Layer 6 corticocortical cells dominate the anatomical organization of intra and interhemispheric feedback

Reviewer #1 (Public review):

Weiler, Teichert, and Margrie systematically analyzed long-range cortical connectivity, using a retrograde viral tracing strategy to identify layer and region-specific cortical projections onto the primary visual, primary somatosensory, and primary motor cortices. Their analysis revealed several hundred thousand inputs into each region, with inputs originating from almost all cortical regions but dominated in number by connections within cortical sub-networks (e.g. anatomical modules). Generally, the relative areal distribution of contralateral inputs followed the distribution of corresponding ipsilateral inputs. The largest proportion of inputs originated from layer 6a cells, and this layer 6 dominance was more pronounced for contralateral than ipsilateral inputs, which suggests that these connections provide predominantly feedback inputs. The hierarchical organization of input regions was similar between ipsi- and contralateral regions, except for within-module connections, where ipsilateral connections were much more feed-forward than contralateral. These results contrast earlier studies which suggested that contralateral inputs only come from the same region (e.g. V1 to V1) and from L2/3 neurons. Thus, these results provide valuable data supporting a view of interhemispheric connectivity in which layer 6 neurons play an important role in providing modulatory feedback.

The conclusions of this paper are mostly well-supported by the data and analysis, but additional consideration of possible experimental biases is needed.

Further discussion or analysis is needed about possible biases in uptake efficiency for different cell types. Is it possible that the nuclear retro-AAV has a tropism for layer 6 axons? Quantitative comparisons with results obtained with alternative methods such as rabies virus (Yao et al., 2023) or anterograde tracing (Harris et al., 2019) may be helpful for this.

Quantitative analysis of the injection sites should be included to account for possible biases. For example, L6 neurons are known to be the main target of contralateral inputs into the visual cortex (Yao et al., 2023). Thus, if the injections are biased towards or against layer 6 neurons, this may change the layer distribution of retrogradely labeled input cells. Comparison across biological replicates may help reveal sensitivity to particular characteristics of the injections.

The possibility of labeling axons of passage within the white matter should be addressed. This could potentially lead to false positive connections, contributing to the broad connectivity from most cortical regions that were observed.

Reviewer #2 (Public review):

Summary:

Weiler et al use retrograde tracers, two-photon tomography, and automatic cell detection to provide a detailed quantitative description of the laminar and area sources of ipsi- and contralateral cortico-cortical inputs to two primary sensory areas and a primary motor area. They found considerable bilateral symmetry in the areas providing cortico-cortical inputs. However, although the same regions in both hemispheres tended to supply inputs, a larger proportion of inputs from contralateral areas originated from deeper layers (L5 and L6).

Strengths:

The study applies state-of-the-art anatomical methods, and the data is very effectively presented and carefully analyzed. The results provide many novel insights into the similarities and differences of inputs from the two hemispheres. While over the past decade there have been many studies quantitively and comprehensively describing cortico-cortical connections, by directly comparing inputs from the ipsi and contralateral hemispheres, this study fills in an important gap in the field. It should be of great utility and an important reference for future studies on inter-hemispheric interactions.

Weaknesses:

Overall, I do not find any major weakness in the analyses or their interpretation. However, one must keep in mind that the study only analyses inputs projecting to three areas. This is not an inherent flaw of the study; however, it warrants caution when extrapolating the results to callosal projections terminating in other areas. As inputs to two primary sensory areas and one is the primary motor cortex are studied, some of the conclusions could potentially be different for inputs terminating in high-order sensory and motor areas. Given that primary areas were injected, there are few instances of feedforward connections sampled in the ipsilateral hemisphere. The study finds that while ipsi-projections from the visual cortex to the barrel cortex are feedforward given its fILN values, those from the contralateral visual cortex are feedback instead. One is left to wonder whether this is due to the cross-modal nature of these particular inputs and whether the same rule (that contralateral inputs consistently exhibit feedback characteristics regardless of the hierarchical relationship of their ipsilateral counterparts with the target area,) would also apply to feedforward inputs within the same sensory cortices.

Another issue that is left unexplored is that, in the current analyses the barrel and primary visual cortex are analyzed as a uniform structure. It is well established that both the laminar sources of callosal inputs and their terminations differ in the monocular and binocular areas of the visual cortex (border with V2L). Similarly, callosal projections differ when terminating the border of S1 (a row of whiskers), and then in other parts of S1. Thus, some of the conclusions regarding the laminar sources of callosal inputs might depend on whether one is analyzing inputs terminating or originating in these border regions.

Finally, while the paper emphasizes that projections from L6 "dominate" intra and contralateral cortico-cortical inputs, the data shows a more nuanced scenario. While it is true that the areas for which L6 neurons are the most common source of cortico-cortical projections are the most abundant, the picture becomes less clear when considering the number of neurons sending these connections. In fact, inputs from L2/3 and L5 combined are more abundant than those from L6 (Figure 3B), challenging the view that projections from L6 dominate ipsi- and contralateral projecting cortico-cortical inputs.