Further information and requests for reagents and resources should be directed to and will be fulfilled by the Lead Contact, Dr. Joseph F. Cheer ( jcheer@som.umaryland.edu ).

Rats were fitted with chronic indwelling jugular catheters (13cm of polyethylene tubing, 0.3mm inner diameter, 0.64 outer diameter; Dow Corning Corporation) for IV drug delivery. One end of the catheter was inserted into the jugular vein and secured in place by silk sutures, the other end passed subcutaneously to a stainless steel guide cannula (Plastics One) that exited the animal’s back. Catheters were flushed once daily with 0.1mL of enrofloxacin antibiotic (Baytril, Bayer DVM; 5mg/mL) followed by 0.1mL of heparinized 0.9% physiological saline (50USP/mL).

In order to deliver both drug and laser light to the VTA, we epoxied an optical fiber to a 26GA cannula (McMasters-Carr) at a 10° angle []. The cannula terminated 0.5 mm above the tip of the optical fiber so as not to obscure light from the fiber. This cannula+fiber was aimed at the VTA (−5.8 AP, +/−0.7 ML, and −7.7 ventral to brain surface).

Bilateral guide cannula (Plastics One, 26 GA) for IC drug infusion were implanted at the following coordinates: VTA, −5.8 AP, +/− 0.7 ML, −5.7 DV; NAcC, +1.3 AP, +/− 1.4 ML, −5 DV; PFC, +3.2 AP, +/− 0.5 ML, −3.0 DV. A stainless steel obdurator (33GA, Plastics One) was placed inside each cannula.

Rats received chronic electrodes [] aimed at the NAcC (+1.3 AP; +1.4 ML; −6.9 DV). A bipolar stimulating electrode (Plastics One) was aimed at the ipsilateral medial forebrain bundle (−2.8 AP; +1.7 ML; −8.8 DV), and an Ag/AgCl reference electrode was placed in the contralateral hemisphere. A triangular voltammetric input waveform (initial ramp, −0.4–1.3V, 400V/s) [] was applied to the working electrode at 10Hz, while subsecond DA release was monitored. Electrical stimulation (60 pulses, 60Hz, 300 μA, 2ms/phase) was applied to the stimulating electrode via a constant-current isolator (A-M Systems). The working electrode was moved ventrally until electrically evoked DA release was detected, dental cement and screws were use to secure the assembly. Subjects were allowed 3 weeks to recover.

Four small holes were drilled over the VTA at the following coordinates: −5.4 and −6.4 AP; ± 0.7 ML. A 5μL Hamilton syringe in a motorized syringe pump was used to deliver 0.5 μL of virus (0.05 μL per minute) at two depths in each hole (−8.4 and −7.4 DV, from brain surface). The needle was left in place for an additional 5min following injection. Bilateral optical fibers were targeted above the VTA (−5.8 AP; +/−0.7 ML; −7.7 DV), the NAcC (+1.7 AP; +/−1.7 ML; −6.6 DV), or the PFC (+3.2 AP; +/−0.5 ML; −3.5 DV). Fibers were made in-house with optical fiber (HUV 200/230 T 48, Ceramoptec) and a zirconia ferrule (FZ1-LC-235, Kientec Systems).

Subjects were male transgenic rats (Long-Evans) expressing Cre-recombinase under the control of the tyrosine hydroxylase promoter (heterozygous, TH::Cre+; n = 44) and wild-type litter mates (TH::Cre-; n = 66). Rats were bred on-site and were group-housed to 275–350 g when they received surgery and were singly housed thereafter in plastic tubs in a 22°C vivarium on a 12-hour light/dark cycle (lights on at 0800 hours). Subjects had ad libitum access to food (Purina Rat Chow) and water unless otherwise stated. All methods and procedures were conducted in strict adherence to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. Our TH::Cre rat colony could not have been established without Dr. Karl Deisseroth’s assistance and for his donation of our founder animals.

