Enhancement of encoding and retrieval functions through theta phase-specific manipulation of hippocampus

Thank you for sending your work entitled “Enhancement of encoding and retrieval functions through theta-phase-specific manipulation of hippocampus” for consideration at eLife. Your article has been favorably evaluated by Eve Marder (Senior editor) and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

This is a fascinating and important article showing a striking double dissociation of behavioral effects caused by optogenetic activation of parvalbumin-positive interneurons in hippocampal region CA1. The activation of PV neurons causes highly temporally specific spiking in the PV cells and a period of about 25 msec of inhibition of spiking in pyramidal cells. The behavior was tested in an end to end T maze where rats had to match the start location A vs. B (described as the encoding segment) with a response to A or B arm after running along a shared stem (described as the retrieval segment). Effects of different stimulation on the peak or trough of theta were evaluated when a rat was in the encoding or retrieval portion of the task. Behavioral performance was enhanced when peak-triggered stimulation was delivered in the encoding segment (falling on the falling slope of theta and presumably reducing CA3 influence during encoding, thereby reducing interference from retrieval during encoding). Behavioral performance was also improved when stimulation was trough triggered in the retrieval segment (falling on the rising slope of theta and presumably reducing EC influence, thereby reducing distraction from external input during retrieval). This double dissociation is very striking and theoretically significant and highly deserving of publication in a high impact journal. The experiment is very technically sophisticated and has addressed important questions of behavioral controls (determining whether behavior changed during stimulation) and questions about neurophysiological changes during stimulation (primarily there was a strong reduction in pyramidal cell spiking activity during the optogenetic activity). The specificity of their effect on behavior depends not on the degree of activity in CA1, but the content of the activity, as they note in the Discussion.

Major concerns:

1) The main issue is that there is some confusion about the phase of theta at which the authors delivered the light stimulation. First, there is a bit of a disconnect between the author's review of the literature, which emphasizes differences between the peak and the trough of theta oscillations as recorded in the hippocampal fissure, and the author's stimulation protocol, which targeted stimulation at either the falling or rising phase of theta at sites other than the hippocampal fissure. The authors should more explicitly link their protocol to the literature and provide the rationale for why their stimulation protocol differed from what they led the reader to expect. Second, the anatomical position of the electrode that the authors used to record theta differed across the four mice (one each in: stratum radiatum at the CA1/CA3 border, stratum pyramidale in CA1, unspecified layer of dentate gyrus, and unspecified area and layer of cortex above CA1). As a result, the phase of theta at which the authors delivered light stimulation would differ across mice. For example, the “peak” of theta would be similar between the cortex above CA1 and CA1 pyramidal layer, but these peaks would not align with the peaks of theta from the electrode in stratum radiatum. None of these peaks would align with the peaks of theta recorded in dentate gyrus. The authors discuss this issue to some extent in the discussion, but they give the impression that only one mouse would differ from the other three. Instead, across the four mice, the authors effectively used three different definitions of theta phase. Indeed, given this variability, the relative consistency of the effects of stimulation on behavior are puzzling. Meaningful interpretations of the results will depend on the extent to which the authors can resolve this puzzle.

2) A potentially related concern is that the straightforward predictions should have been that inactivation of principal cell activity at the theta trough (the putative “encoding phase” of theta) during the encoding phase would impair memory, whereas inactivation at the peak (the putative “retrieval phase” of theta) during retrieval would impair memory. These were not observed. Instead, they observed somewhat of the converse. We think they should acknowledge, rather than avoid, the straightforward predictions.

3) Some discussion of the marginal performance accuracy of the mice should be included. After substantial training, they perform barely above 60% correct, hardly compelling evidence that mice really learn this task. On the other hand, this is a good baseline for improvement by stimulation, as observed. Also, the authors should consider that the poor performance is likely because of the high degree of interference between the many repeated left- and right-turn trials, consistent with the favored interpretation that stimulation at the right time may reduce interference of competing memories, which could be viewed as the principal variable in controlling performance.

