Circuit mechanisms underlying embryonic retinal waves
Curation statements for this article:-
Curated by eLife
eLife Assessment:
This paper should be of high interest to scientists within the field of developmental neuroscience. The authors characterize the earliest spontaneous waves of the retina - a topic that is poorly understood. The ability to monitor waves over the entire retina at high resolution is a strength of the work. Weaknesses include reliance on pharmacology and some missing details in the analysis.
(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 #1 and Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. Blocking gap junctions reduced the frequency and size of stage 1 waves, nearly abolishing them. Global blockade of nAChRs similarly nearly abolished stage 1 waves. Thus, stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the β2 subunit of the nAChRs (β2-nAChR-KO) persisted with altered propagation properties and were abolished by a gap junction blocker. To assay the impact of stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and β2-nAChR-KO mice. We found that the developmental decrease in ipRGC density is preserved between WT and β2-nAChR-KO mice, indicating that processes regulating ipRGC numbers and distributions are not influenced by spontaneous activity.
Article activity feed
-
-
eLife Assessment:
This paper should be of high interest to scientists within the field of developmental neuroscience. The authors characterize the earliest spontaneous waves of the retina - a topic that is poorly understood. The ability to monitor waves over the entire retina at high resolution is a strength of the work. Weaknesses include reliance on pharmacology and some missing details in the analysis.
(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 #1 and Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This paper investigates waves in embryonic mouse retinas. These stage 1 waves have been studied less than the post-natal (stage 2) waves. The paper uses calcium imaging in whole retinas to determine the properties of the waves and their dependence on cholinergic and electrical synapses. A strength of the work is the ability to monitor waves over the entire retina at high resolution and weaknesses include reliance on pharmacology and some missing details in analysis.
Reliance on pharmacology
The results in the paper depend largely on pharmacological manipulations. Not enough consideration is given to the possible unintended effects of those manipulations. This is particularly true for the gap junction inhibitors. The Discussion brings up the possibility of such effects, but they need to come up much earlier. …Reviewer #1 (Public Review):
This paper investigates waves in embryonic mouse retinas. These stage 1 waves have been studied less than the post-natal (stage 2) waves. The paper uses calcium imaging in whole retinas to determine the properties of the waves and their dependence on cholinergic and electrical synapses. A strength of the work is the ability to monitor waves over the entire retina at high resolution and weaknesses include reliance on pharmacology and some missing details in analysis.
Reliance on pharmacology
The results in the paper depend largely on pharmacological manipulations. Not enough consideration is given to the possible unintended effects of those manipulations. This is particularly true for the gap junction inhibitors. The Discussion brings up the possibility of such effects, but they need to come up much earlier. Is there anything else that can be done to mitigate concerns about the drugs - e.g. does MFA affect waves in Cx36 KO mice?Comparison of ACh receptor block and knockout mice
The ACh receptor knockout mouse provides a useful alternative to the pharmacological block of ACh receptors. But different features are described in Figures 2 and 3, preventing direct comparison of the two. A related point is the apparent increased role of gap junctions in mediating waves in the absence of ACh receptors. On this point, the description of the effect of MFA (page 8, second paragraph, 3rd sentence) was confusing. It looks to me like MFA almost completely eliminates waves in both WT and KO - so the connection to an altered role of gap junctions was not clear.ipRGC densities
The goal of the measurements of ipRGC densities was not entirely clear. Why are ipRGCs a reasonable way to determine the importance of waves for development? For example, the introduction raises the issue that changes in RGC proliferation depend on RGC type. Is there reason to think ipRGCs are "special" or, alternatively, that they are following the same developmental trajectory as other RGCs? Is it possible to track another RGC type (e.g. using SMI32 staining)? Related to this general point, page 9 (top) sets up the need to identify the mechanism of RGC cell death but then jumps to waves without a clear connection between the two. It would also be good to mention early that the measurements include multiple ipRGC types, so that issue does not come up only as an explanation for why the ipRGCs are not organized spatially (page 10 top).Analysis
Quantitative analysis of the calcium measurements relies on the discretization of the signals measured in small ROIs. It was not clear how closely the discretized signals represented the original recordings. The traces illustrated in Figures 1 and 2, for example, appear to be measured in larger ROIs. Two things would be helpful here: (1) an illustration of several original recorded traces in the small ROIs plotted with the discretized versions of those traces; (2) a determination of how sensitive the results are to specifics of the discretization process. -
Reviewer #2 (Public Review):
