TITLE: Chronic Vagus-Nerve Activity with Carbon Nanotube Sensors in Freely Moving Rodents
AUTHORS: Grant Mccallum, Joseph Marmerstein, Dominique Durand

[DESCRIPTION]
This study presents the first chronic recording and decoding of activity in the vagus nerve of freely moving animals. The majority of vagal afferent fibers come from the gut and abnormal vagal activity has been linked to eating and metabolic disorders. The study uses carbon nanotube yarn electrodes, which have small size, high flexibility, and low impedance, to provide a stable, high-signal-to-noise ratio interface for chronic recording in small autonomic nerves in rats, with high-quality signals continuing up to four months after implantation. The technology was successfully applied to make the first direct measurements of vagal tone in freely moving animals, and the neural-recording data was synchronized with continuous video recording of the subjects. The results show several spike clusters that show tuning to animal eating, and the firing dynamics of multiple decoded spike clusters can be used to classify eating compared to drinking, grooming, and resting behaviors.

[STEPS]
SECTION: Protocol for CNTY Electrode Manufacture:
1. Carbon nanotube yarns (CNTYs) were manufactured at Case Western Reserve University using a previously described method (https://doi.org/10.1038/s41598-017-10639-w).
SECTION: Protocol for CNTY Electrode Manufacture:
2. CNTYs were connected to 35NLT®-DFT® wire using silver conductive epoxy, creating a CNTYDFT® junction.
SECTION: Protocol for CNTY Electrode Manufacture:
3. Dacron mesh and silicone elastomer were added to seal the junction, and the impedance of the junction was measured at 1kHz in a saline bath to confirm the seal.
SECTION: Protocol for CNTY Electrode Manufacture:
4. The free end of the CNTY was tied to the end of an 11-0 nylon suture using a fisherman’s knot, as shown in Figure 1A.

   

SECTION: Protocol for CNTY Electrode Manufacture:
5. The entire CNTY was coated with parylene-C (5 μm thick vapor deposition coating) on a custom rack.
SECTION: Protocol for CNTY Electrode Manufacture:
6. A small section (~200 μm long) of parylene-C was removed approximately 500 μm behind the CNTY-suture knot using a laser spot welder, as shown in Figure 1B.
SECTION: Protocol for CNTY Electrode Manufacture:
7. Electrode viability was confirmed by measuring the impedance of the recording site before and after using the laser.
SECTION: Surgical procedure for implanting the electrodes in rats:
8. All surgical and experimental procedures were done with the approval and oversight of the Case Western Reserve University, Institutional Animal Care and Use Committee to ensure compliance with all federal, state and local animal welfare laws and regulations.
SECTION: Surgical procedure for implanting the electrodes in rats:
9. Electrodes were implanted in male Sprague Dawley rats between 7–12 weeks of age
SECTION: Surgical procedure for implanting the electrodes in rats:
10. A midline incision was made along the neck to expose the left cervical vagus nerve.
SECTION: Surgical procedure for implanting the electrodes in rats:
11. The muscles and salivary glands were separated and held in place, revealing the carotid sheath which contains the carotid artery and vagus nerve.
SECTION: Surgical procedure for implanting the electrodes in rats:
12. The vagus nerve was carefully separated from the carotid artery using blunt dissection and held in slight tension using a glass hook.
SECTION: Surgical procedure for implanting the electrodes in rats:
13. CNTY electrodes were implanted by sewing the suture through the nerve for ~2 mm.
SECTION: Surgical procedure for implanting the electrodes in rats:
14. After the implantation, the signals travel from the implants to the headcap connector mounted on the animal’s skull, where they are digitized and amplified by the custom amplifier board. These signals are then routed through a commutator, which allows the animal to move freely without twisting or pulling on the cable.
SECTION: Surgical procedure for implanting the electrodes in rats:
15. The signals are sent to an Intan USB interface board, which sends the signals to a computer, where they are saved and can be viewed in real time.