False memories
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A presentation discussing the science and neuroethic implications of a paper published in Science by Ramirez, S. et al. in late 2013.
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False memories
- 1. Creating False Memories in the Hippocampus Katie Strong December 9, 2013 Liotta Group Meeting 1 “Memory is not all that we are, but almost. We are the entire set of memories that we acquire. Every one of our memories changes who we are.” – Alcino Silva, Ph.D.
- 2. “Finding the Engram” 2 An engram is a physical representation or location in the brain of a memory 1920 – 1950 Karl Lashley spent 30 years trying to find the elusive engram and in his seminal paper, “In Search for the Engram,” he concluded that memories are distributed – not localized. 1984 Richard Thompson showed that after conditioning rabbits to fear a tone with an airpuff to the eye, removal of just a few hundred neurons from the interpositus nucleus (a section of the cerebellum) led to fear extinction. Josselyn, S.A. Continuing the search for the engram: examining the mechanism of fear memories.J Psychiatry Neurosci 2010, 35, 221. Lanshley, K. In Search of the Engram. Symp Soc Exp Biol 1950, 4, 452. McCormick, D.A.; Thompson, R.F. Cerebellum: essential involvement in the classically conditioned eyelid response. Science 1984, 20, 296.
- 3. 3 Xu, L. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 2012, 484, 381. Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387. 2012 Susuma Tonegawa labeled and artificially activated specific hippocampal dentate gyrus cells, which lead to a behavioral response associated with the fear memory of foot shocking. 2013 Researchers from the Tonegawa lab next set out to determine if labeling and “artificially activating a previously formed contextual memory engram while delivering foot shocks can result in the creation of a false fear memory.” “Finding the Engram”
- 4. Labeling dentate gyrus (DG) cells using the Tet-Off System 4Figure adapted from the Tet-Off® and Tet-On® Gene Expression Systems User Manual, ClonTech Laboratories, 2012 Tet-Off Systems: tTA binds to TRE in the absence of DOX, and this leads to increased expression of the gene of interest.
- 5. Labeling dentate gyrus (DG) cells using the Tet-Off System 5 Xu, L. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 2012, 484, 381. Researchers used transgenic c-fos-tTA mice and the gene of interest contained ChR2, a photosensitive protein that could be activated with light. • Off Dox, tTA would bind to TRE and promote the expression of ChR2 protein • On Dox, the binding of tTA to TRE would be inhibited. Off Dox On Dox X
- 6. Labeling dentate gyrus (DG) cells using the Tet-Off System 6 Xu, L. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 2012, 484, 381. Researchers used transgenic c-fos-tTA mice and the gene of interest contained ChR2, a photosensitive protein that could be activated with light. • Off Dox, tTA would bind to TRE and promote the expression of ChR2 protein • On Dox, the binding of tTA to TRE would be inhibited. Off Dox On Dox X
- 7. Basic Elemental Scheme 7Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387. Off Dox: Cells are selectively labeled with ChR2-mCherry, a photosensitive protein. ChR2- mCherry is then reactivated by light during the On Dox stage.
- 8. Creation of a False Contextual Fear Memory 8Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 9. 9 Creation of a False Contextual Fear Memory Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 10. 10 Distortion of Real and False Memories False memory interferes with genuine memory Memory recall can be induced for a false memory Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 11. 11 Conditioned Place Avoidance (CPA) Paradigm Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 12. 12Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387. Conditioned Place Avoidance (CPA) Paradigm
- 13. 13 True and False Memory Formations Activate Same Amygdala Cell Populations B’ A’ C Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387. The level of c-fos expression in the basolateral amygdala (BLA) and the central amygdala (CeA), two regions involved in fear learning and memory, were measured during the false and natural recall.
- 14. 14 Creating a False Memory in CA1 cells Researchers redid the experiments with CA1 cells, instead of DG cells, and found no formation of a false memory. Xu, L. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 2012, 484, 381. Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science 2013, 341, 387. Only after activating a small, specific population of cells did a false memory occur involving a foot shock – a memory very simplistic compared to our complex memories and experiences.
