FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes

Miyoshi, Goichi; Ueta, Yoshifumi; Natsubori, Akiyo; Hiraga, Kou; Osaki, Hironobu; Yagasaki, Yuki; Kishi, Yusuke; Yanagawa, Yuchio; Fishell, Gord; Machold, Robert P.; Miyata, Mariko

doi:10.1038/s41467-021-23987-z

Download PDF

Article
Open access
Published: 18 June 2021

FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes

Nature Communications volume 12, Article number: 3773 (2021) Cite this article

8779 Accesses
23 Citations
16 Altmetric
Metrics details

Subjects

Abstract

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain

Article 10 April 2024

Control of neuronal excitation–inhibition balance by BMP–SMAD1 signalling

Article Open access 17 April 2024

Molecular and cellular mechanisms of selective vulnerability in neurodegenerative diseases

Article 04 April 2024

Introduction

ASDs are characterized by social communication deficits and restricted/repetitive behavioral patterns (DSM-5), with a prevalence around 1–2% of all children (USA: CDC estimate). While the vast majority of ASDs are thought to emerge through a combination of genetic and environmental risk factors, a percentage (~5%) of ASD cases are syndromic, in that they are caused by highly penetrant mutations in a single gene, such as MECP2 (Rett or duplication), FMR1 (Fragile X) or SHANK3 (Phelan-McDermid)¹. Despite their relative rarity, these syndromic forms of ASD have provided key insights into the broader etiological basis of ASD. Of particular interest, many syndromic ASD models show an increase in the cortical synaptic excitation-inhibition (E/I) ratio, a phenomenon that has been hypothesized to underlie the cognitive and behavioral symptoms of ASD^2,3.

Inhibition mediated by cortical GABAergic interneurons is known to regulate animal social behaviors⁴ and abnormal GABAergic signaling has been implicated in both schizophrenia and ASD^2,3,5,6. Loss of inhibitory neurons has been observed in postmortem ASD patient studies⁷ and consistent with this, children with ASD are more likely to have epilepsy. In ASD modeling studies, conditional mutations of syndromic ASD genes in GABAergic populations often reproduce the ASD phenotypes found in the respective null animals^8,9. Developmentally, GABAergic systems are known to regulate circuit plasticity and are capable of accelerating or delaying the onset of the visual critical period¹⁰. Furthermore, transplantation of GABAergic neuronal precursors has been shown to induce plasticity in the mature visual cortex¹¹. However, how GABAergic circuit dysfunction during development contributes to adult ASD phenotypes is still not well understood. Indeed, the elevated cortical excitatory-inhibitory ratio observed in syndromic ASD models has been recently reported not to reflect network hyperexcitability per se, but rather is a homeostatic compensation of synaptic drive in the adult circuits¹². Thus, investigating how perturbations in inhibitory circuit development contribute to the etiology of ASD should help innovate therapeutic approaches.

Recently, FOXG1, a critical transcription factor for forebrain development^{13,14,15,16,17,18,19,20,21} has been linked to ASD and other neurodevelopmental disorders such as schizophrenia²². Studies in patient-derived iPS cells have suggested that FOXG1 dysregulation broadly contributes to the etiology of idiopathic ASD²³. Furthermore, perturbation of FOXG1 has been implicated in a new type of syndromic ASD (FOXG1 syndrome), with gene duplications (gain-of-function)^24,25 or loss-of-function mutations (heterozygous haploinsufficiency)^26,27,28,29 observed in affected patients^30,31,32. Moreover, recent advances in sequencing techniques have revealed FOXG1 point-mutations in idiopathic ASD children (FOXG1 Research Foundation), suggesting that there are more undiscovered FOXG1 syndrome patients world-wide. However, both when during development and in which brain circuits FOXG1 dysregulation contributes to ASD etiology have not been elucidated.

