Journal Scan – Transynaptic tracing, fly olfaction, fast super-resolution, localization of perception

Here’s a group of four recent papers that are worth checking out but I don’t have the time to cover.  The first provides a set of tools for neuronal circuit tracing. The second pushes super-resolution imaging into fast, live-cell imaging.  The third, by a friend from graduate school, uses G-CaMP to make strong claims about olfactory coding in fruit flies. The last reports remarkable data pointing to the distributed nature of conscious perception in humans, which would have been a great data set to reference in my recent talk on free will.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools 

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

Super-resolution video microscopy of live cells by structured illumination

Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

Select Drosophila glomeruli mediate innate olfactory attraction and aversion.

Fruitflies show robust attraction to food odours, which usually excite several glomeruli. To understand how the representation of such odours leads to behaviour, we used genetic tools to dissect the contribution of each activated glomerulus. Apple cider vinegar triggers robust innate attraction at a relatively low concentration, which activates six glomeruli. By silencing individual glomeruli, here we show that the absence of activity in two glomeruli, DM1 and VA2, markedly reduces attraction. Conversely, when each of these two glomeruli was selectively activated, flies showed as robust an attraction to vinegar as wild-type flies. Notably, a higher concentration of vinegar excites an additional glomerulus and is less attractive to flies. We show that activation of the extra glomerulus is necessary and sufficient to mediate the behavioural switch. Together, these results indicate that individual glomeruli, rather than the entire pattern of active glomeruli, mediate innate behavioural output.

Movement Intention After Parietal Cortex Stimulation in Humans

Parietal and premotor cortex regions are serious contenders for bringing motor intentions and motor responses into awareness. We used electrical stimulation in seven patients undergoing awake brain surgery. Stimulating the right inferior parietal regions triggered a strong intention and desire to move the contralateral hand, arm, or foot, whereas stimulating the left inferior parietal region provoked the intention to move the lips and to talk. When stimulation intensity was increased in parietal areas, participants believed they had really performed these movements, although no electromyographic activity was detected. Stimulation of the premotor region triggered overt mouth and contralateral limb movements. Yet, patients firmly denied that they had moved. Conscious intention and motor awareness thus arise from increased parietal activity before movement execution.

Infrared fluorescent proteins

Hunting for new fluorescent proteins in the coral reefs of the Caribbean and Australia is a task that a lucky few researchers have managed to get funding for. Scuba diving in some of the world’s most beautiful places; it’s not a bad gig, if you can get it.  Most fluorescent protein scientists are confined to a lab, mutating existing fluorescent proteins from jellyfish and coral. Shifting their excitation and emission spectra has allowed multiple fluorescent proteins to be used as molecular highlighters at the same time, since their colors are distinct from each other. Some members of this palette are shown in Brain Windows top image bar.  After over a decade of research, the spectrum is pretty well covered.  Except for one area…  The infrared.

The near-infrared band is an area of enormous importance to scientific researchers, because is contains the spectral window where the body becomes transparent. Hemoglobin in the blood strongly absorbs visible wavelengths shorter than 650nm, while water absorbs wavelengths above 900nm.  If a fluorescent protein could be found, or engineered to have excitation and emission within this window, we could use it to peer much deeper into the body. The near-IR light penetrates much more easily into and out of the tissue.  This is easily seen by pressing a flashlight against your hand.  Only the deep red light passes through. The quest for an infrared fluorescent protein has preoccupied several labs for a decade.

Efforts to push FPs out to the infrared resulted in mCherry, mPlum, mKate, among others.  The further red-shifting of these proteins is constrained by the space limitations of the beta-barrel structure of GFP-like proteins.  In general, the longer the resonance chain of the chromophore, the longer the wavelength of the chromophore’s excitation and emission spectra.  It has been difficult to extend the FP spectra beyond 650nm without adding an additional bond to the resonance structure, for which there is little space left in the protected center of the barrel.  

 

Wavelength of excitation and emission is longer in larger resonance structures.

In Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome Shu et al. of Roger Tsien’s lab, looked beyond traditionally used fluorescent proteins to extend the palette into the near-IR.  They engineered a protein, IFP, which binds biliverdin, a natural product involved in aerobic respiration (and similar in structure to the phytocyanobilins discussed in our Photoactivated Transcription journal club post). Biliverdin is non-fluorescent in solution, but when bound to IFP, it is rigidized and becomes fluorescent, with excitation at 684nm and emission at 708nm.  IFP can then be fused to the protein of interest and visualized through thick absorbent tissue.  Even the liver, which is dense with heme, is easily seen through the skin when labelled with IFP.  

