Does TRH increase LSNA or BAT SNA via actions in the DMH and/or RPa?

Induce a surgical depth of anesthesia with 5% isoflurane; once established, maintain with 1.5%–2.5% isoflurane. 

Place the rats in a stereotaxic instrument, and make a midline skin incision to expose the surface of the skull.

Level the top of the skull in the sagittal plane by adjusting the mouth-bar until Lambda and Bregma are at the same height.

Drill window (~3x3 mm) into skull just caudal to Bregma until there is a thin layer of bone.

Use fine forceps to pull out the rest of the bone, exposing the brain.

Insert arterial and venous catheters (PE50 tubing).

Identify the lumbar (via a midline incision) or brown adipose tissue (BAT) nerve using a dissecting scope, mount on bipolar stainless-steel electrodes, and then embed with silicone gel (Kwik-Sil, World Precision Instruments). For the BAT nerve, isolate the central cut end of a small-diameter nerve bundle from the ventral surface of the right interscapular fat pad after dividing it along the midline and reflecting it laterally.

For measurements of BAT temperature, insert a thermocouple meter (4134; Control Company, Friendswood, Texas) with a Type T needle style microprobe thermocouple (Physitemp, Clifton, NJ) in the intact, left interscapular BAT fat pad. 

Use a water‐perfused thermal blanket to maintain the skin and body temperature. Record expired CO₂ using a CapStar-100 Carbon Dioxide Analyzer and maintain at 30-40 mmHg via artificial ventilation and by adjusting the respiratory rate and tidal volume of 100% oxygen. Continuously record pulsatile and mean arterial pressure, heart rate, and raw sympathetic nerve activity (SNA) throughout the experiment with Grass amplifiers (model 79D, Grass Instrument Co.) and a Biopac MP100 data acquisition and analysis system (BIOPAC Systems Inc.), sampling at 2,000 Hz. SNA is bandpass filtered (20–3,000 Hz) and amplified (×10,000). The SNA signal is then rectified and integrated in 1-second bins. After data collection, background postmortem SNA is subtracted from values of SNA recorded during the experiment.

After completion of surgery, transition from isoflurane to α-chloralose anesthesia (Sigma; 50 mg/kg loading dose followed by 25 mg/kg hr continuous infusion for the duration of the experiment) over 30 min while slowly withdrawing the isoflurane. Anesthetic depth is regularly confirmed by the lack of a pressor or withdrawal response to a foot or tail pinch.

Pull capillary tubes (I.D. 0.5mm, O.D. 1mm) to produce long but sturdy (unbendable) single‐barreled glass micropipettes (20–40 μm tip OD).

Dissolve drugs for microinjection in artificial cerebrospinal fluid (aCSF) containing (in mM): 128 NaCl, 2.6 KCl, 1.3 CaCl2, 0.9 MgCl2, 20 NaHCO3, 1.3 Na2HPO4 and 2 dextrose, pH 7.4 and filter before use to prevent pipette plugging.

Inject TRH (0.5 mM in 60 nl, P1319, Sigma-Aldrich St. Louis MO) or aCSF into the DMH or RVLM using the following coordinates: 

DMH: 3.2 mm caudal from Bregma, 0.5 mm lateral from the midline, and −8.3 to –8.8 mm ventral from the dura.

RVLM: 3.5 mm caudal from Lamda, 1.8–2.0 mm lateral from the midline, and -8.8 mm ventral from the dura.

Nanoinjections are conducted using the following procedures: 

--Use a pressure injection system (Pressure System IIe, Toohey Company, Fairfield, NJ, USA) to inject over approximately 5–10 s. 

--Nanoinjections are made bilaterally (60 nl for DMH or RVLM), with ∼2 min between injections.

--injection volume is controlled by calibrating the volume in the injection tubing and watching the fall of the meniscus level of the injectate under a surgical microscope.

--include fluorescent polystyrene microspheres (FluoSpheres, F8803, 1:200; Molecular Probes) in the injectate to verify the injection sites.

At the conclusion of the experiment, the remove brains and place in 4% paraformaldehyde for at least 48 hours. 

Slice the hypothalamus (or brainstem) into 50-μm sections using a vibrating microtome. 

Confirm correct injection placement using a rat brain atlas (Paxinos & Watson, 2007) and a fluorescent microscope.