Superior Colliculus-Projecting GABAergic Retinal Ganglion Cells Mediate Looming-Evoked Flight Response

  1. Xue Luo
  2. Danrui Cai
  3. Kejiong Shen
  4. Qinqin Deng
  5. Xinlan Lei
  6. Sen Jin
  7. Wen-Bo Zeng
  8. Hua Li
  9. Fuqiang Xu
  10. Lu Huang
  11. Chaoran Ren
  12. Min-Hua Luo
  13. Ting Xie
  14. Yin Shen

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The looming stimulus-evoked flight response is an experimental paradigm for studying innate defensive behaviors. However, how the visual looming stimulus is transmitted from the retina to the brain remains poorly understood. Here, we report that superior colliculus (SC)-projecting RGCs transmit the looming signal from the retina to the brain to mediate the looming-evoked flight behavior by releasing GABA. In the mouse retina, GABAergic RGCs are capable of projecting to many brain areas, including the SC. Superior colliculus (SC)-projecting GABAergic RGCs (spgRGCs) are mono-synaptically connected to the parvalbumin-positive SC neurons known to be required for the looming-evoked flight response. Optogenetic activation of spgRGCs triggers GABA-mediated inhibition in SC neurons. The ablation or silence of spgRGCs compromises looming-evoked flight response but not image-forming functions. Therefore, this study shows that spgRGCs control the looming-evoked flight response by regulating SC neurons via GABA, providing novel insight into the regulation of innate defensive behaviors.

Article activity feed

  1. eLife

    ###Reviewer #3:

    Luo and colleagues use a combination of viral tracing and targeted neuronal manipulation tools to dissect the role of GABAergic retinal ganglion cells (RGC) in mediating aversive responses to looming visual stimuli. This paper reports the existence of a population of GABAergic RGCs projecting to the superior colliculus (SC). These conclusions were based on a set of retrograde and anterograde tracing experiments in two mouse lines that putatively label GABAergic neurons (vGAT-Cre and GAD2-Cre). Targeted ablation of the GABAergic RGCs compromised looming-triggered escape behavior, which suggests a possible involvement of the superior colliculus projecting GABAergic RGCs in mediating the looming-evoked flight response. Although these findings could provide important insights into the neural circuitry that mediates aversive behaviors, tracking the origin of these circuits back to the retina, we have major concerns with the paper in its current format.

    The current data set raises three major concerns that need to be addressed.

    -First, the specificity of the labelling strategies is insufficient to make the current claims. The viral tools used to kill or manipulate RGCs are not specific for either RGCs (shRNA experiment), or SC-projecting RGCs (DTA experiments), and hence do not support the pathway specific claims in the manuscript. Also, the presented data raises questions as to whether all labeled neurons in these mouse lines are functionally inhibitory in the adult retinal.

    -Second, the behavioral tests performed do not check for effects of killing GABAergic RTGCs on thalamic dependent visual processing.

    -Third, the presentation of the data makes it very difficult for the reader to ascertain the claims made by the authors. In addition, the writing needs major revision.

    Major concerns:

    1. Lack of specificity of the labelling strategy. The authors claim that GABAergic RGCs mediate escape responses via GABA release in the SC. The current data does not support this claim since none of the used tools is specific enough. To maintain these claims, the authors need to either test if the SC-projecting GABAergic RGCs have no (or minimal) collateral projections to other targets or if the ganglion cells that collaterally project to other brain areas are not involved in the aversive behavior being tested. Further, it is necessary to narrow down the cell populations affected by their manipulations and to show that GABA-release by RGCs is involved in the tested escape behavior.

    a. We are concerned that the mouse lines used, in particular the vGAT line, do not label cells that are functionally GABAergic in an adult animal. The gene coding for vGAT, Slc32al, is found in amacrine cells, but not in RGCs (Siegert et al. 2011). In Figure 1, the GABAergic positive cell bodies are not confirmed as being RGCs, i.e. having an axon. In Figure S2 the authors do not differentiate between GAD2 and vGAT.

    b. The viral tools used for ablating SC-projecting GABAergic neurons are not specific for the SC, but will also label very common collaterals to brain targets other than the SC including the thalamus (e.g. Ellis et al., 2016).

    c. The shRNA approach to knock down vGAT expression removes vGAT from all types of vGAT-expressing retinal neurons, not only RGCs. It is very likely that vGAT-expressing amacrine cells are affected by this manipulation.

    d. Please remove the claims that the behavior is mediated through the PBGN pathway. The transsynaptic HSV approach to label multisynaptic targets of GABAergic RGCs does not support this claim. It is unknown whether the axon terminals in the PBGN labelled in these experiments stem from the SC. Also, other SC-targets that are known to mediate escape, such as the PAG (Evans et al. 2018), are also labelled in these experiments.

