PMC

Nicotine blocks PPP. A, Representative traces (left) and plots (right) show that the frequency of mEPSC in MSNs from saline-treated mice was greater than amphetamine self-administering mice but far less than those in nonresponding mice. ^$p<0.05, ^$$p<0.01, ^$$$p<0.001, Student’s t test. B, Compared with saline-treated mice, amphetamine self-administration reduced the frequency of low-amplitude mEPSCs, while the frequency of mEPSCs was broadly higher in nonresponding mice. ^$p<0.05, saline-treated versus amphetamine self-administering mice; ^##p<0.01, saline-treated versus nonresponding mice; Bonferroni t test. Inset, The cumulative mEPSC amplitude distribution was unchanged across groups. C, Frequency distributions (left) and the average peak frequency (right) show a prominent low-frequency distribution of mEPSCs from amphetamine self-administering mice (small arrow) and more broadly distributed firing frequencies in MSNs from nonresponding mice (arrowhead; inset). ^$$p<0.01, ^$$$p<0.001, Student’s t test. D, Responses of individual MSNs from saline-treated mice show that amphetamine, and the addition of nicotine, reduced the frequency of mEPSCs. *p<0.05, paired t test. E, Amphetamine with nicotine or without decreased the frequency of low-amplitude mEPSCs. *p<0.05, Bonferroni t test, amphetamine compared with either vehicle or amphetamine with nicotine. Inset, The mEPSC amplitude distribution was unchanged. F, Frequency distributions (left) and the average peak frequency (right) of mEPSCs from saline-treated mice shows that both amphetamine (arrow) and amphetamine with nicotine (double arrow) increased low-frequency events. G, In MSNs from amphetamine self-administering mice, nicotine blocked the increase in mEPSCs that followed amphetamine. *p<0.05, paired t test. H, Amphetamine augmented the frequency of low-amplitude mEPSCs, but had no effect on their amplitude distribution (inset). *p<0.05, Bonferroni t test, amphetamine compared with either saline or amphetamine with nicotine. I, The distribution (left) and the average peak frequency (right) of mEPSCs from amphetamine self-administering mice show that amphetamine reduces 1–2 Hz activity (arrow). The addition of nicotine moderated the effect of amphetamine by increasing 1–2 Hz activity (double arrows). *p<0.05, paired t test. J, The distribution (left) and average peak frequency (right) show that mEPSCs s from saline-treated mice were similar to those from amphetamine self-administering mice exposed to amphetamine. K, Both amphetamine and amphetamine with nicotine reduced the mEPSC frequency in MSNs from nonresponding mice. *p<0.05, **p<0.01, paired t test. L, Amphetamine and nicotine reduced low-amplitude mEPSCs, but had no effect on their amplitude distribution (inset). *p<0.05, amphetamine compared with either vehicle or amphetamine with nicotine; ^&p<0.01, vehicle compared with either amphetamine or amphetamine with nicotine; Bonferroni t test. M, The frequency distribution (left) and average peak frequency (right) of mEPSCs from nonresponding mice show that amphetamine with (double arrow) or without nicotine (arrow) increases low-frequency events. *p<0.05, paired t test.

Nicotine and amphetamine modify burst and pause activity. A, Nicotine blocked the reduction of discrete burst activity by amphetamine in ChIs from saline-treated mice. For all panels, *p<0.05, **p<0.01, paired t test. B, The burstiness of ChIs from saline-treated mice was unaffected by amphetamine or the coadministration of nicotine. C, Amphetamine with nicotine or without did not change discrete pausing or (D) pause strings in ChIs from saline-treated mice. E, In ChIs from amphetamine self-administering mice, amphetamine or coadministered nicotine did not modify discrete bursting (F) but nicotine increased the length of bursting. G, Nicotine blocked the increase in discrete pausing by amphetamine in ChIs from amphetamine self-administering mice. H, In ChIs from amphetamine self-administering mice, an amphetamine challenge produced a nicotine-dependent enhancement of pause strings by boosting the intra-pause frequency and the percentage of time spent pausing.

Effects of argon on amphetamine-induced changes in locomotor activity and mu receptor activity in the nucleus accumbens. (A) When challenged with amphetamine, rats pretreated with amphetamine and argon had lower locomotor activity than rats pretreated with amphetamine and air (AA); in contrast, no significant difference in locomotor activity was found between rats pretreated with saline and argon and those pretreated with saline and air when challenged with amphetamine (SA) or saline (SS). This indicates that argon blocked locomotor sensitization to amphetamine, but had effect neither on locomotor activity induced by acute amphetamine nor on basal locomotor activity. Locomotor activity is expressed in arbitrary units. (B) As assessed immediately after being challenged with amphetamine, rats pretreated with amphetamine and argon had reduced mu receptor activity compared to rats pretreated with amphetamine and air (AA); in contrast, no significant difference in mu receptor activity was found between rats pretreated with saline and argon and those pretreated with saline and air when challenged with amphetamine (SA) or saline (SS). This indicates that argon blocked the increase in mu receptor activity induced by repeated amphetamine, but had effect neither on the increase in mu receptor activity induced by acute amphetamine nor on basal mu receptor activity. The ratio of the dissociation constant (Kd) to the maximal number of binding sites (Bmax) was calculated to assess the activity of mu receptors in the nucleus accumbens; mu receptor activity in controls rats pretreated and challenged with amphetamine was taken as a 100 % value. SS: pretreatment with saline + challenge with saline; SA: pretreatment with saline + challenge with saline; AA: pretreatment with amphetamine + challenge with amphetamine. * P < 0.001 vs AA + Air.

