Journal Club : Classic Single Unit Physiology in Barrel Cortex

This one is for the aficionados. Here is a little review of four classic single-unit physiology papers investigating the response properties and information flow from whisker through thalamus and into cortex.  It’s quite interesting comparing this data taken from sedated or anesthetized rats to my own in awake, behaving animals. That’s a story for another time and publication venue though 🙂

Fukushima soil contamination

I spent the last two nights arguing with a friend about the potential danger of radioactive fallout from Fukushima.  I contended any US fallout was negligible, he disagreed.  This led to reading a Science magazine post outlining the high soil contamination readings near Fukushima, and then to the real raw data, and comparisons to Chernobyl contamination.  We ran the numbers, and still disagree re: the US, but for Japan, they look bad.  If Chernobyl is a valid guide, which is a pretty conservative estimate of the caution of Japan’s government, there will be permanent closure of some towns that are >30km from the plant.

Here is a crude map I whipped up comparing the soil cesium-137 levels that led to restricted access to areas around Chernobyl, with that reported by the Japanese Ministry of Education, Sports, Science and Technology. Here is a directory with the compiled source data, updated daily.  The results are very disturbing.  Anything above 1500 kBq/m^2 was permanently closed near Chernobyl. Anything above 555  kBq/m^2 was permanently restricted.  If my topsoil conversion factors are correct, and they do match the expert’s assumptions in the Science mag story, then there are numerous villages above 555 kBq/m^2 level that are fairly far from the plant.  The 100 kBq/m^2 levels at a distance of 80km are also worryingly high. These aren’t cherry-picked readings to make things look dramatic, they are the rough average of many repeated samples across weeks.  No wonder the government finally upgraded the incident to a level 7. Click the picture for the big view.

Comparison to the whole of Europe is also illuminating, the numbers are the same scale as above.

Commentary : #tigerblood vs. Tiger Mother

This is straying pretty far from the usual blog posts, but its topical. Listening to Bill Simmons’s podcast with Chuck Klosterman while driving home last night got me thinking about the role of confidence in society.  Would be interested in hearing feedback on it.

A common reaction to periods of high anxiety, stress (or hangovers), is negative self-talk.  Muttering things like “I hate myself, I’m worthless” etc.  These reflexive utterances, though not fully contemplated, produce a running monologue that reinforces negative thought processes during the period of anxiety.  With the amount of stress, hangovers and therapy that Charlie Sheen has been through, I suspect that he has trained himself to reflexively substitute negative self-talk phrases with positive phrases, “I’m winning.” “I have tiger blood.”  Does this lead to a better outcome?

Certainly, it seems that he has exerted more control of the public narrative than other celebrity implosions (Mel Gibson, John Galliano). It is difficult for the public to reach a consensus. Is Sheen on drugs?  Is he in a manic episode?  Or is this Sheen’s “new normal”, after his reward circuits have been twisted by heavy stimulant use paired with his conscious effort to train his mind to project a positive attitude? His hyper-confidence, coupled with his success in certain metrics (money, fame, sexual conquest), and his self-promotion through traditional (TV interviews) and social media (1M twitter followers in 24 hours, #winning and #tigerblood hash-tags), enable him to continue his behavior and promote its acceptance.

Compare Sheen’s #tigerblood approach to that of the Tiger Mother, Amy Chua.  Chua used relentless criticism on her children to drill them into technical mastery of academics and musical performance.  Her kids achieved by her metrics, though felt terrible about their perceived failures. She then cleverly tapped into America’s faltering confidence in the face of rising Chinese competition to drive an incredible volume of analysis and publicity for her book on the Tiger Mother approach to parenting.  The most telling quote is from Elizabeth Kolbert’s analysis in the New Yorker

Just about the only category in which American students outperform the competition is self-regard. Researchers at the Brookings Institution, in one of their frequent studies of education policy, compared students’ assessments of their abilities in math with their scores on a standardized test. Nearly forty per cent of American eighth graders agreed “a lot” with the statement “I usually do well in mathematics,” even though only seven per cent of American students actually got enough correct answers on the test to qualify as advanced. Among Singaporean students, eighteen per cent said they usually did well in math; forty-four per cent qualified as advanced.

Implicit in this quote is that academic achievement is of greater value than self-regard.  But as Larry Summers pointed out to Chua at Davos

Which two freshmen at Harvard have arguably been most transformative of the world in the last 25 years?  You can make a reasonable case for Bill Gates and Mark Zuckerberg, neither of whom graduated.

