Age and genetic strain differences in response to chronic methylphenidate administration

Methylphenidate hydrochloride (MPD) is a psychostimulant used in the treatment of attention deficit hyperactive disorder (ADHD) in adolescents and adults alike. Adolescence involves a period of neural development that is highly susceptible to environmental and pharmacological influence. Exposure to a psychostimulant like MPD during this crucial time period may cause permanent changes in neuronal function and formation. Another factor that may influence changes in neuronal function and formation is genetic variability. It has been reported that genetic variability affects both the initial behavioral response to drugs in general and psychostimulants in particular, and subsequently whether tolerance or sensitization is induced. The objective of the present study is to investigate the dose-response effects of repeated MPD administration (0.6, 2.5, or 10.0 mg/kg, i.p.) using an open field assay to investigate if there are differences between adolescent and adult Wistar-Kyoto (WKY), Spontaneously Hyperactive rat (SHR), and Sprague-Dawley (SD) rats, respectively, and if the genetic variability between the strains influences the degree of change in locomotion. The acute and chronic administration of MPD resulted in unique differences in the level of increasing intensity in locomotor activity in each rat strain, with adult rats for the most part having a more intense increase in locomotor activity when compared to their adolescent counterparts. In conclusion, significant response differences among rat strains and age to acute and chronic MPD administration were observed only following the 2.5 and 10.0 mg/kg i.p. doses and not following the lower MPD dose (0.6 mg//kg i.p.). In addition the variability in activity among the rat strain and age suggests that MPD may affect the same neuronal circuit differently in each strain and age. The unique differences among the individual locomotor indices suggest also that each locomotor index is regulated by different neuronal circuits, and each affected differently by MPD. Keywords Locomotor Behavior Ritalin Adolescent Adult Genetic Strain 1 Introduction Attention deficit hyperactive disorder (ADHD), a behavioral disorder, is one of the most commonly diagnosed psychiatric disorders affecting 3–5% of school-aged children in the United States, possibly 17% if subclinical cases are included. ADHD affects 3–5% of adults in the United States, suggesting the disorder persists into adulthood [1,2] . Methylphenidate hydrochloride (MPD), commonly known as Ritalin is one of the most prescribed treatments for ADHD in adolescents and adults alike [3–5] . Its efficacy and safety have been well documented in many studies [6] , but there is still a lack of information regarding the influence of MPD on the developing brain and its long-term effects on neuronal function. In humans, normal neurodevelopment consists of an overproduction of synaptic connections with a subsequent elimination of these synapses by competitive inhibition. Synaptic pruning, usually occurs between 5 and 15 years of age, when the synaptic density of the prefrontal cortex decreases by approximately 40% [7,8] . It is thought that this synaptic reorganization may be a predisposing factor for many behavioral/psychiatric disorders including ADHD [7,9,10] . The treatment of ADHD in children using MPD parallels the timeframe of synaptic pruning, during which environmental and pharmacological influences exert a strong influence on neuronal formation and function [10,11] . Furthermore, rats exposed to MPD at an age approximating human childhood experienced behavioral changes that endured into adulthood, which suggests that MPD has a long-term effect on normal neurodevelopment [10,12] . The response to psychostimulants has been reported to vary with age [12,13] , including the ability to reduce impulsivity when administered to adolescents, while having no effect in adults [14] . Therefore it is essential to investigate whether there are age differences in response to acute and/or chronic MPD administration. Pharmacogenetic research using genetically defined rodent strains has provided insight into the possibility that genetic factors influence drug-related behaviors [15] . Humans as well as experimental animals exhibit considerable individual variability, both in the initial behavioral response to psychostimulants and in the development of tolerance and/or sensitization [15–17] . It has also been reported that different rat strains, comprised of distinct gene pools, show different susceptibility to psychostimulants and a varied chronic response, i.e., tolerance and/or sensitization [14,18–21] . Although the etiology of ADHD is not yet fully understood, there seems to be a strong underlying genetic component, which has been supported by familial, adoptive, and twin studies [22,23] . Furthermore, strain differences in the acute and chronic response to cocaine in the development of behavioral sensitization in mice have been reported [24–26] . However, rat strain comparisons of the acute and chronic effects