3.1 The effects of METH on the expression of CPP and cognitive functions in mice
Two-way ANOVA revealed that a significant effect of session and drug on the CPP scores (Fig. 1B, drug: F (1,28) = 27.87, P < 0.01; session: F(1,28) = 27.84, P < 0.01; drug × session: F(1,28) = 2329, P < 0.01). The CPP scores in the Sal group were not significantly different between the pre-test and post-test phases (Fig. 1B, Sal: t = -0.51, P = 0.63). The CPP scores of the post-test phase in METH group were significantly higher than that of the pre-test phase (Fig. 1B, METH: t = -9.877, P < 0.01). Independent t-test revealed the expression of PSD-95 and SYP in the VTA of the METH group were significantly higher than that of the Sal group (Figs. 1C – 1E, PSD-95: t = 3.53, P < 0.05; SYP: t = 5.39, P < 0.01). The expression of PSD-95 and SYP in the NAc of the METH group was significantly higher than that of the Sal group (Figs. 1F – 1H, PSD-95: t = 4.59, P < 0.05; SYP: t = 3.04, P < 0.05).
The paired t-test revealed no significant difference in the time spent on the old object between the Sal and METH groups (Fig. 2A, Sal: t = 0.099, P = 0.924; METH: t = 1.254, P = 0.128). The Sal group spent significantly more time in the new location than in the old one (Fig. 2B, t = 2.507, P < 0.05). No significant difference was observed in the time spent between on the old and the new positions in the METH group (Fig. 2B, t = -2.158, P = 0.068). A significant difference was observed in the discrimination index between the Sal and METH groups (Fig. 2C, t = 2.967, P < 0.01). The Sal group spent significantly more time on the new objects than on the old objects (Fig. 2D, t = 3.951, P < 0.01). No significant difference was observed in the time spent on between the old and the new objects in the METH group (Fig. 3D, t = 1.276, P = 0.243). There was a significant difference in the discrimination index between the Sal and METH groups (Fig. 2E, t = 2.851, P < 0.05). The correct conversion rate in the Sal group was significantly higher than that of the METH group (Fig. 2F, t = 3.823, P < 0.01). No significant difference was observed in the distance traveled before and after the CPP test in the Sal and METH groups (Fig. 2G, drug, F(1,28) = 3.89, P = 0.06, session, F(1,28) = 3.95, P = 0.06, drug × session: F(1,28) = 0.003, P = 0.96). The expression of PSD-95 and SYP in the Hip of the METH group was significantly lower than those of the Sal group (Figs. 2H – 2J, PSD-95: t = 3.86, P < 0.05; SYP: t = 3.99, P < 0.05).
3.2 The effect of METH on the reinstatement of METH-induced CPP and cognitive functions in mice
Two-way ANOVA revealed a significant effect of session and drug on the CPP scores (Fig. 3B, drug: F (1,20) = 5.07, P < 0.05; session: F(1,20) = 0.24, P = 0.63; group × test: F(1,20) = 9.64, P < 0.01). The CPP scores of the Sal group were not significantly different between the pre-test and post-test phases (Fig. 3B, t = 1.329, P = 0.241). The CPP scores of the post-test phase in the METH group were significantly higher than those of the pre-test phase (Fig. 3B, t=-3.176, P < 0.05). No significant difference was observed in the CPP scores during the last three days of extinction between the METH and Sal groups (Fig. 3C, drug: F (4,40) = 8842, P = 0.3527; session: F (3,40) = 0.9288, P = 0.4357; drug × session: F (3,40) = 0.9250, P = 0.4375; Post-hoc analysis, test 2: t = 0.870, P = 0.405; test 3: t = -0.584, P = 0.572; test 4: t = -0.508, P = 0.622). The t-test of two independent samples showed a significant difference in the CPP scores during the drug-induced reinstatement period between the METH and Sal groups (Fig. 3D, t = 3.122, P < 0.05). The expression of the PSD-95 and SYP in the VTA of the METH group was significantly higher than those of the Sal group (Figs. 3E − 3G, PSD-95: t = 7.53, P < 0.01; SYP: t = 8.26, P < 0.01). The expression of PSD-95 and SYP in the NAc of the METH group was significantly higher than that of the Sal group (Figs. 3H – 3J, PSD-95: t = 3.02, P < 0.05; SYP: t = 5.56, P < 0.01).
