Anatomical and molecular organization of the nociceptive medial thalamus

Abstract The medial thalamus (MTh) is a critical hub for integrating the affective and motivational dimensions of pain, yet its cellular and circuit organization remains poorly defined. Here, we combine activity-dependent genetic tagging, whole-brain clearing and light-sheet imaging, single-cell transcriptomics, and viral circuit tracing to build a comprehensive anatomical and molecular atlas of nociceptive and opioidergic neurons within the MTh. We show that acute and chronic pain activate distinct, spatially localized microdomains across multiple MTh subnuclei, including the central medial, interanteromedial, mediodorsal, and xiphoid nuclei. Activity-dependent tagging reveals a stable ensemble of nociceptive neurons that remains engaged across acute and chronic pain states, suggesting persistent thalamic encoding of affective nociception. Using fluorescent in situ hybridization, we find that most nociceptive-activated neurons express Oprm1, and that nearly all Oprm1+ neurons are activated by nociceptive stimuli, identifying a pain-active, µ-opioid receptor-expressing population in MTh (MThMOR). Rabies-based tracing demonstrates that MThMOR neurons receive convergent inputs from nociceptive and inhibitory regions including the anterior cingulate cortex, periaqueductal gray, parabrachial nucleus, and habenula, while projecting broadly to cortical, subcortical, and brainstem structures involved in pain, arousal, and motivation. Enkephalinergic inputs to MTh arise from distinct, non-nociceptive populations, suggesting parallel pathways for nociceptive transmission and endogenous opioid modulation. Together, these findings define the anatomical and molecular organization of the nociceptive medial thalamus and highlight MThMOR neurons as a cell-type-specific substrate for the development of targeted opioid-based pain therapies. Competing Interest Statement The authors have declared no competing interest.