GFAP-GFP (Stock No. 003257), MOR knockout (KO) (Stock No. 007559) and MOR-mCherry mice (Stock No. 029013) were obtained from Jackson Laboratory. For primary culture of hippocampal astrocytes, P0-P3 C57BL/6J mice were used. For histological analysis, 7 to 9-week-old male mice were used. All animals were maintained in a vivarium with light/dark cycle (8:00 AM~8:00 PM). Animal care and handling were performed according to the directives of the Animal Care and Use Committee and institutional guidelines of KIST (Seoul, Korea). Animals were randomly used for experiments.

Animals were deeply anesthetized using 2% avertin and perfused with 0.1M PBS, followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde at 4℃ for 24 hr and 30% sucrose 4℃ for 48 hr. Brains were then cut in coronal sections of 30 µm on a cryosection. Sections were blocked in 0.1 M PBS containing 0.3% Triton X-100 (Sigma) and 2% donkey serum (Genetex) for 30 min at room temperature. Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-S100β (1:200, NB110-57478, Novus), rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology), rabbit anti-MOR (1:200, RA10104, Neuromics), goat anti-TH (1:500, ab101853, Abcam), mouse anti-NeuN (1:1,000, MAB377, Millipore), and mouse anti-vGAT (1:200, 131011, Synaptic systems). The brain samples with primary antibodies were incubated overnight at 4℃. Then, the sections were washed three times in 0.1 M PBS and incubated in proper secondary antibodies from the Jackson Laboratory for 1.5 hr. After three rinses in 0.1 M PBS and DAPI staining at 1:3000 (PIERCE), the sections were mounted on polysine microscopic slide glass (Thermo Scientific). Images were acquired using a Nikon A1R confocal microscope. We repeated each experiment at least three times.

Confocal microscopic images were analyzed using the ImageJ program (NIH). For analyzing MOR intensity in an astrocyte, the images were converted into binary and every GFAP⁺ astrocyte was picked as region of interest (ROI). Then the mean intensity value of MOR in every ROI was measured. For colocalization analysis, we first converted the MOR-mCherry⁺ and vGAT⁺ images into 8-bit and then calculated the pearson's correlation by utilizing colocalization threshold tool from ImageJ program. Using this method, there was no correlation between MOR-mCherry⁺ and NeuN⁺ at all.

The MOR nucleotides from 1197 to 1215 (5′-gac tgt ttc ctg gca ctt c-3′) was selected for the target region of shRNA. The pSicoR-MOR-shRNA was synthesized using the complementary oligomers, 5′-tga ctg ttt cct ggc act tct tca aga gag aag tgc cag gaa aca gtc ttt ttt c-3′ and 3′-tcg aga aaa aag act gtt tcc tgg cac ttc tct ctt gaa gaa gtg cca gga aac agt ca-5′. The annealed double-stranded oligomer was inserted into HpaI-XhoI restriction enzyme sites of pSicoR lentiviral vector (Addgene) and verified by sequencing. The virus titer was 1.0×10¹² GC. We used stereotaxis to deliver a certain gene into CA1 of dorsal hippocampus (coordinates: AP −1.8 mm, ML±1.5 mm, DV −1.7 mm). We injected 2 µl of virus at a rate of 0.2 µl/min and waited for 1 or 2 weeks for the knockdown to occur for lentivirus or AAV virus, respectively.

The cerebral cortex was dissected free of adherent meninges, minced and dissociated into single cell suspension by trituration through a Pasteur pipette. Dissociated cells were plated onto either 12 mm glass coverslips or six-well plates coated with 0.1 mg/ml poly d-lysine. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 25 mM glucose, 10% heat-inactivated horse serum, 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 1,000 units/ml penicillin-streptomycin. After three days later, cells were vigorously washed with repeated pipetting using medium and the media was replaced to get rid of debris and other floating cell types.

Cultured astrocytes were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, and 1% NP-40) containing a protease-inhibitor cocktail. Total-cell lysates were incubated on ice for 30 min and then cleared at 13,000 rpm for 20 min at 4℃. The protein samples were separated by SDS-PAGE using 10% gels and blotted onto PVDF membranes. The blots incubated overnight at 4℃ with anti-MOR antibody (1:1,000; sc-15310, Santa Cruz Biotechnology) or anti-β-actin antibody (1:2,000; ab133626, Abcam). Blots were then washed and incubated with horseradish peroxidase-conjugated anti-goat IgG or anti-rabbit IgG, followed by washing and detection of immunoreactivity with enhanced chemiluminescence (Amersham Biosciences).