Effects of argon on amphetamine-induced changes in locomotor activity and mu-receptor activity in the nucleus accumbens. (a) When challenged with amphetamine, rats pretreated with amphetamine and argon had lower locomotor activity than rats pretreated with amphetamine and air (AA); in contrast, no significant difference in locomotor activity was found between rats pretreated with saline and argon and those pretreated with saline and air when challenged with amphetamine (SA) or saline (SS). This indicates that argon blocked locomotor sensitization to amphetamine, but had effect neither on locomotor activity induced by acute amphetamine nor on basal locomotor activity. Locomotor activity is expressed in arbitrary units. (b) As assessed postmortem immediately after being challenged with amphetamine, rats pretreated with argon and repeated administration of amphetamine (AA) had reduced mu-receptor constitutive activity in the nucleus accumbens (as estimated by the ratio of Bmax to Kd) compared with rats pretreated with air; in contrast, argon had effect on mu-receptor activity neither in rats pretreated with saline solution and challenged with amphetamine (SA) nor in control rats pretreated and challenged with saline solution (SS). This indicates that argon blocked the increase in mu-receptor activity induced by repeated administration of amphetamine, but had no effect on basal mu-receptor activity and acute amphetamine-induced changes in mu-receptor activity. Opioid mu-receptor activity in control rats pretreated and challenged with amphetamine was taken as a 100% value. Data are given as the median value±25th–75th percentiles; n=7–8 per condition. *P<0.05; **P<0.001. AA, pretreatment+challenge with amphetamine; SA, pretreatment saline+challenge amphetamine; SS, pretreatment+challenge with saline.

Amphetamine self-administration modifies ChI firing. A, Representative traces of cell-attached recordings in ChIs from saline-treated and amphetamine self-administering mice (left). The average baseline firing frequency (right) was lower in ChIs from amphetamine self-administering mice; This is shown in box-and-whisker plots where the median is shown as a solid line, the mean value is dotted red, the ends of the box indicate the 25^th and 75th percentiles, and the bars indicate the 10^th and 90^th percentiles. For all panels, ^$p<0.05, Student’s t test; *p<0.05 and **p<0.01, paired t test. B, The normalized power distribution (left) and the average peak frequency (right) show prominent low-frequency activity in ChIs from amphetamine self-administering mice. C, The ISI distribution for ChIs from saline-treated and amphetamine self-administering mice (arrow). D, Frequency distributions (left) and their average peaks (right) show the prominent low-frequency distribution (1/ISI) of ChI firing from amphetamine self-administering mice (arrow). E, Mean±SE frequencies of ChIs from saline-treated and (F) amphetamine self-administering mice in response to amphetamine (Amph) or amphetamine with nicotine. G, Normalized firing frequencies over time for the experiments shown in E and F. H, The frequency distribution (left) and the average peak frequency (right) of ChIs from saline-treated mice. Amphetamine produced a small increase in activity at 0.5 Hz (arrow). Nicotine blocked the inhibition caused by amphetamine and increased activity at 2.5 Hz (double arrow). I, Frequency distribution (left) and the average peak frequency (right) of ChIs from amphetamine self-administering mice show that amphetamine reduced 1–2 Hz activity (arrow). The addition of nicotine moderated the potentiating effect of amphetamine by reducing activity between 2 and 3 Hz, while increasing 3–4 Hz activity (double arrows). J, When exposed to amphetamine, the bimodal frequency distribution (left) and average peak frequency (right) of ChIs from amphetamine self-administering mice converged with the unimodal frequency distribution of ChIs from saline-treated mice.

