Source: Science Advances
5 Aug 2020 • Vol 6, Issue 32 • DOI: 10.1126/sciadv.abc0413
Intravenous drug self-administration is considered the “gold standard” model to investigate the neurobiology of drug addiction in rodents. However, its use in mice is limited by frequent complications of intravenous catheterization. Given the many advantages of using mice in biomedical research, we developed a noninvasive mouse model of opioid self-administration using vaporized fentanyl. Mice readily self-administered fentanyl vapor, titrated their drug intake, and exhibited addiction-like behaviors, including escalation of drug intake, somatic signs of withdrawal, drug intake despite punishment, and reinstatement of drug seeking. Electrophysiological recordings from ventral tegmental area dopamine neurons showed a lower amplitude of GABAB receptor–dependent currents during protracted abstinence from fentanyl vapor self-administration. This mouse model of fentanyl self-administration recapitulates key features of opioid addiction, overcomes limitations of the intravenous model, and allows investigation of the neurobiology of opioid addiction in unprecedented ways.
Opioid use disorder is a major worldwide public health concern (1). Its prevalence and associated mortality are escalating globally (1, 2), and opioid overdose deaths have reached epidemic proportions (3). Fentanyl, a synthetic opioid that is commonly used clinically for anesthesia and analgesia, accounts for nearly 46% of opioid overdose deaths (3). It is commonly administered intravenously or by inhalation (smoking/vaping), resulting in rapid drug bioavailability in the brain (4).
Currently, intravenous self-administration models are the “gold standard” to study opioid addiction in rodents (5). Rat models of intravenous opioid self-administration and relapse are widely used and have been instrumental in understanding brain circuits that control drug taking and seeking and drug-induced neuroadaptations (6, 7). However, despite major advances in preclinical opioid addiction research, large knowledge gaps in understanding the unique aspects of opioid addiction persist.
Mouse models offer unique advantages compared with rats in neurobiological investigations when considering the numerous behaviorally selected and transgenic mouse strains that allow genetic targeting and manipulation using sophisticated techniques (e.g., imaging, chemogenetics, and optogenetics). Although intravenous self-administration models in mice have been used successfully (8, 9), the use of such models in opioid addiction research remains very scarce and restricts the duration of exposure to drugs because of high catheter failure rates, especially during prolonged access to drugs (8, 10). Intravenous catheters also limit the ability to perform in vivo electrophysiology or calcium imaging experiments in freely moving mice, in part, because of double tethering.
Given the similarities in pharmacokinetics and pharmacodynamics of inhaled and intravenously infused drugs (11), we developed and validated a noninvasive mouse model to study the neurobiology of opioid addiction using vaporized fentanyl, which overcomes many limitations of intravenous models. Mice exhibited several somatic and motivational signs of opioid dependence that resembled opioid use disorder in humans. We also identified long-lasting γ-aminobutyric acid (GABA)ergic neuroadaptations in ventral tegmental area (VTA) dopamine neurons after protracted abstinence.
Vaporized fentanyl-induced analgesia
We first determined a concentration-response function for the analgesic effects of vaporized fentanyl using the hot-plate test. Different concentrations of fentanyl (0 to 30 mg/ml) were vaporized using a modified e-cigarette device (fig. S1). Mice (n = 8 to 24 males) were placed in the behavior chambers and passively exposed to vaporized fentanyl. One minute after the last fentanyl vapor delivery, the mice were placed on a hot plate, and the latency to signs of nociception was recorded. Vaporized fentanyl dose-dependently increased nociception latency, whereas vehicle vapor in the absence of drug had no effect (Fig. 1A).
FIG. 1 Vaporized fentanyl induces analgesia and increases locomotor activity.
