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

SFN Neuroscience Picower MIT Party 2008

Lots of people searching for “SFN MIT party” for this information in google… Here’s the answer you are looking for.  Right next to the convention center. Much more convenient than three years ago at Cobalt.  No excuse to be late…

Someone send me an invite to the Neuron, Nature and Emory parties, pretty please! 

 

Is that a man in a tuxedo wearing stilts?

 

 

The Picower Institute, The McGovern Institute, and the Department of Brain and Cognitive Sciences at MIT

Invite you to the sixth annual party at the 2008 meeting of the Society for Neuroscience

Monday, November 17th, 2008  

9pm – 2am  

Avenue Nightclub
649 New York Ave NW

Washington, DC

UCSD vs. MIT SFN Party Smackdown

The Society for Neuroscience conference starts today in America’s Finest City (San Diego). The question on everyone’s mind is, who is going to throw the best party? Sure there are plenty of themed mixers and socials, but few really stay interesting for long.

The past few years, the Picower Center for Learning and Memory at MIT has consistently had the biggest bash, really peaking in 2006 at the eye-popping Atlanta mega-club Compound. With a big open bar tab that unfortunately gets drained within an hour, and an open invitation, these are always packed with people early on, go strong till last call, and feature plenty of Neuroscience ‘star power’. This year, the party starts Monday at 9pm at Deco’s on 5th Ave. in the Gaslamp. Get there early, as Deco’s is a relatively small place.

Nature and Neuron each throw lower-key parties, with the best hors d’oeuvres and are definitely the place to do serious science/business networking. Security is pretty loose, as long as you let the door know that you know that the party is for Nature or Neuron. When and where these parties might be in San Diego is under intense investigation by BrainWindows staff.

The most exclusive of all are the mysterious Emory parties, where you better bring the printout of your personalized invitation email if you want to get in.

This year, there is a new group that is trying to dethrone the PCLM as hosts of the biggest event. UCSD Neurosciences is hosting an open-invite, open-bar event this Sunday at Aubergine, at 4th & Island in the Gaslamp. The bar tab opens at 9pm, and if the PCLM parties are any guide, I would get there at 9. Bring friends!

Who will impress the community the most? PCLM has a five year reputation, and the experience of Earl Miller and Susumu Tonegawa behind it. But UCSD knows San Diego, and it’s grad-student run social committee has held numerous, very successful local events. As a soon-to-be alum of both UCSD and PCLM, I’m looking forward to finding out who does it best. See you there!

CSHL Meeting – Session VII – Super-Resolution Optical Techniques

Jean-Louis Bessereau – Ultrastructural mapping of functional domains of synapse at the synapse using high pressure imaging
High pressure freezing instantaneously converts up to 0.3mm thick water into amorphous ice. C Elegans only .1mm thick at maximum. HPF entire worm to obtain EM of ‘living’ synapses. Vesicle priming occurs within 100nm of presynaptic density, directly across from post receptors. However, vesicle recycling occurs only at sites >150nm lateral from presynaptic release sites.

Mark Ellisman – Multiscale light and electron microscopic imaging of the nervous system
Two-color correlated light and EM microscopy using FlAsH and ReAsH. Quantum dot immunohistochemistry for multicolor correlated light and EM. QDs of different wavelength are differently sized and can be distinguished on EM.

Eric Betzig – Superresolution optical location of single proteins.
Sparsely photoactivate PA-GFP tagged proteins with very weak laser pulse. Then image with high laser power to collect light from many point sources. Determine point source centers, then repeat process many times to find protein locations to 2-25nm resolution. See the science 2006 paper.

Winfried Denk – Automated circuit reconstruction
EM :
When doing automated serial EM, individual sections can get lost or crumpled before being imaged. Instead image the block face then shave off a section. Resolution is significantly degraded, because lower power 3keV to limit z-penetration. Penetration goes as E^(5/3). Monte Carlo simulation of backscatter at 3 vs 10keV to determine point spreads and depth. Do 30nm sections, which is very [pun alert] cutting edge. Circuit reconstruction fidelity is limited by the dimension of least resolution, so usually Z section thickness. Block face EM actually used in the 1940s. Can still identify synaptic densities. Use backscatter to see heavy things. Constructing whole c. elegans. Demonstrate by hand reconstruction of some axons, dendrites and synapses as proof of principal. Using ion gun to acquire without need of a vacuum.

Staining Technology :
Colloidal lanthanum greatly enhances contrast of extracellular space but poor tissue penetration. Kevin Briggman and Denk like using extracellular HRP currently. Good membrane contrast. Moritz Helmstadter working on automated segmentation, collaborating with Sebastian Seung @ MIT for neural network based methods. Neural network method looks pretty good.