Here we demonstrate that neuronal FoxG1 dysregulation precipitates ASD-related social impairments in our mouse models when FoxG1 expression changes are paired in both excitatory and inhibitory populations. We find that the second postnatal week is a critical period for developing ASD phenotypes upon FoxG1 dysregulation. Indeed, at the end of this critical period, the cortical excitatory-inhibitory ratio is already imbalanced in all three variants of FoxG1 Low and High ASD models. We show that the GABAergic system plays pivotal roles during the critical period by demonstrating that weakening or supplementing cortical GABAergic tone exacerbates or ameliorates the social behavioral impairments of the FoxG1 ASD model, respectively. We thus identify both the critical time-window and inhibitory circuit mechanisms that lead to ASD-related social behavioral impairments upon FoxG1 dysregulation. Our results support the promise of therapeutic approaches to ameliorate ASD phenotypes through the early intervention of cortical GABAergic systems.

Results

A rodent FOXG1 syndrome model exhibiting ASD-like social behavioral impairments

In order to address the developmental circuit mechanisms underlying the social behavioral alterations found in ASD patients, we first examined if ASD FOXG1 syndrome could be recapitulated in rodents to model the disease phenotypes. We utilized the three-chamber assay to test for ASD-related unconditioned social behaviors^{4,8,33,34,35,36,37} in FoxG1 Heterozygous adult animals (6 weeks old)¹³. In this behavioral assay, animals are first allowed to freely explore the three chambers connected by corridors (Fig. 1a, Habituation). Next, an age and gender-matched stranger wild type mouse is introduced in a cage located in one chamber, allowing the test animal to make a choice between exploring a chamber with a novel mouse versus an empty cage (Fig. 1a, Sociability). In the third session, preference of the test animal to explore the chamber with an already familiar mouse versus the chamber with a newly introduced stranger mouse is compared (Fig. 1a, Social Novelty). As expected, wild type animals spent a significantly increased amount of time in the social side of the chambers in both sociability (Fig. 1b, open orange bar) and social novelty assays (filled orange bar). However, FoxG1 Heterozygous animals showed no obvious preference for the social side of the chamber (Fig. 1b, left versus right chamber) and instead spent a significantly increased amount of time in the center chamber (Fig. 1b, compare filled purple bars of Nov), suggesting decreased sociability, consistent with ASD-like social behavioral deficits. Indeed, the sociability score was found to be decreased in the FoxG1 Heterozygous animals (Fig. 1c, scored based on the orange bar graphs in 1b).

**Fig. 1: The FOXG1 syndrome Heterozygous model mouse displays ASD-like social behavioral impairments.**

We carried out a battery of additional behavioral analyses of this FoxG1 Heterozygous mouse model to test whether there were other behavioral abnormalities beyond the impairments in contextual fear memory reported previously³⁸. Briefly, while general motor functions of the Heterozygous animals were comparable to the controls (Fig. 1d), Heterozygous animals showed decreased anxiety (Fig. 1e) and impairments in working memory (Fig. 1f).

Neither the loss of GABAergic cells nor microcephaly per se causes ASD social phenotypes in FoxG1 haploinsufficient animals

Haploinsufficient FOXG1 syndrome ASD patients exhibit microcephaly^27,28, and likewise, reduction in the size of the cerebral hemispheres has been reported in at least three different FoxG1 Heterozygous mouse models^38,39,40 and was observed here (Fig. 2a, b). In addition, consistent with the cases reported in ASD postmortem studies⁷, we further found a reduction in GABAergic neuron numbers within the medial prefrontal (prelimbic and infralimbic) and somatosensory barrel cortices in these animals (Fig. 2d–g, k). We hypothesized that the microcephaly phenotype is due to the role of FoxG1 in progenitor proliferation within the developing forebrain^18,19,41,42. We thus preserved FoxG1 expression in progenitors (Fig. 2c) by utilizing Cre drivers for postmitotic glutamatergic (Nex-Cre)⁴³ and GABAergic (Dlx-Cre)⁴⁴ neurons of the forebrain and used them in conjunction with one copy of a conditional loss-of-function FoxG1 allele²⁰ (Fig. 2l). Indeed, in this postmitotic neuronal Heterozygous model, both cortical thickness as well as GABAergic cell density were rescued to levels comparable to control animals in both medial prefrontal and somatosensory barrel cortices (Fig. 2h–k). Importantly, this neuronal postmitotic FoxG1 haploinsufficiency model also spares glial FoxG1 expression^45,46,47 unlike the Heterozygous model (Fig. 2c).

