Modulation of Vestibular Microphonics: A Historical Note

Round 1

Reviewer 1 Report

The authors contribution to vestibular and cochlear research over several decades been significant, and the novelty of this research alone represents impressive work in the field. However, there are several sections of the manuscript which could benefit from revision.

Minor Comments:

Abstract:

Line 5: Change (MET) to after the word ‘transduction’ to accurately reflect the abbreviation.

Introduction:

Line 9: Mention that this change in utricular sensitivity was during Bone-conducted vibration i.e. “to investigate the sensitivity of the guinea pig utricle to Bone-conducted vibration, by monitoring the utricular microphonic during low-frequency hydrodynamic biasing…”

Line 10: Please correct uricular to utricular.

Line 12: Please clarify the primary stimulus used to evoke the Vestibular Microphonic, i.e. “rotational modulation of the semiciruclar canal microphonic evoked by air-conducted sound stimuli”

Line 14: Recommend adding a line for context such as “Vestibular microphonics have been recorded using a wide array of stimuli, in several different mammalian and non-mammalian models (Brown, Pastras, Curthoys. 2017).

E.g. Brown, D. J., Pastras, C. J. & Curthoys, I. S. (2017). Electrophysiological Measurements of Peripheral Vestibular Function-A Review of Electrovestibulography. Frontiers in Systems Neuroscience 11.

Line 14-15: Consider removing ‘(cochlear microphonics were already known for a longer time)’. Suggest adding something more specific like “Albeit, 19 years after the first recording of the Cochlear Microphonic by Wever and Bray”.

Reference: Wever, E. G. & Bray, C. W. (1930). Action currents in the auditory nerve in response to acoustical stimulation. Proceedings of the National Academy of Sciences 16(5): 344-350.

Line 21: I recommend clarifying which vestibular end-organ the VM is coming from. Such as “modulation of the acoustically-evoked horizontal Semicircular canal (hSCC) microphonic”.

Also, if the VM is evoked by auditory stimuli (sound) and the modulation or bias stimulus is rotation, then please make this more clear, e.g. “acoustically-evoked VM modulated by rotation”.

Line 26: To me ‘Audio range’ just means 20-20,000Hz, not specifically air-conducted sound (ACS). Maybe this could be spelled out clearer. I.e. replace Audio range with acoustic stimuli or ACS.

Line 26: Modify. Add that the vestibular system is also sensitive to Bone-conducted vibration. For example “However, the vestibular system is also sensitive to audio frequency Air-conducted sound [5, 6, 7,8], and Bone-conducted vibration [Pastras et al., 2017] up to 5kHz. However, due to the low-pass filter characteristics of vestibular hair cells, the AC component of Vestibular Microphonic is largely attenuated at the hair cell corner frequency around 1-2kHz [Pastras et al., 2021].

Line 28-30: Suggestion: modify or re-write. I.e. “This current work reanalyses previous data showing the rotational modulation of the hSCC microphonic in pigeons. Results demonstrate a reliable computational approach to approximate peripheral vestibular sensitivity and MET channel gating from the semi-circular canals, analogous to the recent work by Pastras et al. 2020”.

Materials & Methods:

Line 33: Please add the number of pigeons used in the previous study. i.e. 34.

Line 34: Possibly clarify what ‘slightly affects the vestibular system’? E.g. it caused a dehiscent labyrinth and increased vestibular sensitivity possibly via increased hydroacoustic energy through to the vestibular system OR, altered the homeostatic environment of the labyrinth, and altered ionic conditions, and results in reduced sensitivity?

Lines 37-50 are taken somewhat verbatim from Wit et al. 1988. Please rewrite or mention this. For example, “the following methodology was taken directly from [2].

Line 50: Figures 1 and 2 are taken from Wit et al. 1988. Please acknowledge this in the figure legend (e.g. taken from Wit et al. 1988). Does this need approval from the copyright owner via RightsLink® - Copyright Clearance Center?

Analysis of a result:

Figure 2: It is not clear to the reader what the amplitude of the microphonic is in terms of mV or uV? Please add this in if possible from the original work? If not, please state this and possibly add that voltages are not necessary for this method because you are fitting waveforms to a Boltzmann function to determine MET gating and sensitivity. 

