All mice used in experiments were individually housed under a 12 hr light/dark cycle. At the time of the experiments, animals were 8–20 weeks old. Littermates of the same sex were randomly assigned to experimental groups. Animals weighted approximately 25-28 g. All animals were used in scientific experiments for the first time. This includes no previous exposures to pharmacological substances or altered diets. The only pre-experimental intervention was acclimation to crickets, where all mice were food restricted for two consecutive dark cycles (2.5gram of food chow) and presented with five crickets for hunting habituation. All animals captured and consumed all crickets during habituation. Health status was normal for all animals.

All experiments presented in this study were conducted according to the animal research guidelines from NIH and were approved by the Institutional Animal Care and Use Committee of The J.B. Pierce Laboratory.

Method Details

Stereotaxic viral injections and optical fiber implantation For all details on viral and tracer injections for each mouse strain, see Tables S1–S8 . In all cases, preoperative analgesia: 5mg/Kg Carprofen (i.p.), anesthetic: 2% Isoflurane throughout, postoperative analgesia: 30mg/Kg Ibuprofen (in drinking water). Injections were performed with a Hamilton 1.0μL Neuros Model 7001KH syringe. Tables provide details for each strain separately. We list the viral construct/tracer injected, the relevant stereotaxic coordinates, and when applicable the relevant stereotaxic coordinates for optical fiber implants. When optogenetic and chemogenetics and/or caspase lesions were combined, the relevant constructs and coordinates are also listed on the same cell. Optical fibers were obtained from Doric Lenses Inc., and outer diameter is 240 μm; core diameter is 200 μm; numerical aperture is 0.22. Stereotaxic coordinates are with respect to bregma, according to standardized atlases of the mouse brain.

Retrograde tracing from cranial and cervical muscles Mouse strain Chat-Cre × RΦGT Mo5 (AP-5.1 mm, ML: 1.5mm, DV: −5.0mm) was injected unilaterally with SAD-ΔG-GFP 0.5μL. Appropriate location of viral injections was confirmed by injecting the Masseter with 1% FG 10μL, 0.5μL/min. 11N was also injected unilaterally with SAD-ΔG-GFP 0.5μL. To correctly target 11N, the mouse head was lowered; the skin open and blunt dissection of the cervical muscles exposed the cruciate ligament of atlas between the occipital bone and the first cervical vertebra. Ligament was adjusted to horizontal level. 0.5 μL SAD-ΔG-GFP was then injected 2.0mm caudal to the occipital bone, lateral 0.4mm from the midline, ventral 1.7mm from the ligament. Appropriate location of viral injections was confirmed by injecting the Trapezius: 1% FG 10μL, 0.5μL/min. 3 mice for Mo5 and 3 mice for 11N were used.

Histological procedures Mice were deeply anesthetized with a ketamine/xylazine mix (400 mg ketamine + 20 mg xylazine kg body weight−1 I.P.). All animals were perfused with filtered saline, followed by 4% paraformaldehyde. Following perfusion, brains were left in 4% paraformaldehyde for 24 hr and then moved to a 20% sucrose solution in 0.02 M potassium phosphatebuffer (KPBS, pH 7.4) for 2 days. Brains were then frozen and cut into four series 40 μm sections with a sliding microtome equipped with a freezing stage. To identify fiber and electrode locations, relevant sections were identified and mounted on slides. Sections were then photographed under bright field and fluorescence. For SynaptoBrevin visualization, 4 weeks after viral injection, mice were perfused and brains cut at 40μm. The GFP signal was amplified with Goat Anti-GFP antibody (FITC), (ab6662, Abcam, 1:500). For SynaptoBrevin experiments combined with FluoroGold muscle injections, seven days after muscle injections animals were perfused as above and brains sliced in 40μm sections. For verifying the extension of caspase-induced lesions, slices were incubated with Mouse Anti-Neuronal Nuclei (NeuN, MAB377, Millipore, 1:500) followed by TRITC-conjugated affinipure goat anti-mouse (IgG(H+L) 15-025-166, Jackson Immuno, 1:200). For visualizing FG/CTb dual injections, eight days after the injections, perfuse and slices the brain for 40μm. For visualization of rabies expression, ten days after the rabies injections, animals were perfused and expression was observed in coronal sections at ∼160 μm intervals. Visualized cells were overlaid on a mouse brain atlas template.

