Serotonin modulates infraslow oscillation in the dentate gyrus during Non-REM sleep

Reviewer #1 (Public review):

Turi, Teng and the team used state-of-the-art techniques to provide convincing evidence on the infraslow oscillation of DG cells during NREM sleep, and how serotonergic innervation modulates hippocampal activity pattern during sleep and memory. First, they showed that the glutamatergic DG cells become activated following an infraslow rhythm during NREM sleep. In addition, the infraslow oscillation in the DG is correlated with rhythmic serotonin release during sleep. Finally, they found that specific knockdown of 5-HT receptors in the DG impairs the infraslow rhythm and memory, suggesting that serotonergic signaling is crucial for regulating DG activity during sleep. Given that the functional role of infraslow rhythm still remains to be studied, their findings deepen our understanding on the role of DG cells and serotonergic signaling in regulating infraslow rhythm, sleep microarchitecture and memory.

Reviewer #2 (Public review):

Summary:

The authors investigated DG neuronal activity at the population and single cell level across sleep/wake periods. They found an infraslow oscillation (0.01-0.03 Hz) in both granule cells (GC) and mossy cells (MC) during NREM sleep. The important findings are 1) the antiparallel temporal dynamics of DG neuron activities and serotonin neuron activities/extracellular serotonin levels during NREM sleep, and 2) the GC Htr1a-mediated GC infraslow oscillation.

Strengths:

(1) The combination of polysomnography, Ca-fiber photometry, two-photon microscopy and gene depletion is technically sound. The coincidence of microarousals and dips in DG population activity is convincing. The dip in activity in upregulated cells is responsible for the dip at the population level.
(2) DG GCs express excitatory Htr4 and Htr7 in addition to inhibitory Htr1a, but deletion of Htr1a is sufficient to disrupt DG GC infraslow oscillation, supporting the importance of Htr1a in DG activity during NREM sleep.

Weaknesses:

(1) The current data set and analysis are insufficient to interpret the observation correctly.
a. In Fig 1A, during NREM, the peaks and troughs of GC population activities seem to gradually decrease over time. Please address this point.
b. In Fig 1F, about 30% of Ca dips coincided with MA (EMG increase) and 60% of Ca dips did not coincide with EMG increase. If this is true, the readers can find 8 Ca dips which are not associated with MAs from Fig 1E. If MAs were clustered, please describe this properly.
c. In Fig 1F, the legend stated the percentage during NREM. If the authors want to include the percentage of wake and REM, please show the traces with Ca dips during wake and REM. This concern applies to all pie charts provided by the authors.
d. In Fig 1C, please provide line plots connecting the same session. This request applies to all related figures.
e. In Fig 2C, the significant increase during REM and the same level during NREM are not convincing. In Fig 2A, the several EMG increasing bouts do not appear to be MA, but rather wakefulness, because the duration of the EMG increase is greater than 15 seconds. Therefore, it is possible that the wake bouts were mixed with NREM bouts, leading to the decrease of Ca activity during NREM. In fact, In Fig 2E, the 4th MA bout seems to be the wake bout because the EMG increase lasts more than 15 seconds.
f. Fig 5D REM data are interesting because the DRN activity is stably silenced during REM. The varied correlation means the varied DG activity during REM. The authors need to address it.
g. In Fig 6, the authors should show the impact of DG Htr1a knockdown on sleep/wake structure including the frequency of MAs. I agree with the impact of Htr1a on DG ISO, but possible changes in sleep bout may induce the DG ISO disturbance.

(2) It is acceptable that DG Htr1a KO induces the reduced freezing in the CFC test (Fig. 6E, F), but it is too much of a stretch that the disruption of DG ISO causes impaired fear memory. There should be a correlation.

(3) It is necessary to describe the extent of AAV-Cre infection. The authors injected AAV into the dorsal DG (AP -1.9 mm), but the histology shows the ventral DG (Supplementary Fig. 4), which reduces the reliability of this study.