Lukyanov – New fluorescent proteins
Cloned the first RFP from coral, DsRed. DsRed tends to aggregate due to tetramer nature. Comparison of DsRed2, TurboRFP, TagRFP and mCherry. Brightness by e*QY. 100,172,134,44 respectively. TagRFP has shorter emission wavelength (<600nm) than mCherry (618nm). Can distinguish TagRFP from mCherry by the significantly difference in lifetime. [What about mCherry2?]

Redshifted FPs.
Katusha excitation 588, em 635. QY 0.34 e 45,000. Not quite as redshifted as mPlum but significantly brighter. But not monomeric. Made mKate which has similar brightness but is monomeric.

Cyan to green photoswitchable PS-CFP. Also made Dendra, monomeric green to red photoconvertible FP. [How much bleaching to red occurs to get 90% conversion? In our hands no photoswitchable protein allows total conversion without significant bleaching.]

Visualization of targent protein degredation in real time at single cell level using Dendra2. Zhang Biotechniques Apr 2007. IkBa-Dendra2 degredation down to 20% at 5hr with cycloheximide treatment. Fluoresence stays fixed with proteasome inhibitor. 20min protein halflife following PMA treatment. Hydrogen peroxide sensor HyPer of cpYFP inserted into OxyR-RD. OxyR transcription factor forms reversible S-S bonds in bacteria. Around 2-fold ratio change 490/410 between 0 and 250nM H2O2. Showed some small responses in cells to EGF stimulation. [Is this response reversible? Never showed a recovery trace.] cpCitrine145 with m13 on N, calmodulin on C term makes a GCaMP like sensor, 12-16x maximum ratio change. [How EXACTLY is this sensor different from GCaMP2?]

Killer Red, genetically encoded photosensitizer. Screened different natural FPs for phototoxic proteins that kill bacterial colonies under light. Most FPs non-toxic, 2-5fold increase in bacterial phototoxicity, but 1 FP had 1000x increase. In mammlian cells, expression in cytosol is not enough to cause sufficient oxidative stress to kill cell with light. But target to mitochondria and can kill cells with light. Targeted to cell membrane, blebs occur in 3 minutes. Use to kill off specific muscle cell with light in zebrafish. CALI of phospholipase C1-d PH domain by fusion to killer red. However it is still dimeric, so doesn’t fuse well to some things.

Karel Svoboda – Meeting Summary
Many spatial and timescales of neural questions require development of variety techniques. Interface of new imaging techs and genetically targeted probes making lots of progress on addressing these questions. The developmental talks were some of the best applications of the new technologies. These meetings will be measured by the crystallization of new, unexpected directions in research. Of course GECIs have been very exciting, but in the last 2 years also seen big progress on…
EM
-sectioning and data collection, segmentation and reconstruction, EM on targeted neurons.
Light based approaches
– PALM, STED –EM type resolution in far field optical microscope, spectral multi-plexing, synapse specific tracing.
Optical remote control with light.
ChR2 – The silver bullet
HR – hyperpolarlize
Rhodopsin – seconds time-scale modulation of plasticity
Optical switches without transgenes
Applications

See you in 2009!

CSHL Meeting Session VI

Novel Methods to Dissect Neural Circuits – Saturday afternoon

Dmitri Chklovskii, Janelia Farm

Reconstruction of neuronal wiring diagram from automated serial EM. Must be able to track identity of segments between slices, determine synapses and the cells they belong to. Wiring diagram draft was done in c. elegans (~7000 synapses, 279 neurons) in 1986, Mitya’s student finished it in 2006.

How do we do it? Automated alignment of serial sections by translation, slight rotation and elastic stretching. Automated segmentation of color coding, makes a draft that must be reviewed by human editor. State of art is 10x faster than manual tracing, reconstructed complete 10x10x10um^3 volume two man-months. 1000 synapses, 1000 axons, 100 dendrites.

Biological results : If there are an equal # of spines and axons neighboring a single segment of dendrite, no significant wiring rearrangement possible. But connectivity fraction is actually 0.1-0.3 so plenty of room for structural plasticity. For optimal info storage, there should be equal volume of axons and dendrites, which is shown to be true. Axons appear to be concentrated near other axons, dendrites far from other dendrites, but this actually fits random packing of processes.

Questions : Can you see systematic slicing errors from alignment?
A: Only errors from people walking by.
Q: How much shrinkage do you see from fixation?
A: Significant uniform volume shrinkage, but not worried about that. Loss of extracellular space, may effect shape of processes.

