Multisensory integration enhances audiovisual responses in the Mauthner cell

Reviewer #2 (Public review):

Summary:

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Strengths:

The methods applied are challenging and appropriate and appear to be well executed. Open questions about the physiological underpinnings of M-cell function are addressed using sound experimental design and methodology, and convincing results are provided that advance our understanding of how two streams of sensory information can interact to control behavior.

Weaknesses:

Our concerns about the manuscript are captured in the following specific comments, which we hope will provide a useful perspective for readers and actionable suggestions for the authors.

Comments relevant to the revised manuscript:

Our general assessment (above) stands unchanged from the original version. All of our comments and concerns about the original manuscript have been addressed except for two, one very minor and one quite important:

Original Comment 1 (Minor):
"Line 124. Direct stimulation of the tectum to drive M-cell-projecting tectal neurons not only bypasses the retina, it also bypasses intra-tectal processing and inputs to the tectum from other sources (notably the thalamus). This is not an issue with the interpretation of the results, but this description gives the (false) impression that bypassing the retina is sufficient to prevent adaptation. Adding a sentence or two to accurately reflect the complexity of the upstream circuitry (beyond the retina) would be welcome."

The authors have replied:
"The reviewer is right in that direct tectal stimulation bypasses all neural processing upstream, not only that produced in the retina and that the tectum does not exclusively process visual information. The revised version now acknowledges (lines 245-252, revised manuscript) the complexity of the system."

We think that this is sufficient to address our concern. Some citations may be in order to underpin the new text.

Original Comment 5 (Major):
Figure 4C and lines 398-410.
"These are beautiful examples of M-cell firing, but the text suggests that they occurred rarely and nowhere close to significantly above events observed from single modalities. We do not see this a valid result to report because there is insufficient evidence that the phenomenon shown is consistent or representative of your data."

The authors have replied:
"Our experimental conditions required anesthesia and paralysis, conditions designed to reduce neuronal firing and suppress motor output. We think it is valuable to report that we still see that simultaneous presentation subthreshold unisensory stimuli can add up to become suprathreshold, paralleling behavioral observations. We do not claim and acknowledge that those examples are representative of our recording conditions, but are likely to be more representative of the multisensory integration process taking place in freely moving fish. The revised manuscript adds context to these example traces to justify their inclusion (lines 420-426)."

We do not feel that this important concern has been addressed. The stats are definitively negative. There is no statistical evidence from these data that multisensory integration is occurring in this assay. The aesthesia, paralysis, and low n may provide explanations for this negative result, but it is still a negative result (p=0.5269). To show two examples of multisensory integration for subthreshold stimuli fits the narrative, but this result is not supported. Examples where individual stimuli caused APs (and combined stimuli did not) also occurred, presumably, and at a rate that is statistically indistinguishable to the examples shown in Figure 5. As such, if results from this assay are going to be in the manuscript, acoustic-only and tectum-only examples should be shown as well, although they would not fit the narrative. To be meaningful, this experiment would have to show that multisensory integration is happening in this circuit. Frustrating though it must be, the experiment has given a negative result to that question.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

Otero-Coronel et al. address an important question for neuroscience - how does a premotor neuron capable of directly controlling behavior integrate multiple sources of sensory inputs to inform action selection? For this, they focused on the teleost Mauthner cell, long known to be at the core of a fast escape circuit. What is particularly interesting in this work is the naturalistic approach they took. Classically, the M-cell was characterized, both behaviorally and physiologically, using an unimodal sensory space. Here the authors make the effort (substantial!) to study the physiology of the M-cell taking into account both the visual and auditory inputs. They performed well-informed electrophysiological approaches to decipher how the M-cell integrates the information of two sensory modalities depending on the strength and temporal relation between them.

Strengths:

The empirical results are convincing and well-supported. The manuscript is well-written and organized. The experimental approaches and the selection of stimulus parameters are clear and informed by the bibliography. The major finding is that multisensory integration increases the certainty of environmental information in an inherently noisy environment.

Weaknesses:

Even though the manuscript and figures are well organized, I found myself struggling to understand key points of the figures.

For example, in Figure 1 it is not clear what are actually the Tonic and Phasic components. The figure will benefit from more details on this matter. Then, in Figure 4 the label for the traces in panel A is needed since I was not able to pick up that they were coming from different sensory pathways.

We added an inset to Figure 1 showing how the tonic and phasic components are measured. We now use solid colors instead of transparencies, and the color scheme was modified for consistency. We added labels to the traces used as examples in Figure 4 panel A.

In line 338 it should be optic tectum and not "optical tectum".

We replaced two instances of the term “optical tectum” with “optic tectum”.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well-written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength and MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Strengths:

The methods applied are challenging and appropriate and appear to be well executed. Open questions about the physiological underpinnings of M-cell function are addressed using sound experimental design and methodology, and convincing results are provided that advance our understanding of how two streams of sensory information can interact to control behavior.

