Stress and Addiction Part 3: Molecular Changes

Stress-BrainThis is part three on my series of posts looking at Stress and Addiction. To recap: we’ve seen that, in laboratory studies, stress increases susceptibility to drug addiction. Stress not only increases the self-administration of drugs in adult rats but stress during an early age can have a long-lasting effect on drug-taking behavior. Today, we’ll wrap up by looking at some molecular changes that might help to explain why this effect exists. I’ll conclude by addressing some questions that might have occurred during the course of this discussion.

Paper #1 Sorg 1991. Title

The first paper is examining the effects of stress and cocaine on dopamine. Dopamine is a very important neurotransmitter. All drugs of abuse cause increases in dopamine in an important region of the brain called the mesolimbic pathway. I will discuss this system in detail in the next post but for now don’t worry about the details. All you need to know is that drugs can increase dopamine.

Dopamine levels can be measured directly in the brain using the technique microdialysis (I discuss this technique in more detail in my post The Scientist’s Toolbox: Techniques in Addiction). In this first paper, the scientists use a type of stress called foot-shock stress. It is very similar to tail-pinch stress (see Part 1). The animals are placed on a grid that is connected to an electrical supply. The scientists administer a small amount of electric current to the grid, which gives the animals feet a little shock and stresses them out.

Figure 1.
Figure 1.

The microdialysis technique was used on rats that underwent foot shock stress in order to measure dopamine levels (in a region of the brain called the striatum) after the stress test. As you can see in the top graph of Figure 1, foot shock stress causes an immediate increase in the amount of dopamine released and this eventually returns to normal. The different symbols mean different stress intensity with the most intense stress represented as black squares. Interestingly, as you can see in the lower graph of Figure 1, a more intense foot shock (ie a more intense stress) causes more dopamine to be released.

Remember that I said that cocaine also causes dopamine release? So maybe stress makes cocaine feel better because it works together with cocaine to create a larger release in dopamine than cocaine would by itself. Next, the investigators decided to test this idea.

Sorg 1991. Figure 2
Figure 2.

In this experiment, mice were exposed to a weak foot-shock stress then given an injection of cocaine and the amount of dopamine released was measured. Figure 2 shows much more dopamine was released in the striatum in rats that received cocaine + stress (black squares) compared to cocaine + no stress (white squares) or just stress by itself (black circles). Perhaps the hypothesis that stress makes cocaine more pleasurable because its boosts dopamine released might be true?

Paper #2

Zhou 1996. Title

Recall from Part 1, that stress activates the HPA axis, which results in release of the stress hormone cortisol (corticosterone in rats and mice). But do drugs of abuse also activate the HPA axis? This next paper—done in lab that I work in—takes a look at this question.

Figure 1.
Figure 1.

Cocaine was given to rats under a number of different conditions. In the first experiment, cocaine effects on the HPA axis were examined in the short term (acute cocaine use). Rats were injected with either saline for two days, cocaine for 1 day, cocaine for 1 day and saline for 1 day, or cocaine for 2 days. After the injections, blood was drawn from the animals and the corticosterone in the serum was measured.

*Technical notes: 1) Serum is the liquid part of blood and it does not contain the red blood cells and clotting proteins. Serum is often used when measuring hormones in the blood. 2) Corticosterone can be measured multiple ways but this experiment used something called a radioimmunoassay (RIA). I’ll save the explanation of it for a future Scientist’s Toolbox post.

As you can see in Figure 1, immediately after the rats receive cocaine (either 1 day or 2 days) corticosterone increases. This means that cocaine has resulted in activation of the HPA axis. Interestingly, the animals received 1 day of cocaine and 1 day of saline did not show high corticosterone levels which means that the levels have returned to normal after the cocaine.

But what happens with repeated cocaine use (chronic cocaine use)? Addiction develops because of chronic use of the drug so are any changes occurring after many days of cocaine use?

Figure 2.
Figure 2.

Interestingly, in Figure 2, corticosterone is high after 3 days of cocaine but after 14 days of cocaine corticosterone levels are much lower! What’s going on here? What these data suggest is that 14 days of cocaine use has caused a change in the HPA axis activity. The cocaine has activated the HPA axis so frequently the axis has compensated for this over activation. The activity of the HPA axis response has been blunted because of the repeated cocaine use.