 

 

 

IFP1.1 expressed in mouse liver is clearly visible through the skin

 

IFP1.4 should immediately push forward the field of in-vivo imaging of cancer and other diagnostics, at least in animal models.  It’s not clear yet how useful it will be for in-vivo brain imaging, they show cultured neurons expressing IFP1.4 become fluorescent upon biliverdin application, but can biliverdin be effectively delivered to neurons in vivo? Like channelrhodopsin, there may be sufficent amounts of endogenous co-factor to make the protein useful without exogenous application. Perhaps of greater importance is the new engineering avenues IFP opens up.  This is an entirely new, two-component scaffold, with different characteristics from GFP that protein engineers will be able to optimize and exploit.   Over the next decade, IFP may spawn as diverse a set of tools as GFP has over the previous one.

Deisseroth is on fire

Is there any biology lab hotter that Karl Deisseroth’s right now?  In the last TWO WEEKS he’s authored

3 Nature papers

• Parvalbumin neurons and gamma rhythms enhance cortical circuit performance
• Driving fast-spiking cells induces gamma rhythm and controls sensory responses
• Temporally precise in vivo control of intracellular signalling

2 Science papers

• Phasic Firing in Dopaminergic Neurons Is Sufficient for Behavioral Conditioning
• Optical Deconstruction of Parkinsonian Neural Circuitry

and a PLOS One for icing.

• Manipulation of an innate escape response in Drosophila: photoexcitation of acj6 neurons induces the escape response

He’s the Xander Cage of neuroscience, having just triggered an avalanche, he manages to move quickly enough to stay ahead as his pursuers get mowed down by the rapidly accelerating barrage of papers.  Over the next year, we are going to see hundreds of ChR2 papers coming out, making it really hard to stand out from the crowd.

$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/

Philosophy : The Science of Free Will

Over the weekend I had the opportunity to deliver a Message to a Unitarian Universalist congregation in my hometown.  The topic?  The Science of Free Will : What science does and does not tell us about our ability to choose. It was quite a challenge translating some recent scientific results into layperson terms, while tying the results to more philosophical issues.  I think the sermon went well though.  The churchgoers are almost dogmatically anti-dogma and openminded, with many questions that were dificult to answer. It was a great experience to get outside the bubble of science. Below are my prepared remarks, led off by two readings from Hippocrates and David Foster Wallace. An audio link will be up in a few days. The full version of the David Foster Wallace reading is available here, and is well worth the 10 minute reading time.

The Science of Free Will – Andrew Hires

Non-desensitizing Channelrhodopsin

One of the problems with Channelrhodopsin-2 is that it desensitizes during continuous illumination or at high illumination frequencies. This limits the application of ChR2 to systems where high spike rates are not used to code information.

Desensitization of various Channelrhodopsins

In Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics, John Lin reports a new Channelrhodopsin, ChIEF, that has increased single channel conductance, faster kinetics and much less desensitization. This allows much higher rates of action potential generation. The in vitro results look great!

Success of action potential firing for ChR2 and ChIEF for various frequencies

However, ChR2 has a relatively small effective expression window.  Too little expression and you cannot drive spiking.  Too high and the cell gets sick.  Tuning this expression is finicky. For ChIEF, it remains to be seen how the lack of desensitization, which might cause additional current leak in the resting state, effects this expression window in vivo. It certainly looks like its worth testing in your system of choice.

Background : Perceval, the ATP:ADP sensor

Recently, Brain Windows mentioned the report A genetically encoded fluorescent reporter of ATP:ADP ratio. We invited Dr. Jim Berg, the lead author of the study to provide additional background to our readers. Below, Jim provides a fascinating look at rationale behind sensor development.  I really like that they came at this problem with a biological question in mind, something I would recommend before anyone start the development of a genetically encoded indicator.

