Impaired cortico-striatal excitatory transmission triggers epilepsy

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.

-The title states that striatum is a critical node for epilepsy. The results here seem are supposed to show results from 3 different types of inactivations (ssc, cpu, or thalamus). I only see one set of recordings on the left and I cannot tell what to attribute these to -and it is not the local recording of each, as ssc and thalamus are not listed).
Pg 4, line 15 -"assumed to be absence seizures". It is unclear to me what this very important assumption is based upon. The sentence following it might be an attempt to connect it to REM sleep in some fashion, but I don't see a connection.
There is a bounty of facts relayed in this manuscript, but they lack context. The paragraph starting Pg 5, line 14 contains many such facts. I have no idea what connection they have to the study. pg 3, Line 6 -"suggesting a common pathological mechanism". I appreciate what the authors are attempting, though I think the claim needs to be toned down to what logic can permit. Although ridiculous to say, with this logic, having lungs is a common pathological mechanism. Figure 7. Average firing rates for FSI's are typically double this rate (see Berke 2011 or several other papers). Additionally, the implication is that roughly 1/4 of recorded neurons in CPu were FSI. This rate is approaching 10x what is typically found.
Reviewer #2: Remarks to the Author: This is a very exciting and convincingly documented study that identifies a critical role for decreased corticostriatal excitatory transmission in the generation of non-convulsive epilepsies that are characterized by spike-wave-discharges (SWDs). This is a novel and important insight in the field that has long remained with traditional views provided by rat inbred models of spike-and-wave discharges. In these original studies, a focal area of somatosensory cortex was found to typically act as an initiation site for SWDs and to cause hypersynchronous oscillatory activity in reciprocally connected thalamic circuits, generating the SWDs. The outstanding insight provided by the present work is that corticostriatal excitatory projections onto striatal interneurons is causally involved in these SWDs. The work goes beyond the results of a recently published study on the Scn2a-haploinsufficient mice by senior author J Noebels, a world leader in the field of mouse models of absence epilepsy (Commun Biol. 2018;1. pii: 96. doi: 10.1038/s42003-018-0099-2. Epub 2018. The paper also excels in the use of a large diversity of techniques that illuminate the impact of corticostriatal transmission deficiency from many critical points of view.
Here are my questions and suggestions: 1) The authors should come up with a clearer explanation of how they think abnormal corticostriatal activity ultimately leads to full-blown SWDs that also innvolve primary thalamocortical circuits. Are the corticostriatal-thalamic loop and the cortico-thalamocortical loops reruited sequentially or in parallel? Is there a time delay between onset of hypersynchrony in the former compared to the latter?
2) The lack of effects of Scn2a haploinsufficiency on corticostriatal transmission observed in this study is now becoming clarified through the observation that action potentials are broader in these mice (see the above-mentioned paper from the Noebels group). This could lead to excessive glutamate release followed by a depletion upon repetitive activity. The author could add this as a possibility in the discussion.
2) It would be good to mention both mouse lines that were used in the study in the abstract 3) To further validate the newly proposed mechanism, it would be interesting to test whether SWDs can be suppressed in a well-established rat line of SWDs such as the GAERS or the WAG/Rij

Reviewer #1
(Reviewer #1 comments) The manuscript from the Yamakawa lab explores the role of SWDs (spike and wave discharges) on neural circuitry, with a focus on several genetic mutation mouse models and epileptic phenotypes.
The authors generated multiple conditional knockout mice and performed a considerable amount of work covering behavior, pharmacological manipulations, and both in vivo and in vitro physiology.

Our response 1
We are grateful to the reviewer for these helpful comments. We thoroughly revised our manuscript, with the changes highlighted in yellow.   (Fig. 2a, left). In Stxbp1 +/− mice receiving CPu injection, SWDs were well suppressed not only in the SSC but also in the mPFC and CPu (Fig. 2a, right) where strong SWDs were observed before injection (Fig. 1c). These results demonstrate that neural activity in the SSC,

Although the SSC and thalamus have been well recognized as critical nodes for SWD generation, these results indicate that the CPu is also crucial; thereafter it became a focus of our subsequent experiments. In contrast to muscimol, microinjection of bicuculline, a GABAA receptor antagonist, into the CPu of Stxbp1 +/− mice induced myoclonic and subsequent generalized convulsive seizures (Supplementary Fig. 5). These data suggest that the CPu is involved in the generation of both absence-like (non-convulsive) and convulsive seizures."
(Reviewer #1 comments) Pg 4, line 15 -"assumed to be absence seizures". It is unclear to me what this very important assumption is based upon.