**Fig. 2: ASD FOXG1 syndrome social phenotypes are due to neuronal *FoxG1* haploinsufficiency, but not microcephaly or loss of GABAergic cells.**

We first asked if neuronal FoxG1 haploinsufficiency is sufficient to reproduce the ASD-phenotypes observed in FoxG1 Heterozygous animals (Fig. 2m, Glu+GABA Low, traces in Supplementary Fig. 1). Indeed, FoxG1 haploinsufficiency in both pyramidal and GABAergic neurons led to social behavioral impairments in a manner comparable to the Heterozygous model (Fig. 1b). This suggests that neither microcephaly nor loss of GABAergic cells causes social behavioral impairments per se. We next asked if ASD-related social behavioral deficits originate from excitatory or inhibitory circuit mechanisms upon removal of one copy of FoxG1. Reduction of FoxG1 levels in postmitotic pyramidal cells alone did not affect sociability (Fig. 2m, Glu Low). GABA Low littermate animals unexpectedly exhibited an increased preference for social novelty, with animals spending significantly more time in chambers containing mice in comparison to controls (Fig. 2m, GABA Low, orange asterisks). Thus, our results indicate that in FOXG1 haploinsufficiency ASD patients, reduced sociability is due to FOXG1 hypofunction in both excitatory and inhibitory postmitotic neurons.

ASD phenotypes in haploinsufficient and duplication FOXG1 syndrome models arise from paired FoxG1 changes in both excitatory and inhibitory neurons

Increased FOXG1 gene copy number can also result in ASD symptoms^24,25; thus, we sought to examine how FoxG1 gain-of-function could affect social behavior. Considering that FoxG1 gain-of-function can disrupt the migration of pyramidal neurons²⁰, we chose to augment FoxG1 levels during the early postnatal period after most neuronal migration is complete (~1st postnatal week; P7)⁴⁸. We again utilized Cre drivers specific for excitatory and inhibitory lineages in the forebrain, here combined with a conditional transactivator line (R26-stop-tTA) and a transactivator-dependent FoxG1 expression allele (TRE-FoxG1), and FoxG1 gain-of-function was initiated only after P7 (Fig. 3a). Using the three-chamber assay, we found that animals exhibited social behavioral deficits only when FoxG1 is augmented in both Glu+GABA lineages simultaneously (Fig. 3b, traces in Supplementary Fig. 2). This outcome in the sociability scores is similar to that observed upon reduction of FoxG1 expression (Fig. 3c), albeit with a milder phenotype in social novelty sessions compared to the Heterozygous (Fig. 1b) or neuronal Glu+GABA Low models (Fig. 2m). In sum, we conclude that either up- or downregulation of FoxG1 expression levels impairs social behavior, but only when the perturbation occurs in both excitatory and inhibitory populations (Fig. 3c, purple bars).

**Fig. 3: ASD social phenotypes arise from a balanced decrease or increase of *FoxG1* in excitatory and inhibitory populations.**

Upon establishing three ASD FOXG1 syndrome models of Heterozygous, Low (neuronal Glu + GABA Low) and High (1 week Glu + GABA High), in addition to the social behavioral evaluations, we cross-correlated other behavioral features and cortical EEG patterns. In contrast to the FoxG1 Heterozygous animals (Fig. 1f), no working memory deficits were observed in FoxG1 Low and High models (Fig. 3d, see also Supplementary Fig. 3). FoxG1 Heterozygous animals are often aggressive, and attack siblings or partners during mating, but these behaviors were also not evident in the Low and High models. In addition, no clear anxiety phenotype was observed in the FoxG1 Low and High models in the open field and elevated plus maze assays (Fig. 3e, f, see also Supplementary Figs. 1 and 2). These results indicate that working memory impairment, aggression or anxiety does not account for the social behavior abnormalities observed in our FoxG1 Low and High perturbation models.