Locomotor sensitization is dependent on D1Rs. A, B, Interval and mean ± SE (inset) locomotor ambulations over 90 min of saline-treated (A) and amphetamine-treated (B) mice on experiment day 57 (WD 50) tested with either SCH23390 or saline. For all panels, *p < 0.05, Student's t test; ^##p < 0.01, ^###p < 0.001, ANOVA. C, Interval ambulations over 90 min of amphetamine-treated mice on experiment day 57 tested with amphetamine (Amph) or with amphetamine and SCH23390 (Amph+SCH23390) compared with ambulations after their first dose of amphetamine (experiment day 3) and with ambulations of saline-exposed mice treated with saline. Inset: Mean ± SE ambulations of amphetamine-treated mice after a test dose of amphetamine (left) or after amphetamine and SCH23390 (right). D, E, Interval and mean ± SE (inset) ambulations over 90 min of saline-treated (D) and amphetamine-treated (E) mice on experiment day 57 tested with a higher dose of SCH23390 or saline. F, Interval ambulations of amphetamine-treated mice on experiment day 57 tested with amphetamine or with amphetamine and SCH23390 compared with locomotor responses after their first dose of amphetamine and to saline-exposed mice tested with saline. Inset: Mean ± SE ambulations for amphetamine-treated mice after a test dose of amphetamine (left) or after both amphetamine and SCH23390 (right).

Amphetamine challenge in withdrawal causes PPP in D1+ MSNs. A, Locomotor ambulations in response to repeated amphetamine in Drd1- and Drd2-EGFP mice was not significant (n.s.), p = 0.09, Student's t test. B, Representative traces (top) show the average responses to cortically evoked paired pulses in MSNs before (left) and 5 min after bath application of amphetamine (right). In D1+ MSNs from saline-exposed mice, amphetamine in vitro had no effect on the amplitude or PPR. C, In D1+ MSNs from amphetamine-treated mice on WD 10, amphetamine increased the eEPSC amplitude and reduced the PPR. D, In D2+ MSNs from saline-exposed mice, amphetamine decreased the amplitude and increased PPR. E, In D2+ MSNs from amphetamine-treated mice, amphetamine did not change the amplitude or the PPR. Amphetamine increased the eEPSC amplitude and reduced the PPR in a subset of D2+ MSNs from amphetamine-treated mice (F), but reduced the amplitude and increased the PPR in the remainder (G). H, Representative traces (top) show mEPSCs in aCSF (left) and 5 min after bath-applied amphetamine (right). In D1+ MSNs from saline-exposed mice, amphetamine in vitro did not change mEPSC frequency (inset, left) or the cumulative amplitude distribution (inset, right). I, In D1+ MSNs on WD 10, amphetamine in vitro increased the frequency of mEPSCs by augmenting 5–10 pA inward currents, but had no effect on their cumulative amplitude distribution. For I–L, *p < 0.05, **p < 0.01, ***p < 0.001, paired t test. J, In D2+ MSNs from saline-exposed mice, amphetamine in vitro decreased the frequency of mEPSCs, but had no effect on the cumulative amplitude distribution. In a subset of D2+ MSNs examined on WD 10, bath-applied amphetamine increased the frequency of mEPSCs (K), but decreased the frequency of mEPSCs in the remaining cells while having no effect on their cumulative amplitude distributions (L). Scale bars in B–D, F, G, 100 pA, 5 ms; H–L, 10 pA, 1 s.

Amphetamine action in LAB mice interfere with NMDA receptor signaling (A). Pre-treatment (−20 min) with MK-801 abolishes amphetamine effect on the locomotor activity in LAB mice. The dashed lines mark the moments either of saline and amphetamine (1 mg/kg, i.p.) or MK-801 (0.3 mg/kg, i.p.) and amphetamine (1 mg/kg, i.p.) administration. **p < 0.01. (B). Co-treatment with MK-801 counteracts the amphetamine calming effect in LAB mice. The dashed lines mark the moments of either saline + amphetamine (1 mg/kg, i.p.) or MK-801 (0.3 mg/kg, i.p.) + amphetamine (1 mg/kg, i.p.) administration. ***p < 0.001. (C). MK-801 post-treatment (+20 min) did not interfere with amphetamine action. The dashed lines mark the moments of amphetamine (1 mg/kg, i.p.) and MK-801 (0.3 mg/kg, i.p.) injections. (D). Decrease in the phospho-Ser9-GSK3β levels in the mPFC of LAB (n = 5/5) mice 60 min after MK-801 (0.3 mg/kg, i.p) and amphetamine (1 mg/kg, i.p.) co-treatment and coinciding with OF exposure. *p < 0.05 vs. amphetamine + saline. (E). Representative Western blots for the analysis of GSK3β, pGSK3β protein levels in LAB mice. Bands include samples both from amphetamine + saline- and amphetamine + MK-801-treated animals. (F). MK-801 pre-treatment (−20 min) effectively collapses the calming effect of LiCl. The dashed lines mark the moments of MK-801 (0.3 mg/kg, i.p.) and LiCl (100 mg/kg, i.p.) injections. ***p < 0.0001. (G). MK-801 does not interfere with the activity of the pure GSK3β inhibitor TDZD-8 in its ability to modulate locomotor activity in LAB mice. The dashed lines mark the moments of either MK-801 (0.3 mg/kg), saline + TDZD-8 (20 mg/kg, i.p.) or MK-801 (0.3 mg/kg, i.p.) + TDZD-8 (20 mg/kg, i.p.) administration. Since there was no difference between saline and 0.5% DMSO treatment, results for both vehicle-treated groups were pooled together. ***p < 0.001, n.s. non significant. (H). Hypothetical scenario: amphetamine effects on GSK3β comprise NMDA receptor-mediated kinase slowdown (due to glutamate release) rather than any direct effect. This pathway, unidentified in details, is uniquely activated in the mPFC of LAB mice and leads to GSK3β inhibition. Hypofunction of NMDA receptors might be a permissive cause of this pathway activation.