of MPD are limited. Several animal ADHD models are currently used. The rat strain most commonly used as a genetic model for ADHD is the Spontaneous Hypertensive/Hyperactive rat (SHR), since it mimics several key characteristics found in ADHD patients including: motor hyperactivity, decreased sustained attention, motor and cognitive impulsiveness, increased behavioral variability, and decreased dopamine transmission [27,28] . The SHR strain has been bred from its progenitor the Wistar-Kyoto (WKY) rat [29] , which is used as a control to the SHR strain [30] . It is also of importance to compare the effects of MPD with another rat stain that is frequently used in studying the effects of psychostimulants, the Sprague-Dawley rat (SD). This strain has been reported to develop behavioral sensitization after repeated exposure to MPD [12,21,31–36] . In order to better understand the role genetics plays in the treatment of ADHD with MPD, a study using animal models of different strains is required. The objective of the present study is to investigate whether age and genetic variability influences the locomotor response to repeated MPD administration using the open field assay. 2 Materials and methods 2.1 Animals A total of 216 male rats were used for this study as follows: SHR ( N = 71), WKY ( N = 73), and SD ( N = 72). The rats were purchased with a postnatal age of 30 days (P-30) for adolescents and 52 days (P-52) for adults. The rats in groups of four per Plexiglas cage were placed in the experimental room for 5–7 days for acclimation. The ambient temperature of the room was maintained at 21 ± 2 °C and a relative humidity of 37–42%. The rats were maintained on a 12:12 h light/dark cycle(light on starting at 05:30 h) with food and water given ad libitum. After 5–7 days of habituation to the experimental room, rats were randomly divided into groups as summarized in Table 1 . Then rats were placed individually into automated animal activity monitoring chambers (test cage) for an additional 24–36 h for habituation. This test cage then became their home cage during the subsequent 11 consecutive experimental days, i.e., during the locomotor activity recording ( Table 1 ). At the beginning of the recording the animals were at P-40 (adolescent) and P-62 (young adult). All efforts were made to minimize the number of animal used and their suffering. Housing conditions and experimental procedures were approved by our animal welfare committee. This study was done over three years. Only one age, sex and strain were studied at a time to eliminate additional factors that could affect the recording. 2.2 Drugs Methylphenidate hydrochloride (MPD) was obtained from Mallinckrodt Inc. (Hazelwood, MO), and was dissolved in 0.9% isotonic saline solution at 0.6, 2.5, and 10.0 mg/kg. Syringes containing different dosages were equalized to 0.8 ml by adding saline, to ensure that total volume of the injection did not vary from animal to animal. The injections were administered intra-peritoneally (i.p.) around 07:00 h. The dosages used and times of injection were selected based on previous MPD dose dependent response studies which showed that this time is optimal for eliciting dose dependent tolerance and/or behavioral and electrophysiological sensitization [20,21,31,35,37] . In general, the treatment schedule included an initial saline administration (day 1), followed by 6 consecutive days of MPD administration, then 3 days without any treatment (washout) followed by a rechallenge of MPD on the final 11th day ( Table 1 ). 2.3 Apparatus An open field monitoring system was used to record locomotor activity during the duration of the experiment. Testing chambers consisted of a clear acrylic box (40.5 cm × 40.5 cm × 31 .5 cm) fitted with two sets of 16 infrared motion sensors located 6 and 12 cm above the floor (AccuScan Instruments, Inc., Columbus, OH). Briefly, the system records activity by counting the number of interruptions in the beams at a frequency of 100 Hz. The interruption of any beam was transformed it into an activity score. Cumulative counts of scores were compiled and downloaded every 10 min into the OASIS data collection software and organized into various locomotor indices. Total distance traveled (TD), measured the total amount of forward movement in centimeters during a given period; horizontal activity (HA), recorded the total number of beams interrupted at the horizontal sensor (lowest tier) level during a given period a measure of the overall activity; vertical activity (VA) measured the total number of beams interrupted at the vertical (highest tier) level during a given period, which mainly represented rearing; number of stereotypic movements (NOS), measured the number of repetitive purposeless episodes with at least a 1 s interval before the initiation of another episode, which could represent general stereotyped behaviors, such as sniffing and grooming [20,21,31,37–39] . This system has been previously described in detail [21,31,40–42] . 