Experiment 1: Effect of Inhaled Stimulant Administration on Activity and Body Temperature
Activity
The inhalation of MA significantly increased locomotor activity in the 25 and 100 mg/ml concentration test conditions (Figure 1b). The ANOVA confirmed a significant effect of Time (F(7, 84)=6.93; P<0.0001) and Drug (F(2, 24)=19.03; P<0.0001) and of the Time × Drug interaction (F(14, 168)=2.54; P<0.005). Tukey’s post hoc analysis confirmed that locomotor activity was increased over the vehicle PG condition after the start of vapor when the 25 mg/ml (90–180 min after vapor initiation) or 100 mg/ml MA (60–150 min after vapor initiation) concentrations were used. Activity did not differ significantly between 100 and 25 mg/ml concentrations at any time point.
The three different MDPV concentrations similarly increased activity compared with the effect of PG inhalation (Figure 1c). The ANOVA confirmed a significant effect of Time (F(7, 84)=50.83; P<0.0001) and Drug (F(3, 36)=10.61; P<0.0001) and of the Time × Drug interaction (F(21, 252)=3.84; P<0.0001). Tukey’s post hoc analysis confirmed increased activity relative to PG condition for the 25 mg/ml (60–180 min after vapor initiation), 50 mg/ml (60–120 min after vapor initiation), and 100 mg/ml (60–180 min after vapor initiation) MDPV concentrations. Activity was significantly higher in the 60 min time point following 50 mg/ml MDPV compared with the 100 mg/ml MDPV condition.
As shown in Figure 1d, mephedrone (4MMC) increased motor activity at the 200 mg/ml but not the 100 mg/ml concentration. The ANOVA confirmed a significant effect of Time (F(8, 84)=46.02; P<0.0001) and Drug (F(2, 24)=8.54; P<0.005) and of the Time × Drug interaction (F(14, 168)=5.84; P<0.0001). Tukey’s post hoc analysis confirmed that 200 mg/ml mephedrone increased motor activity from 60 to 120 min following exposure compared with either the PG or 100 mg/ml mephedrone condition.
Temperature
Vapor exposure to MA increased body temperature (Table 1), as confirmed by main effects of Vaporized Dose (F(2, 24)=6.83; P<0.005), Time (F(7, 84)=20.06; P<0.0001), and the interaction (F(14, 168)=2.98; P<0.0005). Tukey’s post hoc test confirmed temperature as being significantly elevated compared with the PG exposure for both the MA 25 mg/ml (150–210 min after vapor initiation) and MA 100 mg/ml (60, 210–240 min after vapor initiation) concentrations.
Vapor exposure to MDPV also increased body temperature (Table 1), as confirmed by main effects of Vaporized Dose (F(3, 36)=7.77; P<0.0005), Time (F(7, 84)=27.70; P<0.0001), and the interaction (F(21, 252)=3.73; P<0.0001). Tukey’s post hoc test confirmed temperature as being significantly elevated compared with the PG exposure for 25 mg/ml MDPV (60, 150–210 min after vapor initiation), 50 mg/ml MDPV (60–120 min after vapor initiation), and 100 mg/ml MDPV (60, 120–210 min after vapor initiation).
Vapor exposure to mephedrone did not change body temperature (Table 1). Although the ANOVA confirmed main effects of Time (F(7, 84)=23.04; P<0.0001) and the interaction of Time with Vaporized Dose (F(14, 168)=2.41; P<0.005), Tukey’s post hoc test only confirmed temperature as being significantly different from the PG condition at baseline for either mephedrone concentration.