The paired t-test revealed no significant difference in the time spent on the old object between the Sal and METH groups (Fig. 4A, Sal: t = 1.218, P = 0.277; METH: t = 1.247, P = 0.268). The Sal group spent significantly more time in the new location than in the old one (Fig. 4B, t = 2.941, P < 0.05). No significant difference was observed in the time spent on between the old and the new positions in the METH group (Fig. 4B, t = -0.842, P = 0.438). A significant difference was observed in the new position discrimination indicators between the Sal and METH groups (Fig. 4C, t = 2.941, P < 0.05). The Sal and METH groups spent significantly more time on the new objects than on the old one (Fig. 4D, Sal: t = 3.711, P < 0.05; METH: t = 6.64, P < 0.01). There was a significant difference in the cognitive discrimination indicators of new objects between the Sal and METH groups (Fig. 4E, t = 2.376, P < 0.05). The correct conversion rate in the Sal group was significantly higher than in the METH group (Fig. 4F, t = 4.318, P < 0.01). No significant effect of test or group on the distance traveled (Fig. 4G, drug, F(1,20) = 0.78, P = 0.39, session, F(1,20) = 0.01, P = 0.91, drug × session: F(1,20) = 0.97, P = 0.34). The expressions of PSD-95 and SYP in the Hip of the METH group was significantly lower than that of the Sal group (Figs. 4H – 4J, PSD-95: t = 2.77, P < 0.05; SYP: t = 2.97, P < 0.05).
3.3 The effects of PF on METH-induced CPP and cognitive impairment in mice
Two-way ANOVA revealed no significant effect of session and drug on the CPP scores (Fig. 5B, drug: F(1,44) = 15.84, P < 0.01, session: F(2,44) = 9.29, P < 0.01, drug × session: F(2,44) = 9.79, P < 0.01; Post-hoc analysis, Base vs. Test: Sal + Sal: t = -0.653, P = 0.534; METH + PF: t = -0.392, P = 0.705). The CPP scores of the post-test phase was significantly higher than that of the pre-test phase in the METH + Sal group (Fig. 5B, Base vs. Test: t = -14.748, P < 0.01). There was a significant effect of group on the PSD-95 and SYP (Figs. 5C − 5E, one-way ANOVA: PSD-95: F(2,9) = 6.30, P < 0.05; SYP: F(2,9) = 5.19, P < 0.05). Post-hoc analysis revealed that the expression of PSD-95 and SYP in the VTA of the METH + Sal group was significantly higher than that of the Sal + Sal and METH + PF groups (Figs. 5C − 5E, Sal + Sal vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.01; METH + PF vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.05). There was a significant effect of group on the PSD-95 and SYP (Figs. 5F – 5H, one-way ANOVA: PSD-95: F(2,9) = 14.26, P < 0.01; SYP: F(2,9) = 11.00, P < 0.05). Post-hoc analysis revealed that the expression of PSD-95 and SYP in the NAc of the METH + Sal group was significantly higher than that of the Sal + Sal and METH + PF groups (Figs. 5F – 5H, Sal + Sal vs. METH + Sal, PSD-95: P < 0.01; SYP: P < 0.05; METH + PF vs. METH + Sal, PSD-95: P < 0.01; SYP: P < 0.05).
