Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ilana B. Witten ( iwitten@princeton.edu ).

All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Princeton University Institutional Animal Care and Use Committee (IACUC). For all experiments, male wild-type Long Evans rats (Charles River: 006; n = 90) aged 8 weeks (∼275-325 g) upon arrival were used. Rats were housed individually and maintained on a reverse 12-h light:dark cycle (lights off at 8:00 a.m.). All behavioral testing was performed during the dark phase of the cycle. Food and water were available ad libitum for the ∼7-day period of acclimation to the vivarium and during recovery from all surgical procedures. During behavioral training and drug-free period, animals were restricted to no < 85% of their preoperative body weight by limiting food access to ∼19 g per day.

Method Details

Surgery For all surgical procedures, rats were anesthetized with ketamine and xylazine (100 mg/kg and 7 mg/kg, respectively, i.p.) and the anesthetic plane maintained with isoflurane (1%–2%). Rats received the antibiotic enrofloxacin (5 mg/kg, i.m.) before surgery and the analgesic meloxicam (2 mg/kg, s.c.) before and 24 h after surgery. Rats were allowed a 5- to 7-day recovery period following all surgeries. Calcium imaging 12 parts/ml, IGMM Vector core, France) and in the IL (ML +0.6 mm, AP +3.1 mm relative to bregma, DV −5.5 & −4.7 mm relative to skull) with AAV2/5-CAG-DIO-GCamp6f-WPRE-SV40 (n = 29 rats, 2 μl, titer 1.17 × 1013 parts/ml, UPenn Vector Core) or AAV2/5-CAG-DIO-RatOptimized-GCamp6f-WPRE-SV40 (see Rat Codon Optimization of GCaMP below; n = 4 rats, 2 μl, titer 1.15 × 1013 parts/ml, Princeton Vector Core). One week later, rats were implanted with a GRIN lens (n = 32 rats, 0.5-0.6 mm in diameter, 7.3-8.4 mm in length; Inscopix, Palo Alto, CA, USA, 130-000152, 130-000150) or GRIN lens with prism attached (n = 1 rat, 1mm in diameter, 9.1 mm in length, 1mm x 1mm prism; Inscopix, Palo Alto, CA, USA, 130-000444) in the IL (ML +0.9 mm, AP +2.6 mm relative to bregma, DV −4.4 to −4.7 mm relative to skull). The lens was secured with metabond (Parkell, Edgewood, NY, USA) and fixed to the skull with skull screws and dental cement (Lang Dental, Wheeling, IL, USA). A plastic cap was glued in place to protect the lens. Rats were also implanted with an intravenous catheter using established procedures ( Carelli et al., 2000 Carelli R.M.

Ijames S.G.

