Continuous flashing suppression of V1 responses and the perceptual consequences revealed via two-photon calcium imaging and transformer modeling

Reviewer #1 (Public review):

Disclaimer: While I am familiar with the CFS method and the CFS literature, I am not familiar with primate research or two-photon calcium imaging. Additionally, I may be biased regarding unconscious processing under CFS, as I have extensively investigated this area but have found no compelling evidence in favor of unconscious processing under CFS.

This manuscript reports the results of a nonhuman-primate study (N=2 behaving macaque monkeys) investigating V1 responses under continuous flash suppression (CFS). The results show that CFS substantially suppressed V1 orientation responses, albeit slightly differently in the two monkeys. The authors conclude that CFS-suppressed orientation information "may not suffice for high-level visual and cognitive processing" (abstract).

The manuscript is clearly written and well-organized. The conclusions are supported by the data and analyses presented (but see disclaimer). However, I believe that the manuscript would benefit from a more detailed discussion of the different results observed for monkeys A and B (i.e., inter-individual differences), and how exactly the observed results are related to findings of higher-order cognitive processing under CFS, on the one hand, and the "dorsal-ventral CFS hypothesis", on the other hand.

Major Comments:

(1) Some references are imprecise. For example, l.53: "Nevertheless, two fMRI studies reported that V1 activity is either unaffected or only weakly affected (Watanabe et al., 2011; Yuval-Greenberg & Heeger, 2013)". "To the best of my understanding, the second study reaches a conclusion that is entirely opposite to that of the first, specifically that for low-contrast, invisible stimuli, stimulus-evoked fMRI BOLD activity in the early visual cortex (V1-V3) is statistically indistinguishable from activity observed during stimulus-absent (mask-only) trials. Therefore, high-level unconscious processing under CFS should not be possible if Yuval-Greenberg & Heeger are correct. The two studies contradict each other; they do not imply the same thing.

(2) Line 354: "The flashing masker was a circular white noise pattern with a diameter of 1.89{degree sign}{degree sign}, a contrast of 0.5, and a flickering rate of 10 Hz. The white noise consisted of randomly generated black and white blocks (0.07 × 0.07 each)." Why did the authors choose a white noise stimulus as the CFS mask? It has previously been shown that the depth of suppression engendered by CFS depends jointly on the spatiotemporal composition of the CFS and the stimulus it is competing with (Yang & Blake, 2012). For example, Hesselmann et al. (2016) compared Mondrian versus random dot masks using the probe detection technique (see Supplementary Figure S4 in the reference below) and found only a poor masking performance of the random dot masks.

Yang, E., & Blake, R. (2012). Deconstructing continuous flash suppression. Journal of Vision, 12(3), 8. https://doi.org/10.1167/12.3.8

Hesselmann, G., Darcy, N., Ludwig, K., & Sterzer, P. (2016). Priming in a shape task but not in a category task under continuous flash suppression. Journal of Vision, 16, 1-17.

(3) Related to my previous point: I guess we do not know whether the monkeys saw the CF-suppressed grating stimuli or not? Therefore, could it be that the differences between monkey A and B are due to a different individual visibility of the suppressed stimuli? Interocular suppression has been shown to be extremely variable between participants (see reference below). This inter-individual variability may, in fact, be one of the reasons why the CFS literature is so heterogeneous in terms of unconscious cognitive processing: due to the variability in interocular suppression, a significant amount of data is often excluded prior to analysis, leading to statistical inconsistencies. Moreover, the authors' main conclusion (lines 305-307) builds on the assumption that the stimuli were rendered invisible, but isn't this speculation without a measure of awareness?

Yamashiro, H., Yamamoto, H., Mano, H., Umeda, M., Higuchi, T., & Saiki, J. (2014). Activity in early visual areas predicts interindividual differences in binocular rivalry dynamics. Journal of Neurophysiology, 111(6), 1190-1202. https://doi.org/10.1152/jn.00509.2013

(4) The authors refer to the "tool priming" CFS studies by Almeida et al. (l.33, l.280, and elsewhere) and Sakuraba et al. (l.284). A thorough critique of this line of research can be found here:

Hesselmann, G., Darcy, N., Rothkirch, M., & Sterzer, P. (2018). Investigating Masked Priming Along the "Vision-for-Perception" and "Vision-for-Action" Dimensions of Unconscious Processing. Journal of Experimental Psychology. General. https://doi.org/10.1037/xge0000420

This line of research ("dorsal-ventral CFS hypothesis") has inspired a significant body of behavioral and fMRI/EEG studies (see reference for a review below). The manuscript would benefit from a brief paragraph in the discussion section that addresses how the observed results contribute to this area of research.

