Sex Differences in the Physiological and Behavioral Effects of Chronic Oral Methylphenidate Treatment in Rats

Abstract

Methylphenidate (MP) is a psychostimulant prescribed for Attention Deficit Hyperactivity Disorder. Previously, we developed a dual bottle 8-h-limited-access-drinking-paradigm for oral MP treatment of rats that mimics the pharmacokinetic profile of treated patients. This study assessed sex differences in response to this treatment. Male and female Sprague Dawley rats were assigned to one of three treatment groups at 4 weeks of age (n = 12/group): Control (water), low dose (LD) MP, and high dose (HD) MP. Rats drank 4 mg/kg MP (LD) or 30 mg/kg MP (HD) during the first hour, and 10 mg/kg (LD) or 60 mg/kg MP (HD) for the remaining 7 h each day. Throughout 3 months of treatment, rats were monitored for body weight, food intake, and fluid intake; as well as tested for open field behavior, circadian activity, novel object recognition, and social interaction. Chronic MP treated rats exhibited reduced fluid intake during distinct treatment weeks to a greater extent in males, and reduced total fluid intake in males only. HD MP treatment decreased body weight in both sexes, while HD MP increased total food intake in females only, likely to offset energy deficits resulting from MP-induced hyperactivity. LD and HD MP increased locomotor activity in the open field, particularly in females and during later treatment weeks. MP dose-dependently increased activity during the dark cycle of circadian testing in females, while in males hyperactivity was only exhibited by HD rats. HD MP increased center activity to a greater extent in males, while MP increased rearing behavior in females only. MP had no effect on social behavior or novel object recognition in either sex. This study concludes that chronic oral MP treatment at clinically-relevant dosages has significant effects on food intake, body weight, open field behavior, and wake cycle activity. Particularly marked sex differences were apparent for locomotor activity, with females being significantly more sensitive to the hyperactivating effects of the drug. These findings suggest that chronic MP exposure beginning in adolescence can have significant behavioral effects that are both dose- and sex-dependent, and raise concerns regarding the reversibility of these effects post-discontinuation of treatment.

Keywords: attention deficit hyperactivity disorder; methylphenidate; psychostimulant; ritalin; sensitization; sex differences.

Figure 1

(A) Mean (+SEM) daily fluid intake by treatment week. All groups generally increased fluid intake as they grew from adolescents into adults. Males had greater fluid intake than females. HD male rats drank less than water control rats during treatment weeks 1–5 and 7–12 (^*p < 0.05); HD male rats also drank less than LD rats in treatment weeks 1 and 3 (^$p < 0.05); LD male rats drank less than water control rats for treatment weeks 5, 8, 9, 11, and 12 (^#p < 0.05). HD female rats drank less than water control rats in treatment week 1, 3, and 8 (^%p < 0.05). HD female rats drank less than LD female rats in week 3, but drank more in weeks 6 and 8 (^∧p < 0.05). LD female rats drank less than water control females in week 1 (^&p < 0.05). (B) Mean (+SEM) total fluid consumption throughout the entire experiment. HD MP males drank less than both LD MP (^$p < 0.05) and water (^*p < 0.05) treated males, while LD MP males drank less than water treated males (^#p < 0.05).

Figure 2

(A) Mean (+SEM) daily food intake by treatment week. Rats expectedly increased food intake as they grew from adolescents into adults. Males had greater food intake in comparison to females within all groups of treatment. HD male rats ate less than water control males rats during the first treatment week (^*p < 0.05), and ate less than LD rats in treatment weeks 1–5 (^$p < 0.05). LD male rats ate more than water control rats for treatment weeks 3–5 (^#p < 0.05). HD female rats ate less than water control female rats in treatment week 3, but ate more in comparison of treatment week 4, 8–12 (^%p < 0.05) HD female rats ate less than LD female rats during treatment weeks 1, 3, 8, 11, and 12 (^∧p < 0.05). LD female rats ate more than water control females during treatment weeks 9 and 10 (^&p < 0.05). (B) Mean (+SEM) total food consumption throughout the entire experiment, with HD MP females eating more than water treated females (^%p < 0.05).

