Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep

Reviewer #1 (Public review):

Summary:

Meijer et al. sought to investigate the role of cortical layer 6b (L6b) neurons in modulating sleep-wake states and cortical oscillations under baseline and sleep deprived conditions and in response to orexin A and B. Using chronic EEG recordings in mice with silencing of Drd1a+ neurons (via constitutive Cre-dependent knockout of SNAP25), the authors report that while overall baseline sleep-wake architecture and response to sleep deprivation minimal/unchanged, "L6b silencing" leads to a slowing of theta activity during wakefulness and REM sleep, and a reduction in EEG power during NREM sleep. Additionally, orexin B-induced increases in theta activity were attenuated in L6b silenced mice, which the authors state suggests a modulatory role for L6b in orexin-mediated arousal regulation. The manuscript is generally well written with clarity and transparency. However, a major concern is the lack of specificity in the genetic manipulation, which targets Drd1a+ neurons not exclusive to L6b, undermining the attribution of observed effects solely to L6b. Verification of neuronal silencing is also unclear, and statistical inconsistencies between the main text and figures/tables make it difficult to effectively evaluate the text and stated outcomes.

Strengths:

(1) The text is well written.

(2) The authors are transparent about methodological details.

(3) The stated sleep, circadian, and orexin infusion experiments appear to be well designed, executed, and analyzed (with the exceptions of some statistical analyses detailed below).

Weaknesses:

(1) All outcomes are attributed specifically to L6b neurons, but the genetic manipulation is not specific to L6b neurons. The authors acknowledge this as a limitation, but in my view, this global manipulation is more than a limitation - it affects the overall interpretations of the data. The Hoerder-Suabedissen et al., 2018 paper shows sparse, but also dense, expression of Drd1a+ neurons in brain regions outside of the L6b. Given this issue, the results are largely overstated throughout the paper.

(2) It is not clear to me that the "silencing" of Drd1a+ neurons was verified.

(3) There were various discrepancies (and potentially misattributions) between the stated significant differences in Supplementary Table T1 data and Figure 3a & S2 spectral plots. This issue makes it difficult to effectively evaluate the main text and stated outcomes.

Related, the authors stated that post hoc comparisons of EEG spectral frequency bins were not corrected for multiple testing. Instead, significance was only denoted if changes in at least two consecutive frequency bins were significant. However, there are multiple plots in which a single significance marker is placed over an isolated bin (i.e., 4c, 6, S5, S6). Unless each marker is equivalent to 2 consecutive frequency bins, these markers should be removed from the plots. Otherwise, please define the frequency and size of these markers in the main text.

(4) A rainbow color scale, as in Figure 3, we've now learned, can be misleading and difficult to interpret. The viridis color scale or a different diverging color scale are good alternatives.

(5) How much time elapsed between vehicle/orexin A & B infusions?

(6) For Figure 6, there are statistical discrepancies between the main text and the plots (pg. 10):

a) The text claims post hoc differences for relative ORXA frontal EEG, but there are no significance markers on the plot.
b) The text states that there were no post hoc differences for the relative ORXA occipital EEG, but significance markers are on the plot.
c) The main test for the relative ORXB frontal EEG was not significant, but there are post hoc significance markers on the plot.
d) For relative ORXB occipital EEG, there are significant markers on the plot outside of the stated range in the text.

(7) Some important details are only available in figure captions, making it difficult to understand the main text. For example, when describing Figure 3c in the main text on page 7, it is not clear what type of transitions are being discussed without reading the figure caption. Likewise, a "decrease," "shift," and "change" are mentioned, but relative to what? Similar comment for the EEG theta activity description on pages 7 - 8. Please add relevant details to the main text.

(8) Statistical comparisons for data in Figure 3e, post hoc analyses for data in Figure S7a-b REM data, and post hoc analyses for Figure S7c (not b) occipital EEG should be included to support differences claims. Please denote these differences on the respective plots.

(9) In the subsection titled "Layer 6b mediates effects of orexin on vigilance states (pg. 8)," there does not seem to be any stated differences between control and L6b silenced mice. A more accurate subtitle is needed.

