In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out‐of‐focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out‐of‐focus planes. Confocal microscopy provides a means of rejecting the out‐of‐focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high‐resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction‐limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high‐resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.

Elliott AD, Ustione A, Piston DW. Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreatic ␣-cell by lowering cAMP. Am J Physiol Endocrinol Metab 308: E130 -E143, 2015. First published November 18, 2014 doi:10.1152/ajpendo.00344.2014.-The dysregulation of glucose-inhibited glucagon secretion from the pancreatic islet ␣-cell is a critical component of diabetes pathology and metabolic disease. We show a previously uncharacterized [Ca 2ϩ ]i-independent mechanism of glucagon suppression in human and murine pancreatic islets whereby cAMP and PKA signaling are decreased. This decrease is driven by the combination of somatostatin, which inhibits adenylyl cyclase production of cAMP via the G␣ i subunit of the SSTR2, and insulin, which acts via its receptor to activate phosphodiesterase 3B and degrade cytosolic cAMP. Our data indicate that both somatostatin and insulin signaling are required to suppress cAMP/PKA and glucagon secretion from both human and murine ␣-cells, and the combination of these two signaling mechanisms is sufficient to reduce glucagon secretion from isolated ␣-cells as well as islets. Thus, we conclude that somatostatin and insulin together are critical paracrine mediators of glucoseinhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose. cyclic AMP; glucagon; pancreatic islets; insulin; somatostatin GLUCAGON PLAYS A CRITICAL ROLE in blood glucose homeostasis, and its secretion from pancreatic islet ␣-cells is inhibited with rising glucose. During diabetes, persistent glucagon secretion from ␣-cells leads to hyperglucagonemia, which overproduces glucose, exacerbating hyperglycemia. This makes the ␣-cell an important target for therapeutic intervention, but relatively little is known about how glucagon secretion is regulated under normal physiological conditions (18). There are many hypotheses about how glucagon is suppressed by glucose, including paracrine regulation by islet factors (28) and changes in ion channel activity (44), but they commonly depend on a decrease in intracellular Ca 2ϩ . However, published data show that glucagon inhibition is independent of intracellular Ca 2ϩ activity, and no hypothesis explains the loss of suppression from purified ␣-cells (30,31,38). Progress toward understanding ␣-cell regulation and function has been hampered by a lack of approaches to measure ␣-cell properties without first separating them from the rest of the islet, which we know critically changes their function.Multiple G protein-coupled somatostatin receptors (SSTRs) have been identified in islets. SSTR2 is the most abundantly expressed and functionally dominant isoform in both human and murine ␣-cells (12, 24). Upon SSTR2 activation by somatostatin, the G␣ i subunit inhibits adenylyl cyclase to reduce cAMP. Isolated islets from SSTR2 knockout mice show a twofold increase in glucagon secretion, suggesting a role for somatostatin in glucose-inhibited glucagon secretion (37). In contrast, global somatostatin deletion does not ...

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.

Neural networks are typically defined by their synaptic connectivity, yet synaptic wiring diagrams often provide limited insight into network function. This is due partly to the importance of non-synaptic communication by neuromodulators, which can dynamically reconfigure circuit activity to alter its output. Here, we systematically map the patterns of neuromodulatory connectivity in a network that governs a developmentally critical behavioral sequence in Drosophila. This sequence, which mediates pupal ecdysis, is governed by the serial release of several key factors, which act both somatically as hormones and within the brain as neuromodulators. By identifying and characterizing the functions of the neuronal targets of these factors, we find that they define hierarchically organized layers of the network controlling the pupal ecdysis sequence: a modular input layer, an intermediate central pattern generating layer, and a motor output layer. Mapping neuromodulatory connections in this system thus defines the functional architecture of the network.