 

A pixel-by-pixel ratio of the 490 nm excitation image by the 430 nm excitation image from two cultured HEK293 cells expressing Perceval during control conditions (left) and after 40 min of metabolic inhibition with 5 mM 2-deoxyglucose (right)

 

Here’s a little insight into why we decided to develop a fluorescent sensor for cellular energy, and how Perceval evolved. One of the primary research interests of the Yellen lab is the interaction between diet and epilepsy. The ketogenic diet, a high fat, low carbohydrate regimen, is remarkably effective at reducing seizure number. We are investigating how the transition in brain metabolism from glucose to a mixture of glucose and ketone bodies (the metabolically active byproduct of fat metabolism) could lead to a change in neuronal excitability. Previously, we described how acute application of ketone bodies reduces the excitability of substantia nigra neurons, an effect that relies on the opening of ATP-sensitive potassium (K_ATP) channels. Our hypothesis is that the inhibition of glycolysis by ketone body metabolism leads to a reduction in sub-membrane ATP, resulting in an opening of K_ATP channels and a decrease in neuronal excitability. This relies on the controversial idea that sub-membrane ATP is provided by glycolysis (possibly by glycolytic enzymes tethered to the membrane), and that the diffusion of ATP is restricted between the submembrane space and bulk cytoplasm, and concept known as “compartmentation”. To fully test this hypothesis, we required an optical sensor for ATP levels.

When planning these experiments, our first thought was to use Luciferase to detect different subcellular ATP levels. For a number of reasons, primarily Luciferase’s weak signal, we decided that a fluorescent sensor for ATP would be much more useful for our application. Our initial approach was a FRET-based design, with CFP and YFP tethered to a bacterial periplasmic binding protein that dimerized upon ATP application. Although these sensors gave some encouraging results, we never got the change in signal that would be required for cellular assays. We then adopted the ‘circularly permuted fluorescent protein (cpFP) approach that had previously produced sensors for calcium (pericam) and hydrogen peroxide (HyPer). We inserted the yellow fluorescent protein cpmVenus into the loop of the bacterial ATP binding protein, GlnK1 (involved in the regulation of ammonia transport) and found that application of small amounts of ATP to the purified sensor led to a substantial change in the excitation spectrum of the sensor. The affinity of the sensor for ATP was extremely high, orders of magnitude more sensitive than would be appropriate for cellular assays. We also found that our sensor responded to ADP application, only with a much smaller fluorescence change. It was then that we determined that these two perceived negatives (too high affinity and ADP binding) would lead to a sensor that reports the ratio of ATP to ADP. In a bit of good fortune, our design for an ATP sensor had in fact given us a sensor for the more valuable ATP:ADP ratio. After tinkering with our sensor by semirandom mutagenesis of the GlnK1 portion of the protein, we expressed the improved sensor, which we named Perceval (for permuted reporter of cellular energy value) into cultured cells and monitored a change in fluorescence with metabolic inhibition.

Right now, we are excited to use Perceval to investigate neuronal/glial metabolism in mammals. We may target subcellular ATP by either tethering Perceval to a membrane protein, or by using TIRF microscopy. In addition, we are continuing to design improved versions of Perceval, as well as sensors for other metabolic intermediates. We also hope that these sensors will be useful in applications beyond neuronal metabolism, from studies of cancer cells to bacterial metabolism.

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.

Symposium : A Revolution in Fluorescence Imaging

This coming Tuesday and Wednesday (Feb 17th & 18th) at UCSD, there will be a symposium honoring Roger Tsien, featuring presentations from 32 former and current members of the Tsien Lab. The topics are quite diverse, concentrated in genetically-encoded indicators, but also featuring fluorescent cell penetrating peptides for cancer therapy, photophore ligases for imaging synaptic development, and even a radical new design for the internal combustion engine.

The quality of speakers and subjects looks to be outstanding.  Here is a complete schedule.  You may notice that at 11:15 AM on Tuesday in Price Center East Ballroom, I will be presenting recent progress we have made in the development of genetically-encoded calcium indicators and their application to in vivo imaging.  Don’t miss that one!  :)  Roger’s talk, which will assuredly be equal parts absorbing, humorous, and illuminating, is at 4pm Wednesday in the Price Center Theater.

If you live in Southern California and are interesting in imaging technology, there isn’t a better place to be than this symposium.  If you can’t make it, Brain Windows will have a full write-up following the event.

Here is the un-official schedule.

Tuesday February 17th – Price Center East Ballroom

9:00 -9:05 Varda  Levram -Ellisman Opening

9:05-9:15 Palmer Taylor

Designing the next generation of genetically encoded sensors

9:15-9:30 Roger Heim

FRET for compound screening at Aurora/Vertex

9:30-9:45 Amy Palmer

Designing and using genetically encoded sensors: Lessons I learned from Roger

9:45-10:00 Robert Campbell

Beyond brightness: colony screens for fluorescent protein photo stability and biosensor FRET changes

10:00-10:15 Colette Dooley

GFP sensors for reactive oxygen species: Tying up loose ends and looking forward.

