Retinal circuits driving a non-image forming visual behavior
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Summary: The reviewers all appreciated the potential of the work and felt that the general approach followed was strong. The reviewers, however, raised several important concerns. Discussion among the reviewers emphasized the importance of these. Chief among these was a concern about the extent to which the paper breaks new ground in a way that will appeal to a broad audience. Specifically, several of the results reported are expected based on prior work on the retinal pathways involved, and the results that do not fit with existing knowledge were not pursued in sufficient detail. These, and several other concerns, are detailed in the individual reviews below.
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Abstract
Outer retinal circuits that drive non-image forming vision in mammals are unknown. Rods and cones signal light increments and decrements to the brain through the ON and OFF pathways, respectively. Although their contribution to image-forming vision is known, the contributions of the ON and OFF pathway to the pupillary light response (PLR), a non-image forming behavior, are unexplored. Here we use genetically modified mouse lines, to comprehensively define the outer retinal circuits driving the PLR. The OFF pathway, which mirrors the ON pathway in image-forming vision, plays no role in the PLR. We found that rods use the primary rod pathway to drive the PLR at scotopic light levels. At photopic light levels, the primary and secondary rod pathways drive normal PLR. Importantly, we find that cones are unable to compensate for rods. Thus, retinal circuit dynamics allow rods to drive the PLR across a wide range of light intensities.
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Reviewer #3
This manuscript by Beier et al. has used an impressive array of genetically modified mouse lines to study, which retinal circuits are responsible for driving the pupillary light reflex (PLR). These mouse lines are validated by direct electrophysiological recordings from rods, to rod bipolar cells, to ON and OFF cone bipolar cells. The manuscript makes two key conclusions based on measurements of PLR from darkness to 100 lux and 1 lux light steps: 1) the ON but not the OFF pathways drive PLR, 2) PLR relies on the most sensitive rod pathway - the primary rod pathway. My main concern is that the data shown in the paper does not uniquely support these two key conclusions. There are many issues, some of which may be fixed by better explanations, some of which may require more complete measurements. I outline my main concerns below:
Reviewer #3
This manuscript by Beier et al. has used an impressive array of genetically modified mouse lines to study, which retinal circuits are responsible for driving the pupillary light reflex (PLR). These mouse lines are validated by direct electrophysiological recordings from rods, to rod bipolar cells, to ON and OFF cone bipolar cells. The manuscript makes two key conclusions based on measurements of PLR from darkness to 100 lux and 1 lux light steps: 1) the ON but not the OFF pathways drive PLR, 2) PLR relies on the most sensitive rod pathway - the primary rod pathway. My main concern is that the data shown in the paper does not uniquely support these two key conclusions. There are many issues, some of which may be fixed by better explanations, some of which may require more complete measurements. I outline my main concerns below:
The manuscript uses an incoherent terminology of the retinal pathways. For example, the beginning of the second paragraph of the introduction states that the ON and OFF pathways split in the first synapse, which is not true, for example, for the primary rod pathway (rod bipolar pathway). The latter segments of the same paragraph lay out more clearly the conventional definition of the primary, secondary and tertiary rod pathways. In short, it would be important to use a coherent and conventional terminology of the retinal pathways and relate the experiments and conclusions to these. It would also be important to correlate the used stimuli to the light levels defined to drive signals across different retinal pathways in image forming vision (see Grimes et al. 2014 & Grimes et al. 2018). Now that the light levels for physiological studies are expressed in R* / rod (see Supplementary Table), whereas lux are used as units for PLR. Comparison to the previous literature would require a unified intensity space (preferentially Rs or both luxes and Rs). It would also be important to relate the sensitivity of the primary rod pathway (as the authors claim is driving the PLR) to the signaling levels (extremely low light levels, <10 R*/rod/sec) where this pathway is supposedly driving image forming vision (Murphy & Rieke, 2006). It seems that the current PLR experiments probe much higher light levels than these papers in relation to the primary rod pathway. A wider stimulus space should be tested and/or at least a clear explanation would be needed for the choices made.
One of the two main conclusions of the paper is that the retinal ON pathway drives the PLR and the OFF pathways do not contribute to the PLR. The authors state (see abstract): "The OFF pathway, which mirrors the ON pathway in image forming vision, plays no role in the PLR". The data in Figs. 2A & B and 3 A & B indeed give strong support for the notion that light steps from darkness to 100 lux and 1 lux drive light responses through the ON pathway. However, this finding is not in conflict with the image forming vision. In fact, both the classic papers (Schiller, 1982, photopic ) as well as more recent results (Smeds et al. 2019; scotopic) support the notion that light increments are coded by the ON pathway. Now the circuits controlling PLR seem to fall exactly in this picture. However, the classic papers based on image forming vision (see e.g. Schiller, 1992) propose that the OFF pathways would drive light decrement stimuli. To justify the conclusion that "OFF pathways do not contribute to PLR" the authors should test a wider stimulus space including light decrements across scotopic and photopic light levels or limit their conclusions to light increments and in line with current notion for image forming vision. The reason that OFF pathways do not play a role may just reflect a limitation in the stimulus space probed.
The authors appear to ignore that the division into ON and OFF pathways occurs only after the AII cells along the primary rod pathway. The fact that Cx36 KO mice exhibit a normal PLR thus seems to invalidate the main claim of the paper that the primary ON pathway drives the PLR. The authors state: "These results imply that either the rod to rod bipolar cell pathway, independent of the AII ON pathway, is capable of driving pupil constriction or that cones are playing a role". Both of these conclusions are in contradiction with the main conclusion that the primary rod pathway as defined conventionally would be the underlying mechanism. If indeed cones are driving the PLR in Cx36 KO mice, that would be in contradiction with the previous literature (Keenan et al. 2016). It would be important to test this perhaps by using a different mouse line allowing to eliminate the cone contribution. Alternatively, showing data on Cx36 KO mice at lower light levels could help but this dataset is missing from the Fig. 3. Similarly, the Cone Cx36 KO dataset seems too sparse (n = 3) to justify the current conclusions in Fig. 3D and for some reason, the corresponding data trace is missing completely from Fig. 3C. In fact, the authors as they speculate might have uncovered (see Discussion) an entirely novel mechanism controlling the PLR. However, this now has been left untested even if it could be the most interesting new discovery if properly tested/shown.
