The Scientist’s Toolbox: Techniques in Addiction Research, Part 2

Lab Mice IMG_4102

When a news article starts with the headline “A new study finds…” do you know what that means? The article is (allegedly) referring to a peer-reviewed scientific research paper. Research papers are the heart of the scientific research field and are a report of a series of experiments conducted by a scientist or team of scientists. In a future post, I’ll do a break down what a paper looks like but for now all you need to know is that the heart of the paper is the data. The data are the pieces of information that scientists have acquired from their experiments and are reporting in the scientific paper.

But how do scientists generate data?

This is one of the crucial questions in the scientific field because it refers to experimental design: 1) what is the question the scientists wishes to answer, 2) which experiments does the scientist need to design in order to answer those questions and 3) what are the different techniques and tools needed in those experiments?

This is my second post in series of posts I’m doing to show how scientists actually collect data and the various experimental techniques and tools we have at our disposal. Right now I’m only talking about neuroscience and techniques specific to the addiction field but may discuss more general biological tools and experimental techniques in the future.

In my last post in this series, I discussed the locomotor activity test (also known as the open field test), intravenous self-administration, and microdialysis. Today, I’ll discuss a behavioral technique that’s an alternative to self-administration: conditioned place preference.

 Conditioned Place Preference

Recall our discussion on self-administration. It’s a powerful technique that allows animals to administer drugs to themselves. The technique also has the potential to model initiation of drug taking, maintenance/escalation in drug taking, and even relapse-like behaviors. However, there is one major flaw with this technique. It is extremely difficult and very time consuming! After all, a mouse jugular vein is really small, which makes doing the surgeries not a trivial exercise…

Is there an easier way to study addiction that doesn’t require surgery? Thankfully, there is! Conditioned place preference (CPP) is another model to test whether animals find a drug of abuse pleasurable/rewarding or not pleasurable/aversive.

The technique is based on a Pavlovian or classical conditioning mechanism. Perhaps you’ve heard of the famous Russian scientist Ivan Pavlov? In a series of very famous experiments, he was able to cause dogs to salivate anytime he rang a bell (or any neutral stimulus for that matter). Like most famous discoveries, he wasn’t trying to do this but through careful observations he uncovered one of the basic mechanisms that underlies learning.

Figure 1: Classical Conditioning (http://www.dog-training-excellence.com/classical-conditioning.html)
Figure 1: Classical Conditioning
(http://www.dog-training-excellence.com/classical-conditioning.html)

Pavlov’s conditioning experiment was done by presenting the dogs with an unconditioned stimulus, that is to say something that will cause a response in the animal no matter what, which is called an unconditioned response. In Pavlov’s case, he would present the dog with the unconditioned stimulus of food, which would cause the unconditioned response of salivating (Figure 1). Through careful observation, he was able to identify that dogs would salivate even before he put the food in front of them, sometimes just the site of the food dish was enough to cause the dogs to salivate. He followed up on this intriguing observation.

While the food is the unconditioned stimulus, the food dish or scientist bringing the food served as a neutral stimulus that normally would have no effect on the dogs ability to salivate. Pavlov tested if he could induce this salivating effect with other neutral stimuli. A neutral stimulus that normally has no effect on the animal, called a conditioned stimulus, would become associated with the unconditioned stimulus to produce a response (the conditioned response). In Pavlov’s experiments, the conditioned stimulus (food), when paired with the unconditioned stimulus (bell), would then produce a conditioned response (salivating).

Now let’s see how Pavlov’s conditioning experiment was actually done. If he rang the bell before the conditioning (the conditioned stimulus), it would have no effect. The dogs don’t really care about the noise from the bell because it is not associated with anything in the dog’s brains. But every time the food (unconditioned stimulus) is presented to the dogs, Pavlov would ring the bell (conditioned stimulus). Now the ringing of the bell became associated in the dog’s brain with the presence of the food.

Finally, after the conditioning sessions, Pavlov would ring the bell and would remarkably cause the dogs to salivate (conditioned stimulus)! They had learned to associate the sound of the bell with the presence of the food. Just to clarify, the dogs are not “choosing” to associate the bell with food. This type of conditioning is hard wired into the brain itself—forming these type of associations is one of the things that brain does best. In fact, classical conditioning is a basic mechanism in many types of learning. To this day, Pavlov’s work remains some of the foundational experiments in the biological basis of learning.

