Diverse inhibitory projections from the cerebellar interposed nucleus
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Evaluation Summary:
Judd and colleagues use a combination of mouse genetics and viral marking to expand the extra-cerebellar map of projections. These data will impact our understanding of how the cerebellum contributes to behavior and in general how brain function is packaged at the anatomical level. These data will not only impact cerebellar scientists but also those workers interested in how inter-regional brain connectivity is organized and how fine input-output circuit relationships are structured.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2, and #3 agreed to share their names with the authors.)
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Abstract
The cerebellum consists of parallel circuit modules that contribute to diverse behaviors, spanning motor to cognitive. Recent work employing cell-type-specific tracing has identified circumscribed output channels of the cerebellar nuclei (CbN) that could confer tight functional specificity. These studies have largely focused on excitatory projections of the CbN, however, leaving open the question of whether inhibitory neurons also constitute multiple output modules. We mapped output and input patterns to intersectionally restricted cell types of the interposed and adjacent interstitial nuclei in mice. In contrast to the widespread assumption of primarily excitatory outputs and restricted inferior olive-targeting inhibitory output, we found that inhibitory neurons from this region ramified widely within the brainstem, targeting both motor- and sensory-related nuclei, distinct from excitatory output targets. Despite differences in output targeting, monosynaptic rabies tracing revealed largely shared afferents to both cell classes. We discuss the potential novel functional roles for inhibitory outputs in the context of cerebellar theory.
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Author Response:
Reviewer #1 (Public Review):
In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make …
Author Response:
Reviewer #1 (Public Review):
In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make widespread extra-cerebellar projections relies on the assumption that the Cre-lines used in the study are able to specifically/exclusively mark to those inhibitory neurons – these details were not fully worked out in this study.
We thank the reviewer for recognizing the importance of the study and pointing out important caveats. We have included a variety of validation methods to address the major concerns.
Reviewer #3 (Public Review):
By applying modern viral tracing methods, this paper described in detail extensive input-output connections of Gad1Cre+, VgatCre+, or Ntsr1Cre+ IntA projection neurons.
Because diverse neurons are intermingled in a small region, it is generally challenging to isolate specific excitatory or inhibitory neurons and their circuits in the cerebellar nucleus.
The authors focused on IntA of CN and demonstrated that 1) both inhibitory (Gad1Cre+ and/or VgatCre+) and excitatory (Ntsr1Cre+) neurons comprise extensive input-output connections with many extracerebellar regions, and 2) inhibitory circuits are functionally distinct from excitatory circuits on the basis of projection targets. This work could provide insights into diversity of inhibitory IntA neurons, and thus could be an interesting addition to the field's expanding efforts to identify cell types of CN, their input-output connections, and their functions.
However, interpreting the data is difficult because of technical challenges. Critically, the main conclusion could be compromised by experimental artifacts, which need better characterization. In addition, the text could be revised to make it more accessible to a broad audience.
We appreciate the reviewer’s recognition of the value of the questions addressed in this study and for raising the important technical points that are addressed in this revision.
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Reviewer #3 (Public Review):
By applying modern viral tracing methods, this paper described in detail extensive input-output connections of Gad1Cre+, VgatCre+, or Ntsr1Cre+ IntA projection neurons.
Because diverse neurons are intermingled in a small region, it is generally challenging to isolate specific excitatory or inhibitory neurons and their circuits in the cerebellar nucleus.
The authors focused on IntA of CN and demonstrated that 1) both inhibitory (Gad1Cre+ and/or VgatCre+) and excitatory (Ntsr1Cre+) neurons comprise extensive input-output connections with many extracerebellar regions, and 2) inhibitory circuits are functionally distinct from excitatory circuits on the basis of projection targets. This work could provide insights into diversity of inhibitory IntA neurons, and thus could be an interesting addition to the field's …
Reviewer #3 (Public Review):
By applying modern viral tracing methods, this paper described in detail extensive input-output connections of Gad1Cre+, VgatCre+, or Ntsr1Cre+ IntA projection neurons.
