Three ways of looking at touch coding

20 09 2012

At SfN, a block of three posters by myself, Simon Peron and Daniel O’Connor will showcase three ways to approach the problem of touch coding.

My work on whisker force measurements, and single cell and silicon probe based cortical recordings during active objection localization :

Program#/Poster#: 677.18/KK18
Presentation Title: Encoding whisking-related variables in the mouse barrel cortex during object localization
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Authors: *S. A. HIRES, D. O’CONNOR, D. GUTNISKY, K. SVOBODA;
Janelia Farm Res. Campus, ASHBURN, VA

Simon Peron’s work on recording a complete representation of touch using in-vivo imaging with new G-CaMP variants during a similar behavior :

Program#/Poster#: 677.12/KK12
Presentation Title: Towards imaging complete representations of whisker touch in the mouse barrel cortex
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 4:00 PM – 5:00 PM
Authors: *S. P. PERON1, V. IYER2, Z. GUO2, T.-W. CHEN2, D. KIM2, D. HUBER3, K. SVOBODA2;

Daniel O’Connor’s work on constructing synthetic perception of touch and object localization via cortical cell-type specific optogenetic stimulation during behavior :

Program#/Poster#: 677.06/KK6
Presentation Title: Neural coding for object location revealed using synthetic touch
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Authors: *D. H. O’CONNOR1, S. A. HIRES1, Z. GUO1, Q.-Q. SUN2, D. HUBER1, K. SVOBODA1;

This is a must-see session for people interested in touch coding, the whisker system, in-vivo cortical imaging, or synthetic perception via optogenetics.

I hope to see you there.

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UPDATE : Bi-Directional Optogenetic Control

26 03 2010

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).


ResearchBlogging.org
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

ResearchBlogging.org

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

19 01 2010

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

13 01 2010

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.





Three Cheers for GCaMP : Optogenetic Brain Reading

9 11 2009

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.

Crystal structure of GCaMP2

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.

Screen shot 2009-11-09 at 7.19.27 PM

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.

Screen shot 2009-11-09 at 7.20.12 PM

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.

For further info about strategies for GECI use and optimization, check out our previous paper, Reporting neural activity with genetically encoded calcium indicators in Brain Cell Biology.

The official press release from HHMI regarding GCaMP3 is available here.





Optogenetic induction of memory recall

18 09 2009

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.

Get your ass to Mars!

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

16 07 2009

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).

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.