4) How can they exclude stimulation produced alterations in high frequency oscillations as a confound?

We appreciate the reviewers’ enthusiasm for our study, and thank them for their helpful feedback. The changes made in response to their comments have substantially improved the quality of our manuscript. Most importantly, we have updated our analysis of stimulation times to use absolute, rather than relative, phase. A new data panel, which shows consistent phase of stimulation across animals, makes the main result of our experiments much more interpretable. We have also added additional figure panels that document the types of errors mice make in our task. This helps to clarify the reasons our optogenetic stimulation benefitted behavior. Below, we describe how we have addressed the specific concerns put forward by the reviewers.

1) The main issue is that there is some confusion about the phase of theta at which the authors delivered the light stimulation. First, there is a bit of a disconnect between the author's review of the literature, which emphasizes differences between the peak and the trough of theta oscillations as recorded in the hippocampal fissure, and the author's stimulation protocol, which targeted stimulation at either the falling or rising phase of theta at sites other than the hippocampal fissure. The authors should more explicitly link their protocol to the literature and provide the rationale for why their stimulation protocol differed from what they led the reader to expect.

We have added two sentences to the Introduction that address this point:

“Our stimulation occurred relative to the phase of locally recorded theta on the trigger electrodes, rather than the phase at the hippocampal fissure, to which much of the previous literature uses as a landmark (Brankack et al., 1993; Kamondi et al., 1998; Hasselmo et al., 2002). However, post-hoc analysis revealed that light pulses were delivered at similar absolute phases across animals.”

Second, the anatomical position of the electrode that the authors used to record theta differed across the four mice (one each in: stratum radiatum at the CA1/CA3 border, stratum pyramidale in CA1, unspecified layer of dentate gyrus, and unspecified area and layer of cortex above CA1). As a result, the phase of theta at which the authors delivered light stimulation would differ across mice. For example, the “peak” of theta would be similar between the cortex above CA1 and CA1 pyramidal layer, but these peaks would not align with the peaks of theta from the electrode in stratum radiatum. None of these peaks would align with the peaks of theta recorded in dentate gyrus. The authors discuss this issue to some extent in the discussion, but they give the impression that only one mouse would differ from the other three. Instead, across the four mice, the authors effectively used three different definitions of theta phase. Indeed, given this variability, the relative consistency of the effects of stimulation on behavior are puzzling. Meaningful interpretations of the results will depend on the extent to which the authors can resolve this puzzle.

The reviewers are correct to point out that meaningful interpretation of our behavioral results depends on our ability to measure stimulation phase relative to some absolute landmark within the theta cycle. In response, we have completed a more comprehensive analysis of both the histological and electrophysiological data that shed light on this issue. These efforts have produced a new data panel (Figure 4c), as well as additional text in the Results section.

The general conclusion is that, in all instances we can measure, the phase of peak- and trough-triggered stimulation relative to a ’landmark’ event; the peak of high gamma power is remarkably consistent across mice. In addition to showing the relationship between stimulation times and high gamma power for these two conditions, we also show images of the precise locations of each electrode lesion. Because absolute theta phase was not captured by our previous descriptions and analysis, we think readers will find the current presentation much more informative. In addition, the similarity of stimulation times relative to the high gamma peak (except in one animal, in which we were unable to measure this variable) supports the hypothesis that our consistent behavioral results are due to the precision of our phase-specific intervention.

2) A potentially related concern is that the straightforward predictions should have been that inactivation of principal cell activity at the theta trough (the putative “encoding phase” of theta) during the encoding phase would impair memory, whereas inactivation at the peak (the putative “retrieval phase” of theta) during retrieval would impair memory. These were not observed. Instead, they observed somewhat of the converse. We think they should acknowledge, rather than avoid, the straightforward predictions.

