Three Cheers for GCaMP : Optogenetic Brain Reading

Three papers are out online in Nature Methods that show big improvements in calcium imaging with genetically encoded sensors.  They are are based on the fluorescence intensity indicator, GCaMP.   GCaMP, first developed by Junichi Nakai, consists of a GFP that has been circularly permuted so that the N and C termini are fused and new termini are made in the middle of the protein.  Fused to one terminus is calmodulin and the other is a peptide, M13, that calmodulin (CaM) binds to in the presence of calcium. The name is supposed to look like GFP with a CaM inserted into it, G-CaM-P.  Normally the GFP is dim, as there is a hole from the outside of its barrel into the chromophore.  Upon binding calcium, this hole is plugged and fluorescence increases.

The first paper, A genetically encoded reporter of synaptic activity in vivo, from Leon Lagnado’s group, targets GCaMP2 to the outer surface of synaptic vesicles. This localization allows the fluorescence signal to be confined to the presynaptic terminal, where calcium fluxes in response to action potentials are high.  This targeting improves the response magnitude of GCaMP2 and permits the optical recording of synaptic inputs into whatever region of the brain one looks at.  They demonstrate the technique in live zebrafish.

In the second paper, Optical interrogation of neural circuits in Caenorhabditis elegans, from Sharad Ramanathan’s group, GCaMP2 has been combined with Channelrhodopsin-2 to perform functional circuit mapping in the worm.   Since the worm’s structural wiring diagram has been essentially solved, functional data could say much about how “thick” the wires between each cell are.  Unfortunately, with GCaMP2, the responses are too slow and weak to distinguish direct from indirect connections.

Finally, we have published a paper, Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators, describing the improved GCaMP3.  This indicator has between 2-10x better signal to noise than GCaMP2, D3cpv and TN-XXL, depending on the system you are using.  It’s kinetics are faster and it is more photostable than FRET indicators, and the responses are huge.  When expressed in motor cortex of the mouse, neuronal activity is easily seen directly in the raw data.  Furthermore, the sensor can be expressed stably for months, making it a potential tool for observing how learning reshapes the patterns of activity in the cortex.

Imaging of mouse motor cortex (M1) expressing the genetically-encoded calcium indicator GCaMP3 through a cortical window. After 72 days of GCaMP3 expression, large fluorescence transients can be seen in many neurons that are highly correlated with mouse running.

GCaMP3 is not perfect. It cannot reliably detect single action potential in vivo in mammals, though I doubt that any existing GECI can. Work continues on future generations of GCaMP that may achieve 100% fidelity in optical reading of the bits in the brain. However, there is considerable evidence from a number of groups that have been beta-testing the sensor, including the Tank lab of “quake mouse” fame, that it is a significant leap forward and unlocks much of the fantastic and fantasized potential of genetically-encoded calcium indicators.

Comparison of fluorescence changes in response to trains of action potentials in acute cortical slices.

I will try to post a more complete writeup of GCaMP3 for Brain Windows soon, with an unbiased eye to its strengths and weaknesses.  We worked very hard to carefully characterize this sensor’s effects on cellular and circuit properties.  If you have any questions about GCaMP3, please post them to the comments.

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

Neurons controlled by DREADD

A big advance in non-invasive neuronal remote control was published today in Neuron. Several groups have been working on expressing non-endogenous or customized receptors into neurons so that specific genetically selected neurons can be turned on or off.  Channelrhodopsin, halorhodopsin and Opto-XRs do this via a light-gated membrane channels or receptors. Ligand-gated alternatives are the Drosophila allatostatin receptor, and RASSLs, GPCRs with customized binding sites. Each one of these has particular drawbacks.  The opsins require coupling of a light fiber into the brain, and high expression of some opsins can cause cytotoxicity.  The allatostatin ligand needs to be perfused directly into the brain. RASSLs show background activity in the absence of the applied ligand, which also can cause toxicity. Bryan Roth’s group has been pioneering RASSLs and has produced second-generation receptors which avoids these drawbacks.  In these new receptors, DREADDs, the background activity is completely abolished and the ligand has no off target effects.  

 

DREADDs have no effect on the spiking activity of the hippocampus in the absence of CNO (top). Subcutaneous injection of CNO causes bursts of action potentials in DREADD expressing hippocampus (bottom).