2.4 Data analysis Locomotor activity counts were collected every 10 min for 2 h. These recordings were used to produce a sequential temporal graph recording the first 2 h after injection and a histogram comparing the average sums of 2 h activity under the temporal graph. For the temporal graph, each 10 min recording bin was plotted sequentially and the standard error (S.E.) was used to calculate the significance of changes between experimental days using two-way ANOVA and Post hoc analysis with LSD test ( [20,21,31,33,37–42] ). Statistically significant changes in two or more consecutive recording points (bins) were considered to be due to the drug treatment. Differences in the temporal effect for each drug dose was qualitatively described using the 10 min bin data, in order to establish the latency, time to maximum effect, and duration of the effect for each dose and locomotor index [38,43–45] . The histograms were used to calculate the average activity level for each test group during the 2 h post treatment. The t test was used to compare the difference between two given experimental groups. The evaluation was divided into three phases: acute, induction, and expression. The acute effect of MPD was determined by comparing the difference in activity during the initial 2 h post injection of MPD on experimental day 2 to the activity during the initial 2 h post injection of saline in the same rat on experimental day 1 ( Table 1 ). The chronic effect of MPD, i.e., sensitization and/or tolerance was determined by comparing the activity scores post MPD injection on experimental days 7 and 11 to the activity score at 2 h post injection of the initial MPD challenge on experimental day 2 ( Table 1 ). For the induction and expression phase, statistical significance was set at p < 0.05. 3 Results 3.1 Saline control Six control groups were used (groups A 1-4; A 2-4; B 1-4; B 2-4; C 1-4; and C 2-4) ( Table 1 ) to determine the effects of handling and injection. Mild increases in activity for about 5–7 min post injection were observed, but within 5–7 min after saline injection activities returned to baseline. All the experimental days post saline injection in each of the six groups exhibited similar activities with minor non-significant fluctuation. Since experimental day one (ED 1) activity post injection was similar to that obtained on all the other experimental days (ED's) post saline injection, ED 1 post saline injection activity was used as control for the other ED's post saline injection. Moreover, since in the MPD groups ED 1 activity is the recording post saline injection, this activity (ED 1 post saline) was used as control for the MPD acute effect injection given on ED 2. 3.2 Acute effect of MPD in adult WKY, SHR, and SD rats – (Comparing ED 2 to ED 1) Using the dose-response protocol, three MPD doses were injected into adult rats from three genetically different strains to investigate possible differences in their response to the drug. Fig. 1 summarizes the horizontal activity (HA), total distance (TD) traveled, vertical activity (VA), and number of stereotypic movements (NOS) of the three adult rat strains following saline (ED 1) and 0.6, 2.5, and 10.0 mg/kg MPD, i.p. (ED 2) administration. Fig. 1 A summarizes the horizontal activity of all three strains, and shows an equal baseline (ED 1) among the three rat strains. The 0.6 mg/kg MPD dose given at ED 2 failed to significantly alter the horizontal activity of the WKY or SHR rats when compared to ED 1. However, the same dose of MPD at ED 2 caused a significant ( p < 0.05; F 1,27 = 5.01) increase in horizontal activity in the SD rats when compared to saline administration (ED 1). The 2.5 mg/kg MPD dose significantly ( p < 0.05; F 1,27 = 4.93 and F 1,27 = 5.11) increased the horizontal activity of the WKY and SD rats, respectively; however, it elicited a non-significant increase in horizontal activity in the SHR rats. The 10.0 mg/kg MPD dose elicited a significant ( p < 0.05; F 1,27 = 4.82; F 1,27 = 5.13 and F 1,27 = 5.22) and robust increase in the horizontal activity of all three rat strains (WKY, SHR, and SD), respectively, with the WKY rats exhibiting the most intense increase in activity. Fig. 1 B summarizes the total distance traveled (TD) by the three rat strains. Unlike the horizontal activity, the total distance traveled showed that each strain exhibits different levels of baseline activity (ED 1), i.e. the WKY, SHR, and SD rats traveled 1215, 620 and 505 cm, respectively in the 2 h post saline injection. The 0.6 mg/kg MPD dose slightly decreased the total distance traveled of the WKY rats and slightly increased the total distance traveling of the SHR and SD rats. However, these changes were not significant when compared to the saline control (ED 1). The 2.5 mg/kg MPD dose increased the total distance of all three strains. However, only the SD rats exhibited a significant ( p < 0.05; F 1,27 = 4.93) increase. The 10.0 mg/kg MPD dose produced a significant ( p < 0.05; F 1,27 = 5.14; F 1,27 = 4.79; F 1,27 = 4.90) robust increase in the total distance traveled by all three strains (WKY, SHR, and SD), respectively. The WKY rats showed the most intense increase in total distance traveling following the 10.0 mg/kg MPD administration. Fig. 1 C summarizes the vertical