Almost Creating a Fake Memory Trace

Mouse Memories (source)

Last post, we talked about the fallibility of flashbulb memories. Today we're going to discuss a new paper in which scientists claim to have created a fake memory in a mouse. 

Garner et al., (2012) use the same kind of genetic trickery that Han et al. (2009) used to erase memories.  They genetically modified mice to express a foreign receptor that mice don't normally express. These kinds of receptors are called DREADDs which stands for "Designer Receptor Exclusively Activated by a Designer Drug." A DREADD can activate the cell, inactivate the cell, or even kill the cell. (Han et al., added a receptor that killed the the cells, but Garner et al. add a receptor than activates the cells.)

But, here's the real genetic trickery, the DREADD is promoted only in the cells that are active at a certain time. When something happens, the cells that are active during the event will express the DREADD. So later, when the designer drug is applied, only the cells which were active during the event will respond.

Using this DREADD system, Garner et al. try to trick mice into thinking that they were shocked in one room, when really they were shocked in another room.  They call this a 'generating a synthetic memory trace' and this is how they do it:

Garner et al., 2012 Figure 2A

First of all the kind of memory the authors are synthesizing is the association between a room (or context) and an electric shock. If you put a mouse in a room and then give it an electric shock, the next time it is in that same room it will 'remember' that that room is scary and will show a freezing behavior. The measurement of how good this memory is is simply counting the percent of the time that the mouse spends freezing in the room.

They have two rooms, context A (Ctx A) and context B (Ctx B).  First they take the mouse and put it in context A (but don't give it an electric shock).  This activates a certain subset of neurons and so the DREADD will get expressed in those neurons. Let's call them the "Context A neurons." Then they stop the creation of new DREADDs by adding in doxycyclin, which turns off the DREADD gene expression. This makes it so (in theory) the only cells that have DREADDs are the "Context A neurons." 

Then they put the mouse in context B, but at the same time they apply the "designer drug" to activate the DREADDs.  Since the DREADDs are (supposedly) only in the "Context A neurons," the neurons that the drug activates should trick the mouse into thinking it is actually in context A, when really it is in context B. Then they apply the shock to the mouse.

To see if they have 'generated a synthetic memory trace' the authors test whether the mouse freezes in context A (where it thinks it was shocked) or context B (where it was actually shocked). 

Garner et al., 2012 Figure 2B&C

Unfortunately the authors don't find something simple.  First of all, they find that the mice with the DREADDs (the filled black circles above) almost always freeze less than the normal control mice (grey triangles), and they don't really explain why that might be. Second of all, they find that the application of the designer drug (+CNO) increases freezing for the DREADD mice in both context A and context B. 

The mouse didn't learn that Context A is where it got shocked.  Instead it learned that Context B with the "Context A neurons" is where it got shocked.  It's like the "Context A neurons" become part of context B. 

The authors call this a 'hybrid memory trace' where the mouse learns to associate a combination of the "Context A neurons" and the actual context B environment with the shock.

So what if just adding this drug is enough to create a hybrid memory? The authors did a nice control experiment to test this. They did the exact same protocol, but put the mouse in context B every single time (never in context A). That way the neurons expressing the DREADD are the "Context B neurons" and should basically be the same set of neurons that are active anyway when the mouse is shocked in Context B. In this case, the mouse froze a lot to context B without the drug, and it froze the same amount to context B with the drug.  The drug caused no enhancement when it was activating the "Context B neurons." This is strong evidence that the hybrid memory trace has to involve the activation of a new set of neurons.

This is a really nice experimental design, but I think that the authors oversold their result a little bit in the title "Generation of a Synthetic Memory Trace." They didn't create a totally fake memory, they created a hybrid memory by adding in new neurons to the 'context' that the animal associated with the shock.  There is no evidence that  the mouse thought it was in context A or even that having a context A is important. If they had just stimulated a random, but new, set of neurons in context B and then stimulated that same random set of neurons when testing the mouse for freezing behavior, they might have seen the same results.

© TheCellularScale


Garner AR, Rowland DC, Hwang SY, Baumgaertel K, Roth BL, Kentros C, & Mayford M (2012). Generation of a synthetic memory trace. Science (New York, N.Y.), 335 (6075), 1513-6 PMID: 22442487

LMAYQ: Safety First

Welcome to part 3 of the million part series "Let Me Answer Your Questions" (LMAYQ).  Here I address questions that people have asked The Internet.  The Internet, in turn, directed these people to The Cellular Scale, where their questions were... not answered. 

