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Antidepressant Treatment Can Normalize Adult Behavioral Deficits Induced by Early-Life Exposure to Methylphenidate
Antidepressant Treatment Can Normalize Adult Behavioral Deficits Induced by Early-Life Exposure to Methylphenidate Carlos A. Bolaños, Matthew D. Willey, Melissa L. Maffeo, Kyle D. Powers, Daniel W. Kinka, Katie B. Grausam, and Ross P. Henderson Background: Methylphenidate (MPH) is prescribed for the treatment of attention-deficit/hyperactivity disorder. Exposure to MPH before adulthood causes behavioral deficits later in life, including anxiety- and depression-like behaviors and decreased responding to natural and drug rewards. We examined the ability of fluoxetine (FLX), a selective serotonin reuptake blocker, to normalize these MPH-induced behavioral deficits. Methods: Male rats received MPH (2.0 mg/kg) or saline (VEH) during preadolescence (postnatal day [PD] 20 –35). When adults, rats were divided into groups receiving no treatment, acute or chronic FLX, and behavioral reactivity to several emotion-eliciting stimuli were assessed. Results: The MPH-treated rats were significantly less responsive to natural (i.e., sucrose) and drug (i.e., morphine) rewards and more sensitive to stress- and anxiety-eliciting situations. These MPH-induced deficits were reversed by exposure to FLX. Conclusions: These results indicate that exposure to MPH during preadolescence leads to behavioral alterations that endure into adulthood and that these behavioral deficits can be normalized by antidepressant treatment. These results highlight the need for further research to better understand the effects of stimulants on the developing nervous system and the potential enduring effects resulting from early-life drug exposure. Key Words: Adolescence, antidepressant treatment, development, emotion, fluoxetine, methylphenidate, morphine, rat
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timulant medications are effective for the treatment of attention-deficit/hyperactivity disorder (ADHD) (1,2), and methylphenidate (MPH) is the most prescribed therapeutic agent for ADHD in the United States (3–5). Although ADHD can be difficult to diagnose (6,7), prescription use of stimulants for its treatment has risen within the past decade (8,9), thus increasing the likelihood for stimulant exposure in children who do not meet ADHD criteria (8,10). Despite increases in stimulant prescriptions and the fact that affected children may be treated with MPH for years (11,12), little is known about the long-term neurobiological adaptations resulting from MPH exposure during juvenile periods. Basic and clinical studies show that MPH has psychomotor stimulant-like properties similar to those observed with cocaine and amphetamine (13,14). Methylphenidate blocks the dopamine transporter (15), increases dopamine levels in brain reward pathways (16,17), and enhances locomotion after its repeated administration (18,19). Because exposure to stimulants can result in long-lasting cellular, molecular, and behavioral adaptations implicated in the transition from drug use to abuse (13,20), most research has focused on assessing MPH’s potential to predispose individuals toward drug addiction (21,22). Longitudinal studies indicate that children treated with MPH may not be at risk of developing substance abuse disorders (23,24). In line with these findings, studies show that exposing rodents to MPH during preadolescence results in decreased
From the Department of Psychology and Program in Neuroscience, Florida State University, Tallahassee. Address reprint requests to Carlos A. Bolaños, Ph.D., Department of Psychology and Program in Neuroscience, Florida State University, 1107 West Call Street, Tallahassee, FL 32306-4301; e-mail: [email protected]. Received January 24, 2007; revised June 1, 2007; accepted June 23, 2007.
0006-3223/08/$34.00 doi:10.1016/j.biopsych.2007.06.024
sensitivity to cocaine during adulthood (25–27). Interestingly, these rats also show increases in depression-like behaviors manifested in enhanced vulnerability to stressful environments and anxiety-eliciting situations, as well as reduced reactivity to natural rewards (27,28). Given the degree of prescribed use of MPH and reports indicating that in some pediatric populations up to 66% of those treated with stimulants for ADHD do not meet the criteria for this disorder (8,10,29), it is essential that the long-term neurobiological consequences associated with developmental psychostimulant exposure be characterized. Accordingly, the studies presented here were designed to assess whether the long-term behavioral effects induced by early-life MPH exposure could be reversed by subsequent antidepressant treatment during adulthood. To this end, we treated rats with MPH or saline during preadolescence (postnatal day [PD] 20 –35). After treatment, rats were left undisturbed until adulthood (PD90⫹), at which point they were separated into several groups: those receiving no treatment, and those subsequently exposed to fluoxetine before assessing their behavioral reactivity to various emotion-eliciting stimuli, including responsivity to the rewarding effects of morphine.
Methods and Materials Drugs Methylphenidate hydrochloride, fluoxetine hydrochloride (FLX), and morphine sulfate were obtained from Sigma (St. Louis, Missouri). Each drug was dissolved in .9% saline (VEH) and administered in a volume of 1 mL/kg for MPH and morphine and 2mL/kg for FLX. Doses are based on the salt form of each drug. Subjects and Drug Treatments Litters containing Sprague-Dawley male rat pups with their dams were obtained from Charles River Laboratories (Raleigh, North Carolina) at PD14. Rats were housed in clear polyproBIOL PSYCHIATRY 2008;63:309 –316 © 2008 Society of Biological Psychiatry
310 BIOL PSYCHIATRY 2008;63:309 –316 pylene boxes containing wood shavings in an animal colony maintained at 23–25°C on a 12-hour light-dark cycle in which lights were on between 7:00 and 19:00 hour. Injections of MPH (2.0 mg/kg, intraperitoneally [IP]) or VEH were given to pups starting on PD20 twice daily (4 hours apart) for 15 days. From PD20 to PD23, MPH- and VEH-treated rats were kept together in same cage with their dams. At PD24 (weaning), rats were separated by treatment condition in groups of four per cage. At PD45, rats were further separated into groups of two per cage and were left undisturbed until adulthood. Testing started 8 weeks after the last MPH injection. In addition, separate groups of rats were divided into subgroups receiving acute (one injection) or chronic (one daily injection for 7 days) FLX (5.0 or 10.0 mg/kg IP), 8 weeks after MPH exposure. Testing began 24 hours, 1 week, or 4 weeks after the last FLX injection (see Supplement 1). The period between PD20 and PD35 in rats was chosen because it approximates preadolescence (i.e., age 4 –12 years) in humans (30). The dose of MPH chosen approximates clinically relevant doses in pediatric patient population (31,32). All behaviors, except for place conditioning (CPP) and sucrose preference, were recorded with a video camera located on the ceiling of separate testing rooms. Behavioral observations and analyses were done by observers with no knowledge of the treatment conditions of each rat. Rats were provided with food and water ad libitum. Experiments were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, 1996) and approved by Florida State University animal care and use committee. Sucrose Preference The sucrose preference test consisted of a two-bottle choice paradigm. In this test, rats are given the choice between consuming water versus sucrose. This paradigm has been used extensively to assess the effects of stress-induced anhedonia (33). Rats were habituated to drink water from two bottles for 5 days. At the start of the experiment, rats were exposed to ascending concentrations of sucrose (.0, and .25–1%) for 2 days per sucrose concentration. Water and sucrose consumption were measured at 8:00 and 17:00 hours each testing day at which time the position of the sucrose bottle (left or right) was balanced between the MPH and VEH groups. The preference for sucrose over water was used as a measure for rats’ sensitivity to reward. Place Preference Conditioning Place conditioning (CPP) was carried out as described previously (27). This paradigm has been widely used to assess the rewarding or aversive properties of drugs. In this behavioral assay, rats learn to prefer environments previously associated with rewarding drug effects while avoiding environments associated with aversive drug effects (34,35). Briefly, CPP to morphine was performed in a three-compartment apparatus (FSU Psychology engineering group). Compartments differed in floor texture, wall coloring, and lightning. On the preconditioning day (day 0), rats were allowed to explore the entire apparatus freely for 30 min to obtain baseline preference to any of the three compartments (side compartments: 35 ⫻ 27 ⫻ 25 cm; middle compartment: 10 ⫻ 27 ⫻ 25 cm, L ⫻ W ⫻ H). Only rats showing no preference (before drug training) to either side compartment were used (see Supplement 2); this accounted for more than 90% of all of the rats tested. Conditioning trials occurred on 2 consecutive days (days 1 and 2). During conditioning, rats received VEH (1.0 mL/kg, subcutaneously [SC]) and were confined to one of the side compartments of the apparatus for www.sobp.org/journal
C.A. Bolaños et al. 1 hour. After 3 hours, rats received morphine (0, .25, .50, or 1.0 mg/kg SC) and were confined to the opposite side compartment for 1 hour. On the test day (day 3), rats received VEH (SC) and were again allowed to explore the entire apparatus freely for 30 min; the time spent in the drug-paired compartment(s) was assessed. Elevated-Plus Maze and Self-Grooming Behavior The MPH- and VEH-treated rats were tested for 5 min on the elevated-plus maze (EPM), a behavioral model of anxiety-like behavior. The maze was made of gray plastic and consisted of two perpendicular, intersecting runways (12 cm wide ⫻ 100 cm long; FSU Psychology engineering group). One runway had tall walls (40 cm high), or “closed arms,” and the other had no walls, or “open arms.” The arms were connected together by a central area, and the maze was elevated 1 m from the floor. Testing was conducted between 9:00 and 13:00 hours under controlled light conditions (⬃90 lux). At the beginning of the test, rats were placed in the central area, facing one of the open arms, and the cumulative time spent in the open arms was videotaped. We also assessed self-grooming in the “closed arms” because rats engage in repetitive grooming in response to anxiogenic stimuli (36). Forced Swimming The forced swim test (FST) is a 2-day procedure in which rats are forced to swim under conditions in which they cannot escape. On the first day, rats are forced to swim. Initially, they engage in escapelike behaviors but eventually adopt a posture of immobility in which they make only the movements necessary to maintain their head above water. When retested 24 hours later, rats become immobile more quickly; however, antidepressant treatment between the forced swim exposures can significantly increase their escapelike behaviors, an effect that has been correlated with antidepressant activity in humans (37). At the start of the experiment, rats were placed in plastic cylinders (30 ⫻ 45 cm) filled to 30 cm depth (so that their paws and tail do not touch the bottom) with 25°C water and forced to swim for 15 min. At the end of this period, rats were removed from the water, dried with towels, and kept in a warm enclosure for 30 min. All cylinders were emptied and cleaned between rats. Twenty-four hours after the forced swim, rats were retested for 5 min under identical conditions, and sessions were videotaped. In this study, the latency to become immobile was the dependent variable. Latency to immobility was defined as the time at which the rat first initiated a stationary posture that did not reflect attempts to escape from the water (38). To qualify as immobility, this posture had to be clearly visible and maintained for ⱖ2.0 sec. We also assessed immobility, swimming, and climbing counts in the FST (see Supplement 3). Statistical Analysis Statistical significance was measured using mixed-design (between and within variables) analysis of variance (ANOVA) followed by Tukey post hoc test. When appropriate, Student’s t and F tests were used to determine statistical significance of preplanned comparisons. Data are expressed as the mean ⫾ SEM. Statistical significance was defined as p ⬍ .05.
Results Effects of MPH and Subsequent FLX Exposure on Sucrose Preference in Adulthood Overall repeated-measures ANOVA indicated that exposure to MPH did not affect the rats’ total fluid intake (water ⫹ sucrose;
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BIOL PSYCHIATRY 2008;63:309 –316 311 preference as a function of treatment frequency and dose [F (7,60) ⫽ 4.79; p ⫽ .0002], without affecting water intake. Acute FLX (5 or 10 mg/kg) did not reverse the effects of MPH on sucrose preference (Figure 1C and 1D). Although there was a trend toward decreases in sucrose preference in rats exposed to 10 mg/kg compared with 5 mg/kg FLX, this difference was not statistically significant. However, acute FLX (10 mg/kg) decreased preference for sucrose in the VEH group (Figure 1D) compared with the VEH-exposed rats from Figure 1A at the .5% solution. Rats repeatedly exposed to 5 mg/kg FLX showed a nonsignificant trend toward an increase in sucrose preference (VEH vs. MPH: p ⫽ .07), whereas exposure to 10 mg/kg FLX reversed the effects of early-life MPH exposure on sucrose preference.
Figure 1. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate adult responses to sucrose reward (n ⫽ 128). (A) Developmental exposure to MPH significantly decreased sensitivity to the rewarding effects of sucrose when compared to vehicle (VEH)-treated rats. (B) MPH pretreatment did not affect total fluid (sucrose ⫹ water) intake. *Significantly different at .25% (p ⫽ .001) and .50% sucrose (p ⫽ .018). (C and D) A single injection of FLX (5 or 10 mg/kg) did not reverse MPH-induced decreases on sucrose preference. (E) Rats treated with chronic FLX (5 mg/kg) show a trend († p ⫽ .078) toward reversal of MPH effects, whereas chronic exposure to 10 mg/kg FLX did reverse MPH-induced effects of sucrose preference (F). Data are presented as percentage preference between MPH- and VEH-treated rats.
