Frontiers in Biophysics

In March I had the opportunity to attend the annual Biophysical Society meeting here in Boston.  I've been meaning to write about it for a weeks now, because I did attend an interesting session at the meeting entitled 'Frontiers in Biophysics'.  Hagan Bayley from Oxford presented some unusual work using what he termed 'droplets'.  These droplets are small aqueous drops surrounded by a monolayer (or single layer) of lipid that can be held on the end of a movable pipette, or anchored to a tether of some sort.  When two of these droplets are brought into close proximity, a lipid bilayer forms at the interface between them.  The lipid bilayer is the double layer of lipid that surrounds all living cells and provides cells with features fundamental to their function and survival.  For example, a bilayer is necessary for the proper function of many of the proteins that allow the cell to communicate with its surroundings, or with other cells.  

Any purified membrane proteins present in the droplets will self-insert into the newly formed bilayer.  Ion channels and pumps, responsible for controlling the electrical properties of cells, can be inserted and their activity measured; Bayley claims this system is more stable than conventional bilayer recording techniques.  However, the interesting part comes when you start to take advantage of the scalability of the technique.

Many of these droplets can be assembled in an array with different proteins in each droplet.  Using a combination of ion channels and pumps, like the components of an electrical circuit board, actual circuits performing computations and even acting as batteries can be assembled from these droplets.  These circuits are really something of a novelty, as they would be much larger and difficult to maintain than current circuitry.  But Bayley's aim is larger than mere circuits; he is actually hoping to recreate entire cellular functions from the droplets as well, such as signal transduction, production and secretion of factors (proteins or other molecules) in a highly controlled manner.  Potential applications might be a more stable or more realistic assay system for various biological processes, testing the functions of new proteins associated with those processes, and perhaps the ability to create a responsive secretion apparatus (insulin, perhaps?) comprised entirely of biological material.

Yeah, I said it was 'Frontiers'.  I enjoy this kind of work for its own sake. 

The fine line between life and death for the neuron

We tend to consider the brain of an adult to be like a complex piece of electronic equipment, in which static circuits comprised of neuronal cells, or neurons, perform calculations to enable thought and action.  After adolescence, it was previously thought that these circuits and wiring are complete and can not be further modified.  However, thanks to the burgeoning field of adult neurogenesis, it is now well accepted that new neurons can be born in the brain's subventricular zone and migrate to two distinct areas:  the hippocampus, or memory center of the brain; and the olfactory bulb, the first odor processing center in the brain.  Once they migrate to these regions, these neurons form connections with their pre-existing neighbors, presumably replacing lost neurons, or perhaps modifying existing circuits.  How does this happen?

Carlos Lois, whose lecture I recently attended at Children's Hospital, has been studying this question for some time and presented some interesting evidence in favor of the idea that new neurons integrate into existing adult circuits in much the same way that the brain develops during adolescence.  This is to say that more neurons than are actually needed are birthed and migrate to the region of interest (in his case, the olfactory bulb).  There they attempt to integrate into the existing circuitry.  Those that succeed survive and become functional; those that do not integrate well die.  On the particular topic of survival, he presented an interesting experiment.

His lab introduced one of two proteins into the subventrical zone of mice using a viral expression technique.  The first protein, called NachBac, is an excitatory ion channel that tends to polarize the voltage of the cell's membrane towards a positive potential.  The second protein was a potassium channel that has the opposite effect, making the cell's membrane potential more negative.  Interestingly, both proteins increased the apparent activity of the neuron, as assessed by the number of times it fired an action potential at rest (action potentials are the electrical impulses that transmit information along nerves and allow neurons to communicate).  Despite the increase in activity under both conditions, the cells with a more negative resting membrane potential died more than normal neurons, while those that were more skewed towards positive potentials survived more readily.  This makes the argument that the only deciding factor in the survival of new neurons is the resting membrane potential.  This likely means that spontaneous activity is not important for neuron surival, but coordinated or responsive activity is, as the neurons expressing NachBac would be more responsive to incoming signals.

