Hippocampal place cell remapping occurs with memory storage of aversive experiences

  1. Garrett J Blair
  2. Changliang Guo
  3. Shiyun Wang
  4. Michael S Fanselow
  5. Peyman Golshani
  6. Daniel Aharoni
  7. Hugh T Blair

  • Curated by eLife

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    Evaluation Summary:

    This paper describes results obtained from multi-cellular imaging of CA1 cells using large-field-of-view miniscopes in rats performing a shock avoidance task. By exploiting behavioral (barriers) and pharmacological (scopolamine) manipulations the authors explore cell remapping dynamics during aversive learning. This work will be of interest to the neuroscience community by setting new methodological standards and providing data for across-species comparisons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer 3 agreed to share their name with the authors.)

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Abstract

Aversive stimuli can cause hippocampal place cells to remap their firing fields, but it is not known whether remapping plays a role in storing memories of aversive experiences. Here, we addressed this question by performing in vivo calcium imaging of CA1 place cells in freely behaving rats (n = 14). Rats were first trained to prefer a short path over a long path for obtaining food reward, then trained to avoid the short path by delivering a mild footshock. Remapping was assessed by comparing place cell population vector similarity before acquisition versus after extinction of avoidance. Some rats received shock after systemic injections of the amnestic drug scopolamine at a dose (1 mg/kg) that impaired avoidance learning but spared spatial tuning and shock-evoked responses of CA1 neurons. Place cells remapped significantly more following remembered than forgotten shocks (drug-free versus scopolamine conditions); shock-induced remapping did not cause place fields to migrate toward or away from the shocked location and was similarly prevalent in cells that were responsive versus non-responsive to shocks. When rats were exposed to a neutral barrier rather than aversive shock, place cells remapped significantly less in response to the barrier. We conclude that place cell remapping occurs in response to events that are remembered rather than merely perceived and forgotten, suggesting that reorganization of hippocampal population codes may play a role in storing memories for aversive events.

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  1. Version published to 10.7554/elife.80661 on eLife
  2. Version published to 10.1101/2022.05.26.493592v4 on bioRxiv
  3. eLife

    Author Response

    Reviewer #2 (Public Review):

    This manuscript reports an experiment involving learning and imaging of neural activity in rats. The goal was to test if scopolamine, which is an antagonist of acetylcholine receptors, could cause memory loss (amnesia). Two types of learning were tested: first, rats learned to prefer a short path, compared to a detour path, between two rewarded locations in a linear maze; second, in a subset of the experimental sessions, a shock zone was activated in the middle of the short path, and rats had to learn to avoid it. As a control, some sessions had a clear plexiglass barrier placed in the middle of the short path, which should not have aversive properties. The order of sessions was different for different groups of rats, but shock learning was always followed by a number of 'extinction' sessions without shock. In some groups, shock learning was accompanied by a systemic (intraperitoneal) scopolamine injection, 30 min before the start of the session. This manipulation was performed on most rats once but at a different slot in the sequence of sessions (sometimes before the drug-free shock learning, sometimes after it, sometimes in the absence of preceding barrier sessions, sometimes after them). In what follows, I might use the terms 'control group' and 'test group' to refer to sessions without scopolamine and sessions with it, respectively.

    The main behavioural results are that rats increase their visits to the short path with learning, then visit less the short path once the shock zone is active or when the barrier is there. When re-tested in later sessions, rats trained in the absence of the scopolamine injection still avoid the short path, while most of the rats that were given the scopolamine injection do not avoid it, suggesting a deficit in encoding or recall of the shock zone memory.

    In addition to these behavioural manipulations, the authors image the activity of dorsal CA1 hippocampal neurons using calcium imaging. They detect the existence of place cells, which increase their firing on specific portions of the short path of the maze (the long path data is not analysed). When comparing data before the shock training to during shock training, control place cells were more stable (i.e. had increased between-session correlations) and had more recurrent place fields (i.e. spatially active in one session then still active in another session) with respect to test data (rats injected with scopolamine). When comparing the pre-shock session and the first extinction session, place cell activity was less similar in the control rats (no scopolamine) compared to the scopolamine rats; but note that for scopolamine rats, extinction occurred earlier, so instead of using the first extinction session, the 4th session post-shock training was used to match the control data. Place cell activity has been shown to allow decoding of the animal's position; here, position decoding accuracy was lower around the shock zone in the control group compared to the scopolamine group.

