neurodudes

This is kind of awesome. Go Stanford Hum Bio!

I finally got around to reading the Nature Methods that has been sitting on my coffee table for a few weeks and I was surprised to see an article on photoactivatable transcription using caged doxycycline with standard Tet On/Off genetics. A postdoc in my lab has been suggesting this type of technology for the last few years and speculating about different ways to possibly implement it. What’s so remarkable about this work is the simplicity of the implementation. In our lab (and others), the common assumption has been that the photoactivatable mechanism should be designed such that a recombinase is directly light-activated. (For example, a membrane tethered Cre recombinase where the tether is broken by light and releases the recombinase to the nucleus after illumination.) But that seems a bit overengineered. Is there a simpler way? Yes!

In this work, Cambridge et al. generated a dox analog, cyanodoxycycline, that is better retained inside cells (reduced membrane permeability) and put it inside a stable photoactivatable (UV) cage. Instead of making the system entirely genetic, they use small molecule uncaging (an established technology) to make problem simpler. In this case, standard Tet genetics can be used (thus leveraging existing model organism Tet lines) and the novel caged Dox can be easily added to food, etc. I think this is a great example of finding the simplest solution to a problem that at first seems very complex.

And the uncaging results are quite impressive. Here are two spatial patterns of gene expression in hippocampal slice culture, a smiley face and a single neuron, achieved by clamping down the field stop before illumination:

Neurodudes reader Jason M. sent me some information about a funding agency, IARPA, or Intelligence Advanced Research Projects Activity, that is funding neuroscience-related research. I had never heard of IARPA before but it has existed since 2006 as something of an intelligence-focused DARPA. There upcoming funding deadline (Aug 21) is for projects on detecting trust signals between humans.

Just last night, I watched the tense but amazing film The Hurt Locker (don’t let the name disuade you, see the phenomenal Metacritic rating), which is about a bomb disposal squad during the recent Iraq War. There is one particularly stirring scene with a suicide bomber who claims that he was forced to wear a vest with explosives and doesn’t want to go through with it. The difficulty in the limited time before the bomb explosion revolves around whether to actually trust the man and the challenge of trusting someone when neither party speaks the other’s language. You can certainly at least understand (putting aside the ethics of war itself) why governments are interested in detecting nonverbal trust cues.

Details about the IARPA call for proposals are after the jump. (more…)

Recently, Alexander et al. published Remote Control of Neuronal Activity in Transgenic Mice Expressing Evolved G Protein-Coupled Receptors [Neuron Neurotechniques], in which they use directed evolution techniques to modify a muscarinic GPCR to selectively bind an orally-deliverable small molecule that is otherwise inert. Apparently, this is the first time a channel has been engineered such that is selective for a biologically inert molecule, providing specificity of action. (They compare their technology with the hyperpolarizing allatostatin receptor which can have off-target effects.) Because the channel is specified genetically and the drug circulates systemically, it is easier to activate large populations of neurons (viz. optogenetic methods which are constrained to neurons in the light delivery volume) without implanted devices (eg. cannulas for AlstR, fiber optics for optogenetics, etc.) Another new technique/neurotechnology… onwards marches the innovation of new circuit-cracking tools!

A wonderful illustrated piece on Benjamin Franklin in the NYT. I especially love his daily schedule (complete with morning self-analysis!)

Last night was not a restful one for me. Can neurotechnology help make us more aware of our sleep problems? Over at the NYT, David Pogue thinks so. He recently reviewed an alarm clock with an EEG headband transmitter that analyzes sleep (“To Sleep, Perchance to Analyze Data“).

As he says in the article, the initial reaction to this kind of product might be, I don’t need something to tell me when I didn’t sleep well. I know when I haven’t slept well! As he says in his nice video review, there are some advantages to all this technology (automation is good!… I certainly don’t keep a daily journal of sleep quality…):

But as my wife said, “If I wake up and feel lousy, I don’t need a $400 gadget to tell me it’s because I didn’t sleep well.”

Ah, but that’s where the coaching comes in.

The Zeo stores your sleep records on a memory card. As often as you can, you’re supposed to pop it out and insert it into a U.S.B. card reader (also included) on your computer. At this point, you can go to MyZeo.com and upload your data to the Web.

