Article Figures & Data
Figures
- Figure 1.
Tissue preparation and analysis. A, The cryosectioned coronal tissue was collected on electrostatic microscope slides (Fisherbrand Superfrost Plus Microscope Slides, Thermo Fisher Scientific) and stored at −80°C. B, A hydrophobic barrier (blue oval) was drawn on the microscope slides around the coronal section on the day of assaying. This allowed the reagents to be applied directly to the microscope slide for better control and waste management. After the assay was completed, the tissue was imaged and processed. C–E, Illustrative images are from Imaris. DAPI stain (blue), oprm1 (cyan), oxtr (yellow), and oprk1 (pink). DAPI-stained cells without (C) and with (D) all puncta present. Imaris was used to determine nonexclusionary cell populations, such as “DAPI × oprm1 × oprk1 spots” (E) from which exclusionary cell populations (e.g., “oprm1 + oprk1-only cells” and “oprm1 + oxtr + oprk1-only cells”) were derived.
- Figure 2.
mRNA density throughout the central amygdala. The density of oprm1 puncta was higher than oxtr and oprk1 puncta. The number of puncta per cell for each mRNA was the following: central amygdala [oprm1, 9.3 ± 0.6; oxtr, 3.4 ± 0.3; oprk1, 2.6 ± 1.0 (one-way ANOVA, F(2,54) = 84.62; p < 0.0001; oprm1 vs oxtr, p < 0.0001; oprm1 vs oprk1, p < 0.0001; oxtr vs oprk1, p = 0.1415)] (A), CeC [oprm1, 8.4 ± 0.8; oxtr, 3.8 ± 0.5; oprk1, 2.0 ± 0.3 (one-way ANOVA, F(2,54) = 35.14; p < 0.0001; oprm1 vs oxtr, p < 0.0001; oprm1 vs oprk1, p < 0.0001; oxtr vs oprk1, p = 0.0233)] (B), CeL [oprm1, 7.7 ± 0.7; oxtr, 2.9 ± 0.3; oprk1, 4.3 ± 0.4 (one-way ANOVA, F(2,42) = 26.09; p < 0.0001; oprm1 vs oxtr, p < 0.0001; oprm1 vs oprk1, p < 0.0001; oxtr vs oprk1, p = 0.0500)] (C), CeM [oprm1, 10.3 ± 0.7; oxtr, 3.5 ± 0.4; oprk1, 2.2 ± 0.3 (one-way ANOVA, F(2,54) = 69.47; p < 0.0001; oprm1 vs oxtr, p < 0.0001; oprm1 vs oprk1, p < 0.0001; oxtr vs oprk1, p = 0.0782)] (D). *p ≤ 0.05, relative density of oprm1 puncta compared with other puncta; ☨p < 0.05, relative density of oprk1 puncta compared with other puncta.
- Figure 3.
A large proportion of cells in the central amygdala are positive for oprm1 and oxtr. A, The donut chart shows the proportion of central amygdala cells that expressed oprm1, oxtr, and oprk1 across five naive B6 mice. The proportion of each cell type throughout the central amygdala was the following: oprm1 + oxtr-only (26.1 ± 1.4%), oprm1 + oxtr + oprk1 (17.7 ± 0.9%), oprm1-only (28.8 ± 1.9%), oxtr-only (2.5 ± 0.9%), oprm1 + oprk1-only (10.7 ± 1.1%), oxtr + oprk1-only (0.7 ± 0.2%), oprk1-only (1.1 ± 0.2%), and no transcript (12.3 ± 1.1%). B, The bar chart shows the same cell proportions as a factor of sex (i.e., two males vs three females). Proportions of cell types not listed were the following: males (oxtr-only cells comprised 2.9 ± 1.6%, oxtr + oprk1 cells comprised 0.8 ± 0.3%, and oprk1-only cells comprised 1.0 ± 0.3% of the total central amygdala cell population) and females (oxtr-only cells comprised 2.1 ± 0.3%, oxtr + oprk1-only cells comprised 0.7 ± 0.2%, and oprk1-only cells comprised 1.2 ± 0.3% of the total central amygdala cell population). Experimental male (n = 3) and female (n = 2) groups were underpowered for sex-based statistical comparison. C, The Venn diagram shows the population of each cell type within subsets of oprm1-, oxtr-, and oprk1-expressing cells. Cell-type proportions are reported as the mean ± standard error of the mean, representing an average of cell-type proportions of each section that was analyzed. Cell-type proportions for each section were calculated by dividing the number of cells of a particular cell type (e.g., oxtr-only cells, oxtr +oprk1-only cells, and no transcript) by the total cell count for that coronal section. See Extended Data Figure 3-1 for a distribution of each cell types across the experimental animal used in this study.
