The self-administration chamber ENV-307W (21.6 cm × 17.8 cm × 12.7 cm; Med Associates, St Albans, VT) was located inside a larger box (Med Associates). The front, back, and top were constructed of 5.6 mm polycarbonate. Each chamber contained a wall with two small holes (0.9 cm diameter, 4.2 cm apart, 1.5 cm from the floor of the chamber). One hole was defined as active, the other was inactive. When the photocell in the active hole was triggered by a nose poke, the infusion pump (Med Associates) delivered an infusion of 20 μl / 3 s from a 5 ml syringe. The syringe was connected by a swivel via Tygon tubing. The infusion pump and syringe were outside the chamber. During infusion, a cue light above the active hole was illuminated. Each injection was followed by a 20-s ‘time-out’ period during which poking responses were recorded but had no programmed consequences. All responses at the inactive hole were also recorded. Mice were tested during the light phase of the diurnal cycle (all experiments were performed between 0800 and 1400 hours).
1 The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA.
Typical patterns of responding under the FR3 schedule for adolescent and adult mice are shown in Figure 2. Each vertical mark on the horizontal time line indicates delivery of a single oxycodone infusion.
Striatal dopamine is involved in the reinforcing effects of drugs of abuse including opioids. Acute opioid administration in the adult rodent results in increases in striatal dopamine levels ( Hemby et al, 1995 ; Wise et al, 1995 ), whereas repeated opioid administration leads to decreases in basal striatal dopamine levels ( Rossetti et al, 1992 ; Diana et al, 1995 ; Gerrits et al, 2002 ). It is not clear yet whether effects of oxycodone on adolescent rodents are similar to those on adults.
As with other drugs of abuse, the onset of abuse of the prescription opioid oxycodone occurs mostly in adolescents and young adults. Oxycodone abuse in the adolescent is especially worrisome as adolescence is a unique developmental stage during which the central nervous system undergoes marked alteration. For example, dopamine receptors in the nigrostriatal and mesolimbic dopaminergic systems are overproduced in early adolescence ( Teicher et al, 1995 ; Tarazi et al, 1998, 1999, 2000 ). μ -Opioid receptor-stimulating [ 35S ] GTP γ S binding increased from 0.13 to 3.6 fmol / mg tissue between postnatal day (P or PND) five and adulthood ( Talbot et al, 2005 ). During this period, exposure to oxycodone may result in neurobiological changes in the adolescent brain that may predispose an adolescent to take more opioids and increase the risk of developing addiction on subsequent re-exposure. This is in accord with reports showing that early onset of cigarettes or alcohol use increased the likelihood of using the same or other drugs of abuse ( Yu and Williford, 1992 ; Merrill et al, 1999 ).
Neuropsychopharmacology (2009) 34, 912–922; doi:10.1038/npp.2008.134; published online 10 September 2008.
Male adolescent and adult (4 or 10 weeks old on arrival) C57BL / 6J mice (Jackson Laboratory, Bar Harbor, ME) were housed in groups of five with free access to food and water in a light (12 : 12 h light / dark cycle, lights on at 0700 hours) and temperature (25°C) controlled room. Animal care and experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences, 1996). The experimental protocols used were approved by the Institutional Animal Care and Use Committee of The Rockefeller University. The time line of self-administration and microdialysis studies and the age of mice are shown in Table 1.
It should be noted that, when the schedule was changed from FR1 to FR3, the total intake for adults was reduced from 2.5, 4, 3, 4.4, and 4.4 mg / kg throughout the five sessions during FR1 to 2.2, 2.5, 2.7, and 2.8 mg / kg during FR3, a reduction in the total intake in adult animals. In contrast, the total intake of oxycodone by adolescent mice was from 1.7, 1.3, 1.7, 1.7 to 2.1 mg / kg in FR1 to 2.1, 1.3, 2.3, 2.5 to 2.4 mg / kg in FR3, showing an increase.
