An inhibitory circuit from central amygdala to zona incerta drives pain-related behaviors in mice
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Evaluation Summary:
This manuscript from Singh and colleagues investigates neural connections between the central amygdala and the zona incerta, two subcortical brain regions previously implicated in pain, and further describes the role of the zona incerta to preclinical pain-related behavior in mice. This study employed anatomical tracing, electrophysiology, optogenetics, chemogenetics, and behavioral assays in various pain modalities to link the zona incerta to pain modulation by providing new evidence for a direct inhibitory connection from the central amygdala to the zona incerta that could explain neuropathic pain hypersensitivity. While rigorous, well written, and well executed, the study in its current form lacked evidence to directly support the PKCδ neurons in the central amygdala projecting to the zona incerta as being explicitly involved in this process.
(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. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Central amygdala neurons expressing protein kinase C-delta (CeA-PKCδ) are sensitized following nerve injury and promote pain-related responses in mice. The neural circuits underlying modulation of pain-related behaviors by CeA-PKCδ neurons, however, remain unknown. In this study, we identified a neural circuit that originates in CeA-PKCδ neurons and terminates in the ventral region of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induced bilateral hypersensitivity in uninjured mice and contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reversed nerve injury-induced hypersensitivity. Optogenetic manipulations of CeA-PKCδ axonal terminals in the ZI further showed that inhibition of this pathway reduces nerve injury-induced hypersensitivity whereas activation of the pathway produces hypersensitivity in the uninjured paws. Altogether, our results identify a novel nociceptive inhibitory efferent pathway from CeA-PKCδ neurons to the ZI that bidirectionally modulates pain-related behaviors in mice.
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Evaluation Summary:
This manuscript from Singh and colleagues investigates neural connections between the central amygdala and the zona incerta, two subcortical brain regions previously implicated in pain, and further describes the role of the zona incerta to preclinical pain-related behavior in mice. This study employed anatomical tracing, electrophysiology, optogenetics, chemogenetics, and behavioral assays in various pain modalities to link the zona incerta to pain modulation by providing new evidence for a direct inhibitory connection from the central amygdala to the zona incerta that could explain neuropathic pain hypersensitivity. While rigorous, well written, and well executed, the study in its current form lacked evidence to directly support the PKCδ neurons in the central amygdala projecting to the zona incerta as being …
Evaluation Summary:
This manuscript from Singh and colleagues investigates neural connections between the central amygdala and the zona incerta, two subcortical brain regions previously implicated in pain, and further describes the role of the zona incerta to preclinical pain-related behavior in mice. This study employed anatomical tracing, electrophysiology, optogenetics, chemogenetics, and behavioral assays in various pain modalities to link the zona incerta to pain modulation by providing new evidence for a direct inhibitory connection from the central amygdala to the zona incerta that could explain neuropathic pain hypersensitivity. While rigorous, well written, and well executed, the study in its current form lacked evidence to directly support the PKCδ neurons in the central amygdala projecting to the zona incerta as being explicitly involved in this process.
(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. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript the authors found a direct synaptic connection between inhibitory neurons in the central nucleus of the amygdala and inhibitory and other neurons in the zona incerta. They conducted a rigorous and detailed anatomical study of both the anterograde and retrograde connections between PKCdelta CeA neurons and the zona incerta. Furthermore they conducted rigorous chemogenetic investigation of the zona incerta inhibitory neurons across pain modalities. This led to the overall conclusion that PKCdelta neurons inhibit zona incerta inhibitory neurons leading to enhanced pain processing. While the results mainly support the conclusions, there is a lack of direct support for the CeA-PKCdelta-->vGAT-ZI hypothesis.
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Reviewer #2 (Public Review):
This manuscript from Singh and colleagues investigates neural connections between the central amygdala and the zona incerta (ZI), two subcortical brain regions previously implicated in pain, and further describes the role of the ZI to preclinical pain-related behavior in mice. The work builds directly from prior work published by the corresponding author on the role of specific and functionally-opposing cell-types in the central amygdala to pain-the somatostatin (Sst) and protein kinase C delta (Pkcd) GABAergic cell-types. However the evidence to support the direct link between the CeA cell-type expressing Pkcd+ and the functional consequences of ZI processes during pain is mild and not fully supported for conclusions made in the Discussion, specifically around potential shifts to Pkcd-neuron dominate …
Reviewer #2 (Public Review):
This manuscript from Singh and colleagues investigates neural connections between the central amygdala and the zona incerta (ZI), two subcortical brain regions previously implicated in pain, and further describes the role of the ZI to preclinical pain-related behavior in mice. The work builds directly from prior work published by the corresponding author on the role of specific and functionally-opposing cell-types in the central amygdala to pain-the somatostatin (Sst) and protein kinase C delta (Pkcd) GABAergic cell-types. However the evidence to support the direct link between the CeA cell-type expressing Pkcd+ and the functional consequences of ZI processes during pain is mild and not fully supported for conclusions made in the Discussion, specifically around potential shifts to Pkcd-neuron dominate signaling following sever injury. Furthermore, the cell-type manipulations of theZI, which cover a large population of inhibitory cells, contrasts with recent published findings on a small subpopulation of these inhibitory neurons and warrants further exploration to provide readers with a clearer understanding of this brain regions function to pain-related behaviors in normal and persistence pain states.
