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Monday, August 1, 2016

Claims of Mouse Memory Manipulation by Optogenetics Are Unproven

Contrary to claims sometimes made, scientists have no solid evidence for any such thing as a memory trace (also called an engram), a physical change in a particular part of the brain that corresponds to the storage of a memory. How memory works is a great mystery. There are some scientists that have claimed to have learned or discovered something about memory traces or engrams, but such claims are not well founded. An engram is a hypothetical group of cells that might store a memory.

In 2013 we had an example of a very dubious scientific paper claiming to have found something relevant to this issue. Two scientists (Xu Liu and Steve Ramirez) claimed to have created a false memory in a mouse. Their paper was entitled “Inception of a false memory by optogenetic manipulation of a hippocampal memory engram.” Their claim was picked up by countless mainstream news sources, which failed to apply critical scrutiny to the very dubious claim. 

The experiment was done using some mice that were genetically engineered to be light sensitive.  An optical device was connected to part of their brains.  Mice were put into a box and given an electric shock. This, the scientists claimed, created a memory in part of the brains of the mice. 

Then later, when the mice were in a different room, light was transmitted over the optical device, into the brains of the mice. This, the scientists claimed, activated the memory that had been stored in the brain of the mice – supposedly because the mice “froze.” I may note that the use of the term “froze” and “freezing” in the study is kind of loaded terminology, a kind of non-objective “assuming what you are trying to prove” terminology. The correct objective way to describe a mouse that is not moving is to say the mouse was temporarily not moving. “Froze” is a loaded term specifically designed to get someone to think that a mouse stopped moving because of fear, but you can't tell how a mouse is feeling or what it is recalling merely by the fact that it stops moving.

We are told that this was an “implanted memory,” because the original memory was created not in the second box but the first box. This term is inaccurate. If an experiment like this were ever done in a convincing manner, the most it would demonstrate is an electronic activation of a memory. It is also inaccurate to describe the result as an inception or implant of a false memory (as their scientists did in their scientific paper). If I have a memory of being punched on Fifth Avenue, and then I recall or relive that memory while on Lexington Avenue, that is not a false memory. It is a true memory of something that happened in a different place.

A more serious objection to the research is that is did not provide convincing evidence of a memory activation or any type of unusual memory effect at all in the mice being studied. There are three reasons why I make this claim.

The first reason is that the number of mice tested was very small. When you read the paper, you will find the number of mice used was only about 6. That's way too small a sample size to be drawing any reliable conclusions. With a sample size that small, the results could easily have been due to coincidence. An experimenter wishing to show some particular effect could just keep trying until some round of experiments showed the desired effect. That would be hard to do with a large sample size, but easy to do with a very small sample size such as only 6 or 8 mice.

The second reason is that the conclusion about whether the memory was being remembered was presumably based on an observer judging whether a mouse froze, or stopped moving. The authors did not explain how it was determined that particular mice had “frozen,” and we can only assume that such a determination was reached from a subjective human judgment. Given the start-stop, helter-skelter way in which mice move, any judgment about whether a mouse froze is going to be a subjective judgment. So there is too much of a possibility of observational bias here, one in which an observer subjectively reports the effect he is hoping to find. Similarly, you might subjectively report that your goldfish in a goldfish bowl tends to move towards you when you are looking into the bowl, but that would probably tell us more about your desire to see something than about the goldfish.

The third reason is that the freezing effect could have been produced not by a recall of memories, but by the very fact that the energy was being transmitted into the brain of the mice. Imagine you are running along, and suddenly a scientist switches on some weird thing that causes some energy to pour into your brain. This all by itself might cause you to stop, even if it didn't cause you to recall some memory that caused you to stop. What could have been going on in the mice was just a kind of pausing effect caused by a novel stimulus rather than a recalled fear effect.

We have no idea what was going on in the minds of these mice. It is not sound to assume that a mouse is “frozen in fear” merely because it stops moving, or to assume that the mouse is remembering something when it stops moving. We have no way of knowing what mouse are remembering at any particular moments. We can also ask: why didn't the scientists try to use dissection to confirm their claim of a memory stored in some particular spot of the brain? The technique would be simple: train a mouse to fear some particular stimulus, then dissect some little part of the brain where you think the memory is stored, and see whether the mouse still fears the stimulus. 