The DREADD, dubbed hM3Dq, in the paper, Remote Control of Neuronal Activity in Transgenic Mice Expressing Evolved G Protein-Coupled Receptors, allows selective activation of a genetically targeted population of neuron in a totally non-invasive way.  Simply inject the ligand, CNO, and the activity of the expressing neurons will rise in a dose dependent manner.  Onset is rather slow, starting around 10 minutes post-injection and peaking within 45 minutes.  Offset takes hours, so this isn’t the right technology to explore precise temporal coding of spike trains. But, when combined with the genetic targeting information from the Allen Brain Atlas, this tool will find great use in demonstrating the function of specific brain regions and even specific cell types within a brain region. 

The authors have also published an inhibiting DREADD, hM4Di, which can turn off targeted neurons.  I’ve personally tested a variety of neuronal silencing technologies in the last 6 months, including the hM4Di inactivating DREADD.  In in utereo electroporated cortical slices, the expression of these receptors had no discernible effect on the morphology, eletrophysiological parameters or cell health.  When CNO was puffed onto the slice, the amount of current injection required to elicit a spike doubled or tripled. CNO did nothing to non-expressing neurons. The cell returned to normal within seconds of washout of the drug.  I haven’t tested the hM3Dq activating DREADD, but from my experience with hM4Di, I highly recommend these tools for getting the control you want with minimal fiddling with light fibers or expression levels.

Photoactivated Transcription Revisted

Looks like there has been some new results in the field of photoactivated transcription.  Unlike the fully genetically-encoded systems reviewed in a Journal Club, this uses a hybrid genetic and small molecule approach. In Doxycycline-dependent photoactivated gene expression in eukaryotic systems, Cambridge et al. add the photolabile protecting groups to doxycyclin derivatives, which then function as photoactivatable switches in the commonly used Tet-on system. Dr. Dan O’Connor described the technique as “the path of least resistance to photoactivated transcription.” 

 

Local (left) and global (right) GFP expression following optical uncaging of cyanodoxycyclin

 

 

      The authors were able to get robust gene expression with standard UV irradiation, but also were able to uncage sufficient cyanodoxycycline with two-photon illumination to cause highly localized gene expression in cultures.  In live tadpoles, they stuck to UV for the greater efficiency.

     The standard caveats of the tet system apply. The off-state still has a bit of residual gene expression, which is fine for some applications (like fluorescent tagging), and a dealbreaker for others (cre induction). Drug delivery takes time and comes with diffusion, penetration and clearance issues.  UV penetration through deep tissue is going to be a big technical hurdle to overcome to apply this to full-grown mammals. Blasting living tissue with high power UV usually isn’t a good idea. Despite these caveats, the system clearly works and I’d bet the authors are already applying the system to some next-step applications and biological questions. The potential of selectively turning on genes in functionally identified neurons via light is enormous.  It is one of the most likely eventual avenues into possible optical activation or suppression of specific thought patterns (at least if you are willing to squirt virus into your brain and eat a bunch of nasty antibiotics).

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 10×10x10um^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.

Two-Color, Bi-Directional Optical Voltage Control of Genetically-Targeted Neurons

Looks like Ed Boyden may have another hit on his hands. He and Xue Han reported at the recent Cosyne 2007 meeting a new method of controlling neural circuits. You may remember his work on Channelrhodopsin-2, which gave us millisecond optical control of neuronal spiking. Now they have reported a complementary technique with millisecond optical control of neuronal silencing. From the abstract :

We report that cultured hippocampal pyramidal neurons expressing Halo-GFP under the CaMKII promoter experienced strong hyperpolarizations (> -20 mV) upon exposure to brief pulses of moderate-intensity yellow light (~565 nm). In the absence of light, Halo-expressing neurons were physiologically indistinguishable from wild-type neurons.

But wait! There’s more :

We also demonstrated that in individual neurons expressing both yellow-light driven Halo and the blue-light driven cation channel ChR2, neural inhibition and excitation could be efficiently and independently controlled at the millisecond timescale, by interleaving brief pulses of yellow and blue light.

Hey Ed! Leave some neuroengineering projects for the rest of us please.