An endogenous GLP-1 circuit engages VTA GABA neurons to regulate mesolimbic dopamine neurons and attenuate cocaine seeking

Abstract

Recent studies show that systemic administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist is sufficient to attenuate cocaine seeking. However, the neural mechanisms mediating these effects and the role of endogenous central GLP-1 signaling in cocaine seeking remain unknown. Here, we show that voluntary cocaine taking decreased plasma GLP-1 levels in rats and that chemogenetic activation of GLP-1-producing neurons in the nucleus tractus solitarius that project to the ventral tegmental area (VTA) decreased cocaine seeking. Single-nuclei transcriptomics and FISH studies revealed that GLP-1Rs are expressed primarily on GABA neurons in the VTA. Using in vivo fiber photometry, we found that the efficacy of a systemic GLP-1R agonist to attenuate cocaine seeking was associated with increased activity of VTA GABA neurons and decreased activity of VTA dopamine neurons. Together, these findings suggest that targeting central GLP-1 circuits may be an effective strategy toward reducing cocaine relapse and highlight a functional role of GABAergic GLP-1R-expressing midbrain neurons in drug seeking.

Fig. 1.. Cocaine self-administration decreases circulating GLP-1 levels.

(A) Schematic illustrating the experimental timeline and blood collection time points [yoked saline: n = 15 (6 female and 9 male rats); cocaine self-administration: n = 15 (6 female and 9 male rats)]. (B and C) Plasma GLP-1 concentrations were significantly decreased in cocaine-experienced rats following 21 days of cocaine self-administration (unpaired t test: t₂₈ = 3.604, **P = 0.0012) and 1 day of extinction (unpaired t test: t₂₈ = 2.474, *P = 0.0197). (D) There were no significant differences in plasma GLP-1 levels between cocaine-experienced rats and yoked saline controls following 7 days of extinction (unpaired t test: t₂₈ = 0.4042, P = 0.6891). Data are mean ± SEM. SA, self-administration.

Fig. 2.. Activating NTS➔VTA projections attenuates cocaine seeking.

(A and B) Illustration of viral approach and experimental timeline. (C) Representative images showing increased c-Fos expression in hM3D(Gq)-expressing NTS neurons of rats treated with CNO versus mCherry controls. (D) CNO significantly increased c-Fos expression in ~55% of hM3D(Gq)-expressing NTS neurons versus <5% of mCherry-expressing NTS neurons (n = 3 per treatment; unpaired t test: t₄ = 4.911, **P = 0.0080). (E and F) Representative whole-cell current-clamp traces showing increased firing of hM3D(Gq)-expressing NTS neurons and no change in firing of mCherry-expressing NTS neurons following CNO administration. (G) CNO dose-dependently decreased active lever presses in rats expressing hM3D(Gq) in NTS neurons that project to the VTA [n = 9 (three female and six male rats); two-way RM analysis of variance (ANOVA), treatment × lever interaction: F_2,16 = 5.362, P = 0.0165; Bonferroni’s test: vehicle versus CNO (0.1 mg/kg), *P = 0.025; vehicle versus CNO (1.0 mg/kg), ***P = 0.0009]. (H and I) CNO did not reduce cumulative chow intake (two-way RM ANOVA, treatment: time × treatment: F_3,21 = 0.6343, P = 0.6012) or 24-hour body weight gain (paired t test: t₇ = 1.838, P = 0.1087) in rats expressing hM3D(Gq) in NTS neurons that project to the VTA [n = 8 (three female and five male rats)]. (J to L) CNO had no effects on cocaine seeking (two-way RM ANOVA, treatment × lever: F_1,4 = 0.6170, P = 0.4761), cumulative chow intake (two-way RM ANOVA, treatment × lever: F_3,12 = 0.421, P = 0.7419), or 24-hour body weight gain (paired t test: t₄ = 2.400, P = 0.0743) in mCherry-expressing control rats [n = 5 (three female and two male rats)]. Data are mean ± SEM.

