1. Author Response:

    Reviewer #2 (Public Review):

    Mattis et al have used a hemizygous mutant of the gene Scn1a to study changes underlying the severe epilepsy disorder Dravet syndrome. They describe a change in activation of the dentate gyrus in this mouse model, due to altered excitatory synaptic input. They show that this occurs in the age range after normalization of early inhibitory interneuron dysfunction. This provides an interesting potential mechanism by which neural circuit function is altered even after deficits in inhibition are seemingly corrected. They also report that stimulation of inputs to the dentate gyrus increase seizure susceptibility when body temperature is elevated. Overall these findings indicate a new form of circuit dysfunction that may underlie the etiology of this severe genetic epilepsy disorder.

    These findings are not fully complete, and the manuscript suffers from some flaws in experimental design.

    The most pressing issue is the lack of a counter-balanced design in experiments testing the ictogenicity of DG stimulation. The authors attempt to justify this stating "there is a theoretical concern that seizure threshold on Day 2 (the second consecutive day of stimulation) could be lowered by a seizure 24 hours prior (a "kindling"-like phenomenon)". In the very next sentence, they cite a study in which this phenomenon has been shown (thus the concern is not theoretical). That said, this is not a semantic argument, but a flaw in experimental design. On day 1, the authors perform experiment A. On day 2, they perform experiment A+B. In an attempt to show that performing experiment A on day 1 does not by itself lead to changes in experiment A+B, they use a separate cohort and show that experiment A does not lead to changes in a repetition of experiment A. Unfortunately, this is not an adequate control. Experiment A+B involves a different set of stimuli, to which the response could very well be altered by the day 1 experiment, but this change would not be revealed with the described experimental design. To determine whether the effect shown in experiment A+B requires a more rigorous, counter-balanced experimental design where one group undergoes experiment A followed by experiment A+B, and a second group undergoes experiment A+B followed by experiment A.

    Thank you for this important critique.

    → We agree with these points and have repeated this experiment using an improved experimental design (Figure 6). We now present data from three groups of mice: Scn1a-ChR2 (experimental mice), Scn1a-YFP (photostimulation control), and WT-ChR2 (genotype control), tested on a single day (obviating concerns about day 2).

    → Please note that this revised manuscript includes an additional ictogenicity experiment (Figure 7), in which we employ the proposed counter-balanced experimental design.

    The second major issue is a lack of wild type control groups for several experiments. The experiments presented in Figures 4, 6C and F, and 7 all lack the necessary wild type control measures. Wild type controls were done for Figure 6E, but the data are not presented in the figure.

    This is also an important point.

    → For the Hm1a experiment (Figure 4), we now present wild-type control data for both PV-IN electrophysiology and 2P circuit-level imaging (Figure 4 – figure supplement 1).

    → We have removed the optogenetic imaging data (previously Figure 6C).

    → The entorhinal cortex ictogenicity experiment (Figure 6) has been re-designed, as per above, and includes appropriate controls.

    → For the experiment demonstrating a decrease in circuit activation in response to PV-IN stimulation (now Figure 8), we were not able to perform a wild-type control due to very low levels of wild-type activation under those conditions (see Figure 2 panel A3 – response to 1 pulse in young adult wild-type mice), as noted in the comments in response to the critique of Reviewer #1. In other words, in the wild-type mice, there was essentially no signal to block. In this experiment we in fact conceptualize the SST activation as the control group (for the PV activation), which we clarify in the text.

    Some of the cell physiology experiments presented were not optimally designed to provide a relevant mechanistic follow-up to the major findings. For the first major finding of the paper, Figure 2 shows clear and interesting changes in DG activation in the mouse model, and Figure 5 reveals changes to synaptic excitation and inhibition in these neurons. Figure 3 and 4 present data showing changes to PV-interneuron intrinsic properties that only reveal themselves under very intense stimulation. While these findings are interesting and worthy of follow-up, the changes aren't relevant to the synaptic stimulation used in Figure 2.

    Thank you for this important comment. We now include additional data, as follows:

    → A parallel dataset quantifying intrinsic properties in the early postnatal timepoint (Figure 3 – figure supplement 1; Table 2). We find that the PV-INs are much more profoundly impaired at this younger timepoint, which further argues against PV-IN dysfunction as the cause of the increased DG activation seen in young adult Scn1a mice relative to wild-type; i.e., PV-IN excitability partially normalizes with development in Scn1a+/- mice, whereas the DG hyperactivation becomes more severe.

    → Synaptic data from the early postnatal timepoint (Figure 5 – figure supplement 2), in which we find no genotype difference in the E/I ratio or EPSC magnitude.

