A Novel Assay Allowing Drug Self-Administration, Extinction, and Reinstatement Testing in Head-Restrained Mice

Abstract

Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.

Keywords: addiction; calcium imaging; ensembles; longitudinal tracking of individual cells; two-photon (2P).

FIGURE 1

Head-restrained mice self-administer heroin and display extinction and reinstatement of heroin seeking. (A) An experimental timeline demonstrating the behavioral procedure used for head-restrained heroin self-administration. (B) Illustration of head fixation for heroin self-administration experiments. Active lever presses resulted in a tone cue that predicted intravenous heroin [at doses of 0.1 mg/kg (day 1–2), 0.05 mg/kg (day 3–4), or 0.025 mg/kg (day 5–14)] or intravenous saline. (C) Schematic for heroin/saline self-administration experiments, where active but not inactive lever presses resulted in a tone cue (2s), a gap in time (TI, trace interval; 1s), an infusion of heroin or saline, and a timeout period (20s). (D) Data showing that heroin self-administering mice pressed the active lever more than the inactive lever (****p < 0.001), whereas saline self-administering mice did not. (E) A comparison of active and inactive lever pressing across sexes. Male and female heroin self-administering mice showed no differences in lever pressing across acquisition. (F) During extinction, heroin self-administering mice significantly decreased active lever pressing from the first day of extinction to the last (****p < 0.001). (G) Data showing that mice displayed cue- (****p < 0.001), heroin- (***p = 0.001), yohimbine- (**p = 0.002), and predator odor (TMT; *p = 0.048)-induced reinstatement of active lever pressing as compared with the previous extinction test. Mice did not reinstate following an injection of saline.

FIGURE 2

Head-restrained mice self-administer cocaine and display extinction and reinstatement of cocaine seeking. (A) An experimental timeline demonstrating the behavioral procedure used for head-restrained cocaine self-administration. (B) Illustration of head fixation for cocaine self-administration experiments. Active lever presses resulted in a tone cue that predicted intravenous cocaine (0.75 mg/kg on all days) or saline. (C) Schematic for cocaine self-administration experiments, where active but not inactive lever presses resulted in a tone cue (2s), a gap in time (TI, trace interval; 1s), an infusion of cocaine or saline, and a timeout period (20s). (D) Data showing that cocaine self-administering mice pressed the active lever more than the inactive lever (**p = 0.002). (E) A comparison of active and inactive lever pressing across sexes. Male and female cocaine self-administering mice showed no differences in lever pressing across acquisition. (F) All cocaine self-administering mice significantly decreased active lever pressing from the first day of extinction to the last (**p = 0.006). (G) Mice displayed cue- (**p = 0.007), cocaine- (**p = 0.002), yohimbine- (*p = 0.017), and predator odor (TMT; *p = 0.019)- induced reinstatement of active lever pressing as compared with the previous extinction test. Mice did not reinstate following an injection of saline.

FIGURE 3

Head-restrained mice self-administer sucrose and display reinstatement of sucrose seeking to the reward-associated cue. (A) An experimental timeline demonstrating the behavioral procedure used for head-restrained sucrose self-administration. (B) Illustration of head fixation for sucrose self-administration experiments. Active lever presses resulted in a tone cue that predicted a sucrose droplet (12.5 μl of 12.5% sucrose). (C) Schematic for sucrose self-administration experiments, where active but not inactive lever presses resulted in a tone cue (2s), a gap in time (TI, trace interval; 1s), a palatable sucrose droplet, and a timeout period (20s). (D) Sucrose self-administering mice pressed the active lever significantly more than the inactive lever on each day of acquisition (*ps < 0.05). (E) A comparison of active and inactive lever pressing across sexes. Male and female sucrose self-administering mice showed no differences in lever pressing across acquisition. (F) All sucrose self-administering mice significantly decreased active lever pressing from the first day of extinction to the last (****p < 0.001). (G) Sucrose self-administering mice displayed an increase in active lever pressing to cue- (****p < 0.001), but not heroin-, yohimbine-, predator odor (TMT)-, or saline-induced reinstatement tests.