Auditory Cortex Learns to Discriminate Audiovisual Cues through Selective Multisensory Enhancement

Reviewer #1 (Public review):

Summary:

Chang and colleagues used tetrode recordings in behaving rats to study how learning an audiovisual discrimination task shapes multisensory interactions in the auditory cortex. They found that a significant fraction of neurons in the auditory cortex responded to visual (crossmodal) and audiovisual stimuli. Both auditory-responsive and visually-responsive neurons preferentially responded to the cue signaling the contralateral choice in the two-alternative forced choice task. Importantly, multisensory interactions were similarly specific for the congruent audiovisual pairing for the contralateral side.

Strengths:

The experiments were conducted in a rigorous manner. Particularly thorough are the comparisons across cohorts of rats trained in a control task, in a unisensory auditory discrimination task, and the multisensory task, while also varying the recording hemisphere and behavioral state (engaged vs. anesthesia). The resulting contrasts strengthen the authors' findings and rule out important alternative explanations. Through the comparisons, they show that the enhancements of multisensory responses in the auditory cortex are specific to the paired audiovisual stimulus and specific to contralateral choices in correct trials and thus dependent on learned associations in a task-engaged state.

Weaknesses:

The main result is that multisensory interactions are specific for contralateral paired audiovisual stimuli, which is consistent across experiments and interpretable as a learned task-dependent effect. However, the alternative interpretation of behavioral signals is crucial to rule out, which would also be specific to contralateral, correct trials in trained animals. Although the authors focus on the first 150 ms after cue onset, some of the temporal profiles of activity suggest that choice-related activity could confound some of the results.

The auditory stimuli appear to be encoded by short transient activity (in line with much of what we know about the auditory system), likely with onset latencies (not reported) of 15-30 ms. Stimulus identity can be decoded (Figure 2j) apparently with an onset latency around 50-75 ms (only the difference between A and AV groups is reported) and can be decoded near perfectly for an extended time window, without a dip in decoding performance that is observed in the mean activity Figure 2e. The dynamics of the response of the example neurons presented in Figures 2c and d and the average in 2e therefore do not entirely match the population decoding profile in 2j. Population decoding uses the population activity distribution, rather than the mean, so this is not inherently problematic. It suggests however that the stimulus identity can be decoded from later (choice-related?) activity. The dynamics of the population decoding accuracy are in line with the dynamics one could expect based on choice-related activity. Also the results in Figures S2e,f suggest differences between the two learned stimuli can be in the late phase of the response, not in the early phase.

First, it would help to have the same time axis across panels 2,c,d,e,j,k. Second, a careful temporal dissociation of when the central result of multisensory enhancements occurs in time would discriminate better early sensory processing-related effects versus later decision-related modulations.

In the abstract, the authors mention "a unique integration model", "selective multisensory enhancement for specific auditory-visual pairings", and "using this distinct integrative mechanisms". I would strongly recommend that the authors try to phrase their results more concretely, which I believe would benefit many readers, i.e. selective how (which neurons) and specific for which pairings?

Reviewer #2 (Public review):

Summary

In this study, rats were trained to discriminate auditory frequency and visual form/orientation for both unisensory and coherently presented AV stimuli. Recordings were made in the auditory cortex during behaviour and compared to those obtained in various control animals/conditions. The central finding is that AC neurons preferentially represent the contralateral-conditioned stimulus - for the main animal cohort this was a 10k tone and a vertically oriented bar. Over 1/3rd of neurons in AC were either AV/V/A+V and while a variety of multisensory neurons were recorded, the dominant response was excitation by the correctly oriented visual stimulus (interestingly this preference was absent in the visual-only neurons). Animals performing a simple version of the task in which responses were contingent on the presence of a stimulus rather than its identity showed a smaller proportion of AV stimuli and did not exhibit a preference for contralateral conditioned stimuli. The contralateral conditioned dominance was substantially less under anesthesia in the trained animals and was present in a cohort of animals trained with the reverse left/right contingency. Population decoding showed that visual cues did not increase the performance of the decoder but accelerated the rate at which it saturated. Rats trained on auditory and then visual stimuli (rather than simultaneously with A/V/AV) showed many fewer integrative neurons.

Strengths

There is a lot that I like about this paper - the study is well-powered with multiple groups (free choice, reversed contingency, unisensory trained, anesthesia) which provides a lot of strength to their conclusions and there are many interesting details within the paper itself. Surprisingly few studies have attempted to address whether multisensory responses in the unisensory cortex contribute to behaviour - and the main one that attempted to address this question (Lemus et al., 2010, uncited by this study) showed that while present in AC, somatosensory responses did not appear to contribute to perception. The present manuscript suggests otherwise and critically does so in the context of a task in which animals exhibit a multisensory advantage (this was lacking in Lemus et al.,). The behaviour is robust, with AV stimuli eliciting superior performance to either auditory or visual unisensory stimuli (visual were slightly worse than auditory but both were well above chance).