Figure 2: Onset responses evoked by transient stimuli such as monophasic square wave pulses are complex and difficult to interpret. This is evident in the complex differences between onset microphonics, between the 20deg/sec2 and the 200deg/sec2 stimuli. I suggest using figure 3 from Wit et al. 1988, which shows the modulation of the microphonic to sinusoidal stimuli. This is more easy to interpret, and clearly shows the sinusoidal vestibular microphonic saturating nonlinearly at higher simulation levels at the positive excursion of the waveform (in the open direction of the MET channels) – i.e. the stereocilia are biased in the OPEN direction.

Line 54: It’s interesting that x10 larger angular acceleration (200deg/sec2) resulted in a double-peaked onset microphonic (with 2 different time constants) – what do you think is happening at the level of the stereocilia and MET channels compared to the ‘monophasic’ onset response evoked by 20deg/sec2? Maybe you could comment on this? Do you think there are different mechanics of the canal hair cells with different stimulation levels, i.e. a more complex micro- (or macromechanical) activation of the stereocilia with a higher stimulation level – with abrupt opening and closing of the MET channels, followed by more sustained opening and closing of these channels. Another reason, why sinusoidal stimuli is more appropriate to use – because you avoid this complexity.

Line 56-58: Consider re-writing.

Line 58: Geleoc measured in vitro hair cell responses in much different physiological conditions to these experiments – you could mention that it was ‘in vitro’ to aid the reader.

Geleoc et al used a ‘second order Boltzmann function’, which is more asymmetric about the zero-crossing – please note this.

It seems Fig. 3 shows a first-order Boltzmann function, whereas Geleoc used a 2^nd order function – please justify why you are using a 1^st order symmetrical Boltzmann function here when the mathematical fit was that of a 2^nd order?

Although experiments by Geleoc et al. in vitro suggest isolated cochlear and vestibular Hair cell transduction is best characterised by a 2^nd order curve (which is asymmetric), Patuzzi & Moleirinho and recently Pastras et al., 2020 determined that the cochlear microphonic and utricular microphonic can be adequately characterized by using a 1^st order Boltzmann function. There is no good evidence to suggest that the MET transfer curve is second order under the conditions the cells encounter in vivo, or at the >200Hz frequency used in these experiments. In particular, the waveforms in vivo are filtered by the electroanatomy of cochlea and vestibular organs, in vivo, and the complex extracellular environment between the recording electrode and the excitable cells. Patuzzi and Pastras both determined that the ‘filtered’ in vivo potentials are most accurately fit by using a 1^st order Boltzmann function. Looking at the waveform in Fig. 3 (Wit et al. 1988) it also looks like these microphonics could be modelled using a 1^st order Boltz function.

Consider discussing these differences and describe why you have chosen to go with a 2^nd order function, which has been used in the in vitro setting.

Figure 3: Please remove the visible textboxes in the figure (visible in PDF form). Make these transparent in their original format.

Line 61-65: Good work. No one has done this numerical analysis of the vestibular system to date – especially with in vivo data and with the microphonic.

Figure 4: Please remove textbox around y-axis label.

Figure 5: b) Possibly position the dot on the Boltzmann function so the reader can both visualize MET current and VM amplitude simultaneously.

Figure 5: Consider switching a and b – i.e. have MET current first since it’s modulation is dependent factor which changes VM sensitivity.

Consider having a paragraph directly after figure 5, i.e. following on from line 76, describing the potential utility of the tool in vivo. For example “The vestibular microphonic provides an objective measure of vestibular hair cell function in various experimental animals models (Wit et al, 1986; Pastras et al, 2017). Like the cochlear microphonic, the technique also has the ability to differentially diagnose peripheral nerve from hair cell dysfunction. In order to do this reliably, there needs to be accurate assessment of peripheral vestibular hair cell sensitivity. This tool demonstrates a novel technique to approximate MET channel conductance and VM sensitivity during longitudinal experimental manipulations, similar to recent work by Pastras et al. 2020”.

Overall, your work in the field is admirable and a constant inspiration to the reviewer. This work is worthy of publication following some minor revisions. 

Best, 

Chris Pastras

Author Response

Almost all comments and suggestions from reviewer 2 are used to improve the manuscript.

