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1.
Sci Rep ; 14(1): 15195, 2024 07 02.
Article in English | MEDLINE | ID: mdl-38956443

ABSTRACT

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.


Subject(s)
Cell Cycle , Epithelial Cells , Intestinal Mucosa , Humans , Epithelial Cells/cytology , Epithelial Cells/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Oligomycins/pharmacology , Cells, Cultured
2.
J Cardiovasc Dev Dis ; 10(12)2023 Nov 29.
Article in English | MEDLINE | ID: mdl-38132647

ABSTRACT

Males and females differ in the basic anatomy and physiology of the heart. Sex differences are evident in cardiac repolarization in humans; women have longer corrected QT and JT intervals. However, the molecular mechanisms that lead to these differences are incompletely understood. Here, we present that, like in humans, sex differences in QT and JT intervals exist in mouse models; female mice had longer corrected QT and JT intervals compared with age-matched males. To further understand the molecular underpinning of these sex differences, we developed a novel technology using fluorescent confocal microscopy that allows the simultaneous visualization of action potential, Ca2+ transients, and contractions in isolated cardiomyocytes at a high temporal resolution. From this approach, we uncovered that females at baseline have increased action potential duration, decreased Ca2+ release and reuptake rates, and decreased contraction and relaxation velocities compared with males. Additionally, males had a shorter overall time from action potential onset to peak contraction. In aggregate, our studies uncovered male and female differences in excitation-contraction coupling that account for differences observed in the EKG. Overall, a better understanding of sex differences in electrophysiology is essential for equitably treating cardiac disease.

3.
bioRxiv ; 2023 Oct 10.
Article in English | MEDLINE | ID: mdl-37873351

ABSTRACT

Background and Aims: The intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods: 3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. Results: Chamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. Conclusions: We present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.

4.
BMJ Open ; 11(3): e040522, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33722864

ABSTRACT

INTRODUCTION: Mechanical thrombectomy (MT) increases functional independence in patients with acute ischaemic stroke with anterior circulation large vessel occlusion (LVO), and the probability to achieve functional independence decreases by 20% for each 1-hour delay to reperfusion. Therefore, we aim to investigate whether direct angiosuite transfer (DAT) is superior to standard imaging/emergency department-based management in achieving 90-day functional independence in patients presenting with an acute severe neurological deficit likely due to LVO and requiring emergent treatment with MT. METHODS AND ANALYSIS: DIRECT ANGIO (Effect of DIRECT transfer to ANGIOsuite on functional outcome in patient with severe acute stroke treated with thrombectomy: the randomised DIRECT ANGIO Trial) trial is an investigator-initiated, multicentre, prospective, randomised, open-label, blinded endpoint (PROBE) study. Eligibility requires a patient ≤75 years, pre-stroke modified Rankin Scale (mRS) 0-2, presenting an acute severe neurological deficit and admitted within 5 hours of symptoms onset in an endovascular-capable centre. A total of 208 patients are randomly allocated in a 1:1 ratio to DAT or standard management. The primary outcome is the rate of patients achieving a functional independence, assessed as mRS 0-2 at 90 days. Secondary endpoints include patients presenting confirmed LVO, patients eligible to intravenous thrombolysis alone, patients with intracerebral haemorrhage and stroke-mimics, intrahospital time metrics, early neurological improvement (reduction in National Institutes of Health Stroke Scale by ≥8 points or reaching 0-1 at 24 hours) and mRS overall distribution at 90 days and 12 months. Safety outcomes are death and intracerebral haemorrhage transformation. Medico-economics analyses include health-related quality of life and cost utility assessment. ETHICS AND DISSEMINATION: The DIRECT ANGIO trial was approved by the ethics committee of Ile de France 1. Study began in April 2020. Results will be published in an international peer-reviewed medical journal. TRIAL REGISTRATION NUMBER: NCT03969511.


Subject(s)
Brain Ischemia , Endovascular Procedures , Stroke , Brain Ischemia/surgery , France , Humans , Multicenter Studies as Topic , Prospective Studies , Quality of Life , Randomized Controlled Trials as Topic , Stroke/surgery , Thrombectomy , Treatment Outcome
5.
Cardiovasc Res ; 117(2): 520-532, 2021 01 21.
Article in English | MEDLINE | ID: mdl-32053173

ABSTRACT

AIMS: Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. METHODS AND RESULTS: The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. CONCLUSION: LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.


