Beta funaltrexamine β fna

The hot water (50°C ± 0.5) tail-flick assay was carried out as previously described using a cut off time of 10 s.^{26 (link), 40 (link)} Briefly, mice were habituated to the restrainer for 15 min over 3 days. On the test day baseline measurements were recorded in triplicate and the average tail-flick latency recorded. Latencies were measured at 1, 5, 10, 15, 30, 45, 60, 90, and 120 min post-intraperitoneal (i.p.) injection of 25, Morphine Sulfate (Hospira, New Zealand Limited), or vehicle (2:1:7; Dimethyl Sulfoxide (DMSO), Tween-80, Saline). To evaluate whether the analgesic effects of 25 were MOR mediated, mice were pretreated with the selective MOR antagonist beta-funaltrexamine (β-FNA) (Tocris) (5 mg/kg, s.c.) 24 hrs prior to administration of 25 (5 mg/kg, i.p.). The maximum possible effect (MPE) was calculated as follows: % MPE = 100 × (test latency − control latency) / (10 − control latency).
To determine dose-response effects and analgesic tolerance, a within animal cumulative dose-response tail-flick assay was carried out on day 1 and following 9 days of subcutaneous (s.c.) administration of morphine (10 mg/kg) or 25 (5 mg/kg) as previously described.^{17 (link), 26 (link)} The induction of tolerance was also observed on days 3, 5, 7 and 9 by measuring tail-flick latencies 30 min post-injection.

Beta-funaltrexamine (β-FNA) (Tocris), a selective irreversible µ-opioid receptor antagonist, was solubilized in saline for a final concentration of 3 µg/µL. The compound was sonicated for several minutes and kept in 52°C water bath to maintain its soluble state. Prior to drug administration, rats were anaesthetized with a 2% isoflurane O₂ mixture. Using a Quintessential Stereotaxic Injector (Stoelting), a bilateral microinjection (1 µL/hemisphere) of β-FNA was administered over a 5-min period to allow for diffusion of the drug. Rats received β-FNA 20-hours prior to the start the morphine hourly microdialysis study.