(A) Passive administration of vaporized fentanyl (four vapor deliveries over 8 min) increased the latency to nociception in the hot-plate test [one-way analysis of variance (ANOVA); F7,112 = 38.45; P < 0.0001] in a concentration-dependent manner. Sidak’s multiple comparisons test shows that the 1, 3, 10, and 30 mg/ml groups were significantly different from vehicle. “NV” indicates mice that were not exposed to fentanyl or vehicle vapor. (B) Locomotor activity in meters was measured at baseline and after passive exposure to different concentrations of vaporized fentanyl (five vapor deliveries over 10 min) versus vehicle vapor (0 mg/ml). Locomotor activity increased with the concentration of vaporized fentanyl [two-way repeated-measures (RM) ANOVA; concentration × time interaction F15,150 = 10.14; P < 0.0001]. Sidak’s multiple comparisons test shows significant differences in locomotion between the different concentrations. (C) Blood fentanyl levels in response to five fentanyl vapor deliveries (2.5 mg/ml: 1.91 ± 0.39 ng/ml; 10 mg/ml: 8.27 ± 0.95 ng/ml) that were passively delivered over 1 hour (unpaired t test; t17 = 5.93; P < 0.0001). The ratio of fentanyl blood levels at 10 and 2.5 mg/ml (8.27/1.91 = 4.3) is a close approximation of the ratio of fentanyl concentrations (10/2.5 = 4). The regression analysis of measured blood fentanyl levels yields a slope a = 0.85 (CI: 0.55 to 1.15; F1,17 = 35.14; r2 = 0.67; P < 0.0001), which is not different from the presumed linear metabolism slope (𝑎=(1.91×4)−1.9110−2.5=0.76; test of equal slopes F1,17 = 0.033; P = 0.86), suggesting that fentanyl metabolism was linear within the range of five vapor deliveries over 1 hour at 2.5 and 10 mg/ml, equivalent to a range of 12.5 to 50 mg/ml per hour. The number of mice (n) is shown in the graphs. The data are expressed as means ± SEM. *P < 0.05.
Vaporized fentanyl-induced locomotion
On the basis of the concentration-dependent analgesic effects of fentanyl, we investigated the effects of vaporized fentanyl (2.5, 5, and 10 mg/ml) on locomotor activity, a function of the fentanyl effects on the mesolimbic dopaminergic system (12). A separate cohort of mice (n = 34, F/M = 18/16) was placed in locomotor activity boxes for 30 min to measure baseline locomotion. The mice were then passively exposed to vaporized fentanyl. One minute after the last fentanyl vapor delivery, the mice were returned to the locomotor activity boxes. Fentanyl significantly increased locomotor activity in a concentration-dependent manner (Fig. 1B).
Blood fentanyl levels
To confirm that exposure to different concentrations of vaporized fentanyl correlates with blood fentanyl levels, drug-naïve mice were passively exposed to fentanyl vapor (five vapor deliveries over 1 hour) at 2.5 mg/ml (n = 9, F/M = 4/5) or 10 mg/ml (n = 10, F/M = 5/5). Blood samples were collected 2 min after the last vapor delivery. Blood fentanyl levels were significantly higher after vaporized fentanyl at 10 mg/ml compared with 2.5 mg/ml and reflected the linear metabolism of fentanyl within the experimental range of fentanyl concentrations and number of vapor deliveries (Fig. 1C). Blood fentanyl levels were higher in females than in males in response to fentanyl (10 mg/ml) (fig. S2A). This difference may be attributable to the lower body weight of females (females, 19.6 ± 0.7 g; males, 28.2 ±1.3 g; t8 = 5.9, P = 0.0004), which was supported by a negative correlation between body weight and blood fentanyl levels at 10 mg/ml (fig. S2B). Another possibility is that fentanyl is metabolized more slowly in females than in males. An intraperitoneal injection of 0.2 mg/kg fentanyl, a dose that was previously reported to induce analgesia, conditioned hyperlocomotion, and conditioned place preference in mice (13), resulted in comparable blood fentanyl levels and locomotor activity to vaporized fentanyl (2.5 mg/ml) (fig. S2, C and D).
Fentanyl vapor self-administration at different fentanyl concentrations
Using a fixed-ratio 1 (FR1) schedule of reinforcement, a separate group of mice (n = 16 males) quickly learned to nosepoke for fentanyl vapor in 1-hour sessions (movie S1). The mice initially self-administered vaporized fentanyl (10 mg/ml) for eight sessions, followed by eight sessions at 5 mg/ml and then eight sessions at 2.5 mg/ml (Fig. 2A). This experiment was conducted with two groups of mice (n = 8 per group) under different vaporizer power settings that were adjusted to allow exposure to fentanyl vapor for ~1 min (60 W for 1.5 s versus 20 W for 5 s). The data were pooled because both groups exhibited similar results. Mice titrated their fentanyl vapor self-administration based on the fentanyl concentration (i.e., increased active responding with a lower fentanyl concentration) (Fig. 2, A and B). Inactive nosepokes were not different at the different fentanyl concentrations, and the number of active nosepokes was higher than the number of inactive nosepokes, indicating clear discrimination between the two nosepoke ports (Fig. 2B).