    From these analyses, the manuscript proposes the following findings: 1) scopolamine injections impair avoidance learning, and 2) scopolamine affects the long-term response of place cells to the aversive experience (less "remapping"). These findings are interpreted to support the idea that 3) place cell remapping is involved in avoidance/aversive learning and 4) that scopolamine, as an antagonist of the muscarinic acetylcholine receptors in the hippocampus (or elsewhere?), produces amnesia.

    These findings, if properly supported, would be very interesting to a wide range of researchers interested in the neural bases of memory and learning, specifically aversive memory and spatial learning. The manuscript is well-written, has the advantage of using both male and female rats (which have consistent results), is one of the rare studies to date to perform calcium imaging of the hippocampus in rats, and records along learning of two simple tasks which seems relatively ecological and produce robust learning effects, and uses an experimental manipulation (injection of scopolamine) instead of purely correlational measures. The authors make some analytical effort in equalizing the number of trials across different sessions (even though I do not believe this fixes the existing confounds). I particularly appreciated the nice '3D' trajectory plots that show the unfolding of behaviour along a given session and that each individual's data are generally shown in the figures.

    However, the experiment and its analysis seem to have some major flaws both in the experimental design (which may be difficult to fix) as well as in the analysis (which might be easier to fix), which prevent proper interpretation of the results. Specifically:

    • To demonstrate finding 1 (that scopolamine specifically impairs avoidance learning), a control would be needed to show that the injection procedures do not impair general behaviour (e.g. motivation, attention, level of stress) as well as other forms of learning. Indeed, the control rats - as far as I understood - are not injected with saline, which would have been an appropriate control. One example of non-specific confounding effects of scopolamine is that it could, for example, reduce sensitivity to pain, thus to some extent decreasing the relevance of the shock to the rats, but many other interpretations are possible; most revolve around the idea that instead of scopolamine impairing learning, scopolamine might impair behaviour, which might, in turn, impair learning. Indeed, the scopolamine injection is shown to decrease running speed and the number of trials run, even before exposure to the shock. Related to this, the "short path preference" does not seem to be quantified properly: instead of simply using the number of visits to the short path, a better measure would be to compute a relative preference index quantifying visits to the short path with respect to visits to the long path [e.g. (num short - num long) / num total], to focus on the preference regardless of global changes in behavioural activity levels. In summary: the proposition that scopolamine specifically impairs avoidance learning has not been convincingly demonstrated; the possibility that it even impairs any form of learning is not currently demonstrated either.

    We have run an additional saline-only control group (along with additional scopolamine groups) to demonstrate that scopolamine does impair avoidance learning. As shown in the Supplement to Fig. 1, rats receiving saline alone show significantly greater post-training avoidance of the short path than mice receiving scopolamine.

    • For similar reasons, finding 2 (that the place cell response to aversive learning is affected by the scopolamine injection) is subject to the same lack of controls and existence of possible confounds noted above. Specifically, running more slowly and running fewer laps would have affected the overall amount of excitation of place cells during the session, which might affect plasticity, as well as the amount of reactivations/replay at the reward sites, which is likely to have effects in terms of memory consolidation. One way to potentially control for this would be to have the control group run the same amount of trials as the test group (but then session durations would be different); it is unclear how to prevent the difference in running speed. To be able to claim that the effects of scopolamine are specific to aversive learning, a control with either no learning (perhaps the long path data would be useful for this?) or appetitive learning (e.g. of a reward location, which also involves place field reorganization in some cases) would be useful.

    We recognize and understand the referee’s valid concerns about these potential confounds. The revised paper makes more clear that the scopolamine condition is designed as a control for whether an aversive stimulus is remembered or forgotten, and the barrier condition is a control for whether a novel path-blocking stimulus is aversive or neutral. As the referee points out, there are other confounding variables that may covary with these manipulated factors. The revised discussion (ll. 544-559) acknowledges potential confounds arising from learning-induced behavior changes, and argues that even though it is not possible to perfectly control for such changes, our current study does so more effectively than most prior studies because the rat’s behavior during isolated beeline trials is highly stereotyped and thus more similar across experimental conditions than in any prior study that we know of. The revised discussion also acknowledges the difficulty of dissociating acquisition versus extinction effects upon remapping (ll. 633-644). The significance of the barrier manipulation is now acknowledged more clearly in the abstract, introduction, and discussion.