This is another step along the way to understanding how we might one day be able to replace regions of the brain lost to stroke, disease, or injury.

Reworking

Hi folks, here's a quick update for you. I've received a lot of feedback on the blog, and it seems like people really don't have the time or inclination to read my titanic posts. So, I'm in the process of re-working the format to be something shorter and more to the point. I will also be including some commentary of seminars or lectures that I attend. Hopefully this will keep people interested and engaged! I'll see you soon.

Bio-electric slide

Microbial ecology meets electrochemistry: electricity-driven and driving communities.
http://www.ncbi.nlm.nih.gov/pubmed/18043609

I'm going to explore a different tactic for this week, which is provide a review of... a review! This field is fascinating but still nascent and therefore highly technical. Therefore I'll try to boil down an overview of this topic so something even shorter, simpler and, with luck, sweeter. You may ask about its relation to neuroscience but if you get to the end you'll see my plug for why neurons might be useful to consider.

The future of energy
As oil prices rise and supplies of fossil fuels dwindle, it has become clear the the future of energy lies in currently underutilized sources. In terms of generating electricity, probably the most important form of energy our world needs, we have heard a lot about solar and wind power lately. Hydro-electric power is seeing heavy use in developing countries such as China, but does have some significant ecological side-effects, especially if its implementation is not well thought-out. You may be aware of even newer and more unconventional technologies for energy generation, such as bio-diesel, ethanol, and methane extraction from farm waste (all of these methods rely on combustion).

The bottom line is, we are going to need to harness ALL available sources of energy that we can conceive of in order to meet our future energy needs, both static (the electric grid) and portable (vehicles). One rapidly developing area of research involves harnessing bacteria to generate power. Some early designs include the use of methane-producing bacteria to consume sewage or other waste water and release methane or other combustible gases.

Bio-electricity as an energy source
A newer direction for biological research in the field of energy generation is harnessing bio-electricity. All living cells are constantly generating a little bit of electricity in the form of a voltage across the membrane that separates the inside of the cell from the outside world. This lipid membrane forms what we call a 'capacitor'; that is, it separates charges and stores energy. The capacitor is charged by small currents that cross the cell's membrane, carried by ions (such as sodium and potassium) in water. This is as opposed to the system of electricity we typically think of, which involves (roughly) the flow of electrons through an organized metal lattice - a copper wire, for instance. It would be great if we could access that energy - however, this would require us to hook up wires outside the cell (not too much of a problem) and also inside the cell (well, that is a problem). How can you get access to the inside of a cell without killing it? The answer is, you can, but it's difficult and time-consuming. Some people are actually thinking about it, but we can come back to that later.

However, there are also bacteria, discovered in the early 20th century, that can actually generate electric currents outside of themselves, without any involvement (or at least minimal involvement) of the cell's interior. Now we're talking. How does this work? Interestingly, these bacteria facilitate the transfer of electrons from organic matter (ie. sewage or other organic waste) to metals like iron - or indeed, to an electrical anode. In a battery, the anode is the metal contact that receives electrons. Therefore, these bacteria, when mixed with organic matter, and grown on an anode (the bacteria tend to grow as a thin film, or biofilm) within a battery will actual power that battery. So far researchers have achieved near 1 Volt and several milliamps of power from a small bacteria-fueled battery. In larger installations, up to 1 kWh of electricity can be gleaned from 1 kg of waste (1 kWh, or kilowatt-hour, is the amount of power that ten 100-watt light bulbs use in an hour).

Although these amounts of power are small, and currently somewhat inefficient, this design is in its infancy. A further amazing feature of bio-electric batteries is that if the bacteria inside are grown over time (unclear from the article but I would guess days to weeks), the circuit becomes MORE efficient as the bacterial community develops and interacts. Perhaps the bacteria are trying to help us out with this energy problem?