Now the real fun begins. This Web site lets you slice, dice and cross-compare your sleep data in a million ways.

Malcolm Gladwell provides an interesting take on how human psychology contributed to the demise of Bear Stearns. [New Yorker]

Twin-spot MARCM to reveal the developmental origin and identity of neurons [Nature Neuro]

We mentioned the innovative MARCM technique in a previous post. Here, Lee and colleagues extend MARCM (Mosaic Analysis with a Repressible Cell Marker, pronounced mark-em) to twin-spot MARCM, where both cells from a mitotic event are labeled with different colors fluorescent proteins. In regular MARCM, only one cell is labeled and the other daughter cell remained unlabeled. Like the original MARCM, this technique lets you distinguish between what would otherwise be identical pairs/clonal populations of cells during development and gain insight into the (lack of) stereotypy in development. Under the hood, twin-spot MARCM is a bit different: Instead of relying on GAL80 suppression of GAL4-driven transcription (regular MARCM), twin-spot MARCM uses RNAi directed against the protein-coding transcripts.

Since MARCM can be difficult to understand, here’s an excellent, detailed yet easy-to-understand description written for a bio lab class from Richard Vogt at the University of South Carolina:

1. A fly is constructed with the following genotype: (promotor)Gal4; UAS-GFP. In this fly, the promoter drives the expression of a transcription factor called Gal4, and Gal4 binds to and activates a regulatory site referred to as “UAS” (upstream activating sequence). Activation of the UAS site drives expression of GFP (green fluorescent protein) which fluoresces green when stimulated by blue light.
2. This fly also contains a gene encoding and expressing a protein called “Gal80″; Gal80 suppresses the action of Gal4. If Gal80 is expressed, no GFP is made and no green fluorescence can occur.
3. This fly also contains a complex of genes referred to as the FLP/FRT system; FLP is a transcription factor that activates the FRT site, which is situated adjacent to the Gal80 site. Further more, at least in our case, the FLP is driven by a “heat shock” promoter (hs). All this means is that when you raise the temperature of the animal to 37^oC, this activates the hs promoter which activates the expression of FLP which activates the FRT site.

           Something I’ve not mentioned yet… there is also an FRT site adjacent to the UAS-GFP site. Something else I’ve not mentioned yet, the FRT-UAS-GFP site and the FRT-Gal80 site are on the same chromosome, but importantly on different chromatids.

4. So we make a bunch of fly embryos that have all this stuff in them. Procedurally this is really easy, since the genes have already been put in the flies, and all we have to do is take virgin females of one stain (FRT-Gal80) and mate them to males of another strain (FRT-UAS-GFP) and… POW… we have fly embryos that have all this stuff in them.
5. All the cells in the embryos we now have are capable of expressing GFP except for the one problem… all the cells are expressing Gal80 which is blocking the expression of GFP. We need to turn off Gal80 expression. We do this by activating the FLP/FRT system.
6. Normally, a cell has two copies of each chromosome called chromatids. In our case, the chromatids are different, one containing by FRT-Gal80 and the other containing FRT-UAS-GFP. This cell can not express GFP because Gal80 is present. During mitosis, the chromatids are duplicated and sort to produce two identical chromatid pairs, both pairs consisting of a FRT-Gal80 chromatid and a FRT-UAS-GFP chromatid. Like their mother, neither daughter cell would be able to express GFP, again because Gal80 is present.

           HOWEVER, AND HERE IS THE TRICK… if the FRT is activated during mitosis, it induces a recombination event (recombination normally only occurs during meiosis), creating one chromatid pair that contains only UAS-GFP and another chromatid pair that contains only Gal80. One of the resulting daughter cells now contains no Gal80, and suddenly is able to express GFP and fluoresce green light. And any additional cells produced by this daughter will also express GFP.