Our response 4
In response to the reviewer's comment, we eliminated the phrase "…assumed to be absence seizures", and revised this area at page 4, line 20, as follows:

Scn2a +/− mice (Ogiwara 2018), Stxbp1 knockout mice showed synchronous bilateral cortical
SWDs during behavioral quiescence (Fig. 1a) and effective suppression of SWDs following ethosuximide administration (Fig. 1b), therefore they were regarded as experiencing absence seizures." (Reviewer #1 comments) The sentence following it might be an attempt to connect it to REM sleep in some fashion, but I don't see a connection.

Our response 5
In response to the reviewer's comment, we revised the manuscript at page 5, line 19, as follows:

"We observed SWDs not only during quiet waking but also during non-rapid eye movement (REM) and REM sleep in Stxbp1 +/− mice (Supplementary Fig. 4)."
(Reviewer #1 comments) There is a bounty of facts relayed in this manuscript, but they lack context. The paragraph starting Pg 5, line 14 contains many such facts. I have no idea what connection they have to the study.

Our response 6
To clarify the context, we added a new figure (Supplementary Fig. 3) and revised the manuscript at page 7, line 12, as follows:

Stxbp1 flox/+ /Vgat-Cre (Stxbp1 fl/+ /Vgat) mice showed a normal survival rate, normal growth and locomotor ability (Miyamoto, 2017). Our results clearly indicate that Stxbp1-haploinsufficiency in dorsal-telencephalic excitatory neurons is responsible for SWDs during behavioral quiescence, while the same condition in GABAergic neurons is responsible for the twitches/jumps."
(Reviewer #1 comments) pg 3, Line 6 -"suggesting a common pathological mechanism". I appreciate what the authors are attempting, though I think the claim needs to be toned down to what logic can permit. Although ridiculous to say, with this logic, having lungs is a common pathological mechanism. Our response 7 In response to the reviewer's comment, we revised the manuscript at page 3, line 5, as follows:  Fig. 13h). The decrease in pFSI firing rates at the onset of SWD was statistically significant at their higher firing rates (>6 Hz; mean firing rate: 10.5 Hz) ( Fig. 7c and d), whereas changes in cells with lower firing rates (<6 Hz; mean firing rate: 2.3 Hz) were not significant. These results suggest that cells with higher firing rates and assumed to be FSIs specifically decrease firing at the onset of SWDs. We revised the manuscript accordingly:

"In particular, STXBP1 and SCN2A mutations are common in patients with early-infantile epileptic encephalopathy (Ohtahara syndrome), West syndrome and Lennox-
At the Results section, page 12, line 8;

"The recorded cells were classified into two neuron types, putative FSIs and MSNs (pFSIs and
pMSNs, respectively), based on waveform characteristics (Fig. 7a and Supplementary Fig. 13a-

d) and firing rates (Thorn 2014)."
We also updated Fig. 7c and d and Supplementary Fig. 13d-f, h accordingly.
(Reviewer #1 comments) Additionally, the implication is that roughly 1/4 of recorded neurons in CPu were FSI. This rate is approaching 10x what is typically found. Our response 9 In response to the reviewer's comment, we revised the manuscript at page 12, line 12, as follows:

Reviewer #2
Reviewer #2 (Remarks to the Author): This is a very exciting and convincingly documented study that identifies a critical role for decreased corticostriatal excitatory transmission in the generation of non-convulsive epilepsies that are characterized by spike-wave-discharges (SWDs). This is a novel and important insight in the field that has long remained with traditional views provided by rat inbred models of spike-and-wave discharges. In these original studies, a focal area of somatosensory cortex was found to typically act as an initiation site for SWDs and to cause hypersynchronous oscillatory activity in reciprocally connected thalamic circuits, generating the SWDs. The paper also excels in the use of a large diversity of techniques that illuminate the impact of corticostriatal transmission deficiency from many critical points of view.