Experiment 2: Effect of Intraperitoneal Stimulant Administration on Activity and Body Temperature
A follow-up study conducted to assess activity after a second round of PG, MA (100 mg/ml), or MDPV (100 mg/ml) vapor inhalation found that the activity response of the group was slightly attenuated from first (experiment 1, Figures 1a and b) to final drug challenges (experiment 2, Figure 2d). The first analysis compared the effects of 40 min exposure to MA (100 mg/ml, 4 puffs every 5 min) and PG across the two determinations and the ANOVA confirmed a significant effect of Time (F(7, 84)=17.02; P<0.0001) and Vaporized Drug Condition (F(3, 36)=15.38; P<0.0001) and of the Time × Vaporized Drug Condition interaction (F(21, 252)=2.45; P<0.001). Tukey’s post hoc analysis confirmed a significant difference between MA and PG vehicle from 60–240 min for the first run (experiment 1), and from 60–120 for the second run (experiment 2). Activity was significantly different between the first and second MA sessions for the 60 min time bin only and did not differ between the first and second PG sessions for any time bin.
Mean (N=13; +SEM) activity rates after intraperitoneal (i.p.) injection of a single vehicle condition compared with doses of (a) MA (0.5, 1.0 mg/kg), (b) MDPV (0.5, 1.0 mg/kg), and (c) 4MMC (1.0, 5.0 mg/kg). Activity rates after inhalation exposure to the PG vehicle, MA (100 mg/ml), or MDPV (100 mg/ml) for the same group are presented in (d). Gray shaded symbols indicate a significant difference from vehicle at the corresponding time point and the symbol # indicates a significant difference between routes of administration of a given drug at a given time point. Base=preinhalation baseline.
Inhalation of MDPV also significantly increased locomotor activity in both the first (experiment 1) and second (experiment 2) tests. The ANOVA confirmed a significant effect of Time (F(7, 84)=52.48; P<0.0001) and Vaporized Drug Condition (F(3, 36)=30.29; P<0.0001) and of the Time × Vaporized Drug Condition interaction (F(21, 252)=5.588; P<0.0001). The post hoc analysis (Tukey) confirmed a significant difference between MDPV and PG vehicle from 60–180 min for the first run and 60 min after vapor initiation for the second run. Activity was significantly different between the first and second MDPV sessions for the 60–150 min time bins but, again, did not differ between the first and second PG sessions for any time bin.
The i.p. administration of MA, MDPV, and mephedrone increased locomotor activity (Figures 2a–c) and the ANOVA for all i.p. conditions confirmed that there were significant effects of Time after injection (F(7, 84)=34.59; P<0.0001) and drug i.p. Dose Condition (F(6, 72)=6.89; P<0.0001) and of the interaction of Time and i.p. Dose Condition (F(42, 504)=1.62; P<0.05). Tukey’s post hoc test confirmed that activity rates were significantly higher following 0.5 mg/kg (90 min after injection) and 1.0 mg/kg (60–120 min after injection) MA doses, following 0.5 mg/kg (90 min after injection) and 1.0 mg/kg (90–120 min after injection) MDPV doses, as well as after the 5.0 mg/kg (60–90 min after injection) mephedrone dose as compared with the vehicle injection.
The i.p. administration of the three drugs also altered body temperature (Table 2); the ANOVA confirmed main effects of Time (F(7, 84)=15.24; P<0.0001) and i.p. Drug Condition (F(6, 72)=3.40; P<0.01) and of the Time × i.p. Drug Condition interaction (F(42, 504)=1.64; P<0.01). Dunnett’s post hoc test confirmed significant elevations of body temperature relative to vehicle for 0.5 mg/kg MA (120–150 min after injection), 0.5 mg/kg MDPV (120–180 min after injection), and 1.0 mg/kg MDPV (120–150 min after injection).
