Cell-to-cell signalling mediated via CO₂: activity dependent CO₂ production in the axonal node opens Cx32 in the Schwann cell paranode

Reviewer #1 (Public review):

The manuscript by Butler et al. explores a novel physiological role for connexin 32 (Cx32) hemichannels in Schwann cells at peripheral nerves. Building on the authors' prior work on CO₂-sensitive gating of connexins, this study proposes that mitochondrial CO₂ production dependent on neuronal activity promotes the opening of Cx32 hemichannels in the paranode, which in turn modulates neuronal activity by reducing conduction velocity. This hypothesis is addressed using a multifaceted approach that includes immunofluorescence microscopy, dye uptake assays, calcium imaging, computational modeling, and extracellular recordings in isolated sciatic nerves.

Among the strengths of the study are the interdisciplinary integration of imaging, in silico approaches, and functional data. Also, this study proposes a new mechanism with profound physiological relevance. Specifically, Butler et al. provide new insights into glial modulation of electrical conduction in sensory/motor myelinated nerves.

In the current state, the study has some limitations. The evidence linking Cx32 to the observed dye uptake and conduction velocity changes relies primarily on pharmacological inhibition with carbenoxolone, which lacks specificity. The imaging data show overlapping marker signals that preclude the anatomical distinction between nodes and paranodes. FITC uptake, while convincing to test Cx32 hemichannel gating, lacks spatial-temporal information and validation of distribution and localization to viable intracellular compartments. Moreover, while the findings are intriguing, functional proof that Cx32 regulates conduction velocity through ATP release or other downstream effects remains incomplete. Further work using targeted genetic tools, live-tissue imaging, and additional controls would strengthen the mechanistic conclusions.

Overall, the manuscript offers compelling preliminary evidence that supports a new role for Cx32 in peripheral nerve physiology and raises important questions for future investigation.

Reviewer #2 (Public review):

Summary:

This article aims to demonstrate that local production of CO₂ at the axonal node opens Cx32 hemichannels in the Schwann cell paranode, and that CO₂ diffuses through the AQP1 channel to reach Cx32 and trigger its opening. The authors also present evidence supporting a physiological role for this regulatory mechanism. They propose that CO₂-dependent Cx32 activation mediates activity-dependent Ca²⁺ influx into the paranode, and by increasing the leak current across the myelin sheath, it contributes to a slowing of action potential conduction velocity.

The study presents a very interesting and novel mechanism for the physiological regulation of Cx32 hemichannels. The findings are relevant to the field, and the methods and results are of good quality, with some improvements in interpretation and explanation required, and some minor experimental suggestions.

Strengths:

The article is solid in terms of the novelty of the findings and relevance for the physiology of myelinated axons. In addition, it is of major interest for the Connexin field because it explores a physiological way to open Cx32 hemichannels. The experiments are well elaborated, and most of them are sufficient for the main points described by the authors. The finding that nervous activity will trigger the mechanism of hemichannel opening by CO2 is probably the most relevant biological mechanism derived from this article.

Weaknesses:

Throughout the manuscript, the authors interpret their findings as if the described mechanism specifically occurs in the node and paranode regions. However, there is no direct evidence identifying the precise site of CO₂ production or the activation site of Cx32 hemichannels. Therefore, statements such as the one in the title ("activity-dependent CO₂ production in the axonal node opens Cx32 in the Schwann cell paranode") should be reconsidered or removed, as they may be misleading and are not essential to the interpretation of the data. In addition, the participation of aquaporin AQP1 as the main conduit for CO2 diffusion through the plasma membrane could have another interpretation.

Author response:

Reviewer #1 (Public review):

The manuscript by Butler et al. explores a novel physiological role for connexin 32 (Cx32) hemichannels in Schwann cells at peripheral nerves. Building on the authors' prior work on CO₂-sensitive gating of connexins, this study proposes that mitochondrial CO₂ production dependent on neuronal activity promotes the opening of Cx32 hemichannels in the paranode, which in turn modulates neuronal activity by reducing conduction velocity. This hypothesis is addressed using a multifaceted approach that includes immunofluorescence microscopy, dye uptake assays, calcium imaging, computational modeling, and extracellular recordings in isolated sciatic nerves.