Figure 1B) at any of the sucrose concentrations tested (p ⬎ .05). In addition, sucrose preference varied as a function of MPH treatment and sucrose concentration [interaction: F (5,290) ⫽ 10.52; p ⫽ .0001]. On the basis of previous observations (28), we hypothesized that MPH-treated rats would show a decrease in preference for sucrose compared with VEH-treated control rats (preplanned comparison). Unpaired t tests demonstrated that prior MPH treatment decreased preference for a .25 [t (58) ⫽ 4.15; p ⫽ .001], and .5% [t (58) ⫽ 2.42; p ⫽ .018] sucrose solution compared with the VEH group (Figure 1A). Differences in sucrose preference between groups were not present at the 1% concentration. Decreased sensitivity to rewarding stimuli can be interpreted as anhedonia (decreased ability to experience pleasure), a common symptom of depression. Thus, we assessed the ability of FLX to reverse MPH-induced effects on sucrose preference. Figure 1C–1F shows that FLX differentially regulated sucrose
Effects of MPH and Subsequent FLX Exposure on MorphineInduced Place Preference Conditioning in Adulthood No conditioning effects were observed in VEH- or MPHpretreated rats conditioned with saline (Figure 2A). Time spent in the morphine-paired compartments varied as a function of developmental [F (1,46) ⫽ 9.674; p ⫽ .0032] and morphine posttreatment [F (2,46) ⫽ 7.084; p ⫽ .002], respectively (Figure 2A). Rats treated with VEH during development spent significantly more time in environments paired with morphine (.25 [p ⫽ .06], .50 [p ⫽ .043], and 1.0 [p ⫽ .035] mg/kg), whereas rats exposed to MPH did not condition to morphine (p ⬎ .05). We assessed the ability of FLX (0, 5, or 10 mg/kg) to reverse MPH-induced effects on morphine conditioning. Figure 2B shows that time spent in morphine-paired (.5 mg/kg) environments varied as a function of developmental treatment [F (1,36) ⫽ 22.06; p ⫽ .0001]. Rats treated with VEH during development and subsequently exposed to acute FLX spent significantly more time in environments paired with .5 mg/kg morphine (0 [p ⫽ .0066], 5.0 [p ⫽ .0025], and 10 [p ⫽ .04] mg/kg FLX). However, acute FLX did not influence conditioning in MPH-pretreated rats because these showed no preference for the morphine-paired environments. Figure 2C shows time spent in morphine-paired compartments of rats treated with chronic FLX (0, 5, or 10 mg/kg). Times varied as a function of developmental treatment and FLX exposure [F (2,37) ⫽ 3.28; p ⫽ .05]. As expected, MPH rats chronically treated with .0 mg/kg FLX did not show CPP, and those treated with 5 mg/kg showed a weak trend (p ⫽ .09) toward developing morphine CPP. However, chronic exposure to 10 mg/kg FLX reversed the effects of MPH pretreatment on CPP (Figure 2C) because time spent in the morphine-paired compartments did not differ from time observed in the VEH-pretreated rats (p ⬎ .05). Effects of Withdrawal Time after Chronic FLX Exposure on Sucrose Preference and Morphine-Induced Place Preference Conditioning in Adulthood Rats exposed to chronic FLX (10 mg/kg) did not show significant differences in their preference for sucrose (.5 mg/kg) 1 week after FLX treatment (p ⬎ .05; Figure 3A). Although MPH-treated rats showed a tendency toward decreased sucrose preference 4 weeks after FLX treatment, this trend was not statistically significant (p ⬎ .05). Conversely, Figure 3B shows that CPP to morphine varied as a function of FLX (10 mg/kg) withdrawal [F (3,22) ⫽ 4.52; p ⫽ .013]. Specifically, VEH- and MPH-exposed rats readily conditioned to morphine-paired environments 1 week after the last FLX injection (Figure 3B). However, MPH-exposed rats tested for CPP 4 weeks after the last FLX injection did not reliably condition to the morphine-paired environments compared with VEH-exposed rats (p ⬍ .05; Figure 3B). In www.sobp.org/journal
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C.A. Bolaños et al. counts than VEH control rats [t(34) ⫽ 4.96; p ⫽ .0001; Figure 4B]. Acute FLX (5 or 10 mg/kg) did not reverse the effects of MPH on the EPM (Figure 4C and 4D). In fact, FLX, at 10 mg/kg, further decreased time spent in the open arms in VEH and MPH rats (p ⬍ .05, Figure 4D, compared with VEH and MPH groups in Figure 4A). Additionally, this dose of FLX increased self-grooming in VEH-treated rats (p ⬍ .05, Figure 4E, compared with VEH rats in Figure 4A). Chronic exposure to 5 mg/kg FLX eliminated the differences in time spent in the open arms between VEH and MPH groups (Figure 4F); however, time spent in open arms by the VEH group remained significantly lower than that observed by the VEH-treated group from Figure 4A (p ⬍ .05). Chronic exposure to 10 mg/kg FLX reversed the effects of MPH pretreatment on the EPM (Figure 4G), and time spent in the open arms by these rats was significantly higher than time observed in the MPH group from Figure 4A (p ⬍ .05). There were no differences in self-
Figure 2. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate morphine-induced place preference conditioning (n ⫽ 137). (A) Adult rats exposed to MPH during development spent significantly less time in morphine-paired environments, whereas rats exposed to vehicle (VEH) spent significantly more time in morphine-associated environments in a dose-dependent manner. (B) Exposure to acute FLX (5 or 10 mg/kg) did not reverse MPH-induced effects on morphine (.5 mg/kg) place conditioning, whereas VEH-pretreated rats do show robust morphine CPP. (C) MPH rats treated with chronic FLX (5 mg/kg) show a trend toward reversal of MPH-induced effects on place conditioning (†p ⫽ .090). Chronic exposure to the higher dose of FLX (10 mg/kg) reversed the behavioral effects induced by developmental MPH exposure on morphine (.5 mg/kg) place conditioning. Data are presented as time spent in the morphine-paired minus time spent in the VEH-paired compartments after conditioning (mean ⫾ SEM, in sec).
fact, the magnitude of morphine conditioning exhibited by these MPH rats was somewhat lower than the conditioning observed in the MPH rats in the 1 week withdrawal FLX condition (p ⫽ .057). Effects of FLX on MPH-Induced Anxiety-like Behaviors in the Elevated-Plus Maze We also studied the effects of MPH, and subsequent FLX treatment, on anxiety-like behavior using the EPM (Figure 4A– 4F). Time spent in the open arms of the EPM was differentially affected by drug treatment and exposure frequency [F (9,96) ⫽ 4.98; p ⫽ .0001]. Figure 4A shows that MPH rats spent significantly less time in the open arms than the VEH control rats [t (34) ⫽ ⫺2.73; p ⫽ .01]. In addition, the MPH group had higher self-grooming www.sobp.org/journal
Figure 3. Sucrose preference (n ⫽ 20) and morphine-induced place conditioning (n ⫽ 26) in vehicle (VEH)- and methylphenidate (MPH)-treated rats 1 and 4 weeks after the last FLX injection. (A) Both VEH- and MPH-exposed rats show no significant differences in sucrose preference (.5%) 1 or 4 weeks after chronic FLX (10 mg/kg). (B) VEH- and MPH-treated rats readily conditioned to environments previously paired with morphine (.5 mg/kg) 1 week after the last FLX exposure (left panel), whereas animals conditioned 4 weeks after the last FLX injection did not show reliable place conditioning to morphine (*p ⬍ .05 when comparing VEH with MPH 4 weeks after FLX; †p ⫽ .057 when comparing MPH at 4 weeks versus MPH at 1 week after FLX). Data are presented as time spent in the morphine-paired minus time spent in the VEH-paired compartments after conditioning (mean ⫾ SEM, in sec).
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10 mg/kg) did not show differences in latency to immobility on day 1 or 2 of the FST (Figure 5F and 5G and 5H and 5I).
Discussion
Figure 4. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate responses to anxiogenic stimuli (n ⫽ 106). (A) The MPH-treated rats spent significantly less time in the open arms of the elevated-plus maze (EPM) than the VEH-treated control rats (*p ⫽ .01). (B) The MPH-treated rats spent significantly more time engaged in self-grooming behavior in the closed arms of the EPM than the VEH-treated controls (*p ⫽ .0001). (C) Acute FLX (5 mg/kg) did not reverse the MPH-induced effects. (D) Acute FLX (10 mg/kg) significantly decreased time spent in open arms of both VEH- and MPHtreated rats (*p ⬍ .05). (E) Acute FLX (10 mg/kg) increased self-grooming in VEH- and MPH-pretreated rats. (F–H) Chronic FLX (5 or 10 mg/kg) reversed the effects of MPH (time spent on open arms and self-grooming) on the EPM. Data are presented as percent time spent (mean ⫾ SEM) in the open arms and as self-grooming counts (mean ⫾ SEM) in the closed arms.
