Schematic representation of the positions of all microdialysis probes and microinjection cannulae in the rat mPFC and VTA. Coronal brain sections were analyzed to verify the position of the membrane segment of each microdialysis probe and microinjection cannulae (the membrane regions of probes are indicated by line segments and the tips of microinjection cannulae by dots). All animals used in these investigations are represented. mPFC and VTA diagrams are 3.0 mm anterior and 5.95 mm posterior to bregma, respectively (adapted from Paxinos and Watson 1986).

GABA responses in the mPFC (panel a) and VTA (panel b), induced by intra-mPFC NMDA, were not enhanced by prior nicotine SA. GABA levels were measured on the microdialysates obtained in the experiments shown in Fig. 3. Two-way ANOVA with repeated measures showed that the mPFC and VTA GABA responses to 200 or 500 μM NMDA were not affected by nicotine SA [mPFC (panel a): F_1,6 = 0.45, p > 0.05; VTA (panel b): F_1,4 = 0.51, p > 0.05; n = 3–5).

After extinction from chronic nicotine SA, glutamate release in the mPFC (panel a) and VTA (panel b), induced by intra-mPFC NMDA, was not enhanced by prior nicotine SA. Glutamate was measured in concurrent microdialysates obtained from both brain regions. Basal levels of glutamate in mPFC and VTA were unaffected by prior nicotine SA. After 10 days of extinction, two-way ANOVA with repeated measures indicated that both the mPFC and VTA glutamate responses induced by intra-mPFC NMDA (200 or 500 μM; administered by reverse microdialysis) or KCl were not affected by prior nicotine SA [mPFC (panel a):F_1,6 = 0.50, p > 0.05; VTA (panel b): F_1,6 = 0.02, p > 0.05; n = 4 for Nic and Sal].

After extinction of nicotine SA, the mPFC and VTA levels of the ionotropic glutamate receptor subunits, NR1, NR2A, NR2B, GluR1 and GluR2/3 were unaffected by prior nicotine SA. Following regional brain microdissection and punching, tissue extracts were prepared in SDS, separated by electrophoresis, and then subjected to western immunoblotting and quantitation, as described in the Methods. For all subunits, data were normalized to β-actin levels and then converted to mean percentage of saline (± SEM) for each subunit. Representative blots comparing saline (S) vs. nicotine (N)-treated animals are shown for each subunit. (panel a) In mPFC, the previously reported enhancement of NMDA receptor subunit expression (e.g., NR2A and NR2B) by nicotine SA was no longer detectable (unpaired student t-test, p > 0.05 for each subunit; n = 4 per group) (Wang et al. 2007). (panel b) Similarly, in the VTA, the reported elevation of the AMPA receptor subunit, GluR2/3, during nicotine SA, was not detected after extinction (unpaired student t-test, p > 0.05 for each subunit; n = 4 per group).

The NMDA receptor antagonist, AP-5, did not affect the maintenance of chronic nicotine or sucrose SA. Using a counterbalanced design in two separate cohorts of animals (nicotine SA, panel a; sucrose SA, panel b), each animal received AP-5 (1.5 and 5 μg/side) and KRB microinjected into mPFC 10 min before the initiation of daily test sessions on days 19, 21, and 23, after 18 days of nicotine or sucrose SA. Animals self-administered nicotine or sucrose on days 20 and 22 without receiving mPFC injections. (panel a) Active lever presses, expressed as the ratio of presses on the treatment day/presses on the preceding day without treatment, were not altered by AP-5 in the nicotine SA group (one way ANOVA: F_2,15 = 0.25, p > 0.05, n = 6). (panel b) The time required to obtain 100 sucrose pellets in response to lever pressing (FR1) was expressed as the ratio of time required on the treatment day/time required on the preceding day without treatment. This ratio was not affected by intra-mPFC AP-5 (one way ANOVA, F_2,25 = 0.44, p > 0.05, n = 8–10 per group).

