Behavioral Mechanisms of Oxycodone’s Effects in Female and Male Rats: Reinforcement Delay and Impulsive Choice

Abstract

μ-Opioid agonists (e.g., morphine) typically increase impulsive choice, which has been interpreted as an opioid-induced increase in sensitivity to reinforcement delay. Relatively little research has been done with opioids other than morphine (e.g., oxycodone) or on sex differences in opioid effects on impulsive choice. The present study investigated effects of acute (0.1–1.0 mg/kg) and chronic (1.0 mg/kg twice/day) administration of oxycodone on choice controlled by reinforcement delay, a primary mechanism implicated in impulsive choice, in female and male rats. Rats responded under a concurrent-chains procedure designed to quantify effects of reinforcement delay on choice within each session. For both sexes, choice was sensitive to delay under this procedure. Sensitivity to delay under baseline was slightly higher for males than females, suggesting more impulsive choice with males. When given acutely, intermediate and higher doses of oxycodone decreased sensitivity to delay; this effect was larger and more reliable in males than in females. When given chronically, sex differences were also observed: tolerance developed to the sensitivity-decreasing effects in females, whereas sensitization developed in males. These data suggest that reinforcement delay may play an important role in sex differences in impulsive choice, as well as in the effects of acute and chronic administration of opioids in impulsive choice. However, drug-induced changes in impulsive choice could be related to at least two potential behavioral mechanisms: reinforcement delay and/or reinforcement magnitude. Effects of oxycodone on sensitivity to reinforcement magnitude remain to be fully characterized.

Substance use, including misuse of prescription opioids, is associated with a variety of behavior patterns described as impulsive¹ (Carroll et al. 2010; Madden et al., 1997; Perry & Carroll, 2008; Yi et al., 2010). These behavior patterns may place individuals at risk of contracting and transmitting diseases such as HIV/AIDS and at risk for continued substance use, which can increase the chances of dependence and overdose. Although evidence indicates that impulsive behavior is a pre-existing factor related to substance use (Odum, 2011; Perry & Carroll, 2008), evidence also suggests that substance use may increase the likelihood of such behavior (see de Wit, 2009; de Wit & Mitchell, 2010; Simon et al., 2007; Weafer et al., 2014). Unfortunately, it is not well understood how opioids, particularly prescription opioids, affect impulsive behavior, creating a need for preclinical evaluation in nonhuman models. One common way to study impulsive choice is with delay-discounting procedures, in which subjects are presented with a series of choices between a smaller-sooner reinforcer (SSR) and a larger-later reinforcer (LLR; Ainslie, 1974; Logue, 1988; Madden & Johnson, 2010). For example, Pitts and McKinney (2005) arranged a discrete-trial procedure similar to one originally developed by Evenden and Ryan (1996) and found that morphine decreased choice of the LLR (i.e., increased impulsive choice). Similar findings with morphine have been obtained in other studies (Eppolito et al., 2013; Kieres et al., 2004; Maguire et al., 2016; Pattij et al., 2009).

Drug-related increases in impulsive choice on delay-discounting procedures could result from at least two different behavioral mechanisms (for more on behavioral mechanisms of drug action, see Branch, 1991; Thompson & Schuster, 1968). Opioids could decrease sensitivity to reinforcement magnitude (henceforward, sensitivity to magnitude), such that the difference between the small and large reinforcer has less impact on choice. Alternatively, opioids could increase sensitivity to reinforcement delay (henceforward, sensitivity to delay), thereby shifting preference toward the more immediate reinforcer (Pitts, 2014). In typical impulsive-choice procedures, these two mechanisms are confounded; most choices within a session differ in terms of both reinforcement magnitude and delay. As a result, the response allocation of the subjects may be a result of a tradeoff between conflicting reinforcer dimensions (e.g., Logue, 1988; Rodriguez & Logue, 1986; see also Kyonka & Grace, 2008). Our approach has been to start by characterizing effects of drugs on choice controlled by magnitude and delay in isolation to determine how each is affected when the other is held constant (see Pitts et al., 2016). A more thorough understanding can then be developed by characterizing effects of drugs when these reinforcer dimensions are manipulated in combination (see Hughes et al., 2022).

Concurrent-chains procedures (see Autor, 1969; Herrnstein, 1964) have been used extensively to study how choice is affected by different reinforcement parameters in isolation, including magnitude (Hunt et al., 2020; Neuringer, 1967; Pitts et al., 2016, Experiment 2) and delay (Grace et al., 2003; Grace & Nevin, 1999; Ito & Asaki, 1982; Oliveira et al., 2014; Orduña et al. 2013; Pitts et al., Experiment 1). An advantage of concurrent-chains procedures, over typical discrete-trial procedures, is that subjects respond to each alternative multiple times prior to the presentation of the outcome. Thus, preference for a particular outcome can be quantified more precisely and exclusive preference for one outcome is less likely (de Villiers, 1977; Hughes et al., 2022; Mazur, 1987). To that end, Hunt et al. (2020) used a concurrent-chains procedure to examine the effects of oxycodone on sensitivity to magnitude, while holding delay constant, in male Sprague-Dawley rats. They found that oxycodone decreased sensitivity to magnitude, which would suggest an increase in impulsive choice (i.e., increase choice of the SSR).

The primary purpose of the present study was to extend, and complement, the work of Hunt et al. (2020) by examining effects of oxycodone on sensitivity to delay, while holding magnitude constant, in a concurrent-chains procedure. An increase in sensitivity to delay suggests a shift in preference for the SSR (impulsive choice), whereas a decrease suggests a shift in preference for the LLR (“self-control” choice). Additionally, effects of oxycodone in combination with naloxone on sensitivity to delay and overall response rates were assessed to determine the extent to which effects of oxycodone could be reversed by selective antagonism of the μ-opioid receptor.

A second purpose of the present study was to compare oxycodone’s effects in female and male Sprague-Dawley rats. Most of the animal research on impulsive choice has used male subjects. Evidence of potential sex differences in substance use and misuse warrant further research with female subjects (see Becker & Hu, 2008; Carroll et al., 2004; Festa & Quinones-Jenab, 2004 for reviews); for example, opioids are prescribed more frequently in women to treat pain, and women may become dependent more quickly than men (Health Resources and Services Administration, 2021). Additionally, there are conflicting data regarding sex differences in impulsive choice across delay-discounting studies. In some studies, female rats (e.g., Eubig et al., 2014; Lukkes et al., 2016; van Haaren et al., 1988) and mice (e.g., Koot et al., 2009) tend to make more impulsive choices than males; however, this is not always the case (Panfil et al., 2020; see Weafer & de Wit, 2014 for a review).

A third purpose of the present experiment was to examine effects of oxycodone on sensitivity to delay when rats were administered oxycodone chronically (i.e., twice daily) in an attempt to produce mild dependence (Beardsley et al., 2004). Effects of naloxone- and saline-induced withdrawal on sensitivity to delay were also evaluated. Opioid-dependent humans have consistently made more impulsive choices in delay-discounting procedures (e.g., Yi et al., 2010 for a review), which suggests that if sensitivity to delayed reinforcement plays a role in impulsive choice, oxycodone could increase such sensitivity during chronic administration.

Method

Subjects

Fourteen Sprague-Dawley rats (6 females and 8 males; the difference in number is due to the number of operant chambers available for each sex) from Envigo Laboratories served as subjects. According to a power analysis with a target power of 0.8 and using effect sizes from our previous studies with opioids yielded n = 12, with n = 6 for each sex. Thus, the sample sizes used here were deemed sufficient. Eleven rats were approximately 2 months old and experimentally naive when the experiments began; Male Rats C5, C6, C7 were approximately 7 months old and had experience responding under fixed-interval schedules of reinforcement. Rats were individually housed in a temperature- and humidity-controlled colony room which operated on a 12-12 hr reverse light-dark cycle; lights turned off at 7:00 a.m. About 15 min after each experimental session (or at a similar time on days when sessions were not run), females and males were fed approximately 10 g and 15 g of food (Lab Diet 5001), respectively. Water was also provided at this time and removed 2 hr later so the rats were without water for 21 hr prior to the next experimental session to help ensure sucrose water (25% sucrose solution) served as an effective reinforcer. All procedures conformed to the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th Edition) and were approved by the Institutional Animal Care and Use Committee at the University of North Carolina Wilmington.

Apparatus

Ten Med Associates (24.5 X 29.5 X 25.5 cm; Model ENV-008) and four Coulbourn Instruments (32.5 X 30.5 X 25.5 cm; Model H10-11R-TC) operant chambers were enclosed in sound-attenuating cubicles. Six chambers were in one room for females and eight chambers were in a second room for males. Additionally, white noise was broadcast into each room via speakers to attenuate external sounds during experimental sessions. The floor grid of each chamber consisted of stainless-steel rods that sat approximately 3.65 cm above the bottom of the chambers. Two retractable levers, which required approximately 0.30 N of force for a response to be recorded, were located on the left and right of the front wall (3.5 cm from the floor and 1 cm from the adjacent wall closest to each lever); a stimulus light was located 2 cm above each lever. A house light was centered on the front wall approximately 2.5 cm from the ceiling. During experimental sessions, sucrose solution (described above) was delivered by raising a 0.02 cc dipper (Med Associates model ENV-202M-S) into a 5 X 5 cm opening, which was centered between the two front levers and located approximately 2.5 cm from the floor grid. A Windows operated computer controlled the experimental session using Med PC-IV software and interfacing.