Today our topic is:

 

These 3 questions inspire me to warn you readers about a couple dangerous things.


1. "Will Parkinson's doctor let me have DBS?"

This is a really really important question because I absolutely do not know the answer to this. It must have brought someone to my post lauding the amazing power of Deep Brain Stimulation and its ability to stop Parkinson's Disease symptoms in their tracks. This is such an important question because it has prompted me to make this point:

This blog is not giving medical advice.

 

 

I am not a clinical doctor and more importantly The Internet is not your doctor and you should be careful when you ask The Internet medical questions. Yes DBS has shown amazing results, but its long term side effects are not even known yet and it requires a very serious surgical procedure. Just because I think its an amazing step forward, doesn't mean I think you should get it.  I don't know any thing about you or your condition (and neither does anyone else on The Internet). 


2. "Is DBS a cure for Parkinson's Disease?"

Following up on question 1, I titled my blog post "How does DBS cure Parkinson' Disease?", and focused on some new research looking into its mechanism of action. It was somewhat careless of me to use the word 'cure' all throughout the post, when really it should have been 'treatment'.  The most accurate answer to your question is "DBS treats Parkinson's Disease symptoms"


3. "What happens when you give a mouse cocaine?"
 
This is a good and interesting question, but also inspires me to make a warning statement:

Do not try this at home. 

Do NOT give your pet mice random drugs (even legal ones). It is not healthy for them.

This question brought someone to the post "If you give a mouse a placebo..." which discusses how to trick a mouse into thinking it is getting cocaine. This is really important research isolating the effect of the actual cocaine directly acting on the brain compared to the effect of the inactive cocaine acting indirectly on the brain.

The answer is: If you give a mouse cocaine, it can get addicted to it, which is why mice are used to study the effects of cocaine addiction and the efficacy of possible treatments.

But really I want to use this question as an opportunity to explain how research on animals is different from just giving animals drugs.

All the research I report on here is peer-reviewed.  This means other scientists besides the authors have read it carefully, and had an opportunity to point out any flaws or weak points in the research design or execution. In addition, research that uses animals adheres to extensive animal usage and handling regulations. It's not like someone just thinks 'dude, I wonder what would happen if you gave a mouse this or that' and then does and calls it research. Before scientists uses a single animal for any research, they have to write a thorough (usually 6 pages or so) explanation for exactly how many animals they are using and why they have to use that many and what they are going to use them for.  They have to list the steps they will take to minimize pain and explain how they will ensure that the animals are treated humanely. Then that document is read and discussed by a whole committee whose job it is to make sure that animals are being treated well at that institute. The committee then can either approve the animal use, or reject it and ask the scientist to only use this many instead of that many or use this kind of surgery instead of that kind of surgery. 

So there you have it, some warnings about what you should and shouldn't draw from this blog (and The Internet in general). Stay tuned for more fantastic answers to your fantastic questions.

© TheCellularScale

 

A little stress goes a long way

.... toward preventing PTSD symptoms.

Post Traumatic Stress Disorder

This may surprise you as the S in PTSD stands for STRESS.  How on earth could stress prevent it? But you heard correctly. A new paper by Rao et al., (2012) from Biological Psychiatry shows that a little stress in the form of glucocorticoids, prior to an acute stress event actually prevents PTSD-like symptoms in rats.

First of all how do you tell if a rat has PTSD?
This study uses two measures: one behavioral and one cellular.

To test anxiety in a rat, you can put in on an Elevated Plus Maze (EPM). Rats don't love heights, and they do love dark corners. But, they are also somewhat naturally curious. The EPM makes use of these rat characteristics to test how anxious the rat is.

Elevated Plus Maze (source)

The EPM has four arms, two are open (but far enough off the ground that the rat can't just step off the maze) and two are enclosed with walls. Normal rats tend to explore all the arms of the maze roughly equally, but anxious rats tend to strongly avoid the open arms. The amount of time spent in the open arm area is a generally accepted measure of how anxious the rat is.

An earlier paper from the same lab, found that rats who had undergone the single stress event were more anxious (spent less time in the open arms of the EPM) 10 days after the event, but NOT 1 day after the event.  The single event stress and the delay of symptom onset are why this study is more relevant for PTSD than for chronic stress. 

Rao et al., 2012 Fig 4B

As interesting as the behavioral experiments are, the cellular level experiments are where it gets really cool (The Cellular Scale is not biased or anything). They used the Golgi stain to visualize neurons in the Amygdala. They measured how long the dendrites were and also how many spines they had on them. (Spines are the little protrusions that come of dendrites to receive synaptic inputs).