Method Details

Avoidance task All behavioral procedures were conducted in operant chambers (12.0” L x 9.5” W x 8.25”; Med Associates) inside sound-attenuating cabinets. Chambers were fitted with footshock grids, retractable levers, cue lights above the levers, a house light, and speakers for cue tone and white noise. Behavioral programs were controlled by Med PC software. Rats were initially shaped to press a lever to terminate footshock in single daily 30min sessions. At the start of each session, subjects were presented with a lever, white noise (70dB), and a cue light paired with continuous footshock (0.56mA). A response on the lever resulted in termination of footshock for a 20 s “safety” period paired with its own unique discrete cues: the retraction of the lever, dimming of the cue light, illumination of the house light, silencing of white noise, and presentation of a tone (70dB). Subjects were gradually shaped toward the lever by the experimenter until acquisition, upon which an avoidance contingency was introduced. Subjects received single daily (30min) avoidance training sessions. At trial onset, the response lever was extended and a WS (cue light + white noise) was presented. A response on the lever during the 2 s before the initiation of footshock, was considered an avoidance response and resulted in the retraction of the lever, dimming of the cue light, silencing of white noise, and a 20 s safety period during which the house light was illuminated, a tone persisted and no footshock was delivered. If animals failed to press within this 2 s, recurring footshock was applied (0.5ms, 0.56mA, delivered at 2 s intervals) until the animal responded at which point footshock was terminated and the 20 s safety period was initiated; this was considered an escape response. “Escape” and “avoidance” responses were tallied and data are presented as the percentage of trials on which rats emitted an avoidance response [(# of avoidance responses per session/ total number of responses in session) ∗100]. Rats were trained until they reached 50+/−15% (or 80+/−15%) avoidance for two consecutive sessions. Once subjects reached criteria, and at least three weeks after viral transduction, they were tested under the same conditions in conjunction with optogenetic or pharmacological manipulations, and/or voltammetric recording of DA release in the NAcC.

Optogenetic manipulation of avoidance Before each test session rats were attached to an optical fiber patch cable (MMC28550122C, Fiber Optics for Sale). The cable terminated with bilateral ferrules that were secured to the rat’s cranial implant with fitted ceramic split sleeves (SM-CS125S, Precision Fiber Products). The other end of the patch cable attached to a 473nm (for ChR2, 10-15mW; MBL-III-473, Opto Engine) or 532nm (for NPHR, 10-15mW; MGL-III-532, Opto Engine) DPSS laser. Optical stimulation was controlled by Med PC IV (Med Associates) and Tarheel CV software. Each test session was divided into two segments: a 30min baseline during which animals performed the avoidance task with no laser stimulation, and a 30min laser segment during which each presentation of the WS was accompanied by laser (ChR2: 10 pulses at 20Hz, 5ms pulse width; NPHR: 3 s of stimulation beginning 2 s before WS presentation). The order of baseline and laser stimulation segments were counterbalanced across subjects and days.

Pharmacological manipulation of avoidance Analysis of performance during our previously implemented 60min optical manipulation avoidance sessions showed that there are no within-session gains in performance (p > 0.05; Figure S4 ). Therefore, we halved the test session length to minimize discomfort and better suit pharmacological manipulations. IV drug drug delivery was achieved by attaching the animal’s catheter cannula to a syringe filled with drug via PE20 tubing. Rimonabant or vehicle (VEH) were delivered IV over 4 s and animals performed on the avoidance task 5min after infusion. For IC infusions, obdurators were removed and replaced by bilateral internal infusion cannula (33GA, Plastics One; VTA: 1mm projection; PFC: 0.5mm projection; NAcc: 1.5mm projection), which were connected to a 5 μL Hamilton syringe via PE50 tubing back-filled with drug. Infusion cannula were inserted 1min prior to drug delivery, drug was delivered in a volume of 0.5 μl/side over 2min using a motorized syringe pump. After each infusion, infusion cannula were left in place for an additional 5min before obdurators were replaced and animals performed in the avoidance task.

In vivo voltammetry 8 Oleson E.B.

Gentry R.N.

Chioma V.C.

Cheer J.F. Subsecond dopamine release in the nucleus accumbens predicts conditioned punishment and its successful avoidance. 37 Oleson E.B.

Beckert M.V.

Morra J.T.

Lansink C.S.

Cachope R.

Abdullah R.A.

Loriaux A.L.

Schetters D.

Pattij T.

Roitman M.F.

et al. Endocannabinoids shape accumbal encoding of cue-motivated behavior via CB1 receptor activation in the ventral tegmentum. Voltammetric recordings (versus an Ag/AgCl reference electrode) and data acquisition were performed using TarHeel CV software. Animals’ electrode implants were attached to custom FSCV equipment via a custom head-stage. Rats received IV VEH and performed the avoidance task for 20min. Next, an IV infusion of 0.56mg/kg rimonabant was delivered and rats performed for an additional 20min, rats then received a final infusion of 0.44mg/kg rimonabant to achieve a cumulative dose of 1.0mg/kg, and performed the task for a final 20min. FSCV procedures and DA signal calibration employed here have been previously described []. 8 Oleson E.B.

