For experiments using rats, adult (150–250 g) male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were used. Details of the method of slice preparation and recording have been published previously (Williams et al., 1984). Briefly, animals were anesthetized with isoflurane and killed. The brain was dissected, blocked and mounted in a vibratome chamber in order to cut horizontal slices (200–260 μm thick) containing locus coeruleus (LC). Slices were stored at 35°C in an artificial cerebro-spinal fluid (aCSF) containing (in mM) 126 NaCl, 2.5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and 11 D-glucose while being continuously equilibrated with 95% O2/5% CO2. Slices were incubated for a minimum of 1 hour in order to wash out drugs used in chronic treatment protocols that may have remained in brain tissue.
Slices (260 μm) were hemisected and transferred to the recording chamber (0.5 ml) where they were superfused with 35°C aCSF at a rate of 1.5 ml/min. Whole-cell recordings were made from rat LC neurons with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) in the voltage-clamp mode (cells held at −55 mV). Pipettes (1.7–2.1 MΩ) were filled with an internal solution containing the following (in mM): 115 Methyl Potassium Sulfate, 20 NaCl, 1.5 MgCl2, 10 HEPES, 10 BAPTA, 2 Mg-ATP, 0.5 Na-GTP, and 10 phosphocreatine, pH 7.3. Intracellular recording in mouse LC were made with an Axoclamp 2A using sharp electrodes (50–60 MΩ) filled with KCl (2 M). DC current (0–200 pA) was applied to inhibit spontaneous firing and hold the membrane potential at approximately −60 mV. Data was collected with PowerLab (Chart version 4.2.3). Analysis was performed with Prism and Kaleidagraph software. Values are presented as arithmetic mean±SEM. Comparisons of ME-induced currents and hyperpolarizations and of acute desensitization between treatment groups was performed using one-way ANOVA followed by Dunnett’s post-hoc test. The interaction between the time and extent of recovery between treatment groups was examined using two-way ANOVA followed by bonferonni’s adjusted-alpha multiple comparison test to compare recovery at each time point. P values less than 0.05 were considered significant.
Rats were implanted with osmotic minipumps (Alzet, 2ML1) in order to deliver morphine (NIDA–Neuroscience Center), methadone (NIDA–Neuroscience Center), or carrier (control). The minipumps have a 2 ml reservoir and deliver their contents for 7 days at the rate of 10 μl/hour. Pumps were filled with the required concentration of drug, dissolved in water, based on the weight of the rat and the desired dosing parameter (morphine: 60, 30, 15 mg/kg/day; methadone: 60, 30, 5 mg/kg/day). Mice received 45 mg/kg/day of either morphine or methadone via osmotic minipumps that have a 200 μl reservoir and release at a rate of 1 μl/hour for 7 days. Animals were anesthetized with isoflurane and an incision was made in the mid-scapular region to insert the pump subcutaneously. Rats receiving 60 mg/kg/day of either morphine or methadone were first given IP injections of 5 mg/kg at 9 am and 7 mg/kg at 6 pm on Day 1. On Day 2, they received 7 mg/kg IP at 9 am and the osmotic minipump implanted at 6 pm. Animals were returned to their housing facility upon recovery. Experiments were performed on day 6 or 7 following minipump implantation. Control animals consisted of naïve animals and those implanted with vehicle-filled pumps.
Drug Concentration Analysis
Brain and plasma samples were analyzed at the University of Utah, Center for Human Toxicology under the supervision of Dr. Roger Foltz in conjunction with NIDA. Plasma and whole brain samples were obtained for drug (morphine or methadone) concentration analysis at the time of brain slice preparation. Following isoflurane anesthesia, 3 ml whole blood was obtained via cardiac puncture with a heparinized syringe. Blood was centrifuged and plasma was collected. Brain tissue removed after blocking the LC was collected and homogenized in water. Samples were frozen at −20°C and shipped to University of Utah, Center for Human Toxicology for analysis. Samples were analyzed by liquid chromatography/tandem mass spectrometry using electrospray ionization and selected reaction monitoring. Samples from morphine treated animals were analyzed for morphine and the metabolites morphine-3-glucuronide, and morphine-6-glucuronide. The quantification range for these compounds was between 1.0 and 1,000 ng/ml. Samples from methadone treated animals were analyzed for R- and S-methadone and their respective metabolites R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The quantification range for these compounds was between 2.5 and 500 ng/ml.