Comments on revisions:

In the first revision, I pointed out the inappropriate analysis of the EEG/EMG/photometry data and gave examples. The authors responded only to the points raised and did not seem to see the need to improve the overall analysis and description. In this second revision, I would like to ask the authors to improve them. The biggest problem is that the detection criteria and the quantification of the specific event are not described at all in Methods and it is extremely difficult to follow the statement. All interpretations are made by the inappropriate data analysis; therefore, I have to say that the statement is not supported by the data.

Please read my following concerns carefully and improve them.

(1) The definition of the event is critical to the detection of the event and the subsequent analysis. In particular, the authors explicitly describe the definition of MA (microarousal), the trough and peak of the population level of intracellular Ca concentrations, or the onset of the decline and surge of Ca levels.

(1-1) The authors categorized wake bouts of <15 seconds with high EMG activity as MA (in Methods). What degree of high EMG is relevant to MA and what is the lower limit of high EMG? In Fig 1E, there are some EMG spikes, but it was unclear which spike/wave (amplitude/duration) was detected as MA-relevant spike and which spike was not detected. In Fig 2E, the 3rd MA coincides with the EMG spike, but other EMG spikes have comparable amplitude to the 3rd MA-relevant EMG spike. Correct counting of MA events is critical in Fig 1F, 2F, 4C.

(1-2) Please describe the definition of Ca trough in your experiments. In Fig 1G, the averaged trough time is clear (~2.5 s), so I can acknowledge that MA is followed by Ca trough. However, the authors state on page 4 that "30% of the calcium troughs during NREM sleep were followed by an MA epoch". This discrepancy should be corrected.

(1-3) Relating comment 1-2, I agree that the latency is between MA and Ca through in page 4, as the authors explain in the methods, but, in Fig 1G, t (latency) is labeled at incorrect position. Please correct this.

(1-4) The authors may want to determine the onset of the decline in population Ca activity and the latency between onset and trough (Fig 1G, latency t). If so, please describe how the onset of the decline is determined. In Fig 1G, 2G, S6, I can find the horizontal dashed line and infer that the intersection of the horizontal line and the Ca curve is considered the onset. However, I have to say that the placement of this horizontal line is super arbitrary. The results (t and Drop) are highly dependent on the position of horizontal line, so the authors need to describe how to set the horizontal line.

(1-5) In order to follow Fig 1F correctly, the authors need to indicate the detection criteria of "Ca dip (in legend)". Please indicate "each Ca dip" in Fig 1E. As a reader, I would like to agree with the Ca dip detection of this Ca curve based on the criteria. Please also indicate "each Ca dip" in Fig 2E and 2F. In the case of the 2nd and 3rd MAs, do they follow a single Ca dip or does each MA follow each Ca dip? This chart is highly dependent on the detection criteria of Ca dip.

As I mentioned above, most of the quantifications are not based on the clear detection criteria. The authors need to re-analyze the data and fix the quantification. Please interpret data and discuss the cellular mechanism of ISO based on the re-analyzed quantification.

Reviewer #3 (Public review):

Summary:

The authors employ a series of well-conceived and well-executed experiments involving photometric imaging of the dentate gyrus and raphe nucleus, as well as cell-type specific genetic manipulations of serotonergic receptors that together serve to directly implicate serotonergic regulation of dentate gyrus (DG) granule (GC) and mossy cell (MC) activity in association with an infra slow oscillation (ISO) of neural activity has been previously linked to general cortical regulation during NREM sleep and microarousals.

Strengths:

There are a number of novel and important results, including the modulation of dentage granule cell activity by the infraslow oscillation during NREM sleep, the selective association of different subpopulations of granule cells to microarousals (MA), the anticorrelation of raphe activity with infraslow dentate activity.

