p11 in Cholinergic Interneurons of the Nucleus Accumbens Is Essential for Dopamine Responses to Rewarding Stimuli

Abstract

A recent study showed that p11 expressed in cholinergic interneurons (CINs) of the nucleus accumbens (NAc) is a key regulator of depression-like behaviors. Dopaminergic neurons projecting to the NAc are responsible for reward-related behaviors, and their function is impaired in depression. The present study investigated the role of p11 in NAc CINs in dopamine responses to rewarding stimuli. The extracellular dopamine and acetylcholine (ACh) levels in the NAc were determined in freely moving male mice using in vivo microdialysis. Rewarding stimuli (cocaine, palatable food, and female mouse encounter) induced an increase in dopamine efflux in the NAc of wild-type (WT) mice. The dopamine responses were attenuated (cocaine) or abolished (food and female mouse encounter) in constitutive p11 knock-out (KO) mice. The dopamine response to cocaine was accompanied by an increase in ACh NAc efflux, whereas the attenuated dopamine response to cocaine in p11 KO mice was restored by activation of nicotinic or muscarinic ACh receptors in the NAc. Dopamine responses to rewarding stimuli and ACh release in the NAc were attenuated in mice with deletion of p11 from cholinergic neurons (ChAT-p11 cKO mice), whereas gene delivery of p11 to CINs restored the dopamine responses. Furthermore, chemogenetic studies revealed that p11 is required for activation of CINs in response to rewarding stimuli. Thus, p11 in NAc CINs plays a critical role in activating these neurons to mediate dopamine responses to rewarding stimuli. The dysregulation of mesolimbic dopamine system by dysfunction of p11 in NAc CINs may be involved in pathogenesis of depressive states.

Keywords: acetylcholine; anhedonia; cholinergic interneuron; dopamine; nucleus accumbens; p11.

Graphical abstract

Figure 1.

The dopamine (DA) response to rewarding stimuli in the NAc and PFC of constitutive p11 KO mice. A, B, Representative location of a microdialysis probe placed in the mouse NAc (A) and PFC (B) (Paxinos and Franklin, 2001). The position of dialysis membrane is indicated with yellow color. C–H, The effects of cocaine infusion (1 µM) into the NAc (C) or PFC (F), exposure to palatable food (D, G), and exposure to female mice (E, H) on the extracellular levels of DA in the NAc (C–E) and PFC (F–H) of WT and constitutive p11 KO mice. The DA levels were determined with in vivo microdialysis. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT mice; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses.

Figure 2.

The dopamine (DA) response to cocaine infusion in the NAc in constitutive p11 KO mice is restored by nicotinic or muscarinic receptor stimulation in the NAc. Effects of local infusion of cocaine (1 µM) and/or nicotine (1 µM) (A) or cocaine (1 µM) and/or non-selective muscarinic receptor agonist, oxotremorine (0.1 µM) (B) into the NAc on the extracellular levels of DA in the NAc of constitutive p11 KO mice. The dose of nicotine or oxotremorine without effects on the dopamine levels was used. Data for cocaine infusion alone were reproduced from Figure 1C for comparison. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. **p < 0.01, ***p < 0.001 versus the cocaine group; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses under each experimental condition.

Figure 3.

The dopamine (DA) response to rewarding stimuli in the NAc of ChAT-p11 conditional KO (cKO) mice. The effects of cocaine infusion (1 µM) into the NAc (A), exposure to palatable food (B), and exposure to female mice (C) on the extracellular levels of DA in the NAc of WT (ChAT-Cre^-/- p11^flox/flox) and ChAT-p11 cKO (ChAT-Cre⁺ p11^flox/flox) mice. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT mice; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses.

Figure 4.