Both of these people had extreme self-confidence, though also significant intellectual candlepower. Charlie can’t match on the brains front, but he possesses a certain charisma that is perhaps the Hollywood equivalent of book smarts.

The real fascination with Charlie Sheen saga is what it says about the role of confidence in society. Sheen represents an extreme test case to the question, “Is confidence the key to success?”

Rapid warping of two-photon illumination wavefronts

A short paper in Optics Express looks interesting.  In A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media, Meng Cui presents a method for rapidly determining the optimal wavefront to ‘cancel out’ the scattering when 785nm light passes through turbid media.  In his example, a glass diffuser was used, but the clear goal for this work is to replace the glass with a brain.

To understand why this is so important for in vivo two-photon imaging, let’s review how 2-p imaging works. Light from a laser is focused to a point and swept across the field in a raster. The resulting fluorescence is of a different wavelength and can thus be filtered out from the excitation light. For each voxel, all the fluorescence that re-enters the objective is collected, regardless of its source.  The total amount of fluorescence collected for that timepoint in the sweep is assigned as the brightness of that voxel. Since the user knows where the laser was being aimed, scattering of fluorescence emission may reduce the brightness but will not blur the image.  However, scattering of the excitation light can dramatically reduce the excitation at the target voxel while increasing the off-target excitation of its neighbors. This causes a rapid increase in background fluorescence and blur at increasing brain depth.

The vasculature was labeled by injecting flourescein dextran into the circulatory stream. The light source was a regenerative amplifier. ‘‘0 mm’’ corresponds to the top of the brain. Left, XZ projection. Right, examples of XY projections. Note the increase in background fluo- rescence deeper than 600 mm in the brain due to out-of-focus 2PE. (Theer et al., 2003)

Previous reports work has shown that one can use adaptive optics to adjust the phase of the wavefront of the excitation light to correct for the scattering of the excitation.  However, determination of the optimal wavefront for a field of view took minutes, which could be problematic for imaging in an awake animal.  Any changes in the precise position of the brain might change the optimal wavefront.  Ideally, one would want a system that could optimize the wavefront every second, or even before every frame of acquisition (typically 4-8 Hz in a raster scan in vivo experiment)

Scattering in the brain warps two-photon excitation light, but adaptive optics can correct this.

I’ll let Meng Cui explain the technique in his own words

Elastic scattering is the dominant factor limiting the optical imaging depth in tissues. Take gray matter as an example, at 800 nm the scattering coefficient is 77 /cm and the absorption coefficient is 0.2 / cm. If there is a way to suppress scattering, the optical imaging depth could be greatly improved. Despite the apparent randomness, scattering is a deterministic process. A properly engineered wave can propagate inside scattering media and form a focus, a well understood phenomenon in the time reversal and optical phase conjugation (OPC) studies…

For applications on biological tissues, acquisition time on the order of one millisecond (ms) per degree of freedom is desired. Deformable mirrors can provide a high modulation speed. However the degrees of freedom are rather limited. A phase-only SLM can provide about one million degrees of freedom at a much lower modulation speed. In this work, I present a novel method, capable of providing as many degrees of freedom as a SLM with a data acquisition time of one ms per degree of freedom. The method was employed to focus light through a random scattering medium with a 400 ms total data acquisition time, ~three orders of magnitude faster than the previous report [25].

The essence of a COAT system is to phase modulate different input spatial modes while detecting the output signal from the target. To greatly improve the operation speed, the experiment requires a device that can provide fast phase modulation and can access a large number of spatial modes very quickly. To meet these two requirements, a pair of scanning Galvanometer mirrors was used to quickly visit different modes in the spatial frequency domain or k space, and a frequency shifted reference beam was provided for a heterodyne detection. The wavefront profile was first determined in k space and then transformed to the spatial domain. The spatial phase profile was displayed on a SLM to focus light onto the target. In such a design, the number of degrees of freedom is limited by the number of pixels on the SLM and the experiment speed is determined by the scanning mirror speed…

Compared to existing techniques, the reported method can provide both a high operation speed and a large number of degrees of freedom. In the current design, the operation speed is limited by the scanning mirror speed and the maximum number of degrees of freedom is limited by the SLM pixel number. In this demonstration, 400 spatial modes in k space were visited and the determined phase profile was displayed on the SLM. Depending on the scattering property of the media, more (up to 1920 x 1080) or less number of degrees of freedom can be used to optimize the focus quality and the operation speed.

