Han et al vs Yiu et al - Joe

Han et al vs Yiu et al

Seminar in Biopsych

Fall 2016

Rebecca Shansky

Diphtheria toxin receptors (DTRs) are not endogenously expressed in mice, and neither is diphtheria toxin (DT). Thus, inducing expression of DTRs in the LA in a cre-dependent manner allows for specific ablation of neurons in the LA through injection of DT anytime after ample infection of the virus. This is what Han et al did. Transgenic mice (iDTR mice) were injected stereotaxically with a cre-dependent virus. In the population of cells that the virus infected, cre was expressed, which means that DTR was also expressed. Thus, (intraperitoneal?) injection of DT anytime after the cre had had enough time to be expressed would induce apoptosis in the susceptible (DTR-positive) population. Up to this point, all the cells in the LA injection site expressed the DTR; however, if the cre virus also encoded cDNA for CREB, only the LA neurons that expressed high levels of CREB would also be infected with cre, thus inducing DTRs (introducing apoptosis susceptibility). This is interesting because they mentioned that the CREB-cre group and the control-cre group showed similar levels of cell death — if the CREB-cre group is supposed to infect a narrower subset of neurons and the same volume of virus is injected into both groups, one would imagine that DT injection would kill a smaller population of cells in the CREB-cre group.

It was convincing that there were several groups of controls in this paper. Firstly, there was the experimental group (CREB-cre with DT in iDTR mice), and the subsequent iDTR mouse controls (control-cre with DT and CREB-cre with vehicle). Another control could have been control-cre with vehicle, but if there was no experimental effect in the control-cre with DT group, the control-cre with vehicle probably would not have been very informative. Furthermore, there were wild type controls as a proof of principle that the CREB-cre virus was infecting cells that were preferentially active during fear conditioning (the cells that were encoding the memory). With all the controls, only the experimental group showed a significant change before and after DT administration. However, this entire paper is based on cell-type specific apoptosis and concurrent freezing behavior change, and no control was done to account for crude locomotor changes. The LA is just medial to the cortex, and CREB is expressed throughout the entire brain, and so ablation of cells expressing high levels of CREB can precipitate a number of confounds outside of the LA. This is without considering that probably not every cell in the LA that expresses high levels of CREB is recruited in a particular memory trace. Still, it controls for impairments in learning after ablation and impairments in other memories prior to ablation, which are very convincing. Still, this change in behavior could be due to a change in locomotor activity around ablation, which is accounted for by the brain days later.

An interesting question to ask is if the title of the paper suits the findings. The most convincing experiments in this paper were the controls for inabilities to learn after ablation and impairments to learn prior to ablation. This showed that if ablation is targeting a memory trace, it is not affecting other memories learned prior to the auditory fear learning, and it also did not actually damage the memory-making system in the brain for future use. Still, it is unclear whether the neuronal population annihilated specifically encoded that memory.

Yiu et al attempted to follow up the findings of Han et al and others to conclusively say how neurons in the LA are recruited to be a part of a particular memory trace. To test the claim that LA neurons are recruited based on intrinsic properties (their current excitability), Yiu et al employed similar methods to those that Han et al did; however, instead of completely eliminating a particular subset of neurons in the LA, Yiu et al modulated their neuronal excitability through various viral techniques. The best part about these first few experiments was that before they began to show how excitability ties into the recruitment of neurons for the memory trace, they did a series of proof of concept experiments showing that their methods indeed increase/decrease excitability reliably — good controls.

