neurodudes

Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement

Just the optical engineering alone here deserves mention: 28 frames per second at 62nm resolution (well below the diffraction limit of 260nm for light of the wavelength used)! STED (or stimulated emission depletion, developed in Stefan Hell’s group) is ideal for visualizing synaptic vesicles, whose small size (~50nm) has typically confined them to the domain of electron microscopists. The ability to get high-speed STED allowed the researchers to track individual vesicles and their path dynamics. They conclude that vesicle movement has both motor-driven and diffusive components (ie. a biased random walk). I’m sure with more time and more analysis there will be a lot of interesting applications for this kind of real-time vesicle tracking. Perhaps in the near future we will have single vesicle “minis” monitored at multiple sites through microscopy instead of just one or two sites electrophysiologically…

Here’s the resolution difference between STED and confocal for a single vesicle:

And, for those of you with ~$1.25M lying around, you can now purchase a STED setup directly from Leica!

This week’s Nature has quite a few additional halorhodopsin articles for photochannel fans.

Halorhodopsin article from Deisseroth’s lab:
Multimodal fast optical interrogation of neural circuitry [News & Views]

Also, there is an intriguing article on both the general excitement in the neuroscience community with this new technology and a possible intellectual property dispute over it.

Im a molecular/cellular neurobiologist. I do however, have a deep interst in neural prosthetics, bionics research. Is there a place for me in this field?

ScienceDaily: Semiconductor Brain: Nerve Tissue Interfaced With A Computer Chip

From the article:

16384 transistors on an area of one square millimeter record the neural activity in the brain.

Hmmm, that sounds like a lot of transistors… what kind of voltage sensing resolution can a device like that provide? Well, that works out to 1.6 transistors per 10 square microns, which is arguably the relevant area for a neuron. Although these are extracellular signals, this high-resolution tool is going to have quite a large impact.

From the abstract:

We report on the recording of electrical activity in cultured hippocampal slices by a multi-transistor array (MTA) with 16384 elements. Time-resolved imaging is achieved with a resolution of 7.8 µm on an area of 1 mm2 at 2 kHz. A read-out of fewer elements allows an enhanced time resolution. Individual transistor signals are caused by local evoked field potentials. They agree with micropipette measurements in amplitude and shape. The spatial continuity of the records provides time-resolved images of evoked field potentials and allows the detection of functional correlations over large distances. As examples, fast propagating waves of presynaptic action potentials are recorded as well as patterns of excitatory postsynaptic potentials across and along cornu ammonis.

M. Hutzler, A. Lambacher, B. Eversmann, M. Jenkner, R. Thewes, and P. Fromherz: High- resolution multi-transistor array recording of electrical field potentials in cultured brain slices. Journal of Neuropyhsiology. Preprint online (May 10, 2006).

The original article (whichs seems to online in a preprint form) has excellent photos of the array (showing how it can cover a lot of a hippocampal slice), the tight correspondence between the transistor signal and a microelectrode field signal, and some cool readouts of the “whole hippocampus” with various blockers. I doubt anyone has ever been able to simultaneously do such fine scale electrophysiology on such a large portion of the mammalian brain ever before.

Synaptic tuning : Nature Reviews Neuroscience

For those interested in neuromodulators:

Treatment of striatal neurons with a D1 receptor agonist led to an increase in the dendritic staining intensity of NMDA receptor NR2B subunits. There was also an increase in the association of NR2B subunits with PSD-95 — a scaffold protein required for the assembly of NMDA receptors — and in the surface localization of NR2B-containing receptors.

Original article in J. Neurosci. from Dunah and colleagues. An excerpt from the original aricle of a neat application of FRET continues after the jump.
(more…)

A FRET-Based Calcium Biosensor with Fast Signal Kinetics and High Fluorescence Change — Mank et al. 90 (5): 1790 — Biophysical Journal

Relevant details (from the discussion):

Above we reported the generation of a FRET-based calcium biosensor employing TnC as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. Its off-rate is significantly faster than those of previous double chromophore sensors and even outmatches the fastest single fluorophore sensors to date.