On the day before each dialysis experiment, mice were placed into individual microdialysis chambers with free access to food and water. Dialysis probes (2.0 mm; BAS) were then lowered into the striatum. Probes were perfused with artificial cerebrospinal fluid (CMA, North Chelmsford, MA) overnight at a rate of 1.0 μl / min. Following the overnight stabilization period (15–16 h), basal levels of dialysate were collected from the freely moving mice every 20 min for 1 h at a 1.0 μl / min flow rate. After collection of baseline samples, the mice that had previously self-administered oxycodone received oxycodone injections i.p. in an ascending order (1.25, 2.5, and 5.0 mg / kg at hourly intervals), whereas yoked saline controls received saline injections in the same pattern. Dialysate samples were collected every 20 min throughout the oxycodone administration period and for 1 h after the last injection. Samples were frozen and stored at −80°C before dopamine analysis ( Zhang et al, 2001 ).
Acquisition: The acquisition testing was also conducted as described in Experiment 1. Subsequently, an oxycodone dose–response curve was determined with a Latin square design. The testing doses of oxycodone were the same as in Experiment 1, but each dose was tested once before extinction sessions.
The clinical problem of nonmedical use of opioids, such as oxycodone and hydrocodone, has become a major health concern in the United States. According to the National Survey on Drug Use and Health, this problem has escalated in recent years. Although the overall use of illicit drugs by young people has dropped by approximay 24 % in recent years, an exception to this trend is the rise in nonprescription use of oxycontin (oxycodone) and vicodin (hydrocodone), with approximay 10 % of high school seniors reporting illicit use of these prescription opioids ( Johnston et al, 2006 ). Furthermore, prescription opioid analgesics (eg oxycodone and the prototypical compound, morphine) are the most commonly used and most powerful medications in pain management ( Cherny, 1996 ; Nicholson, 2003 ; Shelley and Paech, 2008 ). Nonmedical use of prescription opioids has been associated with opioid abuse and the development of addiction in some individuals.
Nonmedical use of the prescription opioid analgesic oxycodone is a major problem in the United States, particularly among adolescents and young adults. This study characterized self-administration of oxycodone by adolescent and adult mice, and how this affects striatal dopamine levels. Male C57BL / 6J mice (4 or 10 weeks old) were allowed to acquire oxycodone self-administration (0.25 mg / kg per infusion) for 9 days, and then tested with varying doses of oxycodone (0, 0.125, 0.25, 0.5, and 0.75 mg / kg per infusion). On completion of the self-administration study, a guide cannula was implanted into the striatum of these mice. Six days later, microdialysis was conducted on the freely moving mouse. After collection of baseline samples, oxycodone was administered i.p. (1.25, 2.5, and 5.0 mg / kg) and samples were collected for 1 h after each dose. Adult mice self-administered significantly more oxycodone across the doses tested. After 1 week, basal striatal dopamine levels were lower in mice of both ages that had self-administered oxycodone than in yoked saline controls. Oxycodone challenge increased striatal dopamine levels in a dose-dependent manner in both age groups. Of interest, the lowest dose of oxycodone led to increased striatal dopamine levels in the mice that had self-administered oxycodone during adolescence but not those that self-administered it as adults. The lower number of infusions of oxycodone self-administered by adolescent mice, and their later increased striatal dopamine in response to the lowest dose of oxycodone (not found in adults), suggest differential sensitivity to the reinforcing and neurobiological effects of oxycodone in the younger mice.
Following acclimation for 5 days, the mice were anesthetized with a combination of xylazine (8.0 mg / kg, i.p.) and ketamine (80 mg / kg, i.p.). After shaving and application of a 70 % alcohol and iodine preparatory solution, incisions were made in the midscapular region and anteromedial to the forearm. A catheter approximay 6 cm in length (i.d.: 0.31 mm, o.d.: 0.64 mm) (Helix Medical Inc., CA) was passed subcutaneously from the dorsal to the ventral incision. After exposure of the right jugular vein, a 22-gauge needle was inserted into the vein to guide the catheter into the jugular vein. Once the catheter was inside the vein, the needle was removed and the catheter was inserted to the level of a silicone ball marker 1.1 cm from the end. The catheter was tied to the vein with surgical silk. Physiological saline then was flushed through the catheter to avoid clotting and the catheter then capped with a stopper. Antibiotic ointment was applied to the catheter exit wounds on the animal's back and forearm. Mice were individually housed after the surgery and were allowed 3 days of recovery (due to the limited period of adolescence in the mouse) before being placed in operant test chambers for the self-administration procedure ( Zhang et al, 2006 ).
