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Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice - PubMed

Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice

Lauriane Harrington et al. Neuropsychopharmacology. 2016 Jun.

Abstract

Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological, and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was lower in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30, and 60 μg/kg/infusion) compared with wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens than in WT mice, and exhibit a differential sensitivity to nicotine-induced DA outflow. Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to lower doses of nicotine than that of WT mice. Re-expression of β4* nAChRs in IPN neurons fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. These findings suggest that β4* nAChRs in the IPN have a role in maintaining nicotine IVSA.

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Figures

Figure 1
Figure 1

Operant responding for food pellets and intravenous nicotine in WT and β4KO mice. (a) Food pellets earned on an FR1 schedule of reinforcement during 8 days followed by an FR3 during 6 days in WT (n=41) and β4KO (n=36) mice, and 3 days of re-training after catheter surgery (WT n=38, β4KO n=27). Results are expressed as mean number of food pellets in 1 h±SEM. (b) Nicotine IVSA in WT (n=22) and β4KO (n=15) mice. Mice were trained on an FR3 schedule of reinforcement during 10 days to respond for nicotine at a dose of 30 μg/kg/infusion. Results are expressed as mean number nicotine infusions in 1 h±SEM. Two-way repeated measures ANOVA, **p<0.01 genotype effect. The inset shows the area under the curve for active nose-poke responding in WT and β4KO mice (**p<0.01 WT vs KO). (c) Progressive ratio schedule of reinforcement in WT (n=22) and β4KO (n=15) mice. The data are expressed as the mean breaking point+SEM for nicotine IVSA (30.0 μg/kg/infusion, F(1,36) = 6.9, *p<0.05). (d) Dose-response curve for nicotine reinforcement in WT (n=11) and β4KO (n=9) mice. Mice were trained to respond for ascending doses of nicotine (7.5, 15, 30, and 60 μg/ kg/ infusion) and saline on an FR3 schedule of reinforcement. Results are expressed as mean number of nicotine infusions earned in 1 h±SEM for each dose during 3 days of training. Two-way repeated measures ANOVA followed by LSD post hoc (*p<0.05, **p<0.01).

Figure 2
Figure 2

Basal and nicotine-stimulated extracellular DA levels in the NAC of β4KO and WT mice. (a) Basal extracellular DA levels in WT (n=21) and β4KO (n=21) mice, #p<0.05 (left panel). Change in NAC levels relative to baseline following an injection of saline (s.c.) in WT (n=10) and β4KO (n=9) mice, (right panel). (b–d) Change in NAC DA levels relative to baseline following an acute injection of nicotine (s.c.), at 0.17 mg/kg (b; WT n=10, KO n=9), 0.24 mg/kg (c; WT n=5, KO n=5), or 0.33 mg/kg (d; WT n=6, KO=7), at 15 min intervals up to 120 min post injection. Data represented as mean±SEM. Two-way repeated measures ANOVA followed by LSD post hoc test. *p<0.05 vs baseline; #p<0.05 genotype difference.

Figure 3
Figure 3

Nicotine-elicited VTA DAergic neuron responses in vivo in WT and β4KO mice. (a) Spontaneous firing frequency (left panel), and spontaneous bursting activity (right panel) in WT (n=90) and β4KO mice VTA DA neurons (n=101). The data represent mean±SEM. (b) Top—a representative trace at 10 μg/kg/injection nicotine in a VTA DA neuron of a WT and β4KO mouse. Bottom—Increased variation from baseline in firing frequency for WT and β4KO mice injected with nicotine (0, 5, 10, 15, 30, and 60 μg/kg/injection). Paired t-test vs saline *p<0.05, ***p<0.001. (c) Changes in proportion of spikes within bursts (SWB) in WT and β4KO mice injected with nicotine (0, 5, 10, 15, 30, and 60 μg/kg/injection). Wilcoxon paired test vs saline *p<0.05. Data represented as median±interquartile intervals. Dots represent outliers. The number of cells recorded is indicated on the graph.