grooming between the VEH and MPH groups after 10 mg/kg FLX (Figure 4H). Effects of FLX on MPH-Induced Effects on Forced Swimming Behavior We used the FST to study rats’ responses to stressful conditions. The MPH-treated rats had significantly shorter latency to immobility on day 2 of the FST [F (1,21) ⫽ 14.6; p ⫽ .0010] than the VEH group (Figure 5A). The effects of FLX on latency to immobility varied as a function of test day, frequency of exposure, and dose [F (7,51) ⫽ 3.176; p ⫽ .0073; Figure 5B–5I]. The MPH-treated rats receiving acute FLX (5 mg/kg) showed significantly shorter latencies to immobility on day 1 of the FST compared with the VEH control rats (p ⬍ .05; Figure 5B), and this persisted on day 2 (p ⬍ .05; Figure 5C). Rats treated with acute FLX (10 mg/kg) showed no changes in immobility on day 1 regardless of developmental pretreatment (Figure 5D); however, these rats showed a significant decrease in latency to immobility on day 2 (p ⬍ .05; Figure 5E). Rats treated with chronic FLX (5 or
Recent studies demonstrate that exposing preadolescent rats to MPH results in profound changes in their behavioral reactivity to various emotion-eliciting stimuli indicative of a depressionlike state in adulthood (27,28,39). Thus, the main goal of our studies was to determine whether treatment with the selective serotonin reuptake inhibitor FLX could reverse these enduring MPH-induced effects. Here we show that exposure to MPH during preadolescence results in altered responses to rewarding and aversive stimuli in adulthood and that these behavioral deficits can be reversed by subsequent antidepressant treatment in adulthood. Our findings show that exposure to MPH results in significant decreases in rats’ normal sensitivity to the rewarding properties of sucrose (a natural reward) and morphine during adulthood. Decreased sucrose preference after MPH exposure is likely due to MPH’s ability to alter responsiveness to the rewarding effects of the solution, because the overall liquid intake during sucrose testing did not differ between the treatment groups. In addition, MPH-exposed rats did not consistently approach environments associated with doses of morphine that readily induced CPP in VEH-exposed rats. It is well established that the brain’s reward pathways, such as the nucleus accumbens (NAc) and its dopaminergic input from the ventral tegmental area (VTA), are involved in regulating responses to natural and drug rewards (40,41). Exposure to sweet solutions and drugs of abuse are rewarding: they activate the mesolimbic dopamine system and cause increases in dopamine release in the NAc, whereas disruptions in functioning of these pathways results in decreased interest for sucrose and drugs of abuse (41– 44). Together our results are in agreement with findings indicating that early-life exposure to MPH leads to long-lasting alterations in brain reward pathways resulting in decreased interest for natural rewards and diminished sensitivity to the rewarding effects of cocaine (25,26,28,39), and we now extend these findings to morphine reward. It must be noted, however, that under different experimental conditions (i.e., different developmental stage, dose, and treatment duration), MPH exposure results in sensitized responses to cocaine and morphine (45– 47). Within this ontogenetic context, it is not surprising to encounter different and, at times, conflicting results given that qualitative differences frequently emerge when manipulating the nervous system across development (48 –50). Decreased sensitivity to rewarding stimuli has been interpreted as a sign of anhedonia (decreased ability to experience pleasure, a core symptom in human depression) in animal models of depression (51–53). We demonstrate here that the MPH-induced deficits in sucrose and morphine reward can be restored to normal levels by chronic, not acute, treatment with FLX. Chronic FLX (10 mg/kg) normalized MPH-exposed rats’ preference for sucrose, and for environments previously paired with morphine, to comparable levels as shown by the VEHexposed rats. These findings are in agreement with studies showing that reductions in appetitive motivation can be reversed with antidepressant treatment (54 –56). The acute effects of antidepressant medications have been well delineated: they increase brain’s serotonergic or noradrenergic neurotransmission, and they exert their mood-elevating effects after prolonged (i.e., weeks) administration (57,58). Prewww.sobp.org/journal
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Figure 5. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate responses to forced swim stress (FST; n ⫽ 82). (A) Latencies to become immobile varied as a function of developmental drug treatment. Latencies were significantly decreased in rats treated with MPH during development when compared to the vehicle (VEH)-treated control rats on day 2 of the FST (*p ⫽ .0010). (B and C) Exposure to acute FLX (5 mg/kg) did not reverse MPH-induced effects on forced swimming on day 1 or day 2 of the FST (*p ⬍ .05). (D) No differences in the latency to immobility of MPH and VEH rats receiving acute FLX (10 mg/ kg) on day 1 of the FST. (E) MPH rats receiving same dose of FLX had a significant decrease in latency to immobility of day 2 of the FST (*p ⬍ .05). (F–I) Rats exposed to chronic FLX (5 or 10 mg/kg) show no significant differences in their latency to immobility on either day 1 or day 2 of the FST. Data are presented as latencies (mean ⫾ SEM, in sec) to become immobile.
clinical studies also show that depression-like behaviors can be reversed after prolonged antidepressant treatment (59 – 61). Interestingly, we show that the MPH-induced behavioral effects tend to dissipate after 1 week of once-daily FLX (10 mg/kg) treatment. Thus, to assess further whether the FLX-induced normalization for sucrose and morphine reward would be long lasting, we assessed sucrose and morphine preference in separate groups of rats 1 or 4 weeks after FLX (10 mg/kg) treatment (i.e., FLX withdrawal). Our results show normal levels of sucrose preference and morphine CPP 1 week after FLX. However, FLX-induced effects on sucrose preference showed a trend toward a decrease, whereas its effects on morphine CPP were not reliably present 4 weeks after FLX exposure. Although outside of the main objective of our study, these results indicate that long-term antidepressant treatment may perhaps be necessary for neurobiological adaptations to take place and mediate longterm maintenance of antidepressant effects (58,62). The mechanism(s) of antidepressant action are complex, and much more detailed assessments of these phenomena, accounting for length of antidepressant treatment and discontinuation, are clearly needed. The cellular mechanism(s) underlying these FLX-induced effects are unknown. Antidepressants increase the firing activity of VTA dopamine neurons (63), increase dopamine neurotransmission in the striatum (64 – 66), and enhance cocaine and morphine CPP (67, 68). Thus, given that depression and its deficits in appetitive motivation (anhedonia) are also associated www.sobp.org/journal
with impaired functioning of dopamine systems (52,53,69), it is conceivable that FLX treatment enhances dopamine transmission in reward-related brain areas to reverse depression-like behaviors after preadolescence MPH exposure. Our findings also indicate that MPH treatment renders rats significantly more vulnerable to anxiety- and stress-eliciting situations (27,28). Rats pretreated with MPH exhibited increased reactivity to anxiogenic stimuli because they spend significantly less time in the open arms of the EPM and significantly more time engaged in self-grooming behavior, a well-known behavioral response to anxiety-eliciting situations (36,70). Further studies with the FST found that MPH exposure also decreased latency to immobility, an effect opposite to that of antidepressant treatments (37,38,71). Similar to our findings with reward-related behaviors, treating rats with chronic FLX reversed the anxiogenic and pro-depressive behaviors induced by MPH. Interestingly, acute FLX (10 mg/kg) exacerbated anxiogenic-like behaviors in both VEH- and MPH-exposed groups because these rats spent less time in the open arms of the EPM than those not treated with FLX. These findings are in agreement with studies showing that initial exposure to antidepressants, which have been used successfully in the management of several anxiety disorders, exacerbates anxiogenic-like behaviors in humans (72–74) and animals (75–77) and that these anxiety-like behaviors dissipate after prolonged exposure to antidepressants (59,78). Our findings are in agreement with those showing that early-life exposure to MPH and cocaine has long-lasting effects:
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C.A. Bolaños et al. decreased responsiveness to rewards while enhancing sensitivity to aversive stimuli (depression-like behaviors) (25–28,39). The mechanism(s) underlying these effects are unknown. It has been suggested that increased activity of the transcription factor CREB (cAMP response element-binding protein) within the NAc (79) and deficits in hippocampal neurogenesis (80) may mediate these MPH-induced effects. This is likely because experimental manipulations similar to our study increase CREB expression in the NAc and disrupt hippocampal neurogenesis (25,80), and increasing CREB within the NAc causes depression-like behaviors (81,82). Within this context, it is tempting to suggest that early-life exposure to psychostimulants (see 25,27,28,30,39,80) could serve as a potential model for depression. Our demonstration that these MPH-induced effects can be normalized by antidepressant treatment lends support to this notion. To summarize, our study shows that antidepressant treatment can normalize depression-like behaviors induced by preadolescence exposure to MPH. Our findings underscore the need for further assessment of potential long-term neurobiological adaptations induced by early-life experiences that may contribute to the pathophysiology of neuropsychiatric disorders later in life. This work was supported by R03 Grant No. 1R03DA020089-01 from the National Institute on Drug Abuse, a NARSAD Young Investigator Award, and a FYAP grant from Florida State University to CAB. The authors have no financial or competing interests to declare. Supplementary material cited in this article is available online.