Behavioral Procedure

Preliminary Training

Prior to experimental sessions, each subject underwent adaptation, dipper-training, and lever-training phases. During the adaptation phase, each subject was placed in the chamber with an illuminated house light for 15 min. Following each session, subjects were given a sucrose solution to drink from a syringe. Once three adaptation sessions were completed and subjects reliably drank from the syringe, they were trained to drink from the dipper. During dipper-training sessions, the house light was illuminated, and the dipper was raised for 5 s according to a variable-time 15-s schedule (this schedule was then decreased to 8 s and 5 s). This continued until each subject drank from the dipper within 1 s of the dipper raising.

An auto-shaping procedure was used during the lever-training phase. Within a session, the left or right lever was extended into the chamber for 8 s. If the lever was pressed while extended, it was retracted, and the dipper was presented immediately for 5 s. If the lever was not pressed within 8 s, the lever retracted, and the dipper was presented. After each dipper presentation all lights were turned off for a variable-duration intertrial interval which averaged 15 s. Sessions were terminated after 40 trials, and lever training ended after subjects responded consistently on both levers.

Next, subjects were trained on a multiple fixed-ratio (FR; left lever) FR (right lever) schedule. During these sessions, one lever was extended and the corresponding stimulus light was turned on. Contingent on the completion of the FR, the lever was retracted, and the dipper was presented for 3 s (it remained at 3 s for the remainder of the study). Each session ended after 40 reinforcer presentations (20 from pressing each lever). Presentations of the left and right lever alternated irregularly with one lever presented during each component. The schedule was gradually increased from FR1 to FR 5 simultaneously for both components. Experimental sessions began once subjects consistently pressed both levers at approximately equal rates on the multiple FR 5 FR 5 schedule.

Experimental Procedure

A concurrent-chains procedure was arranged such that responding during the initial links resulted in access to a terminal link, which ended with a reinforcer. Each experimental session consisted of 66 initial link-terminal link cycles divided across 3 blocks; each block consisted of 2 forced and 20 choice cycles. Sessions ended after the 60 choice cycles were completed or 50 min had elapsed, whichever occurred first. Figure 1 shows a diagram of the procedure.

Figure 1. Concurrent-chains Cycle.

Note. Outlined at the top is the initial links, in the middle is the terminal link, and at the bottom are the outcomes associated with each alternative. On the left is the procedure as it occurs when the standard-alternative is selected and on the right is the process as it occurs when the variable-alternative is selected.

During the initial links (the top section of Figure 1), both levers were extended, the lights above them (lever lights) were turned on, and one of the levers was pre-assigned. Assignment of the levers was done pseudo-randomly such that for every 10 cycles, there were 5 entries into the terminal link associated with the left lever, and 5 entries into the terminal link associated with the right lever, the lever assignment was not signaled to the subject. This dependent schedule arranged equal exposure to the terminal link associated with each lever and required responding on both levers for completion of a session. Responses on the unassigned lever were recorded but produced no programmed consequences. Satisfying a conjunctive variable-interval (VI) 5-s FR 5 schedule on the assigned lever produced entry into the associated terminal link. Under this schedule, both the VI and FR requirements had to be satisfied for terminal-link entry, but in any order. For example, if the left lever was the assigned lever, the subject advanced to the associated terminal link when at least 1 response occurred on that lever after an average of 5 s had elapsed and at least a total 5 responses had occurred on that lever (See Hughes et al., 2022 for the rationale of this and other features of the procedure). During the initial link, the lever light associated with the more-favorable outcome (i.e., the terminal link that provided the shorter delay) blinked on-and-off (1-s period) and the lever light associated with the less-favorable outcome (i.e., the terminal link that provided the longer delay) was on continuously. When the delays provided by the terminal links were equal, both lever lights were on continuously.

Upon entry into the terminal link (the middle section of Figure 1), both levers were retracted, and a delay was in place during which the light associated with the assigned lever remained illuminated. After the delay elapsed, the assigned lever extended, and the reinforcer (three dipper presentations) was provided contingent on one response on that lever (the bottom section of Figure 1).

Across blocks of cycles within each session, the delay associated with one of the levers (the variable lever) changed within the session, and the delay associated with the other lever (the standard lever) remained constant. The assignment of the standard and variable levers (left or right) was counterbalanced across subjects. Selection of the standard lever always resulted in the opportunity for reinforcement after a 9-s delay, whereas selection of the variable lever resulted in the opportunity for reinforcement after either a 3-, 9-, or 27-s delay. These delays were arranged in three different block types: variable-sooner (9 vs. 3), variable-equal-standard (9 vs. 9), and standard-sooner (9 vs. 27). Each session, the order of the block types was randomly selected, without replacement, from a list of all six possible sequences.

The first two cycles of each block were forced cycles to ensure subjects contacted the delays associated with each lever prior to choice cycles. Forced cycles involved the presentation of one lever with the contingencies associated with that lever the same as those in the subsequent choice trials.

Responding on the concurrent-chains procedure was considered stable when the following criteria were met. First, all cycles were completed for 12 consecutive sessions. Second, each session type had to be experienced at least twice. Third, sensitivity values (see Data Analysis for information on how this was calculated) across the last 12 sessions could not show a consistent trend based on visual inspection. The pharmacological procedure began once performance was considered stable or after a minimum of 30 sessions on the Experimental Procedure.

Pharmacological Procedure

Oxycodone hydrochloride (Spectrum Chemical, New Jersey, USA) was dissolved in 0.9% sodium chloride (i.e., saline) and injected s.c. in a volume of 0.5 ml/kg 15 min prior to predetermined test sessions (typically on Tuesdays and Fridays; injections always were separated by at least 2 days). A subject was injected only when its sensitivity value on the previous session was comparable to that of the five previous non-injection sessions. Test doses included 0.1, 0.3, 0.56, and 1.0 mg/kg (expressed as salt); doses larger than 1.0 mg/kg were not tested, as Hunt et al. (2020) found that the administration of 1.7 mg/kg sometimes resulted in dermatophagia (i.e., excessive chewing of one’s skin). Effects of each dose and saline were determined at least three times, such that three different sequences (out of the possible six) for each subject were arranged. It was ensured that each of the three sessions at each dose started with a different one of the three block-types (i.e., standard-sooner, variable-equal-standard, or variable-sooner); the order of the remaining two block-types in these sessions was counterbalanced across subjects. Upon completion of the acute dosing regimen, naloxone antagonism tests were conducted. For these tests, subjects were injected with naloxone (3.0 mg/kg) 20 min prior to the session, followed 5 min later with an injection of an effective dose of oxycodone (i.e., oxycodone was given at the usual pre-session time). These tests were conducted to determine if the effects of oxycodone could be attenuated by naloxone, which would suggest that these effects were related to activity at opioid receptors. Larger doses of naloxone were not tested in these rats because 1) 3.0 mg/kg produced some behavioral effects when administered with saline (see Figure 2) and 2) 10.0 mg/kg produced greater effects in another group of rats in our laboratory.

Figure 2. Group Sensitivity, Bias, and Response Rates.

Note. Mean (of individual medians) sensitivity, bias, and initial-link response rates for female (open circles; n = 5) and male (filled circles, n = 6) rats. Data points above C represent data from non-injection control sessions immediately prior to an injection; data points above S represent data from sessions before which saline was administered. Triangles above saline and 0.56 mg/kg represent data from sessions before which 3.0 mg/kg naloxone was administered in combination with saline and 0.56 mg/kg, respectively. Error bars show standard error. Top panel shows mean sensitivity values in the left graph and those values expressed as a percent of control values in the right graph.

After the naloxone tests were conducted, a chronic regimen of oxycodone administration based on that conducted by Wiebelhaus et al. (2016) began. During this regimen, subjects were injected twice daily, 7 days a week. Presession injections occurred 15 min prior to each session and postsession injections occurred approximately 8-9 hr later. Injections occurred at approximately the same times when sessions were not conducted (e.g., weekends). Subjects first received injections of saline, then received injections of 0.3 mg/kg for at least 3 days and then 0.56 mg/kg for at least 5 days before receiving the terminal chronic dose of 1.0 mg/kg. Although this dose was lower than that used by Wiebelhaus et al., preliminary studies in our lab have shown that chronic administration of 1.0 mg/kg (or higher) occasionally results in the development of dermatophagia, so the rats were monitored carefully for this, and for any other signs of distress (e.g., failing to eat or drink, weight loss). We have found that providing wooden chew toys can reduce the likelihood of dermatophagia.