The discussion includes a general survey of ISOs and recent work relating to their expression in other brain areas and other potential neuromodulatory system involvement, as well as possible connections with infraslow oscillations, micro arousals, and sensory sensitivity.

Weaknesses:

- The behavioral results showing contextual memory impairment resulting from 5-HT1a knockdown are fine, but are over-interpreted. The term memory consolidation is used several times, as well as references to sleep-dependence. This is not what was tested. The receptor was knocked down, and then 2 weeks later animals were found to have fear conditioning deficits. They can certainly describe this result as indicating a connection between 5-HT1a receptor function and memory performance, but the connection to sleep and consolidation would just be speculation. The fact that 5-HT1a knockdown also impacted DG ISOs does not establish dependency. Some examples of this are:
o The final conclusion asserts "Together, our study highlights the role of neuromodulation in organizing neuronal activity during sleep and sleep-dependent brain functions, such as memory.", but the reported memory effects (impairment of fear conditioning) were not shown to be explicitly sleep-dependent.
o Earlier in the discussion it mentions "Finally, we showed that local genetic ablation of 5-HT1a receptors in GCs impaired the ISO and memory consolidation". The effect shown was on general memory performance - consolidation was not specifically implicated.

- The assertion on page 9 that the results demonstrate "that the 5-HT is directly acting in the DG to gate the oscillations" is a bit strong given the magnitude of effect shown in Fig. 6D, and the absence of demonstration of negative effect on cortical areas that also show ISO activity and could impact DG activity (see requested cortical sigma power analysis).

- Recent work has shown that abnormal DG GC activity can result from the use of the specific Ca indicator being used (GCaMP6s). (Teng, S., Wang, W., Wen, J.J.J. et al. Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures. Sci Rep 14, 8104 (2024). https://doi.org/10.1038/s41598-024-58819-9). The authors of that study found that the effect seemed to be specific to GCaMP6s and that GCaMP6f did not lead to abnormal excitability. Note this is of particular concern given similar infraslow variation of cortical excitability in epilepsy (cf Vanhatalo et al. PNAS 2004). While I don't think that the experiments need to be repeated with a different indicator to address this concern, you should be able to use the 2p GCaMP7 experiments that have already been done to provide additional validation by repeating the analyses done for the GCaMP6s photometry experiments. This should be done anyway to allow appropriate comparison of the 2p and photometry results.

- While the discussion mentions previous work that has linked ISOs during sleep with regulation of cortical oscillations in the sigma band, oddly no such analysis is performed in the current work even though it is presumably available and would be highly relevant to the interpretation of a number of primary results including the relationship between the ISOs and MAs observed in the DG and similar results reported in other areas, as well as the selective impact of DG 5-HT1a knockdown on DG ISOs. For example, in the initial results describing the cross correlation of calcium activity and EMG/EEG with MA episodes (paragraph 1, page 4), similar results relating brief arousals to the infraslow fluctuation in sleep spindles (sigma band) have been reported also at .02 Hz associated with variation in sensory arousability (cf. Cardis et al., "Cortico-autonomic local arousals and heightened somatosensory arousability during NREMS of mice in neuropathic pain", eLife 2021). It would be important to know whether the current results show similar cortical sigma band correlations. Also, in the results on ISO attenuation following 5-HT1 knockdown on page 7 (fig. 6), how is cortical EEG affected? is ISO still seen in EEG but attenuated in DG?

- The illustrations of the effect of 5-HT1a knockdown shown in Figure 6 are somewhat misleading. The examples in panels B and C show an effect that is much more dramatic than the overall effect shown in panel D. Panels B and C do not appear to be representative examples. Which of the sample points in panel D are illustrated in panels B, C? it is not appropriate to arbitrarily select two points from different animals for comparison, or worse, to take points from the extremes of the distributions. If the intent is to illustrate what the effect shown in D looks like in the raw data, then you need to select examples that reflect the means shown in panel D. It is also important to show the effect on cortical EEG, particularly in sigma band to see if the effects are restricted to the DG ISOs. It would also be helpful to show that MAs and their correlations as shown in Fig 1 or G as well as broader sleep architecture are not affected.

