Western Blot and Thursday Activities of Daily Living

Sorry I missed posting yesterday. It was a bit of a crazy day.

This morning when I got to the lab, Kate was preparing for a scheduled kill. Unfortunately, the lab rats cannot live forever, and as a result, they must eventually die. I know this might seem very sad, but it is important to remember several things. First of all, if it wasn't for the purposes of science, then these rats wouldn't have been born in the first place. Secondly, this is a fact that goes along with this type of animal research. Lastly, and fortunately, although the rats must be killed, the researchers in the lab make every effort for their death to be as quick and painless as possible. That is exactly what I saw today, and I can report to you that no rat was stressed out before its death. 

The rest of the day was spent confirming that I did not have TB, finishing up some training for OSHA, and learning about a Western Blot. Wikipedia:

The Western blot (alternatively, protein immunoblot) is an extremely useful analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose orPVDF), where they are probed (detected) using antibodies specific to the target protein.^[2][3]

A western blot's name comes from the fact that the first sort of immunoblotting technique 

was invented by a man of the last name Southern. This allowed scientists to detect DNA. 

Just to be witty, the scientists who invented the technique for detecting RNA decided to call 

the stain a Northern blot. Similarly, the Western blot was named to play on these already 

established techniques.

Step 1:

Step 2:

Step 3:

Step 4:

And lastly:

Sorry...

I totally forgot to blog yesterday. It was a little crazy of a day and it slipped my mind. I haven't forgotten, and just wanted to say come back soon to learn about Western blots.

Slow day

Today has been a fairly slow day. I suppose it fits with the weather here in Charleston, which is gray and overcast. After arriving at work this morning, I watched a few rat surgeries (putting in cathedars and cannulas). [I want to add a note that the photos that you have seen of the rats on the blog have not been my own pictures. Those are ones from Google, and I simply use them to try to illustrate my point a little bit.]

Next stop: at 1:00 I had to go to employee health to get my TB test and sign a bunch of paperwork. Following that, I ate lunch, then walked back to the lab. Now that I am back, I am writing my blog post for today while my mentor, Kate, is doing some computer paperwork. I think later on today we are developing a Western blot. After I learn a little bit more about Western blots, besides the fact that they are specific for proteins (instead of DNA or RNA), I will write a post about that.

Overall, there is not as much going on today as there is on most days, so I hope you will forgive my short post.

Histology and slicing

Good morning and Happy MLK Day! Unfortunately I did not get to sleep in this morning like many people because I am at work today. Last friday I learned slicing techniques to prepare slides of specimens (rats' brains, specifically the Nucleus Accumbens) for histology. I will be doing more of that today, and so I decided that I would write my post about that. Here are some pictures to get started. 

I'm going to use wikipedia to help me to describe some of the processes here, and since I am giving a broad overview, and not writing a paper, I think that should suffice for now.

We fix tissues via perfusion (I'll tell you more about that another day.) According to wikipedia,  

Perfusion: Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)

After the tissues have been placed in a formalin solution for an amount of time, and you are ready to actually section the brain, we use a vibratome which is a microtome sectioning machine to cut sections of the brain and place them onto a slide to examine them under the microscope. Here is a machine like the one we use.


Here's what wikipedia has to say about microtome sectioning machines: 

A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device inmicroscopy preparation, allowing for the preparation of samples for observation under transmitted light or electronradiation. Microtomes use steel, glass, or diamond blades depending upon the specimen being sliced and the desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissues for light microscopy histology. Glass knives are used to slice sections for light microscopy and to slice very thin sections forelectron microscopy. Industrial grade diamond knives are used to slice hard materials such as bone, teeth and plant matter for both light microscopy and for electron microscopy. Gem quality diamond knives are used for slicing thin sections for electron microscopy.

Microtomy is a method for the preparation of thin sections for materials such as bones, minerals and teeth, and an alternative to electropolishing and ion milling. Microtome sections can be made thin enough to section a human hair across its breadth, with section thickness between 0.05 and 100 µm.

After placing the rat brain on a metal piece to place in the formalin mixture, you are ready to turn on the machine and get thin sections of the brain. An ideal slice on this machine is 100 microns, which is is 1/10 of a millimeter. It takes you a while to be able to do this, and so when I was learning I started by sectioning thicker slices. As I got progressively better at it, I was able to do the 100 micron slice without having the specimen tear. While there is a technique to learn before you are able to be good at it, it is not very hard to do quickly and correctly once you master the skill.

If you ignore the part about freezing, then this is a good video to watch to learn more, in case my explanation is unclear.

Have a great Monday!

How do rats become addicted to cocaine?

Cocaine

You guessed it: Cocaine.

The title says it all. Sure, people might do cocaine for a variety of reasons, such as availability, curiosity, or for social acceptance within a group of peers that uses the substance. In my post from Wednesday, I discussed the effects of cocaine on the brain, and the changes that occur as a result of using the drug. Today I want to discuss how rats in a lab "do" cocaine, since it's clear they can't do this:

So here is the deal. If you know anything about or operant conditioning, then you will follow what I am about to describe. If you are not familiar with operant conditioning, here is a good link for a refresher course on behaviorism. Operant Conditioning

First, rats are not given food for approximately one day, so that they will be hungry when first placed in the operant box for what we call "food training." Here is a picture of the operant box:

A lever is extended out from both sides, but only one side actually does anything. Here, we are saying that is the right side. So, the rat is curious and moving about in the cage, and presses the lever on the right side. A light comes on above the lever, a tone sounds, and most importantly, he is rewarded by a pellet of food that is released for him. He thinks to himself how cool that was, and tries it again on the left side. No such luck. Tries the right side again, and sure enough: a pellet of food is released following the tone and light paired cue.

