Publications by authors named "Peter O Bayguinov"

24 Publications

  • Page 1 of 1

Liver specific deletion of mouse Tm6sf2 promotes steatosis, fibrosis and hepatocellular cancer.

Hepatology 2021 Feb 27. Epub 2021 Feb 27.

Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, United States.

Background And Aims: Human TM6SF2 variant rs58542926 is associated with nonalcoholic fatty liver disease (NAFLD) and hepatocellular cancer (HCC). However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased VLDL secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD.

Approach And Results: Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride (TG) species whose distribution and abundance phenocopied findings in mice with liver specific deletion of microsomal triglyceride transfer protein. VLDL TG secretion was reduced, with small, underlipidated particles and unchanged or increased APOB. Liver-specific adeno-associated viral (AAV8-TBG) rescue using either wild type (WT) or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. Tm6 LKO mice fed a high milk-fat diet for 3 weeks exhibited increased steatosis and fibrosis and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20 weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high fat fed or with diethylnitrosamine (DEN) injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden and increased tumor area versus Tm6 flox controls. Additionally, DEN-injected and fibrogenic diet fed Tm6 LKO mice administered WT Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels.

Conclusions: Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis and accelerated development of HCC, which was mitigated with AAV8- mediated rescue.
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http://dx.doi.org/10.1002/hep.31771DOI Listing
February 2021

Tropism of Severe Acute Respiratory Syndrome Coronavirus 2 for Barrett's Esophagus May Increase Susceptibility to Developing Coronavirus Disease 2019.

Gastroenterology 2021 Jan 20. Epub 2021 Jan 20.

Division of Gastroenterology, Department of Medicine, Washington University in St Louis, School of Medicine, St Louis, Missouri; Department of Pathology and Immunology, Washington University in St Louis, School of Medicine, St Louis, Missouri; Department of Developmental Biology, Washington University in St Louis, School of Medicine, St Louis, Missouri. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2021.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816599PMC
January 2021

Pancreatic β-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Diabetes 2021 02 9;70(2):436-448. Epub 2020 Nov 9.

Department of Biological Sciences, University of Denver, Denver, CO

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic β-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in β-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.
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http://dx.doi.org/10.2337/db20-0454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881869PMC
February 2021

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches.

J Biol Chem 2020 11 16;295(46):15782-15793. Epub 2020 Sep 16.

Washington University Center for Cellular Imaging, Washington University School of Medicine, Saint Louis, Missouri, USA; Departments of Cell Biology and Physiology and Neuroscience, Washington University School of Medicine, Saint Louis, Missouri, USA; Department of Biomedical Engineering, Washington University in Saint Louis, Saint Louis, Missouri, USA. Electronic address:

Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes has been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, while insightful, are nonetheless limited either by restrictions in resolution or by the sheer complexity of generating multidimensional data. Recent advances in the use and application of X-rays to acquire micro- and nanotomographic data sets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the subcellular analyses of unstained vitrified cells and three-dimensional localization of specific protein targets and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles, and applications of both soft X-ray tomography and X-ray microscopy and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches and detail how they have become integral in correlative microscopy workflows.
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http://dx.doi.org/10.1074/jbc.REV120.009633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667966PMC
November 2020

Optical Studies of Action Potential Dynamics with hVOS probes.

Curr Opin Biomed Eng 2019 Dec 23;12:51-58. Epub 2019 Sep 23.

Department of Neuroscience, University of Wisconsin - Madison.

The detection of action potentials and the characterization of their waveform represent basic benchmarks for evaluating optical sensors of voltage. The effectiveness of a voltage sensor in reporting action potentials will determine its usefulness in voltage imaging experiments designed for the study of neural circuitry. The hybrid voltage sensor (hVOS) technique is based on a sensing mechanism with a rapid response to voltage changes. hVOS imaging is thus well suited for optical studies of action potentials. This technique detects action potentials in intact brain slices with an excellent signal-to-noise ratio. These optical action potentials recapitulate voltage recordings with high temporal fidelity. In different genetically-defined types of neurons targeted by cre-lox technology, hVOS recordings of action potentials recapitulate the expected differences in duration. Furthermore, by targeting an hVOS probe to axons, imaging experiments can follow action potential propagation and document dynamic changes in waveform resulting from use-dependent plasticity.
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http://dx.doi.org/10.1016/j.cobme.2019.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449517PMC
December 2019

translational machinery condones polyadenosine repeats.