Behavioral alterations are similar in wild-type and NOX2-deficient mice after amphetamine exposure. Thirty minutes after injection with amphetamine (1 mg/kg, i.p.) or saline, mice were placed in the arena for open-field test. A–D, Bar graphs represent the frequency of groomings (A), rearings (B), crossings (C), and sniffings (D) recorded during the 20 min of the test in WT and KO NOX2 mice. For groomings: Ftr×gen_(1,11) = 2.299, p = 0.147; Ftr_(1,11) = 415.537, p < 0.001; Fgen_(1,11) = 1.123, p = 0.312. ***p < 0.001 WT saline versus WT amphetamine and KO NOX2 saline versus KO NOX2 amphetamine; NS: WT saline versus KO NOX2 saline, p = 0.837; WT amphetamine versus KO NOX2 amphetamine, p = 1.000. For rearings: Ftr×gen_(1,11) = 0.363, p = 0.703; Ftr_(1,11) = 21.464, p < 0.001; Fgen_(1,11) = 0.0546, p = 0.819; **p < 0.01 WT saline versus WT amphetamine; *p < 0.05 KO NOX2 saline versus KO NOX2 amphetamine; NS: WT saline versus KO NOX2 saline, p = 0.485; WT amphetamine versus KO NOX2 amphetamine, p = 0.862. For crossings: Ftr×gen_(1,11) = 0.00361, p = 0.996; Ftr_(1,11) = 13.164, p = 0.001; Fgen_(1,11) = 0.114, p < 0.741; **p < 0.01 WT saline versus WT amphetamine; *p < 0.05 KO NOX2 saline versus KO NOX2 amphetamine; NS: WT saline versus KO NOX2 saline, p = 0.796; WT amphetamine versus KO NOX2 amphetamine, p = 0.854. For sniffings: Ftr×gen_(1,11) = 1.028, p = 0.327; Ftr_(1,11) = 20.887, p < 0.001; Fgen_(1,11) = 0.785, p = 0.390 NS: WT saline versus KO NOX2 saline, p = 0.927; KO NOX2 saline versus KO NOX2 ketamine, p = 0.211. ***p < 0.001; **p < 0.01; *p < 0.05 using two-way ANOVA followed by Tukey's post hoc test (n = 5 WT saline; n = 5 WT amphetamine; n = 4 KO NOX2 saline; n = 5 KO NOX2 amphetamine).

(A) Time course of horizontal activity per 5 min bin of Dat:Ift88 WT (WT, filled symbols) and Dat:Ift88 KO (KO, open symbols) mice, before and after 3.0 (dark red) or 1.0 (light red) mg/kg amphetamine (injection time-point marked with arrow). Dat:Ift88 KO mice (n=18) traveled a shorter distance than Dat:Ift88 WT mice (n=11) when treated with 3.0 mg/kg (3-Way ANOVA main effect of genotype, F_(1,25)=12.85, p=.001; genotype x time bin interaction, F_(11,275)=3.32, p<.001). There was also a significant genotype x bin interaction between Dat:Ift88 KO mice (n=11) Dat:Ift88 WT mice (n=14) following 1.0 mg/kg amphetamine. (B-C) Time course of horizontal activity per 5 min bin before and after 3.0 or 1.0 mg/kg amphetamine shown by sex. Both female KO (B) and male KO (C) mice showed a significant reduction in distance traveled following injection of 3.0 mg/kg amphetamine, but no sex differences were detected (main effect of sex F_(1,25)=0.05, p=.83). 1.0 mg/kg amphetamine resulted in no differences in locomotor activity following injection in either sex. (D) Time course of repeated beam break counts (counts) per 5 min bin before and after 3.0mg/kg and 1.0mg/kg amphetamine injection (arrow) as determined by genotype analysis. Unlike distance traveled, there was no statistically significant differences in stereotypy counts for 3.0mg/kg amphetamine (main effect of genotype, F_(1,25)=3.81, p=.06). For 1.0mg/kg amphetamine there was a main effect of time bin (F_(11,550)=67.11, p<.001) as well as a time bin x sex interaction (F_(11,550)=2.04, p=.02), but no main effects or interactions involving genotype (Fs<2.37, ps>.13) during the baseline analysis. Following 1.0mg/kg amphetamine there were main effects of time bin (F_(11,231)=23.93, p<.001), sex x time bin (F_(11,231)=1.85, p=.047), and genotype x time bin (F_(11,231)=2.17, p=.02) interactions (E-F) Time course of repeated beam break counts of each genotype, by sex, per 5 min bin before and after amphetamine injection (arrow).