    1. Inadequate test of visual circuitry function. The approaches used by the authors to test for different aspects of visual processing focus on a few known reflex pathways, but do not directly test for thalamic/cortical dependent visual behaviors, i.e. image formation. This needs to be changed in the description of the tests and the interpretation of the results. The description in the figure itself is good, but the conclusions drawn are misleading. The authors use four different approaches to test for visual function and draw the following conclusions:

    a. Looming-evoked escape is decreased in two experimental conditions where vGAT-expressing neurons are impaired. The authors conclude that GABAergic RGCs projecting to the SC mediate escape. As described in point 1, these experiments are not specific enough to make those claims.

    b. Electroretinograms (ERG) have unchanged a- and b- waves, which leads to the claim that GABAergic RGCs are not necessary for normal retina function. This is misleading as the largest contributors to an ERG signal are the photoreceptors and bipolar cells (e.g. Smith, Wang ... and Trembley, 2014).

    c. Optokinetic reflex is unchanged after vGAT ablation. The authors conclude from these experiments that image formation is independent of GABAergic RGCs. This approach only tests for very specific retinal projections, probably to the AOS, but does not test for the function of the LGN-cortex projections, which form the major image formation pathway.

    1. Insufficient data presentation. The currently presented histological sections are not sufficient to draw the same conclusions as the authors. In addition, several of the experiments lack clear quantifications to back up the claims in the text. The authors need to provide complete and readable pictures, and add quantifications to support their claims.

    a. The role of PV+ SC neurons in the present study is unclear. The authors do not provide adequate evidence that GABAergic RGC-recipient neurons in the colliculus are PV+ (line 196). The referenced figure (Fig. 4B) shows only a single example of a staining. Quantification of the % of labeled neurons that are PV positive is required to strengthen this claim.

    b. Please provide complete images of the histology at a readable size for all figures and add outlines of brain areas where necessary. Importantly, in Fig. 1G it looks as if the RGC axon terminals are only present in the intermediate layers of the medial SC. Since it is claimed that the GABAergic RGCs include many different types, one would expect axon terminals across all depths of the superficial SC.

    c. The re-analysis of the published scRNA results from Rheaume et al 2018 should be made clear in the manuscript. Currently there is no information in the main text about the underlying data set or analysis.

    d. It is not clear in the text what the differences/similarities are between vGAT- and GAD2-Cre mouselines. Please make clear in the narrative and conclusion how each mousseline was used.

  2. eLife

    ###Reviewer #2:

    In this study, Luo et al. report that GABAergic retinal ganglion cells projecting to the superior colliculus (spgRGCs) drive defensive responses to looming. Previous studies demonstrated that neurons in the superior colliculus (SC) mediate behavioral responses to looming. Most of the >40 ganglion cell types in mice project to SC. Which of these convey the relevant looming signals from the retina is unclear. Most ganglion cells express VGLUT2 and release glutamate from their axon terminals, but anatomical evidence suggested that a subset of ganglion cells may contain GABA. Furthermore, a recent study (Sonoda et al., 2020) showed that some intrinsically photosensitive retinal ganglion cells are labeled in Gad2-Cre mice and reduce the light sensitivity of photoentrainment and pupillary light responses.

    Here, Luo et al. find that intravitreal injections of Cre-dependent adeno-associated virus (AAVs) reporters in Vgat-Cre and Gad2-Cre transgenic mice label ganglion cell axons in many retinorecipient brain areas, including SC. The authors reanalyze a published single-cell RNA sequencing dataset (Rheaume et al., 2018), which suggest that subsets of ganglion cells of most types express Gad2. Using optogenetics, the authors confirm that activation of Gad2-Cre- and Vgat-Cre-positive ganglion cells elicit inhibitory postsynaptic currents (IPSCs) in SC neurons. Based on retrograde tracing, the authors suggest that these ganglion cells belong to a variety of types. Next, Luo et al. demonstrate that the deletion of spgRGCs abolishes defensive responses to looming stimuli and looming-evoked cFos expression in SC. The authors illustrate the selectivity of these effects by showing that electroretinograms, optomotor responses, and pupillary light responses are unaffected by spgRGC deletion. Finally, the authors use an AAV-based shRNA strategy to knock down Vgat in the retina and show that this abolishes looming responses.

    Overall, this study reports a surprising and potentially transformative finding (i.e., that a small subset of many RGC types uses GABA to drive looming responses in SC and behavior). The authors leverage a wide range of techniques to study spgRGCs, but some results and interpretations are confusing, and some conclusions are insufficiently supported evidence.

    Specific comments:

    1. The Vgat knockdown experiments are critical to show that GABAergic transmission matters. The current strategy targets all Vgat-expressing neurons in the retina. The vast majority of these are amacrine cells. Silencing amacrine cells will likely have widespread effects among ganglion cells. The authors should use a dual AAV strategy similar to the one they employed for DTA to restrict Vgat-shRNA expression to spgRGCs and show that ganglion cells' responses to looming are unchanged.

    2. Previous studies show that activation of SC neurons (particularly PV+cells) promotes defensive responses to looming, and the cFos labeling in this study suggest that spgRGCs activate SC neurons. Yet, optogenetics experiments indicate that spgRGCs elicit IPSCs in SC neurons. These findings seem at odds. Although the authors show that some spgRGCs elicit a mixture of EPSCs and IPSCs, the Vgat knockdown experiments suggest that the GABAergic transmission mediates looming signals and elicits behavioral responses. The authors should characterize looming responses in SC by electrophysiology or optical recordings (as they have done in previous studies) to clarify the contributions of spgRGCs.