Gentry R.N.

Chioma V.C.

Cheer J.F. Subsecond dopamine release in the nucleus accumbens predicts conditioned punishment and its successful avoidance. For FSCV confirmation of optically-evoked DA, rats were transduced with ChR2 and implanted with bilateral optical fibers (as described above). At least three weeks after transduction, animals were anesthetized with isoflurane and implanted with an Ag/AgCl reference electrode and a a glass carbon fiber electrode was lowered into the NAcC (AP:+1.4, ML: −1.4) of the contralateral hemisphere []. The glass electrode was driven ventrally until optical stimulation elicited robust DA release (30 pulses; 30Hz). Animals then received trains of optical stimulation consisting of varying pulse number and frequency in a random order (10 pulses at 20, 30, 60 and 90Hz; and 10, 20, and 30 pulses at 30Hz), with at least 5min between each stimulation. Data was collected and compiled with Tarheel CV software and current was translated to DA concentration utilizing a laboratory-generated calibration set.

Drugs for in vivo use 46 Cheer J.F.

Wassum K.M.

Sombers L.A.

Heien M.L.A.V.

Ariansen J.L.

Aragona B.J.

Phillips P.E.

Wightman R.M. Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation. 59 Nowend K.L.

Arizzi M.

Carlson B.B.

Salamone J.D. D1 or D2 antagonism in nucleus accumbens core or dorsomedial shell suppresses lever pressing for food but leads to compensatory increases in chow consumption. 60 Saunders B.T.

Robinson T.E. The role of dopamine in the accumbens core in the expression of Pavlovian-conditioned responses. 61 Yun I.A.

Nicola S.M.

Fields H.L. Contrasting effects of dopamine and glutamate receptor antagonist injection in the nucleus accumbens suggest a neural mechanism underlying cue-evoked goal-directed behavior. 37 Oleson E.B.

Beckert M.V.

Morra J.T.

Lansink C.S.

Cachope R.

Abdullah R.A.

Loriaux A.L.

Schetters D.

Pattij T.

Roitman M.F.

et al. Endocannabinoids shape accumbal encoding of cue-motivated behavior via CB1 receptor activation in the ventral tegmentum. 62 Hernandez G.

Cheer J.F. Extinction learning of rewards in the rat: is there a role for CB1 receptors?. 63 Hernandez G.

Cheer J.F. Effect of CB1 receptor blockade on food-reinforced responding and associated nucleus accumbens neuronal activity in rats. 64 Hernandez G.

Oleson E.B.

Gentry R.N.

Abbas Z.

Bernstein D.L.

Arvanitogiannis A.

Cheer J.F. Endocannabinoids promote cocaine-induced impulsivity and its rapid dopaminergic correlates. 65 Morra J.T.

Glick S.D.

Cheer J.F. Neural encoding of psychomotor activation in the nucleus accumbens core, but not the shell, requires cannabinoid receptor signaling. 31 Wang H.

Treadway T.

Covey D.P.

Cheer J.F.

Lupica C.R. Cocaine-induced endocannabinoid mobilization in the ventral tegmental area. 66 Gregg L.C.

Jung K.M.

Spradley J.M.

Nyilas R.

Suplita 2nd, R.L.

Zimmer A.

Watanabe M.

Mackie K.

Katona I.

Piomelli D.

Hohmann A.G. Activation of type 5 metabotropic glutamate receptors and diacylglycerol lipase-α initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated analgesia. The DA D1 receptor antagonist SCH23390 (SCH; 125941-87-9, Sigma-Aldrich) and the DA D2 receptor antagonist Eticlopride (97612-24-3, Sigma-Alrich) were each dissolved in aCSF to one of three doses (0.25 μg/0.5 μl; 0.5 μg/0.5 μl; 1.0 μg/0.5 μl) for IC delivery. Similar doses of SCH diminish responding for positive reinforcers and do not result in locomotor impairments []. The CB1 antagonist rimonabant (National Institute on Drug Abuse Drug Supply Program) was dissolved in a 1:1:18 mixture of ETOH, alkamuls (EL620, Rhodia Group), and 0.9%physiological saline to either of two doses (0.56mg/0.1mL; 1mg/0.1mL) for IV delivery. Rimonabant was dissolved in a 1:1:18 mixture of ETOH, alkamuls, and aCSF to one of two doses (0.2 μg/0.5 μl; 1.0 μg/0.5 μl) for IC delivery. Similar doses of rimonabant have been utilized by our laboratory to investigate food-maintained responding, and have not been found to affect locomotor behavior []. The diacylglycerol lipase inhibitor tetrahydrolipstatin (Tocris Bioscience) was dissolved in a 1:1:18 mixture of ETOH, alkamuls, and aCSF to a dose of 5 μg/0.5 μL or 0.5 μg/0.5 μL [].

