Artificially Enhancing and Suppressing Hippocampus-Mediated Memories
Authors: Emily Merfeld
Abby Finkelstein
Monika Shpokayte
Anahita B Hamidi
Yosif Zaki
Steve Ramirez
Olivia McKissick
Stephanie L Grella
Nathen J Murawski
Briana K Chen
Amanda Fortin
Christine Cincotta
Emily Doucette
Journal: Current Biology (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: emotion; engram; hippocampus; longitudinal axis; memory
Affiliations: Doctoral Program in Neurobiology and Behavior, Columbia University, New York, NY 10032, USA., Department of Psychological and Brain Sciences, Boston University, Boston, MA 02215, USA., Department of Psychological and Brain Sciences, Boston University, Boston, MA 02215, USA. Electronic address: .

Abstract:  Emerging evidence indicates that distinct hippocampal domains differentially drive cognition and emotion [1, 2]; dorsal regions encode spatial, temporal, and contextual information [3-5], whereas ventral regions regulate stress responses [6], anxiety-related behaviors [7, 8], and emotional states [8-10]. Although previous studies demonstrate that optically manipulating cells in the dorsal hippocampus can drive the behavioral expression of positive and negative memories, it is unknown whether changes in cellular activity in the ventral hippocampus can drive such behaviors [11-14]. Investigating the extent to which distinct hippocampal memories across the longitudinal axis modulate behavior could aid in the understanding of stress-related psychiatric disorders known to affect emotion, memory, and cognition [15]. Here, we asked whether tagging and stimulating cells along the dorsoventral axis of the hippocampus could acutely, chronically, and differentially promote context-specific behaviors. Acute reactivation of both dorsal and ventral hippocampus cells that were previously active during memory formation drove freezing behavior, place avoidance, and place preference. Moreover, chronic stimulation of dorsal or ventral hippocampal fear memories produced a context-specific reduction or enhancement of fear responses, respectively, thus demonstrating bi-directional and context-specific modulation of memories along the longitudinal axis of the hippocampus. Fear memory suppression was associated with a reduction in hippocampal cells active during retrieval, while fear memory enhancement was associated with an increase in basolateral amygdala activity. Together, our data demonstrate that discrete sets of cells throughout the hippocampus provide key nodes sufficient to bi-directionally reprogram both the neural and behavioral expression of memory. 

Retracted: No

PubPeer comments:
 [See  Update section below for author responses.] I can t find where the authors provide sample sizes for the numbers of animals used in their various manipulations. Am I missing it? Update: Yes, I was missing something; the info is in the figure captions, samples ranging from 5-12 animals. My follow-up q is this: Why the variety, and how were sample sizes chosen? The authors say that:  No statistical methods were used to determine sample size; the number of subjects per group were based on those in previously published studies and are reported in figure captions. To which previously published studies the researchers are referring, and on what basis do they consider those studies sample sizes to be valid? If they (or their editors) don t feel that sample sizes should be selected based on some type of statistical test, then why mention this issue at all? If it is important, then the reference to other, unnamed previous publications is rather inadequate.  Exploration of the context while off Dox increased eYFP-expressing (eYFP+) cells in both the dorsal and ventral DG relative to on-Dox controls (Figures 1G, 1H, 1J, and 1K). The following day, mice that explored the same context showed a significant increase in the number of overlapping eYFP+ (i.e., cells labeled by the 1st exposure) and c-Fos+ cells (i.e., cells labeled by the 2nd exposure) in the dorsal but not ventral DG (relative to chance overlap)... How is  chance ascertained here? The brain is highly-condition-sensitive, to both external and internal events, in ways we don t understand. There s really no possible  no context condition; and there was no  explored a different context control. When the mice are returned to their normal cages, they are also returning to familiar territory; when they are being moved from one place to another, these may also be familiar experiences. All of this affects the brain. Given our level of ignorance about the brain and the countless confounds, I don t see what the authors could validly be using as their  chance baseline.  Together, these data demonstrate that the dorsal DG is reactivated following retrieval of both a neutral or aversive context memory, whereas cells in the ventral DG show reactivation only in a shock-paired environment. Sentences like this should raise alarm bells. They are passive (they don t corroborate or falsify predictions made before examining the data) descriptions of correlations in a sample examined post hoc, meaning they can t count as evidence of causal connections. In other words, data analyzed in this way can t be taken to  demonstrate anything. Claims are as speculative post as they were prior to experiment. In addition:  Post hoc analyses (Newman-Keuls) were used to characterize treatment and interaction effects, when statistically significant (alpha set at p 0.05, two-tailed). According to graphpad.com, maker of Prism, one of the software packages employed in this study: [ It is difficult to articulate exactly what null hypotheses the Newman-Keuls test actually tests, so difficult to interpret its results. ] And from the same source:  Although the whole point of multiple comparison post tests is to keep the chance of a Type I error in any comparison to be 5%, in fact the Newman-Keuls test doesn t do this.  