The changes are in red in the manuscript.

Reply to a few comments from reviewer 2:

1. Figure 2. The text amplitude (n) was added to fig. 2. 

2. The responses to sinusoidal stimuli was not chosen. After the square acceleration pulse has ended, the system returns “freely” towards its equilibrium position, leading to x[t] = e ^ {-beta * t ( line 73 in the revised manuscript). This relation is an essential assumption in the fit. 

This stimulus gives - furthermore - by far the largest excursions, covering a large part of the MET curve (see figure 5). 

3. Figure 3 is asymmetric. It illustrates formula (1). (Line 69 in the revised manuscript). A comparison of an analysis with a first order and a second order Boltzmann function is beyond the scope of this note, which is just a description of a method.

4. There are no visible textboxes in my figures and pdf.

5. A dot was added to figure 5b. Figures 5a and b were not switched. Figure 5a “naturally” follows figure 4. See text lines 79-83 in the revised manuscript. Figure 5b then “explains” figure 5a.

Reviewer 2 Report

This is an interesting short reanalysis of previous data. This reviewer believes that the manuscript could be improved following the comments below. 

MAJOR COMMENTS:

Comment #1- The introduction section needs to be modified.

First, basic information about the vestibular microphonic should be found in the first lines of the introduction in order to provide guidance to the reader about what is vestibular microphonic and what is the value of developing methods to assess this specific evoked potential. Part of this could be answered by moving the paragraph found at page 5 line 85 to 91 to the introduction section.

Moreover, the paper is lacking a clear and concise objective. Why is it important to reanalyze these data? What was in Pastras et al. paper that drove the author to reanalyze his data?

Comment #2- Methods and material: information is missing

1. How many pigeons were analyzed?
2. Was the test performed on both peripheral vestibular systems?
3. What were the parameters of the rotational stimuli (frequency, direction [ipsilateral to labyrinth or control lateral], etc)
4. Why these specific parameters were selected for the auditory stimuli? Any reference to support its use?

MINOR COMMENTS:

Comment #3- Title could be modify. The use of the term historical note is not clear. The author is invited to modify this term. A suggestion would be: "Modulation of vestibular microphonics: revisiting the use of rotational stimuli”

Comment#4- modification to the text page 1, line 29-30. Please change: “in the vestibular system [4].”for “in the peripheral vestibular system [4].”

Comment#5- modification to the text page 2, line 44 please cite the study. Instead of “[…] was taken directly from reference 2” write  "[...] directly from Wit et al. [2]:"

Comment#6- Figures. Figures have several undesired box and bars across. Please clear these lines in figure 3(lots of them), beside ylabel in figure 4, around arrow in figure 5a.

Comment#7- Future studies. It could be very interesting that the author suggest what the data provided by this study would bring or help future studies to do. 

Author Response

Dear Editor,

This is a revised manuscript. Changes in it are based on the (delayed) comments from reviewer 2, that I received on August 2.

Reviewer 2 comments on undesired boxes and bars in the figures. Are these also seen in the figures received at the editorial office?

Reply to the comments from reviewer 2:

Changes in the text are in blue.

#1.

Importance of studying the vestibular microphonic: see lines 15-18.

What is the importance of reanalyzing old data: The importance (for this author) is that attention is drawn to the fact that modulation of microphonics was described more than 30 years before it was again used by Pastras et al. The original work was only published in the conference proceedings of the Barany Society Meeting in Bologna (1987).

#2.

1. line 42 (based on the memory of the author).

2. line 57.

3. line 61. Because the rotational stimuli are symmetric around zero velocity the stimuli are alternately in clockwise and counter-clockwise direction. The trapezoidal velocity profile gives rectangular acceleration pulses, with a time course as shown in figure 2.

4. line 51. (Stimulation with this frequency gives an easily measurable microphonic).

#3. The suggestion was not taken. The history is also in mentioning that the affiliation of the author has a long tradition in investigating the sensitivity of the vestibular system to sound. It is the place where the vestibular microphonic was discovered.

#4. line 32.

#5. line 47.

#6. The figures in the pdf of the author do not show these undesired boxes and bars. See the question for the editor above.

#7. lines 15-18 and 89-91.

With regards,

Hero Wit