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.