Among the strengths of the study are the interdisciplinary integration of imaging, in silico approaches, and functional data. Also, this study proposes a new mechanism with profound physiological relevance. Specifically, Butler et al. provide new insights into glial modulation of electrical conduction in sensory/motor myelinated nerves.

In the current state, the study has some limitations. The evidence linking Cx32 to the observed dye uptake and conduction velocity changes relies primarily on pharmacological inhibition with carbenoxolone, which lacks specificity. The imaging data show overlapping marker signals that preclude the anatomical distinction between nodes and paranodes. FITC uptake, while convincing to test Cx32 hemichannel gating, lacks spatial-temporal information and validation of distribution and localization to viable intracellular compartments. Moreover, while the findings are intriguing, functional proof that Cx32 regulates conduction velocity through ATP release or other downstream effects remains incomplete. Further work using targeted genetic tools, live-tissue imaging, and additional controls would strengthen the mechanistic conclusions.

Overall, the manuscript offers compelling preliminary evidence that supports a new role for Cx32 in peripheral nerve physiology and raises important questions for future investigation.

We thank the reviewer for their comments and agree that the evidence for involvement of Cx32 is indirect. We are planning to perform genetic manipulations to strengthen this link. We shall review our presentation of the morphology in terms of the node/paranode/juxtaparanode distribution and adjust accordingly. We have in the interim generated new data using GCaMP transduced into Schwann cells that provides the live-tissue imaging that the reviewer requests. This strengthens our conclusions, and we will add these data into the paper.

Reviewer #2 (Public review):

Summary:

This article aims to demonstrate that local production of CO₂ at the axonal node opens Cx32 hemichannels in the Schwann cell paranode, and that CO₂ diffuses through the AQP1 channel to reach Cx32 and trigger its opening. The authors also present evidence supporting a physiological role for this regulatory mechanism. They propose that CO₂-dependent Cx32 activation mediates activity-dependent Ca²⁺ influx into the paranode, and by increasing the leak current across the myelin sheath, it contributes to a slowing of action potential conduction velocity.

The study presents a very interesting and novel mechanism for the physiological regulation of Cx32 hemichannels. The findings are relevant to the field, and the methods and results are of good quality, with some improvements in interpretation and explanation required, and some minor experimental suggestions.

Strengths:

The article is solid in terms of the novelty of the findings and relevance for the physiology of myelinated axons. In addition, it is of major interest for the Connexin field because it explores a physiological way to open Cx32 hemichannels. The experiments are well elaborated, and most of them are sufficient for the main points described by the authors. The finding that nervous activity will trigger the mechanism of hemichannel opening by CO2 is probably the most relevant biological mechanism derived from this article.

Weaknesses:

Throughout the manuscript, the authors interpret their findings as if the described mechanism specifically occurs in the node and paranode regions. However, there is no direct evidence identifying the precise site of CO₂ production or the activation site of Cx32 hemichannels. Therefore, statements such as the one in the title ("activity-dependent CO₂ production in the axonal node opens Cx32 in the Schwann cell paranode") should be reconsidered or removed, as they may be misleading and are not essential to the interpretation of the data. In addition, the participation of aquaporin AQP1 as the main conduit for CO2 diffusion through the plasma membrane could have another interpretation.

We thank the reviewer for their comments and agree that we do not have direct evidence for the site of CO2 production or the site of activation of Cx32 hemichannels. This direct evidence is extremely difficult to obtain, and we therefore depend on indirect arguments. Mitochondria represent the major source of CO2, and their distribution will therefore indicate where CO2 is likely to be produced. We agree that this is not essential to the interpretation of the data and will adjust the text as recommended. We will add a section to the Discussion to consider this point in more detail.