Dopaminergic activation mediates the effects induced by amphetamine, but not MDPV, on memory generalization. On the 48-h retention test, rats were sequentially tested in all three contextually modified inhibitory avoidance apparatuses in a random order and their retention latencies were analyzed. (A) Retention latencies of rats treated with cis-flupenthixol or saline 30 min prior to training together with amphetamine or saline administered immediately after training. Saline alone-treated animals showed longer retention latencies in the Shock box and Non-Shock box compared to those induced in the Novel box. Cis-flupenthixol alone-treated animals showed higher retention latencies in Shock box compared only to those showed in the Novel box. Cis-flupenthixol together with amphetamine treated-rats showed longer retention latencies in the Shock box and Non-Shock box compared to those induced in the Novel box. In all three boxes, amphetamine alone-treated rats showed higher retention latencies than saline alone-treated rats and cis-flupenthixol alone-treated rats. Retention latencies of rats treated with cis-flupenthixol together with amphetamine were significantly lower than those of amphetamine alone-treated rats, only in the Novel box. ^#P < 0.05 saline group latencies in the Shock box or Non-Shock box vs. saline group latencies in the Novel box; ^∧P < 0.05 cis-flupenthixol alone latencies in the Shock box vs. cis-flupenthixol alone latencies in the Novel box; ⁺P < 0.05, cis-flupenthixol together with amphetamine latencies in the Shock or Non-Shock box vs. cis-flupenthixol together with amphetamine latencies in the Novel box; *P < 0.05, **P < 0.01, amphetamine alone-treated group latencies in the Shock box, Non-Shock box or Novel box vs. saline group latencies in the Shock box, Non-Shock box or Novel box; ^§P < 0.05, ^§§P < 0.01, amphetamine alone group latencies in the Shock box, Non-Shock box or Novel box vs. cis-flupenthixol alone-treated group latencies in the Shock box, Non-Shock box or Novel box; ^°°P < 0.01, cis-flupenthixol and amphetamine-treated group latencies in the Novel box vs. amphetamine alone-treated group in the Novel box; NS, no significant differences (n = 9–10 rats). (B) Retention latencies of rats treated with cis-flupenthixol or saline 30 min prior to training together with MDPV or saline administered immediately after training. Saline alone-treated animals showed longer retention latencies in the Shock box and Non-Shock box compared to those induced in the Novel box, the same happens to cis-flupenthixol alone-treated animals. In the Novel box, MDPV alone-treated rats showed higher latencies with respect to saline-treated rats and cis-flupenthixol alone-treated rats; cis-flupenthixol and MDPV-treated rats showed higher latencies with respect to cis-flupenthixol alone-treated rats and with respect to cis-flupenthixol alone-treated. ^#P < 0.05 saline group latencies in the Shock box or Non-Shock box vs. saline group latencies in the Novel box; ^∧P < 0.05, ^∧∧P < 0.01, cis-flupenthixol alone latencies in the Shock box or Non-shock box vs. cis-flupenthixol alone latencies in the Novel box; **P < 0.01, MDPV alone-treated group latencies in the Novel box vs. saline group latencies in the Novel box; ^§§P < 0.01, MDPV alone-treated group latencies in the Novel box vs. cis-flupenthixol alone-treated group in the Novel box; ^&P < 0.05, cis-flupenthixol together with MDPV retention latencies in the Novel box vs. cis-flupenthixol alone latencies in the Novel box; NS, no significant differences (n = 8–11 rats).

