Direction and orientation preferences in mouse superior colliculus and its retinal inputs exhibit a topography of cardinal biases atop locally mixed tuning

Reviewer #1 (Public review):

Summary:

When contemplating the role of any sensory area in the brain, an essential question is: How much of the neural code is inherited from the inputs versus constructed de novo by the local circuitry? This study tackles that important question for the case of the mouse superior colliculus (SC), a visual brain area that receives direct input from the retina. The specific aspects of the neural code are the representation of line orientation and direction of motion in the visual image. Over the past 10 years or so, there have been reports that the preferred directions and orientations of neurons vary systematically across the SC in a global map that is not present in the retina, and therefore computed locally.

Here, the authors revisit this question by expanding the range of measurements: They record from the axonal boutons of retinal ganglion cells in the input layer of the SC, from the post-synaptic neurons there, and from neurons in deeper layers of the SC. They conclude that at any given location in the SC, the signals in retinal boutons recapitulate the tuning of retinal ganglion cells, and that SC neurons follow that organization, though it is lost in the deeper layers. Notably, they find no evidence for a global map of these response properties other than what is contributed by retinal input.

Strengths:

The study combines multiple recording methods - calcium imaging and electrical recording - to capture the activity of retinal inputs to the colliculus, the tuning of neurons in the superficial layers close to the input, as well as neurons in deeper layers. Furthermore, the work connects to the recent literature on gradients of tuning properties among retinal ganglion cells. All these set the stage for testing the correspondence between retinal inputs and collicular outputs.

Weaknesses:

The methods used to identify direction-selective and orientation-selective neurons based on visual responses are overly permissive and don't match common usage in this research area. Furthermore, the measurements covered only a small fraction of the visual field, which limits their ability to distinguish between different hypotheses for the global map of visual response properties. Relatedly, the claim that retinal input patterns explain much of the layout in the superior colliculus should be made more quantitative.

Reviewer #2 (Public review):

In this study, the authors investigate the spatial organization of direction and orientation selectivity in the mouse superior colliculus (SC) and its retinal inputs. By combining two-photon imaging of retinal boutons and SC neurons with Neuropixels recordings, they assess whether tuning preferences form structured maps or are arranged in a salt-and-pepper fashion. They further compare SC tuning organization to previously described retinal geometric models to determine the extent to which collicular responses inherit retinal topography. The authors conclude that SC inherits a cardinally biased topographic scaffold from the retina, which progressively weakens with depth, and that strong global maps are absent.

A major strength of the study is the impressive combination of methodologies, including imaging of retinal boutons, imaging of SC neurons, and large-scale electrophysiological recordings across SC depth. The direct comparison to retinal geometric models is particularly valuable, as it situates the SC within a broader framework of retinotopic information transfer and advances our understanding of how retinal computations are transformed in downstream targets.

A limitation of the study, however, is that the imaging experiments sample only a relatively small and spatially homogeneous region of the visual field, whereas the electrophysiological recordings cover a different portion of SC. This separation makes it difficult to form a fully integrated, global picture of the spatial organization of direction and orientation selectivity across the entire collicular map.

Reviewer #3 (Public review):

Summary:

The authors studied the organisation of orientation and direction-selective retinal ganglion cells' boutons in the mouse superior colliculus. They confirmed the results already published (Molotkov, 2023; de Malmazet, 2024; Vita, 2024; Laniado, 2025), that retinal ganglion cells' boutons in the superior colliculus conserve the retinal organisation. Thereby, orientation and direction preferences of retinal boutons at each collicular location reflect the tuning of retinal ganglion cells found at the corresponding retinal location, that is covering a matching receptive field location.

The authors also studied the organization of orientation and direction-selective neurons in the superior colliculus. They report a lack of functional organisation in the superior colliculus for neurons preferring the same stimulus orientation or direction of movement. This goes against several published reports (Ahmadlou and Heimel, 2015; Liang et al., 2023; De Malmazet et al., 2018; Feinberg and Meister, 2014; Kasai and Isa, 2021; Li et al., 2020) but echoes a study from Chen et al. (Chen, 2021). The latter authors contested the strength of the anatomical clustering of tuned alike direction-selective neurons. They found, however, that in about a quarter of their recordings, direction-selective cells with similar preferred directions did cluster anatomically in the superior colliculus.

Here, the authors of the current manuscript under review report that local clustering of tuning was weak in all neural populations and confined to very small spatial scales (10-20 μm). This is one order of magnitude smaller than previously reported clusters of around 100-300μm wide. Therefore, the authors conclude that orientation and direction tuning in the mouse superior colliculus follows a salt and pepper organisation.

Strengths & Weaknesses:

Although the authors performed a solid analysis contesting the functional clustering of direction and orientation selective neurons, there seemed to be some elements in their data in favour of a functional clustering of neurons.

As an illustration, the authors plotted in Figure 1Q the distribution of preferred orientations from all their recorded orientation-selective cells. The curve shows a clear bias, indicating that neurons preferring horizontal orientations were found two times more often than neurons encoding any other orientations. Moreover, the authors recorded all their neurons from a defined anatomical location of the colliculus, marked by the dotted rectangle in Figure 3A-C. Therefore, this suggests that orientation-selective cells in this particular collicular location are biased towards preferring horizontal orientations. This supports an anatomical clustering of tuned alike orientation-selective cells in the superior colliculus.