Subject(s)
Carotid Arteries/pathology , Carotid Artery Injuries/pathology , Carotid Stenosis/pathology , Imaging, Three-Dimensional , Microscopy, Fluorescence , Angioplasty, Balloon , Animals , Disease Models, Animal , Ligation , Male , Mice, Inbred C57BL , Neointima , Rats , Reproducibility of Results , Vascular Remodeling
6.
J Comp Neurol ; 529(4): 757-785, 2021 03.
Article in English | MEDLINE | ID: mdl-32632959

ABSTRACT

Light sheet fluorescence microscopy (LSFM) provides a rapid and complete three-dimensional image of the cochlea. The method retains anatomical relationships-on a micrometer scale-between internal structures such as hair cells, basilar membrane (BM), and modiolus with external surface structures such as the round and oval windows. Immunolabeled hair cells were used to visualize the spiraling BM in the intact cochlea without time intensive dissections or additional histological processing; yet material prepared for LSFM could be rehydrated, the BM dissected out and reimaged at higher resolution with the confocal microscope. In immersion-fixed material, details of the cochlear vasculature were seen throughout the cochlea. Hair cell counts (both inner and outer) as well as frequency maps of the BM were comparable to those obtained by other methods, but with the added dimension of depth. The material provided measures of angular, linear, and vector distance between characteristic frequency regions along the BM. Thus, LSFM provides a unique ability to rapidly image the entire cochlea in a manner applicable to model and interpret physiological results. Furthermore, the three-dimensional organization of the cochlea can be studied at the organ and cellular level with LSFM, and this same material can be taken to the confocal microscope for detailed analysis at the subcellular level.


Subject(s)
Cochlea/anatomy & histology , Cochlea/chemistry , Imaging, Three-Dimensional/methods , Animals , Cochlea/cytology , Gerbillinae , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods
7.
Toxicol Sci ; 177(1): 140-155, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32525552

ABSTRACT

In vitro bronchial epithelial monoculture models have been pivotal in defining the adverse effects of inhaled toxicant exposures; however, they are only representative of one cellular compartment and may not accurately reflect the effects of exposures on other cell types. Lung fibroblasts exist immediately beneath the bronchial epithelial barrier and play a central role in lung structure and function, as well as disease development and progression. We tested the hypothesis that in vitro exposure of a human bronchial epithelial cell barrier to the model oxidant diesel exhaust particulates caused transepithelial oxidative stress in the underlying lung fibroblasts using a human bronchial epithelial cell and lung fibroblast coculture model. We observed that diesel exhaust particulates caused transepithelial oxidative stress in underlying lung fibroblasts as indicated by intracellular accumulation of the reactive oxygen species hydrogen peroxide, oxidation of the cellular antioxidant glutathione, activation of NRF2, and induction of oxidative stress-responsive genes. Further, targeted antioxidant treatment of lung fibroblasts partially mitigated the oxidative stress response gene expression in adjacent human bronchial epithelial cells during diesel exhaust particulate exposure. This indicates that exposure-induced oxidative stress in the airway extends beyond the bronchial epithelial barrier and that lung fibroblasts are both a target and a mediator of the adverse effects of inhaled chemical exposures despite being separated from the inhaled material by an epithelial barrier. These findings illustrate the value of coculture models and suggest that transepithelial exposure effects should be considered in inhalation toxicology research and testing.


Subject(s)
Lung , Vehicle Emissions , Bronchi , Epithelial Cells , Fibroblasts , Humans , Oxidative Stress , Vehicle Emissions/toxicity
8.
Mol Cell ; 75(3): 523-537.e10, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31256989

ABSTRACT

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.


Subject(s)
RNA, Long Noncoding/genetics , Animals , Chromatin/genetics , Chromatin Assembly and Disassembly , CpG Islands/genetics , Genome/genetics , Genomic Imprinting/genetics , Humans , Mice , Polycomb Repressive Complex 1/genetics , Promoter Regions, Genetic , Stem Cells/metabolism , Trophoblasts/metabolism
9.
Immunity ; 50(3): 591-599.e6, 2019 03 19.
Article in English | MEDLINE | ID: mdl-30893587

ABSTRACT

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.


Subject(s)
DNA, Viral/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interferon Type I/metabolism , Membrane Proteins/metabolism , Animals , Cell Line , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Nucleic Acids/metabolism , Protein Binding/immunology
10.
PLoS One ; 12(12): e0190528, 2017.
Article in English | MEDLINE | ID: mdl-29284058