Figure 4
Figure 4

Endogenous and exogenous expression of β4 in the mouse brain. (a) PGK-β4 lentiviral vector for expression of β4 nAChR subunits and enhanced green fluorescent protein (eGFP). Sub-cloning by XhoI/BsrGI restriction enzyme digestion. ΔU3, deletion in U3 of 3'LTR; LTR, long terminal repeat; PGK, phosphoglycerate kinase promotor; β4–β4 WT mouse cDNA sequence; IRES, internal ribosome entry sequence; WPRE, woodchuck hepatitis B virus posttranscriptional response element. (b) Autoradiography of heteromeric nAChRs (total I125-epibatidine) and β4* nAChRs (I125-epibatidine+5-I-A85380) of MHb-injected (left) and IPN-injected (right) mice. WT-GFP and KO-GFP mice were injected with control GFP-only expressing lentivirus. (c) Immunofluorescence staining of the IPN. Transgenic β4-cre mouse injected with an AAV for cre-dependent fluorophore expression (green). GFP (green), green fluorescent protein; TH (blue), tyrosine hydroxylase; ChAT (red), choline acetyl transferase. (d) Neuro-anatomical localization of lentiviral infection and the corresponding Paxinos and Franklin (2004) brain atlas images, visualized by the eGFP reporter protein in the MHb and the IPN.

Figure 5
Figure 5

Effects of β4* nAChR re-expression in the MHb (a, b) and IPN (c–f) on nicotine IVSA and VTA DAergic neuron responses to nicotine. (a) Mean±SEM. number of intravenous nicotine infusions during 3 days for each dose of nicotine tested (7.5, 15.0, 30.0, and 60.0 μg/kg/infusion) on an FR3 reinforcement schedule in WT-GFPMHb (n=9), KO-GFPMHb (n=8), and KO-β4MHb (n=7) mice. Two-way repeated measures ANOVA with Tukey's post hoc test (**p<0.01, group effect). The inset represents the quantification of 5-I-A85380-resistant I125-epibatidine autoradiographic intensity in the MHb of KO-β4MHb mice. Data are the normalized mean inverse luminosity+SEM relative to WT-GFPMHb (n=6), KO-GFPMHb (n=6), and KO-β4MHb (n=14). (b) The mean+SEM amount of nicotine consumed during the IVSA experiment (μg/kg), at each dose tested. One-way ANOVA with Tukey's post hoc test. *p<0.05, **p<0.01, ns p>0.05. (c) Mean±SEM number of intravenous nicotine infusions during 3 days for each dose of nicotine tested (7.50, 15.0, 30.0, and 60.0 μg/kg/infusion) on an FR3 reinforcement schedule in WT-GFPIPN (n=5), KO-GFPIPN (n=9), and KO-β4IPN (n=9) mice. Two-way repeated measures ANOVA with Tukey's post hoc test. ***p< 0.001 KO-GFPIPN vs WT-GFPIPN, $$p<0.01, $$$p<0.001 KO-β4IPN vs KO-GFPIPN, #p<0.05 KO-β4IPN vs WT-GFPIPN. The inset represents the quantification of 5-I-A85380-resistant I125-epibatidine autoradiographic intensity in the IPN. Data show the normalized mean inverse luminosity+SEM relative to WT-GFPIPN (n=4), KO-GFPIPN (n=4), and KO-β4IPN (n=7). (d) The mean amount of nicotine consumed during the IVSA experiment (μg/kg), at each dose tested. One-way ANOVA with Tukey's post hoc test *p<0.05, **p<0.01, ***p<0.001, ns p>0.05. (e) Variation from baseline in firing frequency for WT-GFPIPN, KO-GFPIPN, and KO-β4IPN mice in response to nicotine (0, 10, and 30 μg/kg/injection). Mean+SEM, t-test vs saline *p<0.05, **p<0.01, ***p<0.001. (f) Changes in the proportion of spikes within bursts (SWB) in WT-GFPIPN, KO-GFPIPN, and KO-β4IPN mice in response to nicotine (0, 10, and 30 μg/kg/injection). Median±interquartile intervals. Dots represent outliers. Wilcoxon paired test vs saline. *p<0.05, **p<0.01. The number of cells recorded is annotated.

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