Regarding AAV vectors for imaging and optogenetic manipulation: AAV vectors carrying DIO-ChR2-mCherry, DIO-GtACR1-P2A-GFP, DIO-hM3Dq-2A-mCherry, DIO-mGFP, or DIO-GCaMP6m constructs were packaged into AAV2/9 serotype with titers of 1-5×10 12 viral particles/ml. We obtained the enhanced membrane GFP (Addgene Plasmid #14757) and GCaMP6m (Addgene Plasmid #40754) from Addgene. The gene encoding the Guillardia theta anion channel rhodopsins 1 (GtACR1) was synthesized according to the sequence from GenBank (KP171708.1). We built a fusion construct that was composed of the sequences for GtACR1, the P2A peptide, and an EGFP reporter(GtACR1-P2A-GFP); this fusion construct, or mGFP, or GCaMP6m was inserted into a pAAV-EF1α-DIO backbone derived from the pAAV-EF1α-DIO-hChR2(H134R)-mCherry plasmid (a gift from K. Deisseroth) in an inverted orientation. These were then packaged into AAV vectors (1-5 × 10 12 v.g./mL). AAV2-retro-hSyn-Cre (2.7 × 10 13 v.g./mL), AAV2-retro-hSyn-Flp (1.75 × 10 13 v.g./mL), AAV2-EF1α-fDIO-hChR2(H134R)-EYFP (1.71 × 10 13 v.g./mL), AAV2-EF1α-fDIO-GtACR1-EGFP (1.57 × 10 13 v.g./mL) and AAV2-retro-CAG-DIO-Flp (1.7 × 10 13 v.g./mL) were purchased from Shanghai Taitool Bioscience Co. (China).

Injections were performed using a microsyringe pump (Nanoliter 2010 Injector, WPI). A Micro4 controller (WPI) was used to deliver the virus at a rate of 46 nL/min. For optogenetic manipulation and fiber photometry of LH, a total volume of 300-500 nL virus was injected into the LH. To specifically manipulate periaqueductal gray (PAG)-projecting LH neurons, we first bilaterally injected AAV-retro-Cre (300 nL) into the PAG using the following coordinates: −4.1 mm posterior to the bregma, ± 1.05 mm lateral to the midline, and −2.55 mm ventral to the skull surface with a 14° angle (lateral to middle). Cre-dependent or Flp-dependent AAV viruses were then injected to the LH in the same surgery. After completion of the injection, two minutes were allowed to pass before withdrawing the glass needle by a distance of 50 μm; the glass pipette was left in place for five additional minutes and then slowly withdrawn completely. Optical fiber implantation was carried out immediately after virus injection. A piece of optical fiber (230 μm) was fit into an LC-sized ceramic fiber ferrule. The optical fiber (230 μm O.D., 0.37 NA; Shanghai Fiblaser, China) was implanted in specific brain regions (LH and PAG). The ceramic ferrule was supported with a skull-penetrating M1 screw and dental acrylic. Following surgery, animals were allowed to recover from anesthesia under a heat lamp. Lincomycin hydrochloride and lidocaine hydrochloride gel (Shandong Fangming Pharmaceutical Group, China) was applied to the sterilized incision site as an analgesic and anti-inflammatory drug. Animals were allowed to recover for two to three weeks after surgery.

Adult mice were anesthetized with pentobarbital (i.p. 80 mg/kg) and mounted on a stereotaxic apparatus (Nanjing ThinkerTech, China). Body temperature was maintained at around 37°C with an electric heating pad, and erythromycin eye ointment (Beijing Shuangji Pharmacy Limited Company, China) was used to maintain eye lubrication. After disinfection with 0.3% hydrogen peroxide, a small incision of the scalp was created to expose the skull. Then, 0.3% hydrogen peroxide was again applied to clean the skull, and craniotomy was conducted. In order to implant two optical fibers bilaterally, we used two stereotactic coordinates to target the lateral hypothalamus (LH). On the left side, we used the following stereotactic coordinates: −0.70 mm posterior to the bregma, 1.0 mm lateral to the midline, and −4.75 mm ventral to the skull surface. On the contralateral (right) side, we targeted the LH at 2.2 mm lateral to the midline with a 14° angle (lateral to middle) with the same anterior-posterior and dorsal-ventral as the left side.

To record fluorescence signals, a beam from a 488 nm laser (OBIS 488LS; Coherent, USA) was reflected via a dichroic mirror (MD498; Thorlabs, USA), focused by a 10 × objective lens (NA = 0.3; Olympus, Japan), and then coupled to a rotary joint (FRJ_1x1_FC-FC, Doric Lenses, Canada). An optical fiber (230 μm O.D., NA = 0.37; 2-4 m long) guided the light between the rotary joint and the implanted optical fiber. The laser power was adjusted at the tip of the optical fiber to a low level (0.01 - 0.02 mW) to minimize bleaching. The GCaMP fluorescence was bandpass filtered (MF525-39, Thorlabs) and collected by a photomultiplier tube (R3896; Hamamatsu, Japan). An amplifier (C7319; Hamamatsu) was used to convert the PMT current output to voltage signals, which was further filtered through a low-pass filter (40 Hz cut-off). The analog voltage signals were digitalized at 200 Hz and recorded with custom software developed in house using MATLAB or LabView.

