Developmental changes in brain activity of heterozygous Scn1a knockout rats

Introduction Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to loss-of-function mutation of SCN1A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of different brain regions in Scn1a knockout rats at each developmental stage. Methods We established an Scn1a knockout rat model and examined brain activity from postnatal day (P) 15 to 38 using a manganese-enhanced magnetic resonance imaging technique (MEMRI). Results Scn1a heterozygous knockout (Scn1a+/−) rats showed a reduced expression of voltage-gated sodium channel alpha subunit 1 protein in the brain and heat-induced seizures. Neural activity was significantly higher in widespread brain regions of Scn1a+/− rats than in wild-type rats from P19 to P22, but this difference did not persist thereafter. Bumetanide, a Na+-K+-2Cl− cotransporter 1 inhibitor, mitigated hyperactivity to the wild-type level, although no change was observed in the fourth postnatal week. Bumetanide also increased heat-induced seizure thresholds of Scn1a+/− rats at P21. Conclusions In Scn1a+/− rats, neural activity in widespread brain regions increased during the third postnatal week, corresponding to approximately 6 months of age in humans, when seizures most commonly develop in DS. In addition to impairment of GABAergic interneurons, the effects of bumetanide suggest a possible contribution of immature type A gamma-aminobutyric acid receptor signaling to transient hyperactivity and seizure susceptibility during the early stage of DS. This hypothesis should be addressed in the future. MEMRI is a potential technique for visualizing changes in basal brain activity in developmental and epileptic encephalopathies.

Introduction: Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to lossof-function mutation of SCN A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of di erent brain regions in Scn a knockout rats at each developmental stage.

Methods:
We established an Scn a knockout rat model and examined brain activity from postnatal day (P) to using a manganese-enhanced magnetic resonance imaging technique (MEMRI).
Results: Scn a heterozygous knockout (Scn a +/− ) rats showed a reduced expression of voltage-gated sodium channel alpha subunit protein in the brain and heat-induced seizures. Neural activity was significantly higher in widespread brain regions of Scn a +/− rats than in wild-type rats from P to P , but this di erence did not persist thereafter. Bumetanide, a Na + -K + -Cl − cotransporter inhibitor, mitigated hyperactivity to the wild-type level, although no change was observed in the fourth postnatal week. Bumetanide also increased heat-induced seizure thresholds of Scn a +/− rats at P .

Conclusions:
In Scn a +/− rats, neural activity in widespread brain regions increased during the third postnatal week, corresponding to approximately months of age in humans, when seizures most commonly develop in DS. In addition to impairment of GABAergic interneurons, the e ects of bumetanide suggest a possible contribution of immature type A gamma-aminobutyric acid receptor signaling to transient hyperactivity and seizure susceptibility during the early stage of DS. This hypothesis should be addressed in the future. MEMRI is a potential technique for visualizing changes in basal brain activity in developmental and epileptic encephalopathies.