    • Statement 3 implies a causal link between the two first statements, suggesting that place cell remapping would be necessary for the memory of an aversive experience or aversive location. Given the weakness of the arguments supporting the first 2 statements, this is also non convincingly demonstrated. In any case, the current paradigm would not be sufficient to make a causal link, but it might be sufficient to show a correlational link by showing a correlation between the amount of remapping and memory performance, such as presented in supplementary figure 5 (which would still be informative even if results from the scopolamine sessions were removed?).

    We agree with the referee’s point that our study’s evidence is correlational in nature. The revised manuscript prominently acknowledges this in the concluding sentence of the discussion’s opening paragraph (ll. 500-503), which now reads: “While these results do not definitively prove that place cell remapping is causally necessary for storing memories of aversive encounters, they provide correlational evidence that remapping occurs selectively under conditions where a motivationally significant (rather than neutral) stimulus occurs and is subsequently remembered rather than forgotten.”

    • Statement 4 - that scopolamine causes amnesia - is both not fully defined (what form of amnesia?) and not supported by the findings for the reasons mentioned above.

    It is unclear what remedy the referee is recommending for this concern. We do not present the idea that scopolamine is an amnesic drug as a novel conclusion of our study; rather, this is a widely accepted view in the literature that motivated our experimental design decision to use scopolamine as a tool for dissociating whether an aversive event was remembered or forgotten. We recognize that scopolamine’s effects on memory may vary with experimental conditions. In the revised discussion, we extensively compare and contrast our experimental findings with acute and chronic results from numerous prior studies using scopolamine and other cholinergic drugs (ll. 561-678). We hope this is sufficient to address the referees concerns.

    • In addition to these concerns, a study cited here (Sun et al 2021) mentions a few references in the discussion regarding how muscarinic receptor agonists might affect the link between spikes and calcium signals. Scopolamine is a muscarinic receptor antagonist and might thus have related/reversed effects. Thus, the technique used (calcium imaging) does not seem the best to address questions related to scopolamine. The current manuscript also mentions some findings that were not replicated (e.g. lack of over-representation of the shock zone) which are probably due to the fact that the finding relied on extra-field isolated spikes, which are less likely to be detected via calcium imaging.

    This is an excellent point which is now addressed in the revised discussion; it is acknowledged (ll. 536-539) that mAChRs can regulate calcium signals and thus that scopolamine may have different effects on spikes detected from calcium imaging versus electrophysiology, which in turn could account for discrepancies between our finding that place fields do not migrate to aversively reinforced locations and Milad et al.’s (2019) findings that they do. The Sun et al. (2021) paper used calcium imaging methods similar to ours, so this factor is less likely to account for the discrepancy between their prior finding that place fields were acutely disrupted by scopolamine and our current finding that they were not.

    • If anything, perhaps targeted injections in a specific brain region (e.g. dorsal CA1), instead of systemic injection, might give a more precise picture of the effects of scopolamine on place cells and spatial memory, but I do not know if this is technically possible.

    Unfortunately the necessary placement of the GRIN lens above the recording location prevented the direct application of scopolamine there via cannulae. To date only one series of experiments has demonstrated single unit place cell recordings with direct microdialysis (Brazhnik et al. 2003, 2004). To study the effects of aversive learning across many days, we needed to utilize a recording method capable of tracking many cells across long time periods. However, systemic scopolamine has been widely used to study both learning (Anagnostaras et al. 1999; Huang et al. 2011; Svoboda et al. 2017) and its effects on place cells (Douchamps et al. 2013; Newman et al. 2017; Sun et al. 2021), thus by utilizing this method we can directly compare our findings with previous work. We have added a paragraph to the discussion (ll. 665-678) in which it is explained why the main conclusions of our study (namely, that place cell cell remapping is related to storage of memories for aversive events) do not depend upon whether or not scopolamine’s pharmacological actions were localized to the hippocampus (almost certainly they were not).