FIG. 2 Mice self-administer fentanyl vapor and titrate their intake in response to different vaporized fentanyl concentrations and reinforcement schedules.
(A) Mice self-administered fentanyl vapor in 1-hour sessions on an FR1 schedule and increased their responding when the concentration of vaporized fentanyl was reduced from 10 to 5 to 2.5 mg/ml. The graph shows the number of active and inactive nosepokes (NP) (left y axis) and vapor deliveries (VD) (right y axis) in each self-administration session. Two-way RM ANOVA shows a significant concentration × session interaction (F3.73,55.98 = 4.26; P = 0.005) and significant effect of concentration on the number of VD (F1.38,20.65 = 23.84; P < 0.0001). A similar analysis shows a significant effect of fentanyl concentration on the number of active NP (F1.15,17.21 = 9.73; P = 0.005). The number of inactive NP did not change (P = 0.38). (B) Average of all self-administration sessions at each concentration (the first session at 10 mg/ml was a significant outlier per Grubbs’ test likely because of a novelty effect and was not included in the data analyses). Mice discriminated between active and inactive NP operandum as the fentanyl concentration changed. Two-way RM ANOVA shows a significant concentration × NP interaction (F1.16,17.39 = 10.88; P = 0.003). (C) Mice exhibited an increase in the number of active NP when they were switched from an FR1 to FR5 schedule and then to an FR10 schedule (two-way RM ANOVA; F1.48,19.20 = 19.20; P = 0.0002). The number of inactive NP did not change. The number of VD decreased with increasing FR (two-way RM ANOVA; F1.27,16.51 = 16.35; P = 0.0005). (D) Averaged data from (C) at each FR. (E) The discrimination index of fentanyl vapor self-administration was greater than 0 for FR1, FR5, and FR10 (one-sample t test; FR1: t13 = 4.28, P = 0.0009; FR5: t13 = 6.82, P < 0.0001; FR10: t13 = 9.50, P < 0.0001). The discrimination index increased with increasing FR schedule (one-way RM ANOVA; F1.65,21.39 = 4.17; P = 0.036). The number of mice (n) is shown in the graphs. The data are expressed as means ± SEM. *P < 0.05.
To estimate blood fentanyl levels after the self-administration of different fentanyl concentrations, we used data from Fig. 1C to generate a regression equation (y = 0.6231x). These data reflect blood fentanyl levels after five vapor deliveries that were passively delivered over 1 hour. We calculated the average blood fentanyl level for each vapor delivery at 10, 5, and 2.5 mg/ml (y/5), multiplied by the average number of vapor deliveries at each fentanyl concentration (data from Fig. 2A). The results showed equivalent blood fentanyl levels after vapor self-administration at different fentanyl concentrations (fig. S3A), again indicating that the mice titrated their drug self-administration to their preferred levels.
We also tested the self-administration of vaporized fentanyl at 1 mg/ml in a separate cohort of mice (n = 12 males). The mice did not maintain a stable level of fentanyl vapor self-administration at this concentration (fig. S3, B and C). Although the 2.5 mg/ml concentration elicited a peak self-administration response relative to 10, 5, and 1 mg/ml, the variability in the number of active nosepokes and vapor deliveries at 2.5 mg/ml was higher than at 10 and 5 mg/ml, as measured by the coefficient of variation that was averaged across sessions and animals at each concentration. The coefficient of variation at 10, 5, 2.5, and 1 mg/ml was 16.0, 16.6, 41.0, and 30.3% for active nosepokes, 16.0, 17.8, 22.6, and 27.1% for vapor deliveries, and 25.1, 28.5, 24.4, and 27.2% for inactive nosepokes, respectively. Therefore, we chose the 5 mg/ml fentanyl concentration for the subsequent experiments.
