Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices
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eLife assessment
This study offers a valuable description of the layer-and sublayer specific outputs of the somatosensory cortex based on convincing evidence obtained with modern tools for the analysis of brain connectivity, together with functional validation of the connectivity using optogenetic approaches in vivo. Beyond bridging together, in one dataset, the results of disparate studies, this effort brings new insights on layer specific outputs, and on differences between primary and secondary somatosensory areas. This study will be of interest to neuroanatomists and neurophysiologists.
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Abstract
The extensive long-range axonal projections of various classes of neocortical excitatory neurons are thought to contribute importantly to the highly integrative brain-wide interactions underlying the processing of sensory, cognitive and motor signals. Here, we investigated the long-range axonal output of various classes of genetically-defined projection neurons with cell bodies located in the whisker-related somatosensory cortices of the mouse through brain-wide light-sheet imaging of fluorescently-labeled axons segmented by specifically-trained convolutional networks quantified within the Allen Mouse Brain Atlas Common Coordinate Framework. We injected Cre-dependent virus to express GFP or tdTomato in the posterior primary somatosensory barrel cortex and the posterior supplemental somatosensory cortex, which contain the representations of the large posterior mystacial whiskers. We investigated the six following transgenic mouse lines: Rasgrf2-dCre, Scnn1a-Cre, Tlx3-Cre, Sim1-Cre, Rbp4-Cre and Ntsr1-Cre. We found long-range axonal projections in many diverse downstream brain areas with genetically-defined cell classes showing distinct innervation patterns. To test whether the revealed axonal projections might underpin functional circuits, we compared the spatial organization of the axonal innervation with functional connectivity maps obtained from optogenetic stimulation of sensory cortex and wide-field imaging of the activity propagation to frontal cortices. Both methods indicated that neurons located more laterally in somatosensory cortex topographically signaled to more anteriorly located regions in motor cortex. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling, and, together with further technological advances, this will help provide increasingly detailed connectivity information of the mouse brain, essential for understanding the complex neuronal circuitry underlying even simple goal-directed behaviors.
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eLife assessment
This study offers a valuable description of the layer-and sublayer specific outputs of the somatosensory cortex based on convincing evidence obtained with modern tools for the analysis of brain connectivity, together with functional validation of the connectivity using optogenetic approaches in vivo. Beyond bridging together, in one dataset, the results of disparate studies, this effort brings new insights on layer specific outputs, and on differences between primary and secondary somatosensory areas. This study will be of interest to neuroanatomists and neurophysiologists.
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Reviewer #1 (Public Review):
Summary:
This is a fine paper that serves the purpose to show that the use of light sheet imaging may be used to provide whole brain imaging of axonal projections. The data provided suggest that at this point the technique provides lower resolution than with other techniques. Nonetheless, the technique does provide useful, if not novel, information about particular brain systems.
Strengths:
The manuscript is well written. In the introduction a clear description of the functional organization of the barrel cortex is provided provides the context for applying the use of specific Cre-driver lines to map the projections of the main cortical projection types using whole brain neuroanatomical tracing techniques. The results provided are also well written, with sufficient detail describing the specifics of how …
Reviewer #1 (Public Review):
Summary:
This is a fine paper that serves the purpose to show that the use of light sheet imaging may be used to provide whole brain imaging of axonal projections. The data provided suggest that at this point the technique provides lower resolution than with other techniques. Nonetheless, the technique does provide useful, if not novel, information about particular brain systems.
Strengths:
The manuscript is well written. In the introduction a clear description of the functional organization of the barrel cortex is provided provides the context for applying the use of specific Cre-driver lines to map the projections of the main cortical projection types using whole brain neuroanatomical tracing techniques. The results provided are also well written, with sufficient detail describing the specifics of how techniques were used to obtain relevant data. Appropriate controls were done, including the identification of whisker fields for viral injections and determination of the laminar pattern of Cre expression. The mapping of the data provides a good way to visualize low resolution patterns of projections.
Weaknesses:
(1) The results provided are, as stated in the discussion, "largely in agreement with previously reported studies of the major projection targets". However it must be stated that the study does not "extend current knowledge through the high sensitivity for detecting sparse axons, the high specificity of labeling of genetically defined classes of neurons and the brain wide analysis for assigning axons to detailed brain regions" which have all been published in numerous other studies. ( the allen connectivity project and related papers, along with others). If anything the labeling of axons obtained with light sheet imaging in this study does not provide as detailed mapping obtained with other techniques. Some detail is provided of how the raw images are processed to resolve labeled axons, but the images shown in the figures do not demonstrate how well individual axons may be resolved, of particular interest would be to see labeling in terminal areas such as other cortical areas, striatum and thalamus. As presented the light sheet imaging appears to be rather low resolution compared to the many studies that have used viral tracing to look at cortical projections from genetically identified cortical neurons.
(2) Amongst the limitations of this study is the inability to resolve axons of passage and terminal fields. This has been done in other studies with viral constructs labeling synaptophysin. This should be mentioned.
(3) There is no quantitative analysis of differences between the genetically defined neurons projecting to the striatum, what is the relative area innervated by, density of terminals, other measures.