Figure 3

(A) Mean (+SEM) body weight by treatment week. Rats expectedly gained weight as they grew from adolescents into adults. Males expectedly gained more weight than females. HD MP treatment dose-dependently attenuated body weight throughout most of the treatment period. HD male rats weighed significantly less than water control males (^*p < 0.05) and LD males (^$p < 0.05) in treatment weeks 1–12. HD MP female rats weighed less than water control females in treatment weeks 4–12 (^%p < 0.05) and less than LD MP female rats in treatment weeks 3–12 (^∧p < 0.05). (B) Mean (+SEM) percent change in body weight from pretreatment to the final week of the experiment. HD MP attenuated weight gain, regardless of sex.

Figure 4

Horizontal activity in the open field. (A) Mean (+SEM) distance traveled in the open field. There was an overall increase in distance traveled as time passed. Male HD MP rats exhibited greater activity than male water (^*p < 0.05) and LD MP (^$p < 0.05) rats in weeks 6 and 8–12. Female LD MP rats traveled a greater distance than female water rats in weeks 1, 5, 7–12 (^&p < 0.05) while female HD MP rats were more active than water rats in all MP treatment weeks (%p < 0.001). Female HD MP rats were also more active than female LD MP rats in weeks 2–12 (^∧p < 0.001). Females were more active than males on HD (weeks 2–12, ^@p < 0.05) and LD (weeks 6–12, +p < 0.05) MP treatment. Insert graph shows area under the curve by treatment group for all treatment weeks. (B) Mean (+SEM) velocity in the open field. There was an overall increase in velocity as time passed. Male HD MP rats had greater velocity than water rats in weeks 6, 8, and 10–12 (^*p < 0.05) and greater velocity than LD MP rats in weeks 6–8 and 10–12 (^$p < 0.05). Female LD MP rats had significantly greater velocity than female water rats in weeks 5 and 7–12 (^&p < 0.05). Female HD MP rats had significantly greater velocity than female water rats in all MP treatment weeks (^%p < 0.001) and also greater than female LD MP rats in weeks 2–12 (^∧p < 0.001). Females moved at a greater velocity than males on HD (weeks 2–12, ^@p < 0.05) and LD (weeks 5–12, ⁺p < 0.05) MP treatment, although LD MP females moved at a slower velocity water treated females in week 1 (^&p < 0.05). Insert graph shows area under the curve by treatment group for all treatment weeks.

Figure 5

Center activity in the open field. (A) Mean (+SEM) relative center distance. There was an overall increase in center activity as time passed, and over time males exhibited greater center activity than females. HD MP treatment increased center activity compared to water and LD MP rats in males only. Insert graph shows area under the curve by treatment group for all treatment weeks. (B) Mean (+SEM) center time in the open field. There was an overall increase in center time as time passed. Overall, HD MP rats spent more time in the center of the arena than water and LD MP treated rats. Additionally, males exhibited more center time than females. Insert graph shows area under the curve by treatment group for all treatment weeks.

Figure 6

Rearing activity in the open field. (A) Mean (+SEM) rearing events in the open field. There was an overall increase in rearing events as time passed. HD rats exhibited more rearing events than both water and LD MP treated rats in both males and females, and throughout most treatment weeks. LD MP increased rearing events in females only. LD and HD MP treated females exhibited greater rearing events than their male counterparts. Insert graph shows area under the curve by treatment group for all treatment weeks. (B) Mean (+SEM) rearing time in the open field. There was an overall increase in rearing time as time passed. Females of all treatment groups exhibited greater rearing time than their male counterparts. LD and HD MP increased rearing time in several weeks, however both MP doses increased rearing time overall in female rats only. Insert graph shows area under the curve by treatment group for all treatment weeks.

Figure 7

Circadian locomotor activity during the last week of treatment. (A) Mean (+SEM) hourly activity over the circadian cycle. A normal circadian cycle was exhibited by all groups, with no apparent shift in cycle. In females, HD MP treatment resulted in hyperactivity compared to both LD MP (^∧p < 0.05) and water (^%p < 0.05) treatment. HD males showed an increase in activity over water control males at 16:00 (^*p < 0.05). HD females exhibited more activity in comparison to HD males at 17:00 to 18:00 (^@p < 0.05). (B) Mean (+SEM) total activity throughout the dark cycle. In females, MP dose-dependently increased circadian activity [HD > Water (^%p < 0.05); HD > LD (^∧p < 0.05); LD > Water (^&p < 0.05)], while in males HD MP increased activity compared to water (^*p < 0.05) and LD MP ($p < 0.05) treated rats. Additionally, HD treated females were more active than male HD rats (^@p < 0.05).