Crumling A.J. Evidence that separate neural circuits in the nucleus accumbens encode cocaine versus “natural” (water and food) reward. Rats (n = 33; aged 8-9 weeks) were placed in a small animal stereotax (Kopf Instruments, Tujunga, CA, USA) and unilaterally injected in the NAc (ML +1.0 mm, AP +1.7 mm relative to bregma, DV −7.4 mm relative to skull) with a retrogradely transporting CAV2-Cre virus (n = 33 rats, 1 μl, titer 2.5 × 10parts/ml, IGMM Vector core, France) and in the IL (ML +0.6 mm, AP +3.1 mm relative to bregma, DV −5.5 & −4.7 mm relative to skull) with AAV2/5-CAG-DIO-GCamp6f-WPRE-SV40 (n = 29 rats, 2 μl, titer 1.17 × 10parts/ml, UPenn Vector Core) or AAV2/5-CAG-DIO-RatOptimized-GCamp6f-WPRE-SV40 (see Rat Codon Optimization of GCaMP below; n = 4 rats, 2 μl, titer 1.15 × 10parts/ml, Princeton Vector Core). One week later, rats were implanted with a GRIN lens (n = 32 rats, 0.5-0.6 mm in diameter, 7.3-8.4 mm in length; Inscopix, Palo Alto, CA, USA, 130-000152, 130-000150) or GRIN lens with prism attached (n = 1 rat, 1mm in diameter, 9.1 mm in length, 1mm x 1mm prism; Inscopix, Palo Alto, CA, USA, 130-000444) in the IL (ML +0.9 mm, AP +2.6 mm relative to bregma, DV −4.4 to −4.7 mm relative to skull). The lens was secured with metabond (Parkell, Edgewood, NY, USA) and fixed to the skull with skull screws and dental cement (Lang Dental, Wheeling, IL, USA). A plastic cap was glued in place to protect the lens. Rats were also implanted with an intravenous catheter using established procedures (). Briefly, a custom-made catheter (Access Technologies and Norfolk Medical, Skokie, IL, USA) was inserted into the right jugular vein and passed subcutaneously to the back of the animal between the shoulder blades. During the recovery period, catheters were flushed daily with heparin (1USP unit/ml) dissolved in 0.9% sterile saline (0.1ml, i.v.). Two to three weeks after lens and catheter implantation, a baseplate (Inscopix, Palo Alto, CA, USA, 100-000279) which supported attachment of a miniaturized microscope (epifluorescence, 475 nm LED; Inscopix, Palo Alto, CA, USA) was cemented in place above the GRIN lens. A baseplate cover (Inscopix, Palo Alto, CA, USA, 100-000241) was attached to protect the lens. Some rats (n = 11) did not have any recorded neurons and were included only in behavioral analysis ( Figure 1 ). Of rats included in imaging analysis (n = 22), 2 were implanted in the left hemisphere and 20 were implanted in the right hemisphere. Optogenetic terminal activation 12 parts/ml, UNC vector core; n = 7 rats, 2 μL per hemisphere, 9.6 × 1013 titer parts/ml, UPenn vector core) or AAV2/5-CamKIIa-eYFP (n = 21 rats, 2 μL per hemisphere, titer 7.5 × 1012 parts/ml, UNC vector core). ChR2-YFP is thought to diffuse passively to axon terminals, and previous work indicates that a 3-4 month time frame is appropriate for terminal expression in rats, given the length of long-range axons in this species ( Warden et al., 2012 Warden M.R.

Selimbeyoglu A.

Mirzabekov J.J.

Lo M.

Thompson K.R.

Kim S.Y.

Adhikari A.

Tye K.M.

Frank L.M.

Deisseroth K. A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Rats (n = 57; aged 8-9 weeks) were placed in a stereotax and bilaterally injected in the IL (ML ± 0.6 mm, AP +3.1 mm relative to bregma, DV −5.6 & −4.8 mm relative to skull) with either AAV2/5-CamKII-hChR2(H134R)-eYFP (n = 29 rats, 2 μL per hemisphere, titer 4 × 10parts/ml, UNC vector core; n = 7 rats, 2 μL per hemisphere, 9.6 × 10titer parts/ml, UPenn vector core) or AAV2/5-CamKIIa-eYFP (n = 21 rats, 2 μL per hemisphere, titer 7.5 × 10parts/ml, UNC vector core). ChR2-YFP is thought to diffuse passively to axon terminals, and previous work indicates that a 3-4 month time frame is appropriate for terminal expression in rats, given the length of long-range axons in this species (). Therefore, optical fibers and catheters were implanted during a second surgery performed 3-4 months after virus injection. Optical fibers (300 μm core diameter, 0.37 NA; Thor Labs, BFL37-300) attached to stainless steel ferrules (PFP, Milpitas, CA, USA, MM-FER-2006SS-3300) were implanted bilaterally above the NAc shell (ML ± 2.46 mm, AP +1.8 mm relative to bregma, DV −7.08 mm relative to skull, 10° angle).