Jeff Lichtman, Harvard
Connectomics : Brief definition – Map neural circuits.
Naturally occurring synapse elimination in the developing brain.
Three changes in synaptic connectivity:
1. Decreased axon connectivity
a. Imaging NMJ, decreased convergence with compensatory synaptic takeover by the remaining input
b. Non-monotonic process, appears to be competition
c. Axons are branches, other branches of same axon innervates other targets, these effect the competition
2. Decreased axonal divergance
a. E18 – 80% NMJ innervation. P13 – 4.2% innervation
3. “Synchronization” or rewiring process
a. When two axons compete on multiple terminals, same axon loses in both
b. Is there a deeper hierarchical structure?
Each outcome of synapse elimination causes unique pattern of synapse innervation in each axon.
Automated Tape-collecting lathe ultramicrotome (ATLUM). Grad student made a homemade one with 15uM thickness. Now building 50nM thickness with $200K McKnight. http://www.extremeneuroanatomy.com

Clay Reid, Harvard Med – New tools for imaging the functional anatomy of the visual system.
Originally an electophysiologist only, mapping functional connectivity with electrodes. Now doing functional imaging.
Calcium imaging of the visual cortex. Bulk loading of calcium indications in cerebral cortex, look at 300uM cubes. What is the function of each of these cells? Excite with visual stimuli of anesthetized animal, 2p imaging of 2/3 rat visual cortex. Find orientation selectivity without clustering : salt and pepper. No apparent functional microorginaization. However, in the cat, similar neurons types (horizantal, vertical) cluster together with sharp cutoff between cells in orientation pinwheels. How do they do this?

Are functionally similar groups of cells:
Spontaneously co-active?
Correlated with cell type?
Anatomically/functionally connected?
What is the wiring diagram?
Tracing individual connections with viruses
Tracing many/all connections with serial electron microscopy
Use conventional sectioning and imaging with high throughput camera array.
Large volumes up to 500uM cubes at 5nm x-y resolution
Large datasets of 10-100 terabytes
Record everything but analyze only a bit, a very relevant bit
Showing preliminary data of automated serial em collection and analysis

Andre Fiala – Optophysiological techniques for the dissection of neuronal circuits underlying learning and memory in Drosophila
[Great talk content, but my notes are poor.]
In vivo monitoring of neural activity
Glue fly under coverslip. 1p DualView with Cameleon expressed in dopaminergic neurons, which have extensive innervation throughout brain. Following 8 training sessions, dopamine neurons show prolonged activity that persists thru conditioned stimulus, suggesting predictive abilities.
Expresses ChR2 in fly larva and can control contraction on larva with light. Can substitute light stimulation in octopamine neurons for appetitive odor stimulus in learning paradigm. Substitute ChR2 dopamine light stimulation for aversive stimulus. Express ChR2 in gustatory neurons, flash light, proboscis extends. “The light tastes sweet.”

Tamily Weissman, Harvard – Mapping neural circuitry in the cerebellum using multicolor fluorescent “Brainbow” mice
Gain neuronal identity in labeling by using combinations of fluorescent proteins “Technicolor Golgi stain”.
Thy-1 promoter-L1-L2-RFP-L1-mYFP-L2-mCFP with incompatible Lox sites. PreCre get RFP, Post Cre get YFP or CFP. Since multiple copies per cell, get blends of colors. [I doubt there is any FRET since they are using monomeric (A206K) mutants of C/YFP.] How many colors? Hard to say, conservative estimate for 100% confidence by eye is 78 colors eye can descriminate. [Why limit by eye? What is the limit using spectral deconvolution?] Limiting 20% mossy fiber, 5% granual cell and can do total reconstruction of this fairly dense labeling. Appears there is some convergence in circuit of mossy fibers onto granual cells by looking at ratio of filled terminals. Granual cells sometimes innervate same presynaptic mossy fiber at two distinct terminals on different dendrites.

Wei Chen – In vivo two photon imaging of firing and wiring of local neuronal circuits.
[Speaker is the lead author on the in vivo electroporation paper we recently covered, see the paper for more details.] Understanding the brain depends on sparse labeling of neurons. Konnerth, Reid using bulk loading, but this obscures fine neuronal structures. Tried bulk loading, G-CaMP2 mouse, now trying local electroporation. Following electroporation, only very small change in field recording. Hey but aren’t only a small proportion of the neurons electroporated? Hmm….

Ian Wickersham, Salk – Transcomplemented transsynaptic tracing : mediation by helper viruses
[I was planning on covering this work in the recent publication in Neuron, but will just do it here.] How do we determine what cell is monosynaptically connected to other cell types? Classic transsynaptic tracers pass at different rates due to connection strength and can move through strong polysynaptic connection steps. Enter transcomplemented tracing.

Component 1 – Deletion mutant tracing virus
Component 2 – Complement of the deletion, activates virus.

