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  1. Author Response

    Reviewer #1 (Public Review):

    In this paper, the authors ask a key question in the field of adult plasticity, and in particular, amblyopia treatment: whether transient dark exposure followed by light re-introduction disrupts neural representation for basic stimulus attributes in a manner that could negatively impact vision. Prior work by Rose and colleagues using calcium imaging showed that closing one eye in adult mice leaves the responsiveness of V1 neurons unchanged but alters their orientation preference and pairwise correlations; such representational drift may require downstream areas to adjust how they readout V1 signals. The question posed here is whether binocular visual deprivation in adult mice does the same. The authors use 2-photon calcium imaging in 6 awake, head-fixed [transgenic - GCaMP6f driven by the EMX1 promoter] mice before and after transient dark exposure to record ensemble responses of layer 2/3 excitatory V1 neurons to oriented gratings of varying spatial frequencies. Data were acquired twice at baseline (allowing for an assessment of representational drift during exposure to the natural [cage] environment), once immediately after 8 days of dark exposure and once about 8 days after animals were once again exposed to their natural [cage] environment.

    The study appears to be generally well designed with multiple analytical approaches trained on the same questions. Major strengths include the ability to analyze a large number of neuronal responses simultaneously in the awake-behaving state using calcium imaging in transgenic mice, and the ability to record activity in the same neurons across several weeks and following different behavioral manipulations. A relative weakness was the implication of only being able to elicit relevant visual responses from a small fraction of V1 neurons for comparison purposes. This begs the question of what may have happened to the neurons that were not tracked, and whether this in fact may have been significant.

    A consist finding across laboratories is that 30-50% of the neural population in rodent V1 is visually responsive to grating stimuli, and drifting gratings recruit neurons to a greater extent that static gratings1–5. This is unrelated to tracking, as it is the case for single-session analysis. The reviewer brings up an interesting question, given we are tracking neurons across sessions we are in a unique position to gain insight into properties that might correlate with responsiveness. To that end, we performed additional analysis to determine whether low trial reliability is predictive of whether a specific neuron will ‘drop out’ from being visually responsive on a subsequent session. The new analysis shows that under control conditions, trial reliability is correlated with reliability on the subsequent session. Consistent with our observation that reliability across the population decreases following dark exposure and then improves during light reintroduction, the new analysis also shows that the change in reliability for individual neurons is significantly skewed to lower values in the DE condition (single-sample KS test), while in the light reintroduction condition values are significantly skewed in the positive direction (Figure 3 – figure supplement 3A).

    1. Ohki, K., Chung, S., Ch’ng, Y. H., Kara, P. & Reid, R. C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).
    2. Montijn, J. S., Meijer, G. T., Lansink, C. S. & Pennartz, C. M. A. Population-Level Neural Codes Are Robust to Single-Neuron Variability from a Multidimensional Coding Perspective. Cell Rep. 16, 2486–2498 (2016).
    3. Ko, H., Mrsic-Flogel, T. D. & Hofer, S. B. Emergence of feature-specific connectivity in cortical microcircuits in the absence of visual experience. J. Neurosci. 34, 9812–9816 (2014).
    4. Jeon, B. B., Swain, A. D., Good, J. T., Chase, S. M. & Kuhlman, S. J. Feature selectivity is stable in primary visual cortex across a range of spatial frequencies. Sci. Rep. 8, 15288 (2018).
    5. de Vries, S. E. J. et al. A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex. Nat. Neurosci. 23, 138–151 (2020).

    Reviewer #3 (Public Review):

    This paper uses transient dark exposure to induce plasticity in the adult visual cortex. It shows that transient dark exposure in the adult mice has opposing effects at the single neuronal level versus the population level. At the population level, the stimulus representation is degraded following dark exposure but rebounds back to normal within 8 days of light re-introduction. Thus, dark exposure does not have a lasting negative impact on the visual cortex. Unexpectedly, at the single neuronal level, following dark exposure a fraction of neurons show more stable responses and higher correlations among pairs of neurons. It is inspiring to hypothesize that this fraction of neurons may form a plastic substrate for representation of complex natural scenes.

    Strengths:

    The paper uses a combination of single neuron and population analyses to identify the effects of transient dark exposure on visual responses in the adult mouse visual cortex. It succeeds in identifying degradation of stimulus representation at the population level following dark exposure, and stabilization of visual stimulus preference at the single neuron level as well as stabilization of stimulus correlations among pairs of neurons. This success is in part due to an impressively large set of simple visual stimuli used (180 different stimuli). This large set allows the authors to identify even small changes in stimulus preferences at the single neuronal level. This paper uses transient dark exposure to induce plasticity. An alternative and commonly used method to induce plasticity is monocular deprivation. This paper shows that at the single neuron level, the effects of transient dark exposure are different from the previously reported effects of monocular deprivation. This is an important finding for the field.

    Weaknesses:

    The analysis methods used are thoughtful and complementary. The statistical tests are mostly performed on visual responses pooled across 6 mice. These statistical tests support the claims of the paper. However, we are left wondering whether the effects identified would also be significant for visual responses of each individual mouse.

    Further analysis of individual mice is now included. From this analysis we can verify that the effects observed are not driven by one or two animals, rather are representative of the majority of the animals included in the study.