Approximately 4 weeks after the start of the chronic regimen, three different types of test sessions were conducted: tolerance/sensitization, antagonist-induced withdrawal, and abstinence-induced withdrawal. During the tolerance/sensitization tests, an injection of a different dose of oxycodone was substituted for the chronic dose prior to the session. During the antagonist-induced withdrawal tests, 1.0 and 3.0 mg/kg of naloxone was substituted for the chronic dose prior to the session in an attempt to precipitate withdrawal. These doses were used for these tests because 3.0 mg/kg of naloxone did not substantially alter behavior when administered with saline prechronically. During the abstinence-induced withdrawal tests, saline was substituted for the presession dose of oxycodone. During all tests, the usual postsession dose of oxycodone continued to be given in the evening. These procedures were based in part on the regimen used by Wiebelhaus et al. (2016) and by preliminary studies conducted in our laboratory.

Data Analysis

During the initial links, responses on each lever within each session block were collected for individual subjects. For each block, the number of responses on the variable lever was then divided by the number of responses on the standard lever to produce a ratio of initial-links responding (variable/standard). Initial-link response ratios were used to calculate sensitivity and bias using the following version of the generalized matching law (e.g., Grace et al., 2003):

$$log\left(R_V/R_S\right)=alog\left(I_V/I_S\right)+\log b,$$

in which R represents lever presses, I represents immediacy of reinforcement (i.e., the reciprocal of delay), and the subscripts V and S represent the variable and standard levers, respectively. The parameters a (sensitivity) and b (bias) were derived from the data collected each session using equation 1. Sensitivity and bias were only calculated if at least 40 choice cycles were completed.

Overall response rates in the initial links (henceforward, response rates) were calculated for each subject by dividing the total number of responses in the initial links (i.e., the sum of responses across both the standard and variable levers) by the duration, in minutes, spent in the initial links. Dose-effect functions for sensitivity, bias, and response rates were then constructed for individual subjects during the acute regimen. Of interest were selective effects of oxycodone on sensitivity to delay, which is illustrated whenever there was a reliable effect on sensitivity at a dose that did not substantially alter bias or response rate. A reliable effect in an individual subject is concluded when the mean of a measure at a given dose is outside the 95% CI of the data from saline.

To analyze potential sex differences, a mixed-factor analysis of variance (ANOVA) was conducted with sex as a between-group factor and dose as a within-subjects factor. Effects of oxycodone within each group then were analyzed using separate one-way, repeated-measures ANOVAs upon each dependent variable (sensitivity, bias, response rate, and percentage of cycles completed). To provide a reasonable estimate of sensitivity and bias, a subject had to complete a minimum of 40 choice cycles in a session; thus, these ANOVAs included only those sessions in which subjects met this criterion. For response rate and percentage of cycles completed, this analysis included the data at saline and at all doses given (including doses that completely suppressed lever pressing). Because responding in one female (E16) and two males (D7 and D10) was substantially disrupted by at least one dose (i.e., these subjects failed to meet this criterion for all sessions under at least one dose), these subjects’ data were not included in the ANOVAs or in the corresponding figures; their results are presented in Appendix A.

Male rats, D7 and D10, completed the chronic phase, and their data are included in those analyses. During chronic administration of oxycodone, effects of an oxycodone dose, which was substituted for the usual chronic dose, on sensitivity and response rates were compared to the acute effects of that dose. Tolerance was indicated by a smaller effect relative to acute administration; sensitization was indicated by a larger effect relative to acute administration. In addition, data following administration of naloxone or saline were compared to those from the chronic regimen. Analyses of the chronic and withdrawal effects were conducted for both the individual-subject and the group data. Additionally, analyses were conducted to determine any potential sex differences.

Some additional effects of antagonist-induced withdrawal were examined via 5-min videos. Two video-recording devices (set up at two opposite angles) recorded rats immediately after an administration of chronic oxycodone or naloxone substitution (1.0 and 3.0 mg/kg). In each video, two to three rats were recorded from one side of their cages, and the cages were arranged such that all rats were in view of both cameras. Observers recorded behavior by viewing a video from each angle. Instances of jumping, wet dog shakes, and coprophagia were counted throughout the 5 min from each angle; counts were also recorded within 5-s intervals for purposes of interobserver agreement (IOA; see more below). During the 5-s intervals, the presence of ptosis, chewing, non-genital grooming, genital grooming, writhing (using partial-interval time sampling) and/or stationary behavior (using whole-interval time sampling) was also recorded (see Appendix B for operational definitions).

Records of the count and interval measures were included in the overall calculations of each respective measure if a behavior was recorded in an interval of only one video or if a behavior was recorded in the same interval of both videos; however, note that for the count measures, the largest count from one interval was the count which was reported (e.g., if 2 counts were recorded in an interval of one video, but 1 count in the same interval of the other video, then 2 counts were reported). Estimates of interval-by-interval IOA (for the interval measures) and total count per interval IOA (for the count measures) were calculated for 44.4% of the sessions; IOA was only scored for measures from sessions in which at least one observer recorded at least one instance of the behavior.

To measure stationary behavior, grids were overlayed on the video using Animotica (Mixilab, 2017). In each video, approximately 2.5 rows and 3 columns of grids were arranged per cage of rats. When a rat’s nose was recorded as either not visible at any point in an interval or as located in-between grids, that interval was not utilized in (i.e., was not legitimate for) the calculation of stationary behavior. Further, only intervals recorded as legitimate by the primary data recorder were utilized in the IOA calculation of the stationary measure.

Additional measures (fecal boli, diarrhea, and irritability; see Appendix B) were recorded by direct observation immediately after rats were placed into their chambers for experimental sessions. Total count IOA estimates were calculated for fecal boli and diarrhea for 83.3% of these sessions.

Transparency and Openness

We report how we determined our sample size, why we excluded some data, all manipulations, and measures in the Method section. All data, data analyses, and research are available from the corresponding author. Data were analyzed using R Statistical Software (version 4.2.1) via the package ez (version 4.4.0); VassarStats (http://vassarstats.net/) was also utilized for data analysis. This study’s design and its analysis were not preregistered.

Results

For both females and males, choice within each session was well-controlled by reinforcement delay under the concurrent-chains procedure. Average sensitivity under nondrug conditions for both sexes was above 0.5 (see data points above C and S in Panel A of Figure 2). Mean sensitivity for males (0.82, SEM = 0.08; filled symbols) was higher than for females (0.60, SEM = 0.08; unfilled symbols); non-parametric statistical analyses (Mann-Whitney U) indicated a significant difference under control (p = .03) and after administration of saline (p = .04)².

Acute Effects of Oxycodone

Figure 2 presents oxycodone dose-effect functions for sensitivity (Panels A and B), bias (Panel C), and response rates (Panel D); data are shown for both females (unfilled symbols) and males (filled symbols). Panel A in Figure 2 shows that oxycodone produced dose-related decreases in sensitivity for both females and males, although the shapes of the functions differ somewhat across the sexes (see top row of Appendix C for data from selected individual subjects). The function for females shows a slight, and relatively gradual, decrease across doses. In contrast, the function for males is shallow across the two lower doses but shows much sharper decreases at 0.56 and at 1.0 mg/kg. A 2 (sex: F, M) x 5 (dose: S, 0.1-1.0 mg/kg) mixed-factor ANOVA revealed a significant main effect of dose (F(4, 36) = 11.83, p < .01), no main effect of sex (F(1, 9) = 0.69, p = .42), and a significant sex by dose interaction (F(4, 36) = 2.80, p = .04). Separate one-factor ANOVAs for each sex (using saline and each dose) indicated that oxycodone produced a significant decrease in sensitivity for males (F(4) = 11.90, p < .01), but not for females (F(4) = 1.66, p = .21). For males, post-hoc analyses indicated that both 0.56 and 1.0 mg/kg significantly decreased sensitivity. Because of the different sensitivity values for females and males under nondrug conditions, dose-effect functions for sensitivity are plotted as a percentage of control value in Panel B of Figure 2. These data provide a clearer illustration that 0.56 and 1.0 mg/kg oxycodone produced larger decreases in sensitivity for males than for females.

The data in Panel C of Figure 2 show that, on average, oxycodone shifted bias more toward the standard option (i.e., became more negative). A 2 (sex) x 5 (dose) mixed-factor ANOVA yielded a significant main effect of dose (F(4, 36) = 2.95, p < .03) but no main effect of sex (F(1, 9) = 0.71, p = .42). Somewhat surprisingly, there was no sex by dose interaction (F(4, 36) = 2.31, p = .07).