This one small example of how drugs can cause long lasting molecular adaptations and changes in the brain. Perhaps this is why stress helps to make someone more vulnerable to addiction, because changes occur both at the level of dopamine release (paper #1) and in HPA axis activity (paper #2). Both drugs and stress have similar molecular effects that may work together! I’d like to very briefly discuss one more paper that combines both of these concepts.

Paper #3

Boyson 2014. Title

This paper is complicated but I’m just going to present a small amount of the data. The key points you need to know is that the scientists are using social stress (see Part 1) in this paper for two key experiments 1) self-administration to measure cocaine taking behavior and 2) microdialysis to measure dopamine release. However, they also inject a chemical compound directly into the rat’s brain that blocks HPA axis activity. This chemical acts at the starting point in the HPA axis: the activity of CRF is blocked (the chemical name is abbreviated as CP). Let’s see what happens in this experiment!

*Technical notes: 1) the drug actually prevents the action of CRF interacting with its receptor. Chemicals that do this are called antagonists. Therefore, the scientists are injecting a CRF Receptor antagonist into the rat brains. 2) as a control, an inactive solution is also injected into some animals. This is called artificial cerebral spinal fluid (aCSF). For the drug studies, the correct comparision is CP vs aCSF.

Figure 1.
Figure 1.

Like we saw in other papers, stress increases self-administration (Figure 1, black circles) compared to no stress (white triangles). However, when you give the CP at a high dose (light grey circles) compared to a low dose (dark grey circles) it reduces the self-administration! This means that blocking HPA axis activity reduces the effects of the stress on the cocaine self-administration. Cool!

Figure 2.
Figure 2.

Next, they did a very similar experiment but only this time measure the interaction between stress, cocaine, and the CRF antagonist on dopamine release. The results are presented in Figure 2. Animals that were stressed and than given a dose of cocaine but not the CP (stress + cocaine + aCSF, black circles) released a large amount of dopamine compared to animals that were only given the cocaine injection (white triangles), which is consistent with findings from Paper #1. Amazingly animals that were stressed and then given cocaine + the anti-HPA axis drug CP showed reduced amounts of dopamine released at bot a low dose of CP (dark grey circles) and high dose (light grey circles). These experiments show that the effect of stress on cocaine taking behavior might be because the stress activates the HPA axis which causes more dopamine to be released.

*Technical note: I described this experiments very briefly but they are extremely technically challenging and probably required months of hard work just to make the two little graphs!

Summary

Finally, if we summarize the papers from Part 1, 2 and 3 we can come up with a little mechanism to help explain the different results from the different papers. Based on the data, stress can contribute to the vulnerability of becoming an addict because it activates the HPA axis and increase the dopamine released, which may cause the drug to feel better to a person and make them want to take more of it. There may be a synergy between stress and drugs that changes brain function so that addictive drugs feel more addictive.

You probably noticed I used the word “may” many times and this is because our proposed mechanism requires a lot more testing. In fact, we barely even scratched the surface with this discussion! There are literally hundreds more papers looking at many other details just on stress and addiction. Hopefully this post and the previous two can give you a little appreciation for the difficultly in learning anything about how addiction really works and what specific changes occur in the brain from drug use! Science is a challenging and time-consuming pursuit but also totally worth it!

To wrap up our discussion on stress and addiction, I’ll address some questions/criticisms that you might have with the research papers in this and previous two posts.

Q & A

Some questions about the research you might have and my answers:

Q: Only the psychostimulants cocaine and amphetamine were looked at in these papers. Does stress have the same effects on other drugs of abuse?

A: Yes. The effect of stress is the same with nearly all drugs of abuse tested including the opioid morphine and heroin, alcohol, and nicotine. The neural machinery that is responsible for enhancing the addictive powers of drugs is common to all drugs of abuse.

Q: Only the initial stages of drug taking were looked at in these papers. That is to say, the role of stress was only discussed in the initiation of addiction. How does this translate into progression to full blown addiction?

A: The effect of stress is consistent regardless of where you are on the addiction continuum: stress enhances the reinforcing properties of drugs of abuse. That is to say, stress makes the pleasurable feeling from drugs more pleasurable. However, in humans, you will never get as clear of an effect (that means, easily testable) as you will in laboratory animals. Humans experience many different types of stress throughout a single day and the specific effect of stress on drug taking depends on the type/length/frequency of the stress and other environmental factors. Nevertheless, in controlled clinical studies, changes in HPA axis function as a result of drug use have been widely reported. A feed-forward mechanism exists in which stress promotes drug taking and then drug effects the stress response so that the next stressor has a greater effect on drug taking, etc.