- 15. 15 Similar Mechanism behind False and True Memories “Whether it’s a false or genuine memory, the brain’s neural mechanism underlying the recall of the memory is the same” - Susumu Tonegawa, Ph.D. Trafton, A. Neuroscientists plant false memories in the brain. MIT News (accessed November 2013).
- 16. 16 Similar Mechanism behind False and True Memories “Whether it’s a false or genuine memory, the brain’s neural mechanism underlying the recall of the memory is the same” - Susumu Tonegawa, Ph.D. How reliable is our memory? Trafton, A. Neuroscientists plant false memories in the brain. MIT News (accessed November 2013). DNA Exonerations Nationwide. The Innocence Project (accessed November 2013). Lacy, J.W.; Stark, C.E. The neuroscience of memory: implications for the courtroom. Nature Reviews Neuroscience 2013, 14, 649. • 311 people in the United States have been exonerated based on DNA evidence since 1989, and in 72% of those cases eyewitness misidentification testimony was the leading reason for a wrongful conviction. • At many points in the justice system eyewitness testimony can become distorted, but these eyewitnesses can also become more confident. • In a mock trial, jurors “recalled” 15% of information that was not stated, but that could fit a typical crime scene. • Mock jurors also “recalled” 25.8% of information that was not stated by an eyewitness, but was implied by the prosecutor in a leading question.
- 17. 17 State v. Henderson: An Update to Eyewitness Identification and Jury Instructions Cpgo Wrong, New Jersey Supreme Court Uses Psychological Research to Update Admissibility Standards for Out-of-Court Identifications, 125, Harv. L. Rev. 1514 (2012). Rodney Harper and James Womble were celebrating by drinking and smoking when Harper was shot and Womble was held by the shooter’s armed accomplice. Womble later identified Larry Henderson as the accomplice 13 days after the murder and Henderson was convicted of first-degree murder. • During the photo lineup, primary investigators interrupted and encouraged Womble to make a decision. • After conviction, Henderson was grated a Wade hearing to determine the admissibility of the identification. The court then applied the Manson/Madison test, but found that “nothing in the case was improper, and certainly nothing that was so suggestive as to result in the likelihood of misidentification.” The Manson/Madison test considers • witness' opportunity to view the person at the time of the crime • witness' degree of attention • accuracy of the witness' prior description • level of certainty at the time of the confrontation • time between the crime and confrontation
- 18. 18 State v. Henderson: An Update to Eyewitness Identification and Jury Instructions Reinhart, C. Summary of New Jersey Case on Eyewitness Identification. <http://www.cga.ct.gov/2011/rpt/2011-R-0334.htm> Office of Legislative Research. (accessed December 2013). On appeal, the Appellate Division of the Superior Court of New Jersey decided that the identification procedure was “impermissibly suggestive.” The court ruled that the legal standard for eyewitness testimony must change because it • does not offer an adequate measure of reliability • does not sufficiently deter inappropriate police conduct • relies too heavily on the jury's ability to evaluate identification evidence. Now, the initial burden of proof for suggestive evidence lies with the defendant, and this evidence is usually tied to a systems variable: • Blind administration • Pre-identification instructions • Feedback • Lineup construction The state then offers proof that the identification is reliable accounting for systems and estimator variables: • Stress and weapon focus • Witness and perpetrator characteristics • Race-bias
- 19. 19 State v. Henderson: An Update to Eyewitness Identification and Jury Instructions Reinhart, C. Summary of New Jersey Case on Eyewitness Identification. <http://www.cga.ct.gov/2011/rpt/2011-R-0334.htm> Office of Legislative Research. (accessed December 2013). Lacy, J.W.; Stark, C.E. The neuroscience of memory: implications for the courtroom. Nature Reviews Neuroscience 2013, 14, 649. The court can then suppress eyewitness identification if sufficient evidence for suggestiveness is presented. When evidence is submitted though, tailored jury instructions must be provided.