The behavioral and neurobiological changes that occur during acute or chronic re-exposure to the widely abused opioid oxycodone are very difficult to study in human adolescents, as patients in this early stage do not often seek treatment. The current experiments were designed to study oxycodone-induced behavioral changes in adolescent and in adult mice in a self-administration model. We then examined oxycodone-induced alterations in striatal dopamine levels in these mice when they were young adults and compared the effects to those of the older mice.
Correspondence: Dr Y Zhang, The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, 1230 York Avenue, Box 171, New York, NY 10065, USA. +1 212 327 8247; +1 212 327 8574;
Once the mice reached the full self-administration criteria, the doses of oxycodone were presented in an ascending (0, 0.125, 0.25, 0.5, and 0.75 mg / kg per infusion), then a descending order (0.75, 0.5, 0.25, 0.125, and 0 mg / kg per infusion), one dose per session with each dose tested in two separate sessions. A Latin square design was not used in this experiment because neurochemical measurements were to be conducted in the same mice following the self-administration study (but see Experiment 2). Mice in the control groups received yoked saline infusions during all sessions (saline was infused in the control mouse whenever the oxycodone mouse self-administered oxycodone).
self-administration, adolescent, adult, mice, oxycodone, microdialysis.
The numbers of self-administered infusions of oxycodone (0.25 mg / kg per infusion) by both adolescent and adult mice during the acquisition phase FR1 and FR3 are shown in Figure 1. During the FR1 phase, three-way ANOVA, age × hole × session, revealed a significant main effect of age, F(1, 12) = 19.01, p <0.001; adolescent mice self-administered significantly less oxycodone (nose pokes at the active hole) than the adult mice. Nose poking at the active hole was significantly more than at the inactive hole across both age groups, F(1, 12) = 88.62, p <0.000001. There were no significant differences across the sessions. However, there was a significant age × hole interaction, F(1, 12) = 10.93, p <0.01 and a significant hole × session interaction, F(4, 48) = 5.25, p <0.005. During the FR3 phase, three-way ANOVA, age × hole × session, revealed no significant difference between the two age groups, F(1, 12) = 4.19, p = 0.063. There was a significant difference between nose poke at the active hole vs the inactive hole, F(1, 12) = 151.85, p <0.000001. There were significant differences across the sessions, F(3, 36) = 16.29, p <0.000001 and a significant hole × session interaction, F(3, 36) = 20.41, p <0.000001.
Experiment 2 was conducted to determine whether there were any differences in a dose–response study with a Latin square design, and whether there were differences during extinction between adolescent and adult mice in oxycodone self-administration. Adolescent ( n = 6) and adult ( n = 6) mice received the same surgical procedure described above in Experiment 1.
Yong Zhang 1, Roberto Picetti 1, Eduardo R Buman 1, Stefan D Schlussman 1, Ann Ho 1 and Mary Jeanne Kreek 1.
Mice that finished the self-administration study were then studied by in vivo microdialysis. A total of 20 mice received guide cannula implantation, and 18 mice were used in the final analyses; two were not included due to loss of their cannula.
To evaluate the significance of differences between adolescent and adult mice in the acquisition of oxycodone self-administration, a three-way analysis of variance (ANOVA), age × hole × session, with repeated measures on the last factor, was conducted separay for the FR1 and FR3 schedules. To examine the differences between age groups in self-administration across the five doses of oxycodone, from 0 to 0.75 mg / kg per infusion, a two-way ANOVA, age × dose, was used with repeated measures on the second factor. To evaluate the differences in basal levels of extracellular dopamine induced by prior oxycodone self-administration, a two-way ANOVA was used, preexposure (oxycodone self-administration vs yoked saline) × age. Finally, to examine the effect of age during oxycodone self-administration on changes in dopamine levels after oxydocone challenge, a two-way ANOVA, age × dose, with repeated measures on the second factor, was used, followed by a planned comparison of age groups at the first dose of the oxycodone challenge (1.25 mg / kg). Regression was used to examine the relationship between total amounts of oxycodone self-administration with increases in dopamine levels induced by subsequent experimenter administration of oxycodone. The self-administration behavior and nose pokes during extinction in Experiment 2 were examined by two-way ANOVA with repeated measures. The criterion for significance is p <0.05.