A more recent paper by Ramirez and Liu was published in Nature, and was entitled, “Activating positive memory engrams suppresses depression-like behaviour.” But the paper shows the same type of methodological problems of their earlier paper. Figure 2 of the paper says that in one group there were only 6 mice used, and elsewhere the paper states that a control group had only 3 mice. These sizes are way below the 15 animals per study group (control and non-control) recommended for a reliable experimental result. The authors claim to have counted differences in the degree to which mice “struggled” when presented with a maze – again something involving a subjective interpretation in which a researcher might tend to see whatever he wants to see. The authors' interpretation of what is going on is speculative. The authors do not present any solid evidence that they actually activated a memory by optogenetic stimulation.

But to its credit, Nature did publish an article entitled “Brain-manipulation studies may produce spurious links to behaviour,” pointing out that shooting light into one part of a brain (the technique used by Ramirez and Liu) may cause other parts of the brain to fire off, resulting in unpredictable effects. “Manipulating brain circuits with light and drugs can cause ripple effects that could muddy experimental results,” the article cautions. That's another reason for doubting these mouse memory studies based on optogenetic brain stimulation, since it undermines the whole simplistic idea of “stimulate just this area and activate just this memory.”

The “mouse memory implant” research described above is inconsistent with a body of memory research produced over a much larger period of time: the memory research of
Karl Spencer Lashley. Over many years, Lashley did extensive research in which he tested how memory and learning is affected when you take out various parts of an animal's brain. In one extensive set of experiments, Lashley trained rats to run a maze. The rats then had parts of their brains removed. Lashley found the rats were able to run the maze just as well regardless of which part of the brain was removed. Strongly indicating that particular memories are not localized in one particular part of the brain, this research directly contradicts the “mouse memory implant” work that tried to suggest that a memory was stored in one particular part of the brain.

Lashley tested using three types of mazes of varying difficulty. Astonishingly, Lashley found that you could remove half of a rat's brain, and it had very little effect on the rats ability to remember either of the two simpler types of mazes.

Here are some startling results listed by Lashley (and discussed here):
  1. Rats, trained to have a differential reaction to light, showed no reduction in accuracy of performance when the entire motor cortex of the brain, along with the frontal poles of the brain, was removed.
  2. Monkeys were trained to open various latch boxes. The entire motor areas of the monkeys' brains were removed. After 8 to 12 weeks of paralysis, during which they had no access to the latch boxes, the monkeys were then able to open the boxes “promptly” and “without random exploratory movements.”
  3. Rats were trained to solve mazes, and the rats then had incisions made separating different parts of their brains. This produced no effect in memory retention.
  4. Monkeys were trained to unlatch latch boxes. After having their prefrontal cortex removed, there was “perfect retention of the manipulative habits.”
  5. A number of experiments with rats have shown that habits of visual discrimination survive the destruction of any part of the cerebral cortex except the primary visual projection area.”

After discussing these and many other experiments he did for many years, Lashley said this about the idea of an engram or memory trace: “It is not possible to demonstrate the isolated localization of a memory trace anywhere within the nervous system.”

Lashley's research is completely inconsistent with the research claim of Ramirez and Liu. Lashley's research provides compelling evidence that particular memories are not stored in particular parts of a brain. Conducted over more than 30 years with a huge number of animals, Lashley's research was many times more extensive than the scanty 6-mouse research of Ramirez and Liu that got so much press coverage. Given a conflict between the two lines of research, we should believe Lashley's research, which is so much more voluminous. Contrary to the claims of some optogenetic researchers using dubious methodology, there is no compelling evidence that particular memories are stored in particular parts of the brain, and no convincing evidence that specific memories can be recreated by stimulating particular parts of the brain. There is no good evidence for any such thing as a memory engram, a particular set of cells that stores a particular memory. Lashley's many years of research strongly indicates that such ideas are not valid, as does the research of John Lorber (who, as described here,  documented many cases of humans who functioned very well, despite having most of their brains destroyed through disease). 

Postscript: In 2014 our credulous and exaggeration-prone news media reported that researchers Wiltgen and Tanaka had erased specific memories in a mouse. But the reports were based on a research paper that justified no such conclusions.  The paper states in its Figure 2 that only 6 mice were used for a study group, and 6 mice for the control group. This is way below the standard of 15 animals per study group (control and non-control) needed for a reliable experimental result. The same problem is shown in Figure 3 and Figure 4 of the paper. The paper reported borderline results, bordering on statistical insignificance.  It gave no clear evidence of having erased any memory in a mouse.  The paper had some of the same methodological problems as discussed above, such as relying on judgments of a mouse's "freezing rate" that is very hard to objectively quantify. 

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