Fentanyl vapor self-administration under different schedules of reinforcement
To test whether mice would expend more effort (“cost”) to obtain fentanyl, a separate cohort of mice was tested for fentanyl vapor self-administration under different schedules of reinforcement. Mice (n = 14, F/M = 6/8) self-administered fentanyl vapor (5 mg/ml) on an FR1 schedule in four sessions, followed by four sessions on an FR5 schedule and four sessions on an FR10 schedule. The mice exhibited a significant increase in the number of active nosepokes at higher FR schedules (Fig. 2, C and D). The number of inactive nosepokes did not change with FR schedule. Although the number of vapor deliveries was lower at higher FR schedules, the discrimination index ([active nosepokes − inactive nosepokes]/[active nosepokes + inactive nosepokes]) was significantly greater than 0 for all FR schedules and increased at higher FR schedules, thus reflecting the specificity of active nosepoke responses (Fig. 2E).
A cohort of mice (n = 14, F/M = 7/7) that self-administered vehicle vapor in the absence of fentanyl did not increase their active nosepoke responding when the reinforcement schedule was increased from FR1 to FR5 and FR10, and the number of vapor deliveries decreased significantly at higher FRs (fig. S4, A and B). Similarly, a separate cohort of mice (n = 8, F/M = 4/4) responded for the light cue in the absence of vehicle or fentanyl vapor, but the number of nosepokes did not change with FR, and the number of light cue presentations decreased significantly at higher FRs (fig. S4, C and D).
When the data were normalized, the relative increase in the number of active nosepokes between FR10 and FR1 was significantly greater in the fentanyl vapor group than in the vehicle vapor and light cue groups (fig. S4E). The relative decrease in the number of vapor deliveries between FR10 and FR1 was significantly smaller in the fentanyl vapor group than in the vehicle vapor and light cue groups (fig. S4F). Furthermore, the discrimination index at FR1 was significantly larger in the fentanyl vapor group than in the vehicle vapor group (fig. S4G). These data indicate that fentanyl vapor was more reinforcing than vehicle vapor or the light cue.
Extinction and reinstatement of fentanyl vapor self-administration
We used a paradigm of extinction followed by cue-induced reinstatement to model relapse. A separate cohort of mice (n = 7 males) self-administered fentanyl vapor (5 mg/ml) in eight 1-hour sessions (FR1), in which vapor deliveries were associated with light cue presentation. On day 9, the mice began extinction training in the absence of cues, during which they were placed in the vapor chambers in 1-hour sessions, but both active and inactive nosepoke responses had no scheduled consequences (Fig. 3A). The number of active nosepokes significantly increased on day 1 of extinction, reflecting drug seeking (Fig. 3B). The mice continued extinction training for 30 sessions, during which the number of active nosepokes significantly decreased, demonstrating the extinction of drug seeking (Fig. 3A). The number of active nosepokes at the end of extinction training remained higher than during the self-administration sessions, an effect that was previously observed in rats (14). This is likely because nosepoking is a prepotent response in rodents that was suppressed during fentanyl self-administration because of fentanyl’s pharmacological effect but was unfettered in the absence of fentanyl.
FIG. 3 Mice extinguish and reinstate fentanyl vapor seeking.
(A) After 8 days of fentanyl vapor self-administration sessions, mice underwent extinction training in the absence of cues for 30 sessions. The number of active NP decreased over sessions, reflecting the extinction of drug seeking. Two-way RM ANOVA shows a significant sessions × NP interaction (F29,348 = 2.23; P = 0.0004) and significant effect of session on the number of NP (F3.24,38.84 = 4.70; P = 0.006). (B) The number of active NP increased significantly on day 1 of extinction training compared with the last day of fentanyl self-administration. Two-way RM ANOVA shows a significant training days × NP interaction (F1,12 = 25.92; P = 0.0003), indicating that day 1 of extinction affected active and inactive NP differently. (C) Mice showed robust light cue–induced reinstatement. Two-way RM ANOVA shows a significant reinstatement × NP interaction (F1,12 = 17.44; P = 0.001). SA, self-administration; n, number of mice. The data are expressed as means ± SEM. *P < 0.05.