We next tested if the gamma-band frequency of EEG is attenuated in a manner similar to ASD patients^49,50 or in rodent models^4,51,52. We performed EEG recordings during the awake free-moving state with the electrodes aligned nearby the medial prefrontal cortex (Fig. 3g to j, Supplementary Fig. 4). In the control 8 weeks old wild type mice, the power spectrum peak in the high-frequency range was found nearby 58 Hz (Fig. 3k, orange trace). However, this peak was reduced as well as shifted to a lower frequency range in the Heterozygous model (Fig. 3k, black trace), with peak amplitude reduced in the Low model (Fig. 3k, gray trace), or the peak shifted to lower frequency in the High model (Fig. 3k, blue trace), resulting in overall reduction in high-gamma frequency power (55–80 Hz) in these socially impaired FoxG1 models (Fig. 3l). While epilepsies have been observed in some human FOXG1 syndrome patients^{24,26,27,28,30,31}, we did not detect any obvious epileptic behavior in any of our FoxG1 perturbation models.

In order to verify the expected alterations in FoxG1 expression in our socially impaired FoxG1 mutant models, we measured the FoxG1 protein levels within the medial prefrontal cortex at postnatal 2 and 6 weeks by western blotting (Fig. 3m–o). By removing one copy of FoxG1, FoxG1 protein levels were reduced to approximately half of that in the control animals in both Heterozygous and Low models (Fig. 3o). This result indicates that a compensatory upregulation of FoxG1 mRNA transcription likely does not occur in haploinsufficient FOXG1 patients either. Already at postnatal 2 weeks, FoxG1 High manipulation at 1 week was sufficient to increase FoxG1 protein levels to that observed in 6-week old animals (Fig. 3o). We further validated our gain-of-function method by confirming that the increase of FoxG1 is suppressed by continuous feeding of doxycycline (Fig. 3o, +Dox). We conclude that our genetic strategies enabled precise manipulations in FoxG1 protein levels in our model animals.

An early juvenile ASD critical period identified through temporal FoxG1 dysregulation and imbalanced excitatory-inhibitory circuits

While excess copy number of FOXG1 has been identified in syndromic forms of ASD, the ectopic upregulation of FOXG1 expression has been suggested to be a key contributor to disease etiology in idiopathic ASD patients²³. We thus took advantage of our doxycycline-repressible gain-of-function system (Fig. 3a) and carried out FoxG1 High (Glu + GABA) manipulations during selective postnatal time-windows (Fig. 4a). When FoxG1 augmentation is delayed for 2 weeks (Fig. 4a, 3w High), instead of 1 week (Figs. 4a and 3b), this no longer resulted in social behavioral impairments (Fig. 4a), demonstrating that FoxG1 augmentation per se does not affect animal social behavior. Thus, we performed FoxG1 augmentation exclusively between 1 and 3 weeks by replacing the doxycycline diet with a regular one only during this period and found that these animals (1–3w High) showed alterations in sociability to a similar extent as 1 week High animals (Fig. 4a, scores compared in Fig. 4b). We further carried out FoxG1 augmentation between 1 and 2 weeks or 2–3 weeks (Fig. 4a, 1–2w and 2–3w High) and discovered that between 1 and 2 weeks is the critical time-window for FoxG1 augmentation (Fig. 4b). Western blotting analysis revealed that the changes in FoxG1 protein levels occur within 2 days upon addition or removal of the doxycycline-containing diet (Supplementary Fig. 5d).

