Author response:
The following is the authors’ response to the previous reviews
Public Reviews:
Reviewer #2 (Public review):
Summary:
The manuscript by Wang and colleagues aims to determine whether hepatic glucose metabolism is differentially regulated by the left and right sides of the LPGi and to reveal decussation of hepatic sympathetic nerves.
The authors used tissue clearing to identify sympathetic fibers in the liver lobes, then injected PRV into the hepatic lobes. Five days post-injection, PRV-labeled neurons in the LPGi, which were identified. The results indicated contralateral dominance of premotor neurons and partial innervation of more than one lobe. Then the authors activated each side of the LPGi, resulting in a greater increase in blood glucose levels after right-sided activation than after left-sided activation, and in changes in protein expression in the liver lobes. These data suggested lobe-specific modulation of HGP. Chemical denervation of a particular lobe did not affect glucose levels due to compensation by the other lobes. In addition, nerve bundles decussate in the hepatic portal region.
Strengths:
The manuscript is timely and relevant. It is important to understand the sympathetic regulation of the liver and the contribution of each lobe to hepatic glucose production. The authors use state-of-the-art methodology.
Weaknesses:
(1) Image clarity was improved in some cases, but not in others. For example, Figure 3I, showing c-Fos expression, is not convincing due to the image quality and lack of orientation.
We sincerely apologize for the insufficient image clarity and anatomical orientation in the original Figure 3I. To resolve this issue, we have performed the following revisions in the revised Figure 3I:
(1) Replaced the original panels with the high-resolution confocal images showing clear c-FOS immunofluorescence in the LPGi.
(2) Included explicit anatomical orientation indicators (Bregma −6.75 mm) to clearly demarcate the boundaries of the LPGi.
(3) Added ROI outlines surrounding the LPGi region.
(2) The methods section states that 8-weeks-old male mice were used in the experiments without specifying the experiments (e.g., brain injection with AAVs or PRV organ inoculation). The authors should include these details.
We thank the reviewer pointing out this oversight. We have updated the Methods section under "Animals" and specific procedure subsections to clearly state the exact age of animals.
(1) For retrograde trans-synaptic PRV tracing, 8-week-old mice received intrahepatic viral injections and were sacrificed 5 days post-injection.
(2) For chemogenetic and optogenetic manipulations, stereotaxic AAV injections were performed at 8 weeks of age. Mice were allowed 4 weeks for viral expression and recovery before undergoing metabolic tests or light stimulation at 12 weeks of age.
(3) For chemical denervation (6-OHDA), 8-week-old mice were injected into targeted lobes and examined 7 days post-denervation.
(4) For postnatal innervation mapping, neonatal mice at postnatal week 0 (P0), week 1 (P7), and week 2 (P14) were harvested for tissue clearing.
(3) The authors should use the exact location of pre- and postganglionic neurons as they often refer to neurons in the sympathetic chain. Their findings should be compared with the existing literature on the location of preganglionic cells.
We appreciate the reviewer for this feedback. We agree that our original description lacked precise anatomical localization regarding the pre- and postganglionic neurons, and it was inaccurate to state that descending fibers pass through the sympathetic chain (SyC).
Based on our whole-mount tissue clearing data, we observed that the preganglionic neurons of the brain-liver sympathetic circuit are primarily located in the T6–T12 segments of the thoracic spinal cord. Accordingly, we have revised the text in Results 4 to specify these exact locations.
Manuscript Revision (Results 4):
"Using whole-mount clearing, we visualized the brain–liver sympathetic circuit and found that preganglionic neurons in the thoracic spinal cord (T6–T12) send descending fibers via the splanchnic nerves to innervate postganglionic neurons in the CG-SMG (Figure 4A)."
Furthermore, following your valuable suggestion to compare our findings with existing literature, we reviewed a recent study published in Nature Communications (Harima, Yukiko et al. Parallel labeled-line organization of sympathetic outflow for selective organ regulation in mice. Nat Commun. 2024;15(1):10478). In that study, researchers injected retrogradely transducible AAVs directly into the CG-SMG and traced the preganglionic neurons predominantly to the T8–T13 segments. Their results are largely consistent with our findings. Interestingly, the broader anatomical range observed in our trans-synaptic liver-to-brain mapping (T6–T12) compared to their CG-SMG-specific tracing (T8–T13) reveals a slight discrepancy. This observation suggests an intriguing anatomical hypothesis: a subset of sympathetic preganglionic nerves may bypass the CG-SMG relay entirely and project directly to the liver.
(4) Figure legends should be revised and matched with the text.
We apologize for the oversight. We have conducted a comprehensive audit of all figure and legends to ensure precise matching between the main text and the figures.
Specifically, we have corrected a typographical error in the Figure 1 Legend where panel (C) was mistakenly labeled as a second panel (B), and we fixed a spelling error ("LPG" corrected to "LPGi"). Additionally, we corrected a miscitation in Results (Section 3) regarding Figure 3. In the original text, Figure 3C was incorrectly grouped with blood glucose data, whereas it actually displays the Western blot validation of sympathetic denervation.
We have revised the corresponding sections in the manuscript as follows:
Manuscript Revision (Figure 1 Legend):
“(C) Quantification of PRV-labeled neurons in left and right LPGi across different hepatic lobes: left lateral, median, right posterior, right anterior, caudate, and porta hepatis (n = 3).
