TITLE: The Acute Effects of Gastric Electrical Stimulation on Nucleus of the Solitary Tract Neural Activity Assessed with Electrophysiological Recording
AUTHORS: Jiayue  Cao, Kun-Han Lu, Xiaokai  Wang, Zhongming Liu

[DESCRIPTION]
Brief description
This protocol describes the methods used to evaluate the effects of gastric electrical stimulation (GES) on neural activity in the nucleus of solitary tract (NTS). Briefly, the multi-unit activity (MUA) and local field potential are accessed through multi-channel electrophysiological recording. The stimulation is applied during the imaging acquisition. The configuration of VNS was varied in terms of pulse parameters (amplitude, width) and polarity (proximal or distal coil of a bipolar electrode connected as the cathode).

Experiment design
The purpose of this study is to study how NTS neurons respond to GES with different parameter settings. To do so, we varied the stimulus parameters in terms of pulse amplitude (PA: 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6mA), width (PW: 0.1, 0.2, 0.3, 0.4, 0.5ms), and the pulse polarity. The pulse polarity can also be viewed as the current direction of the stimulus. For example, when delivering current through the bipolar electrode, the anode and cathode setting has two options which can result in two polarities of the stimulus. We altered the amplitude, pulse width, and pulse polarity. A total of 100 different parameter settings were tested for a single trail. The recording electrode was moved around to record from different groups of neurons in a single animal. The stimulation trail can be repeated multiple times on each animal but for different neurons.

[STEPS]
SECTION: Anesthesia Procedure
1. Anesthetize the animal with 5% Isoflurane mixed with 100% oxygen at a flow rate of 500-1000 mL/min for 5 minutes (or till the animal loses consciousness).
SECTION: Anesthesia Procedure
2. After the animal loses consciousness, deliver a subcutaneous bolus injection of 0.075 mg/kg dexmedetomidine solution (0.05 mg/mL, Zoetis, NJ, USA).
SECTION: Anesthesia Procedure
3. After five minutes, continuously deliver 0.1-0.5% isoflurane through a nose cone.
SECTION: Anesthesia Procedure
4. Fifteen minutes after the bolus, deliver a continuous subcutaneous infusion of the dexmedetomidine solution at 0.015 mg/kg/hour through an infusion line by a syringe pump.
SECTION: Anesthesia Procedure
5. Adjust the dose of isoflurane and dexmedetomidine to keep the heart rate between 250 beats/min and 350 beats/min, and respiratory rate between 30 times/min and 60 times/min (Lu, et al., 2012, https://doi.org/10.1073/pnas.1200506109).
SECTION: Surgical preparation
6. 
 

SECTION: Surgical preparation
7. Following a toe-pinch test, made a ∼3 cm incision starting at 1 cm caudal to the xiphoid and moving 3 cm caudally.
SECTION: Surgical preparation
8. After separating and retracting the skin and muscle layers, expose the ventral stomach such that the intersection of the greater curvature and the limiting ridge can be identified.
SECTION: Surgical preparation
9. Suture a pair of electrodes (Microprobes, Gaithersburg, MD, USA) directly onto the forestomach wall.


 

SECTION: Surgical preparation
10. Close the muscle and skin layers at the incision site with sutures.
SECTION: Electrical Stimulation
11. After the electrode implantation, secure the animal in the stereotaxic frame (Stoelting Co, Wood Dale, IL, USA).
SECTION: Electrical Stimulation
12. Made a midline incision through the scalp.
SECTION: Electrical Stimulation
13. Localize the nucleus of solitary tract (NTS) at the connection between the occipital bone and spinal cord.
SECTION: Electrical Stimulation
14. Open a 3 × 3 mm window at the end of the occipital bone, and dissect the tissue above the spinal cord to expose the dorsal surface of the brainstem.
SECTION: Electrical Stimulation
15. Identify the obex was, and place the recording electrode on the left side of obex at AP from 50 µm to -10 µm

SECTION: Electrical Stimulation
16. Secure micro-screws above the olfactory bulb, and use them as reference and ground.
SECTION: Electrical Stimulation
17. Connect the recording electrode to a broadband recording system (Tucker Davis Technologies, Alachua, FL, USA) that amplifies and digitizes (at 25 kHz) the voltage between the recording site and the reference site (i.e., a micro-screw secured over the olfactory bulb).


 

SECTION: Electrical Stimulation
18. Recording setup:

1. Recording details
Sampling rate = 24 kHz
AC coupling
No band-pass filter

2. Data saving
Raw data
Local field potential (optional)
Stimulation triggers
SECTION: Electrical Stimulation
19. Stimulation parameters:
Waveform: biphasic pulses with delay
Stimulation amplitude: 0.2 mA, 0.4 mA, 0.6 mA, 0.8 mA, 1.0 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA
Stimulation pulse width: 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms
Inter-pulse duration: 50 m