G-CaMP5 is finally published!

The paper on G-CaMP5 has been published.

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 Caenorhabditischemosensory 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 vivoimaging 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.

This is the best fully-characterized GECI available, but publication of the paper was repeatedly delayed. Why? Because reviewers viewed it as ‘too incremental’ of an upgrade, and not worth publishing in a prominent journal (no, I’m not talking Nature or Science level) when the plasmids are already available.

A friendly suggestion for authors and future GCaMP6+ reviewers : You can’t have it both ways. If you want access to the best molecular tools before publication (GCaMP5 has been available for over a YEAR), you cannot turn around and say its not worth publishing because you already have the plasmid. Multiple post-docs spent years of their lives developing and carefully testing this tool. They deserve a quality publication for their efforts. Furthermore, the rigorous performance data collected NEEDS to be available to current and future users. Finally, there is no doubt that this will be a highly cited and viewed paper in whatever journal were to publish it. Our GCaMP3 paper already has 182 citations in less than 3 years, this may do even better.

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GCaMP6 candidate presentations

The first public presentation of data on candidates for GCaMP6 will be presented here.  This is obviously a big deal for brain imaging.

Reliable detection of single action potentials and synaptic calcium signals using improved genetically-encoded calcium indicators

*T.-W. CHEN, J. YU, R. A. KERR, V. JAYARAMAN, L. L. LOOGER, K. SVOBODA, D. S. KIM

FFF77 / 927.08Oct 17 – 4:00pm/5:00pm

and here

Engineering next generation GCaMP calcium indicators using neuron-based screening

T.-W. CHEN, T. J. WARDILL, J. P. HASSEMAN, G. TSEGAYE, B. F. FOSQUE, E. R. SCHREITER, B. E. KIMMEL, R. A. KERR, V. JAYARAMAN, K. SVOBODA, L. L. LOOGER, *D. S. KIM

DDD38 / 207.14Oct 14 – 9:00am/10:00am

Where to get new BrainWindows content

It’s been pretty quiet around here lately and will likely continue that way for some time. BrainWindows is not dead, just resting. Experiments, paper writing, new baby, etc. However, if you still need a fix of BrainWindows analysis, please follow me, @AndrewHires, on Twitter. If you don’t have a Twitter account, get one! It’s the easiest way to get published in the Library of Congress. I’ll be honing the analysis to fit into 140 character snippets. There will also be a much broader range of topics. Hopefully we can continue a quality scientific discourse publicly, in near-real time.

Cortical encoding of somatosensory input

Wednesday morning, I’m giving the first public presentation of my current work on sensory coding in the barrel cortex. The big picture question is, How does the cortex translate sensory input into perception and recognition of objects and their position?

To study this cortical function, we used a methodology with 3 critical components

1 – Goal-directed discrimination task

The activity of an awake cortex is profoundly different in character from an anesthetized cortex.  There is also gathering evidence that the activity of sensory cortex when attending to and using its sensory input to perform a discrimiation is quite diffent from periods of inattention.  We use a goal-directed discrimination task to enforce attention and to ensure the recorded activity is relevant to the fundamental function of the area.

2 – High speed neural recording techniques

Spikes are the words in the language of cortex. We must record them with high fidelity and temporal precision.  We can then use correlation of neural activity to sensory input to try to understand what the cells are saying.

3 – Precise measurement of sensory input

If we are to use correlational techniques to describe the language of cortex, we must match the millisecond precision of cortical communication with millisecond precision in measuring the exact sensory input.

Within the rodent, no study has yet combined all three components, goal directed discrimination behavior, millisecond precision neuronal recordings and millisecond precision sensory input recordings.

Anyway, if you want to hear more, try to get up in time to make it to the conference by 8:45AM.

Program#/Poster#:	750.04
Presentation Title:	Encoding of vibrissal sensory input and task-related variables in the mouse barrel cortex during whisker-based object localization
Location:	152B
Presentation time:	Wednesday, Nov 16, 2011, 8:45 AM – 9:00 AM

Useful ChR2 mouse lines

Guoping Feng’s group has released what looks to be a very useful set of BAC transgenic Channelrhodopsin mice.  The four lines specifically express YFP-tagged ChR2 in GABAergic, cholinergic, serotonergic and parvalbumin-expressing neurons.
They are well characterized and available from Jackson labs.