The overall goal of this study is to determine the mechanism of early retinal wave initiation and propagation. Despite a number of earlier studies, the precise mechanism of Stage1 waves and how they differ from Stage 2 waves remained controversial. To address this, the authors describe the timing and character of Stage 1 retinal waves using a custom build imaging system allowing for wide-field monitoring of neuronal activity while preserving high spatial resolution. In a set of elegantly designed experiments, they reveal that the initiation and propagation of Stage 1 waves are driven by distinct mechanisms involving complex circuitry of nAChRs and gap junctions. Interestingly, the data also demonstrate that Stage 1 and Stage 2 waves rely on different subtypes of AChRs. The signaling via beta2AChRs appears to …
Reviewer #2 (Public Review):
The overall goal of this study is to determine the mechanism of early retinal wave initiation and propagation. Despite a number of earlier studies, the precise mechanism of Stage1 waves and how they differ from Stage 2 waves remained controversial. To address this, the authors describe the timing and character of Stage 1 retinal waves using a custom build imaging system allowing for wide-field monitoring of neuronal activity while preserving high spatial resolution. In a set of elegantly designed experiments, they reveal that the initiation and propagation of Stage 1 waves are driven by distinct mechanisms involving complex circuitry of nAChRs and gap junctions. Interestingly, the data also demonstrate that Stage 1 and Stage 2 waves rely on different subtypes of AChRs. The signaling via beta2AChRs appears to be the driver of Stage 2 waves. However, the precise identity of nAChRs and GJs contributing to Stage 1 waves remains a mystery. Next, to determine the impact of early retinal waves on retinal circuit formation, the authors evaluate their impact on the survival of ipRGC. They show that ipRGC cell survival and their distribution mosaics are not influenced by spontaneous activity. While the observation of ipRGC data and their mosaic are interesting, the rationale for these experiments in the context of this study is not well presented.
-
Reviewer #3 (Public Review):
The manuscript by Voufo et al. aims to advance our understanding of the mechanisms responsible for the earliest pattern of spontaneous activity in the mouse retina, stage I retinal waves. These waves occur during embryonic development (E16-18) and are the least known form of activity in the immature retina.
The authors show that stage I waves have broad spatiotemporal features and are mediated by circuitry involving subtypes of nicotinic acetylcholine receptors (nAChRs) and gap junctions. The authors also found that the developmental decrease of intrinsic photoreceptor retinal ganglion cells (ipRGCs) density is preserved between control and ß2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by early spontaneous activity.
The quality of the data is excellent, and the …Reviewer #3 (Public Review):
The manuscript by Voufo et al. aims to advance our understanding of the mechanisms responsible for the earliest pattern of spontaneous activity in the mouse retina, stage I retinal waves. These waves occur during embryonic development (E16-18) and are the least known form of activity in the immature retina.
The authors show that stage I waves have broad spatiotemporal features and are mediated by circuitry involving subtypes of nicotinic acetylcholine receptors (nAChRs) and gap junctions. The authors also found that the developmental decrease of intrinsic photoreceptor retinal ganglion cells (ipRGCs) density is preserved between control and ß2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by early spontaneous activity.
The quality of the data is excellent, and the conclusions of this paper are mostly well supported by data, but the presentation of the data and the analysis need to be clarified and extended.Strengths:
The earliest patterns of spontaneous activity are crucial for the correct development of sensory circuits. In the visual system, most studies focus on postnatal activity (stage 2 and 3 retinal waves) overlooking embryonic stages, likely due to challenges related to methods and animal handling. Therefore, in this manuscript, the authors from a laboratory pioneer in studying retinal waves in the mouse, tackle a very relevant subject that has not been explored in detail. The bibliography that encompasses most of the current knowledge about stage 1 retinal waves in mammals is compressed into three fairly dated publications: Galli and Maffei 1988, Bansal et al 2000, and Syed et al 2004. These publications were pioneering attempts to describe early spontaneous activity; however, much work remained to be done regarding the molecular and cellular mechanisms involved. Here, Voufo and colleagues provide additional fundamental details about the properties and components of stage 1 waves. The dataset has excellent quality and plenty of information could be extracted from it. The authors used a macroscope that allows the acquisition of images from the entire retina while preserving a good spatial resolution.Weakness:
The authors distinguish different subtypes of activity during embryonic stages in the retina of mice. However, they do not provide a detailed characterization that allows a clear definition of these subtypes (and specifically stage 1 waves). Moreover, throughout the manuscript, there are many technical details of the analysis that are missing and preclude a complete understanding of the robustness of the data. The authors have an excellent dataset that needs more analysis and an improvement in the presentation of the results. -
Author Response:
Reviewer #1 (Public Review):
This paper investigates waves in embryonic mouse retinas. These stage 1 waves have been studied less than the post-natal (stage 2) waves. The paper uses calcium imaging in whole retinas to determine the properties of the waves and their dependence on cholinergic and electrical synapses. A strength of the work is the ability to monitor waves over the entire retina at high resolution and weaknesses include reliance on pharmacology and some missing details in analysis.
