Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses

Abstract

Background: Mu-opioid receptors (MORs) are critical regulators mediating the modulation of several behavioral reactions, including analgesia, addiction, and sedation. Recent studies have reported that MORs are closely associated with mood disorders or anxiety behaviors; however, the underlying neural mechanisms remain unclear. The periaqueductal gray (PAG), a key brain area, participates in the modulation of aversive emotional behaviors. MORs show a high expression in the ventrolateral PAG (vlPAG) region. This study explored the preliminary role of MORs expressed in the vlPAG in modulating emotional behaviors.

Results: Bilateral administration of DAMGO, an MOR-specific agonist, into the vlPAG of male mice elicited anxiety-like behaviors in elevated plus maze tests. This phenotype was reversed by conditional knockdown of astrocytic MORs. In contrast, glutamatergic or GABAergic MORs were not involved in vlPAG MOR-dependent anxiety-like behaviors. By using in vitro calcium imaging of vlPAG astrocytes and chemical genetic technologies, we found that vlPAG astrocytic MORs can promote astrocytic calcium signaling, which can efficiently induce anxiety-like behaviors. Accordingly, the interference of astrocytic calcium signaling by viral infection reversed vlPAG-dependent anxiety-like behaviors.

Conclusion: Our findings demonstrated that vlPAG astrocytic, but not glutamatergic or GABAergic, MORs are involved in modulating emotional reactions, and these effects are accomplished by MOR-elicited astrocytic calcium signaling mechanisms. The present study provides a theoretical basis for treating emotional dysfunctions during MOR-targeted management.

Keywords: Anxiety; Astrocyte; Calcium signaling; Mu-opioid receptors; Periaqueductal gray.

Fig. 1

Activation of ventrolateral periaqueductal gray (vlPAG) mu-opioid receptors (MORs) elicited anxiety-like behavior. A Schematic showing the implantation and morphological confirmation of the guide cannulas in the vlPAG. Scale bar: 400 μm. B Experimental schedule. C Typical representative activity tracking in EPM tests. Summary plots of the total distance (D), percentage of open arm entries (E), and percentage of time spent in the open arms (F) during EPM tests. Saline-treated mice: n = 8; DAMGO-treated mice: n = 8; DAMGO+CTAP-treated mice: n = 8. Total distance: ordinary one-way ANOVA measures, F_(2,21) = 0.9410, p = 0.4061. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(2,21) = 12.85, p = 0.0002 (**), saline vs. DAMGO, p = 0.0005 (**); saline vs. DAMGO + CTAP, p = 0.9358; DAMGO vs. DAMGO + CTAP, p = 0.0011 (**). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(2,21) = 12.53, p = 0.0003 (**), saline vs. DAMGO, p = 0.0002 (**); saline vs. DAMGO + CTAP, p = 0.3128; DAMGO vs. DAMGO + CTAP, p = 0.0075 (**)

Fig. 2

MOR_Astro are involved in vlPAG-MOR-elicited anxiety-like behavior. A Schematic of in situ hybridization for Oprm1 mRNA and immunofluorescence for the GFAP protein in the vlPAG areas in MOR_Astro−/− and MOR_Astro+/+ mice. The nucleus is stained in blue (DAPI), GFAP is stained in green, and Oprm1 mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with Oprm1 mRNA and GFAP; the purple arrowheads represent Oprm1 mRNA localization in GFAP-negative cells; and the yellow arrowheads represent GFAP-positive cells without Oprm1 mRNA. B Quantitative analysis of the percentage of double-positive cells (Oprm1 and GFAP) against GFAP-positive cells. t₄ = 16.01, p < 0.0001, unpaired Student’s t test, n = 3 mice per group. C Schematic showing the timeline of the experimental procedures. Summary plots of the total distance (D), percentage of open arm entries (E), and percentage of time spent in the open arms (F) during EPM tests. MOR_Astro+/+ + saline-treated mice: n = 7; MOR_Astro−/− + saline-treated mice: n = 7; MOR_Astro+/+ + DAMGO-treated mice: n = 7; MOR_Astro−/− + DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, F_(3,24) = 2.177, p = 0.1169. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 6.856, p = 0.0017 (**), MOR_Astro+/+ + saline vs. MOR_Astro+/+ + DAMGO, p = 0.0027 (**); MOR_Astro−/− + saline vs. MOR_Astro−/− + DAMGO, p = 0.9988; MOR_Astro+/+ + DAMGO vs. MOR_Astro−/− + DAMGO, p = 0.0221 (*). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 10.69, p = 0.0001 (**), MOR_Astro+/+ + saline vs. MOR_Astro+/+ + DAMGO, p = 0.0028 (**); MOR_Astro−/− + saline vs. MOR_Astro−/− + DAMGO, p = 0.9999; MOR_Astro+/+ + DAMGO vs. MOR_Astro−/− + DAMGO, p = 0.0003 (**)