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Reviewer #2:
This work from Beier et al. elegantly dissects the rod circuits contributing to the mouse pupillary light reflex through ipRGCs. The authors combine multiple genetic mouse models with electrophysiology and behavior to demonstrate that the primary rod pathway is the primary driver of the dim light pupillary light reflex, and that the secondary pupillary light reflex cannot effectively compensate for this pathway if it is lost. My technical comments are minor. This will be a welcome addition to the field of ipRGC research. My main concern, which I will leave to the editors, is that the actual advance may not be substantial enough.
This is the first study to attribute the rod contribution to the PLR to the primary rod pathway. Though elegant, the fact that the primary rod pathway through ipRGCs is the major contributor in low …
Reviewer #2:
This work from Beier et al. elegantly dissects the rod circuits contributing to the mouse pupillary light reflex through ipRGCs. The authors combine multiple genetic mouse models with electrophysiology and behavior to demonstrate that the primary rod pathway is the primary driver of the dim light pupillary light reflex, and that the secondary pupillary light reflex cannot effectively compensate for this pathway if it is lost. My technical comments are minor. This will be a welcome addition to the field of ipRGC research. My main concern, which I will leave to the editors, is that the actual advance may not be substantial enough.
This is the first study to attribute the rod contribution to the PLR to the primary rod pathway. Though elegant, the fact that the primary rod pathway through ipRGCs is the major contributor in low light and that both primary and secondary pathways contribute to the photopic light PLR is not particularly surprising given the previous clear demonstration by the Hattar group that the rod pathway itself is required for the pupillary light reflex (Keenan et al., 2016).
The authors do convincingly show that the OFF pathway cannot drive the PLR, but again this is in agreement with data showing ipRGCs are the sole conduit for light to drive the PLR (Güler 2008; Chew 2015) and that all ipRGCs get info primarily or solely from the ON pathway (Dumitrescu et al. 2009 and Schmidt 2010, etc.).
Is 1 lux of mixed wavelength light truly in the scotopic regime? How was this calculation/determination made?
Was there any difference in the dark adapted pupil diameter between each of the mouse lines?
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Reviewer #1:
This paper uses a variety of mouse lines to investigate what retinal circuits control the pupillary light reflex (PLR). Recordings from rods and bipolar cells confirm that the manipulations work as expected - at least at the level of the bipolars. Measurements of the PLR in these mice then are used to draw inferences about the relevant pathways. The main conclusions are that cones contribute little to the PLR across light levels, that signaling in Off retinal circuits contributes little, and that both primary and secondary rod pathways contribute.
I have several concerns about the work as presented:
Use of mouse lines. The mouse lines are interpreted as cleanly dissecting different retinal pathways, but this may not be the case. For example, deletion of one pathway may alter signaling in another pathway - either through …
Reviewer #1:
This paper uses a variety of mouse lines to investigate what retinal circuits control the pupillary light reflex (PLR). Recordings from rods and bipolar cells confirm that the manipulations work as expected - at least at the level of the bipolars. Measurements of the PLR in these mice then are used to draw inferences about the relevant pathways. The main conclusions are that cones contribute little to the PLR across light levels, that signaling in Off retinal circuits contributes little, and that both primary and secondary rod pathways contribute.
I have several concerns about the work as presented:
Use of mouse lines. The mouse lines are interpreted as cleanly dissecting different retinal pathways, but this may not be the case. For example, deletion of one pathway may alter signaling in another pathway - either through compensatory effects, or from interactions between the pathways that are missing when one is removed. One way to address this concern would be to record from RGCs to test for such effects. For example, the cone sensitivity in the RGCs in Cx36-/- mice should not be altered. The bipolar recordings are helpful in this regard, but they do not represent the circuit output and hence could miss key interactions or compensation.
Interpretation. The results are interpreted in the context of a standard model of retinal circuitry. Yet several aspects of the results suggest that such a model is incomplete. One example mentioned in the text is the possibility of direct RBC to RGC connections. A specific concern in this regard is that it is unclear how the secondary pathway could control the PLR but cones could not - since rod and cone signals are mixed in the secondary pathway. Accounting for the results in the paper would appear to require revisiting our understanding of retinal circuits - but more direct tests of the circuits are needed for such a conclusion.
Relation with past work. The paper is short and suffers from short or missing descriptions of related past work. For example, a good deal is known about how signals from the primary and secondary pathways modulate cone bipolar and RGC responses. This is directly relevant to what is expected and unexpected in the present work. Recent work (Lee et al., 2019) also shows a contribution of melanopsin to ipRGC responses at low light levels - but this is mentioned only in passing in the present paper. This work appears highly relevant to the present study.
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Summary: The reviewers all appreciated the potential of the work and felt that the general approach followed was strong. The reviewers, however, raised several important concerns. Discussion among the reviewers emphasized the importance of these. Chief among these was a concern about the extent to which the paper breaks new ground in a way that will appeal to a broad audience. Specifically, several of the results reported are expected based on prior work on the retinal pathways involved, and the results that do not fit with existing knowledge were not pursued in sufficient detail. These, and several other concerns, are detailed in the individual reviews below.
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