They found that the stressed rats had more dendritic spines on the amygdala neurons than the non-stressed rats.  Not only that, but this increase in spine density was apparent 10 days after the stress event, but not 1 day after.  


You might think dendritic spine growth is a good thing, and likely signifies synaptic plasticity and pathway strengthening... but remember this is the amygdala, a structure critical for FEAR learning, more spines here may not be beneficial. Stronger pathways to these amygdala neurons likely means that they fire more easily.


Now that we understand how PTSD is measured in a rat, we can move on to how they 'cured' it in this paper.  

Rao et al found that when they injected vehicle (a fancy science term for 'nothing' or 'placebo' or 'saline') into the rat 30 minutes before the 2 hour stress event, the rat no longer showed either the increased in anxiety (fewer open arm entries on the EPM) or the increase in dendritic spine density.

Pretty weird, considering they were injecting vehicle prior to the stress event.  How could inactive saline (essentially nothing) cure PTSD symptoms?

They figured out that the actual injection process was stressing the rat out a little bit. When animals (including humans) are stressed, they release a hormone called cortisol.

Rao et al., 2012 Fig 1C,D,E

They found that the 2 hour stress event caused a huge rise in corticosterone (right and left panels), while the injection (vehicle) alone caused a small rise (middle panel). 

Because they were injecting nothing, they hypothesized that the corticosterone produced by the small stress of being injected was somehow protecting against the large 2 hour stress event.

The rest of their paper is basically confirming this. They add corticosterone to the water of the rats and this also prevents the PTSD-like symptoms.  They find that all their manipulations isolating the corticosterone confirm that this is what is protecting the rats from the delayed impact of the stress event.  

Interestingly there is evidence that 'small stress' can help prevent 'big stress' in humans too. They cite clinical studies reporting that intensive care unit (ICU) patients who receive injections of stress-level cortisol during treatment are less likely to develop ICU-related PTSD symptoms.

It is a puzzling paradox at the moment, but the next step is to figure out how exactly this little stress can reduce big stress.


Epilogue: 

I was lucky enough to see Dr. Chattarji, the principle investigator of this study, give a talk at a conference a few months ago.  And one interesting piece of information that you can get from a talk, but will never read in a paper is how the scientists originally stumbled upon their finding.  In this case, Chattarji's lab didn't start their study by injecting vehicle. They were actually testing a real drug that they thought might help alleviate PTSD.  They had a beautiful result showing that when you injected "drug X" before the 2 hour stress event, you eliminated the PTSD symptoms. The natural conclusion is to think that "drug X" is a new cure for PTSD.

 But therein lies the importance of the control group. To control for any effects of simply injecting the rat, they injected vehicle. When they saw that the vehicle prevented the PTSD symptoms just like the actual drug, they were crushed! This is the ultimate demise of an experiment.  The control group shows the same thing as the drug group, which means that the drug does not work! Luckily they were flexible and smart enough to investigate what they did see, that the injection alone could protect against the PTSD symptoms.

Also, if someone would like to explain the difference between cortisol and corticosterone, please do. I clearly do not have a full understanding here.

© TheCellularScale




Rao RP, Anilkumar S, McEwen BS, & Chattarji S (2012). Glucocorticoids Protect Against the Delayed Behavioral and Cellular Effects of Acute Stress on the Amygdala. Biological psychiatry PMID: 22572034

Mitra R, Jadhav S, McEwen BS, Vyas A, & Chattarji S (2005). Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala. Proceedings of the National Academy of Sciences of the United States of America, 102 (26), 9371-6 PMID: 15967994

If you give a mouse a placebo...

...It might ask for some cocaine.  Or it might feel the effects of cocaine anyway. 

Just say no, Rat (source)

The "Placebo Effect" occurs when someone takes a functionally ineffectual drug, but feels the effects anyway. There are many examples of this: Someone in pain takes a sugar pill, but is told that it is a painkiller might report 'feeling much less pain'.  A Parkinson's patient takes a sugar pill having been told it was their 'L-dopa' medication and can suddenly move more fluidly. The "Placebo Effect" is so strong that most experiments testing the effectiveness of a drug in healing anything use a placebo control.  The researchers want to make sure that the drug has an actual effect that is greater than the placebo effect.  (It has been proposed that homeopathic remedies are entirely due to the placebo effect)

One problem with deeply understanding the physical mechanisms which underlie the placebo effect is that all the experiments must be on humans.  You can't simply tell a mouse it's getting a 'cure' and give it a fake pill.  However, scientists at the National Institute on Drug Abuse (NIDA) have conducted an ingenious experiment that involves giving a mouse what is essentially a placebo.  Better still, they published it in PLoS One, so everyone can read the paper for free!