Using a stepwise position scanning, the method achieves an operation speed of one ms (400 μs transition time + 600 μs recording time) per spatial mode, ~three orders of magnitude faster than the previous report. Using a continuous position scanning and a faster position scanner such as resonant scanning mirrors, polygon mirror scanners, or acousto-optic deflectors, the operation speed can be potentially increased by at least one order of magnitude. It is anticipated that the reported technique will find a broad range of applications in biomedical deep tissue imaging.

Quick Picks : Brainbow flies

Nature methods published two papers which extend brainbow-like techniques of stochastic multicolored neuronal labeling into fruit flies.  Nature’s summary explains the two methods.

 

dBrainbow expression examples

 

 

The first technique, called dBrainbow, was developed by Julie Simpson, a neuroscientist at the Howard Hughes Medical Institute’s Janelia Farm Research Campus in Ashburn, Virginia, and her colleagues². This method uses enzymes called recombinases to randomly delete some of the colour-producing genes from the string, leaving different genes next to the promoter regions in different cells. Individual cells are therefore uniquely coloured and so can be easily distinguished…

The second technique, called Flybow, was developed by Salecker and her colleagues³. They used an enzyme that ‘flips’ pairs of colour-producing genes on the string, leaving different genes next to the promoter region. The ‘flipping’ enzyme is also a recombinase, and so after being inverted, some of the colour-producing genes are randomly deleted. This ensures that all the different genes on the string can potentially end up next to the promoter, and be displayed by individual modified neurons.. Flybow uses a single string of four colours — red, green, blue and yellow.

These techniques will find use in building the structural and functional connectome of the fly.

 

Optical control of gene expression in mammalian cells

Trying to start a reboot of the posts here on Brain Windows. Lots of great stuff has come out since the last regular posting period, and unfortunately I don’t have the time to cover it all. One of the most exciting papers of the last few months was Rapid blue-light–mediated induction of protein interactions in living cells published in Nature Methods. This paper reports the  logical extension of previous technologies for photoactivatable transcription we previously covered here, here, and here.

There are two key technical improvements in the system from the Tucker Lab.  First, the genetic light switch, a cryptochrome 2 (CRY2) interaction with cryptochrome-interacting basic-helix-loop-helix protien (CIB1), is activated by blue light rather than the red light of previous switches based on phycocyanobilins.  Second, and more importantly, the cofactors necessary for the switch action (flavin and pterin chromophores), are endogenously expressed in mammalian tissues.  Thus, these switches should be usable in vivo without potentially tricky loading of the cofactors.

Upon illumination, the authors observed rapid translocation (in 1 second!) of fluorescent proteins tagged with CRY2 to cell membranes with CIB1 anchored to it.  They also were able to couple it to Gal4-UAS and Split-Cre expression systems, which let them drive reporter genes such as GFP by blue-light illumination.  I was a bit underwhelmed by the efficacy of the cre-induction, only around 15% of cells expressed the cre-driven EGFP after 24 hours of illumination, but maybe that is due to my ignorance of the current limits of the split-cre system.  That efficacy will certainly need to be improved for the REALLY cool stuff one can imagine doing with this.

What are the cool things?  Well, say you are doing some GCAMP3 imaging of a few hundred cells in the cortex during an awake behavior.  You see an ensemble of neurons whose activity is correlated to some aspect of the behavior, like a motor command, a perception or a decision. You want to prove the function of these neurons, to investigate their coding by subtracting or adding activity directly into this specific functional group. How do we control ONLY this group?

A pan-neuronal channelrhodopsin, or even one packaged in a cre-dependent virus injected into a cre reporter line will not allow you to change the spike patterns of only this ensemble. This ensemble is not differentiable from its neighbors by genetic type, only by functional relevance.  You have to hit its neighbors or shared genetic subtype with the same hammer.  But if you have one of these CRY2-CIB1 split cre switches that drive ChR2 expressed across the cortex, you could shine a blue laser (or presumably a two-photon laser) onto the members of the ensemble and turn on optical control of only that functional group.

Details of course still need to be worked out. What is the 2p cross-section of the system? How do you make it compatible with optical imaging and optical control?  How do you improve the speed and efficacy of the switch? These are things that will come with time.  The power of this technique is even recognized by apparent competitors in the field; Anselm Levskaya closed his packed SfN talk on phycocyanobilin-based optical switches with a shout out to this work.

