Reviewer #2:
This manuscript by Diamanti et al. describes their study on how visual neurons responded to identical visual stimuli at two different locations along a virtual linear track. Extending their previous result that spatial location modulates the neuronal activities in the primary visual cortex (V1), they now demonstrate that similar spatial modulation also occurred in the higher visual areas (HVAs), but not so much in a lower visual area, the lateral geniculate nucleus (LGN). In addition, they show that the modulation, measured by a spatial modulation index (SMI), was stronger when animals had more experience in the track and when the animals were actively performing a task rather than passively viewing the same virtual track. The authors have been responsive to comments by previous reviewers at a different journal. Data are appropriately analyzed and clearly presented.
Since the finding that visual neurons are spatially modulated similarly as hippocampal place cells in spatial navigation tasks (Ji and Wilson, 2007; Haggerty and Ji, 2015; Fiser at al, 2016; Saleem at al, 2018), there has been increasing interest in identifying the source(s) of this modulation. This study adds new evidence to this puzzle, suggesting that it is more likely either generated within the visual cortex or top-down propagated from higher brain areas, rather than bottom-up propagated from the thalamus. This is an important contribution. However, there are concerns, mainly on the data interpretation and the clarification of the main conclusion, as elaborated below.
1) Because experience and task engagement enhanced spatial modulation, the authors concluded in the abstract that "Active navigation in a familiar environment, therefore, determines spatial modulation...". This conclusion is too strong and not well-supported by the data. First, spatial modulation on Day 1, when the task was novel, was lower than on later days, but it was already much higher than 0 (Fig. 1h). Also the individual neuron data (Fig. 1e) display clear spatial modulation on Day 1. Therefore, "familiar environment" is not a requirement. Second, spatial modulation during passive viewing was much higher than 0 and was correlated with that during active navigation, as shown in Fig. 4e - Fig. 4l. Therefore, "active navigation" is not a requirement either. It is true that both active navigation and familiar environment enhanced spatial modulation. They did not "determine" spatial modulation.
2) Related to the point above, the presence of spatial modulation in passive viewing reminds us that these cells in the visual system were still mainly driven by visual stimuli. The data in Fig. 4e,f are especially telling: the modulation in V1 was similar and highly correlated between active navigation and running replay. In addition, it is clear from all the raw traces in Fig. 1 and Fig. 2 that these cells did respond to the two segments with identical stimuli reliably with two peaks. The spatial modulation was just a change in one of the peaks. So the nature of the modulation is a "rate remapping" of the expected, classical visual responses. I believe, in order to maintain the big picture of what drives the activities of these neurons, it is beneficial to clarify that the "spatial modulation" is a modulation on top of the expected visual responses. This message is not explicitly conveyed in the current manuscript.
3) The authors stated that spatial modulation is "largely absent in the main thalamic pathway into V1". This was based on the significantly weaker SMIs in LGN than those in V1 and HVAs. However, it is unclear whether the SMIs in LGN were still significant. The SMI values for both LGN buttons (Line #100) and LGN units (Line# 130) might be statistically significant from zero. The statistical comparison p-values should be given in both cases. Second, Figure 3 - figure supplement 1 b,f show that the SMI values in LGN could be predicted by spatial modulation, but not by visual stimuli alone or behavioral variations, just like those in V1 and HVAs. This seems to me good evidence for the presence of spatial modulation in LGN. Therefore, it is my opinion that the data do not support the complete lack of spatial modulation in LGN, but do clearly demonstrate weaker spatial modulation in LGN than in V1 and HVAs.