scholarly journals Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii

2015 ◽  
Vol 113 (4) ◽  
pp. 1165-1174 ◽  
Author(s):  
Hong Xu ◽  
Jeffery A. Boychuk ◽  
Carie R. Boychuk ◽  
Victor V. Uteshev ◽  
Bret N. Smith
Keyword(s):  
Motor Neurons ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Gabaergic Neurons ◽  
Therapeutic Interventions ◽  
Motor Nucleus ◽  
Dorsal Motor Nucleus ◽  
Green Fluorescent

The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments.

scholarly journals Responsiveness to nicotine of neurons of the caudal nucleus of the solitary tract correlates with the neuronal projection target

2012 ◽  
Vol 108 (7) ◽  
pp. 1884-1894 ◽  
Author(s):  
Lin Feng ◽  
Evgeny A. Sametsky ◽  
Alexander G. Gusev ◽  
Victor V. Uteshev
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Ex Vivo ◽  
Motor Nucleus ◽  
Solitary Tract ◽  
Axonal Projections ◽  
Dorsal Motor Nucleus ◽  
Electrophysiological Recordings ◽  
Neuronal Projection

The caudal nucleus of the solitary tract (NTS) is the key integrating center of visceral sensory-motor signaling supporting autonomic homeostasis. Two key projections of this nucleus are the parabrachial nucleus (PbN) and the dorsal motor nucleus of the vagus (DMV). The PbN integrates and relays viscerosensory information primarily to the forebrain, supporting behavioral, emotional, and endocrine responses to visceral events, while the DMV contains parasympathetic preganglionic cholinergic motoneurons that support primarily gastrointestinal reflexes. Subsets of caudal NTS neurons express presynaptic and somatodendritic nicotinic acetylcholine receptors (nAChRs). However, the anatomical identification of nicotine-responsive caudal NTS neurons has not been determined. This study used in vivo and ex vivo fluorescent tracing and slice patch-clamp electrophysiological recordings from anatomically identified caudal NTS neurons to test the hypothesis that the responsiveness of these cells to nicotine correlates with the target of their axonal projections. The results demonstrate that the majority of glutamatergic terminals that synapse on PbN-projecting caudal NTS neurons are unaffected by nicotine. Moreover, only a fraction of these cells express somatodendritic nAChRs. In contrast, the majority of DMV-projecting caudal NTS neurons exhibit robust presynaptic and somatodendritic responsiveness to nicotine. However, PbN-projecting neurons also exhibit significantly lower background frequencies of glutamatergic miniature postsynaptic currents than DMV-projecting neurons. Therefore, presynaptic unresponsiveness to nicotine may result from deficient glutamatergic innervation of PbN-projecting neurons. Nevertheless, the caudal NTS contains function-specific subsets of cells with target-specific responsiveness to nicotine. These results may support development of therapeutic strategies for selective targeting of specific autonomic pathways and impaired autonomic homeostasis.

scholarly journals Effects of acute and chronic nicotine on catecholamine neurons of the nucleus of the solitary tract

2019 ◽  
Vol 316 (1) ◽  
pp. R38-R49
Author(s):  
Stephen J. Page ◽  
Mingyan Zhu ◽  
Suzanne M. Appleyard
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Nicotine Withdrawal ◽  
Cardiovascular Reflexes ◽  
Solitary Tract ◽  
Catecholamine Neurons ◽  
Green Fluorescent ◽  
Tyrosine Hydroxylase Promoter

Nicotine is an addictive drug that has broad effects throughout the brain. One site of action is the nucleus of the solitary tract (NTS), where nicotine initiates a stress response and modulates cardiovascular and gastric function through nicotinic acetylcholine receptors (nAChRs). Catecholamine (CA) neurons in the NTS influence stress and gastric and cardiovascular reflexes, making them potential mediators of nicotine’s effects; however nicotine’s effect on these neurons is unknown. Here, we determined nicotine’s actions on NTS-CA neurons by use of patch-clamp techniques in brain slices from transgenic mice expressing enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). Picospritzing nicotine both induced a direct inward current and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in NTS-CA neurons, effects blocked by nonselective nAChR antagonists TMPH and MLA. The increase in sEPSC frequency was mimicked by nAChRα7 agonist AR-R17779 and blocked by nAChRα7 antagonist MG624. AR-R17779 also increased the firing of TH-EGFP neurons, an effect dependent on glutamate inputs, as it was blocked by the glutamate antagonist NBQX. In contrast, the nicotine-induced current was mimicked by nAChRα4β2 agonist RJR2403 and blocked by nAChRα4β2 antagonist DHβE. RJR2403 also increased the firing rate of TH-EGFP neurons independently of glutamate. Finally, both somatodendritic and sEPSC nicotine responses from NTS-CA neurons were larger in nicotine-dependent mice that had under gone spontaneous nicotine withdrawal. These results demonstrate that 1) nicotine activates NTS-CA neurons both directly, by inducing a direct current, and indirectly, by increasing glutamate inputs, and 2) NTS-CA nicotine responsiveness is altered during nicotine withdrawal.

scholarly journals Generation of a Novel Transgenic Zebrafish for Studying Adipocyte Development and Metabolic Control

2021 ◽  
Vol 22 (8) ◽  
pp. 3994
Author(s):  
Yousheng Mao ◽  
Kwang-Heum Hong ◽  
Weifang Liao ◽  
Li Li ◽  
Seong-Jin Kim ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Metabolic Diseases ◽  
Fluorescent Protein ◽  
Therapeutic Interventions ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Adipose Tissues ◽  
Green Fluorescent

Zebrafish have become a popular animal model for studying various biological processes and human diseases. The metabolic pathways and players conserved among zebrafish and mammals facilitate the use of zebrafish to understand the pathological mechanisms underlying various metabolic disorders in humans. Adipocytes play an important role in metabolic homeostasis, and zebrafish adipocytes have been characterized. However, a versatile and reliable zebrafish model for long-term monitoring of adipose tissues has not been reported. In this study, we generated stable transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) in adipocytes. The transgenic zebrafish harbored adipose tissues that could be detected using GFP fluorescence and the morphology of single adipocyte could be investigated in vivo. In addition, we demonstrated the applicability of this model to the long-term in vivo imaging of adipose tissue development and regulation based on nutrition. The transgenic zebrafish established in this study may serve as an excellent tool to advance the characterization of white adipose tissue in zebrafish, thereby aiding the development of therapeutic interventions to treat metabolic diseases in humans.

scholarly journals Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors

2021 ◽  
Vol 8 ◽  
Author(s):  
Igor E. Kasheverov ◽  
Alexey I. Kuzmenkov ◽  
Denis S. Kudryavtsev ◽  
Ivan S. Chudetskiy ◽  
Irina V. Shelukhina ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Full Length ◽  
Fluorescent Label ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Gfp Chromophore ◽  
Green Fluorescent

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

scholarly journals Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure

2012 ◽  
Vol 107 (1) ◽  
pp. 257-264 ◽  
Author(s):  
Jason Q. Pilarski ◽  
Hilary E. Wakefield ◽  
Andrew J. Fuglevand ◽  
Richard B. Levine ◽  
Ralph F. Fregosi
Keyword(s):  
Motor Neurons ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Upper Airway ◽  
Motor Nucleus ◽  
Nicotine Exposure ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Whole Cell Patch Clamp ◽  
Nachr Subtype ◽  
Inward Currents

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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