Experimental Studies

Male Wistar rats (4 weeks old) were anaesthetized through i.p. injection of 160 mg kg⁻¹ ketamine and 20 mg kg⁻¹ xylazine and then decapitated. Brains were then removed and submerged in an ice-cold cutting solution containing (in mM): 20 NaCl, 2.5 KCl, 1.6 NaH₂PO₄, 7 MgCl₂, 85 sucrose, 25 D-glucose, 60 NaHCO₃ and 0.5 CaCl₂, saturated with 95% O₂/5% CO₂. Horizontal 230 μm thick brain slices containing the locus coeruleus (LC) were then prepared using a vibratome. Slices were subsequently incubated in a warm (32°C) artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 11.1 D-glucose, 21.4 NaHCO₃, 2.4 CaCl₂ and 0.1 ascorbic acid, saturated with 95% O₂/5% CO₂ and were left to equilibrate for at least 1 h.

All animal care and experimental procedures were in accordance with the UK Animals (Scientific Procedures) Act 1986, the European Communities Council Directive (2010/63/EU), the ARRIVE guidelines (58) and the University of Bath ethical review document.

Rat brain slices were transferred to a recording chamber and superfused with continuous flow (2.5 ml min⁻¹) of warm (32°C) aCSF. Whole-cell recordings were made using recording electrodes (3–5 MΩ) containing an internal solution of (in mM): 115 potassium gluconate, 10 HEPES, 11 EGTA, 2 MgCl₂, 10 NaCl, 2 MgATP, and 0.25 Na₂GTP, and pH 7.3 and with an osmolarity of 270 mOsm.L⁻¹. LC neurones were voltage-clamped at −60 mV, with a correction made for a −12 mV junction potential.

All drugs were applied in the superfusing solution at known concentrations. Fentanyl and morphine were applied at concentrations determined to evoke equivalent submaximal responses (EC₈₀) in rat LC neurones (100 nM and 1 μM respectively, data not shown). Opioids were applied for 10 min to allow for evoked outward GIRK currents to rise to a steady state. Subsequently, naloxone (30 nM) was applied in superfusing solution in combination with fentanyl or morphine for 15 min. At this concentration, naloxone was demonstrated to partially reverse GIRK currents evoked by morphine (1 μM) and fentanyl (100 nM) in LC neurones to similar levels. Drug-free aCSF was then superfused over the slice and the GIRK current was tracked for 10 min, before 10 μM naloxone was applied to fully reverse opioid-induced GIRK currents.

The data were tested for normality by the Shapiro-Wilk test (passed, W = 0.9583, p = 0.7962) and visual examination of the QQ plot. Therefore we used the parametric paired two-tailed t-test to determine statistical differences between conditions. Values are presented as mean ± SEM where N = 5. Each experimental replicate (N) was run in brain slices derived from separate animals.

Wild type AtT20 cells stably expressing human MOPr were a gift from Marina Santiago (Macquarie University, Australia). An AtT20 stable cell line expressing a MOPr double mutant, MOPr^{P309R−E310R} was generated using the Invitrogen Flp-In protocol. Hygromycin-resistant and zeocin-sensitive clones were selected and expanded.

Cells were cultured in DMEM supplemented with 10% FBS, 50 U/mL penicillin, 0.5 mg/ml streptomycin (P/S) and 80 μg/ml hygromycin B for the maintenance of transfected cells. Incubator conditions were maintained at 5% CO2, 37°C and high relative humidity.

The protocol followed was as previously described (59). AtT20 cells at ~90% confluency were detached using trypsin/EDTA and resuspended in Leibovitz’s L-15 media supplemented with P/S 1%, FBS 1% and 15 mM glucose. In poly-L-lysine coated black 96-well clear flat-bottom plates, 90 μL of the cell suspension were seeded in each well and incubated overnight in an air-only incubator. One hour prior to the experiment, 90 μL of the fluorescent blue membrane potential dye was loaded into each well. Blue dye as well as all drug dilutions were prepared in a low potassium buffer. Fentanyl hydrochloride was purchased from Tocris, morphine hydrochloride from Macfarlan Smith, and naloxone hydrochloride was from Sigma-Aldrich.

Fluorescence was measured using the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) where cells were excited at a wavelength of 530 nm, emission measured at 565 nm and readings were taken every 2 s and continued until agonist or antagonist responses had reached a steady state. The amplitude of responses was calculated as the percentage change from baseline fluorescence readings. Baseline readings were taken for 30 s before 10 μL of agonist or buffer was injected. The response was measured at the lowest reduction in signal. Responses from wells that received buffer only were subtracted. The change in the signal produced by the addition of buffer alone was less than 5% of the baseline. Background fluorescence in wells with cells only or dye only was very low and regarded as negligible. For the antagonist reversal experiments, baseline readings were taken for 30 s prior to the addition of 10 μL of the submaximal concentration of each agonist (morphine 1 μM and fentanyl 20 nM). These agonist concentrations were chosen to produce comparable amplitudes of response for morphine and fentanyl in wildtype MOPr cells (see Figure 7C and Figure 7E). When agonist response reached steady state (60 s post agonist addition), 10 μL of naloxone (final concentration 10 μM) was used to reverse the signal. Assays were conducted in duplicate and mean data from 5 separate experiments are presented. The concentration-response data were analysed by non-linear regression (GraphPad Prism v8).

(A,B). Concentration response curves for fentanyl (green) and morphine (orange) in a membrane potential assay of AtT20 cells expressing (A) WT-MOPr and (B) the MOPr^{P309R−E310R} double mutant. (C,D). Naloxone reversal of the morphine (1 μM) response in (C) WT-MOPr and (D) MOPr^{P309R−E310R} double mutant expressing cells. (E,F) Naloxone reversal of the fentanyl (20 nM) response in (E) WT-MOPr and (F) MOPr^{P309R−E310R} double mutant expressing cells. All data are shown as mean ± SEM, N = 5–6.