Two-way ANOVA showed neither group nor group was a major effect on the time spent on the old objects among the Sal + Sal, METH + PF and METH + Sal groups (Fig. 6A, test: F(1,44) = 0.003, P = 0.95, group: F(2,44) = 0.05, P = 0.95, test × group: F(2,44) = 0.007, P = 0.99). The Sal + Sal and METH + PF groups spent significantly more time in the new location than in the old one (Fig. 6B, Sal + Sal: t = 2.523, P < 0.05; METH + PF: t = 2.738, P < 0.05). No significant difference was observed in the time spent between on the old and the new positions in the METH + Sal group (Fig. 6B, t = -0.713, P = 0.499). A significant difference was observed in the discrimination index between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 6C, one-way ANOVA: F(2,22) = 6.32, P < 0.01). Post-hoc analysis revealed that the new position discrimination indicts of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 6C, Sal + Sal vs. METH + Sal, P < 0.01; METH + PF vs. METH + Sal, P < 0.05). The Sal + Sal and METH + PF groups spent significantly more time on the new objects than on the old objects (Fig. 6D, Sal + Sal: t = 3.184, P < 0.05; METH + PF: t = 5.067, P < 0.01). No significant difference was observed in the time spent on the old and new objects in the METH + Sal group (Fig. 6D, t = -1.790, P = 0.117). There was a significant difference in the discrimination index between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 6E, one-way ANOVA: F(2,22) = 13.43, P < 0.01). Post-hoc analysis revealed that the cognitive discrimination indicts of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 6E, Sal + Sal vs. METH + Sal, P < 0.01; METH + PF vs. METH + Sal, P < 0.01). There was a significant difference in the correct conversion rate between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 6F, one-way ANOVA: F(2,22) = 9.35, P < 0.01). Post-hoc analysis revealed that the correct conversion rate of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 6F, Sal + Sal vs. METH + Sal, P < 0.05; METH + PF vs. METH + Sal, P < 0.01). No significant difference was observed in the distance traveled before and after the CPP test between in the Sal + Sal, METH + Sal and METH + PF groups (Fig. 6G, test: F(1,44) = 1.41, P = 0.24, group: F(2,44) = 3.04, P = 0.06, test × group: F(2,44) = 0.91, P = 0.41). There was a significant effect of group on the PSD-95 and SYP (Figs. 6H – 6J, one-way ANOVA: PSD-95: F(2,9) = 10.40, P < 0.01; SYP: F(2,9) = 57.18, P < 0.01). Post-hoc analysis revealed that the expressions of PSD-95 and SYP in the Hip of the METH + Sal group were significantly lower than those of the Sal + Sal and METH + PF groups (Figs. 6H – 6J, Sal + Sal vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.01; METH + PF vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.01).
3.4 The effects of PF on the reinstatement of METH-induced CPP and cognitive functions in mice
Two-way ANOVA revealed no significant difference of session and drug in the CPP scores (Fig. 7B, drug: F(1,60) = 24.11, P < 0.01, session: F(2,60) = 10.07, P < 0.01, drug × session: F(2,60) = 4.845, P < 0.05). The CPP scores of the post-test phase were significantly higher than those of the pre-test phase in the METH + PF and METH + Sal groups (Fig. 7B, Base vs. Test: METH + Sal: t = -3.422, P < 0.01; METH + PF: t = -3.694, P < 0.01). No significant difference in CPP scores was observed in the last three days of extinction period among the METH + Sal, Sal + Sal and METH + PF groups (Fig. 7C, drug: F(6,120) = 0.4595, P = 0.8370; session: F(6,120) = 0.7630, P = 0.5170; drug × session: F(6,120) = 2.851, P = 0.0617). One-way ANOVA showed a significant differences in the CPP scores during the drug-induced reinstatement period between the METH + Sal, Sal + Sal and METH + PF groups (Fig. 7D, F(2,30) = 6.25, P < 0.01) Post-hoc analysis revealed that the CPP scores of the METH + Sal group were significantly higher than those of the Sal + Sal and METH + PF groups(Fig. 7D, Sal + Sal vs. METH + Sal, P < 0.01; METH + PF vs. METH + Sal, P < 0.01). There was a significant effect of group on the PSD-95 and SYP (Figs. 7E – 7G, one-way ANOVA: PSD-95: F(2,6) = 9.34, P < 0.05; SYP: F(2,6) = 8.38, P < 0.05). Post-hoc analysis revealed that the expression of PSD-95 and SYP in the VTA of the METH + Sal group was significantly higher than those of the Sal + Sal and METH + PF groups (Figs. 7E – 7G, Sal + Sal vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.05; METH + PF vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.05). There was a significant effect of group on the PSD-95 and SYP (Figs. 7H – 7J, one-way ANOVA: PSD-95: F(2,9) = 20.98, P < 0.01; SYP: F(2,9) = 9.33, P < 0.01). Post-hoc analysis revealed that the expression of the PSD-95 and SYP in the NAc of the METH + Sal group was significantly higher than those of the Sal + Sal and METH + PF groups (Figs. 7H – 7J, Sal + Sal vs. METH + Sal, PSD-95: P < 0.01; SYP: P < 0.01; METH + PF vs. METH + Sal, PSD-95: P < 0.01; SYP: P < 0.05).