Rabies virus, RNA virus (can’t use Cre recombinase)
Replace the glycoprotein of rabies virus with GFP. Virus can replicate core but cannot cross membrane. Pseudotype virus with coat glycoprotein to avian ASLV’s membrane protein. Express gene of ASLV receptor, dsRed and native virus coat protein complementation gene in single neuron in the brain. Then pseudotyped virus infects that single cell and can cross 1 step. But, since complementation gene only exists in single cell, virus stops crossing after 1 step.

Day 1 : shoot in triple gene coated particles with genegun
Day 2 : Apply pseudotyped rabies virus
Get 1 red cell, and many sparse green cells that are monosynaptically connected.

Aravinthan Samuel, Harvard – Brain and behavior in freely moving worms
Thermotaxis exhibits long-term plasticity. Thermosensation occurs at tip of nose. Side to side wiggles and net forward movements could contribute to perception of thermogradients. Express cameleon in AFD neuron using cell-specific promoters.

Worm wants 2 pieces of info:
Is temp higher than it likes?
Is temp rising or falling?

Immobilized worm subjected to defined thermosensory inputs. Increasing T in a linear rate with wiggle induces a phased locked ratio change to the wiggle that starts above about 18C. Getting 150% dR with YC3.60 in response to wiggles. Ratio in AFD is directly correlated to T in tail fixed worms moving head around on a temp gradient. Turning off gradient kills correlation, reversing grad reverses side correlation.
AFD detects the temp variations driven by self-movement in a spatial gradient.

CSHL Meeting Session IV

Evening Session – Optical Measures of Neuronal Excitation And Signaling

Rainer Friedrich – Measuring spatio-temporal activity patterns by temporally deconvolved 2-photon calcium imaging
Bulk loading of olfactory cortex with AM Ca dyes. Calcium signal is much slower than action potentials because the signal is a convolution of Ca trace with binary spikes. Deconvolution of the raw Ca signal with a canonical Ca response derives the firing rate. Reconstruction of population activity pattern improves only a little bit with letting tau or spike amplitude vary. This technique requires low noise. Published in nature methods. Application in olfactory bulb of zebrafish. Factor analysis of spatially clustered activity in OB maps regions of activity to amino acid properties (aromatic, long-chain, basic). Canonical trace is average of single APs. Little contribution of intracellular stores or synaptic input due to the localization of those calcium transients. They image Ca flux in nucleus that has slow timeconstant, but that doesn’t have a negative impact with deconvolution.

Wen-hong Li, UT Southwestern – Dynamic gap junctional communication in developing nervous systems

Vivek Jayaraman – Calibrating a genetically encoded optical sensor of neural activity with electrophysiological techniques in intact fruit flies
1. Does the GECI affect the system’s normal development and function?
How are odors in PNs in flys generally represented? Simultaneous loose patch and 2p imaging with GCaMP1.3 in PN neurons. GCaMP+ neurons not significantly different from matching EGFP+ neurons.

2. Is GECI signal a good proxy for spiking activity? If not what is threshold?
It is OK if you have a high number and rate of spikes. Mean spike rate of 30Hz is the minimum to see GCaMP1.3 response.

3. Can we find a GECI -> spike transfer function?
GCaMP singal rises faster in glomeruli than in PNs. However it still doesn’t track the temporal dynamics. So No.

4. How does expression level and compartment imaged effect the GECI signal?
GCaMP singal rises faster in glomeruli than in PNs. Must calibrate in the specific structure that is being imaged.

Venky Murthy, Harvard – Studying neurovascular coupling using multi-photon microscopy of olfactory glomeruli in vivo
What are noninvasive methods for brain imaging? fMRI measures bloodflow. Olfactory bulb as a model system for studying neurovascular coupling. Can isolate post vs. presynaptic activity.
Measure neural activity using the oldskool (2004) synaptopHluorin mouse from Mombaerts. Look at dF/F changes in olfactory glomerulus. Very slow timecourse of response with 15s odor pulse, increases in a dose dependent manner.
Measure bloodflow by injecting dextran conjugated dye into the bloodstream.
Simultaneous imaging of presynatpic activity and blood flow by linescanning along a vessel and beyond on both sides. Odor stimulation increases bloodflow, which is also correlated with vesicle release. Vasoregulation is likely coupled through astrocytes. Coupled via glutamate, ATP or COX-2. Applying MK-801 and others to block postsynaptic activity does not change blood flow. MCPG, a broad mGluR blocker reduces flow increase by 50% with no affect on the presynaptic activity seen with SpH. What is the other 50% of the coupling from? TBOA (general) and DHK (astrocyte specific) glutamate reuptake blockers also reduce flow increase by about 50%. This is very interesting as this might be expected to increase postsynaptic activation due to greater spillover glutamate levels. Therefore direct activation of mGluRs and glutamate reuptake into astrocytes both regulate cerebral blood flow. COX-2 inhibitors occlude the mGluR effect, but not the TBOA effect suggesting the two pathways are independent.