    → PPR at both timepoints, showing no genotype difference in the early postnatal mice, but a higher release probability in the young adult mice.

    Finally, Figure 2 has missing data points, seemingly due to cropping of panels. Data visualization is problematic for this vital figure. The fit lines for individual experiments overwhelm the color-filled variance of the mean. Thus, the data in this figure are very difficult to read and interpret. The figure would benefit from including all the individual data points and summary data, but removing the individual fits or putting them into a supplement.

    We appreciate this very helpful feedback. We now present a “cleaner” version of this main Figure (Figure 2), with the individual fit lines shown in a supplemental Figure (Figure 2 – figure supplement 1).

    Reviewer #3 (Public Review):

    The authors tackle an interesting question - whether the dentate gyrus is a locus of pathology in Scn1a+/- mice and uncover a strong phenotype - the granule cells of the dentate gyrus are over-activated and the EC to dentate pathway is prone to seizure genesis. In the discussion, they suggest that their results support the idea that the DG may be a common locus to several different types of epilepsy… an attractive hypothesis! There are several strengths of the paper. The team has done a nice job of presenting 'ground-truth' data that their measurements of dF/F across a large population of granule cells correlates with action potentials in these cells. As the authors point out, this is especially important when working in disease models in which the dF/F-action potential relationship may be altered. Throughout, the authors were also careful about considering the limitations of their various techniques and analyze the data in several ways to account for possible artifacts (e.g. ensuring that differences in activation are not arising because of slicing and consideration of kindling in later in vivo seizure threshold experiments). The experiments were well designed and appropriately interpreted.

    One of most intriguing results of the work is that PV interneurons in the DG of Scn1a+/- show only very minor impairments in young adult animals (they show more spike accommodation than in control animals). Rather, it seems that the GCs receive enhanced excitation from the entorhinal cortex. They perform a set of pharmacological experiments to prove that PV interneurons (and more generally inhibition) do not account for the difference in granule cell activation - however, here it would be useful to see the data summarized more consistently. It is difficult to interpret the pharmacological results (both of which are presented as changes in dF/F0) with respect to the initial findings of the manuscript (presented as estimated activation across the entire population).

    We appreciate this helpful suggestion. We agree that the presentation of the calcium imaging data in the initial submission made data interpretation more difficult for the reader. In this revised manuscript we have improved the consistency of presentation of the calcium imaging data. Please note however that we conceptualize this imaging data as fitting into two categories, which do require different graphical depiction:

    1. Unpaired data in which we analyze responses across a range of stimulation conditions, shown in Figure 2 and associated Figure 2 – figure supplement 1 and Figure 2 – figure supplement 3; and
    2. Paired data in which we assess the response within a given imaging field to a manipulation performed at a single stimulation condition (Hm1a data in Figure 4 and Figure 4 – figure supplement 1; PTX data in Figure 5 and Figure 5 – figure supplement 2; PV-IN data in Figure 8)

    A beautiful aspect of this work is that it goes from cells to circuits to intact brain (in vivo). They nicely show that the heightened excitation from the EC to the DG is sufficient to drive seizures in the Scn1a+/- mice, and finally that since PVs are intact, they can be harnessed to balance out the over activation of GC via optogenetic stimulation of PVs.

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  2. Reviewer #3 (Public Review):

    The authors tackle an interesting question - whether the dentate gyrus is a locus of pathology in Scn1a+/- mice and uncover a strong phenotype - the granule cells of the dentate gyrus are over-activated and the EC to dentate pathway is prone to seizure genesis. In the discussion, they suggest that their results support the idea that the DG may be a common locus to several different types of epilepsy... an attractive hypothesis! There are several strengths of the paper. The team has done a nice job of presenting 'ground-truth' data that their measurements of dF/F across a large population of granule cells correlates with action potentials in these cells. As the authors point out, this is especially important when working in disease models in which the dF/F-action potential relationship may be altered. Throughout, the authors were also careful about considering the limitations of their various techniques and analyze the data in several ways to account for possible artifacts (e.g. ensuring that differences in activation are not arising because of slicing and consideration of kindling in later in vivo seizure threshold experiments). The experiments were well designed and appropriately interpreted.

    One of most intriguing results of the work is that PV interneurons in the DG of Scn1a+/- show only very minor impairments in young adult animals (they show more spike accommodation than in control animals). Rather, it seems that the GCs receive enhanced excitation from the entorhinal cortex. They perform a set of pharmacological experiments to prove that PV interneurons (and more generally inhibition) do not account for the difference in granule cell activation - however, here it would be useful to see the data summarized more consistently. It is difficult to interpret the pharmacological results (both of which are presented as changes in dF/F0) with respect to the initial findings of the manuscript (presented as estimated activation across the entire population). A beautiful aspect of this work is that it goes from cells to circuits to intact brain (in vivo). They nicely show that the heightened excitation from the EC to the DG is sufficient to drive seizures in the Scn1a+/- mice, and finally that since PVs are intact, they can be harnessed to balance out the over activation of GC via optogenetic stimulation of PVs.