**Fig. 4: An early juvenile ASD critical period is revealed by timed *FoxG1* dysregulation and imbalanced excitatory-inhibitory circuits.**

Upon identifying a critical period for FoxG1 dysregulation, we hypothesized that there would be a shared circuit deficit found across all three FoxG1 ASD models particularly during this time-window. To this end, we evaluated excitatory-inhibitory (E/I) balance by performing slice electrophysiological recordings of mEPSC and mIPSC events in layer 2/3 pyramidal neurons of the medial prefrontal cortex (prelimbic and infralimbic, Fig. 4c, right) in our three ASD FOXG1 syndrome models of Heterozygous, Low (neuronal Glu + GABA Low) and High (1 week Glu + GABA High). Upon examining 2-week old juvenile animals, we found that the E/I ratio within the medial prefrontal cortex was altered in all of these ASD models (Fig. 4d, e). However, to our surprise, and distinct from other rodent ASD models¹², we found no obvious change in the E/I balance within the adult medial prefrontal cortex in our FoxG1 ASD models (Fig. 4f–h). In fact, not just the E/I ratio but the values for the mEPSC and mIPSC were comparable to that of controls (Fig. 4g, h). The exception was a slight decrease in the E/I frequency ratio in the Heterozygous medial prefrontal cortex due to a reduced mEPSC frequency (Fig. 4g), although this was not the case in the barrel field (Supplementary Fig. 6). These data suggest that, in our FoxG1 perturbation ASD models, although the transiently imbalanced E/I ratio within the juvenile medial prefrontal cortex normalizes by adult ages, this results in permanent circuit changes as indicated by altered gamma EEG power (Fig. 3l) and impaired social behavior (Fig. 3c).

Decreased GABAergic activity exacerbates ASD social phenotypes in the FOXG1 syndrome model

In the FoxG1 Heterozygous model, the E/I ratio in the cortical circuit is transiently increased immediately after the critical period (Fig. 4d). We thus hypothesized that this model precipitates ASD phenotypes through a decrease in inhibitory GABAergic tone. In order to test this, we first addressed the roles of GABAergic transmission for the development of ASD phenotypes by analyzing Gad2 mutant animals^53,54 in the three-chamber assay (Fig. 5a). In contrast to its isoform Gad1, Gad2 predominantly resides at presynaptic terminals to synthesize GABA locally. Thus, removal of the Gad2 gene effectively reduces overall GABAergic synaptic tone and its loss has been shown to modify the visual critical period^10,55. In the three-chamber sociability assay, while we found no obvious changes in the Gad2 heterozygotes (Fig. 5b), Gad2 nulls did not show a preference for the social side of the chamber (Fig. 5b, NS purple). During the social novelty session, although Gad2 null animals still preferred the chamber with a novel animal, they spent more time in the center chamber (Fig. 5b, purple asterisks) and thus their overall sociability score was reduced (Fig. 5d). These data suggest that the reduction of GABAergic tone in Gad2 nulls leads animals to develop ASD-like social behavioral impairments. Interestingly, the social behavior score of the Gad2 nulls was decreased to a level comparable to the FoxG1 Heterozygous model (Fig. 5d purple bars).

**Fig. 5: Decrease in GABAergic tone exacerbates the ASD social phenotypes of the *FoxG1* Heterozygous model.**

We next analyzed the consequence of reducing GABAergic transmission in the FoxG1 Heterozygous background by cross-comparing the Gad2 littermate animals (Fig. 5c, d). In the FoxG1 Heterozygous background, both Gad2 heterozygous and null animals showed no preference to the novel animal (Fig. 5c) and preferred the center chamber in the social novelty session (Fig. 5c), suggesting that they exhibit ASD-like social impairments. Moreover, both Gad2 heterozygous and null mutations further reduced the sociability score of the FoxG1 Heterozygotes (Fig. 5d dark bars). We further found that reduction of GABAergic tone does not affect the working memory of the animals (Fig. 5e) in contrast to the sociability (Fig. 5d). These data suggest that reducing GABAergic tone exacerbates the social behavioral alterations found in the ASD FoxG1 haploinsufficiency mouse model. This further supports the idea that the FoxG1 Heterozygous model develops ASD phenotypes through alterations in GABAergic systems during the early juvenile critical period.

