DOI: 10.5061/dryad.djh9w0w8v
title: An endogenous GLP-1 circuit engages VTA GABA neurons to regulate mesolimbic dopamine neurons and attenuate cocaine seeking

description: Recent studies show that systemic administration of a glucagon-like
 peptide-1 receptor (GLP-1R) agonist is sufficient to attenuate the
 reinstatement of cocaine-seeking behavior, an animal model of relapse.
 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 (NTS) that project to the ventral tegmental
 area (VTA) decreased cocaine reinstatement. Single nuclei transcriptomics
 and FISH studies revealed 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 novel functional role of GABAergic
 GLP-1R-expressing midbrain neurons in drug seeking.
description: ## An endogenous GLP-1 circuit engages VTA GABA neurons to regulate
 mesolimbic dopamine neurons and attenuate cocaine seeking We have uploaded
 all the data associated with this manuscript on Dryad
 (**DOI:10.5061/dryad.djh9w0w8v**). Single cell RNA-sequencing data sets
 can be found at GSE284465. ## Description of the data and file structure
 All datasets from this manuscript are compiled in the file (**Auxiliary
 Supplementary Data File.CSV**). Each sheet in the file includes the data
 from each of figure panels. Below details a summary of the data and
 variables present on each sheet of the .CSV file. Figure 1B: GLP-1 blood
 plasma levels on day 21 of cocaine self-admninstration * *Animal ID*: Rat
 subject ID name * *cocaine*: animals that self-admnistered cocaine (even
 animal IDs) * *yoked-saline*: yoked-saline control animals (odd animal
 IDs); yoked-saline animals are paired to the even number cocaine animal
 following (i.e. cocaine 2's yoked-saline control is 1) * *Blood
 plasma*: Value of GLP-1 blood plasma expression in (pg/ml) Figure 1C:
 GLP-1 blood plasma levels on day 1 of extinction Same variables as 1B
 Figure 1D: GLP-1 blood plasma levels on day 7 of extinction Same variables
 as 1B Figure 2D * *virus*: virus injected into the NTS (either hM3D(Gq) or
 mCherry control) * *% cFOS + Cherry/total mCherry*: (# of cells
 co-expressing cfos and mCherry) / (total number of mCherry expressing
 cells)*100% Figure 2G: Lever responses during reinstatement for hM3D(Gq)
 injected rats self-admnistering cocaine * *Animal ID*: Rat subject ID name
 * *Treatment*: Dose of CNO administered prior to cocaine prime (e.g. 0
 mg/kg; 0.1 mg/kg; or 1.0 mg/kg) * *Lever*: # of active lever or inactive
 lever responses during reinstatement test session Figure 2H: Cummulative
 chow intake (in grams) following reinstatement tests for hM3D(Gq) injected
 rats * *Animal ID*: Rat subject ID name * *Treatment*: Dose of CNO
 administered prior to cocaine prime (e.g. 0 mg/kg or 1.0 mg/kg) * *Time*:
 Time in hours following end of reinstatement test session Figure 2I: 24
 hour bodyweight change (in grams) following reinstatement tests for
 hM3D(Gq) injected rats Bodyweight change is reported in grams per 24 hours
 * *Animal ID*: Rat subject ID name * *Treatment*: Dose of CNO administered
 prior to cocaine prime (e.g. 0 mg/kg or 1.0 mg/kg) Figure 2J: Lever
 responses during reinstatement for mCherry injected rats self-admnistering
 cocaine * *Animal ID*: Rat subject ID name * *Treatment*: Dose of CNO
 administered prior to cocaine prime (e.g. 0 mg/kg or 1.0 mg/kg) * *Lever*:
 # of active lever or inactive lever responses during reinstatement test
 session Figure 2K: Cummulative chow intake (in grams) following
 reinstatement tests for mCherry injected rats * *Animal ID*: Rat subject
