Background & Aims

Noxious stimuli are received by the superficial dorsal horn (DH) but sensory afferents conveying innocuous information mostly terminate in the deep DH. Under certain conditions, innocuous mechanosensory information can be perceived as painful (i.e. allodynia). Parvalbumin (PV) neurons have been shown to function as gate keepers preventing allodynia. However, more recently we have shown that ablating the inhibitory PV neurons results not only in allodynia, but also in increased biting, which is an aversive behaviour indicative of pruritus. A similar phenotype was found when spinal Gbx1 neurons were ablated. We found a significant overlap between inhibitory spinal interneurons expressing Gbx1 and PV and are now investigating whether Gbx1;PV positive subset of DH neurons may specifically be involved in suppressing itch evoked by innocuous mechanical input and are therefore critical to prevent mechanical or spontaneous pruritus.

Methods

We used intersectional genetics and AAV mediated gene delivery to silence spinal Gbx1;PV neurons and dissect if and how they modulate neuronal activity and behaviour. To specifically address the function of Gbx1;PV neurons in processing innocuous peripheral input we expressed ChR2-YFP in non-nociceptive fibres employing an AAV based cre-off system. Combining this approach with silencing Gbx1;PV neurons we studied light induced place aversion. To analyse non-nociceptive fibre mediated activity in DH neurons with or without ablation of Gbx1;PV cells we expressed ChR2-YFP in non-nociceptive fibres and an unconditional red shifted calcium indicator (RCamP) in DH neurons. Lastly, we tested whether non-nociceptive fibres and/or the Gbx1;PV neurons are directly connected to crucial neuronal components of known DH itch pathways (i.e. Grp and GRPR neurons). To this end we labelled spinal terminals of Gbx1;PV neurons and LTMRs/proprioceptor terminals and analysed contacts on Grp and GRPR neurons

Results

Ablation of the Gbx1;PV double positive subset of spinal interneurons resulted in significantly increased biting behaviors as seen after ablation of either Gbx1 or PV neurons but did not result in allodynia. Blue light stimulation of ChR2-YFP expressing non-nociceptive fibres together with ablation of the Gbx1;PV neurons induced light evoked place aversion. Using calcium imaging in the DH, we observed a trend of increased LTMR/proprioceptor evoked activity in the superficial DH after ablation of Gbx1;PV neurons. Analysis of sections with labelled GRPR neurons, Gbx1;PV terminals and LTMRs/proprioceptor terminals suggests that GRPR neurons are vertical cells extending their dendrites into the deep dorsal horn where they form connections with vGluT1 labelled non-nociceptive primary afferents and receive inhibitory input from Gbx1;PV neurons.

Conclusions

Loss of Gbx1;PV double spinal interneurons increases the aversiveness to stimulating non-nociceptive peripheral fibres. The fact that biting behaviour increases but little/no mechanical hypersensitivity or allodynia was observed after ablation of spinal Gbx1;PV neurons suggests that they are required to prevent mechanical or spontaneous pruritus and not pain. Our preliminary immunohistochemical circuit analysis suggests that Gbx1;PV neurons may act via inhibiting mechanoreceptive input onto GRPR neurons.

References

Takazawa T, MacDermott AB. Synaptic pathways and inhibitory gates in the spinal cord dorsal horn. Ann N Y Acad Sci. 2010 Jun;1198:153-8
Foster E, et al. Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch. Neuron. 2015 Mar 18;85(6):1289-304
Petitjean H, et al. Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury. Cell Rep. 2015 Nov 10;13(6):1246-1257
Boyle KA, et al. Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia. Cell Rep. 2019 Jul 9;28(2):526-540.e6
Peirs C, et al. Mechanical Allodynia Circuitry in the Dorsal Horn Is Defined by the Nature of the Injury. Neuron. 2021 Jan 6;109(1):73-90.e7
Frezel N, et al. c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy. Cell Rep. 2023 Apr 25;42(4):112295

Presenting Author

Raquel Mendes

Poster Authors

Raquel Mendes

MSc

University of Zurich

Lead Author

Robin Portmann

MSc

Lead Author

Simon D'Aquin

PhD

Lead Author

Hendrik Wildner

PhD

University of Zurich, Institute of Pharmacology and Toxicology

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology