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The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons., Competing Interests: Competing interests statement:The authors declare no competing interest.

Allodynia is a state in which pain is elicited by innocuous stimuli. Capsaicin applied to the skin results in an allodynia that extends to a broad region beyond the application site. This sensitization is thought to be mediated by spinal networks; however, we do not have a clear picture of which spinal neurons mediate this phenomenon. To address this gap, we used two-photon calcium imaging of excitatory interneurons and spinal projection neurons in the mouse spinal dorsal horn. To distinguish among neuronal subtypes, we developed CICADA, a cell profiling approach to identify cell types during calcium imaging. We then identified capsaicin-responsive and capsaicin-sensitized neuronal populations. Capsaicin-sensitized neurons showed emergent responses to innocuous input and increased receptive field sizes consistent with psychophysical reports. Finally, we identified spinal output neurons that showed enhanced responses from innocuous input. These experiments provide a population-level view of central sensitization and a framework with which to model somatosensory integration in the dorsal horn., (© 2022. The Author(s).)