(4) Figure 5 is an example of the type of large sets of data that can be generated with whole brain mapping and registration to the Allen CCF that provides information of questionable value. Ordering the 50 plus structures by the density of labeling does not provide much in terms of relative input to different types of areas. There are multiple subregions for different functional types ( ie, different visual areas and different motor subregions are scattered not grouped together. Makes it difficult to understand any organizing principles.
(5) The GENSAT Cre driver lines used must have the specific line name used, not just the gene name as the GENSAT BAC-Cre lines had multiple lines for each gene and often with very different expression patterns. Rbp4_KL100, Tlx3_PL56, Sim1_KJ18, Ntsr1_ GN220. -
Reviewer #2 (Public Review):
Summary:
This study takes advantage of multiple methodological advances to perform layer-specific staining of cortical neurons and tracking of their axons to identify the pattern of their projections. This publication offers a mesoscale view of the projection patterns of neurons in the whisker primary and secondary somatosensory cortex. The authors report that, consistent with the literature, the pattern of projection is highly different across cortical layers and subtype, with targets being located around the whole brain. This was tested across 6 different mouse types that expressed a marker in layer 2/3, layer 4, layer 5 (3 sub-types) and layer 6.
Looking more closely at the projections from primary somatosensory cortex into the primary motor cortex, they found that there was a significant spatial …Reviewer #2 (Public Review):
Summary:
This study takes advantage of multiple methodological advances to perform layer-specific staining of cortical neurons and tracking of their axons to identify the pattern of their projections. This publication offers a mesoscale view of the projection patterns of neurons in the whisker primary and secondary somatosensory cortex. The authors report that, consistent with the literature, the pattern of projection is highly different across cortical layers and subtype, with targets being located around the whole brain. This was tested across 6 different mouse types that expressed a marker in layer 2/3, layer 4, layer 5 (3 sub-types) and layer 6.
Looking more closely at the projections from primary somatosensory cortex into the primary motor cortex, they found that there was a significant spatial clustering of projections from topographically separated neurons across the primary somatosensory cortex. This was true for neurons with cell bodies located across all tested layers/types.Strengths:
This study successfully looks at the relevant scale to study projection patterns, which is the whole brain. This is achieved thanks to an ambitious combination of mouse lines, immuno-histochemistry, imaging and image processing, which results in a standardized histological pipeline that processes the whole-brain projection patterns of layer-selected neurons of the primary and secondary somatosensory cortex.
This standardization means that comparisons between cell-types projection patterns are possible and that both the large-scale structure of the pattern and the minute details of the intra-areas pattern are available.
This reference dataset and the corresponding analysis code are made available to the research community.Weaknesses:
One major question raised by this dataset is the risk of missing axons during the post-processing step. Indeed, it appears that the control and training efforts have focused on the risk of false positives (see Figure 1 supplementary panels). And indeed, the risk of overlooking existing axons in the raw fluorescence data id discussed in the article.
Based on the data reported in the article, this is more than a risk. In particular, Figure 2 shows an example Rbp4-L5 mouse where axonal spread seems massive in Hippocampus, while there is no mention of this area in the processed projection data for this mouse line.
Similarily, the Ntsr1-L6CT example shows a striking level of fluorescence in Striatum, that does not reflect in the amount of axons that are detected by the algorithms in the next figures.
These apparent discrepancies may be due to non axonal-specific fluorescence in the samples. In any case, further analysis of such anatomical areas would be useful to consolidate the valuable dataset provided by the article. -
Reviewer #3 (Public Review):
Summary:
-The paper offers a systematic and rigorous description of the layer-and sublayer specific outputs of the somatosensory cortex using a modern toolbox for the analysis of brain connectivity which combines: 1) Layer-specific genetic drivers for conditional viral tracing; 2) whole brain analyses of axon tracts using tissue clearing and imaging; 3) Segmentation and quantification of axons with normalization to the number of transduced neurons; 4) registration of connectivity to a widely used anatomical reference atlas; 5) functional validation of the connectivity using optogenetic approaches in vivo.
Strengths:
- Although the connectivity of the somatosensory cortex is already known, precise data are dispersed in different accounts (papers, online resources,) using different methods. So the present …
Reviewer #3 (Public Review):
Summary:
-The paper offers a systematic and rigorous description of the layer-and sublayer specific outputs of the somatosensory cortex using a modern toolbox for the analysis of brain connectivity which combines: 1) Layer-specific genetic drivers for conditional viral tracing; 2) whole brain analyses of axon tracts using tissue clearing and imaging; 3) Segmentation and quantification of axons with normalization to the number of transduced neurons; 4) registration of connectivity to a widely used anatomical reference atlas; 5) functional validation of the connectivity using optogenetic approaches in vivo.
Strengths:
- Although the connectivity of the somatosensory cortex is already known, precise data are dispersed in different accounts (papers, online resources,) using different methods. So the present account has the merit of condensing this information in one very precisely documented report. It also brings new insights on the connectivity, such as the precise comparison of layer specific outputs, and of the primary and secondary somatosensory areas. It also shows a topographic organization of the circuits linking the somatosensory and motor cortices. The paper also offers a clear description of the methodology and of a rigorous approach to quantitative anatomy.
Weaknesses:
The weakness relates to the intrinsic limitations of the in toto approaches, that currently lack the precision and resolution allowing to identify single axons, axon branching or synaptic connectivity. These limitations are identified and discussed by the authors.
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