Optogenetic inhibition/activation of ERαvlVMH → DRN neurons

Esr1-Cre female mice at eight weeks of age were bilaterally injected with 200 nl AAV-DIO-iC++-EYFP into the vlVMH. Simultaneously, a light fiber (200 nm diameter core, N.A. 0.22, Thor Laboratories, Newton, NJ, USA) was implanted to target the DRN (4.65 mm posterior, 0.00 mm lateral and 3.25 mm ventral to the Bregma). Under the same anesthesia, an emitter was intra-abdominally implanted and a temperature transponder (IPTT-300, Bio Medic Data Systems, Seaford, DE, United States) was implanted under the BAT. Two weeks after surgeries, blue light stimulation (473 nm, 10 ms/pulse, 20 Hz, 3 s on and 2 s off for 1 h) was used to inhibit ERα^vlVMH→DRN neurons during the lights-on period between 8 am to 9 am. BAT thermogenesis, core temperature, and physical activity were recorded before, during and 1 hour after stimulation. As a control experiment, yellow light with the same modulation was applied. The intensity of light power exiting the fiber tip corresponds to 5 mW for blue and yellow light. 

                                                                                        IPTT-300 implantation under BAT

For IPTT-300 implantation, the following skin preparation will be done. The sterile, bland ophthalmic ointment will be placed in the eyes after animals become unconscious to prevent corneal drying and to keep solutions used in surgical scrub out of the eyes. After being anesthetized, mice will be shaved to expose the skin of interscapular areas. Then, we will sterilize this area by cleansing with surgical soap, followed by wiping with 70% alcohol (3 times). The surgeon wears a mask and sterile gloves.

BAT IPTT-300 sc implantation: 

(1) Absence of withdrawal reflex to toe pinch will be used to monitor the depth of anesthesia. 

(2) A 0.5cm incision will be made between the scapulae for implantation. 

(3) IPTT-300 transponder is preloaded on a syringe-based device. Before use, unlock the plunger to make it ready for implanting. Holding the device like a hypodermic syringe with a thumb at the rear of the implanter, apply slight forward force on the plunger to unlock. Insert the entire cannula through the incision and place it along the midline of the upper dorsal surface between two BAT pads. 

(4) The whole procedure will take about 5 mins. 

(5) The skin incision will be sutured by sterile Prolene thread. The suture will be removed 7 days after surgery.

(6) Meloxicam (2mg/kg) will be sc injected 0.5 hours before the surgery.

Emitter abdominal implantation

1. Keep the probes in 17% Benzalkonium chloride solution the day before implantation, and rinsed them with two changes of sterilized MilliQ water (50mL each) just before use.
2. Anesthetize the animal prior to preparation. Depth of anesthesia may be ascertained by using the pedal reflex technique. Pinch the toe of the animal. The lack of a withdrawal reaction means the depth of anesthesia is adequate.
3. Shave the ventral surface of the abdomen, shave the thorax to the area of the axilla, slightly cranial on the right side (light gray areas illustrated below).
4. Scrub the shaved area with Betadine or other surgical scrub.
5. Secure the animal to a sterile surgical surface with adhesive tape. Make sure the surgical surface is heated.
6. Make a midline abdominal skin incision 1 cm below the diaphragm, and no more than 2cm in length.
7. Open the abdomen by making a 2 cm incision along the linea abla (the “white line” of fascia where the abdominal muscles join on the midline).
8. Positioning of the E-Mitter in the abdominal cavity is important to acquire accurate temperature data. If it is too close to the surface of the skin, it may be affected by room temperature.
9. Gently reflect the intestines and colon. Slip the body of the E-Mitter into the abdominal cavity along the sagittal plane, placing it in front of the caudal arteries and veins, but dorsal to the digestive organs.
10. Pass suture material through the silastic tubing that is attached to the outer wall of the E-Mitter capsule.
11. Suture the capsule to the body wall.

Making use of the sleeve to anchor the E-Mitter to the body wall will reduce the likelihood of E-Mitter moving freely within the peritoneal cavity. This may also decrease the need of the ER-4000 Receiver to re-acquire a signal lock from the implanted transponder.

1. Replace the organs, effectively “burying” the E-Mitter.
2. Massage and lightly jostle the abdominal cavity to allow the internal organs to settle. Examine the small intestine and colon for kinks.
3. Place simple interrupted absorbable sutures as needed to close the abdominal incision. See “Closure” in this Section for typical closure procedure.
4. After the study, use 1% Targazyme solution to remove blood and tissue residue.