Fig. 3.. Pharmacological inhibition of VTA GLP-1Rs blocks the suppressive effects of activating NTS➔VTA projections on cocaine seeking.

(A and B) Illustration of viral approach and experimental timeline wherein CAV2-Cre and a Cre-dependent AAV-expressing hM3D(Gq) were infused into the VTA and NTS, respectively, before the cocaine self-administration phase of the experiment. (C) Representative images of hM3D(Gq)-expressing NTS neurons that coexpress GLP-1. (D) Schematic depicting reinstatement test session treatment conditions. Exendin-(9-39) was infused into the VTA before activating endogenous NTS➔VTA projections with CNO. (E) Intra-VTA exendin-(9-39) (Ex-9) prevented the ability of CNO to suppress active lever presses during reinstatement test sessions [n = 8 (three female and five male rats); two-way RM ANOVA, systemic treatment × intra-VTA treatment: F_1,7 = 8.775, P = 0.0210; Bonferroni’s test: vehicle (veh)/vehicle versus vehicle/CNO, *P = 0.0132; vehicle/CNO versus Ex-(9-39)/vehicle, *P = 0.0249; vehicle/CNO versus Ex-(9-39)/CNO, *P = 0.0441]. (F) Intra-VTA exendin-(9-39) had no effect on inactive lever responses (n = 8; two-way RM ANOVA, systemic treatment: F_1,7 = 2.333, P = 0.1705; intra-VTA treatment: F_1,7 = 0.320, P = 0.5891; systemic treatment × intra-VTA treatment: F_1,7 = 0.138, P = 0.7207). Data are mean ± SEM.

Fig. 4.. GLP-1Rs are expressed primarily on GABAergic neurons in the VTA.

(A) FISH revealed Glp1r transcripts coexpressed with Gad1 transcripts in the VTA [4′,6-diamidino-2-phenylindole (DAPI; blue), Glp1r (red), Th (green), and Gad1 (white)]. (B) About 90% of all Glp1r-expressing neurons in the VTA coexpress Gad1. No Glp1r transcripts were detected in Th-positive cells (n = 3 rats; four slices per rat). (C and D) Both the total percentage and number of Glp1r-positive cells coexpressing Gad1 are greater in posterior regions of the VTA (n = 3 rats). (E) Uniform manifold approximation and projection dimension reduction plot for snRNA-seq from the VTA of drug-naïve rats (n = 5). Nuclei are colored by major cell type. (F) Violin plot showing normalized expression of marker genes for major VTA cell types. OPCs, oligodendrocyte precursor cells. (G) Pie charts displaying the percentage of Glp1r-expressing cells (top) or neurons (bottom) that coexpress Gad1, Th, or neither marker in the snRNA-seq dataset. Data are mean ± SEM.

Fig. 5.. Systemic GLP-1R agonist pharmacotherapy increases VTA GABA neuron activity and decreases cocaine seeking.

(A) Schematic illustrating the viral approach and experimental timeline wherein a Cre-dependent AAV-expressing GCaMP8f virus was infused into the VTA of GAD-Cre rats to measure intracellular calcium dynamics in VTA GABA neurons during reinstatement tests sessions. (B) Representative image of GCaMP8f viral expression in the VTA. FISH confirmed selective GCaMP8f expression in Gad1-positive cells [DAPI (blue), eGfp (green), Th (red), and Gad1 (white)]. IPN, Interpeduncular nucleus. (C) Systemic exendin-4 administration decreased active lever responses during cocaine reinstatement test sessions [n = 5 (three female and two male rats); two-way RM ANOVA, treatment × lever: F_1,4 = 19.64, P = 0.0114; Bonferroni’s test: vehicle versus exendin-4, *P = 0.0129]. (D) Normalized z score traces during cocaine seeking in rats pretreated with vehicle or exendin-4 (n = 5 rats; three active lever presses per rat per treatment). (E) Cocaine seeking significantly increased the AUC of recorded Ca²⁺ signals from VTA GABA neurons in rats treated with exendin-4, but not vehicle (n = 15; two-way RM ANOVA, treatment × time: F_1,28 = 9.652, P = 0.0043; Bonferroni’s test: baseline versus cocaine seeking in rats treated with exendin-4, **P = 0.0099). The AUC from rats treated with exendin-4 was also significantly increased during cocaine seeking compared to vehicle-treated controls (Bonferroni’s test: 0.0 versus 2.0 μg/kg exendin-4 during cocaine seeking, ***P = 0.0001). (F) Exendin-4 significantly increased maximum z scores in VTA GABA neurons during cocaine seeking compared to vehicle-treated controls (n = 15; two-way RM ANOVA, treatment × time: F_1,28 = 4.110, P = 0.0522; Bonferroni’s test: 0.0 versus 2.0 μg/kg exendin-4 during cocaine seeking, **P = 0.0036). Data are mean ± SEM.

