Neuropharmacology Poster Session
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Introduction
The chronic use of stimulant drugs often leads to adaptations to their behavioural effects. Behavioural sensitization is an augmentation of the behavioural effects of psychomotor stimulants, or a shift to the left of the dose response curve, over a course of repeated injections. It has been proposed as an animal model of stimulant psychosis (Robinson & Becker 1986) and of addiction (Robinson & Berridge 1993). Tolerance is a decrease in the drug effect, or a shift to the right of the dose response curve, with repeated administration. Behavioural sensitization to psychomotor stimulants is dependent on a range of factors including treatment regimen, context, stress, gender and circadian rhythms (Martin-Iverson, 1991).
(+)-4-propyl-9-hydroxynaphthoxazine (PHNO) is a potent, direct-acting DA agonist with marked selectivity for the D2 receptor subtype (Martin et al. 1984). When rats maintained on a 12-hour light-dark cycle are given continuous infusions of PHNO, they exhibit tolerance to PHNO’s motor stimulant effects during the light hours of the cycle and sensitization during the dark hours (Martin-Iverson et al. 1988a, 1988b). Mild environmental stress temporarily reverses diurnal tolerance (Martin-Iverson et al. 1988a, 1988b). This novel circadian pattern of tolerance and sensitization provides an opportunity to investigate possible circadian factors mediating adaptation to the behavioural effects of DA D2 agonists.
There is evidence that the development of sensitization to the motor stimulant effects of PHNO are due to an interaction between D1 and D2 subtypes and that tolerance develops because of a loss of activation of D1 receptors (Martin-Iverson et al. 1988a, 1988b). The hypothesis is that at night (sensitization) there is an increase in endogenous DA release that can act on D1 receptors in chronic PHNO-infused rats, and during the day (tolerance) there is a decrease in endogenous DA leaving D1 receptors unoccupied. Corticosterone could regulate sensitization and tolerance via actions on endogenous DA release.
Corticosterone, the major stress-related glucocorticoid of the rat, exhibits circadian rhythms in plasma levels, which are highest during the dark hours (Abe et al. 1979). Stress- and drug-induced alterations in corticosterone level influences the behavioural effects of some DA agonists (Marinelli et al. 1996). Glucocorticoids can alter the metabolism and plasma concentration of mesolimbic DA and treatment with DA agonists can increase corticosterone release (Damianapoulos & Carey 1995; Mittleman et al. 1992; Rothschild et al. 1985).
This study replicated the circadian pattern of tolerance and sensitization to the motor effects of PHNO and investigated the putative role of corticosterone in this pattern of drug adaptation. It was hypothesised that circadian corticosterone release mediates the synergistic relationship between D1 and D2 DA receptor subtypes in producing tolerance and sensitization to the motor stimulant effects of PHNO. It was predicted that nocturnal injections of a corticosterone synthesis inhibitor (metyrapone) would block sensitization in PHNO-treated rats. Daytime treatment of PHNO-tolerant rats with a synthetic glucocorticoid (dexamethasone) was expected to reverse tolerance to sensitization. Furthermore, it was hypothesised that the stress-induced reversal of tolerance to PHNO is due the effect of stress-induced corticosterone release on DA levels and/or D1 activation. Therefore, diurnal treatment of PHNO-tolerant rats with metyrapone was expected to block the stress-induced reversal of diurnal tolerance.
Materials and Methods
48 Male Wistar rats (Animal Resources Centre of Western Australia), weighing 300-460 g, were housed in individual cages in a room maintained on a 12-hour light-dark cycle. Motor activity within each cage was monitored continuously via four infrared photoelectric cells located evenly along the bottom of each cage. Photocell interruptions were automatically recorded in one-hour blocks by a PC, measuring locomotion as all photobeam interruptions that were not sequential interruptions of the same beam.
The rats were allowed 19 days to habituate to their home cages and the 12-hour light-dark cycle. This time also allowed for habituation to a housekeeping routine, involving the changing of food, water and litter, on Mondays, Wednesdays and Fridays. The disturbance created by this housekeeping, which lasted for an average of 40 minutes, allowed an ‘environmental stress’ variable to be included in the analysis. Mean hourly motor activity for each 24-hour period was determined over a 12-hour nocturnal period, a 10-hour diurnal period and a 2-hour housekeeping or no housekeeping period.
Following habituation, all animals were surgically implanted with an Alzet Osmotic pump (Model #2002), containing either PHNO (0.005 mg/hour, n = 24, drug courtesy of Merck Sharp & Dohme) or vehicle (distilled water, n = 24), subcutaneously between the scapulae. On the ninth night following surgery each animal received a single s.c. injection of either metyrapone (100 mg/kg) or vehicle (7% Tween 80), forming four groups (vehicle + vehicle, vehicle + metyrapone, PHNO + vehicle, PHNO + metyrapone, all n = 12). On Day 10, animals received a second s.c. injection of metyrapone (100 mg/kg) or vehicle prior to a scheduled housekeeping routine. On Day 12 the animals received a single i.p. injection of either soluble dexamethasone (0.5 mg/kg, weight expressed as available dexamethasone) or vehicle (distilled water). Four new drug groups were formed (vehicle + vehicle, vehicle + dexamethasone, PHNO + vehicle, PHNO + dexamethasone, all n = 12), these being balanced by animal drug history, with half the animals in each of the new groups having previously received metyrapone and half vehicle.