The data in Panel D of Figure 2 show that oxycodone produced dose-related decreases in overall initial-link response rates. A 2 (sex) x 5 (dose) mixed-factor ANOVA yielded a main effect of dose (F(4, 36) = 7.35, p < .01), but no main effect of sex (F(1, 9) = 1.35, p = .28), and no sex by dose interaction (F(4, 36) = 0.75, p = .57). It appears, however, that effects of 0.56 mg/kg oxycodone differed for females and males. Separate one-factor ANOVAs (saline and all doses) yielded significant effects of dose on response rates for both sexes, but post-hoc analyses indicated a significant decrease at 0.56 mg/kg for males, but not for females, when compared with saline data (1.0 mg/kg significantly decreased rates in both sexes).

Comparison of the data in Panels B (sensitivity) and D (response rate) of Figure 2 indicates that oxycodone-induced decreases in sensitivity at 0.56 mg/kg (males) and at 1.0 mg/kg (males, and somewhat in females), on average, were accompanied by small, but relatively consistent, decreases in response rates. An examination of the data presented in Appendix A indicates that the group averages in Figure 2 do not completely capture what occurred at the individual-subject level regarding the association between effects of oxycodone on sensitivity to delay and on response rates (and on the percentage of cycles completed). In some cases, oxycodone decreased sensitivity to delay without affecting response rates or the percentage of cycles completed (e.g., female Rat D29 and male Rat C7 at 0.56 mg/kg). Furthermore, on occasion, a dose of oxycodone decreased response rates without affecting sensitivity (e.g., female Rat D31 at 0.3 mg/kg). Finally, although 0.56 (males) and 1.0 mg/kg (both sexes) decreased sensitivity in most, or all, of the rats, close examination of Appendix A indicates that, for some of the rats, reliable decreases in sensitivity occurred at lower doses (e.g., female Rats D29 and D30 at 0.1 and 0.3 mg/kg, respectively; male Rats C6 and D9 at 0.3 mg/kg).

Figure 2 shows that co-administration of 3.0 mg/kg naloxone attenuated some of the effects produced by 0.56 mg/kg oxycodone (triangles in each panel). In particular, sensitivity at 0.56 mg/kg oxycodone in the presence of naloxone was substantially higher than when oxycodone was given alone. It should be noted, however, that 3.0 mg/kg naloxone when co-administered with saline: a) increased sensitivity in females, but not males, and b) slightly decreased response rates in both sexes.

Figure 3 presents a more fine-grained analysis than shown in Figure 2. These graphs show cycle-by-cycle (i.e., reinforcer-by-reinforcer) log response ratios (V/S) for each block type for females (left column) and males (right column) under control conditions (top row) and following administration of 0.56 mg/kg (middle row) and 1.0 mg/kg oxycodone (bottom row). In these graphs, the first data point in each block type shows the average log response ratio for the first choice cycle in that block type, the second data point shows the response ratio for the second choice cycle, and so on. In each graph, block type is organized from left to right on the x-axis as a function of immediacy ratio. Points below S show data from cycles in which the immediacy ratio favored the standard alternative (log (I_v/I_s) = −0.48), points below E show data from cycles in which the immediacy ratio for the variable and standard alternatives were equal (log (I_v/I_s) = 0), and points below V show data from cycles in which the immediacy ratio favored the variable alternative (log (I_v/I_s) = 0.48). It is important to note that in this figure, the data for each block type are averages across all occurrences of that block type, regardless of the position within the session (first, second, or third).

Figure 3. Cycle-by-Cycle Log Response Ratios.

Note. Log response ratios (variable/standard) across cycles. Data are group averages for females in the left panels and males in the right panels. The top panels show averages for control sessions, the middle panels show averages for the 0.56 dose of oxycodone, and the bottom panels show averages for the 1.0 dose of oxycodone. Control session graphs are averaged across six control sessions (one of each possible within-session orders, see Method for additional information on within-session orders), and the 0.56 and 1.0 graphs are averaged across each determination of the dose. Filled circles show the log response ratio from each cycle and grey bars represent the log reinforcer ratio (i.e., variable delay/standard delay) for each given block of cycles. Black lines across the x-axis denote a block of 20 cycles for each type of session block: standard-sooner (S), variable-equal-standard (E), and variable-sooner (V). Note that these data are taken from when each block type appears in any of the three positions and not when each block type is only in the order displayed in the graph.

Figure 3 illustrates several noteworthy features of behavior under this procedure. First, within each block, choice under nondrug control conditions (top row) came under control of the arranged immediacy ratio relatively rapidly (usually within the first two or three choice cycles). Second, for both sexes, but particularly for males, the shift in the log ratio toward the more favorable option was slowest for the standard-favorable block and reached its maximum after the fifth reinforcer in this block type. Third, 0.56 mg/kg oxycodone (middle row) attenuated the degree of preference by a) shifting the data points from S and the V blocks toward indifference (i.e., closer to the dashed line at 0), and b) increasing the variability in response ratios within each block type. Fourth, 1.0 mg/kg oxycodone (bottom row) further decreased the overall degree of preference (as shown in Panel A of Figure 2) and further increased variability within each block (see bottom row of Appendix C for an additional way of characterizing choice within blocks for individual subjects).

Chronic and Withdrawal Effects

Table 1 shows sensitivity, bias, and response rate values during the chronic oxycodone regimen, when doses of oxycodone, saline (abstinence-induced withdrawal), and naloxone (antagonist-induced withdrawal) were substituted for the presession chronic oxycodone dose, and from postchronic when chronic injections had stopped. Overall, saline and naloxone substitutions did not disrupt responding relative to the chronic control; indeed, sensitivity measures were comparable to those obtained pre- and postchronic under nondrug conditions.

Table 1.

Sensitivity, Bias, and Response Rate Measures during Chronic Oxycodone, Chronic Substitutions, and Postchronic

	Chronic Control	Substitutions During Chronic				Post Chronic Control	Post Chronic Oxy
		Oxy	Saline	1.0 Naloxone	3.0 Naloxone
Females
Sensitivity
D28	0.09	0.05	0.25	0.52	0.16	0.69	0.14
D29	0.39	0.54	0.65	0.70	0.57	0.89	0.31
D30	0.56	0.81	0.81	0.65	0.67	0.77	0.36
D31	0.45	0.62	0.24	0.39	0.57	0.37	0.25
D32	0.11	0.43	0.58	0.45	0.30	0.33	0.31
Bias
D28	−0.10	−0.11	−0.01	−0.14	−0.13	−0.19	−0.25
D29	−0.23	−0.31	−0.26	−0.09	−0.13	−0.16	−0.26
D30	−0.32	−0.38	−0.01	−0.16	−0.19	−0.06	−0.18
D31	−0.39	−0.29	0.04	−0.02	0.07	−0.04	−0.22
D32	−0.01	0.09	−0.15	−0.02	−0.03	−0.04	0.01
Rates (R/min)
D28	128.37	128.85	84.60	85.84	95.72	94.67	96.53
D29	143.13	147.36	149.35	127.43	127.68	139.96	120.34
D30	86.43	77.38	92.92	69.47	86.90	75.91	50.15
D31	112.49	131.34	104.29	98.57	89.60	112.29	135.06
D32	42.54	121.08	122.36	113.50	100.76	92.56	90.50
Males
Sensitivity
D7	−0.01	0.60	0.60	0.66	0.61	0.80	0.05
D8	0.14	0.42	0.37	0.36	0.74	0.88	0.31
D9	0.26	0.33	0.40	0.70	0.79	0.77	0.38
D10	0.25	0.36	0.55	0.65	0.78	0.66	0.21
D11	0.24	0.26	0.45	0.64	0.70	0.89	−.03
Bias
D7	0.16	−0.31	−0.45	−0.39	−0.37	−0.43	−0.38
D8	−0.14	−0.08	−0.33	0.04	−0.20	−0.18	−0.03
D9	−0.21	−0.09	0.01	−0.30	−0.15	0.04	−0.03
D10	−0.13	−0.29	−0.36	−0.24	−0.41	−0.33	−0.11
D11	−0.02	−0.15	−0.46	−0.13	−0.24	−0.20	−0.29
Rates (R/min)
D7	65.16	99.25	103.48	84.93	84.60	111.43	107.09
D8	57.81	71.68	62.42	74.82	66.42	70.31	73.71
D9	27.87	63.66	40.59	77.21	36.55	58.30	30.15
D10	108.90	19.05	94.02	85.93	91.69	85.10	104.77
D11	100.97	93.92	92.50	69.37	68.02	89.50	80.36

Note. Values below chronic control are averages from sessions during the chronic regimen prior to a substitution dose. Values below oxy are from sessions in which a select dose of oxycodone was substituted for the morning chronic dose; rats received 0.56 mg/kg oxycodone, except Rats D32, D7, and D10 received 0.3 mg/kg. Values below saline, 1.0, and 3.0 naloxone are from sessions in which those were substituted for the morning chronic dose. Measures below postchronic control are averages from 3-4 sessions after chronic injections were stopped. Measures below postchronic oxy are from a session following the chronic control sessions, in which the same chronic substitution dose of oxycodone was administered before the session.