Q: Can stress trigger relapse?

A: Yes, this is one of the most well studied effects of stress on drug taking: stress can trigger drug cravings in abstinent individuals. In the laboratory, an animal can be taught to lose its self-administration behavior by switching the drug to a neutral substance like saline. Therefore, when the animal nose pokes it does not get drug and eventually it doesn’t nose poke at all. This is called extinction. Amazingly, if you stress an animal with foot-shocks or some other phase and then test it’s self-administration behavior the animal will go back to lever pressing again!

Thanks again for reading! If you stuck through all three of Stress and Addiction posts please comment or email me. I would love to know!

Next time: Doping on Dopamine.

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The Science of Stress and Addiction: A Mini-review of the Research, Part 1

Stress-Brain

Why does one person become an addict and another person does not?

The vulnerability/susceptibility to addiction is one of the most important questions in the addiction field and also one of most difficult to answer. Is it genetics, the environment, or the addictive power of the drug itself? Spoiler alert: the answer is all three! But rather than trying to explain the answer in mere blog post (which is impossible), I think it’s better to tackle different aspects of the question in multiple posts (well, I probably could do it in one but I’m scientist: I would be a doing a disservice to you and to myself if I didn’t do a thorough job). This is the first post in this series.

Over the years, a lot of research has been done that has been able to show that stress can contribute to why one person becomes an addict and why another person does not. But how do we know that stress is important? And what is “stress” anyways? Let’s get our information straight from the horse’s mouth so to speak: a review of a few research papers that look at this question.

What is Stress?

Stress is one of those terms that is used often but may not be well understood. At one point or another we’ve all described our day as “stressful” and we all understand what this means but just take a moment and try to describe what “stressful” means in words that apply to ALL “stressful” situations. It’s tough, right? That’s because stress can mean any number of things in a number of different contexts.

In biology, we have a specific definition of stress: a response (usually immediate and automatic) to an environmental condition or factor, a stimulus, or other type of challenge. The body has several systems in place that mediate the stress response. For example, you probably have heard of “fight-or-flight”, which is one of the body’s stress responses.

The Hypothalamic-Pituitary-Adrenal (HPA) Axis
The Hypothalamic-Pituitary-Adrenal (HPA) Axis

Another of the key components of the body’s response to stress is the activation of the hypothalamic-pituitary-adrenal (HPA) axis. See the diagram. The HPA axis is hormonal system that involves chemical communications between several organs.

  • First, something happens that requires the body to respond to it, this could be sudden change in temperature, or an attack by an aggressor, or some other challenge. This factor is called a stressor.
  • Second, the stressor causes the hypothalamus, a region of the brain that controls many of the body’s functions, to release a small protein molecule called corticotropin releasing factor or hormone (CRF or CRH)
  • Third, CRF acts on the anterior pituitary gland, a small organ that secretes many different hormones. CRF stimulates the pituitary to release another small protein called adrenocorticotropic hormone (ACTH). ACTH then enters the blood stream.
  • Fourth, ACTH travel through the bloodstream until it finds its way to the adrenal glands, small organs that are located on top of the kidneys.
  • Finally, ACTH causes the adrenal gland to release cortisol (corticosterone in rodents), the “stress hormone.” Cortisol has many effects on many different organs throughout the body. Cortisol can also act on the hypothalamus and the pituitary gland themselves in order to inhibit their release and turn the HPA axis “off” until the next stressor. This is called a negative feedback loop.

Note: This is of course, a simplified model and there is a whole field of research devoted to working out the precise molecular mechanisms that regulate the HPA axis and how it responds to many different kinds of stressors.

Stress plays an important role in addiction. Stressors can make a drug seem more appetizing or even make it even feel better (more pleasurable). Anecdotally, after a stressful day, did you ever feel like you really needed a drink? Or, for the current and/or former smokers, how a cigarette was especially satisfying after a particularly jarring event? There’s a neurobiological reason for that feeling!

How do we know stress is important in addiction?

