Peer review of: Printable microscale interfaces for long-term peripheral nerve mapping and precision control (2019)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 
 
This paper presents a neural interface device for the interfacing of small peripheral nerves, which 
is a difficult problem. The overall goal of this work is the chronic recording and modulation of 
peripheral nerves for therapeutic control of target organs. The researchers present a extraneural 
device made of the unique combination of thin film technology and a 3D printed nerve holder with 
precise geometry. The study showed in vivo implantation of this device for both acute and chronic 
recording and stimulation of a small nerve controlling the songbird syrinx. They characterize the 
stability of the device through recorded neural activity and consistency of songbird vocalizations 
over time. They also present an in vivo acute experimental set up where stimulation from the 
nanoclip produces artificial vocalization as a method of exhibiting current steering patterns. 
Overall, this appears to be a useful tool of broad applicability, and is it particularly remarkable in 
the context of a very small 150 um nerve. However, several major and minor concerns must be 
addressed prior to publication. 
 
Major criticisms 
 
No comparison is presented to any other device, such that one can judge against the state of the 
art. At minimum historical or literature controls should be quantitatively evaluated. 
 
Quantification of the histological response after chronic implant for all 4 animals should be shown if 
it will be discussed. At present, this is qualitatively described, with only a photo of one animal 
shown, whereas this should be easy to measure from these images. If some data has been lost, 
that should be noted, and it should be clear what the distances and n is for the histological claim. 
 
While the unit data presented appears to be impressive, this is a strong claim that should be 
rigorously evaluated. The best evidence is if the song is tightly linked to the putative unit data, as 
it appears to be in Figure 4c and 4d. However, in 4b at the top, there is no clear relationship 
between the ABC motifs and the putative unit activity, which seems inconsistent, and this is also 
inconsistent with 4D. It also also unclear which birds and how many are used throughout this 
figure. The authors should also provide enough information to understand whether most data is 
from 1 exemplary bird out of the 3 by quantifying the uV amplitudes (i.e. not normalized as 
shown) for all 3 birds. 
 
EMG invasion would seem to be a major concern, given the nearby muscles of the trachea and 
syrinx. This could produce the apparent relationship to the song as shown, despite no signals 
being recorded from the nerve. Nerve cuffs often explicitly incorporate shielding because this is 
such a common issue. The authors should confirm that there is no nearby muscle that could 
produce signals that could be misinterpreted as nerve activity, or else confirm this with 
simultaneous recording. 
 
Minor criticisms 
 
Editing for wordiness and run-on sentences is needed throughout the document. 
 
Other thin film nerve cuffs should be cited and discussed, such as TEENI (Desai et al 2017), tf-
LIFE, TIME, and SELINE (Wurth et al.) 
 
P-values are needed with all statements of significance in the results section. 
 
What was the stimulation threshold current used to produce the minimum evoked response in 
Figure 3? 
Correlation and mean amplitude plots in Figure 4D are unclear in the text and caption. 
 
Can you describe the forces exerted on the nerve when pressed against the door via comparison to 
a similar technique? Similarly, fatigue characterization of the trap doors would be of interest. It 
should also be discussed whether the device can be removed from the nerve and repositioned 
during surgery. 
 
It is unclear if the nano clip can move along the length of the nerve. Is there an anchoring method 
used to stabilize the clip in a certain place? 
 
Five animals were recorded until day 30, but 3 animals were recorded past the first month. For 
completeness, what was the reason for 2 animals not being recorded past the first month? 
 
It is important to see the examples of fictive vocalizations in Figure 5e, but the figure is extremely 
small and hard to see. 
 
Is the reason behind the lower stimulation threshold for the nano clip than the bipolar silver hook 
the better insulation provided by the nano clip? 
 

 
Reviewer #2 (Remarks to the Author): 
 
This paper presents a novel microscale implantable device capable of recording from and 
stimulating small nerves. The engineering feats are coupled with detailed electrophysiological and 
behavioral analysis in the songbird vocal system. While a simpler version of this devices has been 
published earlier (Lissandrello et al., 2017), this device is novel as it incorporates a 
microfabricated, multi-channel thin film electrode substrate, which allows for more precise 
stimulation and recording than the previous version. In addition, this paper presents chronic 
behavioral and electrophysiological data, which has not before been published. 
 
In general, the data presented in this paper constitute a ground-breaking advance in small nerve 
recording and modulation, and provides a tool for more detailed study of nerve signals and 
function than currently exists. However, figures tend to lack detail, particularly in the realm of data 
analysis, and should be improved prior to publication. A list of major concerns is presented below: 
 
1. Figure 2 – Histology is mentioned in the text, but no histological images or quantitative 
assessment is provided. Please provide evidence of nerve integrity with implantation of this device, 
both qualitative and quantitative. 
 
2. Figure 2B- Please provide quantitative analysis of the different in spectral similarity between 
nerve crush, sham and Nanoclip implant from non-injured baseline similarity. 
 
3. Figure 4 presents evidence for the stability of nerve recordings across 30 days. However, the 
metric used to quantify these recordings, a smoothed activity envelope, is atypical for neural 
recordings. While not improper, a more conventional metric would be more meaningful for future 
neurophysiological applications of this technology. For instance, Zanos et al. recently presented 
vagal nerve recordings that used wavelet decomposition to identify unique compound action 
potential signals (CAPS), and then reported event rate, inter-cap-interval histograms and signal-
to-noise ratio for each CAP, metrics that are easily comparable to single unit recordings from the 
brain (Zanos et al., 2018). Given the author’s background in neurophysiological recordings in the 
songbird (ie. (Ölveczky et al., 2011)), it is surprising that a more detailed analysis of 
neurophysiological data was not applied. I would recommend the authors add additional 
neurophysiological analysis to demonstrate that this technology can be meaningful to the songbird 
field, and more broadly. 
4. The longevity data in Figure 4 looked very promising and exciting. Again, I would have 
preferred to see this data quantified more extensively – ie. Signal-to-noise, event rate. Predictive 
value for syllable shape would be amazing, although not required. 
 