C-Fos measurements For determining the effects of optical stimulation on PCRt neuronal activity, unilateral 20Hz stimulation was performed using 10 s-long ON(0.02 s on/0.03 s off cycles were used during the ON cycles)/10 s-long OFF cycles for 10 min. For determining the combined effects of optically activating CeA and concomitantly chemogenetically activating PCRt, 10mg/Kg CNO i.p. were injected 10 min before the CeA 1Hz laser stimulation. Unilateral 1Hz stimulation was performed using 10 s ON (0.5 s on/0.5 s off cycles were used during the ON cycles)/10 s OFF cycles for 10 min. 90 min after the appropriate stimulation, mice were sacrificed and perfused as described before. To visualize Fos immunoreactivity, the ABC/DAB procedure was used. Briefly, brain sections were incubated with Rabbit Anti-c-Fos antibody (PC38, Calbiochem, 1:10000) (Concentration 1:10000), followed with Biotinylated Goat Anti-Rabbit IgG Antibody (BA-1000, Vector Laboratories, 1:200), then reacted with avidin-biotin-peroxidase complex (“ABC” method, Vectastain Elite ABC kit, Vector Laboratories, 1:200). A nickel diaminobenzidine (Nickel-DAB) glucose oxidase reaction was used to visualize Fos-like immunoreactive cells. Fos expression was analyzed and quantified as follows: Coronal sections at ∼160 μm intervals in PCRt near the fiber implantation or Gq injection were photographed at 10 × magnification and montaged with Adobe Photoshopto to preserve anatomical landmarks. Fos+ neurons were counted manually on each slice (3 slices per animal) and expressed as the cumulative sum of Fos+ neurons within the relevant regions for each animal.

Electromyogram electrodes, recordings, and analyses First, two twisted Formvar-Insulated Nichrome Wires (Diameter: Bare 0.002 inch. A-M system) were covered with polyethylene tubing (PE20, 0.15” x 0.45,” Braintree scientific). The tips of the nichrome wires were bared and exposed. One bare wire tip was soldered to a Male Miniature Pin Connector (520200, A-M Systems). The other bare wire tip was inserted through a 30G needle, and the tip bent and used for the implants into the trapezius or masseter muscles. For implants, preoperative analgesia consisted of 5mg/Kg Carprofen, anesthetic was 2% Isoflurane throughout and postoperative analgesia, 30mg/Kg Ibuprofen. The skin of cheek was shaved and open to expose the masseter, or the skin on the back of the neck was open to expose the trapezius. The needle was then used for insertion of the wire into the muscle, with the bare wire hooked into the muscle. A suture was used to fix the wire in place. Skin was closed and a cemented miniature screw inserted into the parietal bone for fixating the remaining the polyethylene. Recordings were performed using the electromyogram module of a multichannel acquisition processor (Tucker-Davis Technologies, 3052Hz sampling rate). The male pin connector was attached to the female connector, which had been soldered to a recording headstage. Laser pulses timestamps were synchronized to the recordings via external TTL pulses into the TDT system. EMG signals from masseter and trapezius were recorded simultaneously in the same animals.

In vivo electrophysiological recordings For array implantation C57BL6/J mice (N = 5) were placed on the stereotaxic apparatus and one electrode array consisting of 16 tungsten microwires (35-μm diameter, OMN1005, TDT systems) was implanted onto CeA (AP:-0.9mm ∼1.4mm ML: 2.5mm ∼2.7mm DV:-4.8mm). Locations of electrodes were confirmed histologically. Recordings were performed in combination with masseter recordings by simultaneously using the spike and EMG modules of the multichannel acquisition processor (Tucker-Davis Technologies). Previous to the neuronal and electromyogram recording sessions, mice were connected to commutators with flexible cables for habituation to recording conditions for one training session.