The results for the vapor-inhalation conditions (PG, MA, MDPV) in this experiment were next compared with the saline and 1.0 mg/kg MA and MDPV, i.p. dose conditions in a single analysis. The ANOVA confirmed that there was a significant effect of Time (F(7, 84)=33.38; P<0.0001) and Dose Condition (F(5, 60)=10.17; P<0.0001) and of the Time × Dose Condition interaction (F(35, 420)=3.01; P<0.0001) on activity rate. Tukey’s post hoc test confirmed first that activity levels were higher than their respective vehicle controls after 1.0 mg/kg MA i.p. (90–150 min after injection), 1.0 mg/kg MDPV i.p. (90 min after injection), MA vapor (60–120 min after initiation), and MDPV vapor (60–90 min after initiation) administration. The post hoc test further confirmed that MA vapor exposure significantly increased activity relative to i.p. administration of 1.0 mg/kg MA (60 min after injection/vapor initiation) and 1.0 mg/kg MDPV (60, 120 min) as well as relative to MDPV vapor (120 min). Activity was higher after 1.0 mg/kg MA i.p. compared with MDPV vapor 150 min after injection/vapor initiation.
The ANOVA comparing body temperature responses to the second round of vapor with the corresponding 1.0 mg/kg MA and MDPV i.p. challenge conditions confirmed that there was a significant effect of Time (F(7, 84)=28.30; P<0.0001) and Dose Condition (F(5, 60)=7.33; P<0.0001) and of the Time × Dose Condition interaction (F(35, 420)=2.26; P<0.0001). Tukey’s post hoc test further confirmed that significantly higher body temperature was observed after MA vapor inhalation compared with MA 1.0 mg/kg i.p. (60–180 min time point) and temperature was higher after MDPV vapor compared with MDPV 1.0 mg/kg i.p. (60 min time point). A significantly higher body temperature was confirmed for MDPV 1.0 mg/kg, i.p. (150 min time point) versus vehicle i.p., but no other differences between drug and the respective vehicle conditions were confirmed.
Experiment 3: D1 Receptor Antagonism of Locomotor Response to Vapor Methamphetamine
The mean locomotor response to MA vapor inhalation was attenuated by pretreatment with the dopamine subtype-1 (D1) receptor antagonist SCH23390 (Figure 3). A naive group of male Wistar rats (N=7) received 30 min exposure to PG, MDPV (100 mg/ml), and mephedrone (200 mg/ml) in randomized order with either saline or 10 μg SCH23390, i.p. administered before the session; this was followed with a 30 min exposure to MA (100 mg/ml) with saline or 10 μg SCH23390, i.p. administered before the session in a balanced order. The ANOVA including all eight treatment conditions confirmed a significant effect of Time (F(7, 42)=17.32; P<0.0001), of Drug Treatment Condition (F(7, 42)=13.22; P<0.0001), and of the Time × Drug Treatment Condition interaction (F(49, 294)=3.12; P<0.0001) on activity. Tukey’s post hoc test confirmed that activity was higher when saline was injected before MDPV (60–90 min after vapor initiation), mephedrone (60 min), or MA (60–90, 150 min) compared with the respective 10 μg SCH23390 pretreatment. Activity was also higher compared with the Sal+PG condition after MDPV (60–90 min after vapor initiation) or MA (60–90, 150–210 min) inhalation. Finally, activity was lower in the Sal+4MMC condition compared with the Sal+MA (60–90, 180 min after vapor initiation) and Sal+MDPV (60 min) conditions.
Mean (N=7; ±SEM) activity rates after inhalation of (a) PG, (b) methamphetamine (MA; 100 mg/ml), (c) MDPV, or (d) mephedrone/4MMC vapor following pretreatment with saline (Sal) or SCH23390 (10 μg/kg, i.p.; 10 SCH). Gray shaded symbols indicate a significant difference from PG vehicle at the corresponding time point and the symbol * indicates difference from corresponding SCH23390 pretreatment condition. The saline+PG control data are included in every panel for comparison.