ABSTRACT

The Metabolic Syndrome (MetS) is a cluster of cardiometabolic risk factors, usually accompanied by the presence of insulin resistance (IR) and a systemic subclinical inflammation state. Metabolically healthy obese (MHO) individuals seem to be protected against cardiometabolic complications. The aim of this work was to characterize phenotypically the low-grade inflammation and the IR in MHO individuals in comparison to obese individuals with MetS and control non obese. We studied two different populations: 940 individuals from the general population of Buenos Aires and 518 individuals from the general population of Venado Tuerto; grouped in three groups: metabolically healthy non-obese individuals (MHNO), MHO and obese individuals with MetS (MSO). Inflammation was measured by the levels of hs-CRP (high-sensitivity C reactive protein), and we found that MHO presented an increase in inflammation when compared with MHNO (Buenos Aires: p<0.001; Venado Tuerto: p<0.001), but they did not differ from MSO. To evaluate IR we analyzed the HOMA (Homoeostatic Model Assessment) values, and we found differences between MHO and MSO (Buenos Aires: p<0.001; Venado Tuerto: p<0.001), but not between MHNO and MHO. In conclusion, MHO group would be defined as a subgroup of obese individuals with an intermediate phenotype between MHNO and MSO individuals considering HOMA, hs-CRP and central obesity.


Subject(s)
Inflammation/metabolism , Insulin Resistance , Metabolic Syndrome/metabolism , Obesity/metabolism , Adult , Chronic Disease , Female , Humans , Male , Young Adult
11.
Int J Biochem Cell Biol ; 84: 35-39, 2017 03.
Article in English | MEDLINE | ID: mdl-28082099

ABSTRACT

The last decade has seen a proliferation of tissue clearing methods that render large biological samples transparent and allow unprecedented three-dimensional views of enormous volumes of tissue. For a scientist wondering whether these methods will be useful to address their research problems, it can be bewildering to sort through the ever-increasing number of papers introducing new clearing methods. Here, I provide a concise summary for the novice describing what tissue clearing is, which research problems it can be applied to, how to decide on a clearing method, and where the field is headed in the future.


Subject(s)
Histological Techniques/methods , Animals , Histological Techniques/trends , Hydrogels , Solutions , Solvents , Staining and Labeling/methods , Tissue Embedding/methods
12.
Diabetes Res Clin Pract ; 118: 38-43, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27289208

ABSTRACT

BACKGROUND: Maturity-onset diabetes of the young 2 (MODY2) is a form of diabetes that is clinically characterized by early age at onset and mild hyperglycemia, and has a low risk of late complications. It is often underdiagnosed due to its mild symptoms. To date, over 600 different GCK/MODY2 mutations have been reported. Despite only a few de novo mutations having been described, recent studies have reported the detection of a higher frequency of this kind of mutation. Therefore, de novo mutations could be more frequent than previously described. Even though common recommendations regarding the diagnosis of monogenic diabetes include the existence of a strong family history of diabetes, here we describe the study of mutations in two families with a symptomatic individual with clear clinical features of MODY2 but without any family history of diabetes. METHODS: Genetic diagnosis in a group of participants with MODY2 characteristics was carried out by direct sequencing of coding regions of the GCK gene and analysis of mutations found using bioinformatics tools. RESULTS: We found two de novo mutations, one of them novel, constituting 14.29% of all the participants who were phenotyped as MODY2. CONCLUSIONS: The number of mutations in GCK/MODY2 or even other MODY-related genes is undoubtedly underestimated, as accepted criteria for performing genetic tests include family history of the pathology. These cases illustrate the value of analyzing the GCK gene in patients with clinical features of MODY2, even in the absence of family history of the condition as it is essential for establishing the correct treatment.


Subject(s)
DNA/genetics , Diabetes Mellitus, Type 2/genetics , Glucokinase/genetics , Mutation , Adolescent , Argentina/epidemiology , DNA Mutational Analysis/methods , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/epidemiology , Female , Glucokinase/metabolism , Humans , Male , Pedigree , Phenotype , Prevalence , Retrospective Studies
13.
Cell ; 159(4): 896-910, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25417164

ABSTRACT

The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:


Subject(s)
Imaging, Three-Dimensional/methods , Immunohistochemistry , Animals , Embryo, Mammalian/cytology , Immunohistochemistry/economics , Mice , Nerve Degeneration/pathology
14.
Physiology (Bethesda) ; 27(1): 15-24, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22311967

ABSTRACT

The strength of a synapse can profoundly influence network function. How this strength is set at the molecular level is a key question in neuroscience. Here, we review a simple model of neurotransmission that serves as a convenient framework to discuss recent studies on RIM and synaptotagmin.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Models, Neurological , Neurotransmitter Agents/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Synaptotagmins/metabolism , Animals
15.
Article in English | MEDLINE | ID: mdl-23335896