activity of all three strains and show that each strain exhibited a different level of baseline activity (ED 1) for vertical activity, highest for the WKY rats followed by the SHR and SD rats, respectively. The 0.6 mg/kg MPD dose elicited a slight increase in vertical activity in all the three rat strains but these changes were not significant. The 2.5 mg/kg MPD dose elicited a significant ( p < 0.05; F 3,49 = 4.01 and F 3,49 = 3.87) increase in the WKY and SD rat groups and the 10 mg/kg MPD dose elicited significant ( p < 0.05; F 3,49 = 3.73; F 3,49 = 4.02 and F 3,49 = 4.11) robust increases in vertical activity in all the strain groups (WKY, SHR and SD), respectively ( Fig. 1 C). Fig. 1 D summarizes the number of stereotypic movements of all three rat strains. The 0.6 mg/kg MPD dose elicited significant increases in stereotypic movement in the SD rat strain group. The 2.5 mg/kg dose elicited significant ( p < 0.05; F 1,27 = 4.65; F 3,49 = 5.01) increases in the WKY and SD rat groups whereas the 10 mg/kg MPD dose elicited significantly ( p < 0.05; F 1,27 = 5.11; F 1,27 = 5.02 and F 1,27 = 5.17) robust increases in all groups (WKY, SHR and SD), respectively ( Fig. 1 D). Fig. 1 summarizes the four locomotor indices studied. To reduce the number of figures and for simplification, in the next section only representative figures of activity are shown with the text. Fig. 2 summarizes and compares the cumulative horizontal activity 2 h post injection in WKY, SHR, and SD rats of both age groups (adolescent and adult) following the administration of saline (ED 1), and the initial administration of 0.6, 2.5, and 10.0 mg/kg MPD (ED 2). The figure shows that acute MPD administration elicited similar effects in all the rat groups (adolescent and adult WKY, SHR and SD) with the exception of the SD group following a dose of 0.6 mg/kg MPD ( Fig. 2 0.6 mg MPD). Interestingly, among all three strains the most intense increase in horizontal activity was exhibited by both the adolescent and adult WKY rats, with the adult WKY rats exhibiting a more intense increase in activity when compared to their adolescent counterparts, following the initial 10.0 mg/kg MPD dose (ED 2) ( Fig. 2 – bottom left). Fig. 3 summarizes and compares the total distance traveled 2 h post injection for all three strains of both adolescent and adult rats following the administration of saline (ED 1), and the initial administration of 0.6, 2.5, and 10.0 mg/kg MPD (ED 2). The 0.6 mg/kg MPD dose did not elicit any significant change in the total distance among the adolescent or adult rats regardless of strain. This observation differed from that observed for horizontal activity ( Fig. 2 ). However, following the administration of 2.5 mg/kg MPD, both adolescent and adult rats of all three strains exhibited a significant ( p < 0.05; F 1,24 = 4.53; F 1,24 = 4.28; F 1,24 = 5.13 for the WKY, SHR and SD adolescent, respectively and F 1,24 = 4.93; F 1,24 = 4.37; F 1,24 = 4.77 for the WKY, SHR and SD respectively) increase in the total distance traveled ( Fig. 3 – middle row). This observation was different from that observed for the horizontal activity ( Fig. 2 ). The 10.0 mg/kg MPD dose exhibited a significantly ( p < 0.05; F 1,17 = 6.96; F 1,17 = 7.11; F 1,17 = 6.73 for the WKY, SHR and SD adolescent and F 1,17 = 7.02; F 1,17 = 7.23; F 1,17 = 7.42 for the WKY, SHR and SD adult, respectively) robust increase in the total distance traveled for adolescent and adult rats of all three strains, with both the adolescent and adult WKY rats showing the most intense increase followed by the SHR and SD rats, respectively. Interestingly, when comparing the two age groups in all three strains, the adolescent and adult WKY and SD rats exhibited similar increases the total distance traveled, while the adolescent SHR rats exhibited a more intense increase in activity when compared to their adult counterparts. However, unlike the increase in total distance elicited following 10.0 mg/kg MPD administration ( Fig. 3 ), the increase in horizontal activity following the same 10.0 mg/kg MPD dose ( Fig. 2 ) elicited a more intense increase in activity in the adult WKY rats when compared to their adolescent counterparts. Similar results were also observed following the initial MPD administration (ED 2) for vertical activity and number of stereotypic movements among the three strains, when compared to the saline control (ED 1) (data not shown). 3.3 Chronic effect of saline in adolescent and adult WKY, SHR, and SD rats Fig. 4 summarizes the horizontal activity during the initial 2 h post saline administration for all 11 experimental days of the adolescent and adult rats of all three strains using the same protocol as used for MPD administration ( Table 1 ). Horizontal activity in the adolescent WKY, SHR, and SD rats exhibited similar levels of baseline activity as the adult WKY, SHR, and SD rats throughout the 11 experimental days. Similar observations were also made for the total distance traveled, vertical activity, and number of stereotypic movements following saline administration (data not shown). In summary, the histograms show that repetitive saline administration does not elicit any significant change in the level of locomotor activity in either adolescent or the adult rats regardless of strain, i.e., the activity level following the saline administration at ED 1 was similar to that obtained at all other EDs. Thereafter in the MPD treated groups the ED 1 post saline recording was used as baseline (control) activity. 