Elife 2020 05 29;9. Epub 2020 May 29.

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States.

is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or , an organism with similar AT-content. Finally, using stalling reporters we show that cells evolved not to have a fully functional NGD pathway.
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http://dx.doi.org/10.7554/eLife.57799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295572PMC
May 2020

Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.

J Biomech Eng 2019 Sep 1. Epub 2019 Sep 1.

Department of Biomedical Engineering, Washington University in St. Louis, Whitaker Hall, 1 Brookings Dr., St. Louis, MO, 63130.

Trans-synovial solute transport plays a critical role in the clearance of intra-articularly delivered drugs. In this study, we present a computational finite element model of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix.
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http://dx.doi.org/10.1115/1.4044892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104772PMC
September 2019

The Zinc Finger Transcription Factor PLAGL2 Enhances Stem Cell Fate and Activates Expression of ASCL2 in Intestinal Epithelial Cells.

Stem Cell Reports 2018 08 12;11(2):410-424. Epub 2018 Jul 12.

Department of Medicine, Division of Gastroenterology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8124, CSRB NT 923, Saint Louis, MO 63110, USA. Electronic address:

Intestinal epithelial stem cell (IESC) fate is promoted by two major transcriptional regulators, the TCF4/β-catenin complex and ASCL2, which drive expression of IESC-specific factors, including Lgr5, Ephb2, and Rnf43. Canonical Wnt signaling via TCF4/β-catenin directly transactivates Ascl2, which in turn auto-regulates its own expression. Conversely, Let-7 microRNAs antagonize the IESC lineage by repressing specific mRNA targets. Here, we identify the zinc finger transcription factor PLAGL2 as a Let-7 target that regulates IESC fate. PLAGL2 drives an IESC expression signature, activates Wnt gene expression, and enhances a TCF/LEF reporter in intestinal organoids. In parallel, via cell-autonomous mechanisms, PLAGL2 is required for lineage clonal expansion and directly enhances expression of ASCL2. PLAGL2 also supports enteroid growth and survival in the context of Wnt ligand depletion. PLAGL2 expression is strongly associated with an IESC signature in colorectal cancer and may be responsible for contributing to the aberrant activation of an immature phenotype.
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http://dx.doi.org/10.1016/j.stemcr.2018.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092695PMC
August 2018

Modern Laser Scanning Confocal Microscopy.

Curr Protoc Cytom 2018 07 20;85(1):e39. Epub 2018 Jun 20.

Center for Cellular Imaging, Washington University in St. Louis, St. Louis, Missouri.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpcy.39DOI Listing
July 2018

Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

J Neurosci 2017 09 23;37(38):9305-9319. Epub 2017 Aug 23.

Department of Neuroscience,

Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information. Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In brain slices from these animals, single-trial hybrid optical voltage sensor recordings revealed voltage changes with submillisecond resolution in multiple neurons simultaneously. This imaging tool will allow for the study of the emergent properties of neural circuits and permit experimental tests of the roles of specific types of neurons in complex circuit activity.
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http://dx.doi.org/10.1523/JNEUROSCI.1363-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607471PMC
September 2017

Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor.

eNeuro 2017 Jul-Aug;4(4). Epub 2017 Jul 25.

Department of Neuroscience, University of Wisconsin Madison, Madison, WI 53705.

The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.
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http://dx.doi.org/10.1523/ENEURO.0146-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526655PMC
April 2018

A sodium afterdepolarization in rat superior colliculus neurons and its contribution to population activity.

J Neurophysiol 2016 07 13;116(1):191-200. Epub 2016 Apr 13.

