Han Z.,John B. Pierce Laboratory Inc. |
Han Z.,Yale University |
Jin L.,Yale University |
Chen F.,University of Connecticut |
And 9 more authors.
PLoS ONE | Year: 2014
ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins. © 2014 Han et al.
Han Z.,Yale University |
Han Z.,John B. Pierce Laboratory Inc. |
Jin L.,Yale University |
Platisa J.,John B. Pierce Laboratory Inc. |
And 7 more authors.
PLoS ONE | Year: 2013
We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (T1-on ∼10 ms, T2-on ∼ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (T) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (∼35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals. © 2013 Han et al.
Gruber D.F.,City University of New York |
Gruber D.F.,American Museum of Natural History |
Gaffney J.P.,City University of New York |
Mehr S.,New York University |
And 6 more authors.
PLoS ONE | Year: 2015
We report the identification and characterization of two new members of a family of bilirubininducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. © 2015 Gruber et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Klein M.,Harvard University |
Afonso B.,Harvard University |
Afonso B.,Janelia Farm Research Campus |
Vonner A.J.,Harvard University |
And 16 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015
Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.
Agency: NSF | Branch: Continuing grant | Program: | Phase: OFFICE OF MULTIDISCIPLINARY AC | Award Amount: 735.32K | Year: 2015
This project was developed at an NSF Ideas Lab on Cracking the Olfactory Code and is jointly funded by the Physics of Living Systems program in the Physics Division, the Mathematical Biology program in the Division of Mathematical Sciences, the Chemistry of Life Processes program in the Chemistry Division, and the Neural Systems Cluster in the Division of Integrative Organismal Systems. The project is a synergistic combination of laboratory experiments and computer modeling that will lead to better understanding of how animals use the sense of smell to navigate in the real world. Almost universally, from flies to mice to dogs, animals use odors to find critical resources, such as food, shelter, and mates. To date, no engineered device can replicate this function and understanding the code used by the brain will lead to many novel applications. Cracking codes, from neural codes to the Enigma code of WWII, is aided by a deep understanding of the content of messages that are being transmitted and how they will be used by their intended receivers. To crack the olfactory code, the team will focus on how odors move in landscapes, how animals extract spatial and temporal cues from odor landscapes, and how they use movement for enhancing these cues while progressing towards their targets. The proposed work encompasses physical measurement of odor plumes, behavioral measurement of animals paths through olfactory environments, electrophysiological and optical measurement of neural activity during olfactory navigation, perturbations of the environment via virtual reality and of neuronal hardware via genetics, and multilevel mathematical modeling. The PIs will teach and work with undergraduate, graduate and postdoctoral students and especially recruit students from underrepresented groups in science. The projects results may lead to improved methods for the detection of explosives, new olfactory robots to replace trained animals, and new theoretically-grounded advances in robotic control. The project will inform the development of technologies that interfere with the ability of flying insects (including disease vectors and crop pests) to locate their odor target, thus opening a new door for developing green technologies to solve problems that are of global economic and humanitarian importance.
This proposal is a synergistic combination of laboratory experiments and computational modeling that will probe how animals use olfaction to navigate in their environment. Specifically, this effort seeks to solve the difficult problem of olfactory navigation through the following aims: (i) Generate and quantify standardized, naturalistic odor environments that can be used to perform empirical and theoretical tests of navigation strategies; (ii) Determine phenomenological algorithms for odor-guided navigation through behavioral experiments in diverse animal species; (iii) Determine how odor cues for navigation are encoded and used in the nervous system by recording neuronal data and simulating putative neural circuits that implement these processes; (iv) Manipulate olfactory environments and neural circuitry, to evaluate model robustness. In contrast to previous attempts to understand olfactory navigation, the present strategy emphasizes mechanisms that are biologically feasible and explores the wide range of temporal and spatial scales in which animals successfully navigate. The project will generate datasets of immediate use and importance to scientists in theoretical biology and mathematics, engineering (fluid mechanics, electronic olfaction, and robotics) and biology (neuroscience, ecology and evolution).
Agency: NSF | Branch: Continuing grant | Program: | Phase: ACTIVATION | Award Amount: 560.80K | Year: 2011
This project will examine how neural activity in the medial frontal (or cingulate) cortex changes as new behavioral tasks are learned for the first time. The central issue is to understand how activity in this part of the brain allows for learning from past mistakes in order to avoid future failures. The experimental approach involves recording from chronically implanted arrays of electrodes in the medial frontal cortex of rats as the animals learn to perform reaction time tasks. These tasks are commonly used to study cognitive processing in human beings. The expected results will include testing a leading hypothesis in neuroscience that persistent activity in medial frontal cortex enables self-control, e.g. the ability to wait calmly before acting. We predict that this activity will develop in the medial frontal cortex during learning and will come to predict how well animals are able to adjust performance after mistakes are made. The potential impact of the study is that it will provide some of the first data on how error-related activity develops in this part of the brain during learning. Broader impact activities include data and computer resource sharing on a website, a multi-lingual childrens program on the brain, and participation by undergraduates from a non-traditional research university in cutting-edge neurobiological research.