Fukushima soil contamination

I spent the last two nights arguing with a friend about the potential danger of radioactive fallout from Fukushima.  I contended any US fallout was negligible, he disagreed.  This led to reading a Science magazine post outlining the high soil contamination readings near Fukushima, and then to the real raw data, and comparisons to Chernobyl contamination.  We ran the numbers, and still disagree re: the US, but for Japan, they look bad.  If Chernobyl is a valid guide, which is a pretty conservative estimate of the caution of Japan’s government, there will be permanent closure of some towns that are >30km from the plant.

Here is a crude map I whipped up comparing the soil cesium-137 levels that led to restricted access to areas around Chernobyl, with that reported by the Japanese Ministry of Education, Sports, Science and Technology. Here is a directory with the compiled source data, updated daily.  The results are very disturbing.  Anything above 1500 kBq/m^2 was permanently closed near Chernobyl. Anything above 555  kBq/m^2 was permanently restricted.  If my topsoil conversion factors are correct, and they do match the expert’s assumptions in the Science mag story, then there are numerous villages above 555 kBq/m^2 level that are fairly far from the plant.  The 100 kBq/m^2 levels at a distance of 80km are also worryingly high. These aren’t cherry-picked readings to make things look dramatic, they are the rough average of many repeated samples across weeks.  No wonder the government finally upgraded the incident to a level 7. Click the picture for the big view.

Comparison to the whole of Europe is also illuminating, the numbers are the same scale as above.

$75,000 Prize for Neuronal Circuit Reconstruction

Want to make $75,000?  Good at algorithm design?  Read on…

Neuroscientists map the tree-like structure of nerve cells to better understand how networks of neurons assemble into circuits to enable complex behavior. Despite the advent of computer technology that enables mapping in three dimensions, neuronal reconstructions are still largely performed by hand and reconstructing a single cell may take months. The vast majority of axons (the long neuronal projections that transmit information to neighboring cells) and dendrites (the branches on nerve cells that receive information from neighboring cells) must be traced manually.

The lack of powerful – and effective – computational tools to automatically reconstruct neuronal arbors has emerged as a major technical bottleneck in neuroscience research.

Organizers of a new competition hope to provide incentives for the development of new computer algorithms to advance the field – including a cash prize of up to $75,000 for the qualifying winner.

The DIADEM Competition – short for Digital Reconstruction of Axonal and Dendritic Morphology – will bring together computational and experimental scientists to test the most promising new approaches against the latest data in a real-world environment.

The competition is open to individuals and teams from the private sector and academic laboratories.

Competitors will have a year to design an algorithm and to test it against the manual gold standard. Up to five finalists will compete in a tournament at the Janelia Farm Research Campus in August 2010.

The prize has been established by the Allen Institute for Brain Science and the Janelia Farm Research Campus of the Howard Hughes Medical Institute. The National Institutes of Health is providing support for a scientific conference that is independent of – but held in conjunction with – the tournament phase of the DIADEM Challenge.

http://www.diademchallenge.org/

Symposium Summary

Unfortunately a hard disk crash prevented detailed note taking at the conference.

Briefly :

Amy Palmer demonstrated a microfluidics device for multiple condition fluorescent assays in single mammalian cells. This will be useful to screen next generation calcium indicators.

Rob Campbell showed off multiplexed FRET imaging in live cells, using new FRET pairs generated in his lab.

Brian Bacskai has been observing how alzheimer’s disease affects astrocyte calcium dynamics in vivo, with bulk loaded dyes. Using Cameleons, he demonstrates the range of intracellular calcium in neuritis in healthy and diseased brain. Alzheimer’s placques cause about 20% of neuritis to have extremely elevated basal calcium levels, with a more pronounced effect closer to the plaque.

I showed some data comparing G-CaMP2, TN-XXL and D3cpv to an improved G-CaMP being developed in the Looger Lab.

Using an improved biotin ligase approach, Alice Ting presented evidence that neurexin/neuroligin is a synaptic stabilizer rather than synapse initiating protein.

Rex Kerr gave a talk on recent progress in planar illumination. In this manner, one can perform calcium imaging (using GECIs) in many neurons in a worm at the same time.