For activation experiments, 5-ms blue laser pulses were triggered from a 473-nm blue solid state laser (MBL-III-473, Changchun New Industries Optoelectronics Technology Co., Ltd., China) using a Master-8 pulse stimulator (A.M.P.I., Israel). The intensity power for activation at the fiber tip was about 15-20 mW. The stimulation frequency and duration varied for the different behavioral tasks (see below). The inhibition experiments used continuous 515-nm green light (MGL-F-515, Changchun New Industries Optoelectronics Technology Co., Ltd., China) or 473-nm blue light. The power at the fiber tip was 3-5 mW. A fiber-optic rotary joint (FRJ_1x1_FC-FC, Doric Lenses) was used to avoid winding of the fiber-optic cable in freely behaving animals. Bilateral stimulation was achieved by dividing light from one optical fiber to two fibers through fusion splicing.

Behavioral tasks

Cricket hunting task We conducted cricket predation experiments in a cylindrical arena with a transparent bottom (diameter 400 mm, height 300 mm). Behaviorally, latency to attack was represented as the time taken from light stimulation onset until a mouse actually attacked a cricket. We also calculated the attack probability which was quantified as the number of attack trials divided by the total light-on or light-off trials in a behavior secession. In the optogenetic activation experiments, ad libitum mice were introduced into the arena with ten crickets of medium size (12-20 mm body long). One optogenetic stimulation session of 15 min consisted of 10 stimulation blocks (5-ms pulses of 473-nm blue light at 20 Hz were delivered over a period of 30 s; 20 mW) with random inter-block intervals (40-80 s). In the optogenetic inhibition and activity monitoring experiments, mice were first deprived of standard chow for 12 hours. During fasting, 3-5 crickets were introduced into the home cage of the animal. For inhibition, chow-restricted mice were placed in the cylinder with ten crickets of small size (8-12 mm body long), and 2 s long pulses of 515-nm green laser or 473-nm blue laser light were delivered at 0.25 Hz for 20 min. The intensity at the tip of optical fiber was about 1-5 mW. For neuronal activity monitoring, animals were placed in the arena, and small crickets were delivered manually (one-by-one) to the field. The behaviors of mice and crickets were videotaped via a camera placed ∼1 m below the bottom of the cylinder. The onset of camera recording, laser delivery, and neuronal activity were controlled and logged using custom MATLAB programs.

Computer-controlled food-chasing task Mice were food restricted for 12 hours and then habituated to consume dustless precision rodent pellets (14 mg per pellet; Product# F05684, Lot # 200271, BioServ, USA) in their food dish. In the food-chasing task experiments, a total of 10-30 pellets were placed in a dish (diameter 30 mm; 5 mm height). The dish and food-restricted animals were placed in an arena (560 × 560 mm) for food chasing. A magnet (diameter 20 mm; height 5 mm) was glued to the bottom of the dish. We used a motor-driven, two-dimensional motion platform to control the movement of the food dish. The food dish was connected to the two-dimensional motion platform by magnetic force. We developed a program in LabView to control the motorized movement of the food dish. The positions of the animal and the food dish were calculated from the real-time video recorded via an overhead camera. The food-chasing task encompassed 4 phases: initiation, chase, retrieval, consumption. In the initiation phase, a defined trigger zone in one of the corners of the platform was used to activate a given trial: when a mouse remained in the trigger zone for 1 s, the food dish was introduced to the platform at the corner diagonal to the trigger zone corner. Note that the positions of both the mouse and the food dish were monitored with the overhead camera. In the chase phase, the food dish was moved along the wall (‘border’) of the arena. The border along which the food dish was moved in each trial was specified in a pseudorandom order. We progressively increased the speed of the moving food dish from low to high (speed 10 and 20 cm/s) as the animals learned the paradigm. In the retrieval phase, the movement of the food dish was stopped for 1 s and the animal would reach to get a food pellet. The real-time position information for the mouse and the food dish was used to select the moment when the food dish was stopped: for each trial, the food dish was stopped the first time that the distance between the centroid of mouse body and the center of food dish was less than 10 cm. Although the food dish was moved again after 1 s, the consumption phase of the experiment refers to the period when the mouse remains stationary and eats a pellet. Fiber photometry of GCaMP signals were performed throughout all four phases. Optogenetic inhibition was conducted at three separate stages. For each of these, the inhibition manipulation consisted of the delivery of 515 nm or 473 nm laser light for 5 s. The two stages at which the 5 s inhibition laser light could be applied were as follows: when a mouse entered the trigger zone (initiation), or after animals was consuming a food pellet (consumption).