What was interesting about this paper was that the same claim was proven three times, using three different approaches; however, all three approaches told the same story: dnKCNQ infection increases neuronal excitability which preferentially recruits infected neurons into the memory trace; excitatory DREADD infection coupled with CNO administration increases neuronal excitability which preferentially recruits infected neurons into the memory trace; and lastly ChR2 infection with light administration increases neuronal excitability which preferentially recruits infected neurons into the memory trace. The only change between the experiments was the increase in temporal sensitivity of the experiments. Were they all necessary to drive that point home? I’m not sure. Nonetheless, the finding is awesome! Simply activating a subset of neurons before a learning task “primes them” and they subsequently are more likely to be a part of the memory trace, as measured by neuronal activity during a retrieval task (coupled with an in situ measuring arc translation). The experiments were well-controlled; they accounted for the active timeframe of HSV by doing behavior several days after injection of the virus, and also accounted for overall anxiety behavior by showing that increasing excitability in the LA doesn’t just alter locomotor behavior — it’s context specific, among several other crucial controls. Overall, it seems convincing that increasing neuronal excitability increases probability that the given neuron will become recruited into the memory trace. And it’s also crazy to get that insight into the underlying mechanisms of neuronal recruitment during learning.

Han et al had a question regarding what happens if the neurons that are recruited into a memory trace are eliminated from the mouse’s memory repertoire after fear conditioning; can they still recall the memory? Yiu et al took it a step back and said, “well what is it about these neurons that are recruited that allows them to be recruited; it could be any collection of neurons that becomes a memory trace, but if you induce excitability, can you artificially recruit those neurons in a memory trace?” Apparently, you can.

10/17: Fear Memories

This week’s papers were both related to the effects of neuronal excitability on fear memory expression. Han et al., 2009 showed that increased CREB levels during fear learning are critical to the stability of that memory by deleting neurons showing overexpressed CREB levels, which blocked expression of fear memories by decreasing transgenic mice’s freezing levels. I thought the researchers did a very impressive job in finding the neurons in the LA that are actually involved in the fear learning process because instead of being in a small cluster they are widely spread out. There could have been more experiments in disrupting CREB, however, to further show that it is indeed involved.

Yiu et al., 2014 used the knowledge from the first paper to show that increasing neuronal excitability biases recruitment into the memory trace, enhancing memory formation. I thought this experiment was more comprehensive than the first one because they not only showed the involvement of CREB, but also numerous other molecules such as dnKCNQ2, hM3Dq + CNO, and ChR2 in neuronal excitability and memory formation enhancement. Furthermore, I like how they used Kir2.1 to show opposite effects in mice. They also used a wide-ranging set of control experiments, including the anxiety test and the anatomical specificity test.

From a clinical aspect, these experiments can be very valuable for the treatment of fear disorders such as PTSD. Clearly, these experiments are not exactly feasible in humans, but knowing that you can manipulate neurons in your lateral amygdala during fear memory expression is very important. These papers show that you need to excite neurons prior to training the fear memory, but perhaps researchers can next dive into ways you can manipulate fear memories after it has been encoded.

CREB Giveth & Genetic Manipulation Taketh Away: The Story of Auditory Fear Memory in the Amygala

The idea that CREB levels can designate which neurons are recruited to the auditory fear memory trace is introduced in the 2009 Han et. al. paper. However, CREB plays a vast variety of roles within the cell, so the reason for this is unclear. It is the 2014 Yiu et. a. paper that finally gives an indication as to which role of CREB is important to this particular function. As it turns out, neuron excitability is the key.

After reading the first paper, I will admit that I had some burning questions left. For example- what role was CREB playing in this process? (thank you Yiu et. al) What happens if you knock out or knock down CREB function? Would it make it impossible for an auditory fear memory to be encoded at all? Is CREB necessary for this memory encoding process? Could this even be answered without making the affected neurons completely dysfunctional? Furthermore, could you allow the memory to be encoded, and temporarily erase it by simply inhibiting the CREB-cre cells instead of killing them all together? Could the memory be rescued by re-activating the same CREB-cre cells later on? After reading the second paper, I still wondered if there are other proteins in the cell that could play the same role in increasing neuronal excitability. Is CREB still necessary or simply some factor to increase excitability? Is CREB directly involved with the voltage dependent potassium channels examined in this paper, or are they independent mechanisms of excitability?

Something I have noticed in both of these papers is that the only measure of fear behavior is freezing. While freezing has been traditionally measured, it does not appear to be the only fear behavior displayed by some animals. An escape-like darting behavior has also been observed which would make it seem as though those animals were not freezing at all. Although the data in these papers seemed convincing, this is an important consideration to make when freezing is the only measurement used for fear behavior.

