Bidirectional in vivo structural dendritic spine plasticity revealed by two-photon glutamate uncaging in the mouse neocortex

Abstract Most excitatory synapses in the brain form on dendritic spines. Two-photon uncaging of glutamate is widely utilized to characterize the structural plasticity of dendritic spines in brain slice preparations in vitro. In the present study, glutamate uncaging was used to investigate spine plasticity, for the first time, in vivo. A caged glutamate compound was applied to the surface of the mouse visual cortex in vivo, revealing the successful induction of spine enlargement by repetitive two-photon uncaging in a magnesium free solution. Notably, this induction occurred in a smaller fraction of spines in the neocortex in vivo (22%) than in hippocampal slices (95%). Once induced, the time course and mean long-term enlargement amplitudes were similar to those found in hippocampal slices. However, low-frequency (1–2 Hz) glutamate uncaging in the presence of magnesium caused spine shrinkage in a similar fraction (35%) of spines as in hippocampal slices, though spread to neighboring spines occurred less frequently than it did in hippocampal slices. Thus, the structural plasticity may occur similarly in the neocortex in vivo as in hippocampal slices, although it happened less frequently in our experimental conditions.

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Stringent structural plasticity of dendritic spines revealed by two-photon glutamate uncaging in adult mouse neocortex in vivo

10.1101/577742 ◽

2019 ◽

Author(s):

Jun Noguchi ◽

Akira Nagaoka ◽

Tatsuya Hayama ◽

Hasan Ucar ◽

Sho Yagishita ◽

...

Keyword(s):

Time Course ◽

Hippocampal Slices ◽

Low Frequency ◽

Structural Plasticity ◽

Two Photon ◽

Low Magnesium ◽

Adult Mice ◽

First Time

AbstractTwo-photon uncaging of glutamate is widely utilized to characterize structural plasticity in brain slice preparations in vitro. In this study, we investigated spine plasticity by using, for the first time, glutamate uncaging in the neocortex of adult mice in vivo. Spine enlargement was successfully induced in a smaller fraction of spines in the neocortex (22%) than in young hippocampal slices (95%), even under a low magnesium condition. Once induced, the time course and mean amplitudes of long-term enlargement were the same (81%) as those in vitro. However, low-frequency (1–2 Hz) glutamate uncaging caused spine shrinkage in a similar fraction (34%) of spines as in vitro, but spread to the neighboring spines less frequently than in vitro. Thus, we found that structural plasticity can occur similarly in the adult neocortex in vivo as in the hippocampus in vitro, although it happens stringently in a smaller subset of spines.

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In Vivo Calcium Imaging of Circuit Activity in Cerebellar Cortex

Journal of Neurophysiology ◽

10.1152/jn.01013.2004 ◽

2005 ◽

Vol 94 (2) ◽

pp. 1636-1644 ◽

Cited By ~ 84

Author(s):

Megan R. Sullivan ◽

Axel Nimmerjahn ◽

Dmitry V. Sarkisov ◽

Fritjof Helmchen ◽

Samuel S.-H. Wang

Keyword(s):

Cerebellar Cortex ◽

Calcium Imaging ◽

Time Course ◽

Molecular Layer ◽

Neuronal Cell ◽

Calcium Transients ◽

Two Photon ◽

Neuronal Cell Bodies

In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-d-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.

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3dSpAn: An interactive software for 3D segmentation and analysis of dendritic spines

10.1101/864587 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nirmal Das ◽

Ewa Baczynska ◽

Monika Bijata ◽

Blazej Ruszczycki ◽

Andre Zeug ◽

...

Keyword(s):

Dendritic Spines ◽

Ex Vivo ◽

Three Dimensional ◽

Interactive Software ◽

Seed Selection ◽

Two Photon Microscopy ◽

Two Photon ◽

Observation Methods

AbstractThree dimensional segmentation and analysis of dendritic spines involve two major challenges: 1) how to segment individual spines from the dendrites and 2) how to quantitatively assess the morphology of individual spines. We developed a software named 3dSpAn to address these two issues by implementing our previously published 3D multiscale opening algorithm in shared intensity space and using effective morphological features for individual dendritic spine plasticity analysis. 3dSpAn consists of four modules: Preprocessing and ROI selection, Intensity thresholding and seed selection, Multiscale segmentation and Quantitative morphological feature extraction. We show the results of segmentation and morphological analysis for different observation methods, including in vitro and ex vivo imaging with confocal microscopy, and in vivo samples, using high-resolution two-photon microscopy. The software is freely available, the source code, windows installer, the software manual and video tutorial can be obtained from: https://sites.google.com/view/3dSpAn/.