    My conclusion would be that the experiment either needs to be redesigned to address the original question (effect of scopolamine on place cell firing and aversive learning) or that some of the data could be still used to address different questions which have not been addressed with calcium imaging before, e.g. learning of the short path, activity on the short path vs long path, effects on behaviour and place cell activity of learning & extinction of the barrier and shock zone avoidance; perhaps without focusing on the scopolamine manipulations, which seem to introduce many confounds.

  4. Version published to 10.1101/2022.05.26.493592v3 on bioRxiv
  5. eLife

    Evaluation Summary:

    This paper describes results obtained from multi-cellular imaging of CA1 cells using large-field-of-view miniscopes in rats performing a shock avoidance task. By exploiting behavioral (barriers) and pharmacological (scopolamine) manipulations the authors explore cell remapping dynamics during aversive learning. This work will be of interest to the neuroscience community by setting new methodological standards and providing data for across-species comparisons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer 3 agreed to share their name with the authors.)

  6. eLife

    Reviewer #1 (Public Review):

    In this paper, Blair et al., analyze hippocampal place cell remapping in rats performing a shock avoidance task using miniscopes to image large-field-of-views. They use behavioral (barriers) and pharmacological (scopolamine) manipulations to interfere with place cell representations during the formation and extinction of aversive memories. By exploiting multi-cellular imaging they examine cell remapping dynamics during learning and extinction induced by the different manipulations and evaluate how they relate to behavioral readouts. The work is carefully planned and analyses successfully control for many potential confounds (but see below).

    Major strengths of the paper include a) using large-field-of-view miniscope imaging to provide place cell data from rats thus favoring inter-species comparisons (most miniscope data is emerging from mouse and rats, especially better in memory tasks); b) an appropriate set of control analysis and experiments to exclude for potential confounds especially when it comes to comparison between groups (speed, number of trials, performance, within- and between-trials differences). Major weaknesses are a) the systemic effect of pharmacological manipulations and specificity regarding memory function; b) the lack of appropriate shuffled contrasted effects. Other inevitable methodological aspects (such as the effect of large GRIN lenses on the integrity of the dorsal, and ubiquitous expression of GCaMP) also require further consideration.

    The paper may be of interest to the neuroscience community by setting new methodological standards and providing new data for across-species comparisons.

  7. eLife

    Reviewer #2 (Public Review):

    This manuscript reports an experiment involving learning and imaging of neural activity in rats. The goal was to test if scopolamine, which is an antagonist of acetylcholine receptors, could cause memory loss (amnesia). Two types of learning were tested: first, rats learned to prefer a short path, compared to a detour path, between two rewarded locations in a linear maze; second, in a subset of the experimental sessions, a shock zone was activated in the middle of the short path, and rats had to learn to avoid it. As a control, some sessions had a clear plexiglass barrier placed in the middle of the short path, which should not have aversive properties. The order of sessions was different for different groups of rats, but shock learning was always followed by a number of 'extinction' sessions without shock. In some groups, shock learning was accompanied by a systemic (intraperitoneal) scopolamine injection, 30 min before the start of the session. This manipulation was performed on most rats once but at a different slot in the sequence of sessions (sometimes before the drug-free shock learning, sometimes after it, sometimes in the absence of preceding barrier sessions, sometimes after them). In what follows, I might use the terms 'control group' and 'test group' to refer to sessions without scopolamine and sessions with it, respectively.

    The main behavioural results are that rats increase their visits to the short path with learning, then visit less the short path once the shock zone is active or when the barrier is there. When re-tested in later sessions, rats trained in the absence of the scopolamine injection still avoid the short path, while most of the rats that were given the scopolamine injection do not avoid it, suggesting a deficit in encoding or recall of the shock zone memory.

    In addition to these behavioural manipulations, the authors image the activity of dorsal CA1 hippocampal neurons using calcium imaging. They detect the existence of place cells, which increase their firing on specific portions of the short path of the maze (the long path data is not analysed). When comparing data before the shock training to during shock training, control place cells were more stable (i.e. had increased between-session correlations) and had more recurrent place fields (i.e. spatially active in one session then still active in another session) with respect to test data (rats injected with scopolamine). When comparing the pre-shock session and the first extinction session, place cell activity was less similar in the control rats (no scopolamine) compared to the scopolamine rats; but note that for scopolamine rats, extinction occurred earlier, so instead of using the first extinction session, the 4th session post-shock training was used to match the control data. Place cell activity has been shown to allow decoding of the animal's position; here, position decoding accuracy was lower around the shock zone in the control group compared to the scopolamine group.