. Introduction
Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by pharmacoresistant seizures, seizure susceptibility at high temperatures, cognitive and behavioral impairments after the first year of life, and motor disorders including ataxia and crouch gait (1)(2)(3). High premature mortality (4-20%) has been reported, and the main causes of death include sudden unexpected death, status epilepticus, and acute encephalopathy (3,4).
DS is characterized by a cluster of age-related electroclinical features.
Unilateral/generalized convulsive status epilepticus (CSE) frequently occurs in the early stage of the disease. Thereafter, multiple seizure types, including focal, myoclonic, absence, and atonic seizures, appear. At later stages, interictal electroencephalography (EEG) shows diffuse and/or focal epileptic discharges, which are usually not observed during infancy (5). After 5 years of age, the occurrence of CSE and other seizure types decreases, whereas brief nocturnal generalized tonic-clonic seizures exhibit life-long occurrence (2, 3).
Heterozygous loss-of-function mutations in the SCN1A gene have been identified in over 80% of patients with DS (2, 6). SCN1A encodes voltage-gated sodium channel alpha subunit 1 (Na V 1.1) and is predominantly expressed in the parvalbuminpositive gamma-aminobutyric acid (GABA)ergic interneurons (7,8). These interneurons are the main source of phasic inhibition in the brain, and hence, impaired cerebral inhibition due to Na V 1.1 haploinsufficiency in these neurons is considered the major mechanism underlying DS epilepsy (7,9,10). However, to establish better treatment strategies and improve the disease outcome, it is essential to comprehensively understand the pathogenesis of the age-dependent phenotypic changes that occur in DS.
To address this issue, we employed manganese-enhanced magnetic resonance imaging (MEMRI) in newly established Scn1a knockout rats, a technique used to visualize in vivo brain activity by using properties of manganese ions (Mn 2+ ). Mn 2+ ions shorten the T1 relaxation time in tissues where they accumulate and act as a calcium ions analog in neuronal tissue. They could also enter the firing neurons via voltage-gated calcium channels, and get transported to the adjacent neurons (11). Accordingly, Mn 2+ accumulate in areas with higher neuronal activity, resulting in hyperintensity on T1-weighted images. Therefore, MEMRI has been widely applied as an in vivo method to understand the brain activity in various neurological diseases (12). The kinetic properties of Mn 2+ , which are slowly distributed in the brain over a 24-h period after systemic administration (11,13,14) suggest that MEMRI is more suitable for evaluating basal brain activity than for transient epileptic activity like seizures. Previous reports have also reported difficulty in detecting brain activity in status epilepticus using MEMRI (15)(16)(17), suggesting the usefulness of MEMRI as a tool for detecting developmental changes in the brain activity of different regions in epilepsy and analyzing the underlying pathogenesis.
Here, we evaluated the age-dependent alteration in the brain activity of different regions in the Scn1a knockout rats using MEMRI. Previous studies on the pathogenesis of DS have mainly used mouse models of Scn1a knockout (18)(19)(20). We used rats because they have larger brains than mice, allowing for more accurate evaluation of brain activity by MRI, even in pups. In addition, when using mice, longer imaging times are required to perform high-resolution MRI experiments, and there are concerns about the effects of prolonged anesthesia and the imaging environment on the images. Coil fitting is also an important factor affecting the results of the analysis when targeting small brains. Furthermore, as compared to mice, the developmental patterns in rats, including developmental changes in the Na + -K + -2Cl − cotransporter 1 (NKCC1) and K + -Cl − cotransporter 2 (KCC2), have been reported to have more similar to those in humans (21).
Although we established the rat model for DS, the phenotype was milder than that of Scn1a knockout mice, in terms of lower frequency of spontaneous seizures and mortality rate. However, we hypothesized these rat models may be better suited to evaluate the pathogenic alterations directly caused by Scn1a defects, while minimizing the influences of spontaneous seizures and malnutrition.
. Materials and methods . . Transcription activator-like e ector nuclease -mediated genome editing in rats A pair of TALENs targeting exon 1 of rat Scn1a (Ensembl: ENSRNOG00000053122) was designed and constructed using a two-step assembly method with a Platinum Gate kit as previously reported (22). Assembled sequence was 5′-TGCAGGATGACAAGATGgagcaaacagtgcttGTACCACCAGGAC CTGA-3′, where uppercase and lowercase letters indicate TALEN target sequences and spacer sequence, respectively. TALENs were microinjected into fertilized eggs of Fisher 344 (F344) rats and transferred into the oviducts of pseudopregnant female Wistar rats, as previously described (23). Genomic DNA was extracted from the tail using the GENEXTRACTOR TA-100 automatic DNA purification system (Takara Bio) and amplified with specific primer sets (forward 5′-TCCTCACTTGTTGGGTCTCA-3′, reverse 5′-TCAGGGTGACTTCAGCATTTC-3′). The polymerase chain reaction (PCR) products were directly sequenced using the BigDye terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). Rats from the fifth generation or later were used in all experiments. Genotypes were assessed by running the PCR products from ear DNA on a Caliper electrophoresis system at postnatal day (P9). Only male rats were used in experiments to eliminate sex differences.

