Multimodal mapping of cell types and projections in the central nucleus of the amygdala
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eLife assessment
This study uses top-notch spatial profiling methods to present a valuable account of the number of different neuron types in the central nucleus of the amygdala of the mouse. The approaches and evidence presented are compelling, but the analysis is incomplete and would benefit from increases in sample size. With this aspect strengthened, this paper would be of interest to neuroscientists investigating the function of the central amygdala.
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Abstract
The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-Seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion asisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatio-molecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.
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eLife assessment
This study uses top-notch spatial profiling methods to present a valuable account of the number of different neuron types in the central nucleus of the amygdala of the mouse. The approaches and evidence presented are compelling, but the analysis is incomplete and would benefit from increases in sample size. With this aspect strengthened, this paper would be of interest to neuroscientists investigating the function of the central amygdala.
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Reviewer #1 (Public Review):
The goal of the present study was to provide the spatial, morphological, and connectional properties of molecularly defined CeA neurons. The authors provide compelling, high-quality profiling of the spatial distribution of up to 29 molecular markers in the CeA and its surrounding. In addition, by combining their EASI-FISH pipeline with retrograde labeling, the authors offer a comprehensive view of the connectional and molecular diversity within the CeA. The spatial resolution and utility of the EASI-fish technique are showcased elegantly in the present study. The authors also develop a new method to combine EASI-FISH with retrograde labeling to provide information on the anatomical connectivity of molecularly defined cells. Overall, the authors do provide a novel resource for the field. However, the scRNAseq …
Reviewer #1 (Public Review):
The goal of the present study was to provide the spatial, morphological, and connectional properties of molecularly defined CeA neurons. The authors provide compelling, high-quality profiling of the spatial distribution of up to 29 molecular markers in the CeA and its surrounding. In addition, by combining their EASI-FISH pipeline with retrograde labeling, the authors offer a comprehensive view of the connectional and molecular diversity within the CeA. The spatial resolution and utility of the EASI-fish technique are showcased elegantly in the present study. The authors also develop a new method to combine EASI-FISH with retrograde labeling to provide information on the anatomical connectivity of molecularly defined cells. Overall, the authors do provide a novel resource for the field. However, the scRNAseq dataset presented at the beginning of the study contains ~1300 cells, which is a low number of cells. From this, the authors report that three of their sequencing clusters (c1, c4, and c12) do not have specific molecular markers and were seemingly excluded from spatial validation. As such, throughout the study, how to interpret the relationship between the sequencing clusters and the molecular clusters may require additional experiments or analyses.
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Reviewer #2 (Public Review):
The central nucleus of the amygdala (CEA) has been the subject of numerous studies related to the processing of both aversive and pleasurable events; however, prior to this study a comprehensive understanding of the number of molecularly distinct neurons, their location in the CEA and their axonal projections have not been available. This paper provides a rich resource that fills this gap. The results will facilitate a more refined analysis of the roles of specific CEA neurons in mediating mouse behavior and physiology. A major strength of the paper is the application of their three-dimensional spatial profiling methodology of molecularly defined neurons to the CEA and then determining the axonal projections of those neurons to five of the most prominent target brain regions. The data are clearly presented …
Reviewer #2 (Public Review):
The central nucleus of the amygdala (CEA) has been the subject of numerous studies related to the processing of both aversive and pleasurable events; however, prior to this study a comprehensive understanding of the number of molecularly distinct neurons, their location in the CEA and their axonal projections have not been available. This paper provides a rich resource that fills this gap. The results will facilitate a more refined analysis of the roles of specific CEA neurons in mediating mouse behavior and physiology. A major strength of the paper is the application of their three-dimensional spatial profiling methodology of molecularly defined neurons to the CEA and then determining the axonal projections of those neurons to five of the most prominent target brain regions. The data are clearly presented and rigorously analyzed. The are no weaknesses in this valuable contribution.
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Reviewer #3 (Public Review):
This study aims at classifying central amygdala neurons based on their expression of marker genes along with their spatial, morphological, and connectivity properties. The use of state-of-the-art experimental and analysis approaches to disentangle the functional complexity and heterogeneity of this brain region is a clear strength of the study. The detailed spatial description, including rostral-caudal dimensions and specific CeA subnuclei in all of their analyses as well as the co-expression of multiple marker genes and description of various long-range projection targets, will be valuable, potentially allowing for the targeting of anatomically distinct CeA subregions in future mechanistic studies. The major weaknesses are the exclusion of female subjects in their experiments and the incomplete …
Reviewer #3 (Public Review):
This study aims at classifying central amygdala neurons based on their expression of marker genes along with their spatial, morphological, and connectivity properties. The use of state-of-the-art experimental and analysis approaches to disentangle the functional complexity and heterogeneity of this brain region is a clear strength of the study. The detailed spatial description, including rostral-caudal dimensions and specific CeA subnuclei in all of their analyses as well as the co-expression of multiple marker genes and description of various long-range projection targets, will be valuable, potentially allowing for the targeting of anatomically distinct CeA subregions in future mechanistic studies. The major weaknesses are the exclusion of female subjects in their experiments and the incomplete acknowledgement of previous studies that have addressed transcript expression and cell-type specific function in the CeA.
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