Cocaine Self-administration and Drug-seeking Test Rats were placed on food restriction (∼19 g of food per day) for 24 h before training began, and food restriction was maintained for the duration of the study. All behavioral experiments were performed in a modular operant chamber (Med. Associates Inc., St. Albans, VT, USA; 30.5 × 24.1 × 21 cm, ENV-008CT, for optogenetic experiments, or 30.5 × 24.1 × 29.2 cm, ENV-007CT, for imaging experiments) housed within a sound-attenuating chamber (Med Associates; ENV-018V). Two retractable levers and a central reward receptacle were confined to one wall of the chamber, and a houselight and speaker were located on the opposite side of the chamber. A computer-controlled syringe pump was located outside the sound-attenuating chamber. Rats first underwent 5 days of lever training. The first day consisted of habituation to the chamber with non-contingent delivery of 20% sweetened condensed milk (0.05ml/reward, 50-60 rewards; 20 min session) to the reward receptacle. Rats were then trained to lever press for milk delivery on a fixed ratio 1 (FR1) schedule of reinforcement for 4 days (30 min sessions). A response on the active lever delivered 0.05ml milk; a response on the inactive lever had no programmed consequence. On the last day of lever training, an active lever press also resulted in lever retraction for 20 s in addition to milk delivery. This training schedule allowed rats to acclimate to lever pressing and lever retraction before beginning cocaine self-administration. Rats then underwent daily 2 h cocaine self-administration sessions for 14 consecutive days. The first 7 days of self-administration training were performed under an FR1 schedule of reinforcement in which an active lever press resulted in cocaine infusion (0.33mg/0.2ml/inf, ∼1mg/kg; 6 s) paired with a tone/houselight conditioned stimulus (CS; 20 s) and lever retraction (20 s). Inactive lever presses had no programmed consequence. The last 7 days of training consisted of a modified FR1 schedule with discrete trials ( Figure 1 B). The start of each trial was signaled by extension of the levers into the chamber, and rats could perform 1 of 3 responses: active lever press, inactive lever press, or omission. Active lever presses resulted in cocaine infusion (6 s), CS presentation (20 s), and lever retraction (20 s). Inactive lever presses resulted in lever retraction and a variable inter-trial interval (ITI; 15-30 s). Omissions occurred if a rat made no response for 30 s and resulted in lever retraction and a variable ITI (15-30 s). Following completion of cocaine self-administration, rats were divided into 2 groups: the D1 group underwent a 1-day drug-free period (equivalent to the normal ∼24h drug-free period that elapses between daily self-administration sessions), and the D15 group underwent a 15-day drug-free period. During the drug-free period, rats remained in their homecage without access to cocaine. After this period, all rats underwent a single test of cue-induced drug-seeking to assess cocaine motivation. This test was identical to the discrete trials self-administration paradigm described above except that an active lever press resulted in CS presentation and lever retraction but no drug delivery. The number of active lever presses rats make under these conditions is the operational measure of cocaine motivation. For the imaging experiment, CS probes (5 s non-contingent presentation of the tone/houselight stimulus previously paired with cocaine delivery) were also given during a randomly selected subset of ITIs (average of 11 probes per session).

Cellular Resolution Calcium Imaging During cocaine self-administration training, rats were acclimated to being tethered by attaching a commutating spring to a “dummy” ferrule cemented to the headcap formed around the baseplate. This mimicked the tether of the miniaturized microscope used during test days. Rats were also habituated by attaching the microscope to the baseplate at least twice prior to testing. Before the start of a drug-seeking test, the miniaturized microscope was attached to the baseplate of awake, unrestrained rats and locked in place with a set screw. During the drug-seeking test (1h), the LED power was maintained at 20 to 30% and the analog gain on the image sensor was set to 1.5. Grayscale tiff images were collected at 10 frames per second using Inscopix nVista HD software (Inscopix, Palo Alto, CA). Coincident with each frame, a signal was sent to a digital signal processor (RZ5D, TDT, Alachua, FL, USA), allowing neural data to be time-stamped and synchronized with behavioral events. Another computer controlled the behavioral events of the experiment (Med Associates Inc., St. Albans, VT, USA) and sent digital outputs corresponding to each event to the digital signal processor. Additionally, head tracking data was obtained by using the TDT RV2 system linked to a camera (frame rate 101.7254 Hz) directly above the operant chamber. The RV2 system was trained to detect the blue LED on the miniaturized microscope mounted on rats’ heads. Shape-preserving piecewise cubic interpolation (pchip function in MATLAB) was used to fill in gaps in tracking for any periods when the LED signal was lost. Finally, position data were downsampled to 10 Hz to match the acquisition rate of neural data. Extraction of activity traces Zhou et al., 2018 Zhou P.

Resendez S.L.

Rodriguez-Romaguera J.

Jimenez J.C.

Neufeld S.Q.

Giovannucci A.

Friedrich J.

Pnevmatikakis E.A.

Stuber G.D.

