Theta-phase-specific modulation of dentate gyrus memory neurons
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eLife assessment
This study represents an important step toward unifying two strains of inquiry, one related to the functional role of hippocampal theta oscillations and one related to the behavioral impact of engram reactivation, and thus the findings have implications for our understanding of memory that will impact multiple subfields. In combination with additional context from the literature, the important findings are supported by solid evidence supporting the conclusion that memory recall operations occur preferentially at a specific phase of theta.
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Abstract
The theta rhythm, a quasi-periodic 4–10 Hz oscillation, is observed during memory processing in the hippocampus, with different phases of theta hypothesized to separate independent streams of information related to the encoding and recall of memories. At the cellular level, the discovery of hippocampal memory cells (engram neurons), as well as the modulation of memory recall through optogenetic activation of these cells, has provided evidence that certain memories are stored, in part, in a sparse ensemble of neurons in the hippocampus. In previous research, however, engram reactivation has been carried out using open-loop stimulation at fixed frequencies; the relationship between engram neuron reactivation and ongoing network oscillations has not been taken into consideration. To address this concern, we implemented a closed-loop reactivation of engram neurons that enabled phase-specific stimulation relative to theta oscillations in the local field potential in CA1. Using this real-time approach, we tested the impact of activating dentate gyrus engram neurons during the peak (encoding phase) and trough (recall phase) of theta oscillations. Consistent with previously hypothesized functions of theta oscillations in memory function, we show that stimulating dentate gyrus engram neurons at the trough of theta is more effective in eliciting behavioral recall than either fixed-frequency stimulation or stimulation at the peak of theta. Moreover, phase-specific trough stimulation is accompanied by an increase in the coupling between gamma and theta oscillations in CA1 hippocampus. Our results provide a causal link between phase-specific activation of engram cells and the behavioral expression of memory.
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Author Response
Reviewer #1 (Public Review):
The authors of this study sought to test whether the optogenetic induction of context-related freezing behavior could be enhanced by synchronizing light pulses to the ongoing hippocampal theta rhythm. Theta is a hippocampus-wide oscillation that strongly modulates almost every cell in this structure, which suggests that causal interventions locked to theta could have a more pronounced impact than open-loop ones. Indeed, the authors found that activating engram-associated dentate gyrus (DG) neurons at the trough of theta resulted in an increase in freezing relative to baseline when averaging across all stimulation epochs. In contrast, open-loop stimulation and peak-locked stimulation had weaker effects. Analysis of local field potentials showed that only the theta-locked stimulation …
Author Response
Reviewer #1 (Public Review):
The authors of this study sought to test whether the optogenetic induction of context-related freezing behavior could be enhanced by synchronizing light pulses to the ongoing hippocampal theta rhythm. Theta is a hippocampus-wide oscillation that strongly modulates almost every cell in this structure, which suggests that causal interventions locked to theta could have a more pronounced impact than open-loop ones. Indeed, the authors found that activating engram-associated dentate gyrus (DG) neurons at the trough of theta resulted in an increase in freezing relative to baseline when averaging across all stimulation epochs. In contrast, open-loop stimulation and peak-locked stimulation had weaker effects. Analysis of local field potentials showed that only the theta-locked stimulation facilitated coupling between theta and mid-gamma, indicating that this manipulation likely enhances the flow of activity from DG to CA1 via CA3 (as opposed to promoting transmission from entorhinal cortex to CA1). Previous results from mice, rats, and humans support the hypothesis that memory encoding and recall occur at distinct phases of theta. This work further strengthens the case for phase-specific segregation of memory-related functions and opens up a path toward more precise clinical interventions that take advantage of intrinsic theta rhythm.
Strengths:
This study recognizes that, when artificially reactivating a context-specific memory, the brain's internal context matters. In contrast to previous attempts at optogenetically inducing recall, this work adds an additional layer of precision by synchronizing the light stimulus to the ongoing theta rhythm. This approach is more challenging, because, in addition to viral expression and bilateral optical fibers, it also requires a recording electrode and real-time signal processing. The results indicate that this additional effort is worth it, as it results in a more effective intervention.
The findings on theta-gamma cross-frequency coupling suggest a possible mechanism underlying the observed behavioral effects: trough stimulation enhances DG to CA1 interactions via CA3. LFP recordings showed that stimulation increases the coupling between theta and mid-gamma (though not in all mice), and the percentage of freezing during reactivation is correlated with the gamma modulation index.
Weaknesses:
Given the precision of the intervention being performed, one might expect to see a stronger behavioral impact. Instead, the overall effect is subtle, and quite variable across mice. Looking at individual data points, the biggest overall increase in freezing actually occurred in 2 mice during the 6 Hz stimulation condition. Furthermore, trough stimulation decreased freezing in 3 mice. This is not a weakness in itself; rather, the weakness lies in the lack of an attempt to make sense of this variability. There are a number of factors that could explain these differences, such as viral expression levels, electrode/fiber placement, and behavior during baseline. There is of course a risk of over-interpreting results from a few mice, but there is also a chance that the results will appear more consistent after accounting for these additional sources of variation.