3.4 Chronic effects of MPD in adolescent and adult WKY rats Fig. 5 summarizes the chronic MPD dose-response (0.6, 2.5, and 10.0 mg/kg) on the total distance traveled by adolescent and adult WKY rats. The line graphs represent the temporal total distance traveled in 10 min bins for 120 min post injection for the administration of saline on experimental days 1 and injection of 0.6, 2.5, or 10.0 mg/kg MPD on experimental days 2 and 11 ( Table 1 ). The bar graphs represent the 2 h cumulative activity post injection for all 11 experimental days, i.e., area under the temporal graphs per day. Both acute (ED 2) and chronic (ED 7 and ED 11) administration of 0.6 mg/kg MPD failed to elicit any significant changes in the total distance traveled by either the adolescent or the adult WKY rats. The 2.5 mg/kg MPD dose did elicit an increase in the total distance traveled by both the adolescent and adult rats. However, differences in response patterns were observed between the adolescent and adult WKY rats. For example, the 2.5 mg/kg MPD dose elicited a significant ( p < 0.05; F 2,32 = 2.48) increase in total distance traveled that lasted for 120 min post injection in the adult rats, while a significant ( p < 0.05; F 2,32 = 2.34) increase only lasted for 40 min in the adolescent WKY rats ( Fig. 5 – middle row, temporal graph). The adolescent bar graph demonstrates that the initial 2.5 mg/kg MPD (ED 2) dose elicited a significant ( p < 0.05; F 2,32 = 2.53) increase in the total distance traveled when compared to the baseline activity (ED 1). The bar graph also demonstrates a progressive increase in the daily level of activity elicited by subsequent MPD administration. In contrast, the adult WKY rats exhibited a non-significant increase in activity following the initial 2.5 mg/kg MPD administration (ED 2), and did not exhibit any significant increase in the level of activity until the third day of MPD administration (ED 4). Only the adult WKY rats exhibited a significant ( p < 0.05; F 2,32 = 2.47) increase in the total distance traveled following the MPD rechallenge at this dosage on ED 11 when compared to that elicited by the initial MPD administration (ED 2), i.e., behavioral sensitization was exhibited only by the adult WKY rats ( Fig. 5 – middle right). The 10.0 mg/kg MPD dose exhibited a significantly ( p < 0.05; F 2,32 = 4.44 and F 2,32 = 3.76) robust increase in the total distance traveled by both MPD naïve adolescent and adult WKY rats (ED 2), respectively, with a decrease in intensity following each subsequent MPD administration. By experimental day 11, a significant ( p < 0.05; F 2,32 = 4.08) decrease in total distance was exhibited when compared to the initial MPD administration (ED 2), i.e., tolerance was exhibited in both the adolescent and adult WKY rats at this dosage. Interestingly, the adult WKY rats exhibited tolerance at a much earlier stage (ED 5–ED 7), while the adolescent WKY rats exhibited tolerance only following MPD rechallenge on experimental day 11 ( Fig. 5 – bottom). Similar observations were obtained for the other locomotor indices (data not shown). 3.5 Chronic effect of MPD in the adolescent and adult SHR rats Fig. 6 summarizes the chronic dose-response elicited by 0.6, 2.5, and 10.0 mg/kg MPD administration on the total distance traveled by both the adolescent and adult SHR groups. The 0.6 mg/kg MPD did not elicit any significant change in the total distance traveled by either the adolescent or the adult SHR. However, 2.5 mg/kg MPD did elicit an age-specific response pattern among the SHR rats. For example, the bar graph (histogram) shows that 2.5 mg/kg MPD did not elicit any significant increase in the total distance until the second MPD administration (ED 3) in the adult SHR rats, and not until the third MPD administration (ED 4) in the adolescent SHR rats ( Fig. 6 – middle row). Furthermore, the temporal graph shows that the 2.5 mg/kg MPD rechallenge (ED 11) in the adult SHR rats elicited a significant ( p < 0.05; F 2,32 = 2.48) increase in the total distance traveled that lasted for 60 min post injection, while in the adolescent SHR rats a significant ( p < 0.05; F 2,27 = 3.61) increase in the total distance only lasted for 30 min. Only the adult SHR rats exhibited a significant ( p < 0.05; F 2,27 = 3.84) increase in the total distance on ED 11 when compared to the initial MPD administration (ED 2) ( Fig. 6 – middle right), i.e., behavioral sensitization was exhibited only in the adult SHR rats. The 10.0 mg/kg MPD dose elicited a significantly ( p < 0.05; F 2,27 = 4.15 and F 2,27 = 3.93) robust increase in the total distance traveled by both the adolescent and adult SHR rats ( Fig. 6 – bottom), respectively; however, age-specific differences were also observed. For example, the intensity of the increase in the total distance following 10.0 mg/kg MPD appears to be higher in the adolescent SHR rats when compared to their adult counterparts during the initial experimental day. However, no behavioral sensitization or tolerance was observed in either the adolescent or the adult SHR rats following the MPD rechallenge (ED 11 compared to ED 2). Furthermore, the temporal graph shows that following the 10.0 mg/kg MPD rechallenge a significant ( p < 0.05; F 2,27 = 3.61) increase in activity that lasted for the full 120 min post injection was elicited in the adolescent SHR rats, while the adult SHR rats exhibited a significant ( p < 0.05; F 2,27 = 4.17) increase that only lasted for 80 min post injection ( Fig. 6 – bottom). 