Fig 1.
Heroin withdrawal induces notable withdrawal reactions in mice.
A: Experimental flowchart for the establishment of a heroin withdrawal mouse model and assessment of withdrawal symptoms. B-E: Withdrawal symptoms (n = 8). Number of standing positions (B), number of licking hair (C), number of face washing (D) and number of bowel movements (E). The results are presented as mean± standard error of the mean. No statistical significance is indicated by ns P > 0.05, and significant difference is indicated by * P < 0.05, ** P < 0.01, *** P < 0.001 indicates (Independent-sample t test).
Fig 2.
Increased anxiety-related behaviors observed in mice undergoing heroin withdrawal.
A: Flowchart illustrating the modeling of heroin withdrawal in mice. B-E: Open field test (n = 8). Representative heatmaps of trajectories in the open field test (B), time spent exploring the center area (C), distance traveled in the center area (D), and number of entries into the center area (E). F-I: Elevated plus maze test (n = 8). Representative heatmaps of trajectories in the elevated plus maze test (F), time spent in the open arms (G), distance traveled in the open arms (H), and number of entering the open arms (I). J: Immobility time in the forced swim test (n = 8), K: Immobility time in the tail suspension test (n = 8). L-O: Immunofluorescence staining in the BLA brain region. Immunostaining for c-Fos/VGLUT1/DAPI (L), average optical density of c-Fos immunofluorescence (M) (n = 6), proportion of glutamatergic neurons labeled with VGLUT1 and unidentified neurons expressing c-Fos among the total c-Fos-positive neurons labeled with VGLUT1 (N) (n = 10), and number of glutamatergic neurons (O) (n = 6). The results are presented as mean ± standard error of the mean. No statistical significance is indicated by ns p > 0.05, and significant difference is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 indicates (Independent-sample t test and Mann-Whitney U test).
Fig 3.
Heroin dependence leads to a decrease in glutamatergic neurons within the BLA of mice.
A: Flow chart illustrating the heroin-dependent mice model; B-E: Open field test (n = 8). Representative track heat map from the open field test (B), time spent exploring the central area (C), distance traveled in the central area (D), and number of entries into the central area (E); F-I: Elevated plus maze test (n = 8). Representative track heat map from the elevated plus maze test (F), time spent exploring the open arms (G), distance traveled in the open arms (H), and number of entries into the open arms (I); J: Immobility time recorded in the forced swim test (n = 8); K: Immobility time recorded in the tail suspension test (n = 8); L-O: Immunofluorescence staining of the BLA. Immunofluorescence staining for c-Fos/VGLUT1/DAPI (L), average optical density of c-Fos immunofluorescence (M) (n = 6), proportion of glutamatergic and unidentified neurons expressing c-Fos among all c-Fos-positive neurons labeled with VGLUT1 (N) (n = 10), and the number of glutamatergic neurons (O) (n = 6); The results are presented as mean± standard error of the mean. No statistical significance is indicated by ns P > 0.05, and significant difference is indicated by *P < 0.05, ** P < 0.01, ***P < 0.001 indicates (Independent-samples t test and Mann-Whitney U test).
Fig 4.
Projection of NTS PPG neurons to the glutamatergic neurons of BLA in mice.
A: Schematic diagram of viral injection using pAAV-hSyn-EGFP-2A-Cre-WPRE;B: Stereotaxic injection site in the BLA; C: Morphological verification showing targeted NTS-BLA neurons in wild-type mice injected with pAAV-hSyn-EGFP-2A-Cre-WPRE; D-E: WB analysis of GLP-1 protein in the NTS. Representative images of total GLP-1 expression in the NTS (D) and grayscale values of GLP-1 protein (E) (n = 3), indicating increased GLP-1 expression in the NTS of mice undergoing heroin withdrawal; F-G: WB analysis of GLP-1 in the BLA. Representative images of total GLP-1 expression in the BLA (F), grayscale values of GLP-1 protein (n = 5) (G), demonstrating increased GLP-1 content in the BLA of mice during heroin withdrawal; H-I: Immunofluorescence staining. The number of glutamatergic/GABAergic neurons expressing GLP-1R (J) (n = 6). The results are presented as mean± standard error of the mean. No statistical significance is indicated by ns P > 0.05, significance levels indicated as *P < 0.05, **P < 0.01, and ***P < 0.001 (Independent-samples t test).
Fig 5.
Intraperitoneal administration of Exendin-4 significantly alleviated anxiety-like behaviors in mice undergoing withdrawal from heroin.
A: Represents blood glucose levels (n = 6); B: Reflects the body weight of mice treated with 2ug/kg Exendin-4 (n = 6); C: Illustrates the process of modeling heroin-dependent withdrawal in mice; D-G: Open field test (n = 8). Representative track heat map in the open field test (D), time spent exploring in the center area (E), distance traveled in the center area (F), number of entries into the center area (G); H-K: Elevated plus maze test (n = 8). Representative track heat map in the elevated plus maze test (H), time spent exploring in the open arms (I), distance traveled in the open arms (J), number of entries into the open arms (K); The results are presented as mean ± standard error of the mean. No statistical significance is indicated by ns P > 0.05, significance levels indicated as #/*P < 0.05, ##/**P < 0.01, and ###/***P < 0.001 (Paired sample t-test, independent sample t-test and two factor analysis of variance).
Fig 6.
Intraperitoneal administration of Exendin-4 has been demonstrated to enhance the population of glutamatergic neurons in the BLA of mice undergoing heroin withdrawal.
This effect is accompanied by a reduction in c-Fos protein expression. A-F: Western blot analysis of GLP-1R, TrkB, CREB, GLP-1, and BDNF protein in the BLA. Representative immunoblot images of total expression of GLP-1R, TrkB, CREB, GLP-1, and BDNF in the BLA (A), grayscale value of GLP-1R protein (B) (n = 4), grayscale value of BDNF protein (C) (n = 6), grayscale value of TrkB protein (D) (n = 5), grayscale value of CREB protein (E) (n = 7), grayscale value of GLP-1 protein (F) (n = 4). Increased expression of GLP-1R, TrkB, CREB, GLP-1, and BDNF was observed in the BLA of the Her + Sal and Sal + Sal groups mice. The expression of GLP-1, TrkB, and BDNF in the BLA was decreased in the Her + Ex-4 group mice compared to the Her + Sal group. G-I: Immunofluorescence staining. Immunofluorescence staining of c-Fos/VGLUT1/DAPI in the BLA (G), average light density of c-Fos immunofluorescence (H), number of glutamatergic neurons (I) (n = 6). The results are presented as mean ± standard error of the mean. No statistical significance is indicated by ns P > 0.05, significance levels indicated as #/*P < 0.05, ##/**P < 0.01, and ###/***P < 0.001 (One factor analysis of variance and two factor analysis of variance).
Fig 7.
Heroin dependence has been shown to negatively impact the BLA, resulting in a reduction in the population of glutamatergic neurons.
After withdrawal from heroin dependent mice, significant withdrawal symptoms appear. During this period, the synthesis and secretion of GLP-1 increase in NTS pre glucagon neurons, targeting BLA glutamatergic neurons, promoting the synthesis of BDNF, TrkB, CREB, and c-Fos proteins, facilitating neural repair, and leading to increased anxiety. However, upregulation of GLP-1 signaling does not restore the number of glutamatergic neurons in BLA. Our study found that the administration of Exendin-4 can enhance the neural repair effect of GLP-1. Once the glutamatergic neuron population in BLA recovers, GLP-1 projection to BLA subsequently decreases, which is associated with a reduction in anxiety-like behaviors.