Animals

All mice (20–30 g) were male and received from the Jackson Laboratories (Bar Harbor, ME) at 10 or 30 weeks of age. Mice were housed in a temperature-controlled room maintained on a reverse 12 h light/dark cycle (0700–1900h). Following surgery, mice were housed individually and allowed ad libitum access to water and food. Mice were restricted to 85–90% bodyweight for the duration of behavioral measures. Of note, wild-type (WT) and HD mice are of similar bodyweights, as we have previously shown [20]. All experiments were conducted in the animal’s light cycle. Animal care and experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Use and Care Committee at the University of Maryland, Baltimore.

Surgery

Mice were anesthetized with isoflurane in O 2 (4% induction and 1% maintenance) and implanted with a chronic voltammetry electrode targeting the NAc core (+1.2 anteroposterior (AP), +1.1 mediolateral (ML), −3.7 dorsoventral (DV) relative to Bregma) and a Ag/AgCl reference electrode in the contralateral superficial cortex. All components were permanently affixed with dental cement (Grip Cement Dentsply).

In vivo fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) at chronically implantable electrodes [22] was used to monitor dopamine concentration changes during the progressive-ratio (PR) task as previously described [20]. Individual carbon fibers (r = 3.5 µm, Hexcel Corporation) were aspirated into a 5 mm length segment of fused silica. The seal between the silica and carbon fiber was created by applying a two-part epoxy (Super Glue Corporation; TQs12 Epoxy). The exposed carbon fiber was then cut to ~150 µm and a silver connector was attached on the opposite end for conductivity. A triangular waveform (−0.4 V to +1.3 V at 400 V/s) was applied at 10 Hz to implanted carbon fiber microelectrodes. Principal component regression (PCR) was used to statistically extract the dopamine component from the voltammetric recording of current [23, 24]. The training set for PCR consisted of five background-subtracted dopamine, and five basic pH shift voltammograms. Measured current was converted to concentration based on a data set developed in vitro using a flow cell apparatus to quantify dopamine oxidation current versus non-faradaic background current [25]. The reward-evoked dopamine signal was quantified as the maximal change in dopamine concentration during the 5 s period following reward delivery (see dashed lines in Fig. 1d) relative to 0.5 s preceding reward delivery. Voltammetry data from a recording session were included in the analysis if all reward-evoked dopamine measures met the statistical criteria for the PCR analysis.

Fig. 1 Motivation and dopaminergic encoding of reward is compromised in 10-month-old HD mice and facilitated by endocannabinoids. a Representative cumulative responding during the progressive ratio task for a sucrose pellet reward in WT (left panel) and HD (right panel) mice following each drug treatment. Treatments are vehicle (VEH: WT, n = 11; HD, n = 9), AM-251 (AM: WT, n = 10; HD, n = 9), JZL-184 (JZL: WT, n = 10; HD, n = 11), and JZL-184+AM-251 (JZL+AM: WT, n = 10; HD, n = 9). Vertical tick marks demarcate reward receipt. Drug treatment increased b mean (+SEM) breakpoints (main effect treatment; two-way ANOVA: F (3,71) = 16.32, p < 0.001) and c rewards earned (main effect treatment; two-way ANOVA: F (3,71) = 12.74, p < 0.001) during PR sessions, which were both greater in WT versus HD mice (Breakpoint; main effect genotype; two-way ANOVA: F (1,71) = 34.63, ###p < 0.001; Reward count; main effect genotype; two-way ANOVA: F (3,71) = 29.44, ###p < 0.001). d Mean (+SEM) dopamine concentrations (DA) aligned to reward receipt (arrow). Drug treatment increased e mean (+SEM) change (Δ) in reward-evoked (DA) (main effect treatment; two-way ANOVA: F (3,71) = 28.89, p < 0.001), which was greater in WT versus HD mice (main effect genotype; two-way ANOVA: F (3,71) = 84.13, ###p < 0.001). Time period during which [DA] was compared is demarcated by vertical dashed lines in (d). Post hoc t-test; ***p < 0.001, **p < 0.01, *p < 0.05 JZL versus other treatments Full size image