3.6 Chronic effect of MPD in the adolescent and adult SD rats Fig. 7 summarizes the chronic dose-response elicited by administration of 0.6, 2.5, and 10.0 mg/kg MPD on the total distance traveled by both the adolescent and adult SD rats. The 0.6 mg/kg MPD dose did not elicit any significant change in total distance traveled by either the adolescent or adult SD rats. However, 2.5 mg/kg MPD did elicit an age-specific response pattern among the SD rats. For example, the temporal graph shows that the 2.5 mg/kg MPD rechallenge (ED 11) elicited a significant ( p < 0.05; F 2,26 = 3.48) increase in the total distance for 90 min post injection in the adult SD rats, while eliciting a significant ( p < 0.05; F 2,26 = 3.14) increase for only 50 min in the adolescent SD rats. Furthermore, the bar graph shows that the 2.5 mg/kg MPD dose elicited a more intense increase in total distance traveled by the adult SD rats when compared to their adolescent counterparts, during the initial experimental days. Similarly, only the adult SD rats exhibited a significant ( p < 0.05; F 2,26 = 3.12) increase in the total distance traveled upon the 2.5 mg/kg MPD rechallenge (ED 11) when compared to the initial MPD administration (ED 2), i.e., behavioral sensitization was exhibited only in adult SD rats ( Fig. 7 – middle). The 10.0 mg/kg MPD dose elicited a significantly ( p < 0.05; F 2,26 = 4.03) robust increase in total distance traveled by both the adolescent and adult SD rats. However, age-specific responses can be observed in the temporal graph. For example, the 10.0 mg/kg MPD rechallenge elicited a significant ( p < 0.05; F 2,26 = 3.63) increase in total distance that lasted for the full 120 min post injection in the adult SD rats, while the adolescent SD rats exhibited a significant ( p < 0.05; F 2,26 = 3.75) increase that lasted only for 60 min post injection ( Fig. 7 bottom temporal graphs). Furthermore, the temporal graph also shows that only the adult SD rats exhibited a significant ( p < 0.05; F 2,26 = 3.41) increase in total distance traveled on ED 11 (60–100 min post injection) when compared to the initial MPD administration (ED 2), i.e., behavioral sensitization was exhibited only in the adult SD rats, even though the bar graph (total distance under the curve on ED 11) did not show this augmentation ( Fig. 7 – bottom right). 4 Discussion MPD is a mild stimulant of the central nervous system (CNS) with similarities in its structure and neuropharmacological profile to other psychostimulants with a high potential for abuse such as amphetamine, methamphetamine, and cocaine [46,47] . Psychostimulants can elicit adverse behavioral effects after repeated exposure, i.e., dependence, paranoia, schizophrenia, and sensitization [48] . In rodents, behavioral sensitization is characterized by a progressive increase in locomotor activity produced after repeated administration of a psychostimulant ( [15,17,20,31,33,38,48–50] ). The onset of behavioral sensitization and/or tolerance has been used in experimental models to investigate the risk of other behavioral disorders and to predict a drugs ability to elicit dependency ( [18,51–54] ). The objective of the present study is to investigate the acute and chronic effects of MPD among male adult and adolescent rats of three different genetic strains, and whether the effects of MPD are age and/or genetically dependent. In a preliminary MPD dose-response study using male SD rats [31] the starting dose of 0.3 mg/kg MPD was chosen because of its ability to elicit effects in some studies but not in others. The dosage was then progressively doubled from the initial dose, i.e., 0.3, 0.6, 1.2, 2.5, 5.0, 10.0, 20.0, and 40.0 mg/kg MPD. The administration of 0.3 mg/kg MPD failed to elicit any significant change in locomotor activity, when compared to both the saline and time controls. Following chronic 2.5 and 5.0 mg/kg MPD administration behavioral sensitization was observed. However chronic 10.0 mg/kg MPD administration elicited mixed effects, i.e., behavioral sensitization was elicited in some animals while tolerance was elicited in other animals. Following chronic 20.0 and 40.0 mg/kg MPD administration tolerance was elicited [31] . Based on the data obtained in the above study, the doses 0.6, 2.5, and 10.0 mg/kg were selected for further investigation. In humans, the optimum MPD treatment dosage has becoming increasingly