 ID name * *Treatment*: Dose of CNO administered prior to cocaine prime
 (e.g. 0 mg/kg or 1.0 mg/kg) * *Time*: Time in hours following end of
 reinstatement test session Figure 2L: 24 hour bodyweight change (in grams)
 following reinstatement tests for mCherry injected rats Bodyweight change
 is reported in grams per 24 hours * *Animal ID*: Rat subject ID name *
 *Treatment*: Dose of CNO administered prior to cocaine prime (e.g. 0 mg/kg
 or 1.0 mg/kg) Figure 3E: Active lever responses for new cohort of cocaine
 self-administration hM3D(Gq) injected rats during reinstatement Gq_1b to
 Gq_8b refers to the animal ID * *VTA treatment*: Dose of Ex-9 administered
 prior to CNO treatment (e.g. 0 ug/100nl or 10 ug/100nl) * *IP Treatment*:
 Dose of CNO administered prior to cocaine prime (e.g. 0 mg/kg or 1.0
 mg/kg) Figure 3F: Inactive lever responses for new cohort of cocaine
 self-administration hM3D(Gq) injected rats during reinstatement Gq_1b to
 Gq_8b refers to the animal ID * *VTA treatment*: Dose of Ex-9 administered
 prior to CNO treatment (e.g. 0 ug/100nl or 10 ug/100nl) * *IP Treatment*:
 Dose of CNO administered prior to cocaine prime (e.g. 0 mg/kg or 1.0
 mg/kg) Figure 4B,4C, and 4D: Cells expressing glp1r RNA transcripts in the
 VTA * *Animal ID*: Rat subject ID name * *AP (mm)*: Anterior/posterior
 position of slice based off rat brain atlas according to Paxinos and
 Western (mm) * *Brain section*: Location of brain section on slide *
 *GLP-1R*: # of dapi+ cells expressing glp1r * *GLP-1R+/GAD+*: # of dapi+
 cells expressing glp1r and gad * *GLP-1R+/TH+*: # of dapi+ cells
 expressing glp1r and th Figure 5C: Lever responses during reinstatement
 for GCaMP8f injected Gad-cre rats self-administering cocaine VTA1G-VTA5G
 refers to animal ID ("VTA1_GAD") * *Treatment*: Dose of Ex-4
 administered prior to cocaine prime (0 ug/kg Ex-4 or 2.0 ug/kg) * *Active
 Lever Presses*: # of active lever respones during reinstatement test
 session * *Inactive Lever Presses*: # of inactive lever responses during
 reinstatement test session Figure 5D: GCaMP trace during active lever
 press in Gad_cre rats Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) and
 Exendin-4 (2.0 ug/kg) + Cocaine (10mg/kg) refer to reinstatement treatment
 condition under which the photometry data was recorded * *timestamp*: time
 relative to active lever press (0 seconds) for each z-scored GCaMP vlaue *
 *VTA#G_al#*: Animal ID # and active lever press number (e.g. VTA1G_al1
 -&gt; Animal VTA1G active lever press 1) Figure 5E: Net area under Gad_cre
 GCaMP8f traces associated with active lever press (analysis of traces in
 5D) Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) and Exendin-4 (2.0 ug/kg) +
 Cocaine (10mg/kg) refer to reinstatement treatment condition under which
 the photometry data was recorded * *VTA#G_al#*: Animal ID # and active
 lever press number (e.g. VTA1G_al1 -&gt; Animal VTA1G active lever press
 1) * *time bucket*: data points between which area under the curve is
 being calculated. "Pre (-5 to 0s)" refers to 5 seconds before
 active lever press and "Post (0 to 5s)" refers to 5 seconds
 after active lever press Figure 5F: Maximum Gad_cre GCaMP8f z-score
 associated with active lever press (analysis of traces in 5D) Exendin-4
 (0.0 ug/kg) + Cocaine (10mg/kg) and Exendin-4 (2.0 ug/kg) + Cocaine
 (10mg/kg) refer to reinstatement treatment condition under which the
 photometry data was recorded * *VTA#G_al#*: Animal ID # and active lever
 press number (e.g. VTA1G_al1 -&gt; Animal VTA1G active lever press 1) *
