NIH Scientists Identify a Potential New Treatment for Depression: A Metabolite of Ketamine

In a remarkable example of scientific collaboration, a new study produced by scientists at various research centers at the National Institutes of Health (NIH) have identified how ketamine works as powerful and fast-acting anti-depressant. This discovery may lead to an effective and potent new treatment for depression.

Ketamine is normally used as an anesthetic but at low doses, it has been shown to have rapid acting and long-lasting anti-depressant effects in humans. Fast relief of depression is incredibly important because most anti-depressant medications are not very effective or can take weeks (or even months in some cases) for maximal effect, which hurts the recovery of patients suffering from this crippling psychiatric disorder. However, despite its rapid action, ketamine has many side effects such as euphoria (a “high” feeling), dissociative effects (a type of hallucination involving a sense of detachment or separation from the environment and the self), and it is addictive.

If ketamine could be made safe to use without any of its other more dangerous properties, it would be a powerful anti-depressant medication.

With this goal in mind, scientists at the National Institute of Mental Health (NIMH), National Institute on Aging (NIA), National Center for Advancing Translational Sciences (NCATS), University of Maryland, and University of North Carolina-Chapel Hill sought to unravel the mystery of how ketamine works.

When ketamine enters the body it is broken down (metabolized) into many other chemical byproducts (metabolites). The team of scientists identified that it’s not ketamine itself but one of it’s metabolites, called HNK, that is responsible for ketamine’s anti-depressant action Most importantly HNK does not have any of the addictive or hallucinogenic properties of ketamine. What does this mean? This special metabolite can now be produced and can be given to patients while ketamine (and all its unwanted negative side effects) can be bypassed.

depressionOf course, many tests still need to be done in humans to confirm the effectiveness of HNK, but the study is an amazing example of how an observation can be made in the clinic, brought in the lab for detailed analysis, and then brought back to the clinic as a potential effective treatment.

But how did the scientist’s do it and how do they know that this HNK is what’s responsible for ketamine’s depression-fighting power? Keep reading below to find out.

Also, check out the NIH’s press release on the study.

The original study can be found here.

What is ketamine?

(±)-Ketamine_Structural_Formula_V1.svg
Chemical structure of ketamine (wikimedia.org).

Ketamine has traditionally been used an as anesthetic due to it’s pain relieving and consciousness-altering properties [1]. However, at doses too low to induce anesthesia, it has been shown that ketamine has the ability to relieve depression [2]. Even more remarkably, the anti-depressant effects of ketamine occur within a few hours and can last for a week with only a single dose. Most anti-depressant medications can take weeks before they start relieving the symptoms of depression (this is due to how those medications work in the brain).

However, ketamine also has unwanted psychoactive properties, which limits its usefulness in the treatment of depression. Ketamine causes an intense high or sense of euphoria as well as hallucinogenic effects such as dissociation, a bizarre sense of separation of the mind from the self and environment. Ketamine is also addictive and is an abused party drug [3].

A debate has been going about whether ketamine should be used for the treatment of depression and if its risks outweigh its benefits [4]. However, what if ketamine itself is not responsible for the anti-depressant function but a chemical byproduct of ketamine? This is what the scientist’s in this study reported: it’s HNK and not ketamine that are responsible for the powerful anti-depressant functions. This discovery was made in mice but how do scientists even study depression in a mouse?

 

How do scientists study depression in rodents?

mice-162163_960_720

Depression is a complex psychological state that is difficult to study but scientists have developed a number of tests to measure depressive-like behavior in rodents. While any one particular test is probably not good enough to measure depression, the combination of multiple tests—especially if similar results are found for each test—provide an accurate measurement of depression in rodents.

Some of the tests include:

Forced Swim Test

As the name reveals, in this test rodents are place in a cylinder of water in which they cannot escape are a forced to swim. Mice and rats are very good swimmers and when placed in the water will swim around for a while, searching for a way to escape. However, after a certain amount of time, the mouse will “give up” and simply stop swimming and will just float there. This “giving up” is used as a proxy for depression, similar to how people that are depressed often lack perseverance or motivation to keep trying. If you a give drug and the mice swim for much longer than without the drug, then you can make the argument that the drug had an anti-depressant effect. See this video of a Forced Swim Test.

