Nociceptors use multiple neurotransmitters to drive pain

  1. Donald Iain MacDonald
  2. Rakshita Balaji
  3. Alexander T. Chesler

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Abstract

Nociceptors are excitatory neurons that express a range of neuropeptides and have the essential role of detecting noxious mechanical, thermal and chemical stimuli. Ablating these neurons profoundly reduces responses to pain. Here we investigated how nociceptive information is transmitted by developing genetic approaches to suppress glutamate transmission and neuropeptide signaling, individually and in combination. Remarkably, many pain responses persisted in mice where either nociceptor glutamate or neuropeptide signaling was blocked. By contrast, mice lacking both glutamate and neuropeptide transmission in nociceptors displayed profound pain insensitivity closely matching the effects of cell ablation. Together our results establish a role for neuropeptides as bone fide pain transmitters and demonstrate redundancy in nociceptor signaling, resolving long-standing questions about how pain is communicated to the brain.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17991818.

    Proper relay of pain sensation to the brain is critical for health and survival, yet how this is achieved at the molecular levels remains less understood. In this study, McDonald et al. sought to resolve the contributions of glutamate and neuropeptides in transmitting pain signals in mice, which has remained elusive in the field due to limited tools for parsing out the contributions of each. This study employed mouse genetics tools to selectively impair the release of glutamate, neuropeptides, or both, and utilized behavioral assays to assess pain transmission. In doing so, the study drew three major claims:

    (1)     Loss of glutamate transmission from nociceptors does not abolish pain response. The authors generated mice lacking VGLUT2 in Trpv1+ lineage. These mice still showed behavioral responses to heat, cold, and chemical pain stimuli, while Trpv1+ lineage ablation abolished these responses, consistent with previous findings.

    (2)     Loss of neuropeptide signaling from nociceptors does not abolish pain response. The authors employed a strategy to suppress neuropeptide signaling by knocking out PAM, an enzyme required for functionalizing some of the peptides present in nociceptors. Mice lacking PAM or PACAP in Trpv1+ lineage still showed an overall normal behavioral response to painful stimuli.

    (3)     Blocking both glutamate and neuropeptide signaling from nociceptors abolishes pain response. The authors then knocked out both VGLUT2 and PAM in Trpv1+ lineage. This resulted in profound reduction in pain response comparable to that achieved with Trpv1 ablation.

    This elegant study convincingly demonstrates that Trpv1+ cells require both glutamate and neuropeptides to elicit pain response. This has important clinical implications for treatment of pain and highlights how degeneracy of transmitter systems may be employed to relay this critical sensation. Addressing a few remaining questions, however, would significantly strengthen the authors' conclusion that neuropeptides are bona fide pain transmitters and that multiple neurotransmitters drive pain.

    Major points:

    (1)     The study seeks to establish that neuropeptides are bona fide pain transmitters, yet based on the data presented, it still seems possible that neurotransmitters contribute more indirectly (e.g. allodynia) to enhance the behavioral response of Vglut2 KO mice, rather than by being co-released with glutamate as suggested in the text. To further support this claim, could the authors present more direct evidence of co-release of glutamate and neuropeptides in the central terminals, e.g. electron microscopy in the dorsal horn, or address this possibility in the discussion?

    (2)     For knocking out PAM, 3 different Cre drivers were used interchangeably. Hoxb8-Cre was used to validate loss of CGRP and SP expression in the DRG and dorsal horn (Figure 2), Avil1-Cre was used for the cell based "sniffer" assay (Figure 2), and Trpv1-Cre was used for assessing behavioral pain response (Figure 4). Of note, knocking out PAM under Trpv1-Cre did not cause the robust reduction of CGRP and SP (Figure S7) as shown under Hoxb8-Cre, suggesting incomplete penetrance and/or presence of CGRP and SP in non-nociceptor cells. Therefore, in order to fully support the conclusion made from Figure 4, the authors should consider examining a behavioral response after knocking out PAM under Hoxb8 and/or Avil1-Cre.

    (3)     Knocking out Vglut2 under Trpv1 had a limited effect on pain behavior (Figure 1), but the efficacy of Vglut2 deletion has not been demonstrated. The conclusion to Figure 1 would be strengthened by supporting data like the immunostaining used to assess Vglut2 expression in the dorsal horn for PAM KO under Hoxb8-Cre.

    Minor points:

    (1)     For clarity, figure legends could specify which Cre drivers were used, especially since multiple Cre drivers were used to knock out PAM across Figures 2-4.

    (2)     For Figure 3, axon flare response is diminished but not abolished. Can the authors further elaborate on whether this was expected in the discussion, or if it is still possible that PAM does not completely remove the intended neuropeptides?