Slice electrophysiology On the day of the experiments, VGat-Cre mice with selective ChR2 expression in CeA or PCRt neurons were anesthetized with isoflurane and decapitated for electrophysiological identification of ChR2-expressed neurons and circuit mapping. Brains were quickly removed and immersed in an ice-cold high-sucrose solution containing (in mM): 220 sucrose, 2.5 KCl, 6 MgCl2, 1 CaCl2, 1.23 NaH2PO4, 26 NaHCO3, and 10 glucose (gassed with 95% O2 / 5% CO2; 300-305 mOsm). Coronal brain slices 300 μm thick were sectioned using a vibratome. Brain slices were then transferred to an incubation chamber filled with an artificial CSF (ACSF) solution containing (in mM) 124 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.23 NaH2PO4, 26 NaHCO3, and 10 glucose (gassed with 95% O2 / 5% CO2; 300-305 mOsm) at room temperature (22°C). After a 1-2 hr recovery period, slices containing CeA, PCRt, or PAG were selected and transferred to a recording chamber mounted on a BX51WI upright microscope (Olympus, Tokyo, Japan). The recording chamber was perfused with a continuous flow of gassed ACSF. A dual-channel heat controller (Warner Instruments, Hamden, CT) was used to control the temperature of recording solution at 33 ± 1°C. Whole-cell patch-clamp recordings were performed on neurons in CeA, PCRt or PAG that were visualized using an infrared-differential interference contrast (DIC) optical system combined with a monochrome CCD camera and a monitor. Pipettes were pulled from thin-walled borosilicate glass capillary tubes (length 75 mm, outer diameter 1.5 mm, inner diameter 1.1mm, World Precision Instruments) using a P-97 micropipette puller (Sutter Instruments, Novato, CA). Pipette solution containing (in mM) 145 K-gluconate, 1 MgCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, and 5 Na2-phosphocreatine (pH 7.3 with KOH; 290-295 mOsm) were used for whole-cell recording. The pipettes of resistances ranging from 3 to 6 MΩ were used for experiment. EPC-10 patch-clamp amplifier (HEKA Instruments, Bellmore, NY) and PatchMaster 2.20 software (HEKA Elektronik, Lambrecht/Pfalz, Germany) were used to acquire and analyze data. Pipette and cell capacitance were compensated during experiment and neurons for which the series resistance was > 20 MΩ were excluded from the statistics. Traces were processed using Igor Pro 6.36 (Wavemetrics, Lake Oswego, OR). Inhibitory postsynaptic currents were recorded at the holding potential of −40 mV unless otherwise mentioned. A 473 nm blue laser (Doric Lenses) was used to evoke the stimulation for optogenetic activation of ChR2 channels in brain slices. Continuous stimulation and stimulation of 10 ms duration with different frequency (1, 5, 10, 20 Hz or 1Hz, 500ms) were used in the experiment to test photostimulation-evoked response. For recording VGlut2+ neurons in PAG innervated by ChR2+ CeA afferents, VGlut2-ires-Cre mice transfected with AAV5-DIO-mCherry in PAG were used.

Behavioral Studies Video scoring All behavioral sessions were video-recorded using a high-speed (120fps) camera (DMC-FZ200, Panasonic). Video scoring was performed by extracting timestamps associated with the relevant behavioral events (which are listed below). Timestamp extraction made use of the CPU’s clock during video execution and was obtained using custom software available upon request. A number of measurements, including overall locomotion and displacement velocity made use of automated video analyses (EthoVision XT11.5, Noldus). Cricket hunting during optogenetics experiments Mice, either fed ad libitum or food restricted (“deprived” 2.5g chow/day), were placed in a clean empty cage for 30mins before the laser stimulation. 10 min before the laser stimulation, the cage was cleaned again, and at the beginning of each trial. The mouse was located on one corner of the cage whereas the cricket was released into the cage near the diagonally opposite corner. A stationary object or an artificial moving prey also was gently placed in the diagonally opposite corner of the cage. Stationary objects were one of the following: wood stick cut from applicators (diameter 0.15cm length 1.5cm (short) or 5cm (long)); Small bottle cap (1cm diameter, 0.6cm high); Tape roll (5cm diameter, 2.5cm high); Food pellet (3g of regular chow 5001, Labdiet); Soft pellet (3g of 24%Fat pellet D12451, Research Diets, Inc); Hard pellet (3g 35% Sucrose pellet, D12450B, Research Diets, Inc). The moving artificial prey was a miniature battery powered robot (HEXBUG Nano obtained from Amazon.com ). Optical Stimulation regimens Stimulation frequencies were chosen according to the outcome of the slice electrophysiological studies. Although we did not detect major differences between stimulation frequencies in terms of evoking (or failing to) behavior, we used the following frequencies in each case. For CeA laser stimulation: 473-nm blue laser (or 532-nm green laser), stimulation was performed using 1min OFF - 1min ON (1Hz, 0.5 s on/0.5 s off cycles were used during the ON period) - 1min OFF cycle totaling 3 min. PCRt/CeA = > PCRt or CeA = > PAG: 473-nm blue laser (or 532-nm green laser) stimulation was performed using 1min OFF - 1min ON (20Hz, 0.02 s on/0.03 s off cycles were used during the ON period) - 1min OFF cycle for totally 3 min. House crickets (Grillus domesticus) were purchased from pet food providers ( http://www.petco.com/shop/en/petcostore and http://www.flukerfarms.com/ ). All mice were habituated to hunt and eat crickets for two days before the test day. During habituation, the mice were 2.5g chow restricted, and presented with 5 crickets to hunt overnight. During the test, the mice were 2.5g chow restricted. Behaviors were digitally recorded with high-speed (120fps) camera (DMC-FZ200, Panasonic). Cricket specifications are as follows: large crickets, ∼1 inch, 0.5g. Small crickets, ∼0.5 inch, 0.1g. Experiments were performed on standard mouse home cages. Behavioral parameters Latency to hunt : time taken from mice fixating at the crickets until mice actually start the pursuit. Capture duration : time taken from mice starting to pursue the crickets until successfully capturing the crickets using either the fore-paws or the mouth, not necessarily killing the crickets. Eating duration : time taken from mice capturing the crickets until mice stopped eating the crickets. Attempt with mouth : Mouse tries to capture the cricket only using biting or biting with forepaw assistance. Attempt with forepaw : Mouse tries to capture the cricket using only fore-paws. In every trial, once mice stopped eating the crickets, any insect residuals were removed, and another live cricket was then placed into the cage. All the experiments were repeated 5 times per animal and averages taken. For the hunting data specifically shown in the Results section of the manuscript, the experimental conditions were as follows: CeA stimulation: Mice were either fed ad libitum satiated or food restricted at 2.5g chow/day. 1Hz laser. One cricket per trial. All the results shown correspond to averages over five trials. Chemogenetic activation Clozapine-N-Oxide (1mg/kg) was injected i.p. 10mins before the start of the hunting sessions.