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  2. Evaluation Summary:

    The present manuscript examines cortical representations of basic visual attributes following a manipulation shown to enhance plasticity in the adult brain: binocular dark exposure for several days, followed by light re-introduction. The work has fundamental therapeutic and conceptual implications, and will be of potential interest to a broad readership of vision scientists, neuroscientists, clinicians and modelers. The paper is well-written and based on sophisticated experiments. The evidence provided convincingly supports the authors' contention that dark exposure does not have a negative impact on visual representations in V1. The study uses a generally appropriate study design. However, it would benefit from the addition of some key experimental details, and additional analyses and statistical tests to explore alternative interpretations of results.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    In this paper, the authors ask a key question in the field of adult plasticity, and in particular, amblyopia treatment: whether transient dark exposure followed by light re-introduction disrupts neural representation for basic stimulus attributes in a manner that could negatively impact vision. Prior work by Rose and colleagues using calcium imaging showed that closing one eye in adult mice leaves the responsiveness of V1 neurons unchanged but alters their orientation preference and pairwise correlations; such representational drift may require downstream areas to adjust how they readout V1 signals. The question posed here is whether binocular visual deprivation in adult mice does the same. The authors use 2-photon calcium imaging in 6 awake, head-fixed [transgenic - GCaMP6f driven by the EMX1 promoter] mice before and after transient dark exposure to record ensemble responses of layer 2/3 excitatory V1 neurons to oriented gratings of varying spatial frequencies. Data were acquired twice at baseline (allowing for an assessment of representational drift during exposure to the natural [cage] environment), once immediately after 8 days of dark exposure and once about 8 days after animals were once again exposed to their natural [cage] environment.

    The study appears to be generally well designed with multiple analytical approaches trained on the same questions. Major strengths include the ability to analyze a large number of neuronal responses simultaneously in the awake-behaving state using calcium imaging in transgenic mice, and the ability to record activity in the same neurons across several weeks and following different behavioral manipulations. A relative weakness was the implication of only being able to elicit relevant visual responses from a small fraction of V1 neurons for comparison purposes. This begs the question of what may have happened to the neurons that were not tracked, and whether this in fact may have been significant. For the ~30% of V1 neurons which were tracked, the findings appear to be that dark exposure of adult mice for 8 days did not significantly corrupt their orientation or SF tuning. Instead, there were increase pairwise correlations between them, interpreted as increased stability of stimulus representation. However, when the entire neuronal pool was analyzed, a decrease in decoding accuracy was noted, attributed to decreased response reliability. Nonetheless, a recovery back to baseline was noted after mice were re-exposed to light and their natural cage environments for 8 days. The study thus provides a binocular deprivation alternative to the earlier monocular deprivation findings of Rose et al. In addition, it provides some new insights, suggesting that the early visual system (i.e. V1) of adult animals normally exhibits a flexible stimulus representation for simplistic, artificial visual stimuli such as oriented gratings, and that temporary dark exposure decreases this flexibility. Importantly for therapeutic approaches however, this can be reversed upon re-introduction of the natural, complex visual environment.

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  4. Reviewer #2 (Public Review):

    Dark exposure followed by light reintroduction is being explored as a potential treatment for the recovery of vision in amblyopic subjects. However, previous work has not ruled out the possibility that prolonged dark exposure negatively impacts the quality of visual representations in V1. Here Jeon et al., use 2P calcium imaging in awake adult mice to quantify the impact of prolonged dark exposure and subsequent light reintroduction on stimulus selectivity, discriminability and reliability of neurons in primary visual cortex of mice. the quality of visual stimulates representation. They find that dark exposure reversibly reduces neuronal discriminability and reliability, and stabilizes stimulus selectivity. The latter is somewhat surprising and contrasts with changes in visual neuron responses following monocular deprivation. Together these results contribute important, novel observations into how the visual system responds to prolonged derivation, and suggest that dark exposure treatments pose minimal threats to visual perception.

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  5. Reviewer #3 (Public Review):

    This paper uses transient dark exposure to induce plasticity in the adult visual cortex. It shows that transient dark exposure in the adult mice has opposing effects at the single neuronal level versus the population level. At the population level, the stimulus representation is degraded following dark exposure but rebounds back to normal within 8 days of light re-introduction. Thus, dark exposure does not have a lasting negative impact on the visual cortex. Unexpectedly, at the single neuronal level, following dark exposure a fraction of neurons show more stable responses and higher correlations among pairs of neurons. It is inspiring to hypothesize that this fraction of neurons may form a plastic substrate for representation of complex natural scenes.

    Strengths:

    The paper uses a combination of single neuron and population analyses to identify the effects of transient dark exposure on visual responses in the adult mouse visual cortex. It succeeds in identifying degradation of stimulus representation at the population level following dark exposure, and stabilization of visual stimulus preference at the single neuron level as well as stabilization of stimulus correlations among pairs of neurons. This success is in part due to an impressively large set of simple visual stimuli used (180 different stimuli). This large set allows the authors to identify even small changes in stimulus preferences at the single neuronal level.
    This paper uses transient dark exposure to induce plasticity. An alternative and commonly used method to induce plasticity is monocular deprivation. This paper shows that at the single neuron level, the effects of transient dark exposure are different from the previously reported effects of monocular deprivation. This is an important finding for the field.

    Weaknesses:

    The analysis methods used are thoughtful and complementary. The statistical tests are mostly performed on visual responses pooled across 6 mice. These statistical tests support the claims of the paper. However, we are left wondering whether the effects identified would also be significant for visual responses of each individual mouse.

    Was this evaluation helpful?