Hen R.

et al. Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data. Imaging data was spatially downsampled by a factor of 4 and motion corrected with Mosaic software (Inscopix, Palo Alto, CA, USA). The motion correction used a translational correction algorithm based on cross-correlations computed on consecutive frames (motion correction parameters were as follows, motion correction type: translation only; reference image: the mean image; speed/accuracy balance: 0.1; subtract spatial mean [r = 20 pixels], invert, and apply spatial mean [r = 5 pixels]). Custom MATLAB (Mathworks, Natick, MA) scripts were then used for all subsequent imaging data analysis. After motion correction, a CNMFe algorithm () was used to identify individual neurons, obtain their fluorescence traces, and deconvolve the fluorescence signal into firing rate estimates. Fluorescence traces were used for time-locked analysis (see below). Inferred firing rates (based on the CNMFe algorithm) were used for spatial receptive field estimates (see below).

Optogenetic Activation of IL-NAc Neurons At the start of the behavioral test session, ferrules were connected to patch cables via ceramic sleeves. Patch cables were protected with a metal spring and attached to a bilateral optical commutator (Doric Lenses, Quebec, Canada). The commutator was linked to a laser (100 mW, 447 nm, OEM Laser Systems, Draper, UT, USA) with a multimode fiber coupler for an FC/PC connection. Laser output was calibrated to ∼10 mW measured at the tip of the fiber. Rats were habituated to this set-up by attaching ferrules to “dummy” patch cables that were not connected to a laser during all cocaine self-administration sessions. During the drug-seeking test (2 h), laser stimulation (20 Hz, 5 ms pulses) was presented on a random 50% of trials. On stimulation trials, laser onset began at trial start (lever extension) and stimulation lasted for the duration of the trial, including the response (active lever press, inactive lever press, or omission) and subsequent outcome (CS or ITI). The day after the drug-seeking test, all rats underwent a single 4 h test for intracranial self-stimulation (ICSS). This test was performed in the same operant chambers used for self-administration training and the drug-seeking test; however, rats were assigned to a different specific chamber for the ICSS test. A nosepoke at an active hole resulted in 5 s of stimulation (∼10 mW, 20 Hz, 5 ms pulses) followed by a 1 s timeout. Rats could continue to nosepoke during the stimulation and timeout periods. These nosepokes were recorded but did not result in additional stimulation presentations. A nosepoke at an inactive hole had no programmed consequence. Finally, rats were tested for possible non-specific locomotor effects of optogenetic stimulation in a locomotor chamber (52 × 52 × 52 cm). Rats were allowed to explore the locomotor chamber for 3 min with no laser stimulation, followed by 3 min of laser light stimulation (∼10 mW, 20 Hz, 5 ms pulses), and terminating with an additional 3 min of no light. The location and velocity of rats was tracked using Ethovision (Nodulus). Note that only a subset of rats that performed the drug-seeking and ICSS tests also underwent locomotor testing (ChR2 group, n = 17; YFP group, n = 6). An additional 11 YFP control rats were used for locomotor testing but not the drug-seeking/ICSS test.

Histology Rats were deeply anesthetized with pentobarbital sodium (2 mg/kg, i.p.) and transcardially perfused with 1x phosphate-buffered saline (PBS), followed by fixation with 4% paraformaldehyde (PFA) in PBS. Brains were dissected out and post-fixed in 4% PFA overnight before being transferred to 30% sucrose in PBS solution. 40 μm thick coronal sections containing the brain regions of interest were cut on a freezing microtome or cryostat. For immunohistochemistry, slices were blocked in 3% normal donkey serum (NDS) in PBS with 0.25% Triton X-100 for 1 h. Sections were then incubated at 4°C overnight in monoclonal rabbit anti-GFP primary antibody (1:1000, Life Technologies, No. G10362). PBS washes were performed to remove primary antibody, and slices were then incubated for 2 h in AlexaFluor488-conjugated donkey anti–rabbit IgG (1:500, Jackson ImmunoResearch, No. 711-545-152). Following PBS washes, slices were mounted in 1:2,500 DAPI in Fluoromount-G. For the GCaMP6f imaging experiments, coronal sections of IL were imaged with a Nikon Ti2000E epifluorescence microscope to verify targeting of the GRIN lens to IL. A Leica TCS SP8 confocal microscope was used to confirm virus expression and nuclear exclusion of GCaMP6f as well as produce Figure 2 C–G. 11 of 33 rats did not show GCaMP6f expression and were excluded from analysis of calcium data; however, their behavior during the drug-seeking test was included in analysis. For the optogenetic excitation experiments, coronal sections were imaged with a Nikon Ti2000E epifluorescence microscope to confirm expression of the virus in IL cell bodies and NAc terminals, as well as optical fiber targeting to the NAc shell.