(D) Sankey diagram showing projection patterns from left and right LPGi to individual hepatic lobes. (E and F) Representative slices of EGFP+ and mRFP+ neurons in left (top) and right (bottom) LPGi following PRV-EGFP (right anterior lobe) and PRV-mRFP (median lobe) injections. Proportions of EGFP+, mRFP+, and co-labeled neurons in left and right LPGi (F, n = 3). Scale bars, 100 μm.”
Manuscript Revision (Results 3):
“Despite the absence of directly sympathetic input to denervated lobes, systemic blood glucose levels were unchanged compared with controls (Figures 3A-3B, Figure S5A), indicating functional compensation through the remaining intact liver.”
Reviewer #4 (Public review):
Summary of General Strengths & Weaknesses:
The studies here are highly informative for anatomical tracing and sympathetic nerve function in the liver in relation to glucose levels, but because they are conducted in a single species, it is challenging to translate them to humans or determine whether these neural circuits are evolutionarily conserved. Dual-labeling anatomical studies are elegant, and the addition of chemogenetic and optogenetic studies provides mechanistically informative. Denervation studies lack proper controls, and sensory innervation in the liver is overlooked.
We sincerely thank the reviewer for their time and evaluation. We respectfully note that these comments mirror those raised during the previous round of review. We would like to kindly direct the reviewer to the extensive revisions we implemented in our previous resubmission, which directly and comprehensively addressed these exact concerns. These revisions remain intact in the current version of the manuscript. Below, we briefly summarize how each point was previously addressed for your convenience.
Specific Weaknesses - Major:
(1) The species name should be included in the title.
As addressed in our previous revision, we fully agree with this suggestion. We updated the title of the manuscript to explicitly include the species: "Symmetric brain-liver circuits mediate lateralized regulation of hepatic glucose output in mice." We also clarified the species used throughout the main text to ensure accuracy.
(2) Tyrosine hydroxylase was used to mark sympathetic fibers in the liver, but this marker also labels a portion of sensory fibers that need to be ruled out in whole-mount imaging data.
As detailed in our previous response, we acknowledge this important limitation. In our prior revision, we addressed this concern through both additional data analysis and text revisions:
(1) We provided SyGlass 3D reconstruction data demonstrating that the TH-positive nerve fibers originate from the celiac-superior mesenteric ganglia (CG-SMG), a well-established sympathetic ganglion (Figure S5F).
(2) In parallel, we collected dorsal root ganglia (DRG) from spinal segments T1-6 and T7-12 five days after intrahepatic PRV injection. While the T7-12 DRG segments are historically known to contain the sensory neurons that innervate the liver (Anat Rec A Discov Mol Cell Evol Biol. 2004; Auton Neurosci. 2024), we detected only a remarkably sparse number of PRV-positive neurons in these segments. This effectively functionally distinguishes this efferent pathway from primary sensory afferents (Supplementary figure B).
(3) We explicitly added this methodological limitation to the Discussion section (paragraph 6) of the current manuscript, noting that more selective approaches, such as genetic targeting of sympathetic lineages, will be important for future validation."
(3) Chemogenetic and optogenetic data demonstrating hyperglycemia should be described in the context of prior work demonstrating liver nerve involvement in these processes. There is only a brief mention in the Discussion currently, but comparing methods and observations would be helpful.
As outlined in our previous response, we incorporated this crucial context into our revised manuscript. Specifically, we expanded the Discussion section (paragraph 3) to contrast our precise cell-type-specific chemogenetic and optogenetic approaches with historical studies that relied on coarse electrical stimulation. This addition highlights how our current methodology reveals the contralateral and lobe-specific architecture of brain-liver sympathetic control that was previously obscured.
(4) Sympathetic denervation with 6-OHDA can drive compensatory increases in tissue sensory innervation, and this should be measured in the liver denervation studies to implicate potential crosstalk, especially given the increase in LPGi cFOS that may be due to afferent nerve activity. Compensatory sympathetic drive may not be the only culprit, though that is clearly assumed. The sensory or parasympathetic/vagal innervation of the liver is altogether ignored in this paper and could be better described in general.
We appreciate this insightful physiological perspective, which we addressed comprehensively in our previous revision. As we previously agreed, the central nervous system integrates a broad range of afferent signals, and compensatory sensory or parasympathetic mechanisms likely contribute to the observed LPGi activation following hepatic sympathetic denervation.
To address this, we significantly expanded our Discussion section (paragraph 4) in the prior revision. We explicitly proposed a model wherein hepatic glucose production is regulated by an integrated afferent-central-efferent loop, acknowledging that our current study primarily resolves the efferent component. We clearly noted the lack of direct assessment of sensory or parasympathetic innervation as a limitation and highlighted this dynamic crosstalk as a critical avenue for future investigation.
Comments on the revised version.
Across all reviewer comments, the revised resubmission has adequately addressed all concerns.
Recommendations for the authors:
Reviewer #4 (Recommendations for the authors):
No further recommendations aside from tempering the CGRP language, as marking all sensory fibers.
We appreciate the reviewer for pointing out this important anatomical distinction. We entirely agree that CGRP specifically labels peptidergic sensory afferents and does not represent the entirety of the sensory nervous system.
We have carefully reviewed the entire manuscript and tempered our language accordingly. Wherever CGRP is mentioned, we have clarified that it serves as a marker for peptidergic sensory fibers, rather than functioning as a pan-sensory marker.
Manuscript Revision (Results 1):
"Unlike the NTS, a well-established hepatic sensory center served here as a positive control, the LPGi contained few CGRP-positive cell bodies (Figure S1G), indicating a lack of peptidergic sensory projections."