Although two mice that had negative light induced freezing for trough stimulation, the other 15 mice showed a positive result. Stringent inclusion criteria were used to ensure that mice had adequate viral expression levels and behavior during baseline. Mice without at least 5% light induced freezing in at least two of the four epochs were not included in the study. The negative behavior from some mice is further explained through the correlation between MI and light induced freezing (Figure 5D). 6 Hz showed mixed behavioral results across the different behavioral measures quantified. Additionally, 6 Hz did not show the physiological hallmarks of memory reactivation through the theta-gamma modulation index so having an increased number of negative light induced freezing samples is expected.
Finally, the elevated baseline freezing rate relative to previous literature could have masked some of the behavioral effect.
In the revised manuscript, we discuss the effects of exclusion criteria more clearly.
While trough-locked optogenetic stimulation significantly increases freezing, the effects are much weaker than placing the mouse in the actual fear-conditioned context (average time freezing of 15% vs. 50%). The discussion would benefit from additional treatment of ways to further increase the specificity and effectiveness of artificial memory reactivation.
We have content on future directions for artificial memory reactivation to further approach the behavioral response of natural recall. We believe that incorporating time varying stimulation to different cells or parts of the hippocampus could improve the induced recall value as all current methods stimulate the entire sub-region simultaneously.
Using an open-source platform (RTXI) for real-time signal processing is commendable; however, more work could be done to make it easier to adopt these methods and make them compatible with other tools. The RTXI plugin used for closed-loop stimulation should be fully documented and publicly available, to allow others to replicate these results.
The RTXI plugin can be found here: (https://github.com/ndlBU/phase_specific_stim). The URL has been added in the description of Figure 1.
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eLife assessment
This study represents an important step toward unifying two strains of inquiry, one related to the functional role of hippocampal theta oscillations and one related to the behavioral impact of engram reactivation, and thus the findings have implications for our understanding of memory that will impact multiple subfields. In combination with additional context from the literature, the important findings are supported by solid evidence supporting the conclusion that memory recall operations occur preferentially at a specific phase of theta.
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Reviewer #1 (Public Review):
The authors of this study sought to test whether the optogenetic induction of context-related freezing behavior could be enhanced by synchronizing light pulses to the ongoing hippocampal theta rhythm. Theta is a hippocampus-wide oscillation that strongly modulates almost every cell in this structure, which suggests that causal interventions locked to theta could have a more pronounced impact than open-loop ones. Indeed, the authors found that activating engram-associated dentate gyrus (DG) neurons at the trough of theta resulted in an increase in freezing relative to baseline when averaging across all stimulation epochs. In contrast, open-loop stimulation and peak-locked stimulation had weaker effects. Analysis of local field potentials showed that only the theta-locked stimulation facilitated coupling …
Reviewer #1 (Public Review):
The authors of this study sought to test whether the optogenetic induction of context-related freezing behavior could be enhanced by synchronizing light pulses to the ongoing hippocampal theta rhythm. Theta is a hippocampus-wide oscillation that strongly modulates almost every cell in this structure, which suggests that causal interventions locked to theta could have a more pronounced impact than open-loop ones. Indeed, the authors found that activating engram-associated dentate gyrus (DG) neurons at the trough of theta resulted in an increase in freezing relative to baseline when averaging across all stimulation epochs. In contrast, open-loop stimulation and peak-locked stimulation had weaker effects. Analysis of local field potentials showed that only the theta-locked stimulation facilitated coupling between theta and mid-gamma, indicating that this manipulation likely enhances the flow of activity from DG to CA1 via CA3 (as opposed to promoting transmission from entorhinal cortex to CA1). Previous results from mice, rats, and humans support the hypothesis that memory encoding and recall occur at distinct phases of theta. This work further strengthens the case for phase-specific segregation of memory-related functions and opens up a path toward more precise clinical interventions that take advantage of intrinsic theta rhythm.
Strengths:
This study recognizes that, when artificially reactivating a context-specific memory, the brain's internal context matters. In contrast to previous attempts at optogenetically inducing recall, this work adds an additional layer of precision by synchronizing the light stimulus to the ongoing theta rhythm. This approach is more challenging, because, in addition to viral expression and bilateral optical fibers, it also requires a recording electrode and real-time signal processing. The results indicate that this additional effort is worth it, as it results in a more effective intervention.
The findings on theta-gamma cross-frequency coupling suggest a possible mechanism underlying the observed behavioral effects: trough stimulation enhances DG to CA1 interactions via CA3. LFP recordings showed that stimulation increases the coupling between theta and mid-gamma (though not in all mice), and the percentage of freezing during reactivation is correlated with the gamma modulation index.
Weaknesses:
Given the precision of the intervention being performed, one might expect to see a stronger behavioral impact. Instead, the overall effect is subtle, and quite variable across mice. Looking at individual data points, the biggest overall increase in freezing actually occurred in 2 mice during the 6 Hz stimulation condition. Furthermore, trough stimulation decreased freezing in 3 mice This is not a weakness in itself; rather, the weakness lies in the lack of an attempt to make sense of this variability. There are a number of factors that could explain these differences, such as viral expression levels, electrode/fiber placement, and behavior during baseline. There is of course a risk of over-interpreting results from a few mice, but there is also a chance that the results will appear more consistent after accounting for these additional sources of variation.