Fig. 3

MOR_GABA are not involved in vlPAG-MOR-elicited anxiety-like behavior. A Schematic of in situ hybridization for Oprm1 mRNA and GAT mRNA in the vlPAG areas in MOR_GABA−/− and MOR_GABA+/+ mice. The nucleus is stained in blue (DAPI), GAT is stained in green, and Oprm1 mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with Oprm1 mRNA and GAT mRNA; the purple arrowheads represent Oprm1 mRNA localization in GAT-negative cells; and the yellow arrowheads represent GAT-positive cells without Oprm1 mRNA. B Quantitative analysis of the percentage of double-positive cells (Oprm1 and GAT) against GAT-positive cells. t₄ = 37.73, p < 0.0001, unpaired Student’s t test, n = 3 mice per group. C Schematic showing the timeline of the experimental procedures. Summary plots of total distance (D), percentage of open arm entries (E), and percentage of time spent in the open arms (F) during EPM tests. MOR_GABA+/+ + saline-treated mice: n = 7; MOR_GABA−/− + saline-treated mice: n = 7; MOR_GABA+/+ + DAMGO-treated mice: n = 7; MOR_GABA−/− + DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, F_(3,24) = 1.399, p = 0.2675. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 9.688, p = 0.0002 (**), MOR_GABA+/+ + saline vs. MOR_GABA+/+ + DAMGO, p = 0.0012 (**); MOR_GABA−/− + saline vs. MOR_GABA−/− + DAMGO, p = 0.0345(*); MOR_GABA+/+ + DAMGO vs. MOR_GABA−/− + DAMGO, p = 0.9999. Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 23.57, p < 0.0001 (**), MOR_GABA+/+ + saline vs. MOR_GABA+/+ + DAMGO, p = 0.0001 (**); MOR_GABA−/− + saline vs. MOR_GABA−/− + DAMGO, p < 0.0001 (**); MOR_GABA+/+ + DAMGO vs. MOR_GABA−/− + DAMGO, p = 0.8229

Fig. 4

MOR_Glut are not involved in vlPAG-MOR-elicited anxiety-like behavior. A Schematic of in situ hybridization for Oprm1 mRNA and Glut mRNA in the vlPAG areas in MOR_Glut−/− and MOR_Glut+/+ mice. The nucleus is stained in blue (DAPI), Glut is stained in green, and Oprm1 mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with Oprm1 mRNA and Glut mRNA; the purple arrowheads represent Oprm1 mRNA localization in Glut-negative cells; and the yellow arrowheads represent Glut-positive cells without Oprm1 mRNA. B Quantitative analysis of the percentage of double-positive cells (Oprm1 and Glut) against Glut-positive cells. t₄ = 28.29, p < 0.0001, unpaired Student’s t test, n = 3 mice per group. C Schematic showing the timeline of the experimental procedures. Summary plots of the total distance (D), percentage of open arm entries (E), and percentage of time spent in the open arms (F) during EPM tests. MOR_Glut+/+ + saline-treated mice: n = 7; MOR_Glut−/− + saline-treated mice: n = 7; MOR_Glut+/+ + DAMGO-treated mice: n = 7; MOR_Glut−/− + DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, F_(3,24) = 1.985, p = 0.1432. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 12.77, p < 0.0001 (**), MOR_Glut+/+ + saline vs. MOR_Glut+/+ + DAMGO, p = 0.0001 (**); MOR_Glut−/− + saline vs. MOR_Glut−/− + DAMGO, p = 0.0355(*); MOR_Glut+/+ + DAMGO vs. MOR_Glut−/− + DAMGO, p = 0.2730. Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple-comparison test measures, F_(3,24) = 24.90, p < 0.0001 (**), MOR_Glut+/+ + saline vs. MOR_Glut+/+ + DAMGO, p = 0.0001 (**); MOR_Glut−/− + saline vs. MOR_Glut−/− + DAMGO, p < 0.0001 (**); MOR_Glut+/+ + DAMGO vs. MOR_Glut−/−  + DAMGO, p = 0.9475