Weaknesses

I have a number of points that in my opinion require clarification and I have suggestions for ways in which the paper could be strengthened. In addition to these points, I admit to being slightly baffled by the response latencies; while I am not an expert in the rat, usually in the early sensory cortex auditory responses are significantly faster than visual ones (mirroring the relative first spike latencies of A1 and V1 and the different transduction mechanisms in the cochlea and retina). Yet here, the latencies look identical - if I draw a line down the pdf on the population level responses the peak of the visual and auditory is indistinguishable. This makes me wonder whether these are not sensory responses - yet, they look sensory (very tightly stimulus-locked). Are these latencies a consequence of this being AuD and not A1, or ... ? Have the authors performed movement-triggered analysis to illustrate that these responses are not related to movement out of the central port, or is it possible that both sounds and visual stimuli elicit characteristic whisking movements? Lastly, has the latency of the signals been measured (i.e. you generate and play them out synchronously, but is it possible that there is a delay on the audio channel introduced by the amp, which in turn makes it appear as if the neural signals are synchronous? If the latter were the case I wouldn't see it as a problem as many studies use a temporal offset in order to give the best chance of aligning signals in the brain, but this is such an obvious difference from what we would expect in other species that it requires some sort of explanation.

Reaction times were faster in the AV condition - it would be of interest to know whether this acceleration is sufficient to violate a race model, given the arbitrary pairing of these stimuli. This would give some insight into whether the animals are really integrating the sensory information. It would also be good to clarify whether the reaction time is the time taken to leave the center port or respond at the peripheral one.

The manuscript is very vague about the origin or responses - are these in AuD, A1, AuV... ? Some attempts to separate out responses if possible by laminar depth and certainly by field are necessary. It is known from other species that multisensory responses are more numerous, and show greater behavioural modulation in non-primary areas (e.g. Atilgan et al., 2018).

Reviewer #3 (Public review):

Summary:

The manuscript by Chang et al. aims to investigate how the behavioral relevance of auditory and visual stimuli influences the way in which the primary auditory cortex encodes auditory, visual, and audiovisual information. The main result is that behavioral training induces an increase in the encoding of auditory and visual information and in multisensory enhancement that is mainly related to the choice located contralaterally with respect to the recorded hemisphere.

Strengths:

The manuscript reports the results of an elegant and well-planned experiment meant to investigate if the auditory cortex encodes visual information and how learning shapes visual responsiveness in the auditory cortex. Analyses are typically well done and properly address the questions raised

Weaknesses:

Major

(1) The authors apparently primarily focus their analyses of sensory-evoked responses in approximately the first 100 ms following stimulus onset. Even if I could not find an indication of which precise temporal range the authors used for analysis in the manuscript, this is the range where sensory-evoked responses are shown to occur in the manuscript figures. While this is a reasonable range for auditory evoked responses, the same cannot be said for visual responses, which commonly peak around 100-120 ms, in V1. In fact, the latency and overall shape of visual responses are quite different from typical visual responses, that are commonly shown to display a delay of up to 100 ms with respect to auditory responses. All traces that the authors show, instead, display visual responses strikingly overlapping with auditory ones, which is not in line with what one would expect based on our physiological understanding of cortical visually-evoked responses. Similarly, the fact that the onset of decoding accuracy (Figure 2j) anticipates during multisensory compared to auditory-only trials is hard to reconcile with the fact that visual responses have a later onset latency compared to auditory ones. The authors thus need to provide unequivocal evidence that the results they observe are truly visual in origin. This is especially important in view of the ever-growing literature showing that sensory cortices encode signals representing spontaneous motor actions, but also other forms of non-sensory information that can be taken prima facie to be of sensory origin. This is a problem that only now we realize has affected a lot of early literature, especially - but not only - in the field of multisensory processing. It is thus imperative that the authors provide evidence supporting the true visual nature of the activity reported during auditory and multisensory conditions, in both trained, free-choice, and anesthetised conditions. This could for example be achieved causally (e.g. via optogenetics) to provide the strongest evidence about the visual nature of the reported results, but it's up to the authors to identify a viable solution. This also applies to the enhancement of matched stimuli, that could potentially be explained in terms of spontaneous motor activity and/or pre-motor influences. In the absence of this evidence, I would discourage the author from drawing any conclusion about the visual nature of the observed activity in the auditory cortex.

(2) The finding that AC neurons in trained mice preferentially respond - and enhance - auditory and visual responses pertaining to the contralateral choice is interesting, but the study does not show evidence for the functional relevance of this phenomenon. As has become more and more evident over the past few years (see e.g. the literature on mouse PPC), correlated neural activity is not an indication of functional role. Therefore, in the absence of causal evidence, the functional role of the reported AC correlates should not be overstated by the authors. My opinion is that, starting from the title, the authors need to much more carefully discuss the implications of their findings.

MINOR:

(1) The manuscript is lacking what pertains to the revised interpretation of most studies about audiovisual interactions in primary sensory cortices following the recent studies revealing that most of what was considered to be crossmodal actually reflects motor aspects. In particular, recent evidence suggests that sensory-induced spontaneous motor responses may have a surprisingly fast latency (within 40 ms; Clayton et al. 2024). Such responses might also underlie the contralaterally-tuned responses observed by the authors if one assumes that mice learn a stereotypical response that is primed by the upcoming goal-directed, learned response. Given that a full exploration of this issue would require high-speed tracking of orofacial and body motions, the authors should at least revise the discussion and the possible interpretation of their results not just on the basis of the literature, but after carefully revising the literature in view of the most recent findings, that challenge earlier interpretations of experimental results.

(2) The methods section is a bit lacking in details. For instance, information about the temporal window of analysis for sensory-evoked responses is lacking. Another example: for the spike sorting procedure, limited details are given about inclusion/exclusion criteria. This makes it hard to navigate the manuscript and fully understand the experimental paradigm. I would recommend critically revising and expanding the methods section.