- On page 9 of the results it states that GCs and MCs are upregulated during NREM and their activity is abruptly terminated by MAs through a 5-HT mediated mechanism. I didn't see anything showing the 5-HT dependence of the MA activity correlation. The results indicate a reduction in ISO modulation of GC activity but not the MA correlated activity. I would like to see the equivalent of Fig 1,2 G panels with the 5-HT1a manipulation.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study provides convincing evidence on the infraslow oscillation of DG cells during NREM sleep, and how serotonergic innervation modulates hippocampal activity pattern during sleep and memory.

Strengths and Weaknesses:

The authors used state-of-the-art techniques to carry out these experiments. Given that the functional role of infraslow rhythm still remains to be studied, this study provides convincing evidence of the role of DG cells in regulating infraslow rhythm, sleep microarchitecture, and memory.

I have a few minor comments.

(1) Decreased infraslow rhythm during NREMs in the 5ht1a KO mice is striking. It would be helpful to know whether sleep-wake states, MAs, and transitions to REMs are changed.

We agree with the reviewer that serotonin receptors may be involved in sleep regulation therefore it is important to analyze the effect of their manipulation. We would also like to bring to the attention of the reviewer that in this case we restricted the 5ht1a manipulation to the hippocampus which does not have a known impact on sleep-wake regulation. The analysis of our recorded dataset from these mice confirmed this notion, because we did not see any changes in sleep metrics (see: supplementary figure 6A).

(2) It would be interesting to discuss whether the magnitude in changes of infraslow rhythm strength is correlated with memory performance (Figure 6).

We agree with the reviewer that this could be an interesting point. In our experiments we wanted to minimize the impact of the surgical procedures on the behavior, thus we used separate cohorts to record the photometry and to carry out the behavior experiments, therefore we are unable to correlate behavior and infraslow oscillatory amplitudes in our dataset.

However, a similar experiment was carried out in a recent paper where the authors discovered that the norepinephrine system also displays infraslow oscillatory cycles during NREM sleep (Kjaerby et al 2022). The authors of that paper gradually decreased the magnitude of the NE pulses during NREM by optogenetic manipulation of the locus coeruleus which led to a fragmented sleep phenotype characterized by increased micro arousal occurrence, decreased REM and reduced spindle activity. They also tested the memory performance of the mice in a novel object recognition task and found diminished performance level in the opto group. Serotonin has multiple roles in the brain, many of them show overlap with proposed functions of the noradrenergic system including regulation of plasticity, signaling reward or fearful stimuli. Therefore, we speculate that the modification of serotonin dynamics during sleep will most likely interfere with memory performance.

We inserted this paragraph in the discussion part of our paper.

(3) The authors should cite the Oikonomou Neuron paper that describes slow oscillatory activity of DRN SERT neurons during NREM sleep.

Thank you for the suggestion, we inserted this paper in the manuscript.

(4) The authors should clarify how they define the phasic pattern of the photometry signal.

We have added the details in the Methods.

Reviewer #2 (Public review):

Summary:

The authors investigated DG neuronal activity at the population and single-cell level across sleep/wake periods. They found an infraslow oscillation (0.01-0.03 Hz) in both granule cells (GC) and mossy cells (MC) during NREM sleep.

The important findings are:

(1) The antiparallel temporal dynamics of DG neuron activities and serotonin neuron activities/extracellular serotonin levels during NREM sleep, and

(2) The GC Htr1a-mediated GC infraslow oscillation.

Strengths:

(1) The combination of polysomnography, Ca-fiber photometry, two-photon microscopy, and gene depletion is technically sound. The coincidence of microarousals and dips in DG population activity is convincing. The dip in activity in upregulated cells is responsible for the dip at the population level.