Orie Shafer, Wash. U – Live-imaging of cycle AMP signaling in the mushroom bodies and circadian pacemaker network of the D. melanogaster brain
Trying to find target neurons sensitive to pigment dispersing factor based on camp responses to PDF
EpacCamps: FRET-based genetically encoded cAMP reporter. YFP/CFP goes down upon stimulation. Try to image camp increases in the large ventrolateral neurons of circadian pacemaker network. Small, uneven response to PDF. However small vLNs respond strongly to PDF, with a variable delay. He suspected differences in the delay of FRET change was brain dependent. I suspect it is variation in the pipetting and diffusion when applying the PDF bolus to the dish. KO of PDFR in these cells abolishes the response. Does increased PDFr expression result in an increased PDF response in the large vLNs? Yes. Now express EpacCamps across all neurons in pacemaker network and screen for response to PDF.

Leon Reijmers, TSRI – The TetTag mouse—A transgenic mouse that enables long-term tagging of activated neurons
Dual transcription factor strategy. Fos-promoter for tTA, which is blocked by Doxicycline. Second promoter that is activated by tTA that drives a mutant tTA* transcription element that has a feedback loop and drives the gene of choice. Doxicyclin blocks activation. Removal of Dox turns gene on, with a feedback loop so when Dox is re-fed to mouse, gene stays on.

CSHL Meeting II – Imaging of Neurons

Hollis Cline, CSHL – Ultrastructural analysis of synaptic convergence during circuit development
The synaptotrophic hypothesis: synaptic inputs drive dendrite development.
Immature synapses have NMDARs some AMPARs, mature get more AMPA.
Degenration of synapses promote branching of dendritic arbor.
Single cell in vivo electroporation of GFP into optic tectal neurons of zebrafish.
Cisual stimulation increases retinotectal synaptogenesis and increases strength of per-existing synapes. Try to block development of GluR synapses.
From Malinow tech. Whole brain electroporation of AMPAR C-tails. GluR1Ct mini amplitude 11->5, GluR2Ct 11->7. Neurons expressing either make arbors that are longer and less dense. Dynamic (2hr timepoints) imaging of arbor growth and retraction in tectal neurons. Hypotheses : Ct neurons have shorter branch durations.

Correlated in vivo 2p and serial em reconstructions, map all synapses.
1. All dynamic branches have synapses.
2. Retracting branches have very low synaptic density.
3. Synapses on stable branches are more mature
4. Divergence of axon terminals decreases with stable synapses.

Synapse Maturation metric – Area of clustered synaptic vesicles to terminal area

David Kleinfeld, UCSD – Resilience and control of neocortical blood flow revealed by Optical Imaging and Manipulation.

Motivation: Connection between network topology, flow dynamics and control
Tools:
1. Particle tracking w/ 2p imaging to meauser RBC flow
2. Localized occlusion to perturb flow
3. Global occlusion to regulate source flow
Prep: Lightly Anethistized Rodents

Are the penetrating arterioles (PA) the trunk lines that plunge from top of cortex and have a limited specific range?
Sometimes make clot in PAs, still get flow in neighbors, but usually get total stop in neighbors. Need to move 200-300um away to restore flow. PA makes a cylinder of flow. A-hypoxyprobe labeling (mitochondrial stress) shows 400um diameter stain when PA blocked. Microstrokes of PAs cause localized cortical “burn out”.
Surface Vasculature – Many loops in the surface communicating arteiole network, thus many routes to route around blocks. Block between intersections, flow reverses in some places. 1 vessle away, 60% flow rate, OK. 2 vessles away, 100% flow.
Effect of strong forepaw somatotopic stim on vasodynamics in the surface communicating arterioles. Stimulation leads to both vasodialtion and delayed vasoconstriction in SCAs. Reminiscent of +/-BOLD.
Subsurface microvascular – Gated communities in SoCal. 2p imaging, then zap the top off and image the next level. Make 1mm deep map. Making 3d reconstruction of vessles, cell somata. Density of microvessles is flat. Does not increase in layer 4. Somata does pop up in layer 4. When you block flow in deep microvasculature, 1 vessle distance, total blocked flow, 2-3 little blocked flow. Astros are easy to image Ca, so people are looking at them for vasoregulation. But inhibitory cells release peptides too. Tn-XXL Ca indicator expressed w/ exogenous achr gpcr inject HEK cells into animals. See Ca transients due to Ach release.
Questions : Timecourse of vessel changes? Dilation delay of 400ms, 500ms for constriction. How does vasoregulation occurs at distances of mm? Speculation that inhibitory neurons are part of a broad network whose activity spreads widely. Working on imaging spread with the hybrid imaging probes.

