Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons
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Evaluation Summary:
Yamagata et al., present a new transgenic mouse where the cells expressing the gene Scn1a also express green fluorescent protein. This new model is an important contribution to the study of Dravet Syndrome- an epileptic disorder, that is often drug-resistant, where ~80% of patients have loss-of-function mutations in SCN1A. This study confirms the well-known presence of Scn1a in interneurons and identifies Scn1a-expressing subpopulations of cortical neurons that could also potentially contribute to the symptoms of Dravet Syndrome.
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Abstract
Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.
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Evaluation Summary:
Yamagata et al., present a new transgenic mouse where the cells expressing the gene Scn1a also express green fluorescent protein. This new model is an important contribution to the study of Dravet Syndrome- an epileptic disorder, that is often drug-resistant, where ~80% of patients have loss-of-function mutations in SCN1A. This study confirms the well-known presence of Scn1a in interneurons and identifies Scn1a-expressing subpopulations of cortical neurons that could also potentially contribute to the symptoms of Dravet Syndrome.
(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. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In previous work, the authors identified a subpopulation of neocortical layer (L) 5 pyramidal excitatory neurons that were NAV1.1-positive (Ogiwara et al., 2013). However, the sub-identity of those neurons was unclear. Using a novel Scn1a-GFP BAC transgenic mouse, in this manuscript, they characterize these cells at postnatal day(P)15 and P28, to reveal an inverted expression pattern of two genes previously implicated in the determination of a corticospinal (CS) versus corticothalamic (CT) neuronal fate in L5 and L6 i.e. FEZ family zinc finger protein 2 transcriptional factor (Fezf2) and its CS fate repressor, transcription T-box brain 1 transcription factor (Tbr1) (Han et al., 2011). They found that at P15, 54 % of GFP positive neurons in L5 were FEZF2-positive(+), while minimal FEZF2 expression was …
Reviewer #1 (Public Review):
In previous work, the authors identified a subpopulation of neocortical layer (L) 5 pyramidal excitatory neurons that were NAV1.1-positive (Ogiwara et al., 2013). However, the sub-identity of those neurons was unclear. Using a novel Scn1a-GFP BAC transgenic mouse, in this manuscript, they characterize these cells at postnatal day(P)15 and P28, to reveal an inverted expression pattern of two genes previously implicated in the determination of a corticospinal (CS) versus corticothalamic (CT) neuronal fate in L5 and L6 i.e. FEZ family zinc finger protein 2 transcriptional factor (Fezf2) and its CS fate repressor, transcription T-box brain 1 transcription factor (Tbr1) (Han et al., 2011). They found that at P15, 54 % of GFP positive neurons in L5 were FEZF2-positive(+), while minimal FEZF2 expression was observed in L2/3 and L6 i.e. 16% and 12% respectively. In contrast, TBR1+GFP+ neurons were minimal (10%) in L5 and enriched (45% and 41%) respectively in L2/3 and L6.
Largely based on the previously reported frequency distributions for populations of CT, cortico-cortical (CC), and cortico-striatal (iCS) neurons across the cortical layers, and the aforementioned regulatory relationship between Fezf2 and Tbr1, the authors conjecture a mutually exclusive expression of Scn1a and Scn2a amongst these neuronal cell types. The premise of a mutually exclusive sub-population of Scn1a and Scn2a pyramidal neurons is indeed intriguing as it may help substantiate a circuit-based explanation for common Dravet Syndrome phenotypes. However, the manuscript is largely descriptive and can benefit by including quantitative measures such as cell counts to support text conclusions. Evidence for a mutually-exclusive expression of Scn1a and Scn2a amongst populations of CC, CT and iCS neurons can be bolstered by the use of viral-tracing strategies in combination with co-labeling counts of the relevant marker (processed by an insitu and/or protein expression assay). Additionally, subjective terminology such as "dominant", "dense", "less intense", "major" and minor, are prevalent throughout the manuscript and should be clarified by defining these terms based on objective, qualitative measures such as transcript abundance or fluorescence intensity. A substantial portion of (FEZF2+ and TBR1+) excitatory neurons in L5 express Scn1a- driven GFP. These numbers may conflict with a previous report of endogenous Scn1a antibody- expression of minimal (~5%) co-expression among excitatory pyramidal neurons in the cortex (Dutton et al., 2013). Thus, it is unclear to what extent the BAC-GFP mouse featured in this manuscript recapitulates the endogenous expression of Scn1a. Here, the inclusion of correlation plots for in situ hybridization measures for both the endogenous Scn1a and the transgene GFP, along with western blot quantifications of SCN1A and GFP expression between wild type and BAC-transgenic animals may be helpful. Finally, as the manuscript contains too many long sentences, the intent of the authors is often unclear, and the reader's comprehension of the text may be limited.