Stay tuned…
Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, & Tucker CL (2010). Rapid blue-light-mediated induction of protein interactions in living cells. Nature methods, 7 (12), 973-5 PMID: 21037589

UPDATE : DIADEM Final Results

The DIADEM automated neuronal reconstruction contest has finished.  Accurate, fast, and high-resolution automated neuron reconstruction is of vital importance to cracking the mystery of how neural circuits perform. Even with perfect knowledge of the firing patterns of every cell in a circuit, our understanding of how these patterns are produced and how the information is processed would be quite limited.  True understanding requires knowledge of the precise wiring diagram.  This prize is a good first step towards bringing awareness of this tricky problem to the world’s best computer scientists.

$75,000 in prize money was to go to the group that was able to produce high-quality reconstructions of neuronal structures at least 20x faster than by-hand reconstructions.  In the finals, the fastest speed achieved was 10X the by-hand method. Some groups were hindered by slight variances in the source data formatting, which normally isn’t a big deal unless you only have 20 minutes to produce as much reconstruction as possible…

Since no group was able to beat the hard floor, but substantial progress was made, the money was distributed amongst these finalists.

Badrinath Roysam Team, $25,000
“for the better overall generality of their program in producing robust reconstructions by integration of human and machines interactions.”

Armen Stepanyants Team, $25,000
“for the better overall biological results in the spirit of pure automation.”

Eugene Myers Team, $15,000
“for the excellent quality and strength of their algorithm.”

German Gonzalez Team, $10,000
“for their deeper potential, more original approach, and ultimate scalability of their proposed solution.”

Deniz Erdogmus Team
“for elevating themselves above the current state of automated reconstructions…with a deep understanding of the technical and scientific problems.”

Congrats to the placing teams.

Software Update : Ephus, ScanImage & Neuroptikon

Three excellent pieces of neuroscience software have been recently updated or freshly released.  I have used two of them, Ephus and ScanImage, on a daily basis as primary data collection tools. The third, Neuroptikon, is quite useful for post-hoc illustration of neural circuits.

Ephus is a modular Matlab-based electrophysiology program that can control and record many channels of tools and data simultaneously.  Under control of a sophisticated internal looper or external trigger, you can initiate an ephys recording, trigger camera frames, adjust galvo positions, open/close shutters, trigger optical stimulation, punishments, rewards, etc.  It is a workhorse program for non-imaging related in vitro and in vivo electrophysiology experiments.  Ephus is named for the fabled baseball pitch, and pronounced as “EFF-ess”. As with the pitch, it may trick you at first, but eventually you’re sure to hit a home run. Of course, the name also evokes electrophysiology, which is the fundamental orientation of the project, be it optical or electrical.

Ephus 2.1.0 is a major release, and the only official version at this time.  The software is fully described in a publication in Frontiers in Neuroscience. New features include unlimited recording time, with disk streaming, for applications such as EEGs and long traces during in-vivo behavior. A number of additional scripts for in-the-loop control have been added. New configuration/start-up files have been created, with a template to help get up and running quickly. This release also includes a number of bug fixes.

ScanImage is another Matlab-related software program that is used for optical imaging and stimulation of neurons in vitro and in vivo.  It finds much use a control platform for 2-photon imaging, glutamate uncaging and laser-scanning photostimulation.  An early incarnation is described in this paper by Pologruto, et al.  It provides a lot of power right out of the box (bidirectional scanning @ 0.5ms/line, etc) and is easily extensible via custom user function plugins.

Neuroptikon is a sophisticated network visualization tool.  It can build Van Essen-like diagrams of any circuit you like, but it is so much more.  The direction of communication is animated, and subsets of regions and connections can be brought into focus, which greatly eases the clarity of the network.  The diagrams can be built in three-dimensions, to preserve relative topography, or functional grouping.  There is simple GUI-based control, while more complex tasks can use a scripting interface.  This is great software for anyone who needs to imagine information flow in a complex network.

All three tools are released for free use under the HHMI/Janelia Farm open source license.