- 20. Should neurocognitive enhancement such as deep brain stimulation (DBS) or transcranial magnetic stimulation (TMS) be used to enhance the memory of eyewitnesses since memories are easily manipulated? 20 Neurotechnologies to Improve Eyewitness Memory Vedder, A.; Klaming, L. Human Enhancement for the Common Good-Using Neurotechnologies to Improve Eyewitness Memory. AJOB Neuroscience 2010, 1, 22. Ridding, M.C.; Rothwell, J.C. Is there a future for therapeutic use of transcranial magnetic stimulation? Nature Rev Neurosci 2007, 8, 559. • Should the safety of the eyewitness come before justice in a court case? • Should the privacy of the eyewitness be respected? • Would all types of cases warrant eyewitness neurocognitive enhancement or would a selection process take place? • If neurocognitive enhancement is not mandatory, is all testimony equal? • Should refusing neurocognitive enhancement be considered obstructing with justice, a punishable offense?
- 21. 21 Summary • Recent neuroscience work that suggests specific memories are contained in a discrete number of cells also supplements the original idea from Karl Lashley that a distributed network exists. • Researchers created a false memory in mice by optogenetically manipulating a engram-bearing neuron population in the hippocampus, suggesting that the mechanism for false and real memories is very similar. • Many people believe that memories are similar to a video recorder, but our unreliable memories have the potential for unfortunate consequences in courts of law. • The New Jersey Supreme Court has recently taken great strides to overcome the impact of false memories by modifying the standards for eyewitness evidence and jury instructions. • Initial research has shown the potential for neurotechnologies in the law, but enhancing memories of eyewitnesses raises many ethical concerns.
- 22. Degree of DG Cell Population Overlap during Exposure to Context A and C 22 OR Cells activated by context A would be labeled with CHR2-mCherry (red) Cells activated by either context A’ or C would express c-Fos (green) After second exposure to A (A’) After exposure to novel context C (C) Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 23. Creation of a False Contextual Fear Memory 23Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science, 2013, 341, 387.
- 24. 24 Degree of CA1 cell population overlap CA1 cell population overlap after two consecutive exposures to context A was greater than with DG cells. Engrams rely less on population code and more on a temporal code. Ramirez, S.; Liu, X.; Lin, P.; Suh, J.; Pignatelli, M.; Redondo, R.L.; Ryan, T.J.; Tonegawa, S. Creating a False Memory in the Hippocampus. Science 2013, 341, 387. After second exposure to A (A’) with DG cells labeled After second exposure to A (A’) with CA1 cells labeled OR
Editor's Notes
- The great puzzle of memory’s physical embodiment in the brain, Semon asserted, is that whereas an ordinary object reacts to a force only while that force acts upon it, the nervous system is somehow permanently altered and able to show a unique reaction to a long-ago stimulation years later. Consider, for instance, a baby who grabs a dog by the tail and gets bitten: The bite marks heal, but the painful lesson about not grabbing doggy’s tail remains. The capacity for such learning constituted what Semon called the mneme, borrowing the name of the Greek goddess of memory. The practical result of that capacity is what he called the engram, “this permanent change wrought by a stimulus.” If the mneme can be thought of as Semon’s conception of a biological hard drive, the engram is the byte (or, in that baby’s case, bite) of information written onto it. Karl Lashley spent 30 years trying to find the elusive engram by subjecting rats and monkeys to a series of tasks and then before or after the training, certain parts of the brain were removed and the animals were forced to repeat the tests. Here he mapped the monkey’s brain and then labeled certain visual and motor areas. One example would be that he trained the monkeys to open boxes with a latch and then remove portions of the motor area including parts of the eye field. After the monkeys recovered, they were resubjected to the latch opening test, and the monkeys were able to quickly open the boxes. After 30 years of teaching mice and monkeys various visual and motor task, removing parts of the brain, and watching the animals complete the task, he concluded that specific cell populations are not engrams, but instead a distributed network exists for storing memories. All of these tasks were very complicated – a maze, avoiding a white X in a triangle or not depending in the background of the shape. For 30 more years this was the leading idea in the field until 1984, when in a blink of the eye, Richard Thompson changed the field. Richard Thompson, now professor emeritus of psychology, biological sciences, and neuroscience at the University of Southern California, trained rabbits in what is called eyeblink conditioning, in which the sound of a musical tone is paired with a puff of air to the eye. (Pavlovian Conditioning 101: The puff of air is the “unconditioned” stimulus, because it requires no experimental conditions to produce a behavioral response, the blink. The tone is the “conditioned” stimulus, because only when it is paired with the puff of air will the animal learn to associate the two, producing what then becomes known as the “conditioned response”: a reflexive blink in response to the tone alone that has been produced through the conditions of the experiment.) Rabbits and cats and learn and retain the eyeblink after removal of the hippocampus, neocortex. In a landmark paper published in Science in 1984, Thompson demonstrated that after he trained rabbits and then surgically removed just a few hundred neurons from the interpositus nucleus (a section of the cerebellum, located near the brain’s base), the animals no longer blinked in response to the tone. The meaning of Thompson’s finding was clear: He had found an engram encoding the association between the puff of air, the tone, and the eyeblink, showing for the first time that the destruction of one particular set of neurons could wipe out one particular memory. “The whole point,” Thompson told me, “is that the memory is localized. The eyeblink conditioning is stored in a small number of cells in a particular region of the cerebellum.” When we experience something, say a trip to the park, a memory of the event is stored in a constellation of interconnected neurons in our brains called an "engram," or memory trace. When you recall that trip to the park, neurons in the engram become active. Reactivate those neurons artificially, the theory goes, and you can bring the memory bubbling to the surface of someone's psyche.
