Erasing Memories Cell by Cell

3d glass brain
by Kazuhiko Nakamura

We've discussed recent findings about erasing fears from memories, but today we'll be talking about erasing the fear memory itself. This involves actually inhibiting or killing the individual neurons that encode for a particular memory, so for obvious reasons these experiments are done on mice rather than humans. 

Mice can be trained to associate a mild electrical foot shock with a tone.  The tone plays and then a foot shock is given.  Once the mouse has learned this association, it will freeze in place when the tone is played.  This is called an auditory fear memory. 
Using a fear memory paradigm, Sheena Josselyn in her Toronto lab discovered how to visualize the neurons which are active during fear memory formation. They also developed a way to target and delete them, consequently deleting the memory. 

In Han et al. (2009), some beautiful genetic trickery was used to promote a 'kill switch' only in the neurons which are active during the memory formation.  This kill switch is the diphtheria toxin receptor.  Normally cells do not have this receptor, but when they promote this receptor artificially on the cell surface, an injection of diphtheria toxin will kill that cell, but not neighboring (dtr-free) cells.  The real impressive genetics is in promoting the diphtheria toxin receptor only in neurons active during memory formation.  To do this, the Josselyn lab used a marker for cell activity in amygdala neurons during memory formation, CREB.  Specifically, they used a transgenic mouse that expressed the diphtheria toxin receptor only when CREB activates cre.

So now with the memory encoded and the kill switch in place, they pull the trigger and inject diphtheria toxin into the mice. This kills all the amygdala cells that were active during memory formation (about 250 amygdala cells or so, Han et al., 2009 figure 1B).  They then test the mice again for freezing behavior.

Han et al., 2009 Figure 3

The second set of columns (CREB-cre, DT) is the experiment I have described.  Before any drug is injected the mice freeze in response to the tone, but after the diphtheria toxin (DT) injection, the mice freeze much less in response to the tone. What is really essential to this study is the control experiments that they ran. 

They wanted to make sure that just killing any 250 neurons in the amygdala didn't causes memory loss.  So instead of using the CREB promoter to activate cre (and thus the diphtheria toxin receptor) they used a control promoter (cntrl-cre, DT above) to promote cre in about the same number of neurons, but not dependent on neural activity.  In this case, there is no statistical difference in how much the mouse freezes in response to the tone. (compare the first two columns to each other.) 

Similarly, they wanted to make sure that the diphtheria toxin (DT) itself didn't erase the memories. They injected CREB that did not promote cre, and thus did not cause any diphtheria receptors to be expressed (CREB, DT). In this case, there was again no difference between pre and post DT injection.  Finally, they wanted to make sure it wasn't the CREB-cre construct itself, so they added the CREB-cre like normal, but did not inject the diphtheria toxin, so the receptors were expressed on these cells, but were not activated. In this case again, not difference in the amount of freezing. 

Because none of these control groups showed a difference in freezing, Josselyn could be confident that she had really shown that the specific neurons that encoded the memory were necessary for recalling the memory. 

They are also clear that the amygdala is not seriously damaged in this study, as the mice can re-learn the task after the specific neurons have been deleted.

One particularly interesting aspect of this study, which the authors do not discuss, is the number of neurons necessary for encoding a memory.  They delete hundreds of neurons.  I wonder if deleting half of them or even a quarter would result in the same erasure of the memory. How many neurons does it take to encode a memory?

Recently this concept of targeting proteins to only the active cells has been extended to include channel rhodopsin, the protein which allows cells to be activated by light.  Liu et al., (2012) was able to reactivate the neurons that were specifically active during the learning of a fear response. Stimulating these neurons caused the mouse to freeze, suggesting that stimulating these neurons reactivates the memory. This paper is covered thoroughly by Mo Costandi at Neurophilosophy.


© TheCellularScale

Han JH, Kushner SA, Yiu AP, Hsiang HL, Buch T, Waisman A, Bontempi B, Neve RL, Frankland PW, & Josselyn SA (2009). Selective erasure of a fear memory. Science (New York, N.Y.), 323 (5920), 1492-6 PMID: 19286560