5. Figure 5 shows specificity of stimulation across different contact sites. The variability is 
quantified by tSNE plot. While tSNE offers the advantages of being an unsupervised non-linear 
visualization technique, it can be prone to misinterpretation, and can change shape with differing 
perplexity and step values. In particular, distances between and within clusters can change 
dramatically with different iterations. Replication of the results in Fig. 5F,G using more 
conventional syllable variability metrics are necessary to support tSNE measurements, and the 
overall conclusion regarding stimulation specificity. 
 
Minor points: 
 
Fig. 4D – please provide a color scale for the color map. 
Fig. 4D – the text box overlaps the figure labels. 
Fig. 4F – does this refer to the signal envelope, or the raw (processed?) signal? 
 
Lissandrello CA, Gillis WF, Shen J, Pearre BW, Vitale F, Pasquali M, Holinski BJ, Chew DJ, White AE, 
Gardner TJ (2017) A micro-scale printable nanoclip for electrical stimulation and recording in small 
nerves. J Neural Eng 14:036006. 
Ölveczky BP, Otchy TM, Goldberg JH, Aronov D, Fee MS (2011) Changes in the neural control of a 
complex motor sequence during learning. J Neurophysiol 106:386–397. 
Zanos TP, Silverman HA, Levy T, Tsaava T, Battinelli E, Lorraine PW, Ashe JM, Chavan SS, Tracey 
KJ, Bouton CE (2018) Identification of cytokine-specific sensory neural signals by decoding murine 
vagus nerve activity. Proc Natl Acad Sci U S A 115:E4843–E4852. 
 

 
Reviewer #3 (Remarks to the Author): 
 
This exciting manuscript presents a new device/system for recording and manipulating the activity 
of small peripheral nerves, in vivo, during behavior and over periods as long as 1 month. Small-
scale scale engineering and experimental neurobiology are brought together to develop small, 
flexible, electrode arrays that are secured to the surface of an intact nerve such that compound 
action potentials can be recorded and nerve fibers can be stimulated in small, freely behaving 
animals during behavior. The function of this new interface is demonstrated with multiple 
experiments. Because development of such systems are important for understanding how neural 
signals in the brain are transformed by the peripheral nervous system into muscle activity, the 
results of this study will be impactful for experimental and clinical neuroscience. The manuscript is 
well written; the system components, experiments, data and analyses are clearly described. 
Below, I suggest improvements to the manuscript that would further demonstrate the system’s 
safety and broad-scale usefulness. 
 
Major 
1. The choice of the zebra finch for testing the system is wise because the anatomy and physiology 
of the PNS involved in singing are well described and the motor production of song is highly 
stereotyped. Here, the tracheosyringeal nerve, which innervates the muscles of the syrinx, and 
singing behavior are studied to test the usefulness of the system. First, the authors show that they 
reliably obtained interpretable nerve recordings over time (indicating little to no nerve damage) 
and across animals (indicating precise and consistent placement of electrode contacts). Second 
they show that the system can be used to evoke nerve activity via electrical stimulation that is specific to the spatial pattern of stimulating electrode contacts, and that results in vocal output. 
While these demonstrations are strong, they could be improved without collecting more data. First, 
the results of the histological analyses of nerve segments that were housed in the clip for weeks 
should be added. If the quantified analyses of control and clipped nerve segments shows that the 
clip does not damage the nerve fibers even after a month of implantation, then the success of the 
clip design will be strongly supported. Second, the Results should include information on the 
estimated number and spatial extent of fibers that are stimulated by each electrode contact and 
how that relates to the experimental observations of different activity patterns and vocalizations 
when different spatial patterns of stimulation are used. The addition of this information will 
strengthen the argument that the system can be used to manipulate nerve activity in a precise 
manner. 
 
2. The broad scale usefulness of a new approach/system is clearest when the system is tested in 
more than one experimental context, here, a nerve. With the goals in the Introduction in mind, the 
paper would be stronger if clipping, recording and stimulating were demonstrated in a second 
nerve. A candidate is the 8th nerve, which is accessible and reasonably well studied in birds. The 
addition of these data would show the generalizability of implant placement without functional 
damage, accurate recording of nerve activity and the effectiveness of stimulation. 
 
Minor 
 
1. Add detection threshold to Figure 4c. 
2. Add one high resolution plot of the smoothed activity for at least 1 ABC sequence to Figure 4d. 
The data shown as a heatmap over recording days is great, but an intermediate step between 
panels c and d is needed. 
3. If I understand the Figure 5 experiments, panels a – c and d – g describe completely different 
experiments. The top panels show experiments with 2 clips, one recording and one stimulating. 
The bottom panels show experiments using only a stimulating clip, and vocal production with 
stimulation of the nerve and air pressure in the respiratory system. If this is correct, then Figure 5 
should be revised into 2 figures, each showing one of these experiments. 
4. The full paragraph on the second page of the Discussion includes sentences, phrases and words 
that systems neuroscience readers with not understand without explanation. Examples are 
“present elastic moduli substantially above the kilopascal range of the host tissue” and “despite 
the Young’s modulus of their bulk material being within the gigapascal range.” Please add 
explanation. 
5. The Methods section includes a subsection on the quantification of 5 syllable features. But these 
features are not in the Results. 
6. Typo in the Methods “was calculated was calculated” 
7. Typo in the Methods “20 unrelated.” 
8. Add page numbers or line numbers to the ms.