Probes for TDTOMATO

Glucagon-like peptide-1 receptor (GLP-1R) activation is used in the treatment of diabetes and obesity; however, GLP-1 induces many other physiological effects with unclear mechanisms of action. To identify the cellular targets of GLP-1 and GLP-1 analogues, we generated a Glp1r.tdTomato reporter mouse expressing the reporter protein, tdTomato, in Glp1r-expressing cells. The reporter signal is expressed in all cells where GLP-1R promoter was ever active. To complement this, we histologically mapped tdTomato-fluorescence, and performed Glp-1r mRNA in situ hybridization and GLP-1R immunohistochemistry on the same tissues. In male mice, we found tdTomato signal in mucus neck, chief, and parietal cells of the stomach; Brunner's glands; small intestinal enteroendocrine cells and intraepithelial lymphocytes; and myenteric plexus nerve fibers throughout the gastrointestinal tract. Pancreatic acinar-, β-, and δ cells, but rarely α cells, were tdTomato-positive, as were renal arteriolar smooth muscle cells; endothelial cells of the liver, portal vein, and endocardium; aortal tunica media; and lung type 1 and type 2 pneumocytes. Some thyroid follicular and parafollicular cells displayed tdTomato expression, as did tracheal cartilage chondrocytes, skin fibroblasts, and sublingual gland mucus cells. In conclusion, our reporter mouse is a powerful tool for mapping known and novel sites of GLP-1R expression in the mouse, thus enhancing our understanding of the many target cells and effects of GLP-1 and GLP-1R agonists.

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.

Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival1-4. Vagal sensory neurons (VSNs) form an important body-to-brain connection, navigating visceral organs along the rostral-caudal axis of the body and crossing the surface-lumen axis of organs into appropriate tissue layers5,6. The brain can discriminate numerous body signals through VSNs, but the underlying coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer and stimulus modality-three key features of an interoceptive signal-in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs in mice using multiplexed projection barcodes reveals a 'visceral organ' dimension composed of differentially expressed gene modules that code organs along the body's rostral-caudal axis. We discover another 'tissue layer' dimension with gene modules that code the locations of VSN endings along the surface-lumen axis of organs. Using calcium-imaging-guided spatial transcriptomics, we show that VSNs are organized into functional units to sense similar stimuli across organs and tissue layers; this constitutes a third 'stimulus modality' dimension. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial manner and facilitate the complex projection of VSNs in the brainstem. Our study highlights a multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.

Targeting specific opioid receptor types in distinct sensory neurons could lead to safer and more effective treatments against pain. However, the extent to which different DRG neurons that express opioid receptors (MOR, DOR, KOR) innervate distinct organs, and what sensory information is encoded by these neurons, represent long-standing questions in the field. To fill this knowledge gap, we utilized novel knock-in mouse lines in which the DNA recombinases Cre and/or Flp are expressed in opioid receptor-positive DRG neurons. We injected adeno-associated viruses to express tdTomato and analyzed the organization of DRG axon terminals in peripheral tissues using tissue clearing and immunostaining protocols. In hairy skin, we observed circumferential nerve endings around hair follicles that are either MOR+ or DOR+. However, DOR+ circumferential endings were also NFH+ whereas MOR+ circumferential endings were not, suggesting that MOR is expressed by high-threshold mechanoreceptors, while DOR is expressed by low-threshold mechanoreceptors activated by stroking of the skin. In glabrous skin, we found a similar divergent organization, with MOR+ and DOR+ axon terminals co-expressing CRGP and NFH, respectively. In the colon, we observed innervation by both KOR+ and MOR+ axons whereas, in the muscle (soleus) and kidney, we found axons that are either MOR+, DOR+, or KOR+. Remarkably, these MOR+, DOR+, or KOR+ axons innervate different sub-regions within these organs and form distinct nerve-ending structures. Collectively, our findings show that MOR+, DOR+, and KOR+ DRG neurons are expressed in largely non-overlapping DRG neuron types that distinctly innervate tissues and presumably differently contribute to sensory perception. National Institutes of Health grant R01DA044481 New York Stem Cell Foundation.