Fifteen adult male Sprague-Dawley rats (Harlan Sprague Dawley Inc., Indianapolis, IN, USA; 250–270g) housed two to three to a cage (20°C, 12-h light, 12-h dark cycle lights on 0700) were used in this study. They were allowed ad libitum access to standard chow and water. All procedures were approved by The Institutional Animal Care and Use Committee at Thomas Jefferson University according to the revised Guide for the Care and Use of Laboratory Animals (1996), The Health Research Extension Act (1985) and the PHS Policy on Humane Care and Use of Laboratory Animals (1986). All efforts were made to utilize only the minimum number of animals necessary to produce reliable scientific data, and experiments were designed to minimize any animal distress.
Specificity of antisera
Anti-WLS antibody was generated in chickens against a peptide antigen corresponding to the C-terminal 18 amino acids (HVDGPTEIYKLTRKEAQE) of human WLS (Gene-Tel Laboratories, Madison, WI), which is identical to the rat and mouse peptide sequence. Antibodies of the IgY subtype were harvested from egg yolks and affinity purified prior to use. The characterization and specificity of the chicken antiserum against WLS has been previously described (Jin et al., 2010a
). Western blot analysis using WLS antibody showed expression of the WLS in rat brain lysates indicating that WLS antibody recognized endogenous WLS in brain tissue. WLS recognized single band of proteins with approximate molecular weight of about 50kDa (Jin et al., 2010a
). Omission of the primary antibody abolished any detectable immunoreactivity (Reyes et al., 2010
To further characterize specificity of WLS antibody, brain tissue from frontal cortex was harvested isolated, frozen on dry ice and stored at −80C until analyzed. Tissue was weighed, homogenized in TRIzol® Reagent (Invitrogen, Carlsbad, CA) and combined with 5 volumes of chloroform. Centrifugation at 12,000 × g facilitated separation of aqueous (RNA) and organic phases (DNA/Protein). Protein was precipitated with ethanol, pellets were vacuum-dried and re-suspended in 1% SDS. Protein levels were quantified using Pierce BCA Protein Assay Kit (ThermoScientific g of protein were separated on a NuPage 4–12%μ Product No. 23225) and 50 gradient Bis-Tris gel. Separated proteins were transferred onto a nitrocellulose membrane with the use of an iBlot® Dry Blotting System (Invitrogen, Carlsbad, CA). The primary antibody solution contained chicken anti-WLS antibody at a 1:4500 dilution in 5% dry milk. The blocking peptide solution contained chicken anti-WLS antibody (1:4500 dilution) together with the WLS blocking peptide (1:1000 dilution) in 5% dry milk. Solutions were incubated overnight at 4°C with gentle agitation. Nitrocellulose membranes containing blotted proteins were blocked for 1 hour in 10% dry milk at room temperature and incubated with primary antibody solution or blocked (i.e. with peptide) primary antibody solution, also at room temperature for 1 hour. Following primary antibody incubation, membranes were subjected to three 10 minute wash steps in 1x TBS-T, and incubated with horseradish peroxidase-conjugated anti-chicken secondary antibody (ab6753) for 30 minutes at room temperature. Five additional 10 minute washes were performed in 1x TBS-T, and proteins were visualized using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences, Piscataway, NJ).
Adult male rats received intracerebroventricular (i.c.v.) injections of morphine (Sigma-Aldrich Co., St. Louis, MO) dissolved in 0.9% saline to a concentration of 10 mg/ml and administered at 1.0 μg/kg (n =5), 0.9% saline in a volume of 25 μl/kg (n = 5) or DAMGO (Tocris Bioscience, Ellsville, MO) at 5μg/kg body weight (n = 5). Rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL; 0.5–1.0%, in air) via a specialized nose cone affixed to the sterotaxic frame (Stoelting Corp., Wood Dale, IL) and placed in a stereotaxic apparatus for surgery. Micropipettes (Kwik-Fil, 1.2 mm outer diameter; World Precision Instruments, Inc., Sarasota, FL) with tip diameters of 20–25 μm were filled with saline, morphine or DAMGO. The tips of the micropipettes were placed at the following coordinates, 3.5 mm posterior from bregma, 1.4 mm medial/lateral, 3.7 mm ventral from the top of the skull. The stereotaxic coordinates of the injection sites were based on the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986
). Saline, morphine or DAMGO was injected using a Picospritzer (General Valve Corporation, Fairfield, NJ) at 24–26 psi and over a 10 min period. Pipettes were left at the site of injection for 5 min following drug or vehicle administration.
Thirty minutes following i.c.v. injections of saline, morphine or DAMGO, rats were euthanized. The time of euthanasia post-treatment was selected based on previous studies from our group. Using an in vitro technique in MOR expressing human embryonic kidney 293 cells a MOR/WLS complex was detected 1 hour following DAMGO treatment (Jin et al., 2010a
). Moreover, another in vivo study using high resolution electron microscopy in rat brain showed significant MOR internalization 30 min following treatment with the opiate agonist, etorphine (Van Bockstaele and Commons, 2001
). Finally, it is well known that agonist-induced trafficking and up-regulation may occur rapidly from seconds to minutes (Norgauer et al., 1991
; Zigmond et al., 1982
) and the time point of 30 minutes was considered to be optimal for detecting changes in trafficking patterns here.