. . EEG recordings
All surgical procedures were performed under isoflurane anesthesia (4-1.5%) at P14. Epidural screw electrodes for EEG were mounted on the skull bilaterally over the somatosensory cortex (2.0 mm lateral to midline, 2.0 mm posterior to bregma) and the cerebellum (1.0 mm lateral to midline, 2.0 mm posterior to lambda) as reference electrodes. Electromyography (EMG) electrodes were inserted into the suprascapular area. Heat-induced seizures were triggered in rats as described below, and behavioral seizures, video-EEG, and EMG were recorded using a PowerLab system (AD Instruments) at P21. Data analysis was performed with LabChart software (AD Instruments).

. . Animal preparation for MRI
Rats were initially anesthetized with 3% isoflurane in 30/70% O 2 /N 2 in a closed chamber. Rats were laid in the prone position on a dedicated animal bed heated with warm circulating water. Anesthesia with 1.5-2.5% isoflurane in 30/70% O 2 /N 2 was delivered to the spontaneously breathing animals through a snout mask, and the respiratory rate was maintained at 50-60 breaths/min. The rectal temperature was carefully monitored and maintained at 36.5 ± 0.5 • C by circulating warm water under the bed.

. . MEMRI
An isotonic solution of MnCl 2 ·4H 2 O (203734; Sigma-Aldrich) was prepared at a concentration of 100 mM in 100 mM bicine solution, as previously described (24). Then, the MnCl 2 solution was diluted to 50 mM with saline, and the pH was adjusted to 7.4.
We intraperitoneally administered a 50 mM solution of MnCl 2 immediately after the initial MRI acquisition, twice at 1-h intervals, to get a total dose of 66 mg/kg. MEMRI acquisition was performed 24 h after the first administration of MnCl 2 based on the kinetics of intracerebral Mn 2+ concentration (11). The imaging area was adjusted to the same position as in the initial MRI to facilitate comparison of T1 values at the same ROI.

. . Systemic bumetanide or saline administration
The effective systemic dose of bumetanide on neonatal seizures in rats is 0.1-0.5 mg/kg (21,25). We administered 0.2 mg/kg of bumetanide twice a day in repeated doses as previously described (26). Bumetanide (B3023; Sigma-Aldrich) was dissolved in 100% ethyl alcohol and then diluted with 0.9% saline to a final concentration of 0.05 mg/mL. Bumetanide solution (0.2 mg/kg) or equal volumes of 0.9% saline (4 mL/kg) was injected twice daily from P12 to P20 or P26. MEMRI was performed on P21 or P27 for each rat.

. . Induction of heat-induced seizures
Scn1a +/− rats were subjected to heat-induced seizures on P21 or P27 after daily administration of bumetanide or 0.9% saline from P12 to P20 or P26. Hyperthermia was induced by placing the rats in a bath filled 10 cm deep with 45 • C water, as previously described .
/fneur. . . Genotype analysis shows a bp band in the wild-type allele and a bp band in the Scn a em kyo mutant. The upper band of the Scn a em kyo mutant is nonspecific. Genotypes are indicated by +/+ for wild-type, +/for heterozygous, and -/-for homozygous animals. (B) Western blotting of brain membrane proteins from wild-type (+/+), heterozygous (+/-), and homozygous (-/-) Scn a rats at P using an anti-voltage-gated sodium channel alpha subunit (Na V . ) antibody. β-actin was used as the internal control. Original blots/gels are presented in Supplementary Figure . (C) Relative Na V . protein levels normalized to β-actin. (D) Survival curves of wild-type (n = ), Scn a +/− (n = ), and Scn a −/− (n = ) rats. Scn a −/− rats exhibited ataxia and seizures from postnatal day (P) , gradually progressing to weight loss and complete loss of postural control. They became inactive and did not survive beyond P . In wild-type and Scn a +/− rats, there were no spontaneous deaths. (E) Body weight curves of wild-type (blue circle), Scn a +/− (red square), and Scn a −/− (black triangle) rats. The sample size is shown below the graph. Scn a +/− rats showed no di erence in body weight compared to wild-type rats. Data are presented as mean ± standard deviation. (F) Ictal electroencephalography (EEG) recording of heat-induced seizures from Scn a +/− rats at P . Spiking activity was recorded, and generalized tonic-clonic seizures were observed. Open triangle indicates seizure onset. Asterisk indicates the expanded EEG trace of spiking activity. (G) Representative interictal EEG recordings from wild-type and Scn a +/− rats at P . No interictal epileptic discharge was found. (27), for a maximum of 5 min or until a seizure occurred. When a seizure occurred, the rats were removed immediately from the bath and monitored until recovery. For each rat, we recorded the latency from water contact to seizure onset, the seizure duration, and a score based on the most severe seizure observed. Seizure severity was scored based on Racine stages (28): 0, no response; 1, oral and facial movement; 2, head nodding; 3, forelimb clonus; 4, forelimb clonus with rearing; and 5, generalized tonic-clonic seizures and falling. All procedures were recorded using a video camera. We did not measure body temperature because it was difficult and would be inaccurate to measure body temperature in a water bath.