Variable time out (VTO) task TH::Cre+ rats were food restricted to 95% of their free-feeding weight and trained to lever press for food (FR1 with 10 s time out (TO)). Operant chambers were now fitted with a plastic floor with sloped sides, pellet dispenser and pellet receptacle. Food availability following each TO was signaled by illumination of the cue light and the first cued lever press earned the animal one food pellet (14mg chocolate-flavored pellet, Bio-Serv). The session ended when animals reached 25 rewards or after 30min. Rats were trained on this schedule for five sessions after they consistently achieved 25 rewards. Animals were then transferred to an FR1 with a VTO ranging from 32-60 s (average 46 s). Once animals learned the VTO schedule (i.e., consistently earned 25 rewards), they underwent two test sessions, interleaved with baseline sessions. A digital video recorder above each chamber recoded the animal’s behavior. During baseline sessions, animals’ optical fiber implants were attached to a 473nm laser via an optical patch cord, but the laser remained off. On Test Session 1, rats received laser stimulation (10 pulses at 20Hz) at the presentation of each cue (cue light illumination). The next day animals performed another baseline session with no laser stimulation. On the final test day (Test Session 2), animals received laser stimulation at the midpoint of each VTO (i.e., not in conjunction with the cue). The connection between the animals’ optical implants and the patch cable were insulated to prevent light seepage. Data from each test session was viewed as latency to lever press from either cue-onset or from laser stimulation (Med-PC software). Digital videos of each test session were scored for latency to orient toward the lever (“orient” was defined as movement of the animal’s gaze or body). Video scoring was performed by an experimenter blind to the experimental conditions.

Fear conditioning Rats underwent a 3-day fear conditioning protocol. On day 1 (conditioning day), animals were placed into an operant box fitted with a footshock grid. Subjects were given 30min to acclimate, after which they were presented with a tone cue three times (20 s tone, 3min ITI), with each presentation culminating in a 2 s scrambled electric footshock of 0.7mA. Twenty-four hours after conditioning, day 2, rats were placed in a novel cylindrical test chamber (made of plastic and striped radially to provide unique environmental cues) and their optical fiber implants were attached to a 473nm laser via an optical fiber patch cable. Rats were exposed to the tone at 3min intervals for a total of 18 presentations. Throughout each 20 s tone rats received optical stimulation to the VTA (10 pulses at 20Hz, at 2 s intervals). On day 3, 24 hr later, animals were placed back into the cylindrical test chamber and received 18 tone presentations in the absence of shock and laser. All behavioral sessions were recorded, coded to ensure blind analysis, and hand scored for freezing behavior during each tone. Final data reflect the average scores from two experimenters blind to experimental conditions.

Locomotor testing A subset of rats from the fear conditioning experiment underwent a single locomotor testing trial. Rats were placed in an operant chamber fitted with a plastic floor with sloped sides. Rats received 20 s of laser stimulation (20Hz, 10 pulses, at 3 s intervals) every 3min for 30min. Test sessions were video recorded and locomotor behavior (distance traveled in cm) was analyzed using EthoVision video tracking software (Noldus).

Ex vivo voltammetry In vivo FSCV measurements of DA release concurrent with optical stimulation at the electrode tip are beyond the current scope of these technical approaches, so we verified the ability of terminal stimulation to release DA using ex vivo voltammetry. Less than one week after avoidance testing, rats were decapitated and brains were quickly removed. 250 μm thick coronal slices containing the NAcC or PFC were placed in carbogen-bubbled, ice-cold modified artificial cerebral spinal fluid (aCSF) containing (in mM): 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 glucose. All recordings were performed between 290 and 310 mOSm in oxygenated Krebs buffer containing the following (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, 0.4 L-Ascorbic acid, 20 HEPES, 10 Glucose, 25 NaHCO3, and 10 NaOH. A carbon fiber glass electrode was used to record DA release during laser stimulation of NAcC or PFC DA terminals (10 pulses, 20Hz, 473nm laser; 10-15mW).