Some researchers have, at a purely theoretical level, already begun to model the possibility of harnessing the type of electricity I mentioned beforehand. In other words, they are designing models that involve harnessing the voltage and currents that cells use across their membranes. Brain cells are some of the most electrically active, diverse, and efficient cells in the body. If we could find a way to harness the electrical energy of neurons or an artificial system based upon their physiology, we would have a real winner. Another cell type that merits study is the electric organ of the electric eel which can generate several hundred volts within close proximity to the snimal. I'm not suggesting that we have huge tanks full of eels to power our houses (although apparently they do in Japan) - but you certainly keep an eye out for these interesting ideas when it comes to the future of energy, and our world.

Never fear!

What a month! I wanted to give people a chance to catch up on some of the older posts which they apparently didn't have a chance to read. Now that everyone is caught up again, let's proceed! The hiatus certainly wouldn't have anything to do with my committee meeting, vacation, article submission, and broken computer...

Fear not ladies and gents, the next post will be up sometime tomorrow. Until then, cheers!

Things that glow in the night

I'm presenting something a little different this week. As you might know, the Nobel prize in chemistry was awarded to a trio of scientists for their pioneering work in the field of fluorescent proteins. Osamu Shimomura, Roger Y. Tsien, and Martin Chalfie, who shared the prize, have defined the field for the last 50 years. Dr. Shimomura identified the first two members of the ever-expanding family of these molecules, which are called aequorin and the aptly named Green Fluorescent Protein (GFP). Dr. Chalfie performed early work with the GFP gene. Dr. Tsien is the current guru who has purified many more versions of these proteins, and also created many modified versions that fluoresce different colors.

You might find yourself asking, what exactly is a fluorescent protein? A fluorescent protein contains a special chemical structure called a 'chromophore', a fancy term for something that can absorb light (sometimes several colors), and then release, or 'emit', it as one specific color (for our purposes). For example, GFP can absorb light in the UV and visible blue portions of the electromagnetic spectrum, and then emit green light. The actual chemistry is a little more complicated than that, but I'm no chemist so that's the best you're going to get out of me.

Why are these proteins important? They are fantastic laboratory tools to help us locate and track other proteins we might be studying. With the discovery of DNA, the elucidation of genes and now the sequencing of the entire human genome, we now have the capability to combine the sequence of the gene we are studying with the sequence for a fluorescent protein. This creates a hybrid, or 'tagged' protein, that contains the protein we are studying with a nice little light-sensitive tag. We can use this hybrid to determine what type of organs or cells our protein is found in, and with advanced microscope techniques we can now even follow the movements of individual 'tagged' proteins inside a single cell. We do this by flashing the appropriate type of light into the cell and looking for the fluorescent response of the tag. Additionally, some of these fluorescent proteins like aequorin will only fluoresce in the presence of other molecules like calcium. This allows us to use them as indicators, or 'probes', for these molecules within cells. Also, we can use multiple, complimentary fluorescent proteins attached to two different proteins we might be studying to see if these two proteins interact by creating a chain reaction of light. All told, this is a powerful technique because it allows us to use a minimally invasive, genetically encoded system to study our genes of interest.

I thought I would provide a brief review of some of the important discoveries or techniques that hinged upon fluorescent proteins.

• ATP synthase movement [1997]. Researchers attached a long fluorescent protein to a protein called ATP synthase. ATP synthase is responsible for producing ATP, the main energy source used by the cells in our bodies. Think of it like a windmill producing renewable energy - not oil. In fact, the comparison is apt, because it turns out (no pun intended) that ATP rotates as it produces ATP. In fact, the synthase uses electrical energy in the cell to drive a 'crankshaft' that rotates a barrel-like portion on top that produces ATP. The researchers demonstrated this by literally taking very fast freeze-frame photos of the fluorescent marker that showed it turning around. This is one of those beautiful, elegant experiments that convinced me to pursue science as a career. I always find it amazing to think of microscopic proteins functioning as little machines. It really demonstrates that the fundamental laws of mechanics can operate on a minute scale.