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S
timulant medications are effective for the treatment of attention-deficit/hyperactivity disorder (ADHD) (1,2), and methylphenidate (MPH) is the most prescribed therapeutic agent for ADHD in the United States (3–5). Although ADHD can be difficult to diagnose (6,7), prescription use of stimulants for its treatment has risen within the past decade (8,9), thus increasing the likelihood for stimulant exposure in children who do not meet ADHD criteria (8,10). Despite increases in stimulant prescriptions and the fact that affected children may be treated with MPH for years (11,12), little is known about the long-term neurobiological adaptations resulting from MPH exposure during juvenile periods. Basic and clinical studies show that MPH has psychomotor stimulant-like properties similar to those observed with cocaine and amphetamine (13,14). Methylphenidate blocks the dopamine transporter (15), increases dopamine levels in brain reward pathways (16,17), and enhances locomotion after its repeated administration (18,19). Because exposure to stimulants can result in long-lasting cellular, molecular, and behavioral adaptations implicated in the transition from drug use to abuse (13,20), most research has focused on assessing MPH’s potential to predispose individuals toward drug addiction (21,22). Longitudinal studies indicate that children treated with MPH may not be at risk of developing substance abuse disorders (23,24). In line with these findings, studies show that exposing rodents to MPH during preadolescence results in decreased
From the Department of Psychology and Program in Neuroscience, Florida State University, Tallahassee. Address reprint requests to Carlos A. Bolaños, Ph.D., Department of Psychology and Program in Neuroscience, Florida State University, 1107 West Call Street, Tallahassee, FL 32306-4301; e-mail: [email protected]. Received January 24, 2007; revised June 1, 2007; accepted June 23, 2007.
0006-3223/08/$34.00 doi:10.1016/j.biopsych.2007.06.024
sensitivity to cocaine during adulthood (25–27). Interestingly, these rats also show increases in depression-like behaviors manifested in enhanced vulnerability to stressful environments and anxiety-eliciting situations, as well as reduced reactivity to natural rewards (27,28). Given the degree of prescribed use of MPH and reports indicating that in some pediatric populations up to 66% of those treated with stimulants for ADHD do not meet the criteria for this disorder (8,10,29), it is essential that the long-term neurobiological consequences associated with developmental psychostimulant exposure be characterized. Accordingly, the studies presented here were designed to assess whether the long-term behavioral effects induced by early-life MPH exposure could be reversed by subsequent antidepressant treatment during adulthood. To this end, we treated rats with MPH or saline during preadolescence (postnatal day [PD] 20 –35). After treatment, rats were left undisturbed until adulthood (PD90⫹), at which point they were separated into several groups: those receiving no treatment, and those subsequently exposed to fluoxetine before assessing their behavioral reactivity to various emotion-eliciting stimuli, including responsivity to the rewarding effects of morphine.
Methods and Materials Drugs Methylphenidate hydrochloride, fluoxetine hydrochloride (FLX), and morphine sulfate were obtained from Sigma (St. Louis, Missouri). Each drug was dissolved in .9% saline (VEH) and administered in a volume of 1 mL/kg for MPH and morphine and 2mL/kg for FLX. Doses are based on the salt form of each drug. Subjects and Drug Treatments Litters containing Sprague-Dawley male rat pups with their dams were obtained from Charles River Laboratories (Raleigh, North Carolina) at PD14. Rats were housed in clear polyproBIOL PSYCHIATRY 2008;63:309 –316 © 2008 Society of Biological Psychiatry
310 BIOL PSYCHIATRY 2008;63:309 –316 pylene boxes containing wood shavings in an animal colony maintained at 23–25°C on a 12-hour light-dark cycle in which lights were on between 7:00 and 19:00 hour. Injections of MPH (2.0 mg/kg, intraperitoneally [IP]) or VEH were given to pups starting on PD20 twice daily (4 hours apart) for 15 days. From PD20 to PD23, MPH- and VEH-treated rats were kept together in same cage with their dams. At PD24 (weaning), rats were separated by treatment condition in groups of four per cage. At PD45, rats were further separated into groups of two per cage and were left undisturbed until adulthood. Testing started 8 weeks after the last MPH injection. In addition, separate groups of rats were divided into subgroups receiving acute (one injection) or chronic (one daily injection for 7 days) FLX (5.0 or 10.0 mg/kg IP), 8 weeks after MPH exposure. Testing began 24 hours, 1 week, or 4 weeks after the last FLX injection (see Supplement 1). The period between PD20 and PD35 in rats was chosen because it approximates preadolescence (i.e., age 4 –12 years) in humans (30). The dose of MPH chosen approximates clinically relevant doses in pediatric patient population (31,32). All behaviors, except for place conditioning (CPP) and sucrose preference, were recorded with a video camera located on the ceiling of separate testing rooms. Behavioral observations and analyses were done by observers with no knowledge of the treatment conditions of each rat. Rats were provided with food and water ad libitum. Experiments were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, 1996) and approved by Florida State University animal care and use committee. Sucrose Preference The sucrose preference test consisted of a two-bottle choice paradigm. In this test, rats are given the choice between consuming water versus sucrose. This paradigm has been used extensively to assess the effects of stress-induced anhedonia (33). Rats were habituated to drink water from two bottles for 5 days. At the start of the experiment, rats were exposed to ascending concentrations of sucrose (.0, and .25–1%) for 2 days per sucrose concentration. Water and sucrose consumption were measured at 8:00 and 17:00 hours each testing day at which time the position of the sucrose bottle (left or right) was balanced between the MPH and VEH groups. The preference for sucrose over water was used as a measure for rats’ sensitivity to reward. Place Preference Conditioning Place conditioning (CPP) was carried out as described previously (27). This paradigm has been widely used to assess the rewarding or aversive properties of drugs. In this behavioral assay, rats learn to prefer environments previously associated with rewarding drug effects while avoiding environments associated with aversive drug effects (34,35). Briefly, CPP to morphine was performed in a three-compartment apparatus (FSU Psychology engineering group). Compartments differed in floor texture, wall coloring, and lightning. On the preconditioning day (day 0), rats were allowed to explore the entire apparatus freely for 30 min to obtain baseline preference to any of the three compartments (side compartments: 35 ⫻ 27 ⫻ 25 cm; middle compartment: 10 ⫻ 27 ⫻ 25 cm, L ⫻ W ⫻ H). Only rats showing no preference (before drug training) to either side compartment were used (see Supplement 2); this accounted for more than 90% of all of the rats tested. Conditioning trials occurred on 2 consecutive days (days 1 and 2). During conditioning, rats received VEH (1.0 mL/kg, subcutaneously [SC]) and were confined to one of the side compartments of the apparatus for www.sobp.org/journal