Department of Neuroscience and Neuroscience Training Program, The University of Wisconsin, Madison, Wisconsin; and

The mammalian superior colliculus (SC) is a midbrain structure that integrates multimodal sensory inputs and computes commands to initiate rapid eye movements. SC neurons burst with the sudden onset of a visual stimulus, followed by persistent activity that may underlie shifts of attention and decision making. Experiments in vitro suggest that circuit reverberations play a role in the burst activity in the SC, but the origin of persistent activity is unclear. In the present study we characterized an afterdepolarization (ADP) that follows action potentials in slices of rat SC. Population responses seen with voltage-sensitive dye imaging consisted of rapid spikes followed immediately by a second distinct depolarization of lower amplitude and longer duration. Patch-clamp recordings showed qualitatively similar behavior: in nearly all neurons throughout the SC, rapid spikes were followed by an ADP. Ionic and pharmacological manipulations along with experiments with current and voltage steps indicated that the ADP of SC neurons arises from Na(+) current that either persists or resurges following Na(+) channel inactivation at the end of an action potential. Comparisons of pharmacological properties and frequency dependence revealed a clear parallel between patch-clamp recordings and voltage imaging experiments, indicating a common underlying membrane mechanism for the ADP in both single neurons and populations. The ADP can initiate repetitive spiking at intervals consistent with the frequency of persistent activity in the SC. These results indicate that SC neurons have intrinsic membrane properties that can contribute to electrical activity that underlies shifts of attention and decision making.
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http://dx.doi.org/10.1152/jn.01138.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961755PMC
July 2016

A hard-wired priority map in the superior colliculus shaped by asymmetric inhibitory circuitry.

J Neurophysiol 2015 Jul 20;114(1):662-76. Epub 2015 May 20.

Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, The Brain Research Institute, University of California at Los Angeles, Los Angeles, California; and Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California

The mammalian superior colliculus (SC) is a laminar midbrain structure that translates visual signals into commands to shift the focus of attention and gaze. The SC plays an integral role in selecting targets and ultimately generating rapid eye movements to those targets. In all mammals studied to date, neurons in the SC are arranged topographically such that the location of visual stimuli and the endpoints of orienting movements form organized maps in superficial and deeper layers, respectively. The organization of these maps is thought to underlie attentional priority by assessing which regions of the visual field contain behaviorally relevant information. Using voltage imaging and patch-clamp recordings in parasagittal SC slices from the rat, we found the synaptic circuitry of the visuosensory map in the SC imposes a strong bias. Voltage imaging of responses to electrical stimulation revealed more spread in the caudal direction than the rostral direction. Pharmacological experiments demonstrated that this asymmetry arises from GABAA receptor activation rostral to the site of stimulation. Patch-clamp recordings confirmed this rostrally directed inhibitory circuit and showed that it is contained within the visuosensory layers of the SC. Stimulation of two sites showed that initial stimulation of a caudal site can take priority over subsequent stimulation of a rostral site. Taken together, our data indicate that the circuitry of the visuosensory SC is hard-wired to give higher priority to more peripheral targets, and this property is conferred by a uniquely structured, dedicated inhibitory circuit.
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http://dx.doi.org/10.1152/jn.00144.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512250PMC
July 2015

Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor.

J Neurophysiol 2015 Feb 19;113(4):1249-59. Epub 2014 Nov 19.

Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin; Physiology Graduate Training Program, University of Wisconsin, Madison, Wisconsin; and Department of Neuroscience, University of Wisconsin, Madison, Wisconsin

Genetically encoded voltage sensors expand the optogenetics toolkit into the important realm of electrical recording, enabling researchers to study the dynamic activity of complex neural circuits in real time. However, these probes have thus far performed poorly when tested in intact neural circuits. Hybrid voltage sensors (hVOS) enable the imaging of voltage by harnessing the resonant energy transfer that occurs between a genetically encoded component, a membrane-tethered fluorescent protein that serves as a donor, and a small charged molecule, dipicrylamine, which serves as an acceptor. hVOS generates optical signals as a result of voltage-induced changes in donor-acceptor distance. We expressed the hVOS probe in mouse brain by in utero electroporation and in transgenic mice with a neuronal promoter. Under conditions favoring sparse labeling we could visualize single-labeled neurons. hVOS imaging reported electrically evoked fluorescence changes from individual neurons in slices from entorhinal cortex, somatosensory cortex, and hippocampus. These fluorescence signals tracked action potentials in individual neurons in a single trial with excellent temporal fidelity, producing changes that exceeded background noise by as much as 16-fold. Subthreshold synaptic potentials were detected in single trials in multiple distinct cells simultaneously. We followed signal propagation between different cells within one field of view and between dendrites and somata of the same cell. hVOS imaging thus provides a tool for high-resolution recording of electrical activity from genetically targeted cells in intact neuronal circuits.
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http://dx.doi.org/10.1152/jn.00691.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329433PMC
February 2015