Neurotransmitter level elevation is similar in wild-type and NOX2-deficient mice after amphetamine exposure. A–D, Time-dependent effect of amphetamine or saline injection on extracellular dopamine (DA; A, B) and glutamate (GLU; C, D) levels was determined by microdialysis in the prefrontal cortex of WT (A, C) and KO NOX2 (B, D) mice. Data are expressed as the percentage of baseline (as described in Materials and Methods). E, F, Concentration of dopamine (E) and glutamate (F) in WT and KO NOX2 mice at basal level or 30 min after saline or amphetamine injection. ***p < 0.001 using two-way ANOVA for repeated measures followed by Tukey's post hoc test (n = 4 WT saline; n = 4 WT amphetamine; n = 4 KO NOX2 saline; n = 4 KO NOX2 amphetamine). For dopamine analysis: two-way ANOVA for repeated measures in KO NOX2 saline versus KO NOX2 amphetamine: Ftr_(1,66) = 179.126, p < 0.001; Ft_(11,66) = 62.712, p < 0.001; Ftr×t_(11,66) = 63.847, p < 0.001; in WT amphetamine versus KO NOX2 amphetamine: Fgen_(1,66) = 0.282, p = 0.615; Ft_(11,66) = 62.241, p < 0.001; Ft×gen_(11,66) = 0.413, p = 0.945; however, Tukey's post hoc test did not reveal any difference for time; in WT amphetamine versus KO NOX2 saline: Ftr_(1,66) = 28.066, p = 0.002; Ft_(11,66) = 18.176, p < 0.001; Ft×tr_(11,66) = 18.651, p < 0.001; in WT saline versus KO NOX2 amphetamine: Ftr_(1,66) = 198.908, p < 0.001; Ft_(11,66) = 52.993, p < 0.001; Ftr×t_(11,66) = 63.514, p < 0.001; in WT saline versus KO NOX2 saline: Fgen_(1,66) = 0.239, p = 0.642; Ft_(11,66) = 1.854, p = 0.062; Fgen×t_(11,66) = 0.753, p = 0.684; in WT amphetamine versus WT saline: Ftr_(1,66) = 30.049, p = 0.002; Ft_(11,66) = 16.115, p < 0.001; Ftr×t_(11,66) = 19.754, p < 0.001. For glutamate analysis: two-way ANOVA for repeated measures in KO NOX2 saline versus KO NOX2 amphetamine: Ftr_(1,55) = 421.188, p ≤ 0.001; Ft_(11,55) = 14.986, p ≤ 0.001; Ftr×t_(11,55) = 14.751, p ≤ 0.001; in WT amphetamine versus KO NOX2 amphetamine: Fgen_(1,55) = 0.490, p = 0.515; Ft_(11,55) = 20.271, p < 0.001; Ft×gen_(11,55) = 0.246, p < 0.993; however, Tukey's post hoc test did not reveal any difference for time; in WT amphetamine versus KO NOX2 saline: Ftr_(1,66) = 42.943, p < 0.001; Ft_(11,66) = 7.117, p < 0.001; Ft×tr_(11,66) = 7.061, p < 0.001; in WT saline versus KO NOX2 amphetamine: Ftr_(1,55) = 108.426, p < 0.001; Ft_(11,55) = 9.661, p ≤ 0.001; Ftr×t_(11,55) = 10.743, p ≤ 0.001; in WT saline versus KO NOX2 saline: Fgen_(1,66) = 0.0686, p = 0.802; Ft_(11,66) = 0.143, p = 0.999; Fgen×t_(11,66) = 0.362, p = 0.966; in WT amphetamine versus WT saline: Ftr_(1,66) = 35.877, p < 0.001; Ft_(11,66) = 6.078, p < 0.001; Ftr×t_(11,66) = 6.594, p < 0.001.

Mice self-administer amphetamine. A, Acquisition of amphetamine self-administration: Following sucrose pretraining, mice were trained to self-administer amphetamine under increasing schedules of reinforcement. After 10 d on FR1, mice that had not achieved stable responding (<20 reinforcers during the last 3 d of FR1) were designated as nonresponding mice and were excluded from the remainder of the behavioral study. Thereafter the saline-treated mice and amphetamine self-administering mice underwent a further 6 d on FR2 and FR5 schedules of reinforcement. Following this acquisition phase, the amphetamine self-administering mice underwent 7 d of abstinence. This was followed by an amphetamine challenge in the same operant boxes and protocol as used during intravenous self-administration to assess the incubation of drug seeking behaviors. The effect of nicotine on this amphetamine challenge was also assessed. B, In comparison with saline-treated mice, amphetamine self-administering mice increased the number of active lever presses. C, The number of inactive lever presses was similar across all groups. D, Compared with saline-treated mice, the amphetamine self-administering mice showed drug-lever association, as assessed by the ratio of active to total (inactive + active) lever presses (%). E, During FR1, FR2, and FR5 schedules of reinforcement, amphetamine self-administering mice achieved a greater number of reinforcers than mice treated with saline, often reaching 50, the maximum allowed during the 2 h session. F, Following forced abstinence, nicotine reduced the total number of active lever presses observed after an amphetamine challenge (G) without altering the inactive lever presses. H, There was no effect of nicotine on lever discrimination, as shown by the ratio of active/total lever presses. I, The administration of nicotine with amphetamine reduced the number of reward-associated cues. The effect of a saline injection on saline-treated mice is shown for comparison with amphetamine self-administering mice. For all panels, ^$p<0.05, paired t test.