We are going to examine a few research papers that span over two decades (this discussion will be split over two posts). Each paper will reveal a little piece of the puzzle about why stress makes drugs more addictive. However, a Google Scholar search for “stress and addiction” gives you 527,000 hits! Basically, I chose these ones because they are easy to explain and, more or less, fit together in a sequence. Also, they all use one or more of the techniques that I described in my last post: The Scientist’s Toolbox: Techniques in Addiction Research, Part 1. I encourage you to read it before proceeding.

As we go through, try to keep the question we are trying to answer at the back or your mind: Does exposure to stress make it easier to become an addict and, if so, how does it do this? But this is a big question so it’s broken down into little pieces that each paper will try to answer. By the end of the second post, all the little pieces should add up to the bigger story.

Paper #1

Piazza 1990. Title
Paper #1

Both of the papers we’ll go over today look at what effect stress has on the behaviors of rats exposed to psychostimulants, either amphetamine or cocaine.

As I described in The Scientist’s Toolbox, psychostimulants cause an animal to move around more, and subsequent doses, over a period of a few days, increase that movement. Recall that this phenomenon is called locomotor sensitization.

Figure 1: Behavioral sensitization to amphetamine. Locomotor activity test (left panel) and self-administration (right panel).
Figure 1: Behavioral sensitization to amphetamine. Locomotor activity test (left panel) and self-administration (right panel).

In the first paper, rats are given 4 injections of amphetamine, one injection of amphetamine every three days and, sure enough, after the fourth injection exhibit greater locomotor activity; these rats are exhibiting locomotor sensitization to amphetamines. These results are shown in the left panel of Figure 1: black circles (4th dose of amphetamine) vs white circles (1st dose). Similarly, rats were given the same regimen of injections and 24hrs after the fourth injection self-administration of amphetamine was tested. As show in the right panel of Figure 1, only animals that were previously exposed to amphetamine (black circles) compared to saline-exposed rats (white triangles) self-administered amphetamine (nose-poked in order to receive drug infusions).

For the next experiment, there are two groups of rats: one group is exposed to stress and other is not. The type of stressor used in these experiments is called tail-pinch and it is exactly what it sounds like: a device is set to deliver a quick squeeze to the rat’s tail. This causes just a mild amount of pain and is very unexpected to the animals, thus it “stresses them out”. This means, as shown in other studies, that tail-pinch activates the HPA axis (increased cortisol secretion). In this experiment, no apparatus is used so instead the rats are placed in a bowl one at a time and then the scientist pinches the tail using forceps (tweezers).

Figure 2: Impact of stress on the behavioral effects of amphetamine. Locomotor activity (left panel) and self-administration (right panel).
Figure 2: Impact of stress on the behavioral effects of amphetamine. Locomotor activity (left panel) and self-administration (right panel).

Each animal in the stress group is exposed to 1min of tail pinch, 4times/day for 15days. This represents a chronic stress. The non-stress group rats are also placed in the bowl but no tail-pinch is applied. This is important to make sure that simply being handled or being put in the bowl is not having an effect. This non-stress group is an essential part of the experiment because it allows us to compare the effects of the stress test to animals that did not receive the test. It is called a control group. Controls are necessary for every experiment so that the scientist can make a useful comparison and allows him/her to interpret the experimental results.

Back to the experiment: 24hrs after the last tail pinch, both groups of animals are give an injection of amphetamine and their locomotor activity is measured. As you can see in the left panel of Figure 2, amphetamine caused greater movement in the animals that were stressed (black triangles) compared to the non-stressed control group (white triangles). This means, the ability of amphetamine to affect the animal’s movement was enhanced by stress.

In the second part of this experiment, the same stress exposure procedure is done but then the animals undergo a self-administration experiment (if you’re interested in the details, the catheter surgeries are completed before the stress exposure is started). As shown in the right panel of Figure 2, the stress group (black triangles) successfully acquired self-administration, meaning they gradually self-administered more and more amphetamine every day of the experiment. This behavior is similar to how human addiction begins, escalation in the amount of drug taken each time. Interestingly, the non-stress control group (white triangles) self-administered amphetamine for the first two days but gradually stopped and didn’t really seem interested in receiving the drug by day 5.

Figure 3: Comparison of prior exposure to drug (sensitization) to stress: impact on the behavioral effects of amphetamine. Locomotor activity (left panel) and self-administration (right panel).
Figure 3: Comparison of prior exposure to drug (sensitization) to stress: impact on the behavioral effects of amphetamine. Locomotor activity (left panel) and self-administration (right panel).