Learned Helplessness Test

One theory of depression is that it can result from being placed in a bad situation in which we have no control over. This test models this type of scenario.

First, mice are place in chamber where they experience random foot shocks (the learning about the bad, hopeless situation). Next, they are place in a chamber that has two compartments. When a foot shock occurs, a door opens to a “safe” chamber, which gives the mouse an opportunity to escape the bad situation. One measure of depression is that some mice won’t try to escape or will fail to escape. In essence, they’ve given up at trying to escape the bad situation (learned helplessness). You can then take these “depressed” mice, and run the experiment again but this time with the anti-depressant drug you want to test and see how they do at escaping the foot shocks. Read more here.

Chronic Social Defeat Stress

Imagine you had a bully that would beat you up every day but the bully lived next door to you and would stare at you through his bedroom window? It would probably make you feel pretty crummy, wouldn’t it? Well, in essence, that’s what chronic social defeat stress test is all about [5].

A male mouse is placed in a cage with a much larger, older, and meaner male mouse that then attacks it. After the attack session, the “victim” mouse is housed in a cage where it can see and smell the bigger mouse. This induces a sense of hopelessness or depression in the “victim” mouse and it will not try to interact with a “stranger”” mouse if given a choice between the stranger and an empty cage (mice are pretty curious animals and will usually sniff around a cage with a unfamiliar mouse in it). This social avoidance is a measure of depression. In contrast, some mice will be resilient or resistant to this type of stress and will interact normally with the “stranger” mouse. Similar to above, you can test an anti-depressant drug in the “resilient” mice and the “depressed” mice.

There are a few others but these are three of the main ones used in this paper.

How did the NIH scientists figure out how Ketamine works to fight depression?

It was believed that ketamine’s anti-depressant function was due to its ability to inhibit the activity of the neurotransmitter glutamate. Specifically, ketamine inhibits a special target of glutamate called the NMDA receptor [6].

The first thing done is this paper was to study ketamine’s effects in rodent models of depression and sure enough, it was effective at relieving depression-like behavior in the mice.

Ketamine comes in two different chemical varieties or enantiomers, R-ketamine and S-ketamine. Interestingly, the R-version was more effective than the S-version (this will be more important later).

Recall that ketamine is though to work because it inhibits the NMDA receptor, but the scientists found that another drug, MK-801, that also directly inhibits the NMDA receptor, did have the same anti-depressant effects. So what is it about ketamine that makes it a useful anti-depressant then if not it’s ability to inhibit the NMDA receptor?

Ketamine is broken down into multiple different other chemical byproducts or metabolites once it enters the body. The scientists were able to isolate and measure these different metabolites from the brains of mice. For some reason one of the metabolites, (2S,6S;2R,6R)-hydroxynorketamine (HNK) was found to be three times higher in females compared to males. Ketamine was also more effective at relieving depression in female mice compared to male mice and the scientists wondered: could it be because of the difference in the levels of the ketamine metabolite HNK?

To test this, a chemically modified version of ketamine was produced that can’t be metabolized. Amazingly the ketamine that couldn’t be broken down did not have any anti-depressant effects. This finding strongly suggests that it’s really is one of the metabolites, and not ketamine itself, that’s responsible for the anti-depressant activity. The most likely candidate? The HNK compound that showed the unusual elevation in females vs males.

Similar to ketamine, HNK comes in two varieties, (2S,6S)-HNK and (2R,6R)-HNK. The scientists knew that the R-version of ketamine was more potent than the S-version so they wondered if the same was true for HNK. Sure enough, (2R,6R)-HNK was able to relieve depression in mice while the S-version did not. The scientists appeared to have identified the “magic ingredient” of ketamine’s depression-relieving power.

These experiments required a great deal of sophisticated and complex analytical chemistry. However, this is beyond my area of expertise so unfortunately cannot discuss it further.

So now the team had what they thought was the “magic ingredient” from ketamine for fighting depression. But could they support their behavior work with more detailed molecular analyses?