Excitatory synaptic feedback from the motor layer to the sensory layers of the superior colliculus.

J Neurosci 2014 May;34(20):6822-33

Department of Neuroscience, Department of Psychiatry and Biobehavioral Sciences and Semel Institute for Neuroscience and Human Behavior University of California at Los Angeles, Los Angeles, California 90095-7332

Neural circuits that translate sensory information into motor commands are organized in a feedforward manner converting sensory information into motor output. The superior colliculus (SC) follows this pattern as it plays a role in converting visual information from the retina and visual cortex into motor commands for rapid eye movements (saccades). Feedback from movement to sensory regions is hypothesized to play critical roles in attention, visual image stability, and saccadic suppression, but in contrast to feedforward pathways, motor feedback to sensory regions has received much less attention. The present study used voltage imaging and patch-clamp recording in slices of rat SC to test the hypothesis of an excitatory synaptic pathway from the motor layers of the SC back to the sensory superficial layers. Voltage imaging revealed an extensive depolarization of the superficial layers evoked by electrical stimulation of the motor layers. A pharmacologically isolated excitatory synaptic potential in the superficial layers depended on stimulus strength in the motor layers in a manner consistent with orthodromic excitation. Patch-clamp recording from neurons in the sensory layers revealed excitatory synaptic potentials in response to glutamate application in the motor layers. The location, size, and morphology of responsive neurons indicated they were likely to be narrow-field vertical cells. This excitatory projection from motor to sensory layers adds an important element to the circuitry of the SC and reveals a novel feedback pathway that could play a role in enhancing sensory responses to attended targets as well as visual image stabilization.
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http://dx.doi.org/10.1523/JNEUROSCI.3137-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019797PMC
May 2014

Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.

J Physiol 2012 Jan 7;590(2):335-50. Epub 2011 Nov 7.

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

Enteric glia cells (EGCs) form a dense network around myenteric neurons in a ganglia and are likely to have not only a supportive role but may also regulate or be regulated by neural activity. Our aims were to determine if EGCs are activated during the colonic migrating motor complex (CMMC) in the isolated murine colon. Strips of longitudinal muscle were removed and Ca(2+) imaging (Fluo-4) used to study activity in EGCs within myenteric ganglia during CMMCs, followed by post hoc S100 staining to reveal EGCs. The cell bodies of EGCs and their processes formed caps and halos, respectively, around some neighbouring myenteric neurons. Some EGCs (36%), which were largely quiescent between CMMCs, exhibited prolonged tetrodotoxin (TTX; 1 μm)-sensitive Ca(2+) transients that peaked ∼39 s following a mucosal stimulus that generated the CMMC, and often outlasted the CMMC (duration ∼23 s). Ca(2+) transients in EGCs often varied in duration within a ganglion; however, the duration of these transients was closely matched by activity in closely apposed nerve varicosities, suggesting EGCs were not only innervated but the effective innervation was localized. Furthermore, all EGCs, even those that were quiescent, responded with robust Ca(2+) transients to KCl, caffeine, nicotine, substance P and GR 64349 (an NK2 agonist), suggesting they were adequately loaded with indicator and that some EGCs may be inhibited by substances released by neighbouring neurons. Intracellular Ca(2+) waves were visualised propagating between closely apposed glia and from glial cell processes to the soma (velocity 12 μm s(-1)) where they produced an accumulative rise in Ca(2+), suggesting that the soma acts as an integrator of Ca(2+) activity. In conclusion, Ca(2+) transients in EGCs occur secondary to nerve activity; their activation is driven by intrinsic excitatory nerve pathways that generate the CMMC.
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http://dx.doi.org/10.1113/jphysiol.2011.219519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285069PMC
January 2012

Ca2+ imaging of activity in ICC-MY during local mucosal reflexes and the colonic migrating motor complex in the murine large intestine.