While trough-locked optogenetic stimulation significantly increases freezing, the effects are much weaker than placing the mouse in the actual fear-conditioned context (average time freezing of 15% vs. 50%). The discussion would benefit from additional treatment of ways to further increase the specificity and effectiveness of artificial memory reactivation.
Using an open-source platform (RTXI) for real-time signal processing is commendable; however, more work could be done to make it easier to adopt these methods and make them compatible with other tools. The RTXI plugin used for closed-loop stimulation should be fully documented and publicly available, to allow others to replicate these results.
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Reviewer #2 (Public Review):
In this manuscript, Rahsepar et al. test the hypothesis that the precise timing of engram cell activation in relation to the phase of hippocampal theta oscillations plays a causal role in recall. This hypothesis is derived from theories (e.g. the SPEAR model) positing that the hippocampus segregates information for memory encoding and retrieval in time and that separation is organized across the many neurons and subregions of the hippocampus by theta oscillations. They test this hypothesis using stimulation of dentate gyrus neurons active during the encoding of fear memory. Using closed-loop stimulation that they developed, the authors stimulate these dentate engram cells at different phases of theta to measure freezing behavior to determine if the fear memory is recalled. They compare this stimulation to …
Reviewer #2 (Public Review):
In this manuscript, Rahsepar et al. test the hypothesis that the precise timing of engram cell activation in relation to the phase of hippocampal theta oscillations plays a causal role in recall. This hypothesis is derived from theories (e.g. the SPEAR model) positing that the hippocampus segregates information for memory encoding and retrieval in time and that separation is organized across the many neurons and subregions of the hippocampus by theta oscillations. They test this hypothesis using stimulation of dentate gyrus neurons active during the encoding of fear memory. Using closed-loop stimulation that they developed, the authors stimulate these dentate engram cells at different phases of theta to measure freezing behavior to determine if the fear memory is recalled. They compare this stimulation to stimulation at the same average frequency regardless of theta phase, or at a constant 20Hz, in line with prior research, as control conditions. The authors use an elegant within animal design. They find that stimulating at the theta phase when CA3 inputs most strongly influence CA1 leads to significant increases in freezing (relative to baseline), while none of the other stimulation conditions have significant effects on freezing. They then show that this stimulation also causes increases in gamma modulation by theta, which is correlated with learning in prior work. However, the gamma that is theta-modulated appears to be medium gamma which is not associated with CA3 inputs to CA1. Overall, the study is well-designed and well-controlled. The stimulation effects at the "best" theta phase are modest but do appear different than the other conditions. It is unclear why the authors chose to stimulate in dentate and not CA3 as the SPEAR hypothesis centers around CA3 and EC inputs to CA1. Furthermore, I wonder if the freezing behavior itself confounds the detection of the theta phase. Finally, some of the statistical analyses require controlling for multiple comparisons.
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Reviewer #3 (Public Review):
The paper by Rahsepar et al. employed a closed-loop optogenetic approach to stimulate mouse dentate gyrus (DG) 'engram cells' at different phases of the ongoing theta rhythm. While stimulation of DG engram cells in fear conditioning paradigms has been conducted several times before (with similar results to those presented here), the current approach constitutes a significant methodological improvement over typical 'open loop' designs. The authors first characterize the performance of their closed-loop theta phase prediction method and show that it outperforms constant frequency stimulation in achieving a theta phase-specific stimulation, albeit with some limitations. A prominent theory in the field has proposed that memory encoding and recall preferentially take place at the peak and trough of theta …
Reviewer #3 (Public Review):
The paper by Rahsepar et al. employed a closed-loop optogenetic approach to stimulate mouse dentate gyrus (DG) 'engram cells' at different phases of the ongoing theta rhythm. While stimulation of DG engram cells in fear conditioning paradigms has been conducted several times before (with similar results to those presented here), the current approach constitutes a significant methodological improvement over typical 'open loop' designs. The authors first characterize the performance of their closed-loop theta phase prediction method and show that it outperforms constant frequency stimulation in achieving a theta phase-specific stimulation, albeit with some limitations. A prominent theory in the field has proposed that memory encoding and recall preferentially take place at the peak and trough of theta respectively. Based on this framework, the authors compared the behavioral and physiological effects of stimulating engram cells at either the theta peak or trough as well as with constant frequencies. They found that, as predicted by the theory, stimulation at the theta through was the most effective in inducing enhanced fear memory recall (measured as freezing during re-exposure to a neutral context). Finally, the authors examined theta-gamma hippocampal LFP dynamics to provide physiological support for the observed behavioral differences of the different stimulation patterns.
Overall, this work illustrates an interesting methodological development that will be of relevance for future studies conducting manipulations of engram cells and provides additional experimental support for an influential theory in the memory field. Experiments are well conducted and the results presented support the main interpretation of the authors, but several aspects of the interpretation and discussion of the work need to be improved. Likewise, several aspects of data analysis and interpretation, in particular in reference to hippocampal oscillations and regional differences need to be improved.
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