Distinct calcium responses distinguish itch- and pain-associated neurons in the anterior cingulate cortex

Itch and pain are distinct aversive sensations. We previously identified segregated neuronal populations in the anterior cingulate cortex (ACC) that selectively respond to pain and itch stimuli and reported full-trace classification with a mean-frequency difference, leaving the broader calcium-transient properties associated with this separation unresolved. Here, we reanalyzed denoised in vivo calcium traces from stimulus-responsive ACC neurons and quantified interpretable response features, including peak rate, peak interval, rise and decay area under the curve (AUC), and rise and decay slope. Pain- and itch-associated neurons differed across multiple single-neuron calcium features. Trace-based stochastic gradient descent (SGD) classification separated the two groups, indicating that their response profiles contain label-discriminative information. Notably, classification performance was only weakly affected by neuron-wise z -scoring, suggesting that separation is not explained solely by simple amplitude scaling. A mouse-constrained shuffled-label control reduced classification performance, supporting the contribution of within-mouse label-associated structure while also highlighting mouse-level sampling constraints. Paired mouse summaries and mouse-exclusive validation showed limited cross-animal consistency, whereas within-mouse neighborhood analysis retained local label-associated organization. UMAP of full traces provided descriptive visualization of neuron-level response organization. Together, these results show that interpretable calcium response features carry meaningful itch−/pain-associated structure among the analyzed responsive ACC neurons.