10:15-10:30 Peter Wang

Fluorescent Proteins and FRET biosensors for visualizing cell motility and mechanotransduction

Fluorescent proteins in neuroscience

11:00-11:15 Brian Bacskai

Aberrant calcium homeostasis in the Alzheimer mouse brain

11:15-11:30 Andrew Hires

Watching a mouse think: Novel fluorescent genetically-encoded calcium indicators applied to in vivo brain imaging

11:30-11:45 Alice Ting

Imaging synapse development with engineered photophore ligases

11:45-12:00 Rex Kerr

3D calcium imaging in C. elegans

Clinical applications

12:00-12:15 Todd Aguilera

Activatable Cell Penetrating Peptides for use in clinical contrast agent and therapeutic development

12:15-12:30 Quyen Nguyen

Surgery with Molecular Fluorescence Imaging Guidance

Fluorescent probes (Chemistry)

1:30-1:45 Tito Gonzalez

Voltage-Sensitive FRET Probes & Applications

1:45-2:00 Paul Negulescu

From watching ions to moving them

2:00-2:15 Timothy Dore

Roger-Inspired Photochemistry: Releasing Biological Effectors with 2PE

2:00-2:15 Joe Kao

Electron Paramagnetic Resonance Imaging in Living Animals

2:15-2:30 Brent Martin

Chemical probes for studying protein acylation

2:30-2:45 Jianghong Rao

Non-GFP based probes for imaging of the hydrolytic enzyme activity

Cellular research with and without Fluorescent probes

3:15-3:30 Carsten Schultz

Cell membrane repair visualized by GFP fusion proteins

3:30-3:45 David Green

Transcriptomes and Systems Biology: application to early mammalian embryogenesis

3:45-4:00 Clotilde Randriamampita

Paradoxical aspects of T cell activation revealed with fluorescent proteins

4:15-4:30 Wen-Hong Li

Studying dynamic cell-cell communication in vivo by Trojan-LAMP

4:30-4:45 Martin Poenie

Aim and Shoot: Two roles for dynein in T cell effector function

4:45-5:00 Gregor Zlokarnik

From bla to blah, blah in 20 years

5:00-5:15                        James Sharp

President, Zeiss MicroImaging Gmbh

February 18 2009 – Leichtag 107

Cellular research with and without fluorescent proteins

9:00-9:15 David Zacharias

Fluorescent Proteins, Palmitoylation and Cancer: two out of three ain’t bad

9:15-9:30 Jin Zhang

Visualization of Cell Signaling Dynamics: A Tale of MAPK

9:30-9:45 Paul Sammak

Nuclear organization and movement in pluripotent stem cells measured by Histone GFP H2B

Branching out

9:45-10:00 Yong Yao

NIH Toolbox Program

10:00-10:15 Oded Tour

The Tour Engine – A novel Internal Combustion Engine with the potential to boost efficiency and cut emissions

Into the future

10:45-11:00 Xiaokun Shu

Visibly and infrared fluorescent proteins: photophysics and engineering

11:00-11:15 Michael Lin

Engineering fluorescent proteins for visualizing newly synthesized proteins and improving FRET-based biosensors

11:15-11:30 Jeremy Babendure

Training our next generation of Fluorescent Protein Enthusiasts

Main Event – Price Center Theater

4:00-5:00 Roger Tsien

Chancellor invitational lecture 2008 Nobel Prize in Chemistry

Previews : sCRACM, Red PA-FPs, ATP sensor, Deep tissue PALM

Here is a quick list of papers Brain Windows will be covering in greater depth the next two weeks.

The subcellular organization of neocortical excitatory connections : A new technique, subcellular ChR2-assisted circuit mapping (sCRACM), is used to map neuronal circuitry.  

A genetically encoded fluorescent reporter of ATP:ADP ratio : A new single-FP indicator that reports the relative concentration of ATP to ADP in a cells. 

A bright and photostable photoconvertible fluorescent protein : Evolution of monomeric Eos into a fluorescent protein with better properties for super-resolution imaging.

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy : Evolution of mCherry into a photoactivatable fluorescent protein with better properties for super-resolution imaging.

Multilayer three-dimensional super resolution imaging of thick biological samples : Advanced laser pulse shaping techniques to achieve super-resolution imaging in thick samples.