PREreview of Nociceptors use multiple neurotransmitters to drive pain

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.