scholarly journals rsCaMPARI: an erasable marker of neuronal activity

2019 ◽  
Author(s):  
Fern Sha ◽  
Ahmed S. Abdelfattah ◽  
Ronak Patel ◽  
Eric R. Schreiter
Keyword(s):  
Neuronal Activity ◽  
Calcium Concentration ◽  
Fluorescent Protein ◽  
Light Exposure ◽  
Time Windows ◽  
Active Neuron ◽  
Violet Light ◽  
Neuron Populations ◽  
Activity Marker ◽  
Selection Of

AbstractIdentifying and comparing active neuron ensembles underlying complex behaviors is a key challenge in neuroscience. Recent tools such as CaMPARI have enabled the optical marking and selection of active neuron populations. However, CaMPARI photoconversion is permanent and irreversible, limiting its utility when multiple activity snapshots must be compared within the same sample. We sought to overcome these limitations by developing an erasable neuronal activity marker based on a reversibly switchable fluorescent protein. Here we introduce rsCaMPARI, a reversibly switchable calcium marker that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely-swimming zebrafish.

scholarly journals Erasable labeling of neuronal activity using a reversible calcium marker

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fern Sha ◽  
Ahmed S Abdelfattah ◽  
Ronak Patel ◽  
Eric R Schreiter
Keyword(s):  
Neural Activity ◽  
Calcium Concentration ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Light Exposure ◽  
Time Windows ◽  
Active Neuron ◽  
Neuron Populations ◽  
New Type ◽  
The Brain

Understanding how the brain encodes and processes information requires the recording of neural activity that underlies different behaviors. Recent efforts in fluorescent protein engineering have succeeded in developing powerful tools for visualizing neural activity, in general by coupling neural activity to different properties of a fluorescent protein scaffold. Here, we take advantage of a previously unexploited class of reversibly switchable fluorescent proteins to engineer a new type of calcium sensor. We introduce rsCaMPARI, a genetically encoded calcium marker engineered from a reversibly switchable fluorescent protein that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely swimming zebrafish.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

Selection of transgenic Petunia plants using the green fluorescent protein (GFP)

2011 ◽  
Vol 107 (3) ◽  
pp. 483-492 ◽  
Author(s):  
Viola Mußmann ◽  
Margrethe Serek ◽  
Traud Winkelmann
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Selection Of ◽  
Transgenic Petunia

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

Selection of antibody and light exposure regimens alters therapeutic effects of EGFR-targeted near-infrared photoimmunotherapy

2022 ◽  
Author(s):  
Ryuhei Okada ◽  
Takuya Kato ◽  
Aki Furusawa ◽  
Fuyuki Inagaki ◽  
Hiroaki Wakiyama ◽  
...  
Keyword(s):  
Near Infrared ◽  
Light Exposure ◽  
Therapeutic Effects ◽  
Selection Of

Effects of pentetrazol on neuronal activity and on extracellular calcium concentration in rat hippocampal slices

1990 ◽  
Vol 6 (3) ◽  
pp. 187-198 ◽  
Author(s):  
F.M. Leweke ◽  
J. Louvel ◽  
G. Rausche ◽  
U. Heinemann
Keyword(s):  
Neuronal Activity ◽  
Calcium Concentration ◽  
Hippocampal Slices ◽  
Extracellular Calcium ◽  
Extracellular Calcium Concentration

Effects Of Ultra Violet Light Exposure on Gated Sillicon Field Emitter Arrays

2021 ◽  
Author(s):  
Ranajoy Bhattacharya ◽  
Mason Canon ◽  
Nedeljko Karaulac ◽  
Girish Rughoobur ◽  
Winston Chern ◽  
...  
Keyword(s):  
Light Exposure ◽  
Ultra Violet ◽  
Field Emitter ◽  
Ultra Violet Light ◽  
Violet Light ◽  
Field Emitter Arrays

scholarly journals Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Nancy N Nichols ◽  
Ronald E Hector ◽  
Sarah E Frazer
Keyword(s):  
Green Fluorescent Protein ◽  
Genetic Transformation ◽  
Pure Culture ◽  
Ecological Niche ◽  
Fluorescent Protein ◽  
Microbial Consortium ◽  
Transformed Cells ◽  
Antibiotic Selection ◽  
Green Fluorescent ◽  
Selection Of

Abstract Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

scholarly journals Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 333-340 ◽  
Author(s):  
Jing Xu ◽  
Melissa A. Kirigiti ◽  
Michael A. Cowley ◽  
Kevin L. Grove ◽  
M. Susan Smith
Keyword(s):  
Neuropeptide Y ◽  
Neuronal Activity ◽  
Fluorescent Protein ◽  
Resting Membrane Potential ◽  
Inhibitory Effects ◽  
Gnrh Neurons ◽  
Cell Current ◽  
Green Fluorescent ◽  
Hypothalamic Slices

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 ± 2.86 vs. 7.04 ± 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 ± 0.02 vs. 0.37 ± 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from −56.7 ± 1.94 to −62.1 ± 1.83 mV; NPY’s effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity. Neuropeptide Y (NPY) directly hyperpolarizes GnRH neurons via postsynaptic NPY Y5 receptor. Increased inhibitory NPY tone during lactation is an important component of the suppression of GnRH neuronal activity.

scholarly journals Developing a Universal and Efficient Method for the Rapid Selection of Stable Fluorescent Protein-Tagged Pathogenic Vibrio Species

2020 ◽  
Vol 8 (10) ◽  
pp. 804
Author(s):  
Candice A. Thorstenson ◽  
Matthias S. Ullrich
Keyword(s):  
World Wide ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Optimal Growth ◽  
Vibrio Species ◽  
Delivery Vector ◽  
Pathogenic Vibrio ◽  
Human Infections ◽  
Selection Of

World-wide increases in Vibrio-associated diseases have been reported in aquaculture and humans in co-occurrence with increased sea surface temperatures. Twelve species of Vibrio are known to cause disease in humans, but three species dominate the number of human infections world-wide: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Fluorescent protein (FP)-labelled bacteria have been used to make great progress through in situ studies of bacterial behavior in mixed cultures or within host tissues. Currently, FP-labelling methods specific for Vibrio species are still limited by time-consuming counterselection measures that require the use of modified media and temperatures below the optimal growth temperature of many Vibrio species. Within this study, we used a previously reported R6K-based suicide delivery vector and two newly constructed transposon variants to develop a tailored protocol for FP-labelling V. cholerae, V. parahaemolyticus, and V. vulnificus environmental isolates within two days of counterselection against the donor Escherichiacoli. This herein presented protocol worked universally across all tested strains (30) with a conjugation efficiency of at least two transconjugants per 10,000 recipients.

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