    From these analyses, the manuscript proposes the following findings: 1) scopolamine injections impair avoidance learning, and 2) scopolamine affects the long-term response of place cells to the aversive experience (less "remapping"). These findings are interpreted to support the idea that 3) place cell remapping is involved in avoidance/aversive learning and 4) that scopolamine, as an antagonist of the muscarinic acetylcholine receptors in the hippocampus (or elsewhere?), produces amnesia.

    These findings, if properly supported, would be very interesting to a wide range of researchers interested in the neural bases of memory and learning, specifically aversive memory and spatial learning. The manuscript is well-written, has the advantage of using both male and female rats (which have consistent results), is one of the rare studies to date to perform calcium imaging of the hippocampus in rats, and records along learning of two simple tasks which seems relatively ecological and produce robust learning effects, and uses an experimental manipulation (injection of scopolamine) instead of purely correlational measures. The authors make some analytical effort in equalizing the number of trials across different sessions (even though I do not believe this fixes the existing confounds). I particularly appreciated the nice '3D' trajectory plots that show the unfolding of behaviour along a given session and that each individual's data are generally shown in the figures.

    However, the experiment and its analysis seem to have some major flaws both in the experimental design (which may be difficult to fix) as well as in the analysis (which might be easier to fix), which prevent proper interpretation of the results. Specifically:
    - To demonstrate finding 1 (that scopolamine specifically impairs avoidance learning), a control would be needed to show that the injection procedures do not impair general behaviour (e.g. motivation, attention, level of stress) as well as other forms of learning. Indeed, the control rats - as far as I understood - are not injected with saline, which would have been an appropriate control. One example of non-specific confounding effects of scopolamine is that it could, for example, reduce sensitivity to pain, thus to some extent decreasing the relevance of the shock to the rats, but many other interpretations are possible; most revolve around the idea that instead of scopolamine impairing learning, scopolamine might impair behaviour, which might, in turn, impair learning. Indeed, the scopolamine injection is shown to decrease running speed and the number of trials run, even before exposure to the shock. Related to this, the "short path preference" does not seem to be quantified properly: instead of simply using the number of visits to the short path, a better measure would be to compute a relative preference index quantifying visits to the short path with respect to visits to the long path [e.g. (num short - num long) / num total], to focus on the preference regardless of global changes in behavioural activity levels. In summary: the proposition that scopolamine specifically impairs avoidance learning has not been convincingly demonstrated; the possibility that it even impairs any form of learning is not currently demonstrated either.
    - For similar reasons, finding 2 (that the place cell response to aversive learning is affected by the scopolamine injection) is subject to the same lack of controls and existence of possible confounds noted above. Specifically, running more slowly and running fewer laps would have affected the overall amount of excitation of place cells during the session, which might affect plasticity, as well as the amount of reactivations/replay at the reward sites, which is likely to have effects in terms of memory consolidation. One way to potentially control for this would be to have the control group run the same amount of trials as the test group (but then session durations would be different); it is unclear how to prevent the difference in running speed. To be able to claim that the effects of scopolamine are specific to aversive learning, a control with either no learning (perhaps the long path data would be useful for this?) or appetitive learning (e.g. of a reward location, which also involves place field reorganization in some cases) would be useful.
    - Statement 3 implies a causal link between the two first statements, suggesting that place cell remapping would be necessary for the memory of an aversive experience or aversive location. Given the weakness of the arguments supporting the first 2 statements, this is also non convincingly demonstrated. In any case, the current paradigm would not be sufficient to make a causal link, but it might be sufficient to show a correlational link by showing a correlation between the amount of remapping and memory performance, such as presented in supplementary figure 5 (which would still be informative even if results from the scopolamine sessions were removed?).
    - Statement 4 - that scopolamine causes amnesia - is both not fully defined (what form of amnesia?) and not supported by the findings for the reasons mentioned above.
    - In addition to these concerns, a study cited here (Sun et al 2021) mentions a few references in the discussion regarding how muscarinic receptor agonists might affect the link between spikes and calcium signals. Scopolamine is a muscarinic receptor antagonist and might thus have related/reversed effects. Thus, the technique used (calcium imaging) does not seem the best to address questions related to scopolamine. The current manuscript also mentions some findings that were not replicated (e.g. lack of over-representation of the shock zone) which are probably due to the fact that the finding relied on extra-field isolated spikes, which are less likely to be detected via calcium imaging.
    - If anything, perhaps targeted injections in a specific brain region (e.g. dorsal CA1), instead of systemic injection, might give a more precise picture of the effects of scopolamine on place cells and spatial memory, but I do not know if this is technically possible.
    My conclusion would be that the experiment either needs to be redesigned to address the original question (effect of scopolamine on place cell firing and aversive learning) or that some of the data could be still used to address different questions which have not been addressed with calcium imaging before, e.g. learning of the short path, activity on the short path vs long path, effects on behaviour and place cell activity of learning & extinction of the barrier and shock zone avoidance; perhaps without focusing on the scopolamine manipulations, which seem to introduce many confounds.