Three GFAP-GFP transgenic mice (20~25 g) were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused transcardially with heparinized normal saline (10 ml for mouse and 100 ml for rat), followed by freshly prepared mixture (50 ml) of 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Hippocampus was removed and post-fixed in the same fixative for 2 hours at 4℃. Sections were cut sagittally on a Vibratome at 60 µm and cryoprotected in 30% sucrose in PB overnight at 4℃. Sections were frozen on dry ice for 20 minutes, thawed in phosphate-buffered saline (PBS; 0.01 M, pH 7.2) to enhance penetration. They were pretreated with 1% sodium borohydride for 30 minutes to quench glutaraldehyde and then blocked with 3% H₂O₂ for 10 minutes to suppress endogenous peroxidases and with 10% normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA) for 30 minutes to mask secondary antibody binding sites. For double immunostaining for GFP and MOR, sections of hippocampus pretreated as above were incubated overnight in a mixture of antibodies. Primary antibodies used are as follow: mouse anti-GFP (1:400, MAB3580, Millipore, Temecula, CA) and rabbit anti-MOR (1:2,500, RA10104, Neuromics, Edina, MN) [12,13]. After rinsing in PBS, sections were incubated with a mixture of biotinylated donkey anti-mouse (1:200, Jackson ImmunoResearch) and 1 nm gold-conjugated donkey anti-rabbit (1:50, EMS, Hatfield, PA) for 2~3 hours. The sections were post-fixed with 1% glutaraldehyde in PB for 10 minutes, rinsed in PB several times, incubated for 4 minutes with HQ silver enhancement solution (Nanoprobes, Yaphank, NY), and rinsed in 0.1 M sodium acetate and PB. After rinsing in PBS, sections were incubated with a biotinylated donkey anti-rabbit (1:200, Jackson ImmunoResearch) antibody for 2~3 hours. The sections were rinsed in PB several times, and were incubated with ExtrAvidin peroxidase (1:5,000, Sigma, St. Louis, MO) for 1 hour and the immunoperoxidase was visualized by nickel-intensified 3,3′-diaminobenzidine tetrahydrochloride (DAB). Sections were further rinsed in PB, osmicated (in 1% osmium tetroxide in PB) for 1 hour, dehydrated in graded alcohols, flat-embedded in Durcupan ACM (Fluka, Buchs, Switzerland) between strips of Aclar plastic film (EMS), and cured for 48 hours at 60℃. Chips containing prominent staining for MOR in the hippocampus were cut out of the wafers and glued onto blank resin blocks with cyanoacrylate. Serially cut thin sections were collected on Formvar-coated single-slot nickel grids and stained with uranyl acetate and lead citrate. Grids were examined on a Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV accelerating voltage. Images were captured with Digital Micrograph software driving a cooled CCD camera (SC1000; Gatan, Pleasanton, CA, USA) attached to the microscope, and saved as TIFF files.

To determine the astrocytic microdomain, a synapse composed of a presynaptic axon terminal and a postsynaptic dendrite was first identified as colored in the EM image. And then, fine extensions of astrocytes, 1) which are located near the synapse or encircling the synapse, 2) with clear cytoplasm, 3) highly irregular outline, 4) no mitochondria or microtubule endoplasmic reticulum, are considered as astrocytic microdomains.

To investigate the distribution of MOR in CA1 hippocampal neurons and astrocytes, we first utilized MOR-mCherry mice, in which the reporter mCherry was tagged to the C-terminus of MOR (Fig. 1A) [14]. The advantage of using this reporter mouse is that MOR-mCherry is expressed under the native MOR promoter, ensuring the endogenous level of MOR with its native distribution pattern. We determined the cell-type of mCherry expressing cells in the hippocampus after immunostaining with the astrocytic markers, GFAP and S100β, and the neuronal marker NeuN. We found that 100% of the interneurons in the CA1 stratum radiatum expressed MOR-mCherry signals as previously reported [4,6]. In addition to interneurons, we discovered that 83.2% of the S100β-positive astrocytes (yellow arrowheads) in stratum radiatum of hippocampal CA1 showed MOR-mCherry signals (Fig. 1B). These findings provide the very first evidence of astrocytic MOR in the hippocampus.