Acute stimula tion of a fear memory via either the dorsal or the ventral DG drove freezing behavior and promoted place avoidance (Figures 2I, 2J, 2L, and 2M). Acute stimulation in the female exposure groups promoted place preference but did not affect fear behavior (Figures 2I, 2J, 2L, and 2M). While the hippocampus clearly has a role in enabling memory formation and or retrieval, it cannot possibly contain memorie. Even if we take Chen et al s report at face value, all we would be able to say is that certain cells display a certain type of activity when the mouse has a sharp fear reaction, and also may instigate an acute fear reaction when stimulated. The claim that this activity is causing the mouse to experience mental imagery corresponding to some particular  context - out of the infinite number and variety of contexts a normal mouse may experience in its lifetime - is a bridge too far. There aren t enough cells in the hippocampus to accommodate all possible  contexts. The authors need to be a little more modest in their claims. **Update 12 15** Nearly two weeks ago, senior author Steve Ramirez responded to my comments in an email. I replied that I thought it would be useful to incorporate his responses into this PubPeer thread. He didn t raise any objections, so I m copying those responses below. I ve put Ramirez quotes from my comments in italics, and numbered the points for clarity. I ve put some sections in bold for emphasis. I ve put a few new responses to Ramirez responses in brackets. I ll only add that, having been promised the behavioral datasets, which the article states are available on request, I have yet to receive them more than two weeks later; I m starting to wonder whether they will be available after all. 1. *The authors say that:  No statistical methods were used to determine sample size; the number of subjects per group were based on those in previously published studies and are reported in figure captions. * *To which previously published studies the researchers are referring, and on what basis do they consider those studies sample sizes to be valid? If they (or their editors) don t feel that sample sizes should be selected based on some type of statistical test, then why mention this issue at all? If it is important, then the reference to other, unnamed previous publications is rather inadequate.* We posted the individual N, stats, and so on in our figure legends, and chose our N values for the histology and behavior based on previous engram papers that demonstrated such N provided sufficient statistical power (e.g. Liu et al, Nature, 2012; Denny et al. Neuron, 2014; Tanaka et al. Neuron, 2014). These N values and the corresponding stats were also taken as a standard for circuit level behavioral optogenetic papers (e.g. Tye et al. Nature, 2012; Stuber et al. Nature, 2011) in which we compare across animals for histology and utilize, for instance, a T-test, or across animals and across light on-off-on-off epochs for behavioral data and utilize two-way anovas with repeated measures. [In what way did those previous papers  demonstrate such N... ; on what basis were they  taken as a standard... 2. * Exploration of the context while off Dox increased eYFP-expressing (eYFP+) cells in both the dorsal and ventral DG relative to on-Dox controls (Figures 1G, 1H, 1J, and 1K). The following day, mice that explored the same context showed a significant increase in the number of overlapping eYFP+ (i.e., cells labeled by the 1st exposure) and c-Fos+ cells (i.e., cells labeled by the 2nd exposure) in the dorsal but not ventral DG (relative to chance overlap)... How is  chance ascertained here? The brain is highly-condition-sensitive, to both external and internal events, in ways we don t understand. There s really no possible  no context condition; and there was no  explored a different context control. When the mice are returned to their normal cages, they are also returning to familiar territory; when they are being moved from one place to another, these may also be familiar experiences. All of this affects the brain. Given our level of ignorance about the brain and the countless confounds, I don t see what the authors could validly be using as their  chance baseline.* **I 100% agree that chance is a tricky thing when it comes to the brain, since it s a statistical measure applied to a system as complex as the brain, in which we don t know what true chance would look like. So in that sense, we took the next best approach and utilized statistical chance, i.e. the odds of a set of cells being labeled by one fluorophore (N[number of cells labeled] Total number of cells in the area) multiplied by the odds a set of cells are labeled by the second flourophore (N[number of cells labeled] Total number of cells in the area), and we use the resulting number as statistical chance. We think of it as the brain s way of flipping a coin twice and landing at heads twice, but you re spot on that there s no a priori reason that brain indeed operates in such a quantitative manner.** [ Statistical chance isn t a thing. I guess Ramirez means the binomial test. Arbitrary assumptions about initial conditions that even the authors don t believe in render  significance claims invalid. ] 3. * Together, these data demonstrate that the dorsal DG is reactivated following retrieval of both a neutral or aversive context memory, whereas cells in the ventral DG show reactivation only in a shock-paired environment. Sentences like this should raise alarm bells. They are passive (they don t corroborate or falsify predictions made before examining the data) descriptions of correlations in a sample examined post hoc, meaning they can t count as evidence of causal connections. In other words, data analyzed in this way can t be taken to  demonstrate anything. Claims are as speculative post as they were prior to experiment.