Augmentation of GABAergic systems during the juvenile critical period re-balances the cortical E/I ratio and ameliorates the social impairments of the FoxG1 ASD model

In the FOXG1 syndrome haploinsufficiency model, cortical inhibition is reduced during the critical period (Fig. 4d, e), and further attenuation of GABAergic tone exacerbated the social impairments of this model (Fig. 5). We thus hypothesized that a proper E/I ratio during the early juvenile critical period is important for the development of ASD phenotypes, and that augmentation of GABAergic tone during this period might re-adjust the E/I imbalance and improve the behavioral outcomes of FoxG1 Heterozygotes. In order to test this, we focused on the mPFC, a brain region implicated in social behavior⁴, and we took advantage of the GABAergic cell precursor transplantation technique. This method has been shown in several contexts to therapeutically modify recipient mature neural circuits by transplanted donor cells forming new inhibitory synapses and creating circuit plasticity^11,56,57,58. We dissociated EGFP-labeled GABAergic cell precursors (Dlx-Cre; RCE:loxP^59,60) from the E13.5 medial ganglionic eminences (MGE) that are wild type for FoxG1, bilaterally transplanted these cells into the postnatal 1 week medial prefrontal cortices, and analyzed E/I ratios at 2 weeks of age (Fig. 6a, scheme). When the miniature frequencies of mPFC layer 2/3 cells were analyzed one week after the transplantation (Fig. 6b), we found that the increased E/I ratio in the Heterozygous animals was rescued to wild type levels upon receiving GABAergic neuron precursor transplantation (Fig. 6c, NS in orange). Consistent with this, GABAergic precursor transplantation did not attenuate the peak amplitude E/I ratio of the Heterozygous models (Fig. 6d), suggesting that GABAergic cell precursor transplantation at one week normalized the excitatory-inhibitory local circuit balance in the Heterozygous animal model to levels comparable to wild type. We further confirmed that the rescue of E/I balance at 2 weeks in Heterozygotes upon GABAergic cell precursor transplantation restores the E/I balance to normalcy in adulthood (Fig. 6e, f).

**Fig. 6: Normalization of the mPFC E/I balance by incrementing GABAergic tone during the critical period ameliorates social phenotypes in the *FoxG1* ASD model.**

We next tested if correcting the E/I balance of the mPFC at two weeks would rescue the adult ASD-like social behavioral impairments of the Heterozygous animals. In the three-chamber assay (Fig. 6g and j), GABAergic cell transplantation into wild type animals did not overtly affect the sociability of the animals at 6 weeks and was comparable to non-transplanted wild type (Fig. 1b) or other control animals (Figs. 2m and 3b). When FoxG1 Heterozygotes received GABAergic cell precursors into the mPFC at postnatal 1 week (Fig. 6a and h), the animals spent comparable time in the social side of the chamber to the controls during the sociability session (Fig. 6j, NS in orange). During social novelty sessions, cell-transplanted Heterozygotes preferred the stranger side (Fig. 6j, asterisks in orange) as well as spent comparable time in the center chamber to the controls (Fig. 6j, NS in orange). These data suggest that re-balancing the E/I ratio of the mPFC at 2 weeks (Figs. 6c, d) by GABAergic cell precursor transplantation allows FoxG1 Heterozygous animals to recover from the ASD-like socially impaired state. Furthermore, this is consistent with the notion that FoxG1 must be simultaneously disrupted in both excitatory and inhibitory populations to precipitate ASD behavioral phenotypes (Fig. 3c), since the transplanted GABAergic cells are wild type for FoxG1. Next, in order to test the importance of correcting the circuit E/I ratio at postnatal 2 weeks, we further carried out GABAergic cell transplantation at 3 weeks, instead of 1 week (Fig. 6i). We found that the social behavioral phenotypes of the late-transplanted Heterozygous animals at 6 weeks are distinct from the 1 week-transplanted wild type or Heterozygous animals (Fig. 6g–j) and are comparable to the non-transplanted Heterozygotes (Fig. 1b, scores in Supplementary Fig. 7e). This was consistent even later at 8 weeks (Supplementary Fig. 7f), upon matching the differentiation period of the donor cells to the postnatal 1 week transplantation experiments. These data further support our notion that between postnatal 1–2 weeks of age is the critical time-window to mitigate ASD-related social phenotypes. When working memory was tested, cell transplanted Heterozygotes did not show obvious improvements compared to the non-transplanted Heterozygotes (Fig. 6l, NS), suggesting that GABAergic cell precursor transplantation into the mPFC selectively ameliorates the social-related modality but not the general working memory deficits of the Heterozygous model.

