Neurons Caught in Action: Zebrafish Brain Filmed While Firing Signals

It’s the brain like never seen before. A group of researchers – Misha Ahrens, neurobiologist, and Phillipp Keller, microbiologist from Howard Hughes medical Institute’s Janelia Farm Research Campus, have been able to see individual neurons firing in the brain of a larval zebra-fish, recording activities across the entire fish brain.

And they’ve made a cool video of the whole thing (below).

They have mapped the exact firing pattern for 80% of the 100,000 neurons in the brain, suggesting that, should an upscaling of this mapping be done, the human brain might finally be in the imaging line.

The brain map - taken from the paper (link at the bottom)
The brain map – taken from the paper (link at the bottom)

The Imaging Technique

The imaging technique is ingenious, but theoretically simple. The researchers created genetically modified zebrafish, so that the neurons make a protein which fluoresces when there is a change in the concentration of the calcium ions. Calcium ion concentrations change when a neuron fires, meaning that there will be a small fluorescence when a neuron fires.

Now, a thin sheet of light was sent through the brain and this captures any optical activity in the brain and then records it on a screen. This imaging technique is called ‘light-sheet microscopy’ and the Janelia team was able to upgrade it count at a rate tenfold its original rate. The entire brain of the larval fish was mapped every 1.3 seconds. One whole experiment lasted for 10 hours, generating as much a few terabytes of data.

Ahrens commented on his pet method, explaining why it is so much better than conventional techniques. Available techniques allow one to image at most 2000 neurons at one go, but this one can see the entire circuitry in the brain. As Ahrens puts it:

you don’t need to guess what is happening — you can see it.

There is definitely room for improvement. One would be distinguish between one neuron firing and multiple firings in a short interval. Also, researchers would like to try it on other organisms.

Is the human brain up next? Not for quite some time, surely.

The work appeared here: http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2434.html

New Mechanism of Regulating Gene Expression Discovered

A few months ago, much ado was made about results from the ENCODE project on the human genome, publicized as having made the discovery that 80% of the human genome has a biochemical function. While this is true (with ‘biochemical function’ being defined loosely and broadly), we don’t yet know how or why most of the long stretch of DNA in our cells is important. ‘Genes’ as we know them make up less than 2% of the total DNA. What purpose does the rest of it serve?

What Purpose does “Junk DNA” Serve?

The ENCODE project suggested that the rest of the genome had a strong regulatory potential. How do our cells control when to turn on certain genes, when to ramp up production of one protein and when to slow down? A lot of these regulatory mechanisms remain unknown. A team of researchers at Wistar Institute have now discovered one additional mechanism of regulation.

Before we move on, let’s briefly review how genes function. ‘Genes’ are essentially regions of the genome which are processed into intermediate molecules called ‘RNA’, also linear strings. These RNA strings are further processed to yield the protein that performs the gene’s function. Think of the gene as a ‘recipe’ for a protein, with the RNA molecule being the unfinished product halfway along the recipe. There are, however, some regions of the genome which are processed to form RNAs, but do not form proteins. They often have regulatory functions.

ncRNA-a (the region of the genome on upper segment of the loop) helps the mediator protein complex to gain a foothold on the right region, so that the gene (shown by ‘mRNA’ on the lower segment of the loop) can be transcribed. [Image Credit: Nature Publishing Group]

Long Non-Coding RNAs Regulate Gene Expression

Moving back to the research team, they had previously discovered that a class of these ‘non-coding RNAs’, which they have termed ‘ncRNA-a’, serve to activate processing of their neighbouring genes. But how do they do this? There are certain proteins called mediators which facilitate the processing of genes to RNA. They have now discovered that ncRNA-a helps these mediator proteins bind to these genes at the right place. To determine this, the team removed proteins known to be involved in gene processing (called transcription) one by one, and looked for changes in ncRNA-a mediated activation. And voilà, components of the mediator complex came up immediately. They also found that the chromosome forms a loop between the ncRNA-a locus and the gene locus, for the mediator complex to be able to gain a stronghold at the gene locus using the ncRNA-a as a base.

Why is this result important? It gives us a better idea of the factors controlling gene expression. And as importantly, it helps us understand our DNA just a little better. You can read about this research here.

Novel Method To Invade Cells

One of the current paradigms of molecular biology research is to study cells by manipulating them. Insert a piece of DNA and see what changes. Add a protein and see if the cell can now become cancerous. Inactivate a protein and see if a diseased cell becomes normal.

One of the trickiest steps in such processes is often getting the foreign substance inside a cell. Living cells have membranes designed to keep out foreign substances and to absorb just what the cell wants. How do we let particularly large molecules in?

Infiltrating the Cell’s Walls

Pieces of DNA are usually inserted into a longer fragment of DNA called a vector (to keep the DNA stable). The cell is then shocked to jolt the proteins in its membrane and make it temporarily porous. This method can work for small molecules, and another method is the chemical disruption of the membrane temporarily. The problem with these methods is that they change some properties of the cell, and the goal of such experiments is to observe changes in the cell that is ONLY because of the inserted molecule, and not due to other factors (such that the result of a shock might lead to). Another method is to deliver the molecule inside nanoparticles which can then enter the cell, but the nanoparticle is often captured by an organelle of the cell, and the molecule is then not released. The cell’s membrane also allows certain protein to pass through it—think of it like a gatekeeper, letting proteins of certain charges and small enough sizes go through while keeping out the others.

This image gives the workflow of the microfluidics device. Figure A shows cells flowing through a tube with a constriction. After being squeezed, these cells are now have the delivery material inside them. [Image Credit: Sharei et al; PNAS]

Squeezing Cells to Make Them Yield

A general method of insertion would thus be a useful tool in research. Researchers at MIT have come up with the solution mothers use when their children don’t eat food—they press in the child’s cheeks till their mouths open. This team has developed a microfluidics device that does something similar. Cells (and the delivery material) flow through a tubes under external pressure and are forced to pass through a tiny constriction in the tube (see figure). During this phase, the cells are compressed to such tiny sizes that their membranes ‘split’ temporarily, leaving gaps for molecules to enter.

The team used this method to generate stem cells (by inserting the necessary factors into cells) and found that this method had an efficiency 10 to 100 times greater than other existing methods. Their next step is to use this for therapeutic purposes, wherein a patient’s cells could be taken out of his body, injected with the necessary DNA/protein, and re-injected into his/her body.

You can read about this research here.