Figure 4 shows effects on sensitivity from a probe dose of oxycodone for individual rats during (filled) and after (striped) chronic oxycodone administration expressed as a proportion of effects when that same dose was administered acutely. Overall, as indicated in the group filled bars, there were differential effects observed in females and males when a smaller dose of oxycodone was substituted for the chronic dose. In four of the five females, some degree of tolerance to the sensitivity-decreasing effects was evident (filled bars); that is, substituting 0.3 or 0.56 mg/kg oxycodone did not decrease or decreased sensitivity to delay less than it did during acute administration. In contrast, for all males, sensitization to the sensitivity-decreasing effects was evident. The change in oxycodone’s effects on sensitivity were not simply due to changes in overall response rates during the sessions. Although for some females, tolerance to the sensitivity- and response-rate-decreasing effects of oxycodone occurred, that was not always the case. For example, D28 showed tolerance to the response-rate decreasing effects, but sensitization to the sensitivity-decreasing effects. This relation also occurred for four of the five male rats. But for D30, the opposite pattern occurred. Overall, there was a significant negative correlation between change in sensitivity and in response rates during the chronic regimen. Further, the tolerance to the sensitivity-decreasing effects observed in female rats during the chronic regimen was attenuated substantially when the same dose was administered after at least three sessions without any injections. In contrast, sensitization observed in male rats remained or increased. Postchronic changes in the degree of tolerance and sensitization to the sensitivity-decreasing effects did not appear to be related to systematic corresponding changes in the response-rate decreasing effects. During postchronic control sessions, bias shifted toward the standard option for all females and four of the five males and response rates increased in three females and three males.

Figure 4. Change in Sensitivity Values During and Postchronic Oxycodone Administration.

Note. Change in sensitivity values (top panel) and response rates (bottom panel) when a select dose of oxycodone was administered as a probe dose during chronic administration (filled bars) and 3-4 days after chronic injections stopped (striped bars) relative to values during acute (prechronic) in female and male rats. All rats received 0.56 mg/kg oxycodone, expect Rat D32, D7, and D10 received 0.3 mg/kg oxycodone. Bars above 0 indicate tolerance; bars below 0 indicate sensitization. The 2.61 value on the bottom right graph indicates the outlier value for D10 postchronic.

Observed measures for physical dependence of chronic oxycodone and withdrawal by naloxone are described for females and males in Appendix D. Notably, naloxone (1.0 mg/kg for both groups, and 3.0 mg/kg for females), in comparison to oxycodone (1.0 mg/kg), was associated with chewing much less often and stationary behavior much more often. Further, ptosis was observed in a small to moderate percentage of intervals for most females when naloxone, but not oxycodone, was administered; generally, both genital and non-genital grooming also followed this same trend for many rats in both groups. Overall, there were few to no recordings of many measures in both groups across sessions, such as writhing, wet dog shakes, jumping, and coprophagia.

Discussion

Nondrug Control Performance

Baseline performance indicated control over choice by within-session changes in delay under the concurrent-chains procedure: Average sensitivity was greater than 0.5 for all rats except one of each sex. Relative to similar studies with rodents, sensitivity in the current study was comparable to that obtained with mice (Pope et al., 2020) and substantially greater than that obtained with spontaneously hypertensive and Wistar-Kyoto rats (Aparicio et al., 2019). Although procedural differences across studies make direct comparisons difficult, the concurrent-chains procedure used in the present study shows promise as a baseline for characterizing effects of neurobiological manipulation (e.g., drugs). Further, we believe there are two key advantages of the concurrent-chains procedure over discrete-trial choice procedures (e.g., Evenden & Ryan, 1996). First, changes in choice can be scaled more precisely when using response ratios compared to percent choice. Second, general disruptions in performance following neurobiological manipulation can be detected with changes in response rates (for a more thorough discussion, see Hughes et al., 2022).

Sex Differences

One finding of interest was that sensitivity to reinforcement delay was significantly higher for males than females, which suggests that males may be more likely to engage in impulsive choice than females. Such a suggestion would conflict with data showing that female rodents tend to make more impulsive choice than males (Hernandez et al., 2020; Koot et al., 2009; Perry et al., 2007; van Haaren et al., 1988), but would be in line with those reported by Panfil et al. (2020) showing that males tend to engage in more impulsive choice than females (for a review/commentary on the complexities regarding sex differences in impulsive choice vs. impulsive action, see Weafer & de Wit, 2014). The present data on sensitivity to delay alone are insufficient to reconcile these discrepancies. Sensitivity to magnitude may play an equally important role in determining impulsive choice. Previous research has investigated sensitivity to magnitude with male rats (Hunt et al., 2020), but direct comparisons of sensitivity to magnitude between females and males may help identify some conditions under which more impulsive choices occur.

Although no data on hormone levels (e.g., estrous cycles for females) were collected in the current study, one account for the sex differences may indeed be related to hormonal differences. Unfortunately, the degree to which sex hormones modulate impulsive choice are not well understood. There is evidence that sex hormones (e.g., testosterone; estradiol) may affect impulsive choice (Bayless et al., 2013; Hernandez et al., 2020), and likely play a role in sex differences in several effects of opioids (see Craft, 2008; Sharp et al., 2022). Note, however, that Hernandez et al. found no differences in impulsive choice between the four estrous cycle stages (see also Liley et al., 2019). Indeed, in the present study there was no evidence of cyclical trends based on visual inspection of session-by-session sensitivity values. But the relation between sensitivity and specific stages of the estrous cycle remains to be empirically determined.

Acute Effects of Oxycodone

Acute administration of oxycodone produced dose-related decreases in sensitivity to reinforcement delay which differed across sexes. For males, oxycodone produced a significant decrease in sensitivity to delay at two doses (0.56 and 1.0 mg/kg); in contrast, for females, none of the doses significantly affected sensitivity to delay. It should be noted that the decreases in sensitivity to delay in males at 0.56 and 1.0 mg/kg were accompanied by decreases in overall initial-links response rate. Thus, strictly speaking, effects of oxycodone on sensitivity cannot be considered selective, at least at the group level. It also should be noted, however, that for some of the males (e.g., C5, C6, and C7) 0.56 mg/kg oxycodone decreased sensitivity without substantially decreasing overall response rates. Furthermore, the majority of the rats completed over 90% of the choice cycles at this dose. As such, it is possible that the decreases in sensitivity to delay in males represented a direct effect of oxycodone, rather than an indirect outcome of, for example, motivational and/or motoric effects. Even so, the decreases in sensitivity with males in the present study were not particularly robust and suggests a potential role for other behavioral mechanisms (e.g., reinforcement magnitude) in the reported effects of opioids on impulsive choice. Nevertheless, given the overall lack of effect with females, sensitivity to delay may play a key role in any sex differences in opioid effects on impulsive choice, and additional research is necessary to elucidate the conditions under which oxycodone affects sensitivity to delay across sexes.

In typical delay-discounting arrangements, morphine generally increases impulsive choice (Eppolito et al., 2013; Kieres et al., 2004; Maguire et al., 2016; Pattij et al., 2009; Pitts & McKinney, 2005). Effects of oxycodone in the present study seem at odds with the effects of morphine reported in those studies; all else being equal, a decrease in sensitivity to delay would be expected to decrease impulsive choice. It should be noted, however, that choices in typical delay-discounting arrangements differ in both reinforcement magnitude and delay, whereas, in the current study, only reinforcement delay was manipulated. Thus, it is possible that opioids produce different effects on choice depending on which dimensions are manipulated, or that opioids produce greater effects on one dimension compared to another (for more on behavioral mechanisms, see Branch, 1991; Pitts, 2014; Thompson & Schuster, 1968). For example, if oxycodone decreases sensitivity to magnitude more so than sensitivity to delay, then the net result would be an increase in impulsive choice.

Although more research is needed to assess how oxycodone affects the relative contribution of delay and magnitude on choice, there is growing evidence that opioids decrease sensitivity to magnitude. For example, Hunt et al. (2020) found that oxycodone decreased sensitivity to magnitude in a variation of the concurrent-chains procedure in which magnitude was manipulated in isolation. Similarly, in some studies employing Evenden and Ryan’s (1996) within-session escalating-delay procedure, morphine decreased choice of the larger reinforcer when it was not delayed (i.e., during the first block of the session, e.g., Eppolito et al., 2013; Pattij et al. 2009). Taken together, these findings suggest a potential, and perhaps critical, role of reduced sensitivity to magnitude in opioid-produced changes in impulsive choice. Ongoing research in our lab is examining the effects of opioids, such as oxycodone, on sensitivity to magnitude and delay in combination. Although it is important to understand which dimension oxycodone affects more, it also is important to characterize the manner in which they interact.