• Brainbow [2007]. Yes, you read that correctly. From the Center of Brain Science at Harvard, featuring Josh Sanes and Jeff Lichtman, scientists created a rainbow in the brain. Imagine every cell in the brain fluorescing in a different color. Well, that's a slight exageration, but using combinations of genetically encoded fluorescent proteins the researchers were able to generate 90 colors in the mouse brain. Since there were so many colors, it's highly likely that adjacent neurons, or neurons connected to each other, will be different colors. This allows researchers to differentiate between different components of brain circuits when recording electrical currents or imaging brain activity. Beyond just looking cool, it could prove to be a very powerful tool to help us elucidate how individual neurons contribute to the functioning of brain circuits, and complex behavior.

The uses of fluorescent proteins are legion. As scientists, we use them almost every day. They have been critical tools for basic science and drug discovery for many years, and their uses are still expanding.

Fear and forget

Amygdala intercalated neurons are required for expression of fear extinction.
http://www.ncbi.nlm.nih.gov/pubmed/18615014

Disclaimer: I appreciate all the positive feedback on the blog that I've received over the last month. I'm glad you guys are enjoying it! That having been said, I do get the occasional request for a shorter blog entry. So I suppose once a month, you guys deserve something you can read and digest in 5 minutes or less. So here goes nothing. I hope you appreciate the fanservice.

Today's Article
There are many fearful things in this world. Not the least of them is returning home at 10 PM only to realize you have a blog entry due by the next day. In all seriousness, fear is an adaptive mechanism geared towards survival. It allows us to mount a superior response in situations where we require additional attention. For example, fear is that involuntary emotion that causes us to run very, very fast the other way when we see an angry editor (or hungry lion) bearing down on us. Nevertheless, it is critical that we do not become consumed by the long-term effects of fear, which can become crippling and debilitating. Post-traumatic stress disorder (PTSD) affects sufferers with extreme anxiety. The relationship between long-term fear (anxiety) and and acute fear is still poorly understood, but this study attempts to shed further light upon the normal pathways by which fear is handled in healthy individuals.

Today's researchers asked whether the process of reducing fear after a harrowing situation, and removing the emotion of fear from memories, can be traced to a single region of the brain. This process is called 'fear extinction'. In fact, they found it could be traced to a single cell type, named the ITC neuron. These neurons reside in discrete clusters within the central fear processing center of the brain, the amygdala. Using a nifty biochemical trick, the researchers were able to piggyback a toxic molecule onto a chemical signal that these cells normally respond to in the brain, but neighboring cells don't recognize. This technique is also seeing use in the treatment of cancer. They applied this concoction in the vicinity of the ITC cell clusters in a rat's brain. Once inside the cell, this toxic molecule specifically killed the ITC cells.

The researchers then tested the rats for their responses to fear. They found that rats with the ITC neurons missing were able to respond normally to acutely fearful situations, but they continued to show elevated fear much longer than normal rats (up to a week). This result was strongly suggestive that ITC neurons pay a significant role in fear extinction. Although the length of the study was relatively short, it provides hope that specific neurons whose activity might be targeted by drugs or other therapies, are involved in the processes that underly excessive fear. The study does not describe a link between this region and any of the (rather poor) animal models of PTSD. Nevertheless, it is intriguing that one neuron type could be responsible for such a complex behavior as fear extinction. Substantial further work remains to validate these cells as a therapeutic target for anxiety or PTSD, but the discovery of ITC neurons it is a significant milestone. An interesting next step for this research would be to use functional imaging to study these clusters in the amygdala of patients suffering from PTSD. This technique would allow us to determine whether this brain region is functioning abnormally - it could be that a PTSD event causes such a sustained, high level of fear that these circuits are 'overloaded' so to speak. If so we might be able to study these cells more carefully for drug targets. Treatment of deep brain regions is still very difficult, but some intervention and possibly prophylaxis (for soldiers) might be possible.