Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentrations during amphetamine withdrawal in rats

Abstract

This experiment was designed to characterize the withdrawal syndrome produced by discontiuation of treatment with escalating, non-neurotoxic doses of d-amphetamine (AMPH). AMPH withdrawal was associated with both transient and persistent changes in behavior and postmortem brain tissue catecholamine concentrations. During the first week of withdrawal rats showed a significant decrease in spontaneous nocturnal locomotor activity. This behavioral depression was most pronounced on the first 2 days after the discontinuation of AMPH pretreatment, was still evident after 1 week, but had dissipated by 4 weeks. Behavioral depression was not due to a simple motor deficit, because AMPH-pretreated animals showed a normal large increase in locomotion when the lights initially went out, but they did not sustain relatively high levels of locomotor activity throughout the night, or show the early morning rise in activity characteristic of controls. Behavioral depression was associated with the transient decrease in the concentration of norepinephrine (NE) in the hypothalamus, and a transient decrease in the ability of an AMPH challenge to alter dopamine (DA) concentrations in the caudate-putamen and nucleus accumbens. AMPH pretreatment also produced persistent changes in brain and behavior. The persistent effects of AMPH were not evident in spontaneous locomotor activity, but were revealed by a subsequent challenge injection of AMPH. AMPH pretreated animals were markedly hyper-responsive to the stereotypy-producing effects of an AMPH challenge. This behavioral sensitization was not fully developed until 2 weeks after the discontinuation of AMPH pretreatment, but then persisted undiminished for at least 1 year. It is suggested that the transient changes in brain and behavior described here may represent an animal analogue of the "distress syndrome" seen in humans during AMPH withdrawal, which is associated with symptoms of depression and alterations in catecholamine function. On the other hand, persistent behavioral sensitization may be analogous to the enduring hypersensitivity to the psychotogenic effects of AMPH seen in former AMPH addicts.

Fig. 1

Graphic representation of the escalating dose amphetamine (AMPH) pretreatment regimen (see Methods). Each open circle represents: (1) a day on which AMPH pretreated animals received two injections of d-AMPH sulfate with each injection separated by at least 8 h, and (2) the dose of each injection. On days when the dose equals zero (closed circles) animals did not receive injections. Control animals received saline injections according to the same schedule. This regimen mimics to some extent the pattern of “runs” and “crashes” seen in addicts (Kramer et al. 1967)

Fig. 2A–F

Mean (±SEM) ratings for stereotyped head and limb movements for saline (open symbols)- and AMPH (closed symbols)- pretreated rats before (interval 0) and after a challenge injection of 2.6 mg/kg AMPH given 3, 7,14, 28, 90 or 180 days after the discontinuation of pretreatment (N = 5–7/group). Mann-Whitney U tests were used to compare the cumulative rating for each AMPH-pretreated group to its respective control group. There was no difference in AMPH-induced stereotyped behavior between saline and AMPH-pretreated animals 3 days after discontinuation of AMPH pretreatment (U = 14, P = 0.6). By 7 days of withdrawal AMPH-pretreated animals had significantly higher stereotypy ratings than did saline-pretreated animals (U = 13, P < 0.01). Significant group differences were also found 14, 28, 90 and 180 days after the discontinuation of AMPH pretreatment (U’s = 0–3, P < 0.005). ● Amphetamine – pretreatment; ○ control