The subsequent time-course studies in this group confirmed dose-dependent effects of drug inhalation on locomotor activity (Figure 4). MA inhalation dose-dependently altered activity and the ANOVA confirmed significant effects of Time after inhalation (F(7, 42)=24.93; P<0.0001), of Drug Treatment Condition (F(3, 18)=8.57; P<0.001), and of the Time × Drug Treatment Condition interaction (F (21, 126)=4.31; P<0.0001) on activity rate. Tukey’s post hoc test confirmed that activity differed across all three MA inhalation durations at 60 min after the initiation and relative to vehicle after 20 min (60–90, 150 min after initiation) or 30 min (60–90 min after initiation) of MA. Body temperature after MA inhalation was significantly altered by Time after initiation (F(7, 42)=19.65; P<0.0001) and Drug Treatment Condition (F(3, 18)=6.31; P<0.005) but not by the interaction of factors. Tukey’s post hoc test confirmed that body temperature was higher after 20 or 30 min of MA inhalation compared with the PG (60–150 min after initiation) and higher after 30 min of MA inhalation compared with 10 min (60 min after initiation).
Mean (N=7; ±SEM) activity rates (upper panels) and body temperature (lower panels) after inhalation of methamphetamine (left panels) or MDPV (right panels) for 10, 20, or 30 min. The drug concentration was 12.5 mg/ml and the PG vehicle condition was for 30 min (the single PG challenge is repeated in MA and MDPV panels). Significant differences from the PG condition are indicated with gray shaded symbols, from the 10 min condition by the symbol * and between the 20 and 30 min conditions by the symbol # Base=preinhalation baseline.
The ANOVA of the MDPV conditions confirmed significant effects of Time after initiation (F(7, 42)=45.66; P<0.0001), of Drug Treatment Condition (F(2, 12)=9.23; P<0.005), and of the interaction (F(14, 84)=4.93; P<0.0001) on activity rate. Tukey’s post hoc test confirmed that activity differed across all three inhalation durations at 60 min after the initiation of inhalation, the 30 min duration resulted in higher activity than 10 or 20 min exposure at 90 min and 10 min inhalation significantly less activity 150 min after initiation. Body temperature after MDPV inhalation was significantly altered by Time after initiation (F(7, 42)=25.40; P<0.0001), by Drug Treatment Condition (F(3, 18)=6.16; P<0.005), and by the interaction (F(21, 126)=2.55; P<0.001). Tukey’s post hoc test confirmed that body temperature was higher after 30 or 20 min of inhalation compared with PG (60–150 min after-initiation) as well as the 10 min condition (60–90, 150 min).
Experiment 4: Effect of Vapor MA and MDPV Administration on Wheel Activity
The wheel quarter rotations following MA and MDPV vapor inhalation (40 min) were summed across 30 min intervals for analysis. For lower dose conditions of MA and MDPV (25 mg/ml) (Figure 5a), the ANOVA confirmed a significant effect of Time (F(3, 21)=6.385; P<0.01). Wheel activity was significantly higher in the first 30 min bin compared with all subsequent bins following PG exposure and the 120 min interval following MA vapor inhalation. For higher dose conditions of MA and MDPV (100 mg/ml) (Figure 5b), the ANOVA confirmed a significant effect of Vaporized Drug Treatment Condition (F(2, 14)=5.614; P<0.05) and of the Time × Vaporized Drug Treatment Condition interaction (F(6, 42)=8.234; P<0.0001). Tukey’s post hoc test confirmed that MA vapor inhalation resulted in significantly decreased activity relative to the PG inhalation in the first 30 min, and MDPV vapor inhalation resulted in significantly decreased activity relative to the PG in the first 30 min and increased activity in the 60–90 min intervals. Activity following MA and MDPV vapor inhalation was significantly different at 60 min. Wheel activity was significantly higher in the first 30 min bin compared with all subsequent bins following PG exposure.
