UPDATE : Bi-Directional Optogenetic Control

The Deissseroth lab has released an updated version of their optical neuronal silencing gene Natronomonas halorhodopsin. In Molecular and Cellular Approaches for Diversifying and Extending Optogenetics, Gradinaru et al review current optogenetic methodology, and introduce eNpHR3.0-2A-ChR2, a genetic vector whose expression allows both action potential silencing and firing via illumination. This vector uses post-translational cleavage (via cis-acting hydrolase elements) of the 2A peptide to coexpress channelrhodopsin and halorohdopsin at high levels via a single promoter. The use of 2A provides a more balanced level of relative expression compared to the traditional strategy of using an IRES site, though differing degradation rates of the two proteins cause expression to not be truly stoichiometric.

eNpHR3.0 has superior cellular membrane expression

The improved eNpHR 3.0 contains additional trafficking sequences that greatly reduce expression in intracelluar compartments.  This results in enhanced surface expression a 20-fold increase in photocurrents over eNpHR1 and large, near-nanoampere currents at modest 3.5mW/mm^2 light intensities.  The paper implies superior performance over the Boyden group’s Arch optogenetic silencer technology, but shows no head to head data.  As always, testing both in your own system is the best way to evaluate their relative merits.

Activation spectrum for eNPAC (left), and for ChR2(H134R) (right, blue) and eNpHR3.0 (right, yellow) alone. Maximum eNPAC steady-state excitation was 567 ± 49 pA at 427 nm (n = 9), 62% of the value for ChR2(H134R) alone (916 ± 185 pA; n = 5). Similarly, maximum eNPAC inhibition was 679 ± 109 pA at 590 nm (n = 9), 61% of the value for eNpHR3.0 alone (1110 ± 333 pA; n = 4). Output power density for peak eNpHR3.0 current values was 3.5–5 mW/mm2 (3.5 mW/mm2 at 590 nm).

Gradinaru, V., Zhang, F., Ramakrishnan, C., Mattis, J., Prakash, R., Diester, I., Goshen, I., Thompson, K., & Deisseroth, K. (2010). Molecular and Cellular Approaches for Diversifying and Extending Optogenetics Cell DOI: 10.1016/j.cell.2010.02.037

Tang, W., Ehrlich, I., Wolff, S., Michalski, A., Wolfl, S., Hasan, M., Luthi, A., & Sprengel, R. (2009). Faithful Expression of Multiple Proteins via 2A-Peptide Self-Processing: A Versatile and Reliable Method for Manipulating Brain Circuits Journal of Neuroscience, 29 (27), 8621-8629 DOI: 10.1523/JNEUROSCI.0359-09.2009

Ultrafast optogenetic control with ChETA

The Deisseroth and Hegemann groups have just published a newly engineered channelrhodopsin, ChETA, in the Nature Neuroscience paper, Ultrafast optogenetic control.  Gunaydin et al rationally targeted mutations to the opsin pocket of channelrhodopsin-2 to increase the speed of channel deactivation/closing. ChETA provides higher fidelity optical control of spiking at high expression levels or firing frequencies (up to 200Hz!) and eliminates plateau potentials during sustained spike trains.

ChETA improves spike train fidelity

ChETA clearly provides higher precision in optical control of spiking, particularly at high spike rates.  However, a big problem limiting some in vivo channelrhodopsin use has been insufficient conductance. Some groups have sought to increase single channel conductance, but this approach can lead to increased ChR toxicity and/or spurious spikes. At first glance, increasing deactivation rates, and thus decreasing single channel current from a brief light pulse, seems to make life MORE difficult for situations where light and conductance levels are limiting. ChETA produces a very low number of successful spikes at 1ms illumination (Fig 3f), as compared to ChR2. ChETA response peaks within 1ms but requires 2ms illumination and >10Hz trains to induces spikes more reliably than ChR2. Is this due to a decreased peak single channel conductance in ChETA or just the activation/deactivation rate differences?  I couldn’t find a direct single-channel conductance comparison in the paper.

ChETA requires a longer light pulse than ChR2 to generate spikes.

A reduced conductance might not be a bad thing though. ChETA’s increased deactivation rate might make less toxic to cells, allowing a higher expression level, which would compensate for a reduced single channel current flow. It all depends on what causes ChR2 toxicity.  Is toxicity caused by a non-illuminated leak current or something else?  Is the deactivation rate correlated with a leak current and/or toxicity?  I would love to see a quantitative comparison of expression level and toxicity between wt ChR2  and ChETA.  Maybe our readers can post their experiences with it in the coming weeks.

A Better Neuronal Off-Switch

Ed Boyden’s group has published High-performance genetically targetable optical neural silencing by light-driven proton pumps, detailing a set of new optical neuronal off-switches borrowed from various species that appear to be much better than Halorhodopsin for silencing neurons.  Halo works well for preventing action potentials when the nucleus is illuminated, but has a harder time blocking transmission of action potentials down an axon after it has been initiated. Also, previously engineered Halo variants, including eNpHR, suffer from light-dependent inactivation and have an expression sweet spot that could use broadening.  I’m looking forward to finding out if Arch, and the other new switches from Boyden’s group allow more powerful experiments in our hands.

