Probes for INS

Abstract

Objective

Complex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells.

Methods

To resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy.

Results

From these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin.

Conclusions

These results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes.

Cell-specific molecular investigations of the human brain are essential for understanding the neurobiology of diseases, but are hindered by postmortem conditions and technical challenges. To address these issues we developed a multi-label fluorescence in situ hybridization protocol and a novel optical filter device to identify cell types and control for tissue autofluorescence. We show that these methods can be used with laser-capture microdissection for human brain tissue cell-specific molecular analysis.

Striatal locally projecting neurons, or interneurons, act on nearby circuits and shape functional output to the rest of the basal ganglia. We performed single-cell RNA sequencing of striatal cells enriching for interneurons. We find seven discrete interneuron types, six of which are GABAergic. In addition to providing specific markers for the populations previously described, including those expressing Sst/Npy, Th, Npy without Sst, and Chat, we identify two small populations of cells expressing Cck with or without Vip. Surprisingly, the Pvalb-expressing cells do not constitute a discrete cluster but rather are part of a larger group of cells expressing Pthlh with a spatial gradient of Pvalb expression. Using PatchSeq, we show that Pthlh cells exhibit a continuum of electrophysiological properties correlated with expression of Pvalb. Furthermore, we find significant molecular differences that correlate with differences in electrophysiological properties between Pvalb-expressing cells of the striatum and those of the cortex.

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia.

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

Like most homeostatic systems, adiposity in mammals is defended between upper and lower boundary conditions. While leptin and melanocortin-4 receptor (MC4R) signaling are required for defending energy set point, mechanisms controlling upper and lower homeostatic boundaries are less well understood. In contrast to the MC4R, deletion of the MC3R does not produce measurable hyperphagia or hypometabolism under normal conditions. However, we demonstrate that MC3R is required bidirectionally for controlling responses to external homeostatic challenges, such as caloric restriction or calorie-rich diet. MC3R is also required for regulated excursion from set point, or rheostasis, during pregnancy. Further, we demonstrate a molecular mechanism: MC3R provides regulatory inputs to melanocortin signaling, acting presynaptically on agouti-related protein neurons to regulate γ-aminobutyric acid release onto anorexigenic MC4R neurons, exerting boundary control on the activity of MC4R neurons. Thus, the MC3R is a critical regulator of boundary controls on melanocortin signaling, providing rheostatic control on energy storage.