FlagMOR transgenic mice
Mice were bred and genotyped as previously described (Arrtamangkul et al, 2008
). Data were collected from mice that were hemizygous for the FlagMOR transgene (Flag-TG/+) and backcrossed with C57Bl/6 for at least 5 generations. Mice were also bred with MOR−/− or βArr2−/− mice to generate mice that were hemizygous for the transgene and homozygous knock-outs (Flag-TG/+MOR−/− and Flag-TG/+Arr−/−). Transgenic mice and transgenic mice that were crossed with the MOR knockout animal were used. These mice expressed a somewhat greater number of receptors (Arttamankgul et al., 2008
). The Bmax for MOR binding was two fold higher in the transgenic mouse resulting in a 4 fold shift in the concentration response curve to ME. The concentration response curve approached that of the wild type animals after the transgenic animal was crossed with the MOR knockout animal. Given the small shift in the concentration response curve to ME, it is unlikely that the small difference in the level of MOR expression has an impact on the interpretation of the results.
Receptor internalization and recycling
Brain slices (200 μm) were incubated in aCSF containing M1 antibody (Sigma) that was conjugated to 10 μg/ml Alexa-594 (InVitrogen) for 25–45 min. Slices were visualized using an upright microscope with a custom built 2-photon apparatus. A 15 μm z-series was collected in 1 μm sections using ScanImage software. [Met]5enkephalin (ME) and calcium-free aCSF containing EGTA (0.5 mM, calcium-free) were applied by superfusion and all experiments were performed at 35°C. A control image (C) was collected prior to drug application and a second image was collected after perfusion of ME (30 μM, 10 min). This was followed by perfusion of the calcium free solution (10 min) either immediately after washout of ME (I) or after a 30-min wash-out period (R). Images were analyzed off-line using ImageJ software. Images were z-projected using the sum-slices method. Five regions of interest were selected and averaged for background fluorescence. The average background fluorescence was subtracted from total fluorescence to yield the fluorescence intensity. Internalization and was calculated as the fraction of fluorescence intensity remaining after perfusion of ME and the calcium-free solution, compared to control (I/C). The total fluorescence remaining after a 30 min wash and calcium-free solution (R/C) was subtracted from the average internalization to determine the fraction of internalized receptors that recycled to the plasma membrane. One-way ANOVA was used to compare ME-induced internalization between treatment groups. Bi-directional students t-test was used within each treatment group to compare differences in fluorescence intensity remaining following internalization and recycling.
GRK2as5 transgenic mouse
To acutely regulate GRK2 activity in tissue, a “pseudo-knockin” mouse that expresses a previously described analog-sensitive version of GRK2 (Kenski 2005
) in place of the endogenous kinase was generated. This mutant GRK2 is selectively inhibited by the nucleotide analog, 1NaPP1 (1-(1,1-Dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3, 4-d]pyrimidin-4-amine, gift from Kevan Shokat, UCSF). The fosmid M1-1960 containing the entire murine Adrbk1 gene and conserved 5′ region was selected from the EpiFOS library (Bacpac, CHORI) for targeted mutagenesis. Mutations coding for C221V and L271G, which confer sensitivity to the small molecule inhibitor 1NaPP1 (Kenski et al., 2005
), were introduced into Adrbk1 by homologous recombination using the Counter-Selection BAC Modification method (Genebridges). The modified fosmid was linearized and microinjected into C57Bl/6 blastocysts (Gladstone Transgenic Core at UCSF). Founders, identified by Southern blot and the presence of a silent mutation introducing a BstEII site in the transgene detectable by PCR amplification and restriction digest, were crossed to C57Bl/6 WT animals to identify founders with germ-line transmission. Founders with endogenous levels of grk2as5 transgene expression were selected for crosses to Adrbk1+/− heterozygous knockout mice in a C57Bl/6 background (Jaber et al., 1996
) (gift from Marc Caron, Duke University), and additional crosses were continued until mice homozygous for the transgene in a homozygous Adrbk1−/− background were obtained. Verification that starting breeding pairs were double homozygous mice was confirmed by mating each partner to WT mice, and all resulting offspring were double heterozygotes.
Drugs were applied by bath superfusion. ME, bestatin and yohimbine were dissolved in water. 1NaPP1 and UK14304 were dissolved in DMSO. Final concentrations of DMSO did not exceed 0.01%. For experiments with 1NaPP1 slices were incubated prior to recording and were also superfused with the inhibitor. Thiorphan was dissolved in ethanol (applied less than 0.00001%). Morphine sulfate and methadone hydrochloride used for treatment were obtained from NIDA–Neuroscience Center. All other drugs were obtained from Sigma-Aldrich.