- Figure 4.
The proportion of cells that contained oxtr and oprm1 changed significantly across central amygdala subdivisions. The figure shows differences in cell-type proportions across central amygdala subdivisions for the three most abundant cell types. A, oprm1 + oxtr-only cells comprised 32.3 ± 1.7% of cells in the CeC, 20.2 ± 1.2% of cells in the CeL, and 24.7 ± 2.0% of cells in the CeM (one-way ANOVA, F(2,12) = 13.09; p = 0.0010; CeC vs CeL, p = 0.0008; CeC vs CeM, p = 0.0157; CeL vs CeM, p = 0.0850). B, oprm1 + oxtr + oprk1 cells comprised 12.8 ± 1.4% of the CeC, 30.5 ± 3.9% of the CeL, and 15.9 ± 1.6% of the CeM (one-way ANOVA, F(2,12) = 13.25; p = 0.0009; CeC vs CeL, p = 0.0013; CeC vs CeM, p = 0.4134; CeL vs CeM, p = 0.0037). C, oprm1-only cells comprised 27.9 ± 2.4% of the CeC, 25.7 ± 1.9% of the CeL, and 30.1 ± 2.5% of the CeM (one-way ANOVA, F(2,12) = 0.9046; p = 0.4306). D, oxtr-only cells comprised 2.5 ± 0.6% of the CeC, 1.6 ± 0.4% of the CeL, and 3.3 ± 1.8% of the CeM (one-way ANOVA, F(2,12) = 0.4953; p = 0.6213). Cell-type proportions are reported as the mean ± standard error of the mean, representing an average of cell-type proportions for central amygdala subdivisions in each coronal section. Cell-type proportions for each central subdivision were calculated by dividing the number of cells of a particular type (e.g., oxtr + oprk1-only, no transcript, and oxtr-only) by the total cell count for the particular subdivision within each coronal section. *p < 0.05, relative to CeC; ☨p < 0.05, relative to CeL.
- Figure 5.
The distribution of oprm1 + oxtr-only cells and oprm1-only cells changed significantly across the rostrocaudal axis throughout the central amygdala. The figure shows the topographical distribution of oprm1 + oxtr-only (A), oprm1 + oxtr + oprk1 (B), oprm1-only (C), and oxtr-only (D) cells across the rostrocaudal axis in the central amygdala. oprm1 + oxtr-only cells ranged from 7.6 to 35.7%. oprm1 + oxtr + oprk1 cells ranged from 3.9 to 30.7%. oprm1-only cells ranged from 17.6 to 53.1%. oxtr-only cells ranged from 0.0 to 18.1%. A trend toward an increase from 12.6 ± 0.2% to 27.9 ± 0.9% was observed for oprm1 + oxtr-only cells (A; one-way ANOVA, F(6,12) = 2.725; p = 0.0259; −0.83 vs −0.95; p = 0.0627), and a decrease from 44.9 ± 3.7% to 27.3 ± 2.4% was observed for oprm1-only cells (C; one-way ANOVA, F(6,12) = 10.58; p = 0.0003; −0.83 vs −0.95; p = 0.0062) from −0.83 to −0.95 relative to the bregma. No significant changes across the rostrocaudal axis were observed for oprm1 + oxtr + oprk1 cells (one-way ANOVA, F(6,12) = 2.985; p = 0.0506) or oxtr-only cells (one-way ANOVA, F(6,9) = 0.6341; p = 0.7015). Cell-type proportions are reported as the mean ± standard error of the mean, representing an average of cell-type proportions across the rostrocaudal coordinate. Each coronal section aligns with a particular rostrocaudal axis coordinate. Cell-type proportions at each rostrocaudal axis coordinate were calculated by dividing the number of cells of a particular type (e.g., oxtr-only, oxtr + oprk1, and no transcript) by the total cell count at the particular rostrocaudal axis coordinate. *p < 0.05, compared with rostrocaudal distance: −0.83 relative to bregma.