Rusty Landsford, Caltech – Multi-modal imaging of avian development.
Transgenic Japanese quail – avian eq. of mouse, small, related to chicken, fast lifecycle 5-6wk sex maturity. Synapsin.H2B-GFP. Continues 5 day imaging of embryo development. Quail atlas with 11.7T MRI with 3d website.

Yukako Yokota, UNC – Novel aspects of interneuronal migration in the developing cerebral cortex. How do neurons decide where to end up during cerebral cortex development? In vitro electroportation. Look at interneuron-radial glia interaction with live 2pimaging. Example, interneuron changes direction when it hits the endfeet of glia. Pass through ,Turn back, turn towards, attach. Only looking at a small subset of radial glia due to sparse labeling.

Tim Murphy, UBC – Two-photon imaging of acute stroke indicates that a propagating ischemic depolarization produces profound but reversible alteration in the structure and function of spiny dendrites in vivo.
Originally used Kleinfeld technique of photothrombosis, but it’s very local and clots are hard to break up. Wanted reversible ischemia. Which ionic events trigger loss of dendritic structure?
7min bilateral CCA ligation to do global strokes. Structure with YFP.
After 4 minutes, dendrite is a “blebbed ugly mess” of swelling and restrictions. Reperfuse, 10 minutes getting better, 90minutes, structure is almost unchanged from original. 30% spine loss during ischemia, 8% after 90min reperfuse.
Very sharp threshold around 1-2 minutes the brain area turns to mush in about 10 sec. During occulusion quiet EEG, then an AC ripple @ 2min, drop in DC potential. Ischemic depolarization wave increases cortical light scattering. Slow drop then dramatic wave of increased scatter 3mm/sec. Intracellular calcium jumps during the wave. MK-801 had no effect on the ischemia wave structural change, but block NMDA evoked signal. Thinks wiring stays attached during the ballon, though some spine loss and AMPAR internalization.

Adi Mizrahi, Hebrew Univeristy – Imaging synaptic development and plasticity of adult born neurons in the mouse olfactory bulb.
In vivo 2p imaging of lenti-GFP migration of neurons from SVZ to olfactory bulb. Highly dynamic dendritic development. PSD95-GFP_IRES_DsRed. About 65% of PSD punta are synapses by EM. Synaptic puncta do not accumulate during migration. Distributions of synaptic puncta along dendrites sharpen as neurons mature from 14 to 51 DPI. PSD95 puncta are dynamic only when mice are awake. Doing bulk loading of Oregon Green BAPTA – AM and look at response in a single Lenti-RFP neuron. Get fractional percent dF from electrical stim. Primary and secondary dendrites are stable at 3 months, but somas still move around.

Mark Schintzer, Stanford – Chronic microendoscopy.
Insert cannula with a microcoverslip at tip. Long term imaging of cells in hippocampus. Can find same cells DIV 239 v. 241. 1p bloodflow imaging at 100Hz. 423um/sec mean velocity. Micromechanical 2p scanner about the size of a dime, carbon fiber frame for weight. Full frame imaging at video rate. Not on freely moving mice, yet. 1p imaging of blood flow in freely moving mice is working, even 1 month after implantation. 1.8g mount. Freely rotating camera to relieve torsional strain. When will we get a commercial solution??

CSHL Imaging Neurons Meeting – Session I

Wow! A very busy start to the conference on Imaging Neurons and Neural Activity at Cold Spring Harbor Labs. I arrived at 6:30pm Thursday. Since then, I have seen 30 talks with copious note-taking, seen too many posters, given 1 talk, seen Ohio State win, and had lots of informal science talk over some beers. Not a lot of sleep though! There is literally no time to refine my notes, but to be timely with my posts I will be posting raw notes that will be updated and refined over the course of the next week or two.

Session I – Novel photoactivation and tagging methods

Optical Probes

Louis J. DeFelice, Vanderbilt – Neuronal transporters for monoamines analyzed with fluorescent substrates and fluorescently labeled transporters.
The goal – take a small molecule orally and get spatially specific transmitter release. DeFelice demonstrated that MPP+, IDT307, and other similar drugs are taken up by specific monoamine (dopamine, norepinephrine) transporters. This causes depolarizing currents that may be able to induce neuronal excitation and synaptic release in specific monoamine neuron type. However, they still need to demonstrate release, improve specificity and reduce toxicity.