Although effects of oxycodone on sensitivity to delay were of primary interest, a drug may also produce its effects on behavior by weakening control by relevant stimuli. In the present experiment, the lever lights were used as discriminative stimuli for the outcomes associated with each lever; during the initial link, the lever light associated with the less favorable outcome remained illuminated and the lever light associated with the more favorable outcome blinked on and off. If oxycodone disrupted stimulus control in the initial link, then responding may have been more variable which is evidenced in Figure 3 (0.56 and 1.0 mg/kg increased the variability within blocks and across cycles). Although the initial-link stimuli were included to increase the likelihood that responding would come under control of within-session changes in reinforcement delay, it remains important to examine how procedural variables affect choice under nondrug and drug conditions. Preliminary data from our laboratory in which both lever lights remain steady throughout the initial link (i.e., there is no blinking light correlated with the more favorable alternative) indicate that choice is well controlled under this procedure and that effects of oxycodone on sensitivity are similar in the presence and absence of the blinking light.

Another source of stimulus control that may have been disrupted by oxycodone was the differences in the delay parameters. Previous research with pigeons suggests that morphine can diminish discriminative responding to stimuli that are associated with different temporal aspects (e.g., Knealing & Schaal, 2002; Ward & Odum; 2005). Further, Gooch et al. (2012) reported that oxycodone lengthened reproductions of time intervals with humans. Although some data suggest that choice and timing in concurrent chains may be controlled by different variables (e.g., Grace & Nevin, 1999; Kyonka & Grace, 2007), an interpretation of the effects of opioids on impulsive choice in terms of a disruption in temporal stimulus control cannot be ruled out (e.g., see Smith et al., 2015).

Another consideration is that potential differences in nondrug performance between females and males is a determinant of the effects of oxycodone. For example, choice in males was more sensitive to reinforcement delay and there was a bigger, more reliable effect of oxycodone on choice in males compared to females. Thus, if there is greater control (i.e., sensitivity) by reinforcement delay, oxycodone may affect (reduce in this case) sensitivity to delay more. Such baseline-dependent effects have been suggested by others in the context of impulsive choice (see Bickel et al., 2016; Pope et al., 2020). The procedure used within the current study may be an effective way to examine questions related to behavioral mechanisms of drug action (Hughes et al., 2022).

Naloxone

Effects of naloxone, both alone and in combination with oxycodone, warrant further investigation. Immediately following the acute drug regimen, naloxone (3.0 mg/kg) was co-administered with oxycodone and with saline. There was generally an attenuation of the oxycodone-related decreases in sensitivity. These findings are consistent with those in which opioid antagonists have been shown to reverse effects of opioids on impulsive choice (e.g., Kieres et al., 2004; Pattij et al., 2009). However, naloxone generally increased sensitivity for females and decreased response rates for both sexes when given alone (i.e., co-administered with saline). These effects of naloxone alone are surprising; the dose used (3.0 mg/kg) was selected because it generally does not affect schedule-controlled operant behavior (e.g., Gellert & Sparber, 1977; Sanger & McCarthy, 1982; Wiebelhaus et al., 2016). One potential account for these findings is that the female rats may have been mildly opioid dependent during the acute drug phase. Indeed, Harvey-Lewis et al. (2015) found that naloxone alone increased impulsive choice on a delay discounting procedure for morphine-dependent rats, but did not affect impulsive choice for drug-naive rats. Alternatively, perhaps the present choice procedure used is more sensitive to general pharmacological effects (e.g., naloxone’s anorexic effects; Holtzman, 1974, 1975; Weldon et al., 1996) relative to other procedures commonly used in the literature. These conclusions should be considered tentative given the limited literature on naloxone with female rats and the absence of observational measures of opioid dependence prior to the chronic procedure.

Chronic Effects of Oxycodone

During chronic administration of oxycodone, tolerance to the sensitivity-decreasing effects of oxycodone developed in four of the five females whereas sensitization developed in all five males. Interestingly, in the males, sensitization remained when the same dose was tested postchronically compared to acutely. There is evidence of sex differences in the development of tolerance to opioids’ antinociceptive effects in rats (e.g., Barrett et al., 2001); however, it is not clear why different chronic effects on sensitivity to reinforcement delay were seen in the current study. In addition, it is not known whether the shifts in the sensitivity measures in males would have remained as further testing of the rats was precluded due to interruption by the COVID-19 pandemic. However, the effects on response rate are clearer in that there was some degree of tolerance to the response rate-decreasing effects of oxycodone across most rats of both sexes. These results suggest that tolerance or sensitization to effects on sensitivity were not functions of changes in motivation to respond (i.e., reductions in response rate).

The results of the video measures for chronic oxycodone and antagonist-induced withdrawal tests largely do not suggest that physical dependence developed. While chewing has also been observed in other rodents under chronic oxycodone administration (e.g., mice in Enga et al., 2016), other behaviors (e.g., jumping; wet dog shakes) would have been expected at higher frequencies/percentages of intervals in order for physical dependence of the drug to be claimed (for evidence of this with morphine dependence, see Beardsley et al., 2004). It is possible that dependence could have been detected via other methods (e.g., intracranial self-stimulation; Wiebelhaus et al., 2016), but as it stands, the chronic administration regimen in the current study did not seem to substantially produce any dependence-related behaviors.

Conclusions

Baseline (i.e., nondrug) performance by both sexes provides support for the viability of the concurrent-chains procedure used here for examining the effects of neurobiological manipulations (e.g., drugs) on control of choice by relevant dimensions of reinforcement (see Hughes et al., 2022). Overall, oxycodone tended to decrease sensitivity to reinforcement delay, more so for males than females. The implications of these findings for sex differences in impulsive choice need to be explored further. Although a drug-induced decrease in sensitivity to delay would be expected to decrease impulsive choice, these results need to be considered in the context of oxycodone-related changes in sensitivity to magnitude (Hunt et al., 2020). As discussed above, taken together, the results of the present study and those of Hunt et al. would be expected to oppose each other in the context of an impulsive-choice procedure; the former increases impulsive choice and the latter decreases impulsive choice. Ultimately, however, parametric manipulation of both delay and magnitude in combination are necessary to determine the extent to which oxycodone produces interactive changes between these behavioral mechanisms. The procedure used here seems well-suited to that purpose. Also, once a better understanding of these effects is developed for non-human subjects, implications for their effects in humans could be examined. To that end, there appears to be only one study to date of oxycodone and impulsive choice in humans (Zacny & de Wit, 2009), which found no significant effects. Although there are several reasons why this finding differs from those typically reported for opioids with non-humans, it is possible it reflects the opposing effects of opioids on reinforcement magnitude and delay noted above. Clearly, more research is needed to determine what, if any, effects oxycodone may have on impulsive choice in humans.

Public significance statement:

The study presents an important preclinical evaluation of effects of opioids on a critical behavioral mechanism involved in impulsive choice, reinforcement delay. Although baseline sensitivity to delay was lower in females than males, suggesting males may be more impulsive, oxycodone produced comparable decreases in sensitivity in both sexes. Ultimately, these data may inform translational research on risk-factors associated with and negative outcomes of substance use disorder.

Appendix A.

Sensitivity, Bias, Response Rates, and Percentage of Cycles Completed for Individual Rats