My final thoughts on these papers concerns the human disorder of post-traumatic stress disorder (PTSD). Although these methods are obviously not meant as a clinical treatment for humans, it may be interesting to consider what factors would create such an extremely strong fear memory to be encoded. The papers note that the size of the memory trace does not differ in size with the strength of the memory (ie. less neurons in the trace do not cause a weaker memory)- so what does cause a much stronger memory encoding? Is it possible that people who are susceptible to PTSD have a naturally higher level of CREB? Alternatively, are their amygdala neurons naturally more excitable? These are interesting questions to think about as field moves towards answering more questions about the human correlates of the brain regions and processes observed in mice.

10/17 Han and Yiu

            Han et al. deleted neurons activated during auditory fear memory to attempt to erase the memory. They knew that lateral amygdala (LA) neurons with relatively high CREB levels were preferentially activated during auditory fear memory. They labeled these neurons using HSV vectors in iDTR transgenic mice to elevate CREB levels in small populations of LA neurons. When the CREB-cre vector is injected into the mouse, the stop cassette loxP is excised, and DTR is expressed. When DT is administered, apoptosis occurs. The experiments run by Han et al. show that the cell death of the memory trace erases the fear memory.

            The reserachers proved that the deletion was chronic by testing mice up to twelve days after DT administration. On the twelfth day, the freezing levels were still as low as the second day. Furthermore, these mice were able to relearn the fear memory, which showed that the deletion was not an impairment of overall LA function. However, this made me wonder how many times or how often memory trace neurons could be ablated from the LA without any negative effects for the mouse. Since only a small amount of neurons were recruited and deleted, there were not any negative side effects seen in the mice after one time, but if it was repeatedly done, there may be negative effects. Further experiments could be done to investigate this by subjecting these mice to behavioral tests in the future.

            Yiu et al. sought to determine whether relatively higher LA neuronal excitability before training would preferentially allocate these neurons to a memory trace. They knew from previous research that increased CREB expression caused preferential recruitment on neurons to a fear memory, and ablation of these neurons caused the fear memory to be erased. They aimed to uncover a more detailed reason for the memory erasure. One result of increased CREB in a cell is increased excitability, so the researchers used an extensive amount of techniques to test neuronal excitability. The experiments went beyond using just neuronal excitability by overexpression of CREB; excitability through dnKCNQ2, CNO bound to hM3Dq, and ChR2 were also used. The results were consistent across methods.

            If fear memory deletion was deemed ethical for humans and the technique was safe, it would be difficult to use in a clinical setting. The experiments proved that the vector must be injected before the memory occurs, so it wouldn’t be able to be used to erase a fear memory from a patient’s past. Unless the person knew that they were going to encounter something fearful, then it wouldn’t be useful. If there was a way to inject the vector before fear memory occured, memory deletion could be useful in treating PTSD and other mental illnesses.

Ramirez: Now and Then

      One of the things that I found the most interesting between the 2013 and 2015 Ramirez et. al papers is that in the first paper, the "false memory" is created with negative stimuli, where as in the second paper, the dentate gyrus is activated with a positive stimuli. The fact that this subset of engram neurons can code for both positive and negative memories show how widespread their control of memory formation must be. I find it interesting that it seems that these neurons and their pathways are responsible for any kind of memory- not fear specifically. This is especially important to note because the two papers use very different kinds of tests in order to explore the behavioral outcomes of planting "false memories". The 2013 paper measures differences in freezing levels, where as the 2015 paper measured open field tests, elevated plus maze tests, tail suspension tests, and sucrose preference tests. Measuring freezing levels is generally regarded as a test of fear, but poor performance on the other tests from the 2015 paper are indicators of anxiety.