There’s a nice editorial in Nature Neuroscience about the Broad Institute’s PsychHTS initiative. The initiative invites scientists from outside the Broad to suggest new high-throughput screens that the Broad will perform. The Broad has invested heavily in capital equipment and expertise for chemical biology screens (ie. small molecule drug libraries with robotic delivery and automated screening). These libraries are huge: 50,000-500,000 molecules can be screened. Although much science is hypothesis driven, this kind of large-scale hypothesis-free exploration just hasn’t been possible before. And this certainly isn’t the kind of thing that can be done in a single lab; only dedicated facilities like those at the Broad could carry out this type of “big science.” For collaborators hoping to use the Broad platform, the key appears to be in developing a good automated assay:

Readouts may be anything from classical enzymatic reactions, through FRET for changes in protein interaction, up to subcellular changes captured by automated high-content imaging. An investigator may send a group member to the Broad to take advantage of its resources or may entirely ‘outsource’ assay development to the chaperone. Assay development typically takes two to three months, sometimes up to a year. The assay is then used to screen one or more compound libraries, encompassing at present up to 400,000 substances and growing. (PsychHTS pays for screening a 50,000-compound subset.) ‘Hits’—compounds that affect the assay results in a way that indicates potential usefulness in a psychiatric research context—are automatically retested at several concentrations. The resulting collection of typically between 50 and 500 confirmed hits is then evaluated and prioritized according to criteria of scientific interest and potential drug promise, and thereby winnowed down to the top 10 or 20. The Broad Institute’s organic chemists then synthesize and retest these compounds plus a series of their chemical derivatives, with goals such as improved solubility and more specific binding to putative targets. The goal of the entire procedure is to deliver small-molecule probes that modulate a specific cellular function—essentially tools for subsequent research into the initial hypothesis regarding a psychiatric disease mechanism.

At this point, the new small-molecule probes will need to be tested in animal models of mental illness.

The most appealing aspect is that the Broad is opening up the process to anyone with good ideas for potential screens. The next application deadline is in September. Considering both PsychHTS and the Allen Brain Atlas, is neuroscience moving away from an individual lab model and more toward a “big science” model of projects with lots of collaboration?

Recently, the most famous (and most studied) person in neuroscience died. Science has a nice piece on the planning and post-morten examination of this most famous brain:

[Suzanne] Corkin delivered Cryopaks to his nursing home in Windsor Locks, Connecticut. “They kept them in the freezer so that the moment he died they could wrap his head to preserve the brain,” she says. When Molaison [ie. H.M.] died of respiratory failure at 5:05 p.m. on 8 December 2008, the plan sprang into action. A hearse took his body to Massachusetts General Hospital (MGH) in Charlestown, where researchers began collecting anatomical magnetic resonance imaging (MRI) scans of his brain at about 9 p.m.—and continued until 6 a.m. the next day, when Annese arrived on a red-eye flight from San Diego.

Jacopo Annese, a neuroanatomist at UCSD, is planning on putting H.M.’s whole brain online on his website. But before that happens, he has a rather huge task before him:

Using a microtome, he will slice the brain into very thin sections. “Like prosciutto,” he says, but less than 1/20 the thickness and a lot more fragile. Annese aims to slice the brain whole instead of first cutting it into smaller chunks as is more routinely done. Small chunks are much easier to work with, but the resulting slices are hard to keep in register with one another. Whole-brain slices will keep more of the tissue intact and result in a more faithful reconstruction of the brain, he says. Annese estimates he will end up with about 2600 slices of Molaison’s brain. He and his colleagues will mount some of these, perhaps every 12th one to start, on extra-large glass slides—13 by 18 centimeters—and treat them with a stain that colors cell bodies purple. A camera attached to a microscope will photograph each slice at 20x magnification, sufficient to distinguish different cell types. At that magnification, photographing a single slice will require a mosaic of about 40,000 individual images.

And there is some stress that comes from dealing with such a one-of-a-kind specimen:

But a lot could go wrong. The MRI scans reveal deterioration of the white matter, Annese says, which might make the slices especially delicate and prone to tearing. An even more nightmarish scenario is a cracked brain, he says. Sometimes, a brain will freeze unevenly and break apart—destroying it before it can be sliced. Annese is taking every precaution, but he’s not taking anything for granted. “Cutting will make or break the project,” he says. “But if the brain cracks, I go back to Italy.”