- Skipping ahead about 30 years and leaving out a lot of seminal work in the field we come to a paper from 2012 from Tonegawa’s group at MIT. Tonegawa showed that activating, instead of abolishing, a specific set of cells in the hippocampus induced a fear response associated a fear memory. This was the first time that activating a set of cells led to a behavioral change linked to memory, adding to the idea that engrams are specific populations of cells. Then, just early this year, researchers at the same lab took previous research further. Researchers wanted to determine if activating cells associated with one context while delivering a foot shock could result in the formation of a false memory.
- The way that researchers did this was to label the dentate gyrus cells using the Tet-Off system. There are two important portions of the Tet-Off system: tTA (tetracycline trascriptional activator) – this is a combination of Tet-R, a repressor protein that regulates the genes of Dox-resistance, and VP16, this converts the repressor gene to an activator. In this example, CMV is the promotor: the gene that initiates the entire process. Most transcripitonal activators are DNA binding activators, and this is what happens here with the second important piece: TRE: tetracycline response element – a response plasmid that responds to the binding of tTA by increasing expression of the gene of interest. This entire system is based on tetracycline, an antibiotic, but it also works with doxycycline – shown here. In the absence of Dox, this binding takes place, but in the presence of Dox, the binding in inhibited because Dox instead binds to tTA, making it incapable of binding with TRE.
- Researchers used this Tet-Off system, but the promoter in this case was c-fos because c-fos-TTA transgenic mice were used and the gene of interest was ChR2. c-fos is significant because this is early gene used as a marker for neuronal activity. So what researcher did was then used these transgenic mice, so that is where the C-fos-tTA comes from and then they injected the mice with an Adeno-associated virus and an optical fiber. When mice are given Dox (On-Dox): c-fos promoted tTA will bind to the plasmid TRE and this binding will cause expression of the gene of interest, ChR2. Now, where mice are taken off Dox – this binding in inhibited. When off Dox, training induces the expression of tTA, which binds to TRE and drives the expression of ChR2–EYFP, labelling a subpopulation of activated cells (yellow) in the DG. c
- The reason the gene of interest is significant is because ChR2 is photosensitive, so when exposed to light – this protein will turn on and become activated. Enhanced yellow fluorescent protein is a fluorescent marker.
- The only difference between this set-up and the one on the previous slide is the fluorescent marker – EYFP is yellow and mCherry is red. This just makes seeing which cells have been activated easier. Mice have the surgery to implant the AVV and the optical fiber. At this point, mice are ON DOX so that no cells are labeled with ChR2. Then mice are taken off Dox and allowed to explore chamber A. The neuronal activity of remembering chamber A induces the expression of c-fos and then because no Dox is present, the c-fos cells would be labeled with Chr2 now. Then the mice are put back on Dox to prevent any further labeling. Mice are then allowed to explore Chamber B where mice are simultaneously given a footshock and shined with blue light. This blue light activated ChR2 – this links the footshock of Chamber B with the Chr2 labeling in A. Then mice are returned to chamber A or exposed to a novel context C to measure the freezing. Here's the brilliant bit. ChR2 is a light-sensitive protein; shine a light on it with the tip of an optical fiber that's been securely implanted in the brain, and cells that express it become activated. The technique – known as "optogenetics" – is among the most useful to emerge in the field of neuroscience in recent memory, and Tonegawa and his colleagues use it here to great effect. By placing the animal in a second, entirely different environment (Chamber B, the red box) and delivering light to the hippocampus, the researchers could reactivate the engram established in Chamber A, forcing the mouse to recall its experience while situated in the entirely novel environment of Chamber B.