Don Arnold, USC – Using intrabodies generated by phage display to study subcellular trafficking of Kv4.2
Overexpression of proteins (ex. PSD-95:GFP) can cause artifacts in protein localization and function. To gain protein localization information without this potential confound, Arnold has developed the interbody method to label endogenous protein with GFPs. He uses phage display to iteratively screen for single chain Fv antibody fragments that specifically bind selected proteins (ex. T1 domain of the Kv4.2 voltage-gated potassium channel). He then genetically fuses the ScFv gene with GFP and transfects cells. In neurons, these interbodies show labeling that is much more punctate than exogenous Kv4.2-GFP transfection, and thus closer to the real channel distribution. To further enhance specificity, he ubiquitinated the intrabody. Unbound intrabody is rapidly degraded, but binding to the Kv4.2 prevents degradation for unknown reasons. He then finds that in excitatory neurons there is a decreasing gradation of staining intensity outward from the cell body. I suspect this may reflect production, diffusion and distribution of the intrabody rather than the actual channel itself, but Arnold points out that this pattern is not seen in inhibitory neurons.

Andrew Hires, UCSD (that’s me) – Measuring glutamate spillover and uptake with GluSnFRs
Genetically encoded sensors of glutamate concentration have yet to find quantitative applications in neurons due to poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. GluSnFR is made by bracketing CFP and Citrine with a glutamate periplasmic binding protein and then tethering it to the cell surface by fusion to a truncated PDGF receptor. Truncation of 8AA of the N-terminus and 5AA of the C-terminus of the PBP increases the maximum ratio change from 7% to 44% in phyisiological buffers. Rational mutations of the binding pocket give optimized the affinity to 2.5uMkD.
We performed field stimulation of GluSnFR expressing hippocampal cultures on astrocytes. Glutamate release is calcium dependent. The uptake inhibitor TBOA enhances peak [glu] and duration. Single AP stimulations are resolved with spatial averaging. Multiple AP stimulations resolved at bouton level. Spillover from 1 action potential field stimulation peaks near 700nM at around 10ms.
Trains induce steady state spillover >500nM within 4 AP, sufficient to activate NR2B NMDARs. Spillover modulated by stimulation frequency 60% with active uptake, only 10% with uptake blocked. Thus, non-connected neighboring neurons or astros may detect firing rate from spillover glutamate and induce a measured amount of, homeostatic regulation, heterosynaptic LTD or vasoregulation. Differential synaptic independence based on firing patterns.

Optical Stimulation of Neurons

Karl Deisseroth, Stanford – Multimodal fast optical interrogation and control of neurons.
Karl crushed it again. He mostly focused on the results of his article coming out in Nature next week on in vivo optical control of neurons with channelrhodopsin-2 and halorhodopsin. Halorhodopsin is the same yellow light activated chloride channel that Karl’s former postdoc published online in PLoS One this week. We previously covered that technology story here, but Karl’s lab has pushed it further. He showed a pretty demonstration by dual expressing ChR2 and halorhodopsin in motor control neurons of the worm c. elegans. He made the worm expand or contract dependent on the color of light that was flashed. Using lentivirus delivery of ChR2 into the rat motor cortex, they drove whisker movement by piping blue light into the brain with an elegant fiberoptic mount. He also demonstrated a fiber-optic cannula for deep brain optical stimulation. In brain slice, there is the potential for three-color simultaneous calcium imaging with Fura-2, optical stimulation with ChR2 and optical silencing with halorhodopsin. He is interested in using optical stimulation of genetically targeted neurons in humans to treat psychiatric disease. The technology sounds great for potential treatment in humans, except for the lentiviral gene delivery part…

Stefan Herlitze, Case Western – Vertebrate rhodopsin and channelrhodopsin 2 for control of intracellular signaling and physiological response on ion channels, neurons and neuronal circuits
Herlitze expresses vertebrate rhodopsin in the vicinity of presynaptic Ca channels or postsynaptic GIRK potassium channels. Light activation of the rhodopsin induces a G-protein cascade that modulates the function of these channels. Continuous illumination reduces presynaptic Ca-flux while increasing the paired pulse facilitation. Brief 5ms light during 2AP stim also reduces first EPSC while dramatically enhancing the second. On the postsynaptic side, light induces hyperpolarization by modulation of GIRKs. In vivo chick embryo turning off light can synchronize independent rhythmic neuronal networks. Presynaptic optical stimulation with ChR2 does not directly trigger transmitter release. Simulation of motorneurons expressing ChR2 induces muscle contractions.

Dan Huber, Janelia Farm – Channelrhodopsin-2-assisted microstimulation of layer 2/3 barrel cortex neurons detected by freely moving mice

How many 2/3 pyramidal neurons need to be activated to evoke reliable behavior?