Two-way ANOVA revealed no significant difference in the time spent on the old objects between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 8A, test: F(1,66) = 0.001, P = 0.97, group: F(1,66) = 1.86, P = 0.16, test × group: F(2,66) = 0.46, P = 0.63). The Sal + Sal and METH + PF groups spent significantly more time in the new position than in the old position (Fig. 8B, Sal + Sal: t = 2.552, P < 0.05; METH + PF: t = 3.522, P < 0.01). No significant difference was observed in the time spent between the old and new locations in the METH + Sal group (Fig. 8B, t = -0.369, P = 0.719). There was a significant difference in the new position discrimination indicts between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 8C, one-way ANOVA: F(2,33) = 5.58, P < 0.01). Post-hoc analysis revealed that the new position discrimination indicts of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 8C, Sal + Sal vs. METH + Sal, P < 0.05; METH + PF vs. METH + Sal, P < 0.01). The time spent on new objects in the Sal + Sal and METH + PF groups were significantly higher than the time spent on old objects (Fig. 8D, Sal + Sal: t = 4.199, P < 0.01; METH + PF: t = 2.843, P < 0.05). No significant difference was observed in the time spent between the old and new objects in the METH + Sal group (Fig. 8D, t = -1.289, P = 0.224). There was a significant difference in the cognitive discrimination index between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 8E, F(2,33) = 13.3, P < 0.01). Post-hoc analysis revealed that the cognitive discrimination index of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 8E, Sal + Sal vs. METH + Sal, P < 0.01; METH + PF vs. METH + Sal, P < 0.01). There was a significant difference in the correct conversion rate between the Sal + Sal, METH + PF and METH + Sal groups (Fig. 8F, one-way ANOVA: F(2,33) = 5.67, P < 0.01). Post-hoc analysis revealed that the correct conversion rate of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Fig. 8F, Sal + Sal vs. METH + Sal, P < 0.05; METH + PF vs. METH + Sal, P < 0.01). No significant difference was observed in the distance traveled within 10 minutes before and after the CPP test between the Sal + Sal, METH + Sal and METH + PF groups (Fig. 8G, test: F(1,66) = 0.18, P = 0.67, group: F(2,66) = 2.07, P = 0.13, test × group: F(2,66) = 1.92, P = 0.15). There was a significant effect of group on the PSD-95 and SYP (Figs. 8H – 8J, one-way ANOVA: PSD-95: F(2,9) = 7.07, P < 0.05; SYP: F(2,6) = 11.70, P < 0.01). Post-hoc analysis revealed that the expressions of PSD-95 and SYP in the Hip of the METH + Sal group was significantly lower than those of the Sal + Sal and METH + PF groups (Figs. 8H – 8J, Sal + Sal vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.01; METH + PF vs. METH + Sal, PSD-95: P < 0.05; SYP: P < 0.01).