Delay-Dependent Impairments in Memory and Motor Functions After Acute Methadone Overdose in Rats

Abstract

Methadone is used as a substitution drug for the treatment of opioid dependence and chronic pain. Despite its widespread use and availability, there is a serious concern with respect to the relative safety of methadone. The purpose of this study was to characterize how acute methadone overdose affects the cognitive and motor performance of naïve healthy rats. The methadone overdose was induced by administering an acute toxic dose of methadone (15 mg/kg; ip; the equivalent dose of 80% of LD₅₀) to adolescent rats. Resuscitation using a ventilator pump along with a single dose of naloxone (2 mg/kg; ip) was administered following the occurrence of apnea. The animals which were successfully resuscitated divided randomly into three apnea groups that evaluated either on day 1, 5, or 10 post-resuscitation (M/N-Day 1, M/N-Day 5, and M/N-Day 10 groups) in the Y-maze and novel object memory recognition tasks as well as pole and rotarod tests. The data revealed that a single toxic dose of methadone had an adverse effect on spontaneous behavior. In addition, Recognition memory impairment was observed in the M/N-Day 1, 5, and 10 groups after methadone-induced apnea. Further, descending time in the M/N-Day 5 group increased significantly in comparison with its respective Saline control group. The overall results indicate that acute methadone-overdose-induced apnea produced delay-dependent cognitive and motor impairment. We suggest that methadone poisoning should be considered as a possible cause of delayed neurological disorders, which might be transient, in some types of memory or motor performance in naïve healthy rats.

Keywords: learning and memory performance; methadone; motor coordination; naloxone; overdose; rat.

FIGURE 1

Schematic illustration of the experimental schedule. (A) Protocol overview of the study. After 2 days of behavioral training (rotarod and pole tasks), all rats were administered drug injection on day 0. According to the time of the behavioral test, (starting on day 1, 5, or day 10 after drug administration) they represented here in three parts. (B) After drug application, six separated animal groups (Saline, Methadone, Naloxone, M/N-Sedate, Saline control-Day 1, and M/N-Day 1) were used to evaluate different behavioral tests including Y-Maze, Rotarod test and Pole test on day 1 after drug administration and Novel object recognition (NOR) test from day 1 to day 4 post-treatment. (C) In two other separated groups (Saline control-Day 5, M/N-Day 5), after administration of saline alone or methadone + naloxone on day 0, Y-Maze, Pole test, and Rotarod test were carried out on day 5 followed by NOR test from day 5 to day 8. (D) In Saline control-Day 10 and M/N-Day 10 groups, 10 days after administration of saline alone or methadone + naloxone, Y-Maze, Pole test and Rotarod test were carried out on day 10 followed by NOR test from day 10 to day 13.

FIGURE 2

(A) Spontaneous alternation behavior and (B) Total number of arm entries were recorded in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and spontaneous alternation behavior was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 6), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6 right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 12, methadone; n = 8 and naloxone; n = 7 (saline, methadone or naloxone were administered alone in separated groups in which spontaneous alternation behavior and the number of arm entries were recorded only 1 day after drug administration during an 8-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both (apnea groups) in a single dose. Each bar shows the mean ± SEM for 6–12. ^∗P < 0.05 different from the Saline group. ⁺P < 0.05 and ⁺⁺P < 0.01 different from their respective Saline control groups.

FIGURE 3

Performance of recognition memory in the novel object recognition task in three sessions as follow; (A) Familiarization (rats were allowed to explore freely two identical objects A1 and A2 for 3-min), (B) Short-term memory (object A1 or A2 was replaced with object B while rats were allowed to explore for 3-min), (C) long-term memory (object B was replaced with object C which provided rats explored freely two objects for 3-min) in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and recognition memory was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 7), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups (n = 6); right panel), M/N-Sedate (n = 7; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 11, naloxone; n = 8 (Saline, methadone or naloxone were administered alone in separated groups which recognition memory was evaluated only 1 day after the drug administration in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6–11. ^∗P < 0.05 and ^∗∗P < 0.01 different from the Saline group. ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 different from their respective Saline control groups.

FIGURE 4

Evaluation of motor performance in pole test such that three parameters were measured including; (A) time to turn downward (t-turn), (B) descending time (time to move downward to reach the floor and (C) total time (time to turn and descending the pole to reach the floor) in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor behavior was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 11), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 7) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate; n = 9 (a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 8 and naloxone; n = 8 (saline, methadone or naloxone were administered alone in separated groups in which motor function was evaluated 1 day after drug administration during pole test in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6-14. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 different from the Saline group. ^††P < 0.01 and ^†††P < 0.001 different from the M/N-Sedate group. ⁺P < 0.05 different from their respective Saline control groups.

FIGURE 5

Evaluation of motor coordination and balance in rotarod test during a 5-min trial in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor coordination was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 9), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 8) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 9 and naloxone; n = 6 (saline, methadone or naloxone was administered alone in separated groups in which coordination assessment was evaluated only 24 h following the drug administration in a 5-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. The time each rat stay on the rod before falling was recorded. Each bar shows the mean ± SEM for 6–10. ^∗∗P < 0.01 different from the Saline group.

FIGURE 6

Locomotor activity was recorded by using photobeam activity system during a 5-min session in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and locomotor activity was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 10), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 11; a single dose of naloxone was administered after methadone overdose immediately in the initial stage of sedation state, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 12 and naloxone; n = 8 (saline, methadone or naloxone was administered alone in separated groups in which locomotor activity was evaluated 1 day after drug administration) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Administration of toxic dose of methadone did not change locomotor activity in all groups. Each bar shows the mean ± SEM for 6–14.