Altogether, we conclude that precisely regulated FoxG1 expression levels during early juvenile development is crucial, and dysregulation of FoxG1 expression during this period results in a transient perturbation of E/I balance, resulting in ASD-like gamma EEG patterns as well as social behavioral deficits in adult animals. For the ASD FOXG1 syndrome haploinsufficiency model, in which local inhibitory tone is decreased during the early juvenile critical period, a decrease in GABAergic activity exacerbates the social behavior deficits, whereas re-balancing the E/I ratio by enhancing GABAergic tone ameliorates the social impairments (Fig. 6k, scores compared), thus pinpointing this developmental stage as promising for therapeutic intervention.

Discussion

The developing brain possesses a remarkable resilience to genetic and/or environmental insults, and has the intrinsic means to adjust its functional properties by scaling excitatory and inhibitory neuronal activity through homeostatic regulation¹². It has been hypothesized that neurocognitive disorders such as ASD arise when the brain’s capacity to buffer the cumulative effect of genetic and/or environmental perturbations on synaptic homeostasis during development is exceeded⁶¹. Here in this study, we identified that the second postnatal week is the critical period to develop alterations in inhibitory circuits and to precipitate ASD behavioral phenotypes in mature animals. We further demonstrate that behavioral abnormalities in both haploinsufficiency and gene duplication models of FOXG1 syndrome are caused by the simultaneous disruption of FOXG1 levels in excitatory and inhibitory neuronal circuits. To our surprise, while the E/I balance in cortical circuitry was only transiently disrupted during the critical period in all of our socially impaired FoxG1 animal models (Heterozygous, Low and High), this perturbation was sufficient to cause behavioral symptoms in adults. We conclude that inhibitory circuit development during the critical period is a key aspect of ASD etiology, and that weakening or augmenting GABAergic tone during this period exacerbates and ameliorates the respective adult ASD social phenotypes.

It was only recently discovered that FOXG1 duplication and haploinsufficiency both lead to syndromic forms of ASD^{24,25,26,27,28,30,31,32}. Here, we demonstrate that the dose-dependent aspect of FOXG1 syndrome can be modeled in the mouse by implementing our novel genetic strategies. To our surprise, balanced dysregulation of FoxG1 in both excitatory and inhibitory circuits was necessary to develop ASD-related social behavioral alterations. This is in contrast to what has been shown in conditional mutation studies of syndromic ASD genes such as Mecp2 and Ube3a^8,9. Our current hypothesis is that proper FoxG1 levels in both excitatory and inhibitory neurons are necessary to maintain circuit homeostasis. Our cell transplantation study supports this notion since addition of wild type MGE-derived⁶² GABAergic precursors into the FoxG1-Heterozygous neuronal networks ameliorates the social behavioral deficits (Fig. 6). Importantly, the GABAergic populations affected in our FoxG1 Low and High manipulations include the local interneurons in the hippocampus, cortex, amygdala and striatum as well as striatal projection neurons, whose malfunction has been also implicated in ASD etiology^34,63. It will thus be interesting in the future to investigate the contribution of each forebrain GABAergic cell type⁶⁴ to the ASD-like social behavioral phenotypes in our FoxG1 model animals. Considering the central role of FoxG1 in regulating forebrain development, and its dynamic activity during discrete phases of circuit maturation²⁰, it is likely that FoxG1 is an important component of the brain’s homeostasis machinery. Thus, perturbations in FOXG1 expression result in a failure of homeostasis that may represent a common etiological mechanism for ASD. Consistent with this idea, FOXG1 may participate in the regulation of mitochondrial function⁶⁵, which presents another potential link to ASD etiology^66,67. In total, we conclude that FOXG1 likely regulates multiple sets of target genes in a manner analogous to other ASD-related transcriptional regulators such as MECP2^68,69 and CHD8^70,71.