(2) DG GCs express excitatory Htr4 and Htr7 in addition to inhibitory Htr1a, but deletion of Htr1a is sufficient to disrupt DG GC infraslow oscillation, supporting the importance of Htr1a in DG activity during NREM sleep.

Weaknesses:

(1) The current data set and analysis are insufficient to interpret the observation correctly.

a. In Figure 1A, during NREM, the peaks and troughs of GC population activities seem to gradually decrease over time. Please address this point.

Thank you for the suggestion. We have analyzed and compared the magnitude of the oscillatory signals in the first and last minute of the NREM sleep epochs in Dock10-Cre mice and found no significant difference. However, we did observe that the ISO amplitude is smaller in the early stage of the first NREM epochs, defined as those with the prior wakefulness longer than 5 minutes (new supplementary figure 1).

b. In Figure 1F, about 30% of Ca dips coincided with MA (EMG increase) and 60% of Ca dips did not coincide with EMG increase. If this is true, the readers can find 8 Ca dips which are not associated with MAs from Figure 1E. If MAs were clustered, please describe this properly.

We did not find evidence that MAs were clustered in our dataset (see a representative example in supplementary figure 1A). We replaced the example trace with a new one which shows calcium dips with and without MAs. We believe this new trace better represents the data.

c. In Figure 1F, the legend stated the percentage during NREM. If the authors want to include the percentage of wake and REM, please show the traces with Ca dips during wake and REM. This concern applies to all pie charts provided by the authors.

Figure 1F (and all other pie charts) shows the outcome of brain states following a calcium-dip episode. That is, we found that the Ca-dips during NREM were followed by MAs in 30% of the cases, 59% of the Ca-dips led to the maintenance of NREM (no MAs) while in 2% and 9% of the cases we detected either REM state or wakening of the animal. These numbers correspond very well with similar analysis done in a recent paper which looked at the infraslow oscillatory behavior of the norepinephrine system (Kjaerby et al 2022) during NREM sleep. We apologize if the wording in the manuscript was misleading, we modified the figure legends to clarify what the pie charts represent.

d. In Figure 1C, please provide line plots connecting the same session. This request applies to all related figures.

We have replaced the dot plots in all related figures with the line plots.

e. In Figure 2C, the significant increase during REM and the same level during NREM are not convincing. In Figure 2A, the several EMG increasing bouts do not appear to be MA, but rather wakefulness, because the duration of the EMG increase is greater than 15 seconds. Therefore, it is possible that the wake bouts were mixed with NREM bouts, leading to the decrease of Ca activity during NREM. In fact, In Figure 2E, the 4th MA bout seems to be the wake bout because the EMG increase lasts more than 15 seconds.

We have replaced the Figure 2C with line plots as suggested above. It is clear that MC activity during REM sleep is higher, compared to that in NREM sleep, whereas the overall difference between wake and NREM is not significant (some increased, some decreased). Regarding the MAs, we have added a trace of averaged EMG signals in Figure 2G, showing that the averaged EMG bursts during MA are shorter than 5 seconds.

f. Figure 5D REM data are interesting because the DRN activity is stably silenced during REM. The varied correlation means the varied DG activity during REM. The authors need to address it.

We thank the reviewer for this suggestion. We have added this point to the discussion. We speculate that inputs from the supramammillary nucleus or entorhinal cortex to the DG during REM sleep may both contribute to this variability.

g. In Figure 6, the authors should show the impact of DG Htr1a knockdown on sleep/wake structure including the frequency of MAs. I agree with the impact of Htr1a on DG ISO, but possible changes in sleep bout may induce the DG ISO disturbance.

As suggested, we have performed sleep analysis in the Htr1a knockdown experiments including MA quantification. We have found no significant difference between Hrt1-knockdown and control mice in any of the sleep metrics (see: supplemental figure 6). Our interpretation is that the lack of changes in sleep/wake cycles is likely due to the hippocampus not being directly involved in regulating these brain states.