			Dose (mg/kg)
	Control	Saline	0.1	0.3	0.56	0.56 + Nal	1.0
Females
Sensitivity
D28	0.74 (0.62, 0.84)	0.56 (0.49, 0.62)	0.90 (0.83, 0.97)	0.65 (0.61, 0.72)	0.82 (0.68, 0.99)	0.95 (-, -)	0.59 (0.51, 0.67)
D29	0.75 (0.59, 0.86)	0.56 (0.55, 0.57)	0.34 (0.29, 0.50)	0.51 (0.46, 0.71)	0.36 (0.28, 0.43)	0.81 (-, -)	0.60 (0.52, 0.67)
D30	0.65 (0.61, 0.79)	0.77 (0.56, 0.84)	0.62 (0.53, 0.69)	0.53 (0.48, 0.65)	0.31 (0.18, 0.37)	0.54 (-, -)	0.42 (0.41, 0.62)
D31	0.54 (0.49, 0.60)	0.69 (0.60, 0.76)	0.77 (0.53, 0.97)	0.60 (0.58, 0.67)	0.47 (0.45, 0.50)	0.66 (-, -)	0.37 (0.22, 0.57)
D32	0.30 (0.25, 0.37)	0.20 (0.17, 0.25)	0.25 (0.17, 0.30)	0.14 (0.11, 0.24)	0.25 (0.21, 0.28)	0.17 (0.16, 0.18)	−0.10 (−0.19, −0.01)
E16*	0.78 (0.66, 0.94)	0.90 (0.82, 0.93)	0.64 (0.58, 0.81)	0.73 (0.40, 1.07)	0.53 (0.41, 0.66)	0.99 (-, -)	0(-, -)
Group	0.63 (0.54, 0.73)	0.61 (0.53, 0.66)	0.59 (0.49, 0.71)	0.53 (0.44, 0.68)	0.46 (0.37, 0.54)	0.69 (-, -)	0.31 (0.25, 0.42)
Bias
D28	−0.01 (−0.11, 0.08)	0.05 (−0.04, 0.09)	−0.11 (−0.13, −0.10)	−0.15 (−0.21, −0.04)	−0.21 (−0.24, −0.20)	−0.22 (-, -)	−0.20 (−0.24, −0.17)
D29	0.02 (−0.04, 0.05)	0.03 (0.03, 0.07)	0.08 (0.06, 0.12)	−0.10 (−0.13, −0.06)	−0.21 (−0.31, −0.18)	−0.19 (-, -)	−0.12 (−0.21, −0.03)
D30	0.31 (0.25, 0.37)	0.22 (0.22, 0.25)	0.37 (0.35, 0.44)	0.25 (0.16, 0.28)	0.01 (−0.19, 0.19)	0.17 (-, -)	−0.03 (−0.19, 0.25)
D31	0.14 (0.05, 0.23)	0.12 (0.02, 0.19)	0.06 (−0.04, 0.15)	0.09 (0.04, 0.15)	−0.03 (−0.11, 0.05)	−0.01 (-, -)	−0.09 (−0.21, 0.08)
D32	0.11 (0.05, 0.19)	0.20 (0.13, 0.23)	0.20 (0.13, 0.22)	0.08 (0.06, 0.11)	0.23 (0.12, 0.25)	0.20 (0.19, 0.21)	0.07 (0.01, 0.13)
E16*	0.11 (0.03, 0.17)	0.10 (0.01, 0.18)	0.21 (0.09, 0.27)	0.10 (0.01, 0.15)	0.17 (0.04, 0.21)	0.13 (-, -)	-
Group	0.11 (0.04, 0.18)	0.12 (0.06, 0.17)	0.14 (0.08, 0.18)	0.05 (−0.01, 0.10)	−0.01 (−0.12, 0.05)	0.01 (-, -)	−0.07 (−0.17, 0.05)
Response Rate
D28	68.00 (54.63, 77.17)	81.04 (72.57, 85.09)	34.85 (24.88, 45.02)	68.46 (51.22, 75.27)	53.24 (40.30, 70.41)	55.90 (-, -)	52.77 (50.04, 73.96)
D29	75.37 (64.52, 95.51)	91.86 (77.40, 102.50)	60.43 (57.93, 71.13)	74.52 (70.15, 81.33)	92.51 (80.47, 94.15)	87.66 (-, -)	87.82 (75.14, 111.23)
D30	81.41 (39.39, 87.26)	92.78 (66.55, 152.62)	79.19 (69.33, 92.85)	98.02 (90.52, 101.41)	92.96 (74.57, 98.06)	100.71 (-, -)	53.55 (37.90, 69.87)
D31	101.79 (86.66, 108.60)	110.67 (104.02, 112.21)	100.46 (93.13, 107.06)	69.17 (68.64, 93.05)	77.43 (69.94, 84.91)	66.45 (-, -)	78.09 (66.84, 98.95)
D32	84.00 (80.89, 92.58)	105.01 (91.61, 113.37)	115.27 (110.10, 118.42)	69.61 (68.9, 83.55)	77.59 (52.33, 96.12)	94.27 (90.16, 98.39)	27.35 (14.37, 75.18)
E16*	47.59 (33.85, 63.59)	67.61 (60.20, 75.24)	35.03 (27.03, 39.99)	45.25 (39.57, 50.31)	29.16 (21.76, 35.94)	29.74 (-, -)	7.20 (3.60, 10.81)
Group	76.36 (59.99, 87.45)	91.50 (78.72, 106.84)	70.87 (63.74, 79.08)	70.84 (64.83, 80.82)	70.48 (56.56, 79.93)	72.46 (-, -)	51.13 (41.32, 73.33)
% Session Completed
D28	100.00	100.00	87.83	98.41	88.89	100.00	84.13
D29	99.81	100.00	100.00	100.00	100.00	100.00	84.13
D30	100.00	100.00	100.00	100.00	100.00	100.00	85.71
D31	100.00	100.00	100.00	100.00	100.00	100.00	100.00
D32	100.00	100.00	100.00	100.00	85.71	100.00	62.43
E16*	98.49	100.00	86.51	100.00	84.52	100.00	20.63
Group	99.72	100.00	95.72	99.74	93.19	100.00	72.84
Males
Sensitivity
C5	0.49 (0.34, 0.57)	0.40 (0.33, 0.42)	0.39 (0.30, 0.53)	0.34 (0.16, 0.38)	0.23 (0.23, 0.37)	0.56 (-, -)	0.30 (−0.07, 0.46)
C6	0.83 (0.74, 0.93)	0.83 (0.76, 0.86)	0.80 (0.74, 0.84)	0.51 (0.38, 0.60)	0.42 (0.41, 0.54)	0.72 (-, -)	0.31 (0.29, 0.31)
C7	0.91 (0.80, 1.10)	1.03 (0.79, 1.25)	0.68 (0.63, 0.78)	0.89 (0.73, 1.04)	0.55 (0.49, 0.63)	1.00 (-, -)	0.13 (0.09, 0.21)
D7*	0.62 (0.52, 0.75)	0.42 (0.41, 0.45)	0.51 (0.39, 0.59)	0.78 (0.39, 0.59)	-	0.74 (-, -)	-
D8	0.89 (0.67, 1.16)	0.82 (0.61, 1.03)	0.54 (0.45, 0.64)	0.79 (0.61, 0.93)	0.49 (0.42, 0.52)	0.91 (-, -)	0.36 (0.34, 0.37)
D9	0.82 (0.66, 0.94)	0.73 (0.70, 0.77)	0.65 (0.61, 0.70)	0.58 (0.53, 0.62)	0.70 (0.42, 0.83)	0.90 (-, -)	0.14 (0.01, 0.19)
D10*	0.53 (0.37, 0.60)	0.33 (0.30, 0.40)	0.35 (0.24, 0.43)	0.44 (0.37, 0.51)	-	0.62 (-, -)	-
D11	1.02 (0.88, 1.15)	1.04 (0.79, 1.13)	1.01 (0.93, 1.11)	0.84 (0.47, 1.15)	0.60 (0.48, 0.84)	1.43 (-, -)	0.06 (0.02, 0.55)
Group	0.76 (0.62, 0.90)	0.70 (0.57, 0.79)	0.62 (0.54, 0.70)	0.65 (0.50, 0.75)	0.50 (0.41, 0.62)	0.86 (-, -)	0.22 (0.11, 0.35)
Bias
C5	0.14 (0.04, 0.20)	0.04 (0.00, 0.17)	0.18 (0.14, 0.21)	0.19 (0.17, 0.30)	0.11 (0.11, 0.18)	0.04 (-, -)	−0.04 (−0.08, 0.05)
C6	−0.14 (−0.23, −0.07)	−0.14 (−0.21, −0.06)	−0.13 (−0.20, −0.09)	−0.36 (−0.38, −0.34)	−0.43 (−0.48, −0.36)	−0.22 (-, -)	−0.40 (−0.42, −0.31)
C7	−0.18 (−0.23, −0.14)	−0.14 (−0.19, −0.10)	−0.17 (−0.25, −0.17)	−0.24 (−0.32, −0.17)	−0.26 (−0.31, −0.24)	−0.08 (-, -)	−0.11 (−0.20, 0.04)
D7*	−0.27 (−0.35, −0.20)	−0.16 (−0.24, −0.11)	−0.08 (−0.14, −0.06)	−0.28 (−0.28, −0.28)	-	−0.29 (-, -)	-
D8	0.20 (0.11, 0.25)	0.14 (0.10, 0.21)	0.32 (0.27, 0.37)	0.19 (0.17, 0.22)	0.27 (0.07, 0.28)	0.25 (-, -)	0.25 (0.11, 0.29)
D9	−0.09 (−0.14, 0.02)	−0.12 (−0.15, −0.10)	−0.19 (−0.27, −0.05)	−0.15 (−0.21, −0.07)	−0.04 (−0.1, 0.04)	0.04 (-, -)	0.12 (0.00, 0.16)
D10*	−0.11 (−0.17, −0.07)	−0.08 (−0.13, −0.07)	−0.12 (−0.12, −0.02)	−0.03 (−0.05, 0.01)	-	0.08 (-, -)	-
D11	−0.08 (−0.13, −0.01)	−0.11 (−0.18, 0.02)	−0.09 (−0.12, 0.01)	−0.13 (−0.20, −0.05)	−0.04 (−0.18, 0.09)	0.03 (-, -)	−0.09 (−0.14, 0.09)
Group	−0.07 (−0.14, 0.00)	−0.07 (−0.13, −0.01)	−0.04 (−0.09, 0.03)	−0.10 (−0.14, −0.05)	−0.07 (−0.15, 0.00)	−0.02 (-, -)	−0.05 (−0.12, 0.05)
Response Rate
C5	94.47 (85.71, 107.37)	91.32 (90.57, 100.05)	77.63 (49.45, 104.65)	107.82 (106.72, 109.56)	82.99 (80.67, 85.72)	70.49 (-, -)	47.23 (35.96, 53.13)
C6	80.10 (74.44, 89.91)	79.47 (72.33, 87.77)	76.09 (66.38, 83.58)	68.58 (60.61, 77.84)	77.40 (71.48, 81.81)	100.85 (-, -)	63.85 (51.38, 69.31)
C7	80.16 (73.95, 87.13)	79.75 (72.47, 88.75)	86.06 (73.99, 92.19)	111.59 (92.48, 115.90)	82.30 (66.85, 85.23)	76.47 (-, -)	68.22 (61.51, 73.83)
D7*	75.46 (65.82, 86.32)	74.79 (73.79, 75.60)	61.36 (46. 57, 71.20)	39.42 (22.55, 56.11)	5.76 (4.00, 7.31)	35.17 (-, -)	0.00 (0.00, 0.00)
D8	75.84 (69.24, 77.67)	76.52 (73.44, 80.04)	74.50 (66.84, 82.29)	72.23 (63.54, 74.27)	42.59 (37.83, 52.10)	40.65 (-, -)	53.28 (50.86, 53.46)
D9	83.61 (69.21, 93.39)	90.98 (70.22, 107.71)	72.05 (64.45, 81.98)	73.05 (61.27, 76.04)	46.02 (34.64, 61. 49)	43.53 (-, -)	29.59 (23.02, 35.28)
D10*	51.79 (42.19, 64.03)	49.37 (47.39, 61.55)	63.95 (49.63, 67.12)	29.06 (22.01, 42.22)	13.01 (11.11, 14.23)	37.77 (-, -)	13.43 (7.95, 14.90)
D11	79.10 (65.43, 82.85)	78.60 (76.10, 101.81)	71.54 (67.66, 78.70)	53.14 (35.25, 75.92)	41.80 (35.39, 58.55)	68.15 (-, -)	29.98 (21.55, 40.49)
Group	77.57 (68.25, 86.08)	77.60 (72.04, 87.91)	72.90 (60.62, 82.71)	66.97 (54.14, 78.21)	48.98 (42.74, 55.81)	59.13 (-, -)	38.20 (31.53, 42.59)
% Session Completed
C5	100.00	100.00	98.02	100.00	100.00	100.00	94.05
C6	99.13	100.00	100.00	90.48	100.00	100.00	100.00
C7	100.00	100.00	100.00	94.84	100.00	100.00	100.00
D7*	100.00	100.00	98.41	59.52	19.58	100.00	0.00
D8	100.00	100.00	100.00	100.00	100.00	100.00	95.77
D9	99.92	100.00	100.00	100.00	99.60	100.00	80.16
D10*	99.44	100.00	100.00	91.27	49.21	100.00	20.63
D11	100.00	100.00	100.00	98.41	98.41	100.00	79.76
Group	99.81	100.00	99.55	91.82	83.35	100.00	71.30