C.A. Bolaños et al. 1 hour. After 3 hours, rats received morphine (0, .25, .50, or 1.0 mg/kg SC) and were confined to the opposite side compartment for 1 hour. On the test day (day 3), rats received VEH (SC) and were again allowed to explore the entire apparatus freely for 30 min; the time spent in the drug-paired compartment(s) was assessed. Elevated-Plus Maze and Self-Grooming Behavior The MPH- and VEH-treated rats were tested for 5 min on the elevated-plus maze (EPM), a behavioral model of anxiety-like behavior. The maze was made of gray plastic and consisted of two perpendicular, intersecting runways (12 cm wide ⫻ 100 cm long; FSU Psychology engineering group). One runway had tall walls (40 cm high), or “closed arms,” and the other had no walls, or “open arms.” The arms were connected together by a central area, and the maze was elevated 1 m from the floor. Testing was conducted between 9:00 and 13:00 hours under controlled light conditions (⬃90 lux). At the beginning of the test, rats were placed in the central area, facing one of the open arms, and the cumulative time spent in the open arms was videotaped. We also assessed self-grooming in the “closed arms” because rats engage in repetitive grooming in response to anxiogenic stimuli (36). Forced Swimming The forced swim test (FST) is a 2-day procedure in which rats are forced to swim under conditions in which they cannot escape. On the first day, rats are forced to swim. Initially, they engage in escapelike behaviors but eventually adopt a posture of immobility in which they make only the movements necessary to maintain their head above water. When retested 24 hours later, rats become immobile more quickly; however, antidepressant treatment between the forced swim exposures can significantly increase their escapelike behaviors, an effect that has been correlated with antidepressant activity in humans (37). At the start of the experiment, rats were placed in plastic cylinders (30 ⫻ 45 cm) filled to 30 cm depth (so that their paws and tail do not touch the bottom) with 25°C water and forced to swim for 15 min. At the end of this period, rats were removed from the water, dried with towels, and kept in a warm enclosure for 30 min. All cylinders were emptied and cleaned between rats. Twenty-four hours after the forced swim, rats were retested for 5 min under identical conditions, and sessions were videotaped. In this study, the latency to become immobile was the dependent variable. Latency to immobility was defined as the time at which the rat first initiated a stationary posture that did not reflect attempts to escape from the water (38). To qualify as immobility, this posture had to be clearly visible and maintained for ⱖ2.0 sec. We also assessed immobility, swimming, and climbing counts in the FST (see Supplement 3). Statistical Analysis Statistical significance was measured using mixed-design (between and within variables) analysis of variance (ANOVA) followed by Tukey post hoc test. When appropriate, Student’s t and F tests were used to determine statistical significance of preplanned comparisons. Data are expressed as the mean ⫾ SEM. Statistical significance was defined as p ⬍ .05.
Results Effects of MPH and Subsequent FLX Exposure on Sucrose Preference in Adulthood Overall repeated-measures ANOVA indicated that exposure to MPH did not affect the rats’ total fluid intake (water ⫹ sucrose;
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BIOL PSYCHIATRY 2008;63:309 –316 311 preference as a function of treatment frequency and dose [F (7,60) ⫽ 4.79; p ⫽ .0002], without affecting water intake. Acute FLX (5 or 10 mg/kg) did not reverse the effects of MPH on sucrose preference (Figure 1C and 1D). Although there was a trend toward decreases in sucrose preference in rats exposed to 10 mg/kg compared with 5 mg/kg FLX, this difference was not statistically significant. However, acute FLX (10 mg/kg) decreased preference for sucrose in the VEH group (Figure 1D) compared with the VEH-exposed rats from Figure 1A at the .5% solution. Rats repeatedly exposed to 5 mg/kg FLX showed a nonsignificant trend toward an increase in sucrose preference (VEH vs. MPH: p ⫽ .07), whereas exposure to 10 mg/kg FLX reversed the effects of early-life MPH exposure on sucrose preference.
Figure 1. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate adult responses to sucrose reward (n ⫽ 128). (A) Developmental exposure to MPH significantly decreased sensitivity to the rewarding effects of sucrose when compared to vehicle (VEH)-treated rats. (B) MPH pretreatment did not affect total fluid (sucrose ⫹ water) intake. *Significantly different at .25% (p ⫽ .001) and .50% sucrose (p ⫽ .018). (C and D) A single injection of FLX (5 or 10 mg/kg) did not reverse MPH-induced decreases on sucrose preference. (E) Rats treated with chronic FLX (5 mg/kg) show a trend († p ⫽ .078) toward reversal of MPH effects, whereas chronic exposure to 10 mg/kg FLX did reverse MPH-induced effects of sucrose preference (F). Data are presented as percentage preference between MPH- and VEH-treated rats.
Figure 1B) at any of the sucrose concentrations tested (p ⬎ .05). In addition, sucrose preference varied as a function of MPH treatment and sucrose concentration [interaction: F (5,290) ⫽ 10.52; p ⫽ .0001]. On the basis of previous observations (28), we hypothesized that MPH-treated rats would show a decrease in preference for sucrose compared with VEH-treated control rats (preplanned comparison). Unpaired t tests demonstrated that prior MPH treatment decreased preference for a .25 [t (58) ⫽ 4.15; p ⫽ .001], and .5% [t (58) ⫽ 2.42; p ⫽ .018] sucrose solution compared with the VEH group (Figure 1A). Differences in sucrose preference between groups were not present at the 1% concentration. Decreased sensitivity to rewarding stimuli can be interpreted as anhedonia (decreased ability to experience pleasure), a common symptom of depression. Thus, we assessed the ability of FLX to reverse MPH-induced effects on sucrose preference. Figure 1C–1F shows that FLX differentially regulated sucrose