Here’s a video I found on YouTube that summarizes everything that you just read:

 

The taking of a psychoactive drug can actually have a similar type of classical conditioning effect. Think about it this way, a drug is never taken in a vacuum,it is always taken in a particular context. A drug may be frequently taken in a particular location, or under particular circumstances, or even with certain people.

I’m a former cigarette smoker and this is an example of conditioning that I personally experienced. Every time I got in the car I would light up a cigarette. After months and years of smoking, I caused a classical condition effect in myself. The cigarette (unconditioned stimulus) produces that “nicotine high” and relaxing feeling that smokers crave (unconditioned response). However, driving in a car (conditioned stimulu) normally does not cause that feeling. But every time I would need to drive someplace I would smoke. Eventually, simply being in the car would cause a craving for a cigarette! The conditioned stimulus of driving became associated with the unconditioned stimulus of smoking to produce the conditioned response of nicotine craving every time I go into the car.

Classical conditioning is exactly how conditioned place preference works. In the laboratory, we can use this basic mechanism to force mice to experience a conditioned response when placed in a distinctive chamber. The mouse will even seek out that chamber and spend time in it because they know that they received a “good feeling” anytime they were in the chamber before.

This is what the chamber looks like.

CPP Chamber (© Derek Simon 2014)
CPP Apparatus (© Derek Simon 2015)

It consists of three connected boxes: a central grey one with normal flooring, a white-walled one on the left with a mesh grating as the floor, and a black-walled one on the right with steel bars on the floor. There are special trap doors (white knobs in the picture below) that can be opened or closed so that a mouse is allowed to either explore the whole apparatus or be confined to one of the chambers. When the mouse is being conditioned, the trap doors are closed and the mouse stays in only one chamber the whole time.

CPP Apparatus (© Derek Simon 2014)
CPP Apparatus (© Derek Simon 2015)

A CPP experiment consists of four main steps: 1) the pre-test day, 2) the conditioning sessions (multiple of these), 3) the post-test day, and 4) data analysis. (See Figure 2 below).

Step 1: A mouse in placed in the central grey chamber and it is allowed to explore the entire apparatus all it wants want. Both the white and black chambers represent a conditioned stimulus because right now, they have no association with anything in the mouse’s brain. The time spent in each chamber is recorded.

Step 2: Now the mouse receives an injection of drug (or saline as a control substance) and then is placed in either the white or black chamber. The mouse is forced to stay in the chamber for the entire session (usually 15-30min). That way the features of the chamber (wall color and floor texture) become associated the unconditioned stimulus of the drug. One conditioning session occurs a day for several days.

Step 3: The test day. Now the trap doors are raised and the animal is allowed to explore all three chambers again. If the experiment worked, the mouse will spend most of its time in the chamber that it received the drug injections! In other words, the mouse was conditioned to expect the drug in either the white or black chamber and, given the choice, prefers to spend time in that chamber in anticipation of the drug.

Step 4: Analysis. The time spent in the drug or saline-paired chamber on the test day is subtracted from the time spent in that chamber on the pre-test day. This difference in time is considered the quantitative measure of a successful conditioning session.

This figure summarizes a CPP experiment:

Figure 2: Diagram of a CPP Experiment (© Derek Simon 2015)
Figure 2: Diagram of a CPP Experiment (© Derek Simon 2015)

If an animal likes a drug and finds it pleasurable and rewarding, it will spend a lot of time in the conditioning chamber (see the graph). If the mouse hates the drug, it will not spend time in the conditioning chamber. By using this setup, we can test how different drugs and doses of drugs, and other types of experimental manipulations can effect how a the mouse perceives the drug.

If we were to compare self-administration to CPP, a conditioned response in the CPP experiment would be a similar measure as self-administration of a drug. Both experiments reveal that the animal likes the drug and wants to take it.

And as with self-administration, many variations on the basic setup exist but I’ll spare you those details for now…

Thanks for reading!

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