(A) Time course of horizontal activity per 5 min bin of Gad2:Ift88 WT (WT, filled symbols) and Gad2:Ift88 KO (KO, open symbols) mice, before and after 3.0 (dark blue) or 1.0 (light blue) mg/kg amphetamine (injection time-point marked with arrow). A significant genotype x time bin interaction is present, such that Gad2:Ift88 KO (n=17) mice traveled shorter distances than WT (n=17) following 3mg/kg amphetamine (3-Way ANOVA genotype x time bin interaction (F_(11,330)=2.94, p=.001). (B) Time course of horizontal activity of female Gad2:Ift88 WT (n=9) and Gad2:Ift88 KO (n=9) mice with no significant difference. (C) Time course of male WT (n=8) and KO (n=8) mice shows significantly reduced locomotor activity following injection of 3.0 mg/kg amphetamine (3-Way ANOVA, sex x genotype., (F_(1,30)=4.98, p=.03)), whereas 1.0 mg/kg amphetamine resulted in no differences in locomotor activity following injection. (D) Time course of repeated beam break counts (counts) per 5 min bin before and after 3.0mg/kg (dark blue) and 1.0mg/kg (light blue) amphetamine injection (arrow) plotted by genotype. Gad2:Ift88 KO mice (n=17) show significantly more repeated beam breaks than Gad2:Ift88 WT mice following 3.0mg/kg amphetamine (n=17) (3-Way ANOVA genotype (F_(1,30)=9.91, p=.004), genotype x time bin (F_(11,330)=1.96, p=.03)). Gad2:Ift88 KO mice (n=18) also show significantly more repeated beam breaks than Gad2:Ift88 WT (n=14) following 1.0mg/kg amphetamine as well (main effect of genotype (F_(1,28)=4.95, p=.03). (E-F) Time course of repeated beam break counts of each genotype by sex, per 5 min bin before and after amphetamine injection (arrow).

Effects of amphetamine on locomotor activity and mu-receptor neurotransmission in the nucleus accumbens. (a) When challenged with amphetamine, rats pretreated with repeated administration of saline solution (SA) or amphetamine (AA) had higher locomotor responses than rats pretreated and challenged with saline solution (SS). Locomotor activity is expressed in arbitrary units. (b) As assessed postmortem immediately after being challenged with amphetamine, rats pretreated with repeated administration of saline solution (SA) or amphetamine (AA) had increased mu-receptor activity in the nucleus accumbens (as estimated by the ratio of Bmax to Kd) compared with rats pretreated and challenged with saline solution (SS). Opioid mu-receptor activity in control rats pretreated and challenged with amphetamine was taken as a 100% value. Data are expressed as the median value±25th–75th percentiles; n=7–8 per condition. *P<0.005 and **P<0.001 vs SS; ^#P<0.001 vs SA. AA, pretreatment+challenge with amphetamine; SA, pretreatment saline+challenge amphetamine; SS, pretreatment+challenge with saline.

Effects of amphetamine on locomotor activity and mu receptor activity in the nucleus accumbens. (A) When challenged with amphetamine, rats pretreated with repeated administration of saline solution (SA) or amphetamine (AA) had higher locomotor responses than rats pretreated and challenged with saline solution (SS). Locomotor activity is expressed in arbitrary units. (B) As assessed immediately after being challenged with amphetamine, rats pretreated with repeated injection of saline solution (SA) or amphetamine (AA) had higher mu receptor activity in the nucleus accumbens than rats pretreated and challenged with saline solution (SS). The ratio of the dissociation constant (Kd) to the maximal number of binding sites (Bmax) was calculated to assess the activity of mu receptors in the nucleus accumbens; mu receptor activity in controls rats pretreated and challenged with amphetamine was taken as a 100 % value. SS: pretreatment with saline + challenge with saline; SA: pretreatment with saline + challenge with amphetamine; AA: pretreatment with amphetamine + challenge with amphetamine. * P < 0.001 vs SS + Air; ^# P < 0.001 vs SA + Air.

Effects of amphetamine and housing in a complex environment on dendritic branches (a) and spines (b) on medium spiny neurons in the shell of the NAcc. Both experience in the complex environment [saline treated/housed in standard cage (S/S) vs. saline treated/housed in a complex environment (S/C)] and amphetamine [S/S vs. amphetamine treated/housed in a standard environment (A/S) or amphetamine treated/housed in a complex environment (A/C)] significantly increased both dendritic branching and spine density, but in amphetamine-treated animals, there was no incremental effect of housing in a complex environment. Two-way ANOVA for branches: main effect of amphetamine (F = 73.0, P < 0.0001), main effect of environment (F = 27.2, P < 0.0001), and amphetamine by environment interaction (F = 66.3, P < 0.0001). Spines: main effect of amphetamine (F = 142.9, P < 0. 0001), main effect of environment (F = 6.3, P = 0.015), and amphetamine by environment interaction (F = 12.7, P < 0.001). *, Differs from S/S (Fisher's test).