Although it is faster than what was previously available, it would be nice if the off-rate was even faster:

Its off-rate was extremely fast, optimally fitted with a double exponential with a dominating {tau} of 142 ms (A1 = 0.63) and a minor {tau} of 867 ms (A2 = 0.06) (Fig. 2 D). Mutation of the N-cap residue 131 of helix G within TnC from isoleucine to threonine (35Go) yielded an indicator of higher calcium affinity with a Kd of 1.7 µM (Fig. 2 B) and shifted the Hill slope to 1.1, although at reduced maximal fluorescence change of 270%. TN-XL expressed well in primary hippocampal neurons at 37°C. Fluorescence was evenly distributed, filling all neuronal processes, with no signs of aggregation. The nucleus was devoid of fluorescence. Repeated stimulations with high potassium followed by repeated washouts demonstrated stable baselines over long recording sessions and reproducible signals after stimulation. Moreover the signals induced by high potassium were more than doubled compared to TN-L15.

Weak pairwise correlations imply strongly correlated network states in a neural population : Nature

Very few MEA studies make it into Nature, so this definitely got my attention.

Often in neuroscience we are confronted with a small sample measurement of a few neurons from a large population. Although many have assumed, few have actually asked: What are we missing here? What does recording a few neurons really tell you about the entire network?

Using an elegant prep (retina on a MEA viewing defined scenes/stimuli), Segev, Bialek, and students show that statistical physics models that assume pairwise correlations (but disregard any higher order phenomena) perform very well in modeling the data. This indicates a certain redundancy exists in the neural code. The results are also replicated with cultured cortical neurons on a MEA.

Some key ideas from the paper are presented after the jump. (more…)

There’s been some work recently on looking at second harmonic generation for optical readout of action potentials… any opinions on this work?

First a brief primer on SHG (from Yuste’s recent Nature Methods paper on fluorescence microscopy):

In SHG, high-infrared light intensity drives the lowest-order nonlinear polarizability of molecules (or groups of molecules) in the specimen so that coherent light of exactly double frequency (or half the wavelength) is emitted. Because the process can occur away from resonance frequencies, there is no absorption of light, thus avoiding complications of photochemistry. This phenomenon is rare and requires, like two-photon excitation, a high concentration of photons at the focal point, something that also gives it optical sectioning. SHG is particularly interesting because it only occurs where chromophores are oriented in noncentrosymmetric arrays, such as chromophores adsorbed to biological membranes or other chemical interfaces. Thus, SHG is perhaps the only optical technique that is truly sensitive to biological membranes, something which makes it ideal for detecting changes in membrane potential. As many important biological processes, such as electrophysiological communication, detection and transduction of external molecules and cell-cell interactions occur at plasma membranes, SHG is likely to become a very useful tool for biologists.

Seed papers:

• Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials [PNAS Open Access, Feb 2006] , L. Sacconi, D. A. Dombeck, and W. W. Webb
• Imaging membrane potential in dendritic spines [PNAS, Jan 2006] , Mutsuo Nuriya, Jiang Jiang, Boaz Nemet, Kenneth B. Eisenthal, and Rafael Yuste

Berkeley researchers lay groundwork for cell version of DNA chip

This is a little off the beaten path, but I think that the Neurodudes crowd is generally interested in techniques related to neuron-to-silicon interfacing. Here’s some neat surface chemistry from Livermore Labs that facilitates binding of DNA oligos to the cell surface. Then, just like with a gene chip, you can link cells with the right (complementary) oligos to a pre-coated chip.

My first reaction to this was, Wow, another great application of the homologous base pairing machinery of nucleic acids. I’m amazed by the out-of-the-box thinking in this idea — sticking DNA to the outside of the cell. According to the article, the authors estimate that about 270,000 DNA molecules are put on the surface of each cell by their process. (Though I’m sure they’ve looked at it, one does wonder how this impacts membrane trafficking, receptor internalization processes, etc.)

Let me emphasize… This is totally cool! This allows cell-type-specific micropatterning at the level of whatever your chip printing resolution is. (Traditionally, gene chips are “spotted” using precision multi-head inkjet-like printers.) For you cell culture enthusiasts out there, you might imagine a cell culture where you have many different cell types and have full control (down to a single cell!) of where each type of cell is placed. Talk about a co-culture!

In the August PLoS Biology, there is an article showing the production of pure neural stem cells from human embryonic stem cells.

The procedure is quite simple: Add growth factors FGF-2 and EGF to the ES cells and you get pure NS cells, which overcomes several of the limitations of previous neurosphere-based assays [Nature Methods].