not to chronic stress, although there was a trend for lower baseline corticosterone scores in adolescents given chronic RS. Figure 1. Ethanol intake in adolescent and adult rats after restraint stress. Absolute ethanol intake (g/kg) and percent ethanol preference scores (upper and right panels, respectively) in adolescent and adult Wistar rats as a function of day of assessment (intake test sessions 1, 2, 3 and 4) and stress treatment experienced during postnatal days 30-34 or 70-74 [5 days of restraint stress (120 min per day) or non-stressed]. In each daily intake test (duration: 120 min) animals had access to a bottle of water and a bottle of ethanol (ethanol concentration: 3, 4, 5 or 6% v/v, sessions 1 to 4; respectively). The asterisk indicates that stress-exposed adolescents drank significantly more ethanol than non-stress counterparts during the second testing day. The pound sign indicates a significant main effect of stress exposure on ethanol percent preference in adolescents. Vertical lines indicate SEM. the SEM Figure 2: Baseline and acute-stress induced anxiety response in adolescent and adult rats. Upper panels. Response in a 5-min light-dark box (LDB) test [time spent in the white area (s), latency to escape to dark area (s) and frequency of transfers between compartments, panel A, B and C, respectively], in adolescent and adult Wistar rats as a function of stress treatment experienced during postnatal days 30-34 or 70-74 [5 days of restraint stress (120 min per day) or un-manipulated]. Lower panels. Immediately after the light-dark test animals were exposed for 5 min to inescapable acute stress (confinement in the white area of the LDB, illuminated with 1200 lux). The panels depict locomotor activity (s), frequency of wall-climbing and number fecal boli during acute stress exposure (panels D, E and F; respectively). The asterisk indicates a significant difference between a group exposed to chronic stress and its age-specific control group. Vertical lines indicate SEM. Figure 3: Baseline and acute-stress induced hormonal response in adolescent and adult rats. Corticosterone response (ng/ml) in 38-day old adolescent and 78-day old adult Wistar rats before (i.e., baseline) and immediately after a 5 min exposure to inescapable acute stress (confinement in the white area of a light-dark box, illuminated with 1200 lux). Animals had experienced or not chronic stress treatment during postnatal days 30-34 or 70-74 [5 days of restraint stress (120 min per day) or un-manipulated].The asterisk indicates a significant difference between a group exposed to chronic stress and its age-specific control group. Vertical lines indicate SEM. CONCLUSIONS The results are suggestive of stress-induced increases in impulsivity or inadequate risk assessment in adolescents, which may put them at risk for alcohol and drug initiation. Corticosterone release was significantly enhanced by acute stress exposure. This response was similar at both ages and in rats exposed or not to the chronic stressor.