    3. Details of the cFos experiments were missing. The authors should compare cFos labeling and changes in cFos labeling after spgRGC ablation between looming and other visual stimuli, to discern the specificity of these effects.

    4. The characterization of spgRGC types is superficial. The authors should show patch-clamp recordings from a small number of RGCs, which seem to encompass a variety of types. The authors should record light responses characterization (incl. responses to looming stimuli) and reconstruct the morphology of a larger number of ganglion cells to classify types in line with other studies (e.g., Bae et al., 2018, Reinhard et al., 2019).

  3. eLife

    ###Reviewer #1:

    The goal of this study is to identify the retinal ganglion cells that mediate the flight response of mice to a looming stimulus. The candidate they focus on are a subset of RGCs that release GABA at synapses with a particular subtype of neuron in the SC that was previously implicated in this behavior.

    The impact of the paper is limited for two main reasons. First, there was a paper using a similar mouse line that was just published (Sonoda et al, Science 2020) that revealed that GABAergic intrinsically photosensitive RGCs shape several of the non-vision forming behaviors in mice (such as photoentrainment of circadian rhythms and the pupillary light reflex). The authors may not have been aware of this work when they submitted this paper but now that it is published, the authors need to more rigorously compare their results to that study.

    Second, it is not at all clear that the authors have identified a subtype of RGCs that mediate the looming responses. Rather their data (particularly Figure 3) seems to argue that a lot of different RGC subtypes have GABA. So the model is that the 13% of multiple RGC subtypes project to PV cells in the SC and together they mediate the looming response?

    Finally, there is a lack of quantitative description of many of the experiments that undermines many of the conclusions the authors want to make. These are described explicitly below.

    1. The authors make use of both a GAD2-Cre and vGAT-Cre to label cells in the retina, apparently the results from the two lines are combined throughout the paper. The authors need to verify that the same cells are labeled for each line.

    2. In Figure 1, the authors show beautiful images of their labeling but the quantification of RGCs that express GABA is not described. In the mouse retina 50% of the cells in the ganglion cell layer are amacrine cells. They do show some labeling in the optic nerve so clearly some RGCs are labeled but there is no way to know how many. Rather the authors rely on a re-analysis of an RNA-seq data set to estimate that 13% of RGCs across all subtypes! Of course, protein levels and not transcripts are what matter for this sty so at least a co-stain for a RGC marker would strengthen the finding. This would also resolve the issue brought up in point 1 above.

    3. Figure 2. The authors use optogenetics to characterize the synaptic connections with target neurons in the SC. Again, there is a surprising lack of quantification. In Figure 2C they show light activation ChR induces an inward current but they don't say how many times they do that experiment. Most experiments are done in TTX + glutamate receptor blockers to isolate GABAergic currents but there is a subset in which the igluR blockers are absent and excitatory currents were detected. The authors need to clarify in what percent of neurons did they see GABAergic currents and in what percentage excitatory currents and in what percentage did they see both? Basically, the authors need to clarify if these RGCs are releasing both GABA and glutamate. This is critical for interpreting the experiments in which they kill this population of neurons and inhibit the behavior.

    4. Figure 3. The authors use retrograde labeling to begin to identify the GABAergic RGC subtypes that project to SC. Again, the quantification is lacking - what percent of SC projecting cells were positive for GABA? It is really a confusing result because they find an array of RGC subtypes that seem to express GABA. Hence it does not appear that one type of RGC projects to SC but rather all types - but just the subset that release GABA along with glutamate.

    5. Figure 4AB appears quite impressive but the logic is not clear. Does Herpes simplex virus work as an anterograde transsynaptic virus? More explanation is required. Again, there is no quantification of the results - just examples of single neurons found in several retinorecipient brain regions.

    (Note: Figures 4C-G are impressive - killing this subpopulation of cells seems to eliminate the response to looming stimuli while other visually guided behaviors are retained.)

    1. Figure 5H - this is a difference with the Sonoda paper - they find an effect on PLR when they reduce GABA release from ipRGCs.

    2. Figure 6 - the authors use RNAi to reduce vGAT expression in spgRGCs and this also impacts behaviors. There are many controls that need to be done here primarily showing the glutamate release is normal. Otherwise this could just be a synaptic transmission deficit.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    This study reports that a small subset of different RGC neuron types that release GABA at synapses with a particular class of neurons on the super colliculus mediate the looming responses in mice. This result is potentially highly significant and as noted by one reviewer, potentially transformative in our thinking of how RGC cell types mediate behaviors. However, all three reviewers agree that many of the conclusions are insufficiently supported by the evidence. The reviewers offer many details for necessary experiments and clarifications that need to be made in order for the authors to be able to reach their conclusion.

  5. Version published to 10.1101/2020.05.14.090167 on bioRxiv