A flexible carbon nanotube electrode array for acute in vivo EMG recordings

Abstract

Executing complex behaviors requires precise control of muscle activity. Our understanding of how the nervous system learns and controls motor skills relies on recording electromyographic (EMG) signals from multiple muscles that are engaged in the motor task. Despite recent advances in tools for monitoring and manipulating neural activity, methods for recording in situ spiking activity in muscle fibers have changed little in recent decades. Here, we introduce a novel experimental approach to recording high-resolution EMG signals using parylene-coated carbon nanotube fibers (CNTFs). These fibers are fabricated via a wet spinning process and twisted together to create a bipolar electrode. Single CNTFs are strong, extremely flexible, small in diameter (14-24 µm), and have low interface impedance. We present two designs to build bipolar electrode arrays that, due to the small size of CNTF, lead to high spatial resolution EMG recordings. To test the EMG arrays, we recorded the activity of small (4 mm length) vocal muscles in songbirds in an acute setting. CNTF arrays were more flexible and yielded multiunit/bulk EMG recordings with higher SNR compared with stainless steel wire electrodes. Furthermore, we were able to record single-unit recordings not previously reported in these small muscles. CNTF electrodes are therefore well-suited for high-resolution EMG recording in acute settings, and we present both opportunities and challenges for their application in long-term chronic recordings.NEW & NOTEWORTHY We introduce a novel approach to record high-resolution EMG signals in small muscles using extremely strong and flexible carbon nanotube fibers (CNTFs). We test their functionality in songbird vocal muscles. Acute EMG recordings successfully yielded multiunit recordings with high SNR. Furthermore, they successfully isolated single-unit spike trains from CNTF recordings. CNTF electrodes have great potential for chronic EMG studies of small, deep muscles that demand high electrode flexibility and strength.

Keywords: carbon nanotube fibers; electromyography; motor systems; muscles; neurophysiology.

Graphical abstract

Figure 1.

Motor units, EMG, and carbon nanotube fibers (CNTFs) array construction. A, left: schematic of a motor unit, which consists of all muscle fibers (shown in pink) innervated by a single-motor neuron (shown in black). When a motor neuron fires an action potential (light blue waveform), the action potential moves down the axon to the muscle fibers it innervates, causing the innervated muscle fibers to fire a near-synchronous volley of action potentials (dark blue waveforms). Right: example recordings of multiunit EMG activity. B: scanning electron microscopy image of two parylene-coated carbon nanotube fibers twisted together, creating a bipolar electrode. C: each bipolar electrode was labeled with colored dots of ultraviolet (UV) glue. Each electrode array consisted of four CNTF bipolar electrodes and one ground wire. D: CNTF array schematic for design 1. Each proximal end of four CNTF bipolar electrodes and one ground wire were secured into male Omnetics pins with carbon glue, creating a two by five electrode array. E: CNTF array schematic for design 2. Each proximal end of a CNTF bipolar electrode was inserted into side-by-side gold-plated holes on a Neuralynx electrode board interface and secured with a gold attachment pin.

Figure 2.

Illustration of the respiratory and vocal system of songbirds. We measured EMG activity in either the right or left expiratory (Exp) muscle group or the right and/or left ventral syringeal (VS) muscles and/or superficial ventral tracheobronchial (sVTB) muscles. VS and sVTB muscles are part of the songbird vocal organ, syrinx.

Figure 3.

The bending stiffness of carbon nanotube fiber (CNTF) arrays is over eight times lower than stainless steel fine wire. The bending stiffness of a four-channel array electrode bundle (black lines) was 2.4 ± 0.1 mN/mm for CNTF (yellow, P ≪ 0.01, R² = 0.91) and 19.6 ± 0.5 mN/mm for SSW arrays (orange, P ≪ 0.01, R² = 0.99). The CNTF arrays thus required 8.3 times less force to bend the same amount compared with a SSW array. SSW, stainless-steel wire.

Figure 4.

Multiunit data analysis and results. A: an example recording (Table 1: bird 1, recording 1, electrode 3) of multiunit EMG activity from VS muscle. The signal (pink shaded regions) and noise (blue shaded regions) regions were used to calculate the SNR (see methods). B: example recording (Table 1: bird 1, recording 1, electrodes 1, 2, and 3) of the multiunit SNR values for an extended continuous recording in three vocal muscles. All SNR values for each complete breath cycle were averaged across 1 min. C: the SNR of CNTFs (birds 1–8) in vocal muscles ranged from 8.4 ± 3.7 to 43.6 ± 3.3 and was significantly higher (one-way ANOVA, P = 0.032) compared with SSW (birds 9–11) with SNR of 6.2 ± 1.2 to 7.9 ± 1.7. CNTF, carbon nanotube fiber; SNR, signal-to-noise ratio; SSW, stainless-steel wire; VS, ventral syringeal.

Figure 5.

Recording distinct motor unit populations within and across muscles. A: left is an example recording (Table 1: bird 6, recording 2, electrodes 1, 2, and 3) of different multiunit EMG activity within (electrodes 1 and 2) and across (electrode 3) multiple vocal muscles simultaneously in a female ZF bird. B: left is an example recording (Table 1: bird 8, recording 1, electrodes 1 and 2) of different multiunit EMG activity across two vocal muscles simultaneously (VS and sVTB) in a male ZF bird. In both examples, all electrodes are picking up different motor units in the identified muscle. The right panels in both A and B are ventral views of the female and male syrinx (adapted from Ref. 23), respectively, and the corresponding muscles shown in the example recordings. The female and male syrinx have uniform muscle definitions and muscle attachment sites. The red dots on the syrinx represent the location of the recording electrodes. sVTB, superficial ventral tracheobronchial; VS, ventral syringeal; ZF, zebra finch.

Figure 6.

CNTF electrodes successfully record the activity of single-motor units in small muscles. A, top left: example recording (Table 1: bird 3, recording 1, electrode 3) of single-unit EMG activity from expiratory muscle. We used a previously published spike-sorting algorithm (27) that uses principal component analysis to distinguish voltage waveforms belonging to a single motor unit (magenta dots, top right) from background noise (black dots, top right). The peak amplitude of each single unit waveform and the corresponding noise region (blue shaded region) were used to calculate the SNR, as explained in methods. B: example recording (Table 1: bird 3, recording 1, electrode 3) of the SNR values in a 29-min continuous recording in the right expiratory muscle (n = 2,585 breath cycles). All SNR values for each complete breath cycle were averaged across 1 min. C: additional examples of single motor unit recordings (note that top and bottom traces come from different subjects but are plotted on the same time scale; Table 1: bird 7, recording 1 and bird 5, recording 1, electrode 3; top and bottom, respectively). Colored triangles in A and C indicate the spike times of single motor units as determined by our spike-sorting algorithm (see methods). CNTF, carbon nanotube fiber; SNR, signal-to-noise ratio.