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

8 05 2009

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

8 05 2009

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, mPlummKate, 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.

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 


IFP1.1 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.

Symposium : A Revolution in Fluorescence Imaging

11 02 2009


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

Preview : Structure of G-CaMP2

10 12 2008

A high-resolution crystal structure of the genetically-encoded calcium indicator G-CaMP2 would aid in rational design of improved calcium indicators. Crystallization of G-CaMP2 was first reported here :

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

M. M. Rodríguez Guilbe, E. C. Alfaro Malavé, J. Akerboom, J. S. Marvin, L. L. Looger and E. R. Schreiter

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, [beta] = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

High-resolution atomic structures and mutational analysis were presented at SfN 2008 (see this previous post)

However, today a competing group has published an independent report on a similar set of G-CaMP2 structures in Cell Structure.  More details to come…


Structural Basis for Calcium Sensing by GCaMP2

Qi Wang1,Bo Shui2,Michael I. Kotlikoff2andHolger Sondermann1,Go To Corresponding Author,

Genetically encoded Ca2+ indicators are important tools that enable the measurement of Ca2+ dynamics in a physiologically relevant context. GCaMP2, one ofthe most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca2+ fluxes invivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveala sophisticated molecular mechanism for Ca2+ sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate thatthe Ca2+-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca2+-sensitive probes.

Some interesting posters @ SfN

20 11 2008

Here’s a few posters that caught my eye at SfN.  Click the meeting planner for the full abstract

Optimizing two-photon activation of channelrhodopsin-2 for stimulation at cellular resolution


Spiral pattern of 2-photon excitation can drive neurons to spike.  A low NA objective helps. Need to do piezo-based Z-scanning if you use high NA, don’t with low NA.

In vivo two-photon imaging 1 mm deep into cortical brain tissue with novel microprism probe 


A cute method to image 1mm into cortex with 2-photon imaging. They used 2-6 month old mice. The just took a triangular prism whose hypotenuse was silvered and stuck it in the cortex. Then they internally reflected the beam off the prism and fired it sideways into cortex. Got good SNR to 300um lateral distance.  Some clippling of beam at edges of the prism gave somewhat inconsistent spatial resolution.

Self-complementary adeno-associated viral vectors for fast, efficient labeling of neurons and astrocytes in visual cortex in vivo


AAV is the way to go for expression of GECIs and ChR2 in vivo, but it takes a long time to express at high levels (2 weeks). They show that using a double stranded DNA version of AAV rather than single stranded gets protein expression up high much faster. Very high expression after one week. This is because the virus doesn’t need to take the time to make the second strand before expressing the protein.  See Xiao, X J. Virol 1998

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca2+ sensors


Everything in the poster was in the Nature Methods paper.  Conversation reveled that YC3.60 works as well or better than D3cpv. Only have done up to whisker evoked stimulation, no imaging of spontaneous YC3.60 signals yet.

Characterization of improved probes for the hybrid voltage sensor method of voltage imaging


A nice little sensor optimization poster.  They took the hVOS hybrid voltage sensor of dipicrylamine with membrane tethered GFP and improved it by changing the chromophore to Cerulean, and by using the “membrane-staple” strategy. Having membrane anchors on both the N and C-termini gave better quenching. Fast response, ~0.5ms, and 20% dF/F.

Crystal structure of the genetically encoded calcium indicator gcamp2


Jasper made crystal structures of G-CaMP2 in the apo and bound states.  Bound states crystalized as a heterodimer, but he was able to also crystalize the monomer. The structures show a pore to the chromophore in the apo state that is plugged in the Ca-bound state. Thus, the quenched apo state is due to solvent access to the chromophore.  This structural data should help rational design of better G-CaMP sensors.

Styryl dyes may inhibit synaptic release

19 11 2008

A new report in PNAS, Probing synaptic vesicle fusion by altering mechanical properties of the neuronal surface membrane, from Chuck Stevens’s lab, raises a serious concern about using styryl dyes to study release probability of synapses.  Styryl dyes, such as FM 1-43, partition into cell membranes and have been commonly used to measure synaptic release of vesicles in culture and brain slice.  The protocol is simple, bathe the neurons in dye, electrically stimulate to cause massive synaptic release and then dye uptake via vesicular endocytosis, wash off the dye, then observe the rate of destaining of the synapses following electrical stimulation.  This rate is directly related to the number of vesicle fusions during the final stimulation period. There is just one problem, Zhu and Stevens report that the presence of the dye reduces the release probability of the synapse.


FM 4-64 reduces synaptophysin-pHluorin response

FM 4-64 reduces synaptophysin-pHluorin response

They observed this by taking the FM dye measurements in neurons that expressed synaptophysin-pHluorin.  The fluoresence increase from the pHluorins was reduced in a [dye] dependent manner. A 15uM concentration of dye (not atypical for published experiments) reduced the pHluorin signal by 40%.  The dye had no effect on the calcium levels in the presynaptic terminals, indicating it was potentially due to an increased energetic cost of forming a fusion pore. Chuck then weaves together some basic principles with this data to make an estimate of the fusion pore size.  

While this paper may seem to cover a minor technical point, FM dyes have been used to make numerous inferences about presynaptic release properties, modes of vesicle recycling, the locus of LTP expression, and other basics of synaptic physiology. The thing that’s bugging me is if this effect is as prominent as advertised, how did people not notice the change in release probability with electrophysiological techniques?

Raw Data : Vesicular Release from Astrocytes, SynaptopHluorange

15 11 2008

When I was working on my Ph.D. thesis, I was trying to find some biological question to definitively answer with GluSnFR, my glutamate sensitive fluorescent reporter. One possibility was the study of glutamate release from astrocytes. Around that time, 2003/2004, there was increasing evidence that glutamate was not just scavenged by astrocytes, but was also released from astrocytic vesicles. It released in response to calcium elevations within the cell. Existing methods for measuring this release were somewhat crude, so it seemed a great test system for GluSnFR.

Unfortunately, since there seemed to be no specialized areas on the astrocyte where the vesicles fused, and the release rate was relatively slow, we were unable to detect glutamate release with GluSnFR. I thought this might be a problem of not knowing when and where to look. So my collaborator, Yongling Zhu, and I expressed pHluorins fused to VAMP or to synaptophysin in astrocyte cultures. When we looked at them under the microscope, they just looked green, no action…

But then we left the excitation light on for a few minutes. I happened to look back into the scope after they had been bathing in bright blue light and was astonished. I could directly see, by eye, spontaneous bursts of fluorescence across the cells. It was absolutely magnificent. The long application of light had bleached all of the surface expressed, bright pHluorins. But the pH-quenched pHluorins in the vesicles were resistant to bleaching. On this dimmer background, the fusion events were plain as day.

Unfortunately, the green color overlapped with the emission of GluSnFR, so we couldn’t use it for a spatiotemporal marker of when and where to look for glutamate release. We tried using some ph-sensitive precursors to mOrange and mOrange2, developed by Nathan Shaner, but these seemed to block the release events. Since then, others have shown the functional relevance of glutamate release from astrocytes, and I turned the focus of GluSnFR measurements to synaptic spillover. This was one of the projects that was tantilizingly close, but got away. This movie of VAMP-pHluorin is almost five years old now, but it still looks cool… Enjoy!

If you are curious, this is what the Synaptophysin-mOrange looked like when we expressed it in hippocampal neuron cultures. Ammonium Chloride caused a massive fluorescence increase, by alkalizing the synaptic vesicles. Unfortunately, we never were able to see release via electrical stimulation. Details are in my thesis. Maybe someone else wants to give it a shot?