Thirty minutes following injection, rats were deeply anesthetized with sodium pentobarbital (80 mg/kg; Ovation Pharmaceuticals, Inc., Deerfield, IL, USA) and transcardially perfused through the ascending aorta with 10 ml heparinized saline followed by 25 ml of 3.75% acrolein (Electron Microscopy Sciences, Fort Washington, PA, USA), and 50 ml of 2% formaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brains were removed immediately after perfusion fixation, sectioned into 1–3 mm coronal slices and postfixed in the same fixative overnight at 4°C.
Forty micron-thick coronal sections through the rostrocaudal extent of the striatum (Paxinos and Watson 1986
) were cut using a Vibratome (Technical Product International, St Louis, MO, USA) and rinsed extensively in 0.1 M PB and 0.1 M tris-buffered saline (TBS; pH 7.6). Sections were placed for 30 min in 1% sodium borohydride in 0.1 M PB to reduce amine-aldehyde compounds. The tissue sections were then incubated in 0.5% bovine serum albumin (BSA) in 0.1 M TBS for 30 min. Subsequently, sections were incubated in chicken anti-WLS antiserum (Gene-Tel Laboratories, Madison, WI) at 1:1000 in 0.1% BSA and 0.1M TBS. Incubation time was 15–18 hours in a rotary shaker at room temperature. Thorough rinses in 0.1 M TBS were conducted following each incubation procedure.
WLS was visualized using the immunogold-silver enhancement technique. Tissue sections were rinsed three times with 0.1 M TBS, followed by rinses with 0.1 M PB and 0.01 M phosphate buffered saline (PBS; pH 7.4). This was followed by 10-minute incubation in a 0.2% gelatin-PBS and 0.8% BSA buffer and a two-hour incubation in goat anti-rabbit IgG conjugate in 1 nm gold particles (Amersham Bioscience Corp., Piscataway, NJ) at room temperature. Sections were then rinsed in buffer containing the same concentration of gelatin and BSA as above. Following rinses with 0.01 M PBS, sections were then incubated in 2% glutaraldehyde (Electron Microscopy Sciences) in 0.01 M PBS for 10 min. The conjugated gold particles were intensified by incubation in a silver enhancement solution (Amersham Bioscience Corp.). The optimal times for silver enhancement were determined by empirical observation for each experiment and ranged between 8 and 10 min. Following intensification, tissue sections were rinsed in 0.2 M citrate buffer and 0.1 M PB, and fixed in 2% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M PB for 1 h, washed in 0.1 M PB, dehydrated in an ascending series of ethanol followed by propylene oxide and flat embedded in Epon 812 (Electron Microscopy Sciences; Leranth and Pickel, 1989
) between tow sheets of Aclar plastic (Honeywell, Pottsville, PA).
Thin sections of approximately 50–100 nm in thickness were cut with a diamond knife (Diatome-US, Fort Washington, PA) using a Leica Ultracut (Leica Microsystems, Wetzlar, Germany). Captured images of selected sections were compared with captured light microscopic images of the block face before sectioning. Sections were collected on copper mesh grids, examined with an electron microscope (Morgagni, Fei Company, Hillsboro, OR) and digital images were captured using the AMT advantage HR/HR-B CCD camera system (Advance Microscopy Techniques Corp., Danvers, MA, USA). Figures were assembled and adjusted for brightness and contrast in Adobe Photoshop CS4 software (Adobe Systems, Inc., San Jose, CA).
Immunogold-silver labeling for WLS appeared as intense black electron-dense particles and was identified in striatal somata and dendritic processes as we have recently demonstrated (Jin et al., 2010a
; Reyes et al., 2010
). Selective immunogold-silver labeled profiles were identified by the presence, in single thin sections, of at least two immunogold-silver particles within a cellular compartment (Jin et al., 2010a
; Reyes et al., 2010
). The criterion of two gold particles as indicative of WLS labeling is conservative and may have led to an underestimation of the number of WLS-labeled profiles. Another factor that may have led to the underestimation of labeled profiles is the limitation of immunocytochemical methods to detect trace amounts of WLS. To circumvent the caveat of incomplete antibody penetration which is inherent to preembedding technique, tissue sections were collected close to the plastic-tissue interface and to ensure that the immunogold labeling was detectable in all sections analyzed. Additionally, unbiased stereological methods were not used for counting labeled profiles, and the results of the numerical analysis can only be considered to be an estimate of the numbers of synapses and labeled profiles.