The next step was to look at the actual properties of neurons themselves and see if (2R,6R)-HNK changed their function in the short and long term. Using a series of sophisticated electrophysiology experiments in which the activity of individual neurons can be measured, the scientists found that glutamate signaling was indeed disrupted. However, it appeared that a different type of glutamate receptor was involved: the AMPA receptor, and not NMDA receptor. The scientists confirmed this with protein analysis; components of the AMPA receptor increased in concentration in the brain over time. These data suggest that it is alterations in glutamate-AMPA signaling that underlies the long-term effectiveness of HNK.

OK, so great! HNK reduces depression but does it still have all the other nasty side effects of ketamine? If it does, then it’s no better than ketamine itself.

For the final set of experiments, the scientists looked at the psychoactive and addictive properties of ketamine. Using a wide range of behavioral tests that I won’t go into the details of, 2R,6R)-HNK had a much lower profile of side effects than ketamine.

Finally, ketamine is an addictive substance that can and is abused illegally. A standard test of addiction in mouse models is self-administration (I’ve discussed this technique previously). Mouse readily self-administer ketamine, which indicates they want to take more and more of it, just like a human addict. However, rodent’s do not self-administer HNK! This means that HNK is not addictive like ketamine.

mental health

In conclusion, (2R,6R)-HNK appears to be extremely effective at relieving depression in humans, has less side-effects than ketamine, and is not effective. Sounds pretty good to me!

Next step: does HNK work in humans? To be continued….

Selected References

  1. Peltoniemi MA, et al. Ketamine: A Review of Clinical Pharmacokinetics and Pharmacodynamics in Anesthesia and Pain Therapy. Clinical pharmacokinetics. 2016.
  1. Newport DJ, et al. Ketamine and Other NMDA Antagonists: Early Clinical Trials and Possible Mechanisms in Depression. The American journal of psychiatry. 2015;172(10):950-66.
  1. Morgan CJ, et al. Ketamine use: a review. Addiction. 2012;107(1):27-38.
  1. Sanacora G, Schatzberg AF. Ketamine: promising path or false prophecy in the development of novel therapeutics for mood disorders? Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2015;40(5):1307.
  1. Hollis F, Kabbaj M. Social defeat as an animal model for depression. ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 2014;55(2):221-32.
  1. Abdallah CG, et al. Ketamine’s Mechanism of Action: A Path to Rapid-Acting Antidepressants. Depression and anxiety. 2016.

 

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Important Considerations in Optogenetics Behavioral Experiments

Image credit NSF, Inbal Goshen, Karl Deisseroth.
Image credit NSF, Inbal Goshen, Karl Deisseroth.

The third and final part of my three part guest blog series on Optogenetics has been published on the Addgene blog. Addgene is a nonprofit organization dedicated to making it easier for scientists to share plasmids and I’m thrilled to be able to contribute to their blog! This post covers the running behavioral experiments utilizing optogenetics.

Check it out!

http://blog.addgene.org/important-consideration-in-optogenetics-behavioral-experiments

 

The Materials Science of Optogenetics Experiments

(blog.addgene.org)
(blog.addgene.org)

The second part of my three part guest blog series on Optogenetics has been published on the Addgene blog. Addgene is a nonprofit organization dedicated to making it easier for scientists to share plasmids and I’m thrilled to be able to contribute to their blog! This post covers the material science aspects of running optogenetic experiments.

Check it out!

http://blog.addgene.org/the-materials-science-of-optogenetics-experiments

Optogenetics on the Addgene Blog: Part 1

(blog.addgene.org)
(blog.addgene.org)

The first part of my three part guest blog series on Optogenetics has been published on the Addgene blog. Addgene is a nonprofit organization dedicated to making it easier for scientists to share plasmids and I’m thrilled to be able to contribute to their blog!

Check it out!

http://blog.addgene.org/a-primer-on-optogenetics-introduction-and-opsin-delivery

Morphine and Oxycodone Affect the Brain Differently

(Neurons. Image from Ana Milosevic, Rockefeller University)
(Neurons. Image from Ana Milosevic, Rockefeller University)

Why are some drugs of abuse more addictive than others?