The number of self-administered infusions of oxycodone (0.25 mg / kg per infusion) by both adolescent and adult mice during the acquisition phase with FR1 and FR3 schedules of reinforcement. During the FR1 phase, three-way analysis of variance (ANOVA), age × hole × session, revealed a significant main effect of age, F(1, 12) = 19.01, p <0.001; adults self-administered more infusions of oxycodone than adolescents. During the FR3 schedule, there was no significant main effect of age groups, F(1, 12) = 4.19, p = 0.063.
Mice were decapitated following brief CO 2 exposure at the end of the microdialysis study, and their brains were removed for histological evaluation. Frozen sections of 20 μ m were cut to verify the correct placement of dialysis probes following acetylcholinesterase labeling ( Franklin and Paxinos, 1997 ). Figure 4b shows photomicrographs of a tissue section from the brain of the representative adolescent and adult mice used in this study, showing probe placement in the striatum.
One day after the last self-administration session, mice were anesthetized with a combination of xylazine (8.0 mg / kg, i.p.) and ketamine (80 mg / kg, i.p.) and were placed in a stereotaxic frame modified for the mouse (David Kopf, Topanga, CA) for implantation of a guide cannula. The guide cannula (BAS, West Lafayette, IN) was implanted into the striatum (coordinates from Bregma: A = 0.65 mm, L = ±2.00 mm, and V = 3.00 mm ( Franklin and Paxinos, 1997 ) where nigrostriatal and mesolimbic dopaminergic terminals are located, involved in the rewarding effects of drugs of abuse. The guide cannula was fixed to the skull by dental acrylic. Mice were allowed 5 days to recover from surgery before microdialysis.
Extinction: Extinction conditions were the same as the oxycodone self-administration sessions with the exception that nose poking at the active hole resulted in the infusion of saline instead of oxycodone. Mice were given seven extinction sessions.
Received 15 February 2008; Revised 18 July 2008; Accepted 22 July 2008; Published online 10 September 2008.
At the end of the experiment, only data from mice that passed a catheter patency test (defined as loss of muscle tone within a few seconds after administration of a short-acting anesthetic) with injection of 30 μl of ketamine (5 mg / ml) (Fort Dodge, IA) were included in the analysis of self-administration data. Of a total 32 mice that started the study, 24 mice reached full acquisition criteria. Of these 24 mice, 22 finished the dose–response study and passed the catheter patency test.
HPLC with electrochemical detection (ESA, North Chelmsford, MA) was used to measure dopamine concentration in the dialysates. The HPLC system consisted of an ESA 540 autosampler, an ESA 582 solvent delivery system, a reverse phase C18 column, and an ESA microdialysis cell (model 5014B). The MD-TD mobile phase (10 % acetonitrile) was purchased from ESA and was delivered at a rate of 0.5 ml / min. Chromatograms were integrated and compared with standards using the ESA 501 chromatography system ( Zhang et al, 2001 ).
A 2-h self-administration session was conducted once a day. Each day, mice were weighed and heparinized saline (0.01 ml of 30 IU / ml solution) was used to flush the catheter to maintain patency. During self-administration sessions, mice in the oxycodone (Sigma, St Louis, MO) groups were placed in the self-administration chamber and a nose poke through the active hole led to an infusion of oxycodone (0.25 mg / kg per infusion) under an FR1 schedule for 5 days. The FR schedule was then changed from FR1 to FR3. The FR3 schedule was maintained until the mice reached the full self-administration criteria (which was reached within 4 days) of (1) stable intake, defined as three consecutive sessions in which the total number of infusions per session remained within 20 % of the mean of these three consecutive sessions; (2) the percent responses on the active hole were greater than 70 % of the total responses in these three consecutive sessions; and (3) the minimum infusion number was eight per session in the three sessions. Drug doses were titrated every 3 days for individual animals to follow changes in body weight.Oxycodone behavior