Mapping Astrocytic and Neuronal μ-opioid Receptor Expression in Various Brain Regions Using MOR-mCherry Reporter Mouse

Abstract

The μ-opioid receptor (MOR) is a class of opioid receptors characterized by a high affinity for β-endorphin and morphine. MOR is a G protein-coupled receptor (GPCR) that plays a role in reward and analgesic effects. While expression of MOR has been well established in neurons and microglia, astrocytic MOR expression has been less clear. Recently, we have reported that MOR is expressed in hippocampal astrocytes, and its activation has a critical role in the establishment of conditioned place preference. Despite this critical role, the expression and function of astrocytic MOR from other brain regions are still unknown. Here, we report that MOR is significantly expressed in astrocytes and GABAergic neurons from various brain regions including the hippocampus, nucleus accumbens, periaqueductal gray, amygdala, and arcuate nucleus. Using the MOR-mCherry reporter mice and Imaris analysis, we demonstrate that astrocytic MOR expression exceeded 60% in all tested regions. Also, we observed similar MOR expression of GABAergic neurons as shown in the previous distribution studies and it is noteworthy that MOR expression is particularly in parvalbumin (PV)-positive neurons. Furthermore, consistent with the normal MOR function observed in the MOR-mCherry mouse, our study also demonstrates intact MOR functionality in astrocytes through iGluSnFr-mediated glutamate imaging. Finally, we show the sex-difference in the expression pattern of MOR in PV-positive neurons, but not in the GABAergic neurons and astrocytes. Taken together, our findings highlight a substantial astrocytic MOR presence across various brain regions.

Keywords: Astrocytes; Mapping; Mu-opioid receptor; Sex-difference.

Fig. 1

Expression of functional MOR in the hippocampus of MOR-mCherry mice. (A) Schematic images of the construction of the MOR-mCherry reporter gene. (B) Expression pattern of MOR-associated mCherry in the various brain regions. (C) Representative SIM images of MOR-mCherry expressed in astrocytes, GABAergic neurons, and PV neurons of the hippocampus. (D) Representative images of iGluSnFr (glutamate sensor) and MOR-mCherry expression in the hippocampus of MOR-mCherry mice. (E) Summary traces of glutamate signal from iGluSnFr expressing astrocytes with an application of DAMGO (1 μM), TTX (0.5 μM), and glutamic acid (1 mM). Data are presented as the mean±s.e.m. ***p<0.001.

Fig. 2

MOR expression in the hippocampus. (A) Expression of MOR-mCherry, S100β, and GABA in the hippocampus of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate GABAergic neurons). 3D rendering image was constructed with Imaris software. (B~D) Bar graph of S100β and GABA (B), number of cells and percentage of MOR positive cells (C), and summary table (D) in the hippocampus. (E) Bar graph of the percentage of PV positive cells in GABAergic neurons. (F) Expression of MOR-mCherry, S100β, and PV in the hippocampus of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate PV neurons). 3D rendering image was constructed with Imaris software. (G~I) Bar graph of MOR population in S100β and PV (G), number of cells and percentage of MOR positive cells (H), and summary table (I) in the hippocampus. Data are presented as the mean±s.e.m. ***p<0.001, ****p<0.0001.

Fig. 3

MOR expression in the nucleus accumbens. (A) Expression of MOR-mCherry, S100β, and GABA in the nucleus accumbens of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate GABAergic neurons). 3D rendering image was constructed with Imaris software. (B~D) Bar graph of S100β and GABA (B), number of cells and percentage of MOR positive cells (C), and summary table (D) in the nucleus accumbens. (E) Bar graph of the percentage of PV positive cells in GABAergic neurons. (F) Expression of MOR-mCherry, S100β, and PV in the nucleus accumbens of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate PV neurons). 3D rendering image was constructed with Imaris software. (G~I) Bar graph of S100β and PV (G), number of cells and percentage of MOR positive cells (H), and summary table (I) in the nucleus accumbens. Data are presented as the mean±s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Fig. 4

MOR expression in the periaqueductal gray. (A) Expression of MOR-mCherry, S100β, and GABA in the periaqueductal gray of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate GABAergic neurons). 3D rendering image was constructed with Imaris software. (B~D) Bar graph of S100β and GABA (B), number of cells and percentage of MOR positive cells (C), and summary table (D) in the periaqueductal gray. (E) Bar graph of the percentage of PV positive cells in GABAergic neurons. (F) Expression of MOR-mCherry, S100β, and PV in the periaqueductal gray of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate PV neurons). 3D rendering image was constructed with Imaris software. (G~I) Bar graph of S100β and PV (G), number of cells and percentage of MOR positive cells (H), and summary table (I) in the periaqueductal gray. Data are presented as the mean±s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Fig. 5

MOR expression in the amygdala. (A) Expression of MOR-mCherry, S100β, and GABA in the amygdala of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate GABAergic neurons). 3D rendering image was constructed with Imaris software. (B~D) Bar graph of S100β and GABA (B), number of cells and percentage of MOR positive cells (C), and summary table (D) in the amygdala. (E) Bar graph of the percentage of PV-positive cells in GABAergic neurons. (F) Expression of MOR-mCherry, S100β, and PV in the amygdala of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate PV neurons). 3D rendering image was constructed with Imaris software. (G~I) Bar graph of S100β and PV (G), number of cells and percentage of MOR positive cells (H), and summary table (I) in the amygdala. Data are presented as the mean±s.e.m. **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 6

MOR expression in the arcuate nucleus. (A) Expression of MOR-mCherry, S100β, and GABA in the arcuate nucleus of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate GABAergic neurons). 3D rendering image was constructed with Imaris software. (B-D) Bar graph of S100β and GABA (B), number of cells and percentage of MOR positive cells (C), and summary table (D) in the arcuate nucleus. (E) Bar graph of the percentage of PV positive cells in GABAergic neurons. (F) Expression of MOR-mCherry, S100β, and PV in the arcuate nucleus of MOR-mCherry mice (yellow arrowheads indicate astrocytes; white arrowheads indicate PV neurons). 3D rendering image was constructed with Imaris software. (G-I) Bar graph of S100β and PV (G), number of cells and percentage of MOR positive cells (H), and summary table (I) in the arcuate nucleus. Data are presented as the mean±s.e.m. *p<0.05, ***p<0.001, ****p<0.0001.

Fig. 7

Sex-dependent expression of MOR-mCherry. (A) Representative images of sex-dependent MOR expression in the amygdala. (B-F) The number of MOR-positive astrocytes, GABAergic neurons, and PV-positive neurons in the hippocampus (B), nucleus accumbens (C), periaqueductal gray (D), amygdala (E), and arcuate nucleus (F) from male (♂) and female (♀) MOR-mCherry mice.

Fig. 8

Summary of MOR expression in the various brain regions of astrocytes, GABAergic neurons, and PV neurons (red, purple, and gray circles indicate the astrocytes, GABAergic neurons, and PV neurons, respectively; hippocampus, HPC; nucleus accumbens, NAc; periaqueductal gray, PAG; amygdala, AMG; arcuate nucleus, ARC).