. . Statistical analyses
Statistical analysis was performed using SAS 9.4 software (SAS Institute Inc.) and GraphPad Prism 8 software (GraphPad Software Inc.). We used ANCOVA to compare T1 post (T1 values resulting from MEMRI T1 mapping) and adjusted for native T1 values (T1 pre ), applying a split-split-plot design under different genotypes, developmental stages, and regions (29). Genotypes and developmental stages were applied to whole plots (animals), and regions were applied to split-split-plots (regions in an animal) in the MEMRI experiment at each stage of development. In the MEMRI experiment with and without bumetanide, genotype and drug (bumetanide or saline) were applied to whole plots (animals), and regions were applied to split-split-plots (regions in an animal). The replicate effect was combined into wholeplot error, including Error(A) and Error(B) (29), and Type III Sum of Squares in SAS was used as the main result. The Scheffé or Tukey multiple-comparison test was performed with split-split-plot error to compare genotypes in each region at each developmental stage or to compare bumetanide and normal saline at each region in each genotype. Differences in seizure phenotypes were assessed using the Mann-Whitney Utest. The p-values were two-sided, and p < 0.05 was considered statistically significant.

. . Generation of Scn a knockout rats
In this study, we developed a global Scn1a knockout rat model using the TALEN-mediated genome editing technique by generating a frameshift in exon 1 of the Scn1a gene ( Figure 1A). TALEN mRNAs targeting Scn1a exon 1 were microinjected into fertilized eggs of F344 rats, and a 94-base-pair deletion in exon 1 was identified in a resulting founder mutant rat (F344-Scn1a em1Kyo ). Reduced Na V 1. . . Scn a knockout rats exhibited key phenotypic features of Scn a-related epilepsies, but were milder than those of DS As previously reported in Scn1a knockout mice (9,(18)(19)(20), Scn1a −/− rats died at approximately the second postnatal week, but no spontaneous deaths were observed in Scn1a +/− rats until at least P200 ( Figure 1D). The mean body weight of Scn1a +/− rats was not significantly different from that of wild-type rats during the experimental period ( Figure 1E). Heat-induced seizures were observed in all investigated Scn1a +/− rats (n = 26) at 45 • C water, a temperature that did not induce seizures in wild-type rats ( Figure 1F, Supplementary Video 1). Up to the third postnatal week, spontaneous convulsive seizures were rarely observed and EEG recordings showed no interictal epileptiform discharges or ictal activities during recording in the Scn1a +/− rats ( Figure 1G). Notably, in older Scn1a +/− rats, some seizures were provoked by acoustic or vibrational stimuli. These findings indicate that Scn1a +/− rats have milder symptoms than those reported in Scn1a +/− mice, suggesting their phenotype may be closer to the genetic epilepsy with febrile seizures plus phenotype.
