GLP-1 acts on habenular avoidance circuits to control nicotine intake

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Abstract

Tobacco smokers titrate their nicotine intake to avoid its noxious effects, sensitivity to which may influence vulnerability to tobacco dependence, yet mechanisms of nicotine avoidance are poorly understood. Here we show that nicotine activates glucagon-like peptide-1 (GLP-1) neurons in the nucleus tractus solitarius (NTS). The antidiabetic drugs sitagliptin and exenatide, which inhibit GLP-1 breakdown and stimulate GLP-1 receptors, respectively, decreased nicotine intake in mice. Chemogenetic activation of GLP-1 neurons in NTS similarly decreased nicotine intake. Conversely, Glp1r knockout mice consumed greater quantities of nicotine than wild-type mice. Using optogenetic stimulation, we show that GLP-1 excites medial habenular (MHb) projections to the interpeduncular nucleus (IPN). Activation of GLP-1 receptors in the MHb–IPN circuit abolished nicotine reward and decreased nicotine intake, whereas their knockdown or pharmacological blockade increased intake. GLP-1 neurons may therefore serve as 'satiety sensors' for nicotine that stimulate habenular systems to promote nicotine avoidance before its aversive effects are encountered.

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Figure 1: Nicotine activates medullary GLP-1 neurons.
Figure 2: GLP-1 regulates nicotine intake.
Figure 3: Chemogenetic activation of GLP-1 neurons mice decreases nicotine intake.
Figure 4: GLP-1 inputs from NTS stimulate IPN neurons.
Figure 5: GLP-1 activates IPN neurons by stimulating habenular terminals.
Figure 6: GLP-1 transmission in IPN regulates nicotine intake.
Figure 7: GLP-1 in IPN abolishes nicotine reward.

Change history

  • 10 April 2017

    In the version of this article initially published online, the original source of the Glp1r knockout mice, D.J. Drucker (Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto), was not acknowledged. The error has been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We thank D.J. Drucker (Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto) for providing the Glp1r knockout mice. This work was supported by the US National Institutes of Health (DA032225 to L.M.T., DK096139 to M.R.H., DA020686 to P.J.K.).

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L.M.T., A.D., Z.C., C.D.F., B.R.L., X.-A.L., Q.L. and M.I. conducted all experiments. T.M.K., M.C., M.P. and M.R.H. provided essential reagents. L.M.T. and P.J.K. designed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Paul J Kenny.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Nicotine activates non-catecholaminergic neurons in rostral NTS.

(a) Graphical representation of relative distribution of tyrosine hydroxylase-positive (TH+) neurons in the rostral NTS (rNTS). (b) Representative micrographs of Fos (in red) and TH+ (in green) neurons in the cNTS after nicotine (0.25-1.5 mg/kg) injection. The majority of Fos-positive cells were in non=TH+ cells (identified by white arrows), with few TH+ cells demonstrated Fos immunoreactivity in response to nicotine (identified by yellow arrows).

Supplementary Figure 2 GLP-1 neurons in NTS express α5 nAChR subunits.

40x confocal micrograph of caudal NTS depicting GLP-1-producing neurons (red) and α5* nAChRs (green) from Chrna5-EGFP mice, contrasted by nuclear counterstain (DAPI, blue) (identified by yellow arrows). Yellow insert displays a magnified presentation of the selected cell from the left panel. Note that GFP signal in the caudal NTS is not exclusive to GLP-1 neurons and is detected in neurons that are immunonegative GLP-1 (identified by white arrows). Scale bar: 10μm.

Supplementary Figure 3 Ex-4 dose not alter food responding.

(a) Graphical representation of food responding procedure, showing mouse in operant chamber permitted to respond for food pellets (20 mg). Animals respond on an active lever for food rewards according to a FR5TO20 reinforcement schedule. Each reward earned is delivered into a central food dispenser and triggers the activation of a cue light located above the active lever for 20 sec. Responding on the inactive lever has no scheduled consequence. (b) Mean (± s.e.m.) number of food rewards earned by mice after Ex-4 (10 μg/kg) administration.

Supplementary Figure 4 Nicotine suppresses food responding similarly in wild-type and GLP-1 receptor knockout mice

% Baseline number of food rewards earned by WT (n=4) and GLP-1R KO mice (n=9) after systemic delivery of saline or nicotine. Two-way RM ANOVA, Genotype: F(1, 11)= 0.29, p<0.60; Nicotine: F(1, 11)=19.9, ***p<0.001; Genotype x Nicotine: F(1, 11)=1.4, NS.

Supplementary Figure 5 Cre expression colocalizes almost exclusively with GLP-1 in NTS of Phox2b-Cre mice.

tdTom-expressing neurons in the NTS of the Phox2b::ROSA-tdTom mice were almost exclusively detected in GLP-1+ neurons, but was rarely detected also in TH+ neurons. White arrow highlights a putative TH+, tdTom+ neuron.

Supplementary Figure 6 Chemogenetic activation of GLP-1 neurons does not alter food responding in mice.

Mean (± s.e.m.) number of food rewards earned by Phox2b-Cre mice that had received intra-NTS injection of AAV-FLEX-hM3Dq-mCitrine (n=7) (a), AAV-hM3Dq-mCitrine (n=6) (b) or AAV-EGFP (n=6) (c), or after vehicle (saline) or CNO injection. *P<0.05, paired t-test.

Supplementary Figure 7 Chemogenetic activation of GLP-1 neurons increases activity of IPN neurons.

Representative micrographs showing induction of Fos in IPN of Phox2b-Cre mice that had received intra-NTS injection of AAV-EGFP or AAV-FLEX-hM3Dq-mCitrine following CNO injection. Lower panel shows graphical representation of IPN (gray area) where representative micrographs from upper panels were taken. Scale bar: 100μm.

Supplementary Figure 8 Knockdown of GLP-1 receptors in habenular neurons.

Mean (± s.e.m.) Glp1r expression in MHb relative to GAPDH levels after infusion of AAV-sh-Glp1r-GFP or AAV-GFP viruses into MHb. ***P<0.001; t-test.

Supplementary Figure 9 Knockdown of GLP-1 receptors in habenular neurons does not alter food responding in rats.

Mean (± s.e.m.) number of food rewards earned under an FR5TO20 sec schedule of reinforcement by AAV-GFP and AAV-sh-Glp1r-GFP rats before they had access to nicotine infusions. P>0.1, unpaired t-test; n=6-7 per group.

Supplementary Figure 10 GLP-1 signaling in IPN does not alter food responding.

Mean (± s.e.m.) number of food rewards earned by rats after IPN infusion of vehicle (saline, n=8), Ex-4 (0.1 μg/0.5 μl, n=5) or Ex-9 (20μg/0.5 μl, n=6). Two-way RM ANOVA, treatment: F(2, 48)= 0.05942, p<0.5637; time: F(3, 48)=1.612, p<0.1988; interaction: F(6, 48)=1.731, p<0.1342.

Supplementary Figure 11 Activation of cGMP signaling in IPN does not alter nicotine intake.

Mean (± s.e.m.) number of nicotine infusions earned by rats after IPN infusion of vehicle (saline) or 8-Br-cGMP; n=5. One-way RM ANOVA: F(2, 14)=1.036, p=0.3769.

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Tuesta, L., Chen, Z., Duncan, A. et al. GLP-1 acts on habenular avoidance circuits to control nicotine intake. Nat Neurosci 20, 708–716 (2017). https://doi.org/10.1038/nn.4540

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