Our response 1
We are grateful for the reviewer's encouraging comments and helpful suggestions. Our changes in the revised manuscript are highlighted in yellow.
(Reviewer #2 comments) Here are my questions and suggestions: 1) The authors should come up with a clearer explanation of how they think abnormal corticostriatal activity ultimately leads to full-blown SWDs that also innvolve primary thalamocortical circuits.
Are the corticostriatal-thalamic loop and the cortico-thalamocortical loops reruited sequentially or in parallel? Is there a time delay between onset of hypersynchrony in the former compared to the latter? Our response 2 In response to the reviewer's comment, we re-analyzed the data and added a new figure   (Supplementary Fig. 2a,b) to show that SWD peaks in the CPu and thalamus were equally delayed to those in SSC ECoG recordings, which was consistent with the previous observation that SWDs first appeared in the SSC in GARES rats (Polack et a., 2007). However, our results might still support sequential recruitment of corticostriatal and corticothalamic loops. We revised the manuscript accordingly: At page 5, line 9; "SWD peaks in the mPFC, CPu and Thal were equally delayed compared to those observed in SSC ECoG recordings (Supplementary Fig. 2a,b) 2) The lack of effects of Scn2a haploinsufficiency on corticostriatal transmission observed in this study is now becoming clarified through the observation that action potentials are broader in these mice (see the above-mentioned paper from the Noebels group). This could lead to excessive glutamate release followed by a depletion upon repetitive activity. The author could add this as a possibility in the discussion.

Our response 2
We thank the reviewer for raising this point. We added our interpretation in the Discussion section at page 15, line 3, as follows: Fig. 10f,g)

. Because Scn2a haploinsufficiency results in the broadening of action potentials (Ogiwara 2018), this could lead to excessive glutamate release followed by a depletion upon repetitive activity. Reduced glutamate transmission in the cortico-striatal pathway is also a possible mechanism in Scn2a +/− mice."
(Reviewer #2 comments) 2) It would be good to mention both mouse lines that were used in the study in the abstract Our response 3 We mentioned this in the Abstract. In the Results section, page 13, line 3:

"To investigate whether impaired cortico-striatal excitatory transmission is also observed in animal models of typical absence epilepsy, we tested GAERS rats, a well-established rat strain showing robust and spontaneous SWDs (Danober 1998). Occurrence frequency and duration of
SWDs in GAERS rats (Fig. 8a) were larger than those in Stxbp1 +/− mice (Fig.1a, Supplementary   Fig. 1d). Notably, microinjection of CX516 into the CPu of GAERS rats significantly reduced the number of SWDs (Fig. 8b), whereas NASPM microinjections increased the number of SWDs (Fig. 8c). These data might suggest the generality of our hypothesis that impaired excitatory inputs onto striatal FSIs leads to epilepsy ( Fig. 9; see Discussion)."