- When mice were exposed to the A’, levels of freezing were significant. This isn’t just due to generalization because mice only expressing mCherry didn’t freeze significantly. Remember, without the ChR2, those cells would be unaffected by the blue light. Also, the levels of freezing in C were insignificant as well.
- Mice were taken off Dox and allowed to explore chamber A. Then put on Dox and allowed to allow chamber C before fear conditioning. Again, this false memory is specific for A because no freezing was seen in C’ Also, the light is necessary to activate the labeled DG cells because no light causes no freezing.
- False recall leads to distortion of the real memory, and you can see that the experimental group froze more than the nonexperimental group when put back in Context B Also, memory recall can be induced for a memory by activating the cells. Wanted to see if this also applied to a false memory, and it does. Recalled the memory by artificially activating the cells.
- Taken off Dox and allowed to explore only one side of the chamber, so any cells involved in learning about the chamber are labeled with ChR2. Put back on Dox to prevent any further labeling and allowed to explore the other side of the chamber. Fear conditioned in a neutral context with the footshock and blue light Put in the middle and allowed to choose where to go
- At this level of brain activity, he said, “the difference between a mouse and a human is quite small.” In both, memories form in an area of the hippocampus called the dentate gyrus.
- Using the same population of cells in the DG to form the false and true memories, and similar cell populations in the amygdala were activated during false and true memory recall.
- Wade hearing is hearing to determine if the identification procedure is impermissible. This stems from United States v Wade where is was determined that not having an attorney present at a line-up is a 6th amendment rights violation. Manson/Madison: the court must determine if (1) if the identification procedure was impermissibly suggestive and (2) if so, whether the procedure resulted in a “very substantial likelihood of irreparable misidentification.” In the second part of the test, the court considers the (1) witness' opportunity to view the person at the time of the crime, (2) witness' degree of attention, (3) accuracy of the witness' prior description, (4) level of certainty at the time of the confrontation, and (5) time between the crime and confrontation. This was drawn into law in 1977, and science has had many breakthroughs since then.
- Manson/Madison: the court must determine if (1) if the identification procedure was impermissibly suggestive and (2) if so, whether the procedure resulted in a “very substantial likelihood of irreparable misidentification.” In the second part of the test, the court considers the (1) witness' opportunity to view the person at the time of the crime, (2) witness' degree of attention, (3) accuracy of the witness' prior description, (4) level of certainty at the time of the confrontation, and (5) time between the crime and confrontation. This was drawn into law in 1977, and science has had many breakthroughs since then. Line-up Construction: does the suspect stand out? How many fillers are used? Is the suspect the only person who shows up twice in a photo lineup and alineup.
- Members of the general public (n = 1500) and experts (professors with over 10 years of memory research experience). Jurors need to know if these system or estimator variables were violated and understand things like just because someone is confident, does not mean that they are right.