. . MEMRI revealed increased neural activity in widespread brain regions of Scn a +/rats from P to P To identify disease-specific alterations in regional brain activities of developing Scn1a +/− rats, we employed T1 mapping in a MEMRI experiment. This technique represents the T1 relaxation time as the T1 value for each pixel; decrease in the T1 value in MEMRI indicates increase in neural activity. Several MEMRI studies have used T1 mapping to quantitatively compare brain activity as T1 value or R1(1/T1) and successfully identified regions that show disease-specific changes in brain activity (30,31). Therefore, we applied this technique to quantitatively compare the regional activity of wild-type and Scn1a +/− rats. As T1 values might be influenced by myelination (32, 33), we first investigated T1 pre before performing MEMRI in wild-type (n = 44, body weight range 21-118 g) and Scn1a +/− rats (n = 44, 25-124 g) from P15 to P38 (Supplementary Figure 2). MnCl 2 was intraperitoneally administered immediately afterwards, and MEMRI T1 mapping was performed on the same rats to investigate T1 post (Figure 2A). We compared T1 post values, adjusted for T1 pre , under different genotypes, developmental stages, and regions. Region of interests (ROIs) were defined in 17 areas in the cortex, subcortex, and cerebellum ( Figure 2D). The developmental stages of rats were defined according to a previous report (34): P15-18 (neonatal period), P19-22 (infancy), P23-26 (weaning period), P27-30 (early juvenile period), P31-34 (juvenile period), and P35-38 (prepuberty). If T1 post or T1 pre could not be measured in a region, both values were excluded from the analysis. The sample size for each postnatal day and each ROI is shown in Table 1. Analysis of covariance (ANCOVA) for T1 post showed neither a significant main effect of genotype [F (1,76) = 3.19, p = 0.08] nor an interaction among the developmental stages, genotypes, and ROIs [F (80,1194) = 1.28, p = 0.05]. In Scn1a +/− rats, however, a significant decrease in T1 post was observed compared to wild-type rats at P19-22 in all ROIs, except the cerebellar vermis, by the Tukey multiple-comparison test with respect to the interaction (p < 0.001; cerebellar vermis, p = 0.11), while the decrease was not significant at P15-18 or P23-34 ( Figures 3A, B). At P35-38, a significant decrease reappeared in the hippocampal CA3, lateral habenular nucleus, and basal ganglia (CA3, p < 0.001; lateral habenular nucleus, p = 0.007; globus pallidus, p = 0.05; caudate-putamen, p < 0.001). These results demonstrate that neural activity across widespread brain regions is significantly increased in Scn1a +/− rats at P19-22, which corresponds to the peak onset age of DS in humans (1, 4, 35).
< 0.001). Based on these results, we speculated that the immature inhibitory neural network due to delayed decrease in [Cl − ] i is involved in the increased brain activity in Scn1a +/− rats at P19-22.

. . NKCC inhibitor increased the threshold for heat-induced seizures in Scn a +/rats at P
To investigate whether bumetanide also relieves seizures in Scn1a +/− rats, we examined changes in seizure characteristics in bumetanide-treated Scn1a +/− rats. After the administration of bumetanide (n = 6, body weight range 36-39 g) or normal saline (n = 6, 32-41 g) to Scn1a +/− rats as previously described, seizures were induced on P21 by a 45 • C-hot water bath ( Figure 2C). Seizure latency, seizure duration, and Racine's seizure score were compared between bumetanide-and saline-treated Scn1a +/− rats. Although there was no significant difference in seizure duration (166.  Figure 4B). These results indicate that bumetanide increased the seizure threshold but did not mitigate seizure severity in Scn1a +/− rats.