(2) It is acceptable that DG Htr1a KO induces the reduced freezing in the CFC test (Figure 6E, F), but it is too much of a stretch that the disruption of DG ISO causes impaired fear memory. There should be a correlation.

We have modified the discussion accordingly.

(3) It is necessary to describe the extent of AAV-Cre infection. The authors injected AAV into the dorsal DG (AP -1.9 mm), but the histology shows the ventral DG (Supplementary Figure 4), which reduces the reliability of this study.

The histology image shown in the manuscript was taken from the -2.5 mm anteroposterior level, which we still consider to be part of the dorsal DG. For additional clarity, we have replaced the figure with new histology images slightly more anterior position (AP~2.0mm).

Reviewer #3 (Public review):

Summary:

The authors employ a series of well-conceived and well-executed experiments involving photometric imaging of the dentate gyrus and raphe nucleus, as well as cell-type specific genetic manipulations of serotonergic receptors that together serve to directly implicate serotonergic regulation of dentate gyrus (DG) granule (GC) and mossy cell (MC) activity in association with an infra slow oscillation (ISO) of neural activity has been previously linked to general cortical regulation during NREM sleep and microarousals.

Strengths:

There are a number of novel and important results, including the modulation of dentage granule cell activity by the infraslow oscillation during NREM sleep, the selective association of different subpopulations of granule cells to microarousals (MA), the anticorrelation of raphe activity with infraslow dentate activity.

The discussion includes a general survey of ISOs and recent work relating to their expression in other brain areas and other potential neuromodulatory system involvement, as well as possible connections with infraslow oscillations, micro-arousals, and sensory sensitivity.

Weaknesses:

(1) The behavioral results showing contextual memory impairment resulting from 5-HT1a knockdown are fine but are over-interpreted. The term memory consolidation is used several times, as well as references to sleep-dependence. This is not what was tested. The receptor was knocked down, and then 2 weeks later animals were found to have fear conditioning deficits. They can certainly describe this result as indicating a connection between 5-HT1a receptor function and memory performance, but the connection to sleep and consolidation would just be speculation. The fact that 5-HT1a knockdown also impacted DG ISOs does not establish dependency. Some examples of this are:

a. The final conclusion asserts "Together, our study highlights the role of neuromodulation in organizing neuronal activity during sleep and sleep-dependent brain functions, such as memory.". However, the reported memory effects (impairment of fear conditioning) were not shown to be explicitly sleep-dependent.

We thank the reviewer for this comment. We have revised the sentence.

b. Earlier in the discussion it mentions "Finally, we showed that local genetic ablation of 5-HT1a receptors in GCs impaired the ISO and memory consolidation". The effect shown was on general memory performance - consolidation was not specifically implicated.

We have revised the sentence.

(2) The assertion on page 9 that the results demonstrate "that the 5-HT is directly acting in the DG to gate the oscillations" is a bit strong given the magnitude of effect shown in Figure 6D, and the absence of demonstration of negative effect on cortical areas that also show ISO activity and could impact DG activity (see requested cortical sigma power analysis).

We have revised the sentence.

(3) Recent work has shown that abnormal DG GC activity can result from the use of the specific Ca indicator being used (GCaMP6s). (Teng, S., Wang, W., Wen, J.J.J. et al. Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures. Sci Rep 14, 8104 (2024). https://doi.org/10.1038/s41598-024-58819-9). The authors of that study found that the effect seemed to be specific to GCaMP6s and that GCaMP6f did not lead to abnormal excitability. Note this is of particular concern given similar infraslow variation of cortical excitability in epilepsy (cf Vanhatalo et al. PNAS 2004). While I don't think that the experiments need to be repeated with a different indicator to address this concern, you should be able to use the 2p GCaMP7 experiments that have already been done to provide additional validation by repeating the analyses done for the GCaMP6s photometry experiments. This should be done anyway to allow appropriate comparison of the 2p and photometry results.