Food intake during optogenetics and Chemogenetic stimulation Mice were single caged and 2.5g chow/day food restricted. Soft food (chow) or hard food (High sugar pellet: #D12450B, Research Diet, USA) was placed in their home-cage at the same time of the day. After 1 hr free consumption, the pellets were removed and weighted. After 3 days of habituation with either laser cable connection or Saline i.p. injection, on the test day, the mice were exposed to laser (Laser cycle of 5min ON – 5min OFF for 1Hr. During the ON period, 473-nm blue stimulation was performed 1Hz of 0.5 s on/0.5 s off cycles.) or i.p injected with CNO (1mg/kg). The total food intake was weighted and the eating behavior video recorded for further analysis.

Open field tests To assess locomotor activity in response to laser stimulation, animals were placed on a novel Plexiglas arena (Med Associates, 25 cm × 30 cm). The total area was divided into nine equal rectangular subareas (8.3 × 10 cm), demarcated with yellow tape. Immediately above the central subarea a 150-W lamp was activated to induce natural aversion to this particular location, as usually performed. Animals were tested once in this arena. The laser cycle was randomly chosen for 5min ON – 5min OFF or 5min OFF – 5min ON. During the ON period, 473-nm blue laser (or 532-nm green laser) stimulation was performed 1Hz of 0.5 s on/0.5 s off cycles. The sessions were digitally recorded with a Sony HDR-CX440 camera. Data were analyzed by replaying the sessions in slow motion. Outcomes were the number of sequential crossings over different adjacent yellow lines (representing total locomotor activity), and relative time spent within the illuminated central part of the arena.

Pellet reaching task Mice were food-restricted to 2.5g/day. The training chamber was built from clear Plexiglas (4mm thickness; 30cm x 30cm x 30cm). One vertical slit (1 cm wide; 3-cm high) was located on the front wall of the box. Single reachable sugared food pellets (0.02 g, BioServe) were located 1 cm away from the slit, on a platform of 1.5 cm height. Two photodetectors were located on either side of the platform; the centerline of the two photodetectors is 2mm above the pellet. After one day of habituation to the box without presentation of pellets outside the slit, the assay consisted of 2 phases: shaping and training. During the shaping phase (day 1), mice are allowed to reach for multiple pellets presented to them outside the box to determine the preferred limb. During the training phase (day 2–8), individual pellets are placed in front of the slit on the opposite side the preferred limb. When mice can reach 20 pellets within 10mins, we consider the mice learned the performance. For the test day, all the behaviors were recorded with two cameras (one in front of the slit and the other one beside the cage). Once the forepaw crosses the slit and touches the pellet, the photodetector will detect the movement and trigger the laser source via a programmed TTL pulse (20Hz, 0.02 s on/0.03 off cycles for 5 s). All mice performed 20 trials. The photodetector activated the laser source for every other presented pellet. The Reaching accuracy was determined using slow motion video surveillance according to 4 criteria: “miss” (no touch with the pellet during reach), “no grasp” (paw contact with pellet but no correct grasping), “drop” (the pellet is retrieved but falls before taking it into the mouth), “success” (the mouse retrieves the pellet directly to its mouth). Success rate was calculated as the percentage of successful reaches over total reaching attempts.

Locomotion test CeA laser stimulation: 473-nm blue laser 1min (1Hz, 0.5 s on/0.5 s off cycles).

CeA = > PCRt/CeA = > PAG: 473-nm blue laser 1min (20Hz, 0.02 s on/0.03 s off cycles). Mice were either fed ad libitum satiated or food restricted at 2.5g chow/day, were connected to the laser cable and placed in a clean cage or home cage for 30mins before the laser stimulation. All behaviors were videotaped from above the cages. Videos were analyzed with Ethovision XT11.5 (Noldus). Parameters involved defining the arena, define mouse contour versus background contrast. Parameters extracted included distance covered, velocity and mobile frequency.