In Figure 3 the authors of this study compared the effect of prior exposure (sensitization) to stress for both the locomotor and self-administration experiments. They did this by dividing the experimental data by the control data (this is called normalization). There appears to be no difference between prior exposure and stress on locomotor activity and self-administration.

The authors conclude that stress is as potent as prior exposure to enhance the properties of the drug; stress exposure may be a significant factor why some people become addicted while others do not.

So very cool, it looks like stress can cause rats to want to self-administer more amphetamine and enhance the physical effects of the drug. Many other studies have found similar effects of stress. Let’s take a look at one paper that uses a different stress and a different drug.

Paper #2

Paper #2
Paper #2

In this study, the drug studied is the psychostimulant cocaine and the stressor is social stress. There are many variations of the procedure used for social stress but many are similar. In this paper, the rat to be stressed (the intruder) is placed in the home cage of a different rat (the aggressor). Because rats are territorial, this provokes the aggressor to attack the intruder. The intruder is left in the aggressor’s cage until it is bitten 10 times by the aggressor. The intruder rat is then placed in a mesh cage and put back in the home cage of the aggressor for a period of time. This way the intruder can still see and smell its attacker but can’t be physically attacked. This is repeated for several days. Social stress has been shown to be a very potent stressor, probably more so than tail pinch.

Note: The other study looked only at males but this study is interested in both males and females but for what we are interested in, this is a minor detail.

Haney 1995. Fig 1
Figure 1: Corticosterone levels in a novel environment in stresses and un-stressed male and female rats

First, activity of the HPA axis is measured by looking at corticosterone levels (this it the rodent equivalent of cortisol) when exposed to a novel environment (a novel environment is itself a type of mild stress). As you can see in Figure 1, rats that were previously exposed to social stress (black symbols) released higher amounts of coricosterone when placed in the novel environment compared to their unstressed counterparts (white symbols). This means the social stress has resulted in activation of the rat’s stress response, the HPA axis. Interestingly, female rats seemed to have a greater stress response overall.

Figure 2: The effect of social stress on cocaine self-administration.
Figure 2: The effect of social stress on cocaine self-administration.

Next, the effect of social stress on self-administration of cocaine is tested. As we saw with tail pinch stress and amphetamine, social stress caused an enhanced acquisition of cocaine self-administration whereas unstressed animals did not acquire cocaine self-administration. These data are presented in Figure 2, stressed rats (black symbols) and unstressed rats (white symbols).

In this paper, the authors also conclude that social stress—and activation of the HPA axis—makes it easier for a rat to acquire to cocaine self-administration; stress makes the rat want to self-administer cocaine.

 To summarize: these studies have found that two different types of stress have a similar effect on two different kinds of drugs. The first study found that tail-pinch stress increases the amount of locomotor activity induced by amphetamine. This stress also increases the amount of drug that the animals will self-administer. The second paper found that a different kind of stress, social stress, caused an activation of the HPA axis and had the same effect on cocaine self-administration: animals exposed to stress acquired self-administration behavior.

Based on the self-administration data, we conclude that stress caused the drugs to have a greater reinforcing effect. This is measure of the amount of pleasure the animals get from the drug. Therefore, we interpret that the stress made the drugs more pleasurable to the animals because they wanted to self-administer more drug.

However, there are some caveats that need to be briefly discussed. Both of these studies only looked at short term self-administration experiments (5 days) and both used relatively low doses. Many studies have found the rats and mice will self-administer cocaine and amphetamine regardless of whether they were exposed to stress or not. Nevertheless, these two papers are examples of how exposure to stress can cause a drug to be more addictive (technically, more reinforcing).

Next, we’ll look at some more stress studies that try to identify the molecular mechanisms—what stress is actually doing to the brain—of stress and addiction.

If you made it this far, thanks so much for sticking with it!

Just as a last thought: both of these are old papers, from the 90s and both are not very extensive (compared to today). This may sound incredible but it’s just an example of how difficult and time consuming science really is!

Thanks for reading  🙂

The Scientist’s Toolbox: Techniques in Addiction Research, Part 1

Lab Mice IMG_4102
(Image © Derek Simon 2015)

One of the most important questions that every scientist learns to ask is “How do you know that…?” As scientists, we are trained to be skeptical. When we consider a bit of research done by a colleague, before we are inclined to believe the data,  we need to be sure that they conducted the right experiments and that those experiments were done correctly. This doesn’t mean that scientists are stubborn or closed-minded. The reality is quite the opposite. Scientists are ready to incorporate new ideas and new results but first we need to know that the data are real. That’s what being a skeptic is all about: reserving judgment until you know all the facts.