Mean (N=7; ±SEM) wheel activity for male Wistar rats after inhalation of (a) the propylene glycol (PG) vehicle, methamphetamine (MA; 25 mg/ml in PG), or MDPV (25 mg/ml in PG) and (b) methamphetamine (MA; 100 mg/ml in PG), MDPV (100 mg/ml in PG), or the vehicle for 40 min. (c) Mean (N=16; ±SEM) wheel activity for another group of male Wistar rats after inhalation of MA (100 mg/ml in PG vehicle) or the vehicle for 20 min. Gray shaded symbols indicate a significant difference from PG. A significant difference from the 30 min time point within an inhalation condition is indicated with the symbol * and a difference from MA with the symbol # for corresponding time points.
A final study was conducted to test 20 min inhalation of MA at the higher (100 mg/ml) concentration (Figure 5c). The ANOVA confirmed a significant effect of Time (F(3, 45)=9.23; P<0.0001) and of the Vaporized Drug Treatment Condition × Time interaction (F(3, 45)=12.08; P<0.0001) on wheel activity. Sidak’s post hoc test confirmed that MA vapor inhalation resulted in significantly reduced activity relative to the PG inhalation in the first 30 min and increased activity in the 90–120 min intervals. In addition, wheel activity was significantly higher in the first 30 min bin compared with all subsequent bins following PG exposure but no differences were confirmed across time within the MA vapor condition.
Experiment 5: Effect of Vapor and Intraperitoneal Administration on ICSS Threshold
The average threshold obtained across the two inhalation exposures for MA, MDPV, and mephedrone (Figure 6a) was compared with thresholds obtained after i.p. injection (15 min before session start) of mephedrone (1.0 mg/kg), MA (0.5 mg/kg), or MDPV (0.5 mg/kg). The ANOVA confirmed that there was a significant effect of the Drug Condition (F(3, 51)=16.60; P<0.0001), but not of Route or of the Drug Condition × Route interaction, on ICSS thresholds. The post hoc test (Dunnett) confirmed that ICSS thresholds following mephedrone or MA vapor inhalation were significantly lower than those obtained after the PG vehicle condition and thresholds after MDPV or MA i.p. differed significantly from saline i.p.
Inhaled and i.p. exposure to mephedrone (4MMC; 200 mg/ml in PG), methamphetamine (MA; 100 mg/ml), and 3,4-methylenedioxypyrovalerone (MDPV; 100 mg/ml) decreases threshold of intracranial self-stimulation (ICSS) reward. (a) Mean (N=9–10; ±SEM) change in ICSS threshold produced by i.p. administration of saline, 4MMC (1.0 mg/kg), MA (0.5 mg/kg), or MDPV (0.5 mg/kg) is compared with the average of the two exposures to inhaled PG, 4MMC, MA, or MDPV. (b) Mean (N=10; ±SEM) change in ICSS threshold produced by i.p. administration of three doses of 4MMC, MA, and MDPV. In all panels, significant differences from the respective vehicle condition is indicated by the symbol.
Follow-up studies were conducted to test three doses each of mephedrone (0.0, 0.5, 1.0, and 5.0 mg/kg, i.p.), MDPV (0.0, 0.1, 0.5, and 1.0 mg/kg, i.p.), or MA (0.0, 0.1, 0.5, and 1.0 mg/kg, i.p.) to determine whether appropriate doses had been selected for the i.p./Vapor comparison above (Figure 6b). Doses of the three drugs were estimated as roughly comparable as Low, Medium, and High based on prior locomotor studies and were thus analyzed in these categories. The ANOVA confirmed a significant effect of the Drug (F(2, 18)=7.61; P<0.005) and of Dose (F(3, 27)=18.09; P<0.0001) but not of the interaction. Dunnett’s post hoc test confirmed that reward thresholds were significantly lower than the respective vehicle condition after 1.0 mg/kg MDPV i.p. and after 0.5 or 1.0 mg/kg MA i.p. The only significant difference confirmed across drugs at a given dose rank was between the high doses of MA and mephedrone. No lasting effects of drug days on ICSS thresholds obtained on following (nontreatment) days were confirmed in this study.
