Probes for TDTOMATO

The D2 dopamine receptor (Drd2) is implicated in several brain disorders such as schizophrenia, Parkinson's disease, and drug addiction. Drd2 is also the primary target of both antipsychotics and Parkinson's disease medications. Although the expression pattern of Drd2 is relatively well known in mouse brain, the temporal and spatial distribution of Drd2 is lesser clear in rat brain due to the lack of Drd2 reporter rat lines. Here, we used CRISPR/Cas9 techniques to generate two knockin rat lines: Drd2::Cre and Rosa26::loxp-stop-loxp-tdTomato. By crossing these two lines, we produced Drd2 reporter rats expressing the fluorescence protein tdTomato under the control of the endogenous Drd2 promoter. Using fluorescence imaging and unbiased stereology, we revealed the cellular expression pattern of Drd2 in adult and postnatal rat forebrain. Strikingly, the Drd2 expression pattern differs between Drd2 reporter rats and Drd2 reporter mice generated by BAC transgene in prefrontal cortex and hippocampus. These results provide fundamental information needed for the study of Drd2 function in rat forebrain. The Drd2::Cre rats generated here may represent a useful tool to study the function of neuronal populations expressing Drd2.

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.

Background Grooming dysfunction is a hallmark of the obsessive-compulsive spectrum disorder, trichotillomania. Numerous preclinical studies have utilized SAPAP3 deficient mice for understanding the neurobiology of repetitive grooming, suggesting excessive grooming is caused by increased metabotropic glutamate receptor 5 (mGluR5) activity in striatal direct- and indirect pathway medium spiny neurons (dMSNs and iMSNs, respectively). However, MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Here, we investigate the MSN subtype-specific roles of the striatal signaling hub protein, spinophilin, in mediating repetitive motor dysfunction associated with mGluR5 function. Methods Quantitative proteomics and immunoblotting were utilized to identify how spinophilin impacts mGluR5 phosphorylation and protein interaction changes. Plasticity and repetitive motor dysfunction associated with mGluR5 action was measured using our novel conditional spinophilin mouse model that had spinophilin knocked out from striatal dMSNs or/and iMSNs. Results Loss of spinophilin only in iMSNs decreased performance of a novel motor repertoire, but loss of spinophilin in either MSN subtype abrogated striatal plasticity associated with mGluR5 function and prevented excessive grooming caused by SAPAP3 knockout mice or treatment with the mGluR5-specific positive allosteric modulator (VU0360172) without impacting locomotion-relevant behavior. Biochemically, we determined the spinophilin-mGluR5 interaction correlates with grooming behavior and loss of spinophilin shifts mGluR5 interactions from lipid-raft associated proteins toward postsynaptic density (PSD) proteins implicated in psychiatric disorders. Conclusions These results identify spinophilin as a novel striatal signaling hub molecule in MSNs that cell subtype-specifically mediates behavioral, functional, and molecular adaptations associated with repetitive motor dysfunction in psychiatric disorders.

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.