  8. eLife

    Reviewer #3 (Public Review):

    Understanding how neural representations throughout the brain, including the hippocampus, interact with neuromodulators such as acetylcholine to support flexible and lasting episodic memories is a fundamental question of interest to a broad neuroscientific community. Here, Blair et al. build on existing literature to concurrently characterize the relationships among these elements. Using large-population widefield miniscope recordings combined with systemic scopolamine administration in rats, the authors first demonstrate that localized aversive experiences result in lasting avoidance behavior as well as changes to (a.k.a. 'partial remapping of') the hippocampal neural code, with lasting changes occurring predominantly near the aversive experience, all replicating prior work with high precision. Next, the authors show that systemic administration of the acetylcholine antagonist scopolamine during the aversive experience gives rise to a different but reliable hippocampal code during that experience. Moreover, rats on scopolamine did not exhibit lasting avoidance behavior or changes to their hippocampal codes from before or after the experience, suggesting that the instantiation of a different hippocampal code during the aversive experience shielded the existing representation and its associated behavior from experience-induced changes. Together, these results demonstrate novel, provocative links between episodic memory, the plasticity of hippocampal neural codes, and the neuromodulator acetylcholine, with a number of important implications for how this memory system functions.

    In my eyes, this work has a number of strengths. One major strength is the power and precision afforded by the use of the large-field miniscope recordings. While this may leave questions of fine temporal structure unaddressable, many of the questions of interest here are best addressed with large populations of simultaneously-recorded neurons that can be confidently tracked across at least a week, all of which are strengths of this technique. Another strength of this work is the replicate and extend approach to addressing the relationships among this work's components. The links to prior work in all of these cases are well noted, the replications of prior results are often with significantly more statistical power than the original result had, and these replications raise confidence in the quality of the data and the novel results reported here.

    As with all work, this too has its limitations. One fundamental limitation is the inability to speak to functional localization. That is, although this work points to provocative correlational links among acetylcholine, the plasticity of hippocampal codes, and behavioral memory expression (all of which are well-motivated by existing literature) because the administration of scopolamine is systemic and only one region can be monitored it is impossible to draw causal conclusions from this work. While it is tempting to infer that manipulating acetylcholine modulation of hippocampal plasticity is necessary and sufficient to produce these results, it is also possible that the behavioral impact of the acetylcholine manipulation is driven by regions outside of the hippocampus and that changes to the hippocampal plasticity are not behaviorally relevant, or that these changes are necessary but not sufficient to drive memory expression. A specific version of this limitation is referenced by the authors in the discussion when considering the possible impact of the manipulation on amygdala responses.

    Despite its limitations, this work meaningfully complements and extends existing literature probing the links between episodic memory, the plasticity and stability of hippocampal codes, and neuromodulators such as acetylcholine.

  9. Version published to 10.1101/2022.05.26.493592v1 on bioRxiv
  10. Version published to 10.1101/2022.05.26.493592v2 on bioRxiv