Reliance on pharmacology
The results in the paper depend largely on pharmacological manipulations. Not enough consideration is given to the possible unintended effects of those manipulations. This is particularly true for the gap junction inhibitors. The Discussion brings up the possibility of such effects, but they need …
Author Response:
Reviewer #1 (Public Review):
This paper investigates waves in embryonic mouse retinas. These stage 1 waves have been studied less than the post-natal (stage 2) waves. The paper uses calcium imaging in whole retinas to determine the properties of the waves and their dependence on cholinergic and electrical synapses. A strength of the work is the ability to monitor waves over the entire retina at high resolution and weaknesses include reliance on pharmacology and some missing details in analysis.
Reliance on pharmacology
The results in the paper depend largely on pharmacological manipulations. Not enough consideration is given to the possible unintended effects of those manipulations. This is particularly true for the gap junction inhibitors. The Discussion brings up the possibility of such effects, but they need to come up much earlier. Is there anything else that can be done to mitigate concerns about the drugs - e.g. does MFA affect waves in Cx36 KO mice?
We have added additional experiments based on whole cells recordings to address some off target effects of MFA but we do make note of the limitations of these new controls since we observed significant variability of voltage-gated conductances across RGCs at this age as well as the limited ability to maintain stable recordings for the requisite time to have within cell controls for MFA. (see Figure 2 Supplemental Figure 1).
Over the years we have done several experiments assessing different Cx knockouts and retinal waves (e.g. F. Caval-Holme, et al, “The Retinal Basis of Light Avoidance in Neonatal Mice”, Journal of Neuroscience 42:2022; Blankenship A.G., et al “The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina, Journal of Neuroscience, 3, 2011). It appears that there are multiple connexins in RGCs and which regulate stage 1 retina waves beyond Cx 36 and Cx45 and therefore it is difficult to use these mice as controls for general gap junction antagonists.
In the revision, we are more explicit about the caveats of using MFA both in the results (page 5) and discussion (page 10). Notably, we draw attention to past studies where we have done several controls regarding MFA and RGC activity in older retinas in addition to our more limited controls we were able to carry out in E16-E18 retina.
Comparison of ACh receptor block and knockout mice
The ACh receptor knockout mouse provides a useful alternative to the pharmacological block of ACh receptors. But different features are described in Figures 2 and 3, preventing direct comparison of the two.
Our intention was not to use the knockout mice as an alternative to the pharmacological block since we knew that there are compensatory wave mechanisms in the knockout. Rather we are using the β2-nAChR-KO to establish the effectiveness of this KO as a means of testing the role of Stage 1 waves in developmental processes. We do hope the revised manuscript explains this motivation more clearly.
A related point is the apparent increased role of gap junctions in mediating waves in the absence of ACh receptors. On this point, the description of the effect of MFA (page 8, second paragraph, 3rd sentence) was confusing. It looks to me like MFA almost completely eliminates waves in both WT and KO - so the connection to an altered role of gap junctions was not clear.
We clarified our description of the MFA result (page 5):
Application of the gap junction blocker meclofenamic acid (MFA, 50μM) nearly abolished Stage 1 waves, causing a significant reduction in frequency of waves and cell participation during waves (Fig 2A & 2F).
ipRGC densities
The goal of the measurements of ipRGC densities was not entirely clear. Why are ipRGCs a reasonable way to determine the importance of waves for development? For example, the introduction raises the issue that changes in RGC proliferation depend on RGC type. Is there reason to think ipRGCs are "special" or, alternatively, that they are following the same developmental trajectory as other RGCs? Is it possible to track another RGC type (e.g. using SMI32 staining)? Related to this general point, page 9 (top) sets up the need to identify the mechanism of RGC cell death but then jumps to waves without a clear connection between the two. It would also be good to mention early that the measurements include multiple ipRGC types, so that issue does not come up only as an explanation for why the ipRGCs are not organized spatially (page 10 top).
We have revised text extensively to better motivate our selection of ipRGCs (page 6). Our goal was to use an identified differentiated RGC subtype for which we had genetic access to assess the impact of reduced retinal waves on cell number. We settled on ipRGCs because: 1) ipRGCs undergo a significant amount of cell death during the same period there are retinal waves (Chen et al, Elife 2013) and 2) we show ipRGCs participate in retinal waves.
Analysis
Quantitative analysis of the calcium measurements relies on the discretization of the signals measured in small ROIs. It was not clear how closely the discretized signals represented the original recordings. The traces illustrated in Figures 1 and 2, for example, appear to be measured in larger ROIs. Two things would be helpful here: (1) an illustration of several original recorded traces in the small ROIs plotted with the discretized versions of those traces; (2) a determination of how sensitive the results are to specifics of the discretization process.