Reviewer #2 (Public review):

Summary:

In this manuscript, Meijer and colleagues investigated the effects of inactivation (conditional silencing) of cortical layer 6b neurons on sleep-wake states and EEG spectral power under the following three conditions: during natural sleep-wake states, after sleep deprivation, or after intracerebroventricular administration of orexin A and B. The authors report that silencing of L6b neurons did not have a significant effect on the total time spent in sleep-wake states, duration, or number of state epochs, or the response to sleep deprivation. However, silencing of L6b neurons did slow down theta-frequency (6-9 Hz) during wake and REM sleep, and reduced the total EEG power during NREM sleep. Infusion of orexin A in the mice in which cortical layer 6b neurons were inactivated produced an increase in wakefulness. A similar effect was observed after infusion of orexin A in the mice in which these neurons were not silenced, but the effect (i.e., increase in wakefulness) was of a smaller magnitude. Silencing of cortical layer 6b neurons attenuated the effect of orexin B in increasing theta activity, as was observed in the control mice. The authors conclude that the cortical neurons in layer 6b play an essential role in state-dependent dynamics of brain activity, vigilance state control, and sleep regulation.

Strengths:

(1) A focus on cortical layer 6b neurons, which are an understudied neuronal population, especially in the context of brain and behavioral state transitions.

(2) The authors used a well-established mouse model to study the effect of inactivation of cortical layer 6b neurons.

Weaknesses:

(1) Although the authors used a highly selective approach to silence layer 6b neurons, the observed changes in EEG oscillations cannot be solely attributed to layer 6b neurons because of the ICV route for orexin administration.

(2) The rationale for using only male rats is not provided.

Author response:

Public Reviews:

Reviewer #1 (Public review):

(1) All outcomes are attributed specifically to L6b neurons, but the genetic manipulation is not specific to L6b neurons. The authors acknowledge this as a limitation, but in my view, this global manipulation is more than a limitation - it affects the overall interpretations of the data. The Hoerder-Suabedissen et al., 2018 paper shows sparse, but also dense, expression of Drd1a+ neurons in brain regions outside of the L6b. Given this issue, the results are largely overstated throughout the paper.

We appreciate the reviewer’s careful reading and concern that some of our statements may have overstated the implications of our data. The Drd1a Cre mouse model used (FK164) has a relatively selective expression of Drd1a Cre in cortex, especially in layer 6b, but indeed some expression is seen in layer 6a and subcortically. We will nuance our claims throughout the paper to ensure that the conclusions are supported by our findings, and further discuss the impact of this limitation on the overall interpretation of our results. Specifically, we will discuss the potential contribution of relevant subcortical areas and layer 6a in the effects we observed.

(2) It is not clear to me that the "silencing" of Drd1a+ neurons was verified.

In our previous publications, we showed confirmation of the loss of regulated synaptic vesicle release from the Cre positive neuronal population (Marques-Smith et al., 2016; Hoerder-Suabedissen et al., 2018; Messore et al., 2024), which validates our approach to “silence” cortical neurons. We will discuss this further in the revised manuscript.

(3) There were various discrepancies (and potentially misattributions) between the stated significant differences in Supplementary Table T1 data and Figure 3a & S2 spectral plots. This issue makes it difficult to effectively evaluate the main text and stated outcomes.

We thank the reviewer for spotting the inconsistencies in how the statistical comparisons were presented: indeed, in the text we described two-way ANOVAs with posthoc tests but in the figures significance markers were positioned based on multiple t-tests. We have revised Supplementary Table T1, Figure 3a and S2 to ensure that all statistics are presented consistently throughout the manuscript, i.e. with two-way ANOVAs and accompanying posthoc tests.

Related, the authors stated that post hoc comparisons of EEG spectral frequency bins were not corrected for multiple testing. Instead, significance was only denoted if changes in at least two consecutive frequency bins were significant. However, there are multiple plots in which a single significance marker is placed over an isolated bin (i.e., 4c, 6, S5, S6). Unless each marker is equivalent to 2 consecutive frequency bins, these markers should be removed from the plots. Otherwise, please define the frequency and size of these markers in the main text.

In line with the previous comment, we have adjusted markers to reflect the results from posthoc tests after two-way ANOVAs in Figures 6 and supplementary figures S5 and S6.