J Physiol 2010 Nov 27;588(Pt 22):4453-74. Epub 2010 Sep 27.

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

Colonic migrating motor complexes (CMMCs) are neurally mediated, cyclical contractile and electrical events, which typically propagate along the colon every 2-3 min in the mouse. We examined the interactions between myenteric neurons, interstitial cells of Cajal in the myenteric region (ICC-MY) and smooth muscle cells during CMMCs using Ca(2+) imaging. CMMCs occurred spontaneously or were evoked by stimulating the mucosa locally, or by brushing it at either end of the colon. Between CMMCs, most ICC-MY were often quiescent; their lack of activity was correlated with ongoing Ca(2+) transients in varicosities on the axons of presumably inhibitory motor neurons that were on or surrounded ICC-MY. Ca(2+) transients in other varicosities initiated intracellular Ca(2+) waves in adjacent ICC-MY, which were blocked by atropine, suggesting they were on the axons of excitatory motor neurons. Following TTX (1 μM), or blockade of inhibitory neurotransmission with N(ω)-nitro-L-arginine (L-NA, a NO synthesis inhibitor, 10 μM) and MRS 2500 (a P2Y(1) antagonist, 1 μM), ongoing spark/puff like activity and rhythmic intracellular Ca(2+) waves (38.1 ± 2.9 cycles min(-1)) were observed, yet this activity was uncoupled, even between ICC-MY in close apposition. During spontaneous or evoked CMMCs there was an increase in the frequency (62.9 ± 1.4 cycles min(-1)) and amplitude of Ca(2+) transients in ICC-MY and muscle, which often had synchronized activity. At the same time, activity in varicosites along excitatory and inhibitory motor nerve fibres increased and decreased respectively, leading to an overall excitation of ICC-MY. Atropine (1 μM) reduced the evoked responses in ICC-MY, and subsequent addition of an NK1 antagonist (RP 67580, 500 nM) completely blocked the responses to stimulation, as did applying these drugs in reverse order. An NKII antagonist (MEN 10,376, 500 nM) had no effect on the evoked responses in ICC-MY. Following TTX application, carbachol (1 μM), substance P (1 μM) and an NKI agonist (GR73632, 100 nM) produced the fast oscillations superimposed on a slow increase in Ca(2+) in ICC-MY, whereas SNP (an NO donor, 10 μM) abolished all activity in ICC-MY. In conclusion, ICC-MY, which are under tonic inhibition, are pacemakers whose activity can be synchronized by excitatory nerves to couple the longitudinal and circular muscles during the CMMC. ICC-MY receive excitatory input from motor neurons that release acetylcholine and tachykinins acting on muscarinic and NK1 receptors, respectively.
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http://dx.doi.org/10.1113/jphysiol.2010.196824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008851PMC
November 2010

Colonic elongation inhibits pellet propulsion and migrating motor complexes in the murine large bowel.

J Physiol 2010 Aug 14;588(Pt 15):2919-34. Epub 2010 Jun 14.

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an 'all or none' event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.
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http://dx.doi.org/10.1113/jphysiol.2010.191445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956907PMC
August 2010

Calcium activity in different classes of myenteric neurons underlying the migrating motor complex in the murine colon.

J Physiol 2010 Feb 30;588(Pt 3):399-421. Epub 2009 Nov 30.

Department of Physiology and Cell Biology, Anderson Medical Sciences Building/352, University of Nevada School of Medicine, Reno, NV 89557, USA.

