Nicotinic α7 receptors on cholinergic neurons in the striatum mediate cocaine-reinforcement, but not food reward

Abstract

The neurotransmitter acetylcholine has since long been implicated in reward learning and drug addiction. However, the role of specific cholinergic receptor subtypes on different neuronal populations remain elusive. Here, we studied the function of nicotinic acetylcholinergic alpha 7 receptors (α7 nAChRs) in cocaine and food-enforced behaviors. We found that global deletion of α7 nAChRs in mice attenuates cocaine seeking in a Pavlovian conditioned place preference paradigm and decreases operant responding to cocaine in a runway task and in self-administration, without influencing responding to palatable food. This effect can be attributed to alpha 7 receptor signaling in the striatum, as selective deletion of striatal α7 nAChRs using a viral vector approach resulted in a similar decrease in cocaine-preference as that of global deletion. To investigate which type of striatal neurons are responsible for this effect, we selectively targeted Cholinergic (ChAT-expressing) neurons and dopamine D1-receptor (D1R) expressing neurons. Mice with conditional deletion of α7 nAChRs in ChAT-neurons (α7 nAChR-ChATCre) exhibited decreased cocaine place preference and intact place preference for food, while α7 nAChR-D1RCre mice had no changes in reward learning to neither food nor cocaine. Cocaine induction of striatal immediate early gene expression of cFos, FosB, Arc and EGR2 was blocked in α7 nAChR-ChATCre mice, demonstrating the importance of α7 nAChRs on cholinergic neurons for striatal neuronal activity changes. Collectively, our findings show that α7 nAChRs on cholinergic interneurons in the striatum are pivotal for learning processes related to cocaine, but not food reward.

Keywords: Chrna7; cocaine; food reward; immediate early genes; striatum; α7 nicotinic acetylcholine receptor.

Figure 1

α7 nAChRs are mediators of cocaine reward learning. α7 nAChR −/− mice show a blunted cocaine-seeking response in the conditioned place preference test (n = 7 WT, n = 8 −/−) (A). α7 nAChR deletion also affects operant responding to cocaine, as α7 nAChR −/− mice have a delay in acquiring the operant runway task to i.v. cocaine (n = 8 WT, n = 6 −/−) (B) and perform fewer correct nose-pokes and self-administer less cocaine (n = 7 WT, n = 6 −/−) in comparisons to their wild-type littermates (C). Results are displayed as mean ± SEM.*p < 0.05 Student’s unpaired t-test (A,C) and **p < 0.01 two-way ANOVA (group factor) followed by Bonferroni’s post hoc test (B).

Figure 2

Deletion of α7 nAChRs does not affect cocaine induced locomotion, locomotor sensitization or conditioned sensitization. α7 nAChR −/− mice increased their locomotor response to repeated 15 mg/kg cocaine injections i.p. in the same manner as wildtype littermate controls (n = 6 WT, n = 6 −/−) (A). No significant differences were observed between wildtype and α7 nAChR −/− mice in acute cocaine-induced locomotion (B), locomotor sensitization (C) day 5–1 and (D) day 21–1 or conditioned sensitization (E) day 21–20. Results are displayed as mean ± SEM. No significant differences were found after analysis with two-way ANOVA followed by Bonferroni’s post hoc test (A) or Student’s unpaired t-test (B–E), p > 0.05.

Figure 3

Global deletion of α7 nAChRs does not affect food reward learning Conditioned place preference and operant runway responses to Nutella® (A,B), as well as self-administration of sugar pellets (C) do not differ between mice lacking α7 nAChR and wild-type controls (n = 4–7 WT, n = 4–6 −/−). Results are displayed as mean ± SEM. No significant differences were found after analysis with a Student’s unpaired t-test (A,C) or two-way ANOVA followed by Bonferroni’s post hoc test (B), p > 0.05.

Figure 4

α7 nAChRs on striatal neurons expressing ChAT are mediators of cocaine preference Confocal images (20x magnification, scalebars represent 100 μm) from the striatum showing eGFP expression driven by the Chrna7-promoter in turquoise (pseudo colored) (A,B) and medium spiny neuron marker, DARPP-32, staining in orange (A) or cholinergic neuron maker (ChAT) staining in orange (pseudo colored) (B). Examples of co-labelling are delineated by arrows. AAV-Cre-recombinase mediated deletion of α7 nAChRs in the NAc decreases cocaine CPP, compared to control AAV-injected animals (n = 6 control-virus, n = 6 Cre-virus) (C). Representative example (out of n = 6) of selective Cre-expression in the striatum of mice injected with the AAV-vector expressing Cre at 20x magnification (D). Scalebar represents 100 μm. Conditional deletion of α7 nAChR in D1R expressing neurons does not elicit a difference, in neither cocaine CPP (n = 5 WT, n = 7 −/−) (E) or food CPP (F) (n = 6 WT, n = 6) −/− paradigms, in mutant mice compared to control animals. Mice with selective deletion of α7 nAChR in ChAT-expressing neurons display a blunted CPP-response to cocaine (n = 5 WT, n = 7 −/−) (G) similar to that of the global α7 nAChR−/− mice (Figure 1A). Conditional deletion of α7 nAChRs from ChAT-expressing neurons does not affect conditioned place preference towards food (n = 6 WT, n = 6 −/−) (H). Results are displayed as mean ± SEM. **p < 0.01 Student’s unpaired t-test.

Figure 5

Cocaine-induced immediate early gene expression in the striatum is attenuated in mice lacking α7 nAChRs in cholinergic neurons. The immediate early genes cfos (A), fosB (B), arc (C), and egr2 (E) are upregulated in the striatum of wild-type animals after an acute i.p. injection of 15 mg/kg cocaine, but are significantly attenuated in α7 nAChR^floxed-ChATCre mice. Expression of egr1 (D) and cjun (F) was not affected by cocaine (n = 6 in all four experimental groups) nor α7 nAChR deletion. Results are displayed as mean ± SEM. *p < 0.05 Two-way ANOVA followed by Bonferroni’s post hoc test.