Before we dive into the placebo aspect of this paper, we need to back up and learn a little about as the addiction and reward system works in the brain.

In 1954, James Olds and Peter Milner published a paper showing that a rat would press a lever to receive an electrical stimulation in certain areas of its brain.

Olds and Milner, 1954 Figure 2, Xray of rat

This was a huge discovery showing that 'reward' could be activated directly. 

Later studies found that when this electrode stimulates the dopamine system of a rat, the rat will press and press and press this lever, even forgoing food when it is famished.  Incidentally, a mouse/rat will also compulsively press a lever to get injections of cocaine (which acts by stimulating the dopamine system).  You can do all sorts of experiments on drug addiction using this cocaine self-injection system. You can test how long it takes the mouse to become addicted, you can test the effect of drug concentration, you can test how other drugs interact with self-injection of cocaine, and you can even test aspects of withdrawal and relapse.

Which brings up back to our placebo.  Wise et al., (2008) investigated the mechanisms underlying this self-administration.  What was happening in the mouse brain when they got a dose of cocaine? They found that when the mouse pressed the lever and got the cocaine there was a surge of dopamine almost immediately after.  There is a center in the brain called the VTA that contains neurons which release dopamine. When these neurons are active, other areas of the brain are flushed with dopamine and the person/rat/mouse 'feels reward'.  But what makes these neurons active?

This brings up a problem we discussed a while ago, about the never ending cycle of neuronal firing.  The dopamine neurons fire, but why? what neurons are firing onto them to make them fire, and then, what neurons are making those neurons fire and which ones are firing before that...so forth into forever. 
To go one step up in this firing-chain, Wise et al. cleverly looked at 'brain juice' in the VTA and found that when the cocaine is administered, the mouse gets a surge of glutamate there.  (Glutamate activates cells, so this would cause the dopamine neurons of the VTA to fire and release dopamine onto other cells). 

So what does all this mean, and how does it get us to a placebo for a rat?

Here's the thing: the surge of glutamate that stimulates the VTA only shows up in mice that have already learned that a lever press gives them cocaine.  (That is, this glutamate surge doesn't occur the very first time the mouse gets cocaine)

Wise et al., 2008 (figure1B)

 Here is the figure showing this glutamate surge in the VTA.  The vertical dotted gray line is when the mouse presses the lever for the cocaine.  The red and yellow traces are the condition where the mouse actually gets cocaine in response to the lever press.  the blue and green traces are the condition where the mouse gets saline instead (a control), and the gray trace is the first time the rat gets cocaine in response to the lever press.

 Another peculiar aspect of this glutamate surge is that it is probably too fast a response to be a result of the cocaine acting in the brain.  So how is the injection of cocaine causing a glutamate surge if it is not even acting on the brain yet?  This is quite the puzzle.  Wise et al., wanted to make sure that this glutamate surge was absolutely not due to the cocaine reaching the brain, so they invented the rat placebo! They altered the cocaine molecule, so it was mostly the same shape as normal cocaine, except it couldn't cross the blood brain barrier.  That is, when it is injected into the mouse, it can be detected in the blood and in the peripheral body, but it won't be detected in the brain, and the cocaine will not be able to act directly on the neurons.

When they run the test with this molecule injected instead of cocaine, the figure looks like this:

Wise et al., 2008 (figure1E)

Pretty similar! This cocaine molecule can't reach the brain, but it causes the same glutamate surge as the real stuff! This shows that glutamate surge is somehow due to the cocaine being felt by the body, but not being felt by the brain.  Although they don't call it a placebo in the paper, that is essentially what it is.  It is tricking the brain into thinking it has just received cocaine. (In a stronger way than context can, as evidenced by the lack of response to saline.) 

 So there you have it, a way to trick a rat into thinking it has just received a 'real drug' when it has actually received an ineffectual drug. I think this technique could be adapted to actually study the physiological mechanisms governing the placebo effect. 

© TheCellularScale

Wise RA, Wang B, & You ZB (2008). Cocaine serves as a peripheral interoceptive conditioned stimulus for central glutamate and dopamine release. PloS one, 3 (8) PMID: 18682722