The spontaneous colonic migrating motor complex (CMMC) is a cyclical contractile and electrical event that is the primary motor pattern underlying fecal pellet propulsion along the murine colon. We have combined Ca(2+) imaging with immunohistochemistry to determine the role of different classes of myenteric neurons during the CMMC. Between CMMCs, myenteric neurons usually displayed ongoing but uncoordinated activity. Stroking the mucosa at the oral or anal end of the colon resulted in a CMMC (latency: 6 to 10 s; duration: 28 s) that consisted of prolonged increases in activity in many myenteric neurons that was correlated to Ca(2+) transients in and displacement of the muscle. These neurons were likely excitatory motor neurons. Activity in individual neurons during the CMMC was similar regardless of whether the CMMC occurred spontaneously or was evoked by anal or oral mucosal stimulation. This suggests that convergent interneuronal pathways exist which generate CMMCs. Interestingly, Ca(2+) transients in a subset of NOS +ve neurons were substantially reduced during the CMMC. These neurons are likely to be inhibitory motor neurons that reduce their activity during a complex (disinhibition) to allow full excitation of the muscle. Local stimulation of the mucosa evoked synchronized Ca(2+) transients in Dogiel Type II (mitotracker/calbindin-positive) neurons after a short delay (1-2 s), indicating they were the sensory neurons underlying the CMMC. These local responses were observed in hexamethonium, but were blocked by ondansetron (5-HT(3) antagonist), suggesting Dogiel Type II neurons were activated by 5-HT release from enterochromaffin cells in the mucosa. In fact, removal of the mucosa yielded no spontaneous CMMCs, although many neurons (NOS +ve and NOS ve) exhibited ongoing activity, including Dogiel Type II neurons. These results suggest that spontaneous or evoked 5-HT release from the mucosa is necessary for the activation of Dogiel Type II neurons that generate CMMCs.
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http://dx.doi.org/10.1113/jphysiol.2009.181172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825607PMC
February 2010

Heterogeneities in ICC Ca2+ activity within canine large intestine.

Gastroenterology 2009 Jun 5;136(7):2226-36. Epub 2009 Mar 5.

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, USA.

Background & Aims: In human and canine colon, both slow (slow waves, 2-8/min) and fast (myenteric potential oscillations [MPOs]; 16-20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca(2+) imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms.

Methods: Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca(2+) indicator (fluo-4), and activity was monitored at 36.5 +/- 0.5 degrees C using an electron multiplying charge coupled device (EMCCD).

Results: Rhythmic, biphasic Ca(2+) transients (5-8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2-5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca(2+) waves (approximately 16/min) and spontaneous reductions in Ca(2+) were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca(2+) waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca(2+) were observed that inhibited Ca(2+) waves. N omega-nitro-L-arginine (100 micromol/L; nitric oxide antagonist) blocked the reductions in Ca(2+) and increased the frequency (approximately 19/min) of intracellular Ca(2+) waves within ICC-MY.

Conclusions: ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca(2+) transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.
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http://dx.doi.org/10.1053/j.gastro.2009.02.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802971PMC
June 2009

Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon.

Gastroenterology 2009 Apr 10;136(4):1328-38. Epub 2008 Dec 10.

Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada, USA.

Background & Aims: The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity.

Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa.

Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation.

Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.
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http://dx.doi.org/10.1053/j.gastro.2008.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982771PMC
April 2009

Polarized intrinsic neural reflexes in response to colonic elongation.

J Physiol 2008 Sep 17;586(17):4225-40. Epub 2008 Jul 17.

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca(2+) imaging techniques. Full colons were 158.1 +/- 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s(-1)). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca(2+) waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechanosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.
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http://dx.doi.org/10.1113/jphysiol.2008.155630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652183PMC
September 2008

An enteric occult reflex underlies accommodation and slow transit in the distal large bowel.

Gastroenterology 2007 May 23;132(5):1912-24. Epub 2007 Feb 23.

Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.

Background & Aims: Transit of fecal material through the human colon takes > or =30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena.

Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca(2+) imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit.

Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-omega-nitro-L-arginine (100 micromol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca(2+) imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS +ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N-omega-nitro-L-arginine (100 micromol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility.

Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS +ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.
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http://dx.doi.org/10.1053/j.gastro.2007.02.047DOI Listing
May 2007