    (3)     It is unclear which Cre line was used for nociceptor knockout of PACAP, although the reader is led to assume that Trpv1 was used. Can the authors clarify this in the text and figure legends?

    (4)     To better support conclusions from microscopic images, the authors can quantify immunofluorescence images. Also, can the authors check that representative images used reflect the mean fluorescence signals?

    Competing interests

    The author declares that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The author declares that they did not use generative AI to come up with new ideas for their review.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17992410.

    Proper relay of pain sensation to the brain is critical for health and survival, yet how this is achieved at the molecular levels remains less understood. In this study, McDonald et al. sought to resolve the contributions of glutamate and neuropeptides in transmitting pain signals in mice, which has remained elusive in the field due to limited tools for parsing out the contributions of each. This study employed mouse genetics tools to selectively impair the release of glutamate, neuropeptides, or both, and utilized behavioral assays to assess pain transmission. In doing so, the study drew three major claims:

    (1)     Loss of glutamate transmission from nociceptors does not abolish pain response. The authors generated mice lacking VGLUT2 in Trpv1+ lineage. These mice still showed behavioral responses to heat, cold, and chemical pain stimuli, while Trpv1+ lineage ablation abolished these responses, consistent with previous findings.

    (2)     Loss of neuropeptide signaling from nociceptors does not abolish pain response. The authors employed a strategy to suppress neuropeptide signaling by knocking out PAM, an enzyme required for functionalizing some of the peptides present in nociceptors. Mice lacking PAM or PACAP in Trpv1+ lineage still showed an overall normal behavioral response to painful stimuli.

    (3)     Blocking both glutamate and neuropeptide signaling from nociceptors abolishes pain response. The authors then knocked out both VGLUT2 and PAM in Trpv1+ lineage. This resulted in profound reduction in pain response comparable to that achieved with Trpv1 ablation.

    This elegant study convincingly demonstrates that Trpv1+ cells require both glutamate and neuropeptides to elicit pain response. This has important clinical implications for treatment of pain and highlights how degeneracy of transmitter systems may be employed to relay this critical sensation. Addressing a few remaining questions, however, would significantly strengthen the authors' conclusion that neuropeptides are bona fide pain transmitters and that multiple neurotransmitters drive pain.

    Major points:

    (1)     The study seeks to establish that neuropeptides are bona fide pain transmitters, yet based on the data presented, it still seems possible that neurotransmitters contribute more indirectly (e.g. allodynia) to enhance the behavioral response of Vglut2 KO mice, rather than by being co-released with glutamate as suggested in the text. To further support this claim, could the authors present more direct evidence of co-release of glutamate and neuropeptides in the central terminals, e.g. electron microscopy in the dorsal horn, or address this possibility in the discussion?

    (2)     For knocking out PAM, 3 different Cre drivers were used interchangeably. Hoxb8-Cre was used to validate loss of CGRP and SP expression in the DRG and dorsal horn (Figure 2), Avil1-Cre was used for the cell based "sniffer" assay (Figure 2), and Trpv1-Cre was used for assessing behavioral pain response (Figure 4). Of note, knocking out PAM under Trpv1-Cre did not cause the robust reduction of CGRP and SP (Figure S7) as shown under Hoxb8-Cre, suggesting incomplete penetrance and/or presence of CGRP and SP in non-nociceptor cells. Therefore, in order to fully support the conclusion made from Figure 4, the authors should consider examining a behavioral response after knocking out PAM under Hoxb8 and/or Avil1-Cre.

    (3)     Knocking out Vglut2 under Trpv1 had a limited effect on pain behavior (Figure 1), but the efficacy of Vglut2 deletion has not been demonstrated. The conclusion to Figure 1 would be strengthened by supporting data like the immunostaining used to assess Vglut2 expression in the dorsal horn for PAM KO under Hoxb8-Cre.

    Minor points:

    (1)     For clarity, figure legends could specify which Cre drivers were used, especially since multiple Cre drivers were used to knock out PAM across Figures 2-4.

    (2)     For Figure 3, axon flare response is diminished but not abolished. Can the authors further elaborate on whether this was expected in the discussion, or if it is still possible that PAM does not completely remove the intended neuropeptides?

    (3)     It is unclear which Cre line was used for nociceptor knockout of PACAP, although the reader is led to assume that Trpv1 was used. Can the authors clarify this in the text and figure legends?

    (4)     To better support conclusions from microscopic images, the authors can quantify immunofluorescence images. Also, can the authors check that representative images used reflect the mean fluorescence signals?

    Competing interests

    The author declares that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The author declares that they did not use generative AI to come up with new ideas for their review.

  3. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17992531.

