PREreview of Nociceptors use multiple neurotransmitters to drive pain

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.