Note. Median (quartile one, quartile three) sensitivity, bias, and response rates for individual rats. Data for rats E16, D7, and D10 are marked with an asterisk (*) to indicate that the data included here were excluded from the statistical analyses and figures reported in the main text.

Appendix B.

Operational Definitions of Chronic/Withdrawal Measures

Measures
	Partial-Interval
Ptosis	Eyes are squinted or drooping.
Chewing	Rat bites themselves, or any parts of their cage.
Non-genital grooming	Rat licks/scratches any part of themselves besides their genitals.
Genital grooming	Rat licks/scratches their genitals.
Writhing	Initiation in stretching of the abdomen or legs.
	Whole-Interval
Stationary	Rat’s nose does not move outside of (or in-between) video grids and remains at least partially visible. Note: This measure is not counted when subjects are already engaging in grooming, wet dog shakes, jumping, or chewing within an interval.
	Count
Jumping	No limbs contact the floor or walls of the cage.
Wet dog shakes	Shuddering of the head, neck, or trunk.
Coprophagia	Consumption of a fecal unit.
Fecal boli	Number of fecal units in cage.
Diarrhea	Fecal units are of a liquid and softened form.
Irritability	Rat squeaks upon being picked up.

Note. Shown are measures (left columns), with operational definitions (right columns), that were utilized in the observation of video-recorded chronic oxycodone/naloxone substitution sessions; measures that were recorded immediately after the session, rather than from videos, are italicized.

Appendix C. Selected Individual Female and Male Rats: Sensitivity and Within-Session Effects.

Note. Top panel: Median sensitivity to reinforcement delay for a representative female (left) and male (right) rat. These subjects were selected because the shapes of their dose-effect functions, shown in the top row, closely approximated the average functions of their respective groups shown in Figure 2. Data points above C represent data from non-injection control sessions immediately prior to an injection; data points above S represent data from sessions before which saline was administered. Filled triangles above saline and 0.56 mg/kg represent data from sessions before which 3.0 mg/kg naloxone was administered in combination with saline or 0.56 mg/kg oxycodone, respectively. Error bars are interquartile ranges. Bottom panel: Log response ratio (V/S) across fifths of the block when the delay ratio favored the variable (circles), was equal for variable and standard (squares), and favored the standard (triangles) lever during control conditions (filled) and when 0.56 mg/kg oxycodone was administered (open). Data points are means and error bars are 95% CIs.

Appendix D.

Chronic Oxycodone/Abstinence-Induced Withdrawal Measures

Females
Measures	D28	D29	D30	D31	D32
	Oxycodone (1.0 mg/kg)
Ptosis	-	-	-	-	-
Chewing	8.33	100.00	100.00	11.67	100.00
Non-genital grooming	13.33	-	8.33	3.33	1.67
Genital grooming	1.67	-	-	-	-
Writhing	-	-	-	-	-
Stationary	-	-	-	-	2.78
Jumping	3	-	-	-	-
Wet dog shakes	-	-	-	-	1
Coprophagia	-	-	-	-	-
	Naloxone (1.0 mg/kg)
Ptosis	-	-	13.33	33.33	45.00
Chewing	-	-	-	28.33	26.67
Non-genital grooming	33.33	1.67	8.33	18.33	13.33
Genital grooming	1.67	-	8.33	-	-
Writhing	-	-	-	-	-
Stationary	56.25	60.00	68.18	35.29	39.29
Jumping	-	-	-	-	-
Wet dog shakes	-	-	-	-	-
Coprophagia	-	-	-	-	-
Fecal boli	0	1	0	0	0
Diarrhea	No	Yes	No	No	No
Irritability	No	No	No	No	No
	Naloxone (3.0 mg/kg)
Ptosis	-	1.67	8.33	43.33	1.67
Chewing	3.33	6.67	10.00	25.00	3.33
Non-genital grooming	25.00	3.33	-	15.00	15.00
Genital grooming	1.67	1.67	5.00	3.33	1.67
Writhing	-	-	-	-	-
Stationary	14.58	34.78	56.60	30.61	8.89
Jumping	-	-	-	-	-
Wet dog shakes	-	-	-	-	1
Coprophagia	-	-	-	-	-
Fecal boli	0	1	0	0	3
Diarrhea	No	Yes	No	No	No
Irritability	No	No	No	No	No
Males
Measures	D7	D8	D9	D10	D11
	Oxycodone (1.0 mg/kg)
Ptosis	-	-	-
Chewing	100.00	23.33	100.00
Non-genital grooming	1.67	-	-
Genital grooming	-	-	-
Writhing	-	-	-
Stationary	-	-	-
Jumping	-	-	-
Wet dog shakes	-	-	-
Coprophagia	-	-	-
	Naloxone (1.0 mg/kg)
Ptosis	-	-	-	-	-
Chewing	-	-	-	-	6.67
Non-genital grooming	28.33	23.33	6.67	11.67	5.00
Genital grooming	3.33	3.33	-	-	-
Writhing	-	-	-	-	-
Stationary	52.38	19.23	33.33	64.87	3.92
Jumping	-	-	-	-	-
Wet dog shakes	-	-	-	-	-
Coprophagia	-	-	-	-	-
Fecal boli	0	1	1	9	3
Diarrhea	No	No	No	No	No
Irritability	No	No	No	No	No

Note. Male Rats D10 and D11 were not video recorded following 1.0 mg/kg oxycodone administration. Non-video measures, recorded immediately after the session rather than from videos, are italicized; these measures were not recorded following 1.0 mg/kg oxycodone administration. Mean IOA amongst video recorders for each measure, calculated only from sessions in which at least one instance of the behavior was recorded by at least one observer, were as follows: ptosis (91.0%; range: 83.3-98.3%), chewing (91.2%; 71.7-100.0%), non-genital grooming (91.5%; 73.3-98.3%), and stationary behavior (83.3%; 73.0-98.3%). Additionally, mean IOA for non-video measures, calculated for all measures but irritability, were as follows: fecal boli (100%) and diarrhea (88.24; 0-100%).