After the last day of extinction, we tested the light cue–induced reinstatement of drug seeking. During the reinstatement session (1 hour), the light cue was presented the same way as during the previous self-administration sessions (1-min duration in response to active nosepoke). The results indicated that the light cue significantly reinstated drug seeking (Fig. 3C).
In the next experiment, we tested whether mice extinguish fentanyl vapor self-administration when vehicle vapor and the light cue are available during extinction sessions. A subgroup (n = 8) of the cohort that underwent FR1-FR5-FR10 testing (from Fig. 2C) was transitioned to extinction after their last FR10 self-administration session. These sessions were essentially FR10 vehicle vapor self-administration sessions. The mice did not extinguish nosepoke responses for vehicle vapor and the light cue over 30 sessions (fig. S5A), but the number of active nosepokes was similar to the vehicle vapor self-administration experiment in mice that were never exposed to fentanyl (fig. S4, A and B). The maintenance of active nosepoke responding during extinction in the presence of vehicle vapor and the light cue could be attributed to one or more of the following: (i) vehicle vapor and the light cue that were previously conditioned to fentanyl delivery serve as powerful conditioned reinforcers (15, 16), (ii) vehicle vapor has a sweet flavor because of glycerol and may be reinforcing, and (iii) nosepoking is a prepotent response in rodents. Thus, when designing extinction studies, one needs to consider prepotent responding on the operandum (e.g., nosepoke versus lever press) and possible intrinsic reinforcing properties of the cues or the vehicle that is used to dissolve and vaporize the drug of interest (e.g., appetitive sensory properties).
Escalation of fentanyl vapor self-administration
To test whether mice develop tolerance and escalate fentanyl vapor self-administration, three cohorts of mice were trained as follows: (i) short-access fentanyl (ShA-Fen) group (n = 16, F/M = 8/8) that self-administered fentanyl vapor in 1-hour sessions throughout the experiment; (ii) long-access vehicle (LgA-Veh) group (n = 12, F/M = 8/4) that self-administered vehicle vapor in 1-hour sessions (eight sessions), followed by 12-hour sessions (10 sessions, every other day); and (iii) long-access fentanyl (LgA-Fen) group (n = 16, F/M = 8/8) that self-administered fentanyl vapor in 1-hour sessions (eight sessions), followed by 12-hour sessions (10 sessions, every other day; Fig. 4A). Mice in the LgA-Fen group escalated their fentanyl vapor self-administration across sessions, whereas no escalation was observed in the ShA-Fen or LgA-Veh group (Fig. 4A). These findings are consistent with sufentanil vapor self-administration in rats (14) and intravenous rat and mouse self-administration models (9, 17, 18). Linear regression indicated that the escalation slope in the LgA-Fen group [a = 4.0, confidence interval (CI): 2.5 to 5.6] was significantly greater than 0, unlike in the ShA-Fen and LgA-Veh groups, and all of the slopes in the three groups were different from each other (Fig. 4A). The number of vapor deliveries during the first hour of self-administration (Fig. 4B) in the LgA-Fen group also escalated. Both sexes exhibited significant escalation over time, but the escalation slope was significantly higher in male mice (fig. S6), suggesting that male and female mice escalated their intake at different rates. This finding may be related to the higher drug intake in females early during the escalation phase (fig. S7).
FIG. 4 Mice escalate fentanyl vapor self-administration.
(A) The long-access fentanyl (LgA-Fen) group escalated their intake over time. Linear regression analysis shows a positive slope for the LgA-Fen group (a = 4.04; CI, 2.46 to 5.62), which is significantly greater than 0 (F1,158 = 25.53; r2 = 0.14; P < 0.0001). The calculated slopes for the short-access fentanyl (ShA-Fen) group (a = −0.058; CI, −0.33 to 0.22) and long-access vehicle (LgA-Veh) group (a = −0.64; CI, −1.92 to 0.63) were not different from 0. The slopes from the three groups were different from each other (test of equal slopes; F2,434 = 18.77; P < 0.0001). (B) In the LgA-Fen group, the number of VD during the first hour of self-administration increased across escalation days (slope a = 0.56; CI, 0.31 to 0.82; F1,158 = 18.82; r2 = 0.11; P < 0.0001). n, number of mice. The data are expressed as means ± SEM. *P < 0.05.