. Discussion
This study characterized the developmental changes in basal brain activity of Scn1a knockout rats by using MEMRI and identified a transient P19-22 hyperactivity of the brain.
Our newly generated Scn1a knockout rats had a milder phenotype than the Scn1a +/− mice. The Scn1a +/− rats showed a few spontaneous/reflex seizures, but no premature death occurred during the experimental period. Contrastingly, Scn1a +/− mice show spontaneous seizures more frequently from the third postnatal week and showed premature death in about 35% of pups (18)(19)(20), mainly due to status epilepticus and/or malnutrition. These phenotypic differences may be due to species differences and/or epigenetic factors. Generally, rats are reported to have milder epileptic phenotypes than mice (42). However, our knockout rats had a truncating variant in Scn1a, a reduced brain Na V 1.1 protein level, and heat-induced seizures were observed in all Scn1a +/− . /fneur. .  WT rats. n = per group, Sche é multiple-comparison test; *p < . , **p < . , ***p < . . Data are presented as mean ± SEM. (B) Comparison of seizure latency, seizure duration, and seizure score between BTN-treated and NS-treated Scn a +/− rats at P . BTN significantly increased seizure latency of Scn a +/− rats at P . n = per group, Mann-Whitney U test; *p < . . Data are presented as mean ± SEM. BTN, bumetanide; NS, normal saline; P, postnatal day; M , primary motor cortex; CI, confidence interval; M , secondary motor cortex; CPu, caudate-putamen (striatum); LGP, lateral globus pallidus; RSGb, retrosplenial granular b cortex; DG, dentate gyrus; S BF, primary somatosensory cortex, barrel field; Au , primary auditory cortex; VPM, ventral posteromedial thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; nRT, reticular thalamic nucleus; LHb, lateral habenular nucleus; CA , CA field of the hippocampus; V B, primary visual cortex, binocular area; V L, secondary visual cortex, lateral area; Vermis, cerebellar vermis; Hemisphere, cerebellar hemisphere; SEM, standard error of the mean.
rats. Additionally, all Scn1a −/− rats died by the second postnatal week. These findings clearly indicate a significant involvement of the pathogenic process caused by Scn1a defects in our rats. The body weight gain of Scn1a +/− rats was not impaired and their nutritional status was good. Thus, the MEMRI findings in Scn1a +/− rats purely reflect the Scn1a pathology with a minimal influence of spontaneous seizures, status epilepticus, and malnutrition.
MEMRI revealed a significant widespread increase in the basal brain activity of Scn1a +/− rats at P19-22, which corresponds to approximately 6 months of age in humans (34). This is when seizures most commonly develop in DS (1,4,35). Interestingly, the increase in brain activity was not significant in the fourth postnatal week. This temporal pattern of change, with stronger alterations around the onset age, has also been observed in other studies. Favero et al. (43) reported transient firing impairment of parvalbumin-positive GABAergic interneurons in Scn1a +/− mice from P18 to P21, which subsided with age (43). The decrease in background EEG activity in DS mice compared to that in wild-type mice was more significant in the third postnatal week than in any other age (44). These observations indicate that the enhanced functional imbalance during this period due to impaired neuronal development associated with Scn1a defects possibly drives the onset and underlies frequent CSE during the early stage of DS.
The mechanisms underlying the P19-22 brain hyperactivity remain unknown. In wild-type rodents, the second to third postnatal week is the period when dynamic changes in the inhibitory network occurs. The D-H switch is mostly completed by the second postnatal week (36,37,(39)(40)(41). The expression of Na V 1.1 starts to increase during the second postnatal week (7,45). A functional maturation in intrinsic electrophysiological functions of parvalbumin-positive GABAergic interneurons, such as fast spike frequencies and GABAergic synaptogenesis in fast-spiking cells, is achieved during the second to fourth postnatal week in rodents (46-48). The established mechanism of DS, impaired firing of GABAergic interneurons, especially of parvalbumin-positive cells (43), during this period, will be involved in increasing the brain activity. In this study, however, we found that bumetanide improved this hyperactivity and raised the threshold of heatinduced seizures at the third postnatal week but not at the fourth postnatal week. Thus, in the third postnatal week in Scn1a +/− rats may have higher [Cl − ] i compared to wild-type rats, due to a lower expression ratio of KCC2/NKCC1, and attain the same level by the fourth postnatal week. Decrease of [Cl − ] i is a critical maturation step in GABA A receptor signaling represented by D-H switch and the strength of inward Cl − current. Even if the D-H switch is achieved, [Cl − ] i may still remain high as Na V 1.1 gradually increases from the second postnatal week, resulting in a reduced inhibitory response in addition to impaired firing of GABAergic interneurons. This may lead to changes in calcium homeostasis throughout the brain in Scn1a +/− rats. Subsequently, with increased Na V 1.1 expression, [Cl − ] i may decrease and calcium