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  3. Reviewer #2 (Public Review):

    Mattis et al have used a hemizygous mutant of the gene Scn1a to study changes underlying the severe epilepsy disorder Dravet syndrome. They describe a change in activation of the dentate gyrus in this mouse model, due to altered excitatory synaptic input. They show that this occurs in the age range after normalization of early inhibitory interneuron dysfunction. This provides an interesting potential mechanism by which neural circuit function is altered even after deficits in inhibition are seemingly corrected. They also report that stimulation of inputs to the dentate gyrus increase seizure susceptibility when body temperature is elevated. Overall these findings indicate a new form of circuit dysfunction that may underlie the etiology of this severe genetic epilepsy disorder.

    These findings are not fully complete, and the manuscript suffers from some flaws in experimental design.

    The most pressing issue is the lack of a counter-balanced design in experiments testing the ictogenicity of DG stimulation. The authors attempt to justify this stating "there is a theoretical concern that seizure threshold on Day 2 (the second consecutive day of stimulation) could be lowered by a seizure 24 hours prior (a "kindling"-like phenomenon)". In the very next sentence, they cite a study in which this phenomenon has been shown (thus the concern is not theoretical). That said, this is not a semantic argument, but a flaw in experimental design. On day 1, the authors perform experiment A. On day 2, they perform experiment A+B. In an attempt to show that performing experiment A on day 1 does not by itself lead to changes in experiment A+B, they use a separate cohort and show that experiment A does not lead to changes in a repetition of experiment A. Unfortunately, this is not an adequate control. Experiment A+B involves a different set of stimuli, to which the response could very well be altered by the day 1 experiment, but this change would not be revealed with the described experimental design. To determine whether the effect shown in experiment A+B requires a more rigorous, counter-balanced experimental design where one group undergoes experiment A followed by experiment A+B, and a second group undergoes experiment A+B followed by experiment A.

    The second major issue is a lack of wild type control groups for several experiments. The experiments presented in Figures 4, 6C and F, and 7 all lack the necessary wild type control measures. Wild type controls were done for Figure 6E, but the data are not presented in the figure.

    Some of the cell physiology experiments presented were not optimally designed to provide a relevant mechanistic follow-up to the major findings. For the first major finding of the paper, Figure 2 shows clear and interesting changes in DG activation in the mouse model, and Figure 5 reveals changes to synaptic excitation and inhibition in these neurons. Figure 3 and 4 present data showing changes to PV-interneuron intrinsic properties that only reveal themselves under very intense stimulation. While these findings are interesting and worthy of follow-up, the changes aren't relevant to the synaptic stimulation used in Figure 2.

    Finally, Figure 2 has missing data points, seemingly due to cropping of panels. Data visualization is problematic for this vital figure. The fit lines for individual experiments overwhelm the color-filled variance of the mean. Thus, the data in this figure are very difficult to read and interpret. The figure would benefit from including all the individual data points and summary data, but removing the individual fits or putting them into a supplement.

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  4. Reviewer #1 (Public Review):

    Dravet syndrome is a developmental and epileptic encephalopathy resulting from mutations in a sodium channel subunit that is widely thought to cause disease by affecting synaptic inhibition. Here the authors use a well-established mouse model to show that circuit dysfunction results from excess synaptic excitation in the dentate gyrus, potentially providing new insight into the pathological mechanisms underlying seizure activity.

    Strengths of the study include the sophisticated approach of 2P Ca2+ imaging of population activity and whole-cell recording in slices that provide well-supported evidence that circuit dysfunction is independent of GABAergic inhibition. Weaknesses include some oversimplification of the results in the data interpretation such that not all the claims are fully supported and lack of in-depth analysis of the circuit dysfunction with a clear presentation of its developmental time course.

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  5. Evaluation Summary:

    Dravet syndrome, a severe seizure disorder resulting from a sodium channel mutation, is widely thought to result from impaired synaptic inhibition. Here the authors present multi-level evidence that excess synaptic excitation in the dentate gyrus is a locus of pathology. These results provide new insight into pathological mechanisms in Dravet syndrome that will be of interest to a broad range of neuroscientists studying epilepsy, as well as the role of the hippocampus and synaptic alterations in neurological disease.

    (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. The reviewers remained anonymous to the authors.)

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