Probes for INS

Brain-derived 17β-estradiol (E2) confers rapid effects on neural activity. The tubular striatum (TuS, also called the olfactory tubercle) is both capable of local E2 synthesis due to its abundant expression of aromatase and is a critical locus for odor-guided motivated behavior and odor hedonics. TuS neurons also contain mRNA for estrogen receptors α, β, and the G protein-coupled estrogen receptor. We demonstrate here that mRNA for estrogen receptors appears to be expressed upon TuS dopamine 1 receptor-expressing neurons, suggesting that E2 may play a neuromodulatory role in circuits which are important for motivated behavior. Therefore, we reasoned that E2 in the TuS may influence attraction to urinary odors which are highly attractive. Using whole-body plethysmography, we examined odor-evoked high-frequency sniffing as a measure of odor attaction. Bilateral infusion of the aromatase inhibitor letrozole into the TuS of gonadectomized female adult mice induced a resistance to habituation over successive trials in their investigatory sniffing for female mouse urinary odors, indicative of an enhanced attraction. All males displayed resistance to habituation for female urinary odors, indicative of enhanced attraction that is independent from E2 manipulation. Letrozole's effects were not due to group differences in basal respiration, nor changes in the ability to detect or discriminate between odors (both monomolecular odorants and urinary odors). Therefore, de novo E2 synthesis in the TuS impacts females' but not males' attraction to female urinary odors, suggesting a sex-specific influence of E2 in odor hedonics.

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.

We recently developed a mouse model of appetitive operant aggression and reported that adult male outbred CD-1 mice lever-press for the opportunity to attack subordinate male mice and relapse to aggression seeking during abstinence. Here we studied the role of nucleus accumbens (NAc) dopamine D1- and D2-receptor (Drd1 and Drd2) expressing neurons in aggression self-administration and aggression seeking. We trained CD-1 mice to self-administer intruders (9 d, 12 trials/d) and tested them for aggression self-administration and aggression seeking on abstinence day 1. We used immunohistochemistry and in situ hybridization to measure the neuronal activity marker Fos in the NAc, and cell-type specific colocalization of Fos with Drd1- and Drd2-expressing neurons. To test the causal role of Drd1- and Drd2-expressing neurons, we validated a transgenic hybrid breeding strategy crossing inbred Drd1-Cre and Drd2-Cre transgenic mice with outbred CD-1 mice and used cell-type specific Cre-DREADD (hM4Di) to inhibit NAc Drd1- and Drd2-expressing neuron activity. We found that that aggression self-administration and aggression seeking induced higher Fos expression in NAc shell than in core, that Fos colocalized with Drd1 and Drd2 in both subregions, and that chemogenetic inhibition of Drd1-, but not Drd2-, expressing neurons decreased aggression self-administration and aggression seeking. Results indicate a cell-type specific role of Drd1-expressing neurons that is critical for both aggression self-administration and aggression seeking. Our study also validates a simple breeding strategy between outbred CD-1 mice and inbred C57-based Cre lines that can be used to study cell-type and circuit mechanisms of aggression reward and relapse.SIGNIFICANCE STATEMENTAggression is often comorbid with neuropsychiatric diseases, including drug addiction. One form, appetitive aggression, exhibits symptomatology that mimics that of drug addiction and is hypothesized to be due to dysregulation of addiction-related reward circuits. However, our mechanistic understanding of the circuitry modulating appetitive operant aggression is limited. Here we use a novel mouse model of aggression self-administration and relapse, in combination with immunohistochemistry, in situ hybridization, and chemogenetic manipulations to examine how cell-types in the nucleus accumbens are recruited for, and control, operant aggression self-administration and aggression seeking on abstinence day 1. We found that one population, dopamine receptor 1-expressing neurons, act as a critical modulator of operant aggression reward and aggression seeking.

Midbrain dopamine (DA) and serotonin (5-HT) neurons regulate motivated behaviors, including feeding, but less is known about how these circuits may interact. In this study, we found that DA neurons in the mouse ventral tegmental area bidirectionally regulate the activity of 5-HT neurons in the dorsal raphe nucleus (DRN), with weaker stimulation causing DRD2-dependent inhibition and overeating, while stronger stimulation causing DRD1-dependent activation and anorexia. Furthermore, in the activity-based anorexia (ABA) paradigm, which is a mouse model mimicking some clinical features of human anorexia nervosa (AN), we observed a DRD2 to DRD1 shift of DA neurotransmission on 5-HTDRN neurons, which causes constant activation of these neurons and contributes to AN-like behaviors. Finally, we found that systemic administration of a DRD1 antagonist can prevent anorexia and weight loss in ABA. Our results revealed regulation of feeding behavior by stimulation strength-dependent interactions between DA and 5-HT neurons, which may contribute to the pathophysiology of AN.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

