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.