Download Here :

Ephus 2.1.0

ScanImage 2.6.1

Neuroptikon 0.9.9

Cameleon-Nanos : High Affinity GECIs

Takeharu Nagai’s lab has published in Nature Methods, Spontaneous network activity visualized by ultrasensitive Ca²⁺ indicators, yellow Cameleon-Nano, demonstrating a new set of calcium indicators based on yellow cameleon. Back when he was still Take-san, Take’s ability to churn out and manually screen hundreds of cameleon variants was impressive and inspiring. With high-throughput GECI pipelines now ramping up at Janelia, the idea of laboriously screening 200 variations on a theme (be it cameleons or GluSnFRs), seems a bit archaic. However, this paper is a good example of the progress that can still be made by understanding the needed sensor parameters and fiddling with the primary amino acid structure in a relatively low-throughput way. Take-sensei’s results are another example of the pramatic rule in protein design, “when in doubt, tinker with the linker.”

The cameleon-nano family achieves greater apparent calcium affinity than YC2.60-4.60, reaching levels of up to 15nM.  They did this by increasing the flexibility of the linker by extending the standard Tsien/Miyawaki/Baird Gly-Gly-Ser linker with additional glycines.  In this case, the longer the linker between the CaM and M13 segments, the greater the apparent affinity. Interestingly, improvement by increasing linker flexibility is precisely the opposite the advice Atsushi and Take gave me for achieving high ratio changes with FRET reporters.  Back at RIKEN in 2002, they suggested I use short, stiff linkers to restrict the rotational freedom of the fluorescent pairs.  Then one could find orientations where relative rotation of dipole moment gave much greater FRET changes than would be expected from changes in FP distance alone. Take and Atsushi’s big YC2.60/3.60 paper strongly supported this idea!  However, as our understanding of the ideal parameters of calcium sensor’s for in vivo imaging has grown, development directions have adjusted.

Cameleon-Nanos achieve higher signal/noise for sparse action potentials at the expense of linearity.  Like Fluo-4, the signal saturates at relatively low AP frequencies.  I think the absolute affinities measured for this family (15, 30, 50 and 140nM) should be considered very rough estimates. They extrapolated these values from stopped-flow binding experiments, because

Although we would like to measure the koff of YC2.60 and its high affinity variants such as YC-Nano15, we could not do it because it was very difficult to precisely control free Ca2+ concentration at around few tens of nM as far as we used EGTA (Kd for Ca2+ = 151 nM in 0.1 M ionic strength, pH 7.2 at 25 oC). For this purpose, much stronger Ca2+ chelator with a smaller Kd value was required. However there is no such Ca2+ chelator available now.

I’m not sure why they didn’t just use the higher affinity, Mg++ insensitive, chelator BAPTA to make the Kd measurements the right way, with a linear regression of log-log fluorescence/concentration values.  Due to instrument dead time, and the high affinity, I didn’t like stopped-flow based Kd measurements in the early GCaMP papers, and I don’t like them now.  Also, the apparent calcium Kd will be highly dependent on solution ionic strength and [Mg++] which is unreported. Despite these quibbles, which are important only inasmuch as they give insight into the mechanism of improvement and the direction of future development, the cameleon-nano family looks promising for mammalian brain imaging.  I still wonder if, assuming the reported Kd values are relevant in vivo, YC2.60 would be the best of the bunch, since cortical neurons have a resting Kd of ~50nM, which implies that a single AP transient of say 200nM free [Ca++] increase would push the calcium levels right up into the sweet-spot of YC2.60’s sensitivity.

This is all the more interesting given the recent results in YC3.60 imaging from Maz Hasan’s group.  Previously, he had shown that transgenic YC animals were pretty bad for imaging.  However, AAV-mediated gene delivery of YC3.60 has significantly improved the responses of the YC family. I’m not sure if they are really up to GCaMP3 levels under identical in vivo conditions, but they might have better long-term protein stability (or that might depend on which viral serotype is used.) What about cameleon-nanos, what about YC2.60?

Backyard Brains Homebrew Ephys Rigs

News of a cool new toy comes from a colleague’s recent trip out to the MBL @ Woods Hole.  It is the perfect gift to spark the curiosity of a budding young (or old) neuroscientist. Backyard Brains makes the world’s best value electrophysiology rigs. The SpikerBox comes pre-assembled for $100 or build your own from a bunch of parts for $50.

These rigs are surprisingly powerful. You can go out in the yard, catch a bug (or buy a cockroach), strap one on and start recording neuromuscular potentials. The box interfaces with gorgeous iPhone/iPad so you can hear, see and record the action potentials. They don’t make a big point of this, but you can also wire it up so that the piezo-speaker can drive an electrical stimulator of the cockroach’s leg. You can make the leg twitch to whatever signal input you give it. The educational possibilities of this gear is really limitless.