Similarly, Figure 1P shows a bias in the preferred directions of direction-selective neurons in the same recording area. Cells tended to respond more to upward and forward-moving stimuli. The bias is more modest than the one described above for preferred orientations. However, it still seems significant. For example, cells preferring upwards movements appeared to be four times more abundant than cells preferring downward movements. As a consequence, it indicates that preferred directions might not be uniformly distributed and equally represented across the superior colliculus.

These anatomical biases are also visible in the receptive field analysis of the paper. In Figure 3G, the authors plotted the distribution of preferred orientations for every 10-degree bins within the recorded field of view. Out of 26 bins containing more than one neuron, only 6 seemed to include cells not overwhelmingly preferring a single orientation. These were located towards the top right of the figure. Therefore, over almost 80% of the recorded superior colliculus, the data seem in agreement with the view that orientation-selective cells tend to prefer the same orientation at a given receptive location.

The patch analysis in Figures 4G and H also seems to show some degree of coherence in the preferred orientation and direction of neighbouring tuned collicular cells. In both Figures 4 G and H, clear patches of similar preferred orientation and direction appeared to emerge. For example, in Figure 4H, there is a predominance of horizontally tuned patches. This was expected given the recording bias consisting of a majority of horizontally tuned cells. In addition, vertical and 45-degree patches are also visible, in blue and red, respectively. These patches overlap with the corresponding retinotopic locations in Figure 3G, where the histograms show that cells tend to prefer the same orientations, horizontal, vertical or 45 degrees.

It is important to note that in the previous studies on functional clustering of orientation and direction, variability in the tuning of cells within clusters was always reported (Ahmadlou and Heimel, 2015; Chen et al., 2021; De Malmazet et al., 2018; Feinberg and Meister, 2014; Kasai and Isa, 2021; Li et al., 2020). This was more marked for direction-selective cells than for orientation-selective cells. In general, cells preferring all four cardinal directions were often recorded at any given collicular location. Similarly, orientation-selective cells could be found to prefer deviant orientations compared to adjacent cells. Therefore, it is not surprising to see locally mixed tuning in collicular neurons. However, what appeared significant in these studies was the overall proportion of cells with similar tuning in patches of the superior colliculus. As described above, this also seems to be the case in the data of this manuscript.

To conclude, it seems that authors tend to overlook the sources of agreement between their data and previous reports showing functional clustering of cells in the superior colliculus. Instead, the authors tend to emphasise the dissimilarities and variability to put forward a contentious view on the organisation of orientation and direction selectivity in neurons of the superior colliculus. This, I fear, is detrimental to the field because it creates a sort of manufactured chaos that produces unnecessary confusion for readers who do not attentively read the manuscript. It would be valuable for the authors to consider rewriting the manuscript, acknowledging where their data, in fact, support some level of functional clustering.

Author Response:

We thank the reviewers and editors for their thoughtful and constructive assessment. We are encouraged that the reviewers viewed the combination of retinal bouton imaging, collicular neuron imaging, and depth-resolved electrophysiology, together with the comparison to retinal geometric models, as a strength of the study. As the reviewers note, our findings are consistent with previous in vitro studies showing topographic organization of tuning in the retina and with recent work demonstrating the precision of retinotopic mapping from retina to superior colliculus (SC). In revision, we will refine our definition of tuning, sharpen our claims about the spatial organization across SC and its correspondence to retinal topography, and make clearer our aim of reconciling seemingly opposing findings in the literature. In addition, we will provide a detailed response to all other points raised by the reviewers.

A central point raised in the reviews concerns our definition of direction- and orientation-selective cells. We agree that relying only on statistical significance is not sufficient for our purposes, because the resulting classification can depend on recording duration and statistical power. In the revised manuscript, we will therefore introduce thresholding criteria for direction and orientation selectivity indices (DSI and OSI) in addition to significance-based testing. We will also make clearer that our primary question is which stimulus directions and orientations are represented at a given receptive field location, rather than the distribution of preferences among neurons classified as purely direction- or orientation-selective.

We will also revise the text to define more precisely what our data do and do not establish about the large-scale organization across SC. Our intended conclusion is not that we identify a novel global organization, which would require sampling a larger portion of visual space, but rather that the regions we sampled are not well explained by previously proposed global maps in which each visual field location is dominated by a single tuning preference and the same organization is conserved across individuals. Instead, our data are more consistent with a retinal organization of biases toward specific directions and orientations that vary systematically across visual space.

We will further clarify how we quantified the correspondence between our data and the previously established retinal model of direction and orientation tuning. In the current manuscript, we report the errors between model predictions and measured tuning preferences at the corresponding visual field locations. We then assess model performance by comparing the distribution of these errors with the errors obtained from two surrogate datasets: one in which the correspondence between visual field location and tuning preference is destroyed, and one in which the prior distribution of tuning preferences is assumed to be uniform. In the revised manuscript, we will make the interpretation of this comparison more explicit, so that the reported errors are clearly presented as the relevant effect-size measure alongside significance.

Finally, we appreciate the reviewers’ concern that the manuscript may currently emphasize disagreement with previous studies too strongly. We will revise the Discussion to better acknowledge where our data support some degree of local bias or weak clustering, while clarifying that we do not find evidence for a robust, stereotyped global map that is consistent across animals. Our goal is to sharpen the manuscript so that it better reconciles seemingly divergent findings in the literature rather than setting them in opposition.