 *time bucket*: data points between which maximum z-score is being
 calculated. "Pre (-5 to 0s)" refers to 5 seconds before active
 lever press and "Post (0 to 5s)" refers to 5 seconds after
 active lever press Figure 6C: Lever responses during reinstatement for
 GCaMP8f injected Th-cre rats VTA1T-VTA5T refers to animal ID
 ("VTA1_TH") Same variables as Figure 5C Figure 6D: GCaMP trace
 during active lever press in Th_cre rats Same variables as * *VTA#T_al#*:
 Animal ID # and active lever press number (e.g. VTA1T_al1 -&gt; Animal
 VTA1T active lever press 1) Figure 6E: Net area under Th_cre GCaMP8f
 traces associated with active lever press (analysis of traces in 6D) Same
 variables as 5E * *VTA#T_al#*: Animal ID # and active lever press number
 (e.g. VTA1T_al1 -&gt; Animal VTA1T active lever press 1) Figure 6F:
 Maximum Th_cre GCaMP8f z-score associated with active lever press
 (analysis of traces in 6D) Same variables as 5F * *VTA#T_al#*: Animal ID #
 and active lever press number (e.g. VTA1T_al1 -&gt; Animal VTA1T active
 lever press 1) Figure 7A: Z-scored traces for Th_cre and Gad_cre rats
 under Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) treatment condition *
 *time bucket (seconds)*: refers to bucketed second of time for each
 corresponding data point * *TH-Cre Rats*: refers to th_cre rats injected
 with GCaMP8f * *GAD-Cre Rats*: refers to gad_cre rats injected with
 GCaMP8f Figure 7C: Z-scored traces for Th_cre and Gad_cre rats under
 Exendin-4 (2.0 ug/kg) + Cocaine (10mg/kg) treatment condition same
 variables as 7A Figure S1A: Active lever responses for cocaine and
 yoked-saline animals corresponding to cocaine self-administration day 21
 (figure 1b) * *Animal ID*: Rat subject ID name * *cocaine*: animals that
 self-administered cocaine (even animal IDs) * *yoked-saline*: yoked-saline
 control animals (odd animal IDs); yoked-saline animals are paired to the
 even number cocaine animal following (i.e. cocaine 2's yoked-saline
 control is 1) * *active lever responses*: # of active lever responses
 Figure S1B: Active lever responses for cocaine and yoked-saline animals
 corresponding to extinction day 1 (figure 1c) same variables as S1A Figure
 S1C: Active lever responses for cocaine and yoked-saline animals
 corresponding to extinction day 7 (figure 1D) same variables as S1A Figure
 S1D: Intravenous infusions for cocaine and yoked-saline animals
 corresponding to cocaine self-administration day 21 (figure 1b) * *Animal
 ID*: Rat subject ID name * *cocaine*: animals that self-administered
 cocaine (even animal IDs) * *yoked-saline*: yoked-saline control animals
 (odd animal IDs); yoked-saline animals are paired to the even number
 cocaine animal following (i.e. cocaine 2's yoked-saline control is 1)
 * *infusions*: total # of IV infusions Figure S1E: Intravenous infusions
 for cocaine and yoked-saline animals corresponding to cocaine day 1
 (figure 1c) same variables as S1D Figure S1F: Intravenous infusions for
 cocaine and yoked-saline animals corresponding to cocaine day 7 (figure
 1c) same variables as S1D Figure S3A: GCaMP trace during inactive lever
 press in Gad_cre rats Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) and
 Exendin-4 (2.0 ug/kg) + Cocaine (10mg/kg) refer to reinstatement treatment
 condition under which the photometry data was recorded * *timestamp*: time
 relative to inactive lever press (0 seconds) for each z-scored GCaMP value
 * *VTA#G_ial#*: Animal ID # and inactive lever press number (e.g.