Results
Mean hourly activity for PHNO- and vehicle-infused animals over the 7 days following pump implantation is shown in Figure 1.
Click to enlargeFig. 1: Mean hourly locomotor photocell interruptions over the 10-hour diurnal period (6:30-9:30 and 11:30-18:30 hours) for rats treated with continuous infusions of PHNO (0.005 mg/h) or vehicle (n = 24 per group). Locomotor activity in the PHNO group gradually attenuated following an increase from the Day Before Surgery (DBS) to Day 1, while activity in the vehicle group remained relatively constant after Day 1. Error bars represent the critical difference between means for the multiple F-test (p < .05).
There was a significant Drug x Day interaction, F(6,276) = 12.23, p < .0001. Figure 1 shows this interaction to be due to a steady decline in activity in the PHNO group over successive days, following an initial increase in activity on Day 1. By Day 7, the activity level of the PHNO animals approached that of the vehicle animals, whose locomotor activity remained relatively constant over the same period (Figure 1). Nocturnal activity for PHNO- and vehicle-infused animals over the 8 nights following pump implantation is shown in Figure 2.
Click to enlargeFig. 2: Mean hourly locomotor photocell interruptions over the 12-hour nocturnal period (18:30-6:30 hours) for rats treated with continuous infusions of PHNO (0.005 mg/h) or vehicle (n = 24 per group). Locomotor activity in the PHNO group gradually increased from the Night Before Surgery (NBS) while activity in the vehicle group remained relatively constant after Night 2. Error bars represent the critical difference between means for the multiple F-test (p < .05).
A significant Drug x Night interaction, F(7,322) = 6.13, p < .0001, is due to a gradual augmentation of activity level in the PHNO-infused animals over successive nights but a relatively constant level of locomotion in vehicle-infused animals after Night 2 (Figure 2). The difference in activity levels of PHNO- and vehicle-infused animals for a two-hour period of stress and for the two-hour period preceding stress is shown in Figure 3.
Click to enlargeFig. 3: Mean diurnal locomotor activity during a two-hour period of stress due to housekeeping (stress) and the two-hour period preceding stress (no stress) for rats given continuous infusions of PHNO (0.005 mg/h) or vehicle (n = 24 per group). While stress caused an increase in locomotor activity in both groups, the increase in the PHNO group was significantly greater than the increase in the vehicle group. Error bars indicate the critical difference between means for the multiple F-test (p < .05).
While PHNO had no effect pre-stress, the stress-induced increase in activity in the PHNO group was significantly greater than the stress-induced increase in the vehicle group, as indicated by a significant Drug x Stress interaction, F(1,46) = 5.66, p < .05 (Figure 3). Daytime activity for the hour preceding and following a stressful housekeeping session which followed a single metyrapone or vehicle injection is shown in Figure 4.
Click to enlargeFig. 4: Mean diurnal locomotor activity for the hour preceding (pre-stress) and following (post-stress) a stressful housekeeping session, which followed a single metyrapone (100 mg/kg) or vehicle injection to rats given continuous infusions of PHNO (0.005 mg/h) or vehicle. V+V: vehicle minipump + vehicle injection, V+M: vehicle minipump + metyrapone injection, P+V: PHNO minipump + vehicle injection, P+M: PHNO minipump + metyrapone injection (n = 12 per group). Metyrapone blocked the stress-induced increase in activity in vehicle-infused animals, but did not block the stress-induced reversal of tolerance in PHNO-infused animals. Error bars indicate the critical difference between means for the multiple F-test (p < .05).
There was a significant three-way interaction between PHNO, metyrapone and stress, F(1,44) = 4.28, p < .05. Of the animals who received vehicle minipumps, those who received vehicle injections (V+V) showed a stress-induced increase in activity, while those who received metyrapone injections (V+M) did not (p < .05) (Figure 4). However, all PHNO-infused animals showed a stress-induced increase in activity, with the post-stress levels of locomotor activity for the PHNO-infused, vehicle-injection (P+V) and the PHNO-infused, metyrapone-injection (P+M) groups not significantly different (p < .05) (Figure 4). Nocturnal activity for one-hour prior to and one hour following a single metyrapone or vehicle injection is shown in Figure 5.
Click to enlargeFig. 5: Mean nocturnal locomotor activity for the hour preceding (pre-treatment) and following (post-treatment) a single metyrapone (100 mg/kg) or vehicle injection to rats given continuous infusions of PHNO (0.005 mg/h) or vehicle. V+V: vehicle minipump + vehicle injection, V+M: vehicle minipump + metyrapone injection, P+V: PHNO minipump + vehicle injection, P+M: PHNO minipump + metyrapone injection (n = 12 per group). Metyrapone blocked the post-treatment increase in locomotor activity in vehicle-infused animals but caused a post-treatment increase in PHNO-infused animals. Error bars indicate the critical difference between means for the multiple F-test (p < .05).