Fig. 5

Activation of PAG MOR_Astro triggers astrocytic calcium signaling. A Schematic of the rAAVs monitored to specifically exhibit astrocytic calcium levels in the vlPAG. B Double-immunofluorescence staining of GCamp (green) with GFAP (red). Scale bar: 10 μm. The white arrows represent the co-labeled cells. C Statistics of the co-labeling rate of GCamp coupled with GFAP (n = 3 mice). D Typical change and representative traces of Ca²⁺signals of a single PAG astrocyte during DAMGO (1 μM) perfusion (arrow indicates DAMGO application). Summary bar graph of the change in the ΔF/F of the regions of interest during treatment with ACSF (E) (t₇ = 0.8204, p = 0.4353, paired Student’s t test, n = 8 cells from n = 2 mice), 1 μM DAMGO (F) (t₇ = 2.565, p = 0.0373 (*), paired Student’s t test, n = 8 cells from n = 2 mice), 1 μM DAMGO + 10 μM CTAP (G) (t₇ = 0.9734, p = 0.3628, paired Student’s t test, n = 8 cells from n = 2 mice), and 1 μM DAMGO + 1 μM TTX (H) (t₆ = 3.297, p = 0.0165 (*), paired Student’s t test, n = 7 cells from n = 2 mice) perfusion

Fig. 6

Direct activation of vlPAG astrocytic calcium signaling elicits anxiety-like behavior. A Schematic and fluorescent signal image of the rAAVs engineered to specifically activate vlPAG astrocytic calcium signaling. Aq: Aqueduct. Scale bar: 100 μm. B Double-immunofluorescence staining of hM3Dq (red) with GFAP (green). Scale bar: 10 μm. The white arrows represent the co-labeled cells. C Statistics of the co-labeling rate of hM3Dq coupled with GFAP (n = 3 mice). D Schematic showing the timeline of the experimental procedures. i.p. means intraperitoneal injection. Summary plots of the total distance (E), percentage of open arm entries (F), and percentage of time spent in the open arms (G) during EPM tests. Astro_control + CNO-treated mice: n = 6; Astro_hM3Dq + CNO-treated mice: n = 6; Astro_hM3Dq + saline-treated mice: n = 6. Total distance: ordinary one-way ANOVA measures, F_(2,15) = 0.4609, p = 0.6393. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(2,15) = 12.04, p = 0.0008 (**), Astro_control + CNO vs. Astro_hM3Dq + CNO, p = 0.0008 (**); Astro_hM3Dq + CNO vs. Astro_hM3Dq + saline, p = 0.0084 (**); Astro_control + CNO vs. Astro_hM3Dq + saline, p = 0.6171. Percentage of time spent in open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(2,15) = 11.91, p = 0.0013 (**), Astro_control + CNO vs. Astro_hM3Dq + CNO, p = 0.0013 (**); Astro_hM3Dq + CNO vs. Astro_hM3Dq + saline, p = 0.0040 (**); Astro_control + CNO vs. Astro_hM3Dq + saline, p = 0.9323

Fig. 7

Astrocytic calcium signaling is the cellular mechanism underlying vlPAG MOR_Astro-dependent anxiety-like behavior. A Schematic and fluorescent signal image of the rAAVs engineered to specifically extrude cytoplasmic calcium and reduce calcium oscillations in vlPAG astrocytes. Aq: Aqueduct. B Double-immunofluorescence staining of hPMCA2w/b (red) with GFAP (green). Scale bar: 10 μm. C Statistics of the co-labeling rate of hPMCA2w/b coupled with GFAP (n = 3 mice). D Schematic showing the timeline of the experimental procedures. Summary plots of the total distance (E), percentage of open arm entries (F), and percentage of time spent in the open arms (G) during EPM tests. Astro_control + saline-treated mice: n = 7; Astro_hPMCA2w/b + saline-treated mice: n = 7; Astro_control + DAMGO-treated mice: n = 7; Astro_hPMCA2w/b + DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, F_(3,24) = 1.007, p = 0.4068. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 13.21, p < 0.0001 (**), Astro_control + saline vs. Astro_control + DAMGO, p < 0.0001 (**); Astro_hPMCA2w/b + saline vs. Astro_hPMCA2w/b + DAMGO, p = 0.9999; Astro_control + DAMGO vs. Astro_hPMCA2w/b + DAMGO, p = 0.0005 (**). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, F_(3,24) = 13.35, p < 0.0001 (**), Astro_control + saline vs. Astro_control + DAMGO, p = 0.0013 (**); Astro_hPMCA2w/b + saline vs. Astro_hPMCA2w/b + DAMGO, p = 0.6004; Astro_control + DAMGO vs. Astro_hPMCA2w/b + DAMGO, p = 0.0009 (**)