The question “How do you know that..?” is one of the intellectual tools we use when considering whether or not data are real or not. This question has two parts: 1) how do you measure the thing that you interested in and 2) how do you know the effect you are seeing is actually based on what you think it is? What type of comparisons do you need to make in order to test the effect you’re interested in?

The first point of the question relies on special tools, equipment/technology, and experimental setups that are used to take measurements. For example, if you want to know how much a mouse likes taking a drug, then you need a way to measure how much drug it takes and how often it takes the drug (more on this in a bit). Today, I’ll go over a few of the tools that we use in addiction research.

The second part is more important (and more difficult to explain) but is really at the heart of the scientific method. It is all about experimental design and making sure you make the proper comparisons and analyses. I won’t discuss these details any more right now but will save this discussion for a future post.

Instead, let’s take a look at a few of the tools a scientist studying drug addiction has in his/her toolbox.

Locomotor Activity Test

The psychostimulants amphetamine and cocaine act in very similar ways and have very similar effects on the brain. We know that stimulants sort of “amp you up” or make you feel like you have more energy. Think of how you feel after drinking too much coffee. And what do you do when you have more energy? You tend to move around more (maybe you feel a little twitchy/antsy after too much of that coffee…). The same thing happens to mice and rats.

Locomotor Activity Test Chamber with a mouse. Image from UC-Davis Mind Institute (http://www.ucdmc.ucdavis.edu/mindinstitute/).
Locomotor Activity Test Chamber with a mouse. Image from UC-Davis Mind Institute (http://www.ucdmc.ucdavis.edu/mindinstitute/).

We can measure the amount of movement using a locomotor activity test. This test uses a special piece of equipment that uses light beams and a light-sensitive detector. Whenever the animal moves around the test box, the light beams are broken and the detector records that information. One way to analyze the data is by simply plotting beam-breaks (photo-cell counts are the same thing) that occur over the time of the test period. This way you have a measure of how much the animal moves around in a certain amount of time (more beam-breaks/time unit equals greater movement). A more sophisticated analysis of this same data can actually give you information on where in the box the animal spends its time. Does is just pace back and forth in a small area of the box or does it explore the entire chamber? This type of exploratory behavior data is valuable information and can be useful to other fields that may or may not study drug addiction. The general test for this exploratory behavioral analysis, regardless of speed of the movement caused by drugs, is the open field test.

 

Multiple test boxes with a computer that collects the data. Image from Douglas Mental Health Institute (http://www.douglas.qc.ca/page/neurophenotyping-motor-function).
Multiple test boxes with a computer that collects the data. Image from Douglas Mental Health Institute (http://www.douglas.qc.ca/page/neurophenotyping-motor-function).

An interesting phenomenon has been identified with psychostimulants. If you give an animal an injection of cocaine it will move around more compared to regular animals. But if you give it another dose of cocaine the next day it will move around even more than it did on the first day. This is called locomotor sensitization and is an important property of psychostimulants like amphetamine and cocaine.

The graphs below are real data that I took from a figure from one of our lab’s papers so you can see what locomotor sensitization looks like.

Cocaine-induced locomotor sensitization. Unterwald EM et al. J. Pharmacol. Expt. Ther. 1994.
Cocaine-induced locomotor sensitization. (Unterwald EM et al. J. Pharmacol. Expt. Ther. 1994.)

It’s a little hard to read but there are two groups of animals: one that receives cocaine injections (the top line) and the other that receives saline injections (the bottom line). Saline is a saltwater solution that is a standard control solution that has no biological effects. Each data point represents an average of several animals from each group. The baseline graph shows the locomotor activity before injections (no differences). As you can see, at day 1 the cocaine animals are already moving more than the saline group. This increase in movement continues over the 14 days of the experiment, evidence of locomotor sensitization.

This video shows an analysis of locomotor activity using video tracking software instead of light-beam breaks.

