83 research outputs found
A genetically encoded reporter of synaptic activity in vivo
To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses
Circular Permutation of Red Fluorescent Proteins
Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C- terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments
Selective Gene Expression by Postnatal Electroporation during Olfactory Interneuron Neurogenesis
Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis
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
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
Physiology and cell biology of acupuncture observed in calcium signaling activated by acoustic shear wave
Monitoring neural activity with bioluminescence during natural behavior
Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca^(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior
Chemoreception Regulates Chemical Access to Mouse Vomeronasal Organ: Role of Solitary Chemosensory Cells
Controlling stimulus access to sensory organs allows animals to optimize sensory reception and prevent damage. The vomeronasal organ (VNO) detects pheromones and other semiochemicals to regulate innate social and sexual behaviors. This semiochemical detection generally requires the VNO to draw in chemical fluids, such as bodily secretions, which are complex in composition and can be contaminated. Little is known about whether and how chemical constituents are monitored to regulate the fluid access to the VNO. Using transgenic mice and immunolabeling, we found that solitary chemosensory cells (SCCs) reside densely at the entrance duct of the VNO. In this region, most of the intraepithelial trigeminal fibers innervate the SCCs, indicating that SCCs relay sensory information onto the trigeminal fibers. These SCCs express transient receptor potential channel M5 (TRPM5) and the phospholipase C (PLC) β2 signaling pathway. Additionally, the SCCs express choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) for synthesizing and packaging acetylcholine, a potential transmitter. In intracellular Ca2+ imaging, the SCCs responded to various chemical stimuli including high concentrations of odorants and bitter compounds. The responses were suppressed significantly by a PLC inhibitor, suggesting involvement of the PLC pathway. Further, we developed a quantitative dye assay to show that the amount of stimulus fluid that entered the VNOs of behaving mice is inversely correlated to the concentration of odorous and bitter substances in the fluid. Genetic knockout and pharmacological inhibition of TRPM5 resulted in larger amounts of bitter compounds entering the VNOs. Our data uncovered that chemoreception of fluid constituents regulates chemical access to the VNO and plays an important role in limiting the access of non-specific irritating and harmful substances. Our results also provide new insight into the emerging role of SCCs in chemoreception and regulation of physiological actions
Molecular Mechanism of a Green-Shifted, pH-Dependent Red Fluorescent Protein mKate Variant
Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies
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