Artificial prey-chasing task We used a cylindrical disk made of red wax as an artificial prey (diameter 30 mm; height 15 mm). A magnet (diameter 20 mm; height 5 mm) was embedded in the prey disk. The movement of the prey disk was controlled by a motor driven two-dimensional motion platform as in the food-chasing task. The prey disk and ad libitum mice were placed into an arena (560 × 560 mm). The prey disk was connected to the two-dimensional motion platform by magnetic force. Whenever a mouse came within 100 mm of the prey disk (between the centroid of the mouse body and the center of the disk), the prey disk was moved away (speed ∼20 cm/s) from the mouse; note that the evasive maneuvering of the prey disk could occur continuously if the mouse repeatedly approached the prey disk. Optogenetic activation was achieved by delivering 20 Hz pulses of blue laser light to the mouse for 30 s, starting the first time that a mouse came within 100 cm of the prey disk. The weight loss of the wax disk was determined by the weight difference before and after each artificial prey-chasing session.

Artificial attacker evasion task These experiments used the same red wax disk as the artificial prey-chasing experiments, but the disk was used as an artificial attacker. The disk was moved toward the mouse (chasing speed ∼30 cm/s) for 15 s. The mouse would evade from the approaching artificial predator disk several times during the 15 s period. We also used 5 s chasing for simplicity during fiber photometry recording. We tested how optogenetic inhibition of LH glutamate neurons affected the behavior of mice exposed to the chasing of the artificial disk. The inhibition light was turned on 5 s before the artificial disk started chasing the mouse and during the entire chasing phase. The total evasion distance, median evasion number, and median peak evasion speed were analyzed during the 15 s evasion bout. We also analyzed the minimum distance between the escaping mouse and the artificial disk to represent the predictive evasion behavior.

Evasion-to-predation transition task The red wax disk was also used in experiments that tested whether optogenetics stimulation of GABA neurons could switch a mouse’s behavior from evasion to predation. A trial period of 30 s was split into two 15 s halves. The disk was controlled exactly as in the artificial predator evasion task for the whole 30 s. For the first 15 s of predation, mice were not stimulated. However, throughout the second 15 s of the trial, the LH GABA neurons of the mice were activated with 20 Hz blue laser pulses. Note that the computer program controlling the disk movement was not changed throughout the 30 s trial.

Predation-to-evasion transition task We first trained hungry mice that expressed ChR2 in LH glutamate neurons to perform the food-chasing task. A test session consisted of 30 control trials (“Stim. OFF”) and 30 stimulation trials (“Stim. ON”) that were intermixed in the pseudorandom order. In the stimulation trials, we optogenetically activated LH glutamate neurons (5 ms at 50 Hz for 1 s) at the specific moment when a mouse captured the food dish.

Intraspecific aggression task Before the intraspecific aggression tests, mice were housed in groups with food and water ad libitum. On the test day, the experimental animal was introduced into a cylindrical arena (diameter 400 mm, height 300 mm) and habituated for 1 min. Then, a stranger male mouse (6-8 weeks old, housed in a different group than the subject mouse) was placed into the arena as a target. For the activation experiments, in a single block of stimulation, 5-ms pulses of 473-nm blue light (20 Hz) were delivered for over a period of 30 s. The intensity at the tip of optical fiber was around 20 mW. A 15-min experiment session included ten blocks with random inter-block intervals (40-80 s). The behavior of animals was recorded via an overhead camera and later assessed for aggressiveness. The attack latency was represented as the time taken from light stimulation onset until a mouse actually attacked the stranger mouse. The attack probability was quantified as the number of intraspecific attacks divided by the total stimulation blocks within a behavior secession.

Real-time place preference task Lammel et al., 2012 Lammel S.

Lim B.K.

Ran C.

Huang K.W.

Betley M.J.

Tye K.M.

Deisseroth K.

Malenka R.C. Input-specific control of reward and aversion in the ventral tegmental area. These experiments were conducted using the real-time place preference methods with a 3-chamber apparatus as previous study used (). The 3-chamber apparatus contains a left chamber (28 cm × 24 cm × 30 cm, length × width × height) with a metal grill floor, a center chamber (11.5 cm × 24 cm × 30 cm) with white walls and a smooth metal floor, and a right chamber (28 cm × 24 cm × 30 cm) with a punched metal floor. The walls of the left and right chamber alongside the center chamber were half-open for the freely behaving of mouse connected with optical patch cable. We conducted baseline and stimulation sessions, and examined the effect of activation or inhibition of both PAG-projecting LH GABA and glutamate neurons. In the activation experiments, animals that entered the optogenetic-stimulation chamber received 20 Hz blue laser pulses. In the inhibition experiments, the mice received constant 2 s delivery of blue laser light per 4 s when they crossed into the optogenetic-inhibition chamber. Each session lasted for 15 min, and the locations of the mouse were assessed from the video recording data using a custom MATLAB program.