    MacDonald, Balaji, and Chesler investigated molecular mechanisms of pain using mouse genetics, behavior, and cell-based assays. The authors examined a fundamental question in the field: what signaling molecules are necessary for peripheral afferents to transform noxious sensory inputs into pain behavior? They report pain behavior is intact in mice with genetic blockade of glutamate and C-terminal amidated neuropeptide packaging using conditional deletion of Slc17a6 (Vglut2) and Pam, respectively. Pain behaviors were absent after targeting both alleles in TRPV1 neurons, similar to mice lacking Trpv1-Cre cells via DTA-mediated ablation. Overall, the findings present strong evidence to support the authors' model that combined loss of glutamate signaling and a major class of neuropeptides in Trpv1 neurons are critical to blocking pain behavior. A few clarifying experiments, analysis, and minor text revisions would improve the coherence of the overall model and further strengthen the manuscript.

    Major Comments

    1.     Mechanical allodynia also contributes to pain behavior and can result from axonal flares. Rather than capsaicin, the authors use mustard oil, a classical Trpa1 agonist, to experimentally evoke axonal flares (Fig. 3), and Trpa1 is expressed by many non-neuronal cells. Thus, non-Trpv1 afferents or cells may evoke allodynia-mediated pain behavior through a parallel pathway, effectively 'compensating' for loss of signaling mechanisms in other nociceptors. Is the residual neurogenic inflammation in PAM cKO (Fig 3) sufficient to elicit mechanical allodynia and elicit pain behavior? Is the axonal flare reflex present in VGLUT2 KO or PAM/VGLUT2 DKO mice? Additional experiments testing mechanical allodynia would help clarify the relation between physiological and behavioral consequences of activating potentially distinct neuronal pathways.

    2.     Deletion of Pam targets a major class of neuropeptides, and the authors thoughtfully noted not all neuropeptides are targeted by this genetic approach. Expression analysis shows a major population of Trpv1-expressing "NP3" neurons express Pam-independent neuropeptides (Fig S1), where "loss of function" is only captured by Trpv1-DTA mice. Of the three major signaling modalities noted (Vglut2, Pam-dependent, and Pam-independent), is it possible any two classes are sufficient? As suggested in the Discussion, do the NP3 neurons only play a role in itch and not pain? The authors could elaborate further on the role and relations of NP3 neurons, Pam-independent neuropeptides, and self-wounding behaviors. Certain phrases throughout the manuscript imply all neuropeptide signaling is blocked by Pam deletion (Results subtitles, title Fig 4) and may need to be carefully considered by the authors – could quantification of self-wounding or additional manipulations be added to help clarify this?

    3.     Trpv1-marked spinal cord interneurons are also proposed to mediate pain, and Trpv1-Cre may be lineage expressed in other neurons or cell types. As noted in the Discussion, loss of function in key spinal neurons, rather than peripheral afferents, could also explain the observed phenotypes but this is not examined directly. Can the authors use additional analysis, histology, or genetic tools to examine targeting specificity of Trpv1 (or PAM and glutamate packaging) in DRGs, spinal cord neurons, or both?

    Minor

    1.     The authors use a robust battery of biosensor cell lines to study capsaicin-evoked neuropeptide release and validate their genetic loss-of-function tools. Although VGLUT2 is a major glutamate transporter in sensory afferents, other transporters from the VGLUT or EAAT protein families may contribute to glutamate release in some neurons. Could glutamate biosensors like GluSnFR also confirm glutamate release is abolished in Vglut2 cKO mice? Based on the authors' transcriptomic data (Fig S6), is there any evidence for expression of alternate glutamate transporters?

    2.     The authors conclude "nociceptors developed normally" after constitutive PAM deletion. Image quantification is not shown and representative images may not be of sufficient resolution to examine projection neuroanatomy (Fig 2C, Fig S7). Constitutive deletion of PAM or VGLUT2 may also shape activity-dependent development or maintenance of DRG afferent terminals, thereby leading to abnormal morphology and sensory properties (PMID: 40381613). Could the authors add additional images and quantification?

    3.     "PAM cKO" is used interchangeably from different Cre mouselines. To help with clarity, the authors could note the Cre line used in each figure or legend (e.g. present in Fig 2, but not Fig 3).

    4.     Additional details describing quantification in Legends (e.g. Fig 2C-G) or Methods, including whether data shown depicts technical/biological replicates, number of animals, and independent experiments, would strengthen the rigorous experimentation.

    5.     Some text labels on immunofluorescence images are small and challenging to identify, especially where they overlap the image (e.g. Fig 2F, magenta labels). Labels could be moved to improve readability.

    6.     Fig 4 legend appears to contain typos: Figure depicts A-L, but legend repeats "A-F." Please review for accurate in-text references.

    Competing interests

    The author declares that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The author declares that they did not use generative AI to come up with new ideas for their review.

  4. Version published to 10.1101/2025.09.09.675093 on bioRxiv