We also detected MOR-mCherry signals in CA1 pyramidal layer. We found that most of the mCherry signals do not localize in the soma of CA1 pyramidal neurons, but localize around the soma (Fig. 2A). Based on the previous report that MOR is expressed at the inhibitory presynaptic terminals in hippocampus [5], we hypothesized that the MOR-mCherry localizes to axon terminals of inhibitory neurons. To test this idea, we performed immunohistochemistry with hippocampal tissues of MOR-mCherry mice using an antibody against the vesicular GABA transporter, vGAT, which is a well-known marker for GABAergic axon terminals [15]. We found that MOR in the pyramidal layer was in part co-localized with vGAT (Fig. 2), whereas there was no correlation with the NeuN-labeled nuclei of pyramidal neurons at all. This finding indicates that MOR expression in the pyramidal layer is partly due to axon terminals of GABAergic interneurons.

Using the MOR-mCherry mouse, we unexpectedly found the expression of MOR in the CA1 hippocampal astrocytes. To verify the presence of astrocytic MOR by an independent method, we performed immunohistochemistry in wildtype (WT) and MOR KO mice by using two different commercially available MOR antibodies: Ab_N-term (targeting the N-terminus of human MOR, 1~80) and Ab_C-term (targeting the C-terminus of MOR, 1359~1403). We found that most of the hippocampal astrocytes in WT mice showed noticeable MOR immunoreactivity for both antibodies (Fig. 3). On the other hand, MOR KO mice did not show MOR immunoreactivity, implying that these two antibodies specifically bind to MOR protein (Fig. 3). These findings indicate that astrocytes in hippocampal CA1 region highly express MOR.

To further validate the MOR antibodies, we first developed a lentivirus carrying MOR-shRNA, which should significantly reduce the protein level by removing the entire mRNA, thus providing an advantage over the use of KO mouse in which a truncated form of a target protein might exist. Next, we injected the lentivirus carrying MOR-shRNA into the hippocampal CA1 region and investigated the expression of MOR by immunohistochemistry with Ab_N-term, one of the two antibodies. We found that MOR-shRNA virus-infected cells expressed markedly low MOR-positive signals, whereas scrambled-shRNA virus-infected cells did not (Fig. 4A). We measured the knockdown efficiency in cultured astrocytes which were infected with pSicoR-MOR-shRNA by a western blot. We found that MOR-shRNA significantly reduced the protein expression of MOR by 75.2% (Fig. 4B). In addition, we measured the knockdown efficiency in vivo by western blot with the hippocampi which were infected with pSicoR-MOR-shRNA. We found that MOR-shRNA significantly reduced the protein expression of MOR in vivo by 39.9% (Fig. 4C). These findings validate the specificity of the MOR antibody as well as the specificity of MOR shRNA, which is used for the investigation of MOR distribution in the hippocampus.

To further investigate the subcellular localization of MOR in astrocytes, we performed electron microscopy (EM) with hippocampal tissues from GFAP-GFP mice to visualize astrocytes with GFP staining with immunoperoxidase (dark amorphous deposits, arrows in Fig. 5). We stained MOR with immunogold labeling using Ab_C-term (dark specks, arrowheads in Fig. 5). We found that MOR was highly expressed in the soma and processes of astrocytes. On the other hand, we could detect only very few immunogold-MOR signals in the microdomains, the astrocytic compartments close to synapses (Fig. 5). These data indicate that MOR is expressed in astrocytic soma and processes, but not in microdomains.

MOR has been known to be expressed in NAc and VTA, which are the main brain regions of mesocorticolimbic dopaminergic system of the reward circuitry. Like in hippocampus, MOR has been believed to be exclusively expressed in neurons in NAc and VTA [3,16]. To investigate the presence of astrocytic MOR in these regions, we used the MOR-mCherry mouse and performed immunostaining with tyrosine hydroxylase (TH) antibody to identify the neurons of NAc and VTA. Using the antibody against S100β, we also found that the S100β-positive astrocytes in VTA and NAc also expressed MOR-mCherry (Fig. 6). These results indicate that in addition to hippocampal astrocytes, the astrocytes in NAc and VTA also express MOR. These findings suggest that astrocytes might have a role in opioid-associated addictive behaviors.