Fig. 3A–F

The average number of crossovers (locomotion from one side of the cage to the other) cumulated over 5-min intervals during 1 h of baseline and for 3 h after a challenge injection of 2.6 mg/kg AMPH, in saline (open symbols)- and AMPH (closed symbols )-pretreated animals tested 3, 7, 14, 28, 90 or 180 days after the discontinuation of pretreatment (N = 6–7/group). There was no difference in baseline locomotor activity between saline- and AMPH-pretreated animals at any time following the cessation of AMPH pretreatment (2-way ANOVAs). Two-way ANOVAs (with repeated measures) were also used to compare AMPH-stimulated locomotor activity for each AMPH-pretreated group to its respective control group. There was no effect of AMPH pretreatment on AMPH-induced locomotor activity 3 or 7 days after the discontinuation of AMPH pretreatment (F’s < 0.5; NS = nonsignificant). Significant group differences were apparent by 14 days of withdrawal (F = 2.03, P < 0.001), because AMPH-pretreated rats showed a significant decline in locomotor activity during the period from about 20–60 min after drug injection, during which time they engaged in focused stereotyped behavior (all significant F values represent a group by time interaction; see text and Fig. 2). Significant group differences were also found 28 (F = 3.4, P < 0.001), 90 (F = 3.4, P < 0.001) and 180 days (F = 3.67, P < 0.001) after the discontinuation of AMPH pretreatment. ● Amphetamine – pretreated; ○ control

Fig. 4

Summary of the effect of pretreatment with saline or AMPH on stereotypy phase locomotor activity (left) and stereotypy ratings (right) produced by a challenge injection of 2.6 mg/kg AMPH given 3, 7, 14, 28, 90 or 180 days after discontinuation of pretreatment. Left: Bars in the left panel depict the average ( + SEM) number of crossovers cumulated during the stereotypy phase (10–60 min post-injection) for AMPH-pretreated animals withdrawn for 3–180 days. The horizontal dashed line represents the average number of crossovers for the pooled control group (n = 38) and the vertical line to the right ± SEM for the controls. A one-way analysis of variance comparing all groups was significant (F = 3.58, P < 0.004). The asterisks (*) indicate that relative to the control group AMPH-pretreated animals showed a significant decrease in stereotypy phase locomotor activity 14, 28, 90 and 180 days, but not 3 or 7 days, after the cessation of AMPH pretreatment (P < 0.05, Fisher’s LSD tests). No other comparisons were statistically significant. Right: Bars in the right panel depict the average (+ SEM) rating for stereotyped head and limb movements cumulated over the entire test session following an AMPH challenge in AMPH-pretreated rats withdrawn for 3–180 days. For ease of comparison the average stereotypy rating for the pooled control group is indicated by the horizontal dashed line and ± SEM by the vertical line to the far right. The asterisks (*) indicate that AMPH-pretreated rats had significantly higher stereotypy ratings than controls between 7 and 180 days of withdrawal, but not after 3 days of withdrawal (* P < 0.01; ** P < 0.005,; see Fig. 2 for U values). The dagger (†) indicates that animals withdrawn for 3 or 7 days (which did not differ from one another, U =24) had a significantly lower cumulative stereotypy rating than did those withdrawn for 14–180 days (Kruskal-Wallis test across the six AMPH-pretreated groups, H =27, df = 5, P < 0.001: follow-up Mann-Whitney U tests, U’s = 1–9, P < 0.01)

Fig. 5A, B

The effect of pretreatment with saline (open symbols, n = 6) or AMPH (closed symbols, n = 6) on crossovers (locomotor activity) and stereotyped behavior after a challenge injection of 2.6 mg/kg AMPH (IP) given 1 year after the cessation of pretreatment. Left: The left panel shows the average number of crossovers cumulated over 5-min intervals during baseline and for 2 h and 50 min after the AMPH challenge (given when indicated by the arrow). AMPH-pretreated rats differed significantly from saline pretreated rats, primarily because the former group showed a large decrease in locomotion about 30 min after the challenge injection (2-way ANOVA with repeated measures, group by time interaction, F = 2.09, P < 0.001). Right: The right panel depicts the mean (± SEM) stereotypy ratings (head and limb movements) obtained during baseline (interval 0) and then repeatedly after the challenge injection of AMPH (see Methods). AMPH-pretreated animals showed significantly more intense stereotypy than saline-pretreated animals (Mann-Whitney U test on the cumulative ratings, U = 0, P < 0.002). ● Amphetamine – pretreated; ● control

