Extinction training after cocaine self-administration induces glutamatergic plasticity to inhibit cocaine seeking

Abstract

Learning to inhibit drug seeking can be an important strategy for inhibiting relapse, and this can be modeled by extinguishing drug seeking in response to a drug-paired context. Rats were either extinguished or withdrawn without extinction training (abstinence) from cocaine self-administration, and measurements of postsynaptic density proteins in the core and shell subcompartments of the nucleus accumbens were compared with yoked-saline controls. Only extinguished rats had elevations of PSD-95, Homer1b/c, and Narp in the postsynaptic density of the core, whereas no proteins measured were altered in the postsynaptic density of the shell in either extinguished or abstinent rats. Using a biotinylation strategy, it was found that surface expression of mGluR5 was reduced only in the core of extinguished animals. Although both extinguished and abstinent animals showed a reduction in long-term potentiation elicited in the core by stimulating prefrontal cortex, blunted long-term depression was observed only in extinguished rats. These data indicate that the elevation in Homer1b/c in the core may have sequestered mGluR5 away from the membrane surface and that the loss of surface mGluR5 inhibits long-term depression. Accordingly, when Homer1c was overexpressed in the core of cocaine-naive rats with an adenoassociated virus, long-term depression was inhibited. This mechanism may contribute to the inhibition of cocaine seeking by extinction training because overexpression of Homer1c in the core also inhibited cue-induced reinstatement of cocaine seeking. These data identify a cellular mechanism that may contribute to extinction-induced inhibition of cocaine seeking.

Figure 1.

Extinction training (Ex) and abstinence (Ab) after cocaine self-administration produced different patterns of changes in protein expression in the PSD-enriched fraction. A, The mean number of infusions did not differ between Ab and Ex groups. B, Representative Western blots demonstrating the composition of PSD and non-PSD fractions based on the presence of characteristic proteins. C, Sample Western blots showing protein upregulation in the Ex group. D, NAcore PSD-enriched fraction protein expression. Two-tailed Student's t test revealed that PSD-95 (t₍₁₃₎ = 2.50; p = 0.027), Homer1b/c (t₍₁₅₎ = 3.15; p = 0.007), NARP (t₍₁₄₎ = 2.22; p = 0.043), and mGluR5 (t₍₂₀₎ = 2.00; p = 0.059) were increased after extinction training but not abstinence. E, Nucleus accumbens shell PSD-enriched fraction protein expression showed no differences after extinction training or abstinence. N = 6–12 for all groups. *p < 0.05; ⁺0.05 > p < 0.06. Error bars indicate SEM.

Figure 2.

Extinction training and abstinence after cocaine self-administration produced different patterns of changes in protein expression in the non-PSD subfraction (Triton X-100-soluble fraction). A, No change was measured in the NAcore in extinguished subjects, whereas changes after abstinence were observed in levels of GluR2 (t₍₁₂₎ = 2.18; p = 0.050). B, In the NAshell non-PSD fraction, GluR2 (t₍₁₁₎ = 2.13; p = 0.057) was changed in extinguished animals, and in abstinent animals changes were measured in GluR2 (t₍₁₂₎ = 2.38; p = 0.035). N = 6–12 for all groups. C, Representative immunoblots of significant changes in protein level. *p < 0.05; ⁺0.05 > p < 0.06. Error bars indicate SEM.

Figure 3.

Extinction-associated changes in protein require both extinction training and cocaine self-administration. A, Mean active lever responses during the first 12 d of extinction training after sucrose pellet self-administration. Inset, Total number of sucrose pellets earned during self-administration training. N = 9. B, There were no changes in protein expression of PSD-95, Homer1b/c, and NARP in the NAcore after 2 weeks extinction of sucrose-reinforced responding. N = 6–12 in each group. C, Mean active lever responses during the 2 d after cocaine self-administration. Inset, Total number of cocaine infusions earned during self-administration training. N = 9. D, Nucleus accumbens core PSD-fraction protein expression. There were no changes in protein expression of PSD-95, Homer1b/c, and NARP after 2 d of extinction training of cocaine-reinforced responding. N = 6–12 in each group. Error bars indicate SEM.

Figure 4.

Surface expression of mGluR5 is reduced after extinction training but not abstinence from cocaine self-administration. A, Surface expression of mGluR5 was significantly decreased after cocaine self-administration and 3 weeks of extinction (t_(1,14) = 3.31; p = 0.006), whereas expression was unaltered in a whole-cell fraction. B, Surface expression of mGluR5 was not altered after abstinence from cocaine self-administration. N is shown in bars. Error bars indicate SEM.

Figure 5.

Extinction training inhibits the induction of LTD in the NAcore elicited by PFC stimulation. A, Rats trained to self-administer cocaine show blunted LTP regardless of abstinence or extinction training (time, F_(16,32) = 36.94, p < 0.001; treatment, F_(2,32) = 4.67, p = 0.030; interaction, F_(32,208) = 3.24, p < 0.001). The right panel shows the average field amplitude measured between 15 and 30 min after high-frequency stimulation (F_(2,12) = 8.30; p = 0.008). B, Only rats extinguished from cocaine self-administration show blunted LTD (time, F_(16,32) = 5.26, p < 0.001; treatment, F_(2,32) = 8.90, p = 0.006; interaction, F_(32,160) = 5.63, p < 0.001). The right panel shows the average field amplitude measured between 15 and 30 min after low-frequency stimulation (F_(2,13) = 5.22; p = 0.022). C, When Homer1c was overexpressed in the NAcore of cocaine-naive animals, LTD was blunted relative to GFP-infused controls (time, F_(17,136) = 13.37, p < 0.001; treatment, F_(1,136) = 17.49, p = 0.003; interaction, F_(17,136) = 5.658, p < 0.001). The right panel shows the average field amplitude measured between 15 and 30 min after low-frequency stimulation (t_(1,9) = 17.483; p = 0.003). N is shown in bars. *p < 0.05, using Dunnett's t test to compare extinction and abstinent groups to yoked-saline. Error bars indicate SEM.

Figure 6.

Overexpression of Homer1c in the NAcore attenuated reinstatement of cocaine seeking. A, There was no difference in extinction learning between Homer1c-AAV- and GFP-AAV-infected animals when the AAVs were administered 1 d before the initiation of extinction training. B, There was no difference in extinction learning between Homer1c- and GFP-infected animals when the AAVs were infused 2 weeks before initiating extinction training. C, Homer1c-AAV infection immediately before extinction training attenuated cue-primed reinstatement. A two-way ANOVA with repeated measures over trial (i.e., extinction and cue-prime) confirms significant main effects of virus (F_(1,18) = 5.68; p = 0.028) and trial (F_(1,18) = 20.00; p < 0.001), as well as interaction (F_(1,18) = 4.22; p = 0.054). D, Homer1c-AAV infection followed by 2 weeks of abstinence before extinction training attenuated cue-primed reinstatement. A two-way ANOVA revealed significant main effects of virus (F_(1,13) = 7.43; p = 0.017) and trial (F_(1,13) = 28.90; p < 0.001), as well as a significant interaction (F_(1,13) = 6.37; p = 0.025). E, Immune staining of HA-tagged Homer1c reveals cellular labeling 5 weeks after virus infusion into the NAcore. ac, Anterior commissure. *p < 0.05, compared with extinction; ⁺p < 0.05, comparing Homer-AAV to GFP-AAV, using a least significant difference post hoc test (Milliken and Johnson, 1984). Error bars indicate SEM.