Slice electrophysiology TH::Cre+ rats were transduced with ChR2 in the VTA and left undisturbed in their home-cage for 3 weeks or trained on the avoidance task. On test day, rats were decapitated and their brains were rapidly removed and transferred to an oxygenated (95% O 2 / 5% CO 2 ) ice-cold solution containing: 93mM NMDG, 2.5mM KCl, 1.2mM NaH 2 PO 4 , 30mM NaHCO 3 , 20mM HEPES, 25mM Glucose, 5.6mM Ascorbic acid, 3mM Sodium pyruvate, 10mM MgCl 2 , 0.5mM CaCl 2 . Horizontal slices containing VTA (220μm) were transferred to a holding chamber filled with oxygenated solution containing: 109mM NaCl, 4.5mM KCl, 1.2mM NaH 2 PO 4 , 35mM NaHCO 3 , 20mM HEPES, 11mM Glucose, 0.4mM Ascorbic acid, 1mM MgCl 2 , 2.5mM CaCl 2 . Slices were first incubated at 35°C for 10-12 min before transferred to room temperature until the start of the experiments. Slices were transferred to a recording chamber and immersed in oxygenated aCSF containing 126mM NaCl, 3mM KCl, 1.2 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 11mM Glucose, 1.5mM MgCl 2 , 2.4mM CaCl 2 . The aCSF was flowing (2mL/min) and heated (32-34°C). Slices were visualized with an upright microscope (Olympus, BX51WI) equipped with differential interference contrast (DIC) optics. Recorded neurons were located in the lateral VTA, medial to the terminal nucleus of the accessory optic track and anterior to the third cranial nerve. Whole-cell voltage-clamp recording were acquired using an Axopatch 200B (Molecular Devices) amplifier. Recording pipettes (3-5MΩ) were filled with internal solution containing: 140mM K-gluconate, 2mM NaCl, 1.5 mM MgCl 2 , 10mM HEPES, 10 mM Tris-phosphocreatine, 4mM Mg-ATP, 0.3mM Na-GTP, 0.1mM EGTA, pH 7.2, 290mOSM. DNQX (20μM), DL-AP5 (40μM), picrotoxin (100 μM), strychnine (1 μM) were present to block AMPA, NMDA, GABA A , and glycine receptors, respectively. GABA B IPSCs were evoked using optical stimulation (473nm; 3 s; 6-7mW) or electrical stimulation with bipolar tungsten stimulating electrodes with tip separation 300-400μm. A train of six stimuli 100μs duration of 1mA was delivered at 50Hz every 30 s. Stimulation protocols were generated and signals acquired using the WinLTP program. Control GABA B IPSCs were recorded for 10min before the CB1 receptor blocker AM251 (2μM) was applied for an additional 30min. Data are presented as the change in percent from control traces.

LC-MS/MS Rats were anesthetized with isoflurane gas and placed in a stereotaxic frame for intracranial infusion of THL (n = 9) or VEH (n = 9) into the VTA. Five minutes after infusion, animals were decapitated, their brains were removed, quickly chilled and a 1mm punch of VTA tissue was taken. Tissue was frozen with dry ice and stored at −80°C until analysis. Liquid chromatography-tandem mass spectrometry was used to quantify 2-AG in brain tissue. Samples were homogenized in methanol containing a deuterated standard (Cayman Chemicals) and 0.1% formic acid, bath sonicated at 4C for 10 min, incubated at −20°C overnight, and centrifuged. Water was added to the supernatant for a final ratio of 75:25 Methanol:Water. Samples (20 μl) were injected onto a C-18 column (50 × 2 mm, 1.7 μm; Acquity) at 35C under the following gradient: 40% A (water) and 60% B (2:1 Acetonitrile:Methanol) from 0 to 0.25min, increased to 5% A and 95% B from 0.25 to 3.75min held for 2min, and returned to 40% A and 60% B from 5.75 to 6min. Both mobile phase components contained 0.1% formic acid (v/v). A QTrap 6500 mass spectrometer (Sciex) was used to detect analyte via selective reaction monitoring in the positive ion mode using the following reactions (the mass in parentheses represents the mass of the deuterated internal standard): (m/z 379(384) → 287(287)). Quantification was achieved via stable-isotope dilution.