Fig. 6A–C

Mean spontaneous locomotor activity in AMPH-pretreated (closed symbols) and control animals (open symbols): A 2–3 days (N = 32 and 24, respectively), B 4–7 days (N = 16/group) and C 23–28 days (N = 16/group) after the discontinuation of pretreatment. The panels on the left represent the average number of crossovers cumulated over 30-min intervals across the day/night cycle. The 10 h lights off period, which began at 19:00 hours, is illustrated by the solid black bar on the horizontal axis, and daytime hours by the open bar. The panels on the right show the average (+ SEM) number of crossovers cumulated over 7 h before the lights went off (11:00–19:00 hours), the initial 3 h after the lights went off (19:00–22:00 hours), the middle of the night (22:00–3:30 hours), the last 1.5 h prior to the lights going on (3:30–5:00 hours) and the 2 h after the lights came back on (5:00–7:00 hours). Data for the remaining 3.5 daytime hours were excluded because of the disturbance associated with data collection and animal care that occured at this time. A 2–3 days. AMPH-pretreated animals were significantly less active than controls both during the daytime (2-way ANOVA with repeated measures on the initial 10 h lights-on period, effect of group, F =5.38, P < 0.024, effect of time, F = 24.8, P < 0.001, interaction non-significant), and at night (ANOVA on lights-off period, group F = 15.8, P < 0.001, time F = 11.2, P < 0.001 and interaction F = 3.4, P <0.001). The significant group by time interaction for nocturnal activity indicates that AMPH-pretreated animals did not differ from controls throughout the entire night, and this is best illustrated in the bar graph to the right. In the right panel asterisks (*) indicate times at which AMPH-pretreated animals differed significantly from controls. For the initial daytime period t = 2.32, P = 0.024, but during the initial lights-off period (19:00–22:00 hours) there were no significant group differences (t = 1.82). During the rest of the night AMPH-pretreated rats were significantly less active than controls (22:00–3:30 hours, t = 3.93, P < 0.001; 3:30–5:00, t = 5.85, P < 0.001), and they remained less active after the lights came on again (5:00–7:00, t = 3.01, P = 0.004). B 4–7 days. AMPH-pretreated animals were not significantly less active than controls during the lights-on period (ANOVA) but were significantly hypoactive at night (ANOVA on nocturnal activity, group F = 17.7, P < 0.001, time F = 5.42, P < 0.001, interaction F = 2.07, P < 0.004). Again, the panel to the right shows that there were no significant group differences during the initial lights-off period (19:00–22:00 hours, t = 2.01), but during the rest of the night AMPH-pretreated rats were significantly less active than controls (22:00–3:30 hours, t = 4.47, P < 0.001; 3:30–5:00 hours, t = 4.05, P < 0.001). C 24–28 days. By 24–28 days there was no longer any effect of AMPH pretreatment on either daytime or nocturnal activity. For nocturnal activity, the group F < 1.0 and the group by time interaction F = 1.13, P = 0.32. ● ▪ Amphetamine – pretreated; ● □ n control

Fig. 7A–D

The effect of pretreatment with escalating doses of AMPH on the basal (steady state) postmortem tissue concentrations of monoamines and their metabolites in selected brain regions (N = at least 8/group), The height of the bars represents the mean (+ SEM) concentration of compounds plotted as a percent of the control group that received a saline challenge (see methods and Table 1). The bars in each panel, from left to right, indicate control (C) or AMPH-pretreated animals 3, 7, or 28 days after the discontinuation of pretreatment. For each compound in each structure statistically significant group differences were determined by conducting a one-way analysis of variance, and if indicated by a significant F value (P < 0.05), pairwise comparisons were made using the Fisher’s LSD test. A heavy horizontal line above some bars indicates significant group differences based on the ANOVA. A vertical line extending downwards from the heavy horizontal line indicates those groups that differed significantly from control, based on the Fisher’s LSD test (P < 0.05). A dagger (†) indicates those groups that differed significantly from AMPH-pretreated animals withdrawn for 28 days, again based on the Fisher’s test (P < 0.05). A Caudate-putamen: the only significant effect was in the ratio of HVA/DA (F = 2.94, P < 0.05), and only AMPH-pretreated rats withdrawn for 3 or 28 days differed significantly from control (P < 0.05). For HVA, F = 2.1, P = 0.12, and for all other compounds F < 1.0. B Nucleus accumbens: there was no significant effect of AMPH pretreatment for any compound. C Medial frontal cortex: there was no significant effect of AMPH pretreatment for any compound. All F’s < 1.0. D Hypothalamus: the only significant ANOVA was for NE (F = 33.2, P < 0.001). AMPH-pretreated rats withdrawn for 3 or 7 days had significantly lower hypothalamic NE concentrations than either controls or rats withdrawn for 28 days (P < 0.05). Hypothalamic NE in the 3-day group was also significantly lower than in the 7-day group (P < 0.05). The group withdrawn for 28 days did not differ from controls. For all other compounds F < 1.0.

