31: Coding Properties of Spinal Projection Neurons in Awake, Behaving Mice Before and After Nerve Injury

Topic: Basic Science
Subtopic: Pain category (ICD-11), Chronic neuropathic pain, , , , , , , , , , Assessment and Measurement of Pain/Nociception, Animal imaging (includes Ca2+ imaging, fluorescence microscopy), Behavioral studies

Background and Aims: In acute and chronic neuropathic pain settings, spinal cord dorsal horn projection neurons (SCPNs) are key conduits of peripheral and central sensitization, transmitting nociceptive signals to the brain, to generate pain percepts. Here, by monitoring the activity of SCPNs, before and after chronic pain develops, in awake, behaving mice, we address mechanisms underlying the transition from acute to chronic pain. Our studies expand on previous reports characterizing Phox2a neuron coding properties in naïve states (Zhang et al, 2026) and in anesthetized preparations following nerve injury (Yarmolinsky et al, 2025).

Methods: To study the properties of SCPNs, as chronic pain develops, we took advantage of our recent methodological advances (Ahanonu et al, 2024) that allow long-term monitoring of the same population of SCPNs in awake, behaving mice. We used calcium imaging to longitudinally record the activity of genetically defined lamina I SCPNs (using Phox2a-Cre floxed-GCaMP6 mice), during development of the neuropathic pain phenotype in the sural-spared nerve injury (SNI) model. We performed comprehensive multimodal sensory testing during imaging, assessing mechanical and thermal responses at baseline, and at 4-6 hours, and 1, 3, 7, and 21 days after SNI.

Results: We find that, in awake mice, SCPNs are predominantly polymodal compared to considerable labeled line processing in the anesthetized state. Also in contrast to the anesthetized state, SCPNs recorded in the awake state exhibit increased spontaneous and movement-related activity, pointing to an additional contribution of locomotion to SCPN activity. Importantly, prior to injury, single-neuron response profiles were stable over months. Our initial conclusion is that SNI-induced mechanical allodynia and cold hypersensitivity are driven by a sustained population increase in polymodal responsiveness, recruitment of previously silent SCPNs, and elevated spontaneous activity.

Conclusions: By monitoring the activity of the same population of neurons, before and after peripheral nerve injury, we are now addressing longstanding questions about the mechanisms underlying the transition from acute to chronic pain, in the awake, behaving mouse. Of particular interest is our plan to assess the pharmacodynamics of existing and novel analgesics on the activity of these projection neurons during chronic neuropathic pain development.

References: Ahanonu, B., Crowther, A., Kania, A., Rosa-Casillas, M., & Basbaum, A. I. (2024). Long-term optical imaging of the spinal cord in awake behaving mice. Nature Methods, 21, 2363–2375.
Yarmolinsky, D. A., Zeng, X., MacKinnon-Booth, N., Cheng, Y.-T., Turnes, B. L., & Woolf, C. J. (2025). Differential modification of ascending spinal outputs in acute and chronic pain states. Neuron, 113(8), 1223–1239.
Zhang, X., Bourojeni, F., Ferland, S., McFarlane, A., et al. (2026). A neuronal pathway for the supraspinal relay of warm and cool sensation. bioRxiv.

Presenting Author: Mariela Rosa Casillas
Submitting Author: Mariela Rosa Casillas

Senior Author: Allan Basbaum

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• Mariela Rosa Casillas - University of California San Francisco
• Andrew Crowther - Virginia Commonwealth University
• Biafra Ahanonu - Stanford University
• Allan Basbaum - University of California San Francisco