- Figure 6.
The distribution of oprm1-only and oprm1 + oxtr + oprk1 cells changed significantly across the rostrocaudal axis of the CeM. A, In the CeC, we observed a trend toward a decrease from 47.7 ± 2.7% to 28.1 ± 6.5% for oprm1-only cells from −0.83 to −0.95 (one-way ANOVA, F(6,12) = 5.304; p = 0.0069; −0.83 vs −0.95; p = 0.0554). No significant changes were observed across the rostrocaudal axis for oprm1 + oxtr-only cells (one-way ANOVA, F(6,12) = 2.676; p = 0.0693), oprm1 + oxtr + oprk1 cells (one-way ANOVA, F(6,12) = 1.796; p = 0.1826), or oxtr-only cells (one-way ANOVA, F(6,12) = 0.7048; p = 0.6521). B, In the CeL, no significant changes were observed across the rostrocaudal axis for oprm1 + oxtr-only cells (one-way ANOVA, F(5,9) = 3.020; p = 0.0714), oprm1 + oxtr + oprk1 cells (one-way ANOVA, F(5,9) = 1.761; p = 0.2171), oprm1-only cells (one-way ANOVA, F(5,9) = 2.478; p = 0.1121), or oxtr-only cells (one-way ANOVA, F(5,9) = 0.5899; p = 0.7088). Note that the CeL was not present at anterior/posterior coordinate −0.71. C, In the CeM, a significant increase from 9.5 ± 3.2% to 24.1 ± 4.3% for oprm1 + oxtr + oprk1 cells from −0.95 to −1.07 (one-way ANOVA, F(6,12) = 3.403; p = 0.0337; −0.95 vs −1.07; p = 0.0490) and a significant decrease from 46.1 ± 0.7% to 28.4 ± 3.3% for oprm1-only cells from −0.83 to −0.95 (one-way ANOVA, F(6,12) = 6.026; p = 0.0042; −0.83 vs −0.95; p = 0.0395) were also observed. No significant changes were observed across the rostrocaudal axis for oprm1 + oxtr-only cells (one-way ANOVA, F(6,12) = 2.161; p = 0.5193) or oxtr-only cells (one-way ANOVA, F(6,12) = 0.5474; p = 0.7633). All coordinates cited are relative to bregma. *p < 0.05; compared with rostrocaudal distance, −0.95 relative to bregma. See Extended Data Figure 6-1 for distribution of each cell type across the rostrocaudal axis in the central amygdala and each subdivision.
- Figure 7.
A majority of oxtr-expressing cells also express oprm1. A, Cell-type distributions were different across the CeC, CeL, and CeM. Of oxtr-expressing cells throughout the central amygdala, 55.6 ± 1.5% expressed only oprm1 (oprm1 + oxtr cells), 37.8 ± 1.8% expressed both oprm1 and oprk1 (oprm1 + oxtr + oprk1 cells), 1.5 ± 0.3% expressed only oprk1 (oxtr + oprk1 cells), and 5.1 ± 1.5% expressed no other transcript (i.e., oxtr-only cells). B, oprm1 + oxtr-only cell distributions across central amygdala cell divisions were the following CeC (67.7 ± 1.7%), CeL (38.3 ± 2.9%), CeM (55.5 ± 3.6%). C, oprm1 + oxtr + oprk1 cell distributions across central amygdala cell divisions were the following: CeC (26.5 ± 1.2%), CeL (56.3 ± 3.9%), CeM (36.5 ± 4.4%). Significant differences between central amygdala subdivisions were observed for oprm1 + oxtr-only cells (one-way ANOVA, F(2,12) = 27.10; p < 0.0001; CeC vs CeL, p < 0.0001; CeC vs CeM, p = 0.0103; CeL vs CeM, p = 0.0021) and oprm1 + oxtr + oprk1 cells (one-way ANOVA, F(2,12) = 19.00; p = 0.0002; CeC vs CeL, p = 0.0002; CeC vs CeM, p = 0.0659; CeL vs CeM, p = 0.0033). *p < 0.05, relative to CeC; ☨p < 0.05, relative to CeL.
- Figure 8.
MOXI hypothesis. The present finding that most of oxtr-expressing cells colocalized oprm1, whereas a large proportion of oprm1-expressing cells did not colocalize oxtr, supports our hypothesis of how μ-opioid and oxytocin receptors may interact in the central amygdala. Given that the central amygdala is predominantly γ-aminobutyric acid (GABA)-ergic, we propose that μ-opioid receptor activation can inhibit GABA release from cells that express μ-opioid receptors (Neuron A) and cells that express both μ-opioid and oxytocin receptors (Neuron B). A, Conversely, oxytocin receptor activation can promote GABA release. B, As such, μ-opioid receptor antagonism may block the constitutive endorphin-mediated inhibition of GABAergic neurons, thereby disinhibiting μ-opioid receptor-expressing GABAergic neurons and enhancing oxytocin-induced GABAergic neuron stimulation and GABA release. Modified from Nisbett (2024).
Tables
Cell type Expression oprm1 oxtr oprk1 oprm1 + oxtr only + + − oprm1 + oxtr + oprk1 + + + oprm1 only + − − oxtr only − + − oprm1 + oprk1 only + − + oxtr + oprk1 only − + + oprk1 only − − + No transcript − − − + mRNA expression of respective receptor mRNA is positive in the specified cell type; −mRNA expression of respective receptor mRNA is negative in the specified cell type.
Cell type Central amygdala CeC CeL CeM Exclusionary cell populations oprm1 + oxtr-only 6,529 2,796 1,233 2,500 oprm1 + oxtr + oprk1 4,440 1,087 1,720 1,633 oprm1-only 7,029 2,421 1,635 2,973 oxtr-only 641 217 105 319 oprm1 + oprk1-only 2,647 846 690 1,111 oxtr + oprk1-only 197 31 81 85 oprk1-only 284 106 104 74 no transcript 3,090 1,175 565 1, 350 Nonexclusionary cell populations (subsets) All oprm1-expressing cells 20,645 7,150 5,278 8,217 All oxtr-expressing cells 11,807 4,131 3,139 4,537 All oprk1-expressing cells 7,568 2,070 2,595 2,903 Total cell count 24,857 8,679 6,133 10,045 Central amygdala CeC CeL CeM