In vivo studies utilized a transgenic mouse engineered to express the HIV-1 Tat1-86 protein in astrocytes in a doxycyline-inducible manner. As previously described (Bruce-Keller et al. 2008 ), mice expressing the Tat gene under control of a tet responsive element (TRE) in the pTREX vector (Clonetech, Mountain View, CA) were crossed with mice engineered to express the glial fibrillary acidic protein (GFAP) promoter driving the reverse tetracycline transactivator (RTTA). The inducible Tat transgenic mice (Tat+ mice) used in these studies express both GFAP-RTTA and TRE-Tat genes, while the control mice (Tat- mice) express only the GFAP-RTTA gene. Individual animals were genotyped using RNA isolated from ear clip samples, by standard PCR procedures previously described (Bruce-Keller et al. 2008 ). Tat-induction was obtained by replacing standard chow with a formulation containing doxycycline (DOX) at 6 mg/g (Harlan, Indianapolis, IN). Some control groups were also fed DOX-containing chow to control for non-specific actions of DOX intake.
Morphine was delivered by 25 mg, timed-release pellets implanted subcutaneously in the subscapular region (NIDA Drug Supply System) under aseptic conditions using isoflurane anaesthesia. Sham pellets were similarly implanted as controls. The 25 mg morphine pellets reportedly deplete at a rate of 5 mg/day, yet steady state levels of morphine, measured in ng/ml plasma or ng/gm brain tissue reflect lower availability, probably due to the balance of delivery and metabolism (see, e.g., (Feng et al. 2006 (link))). Both C3HeB and C57Bl mice reportedly tolerate delivery from 75 mg/5 day morphine pellets (Peart and Gross 2004 (link); Rahim et al. 2003 (link)). Less than 5% of the mice in our studies died, and there was no relationship between morphine-induced toxicity and either genotype or DOX administration.
In experiments lasting for a total of 7 days, opioid and control pellets were replaced after 5 days. Naltrexone was administered via an Alzet mini-pump (1007D) implanted in the same region. 600 mg/ml naltrexone (NIDA Drug Supply System) was prepared in 50% DMSO, a standard vehicle for mini-pump delivery (Arnot et al. 1996 (link)). 100 μl total volume was loaded per mini-pump, delivering 0.5 μl/hr for up to 7 days. Pumps containing 50% DMSO and sterile saline were used as naltrexone controls. For studies where mice were exposed to both opioids and Tat, mice were given the DOX feed starting on the night before opioid treatment in order for blood levels of DOX to stabilize. For histological studies, mice were deeply anaesthetized with halothane prior to perfusion with Zamboni's fixative (2% paraformaldehyde, pH 7.4, with 0.15% picric acid).
Morphine was delivered by 25 mg, timed-release pellets implanted subcutaneously in the subscapular region (NIDA Drug Supply System) under aseptic conditions using isoflurane anaesthesia. Sham pellets were similarly implanted as controls. The 25 mg morphine pellets reportedly deplete at a rate of 5 mg/day, yet steady state levels of morphine, measured in ng/ml plasma or ng/gm brain tissue reflect lower availability, probably due to the balance of delivery and metabolism (see, e.g., (Feng et al. 2006 (link))). Both C3HeB and C57Bl mice reportedly tolerate delivery from 75 mg/5 day morphine pellets (Peart and Gross 2004 (link); Rahim et al. 2003 (link)). Less than 5% of the mice in our studies died, and there was no relationship between morphine-induced toxicity and either genotype or DOX administration.
In experiments lasting for a total of 7 days, opioid and control pellets were replaced after 5 days. Naltrexone was administered via an Alzet mini-pump (1007D) implanted in the same region. 600 mg/ml naltrexone (NIDA Drug Supply System) was prepared in 50% DMSO, a standard vehicle for mini-pump delivery (Arnot et al. 1996 (link)). 100 μl total volume was loaded per mini-pump, delivering 0.5 μl/hr for up to 7 days. Pumps containing 50% DMSO and sterile saline were used as naltrexone controls. For studies where mice were exposed to both opioids and Tat, mice were given the DOX feed starting on the night before opioid treatment in order for blood levels of DOX to stabilize. For histological studies, mice were deeply anaesthetized with halothane prior to perfusion with Zamboni's fixative (2% paraformaldehyde, pH 7.4, with 0.15% picric acid).