Molecular and anatomical characterization of parabrachial neurons and their axonal projections
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eLife assessment
The parabrachial nuclei are groups of neurons in the brainstem (one on each side) that integrate information about the state of the body to guide appropriate homeostatic responses. The manuscript by Pauli and Chen et al. is a compelling and much-needed study that characterizes the cell types that make up these nuclei and genetic tools to study them. The result is a highly valuable resource to the academic community.
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Abstract
The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, nociceptive responses, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns of dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are enriched within specific PBN subregions with scattered cells in several other regions. We also provide detailed visualization of the axonal projections from 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.
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eLife assessment
The parabrachial nuclei are groups of neurons in the brainstem (one on each side) that integrate information about the state of the body to guide appropriate homeostatic responses. The manuscript by Pauli and Chen et al. is a compelling and much-needed study that characterizes the cell types that make up these nuclei and genetic tools to study them. The result is a highly valuable resource to the academic community.
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Reviewer #1 (Public Review):
The author's stated goal was to determine how many unique populations of neurons there are in PB, whether these populations occupy discrete subnuclei in PB, and whether they project to and receive inputs from specific brain regions. They have succeeded admirably. This work presents the field with a valuable reference tool that will allow us to formulate testable hypotheses about the structure and function of PB, and to design tools to selectively manipulate discrete populations of PB neurons.
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Reviewer #2 (Public Review):
The Parabrachial Nucleus (PBN) of the pons is a hub for sensory processing that receives sensory signals from the periphery and broadcasts these signals to downstream brain regions. It has been known that the PBN is comprised of several subdomains that have distinct input-output connections with other brain areas and sensory streams. This work provides detailed and much-needed cellular and molecular atlas of the PBN using a single-cell RNA sequencing analysis. The HiPlex RNAscope and Cre-driver line analyses confirm this organizational property of the PBN. This paper will serve as a key reference that defines molecularly and anatomically distinct cell types in the PBN.
The strength and novelty of this study are as follows.
This work is the first in-depth single-cell RNA sequencing study of the PBN.
This study …
Reviewer #2 (Public Review):
The Parabrachial Nucleus (PBN) of the pons is a hub for sensory processing that receives sensory signals from the periphery and broadcasts these signals to downstream brain regions. It has been known that the PBN is comprised of several subdomains that have distinct input-output connections with other brain areas and sensory streams. This work provides detailed and much-needed cellular and molecular atlas of the PBN using a single-cell RNA sequencing analysis. The HiPlex RNAscope and Cre-driver line analyses confirm this organizational property of the PBN. This paper will serve as a key reference that defines molecularly and anatomically distinct cell types in the PBN.
The strength and novelty of this study are as follows.
This work is the first in-depth single-cell RNA sequencing study of the PBN.
This study confirms previous findings and adds new information about the cellular landscape of the PBN; the majority of cells in this region are neurons (57.2%), followed by oligodendrocytes (24.1%), astrocytes (8.1%), and microglia (2.6%). Among the neurons in the PBN, ~90% are Glutamatergic, forming 12 molecularly distinct subtypes, whereas ~10% are GABAergic, forming 2 subtypes of neurons.
Another exciting finding of this study is that molecularly defined Glutamatergic neuronal subtypes are distributed within the PBN in a spatially restricted manner (Figure 4) such that these subtypes largely correspond to subdomains of the PBN that were described in prior studies. In contrast, the two GABAergic subtypes are scattered throughout the PBN.
This work comprehensively analyzes 21 different Cre-driver lines that label different classes of neurons in the PBN, many of which are newly characterized and validated in this work. Characterizing these Cre-driver lines provides valuable information for future studies in the field to understand the function of these genetically defined PBN neuronal subtypes.
Although the key claims are well supported by data presented in the paper, clarification of the following concerns would further strengthen the overall conclusion of the study.
There are two undefined yet significant clusters (without any assigned color codes) - one in the center and the other at the bottom of the UMAP space (Figure 2A). The molecular identity of the two clusters needs to be described in the figure and main text.
The authors claim that neuronal populations located in the dorsal PBN mainly innervate brain regions associated with Central Tegmental Tract (CTT), whereas neuronal populations found in the PBle mainly innervate the brain regions associated with Ventral Pathway (VP). However, there are significant overlaps in the brain regions innervated by both PBN populations (Figure 7B). Thus, the axon projection diagram (Figure 7A) can be misleading.
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Reviewer #3 (Public Review):
The manuscript by Pauli and Chen et al. is a beautiful and much-needed study that characterizes the cell types that make up these nuclei.
The major strength of this study is it combines a profiling approach to identify subtypes (single-cell sequencing) with microscopy that reveals where the cells reside within the PBN (multiplex FISH) and a genetic approach to visualize the projection pattern of these cell types throughout the brain (genetic labeling with Cre-lines). The result is important new insight about the cell types in this structure. Highlights include (1) the identification of two inhibitory neuron subtypes and 19 excitatory cell types, (2) the discovery of two output streams, (3) insight about the ontogeny of cell types and transcription factors that may be involved in fate …
Reviewer #3 (Public Review):
The manuscript by Pauli and Chen et al. is a beautiful and much-needed study that characterizes the cell types that make up these nuclei.
The major strength of this study is it combines a profiling approach to identify subtypes (single-cell sequencing) with microscopy that reveals where the cells reside within the PBN (multiplex FISH) and a genetic approach to visualize the projection pattern of these cell types throughout the brain (genetic labeling with Cre-lines). The result is important new insight about the cell types in this structure. Highlights include (1) the identification of two inhibitory neuron subtypes and 19 excitatory cell types, (2) the discovery of two output streams, (3) insight about the ontogeny of cell types and transcription factors that may be involved in fate specification/determination, (4) clarification of which neuropeptides are expressed/not expressed in which cell types, (5) and the identification of suitable genetic markers to target many of the cell types. Importantly, all the data are available for researchers to mine their genes of interest. This information is a major advance in the field.
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