ABSTRACT

The strength of individual synaptic contacts is considered a key modulator of information flow across circuits. Presynaptically the strength can be parsed into two key parameters: the size of the readily releasable pool (RRP) and the probability that a vesicle in that pool will undergo exocytosis when an action potential fires (Pv). How these variables are controlled and the degree to which they vary across individual nerve terminals is crucial to understand synaptic plasticity within neural circuits. Here we report robust measurements of these parameters in rat hippocampal neurons and their variability across populations of individual synapses. We explore the diversity of presynaptic Ca(2+) channel repertoires and evaluate their effect on synaptic strength at single boutons. Finally, we study the degree to which synapses can be differentially modified by a known potentiator of presynaptic function, forskolin. Our experiments revealed that both Pv and RRP spanned a large range, even for synapses made by the same axon, demonstrating that presynaptic efficacy is governed locally at the single synapse level. Synapses varied greatly in their dependence on N or P/Q type Ca(2+) channels for neurotransmission, but there was no association between specific channel repertoires and synaptic efficacy. Increasing cAMP concentration using forskolin enhanced synaptic transmission in a Ca(2+)-independent manner that was inversely related with a synapse's initial Pv, and independent of its RRP size. We propose a simple model based on the relationship between Pv and calcium entry that can account for the variable potentiation of synapses based on initial probability of vesicle fusion.

16.
Diabetes Res Clin Pract ; 91(2): 208-12, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21168233

ABSTRACT

INTRODUCTION: There are at least six subtypes of Maturity Onset Diabetes of the Young (MODY) with distinctive genetic causes. MODY 3 is caused by mutations in HNF1A gene, an insulin transcription factor, so mutations in this gene are associated with impaired insulin secretion. MODY 3 prevalence differs according to the population analyzed, but it is one of the most frequent subtypes. Therefore, our aims in this work were to find mutations present in the HNF1A gene and provide information on their prevalence. MATERIAL AND METHODS: Mutations screening was done in a group of 80 unrelated patients (average age 17.1 years) selected by clinical characterization of MODY, by SSCP electrophoresis followed by sequenciation. RESULTS: We found eight mutations, of which six were novel and four sequence variants, which were all novel. Therefore the prevalence of MODY 3 in this group was 10%. Compared clinical data between the non-MODY 3 patients and the MODY 3 diagnosed patients did not show any significant difference. DISCUSSION: Eight patients were diagnosed as MODY 3 and new data about the prevalence of that subtype is provided. Our results contribute to reveal novel mutations, providing new data about the prevalence of that subtype.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Adolescent , Adult , Argentina , Child , Child, Preschool , Female , Genetic Predisposition to Disease , Humans , Male , Mutation , White People , Young Adult
17.
BMC Neurosci ; 11: 109, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20809979

ABSTRACT

BACKGROUND: Human ß-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the ß-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. RESULTS: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels CONCLUSIONS: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.


Subject(s)
Amyloid beta-Protein Precursor/genetics , Brachyura/metabolism , Central Nervous System/metabolism , Memory/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Immunohistochemistry , Immunoprecipitation , Male , Molecular Sequence Data , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
18.
Article in English | MEDLINE | ID: mdl-20802854

ABSTRACT

Synapses are important functional units that determine how information flows through the brain. Understanding their biophysical properties and the molecules that underpin them is an important goal of cellular neuroscience. Thus, it is of interest to develop protocols that allow easy measurement of synaptic parameters in model systems that permit molecular manipulations. Here, we used a sensitive and high-time resolution optical approach that allowed us to characterize two functional parameters critical to presynaptic efficacy: vesicle fusion probability (Pv) and readily-releasable pool size (RRP). We implemented two different approaches to determine the RRP size that were in broad agreement: depletion of the RRP by high-frequency stimulation and saturation of the calcium sensor during single action potential stimuli. Our methods are based on reporters that provide a robust, quantitative, purely presynaptic readout and present a new avenue to study molecules that affect synaptic vesicle exocytosis.

20.
Science ; 316(5824): 570-4, 2007 Apr 27.
Article in English | MEDLINE | ID: mdl-17463283

ABSTRACT

Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.


Subject(s)
Dynamin I/physiology , Endocytosis , Neurons/physiology , Synapses/physiology , Synaptic Vesicles/physiology , Action Potentials , Animals , Cell Membrane/ultrastructure , Clathrin-Coated Vesicles/metabolism , Clathrin-Coated Vesicles/ultrastructure , Dynamin I/genetics , Dynamin II , Dynamin III/physiology , Electric Stimulation , Excitatory Postsynaptic Potentials , Exocytosis , Inhibitory Postsynaptic Potentials , Mice , Mice, Knockout , Microscopy, Electron , Neurons/ultrastructure , Patch-Clamp Techniques , Presynaptic Terminals/physiology , Presynaptic Terminals/ultrastructure , Synapses/ultrastructure , Synaptic Transmission , Synaptic Vesicles/ultrastructure
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