variable, with one study reporting that the range of doses ingested by patients being treated with MPD ranged between 0.06 and 29.3 mg/kg, while the majority of patients were being treated with ∼3.0 mg/kg MPD [55] . However, the pharmacokinetics of MPD must be taken into account when comparing the dosages between humans and rodents, since rodents require a higher drug dose, on a mg/kg basis, to produce the same effect. This difference is thought to result from the increased metabolic rate in rodents [56] . In rodents, a dose smaller than 5.0 mg/kg MPD is considered low and is comparable to the dosages being used in the clinical setting [12,57,58] . Dosages between 5.0 and 10.0 mg/kg MPD are considered moderate [12,59–62] . Based on this, we selected 0.6, 2.5, and 10.0 mg/kg MPD to represent a low, moderate and high MPD dose, respectively. Variations among different animal strains have been reported regarding their susceptibility to psychostimulants, e.g., behavioral sensitization and/or tolerance [15,19] . The present study also demonstrates strain variation following the acute administration of MPD ( Fig. 1 ). Among all three strains, the SD rats demonstrated the most susceptibility to MPD, since it was the only strain where 0.6 mg/kg MPD elicited a significant increase in horizontal activity and number of stereotypic movements ( Fig. 1 A and D). The WKY rats demonstrated susceptibility only at the higher dosages of MPD, i.e., 2.5 mg/kg MPD elicited a significant increase in horizontal activity, vertical activity, and number of stereotypic movements ( Fig. 1 A, C and D) and the 10.0 mg/kg dose elicited a significant increase in all locomotor indices. The SHR rats were the least susceptible to effects of acute MPD administration, since only the highest dose of 10.0 mg/kg MPD elicited any significant increase in locomotor activity. Furthermore, the intensity of the increase in locomotor activity demonstrated further differences among the strains, especially at the higher dosages. Among adult rats of all three strains, the WKY rats exhibited the most robust increases in all of the locomotor indices following 10.0 mg/kg MPD administration followed by the SD and SHR rats, respectively ( Fig. 1 ). Similarly, following 10.0 mg/kg MPD administration the adolescent WKY rats had the most robust increase in locomotor activity, horizontal activity ( Fig. 2 ) and total distance ( Fig. 3 ). It has also been reported that psychostimulants can elicit an attenuated behavioral response in the adolescent rats, while eliciting an increased behavioral response in their adult counterparts [13,63] . In the present study, similar but not significant patterns were also observed among the three strains following the acute effect of 0.6 mg/kg MPD, i.e., adult rats of all three strains exhibited a greater increase in activity compared to their adolescent counterparts. However, following 10.0 mg/kg MPD, the adult WKY rats exhibited a more substantial increase in total distance ( Fig. 1 ) and horizontal activity ( Fig. 2 ) when compared to their adolescent counterparts. By contrast, at this dose, adolescent rats of the SHR and SD strains exhibited a more substantial increase in locomotor activity when compared to their adult counterparts. This shows that not only does the acute administration of MPD elicit an age-specific response, but it is also strain-specific as well. Chronic administration of MPD further demonstrates the variability between these strains, and among the two age groups. Chronic administration of 0.6 mg/kg MPD exhibited no significant difference among the three strains regardless of age. However, 2.5 mg/kg MPD did exhibit strain-specific differences in response when comparing the temporal activity following the 2.5 mg/kg MPD rechallenge (ED 11) to the baseline activity (ED 1). For example, on ED 11 the adult WKY rats exhibited the longest significant ( p < 0.05) increase in activity post injection (120 min) among the three rat strains, followed by the SD (90 min) and SHR (60 min) rats, respectively ( Figs. 5–7 ). Furthermore, the adolescent rats not only exhibited a shorter duration in activity following 2.5 mg/kg MPD administration on ED 11 when compared to their adult counterparts, but the SD rats exhibited the longest significant increase in activity post injection (50 min) among the adolescent rats followed by the SHR (40 min) and WKY (30 min) rats, respectively ( Figs. 5–7 ). However, when the activity level upon MPD rechallenge (ED 11) was compared to the initial MPD administration (ED 2) only an age-specific response was observed, since only the adult rats of all three strains exhibited behavioral sensitization following the chronic administration of 2.5 mg/kg MPD ( Figs. 5–7 ). Following the chronic administration of 10.0 mg/kg MPD a distinct age and strain-specific response pattern was observed. For example, when the activity upon the 10.0 mg/kg MPD rechallenge (ED 11) was compared to the initial 10.0 mg/kg MPD administration (ED 2) tolerance was exhibited by both the adult and adolescent WKY rats. However, the adult WKY rats appears to be more susceptible to the