Self-Administration

Locomotor activity is all good and well but not all drugs of abuse cause locomotor sensitization. More directly related to addiction in humans, how do we even know if the animal likes the drug or wants to take the drug? Humans addicts crave the drug and compulsively use it, meaning the desire to do the of the drug overpowers the addict’s self-control. Is there a way we can study this type of drug-taking behavior in animals? The answer is yes!

Self-administration is a very versatile and powerful technique used throughout the addiction field. This technique allows the animal to control whenever it takes the drug and however much it wants. We can study many different aspect of drug taking using self-administration.

A diagram for a typical self-administration chamber. Image from Med Associates (http://www.med-associates.com/).
A diagram for a typical self-administration chamber. Image from Med Associates (http://www.med-associates.com/).

 

The basic idea is is simple: The rodent (mouse or rat) is placed in a chamber and presented with two levers. If the mouse the presses one lever (the active lever) it receives a dose of drug but if it presses the other lever (inactive lever) it does not. The self-administration sessions are run for a set period of time and the number of presses is recorded for each lever. Over the course of several days the animal steadily increases the amount of lever presses, thus the amount of drug it takes. Meaning the animal learns how to take drug and then takes more and more of it. Just like a human addict would do!

Alternatively, the mouse can poke its nose at a special hole that acts just like the active lever. I’ll use “lever press” and “nose poke” interchangeably because they essentially mean the same thing.

Here’s a little cartoon I found on YouTube of a rat that is self-administering nicotine.

 

Here’s another video that shows a real mouse self-administering a natural reward (meaning not a drug of abuse but food in this case).

 

There are several important variations to this basic idea that help scientists to not only make the experiments easier to control and data better/easier to analyze, but allow different aspects of drug taking to be studied.

For example if you are studying alcohol addiction, then when the mouse presses the lever a spout may appear that allows the animal to drink the alcohol (the inactive lever produces a bottle of water only). This is perfect for testing alcohol self-administration because both humans and mice drink alcohol. But what if you want to study heroin or cocaine self-administration? Humans (nor mice) drink or eat these drugs. So how does the drug get delivered to the mouse when it presses the lever?

The answer is intravenous self-administration. In this version, a small surgery is performed where a small tube (a cathether) is threaded into the jugular vein of the animal. This tube is fixed to the mouse back and attached to another tube that is part of the self-administration apparatus. This time when the mouse hits the lever, a dose of drug is pumped directly into its vein! See the diagram and videos above for more details.

Intravenous self-administration has several advantages.

  • As explained above, it allows us to deliver drugs to animals that won’t take them orally.
  • It allows the drug to act immediately on the animal because the drug is being delivered directly into the bloodstream.
  • It allows us to control the dose of the drug. When the mouse hits the lever (or nose pokes) it receives a fixed amount of drug that the scientist decides on ahead of time. That way we know how much total drug the mouse takes during a single self-administration session.
  • There is no variability in whether the animal is receiving the full dose or not. For example, if the lever press results in a food pellet, there is no guarantee the animal will eat the whole thing. But if you set the self-administration apparatus to deliver 0.5mg of heroin every time the lever is pressed, then there is no doubt if the full 0.5mg dose is delivered to mouse ever time.

Warning: not for the squeamish! This video shows you how to do the catheter implantation surgery on a mouse that will be used for intravenous self-administration!

Finally, best of all, self-ad can be used to address many different types of questions related to different stages in the addiction cycle. Here I briefly describe some of the more common experimental questions and applications that self-ad can help to address.

  • Initial use and escalation of use. How much will the animal take when it is first exposed to the drug? Will the animal reach a ceiling in the amount of drug it will take in a single session?
  • Maintenance of drug taking. One cool variation is you can make it more difficult for the animal to get the same dose of drug. This is called a progressive ratio self-administration. For example, the animal may need to press the lever 5 times before it receives a dose. You can keep increasing the number of presses during each session to see how hard the animal will work for a dose. One way this experiment can be interpreted is how badly does the animal want the drug? Some animals will press the lever many, many times just to get a small dose. This type of behavior is similar to the intense cravings that human addicts can experience.
  • Extinction and Relapse. You can run a special type of experiment where you run a self-administration experiment like normal and then change it so that the active lever no longer gives the animal a dose of drug. Eventually, the animal presses the lever less and less as it learns that it will no longer get the drug. This is called extinction of self-administration. This is like being in a rehab clinic where you are prevented from taking the drug. However, after the extinction sessions, if the scientist gives the animal another does of drug this will causes animal to start pressing the lever at high rates again. This a called reinstatement of self-administration and is model of relapse. What other types of conditions or factors can cause reinstatement (relapse behavior)? This situation is just like an abstinent cocaine addict who may not be craving cocaine but if he/she takes even a single hit, this can be sufficient for that person to sink back into full-blown addiction.

