Visceral pain is a key feature of functional gastrointestinal (GI) disorders such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and obstructive bowel disorders (OBD). Notably, there is also a high comorbidity of chronic visceral pain with stress-related psychiatric disorders including anxiety, depression, and fatigue, significantly impairing patients' quality of life. However, the molecular and circuit mechanisms of visceral pain are poorly understood, and the resulting lack of effective clinical management represents a major unmet medical problem. Pilot studies showed that activation of the glutamatergic Tacr3+ neurons in the spinal cord dorsal horn (SDH) is sufficient to drive both sensory discriminative (visceromotor response) and affective (aversion) visceral pain responses. Additionally, the parabrachial nucleus of the pons (PBN) in the brainstem had the most abundant projections of the Tacr3+ SDH neurons. These observations led us to frame the hypothesis that the glutamatergic Tacr3+ SDH neurons are excitatory interneurons and projection neurons mediating visceral pain processing from the mouse colon to the brainstem. The objective of this R56 is to elucidate whether the neural circuits from the colon-innervating spinal visceral afferents form functional synaptic connections with the Tacr3+ SDH neurons and dissect the function of Tacr3+ spinal projections in the brainstem. We will demonstrate for the first time that the glutamatergic Tacr3+ neurons receive synaptic inputs from the colon-innervating spinal visceral afferents, and stimulation of the colon-innervating spinal afferents is sufficient to activate the Tacr3+ SDH neurons. We will also investigate whether activating the Tacr3+ spinal projections in the PBN drives aversive behavior. The outcomes of these studies will advance our understanding of the cellular and circuit mechanisms of visceral sensory processing in the lumbosacral