We thank the reviewer for pointing out that in our comparisons of EEG spectra, in some cases single isolated frequency bins, where p-value reached 0.05 were shown as significantly different, which indeed could have occurred by chance given that, in line with previous literature, we have not employed multiple testing comparison. In the revised manuscript we will use an unbiased approach by plotting actual p-values for all bins, and moderate our conclusions accordingly, while giving the readers the opportunity to evaluate the magnitude and extent of the differences directly, rather than relying on an arbitrary threshold for significance.

(4) A rainbow color scale, as in Figure 3, we've now learned, can be misleading and difficult to interpret. The viridis color scale or a different diverging color scale are good alternatives.

Thank you for pointing this out, we have adjusted the colour scale.

(5) How much time elapsed between vehicle/orexin A & B infusions?

There were 2-4 non-infusions days between infusions. We will add this information to methods when revising the manuscript.

(6) For Figure 6, there are statistical discrepancies between the main text and the plots (pg. 10):

a) The text claims post hoc differences for relative ORXA frontal EEG, but there are no significance markers on the plot.

b) The text states that there were no post hoc differences for the relative ORXA occipital EEG, but significance markers are on the plot.

c) The main test for the relative ORXB frontal EEG was not significant, but there are post hoc significance markers on the plot.

d) For relative ORXB occipital EEG, there are significant markers on the plot outside of the stated range in the text.

Thank you for your careful observations, these issues reflect the same inconsistency as raise above, where the text describes two-way ANOVAs and the figures refers to results obtained with multiple t tests. We shall adjust the markers in the figures to be only shown when the ANOVA is significant and show the results of posthoc tests after ANOVAs instead of the results of multiple t tests.

(7) Some important details are only available in figure captions, making it difficult to understand the main text. For example, when describing Figure 3c in the main text on page 7, it is not clear what type of transitions are being discussed without reading the figure caption. Likewise, a "decrease," "shift," and "change" are mentioned, but relative to what? Similar comment for the EEG theta activity description on pages 7 - 8. Please add relevant details to the main text.

We will adjust the wording in the main text to reflect more precisely which comparisons are shown in the figures.

(8) Statistical comparisons for data in Figure 3e, post hoc analyses for data in Figure S7a-b REM data, and post hoc analyses for Figure S7c (not b) occipital EEG should be included to support differences claims. Please denote these differences on the respective plots.

We have added the statistical comparisons for Figure 3e to the results section.

We have added the statistical comparisons for Figure S7A to the results section.

We have added the statistical comparison for Figure S7b to the results section.

In Figure S7c, there was an overall genotype difference, but there was not a time x genotype interaction, so we have not performed posthoc tests and did not plot posthoc significance markers for this figure. We have adjusted the wording in the results section to make this clearer.

We have adjusted the reference to the figure S7c which was incorrect, thank you for your careful attention.

(9) In the subsection titled "Layer 6b mediates effects of orexin on vigilance states (pg. 8)," there does not seem to be any stated differences between control and L6b silenced mice. A more accurate subtitle is needed.

We shall change the subtitle to: “The effects of orexin on vigilance states in L6b silenced mice”. The main finding described in this section is that the increase in EEG theta frequency after ORXB infusion is attenuated in L6b silenced mice, so a statement summarizing this finding could be an alternative title. However, then it would not accurately reflect other, less conspicuous, yet potentially important findings described in this section (during NREM sleep, only in L6b silenced animals there is an increase in power in the lower frequency bins in the frontal derivation; in the occipital derivation, levels of relative SWA during NREM sleep after ORXA infusion were lower in L6b silenced than in control animals).

Reviewer #2 (Public review):

Weaknesses:

(1) Although the authors used a highly selective approach to silence layer 6b neurons, the observed changes in EEG oscillations cannot be solely attributed to layer 6b neurons because of the ICV route for orexin administration.

We completely agree, and did not want to imply that orexin administered through the ICV route reaches cortical Drd1a Cre expressing neurons only. We will re-word the corresponding sentences accordingly throughout the manuscript.

(2) The rationale for using only male rats is not provided.

We agree that this is an important limitation and will acknowledge and discuss it further in the revised manuscript. Unfortunately, our experimental protocol precluded the possibility of monitoring accurately the oestrous cycle, which as well-known has an influence on sleep-wake architecture, brain oscillations as well as orexin signalling and receptor abundance. We therefore decided to use male mice only for the current study, but planning to use both sexes in our follow up work.