The Discussion section now directly addresses the lack of any apparent decrease in behavioral performance as a result of inactivation:

“Our initial hypothesis was that the effects of stimulation would depend on both the task segment and phase, but we were unsure if they would be beneficial or punitive. Given that we are recruiting inhibition, and thereby suppressing CA1 output, one might expect the behavioral impact on a hippocampal-dependent task to be negative. Recruiting inhibition during the ‘retrieval’ phase should impair performance in the retrieval segment, whereas recruiting inhibition during the ‘encoding’ phase should impair performance during the encoding segment.”

We have also expanded our treatment of the supposed ‘floor effect’ that may explain why we observed behavioral enhancement at certain phases, without a corresponding impairment at the opposite phases:

“Mice were strongly influenced by the outcome of the previous trial (Figure 5e), which explains why their accuracy on the trained task is only slightly (but significantly) above chance. Our phase-specific optogenetic intervention helps them overcome this bias, especially in the case of trials in which they are required to switch arms after receiving reward (Figure 5f). However, even for trials in which the reward location was consistent with animals’ intrinsic biases, stimulation did not interfere with performance. It is possible that higher light intensities, alternate fiber placements, or a different target phase could have created the conditions necessary to negatively impact behavior.”

3) Some discussion of the marginal performance accuracy of the mice should be included. After substantial training, they perform barely above 60% correct, hardly compelling evidence that mice really learn this task. On the other hand, this is a good baseline for improvement by stimulation, as observed. Also, the authors should consider that the poor performance is likely because of the high degree of interference between the many repeated left- and right-turn trials, consistent with the favored interpretation that stimulation at the right time may reduce interference of competing memories, which could be viewed as the principal variable in controlling performance.

We have conducted an in-depth analysis of the types of errors to which mice are prone, and have found that, indeed, there is a high degree of interference between adjacent trials. Mice display a striking—but unsurprising—bias toward risk aversion. If they receive reward on one trial, they are more likely to visit the same reward arm on the next trial, regardless of the spatial cue. If they make a mistake, and no reward is given, they are more likely to choose the alternate arm on the next trial. This means the mice are essentially performing at least two tasks concurrently—one that involves the cued reward location, and one that involves the outcome of the previous trial. These findings (which stem from the analysis of baseline trials only) are summarized in a new data panel, Figure 5e.

When we add our optogenetic manipulation, we see that the main effect of stimulation at the optimal phase is to allow mice to overcome their bias toward returning to the rewarded arm (Figure 5f). We do not see a corresponding decrease in performance on trials in which mice correctly choose the previously rewarded arm, indicating that the light pulses are not simply increasing response variability.

We have added new paragraphs to the Results section and Discussion section describing and analyzing these findings. Additional interpretations of our results in light of these findings have been added to the Discussion.

4) How can they exclude stimulation produced alterations in high frequency oscillations as a confound?

It appears that the changes in low-gamma-range oscillations are primarily due to leakage from the beta band (16-25 Hz), which is itself explained by the shape of the evoked response to the light pulse. The duration of this response (∼50 ms) will cause a peak to appear in the beta/low gamma range following spectral analysis. As far as we can tell, this does not imply that stimulation induces a change in the resonant properties of the local circuit. In addition, because we see similar changes in the power spectra for the encoding and retrieval, it does not appear that the oscillations we recorded can explain our behavioral result. There was a double-dissociation for the impact of our manipulation on behavior, but not on locally recorded high-frequency oscillations.

We have added the following sentences to the results section, describing this new interpretation of our spectral analysis:

“There was also an increase in power in the low-gamma band (25-35 Hz) for both peak and trough stimulation, but this was associated with a much stronger peak in the beta band (16-25 Hz), which may have affected the low-gamma band via spectral leakage. Based on the shape of the evoked response to each optogenetic stimulus, it appears that these effects are due to the frequency content of the average waveform, rather than non-phase-aligned induced power in different frequency bands (Figure 4b). Aligning the local field potential to the start of each light pulse revealed a large deflection, 200-400 µV in amplitude. The shape of the average response accounts for both the shifts in theta frequency (based on the location of the subsequent peak), and the beta-range power increases (due to ∼50 ms deflections).”