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Reviewer #2 (Public Review):
The reasons for focusing on Nav1.1 and Nav1.2 by the authors is clear, the genes which code for these channels have been implicated in a multiple neurological disorders. Identifying distribution patterns and shedding light on how these channels contribute pathological neural circuits is a strong step in the right direct. It has previously been shown that Nav1.1 is mostly localized to inhibitory neurons while Nav1.2 is associated with excitatory neurons. The data shown by the authors suggest that some Nav1.1 expression can be found in excitatory neurons. The implication is interesting as it suggests that the lack of Nav1.1 activity could be beneficial in ameliorating seizure symptoms, especially if one could restrict this decreased expression to excitatory neurons alone. The hypothetical neural circuit in …
Reviewer #2 (Public Review):
The reasons for focusing on Nav1.1 and Nav1.2 by the authors is clear, the genes which code for these channels have been implicated in a multiple neurological disorders. Identifying distribution patterns and shedding light on how these channels contribute pathological neural circuits is a strong step in the right direct. It has previously been shown that Nav1.1 is mostly localized to inhibitory neurons while Nav1.2 is associated with excitatory neurons. The data shown by the authors suggest that some Nav1.1 expression can be found in excitatory neurons. The implication is interesting as it suggests that the lack of Nav1.1 activity could be beneficial in ameliorating seizure symptoms, especially if one could restrict this decreased expression to excitatory neurons alone. The hypothetical neural circuit in Figure 7 is great as it gives a conclusive theory that the authors and other interested researchers can test and work with. Underpinning some conclusions on the intensity of GFP expression makes one wonder if using another transgenic line would have led to similar conclusions (e.g. In all neocortical layers of Scn1a-GFP mice, cells with dense GFP signals are generally inhibitory interneurons (Supplemental Figure S1), and FEZF2 or TBR1 signals were found in cells with less intense GFP signals (see below) indicating that these are excitatory projection
neurons).
However, the weakness of this paper is that a few too many conclusions were drawn based on assumptions. -
Reviewer #3 (Public Review):
Yamagata and collaborators describe the generation of an Scn1a-GFP transgenic mouse. Given that mutations in this gene are a frequent cause of epilepsy, including some of the most severe forms, and that the mechanisms how they result in the dysfunction of cerebral cortical networks are still under investigation; this new resource is very welcome and carries the potential to enable novel experimental approaches to the problem. The authors use a combination of protein (Western blot and immunohistochemistry -IHC-) and mRNA expression assays (in situ hybridization) to confirm the successful expression of GFP only in cells that express Nav1.1, the protein product of Scn1a.
Nav1.1 is one of the pore-forming alpha subunits of the voltage-gated sodium channel (VGSC). It has been previously shown that it is present …
Reviewer #3 (Public Review):
Yamagata and collaborators describe the generation of an Scn1a-GFP transgenic mouse. Given that mutations in this gene are a frequent cause of epilepsy, including some of the most severe forms, and that the mechanisms how they result in the dysfunction of cerebral cortical networks are still under investigation; this new resource is very welcome and carries the potential to enable novel experimental approaches to the problem. The authors use a combination of protein (Western blot and immunohistochemistry -IHC-) and mRNA expression assays (in situ hybridization) to confirm the successful expression of GFP only in cells that express Nav1.1, the protein product of Scn1a.
Nav1.1 is one of the pore-forming alpha subunits of the voltage-gated sodium channel (VGSC). It has been previously shown that it is present predominantly in inhibitory interneurons of the cerebral cortex and hippocampus, and a small subpopulation of cortical excitatory neurons. Nav1.2, the product of the gene Scn2a, is the other VGSC alpha subunit abundantly expressed in the brain; and it exhibits a mutually exclusive expression pattern with Nav1.1. The authors confirmed in the Scn1a-GFP mice the presence of Nav1.1 in interneurons and how it is only present in neurons that are negative for Nav1.2 expression; further supporting the accuracy of this new mouse model.
The authors then focus on better characterizing the expression profile of Nav1.1 in cortical excitatory neurons, using a combination of the cortical layer location of the neurons and their expression of the transcription factors TBR1 or FEZF2. The former transcription factor identified neurons that projected within the cerebral hemispheres to the thalamus, striatum, and other cortical areas; while the latter identifies neurons that project to brainstem or spinal cord. Nav1.1 was found to be expressed predominantly in FEZF2 (+) layer V cortical neurons TBR1 (+) layer II/III neurons (which have been shown to be mostly cortico-cortical in previous reports). On the other hand, layer VI TBR1 (+) were for the most part GFP (-) and expressed instead Nav1.2. However, these associations are not absolute, as the authors found in each group cells that are GFP (+) and GFP (-), the latter of which are assumed to be Nav1.2 (+) although this was not specifically documented in IHC experiments with TBR1 and FEZF2 labelling. Future studies trying to extend this characterization will greatly benefit of the availability of this transgenic mouse line.
The paper concludes with a proposed mechanism through which Scn1a haploinsufficiency in mice can result in epilepsy and sudden death (an animal model of the very severe human epilepsy Dravet Syndrome, which is often secondary to SCN1A loss-of-function mutations and is associated with an increased risk of sudden death). The authors theorize that Scn1a haploinsufficiency might result in the net loss of inhibition of cortical neurons projecting to the parasympathetic brainstem centers, which can then trigger a fatal bradycardia after prolonged seizures. This is an intriguing proposal, and given how little we understand about the contributions of the different cortical and hypothalamic inputs to the autonomic cardiac centers in the brainstem (nucleus ambiguous, RVLM and less prominently dorsal nucleus of vagus) no doubt this new mouse model will prove useful in studies testing this hypothesis. -
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