The rat spends 15 hours in the operant box, which is enough time (for most rats) to put two and two together, thus realizing that pressing the right lever results in a reward.

Okay, so he realizes that the right lever equates to a reward. What does this have to do with drug administration? I'm getting there.

The rat has a catheter on its back, which looks like a backpack. Here is a similar, but not exact design:

[I'm getting a little ahead of myself, but as an FYI, in many cases, the rat also has a cannula on its brain, so that after it is killed, the investigator is able to examine the brain histology.]

The rat has a catheter that is hooked up to an infusion pump that is filled with cocaine.

It is similar to the above picture, but that the levers are still present, and the rat only receives an infusion of cocaine when he presses the right lever. Once again, a light and a tone are cues that come on as the infusion of cocaine is being delivered. 

Most of the rats end up liking the infusion, and thus, continue to press the lever and receive more cocaine. I should note that there are certain precautionary measures that are placed to keep the rat from overdosing on the cocaine. For example, after receiving one infusion, the rat has to wait 30 seconds before it is able to get another "hit." Even if the rat presses the lever during this "time out" period, there will be no drug administered. Also, after receiving a certain amount of infusions per session the rat is cut off. As my mentor Kate says, "The party's over." This keeps them from having a cocaine hangover, and thus generating a negative response to the drug. Having an aversive effect to cocaine would hinder the animal in its later cocaine sessions, and as a result, he would not be very enticed to try the drug at a later time.

Thursday

1. My neurobiology class this past semester as well as my two neuroscience seminars have been extremely helpful in allowing me to understand important topics in neuroscience. This was evidenced in our journal club today, when we read about GIRK channels and the agonists that block them, such as morphine.

2. Today I perfused a rat. After the rat was under anesthesia (ketamine), I learned how to open up the body of the rat and inject formalin into its heart to actually "fix" the brain while it is still alive. While I am not entirely sure about all the reasoning for this, I do know that it is important to fix the brain while it is still alive to be able to get good histologic samples.

3. Another thing I learned about today, from talking to another neuroscience researcher in the department, was the development of eyesight and the way in which humans, cats, and monkeys learn to develop and discriminate colors. You can read more about it here: 

More tomorrow.

Why addicts can't quit their drug, and why "willpower" is not the only factor one should consider.

The short answer: Neuroplasticity.

According to Merriam-Webster online, addiction is the “compulsive need for and use of a habit-forming substance (as heroin, nicotine, or alcohol) characterized by tolerance and by well-defined physiological symptoms upon withdrawal.”

Today my blog entry is going to focus on the physiological mechanisms that underlie addiction, as to explain the changes that occur in the brain as a result of substance abuse. Although some of this information was discussed in my neurobiology class this fall with Dr. Hettes, I am using an article that was given to me by my mentor. It comes from Progress in Molecular Biology and Translational Science (Vol 98): The Brain as a Drug Target. Edited by Rahman, the chapter that I will use is entitled Glutamergic Neuroplasticity in Cocaine Addiction, by Joachim D. Uys and Kathryn J. Reissner.

Although the first time one uses a substance (in this example, we are assuming the substance is cocaine) one does not necessarily become addicted, certain changes do take place in the brain. Generally, dopamine may be released from the ventral tegmental area and may target receptors in different areas including the prefrontal cortex, amygdala, hippocampus, and nucleus accumbens. According to Uys and Reissner, “The projection from the prefrontal cortex to the nucleus accumbens is considered to be a common final pathway engaged in drug-seeking behavior induced by exposure to drug-paired cues, stress, or a drug prime and represents and important site of chronic neuroplastic changes induced by drugs of abuse” (371).

(Used from Dr. Hettes Neurobiology class)

All of this information is important because it deals with the motivational aspect that is involved in the drug seeking behavior that is characteristic of an addict. Long term potentiation is a phenomenon in which, according to Dr. Hettes, “NMDA receptor (a type of ionotropic glutamate receptor) activation results in a calcium ion dependent increase in AMPA receptors (also an ionotropic glutamate receptor) at the synapse.” Because NMDA receptors are dependent on AMPA receptors in order to depolarize the neuronal cell, the placement of more AMPA receptors leads to a greater depolarization of the neuronal cell membrane and thus results in an EPSP (excitatory post-synaptic potential), meaning that it will be easier for a neuron to fire an action potential at this synapse.

(Again, thanks Dr. Hettes).

Okay, maybe that was a little complicated, but I feel like I had to explain that in order to give some background on this next part.

So what does it mean that there is an increase of AMPA receptors in certain areas of the brain (specifically, in the Nucleus accumbens)? An increase in receptors in this area means that the substance abuser is developing a strengthened signal transduction pathway in the area of the brain that is involved in reward, pleasure, and addiction (just to name a few). The motivational aspect to continue the behaviors that result from the cocaine-associated feelings is strengthened.

What are the implications of this?

Even though the addict knows that the actions he or she is undertaking is negative and there might be legal, societal, and moral implications that result from using these drugs, the addicts brain is no longer functioning at the same level as you or I. The drive to use cocaine is increased not only at a psychological level (the feelings that are associated with using drugs) but also at a biological level.

However, one must take into account that separating the aspects of addiction into different areas of science is difficult if not altogether reductionist. The biologic mechanism that induces changes in the brain IS exactly that which is responsible for such drug cravings.

I know my explanation is somewhat simplified, and I hope to go further in depth later on in my postings.