There was a significant three-way interaction between PHNO, metyrapone and treatment (pre-/post-), F(1,44) = 13.84, p < .001. Of the vehicle-infused animals, those receiving vehicle injections (V+V) showed an increase in activity following treatment, while those receiving metyrapone injections (V+M) showed no significant increase in activity following treatment (p < .05) (Figure 5). However, for those animals receiving PHNO infusions, those receiving vehicle injections (P+V) did not show a significant post-treatment increase in activity, while those receiving metyrapone injections (P+M) did show a post-treatment increase (p < .05) (Figure 5). Diurnal activity for one hour prior to, and one hour following, a single dexamethasone or vehicle injection is shown in Figure 6.
Click to enlargeFig. 6: Mean diurnal locomotor activity for the hour preceding (pre-treatment) and following (post-treatment) a single dexamethasone (0.5 mg/kg) or vehicle injection to rats given continuous infusions of PHNO (0.005 mg/h) or vehicle. V+V: vehicle minipump + vehicle injection, V+D: vehicle minipump + dexamethasone injection, P+V: PHNO minipump + vehicle injection, P+D: PHNO minipump + dexamethasone injection (n = 12 per group). There were no significant effects of dexamethasone on the locomotor activity of either PHNO- or vehicle-infused animals. Error bars indicate the critical difference between means for the multiple F-test (p < .05).
There was a significant PHNO x Treatment (pre-/post-) interaction, F(1,44) = 7.23, p < .05, with the post-treatment increase being significantly greater for the PHNO-infused animals than for the vehicle-infused animals (Figure 6). However, there were no significant effects of Dexamethasone (x Treatment, or x PHNO x Treatment).
Discussion and Conclusion
The results replicate previous findings that continuous administration of PHNO, a DA D2 agonist, results in the development of diurnal tolerance and nocturnal sensitization to the drug’s motor stimulant effects in rats. Levels of locomotion in rats receiving continuous infusions of PHNO progressively declined over a 7-day period, an attenuation in drug response characteristic of the development of tolerance (Figure 1). The same PHNO-infused animals showed a progressive increase in locomotor activity over the 8 nights following commencement of PHNO infusion, an augmentation in drug response characteristic of the development of sensitization (Figure 2). Over the same period animals receiving continuous infusions of vehicle solution exhibited relatively constant levels of locomotion (Figures 1 and 2).
Nocturnal metyrapone injections were given to test the hypothesis that corticosterone mediates the development of nocturnal sensitization to PHNO. An injection-stress induced post-treatment increase in activity in vehicle-infused, vehicle-injected (V+V) animals was blocked in the vehicle-infused animals who received metyrapone injections (V+M) (Figure 5). Unexpectedly, the PHNO-infused animals who received vehicle injections (P+V) did not show an injection stress-induced increase in activity (Figure 9). However, the PHNO-infused animals given metyrapone (P+M) injections did show a significant increase in locomotion following injection, this being the opposite to the predicted reduction in nocturnal levels of locomotion in PHNO-infused animals following metyrapone treatment (Figure 5). It can be concluded that reducing nocturnal corticosterone levels by metyrapone injection does not block nocturnal sensitization, despite metyrapone reducing the injection stress-induced activity increase in controls.
Diurnal dexamethasone injections were given to test the hypothesis that decreased diurnal corticosterone plasma levels mediates the development of diurnal tolerance to PHNO. Results indicate that dexamethasone treatment had no significant effect on the activity of either vehicle- or PHNO-infused animals (Figure 6). Had dexamethasone reversed daytime tolerance, the PHNO-infused animals receiving dexamethasone injections (P+D) would have exhibited an increase over and above the injection stress-induced increase in the PHNO-infused animals who received vehicle injections (P+V). It can be concluded that activating corticosterone receptors with dexamethasone injections does increase locomotion in either vehicle- or PHNO-infused rats, at least under conditions when the rats experience injection-stress.
Mild environmental stress reversed diurnal tolerance. While stress caused an increase in daytime motor activity in both vehicle- and PHNO-infused animals, the increase was greater in the PHNO-infused animals (Figure 3). Metyrapone blocked the stress-induced increase in activity in the vehicle-infused control animals (Figure 4). However, the PHNO-tolerant animals, injected with either metyrapone (P+M) or vehicle (P+V) did not differ significantly on either pre- or post-stress levels of locomotion (Figure 4). This suggests that stress-induced corticosterone release is involved in the stress-induced increase in locomotion observed in vehicle-infused control animals, but not in the stress-induced reversal of tolerance in PHNO-treated animals.
These data suggest that corticosterone is important for stress-induced locomotion in controls but does not play a role in either circadian rhythms in sensitization and tolerance to PHNO, or in stress-induced sensitization to PHNO.
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