Effects of MPH and Subsequent FLX Exposure on MorphineInduced Place Preference Conditioning in Adulthood No conditioning effects were observed in VEH- or MPHpretreated rats conditioned with saline (Figure 2A). Time spent in the morphine-paired compartments varied as a function of developmental [F (1,46) ⫽ 9.674; p ⫽ .0032] and morphine posttreatment [F (2,46) ⫽ 7.084; p ⫽ .002], respectively (Figure 2A). Rats treated with VEH during development spent significantly more time in environments paired with morphine (.25 [p ⫽ .06], .50 [p ⫽ .043], and 1.0 [p ⫽ .035] mg/kg), whereas rats exposed to MPH did not condition to morphine (p ⬎ .05). We assessed the ability of FLX (0, 5, or 10 mg/kg) to reverse MPH-induced effects on morphine conditioning. Figure 2B shows that time spent in morphine-paired (.5 mg/kg) environments varied as a function of developmental treatment [F (1,36) ⫽ 22.06; p ⫽ .0001]. Rats treated with VEH during development and subsequently exposed to acute FLX spent significantly more time in environments paired with .5 mg/kg morphine (0 [p ⫽ .0066], 5.0 [p ⫽ .0025], and 10 [p ⫽ .04] mg/kg FLX). However, acute FLX did not influence conditioning in MPH-pretreated rats because these showed no preference for the morphine-paired environments. Figure 2C shows time spent in morphine-paired compartments of rats treated with chronic FLX (0, 5, or 10 mg/kg). Times varied as a function of developmental treatment and FLX exposure [F (2,37) ⫽ 3.28; p ⫽ .05]. As expected, MPH rats chronically treated with .0 mg/kg FLX did not show CPP, and those treated with 5 mg/kg showed a weak trend (p ⫽ .09) toward developing morphine CPP. However, chronic exposure to 10 mg/kg FLX reversed the effects of MPH pretreatment on CPP (Figure 2C) because time spent in the morphine-paired compartments did not differ from time observed in the VEH-pretreated rats (p ⬎ .05). Effects of Withdrawal Time after Chronic FLX Exposure on Sucrose Preference and Morphine-Induced Place Preference Conditioning in Adulthood Rats exposed to chronic FLX (10 mg/kg) did not show significant differences in their preference for sucrose (.5 mg/kg) 1 week after FLX treatment (p ⬎ .05; Figure 3A). Although MPH-treated rats showed a tendency toward decreased sucrose preference 4 weeks after FLX treatment, this trend was not statistically significant (p ⬎ .05). Conversely, Figure 3B shows that CPP to morphine varied as a function of FLX (10 mg/kg) withdrawal [F (3,22) ⫽ 4.52; p ⫽ .013]. Specifically, VEH- and MPH-exposed rats readily conditioned to morphine-paired environments 1 week after the last FLX injection (Figure 3B). However, MPH-exposed rats tested for CPP 4 weeks after the last FLX injection did not reliably condition to the morphine-paired environments compared with VEH-exposed rats (p ⬍ .05; Figure 3B). In www.sobp.org/journal
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C.A. Bolaños et al. counts than VEH control rats [t(34) ⫽ 4.96; p ⫽ .0001; Figure 4B]. Acute FLX (5 or 10 mg/kg) did not reverse the effects of MPH on the EPM (Figure 4C and 4D). In fact, FLX, at 10 mg/kg, further decreased time spent in the open arms in VEH and MPH rats (p ⬍ .05, Figure 4D, compared with VEH and MPH groups in Figure 4A). Additionally, this dose of FLX increased self-grooming in VEH-treated rats (p ⬍ .05, Figure 4E, compared with VEH rats in Figure 4A). Chronic exposure to 5 mg/kg FLX eliminated the differences in time spent in the open arms between VEH and MPH groups (Figure 4F); however, time spent in open arms by the VEH group remained significantly lower than that observed by the VEH-treated group from Figure 4A (p ⬍ .05). Chronic exposure to 10 mg/kg FLX reversed the effects of MPH pretreatment on the EPM (Figure 4G), and time spent in the open arms by these rats was significantly higher than time observed in the MPH group from Figure 4A (p ⬍ .05). There were no differences in self-
Figure 2. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate morphine-induced place preference conditioning (n ⫽ 137). (A) Adult rats exposed to MPH during development spent significantly less time in morphine-paired environments, whereas rats exposed to vehicle (VEH) spent significantly more time in morphine-associated environments in a dose-dependent manner. (B) Exposure to acute FLX (5 or 10 mg/kg) did not reverse MPH-induced effects on morphine (.5 mg/kg) place conditioning, whereas VEH-pretreated rats do show robust morphine CPP. (C) MPH rats treated with chronic FLX (5 mg/kg) show a trend toward reversal of MPH-induced effects on place conditioning (†p ⫽ .090). Chronic exposure to the higher dose of FLX (10 mg/kg) reversed the behavioral effects induced by developmental MPH exposure on morphine (.5 mg/kg) place conditioning. Data are presented as time spent in the morphine-paired minus time spent in the VEH-paired compartments after conditioning (mean ⫾ SEM, in sec).
fact, the magnitude of morphine conditioning exhibited by these MPH rats was somewhat lower than the conditioning observed in the MPH rats in the 1 week withdrawal FLX condition (p ⫽ .057). Effects of FLX on MPH-Induced Anxiety-like Behaviors in the Elevated-Plus Maze We also studied the effects of MPH, and subsequent FLX treatment, on anxiety-like behavior using the EPM (Figure 4A– 4F). Time spent in the open arms of the EPM was differentially affected by drug treatment and exposure frequency [F (9,96) ⫽ 4.98; p ⫽ .0001]. Figure 4A shows that MPH rats spent significantly less time in the open arms than the VEH control rats [t (34) ⫽ ⫺2.73; p ⫽ .01]. In addition, the MPH group had higher self-grooming www.sobp.org/journal
Figure 3. Sucrose preference (n ⫽ 20) and morphine-induced place conditioning (n ⫽ 26) in vehicle (VEH)- and methylphenidate (MPH)-treated rats 1 and 4 weeks after the last FLX injection. (A) Both VEH- and MPH-exposed rats show no significant differences in sucrose preference (.5%) 1 or 4 weeks after chronic FLX (10 mg/kg). (B) VEH- and MPH-treated rats readily conditioned to environments previously paired with morphine (.5 mg/kg) 1 week after the last FLX exposure (left panel), whereas animals conditioned 4 weeks after the last FLX injection did not show reliable place conditioning to morphine (*p ⬍ .05 when comparing VEH with MPH 4 weeks after FLX; †p ⫽ .057 when comparing MPH at 4 weeks versus MPH at 1 week after FLX). Data are presented as time spent in the morphine-paired minus time spent in the VEH-paired compartments after conditioning (mean ⫾ SEM, in sec).
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10 mg/kg) did not show differences in latency to immobility on day 1 or 2 of the FST (Figure 5F and 5G and 5H and 5I).
Discussion
Figure 4. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate responses to anxiogenic stimuli (n ⫽ 106). (A) The MPH-treated rats spent significantly less time in the open arms of the elevated-plus maze (EPM) than the VEH-treated control rats (*p ⫽ .01). (B) The MPH-treated rats spent significantly more time engaged in self-grooming behavior in the closed arms of the EPM than the VEH-treated controls (*p ⫽ .0001). (C) Acute FLX (5 mg/kg) did not reverse the MPH-induced effects. (D) Acute FLX (10 mg/kg) significantly decreased time spent in open arms of both VEH- and MPHtreated rats (*p ⬍ .05). (E) Acute FLX (10 mg/kg) increased self-grooming in VEH- and MPH-pretreated rats. (F–H) Chronic FLX (5 or 10 mg/kg) reversed the effects of MPH (time spent on open arms and self-grooming) on the EPM. Data are presented as percent time spent (mean ⫾ SEM) in the open arms and as self-grooming counts (mean ⫾ SEM) in the closed arms.