Human gastric development has not been well studied. The generation of human pluripotent stem cell-derived gastric organoids (hGOs) comprising gastric marker-expressing epithelium without an apparent smooth muscle (SM) structure has been reported. We modified previously reported protocols to generate hGOs with muscularis mucosa (MM) from hiPSCs. Time course analyses revealed that epithelium development occurred prior to MM formation. Sonic hedgehog (SHH) and TGF-β1 were secreted by the epithelium. HH and TGF-β signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-β. TGF-β signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-β signaling in MM formation in recovering gastric ulcers. HH and TGF-β signaling from the epithelium to the stroma and the mechanical properties of the subepithelial environment may influence the emergence of MM in human stomach tissue.

Targeting neurons with light-driven opsins is widely used to investigate cell-specific responses. We transfected midbrain dopamine neurons with the excitatory opsin Chrimson. Extracellular basal and stimulated neurotransmitter levels in the dorsal striatum were measured by microdialysis in awake mice. Optical activation of dopamine cell bodies evoked terminal dopamine release in the striatum. Multiplexed analysis of dialysate samples revealed that the evoked dopamine was accompanied by temporally coupled increases in striatal 3-methoxytyramine, an extracellular dopamine metabolite, and in serotonin. We investigated a mechanism for dopamine-serotonin interactions involving striatal dopamine receptors. However, the evoked serotonin associated with optical stimulation of dopamine neurons was not abolished by striatal D1- or D2-like receptor inhibition. Although the mechanisms underlying the coupling of striatal dopamine and serotonin remain unclear, these findings illustrate advantages of multiplexed measurements for uncovering functional interactions between neurotransmitter systems. Furthermore, they suggest that the output of optogenetic manipulations may extend beyond opsin-expressing neuronal populations.

The orbitofrontal cortex (OFC) and piriform cortex (Pir) play a role in fentanyl relapse after food choice-induced voluntary abstinence, a procedure mimicking abstinence because of availability of alternative nondrug rewards. We used in situ hybridization and pharmacology to determine the role of OFC and Pir cannabinoid and dopamine receptors in fentanyl relapse. We trained male and female rats to self-administer food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 µg/kg/infusion) for 12 d (6 h/d). We assessed fentanyl relapse after 12 discrete choice sessions between fentanyl and food (20 trials/d), in which rats voluntarily reduced fentanyl self-administration. We used RNAscope to determine whether fentanyl relapse is associated with activity (indicated by Fos) in OFC and Pir cells expressing Cnr1 [which encodes cannabinoid 1 (CB1) receptors] or Drd1 and Drd2 (which encode dopamine D1 and D2 receptors). We injected a CB1 receptor antagonist or agonist (0.3 or 1.0 µg AM251 or WIN55,212-2/hemisphere) into OFC or a dopamine D1 receptor antagonist (1.0 or 3.0 µg SCH39166/hemisphere) into Pir to determine the effect on fentanyl relapse. Fentanyl relapse was associated with OFC cells co-expressing Fos and Cnr1 and Pir cells co-expressing Fos and Drd1 However, injections of the CB1 receptor antagonist AM251 or agonist WIN55,212-2 into OFC or the dopamine D1 receptor antagonist SCH39166 into Pir had no effect on fentanyl relapse. Fentanyl relapse is associated with activation of Cnr1-expressing OFC cells and Drd1-expressing Pir cells, but pharmacological manipulations do not support causal roles of OFC CB1 receptors or Pir dopamine D1 receptors in fentanyl relapse.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Obesity comorbidities such as diabetes and cardiovascular disease are pressing public health concerns. Overconsumption of calories leads to weight gain; however, neural mechanisms underlying excessive food consumption are poorly understood. Here, we demonstrate that dopamine receptor D1 (Drd1) expressed in the agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons of the arcuate hypothalamus is required for appropriate responses to a high-fat diet (HFD). Stimulation of Drd1 and AgRP/NPY co-expressing arcuate neurons is sufficient to induce voracious feeding. Delivery of a HFD after food deprivation acutely induces dopamine (DA) release in the ARC, whereas animals that lack Drd1 expression in ARCAgRP/NPY neurons (Drd1AgRP-KO) exhibit attenuated foraging and refeeding of HFD. These results define a role for the DA input to the ARC that encodes acute responses to food and position Drd1 signaling in the ARCAgRP/NPY neurons as an integrator of the hedonic and homeostatic neuronal feeding circuits.