Jin Zhang has a new FRET reporter for JNK Kinase activity. It’s called JNKAR. Get it?

Michael Lin showed new results with bright, monomeric red-shifted fluorescent proteins. They look better then anything else on the market. More importantly, he has improved his TimeSTAMP technology by adding intrinsically fluorescent proteins to it. Bi-molecular fluorescence complementation tags newly synthesized protein be selectively tagged without immunostaining. This should make the in vivo imaging more robust.

Finally, Xiaokun Shu reported a new fluorecent protein that is excited and emits in the near infrared. This will be very powerful for imaging in deep tissues, as the spectra is in the transparency window out beyond the hemoglobin absorbance. Since its coming out in Science soon, I’ll hold off on a complete description of how it works. New scaffold, requires cofactor that is found in mammals.

Madoff fraud closes Picower Foundation

The Bernie Madoff Ponzi scheme has claimed a victim close to Neuroscience.  The Boston Globe reports that the Picower Foundation had a substantial amount of their endowment invested with Madoff and the losses are suffiecient to  shut down the foundation’s operations immediately. In 2002, the Picower Foundation gave the largest single gift MIT had ever received to that time, $50 million, which currently supports neuroscience research at the  Picower Center for Learning and Memory. I did two years of undergraduate research at the Center for Learning and Memory (the pre-gift name) and Brain Windows has featured research by current PCLM faculty.

The PCLM should be ok, as it appears the gift has been financially mangaed by MIT since 2002.  However, a recurring $200,000 annual gift for support of underrepresented minorities in their doctoral studies in science at MIT will not continue.

Top imaging papers in Chemical Biology 2006-08

Generation of new sensors for brain imaging often requires application of chemistry. If you are interested in recent developments in biological imaging tools from a chemical perspective, here is a collection of recent papers on the subject in ACS Chemical Biology.  Quite a few familiar faces in the list.

Editor-Selected Papers in IMAGING
“The imaging of biochemical events inside living cells and in vivo is crucial to our understanding of biological processes. Biomolecular imaging relies on appropriate probes and labeling technologies. And although powerful tools for the visualization of various biochemical activities have been developed over the last years, the majority of the cellular processes cannot yet be visualized. This defines one of the main challenges and opportunities in chemical biology.  “Since its introduction, ACS Chemical Biology has become a leading journal for top research papers and reviews that describe the generation and application of innovative tools for biomolecular imaging. The list below highlights some of these papers and we eagerly await the submission of new articles that report on further advances in this exciting field.”   ~ Kai Johnsson, Member, Board of Editors, ACS Chemical Biology
École Polytechnique Fédérale de Lausanne

Reading Dynamic Kinase Activity in Living Cells for High-throughput Screening
Michael D. Allen, Lisa M. DiPilato, Meghdad Rahdar, Yunzhao R. Ren, Curtis Chong, Jun O. Liu, and Jin Zhang
ACS Chem. Biol., (Letter), 2006, 1(6), 371-376. DOI: 10.1021/cb600202f

HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
Georgyi V. Los, Lance P. Encell, Mark G. McDougall, Danette D. Hartzell, Natasha Karassina, Chad Zimprich, Monika G. Wood, Randy Learish, Rachel Friedman Ohana, Marjeta Urh, Dan Simpson, Jacqui Mendez, Kris Zimmerman, Paul Otto, Gediminas Vidugiris, Ji Zhu, Aldis Darzins, Dieter H. Klaubert, Robert F. Bulleit, and Keith V. Wood
ACS Chem. Biol., (Article), 2008, 3(6), 373-382. DOI: 10.1021/cb800025k

A Comparative Study of Bioorthogonal Reactions with Azides
Nicholas J. Agard, Jeremy M. Baskin, Jennifer A. Prescher, Anderson Lo, and Carolyn R. Bertozzi
ACS Chem. Biol., (Letter), 2006, 1(10), 644-648. DOI: 10.1021/cb6003228

Measuring Picomolar Intracellular Exchangeable Zinc in PC-12 Cells Using a Ratiometric Fluorescence Biosensor
Rebecca A. Bozym, Richard B. Thompson, Andrea K. Stoddard, and Carol A. Fierke
ACS Chem. Biol., (Article), 2006, 1(2), 103-111. DOI: 10.1021/cb500043a