grooming between the VEH and MPH groups after 10 mg/kg FLX (Figure 4H). Effects of FLX on MPH-Induced Effects on Forced Swimming Behavior We used the FST to study rats’ responses to stressful conditions. The MPH-treated rats had significantly shorter latency to immobility on day 2 of the FST [F (1,21) ⫽ 14.6; p ⫽ .0010] than the VEH group (Figure 5A). The effects of FLX on latency to immobility varied as a function of test day, frequency of exposure, and dose [F (7,51) ⫽ 3.176; p ⫽ .0073; Figure 5B–5I]. The MPH-treated rats receiving acute FLX (5 mg/kg) showed significantly shorter latencies to immobility on day 1 of the FST compared with the VEH control rats (p ⬍ .05; Figure 5B), and this persisted on day 2 (p ⬍ .05; Figure 5C). Rats treated with acute FLX (10 mg/kg) showed no changes in immobility on day 1 regardless of developmental pretreatment (Figure 5D); however, these rats showed a significant decrease in latency to immobility on day 2 (p ⬍ .05; Figure 5E). Rats treated with chronic FLX (5 or
Recent studies demonstrate that exposing preadolescent rats to MPH results in profound changes in their behavioral reactivity to various emotion-eliciting stimuli indicative of a depressionlike state in adulthood (27,28,39). Thus, the main goal of our studies was to determine whether treatment with the selective serotonin reuptake inhibitor FLX could reverse these enduring MPH-induced effects. Here we show that exposure to MPH during preadolescence results in altered responses to rewarding and aversive stimuli in adulthood and that these behavioral deficits can be reversed by subsequent antidepressant treatment in adulthood. Our findings show that exposure to MPH results in significant decreases in rats’ normal sensitivity to the rewarding properties of sucrose (a natural reward) and morphine during adulthood. Decreased sucrose preference after MPH exposure is likely due to MPH’s ability to alter responsiveness to the rewarding effects of the solution, because the overall liquid intake during sucrose testing did not differ between the treatment groups. In addition, MPH-exposed rats did not consistently approach environments associated with doses of morphine that readily induced CPP in VEH-exposed rats. It is well established that the brain’s reward pathways, such as the nucleus accumbens (NAc) and its dopaminergic input from the ventral tegmental area (VTA), are involved in regulating responses to natural and drug rewards (40,41). Exposure to sweet solutions and drugs of abuse are rewarding: they activate the mesolimbic dopamine system and cause increases in dopamine release in the NAc, whereas disruptions in functioning of these pathways results in decreased interest for sucrose and drugs of abuse (41– 44). Together our results are in agreement with findings indicating that early-life exposure to MPH leads to long-lasting alterations in brain reward pathways resulting in decreased interest for natural rewards and diminished sensitivity to the rewarding effects of cocaine (25,26,28,39), and we now extend these findings to morphine reward. It must be noted, however, that under different experimental conditions (i.e., different developmental stage, dose, and treatment duration), MPH exposure results in sensitized responses to cocaine and morphine (45– 47). Within this ontogenetic context, it is not surprising to encounter different and, at times, conflicting results given that qualitative differences frequently emerge when manipulating the nervous system across development (48 –50). Decreased sensitivity to rewarding stimuli has been interpreted as a sign of anhedonia (decreased ability to experience pleasure, a core symptom in human depression) in animal models of depression (51–53). We demonstrate here that the MPH-induced deficits in sucrose and morphine reward can be restored to normal levels by chronic, not acute, treatment with FLX. Chronic FLX (10 mg/kg) normalized MPH-exposed rats’ preference for sucrose, and for environments previously paired with morphine, to comparable levels as shown by the VEHexposed rats. These findings are in agreement with studies showing that reductions in appetitive motivation can be reversed with antidepressant treatment (54 –56). The acute effects of antidepressant medications have been well delineated: they increase brain’s serotonergic or noradrenergic neurotransmission, and they exert their mood-elevating effects after prolonged (i.e., weeks) administration (57,58). Prewww.sobp.org/journal
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Figure 5. Developmental exposure to methylphenidate (MPH) and subsequent fluoxetine hydrochloride (FLX) treatment in adulthood regulate responses to forced swim stress (FST; n ⫽ 82). (A) Latencies to become immobile varied as a function of developmental drug treatment. Latencies were significantly decreased in rats treated with MPH during development when compared to the vehicle (VEH)-treated control rats on day 2 of the FST (*p ⫽ .0010). (B and C) Exposure to acute FLX (5 mg/kg) did not reverse MPH-induced effects on forced swimming on day 1 or day 2 of the FST (*p ⬍ .05). (D) No differences in the latency to immobility of MPH and VEH rats receiving acute FLX (10 mg/ kg) on day 1 of the FST. (E) MPH rats receiving same dose of FLX had a significant decrease in latency to immobility of day 2 of the FST (*p ⬍ .05). (F–I) Rats exposed to chronic FLX (5 or 10 mg/kg) show no significant differences in their latency to immobility on either day 1 or day 2 of the FST. Data are presented as latencies (mean ⫾ SEM, in sec) to become immobile.
clinical studies also show that depression-like behaviors can be reversed after prolonged antidepressant treatment (59 – 61). Interestingly, we show that the MPH-induced behavioral effects tend to dissipate after 1 week of once-daily FLX (10 mg/kg) treatment. Thus, to assess further whether the FLX-induced normalization for sucrose and morphine reward would be long lasting, we assessed sucrose and morphine preference in separate groups of rats 1 or 4 weeks after FLX (10 mg/kg) treatment (i.e., FLX withdrawal). Our results show normal levels of sucrose preference and morphine CPP 1 week after FLX. However, FLX-induced effects on sucrose preference showed a trend toward a decrease, whereas its effects on morphine CPP were not reliably present 4 weeks after FLX exposure. Although outside of the main objective of our study, these results indicate that long-term antidepressant treatment may perhaps be necessary for neurobiological adaptations to take place and mediate longterm maintenance of antidepressant effects (58,62). The mechanism(s) of antidepressant action are complex, and much more detailed assessments of these phenomena, accounting for length of antidepressant treatment and discontinuation, are clearly needed. The cellular mechanism(s) underlying these FLX-induced effects are unknown. Antidepressants increase the firing activity of VTA dopamine neurons (63), increase dopamine neurotransmission in the striatum (64 – 66), and enhance cocaine and morphine CPP (67, 68). Thus, given that depression and its deficits in appetitive motivation (anhedonia) are also associated www.sobp.org/journal
with impaired functioning of dopamine systems (52,53,69), it is conceivable that FLX treatment enhances dopamine transmission in reward-related brain areas to reverse depression-like behaviors after preadolescence MPH exposure. Our findings also indicate that MPH treatment renders rats significantly more vulnerable to anxiety- and stress-eliciting situations (27,28). Rats pretreated with MPH exhibited increased reactivity to anxiogenic stimuli because they spend significantly less time in the open arms of the EPM and significantly more time engaged in self-grooming behavior, a well-known behavioral response to anxiety-eliciting situations (36,70). Further studies with the FST found that MPH exposure also decreased latency to immobility, an effect opposite to that of antidepressant treatments (37,38,71). Similar to our findings with reward-related behaviors, treating rats with chronic FLX reversed the anxiogenic and pro-depressive behaviors induced by MPH. Interestingly, acute FLX (10 mg/kg) exacerbated anxiogenic-like behaviors in both VEH- and MPH-exposed groups because these rats spent less time in the open arms of the EPM than those not treated with FLX. These findings are in agreement with studies showing that initial exposure to antidepressants, which have been used successfully in the management of several anxiety disorders, exacerbates anxiogenic-like behaviors in humans (72–74) and animals (75–77) and that these anxiety-like behaviors dissipate after prolonged exposure to antidepressants (59,78). Our findings are in agreement with those showing that early-life exposure to MPH and cocaine has long-lasting effects:
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C.A. Bolaños et al. decreased responsiveness to rewards while enhancing sensitivity to aversive stimuli (depression-like behaviors) (25–28,39). The mechanism(s) underlying these effects are unknown. It has been suggested that increased activity of the transcription factor CREB (cAMP response element-binding protein) within the NAc (79) and deficits in hippocampal neurogenesis (80) may mediate these MPH-induced effects. This is likely because experimental manipulations similar to our study increase CREB expression in the NAc and disrupt hippocampal neurogenesis (25,80), and increasing CREB within the NAc causes depression-like behaviors (81,82). Within this context, it is tempting to suggest that early-life exposure to psychostimulants (see 25,27,28,30,39,80) could serve as a potential model for depression. Our demonstration that these MPH-induced effects can be normalized by antidepressant treatment lends support to this notion. To summarize, our study shows that antidepressant treatment can normalize depression-like behaviors induced by preadolescence exposure to MPH. Our findings underscore the need for further assessment of potential long-term neurobiological adaptations induced by early-life experiences that may contribute to the pathophysiology of neuropsychiatric disorders later in life. This work was supported by R03 Grant No. 1R03DA020089-01 from the National Institute on Drug Abuse, a NARSAD Young Investigator Award, and a FYAP grant from Florida State University to CAB. The authors have no financial or competing interests to declare. Supplementary material cited in this article is available online.
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