We would like to thank the reviewer for this comment. We also analyzed the two-photon data in the same manner as the photometry data. However, the only supportive evidence that might be related to ISO in the two-photon data, recorded at the somatic level, was decreased fluorescence during MAs in the NREM-upregulated cell group (see Figure 3 D, E). We are unsure why this discrepancy exists, but we have discussed it in the manuscript and offered some alternative explanations. One hypothesis we are currently exploring relates to the different subcellular compartments sampled by the two imaging techniques. The photometry probe was implanted above the dentate gyrus, and since light collection efficiency declines sharply with distance from the probe tip (Pisano et al., 2019), we hypothesize that ISO is stronger at the dendritic level which directly receive the inputs from entorhinal cortex, and which is closest to the probe's tip. We are now conducting multiplane two-photon imaging experiments in our labs to test this hypothesis.

(4) While the discussion mentions previous work that has linked ISOs during sleep with regulation of cortical oscillations in the sigma band, oddly no such analysis is performed in the current work even though it is presumably available and would be highly relevant to the interpretation of a number of primary results including the relationship between the ISOs and MAs observed in the DG and similar results reported in other areas, as well as the selective impact of DG 5-HT1a knockdown on DG ISOs. For example, in the initial results describing the cross-correlation of calcium activity and EMG/EEG with MA episodes (paragraph 1, page 4), similar results relating brief arousals to the infraslow fluctuation in sleep spindles (sigma band) have been reported also at .02 Hz associated with variation in sensory arousability (cf. Cardis et al., "Cortico-autonomic local arousals and heightened somatosensory arousability during NREMS of mice in neuropathic pain", eLife 2021). It would be important to know whether the current results show similar cortical sigma band correlations. Also, in the results on ISO attenuation following 5-HT1 knockdown on page 7 (Figure 6), how is cortical EEG affected? Is ISO still seen in EEG but attenuated in DG?

Thank you for this valuable comment. We performed the analysis and found a positive correlation between cortical sigma band activity and DG activity during NREM sleep (see supplementary figure 1C-1E). Additionally, we conducted further analyses using the local 5-HT1a KO mouse model but did not observe significant changes in sleep architecture or MA frequency (see supplementary figure 6A). It is also important to note that ISO was only analyzed using calcium signals, not EEG signals. The standard filtering settings in our EEG data collection (0.5-500 Hz) do not allow us to analyze signals in such a low-frequency range.

(5) The illustrations of the effect of 5-HT1a knockdown shown in Figure 6 are somewhat misleading. The examples in panels B and C show an effect that is much more dramatic than the overall effect shown in panel D. Panels B and C do not appear to be representative examples. Which of the sample points in panel D are illustrated in panels B and C? It is not appropriate to arbitrarily select two points from different animals for comparison, or worse, to take points from the extremes of the distributions. If the intent is to illustrate what the effect shown in D looks like in the raw data, then you need to select examples that reflect the means shown in panel D. It is also important to show the effect on cortical EEG, particularly in sigma band to see if the effects are restricted to the DG ISOs. It would also be helpful to show that MAs and their correlations as shown in Figure 1 or G as well as broader sleep architecture are not affected.

We agree with the reviewer that the chosen example may appear somewhat exaggerated. However, we must point out that visually assessing missing or downregulated frequency components can be challenging. To provide a more objective presentation, we included Supplementary Figure 6B-C, in which we performed analysis similar to that in Fig1G in 5HT1a mice. These figures show a significant decrease in ISO amplitude, though the blockade is not complete, due to the incomplete nature of genetic manipulation with viral injection (see Suppl Fig 5). Furthermore, recent studies (Dong et al., 2023; Zhang et al., 2024; Kjaerby et al., 2022) have identified several other neuromodulatory and peptidergic systems that might affect DG activity during MAs.