Target ChR2 – in utero electroporation of CAGGS-ChR2-GFP. Expresses specifically in 2/3 pyramidals. 530-2364 neurons (mouse dependent) expressing by serial immunohistochemistry. 327-1412 in the window area.

Characterize the light – 1ms of max 7mW/mm^2 max light. Stim with 1ms pulses.

Characterize response – 100% neurons respond up to 20Hz. 50% up to 50Hz, faster than natural spike trains. Very sharp threshold for spiking/light intensity. The threshold is significantly different between cells, can use to reduce total # of cells firing in graded way.

The task – Train rat in a two choice task to find the water. Train rat to perceive 5AP optical train at 20Hz and go right or left for water dependent on stimulus presence. 1AP 65%, 2AP 80%, 5AP 85% correct. Perfomace is enhanced at low AP if you systemically reduce # of APs in stim rather than randomly interleave. Reduce light intensity to 10% of max to stim small subset and still get 65% correct for 5AP.

Working on cleaner fiber optic stimulator (a la Deisseroth), more complex simulation/discrimination tasks with whiskers.

Questions – why barrel cortex? Can you train it to perceive stimuli in arbitrary cortical region?
A: Because we want to do whisker perception experiments in the future. Probably.
Does minimal stimulation activate neighbors through polysynaptic connections?
A: Field recording sees network activation, don’t know if its 2/3s or just downstream.

Richard Kramer, Berkeley – Teathered small-molecule photoswitchable ion channels.
2 parts
SPARKs
Shaker potassium channel blocker teathered to azobenzene. Azobenzene has trans-cis isomerization hinge motion upon 380nm illumination.  Can be driven back by 500nm light. Bound to mouth of constitutively active mutant of K channel.  Several second on/off switching of hyperpolarization with light.  GYGV->GYGQ mutant converts to non-selective ion-channel.  Light on this mutant induces spiking.  [ChR2 appears to have significant response rate and expression advantages over SPARKs, however, both require transfection of exogenous channels.  The second part of the talk focuses on light regulation of endogenous channels]

Photoswitchable Affinity Label (PAL)
Attach a reactive epoxide or acrylamide reactive group to MAQ.  Apply to channels, binds non-specifically, but near mouth due to MAQ affinity for pore.  Hits variety of K channels.  Light turns off firing in hippocampal neurons treated with PAL.  Collaboration with Bill Kristan to modulate firing of HNl HNr neurons in leech [transgenic techniques are undeveloped in the leech.]  Inject PAL into eye in vivo take retina out.  380nm light turns off inhibitory amacrine cells and induces ganglion cells to spike.

Imaging Neurons & Neural Activity Meeting

Next week a major conference, Imaging Neurons & Neural Activity: New Methods, New Results, focusing on new brain imaging technology will be held at Cold Spring Harbor Laboratory. The lineup of invited speakers and topics is quite impressive.

Keynote Address: Richard Axel, Columbia University, HHMI

Topics and Discussion Leaders:

Controlling Neural Activity with Light
Karl Deisseroth, Stanford University
Stefan Herlitze, Case Western Reserve University
Richard Kramer, University of California, Berkeley

Fluorescence Imaging of Neurons
Hollis Cline, Cold Spring Harbor Laboratory
David Kleinfeld, University of California San Diego
Hannah Monyer, University of Heidelberg, Germany

Optical Measures of Neuronal Excitation And Signaling
Rainer Friedrich, Max-Planck-Institute for Med. Res., Germany
Ryohei Yasuda, Duke University Medical Center
Rafael Yuste, Columbia University

Novel Methods to Dissect Neural Circuits
Dmitri Chklovskii, Cold Spring Harbor Laboratory
Jeff Licthman, Harvard University
Clay Reid, Harvard Medical School

Super-Resolution Optical Techniques
Eric Betzig, Janelia Farm Research Campus
Stefan Hell, Max Planck Institute for Biophys. Chem., Germany
Sunney Xie, Harvard University

Novel Approaches to Ultrastructure
Jean-Louis Bessereau, INSERM Ecole Normal Superieure, France
Winfried Denk, Max-Planck-Institute, Germany
Mark Ellisman, University of California, San Diego

New Protein Tagging Strategies
Konstantin Lukyanov, Shemyakin and Ovchinnikov Inst., Russia

The inaugural meeting two years ago had the highest density of innovative, novel, and unpublished research of any conference I have attended. How much more has the field progressed in two years? We will find out soon. I will be posting a daily blog of my notes on the talks and poster sessions, starting Friday, March 23rd. Take a scan through the full abstract list to whet your appetite.