I have the following comments:
- The current link of the small number of Pkcd+ CeA cells to the ZI's function is pain is tenuous to support the overall claims of the paper. It is clear from the semiquantitative anterograde tracing data that the Pkcd neurons project to the ZI, but that this is not the primary target of this neural population. Indeed, the CTB retro tracing shows that the great majority of neurons projecting to ZI are not the Pkcd popualtion. This was the main conclusion from Zhou et al (Tom Sudhof) Nat Neuro 2018: "... most of the CeA neurons projecting to ZI PV+ neurons express SST (90 {plus minus} 3% of GFP+ neurons, n = 3 mice) but not protein kinase C delta (PRKCD, 9 {plus minus} 3% of GFP+ neurons)". The connectivity is confirmed via slice electrophysiology between Pkcd-ChR2 and VGAT;tdT ZI cells. However the PPR recordings are only conducted in a few neurons from uninjured mice. There are multiple speculative claims in the paper on potential role of this connection during injured states / persistent pain. These statements should be supported by experiments showing changes in synaptic connectivity via additional ephys recordings, and behavioral evidence following silencing of the small population of Pkcd+ CeA neurons projecting to ZI.
- Statements relating to the divergent role of Pkcd and Sst CeA projections to the CeA should be supported by behavioral evidence, since the Sst is the presumptive dominant projection to the ZI.
- In Figure 3 - Supp 1, hM4 expression seems to be mostly in the ventral ZI. Do these cells express PV (ventral ZI marker) vs. nNOS (dorsal ZI marker)? This is important given other recent publications on this exact topic by Wang et al., 2020, Neuron 107, 538-551. Specifically, since VGAT-Cre would cover the Pv population previously shown to be "pain responsive" (i.e. noxious stimulus-induced FOS and calcium imaging) and which was required for engagement in aversive pain behavior (opto inhibition alleviates nocifensive behavior), is the interpretation of the current work that the dorsal ZI inhibition subsumes the function role of the ventral ZI in pain behavior expression? A more careful dissection of the ZI is warranted here since other publications ahve already begun to parcel this structure in subregions with specific cell-types and specific functions.
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Reviewer #3 (Public Review):
This study was designed to test the hypothesis that output from a subpopulation of neurons (PKCδ neurons) in the central nucleus of the amygdala (CeA) inhibits ZI neurons in a neuropathic pain condition and this ZI inhibition results in pain-related behaviors (Fig. 5).
First, the targets of CeA-PKCδ neurons were identified using cre-dependent viral vector for anterograde labeling with red-shifted channelrhodopsin (CrimsonR-tdTomato) or mCherry, and cholera toxin B (CTB) in PKCδ-tdTomato mice for retrograde labeling. The ZI was identified as one of the targets with approximately 19% of CTB+ CeA neurons identified as PKCδ- tdTomato positive, which is significant and makes this pathway worth exploring.
Next, electrophysiological (patch-clamp) studies showed monosynaptic inhibitory transmission from CeA to both …
Reviewer #3 (Public Review):
This study was designed to test the hypothesis that output from a subpopulation of neurons (PKCδ neurons) in the central nucleus of the amygdala (CeA) inhibits ZI neurons in a neuropathic pain condition and this ZI inhibition results in pain-related behaviors (Fig. 5).
First, the targets of CeA-PKCδ neurons were identified using cre-dependent viral vector for anterograde labeling with red-shifted channelrhodopsin (CrimsonR-tdTomato) or mCherry, and cholera toxin B (CTB) in PKCδ-tdTomato mice for retrograde labeling. The ZI was identified as one of the targets with approximately 19% of CTB+ CeA neurons identified as PKCδ- tdTomato positive, which is significant and makes this pathway worth exploring.
Next, electrophysiological (patch-clamp) studies showed monosynaptic inhibitory transmission from CeA to both VGAT+ and VGAT- neurons of the ZI and found no significant difference between these projections (from CeA to GABAergic or non-GABAergic ZI neurons).
Finally, chemogenetics are used to activate or silence GABAergic ZI neurons and determine behavioral consequences. Inhibition of GABAergic ZI neurons induced hypersensitivity in naïve mice and activation of these neurons reversed hypersensitivity in a neuropathic pain model. Interestingly, these effects were modality specific.
The combination of tracing techniques, electrophysiology, chemogenetics and behavior is a strength of this study, and so this the impressive amount of high-quality data. The focus on CeA-PKCδ neurons in the modulation of ZI is an important novelty of the present study.
However, slice physiology and behavioral data presented here do not actually link CeA-PKCδ neurons to ZI. Electrophysiological data show inhibitory transmission from CeA to ZI, but not specifically from CeA-PKCδ neurons to ZI. Behavioral studies assess the effects of modulation of ZI neurons but not of CeA-PKCδ to ZI projections. Previous data already showed the effects of activation and inhibition of GABAergic ZI neurons on pain behaviors, including in a neuropathic pain model.
Therefore, although the proposed model of CeA-PKCδ to ZI interactions in pain (Fig. 5) is novel and significant, additional experiments focusing on CeA-PKCδ neurons and their ZI projections would be needed to fully support this concept and enhance impact of the work.
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