Fig. 6.. Systemic GLP-1R agonist pharmacotherapy attenuates VTA dopamine neuron activity and decreases cocaine seeking.

(A) Schematic illustrating viral approach and experiment timeline where a Cre-dependent AAV-expressing GCaMP8f virus was infused into the VTA of TH-Cre rats to measure intracellular calcium dynamics in VTA dopamine neurons during reinstatement tests sessions. (B) Representative image of GCaMP8f viral expression in the VTA. FISH confirmed selective GCaMP8f expression in Th-positive cells [DAPI (blue), eGfp (green), Th (red), and Gad1 (white)]. (C) Systemic exendin-4 administration decreased active lever responses during cocaine reinstatement test sessions [n = 5 (two female and three male rats); two-way RM ANOVA, treatment × lever: F_1,4 = 39.66, P = 0.0032; Bonferroni’s test: vehicle versus exendin-4: **P = 0.0023]. (D) Normalized z score traces during cocaine seeking in rats pretreated with vehicle or exendin-4 (n = 5 rats; three active lever presses per rat per treatment). (E) Cocaine seeking significantly increased the AUC of recorded Ca²⁺ signals from VTA dopamine neurons in rats treated with vehicle (two-way RM ANOVA, treatment × time: F_1,28 = 10.71, P = 0.0028; Bonferroni’s test: baseline versus cocaine seeking in vehicle-treated controls: **P = 0.0011). Exendin-4 pretreatment significantly attenuated cocaine seeking–evoked increases in VTA dopamine neuron activity (Bonferroni’s test: 0.0 versus 2.0 μg/kg exendin-4 during cocaine seeking: ***P = 0.0002). (F) Cocaine seeking significantly increased maximum z scores in VTA dopamine neurons in rats treated with vehicle (two-way RM ANOVA, treatment × time: F_1,28 = 11.37, P = 0.0022; Bonferroni’s test: baseline versus cocaine seeking in vehicle-treated controls: ***P = 0.0002). Exendin-4 pretreatment significantly decreased maximum z scores in VTA dopamine neurons during cocaine seeking compared to vehicle [Bonferroni’s test: cocaine seeking (0.0 μg/kg) versus cocaine seeking (2.0 μg/kg), ****P < 0.0001]. Data are mean ± SEM.

Fig. 7.. GLP-1R agonist pharmacotherapy attenuates cocaine seeking by activating inhibitory VTA GABA neurons and decreasing activity of VTA dopamine neurons.

(A) Normalized and down-sampled dopamine and GABA z score traces during cocaine seeking from rats pretreated with vehicle before cocaine priming–induced reinstatement test sessions. (B) Schematic illustrating cocaine seeking–evoked increases in dopamine cell firing in the VTA. (C) Normalized and down-sampled dopamine and GABA z score traces during cocaine seeking from rats pretreated with exendin-4 before cocaine priming–induced reinstatement test sessions. (D) Schematic illustrating one proposed mechanism by which exendin-4 increases VTA GABA cell firing which, in turn, inhibits VTA dopamine cell firing and suppresses cocaine-seeking behavior. Additional mechanisms likely include regulation of presynaptic GABA and/or glutamate release (see the main text for more details). DAT, dopamine transporter; DA, dopamine.