Let’s take a look at some real data. The graph below is from a paper from our group that looks at oxycodone self-administration in mice.

Oxycodone self-administration by adult and adolescent mice. (Zhang Y et al. Neuropsychopharmacol. 2009.)
Oxycodone self-administration by adult and adolescent mice. (Zhang Y et al. Neuropsychopharmacol. 2009.)

 

This study is interested in comparing oxycodone self-administration between adult mice and adolescent mice. As you can see, the number of nose pokes at the active hole (remember, same thing as a lever presses) increases during the course of the experiment (don’t worry about FR1 vs FR3) while the inactive hole is ignored, because it does not result in drug administration. Note that the nose pokes are plotted over the time of the administration sessions (2 hours) and that 9 sessions are run (one every day).

Microdialysis

The types of experiments I’ve described so far are great ways of studies addictive behaviors but they don’t really tell you about what’s going on in the brain. These behavior experiments are useful in themselves but they are much more powerful if they can be combined with another type of experiment that gives you a window into what’s changing in the brain at the same time as the behaviors.

In my post Synapse to it, I described how neurotransmitters are released by the pre-synaptic neurons into the synaptic cleft so that they can act on receptors located on the post-synaptic neuron. Using microdialysis, you can sample the fluid that exists in the synaptic cleft and actually measure the amount of neurotransmitters being released!

This is an extremely difficult and very technically complicated technique and I will only go into the basics about it. First, the microdialysis probe is surgically placed into a region of the brain that you are interested in studying.

The microdialysis probe itself is like a very thin piece of tubing that allows the experimenter to flow fluid into it one side(inlet) and collect the fluid that flows out of the other side (outlet). At the tip of the probe (the part that’s actually inside the brain) is a special type of material that allows fluid from inside the brain to flow into the tubing (a semi-permeable membrane).

Schematic of a microdialysis probe. Image from Wikipedia.
Schematic of a microdialysis probe. Image from Wikipedia.

After the surgery, you run your behavioral experiment, and while you are doing that you start flowing fluid into the brain. The fluid that the microdialysis probe flows in is of a similar consistency to the fluid that exists naturally in the brain. As the fluid inside the probe moves through the tubing, it causes fluids in the brain to enter into the probe and through the tubing where it can be collected when it flows out of the tubing.

Let’s say you give an animal a drug that causes a neurotransmitter to be released in the brain region you are interested in. Then some of those released neurotransmitters will enter the microdialysis probe because some of the fluid that enters the probe is from the synaptic cleft.

You keep collecting fluid at different time points during your experiment. When the experiment is over, then you can use chemistry to determine what neurotransmitters are in the fluid you collected. Best of all, you can determine how much of those neurotransmitters you have! How you do actually use chemistry to do this is a very technical part of the procedure and is not important to this discussion.

And all that work gives you a nice graph of the neurotransmitters that are released at different times during your experiment.

Now for some real data. Below are figures from a paper that our lab produced that uses microdialysis to study release of the neurotransmitter dopamine.

Evidence of probe placement in the Caudate  Putamen. (Zhang Y et al. Brain Res. 2001.)
Evidence of probe placement in the caudate putamen. (Zhang Y et al. Brain Res. 2001.)

 

Cocaine-induced increase in DA. (Zhang Y et al. Brain Res. 2001.)
Cocaine-induced increase in DA. (Zhang Y et al. Brain Res. 2001.)

 

In this study, the effect of cocaine on dopamine release in a region of the brain called the caudate putamen is being studied. The first image shows you that the microdialysis probe was placed in the right area of the brain (the white line that pierces through the dark area is the tract in the caudate putamen). The graph shows that injection of cocaine (the arrows) causes an increase in dopamine release in this brain region. Interestingly, the dopamine levels have returned to normal by the end of the experiment. Note: C57Bl/6J is the strain of mouse used in this study.

These are just three of the techniques that are used in addiction research. But we scientists have very big toolboxes! I’ll to explain some more in a later post.

Feel free to contact me or comment if you have questions!

Thanks for reading 🙂