445 research outputs found
Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.
Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators
Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics
Faithful reporting of temporal patterns of intracellular Ca
2
+
dynamics requires the working range
of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent
indicators are likely to distort fast Ca
2
+
signals by apparent saturation and integration due to their
limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of
Ca
2
+
affinities and accelerated kinetics by weakening the Ca
2
+
-calmodulin-peptide interactions. At
37
°C, the GCaMP3-derived probe termed GCaMP3
fast
is 40-fold faster than GCaMP3 with Ca
2
+
decay
and rise times,
t
1/2
, of 3.3
ms and 0.9
ms, respectively, making it the fastest to-date. GCaMP3
fast
revealed discreet transients with significantly faster Ca
2
+
dynamics in neonatal cardiac myocytes
than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca
2
+
at 940
nm,
GCaMP3
fast
is suitable for deep tissue studies. The green fluorescent protein serves as a reporter
providing important novel insights into the kinetic mechanism of target recognition by calmodulin.
Our strategy to match the probe to the signal by tuning the affinity and hence the Ca
2
+
kinetics of
the indicator is applicable to the emerging new generations of calmodulin-based probe
A Chemogenetic Approach for the Optical Monitoring of Voltage in Neurons
Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and sub-threshold activity in neurons
Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy
3D functional imaging of neuronal activity in entire organisms at single cell
level and physiologically relevant time scales faces major obstacles due to
trade-offs between the size of the imaged volumes, and spatial and temporal
resolution. Here, using light-field microscopy in combination with 3D
deconvolution, we demonstrate intrinsically simultaneous volumetric functional
imaging of neuronal population activity at single neuron resolution for an
entire organism, the nematode Caenorhabditis elegans. The simplicity of our
technique and possibility of the integration into epi-fluoresence microscopes
makes it an attractive tool for high-speed volumetric calcium imaging.Comment: 25 pages, 7 figures, incl. supplementary informatio
Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.
To uncover the circuit-level alterations that underlie atypical sensory processing associated with autism, we adopted a symptom-to-circuit approach in the Fmr1-knockout (Fmr1-/-) mouse model of Fragile X syndrome. Using a go/no-go task and in vivo two-photon calcium imaging, we find that impaired visual discrimination in Fmr1-/- mice correlates with marked deficits in orientation tuning of principal neurons and with a decrease in the activity of parvalbumin interneurons in primary visual cortex. Restoring visually evoked activity in parvalbumin cells in Fmr1-/- mice with a chemogenetic strategy using designer receptors exclusively activated by designer drugs was sufficient to rescue their behavioral performance. Strikingly, human subjects with Fragile X syndrome exhibit impairments in visual discrimination similar to those in Fmr1-/- mice. These results suggest that manipulating inhibition may help sensory processing in Fragile X syndrome
Politiebestel in balans? Politiek-bestuurlijke sturing, democratische verantwoording, samenwerking en maatschappelijke inbedding in een nationaal politiebestel
Op 1 januari 2013 trad de Politiewet 2012 in werking. Artikel 74 van de wet bepaalt dat de Minister van Justitie en Veiligheid binnen vijf jaar na inwerkingtreding een evaluatie van die wet aan de Staten-Generaal moet sturen over doeltreffendheid en effecten van de wet in de praktijk.In dit rapport staat de vraag centraal hoe de Politiewet 2012 in de praktijk is geïmplementeerd en welke consequenties zich voordoen voor de politieorganisatie en voor de verschillende externe partijen die bij het politiewerk betrokken zijn. De hoofdvraag die aan dit onderzoek ten grondslag ligt, luidt: Hoe kan de werking van de Politiewet 2012 inzake de thema’s (1) politiek bestuurlijke sturing en politiek-democratische verantwoording, (2) samenwerking en (3) maatschappelijke inbedding, worden geëvalueerd?Security and Global Affair
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Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.Molecular and Cellular Biolog
Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling
In drug discovery, prediction of selectivity and toxicity
require the evaluation of cellular calcium homeostasis. The rat
is a preferred laboratory animal for pharmacology and
toxicology studies, while currently no calcium indicator
protein expressing rat model is available. We established a
transgenic rat strain stably expressing the GCaMP2
fluorescent calcium sensor by a transposon-based methodology.
Zygotes were co-injected with mRNA of transposase and a CAG-
GCaMP2 expressing construct, and animals with one
transgene copy were pre-selected by measuring fluorescence in
blood cells. A homozygous rat strain was generated with high
sensor protein expression in the heart, kidney, liver, and
blood cells. No pathological alterations were found in these
animals, and fluorescence measurements in cardiac tissue slices
and primary cultures demonstrated the applicability of this
system for studying calcium signaling. We show here that the
GCaMP2 expressing rat cardiomyocytes allow the
prediction of cardiotoxic drug side-effects, and provide
evidence for the role of Na+/Ca2+ exchanger and its beneficial
pharmacological modulation in cardiac reperfusion. Our data
indicate that drug-induced alterations and pathological
processes can be followed by using this rat model, suggesting
that transgenic rats expressing a calcium-sensitive protein
provide a valuable system for pharmacological and toxicological
studies
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