'Reward pool' genes characterize the transcriptional response to amphetamine-triggered CPP. (a) Diagram of differentially expressed genes from microarray experiments. Individual microarray experiments were combined to reveal a reward pool. A comparison of the differential expression from two experiments showed no bias in the direction of expression. Pool 1 shows the genes differentially expressed in 'wild type with amphetamine versus wild-type without amphetamine'. Pool 2 represents genes differentially expressed in 'mutant with amphetamine versus sibling with amphetamine'. Pool 3 represents genes differentially expressed in 'mutant without amphetamine versus non-mutant siblings without amphetamine'. The genes in pool 3 were subtracted from pool 2 in order to eliminate basal differences between mutants and siblings, not due to amphetamine administration. The intersection of the remaining genes in pool 2 and the genes in pool 1 forms the 'reward pool'. The genes in this pool are differentially expressed in both experiments - that is, they are involved in the wild-type response to amphetamine, as well as the non-response to amphetamine in the mutant. (b) Comparison of the direction of regulation (up or down) of transcripts from the reward pool for the experiments wt+/wt- and mut+/sib+. No bias towards a particular pattern can be observed.

The reduction of KOP receptor function by amphetamine requires DA and can be mediated through enhanced dynorphin release. (a) The diminished KOP receptor-mediated inhibition of glutamate release in amphetamine treated animals is antagonized by systemic administration of flupenthixol (0.5 mg/kg, s.c.; Flup + AMPH). (b) Administration of SCH-23390 (0.1 mg/kg, s.c.) before amphetamine treatment also blocks the effect on KOP receptor function (SCH + AMPH). (c) Administration of naltrexone (1 mg/kg, s.c.) before amphetamine treatment blocks the effect on KOP receptors (NTX + AMPH). (d) Systemic administration of U69593 (0.32 mg/kg, s.c.) mimics the effects of amphetamine in reducing KOP receptor function. (e) Incubating slices in dynorphin (500 nM) for 30 min mimics also the effects of amphetamine administration. (f) Summary of the U69593 inhibition of fEPSPs in slices obtained from animals administered with saline, amphetamine, amphetamine coadministered with flupenthixol, SCH-23390 or naltrexone, systemic U69593, or bath incubation of dynorphin (*p<0.05 compared to amphetamine-treated group; ^#p<0.05 compared to saline-treated group).

Amphetamine (AMPH) exhibited rate-dependent effects on negative priming (NP). Two-way ANOVA detected a significant effect of Trial Type on accuracy [F(1, 72) = 5.22, p < 0.05]; however, post hoc testing did not reveal any significant differences in accuracy between baseline and NP trials overall at any amphetamine dose (A). No main effects of Amphetamine Dose or Trial Type x Amphetamine Dose interaction were observed overall. However, when animals were examined separately based on their initial NP rate in the drug-free state, significant patterns emerged.
A three-way ANOVA of Median Split Group, Trial Type, and Amphetamine Dose revealed a very strong trend towards a significant interaction [F(3, 33) = 2.90, p = 0.05] between these factors. When accuracy priming values were calculated and compared for the two groups, a significant Median Split Group x Amphetamine Dose interaction [F(3, 33) = 2.98, p < 0.05] was observed. Post hoc testing indicated that groups differed in priming values only after saline administration (p < 0.05), while this difference was lost after AMPH administration at any dose.
In high NP rats, two-way ANOVA revealed a significant effect of Trial Type on accuracy [F(1, 42) = 13.47, p < 0.01]. Post hoc testing revealed that animals exhibited significantly lower accuracy in NP trials compared to baseline trials after saline treatment (p < 0.01). This NP effect was absent after amphetamine administration at any dose (B). Examination of accuracy priming values shows that high NP rats exhibited a robustly positive accuracy priming value after saline treatment that was attenuated by amphetamine treatment at every dose (C), although this effect did not reach statistical significance [F<1.7, ns].
In low NP rats, no significant effect of Trial Type was detected [F<1, ns]. However, a tendency toward lower accuracy in baseline trials compared to NP trials after saline administration was observed. This pattern was reversed after the 0.25 mg/kg dose of amphetamine; here, rats exhibited lower accuracy in NP trials compared to baseline trials, suggesting a strengthened NP effect. Accuracy differences between baseline and NP trials were absent after the higher amphetamine doses (D). While no significant differences in accuracy priming values for low NP were found [F<1.3, ns], inspection of the data suggests that rats did not exhibit an NP effect after saline administration, whereas after administration of the 0.25 mg/kg amphetamine dose, the accuracy difference became positive, indicating a stronger NP effect. At higher amphetamine doses, no NP effect was observed (E).
No main effect of Amphetamine Dose and no interaction were found in either high NP or low NP rats. No significant effect of Trial Type or Amphetamine Dose and no Trial Type x Amphetamine Dose interaction on correct response latency were observed (data not shown).
Data were analyzed using two-way ANOVA, with the two factors Trial Type (baseline or NP) and Amphetamine Dose. Post hoc comparisons of significant effects were conducted using Bonferroni tests. Values are expressed as mean ± SEM. Asterisks (**p < 0.01) denote significant differences compared with baseline trials. BL, baseline; NP, negative priming.