Ludwig, K., & Hesselmann, G. (2015). Weighing the evidence for a dorsal processing bias under continuous flash suppression. Consciousness and Cognition, 35, 251-259. https://doi.org/10.1016/j.concog.2014.12.010

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons, preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

While this is an impressive experimental setup, the major weakness of this study is that the experiments don't advance any theoretical account of why CFS occurs or what CFS implies for conscious visual perception. There are two broad camps of thinking with regard to CFS. On the one hand, Watanabe et al. (2011) reported that V1 activity remained intact during CFS, implying that CFS interrupts stimulus processing downstream of V1. On the other hand, Yuval-Greenberg and Heeger (2013) showed that V1 activity is, in fact, reduced during CFS. By using a parametric experimental design, they measured the impact of the mask on the stimulus response as a function of contrast and concluded that the mask reduces the gain of neural responses to the grating stimulus. They presented a theoretical model in which the mask effectively reduced the SNR of the grating, making it invisible in the same way that reducing contrast makes a stimulus invisible.

An important discussion point of Yuval-Greenberg and Heeger is that null results (such as those presented by Watanabe et al.) are difficult to interpret, as the lack of an effect may be simply due to insufficient data. I am afraid that this critique also applies to the present study. Here, the authors report that CFS effectively 'abolishes' tuning for stimuli in neurons preferring the eye with the grating stimulus. The authors would have been in a much stronger position to make this claim if they had varied the contrast of the stimulus to show that the loss of tuning was not simply due to masking. So, while this is an incredibly impressive set of measurements that in many ways raises the bar for in vivo Ca2+ imaging in behaving macaques, there isn't anything in the results that constitutes a real theoretical advance.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. The binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge, and the imaging results are convincing and consistent across animals.

Weaknesses:

I am not totally convinced by the conclusions that the authors draw based on their machine learning models.

Author response:

Reviewer #2

We respectfully disagree with Reviewer 2’s critiques, upon which the eLife assessment of “incomplete evidence” is primarily based. We believe these critiques do not accurately reflect our study and are rooted in a misinterpretation of the evidence. Consequently, we suggest that the conclusion of “incomplete evidence” is not warranted.

On the basis of Reviewer 2’s critiques, the eLife assessment states: “However, the evidence presented is incomplete and, in particular, does not distinguish whether this suppression is due to reduced contrast or due to masking.” We emphasize that the suppression we observed is a consequence of interocular masking, not contrast reduction. Reviewer 2 cites Yuval-Greenberg and Heeger (2013), which proposes that during CFS, the mask reduces the gain of neural responses in V1 in a manner analogous to reducing stimulus contrast. We agree that both CFS masking and contrast reduction can decrease signal-to-noise ratio and thereby reduce visibility. However, in our paradigm, the physical stimulus contrast was held constant, while suppression was induced by interocular competition under CFS. This is a fundamentally different mechanism from lowering stimulus contrast. Our results therefore reflect genuine masking-induced suppression, rather than the effect of physical contrast reduction.

Furthermore, Reviewer 2 cited Yuval-Greenberg and Heeger’s discussion that null results can arise from insufficient data, and suggested that this applies to our study. This main critique from Reviewer 2 is misplaced for two reasons: First, our main result is not a null effect. A null effect would mean that CFS masking had no impact on population orientation responses. Instead, we observed significant suppression, including abolished tuning in some conditions, which clearly indicates a strong effect of masking. Second, our findings are based on large neural populations recorded using two-photon calcium imaging, providing extensive sampling and high statistical power. Thus, concerns about “insufficient data” do not apply to our study.

Finally, we used machine learning approaches to examine the effects of CFS masking on orientation discrimination and recognition, providing new insight into the long-standing debate over whether the brain can perform high-level cognitive processing under CFS. Although it is, to some extent, a matter of personal judgment whether our work represents a theoretical advance, Reviewer 2 made no comment, positive or negative, on this major component of our study while forming his/her judgment. (In response to Reviewer 3’s main concern about the suitability of SVMs, we now performed a multi-way classification analysis, which yielded results largely consistent with those obtained using the SVM approach in the original manuscript, confirming the robustness of our mechine learning results.