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Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646468 ◽

1991 ◽

Vol 66 (05) ◽

pp. 609-613 ◽

Cited By ~ 14

Author(s):

I R MacGregor ◽

J M Ferguson ◽

L F McLaughlin ◽

T Burnouf ◽

C V Prowse

Keyword(s):

High Purity ◽

Time Course ◽

Prothrombin Complex Concentrate ◽

Degradation Products ◽

Canine Model ◽

Factor Ix ◽

In Vitro Tests ◽

Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

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Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650190 ◽

1981 ◽

Vol 45 (03) ◽

pp. 290-293 ◽

Cited By ~ 9

Author(s):

Peter H Levine ◽

Danielle G Sladdin ◽

Norman I Krinsky

Keyword(s):

Superoxide Dismutase ◽

Cytochrome C ◽

Xanthine Oxidase ◽

Direct Effect ◽

Platelet Function ◽

Experimental Conditions ◽

Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Applicability of Instability Index for In vitro Protein Stability Prediction

Protein and Peptide Letters ◽

10.2174/0929866526666190228144219 ◽

2019 ◽

Vol 26 (5) ◽

pp. 339-347 ◽

Cited By ~ 4

Author(s):

Dilani G. Gamage ◽

Ajith Gunaratne ◽

Gopal R. Periyannan ◽

Timothy G. Russell

Keyword(s):

Protein Stability ◽

Caulobacter Crescentus ◽

Effect Of Temperature ◽

Instability Index ◽

Dipeptide Composition ◽

Experimental Conditions ◽

The Stability ◽

Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

Acta Endocrinologica ◽

10.1530/acta.0.1080511 ◽

1985 ◽

Vol 108 (4) ◽

pp. 511-517 ◽

Cited By ~ 12

Author(s):

Nandalal Bagchi ◽

Birdie Shivers ◽

Thomas R. Brown

Keyword(s):

Time Course ◽

Period 2 ◽

Iodotyrosine Deiodinase ◽

Rate Of Hydrolysis ◽

Underlying Mechanisms ◽

Excess Iodide ◽

Sites Of Action ◽

Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

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Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker

Scientific Reports ◽

10.1038/s41598-020-79903-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Skaidre Jankovskaja ◽

Johan Engblom ◽

Melinda Rezeli ◽

György Marko-Varga ◽

Tautgirdas Ruzgas ◽

...

Keyword(s):

Skin Cancer ◽

Relative Increase ◽

Cancer Biomarker ◽

Cancer Diagnostics ◽

Sampling Time ◽

Skin Permeability ◽

Experimental Conditions ◽

Non Invasive

AbstractThe tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

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Factors affecting in vitro and in vivo antioxidant effects. Experimental conditions and nature of oxidants determine antioxidant efficacy

Journal of Berry Research ◽

10.3233/jbr-200695 ◽

2021 ◽

pp. 1-9

Author(s):

Etsuo Niki

Keyword(s):

Biological Molecules ◽

Experimental Conditions ◽

Factors Affecting ◽

Beneficial Effects ◽

Multiple Oxidants ◽

Antioxidant Effects

Reactive oxygen and nitrogen species have been implicated in the onset and progression of various diseases and the role of antioxidants in the maintenance of health and prevention of diseases has received much attention. The action and effect of antioxidants have been studied extensively under different reaction conditions in multiple media. The antioxidant effects are determined by many factors. This review aims to discuss several important issues that should be considered for determination of experimental conditions and interpretation of experimental results in order to understand the beneficial effects and limit of antioxidants against detrimental oxidation of biological molecules. Emphasis was laid on cell culture experiments and effects of diversity of multiple oxidants on antioxidant efficacy.

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