Figure 1.
Synopsis of the experimental design, including treatment schedule and IHC assays.
A. Pre-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 30 min before each METH or SAL injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. B. Post-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 0.5, 12, 24, 36 and 48 h after the last METH or SAL administration, and 3 days (3d) after the last METH injection were perfused and used for IHC analysis. C. Post-treatment + SR treatment: rats received injection of SR (1 mg/kg, i.p.) or VEH 15 min prior each Δ9-THC (1 mg/kg) or VEH post-treatment injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. 0, 2 h, 4 h, 6 h: 1st, 2nd, 3rd and 4th injection of METH (10 mg/kg, s.c.) or SAL; IHC: immunohistochemistry; SR: SR141716A; VEH: vehicle.
Figure 2.
Core body temperature: effect of methamphetamine (METH) in the presence and absence of Δ9-THC (1 and 3 mg/kg).
Rats were given SAL (1 mL/kg) or METH (4×10 mg/kg s.c., every 2 h) with and without Δ9-THC (1 and 3 mg/kg) pre-treatment. Body temperature was measured prior to and 1 h after each METH injection. Values are expressed as means ± SEM. Arrows indicate each injection of METH or SAL. No difference in baseline temperature was detected among groups. METH administration resulted in a significant increase in rectal temperature over time in comparison with SAL-treated rats. Both doses of Δ9-THC did not significantly change rectal temperature in METH-administered rats at any time point. METH: *p<0.05, **p<0.01 and ***p<0.001 vs corresponding SAL group at each time point. Δ9-THC1-METH: ##p<0.01 and ###p<0.001 vs corresponding Δ9-THC1-SAL group at each time point; Δ9-THC3-METH: +p<0.05 and +++p<0.001 vs corresponding Δ9-THC 3-SAL group at each time point.
Figure 3.
METH increases the number of neuronal nitric oxide synthase (nNOS) neurons and GFAP-immunoreactivity (IR).
Values represent means ± SEM of either number of nNOS positive neurons, expressed per mm2 (A) or as percentage of GFAP-IR density (B). **p<0.01 and ***p<0.001 compared to SAL.
Figure 4.
Δ9-THC reduces METH-induced increase of nNOS neurons in the CPu.
A. Rats received injections of 1 or 3/kg of Δ9-THC either 0.5 h before each METH injection (PRE) or 0.5, 12, 24, 36, and 48 h after the last METH administration (POST), and were sacrificed 3 days after the last METH injection. Pre- and Post-treatment with both doses of Δ9-THC significantly decreased the number of nNOS positive neurons in the CPu. *p<0.05 and **p<0.01 vs PRE METH-VEH; #p<0.05 and ###p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of nNOS positive neurons (31±1.03) in SAL-VEH group. B. Representative images of nNOS immunohistochemical staining 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH-Δ9-THC 1 and 3 mg. Scale bar = 100 µm.
Figure 5.
Δ9-THC reduces METH-induced astrogliosis in the CPu.
A. Rats were treated as described in the legend of Figure 4. Two-way ANOVA revealed a significant effect of treatment (F(2,32) = 16.28, p<0.0001) as well as a significant interaction between time of treatment and treatment (F(2,32) = 8.12, p = 0.0014). Post-hoc comparisons showed that GFAP-IR was lower in the CPu of Post Δ9-THC (1 mg/kg) and Pre Δ9-THC (3 mg/kg) treated rats than in controls (METH-VEH). ***p<0.001 vs PRE METH-VEH and ###p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of percentage of GFAP-IR density (0.75±0.07) in SAL-VEH group. B. Representative images of GFAP immunostaining in the CPu 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH-Δ9-THC 1 and 3 mg. Scale bar = 100 µm.
Figure 6.
Δ9-THC reduces METH-induced astrogliosis in the PFC.
A. Rats were treated as described in the legend of Figure 4. Pre- and Post-administration of Δ9-THC attenuated the astrogliosis induced by METH (Pre: −34% and −37%, Post: −47% and −29%, for 1 and 3 mg/kg, respectively) compared to control groups. **p<0.01 vs PRE METH-VEH and #p<0.05, and ###p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of percentage of GFAP-IR density (1.31±0.10) in SAL-VEH group. B. Representative images of GFAP immunostaining in the PFC 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH- Δ9-THC 1 and 3 mg. Scale bar = 100 µm.
Figure 7.
Effects of SR on nNOS and GFAP-IR in the CPu.
A. Rats received injections of 1/kg Δ9-THC or VEH at 0.5, 12, 24, 36 and 48 h after the last METH administration (Post-treatment, POST) and were sacrificed 3 days after the last METH injection. SR (1 mg/kg, i.p.) or VEH were administered 15 min before each Δ9-THC or VEH injection. Two-way ANOVA in the CPu (A) showed a significant Δ9-THC x SR interaction (F(1,40) = 32.45, p<0.0001); the administration of SR blunted the effect of Δ9-THC on METH-induced nNOS over-expression. SR alone decreased nNOS labeled neurons compared to that of control. ***p<0.001 vs METH-VEH (VEH pretreated) and ##p<0.01 vs METH-VEH-Δ9-THC (VEH pretreated). B. Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F(1,35) = 19.86, p<0001). Δ9-THC and SR, alone or in combination, attenuated the METH-induced increase of GFAP-IR in the CPu. ***p<0.001 vs METH-VEH (VEH pretreated).
Figure 8.
Effects of SR on GFAP-IR in the PFC.
Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F(1,33) = 45.91, p<0001). METH-Δ9-THC significantly reduced METH-induced GFAP-IR. Moreover, GFAP-IR was lower in METH-SR-VEH and METH-SR-THC groups as compared to METH-VEH treated rats. ***p<0.001 vs METH-VEH (VEH pretreated).