Physiology Research – My Work/Activity

Aside Posted on Updated on

“It’s a little too morbid…you might offend someone if you include that part in your presentation”, he was right, I decided to cut certain details out of my presentation at Berkeley symposium that year. I never delete slides, I simply hide them with the mighty right click, so in this post I’m going to discuss some of those slides/explanations that never made into my presentation. It’s going to get a little descriptive, and well, it may even offend you a little bit. So, read on at your own risk.

Prior to being accepted into medical school, a study produced by my former lab was recently published (I was not an author, just a contributor). It was an electrophysiology study, and I helped collect data on this project, and several other pilot projects, so I’m pretty happy about this. i started at this lab when my research mentor, my principal investigator (PI) and former physiology professor, offered me a wonderful experience in his new lab following a satisfying performance in lab and lecture. There was a catch he emphasized, the lab wasn’t actually constructed yet (new lab), nor were there many projects lined up (winging it with science), oh and we’d be learning as we go as it’s a new frontier (kiss your *ss goodbye). At first, I thought it rhetorical, wasn’t all research supposedly on the frontier so to speak? Though, I soon learned he meant everything he said, in the literal sense — in fact, once he asked me “do you know how to solder”, I knew I was in trouble. By new lab, he meant new lab, as in nothing but wall, tiles, and cobwebs. Neither the less, our goal was to get a cellular membrane electrophysiology lab up and running from scratch. We were cavalier on obtaining new equipment, it turns out many pharmaceuticals have blowout sales or giveaways every now and then, presumably with a reward of tax reimbursements. While amassing the fancy equipment, we filled the time by critiquing historic literature (Fatz & Katz, Huxley, Bertil Hille) and emerging research. We lowered our self esteem by spending time trying to decipher Maxwell equations, implications of Brownian motion, and the innards of electric fields and all it’s joyous pleasures — I learned, after taking a year of physiology by that time, that I still knew absolutely nothing. We merged weekly lab discussions with the legacy lab across from ours, another membrane studying lab. But, they had several people interested in medicine, so we had to purchase a tomb called Medical Physiology. To sharpen our fangs on theory, we had to take turns presenting and being torn apart by your peers and several physiology professors (it was good times). I suppose it was here that I finally started to get my answers on how medicine, physiology, and physics were all entangled. It was also the first time that I learnt the main problem/fun of science, the closer your examine the problem the fuzzier it gets, and the less adequate is all the tools you used to get to that point.

After several months of theory work, literature review, technique practice I was finally able to conduct an electrophysiology experiment, independently, from start to finish. Without going into a long, and somewhat esoteric background, let me just tell you (or perhaps remind you) that it’s long been known that electrolytes such as sodium, potassium have an important function in organisms. First year physiology teaches that it’s the gradient of charge, intracellular to extracellular, is vital for the function of most cells. And, it’s long been known that these charges can be created by electrolytes, such as sodium and potassium, and for a while only these electrolytes got a day a fame. But, what about chloride? For a long time It was thought that chloride was just an ion that went along for the ride, and the channels that allowed for its flow did so without control — in other words, that chloride was just this channel that sat wide open and didn’t do much. We thought this both weird in evolutionary terms, and although a lot of labs at that time still went against our hypothesis we thought the chloride channel had to be regulated.

*In case you’re curious, the intracellular side gets it’s charge both from the charge gradient and proteins that created the net negative charge within.

Long story short, we verified our beliefs, found out our study had implications with Huntington’s Disease (Nature), and I learned a lot about ion channels. The study also finally gives some leads to some medical mysteries such as some symptoms of certain muscle diseases. Here’s a little bit about how it was done:

A day in an electrophysiology lab:

1. Go to animal facility, sign out mouse to be euthanize. The mice came out of money from lab funds, or research grants brought to the lab.

2. Euthanize mouse with isoflorane. This would asphyxiate the mouse within seconds. You have to be careful about the level of the gas, the dose is very important. Federal regulations mandate a redundancy in euthanization, this is seen as more ethical than proceeding with the rest of the process without being sure that the animal will not be in pain any further. Some labs use a guillotine (no joke), we went with the standard brute cervical dislocation while the mouse was unconscious with it’s heart presumably already stopped.

3. Harvest the muscles you need using a scalpel after skinning the dead mouse, sheers, a dissecting scope, some enzymes, and steady hands. We’d take out 2 muscles usually (sometimes three), two in the palms and one in the neck region (picture not shown).

Image
We took muscle group 8
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We took muscle group 10.

4. We either kept muscles as whole myofibers (easier experimentally, but less accurate results), or we dissociated them (notoriously difficult to work with, but gives very accurate results). After we prepared a sample, we then had to visualize it on a microscope, and impale the cell with ridiculously sharp electrodes. *Not all electrodes need be sharp, some experiments keep a end “broken”, it sucks a section of membrane in, making a seal and it’s a type of patch-clamp, we didn’t do these types of experiments*. So, the picture was my work space for hours on end.

.

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This is what we affectionately called the rig. You could spend up to 8-10 hours with it at any given day, after a while more of those fancy dials make sense.
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The foil you see there houses a copper case, it’s a ghetto-rigged Faraday cage. Our measurements are very sensitive, so we do a lot to isolate signals that might have originated from the equipment itself. The vials are so we can add stuff to keep the tissue alive, or toxins etc.
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That pink blob in the middle is whole muscle tissue, excised from the neck of a mouse. Those giant glass rods plunging into them are the electrodes, impaling the muscle without killing it with the electrodes is an acquired skill. We controlled the electrodes with a machine, it was like playing a background/inverted “claw” catching game, but on steroids.

Last step: find a good muscle cell, photograph it, and measure it’s electrical properties.

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The horizontal strands are muscle, the lightening strike shaped stuff going down are nerves that innervate the fibers. This was taken with my cell phone, and a lot of patience.
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I was sent to Cal Tech to help acquire this image, this is a muscle cell totally dissociated from others. It’s read because it was using a laser to image the image instead of white light.

And at the end of all of that, you analyze a bunch of squiggly lines, and pretend like you know what you’re talking about. A lot of the formulas I used in this lab later showed up on the MCAT, though in a more “useful” form. All experience is cumulative. Before I thought medicine was for me, I learned a lot on the scientific process, and strongly considered a PhD. Now, I’m getting my MD, but I’ll never forget how much I learned what I didn’t know, and how much I probably won’t ever know.

But, at least I know that we were right =)

 

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2 thoughts on “Physiology Research – My Work/Activity

    Rocco Brown said:
    April 6, 2014 at 6:24 am

    Great post, thanks for sharing!

    […] program you can get into. In graduate school, say if you wanted to get a PhD in the field of electrophysiology, then picking X university because of it’s name wouldn’t be a sound strategy because A) […]

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