Sequential immunogold-silver labeling
Following the procedure for dual immunogold-silver labeling that we recently described in another study (Jin et al., 2010
), WLS and MOR were identified using sequential immunogold-silver labeling. Tissue sections were incubated for 15–18 hours in a cocktail containing chicken anti-WLS antiserum (Gene-Tel Laboratories, Madison, WI) at 1:1000 and rabbit anti-MOR (Immunostar Inc., Hudson, WI, USA) at 1:2000 in 0.1%BSA and 0.1M TBS. Thereafter, sections were rinsed extensively in 0.1 M TBS and 0.2% BSA in 0.01 M PBS followed by incubation in goat anti-rabbit IgG ultra-small conjugate (1:100; Amersham Bioscience Corp., Piscataway, NJ, USA) at room temperature for 8 hours. Tissue sections were rinsed six times in 0.2% BSA-0.01 M PBS and twice in 0.1 M PB. Pre-enhancement washings were done with Enhancement Conditioning Solution (ECS; Amersham Bioscience Corp.) followed with the first silver enhancement (300 μl R-Gent SE-EM enhancement mixture; Amersham Bioscience Corp.) for 90 min. Subsequently, tissue sections were rinsed in 0.2 M citrate buffer, four times in ECS and two times in 0.1 M PB and were incubated in goat anti-chicken IgG ultra-small conjugate (1:100; Amersham Bioscience Corp.) at room temperature for 8 hours followed by rinses with 0.2% BSA-0.01 M PBS and 0.1 M PB. Then, sections were incubated in 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.01 M PBS for 2 hours followed by extensive rinses with 0.1 M PB and distilled water. The second silver enhancement (300 μl R-Gent SE-EM enhancement mixture; Amersham Bioscience Corp.) was performed for 60 min. Tissues sections were washed extensively with distilled water and 0.1 M PB. All washes were done at 10 min-intervals. Following washes, tissue sections were incubated in 2% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M PB for 1 h, washed in 0.1 M PB, dehydrated in an ascending series of ethanol followed by propylene oxide and flat embedded in Epon 812. Sectioning with a diamond knife, examining with an electron microscope and obtaining digital images followed standard protocols described earlier.
Control and data analysis
Every fourth adjacent section through the striatum was included in immunohistochemical staining for WLS. Some sections were processed in parallel with the rest of the procedures identical but the primary antiserum was omitted. Sections processed in the absence of primary antibody did not exhibit immunoreactivity. (Jin et al., 2010a
; Reyes et al., 2010
). Tissue sections were taken from three rats per group with the good preservation of ultrastructural morphology and with clearly apparent immunocytochemical labeling. At least 9 grids containing 5 to 8 thin sections each were collected from at least two plastic-embedded sections of the LC from each animal. The quantification of WLS-immunolabeled profiles were carried out at the plastic-tissue interface to ensure that immunolabeling was detectable in all sections used for analysis (Chan et al., 1990
). To determine whether levels of spurious silver grains could contribute to false positives, blood vessels and myelinated axons (structures that should not contain WLS immunolabeling) were counted in random ultrathin sections. Minimal spurious labeling was identified. Therefore, the criteria for considering a process as immunolabeled was defined by the presence of at least 2–3 silver grains in a cellular profile. Only tissue sections that were singly labeled for WLS were used for the electron microscopic analysis. The identification of cellular elements was based on the standard morphological criteria (Peters et al., 1991
; Peters and Palay, 1996
). WLS immunolabeling was identified as either cytoplasmic or plasmalemmal. If the immunogold-silver grains were associated with the plasma membrane they were classified as plasmalemmal and if the immunogold-silver grains were not in contact with the plasma membrane they were classified as cytoplasmic. A total of 1279 dendritic profiles exhibiting WLS immunoreactivity from all groups were used in the analysis. Specifically, the dendritic profiles were randomly obtained from each rat and ranged in number from 119–182 dendritic profiles per animal. Statistical analysis of the number of profiles obtained showed no significant difference in the number of dendritic profiles sampled per group (saline = 121.67 ± 12.3; morphine 141.67 ± 31.78; DAMGO = 163 ± 17.06) examined between groups. The analysis of WLS internalization in various groups studied was quantified by calculating the ratio of cytoplasmic to total immunogold-silver particles for each singly immunolabeled dendritic profile in individual rats. The density of WLS on the plasma membrane was calculated as the number of plasmalemmal immunogold-silver particles per unit perimeter (μm). Similarly, the density of WLS in the cytoplasm was calculated as the number of cytoplasmic immunogold-silver particles per unit area (μm2
). All the data gathered and analyzed were obtained per animal and the average of three animals was calculated. The perimeter and area of each dendritic profle was obtained using Image J software (NIH). In addition, as with previous studies from our group (Reyes et al., 2006
; Reyes et al., 2008
; Wang et al., 2009
), care was taken to ensure that control and experimental groups contained similarly sized profiles. We did not observe any statistical difference in the size of profiles analyzed in any group examined as we have recently reported (Reyes et al., 2010
). For the sequential immunogold-silver labeling, semi-quantitative analysis was carried out by randomly obtaining the ratio of cytoplasmic to total immunogold-silver particles for WLS and MOR in a dendritic profile exhibiting both WLS and MOR immunoreactivities.