In vitro fast-scan cyclic voltammetry

Coronal slices including NAc were prepared from 10-month-old WT and HD mice following methods described previously [26]. Slices were placed in an interface chamber and continually perfused (2 ml/min) with artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 126, KCl 2.5, NaH 2 PO 4 1.2, CaCl 2 2.4, MgCl 2 1.2, NaHCO 3 25, Glucose 11, HEPES 20, l-ascorbic acid 0.4, pH 7.4, temperature 32 °C. FSCV recordings of electrically evoked dopamine release were performed using Demon acquisition and analysis software [27] at a glass-encased cylindrical carbon fiber placed in the NAc. Stimulation pulses (2 ms pulse width) were delivered by a constant current isolated stimulator (A-M Systems, WA) through a bipolar tungsten electrode in contact with the slice. Stimulation pulse duration and timing were controlled by a Master-8 (A.M.P.I., Jerusalem, Israel). Dopamine release was monitored using FSCV by applying a triangular waveform (−0.4 to +1.2 V at 400 V/s) at 10 Hz to the carbon fiber. An input–output response curve was generated by applying increasing stimulation intensities (100–600 µA). Rate of dopamine uptake was determined from the 0.4 mA evoked signal using a first-order rate constant (k) with Demon Analysis [27]. For pharmacological assays, stimulation was controlled using Axon pClamp 9 Electrophysiological Data Acquisition and Analysis. Drugs were applied to the bath as vehicle (VEH), followed by JZL-184 (1 or 2 μM), and then sulpiride (2 μM). Following the onset of drug application, electrical stimulation (0.3 mA) was applied to the slice every 2.5 min. The evoked dopamine concentration was averaged across 3 stimulations and then compared across groups and treatments.

Apparatus

Mice were tested in operant chambers (21.6 cm × 17.6 cm × 14 cm; Med Associates, St Albans, VT) housed within sound-attenuating enclosures. Each chamber was equipped with two retractable levers (located 2 cm above the floor) and one LED stimulus light located above each lever (4.6 cm above the lever). An external food magazine delivered sucrose pellets (14 mg; Bio-Serv, Frenchtown, NJ) to a dispenser centrally located between the two levers. A house light and a white-noise speaker (80 dB, masking noise background) were located on the opposite wall.

Behavior

Mice were trained to lever press for sucrose pellet reinforcement as previously described [20]. Training began with 30 min sessions of a fixed-ratio (FR) 1 schedule that included a 10 s timeout following reward receipt which WT and HD mice perform at similar levels [20]. Mice were switched to PR after stable responding was established (under 15% variation in response rate across three consecutive sessions). The PR schedule of positive reinforcement was used to quantify the effort that animals were willing to expend for a reward [28]. The number of lever presses required to earn each reward (i.e., response ratio) increased exponentially across successive trials (response ratio = [5 × e(0.2 × reward number) – 5]), yielding ratios of 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, etc. after rounding to the closest integer. The final ratio attained (i.e., breakpoint) is considered a metric of inherent motivation to expel effort to gain reward. FSCV recordings and drug treatments took place after individual animals displayed stable responding on the PR task (varying by no more than ±1 response ratio over 3 consecutive sessions). Session onset was signaled by both levers extending and illumination of the house light and cue light above the active lever. Responses on the inactive lever were recorded but had no programmed consequences. Upon reaching the response requirement on each trial, a single pellet was delivered, both levers were retracted, and house and cue lights dimmed for a 20 s period before the next trial began. Sessions were terminated after 20 min passed without reward delivery.

Pharmacology

Mice were treated prior to the PR session with vehicle, AM-251 (0.75 mg/kg), JZL-184 (18 mg/kg), or JZL (18 mg/kg)+AM-251 (0.75 mg/kg). Injections were administered intraperitoneally and assigned using a Latin square design with a minimum of 3 days between treatments [29, 30]. Pretreatment times were 30 min for AM-251 and vehicle, and 120 min for JZL [31]. Drugs were prepared in a 1:1:18 vehicle consisting of emulphor, ethanol, and saline, respectively. For slice voltammetry recordings, JZL-184 was dissolved in the VEH solution consisting of 0.1% dimethyl sulfoxide and 1.5% cyclodextrin dissolved in ACSF. Sulpiride was dissolved in ACSF.

Statistics

Mean dopamine concentration changes and behavioral measures were compared between genotypes and across drug treatments using a two-way analysis of variance (ANOVA). Because drug treatments similarly affected both genotypes (i.e., no genotype × treatment interaction) and we were primarily interested in treatment effects, subsequent drug treatment effects were compared separately within each genotype using two-way repeated measures ANOVAs. Dopamine release in brain slices was compared across groups and stimulation parameters or drug treatments using a two-way repeated measures ANOVA. Holm–Sidak post-hoc tests were used when both main effects or the interaction was significant. Comparisons between two groups were made using two-tailed Student’s t-tests. Correlations were statistically compared using a one-way ANOVA after transforming Pearson’s correlation coefficients (r) to the normally distributed variable z’ using Fisher’s z’ transformation. Statistical tests were performed with SigmaPlot (version 12.5) and significance was set at p < 0.05.