- Using neurotechnologies to improve cognition is referred to as neurocognitive enhancement DBS: invasive method that implants brain pacemakers that then send an electrical current to the areas where the implantations are TMS: Noninvasive and uses a coil to produce a magnetic field. This then induces an electrical current in that target area and this current stimulates axons of neurons. electric current is used routinely by neurosurgeons to define areas that control movements and sensations. However, it is much more difficult to stimulate the brain through the intact skull and scalp because of the high electrical resistance. Large electric currents have to be applied in order for a small proportion to penetrate into the brain, leading to painful contractions of scalp muscles and activation of sensory receptors in the skin. TMS is a method of stimulating the brain through the intact scalp without causing pain at the surface. The stimulator produces a magnetic field of the same size as that of an MRI scanner, but that lasts for only about a millisecond. The magnetic field easily penetrates the scalp and skull, and because it changes so rapidly (from zero to a very high value, then back to zero again in 1 ms), it induces electrical currents in the area of the brain beneath the coil (BOX 1). Effectively the magnetic field ‘carries’ the electrical stimulus across the barrier of the skull and scalp into the brain. The induced current pulse lasts for about 200 μs and is similar in amplitude to that produced by a conventional stimulator applied directly to the surface of the brain. It is thought to activate the axons of neurons in the cortex and subcortical white matter, rather than the cell bodies of cortical neurons (which have a much higher threshold) (BOX 1). The induced electrical stimulus activates a mixture of neurons beneath the coil. Some are local to the area of cortex under the coil, others project axons to or from the site of stimulation; some are excitatory, others inhibitory. The final outcome of such stimulation might be complex and quite unlike the organized patterns of activity that occur in natural behaviours. However, some selectivity arises owing to the fact that different neurons have different thresholds to electrical stimulation. Low stimulation intensities therefore activate a much more limited selection of neurons than higher intensities.
- So it turns out that Karl Lashley’s belief in memory as existing in a distributed network is still alive and well; Silva and Josselyn have not overturned it, only supplemented it, showing that some parts of some memories do exist in a discrete number of neurons.
- Exposed post-surgery mice to Context A off Dox so c-fos expressed cells could be labeled with ChR2. Then immediately placed back on Dox to prevent further labeling. Half of the cells were then exposed to context A again or a new context, C. Cell activated by the first context A would be labeled with ChR2, but A’ and C would be labeled with c-fos only because at this point, the mice were Off-Dox. You can see that the degree of overlap for c-fos and ChR2 is much greater for A-A’ than A-C.
- Mice have the surgery to implant the AVV and the optical fiber. At this point, mice are ON DOX so that no cells are labeled with ChR2. Then mice are taken off Dox and allowed to explore chamber A. The neuronal activity of remembering chamber A induces the expression of c-fos and then because no Dox is present, the c-fos cells would be labeled with Chr2 now. Then the mice are put back on Dox to prevent any further labeling. Mice are then allowed to explore Chamber B where mice are simultaneously given a footshock and shined with blue light. This blue light activated ChR2 – this links the footshock of Chamber B with the Chr2 labeling in A. Then mice are returned to chamber A or exposed to a novel context C to measure the freezing.
- In CA1, the cell overlap between the two A contexts was much greater that with the DG cell populations – additional work is necessary, but researchers hypothesized this is because it isn’t necessary a cell population code, but actually a temporal code. Exposed post-surgery mice to Context A off Dox so c-fos expressed cells could be labeled with ChR2. Then immediately placed back on Dox to prevent further labeling. Half of the cells were then exposed to context A again or a new context, C. Cell activated by the first context A would be labeled with ChR2, but A’ and C would be labeled with c-fos only because at this point, the mice were Off-Dox. You can see that the degree of overlap for c-fos and ChR2 is much greater for A-A’ than A-C. f Ramirez's study sounds familiar, don't worry; you don't have an implanted memory of it. A study published last year by Aleena Garner and her colleagues at UC San Diego followed a very similar experimental protocol, but failed to see increased freezing in mice re-exposed to either Chamber A or Chamber B. Instead, the mice are believed to have formed what Garner and her team call a "hybrid" memory, one that could only be retrieved by combining "elements of both the... artificial stimulation and the natural sensory cues from the [fear-conditioning environment.]" If either condition were presented independently, the mice would carry on about their business — as though they had forgotten to be afraid. "A key difference in [Garner's system]," write Ramirez and Xu Liu, first authors on the present paper, is that "cells in the entire forebrain were labeled and reactivated over an extended period by a synthetic ligand." Ramirez and Liu therefore hypothesize that activating neurons across larger areas of the brain and for longer periods of time may favor the formation of a memory "which may not be easily retrievable by the cues associated with each individual memory." In contrast, they argue, activating smaller populations of neurons for shorter periods of time "may favor the formation of two distinct (false and genuine) memories," as observed in the present study.