Ramirez Papers - Joe

Ramirez papers 2013 and 2015

    The most compelling part of this paper, if not the interpretation of the data, was the novel genetic/behavioral approach that Ramirez et al. took to addressing their question: how can a subset of neurons that are active during a learning task be labelled and selectively activated? In other words, how can a memory be stored so that it’s accessible externally? To address this, they utilized the cfos-tTA transgenic mouse to be able to express a desired gene under the control of doxycycline. When this mouse is off dox (doxycycline is not in their diet), and a neuron is activated (the IEG, cfos is expressed), the tetracycline response element (TRE) is induced and the gene of interest is expressed (in this case ChR2). This provides temporal and spatial sensitivity to express ChR2 in only the neurons that are active during the off-dox period that have been infected with the AAV-ChR2. This population of cells that expresses ChR2 in the dentate gyrus of the hippocampus is considered the engram population; it is said that this population of cells holds the memory of what was learned during the off-dox period.

    Then, by tagging neurons while learning a context (cfos expression while off dox), the memory trace of the then-neutral context is presumably “saved” by expression of ChR2 — presumably because it cannot be clear whether synchronous optogenetic activation of this entire population should mimic the endogenously emanating retrieval of the memory. That is an apparent confound of this approach, and of in vivo optogenetics in general: does optogenetic stimulation of an ensemble of neurons simulate the activity that those neurons exhibit when stimulated endogenously?

    Whether that question is yes or no, they very interestingly found that optogenetically stimulating this ensemble while fear conditioning a mouse in a completely different context that they were naive to was enough to elicit a CS-US link between the first context (the labelled ensemble) and the fear. This fear was also context-dependent — that is, freezing behavior was not observed in a third context, to which they were still naive. That stood as a control experiment to validate their experimental group’s data. This is an important point to bring up; this genetic, behavioral, time-dependent, spatially-dependent approach demands many controls to account for all the potential misinterpretations that is inevitable with this approach. This becomes more evident in Ramirez et al 2015 when specific circuits are probed using the same tet-tag technique. However, a great control that could have very easily been overlooked was when the behavioral paradigm was flipped so that context A was replaced with context C, to show that there was nothing inherent to context A that was causing this change in behavior.

    Lastly, the entire experiment was repeated with conditioned place avoidance in place of contextual fear conditioning. This was an interesting way to show that this artificially induced memory association could be applied to other behavioral paradigms.

    In Ramirez et al 2015, a more clinically translational question was addressed: can recollection of a positive memory lead to amelioration of depressive symptoms? This question was tackled using the same general experimental design as the aforementioned one: tag neurons off dox during a pleasant experience and then express ChR2 in those neurons to later be able to activate that ensemble. Interestingly, the pleasant experience of choice was a male encounter with a female. Firstly, this implies that the entire experiment was carried out using male mice. And secondly, that seems like an arbitrary classification of a pleasant encounter; it was not clear what the basis of their decision was.

    Despite this, the retrieval tests used were measures of both anxiety- and depression-like symptoms, because the distinction can be variable. Furthermore, the control groups utilized were extensive, and tackled a lot of potential misinterpretations of the data. The 6 group all underwent the tail suspension test (TST) and sucrose preference test (SPT) as measures of anhedonia, open field test (OFT) and elevated plus maze test (EPMT) as measures of anxiety, and novelty-suppressed feeding (NSF) as a measure of motivation. In mice that had undergone stress, activation of the pleasant memory engram was enough to rescue the depressive symptoms exhibited in the TST and the SPT. This effect was acute, which was interesting, because it indicates that perhaps perseverant activation of a positive memory trace is necessary to create a strong positive motivational state following a traumatic experience (this was addressed later). There was no change in behavior in the anxiety-related tests, which is an indication of what sorts of pathological states a positive memory might be able to amend.     All of these mice underwent four retrieval tests after the chronic stress they received, and so it is also possible that some learning went on during that time, especially if the engram was activated during each test. I don’t think the order of the retrieval tests was varied, to account for this possibility.

    The most interesting part of this series of experiments was probing of the potentially important circuits that culminate in the manifestation of this behavior rescue. It was made clear that the behavioral rescue due to activation of the memory trace in the DG can be blocked by pharmacologically inhibiting NAc neurotransmission. And moreover, this effect can be blocked by optogenetically inhibiting the axon terminals of BLA projections to the NAc. This is important because it says that there is something important about the connectivity of the BLA and the NAc to exhibit a positive behavioral state following retrieval of a positive memory. It was never shown that DG->BLA connectivity directly plays a role in this behavior, but previous literature has shown that these three regions are crucial for limbic function.

    Lastly, they wanted to show that this acute effect could become sustained if the memory engram were activated multiple times after stress. This is a great example of the Hebbian synapse concept — that multiple innervations of one neuron by another increases the strength of that synapse, which is the basis of neural plasticity. A great follow up to this experiment would be to see if this sustained rescue of behavior would not diminish over days (or even weeks) after retrieval.

    This novel technique is a very clever way to approach questions of the memory engram; however, it is not clear whether activation of a population of neurons simultaneously mimics endogenous retrieval of a memory. Thus, it is hard to say what neurons these actually encode.