Fig. 8A–D

The effect of pretreatment with escalating doses of AMPH on the postmortem tissue concentrations of monoamines and their metabolites in selected brain regions 40 min following a challenge injection of 2.6 mg/kg AMPH. The height of the bars represents the mean (+SEM) concentration of the compounds plotted as a percent of control animals that did not receive an AMPH challenge (see Table 1). The bars in each panel, from left to right, indicate: (1) control animals that did not receive an AMPH challenge (group C; N = 16); (2) control animals that received an AMPH challenge (group 0; N = 15); and AMPH-pretreated animals that received a challenge injection of AMPH, (3) 3 days (group 3; N = 8); (4) 7 days (group 7; N = 8); or (5) 28 days (group 28; N = 8) after the discontinuation of pretreatment. A heavy horizontal line above some bars indicates there were significant group differences for that compound, based on a one-way ANOVA. A vertical line extending downwards from the heavy horizontal line indicates groups that differed significantly from control animals that received saline (Fisher’s test, P < 0.05). The daggers (†) indicate groups that differed significantly from the control animals that received AMPH (group 0). A Caudate-putamen: for NE, F < 1.0 (non-significant). For DA, F =9.06, P < 0.001. The challenge injection of AMPH significantly increased DA concentrations in control animals, and AMPH-pretreated animals withdrawn for 28 days; but not in AMPH-pretreated rats withdrawn for 3 or 7 days. DA concentrations in AMPH-pretreated rats withdrawn for 3 or 7 days were significantly less than in control animals challenged with AMPH. The 3-day group also differed significantly from the 28-day group (P < 0.05). For OOP AC, F = 31.5, P < 0.001. The AMPH challenge significantly decreased DOPAC in all groups (P < 0.05), but in AMPH-pretreated rats withdrawn for 3 days the effect of the AMPH challenge was significantly less than in control animals. B Nucleus accumbens: for NE, F = 2.54, P < 0.05. The AMPH challenge significantly increased NE concentrations in control rats, and in AMPH-pretreated rats withdrawn for 7 or 28 days. There was no significant effect of AMPH pretreatment. For DA, F = 9.33, P < 0.001. The AMPH challenge significantly elevated DA in all groups, but to a significantly lesser extent in the 3-day group than in controls. For DOPAC, F = 31.1, P < 0.001. The AMPH challenge significantly decreased DOPAC in all groups, and there was no effect of AMPH pretreatment. C Medial frontal cortex: there was no significant effect of AMPH pretreatment or the AMPH challenge on any compound. D Hypothalamus: for NE, F = 19.1, P < 0.001. The AMPH challenge did not have a significant effect on NE concentrations in control animals, or the 28-day group. However, NE was significantly decreased in the 3- and 7-day groups, relative to the saline challenge control group, the AMPH challenge control group, and the 28-day withdrawn group (P < 0.05). In addition, the 3- and 7-day groups differed significantly from one another (Fisher’s LSD tests, P < 0.05). There was no significant effect of the AMPH challenge or AMPH pretreatment on DA or 5-HT concentrations in the hypothalamus (F’s < 1.0)