Optogenetic induction of memory recall

Speaking of reactivating specific memories, at the 2009 Society for Neuroscience meeting, Matteo Rizzi of Michael Häusser’s lab is presenting the realization of an idea that has been floating around in some research proposals I’ve read over the last year.  Express channelrhodopsin-2 under control of the immediate early gene c-fos, induce a strong memory formation via fear conditioning, and then drive the recall of that memory by stimulating the neurons that are expressing ChR2. Immediate early genes are activated shorty after high levels activity in neurons, though the precise patterns are different depending on which promoter (c-fos, Zif268, etc) you use, making precisely HOW they reflect recent neuronal activity patterns unclear.  Nevertheless, the activation of the c-fos based pattern seems close enough to trigger an identical behavioral response as the conditioned stimulus.

Not yet, but getting closer...

Electrically-induced fear conditioning is probably the most blunt instrument possible, encoding a very powerful, general ‘fear’ memory, and many things can make a mouse freeze. Thus, this is definitely the low-hanging fruit on the ‘reverse-engineering’ memories tree. Understanding how the information in a memory is distributed across participating neurons is going to take a more sophisticated approach and a lot more work. This result is still incredibly cool, and I’m somewhat surprised it worked by driving ChR2 with c-fos in a hundred cells in the dentate gyrus. That has pretty powerful implications for avenues by which memories can be recalled.  Surely the entire memory is not encoded by only the 100 neurons that were activated! How many other neurons participate, and how does the optical stimulation activate the entire ensemble? Is it even necessary to activate the entire ensemble to drive behavior? The poster will be MOBBED.  I look forward to reading the details.

Program#/Poster#:	388.8/GG103
Title:	Memory recall driven by optical stimulation of functionally identified sub-populations of neurons
Location:	South Hall A
Presentation Time:	Monday, Oct 19, 2009, 10:00 AM -11:00 AM
Authors:	*M. RIZZI, K. POWELL, J. HEFENDEHL, A. FERNANDES, M. HAUSSER; Wolfson Inst. for Biomed. Res., UCL, London, United Kingdom
Abstract:	The mammalian brain is capable of storing information in sparse populations of neurons encompassing several brain areas. Immediate recall of this information is possible upon presentation of a cue or context. Most aspects of this process remain unresolved: are the cells involved in information storage also responsible for its recall? What portion of this distributed circuit needs to be reactivated, in order to achieve successful recall? To answer these questions we selectively expressed a genetically encoded optogenetic probe (Boyden et al., 2005) in neurons engaged during the learning of a specific association. A plasmid encoding channelrhodopsin-2 and EGFP under an immediate early gene promoter (c-fos-ChR2-IRES-EGFP) was electroporated in vivo into granule cells (GCs) of the dorsal dentate gyrus of anaesthetized C57BL/6 mice. Mice were allowed to recover, and then underwent classical delay fear conditioning (consisting of 10-20 pairings of a 5 second auditory tone and a 2 second footshock). An optic fiber was implanted intra-cranially to allow optical stimulation of transfected neurons. Light stimulation (λ = 530 nm; 5 Hz) successfully induced recall of the fear memory, measured as freezing behaviour (n = 27 animals). Post-hoc analysis of the transfected tissue revealed that a remarkably small subpopulation of GCs (<~100 cells) was sufficient to cause this effect. We then tested whether any, comparatively sized, subset of GCs could be equally effective. We transfected neurons with a plasmid encoding ChR2 expression under a general promoter (pCAG-ChR2) to obtain ChR2 expression in a random population of cells. Interestingly, optical stimulation of this population was insufficient to induce memory recall (population data: n=30). Our results therefore suggest that recall of a learned association, sparsely stored in neuronal circuits distributed over several brain areas, can be achieved by the simple reactivation of a very small subset of neurons involved in learning this association. Furthermore, our strategy may also be useful for dissecting the complexities associated with memory storage and recall. Support: Gatsby Charitable Foundation; Wellcome Trust

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.

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.

Deep & local Channelrhodopsin-2 two-photon activation

An interesting paper on two-photon activation of channelrhodopsin-2 is out in Biophysical Journal. In In-depth activation of ChR2 sensitized excitable cells with high spatial resolution using two-photon excitation with near-IR laser microbeam, Mohanty et. al show cellular activation with a fast-scanning two-photon laser.

Action potential generation from Channelrhodopsin-2 with a two-photon beam has been difficult to achieve, presumably due to the small activation volume of the 2p spot. They show similar calcium transients in response to 2p stimulation as with one-photon stimulation. As depth increases, the one-photon response attenuates faster than the two-photon. Unfortunately, the supplemental info with  electrophysiology traces are not yet online.  Presumably, they are generating action potentials, but I’d like to see the raw data.  Interestingly, they also show calcium increases when the laser stays in once place.  This would imply that local depolarization causes local voltage-gated calcium channels to open, or that calcium is getting through the ChR2. I was under the impression that ChR2 has a low conductance for calcium, though this study by Caldwell et. al, in press for JBC, uses ChR2 specifically for its calcium permeability.

I’m not sure what to make of the first paper. Are they really able to fire action potentials with two-photon stimulation, at depth?  Or are the calcium traces they are seeing simply the result of localized calcium flux.  I’ll followup once the Supplemental Data becomes available.  Still worth a look if this is the sort of thing you are interested in.

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 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.