 This is a central question to the addiction field yet it remains largely a mystery. All drugs of abuse have a similar effect on the brain: they all result in increased amounts of the neurotransmitter dopamine (DA) in an important brain region called the mesolimbic pathway (also known as the reward pathway). One of the core components of this pathway is the ventral tegmental area (VTA), which contains many neurons that make and release DA. VTA neurons communicate with neurons in the nucleus accumbens (NAc). This means that the axons of VTA neurons project to and synapse on NAc neurons. When VTA neurons are stimulated, they release DA onto the NAc, and this is a core component of how the brain perceives that something is pleasurable or “feels good.” Many types of pleasurable stimuli (food, sex, drugs, etc.) cause DA to be released from the VTA onto the NAc (See the yellow box in the diagram below). In fact, all drugs of abuse cause this release of DA from VTA neurons onto NAc neurons.

*Important note: many other brain regions are involved in how the brain perceives the pleasurable feelings of drugs besides the VTA and NAc, but these regions represent the core of the pathway.

"Dopamineseratonin". Licensed under Public Domain via Wikipedia.
“Dopamineseratonin”. Licensed under Public Domain via Wikipedia.

Check out these videos for a more detailed discussion of the mesolimbic pathway.

But if all drugs of abuse cause DA release, then why do different drugs make you feel differently? This is a very complicated question but one component of the answer is that different drugs have different mechanisms and dynamics of DA release.

For the opioid drugs like heroin, morphine, and oxycodone, they are able to bind to a special molecule called the Mu Opioid Receptor (MOPR). This action on the MOPR results in an indirect activation of DA neurons in the VTA and a release of DA in the NAc. While all opioid drugs reduce the feeling of pain and induce a pleasurable feeling, they have slightly different properties at the MOPR.

The different properties of the opioids may be a reason why some are more abused than others. For example, a number of studies have suggested that oxycodone may have greater abuse potential than morphine. This means that oxycodone is more likely to be abused morphine.

But do the different properties of morphine and oxycodone on the MOPR affect DA release and is this important to why oxycodone is more likely to be abused than morphine?

Vander Weele et al. 2015 titleThis is the question that scientists at the University of Michigan sought to address. Using several different sophisticated techniques, the scientists looked at differences in DA release in the NAc caused by morphine and oxycodone, two common opioid drugs.

Rats were injected with either morphine or oxycodone and then DA release was measured using either fast-scan cyclic voltammetry or microdialysis. I’ve discussed microdialysis in a previous post but in brief, it involves drawing fluid from a particular brain region at different time points in an experiment and then measuring the neurotransmitters present (using advanced chemistry tools that I won’t explain here).

Voltammetry is a more technically complicated technique. In brief, it uses electrodes to measure sensitive voltage changes. Since a molecule has specific electrochemical properties, these voltage changes can be related back to a specific molecule, such as the neurotransmitter DA as in this study. Voltammetry may even allow greater temporal resolution (easier to detect very precise changes at very short time frames, like seconds), which may make it more accurate than microdialysis (which can only measure neurotransmitter release on the scale of minutes).

Because each technology has its own limitations and potential problems, the authors used both of these techniques to show that they are observing the same changes regardless of the technology being used. Showing the same observation multiple times but in different ways is a common practice in scientific papers: it increases your confidence that your experiment is actually working and what you are observing is real and not just some random fluke.

The authors administered a single dose of either morphine or oxycodone to rats and then measured the DA release in the NAc as described above. What they found were very different patterns!

Morphine resulted in a rapid increase in DA (less than 30 seconds) but by 60 seconds had returned to normal. In contrast, oxycodone took longer to rise (about 20-30 sec before a significant increase was detected) but remained high for the entire 2 minutes that it was measured. The difference in DA release caused by morphine and oxycodone is striking!

Many other changes were observed such as differences in DA release in different sub-regions of the NAc, different effects on phasic release of DA (DA is often released in bursts), and differences in the other neurotransmitters such as GABA (morphine caused an increase in GABA release too while oxycodone did not). I won’t discuss these details here but check out the paper for more details.

Of course, do these differences in DA release explain why oxycodone is more often abused than morphine? Unfortunately no, there are many other factors (for example, oxycodone is more widely available than morphine) to consider. Nevertheless, this is some intriguing neuroscientific evidence that adds one more piece to the addiction puzzle.

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!

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 🙂