Overexpression of p11 in ChAT cells of the NAc restores the dopamine (DA) response to rewarding stimuli in ChAT p11 cKO mice. A, Immunohistochemical detection of RFP (red) and p11 (green) in the NAc of ChAT-p11 cKO mice injected with p11-overexpressing virus [AAV-loxP-RFP/stop-loxP-p11 (AAV-p11)] into the NAc. RFP is expressed in ChAT-Cre^-/- cells, and p11 was expressed in ChAT-Cre⁺ cells. In images with low magnification (left panel), RFP-positive area in the shell of the NAc corresponds to the area of viral injection. In images with high magnification (right panel), p11 is overexpressed in RFP-negative neurons. Arrows indicate cells overexpressing p11. B, Immunohistochemical detection of RFP (red), YFP (green), and ChAT (blue) in the NAc of ChAT-p11 cKO mice injected with control virus [AAV-loxP-RFP/stop-loxP-YFP (AAV-YFP)]. RFP was expressed in ChAT-Cre^-/- cells, and YFP was expressed in ChAT-Cre⁺ cells. YFP expression overlapped with ChAT staining. Arrow indicates ChAT-positive CINs expressing YFP. C–E, The effects of cocaine infusion (1 µM) into the NAc (C), exposure to palatable food (D), and exposure to female mice (E) on the extracellular levels of DA in the NAc of ChAT-p11 cKO mice injected with control (AAV-YFP) or p11-overexpressing (AAV-p11) virus. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus ChAT-p11 cKO mice with control virus injection; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses.

Figure 5.

The ACh responses to cocaine infusion in the NAc of ChAT-p11 cKO mice. The extracellular levels of ACh in the NAc were measured with in vivo microdialysis after infusion of cocaine (1 µM) into the NAc of WT (ChAT-Cre^-/- p11^flox/flox) and ChAT-p11 cKO (ChAT-Cre⁺ p11^flox/flox) mice. The basal values for each group were obtained as the average of six stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. *p < 0.05 versus WT mice; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus the basal levels of ACh in the same group. The number of mice is indicated in parentheses under each experimental condition.

Figure 6.

Activation of ChAT cells in the NAc using a chemogenetic technique restores the dopamine (DA) response in ChAT p11 cKO mice. A, Immunohistochemical detection of mCherry (red) and ChAT (green) in the NAc of ChAT-p11 cKO mice injected with Gs-DREADD virus [AAV-DIO-rM3D(Gs)-mCherry (AAV-rM3D(Gs))] into the NAc. In images with low magnification (left panel), mCherry-positive cells are aparsely dstributted in the NAc (arrow head). In images with high magnification (right panel), mCherry is expressed in ChAT-positive CINs. Arrows indicate ChAT-positive CINs expressing rM3D(Gs). B, The effects of CNO infusion at 3 or 10 µM into the NAc on the extracellular levels of DA in the NAc of ChAT-p11 cKO mice injected with control [AAV-DIO-mCherry (AAV-mCherry)] or Gs-DREADD virus. The DA levels were determined as the average of those at 40, 60, and 80 min of CNO infusion. Data represent mean ± SEM. **p < 0.01; one-way ANOVA and Newman–Keuls multiple comparison test. C, The effects of CNO infusion (3 µM) into the NAc on the cocaine-induced increases in DA in the NAc of ChAT-p11 cKO mice injected with control (AAV-mCherry) or Gs-DREADD virus. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus ChAT-p11 cKO mice with control virus injection; two-way ANOVA and Bonferroni multiple comparison test. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses under each experimental condition.

Figure 7.

Inhibition of ChAT cells in the NAc using a chemogenetic technique suppresses the dopamine (DA) response in control mice. Gi-DREADD virus [AAV-DIO-rM4D(Gi)-mCherry (AAV-rM4D(Gi))] or control virus [AAV-DIO-mCherry (AAV-mCherry)] was injected into the NAc of ChAT-Cre mice. The effects of CNO infusion (3 µM) into the NAc on the cocaine-induced increases in DA in the NAc were examined. The basal values for each group were obtained as the average of three stable baseline samples, and all values are calculated as a percentage of the basal values within the same group (100%). Data represent mean ± SEM. ^††p < 0.01, ^†††p < 0.001 versus the basal levels of DA in the same group. The number of mice is indicated in parentheses under each experimental condition.