To explore this further, we conducted pharmacological experiments. We administered 8-hydroxy-DPAT, a 5-HT1a agonist (i.p. 1 mg/kg) in Dock10-Cre mice injected with AAV-FLEX-GcaMP6s in the DG. Since 5-HT1a receptors act as autoreceptors on raphe 5-HT neurons, this treatment effectively silences the serotonergic system, thereby “removing” 5-HT signaling from the brain. The results, shown in Author response image 1, indicate that pharmacological suppression of 5-HT dampens the ISO in the DG during subsequent sleep intervals, with ISO recovering after the drug is washed out. These findings are consistent with the results obtained with the more specific local genetic manipulation. We have not included this result in the manuscript because we believe that the local downregulation is a cleaner experiment whose interpretation is more straightforward.

Author response image 1.

Finally, we also performed sleep analysis in 5-HT1a KO mice, showing that the local downregulation of 5-HT1a receptors had no significant effect on sleep metrics (Suppl Fig 6A). The hippocampus is not typically involved in regulating sleep-wake cycles, so we believe this result is consistent with that understanding.

(6) On page 9 of the results it states that GCs and MCs are upregulated during NREM and their activity is abruptly terminated by MAs through a 5-HT mediated mechanism. I didn't see anything showing the 5-HT dependence of the MA activity correlation. The results indicate a reduction in ISO modulation of GC activity but not the MA-correlated activity. I would like to see the equivalent of Figure 1,2 G panels with the 5-HT1a manipulation.

We agree with the reviewer on this point. We did not conduct any pharmacological or genetic manipulation in 2-photon calcium imaging experiments. We have removed that statement. As for the suggested analysis, please see our explanation above (Suppl Fig 6B-C).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) Since the authors did not monitor DG neuronal activity with an electrophysiological tool, please rephrase the following sentence: "In this study, we investigated the neuronal activity of the dentate gyrus (DG) with electrophysiological and optical imaging tools during sleep-wake cycles." in the Abstract.

We have rephrased the sentence as suggested.

(2) Since the authors did not manipulate the serotonin release during sleep to investigate whether serotonin release modulates DG ISO, please edit the following sentence: "Further experiments revealed that the infraslow oscillation in the DG is modulated by rhythmic serotonin release during sleep" in the Abstract.

We have rephrased the sentence as suggested.

(3) Single-cell recording in DG with two-photon microscopy may address the issue raised in the 4th paragraph of the Discussion. In addition, in Fig 6C, the photometry has only captured the diminished oscillation in Htr1a KO, but cannot distinguish whether the activity levels of GC remain at high or low, which is a clear disadvantage of photometry.

We agree with the reviewer, and have added text to the discussion.

Reviewer #3 (Recommendations for the authors):

(1) Some of the figures are missing labels in the spectrogram panels (e.g. no freq units in Figures 4 and 6).

We have added information in those figures.

(2) Missing specific locations for EEG electrodes/screws. The text states "we predrilled 2 holes on the right side of the skull (1.5 mm posterior of the Bregma) for implanting recording electrodes". 2 holes on the right side of the skull are pretty vague.

We have added this information in the Methods.

(3) Some additional work that could be cited particularly when discussing the serotonergic impact on hippocampal function as it might relate to sleep and memory would include work linking mesopontine activity (both serotonergic and non-serotonergic) to memory-associated hippocampal sharp-wave ripple activity (e.g. Jelitai et al. Front. Neural Circ. 2021, Wang et al Nat. Neuro. 2015).

We have cited these papers.

(4) The work cited at the beginning of the Results describing higher population calcium activity during sleep states (15,18,30) is generally appropriate but not explicitly related to GCamP imaging. Pilz et al. "Functional Imaging of Dentate Granule Cells in the Adult Mouse Hippocampus", J.Neurosci. 2016 might be a more relevant citation.

We have added the citation.