Because diverse neurons are intermingled in a small region, it is generally challenging to isolate specific excitatory or inhibitory neurons and their circuits in the cerebellar nucleus.
The authors focused on IntA of CN and demonstrated that 1) both inhibitory (Gad1Cre+ and/or VgatCre+) and excitatory (Ntsr1Cre+) neurons comprise extensive input-output connections with many extracerebellar regions, and 2) inhibitory circuits are functionally distinct from excitatory circuits on the basis of projection targets. This work could provide insights into diversity of inhibitory IntA neurons, and thus could be an interesting addition to the field's expanding efforts to identify cell types of CN, their input-output connections, and their functions.
However, interpreting the data is difficult because of technical challenges. Critically, the main conclusion could be compromised by experimental artifacts, which need better characterization. In addition, the text could be revised to make it more accessible to a broad audience.
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Reviewer #2 (Public Review):
Judd et al. systematically examine the input/output connectivity of discrete excitatory and inhibitory neuronal subpopulations in the cerebellar interposed anterior nucleus (IntA) using conditional AAV and rabies virus mapping strategies. The authors first define distinctions in the output connectivity of excitatory and inhibitory neurons in the IntA nucleus, and describe a surprisingly much wider projection pattern by inhibitory neurons than previously thought. They also characterize distinctions in projection pattern between identifiable subtypes of IntA inhibitory neurons as well as distinctions in morphology of their terminal fields. The authors next explore the input connectivity of excitatory and inhibitory neurons in the IntA nucleus and found that excitatory output neurons receive fewer, but more …
Reviewer #2 (Public Review):
Judd et al. systematically examine the input/output connectivity of discrete excitatory and inhibitory neuronal subpopulations in the cerebellar interposed anterior nucleus (IntA) using conditional AAV and rabies virus mapping strategies. The authors first define distinctions in the output connectivity of excitatory and inhibitory neurons in the IntA nucleus, and describe a surprisingly much wider projection pattern by inhibitory neurons than previously thought. They also characterize distinctions in projection pattern between identifiable subtypes of IntA inhibitory neurons as well as distinctions in morphology of their terminal fields. The authors next explore the input connectivity of excitatory and inhibitory neurons in the IntA nucleus and found that excitatory output neurons receive fewer, but more organized inputs than inhibitory output neurons, and that many output targets provide reciprocal connections with the CN.
In general, the output analysis is strong and there are only a few questions about interpretation of the distinctions of projections by different subtypes of IntA inhibitory neurons. For instance, the distribution of the initial targeting within the cerebellar nuclei, cerebellar cortex and outside the cerebellum was not analyzed in Ntsr1-Cre and Gad1-Cre similar to the analysis performed for the intersectional output analysis. Clarification on whether and how the distinctions in projections could be due to variability in the specificity of the initial targeting or recombination ability of the two mouse Cre-lines is needed to strengthen interpretation of the different projections patterns observed. As for the input analysis using rabies, there were two major issues identified.
First, the use of conditional GFP-labeled G protein and the use of rabies that is also GFP potentially confounds analysis of input cells.
Next, the low number of starter cells is a concern and the identity of starter cells outside the cerebellar nuclei in Ntsr1-Cre and Gad1-Cre is vague and needs to be clarified. This is important for interpretation of whether input structures observed project specifically into the CN or also into the cerebellar cortex, and whether distinctions observed in number of input structures may reflect amount of starter cells in each Cre line.
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Reviewer #1 (Public Review):
In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make widespread …
Reviewer #1 (Public Review):
In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make widespread extra-cerebellar projections relies on the assumption that the Cre-lines used in the study are able to specifically/exclusively mark to those inhibitory neurons – these details were not fully worked out in this study.
-
Evaluation Summary:
Judd and colleagues use a combination of mouse genetics and viral marking to expand the extra-cerebellar map of projections. These data will impact our understanding of how the cerebellum contributes to behavior and in general how brain function is packaged at the anatomical level. These data will not only impact cerebellar scientists but also those workers interested in how inter-regional brain connectivity is organized and how fine input-output circuit relationships are structured.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2, and #3 agreed to share their names with the authors.)
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