We tested somatic signs of naloxone-precipitated opioid withdrawal in mice from the escalation experiment after the last self-administration session. LgA-Fen mice exhibited significantly higher withdrawal scores compared with ShA-Fen mice, whereas LgA-Fen and ShA-Fen mice exhibited higher scores than LgA-Veh mice (fig. S8A). Female mice exhibited greater somatic withdrawal scores compared with male mice, and this difference was most pronounced in the ShA-Fen group (fig. S8B), consistent with intravenous heroin self-administration in mice (9). Similar sex differences were observed in humans with opioid use disorder, in which women presented more signs of opioid withdrawal as measured by the Clinical Opiate Withdrawal Scale (19).
Self-administration of capsaicin-adulterated fentanyl vapor
To test whether the mice continue to self-administer fentanyl vapor despite punishment, we added the respiratory irritant capsaicin (0.2%) to the fentanyl solution. LgA-Fen and LgA-Veh mice from the escalation cohort were gradually transitioned to 1-hour self-administration sessions over 5 days. This was followed by two sessions, in which fentanyl (or vehicle) was adulterated with capsaicin. Mice responded differently to capsaicin, depending on their history of fentanyl self-administration. LgA-Veh mice were the most susceptible to the suppressive effect of capsaicin on self-administration, whereas LgA-Fen mice were the most resistant (Fig. 5A). LgA-Veh mice exhibited a lower number of vapor deliveries on the first day of capsaicin exposure, whereas ShA-Fen and LgA-Fen mice did not. Although capsaicin reduced the number of vapor deliveries in the second session in both ShA-Fen and LgA-Fen mice, this effect was smaller in LgA-Fen mice (Fig. 5B). In the LgA-Fen group, 8 of 16 mice exhibited a <25% reduction of vapor deliveries in the second capsaicin session versus 3 of 16 mice in the ShA-Fen group. Only 1 of 16 mice in the LgA-Fen group exhibited a >75% reduction of vapor deliveries compared with 8 of 16 in the ShA-Fen group (Fig. 5B). Overall, these findings suggest that mice in the LgA-Fen group were more resistant to punishment than ShA-Fen mice, and both fentanyl groups were more resistant than LgA-Veh mice.
FIG. 5 Mice with a history of LgA-Fen vapor self-administration are more resistant to capsaicin-adulterated fentanyl. (A) The LgA-Veh group was most susceptible to the suppressive effects of capsaicin vapor, whereas the LgA-Fen group was the most resistant. Two-way RM ANOVA shows a significant capsaicin exposure × group interaction (F4,74 = 3.01; P = 0.02) and a significant effect of capsaicin on the number of VD (F1.98,73.14 = 21.77; P < 0.0001). The number of VD decreased only in the second capsaicin session in the ShA-Fen group (P < 0.0001) and LgA-Fen group (P = 0.01) of mice with a greater reduction in the ShA-Fen group compared with the LgA-Fen group. (B) Data from the second capsaicin session in ShA-Fen and LgA-Fen mice in (A) were normalized to baseline VD to illustrate the greater reduction of VD in the ShA-Fen group compared with the LgA-Fen group in response to the second capsaicin session (unpaired t test; t30 = 3.13; P = 0.004). Half of the LgA-Fen mice exhibited less than a 25% reduction of the number of VD, whereas half of the ShA-Fen mice exhibited greater than a 75% reduction. Dotted lines represent the quartiles. The number of mice (n) is shown in the bars. The data are expressed as means ± SEM. *P < 0.05.
Effects of fentanyl vapor self-administration on GABAB receptor–mediated currents in VTA dopamine neurons
The activation of GABAB receptors on dopamine neurons causes a large hyperpolarization through the activation of G protein–coupled inwardly rectifying potassium (GIRK) channels (20) and results in a reduction of dopamine neuronal firing and dopamine release in several brain areas, including the striatum (21–23). Clinical and preclinical studies showed that striatal dopamine release in response to drugs or drug cues changes after chronic exposure to opioids (24–26). In addition, GABAB receptor currents in VTA dopamine neurons are known to be lower during acute and subacute (≤7 days) withdrawal after repeated, passive opioid administration (27, 28).