 VTA1G_ial1 -&gt; Animal VTA1G inactive lever press 1) Figure S3B: Net area
 under Gad_cre GCaMP8f traces associated with inactive lever press
 (Analysis of traces in S3A) Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) and
 Exendin-4 (2.0 ug/kg) + Cocaine (10mg/kg) refer to reinstatement treatment
 condition under which the photometry data was recorded * *VTA#G_al#*:
 Animal ID # and inactive lever press number (e.g. VTA1G_al1 -&gt; Animal
 VTA1G inactive lever press 1) * *time bucket*: data points between which
 area under the curve is being calculated. "Pre (-5 to 0s)"
 refers to 5 seconds before inactive lever press and "Post (0 to
 5s)" refers to 5 seconds after active lever press Figure S3C: Maximum
 Gad_cre GCaMP8f z-score associated with inactive lever press (analysis of
 traces in S3A) Exendin-4 (0.0 ug/kg) + Cocaine (10mg/kg) and Exendin-4
 (2.0 ug/kg) + Cocaine (10mg/kg) refer to reinstatement treatment condition
 under which the photometry data was recorded * *VTA#G_al#*: Animal ID #
 and inactive lever press number (e.g. VTA1G_al1 -&gt; Animal VTA1G
 inactive lever press 1) * *time bucket*: data points between which area
 under the curve is being calculated. "Pre (-5 to 0s)" refers to
 5 seconds before inactive lever press and "Post (0 to 5s)"
 refers to 5 seconds after active lever press Figure S4A: Lever responses
 during reinstatement for GCaMP8f injected Gad-cre rats self-administering
 saline VTA1GS-VTA3GS refers to animal ID ("VTA1_GADSaline") Same
 as variables in 5C Figure S4B: GCaMP trace during active lever press in
 Gad_cre saline rats Same as variables in 5D Figure S4C: Net area under
 GAD_cre GCaMP8f traces associated with active lever press (analysis of
 traces in S4B) Same variables as 5E Figure S4D: Maximum Gad_cre GCaMP8f
 z-score associated with active lever press (analysis of traces in S4B)
 Same variables as 5F Figure S5A: Lever responses during reinstatement for
 Gad-cre rats infused with hM3D(Gq) into the VTA * *Treatment*: Dose of CNO
 administered prior to cocaine prime (0 mg/kg CNO, 0.1 mg/kg CNO, or 1.0
 mg/kg CNO) * *Active Lever Responses*: # of active lever respones during
 reinstatement test session * *Inactive Lever Responses*: # of inactive
 lever responses during reinstatement test session Figure S6A: GCaMP trace
 during inactive lever press in Th_cre rats same variables as S3A *
 *VTA#T_ial#*: Animal ID # and inactive lever press number (e.g. VTA1G_ial1
 -&gt; Animal VTA1T inactive lever press 1) Figure S6B: Net area under
 Th_cre GCaMP8f traces associated with inactive lever press (Analysis of
 traces in S6A) same variables as S3B * *VTA#T_ial#*: Animal ID # and
 inactive lever press number (e.g. VTA1G_ial1 -&gt; Animal VTA1T inactive
 lever press 1) Figure S6C: Maximum Th_cre GCaMP8f z-score associated with
 inactive lever press (analysis of traces in S6A) same variables as S3C *
 *VTA#T_ial#*: Animal ID # and inactive lever press number (e.g. VTA1G_ial1
 -&gt; Animal VTA1T inactive lever press 1) Figure S7A: Lever responses
 during reinstatement for GCaMP8f injected Th-cre rats self-administering
 saline VTA1TS-VTA3TS refers to animal ID ("VTA1_ThSaline") Same
 as variables in 5C & S4A Figure S7B: GCaMP trace during active lever
 press in Th_cre saline rats Same as variables in 5D & S4B Figure S7C:
 Net area under Th_cre GCaMP8f traces associated with active lever press
 (analysis of traces in S7B) Same variables as 5E & S4C Figure S7D:
 Maximum Gad_cre GCaMP8f z-score associated with active lever press
 (analysis of traces in S7B) Same variables as 5F & S4D Figure 8A: Th
 expression throughout VTA * *Animal ID*: Rat subject ID name * *AP (mm)*:
 Anterior/posterior position of slice based off rat brain atlas according
 to Paxinos and Western (mm) * *# of th+ cells*: # of th+ dapi+ cells
 Figure 8b: GAD expression throughout VTA * *Animal ID*: Rat subject ID
 name * *AP (mm)*: Anterior/posterior position of slice based off rat brain
 atlas according to Paxinos and Western (mm) * *# of Gad1+ cells*: # of
 gad1+ dapi+ cells ## Code/Software Custom R Code used to analyze the
 fiberphotometry data in this study is accessible at:
 [https://zenodo.org/records/13984378](https://zenodo.org/records/13984378)