Background <p>The insular cortex (IC), a pivotal hub for pain signal integration, projects extensively to the thalamus. However, the functional role of its specific projection to the central medial thalamic nucleus (CM) in pain-affective comorbidities remains elusive. Elucidating the IC-CM pathway is thus critical for revealing endogenous pain regulation and pain-emotion comorbidity mechanisms.</p> Methods <p>A multidisciplinary approach combining neuroanatomical tracing, calcium imaging, electrophysiological recordings, optogenetics, chemogenetics and behavioral observations was used to investigate the role of the IC-CM pathway in pain and emotional modulation with the spared nerve injury (SNI) mouse model. The imaging and 3D visualization methods were also utilized to observe the projection patterns of IC-CM pathway neurons.</p> Results <p>Neuropathic pain activated neurons, predominantly excitatory neurons, in the deep layers (L5-6) of the IC (dlIC) and rostral CM (rCM) in SNI mice. The vesicular glutamate transporter type 2-immunoreactive (VGluT2⁺) neurons in the dlIC project to the calcium/calmodulin-dependent protein kinase IIα-immunoreactive (CaMKIIα⁺) neurons in the rCM, forming a functional pathway for nociceptive information transmission. Fiber photometry and electrophysiological recordings revealed the enhanced excitability in the dlIC-rCM pathway in response to noxious stimuli, confirming its involvement in pain signal transmission. Chemogenetic and optogenetic activation of the dlIC^VGluT2-rCM^CaMKIIα neural pathway significantly reduced mechanical and thermal pain thresholds and evoked anxiety-like behaviors in normal mice. Conversely, inhibition of this pathway alleviated mechanical and thermal hypersensitivity and anxiety-like behaviors in SNI mice. Single-neuron projection pattern analyses revealed 2 distinct axonal arborization phenotypes among dlIC neurons forming synapses with CM^CaMKIIα neurons.</p> Conclusion <p>Collectively, these findings identify the dlIC^VGluT2-rCM^CaMKIIα pathway as a contributor to pain-anxiety comorbidity in mice. The observed distinct axonal arborization phenotypes provide morphological evidence suggesting local integration within the rCM and downstream propagation of nociceptive signals. These results advance our understanding of cortico-thalamic mechanisms in central sensitization and suggest this pathway as a candidate for therapeutic intervention in chronic pain states.</p> Clinical trial number <p>Not applicable.</p>