Chronic nicotine SA enhanced glutamate release in the mPFC and VTA induced by intra-mPFC NMDA (administered by reverse microdialysis). Glutamate was measured in microdialysates obtained concurrently from both brain regions. Basal levels of glutamate in mPFC and VTA were unaffected by nicotine SA. (panel a) In mPFC, glutamate responses to NMDA (200 μM and 500 μM) and 100 mM KCl were determined on day 19 of SA. Two-way ANOVA with repeated measures revealed that nicotine SA (Nic; n = 7) amplified the glutamate response to 200 μM NMDA in comparison to saline SA (Sal; n = 5) (F_1,10 = 6.76, p < 0.05). (panel b) In mPFC, the glutamate peak incremental responses were calculated from the data in panel a. One way ANOVA indicated that these responses were enhanced by nicotine SA (200 μM NMDA, F_1,10 = 9.71, p < 0.05; 500 μM NMDA, F_1,10 = 8.74, p < 0.05). (panel c) In VTA, the glutamate responses to NMDA and KCl were determined on day 19 of SA. Nicotine SA (n = 7) amplified the glutamate responses to 200 μM and 500 μM NMDA in comparison to saline (n = 5) (F_1,10 = 6.36, p < 0.05 for 200 μM NMDA; F_1,10 = 5.96, p < 0.05 for 500 μM NMDA). (panel d) In VTA, the glutamate peak incremental responses were calculated from the data in panel c. NMDA dose-dependently stimulated glutamate release (F_1,9 = 18.73, p < 0.01). The glutamate responses to both doses of NMDA were enhanced by nicotine SA (one-way ANOVA: 200 μM NMDA, F_1,9 = 16.99, p < 0.01; 500 μM NMDA F_1,9 = 16.67, p < 0.01). Data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01 comparing Nic vs. Sal SA at the same dose of NMDA.

The AMPA receptor antagonist, NBQX, reduced active lever pressing and increased the time required to obtain 100 sucrose pellets during the maintenance phase of chronic nicotine SA and sucrose SA, respectively, when administered into the VTA. Using a counterbalanced design in two separate cohorts of animals (nicotine SA, panel a; sucrose SA, panel b), each animal received NBQX (0.067 and 0.25 μg/side) and KRB microinjected into VTA 10 min before the initiation of daily test sessions on days 19, 21, and 23, after 18 days of nicotine or sucrose SA. Animals self-administered nicotine or sucrose on days 20 and 22 without receiving VTA injections. (panel a) Active lever presses, expressed as the ratio of presses on the treatment day/presses on the preceding day without treatment, were reduced by intra-VTA NBQX at 0.25 μg/side (one way ANOVA, F_2,27 = 8.90, p < 0.01, n = 10). (panel b) The time required to obtain 100 sucrose pellets in response to lever pressing (FR1) was expressed as the ratio of time required on the treatment day/time required on the preceding day without treatment. This ratio was increased by intra-VTA NBQX at 0.25 μg/side (one way ANOVA, F_2,15 = 35.60, p < 0.001, n = 6). (panel c) Intra-VTA NBQX did not affect locomotion (two-way ANOVA with repeated measures, F_2,15 = 0.04, p > 0.05). *p < 0.01, **p < 0.001, compared to intra-mPFC or -VTA microinjection of KRB; #p < 0.01, ##p < 0.001, compared to intra-mPFC or -VTA microinjection of NBQX 0.067 μg/side.

Lever press activity during chronic nicotine self-administration (SA) and extinction. (panel a) Adult male Lewis rats acquired nicotine SA (0.03mg/kg b.wt./injection, iv) without prior training, priming or food deprivation when the drug was available 23 h/day from days 1–18. Two-way ANOVA with repeated measures revealed that active lever presses were greater than inactive presses in the nicotine (Nic) group (F_1,10 = 38.89, p < 0.001; n = 7), but not in the saline (Sal) group (F_1,10 = 0.31, p > 0.05; n = 5). Comparing treatment groups, active lever presses were greater in the Nic than Sal group (F_1,10 = 6.79, p < 0.05). (panel b) A separate cohort of rats self-administered Nic or Sal for 18 days, followed by 10 days of extinction. Two-way ANOVA with repeated measures showed that, after extinction, active lever presses in the Nic group (n = 4) were no longer different from the Sal (n = 4) group (F_1,6 = 3.71, p > 0.05). In addition, the mean number of active lever presses during the last 3 days of the late maintenance phase (d 16–18) were greater than during the final 3 days of the extinction period (e.g., days 26–28) (F_1,6 = 12.11, p > 0.05). Data are expressed as mean ± SEM.