behavioral effects of MPD, since the adult WKY rats exhibited tolerance by ED 5 ( Fig. 5 – bottom right bar graph). Similarly, only the adult SD rats exhibited behavioral sensitization upon MPD rechallenge, i.e., a significant increase in activity on ED 11 between 60 and 100 min post injection when compared to the initial MPD administration (ED 2) ( Fig. 7 – bottom temporal graph). On the other hand, neither the adolescent nor adult SHR rats exhibited behavioral sensitization following the MPD rechallenge (ED 11) ( Figs. 6 and 7 – bottom bar graph). This shows that there are both age and strain-specific behavioral responses following the chronic administration of MPD. Methylphenidate hydrochloride is a psychostimulant that acts as an indirect dopamine (DA) agonist, inhibiting the reuptake of DA by blocking dopamine transporters (DAT), and to a lesser extent norepinephrine transporters (NET) [64–66] . The therapeutic effect of MPD has been attributed to its ability to bind to DAT, and cause an increase in extracellular DA levels in the mesocorticolimbic system [67–69] . An explanation for the varied effects elicited by acute and chronic MPD administration among the three rat strain could be the genetic variability in the mesocorticolimbic dopamine system which mediates cognitive, emotional, and motivational behaviors [70] . Several studies have reported significant differences in the density of NET, DAT and serotonin transporters (5-HTT) in several different brain regions when comparing the WKY and SD rats [71–73] . Other studies have reported a decreased release of dopamine from the nucleus accumbens and caudate-putamen slices of SHR rats when compared to their control WKY rats. This observation suggests that the different responses to the drug between the different rat strains are due to differences in impairment of the release of dopamine storage vesicles from the nerve terminals following the MPD treatment(s) [19,74] . In a study using SHR and WKY rats, it was reported that MPD treatment affected presynaptic dopamine release differently among the two strains [19] . Russell et al. [74] suggest that the difference in dopamine release between the strains rats after MPD administration is due to an upregulation of D2 autoreceptors in the WKY, which function to reduce the extracellular dopamine level. However, such a change is not seen in the SHR rats. This suggests that in the SHR rats the D2 autoreceptors may have been previously upregulated to their maximum potential due to an impairment in the dopamine storage vesicles. This would result in DA leaking from the vesicles into the cytoplasm and subsequently into the synaptic cleft [75] . Based on the above finding, we postulate that the genetic variations underlying the mesocorticolimbic system in the WKY, SHR, and SD rats contribute substantially to these observed behavioral differences in response to MPD administration. It has been reported that adolescent rats are affected differently by other psychostimulants when compared to their adult counterparts [13,63,76] . Age differences were also observed in the present study using acute and chronic dose response protocols, recording different locomotor indices (HA, TD, VA, and NOS) and using two methods of data evaluation, i.e. temporal analysis of 10 min sequential activity as well as total activity/2 h post injection. A possible reason for these age differences can be due to the fact that brain maturation occurring during adolescence is considered to be highly plastic and highly sensitive to environmental and pharmacological intervention [77] . The prefrontal cortex, nucleus accumbens, and the amygdala are brain areas that are characterized by selective DA innervations. The DAergic innervations of these brain areas undergo a phase of drastic anatomical and physiological late maturation [11,78,79] . It has also been suggested that the pharmacological interactions of MPD with the DA system during neurodevelopment are likely to produce long lasting changes [10,80,81] . Another study suggests that at least some components of the mesocorticolimbic dopamine system respond differently to psychostimulants such as amphetamines during adolescence compared to adulthood [77,82] . Therefore, we interpret the age related diverse effects observed in the present study following MPD administration arising from variations in the mesocorticolimbic dopamine system associated with different stages in the maturation of the CNS. In conclusion, this study has shown that genotype plays a major role in determining an individual's drug susceptibility and behavioral response following the acute and chronic administration of MPD. Furthermore, this study shows that age at the time of MPD exposure also plays a major role in determining the behavioral response to MPD and whether behavioral sensitization and/or tolerance will develop. Most importantly, this study shows that the Spontaneous Hyperactive rat (SHR) group, an ADHD animal model, is the least susceptible to development of behavioral sensitization and/or tolerance among the three strains studied. 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