We have modified Figure 1 to include example traces of the fractional change in fluorescence computed across the small ROIs used for the analysis of waves on the macroscope. They are at the top of Figure 1B. As can be seen by these traces, the signal-to-noise is fantastic.
Reviewer #2 (Public Review):
The overall goal of this study is to determine the mechanism of early retinal wave initiation and propagation. Despite a number of earlier studies, the precise mechanism of Stage1 waves and how they differ from Stage 2 waves remained controversial. To address this, the authors describe the timing and character of Stage 1 retinal waves using a custom build imaging system allowing for wide-field monitoring of neuronal activity while preserving high spatial resolution. In a set of elegantly designed experiments, they reveal that the initiation and propagation of Stage 1 waves are driven by distinct mechanisms involving complex circuitry of nAChRs and gap junctions. Interestingly, the data also demonstrate that Stage 1 and Stage 2 waves rely on different subtypes of AChRs. The signaling via beta2AChRs appears to be the driver of Stage 2 waves. However, the precise identity of nAChRs and GJs contributing to Stage 1 waves remains a mystery. Next, to determine the impact of early retinal waves on retinal circuit formation, the authors evaluate their impact on the survival of ipRGC. They show that ipRGC cell survival and their distribution mosaics are not influenced by spontaneous activity. While the observation of ipRGC data and their mosaic are interesting, the rationale for these experiments in the context of this study is not well presented.
We thank the reviewer for positive comments. We do hope the revised rationale for ipRGC measurements addresses these comments. It is included here for convenience (page 7)
RGCs undergo a period of dramatic cell death during the first two postnatal weeks of development, the majority occurring during the first postnatal week (Abed et al., 2022; Braunger et al., 2014). Whether this cell death process is regulated by retinal waves is unknown. We looked specifically at intrinsically photosensitive ganglion cells (ipRGCs) for several reasons. First, ipRGCs have completed proliferation (Lucas and Schmidt, 2019; McNeill et al., 2011) and appear to be fully differentiated by E16 (Shekhar et al., 2022; Whitney et al., 2022), the onset of Stage 1 waves. ipRGCs undergo a period of dramatic cell death during the first two postnatal weeks of development, the majority occurring during the first postnatal week, prevention of which profoundly disrupts several important developmental processes in the retina – including spacing of ipRGC somas as well as rod and cone mediated circadian entrainment through the activation of ipRGCs (Chen et al., 2013). However, the exact mechanism regulating ipRGC cell death is unknown. Here we assessed the impact of disrupting Stage 1 and Stage 2 waves on the number and distribution of ipRGCs.
Reviewer #3 (Public Review):
The manuscript by Voufo et al. aims to advance our understanding of the mechanisms responsible for the earliest pattern of spontaneous activity in the mouse retina, stage I retinal waves. These waves occur during embryonic development (E16-18) and are the least known form of activity in the immature retina.
The authors show that stage I waves have broad spatiotemporal features and are mediated by circuitry involving subtypes of nicotinic acetylcholine receptors (nAChRs) and gap junctions. The authors also found that the developmental decrease of intrinsic photoreceptor retinal ganglion cells (ipRGCs) density is preserved between control and ß2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by early spontaneous activity.
The quality of the data is excellent, and the conclusions of this paper are mostly well supported by data, but the presentation of the data and the analysis need to be clarified and extended.
Strengths:
The earliest patterns of spontaneous activity are crucial for the correct development of sensory circuits. In the visual system, most studies focus on postnatal activity (stage 2 and 3 retinal waves) overlooking embryonic stages, likely due to challenges related to methods and animal handling. Therefore, in this manuscript, the authors from a laboratory pioneer in studying retinal waves in the mouse, tackle a very relevant subject that has not been explored in detail. The bibliography that encompasses most of the current knowledge about stage 1 retinal waves in mammals is compressed into three fairly dated publications: Galli and Maffei 1988, Bansal et al 2000, and Syed et al 2004. These publications were pioneering attempts to describe early spontaneous activity; however, much work remained to be done regarding the molecular and cellular mechanisms involved. Here, Voufo and colleagues provide additional fundamental details about the properties and components of stage 1 waves. The dataset has excellent quality and plenty of information could be extracted from it. The authors used a macroscope that allows the acquisition of images from the entire retina while preserving a good spatial resolution.
Weakness:
The authors distinguish different subtypes of activity during embryonic stages in the retina of mice. However, they do not provide a detailed characterization that allows a clear definition of these subtypes (and specifically stage 1 waves). Moreover, throughout the manuscript, there are many technical details of the analysis that are missing and preclude a complete understanding of the robustness of the data. The authors have an excellent dataset that needs more analysis and an improvement in the presentation of the results.
We do hope the extensive revisions satisfy reviewer.
-