homeostasis may become identical to that of wild-type. It is unclear whether catch-up is achieved in more severe phenotypes such as DS. However, the pathological changes due to Na V 1.1-haploinsufficiency may result in delayed maturation of inhibitory neural networks in the early stage of the disease. To confirm this hypothesis, elaborate electrophysiological analysis, including postsynaptic potentials and equilibrium potentials of Cl − are necessary. In vitro electrophysiological experiments may be significantly influenced by the effects of non-physiological brain conditions, such as the changes in neuronal properties caused by slicing or pipetting, as well as the components of the internal or extracellular solution (49), making it challenging to detect minor electrophysiological changes. Notably, a prior study using electrophysiological recordings from hippocampal CA3 pyramidal cells at P13-21 found giant depolarizing potentials and a depolarized reversal potential for GABA A -evoked currents in Scn1b −/− mice, although this was less evident in Scn1a +/− mice (50). Therefore, it is not known whether depolarizing GABA A receptor signaling remains in Scn1a-related DS during the third postnatal week. Combined with a decreased firing capacity of GABAergic interneurons, the present results suggest that the immature and still-insufficient inhibitory response due to higher [Cl − ] i of postsynaptic neurons may be involved in the transient brain hyperactivity and seizure susceptibility at P19-22.
In our study, increased neural activity in CA3, habenular nucleus, and caudate-putamen in Scn1a +/− rats at P35-38 was suggested. These findings may be related to some clinical features of DS that occur at this age, including epilepsy and nonepileptic manifestations. To elucidate this, a combination of functional neuroimaging, electrophysiology, and behavioral analysis is necessary. It would be also useful to evaluate changes in neural activity in these regions in Scn1a +/− rats using MEMRI by administering several antiepileptic drugs, including carbamazepine, which is known to aggravate seizures in patients with SCN1A loss-of-function mutations.
The limitations of this study are as follows. First, the pathophysiology behind the transient increase in brain activity  observed in the third postnatal week in Scn1a +/− rats has been discussed based on the results of the bumetanide experiments and previous reports but remains a hypothetical interpretation because this has not been directly verified. As mentioned above, it is necessary to conduct high-precision electrophysiological experiments and analyze [Cl − ] i , GABA reversal potentials, and the NKCC1/KCC2 expression ratio in the neurons at the singlecell level. We intend to verify this in future studies. Second, only male rats were included in this study. This was to avoid the influence of sex differences in cerebral development, including the NKCC1/KCC2 expression ratio, which has been documented (40, 41). However, future analyses in female rats are necessary for an accurate understanding of this pathology. Third, to achieve the initial goal of elucidating the pathology of DS, we generated Scn1a knockout rats for high-resolution MEMRI experiments. However, the phenotype of Scn1a +/− rats was found to be milder than that of DS mice. Therefore, the present results showing that the differences in brain activity were no longer significant during the fourth postnatal week could have been different if MEMRI had been performed in a well-established mouse model for DS. Nevertheless, as mentioned above, the Scn1a knockout rats certainly reflect the pathogenesis of Na V 1.1 haploinsufficiency and are less affected by additional factors such as status epilepticus, frequent spontaneous seizures, malnutrition, and premature death. Therefore, the Scn1a knockout rats are expected to be more suitable for studying the pathogenesis of Scn1a-related developmental encephalopathy. The MEMRI findings in Scn1a +/− rats may lead to a better understanding of the pathology directly related to Scn1a deficiency. The Scn1a +/− rats may also be used in the future to investigate various environmental factors that contribute to the formation of the DS phenotype.
In conclusion, we characterized age-dependent changes in brain activity of developing Scn1a +/− rats using MEMRI that allowed high-resolution analysis at the wholebrain level in vivo and visualization of drug-induced changes in brain activity at each region. We believe that MEMRI can be a potential technique to uncover unrecognized aspects of various developmental and epileptic encephalopathies and can be applied to future rodent research.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: figshare, https://dx.doi.org/ 10.6084/m9.figshare.12894698.

Ethics statement
The animal study was reviewed and approved by the Animal Research Committee of Kyoto University and the Institutional Animal Care and Use Committee of the Jikei University School of Medicine.

Author contributions
MT, NH, and JH contributed to the conception and design of the study. MT, NH, JH, MN, KI, SH, TK, TM, TS, and TY performed data acquisition and analysis. MT, NH, JH, MN, and HJO wrote the manuscript and prepared the figures. All authors contributed to the article and approved the submitted version.