      Another piece of information I found interesting was that the 2015 paper examined the relationship between the dentate gyrus and the nucleus accumbens. They measured the effects of optically reactivating DG cells that were previously labeled by a positive experience in mice that were given a dopamine blockade or a glutamate blockade drug. This is similar to the previous papers which examined the effects of this blockade in the Nacc when the VTA dopamine neurons were manipulated. From that previous paper, we learned that dopamine receptor blockades in the Nacc can be very detrimental on the motor functions of a mouse. I am curious as to why the Ramirez group chose to pursue this route anyways, even when it had the potential to throw off results as a result of decreased motor functioning. 

10/3 Ramirez 2013 and 2015

Ramirez et al. (2013) optogenetically activated memory engrams in the dentate gyrus (DG) of the hippocampus in c-fos-tTA mice. Optic fibers were implanted into the DG and an AAV encoding TRE-ChR2-mCherry was injected into the DG or CA1.

The mice were taken off dox after surgery and exposed to context A for labeling. While the animals were being fear conditioned in context B, the labeled cells from context A were optically stimulated.  When the ChR2-mCherry mice were placed in context A for the second time, they froze significantly more than the mCherry only mice. In neutral and novel context C, the freezing levels were not significantly different than the controls. This showed that the previously neutral context A was successfully linked with the fear memory from being shocked, creating a false memory. A part of the method that I questioned was that the mice were taken off dox for 42 hours to label neurons associated with context A, and then immediately put back on the dox diet. Since the mice were only in context A for the final 10 minutes of the 42 hours, it seems like it would be possible that other neurons active during the rest of the time might be labeled as well. It might be interesting to compare labeled neurons from the context A exposure to labeled neurons while the mice stayed in the home cage for 42 hours.

Ramirez et al. used the same technique to label memory engram cells in the 2015 paper. One of the experiments in the paper started with the c-fos-tTA rats on dox during surgery and recovery, and then off dox to label either a positive or neutral memory and immediately put back on dox afterwards. Then, all groups except the control were subjected to ten-day immobilization stress. The labeled DG cells were reactivated for zero, one, or five days before the TST and SPT, which are used to measure anhedonia. Chronic (5 day) reactivation of DG positive memory engram cells was shown to rescue depression-like behavior after chronic immobilization stress. The results of the TST and SPT showed no difference between the anhedonic behaviors of the non-stressed controls and the stressed chronic positive memory reactivated group. Furthermore, both groups had similar levels of neurogenesis in the DG. The neutral memory and one day positive memory stimulation groups did not exhibit a rescue effect. Exposing the mice to a real positive memory for five days also did not have a rescue effect or increase neurogenesis. It would be interesting to see how long the rescue effects lasted in the mice. If the effects are long lasting, it could be beneficial to find a way to use the technique in treating depression in humans.

10/3 Ramirez

Both Ramirez et al papers for this week involved optogenetically reactivating dentate gyrus cells, and both discussed several innovative and fascinating experiments to show what manipulating the hippocampus can do to downstream targets and behavior. Ramirez et all (2015) discussed how activating positive memory engrams suppresses depression-like behavior, which I found especially interesting because the past weeks have shown different mechanisms that suppress depression. All of these experiments are of equal importance because the mechanisms of depression are so complex and so many people are affected by it. Thus, the more experiments devoted to studying depression, the better. Targeting specific brain areas and/or cells, like in the second paper, is a step above many current antidepressants that affect the entire brain, and can lead to reduced side effects and greater efficacy.

Ramirez et al (2013), who “created a false memory in the hippocampus,” got me thinking to whether or not the researchers had actually created a false memory. While I thought the experiment itself was remarkable, and the results can be very important for future research, I asked myself if it was truly a false memory? Although the mice did freeze in a completely different context than where they got shocked (chambers A and B were very different in design), I don’t know if “false” is the most accurate word because the mice remembered they got shocked, which was a real memory. It’s just the context that was different. An example of what a real “false” memory could be is if the mouse was never shocked to begin with, and the researchers somehow manipulated their brains to make them think they got shocked. So, it was definitely a manipulated and artificial memory, but I am not sure that I would exactly call it a false memory.