Proportion of oprm1-expressing cells that express: Proportion of oprm1-expressing cells that express oxtr (oprm1 + oxtr only, oprm1 + oxtr + oprk1) 52.8 ± 2.6% 54.8 ± 3.9% 58.1 ± 3.2% 50.0 ± 3.1% Proportion of oprm1-expressing cells that express oxtr only (oprm1 + oxtr only) 31.5 ± 1.9% 39.2 ± 2.2% 23.2 ± 1.2% 30.5± 3.2% Proportion of oprm1-expressing cells that express oprk1 (oprm1 + oprk1 only, oprm1 + oxtr + oprk1) 34.0 ± 1.3% 27.1 ± 0.6% 47.1 ± 3.2% 32.8 ± 2.5% Proportion of oprm1-expressing cells that express oprk1 only (oprm1 + oprk1 only) 12.7 ± 1.2% 11.5 ± 1.5% 12.3 ± 1.4% 13.3 ± 1.5% Proportion of oprm1-expressing cells that express oxtr and oprk1 (oprm1 + oxtr + oprk1) 21.3 ± 1.1% 15.6 ± 1.8% 34.8 ± 3.9% 19.4 ± 1.7% Proportion of oprk1-expressing cells that express: Proportion of oprk1-expressing cells that express oprm1 (oprm1 + oprk1 only, oprm1 + oxtr + oprk1) 94.0 ± 0.9% 93.9 ± 1.3% 93.7 ± 1.8% 94.1 ± 2.3% Proportion of oprk1-expressing cells that express oprm1 only (oprm1 + oprk1 only) 35.1 ± 3.1% 40.1 ± 5.5% 24.8 ± 3.1% 38.5 ± 3.9% Proportion of oprk1-expressing cells that express oxtr (oxtr + oprk1 only, oprm1 + oxtr + oprk1) 61.4 ± 3.1% 55.2 ± 5.8% 71.6 ± 3.8% 58.8 ± 3.4% Proportion of oprk1-expressing cells that express oxtr only (oxtr + oprk1 only) 2.5 ± 0.7% 1.5 ± 0.4% 2.7 ± 0.9% 3.2 ± 1.7% Proportion of oprk1-expressing cells that express oprm1 and oxtr (oprm1 + oxtr + oprk1) 58.9 ± 2.5% 53.8 ± 5.6% 68.9 ± 4.5% 55.6 ± 2.1% The data are expressed as mean ± standard error of the mean.
Figure 3-1
The distribution of various cell types across each experimental animal is similar. Download Figure 3-1, TIF file.
Figure 6-1
Distribution of each cell type across the rostrocaudal axis in the central amygdala (A) and each subdivision: CeC (B), CeL (C), and CeM (D). In the central amygdala, the mean cell distribution ranges were the following: oprm1 + oxtr-only (7.6-35.7%), oprm1 + oxtr + oprk1 (3.9-30.7%), oprm1-only (17.6-53.1%), oxtr-only (0-18.1%), oprm1 + oprk1-only (5.4-24.5%), oxtr + oprk1 (0.1-4.7%), oprk1-only (0.3-4.4%), no transcript (5.8-20.8%) (A). In the CeC, the mean cell distribution ranges were the following oprm1 + oxtr-only (7.6-40.9%), oprm1 + oxtr + oprk1 (5.2-17.9%), oprm1-only (21.6-50.9%), oxtr-only (0-4.0%), oprm1 + oprk1-only (6.0-15.1%), oxtr + oprk1 (0.0-1.1%), oprk1-only (0.3-2.6%), no transcript (7.8-20.6%) (B). In the CeL, the mean cell distribution ranges were the following: oprm1 + oxtr-only (6.1-37.0%), oprm1 + oxtr + oprk1 (12.4-55.7%), oprm1-only (14.8-42.4%), oxtr-only (0.3-4.0%), oprm1 + oprk1-only (4.7-27.8%), oxtr + oprk1 (0.0-5.8%), oprk1-only (0.0-9.4%), no transcript (2.9-17.1%). Note that the CeL was not present at anterior/posterior coordinate -0.71 relative to bregma (C). In the CeM, the mean cell distribution ranges were the follow: oprm1 + oxtr-only (12.0-47.4%), oprm1 + oxtr + oprk1 (2.8-20.2%), oprm1-only (24.1-55.2%), oxtr-only (0.0-1.7%), oprm1 + oprk1-only (0.0-15.4%), oxtr + oprk1 (0.0-0.6%), oprk1-only (0.3-1.0%), no transcript (9.7-19.8% (D). Download Figure 6-1, TIF file.
In this issue