Reviewers' Comments:
Reviewer #1: Remarks to the Author: I thank the authors for their efforts -the manuscript is improved. My main concern still comes from the conclusions about the role of FSIs, a central tenant of the paper and one that is fatally flawed in multiple ways. The issues, quickly summarized with details to follow are: 1) FSI suppression is a common technique, and I am unaware of seizures every being reported 2) NASPM, their putative technique for FSI suppression is well documented to have effects on non-FSIs and has never been reported to have induced seizures.
3) The cortico-FSI-MSN model demonstrates an unawareness or disregard for basic striatal anatomy.
1) Given the conclusions, it seems that ablating FSIs should lead to seizures. This would be an excellent positive control, and in fact it's already been done -Xu et al., 2016 ('Ablation of fast-spiking interneurons in the dorsal striatum, recapitulating abnormalities seen post-mortem in Tourette syndrome, produces anxiety and elevated grooming'). The fact that this very relevant study is not mentioned is a bit concerning, but one cannot read everything.
However, FSI ablation and/or suppression is arguably the most common mouse model for Tourette's. I'm not aware of a single case of seizures in the above study or in any other study using FSI suppression. Given their results, it is surprising that no one has shown this, and I personally know several lab heads who use this FSI suppression model. This is a very serious issue that does not seem to be accounted for either in the experiments nor the discussion.
2) Line 232: "we injected 1-naphthyl acetyl spermine (NASPM), a selective blocker of calciumpermeable AMPA receptors and abundantly expressed in striatal FSIs but not in MSNs, into the CPu of WT mice (Fig. 6a)." Any citation would be good to back up this rather critical point. NASPM is well-known to be expressed in CHaT neurons of the striatum as well as FSI's. There are also several papers -including one in Nature ("Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving", 2008 -also see McCutcheon 2011) that DO find an effect of NASPM on MSNs! They also report zero seizure incidents. An explanation is necessary.
3) Their model is based upon a striatum in which direct pathway neurons, which comprise almost half of the neurons of the striatum, play no role (see figure 9 and page 14). The motivation for this was stated: Line 306 to identify either population should the authors wish to suggest this mechanism. It seems odd that the effect is supposed to be mediated through MSNs, but in my reading they show no effect.
In short, the model and motivation for the observed effects has several serious flaws and cannot be considered to be a putative mechanism.
More minor comments: In Supplemental Figure 2 b, with an n of 5, there does not appear to be a normal distribution of mPFC delays thus a t-test should not be used to test the difference between different brain areas.
Where are the data supporting the claims made on lines 163 -166?
Line 60 -recurrent seems odd. Please change to "common", or the like Reviewer #2: Remarks to the Author: This exciting paper has been excellently revised. I appreciate in particular the additional data in Suppl  Fig 2 regarding the timing of SWDs in CPu and Thal. Additionally, the authors now add data on the GAERS model that confirm their hypothesis. This is a true step forward in the field. I have no further comments. Anita Luthi I thank the authors for their efforts -the manuscript is improved. My main concern still comes from the conclusions about the role of FSIs, a central tenant of the paper and one that is fatally flawed in multiple ways. The issues, quickly summarized with details to follow are: 1) FSI suppression is a common technique, and I am unaware of seizures every being reported 2) NASPM, their putative technique for FSI suppression is well documented to have effects on non-FSIs and has never been reported to have induced seizures.
3) The cortico-FSI-MSN model demonstrates an unawareness or disregard for basic striatal anatomy.

Our response 1
We thank Reviewer #1 for their important and thoughtful comments, which have improved our manuscript. We have revised the manuscript to respond to all concerns raised by the reviewer as described below. in Tourette syndrome, produces anxiety and elevated grooming'). The fact that this very relevant study is not mentioned is a bit concerning, but one cannot read everything.

1) Given the conclusions, it seems that ablating
However, FSI ablation and/or suppression is arguably the most common mouse model for Tourette's. I'm not aware of a single case of seizures in the above study or in any other study using FSI suppression. Given their results, it is surprising that no one has shown this, and I personally know several lab heads who use this FSI suppression model. This is a very serious issue that does not seem to be accounted for either in the experiments nor the discussion. 2) Line 232: "we injected 1-naphthyl acetyl spermine (NASPM), a selective blocker of calcium-permeable AMPA receptors and abundantly expressed in striatal FSIs but not in MSNs, into the CPu of WT mice (Fig. 6a)." Any citation would be good to back up this rather critical point.

Our response 3
We only selectively decreased after application of the blocker in vivo. We observed that upregulation of FSI activity using the DREADD system rescued the SWD phenotype of Stxbp1 mice (Fig. 6) and that temporal downregulation of FSI occurred at the onset of SWDs (Fig. 7), supporting the critical role of FSIs in SWDs. However, we could not exclude the possibility that cholinergic interneurons were affected by NASPM.
Based on this, we have revised our manuscript as follows ( Even if FSI's projected only onto indirect pathway neurons, an account of indirect to direct, and the direct pathway's very strong effect on SNr activity, needs to be taken into account.
-Direct and indirect pathway MSNs are considered together in every instance in the paper.
Why are the neurons indirect pathway MSNs now, other than convenience? There are various methods (transgenic (e.g. drd1a, adora2a, or drd2 cre lines) or tracer injections into the different target nuclei) to identify either population should the authors wish to suggest this mechanism. It seems odd that the effect is supposed to be mediated through MSNs, but in my reading they show no effect.
In short, the model and motivation for the observed effects has several serious flaws and cannot be considered to be a putative mechanism.
Our response 5 We would like to note that we are not proposing that FSIs project only onto indirect pathway MSNs, but we are rather simply suggesting that the MSN-globus pallidus externus (GPe) would be a major route for the seizures, according to our experimental results. Based on these, we propose that dysfunction of FSIs is one of the potential causes More minor comments: In Supplemental Figure 2 b, with an n of 5, there does not appear to be a normal distribution of mPFC delays thus a t-test should not be used to test the difference between different brain areas.
Our response 6