Weaknesses:

Our concerns about the manuscript are captured in the following specific comments, which we hope will provide a useful perspective for readers and actionable suggestions for the authors.

Comment 1 (Minor):

Line 124. Direct stimulation of the tectum to drive M-cell-projecting tectal neurons not only bypasses the retina, it also bypasses intra-tectal processing and inputs to the tectum from other sources (notably the thalamus). This is not an issue with the interpretation of the results, but this description gives the (false) impression that bypassing the retina is sufficient to prevent adaptation. Adding a sentence or two to accurately reflect the complexity of the upstream circuitry (beyond the retina) would be welcome.

The reviewer is right in that direct tectal stimulation bypasses all neural processing upstream, not only that produced in the retina and that the tectum does not exclusively process visual information. The revised version now acknowledges (lines 245-252, revised manuscript) the complexity of the system.

Comment 2 (Major): The premise is that stimulation of the tectum is a proxy for a visual stimulus, but the tectum also carries the auditory, lateral line, and vestibular information. This seems like a confound in the interpretation of this preparation as a simple audio-visual paradigm. Minimally, this confound should be noted and addressed. The first heading of the Results should not refer to "visual tectal stimuli".

We changed the heading of the corresponding section of the Results section as requested and also omitted the term “optic” when we did not specifically refer to tectal circuits that process optic information.

Comment 3 (Major): Figure 1 and associated text.

It is unclear and not mentioned in the Methods section how phasic and tonic responses were calculated. It is clear from the example traces that there is a change in tonic responses and the accumulation of subthreshold responses. Depending on how tonic responses were calculated, perhaps the authors could overlay a low-passed filtered trace and/or show calculations based on the filtered trace at each tectal train duration.

The revised version of the manuscript now includes a description of how the phasic and tonic components were calculated (lines 163-172). We also modified the color scheme and the inset of Figure 1A to clarify how these two components were defined. Since we quantified the response in a 12 ms window, we did not include an overlayed low-pass filtered trace since it might be confusing with respect to the metric used.

Comment 4 (Minor): Figure 3 and associated text.

This is a lovely experiment. Although it is not written in text, it provides logic for the next experiment in choosing a 50ms time interval. It would be great if the authors calculated the first timepoint at which the percentage of shunting inhibition is not significantly different from zero. This would provide a convincing basis for picking 50ms for the next experiment. That said, I suspect that this time point would be earlier than 50 ms. This may explain and add further complexity to why the authors found mostly linear or sublinear integration, and perhaps the basis for future experiments to test different stimulus time intervals. Please move calculations to Methods.

We moved calculations to the Methods section (lines 201-208). We mention the rationale for selecting the 50 ms interval in the next experiment (Figure 4, lines 369-371) and discuss in detail the potential contribution of FFI to the complexity of the integration taking place in the M-cell circuit (Discussion, lines 512-535).

Comment 5 (Major): Figure 4C and lines 398-410.

These are beautiful examples of M-cell firing, but the text suggests that they occurred rarely and nowhere close to significantly above events observed from single modalities. We do not see this as a valid result to report because there is insufficient evidence that the phenomenon shown is consistent or representative of your data.

Our experimental conditions required anesthesia and paralysis, conditions designed to reduce neuronal firing and suppress motor output. We think it is valuable to report that we still see that simultaneous presentation subthreshold unisensory stimuli can add up to become suprathreshold, paralleling behavioral observations. We do not claim and acknowledge that those examples are representative of our recording conditions, but are likely to be more representative of the multisensory integration process taking place in freely moving fish. The revised manuscript adds context to these example traces to justify their inclusion (lines 420-426).

Reviewer #2 (Recommendations For The Authors):

Methods

The Methods section on "Auditory stimuli" contains a long background on the biophysics of the M-cell and its inputs. This does not belong in Methods. The same is true, to a lesser degree, in the next heading. The argument that direct stimulation of the tectum is necessary to bypass adaptation should be in Results, not Methods.

Following the reviewer recommendation, we have moved both paragraphs to the Results section.

Figure 1 and associated text.

Visually, the use of transparency to differentiate phasic and tonic calculations is difficult to read. Example traces are also cut off at the top and bottom at random sizes.

We changed the color scheme to avoid the use of transparency and modified the inset of Figure 1A to clarify how the phasic and tonic components were calculated. We also modified the dimensions of the clipping mask used to trim the stimulation artifacts of sample traces to make them more similar while still enabling clear observation of the phasic and tonic components of the response.

Line 338 "optical tectum" is not correct. "optic tectum" is more common, or better still, just "tectum".

We apologize for the error. The two instances of “optical tectum” were replaced by the correct term (“optic tectum”).