* I again totally agree here and thank you for the great point! **I have no idea what causality truly would look like in the brain or what a ground truth looks like when it comes to a principle of the brain**, and we hesitate to use the word  causality for that reason. I believe most of our data our correlative or can be interpreted as a result of a given perturbation, but this by no means has to equate to causal. I do believe that term gets thrown around a lot these days with optogenetic chemogenetic studies, and the reality is that once we perturb an area and networks respond accordingly, perhaps causality can be observed as a brain-wide phenomenon which we re just started to test out thankfully [Causality can be observed? Without knowing or surmising any principles of function? By making arbitrary assumptions about prior states (see point 2)? The brain is never in an  unperturbed state...] 4. *In addition:  Post hoc analyses (Newman-Keuls) were used to characterize treatment and interaction effects, when statistically significant (alpha set at p 0.05, two-tailed). According to graphpad.com, maker of Prism, one of the software packages employed in this study:  It is difficult to articulate exactly what null hypotheses the Newman-Keuls test actually tests, so difficult to interpret its results. And from the same source:  Although the whole point of multiple comparison post tests is to keep the chance of a Type I error in any comparison to be 5%, in fact the Newman-Keuls test doesn t do this.  Acute stimula tion of a fear memory via either the dorsal or the ventral DG drove freezing behavior and promoted place avoidance (Figures 2I, 2J, 2L, and 2M). Acute stimulation in the female exposure groups promoted place preference but did not affect fear behavior (Figures 2I, 2J, 2L, and 2M). While the hippocampus clearly has a role in enabling memory formation and or retrieval, it cannot possibly contain memorie. Even if we take Chen et al s report at face value, all we would be able to say is that certain cells display a certain type of activity when the mouse has a sharp fear reaction, and also may instigate an acute fear reaction when stimulated. The claim that this activity is causing the mouse to experience mental imagery corresponding to some particular  context - out of the infinite number and variety of contexts a normal mouse may experience in its lifetime - is a bridge too far. There aren t enough cells in the hippocampus to accommodate all possible  contexts. The authors need to be a little more modest in their claims.* I couldn t agree more! I personally think that memories are a distributed brain-wide phenomenon in which circuits and networks utilize spatial-temporal codes to process information, as opposed to having a memory localized to a single X-Y-Z coordinate point. Even within the hippocampus with over 1M cells, the permutations possible of a defined set of cells utilizing a temporal code to process contexts is an astronomically, perhaps wonderfully, big number of experiences that it can be involved in -- not to say that therefore memories are located in the hippocampus because it technically can process them, but that it s contribution to enabling numerous memories I don t believe has an upper limit that we know of, but this is total speculation on my end! However, I do actually believe that our set of experiments beginning with Liu et al. 2012 up to 2019 really hint that we can partly predict what the animal s internal  experience (used very loosely here) is actually like (see Joselyn et al. 2015 Nature Neuroscience) for a fantastic review. In short, we  tag cells active during a defined period of time, say, exposure to context A, and we ve done numerous experiments that suggest these cells are specific to that environment with minimal  noise , i.e. without other contexts that the animal experiences spilling over, given the time period of our tagging system. And when we manipulate these sets of cells, the animals show fear responses specific to that context, i.e. Figure 3 of Chen et al., that suggests that at least some aspects of context A are coming back  online, which dovetails which previous data from a 2013 false memory paper we had as well. That said, I m fully on board that we re not at the stage where we can say with certainty what the animal s mental imagery looks like, though a handful of papers from the Deisseroth lab recently have hinted that we can really force a mental  image to come back online and force the animal to behave as those it s experiencing that image. In our hands, we believe that stimulating these cells in the hippocampus has a sort of domino effect in which downstream circuits become activated and this ultimately leads to memory recall, and that the hippocampus is a key node involved in bringing the brain-wide networks involved in memory back online. So it s not that the memory is located in the hippocampus, it s more that the hippocampus contains a set of cells which, when activated, are sufficient to activate memory recall by engaging the rest of the systems in the brain involved in that discrete experience too. I hope this helps and thank you again for the fantastic back and forth! [The issue of the Newman-Keuls test needs to be addressed before anything else here.] , 
 The paper says data are available on request to senior author but, despite an initial promise to do so, the (behavioral) data has not been made available. , 
 #2 I wonder whether Lydia has ever analyzed data in her life. ,