A Survey of Single-Molecule Techniques in Chemical Biology
Peter V. Cornish and Taekjip Ha
ACS Chem. Biol., (Review), 2007, 2(1), 53-61. DOI: 10.1021/cb600342a

Fluorogenic Label for Biomolecular Imaging
Luke D. Lavis, Tzu-Yuan Chao, and Ronald T. Raines
ACS Chem. Biol., (Article), 2006, 1(4), 252-260. DOI: 10.1021/cb600132m

Chemical Tools for Biomolecular Imaging
Nils Johnsson and Kai Johnsson
ACS Chem. Biol., (Review), 2006, 2(1), 31-38. DOI: 10.1021/cb6003977

Mechanism-Based Probe for the Analysis of Cathepsin Cysteine Proteases in Living Cells
Howard C. Hang, Joana Loureiro, Eric Spooner, Adrianus W. M. van der Velden, You-Me Kim, Annette M. Pollington, Rene Maehr, Michael N. Starnbach, and Hidde L. Ploegh
ACS Chem. Biol., (Article), 2006, 1(11), 713-723. DOI: 10.1021/cb600431a

Fluorogenic Phospholipids as Head Group-Selective Reporters of Phospholipase A Activity
Tyler M. Rose and Glenn D. Prestwich
ACS Chem. Biol., (Article), 2006, 1(2), 83-92. DOI: 10.1021/cb5000014

Imaging Distinct Conformational States of Amyloid- Fibrils in Alzheimer’s Disease Using Novel Luminescent Probes
K. Peter R. Nilsson, Andreas Åslund, Ina Berg, Sofie Nyström, Peter Konradsson, Anna Herland, Olle Inganäs, Frantz Stabo-Eeg, Mikael Lindgren, Gunilla T. Westermark, Lars Lannfelt, Lars N. G. Nilsson, and Per Hammarström
ACS Chem. Biol., (Article), 2007, 2(8), 553-560. DOI: 10.1021/cb700116u

Evolving the Substrate Specificity of O6-Alkylguanine-DNA Alkyltransferase through Loop Insertion for Applications in Molecular Imaging
Christian Heinis, Simone Schmitt, Maik Kindermann, Guillaume Godin, and Kai Johnsson
ACS Chem. Biol., (Article), 2006, 1(9), 575-584. DOI: 10.1021/cb6003146

Novel Genetically Encoded Biosensors Using Firefly Luciferase
Frank Fan, Brock F. Binkowski, Braeden L. Butler, Peter F. Stecha, Martin K. Lewis, and Keith V. Wood
ACS Chem. Biol., (Letter), 2008, 3(6), 346-351. DOI: 10.1021/cb8000414

An Integrated-Molecule-Format Multicolor Probe for Monitoring Multiple Activities of a Bioactive Small Molecule
Sung Bae Kim, Yoshio Umezawa, Kira A. Kanno, and Hiroaki Tao
ACS Chem. Biol., (Article), 2008, 3(6), 359-372. DOI: 10.1021/cb800004s

Coiled-Coil Tag–Probe System for Quick Labeling of Membrane Receptors in Living Cells
Yoshiaki Yano, Akiko Yano, Shinya Oishi, Yukihiko Sugimoto, Gozoh Tsujimoto, Nobutaka Fujii, and Katsumi Matsuzaki
ACS Chem. Biol., (Letter), 2008, 3(6), 341-345. DOI: 10.1021/cb8000556

Molecular Electron Microscopy: State of the Art and Current Challenges
Henning Stahlberg and Thomas Walz
ACS Chem. Biol., (Review), 2008, 3(5), 268-281. DOI: 10.1021/cb800037d

Bright Ideas for Chemical Biology
Luke D. Lavis and Ronald T. Raines
ACS Chem. Biol., (Review), 2008, 3(3), 142-155. DOI: 10.1021/cb700248m

Simultaneous Recording of Multiple Cellular Events by FRET
Alen Piljic and Carsten Schultz
ACS Chem. Biol., (Letter), 2008, 3(3), 156-160. DOI: 10.1021/cb700247q