While FOXG1 haploinsufficiency and duplication have both been classified as syndromic forms of ASD, there are differences found in the disease phenotypes^{24,25,26,27,28,30,31,32}. Consistent with this, our mouse models for FoxG1 haploinsufficiency (Het and Low) and augmentation (High) show distinct phenotypic outcomes. While the sociability scores of the Het, Low and High models are comparable (Fig. 3c), High models do not show obvious deficits in social novelty assays (Figs. 3b and 4a). In addition, the High model shows a decrease in the E/I amplitude but not the frequency at juvenile stages (Fig. 4d, e) and the values for gamma EEG are not identical to the haploinsufficiency models (Fig. 3l). Thus, while FOXG1 augmentation has been proposed to be etiologically relevant in idiopathic ASD patients²³, FOXG1 haploinsufficiency could be a distinct syndrome within the spectrum of ASD. Similar to FOXG1, copy number variants in MECP2 with an extra copy (Duplication syndrome) or deletion (Rett) have both been observed in syndromic ASD patients^1,72. While Mecp2 and FoxG1 have been shown to interact directly in neuroprotection studies⁷³, the clinical presentations of FOXG1 and MECP2 related syndromic ASDs are distinct⁷⁴. Consistent with this idea, in contrast to the Mecp2 mutants, FoxG1 adult mouse models do not exhibit increases in cortical E/I activity.

It has long been suggested that GABAergic neuron dysfunction contributes to the pathophysiology of ASD^2,3,5,6. Although a central role for the critical developmental period of inhibitory circuits in ASD etiology has not been proposed, recently, by using a juvenile social isolation paradigm, the proper maturation of Pvalb-positive interneurons in the mPFC has been shown to be important for animal social behavior⁷⁵. While this study highlights the social experience- (3 to 5 weeks) and interneuron-dependent (Pvalb subtype) critical period of mPFC for social behavior, our study defines an innate developmental critical period for mPFC social circuits and ASD. Interestingly, by analyzing intrinsic electrophysiological properties⁷⁶, we found clear alterations in putative Pvalb subtype (Fast-spiking) interneurons of the FoxG1 Heterozygous model already at two weeks, in contrast to pyramidal cells that were found to be abnormal at later timepoints (Supplementary Fig. 8).

Cortical activity during early postnatal development has been shown to facilitate cortico-striatal coupling, and decreasing the cortical hyperactivity observed in the Shank3B mutant ASD model mouse during this period was able to rescue the striatal phenotypes⁶³. Moreover, in rodent ASD models, the connectivity between the mPFC and areas such as the striatum, basolateral amygdala and ventral tegmental area has been proposed to be disrupted⁷⁷. By analyzing Gad2 mutant animals, we provide direct evidence that a decrease in overall GABAergic synaptic tone leads to ASD-like social behavioral impairments, to a comparable extent as observed in the FOXG1 syndrome model. In addition, our Gad2 mutant studies revealed that a reduction in GABAergic tone further exacerbates the ASD social phenotypes of the FoxG1 model. This strongly supports the notion that transient circuit E/I imbalance during the critical period does not simply reflect homeostatic compensation per se, but rather is a causative driver of ASD phenotypes in adult FOXG1 syndrome model animals. Thus, it will be interesting to investigate the E/I balance in juvenile animals of other established ASD models¹². Consistent with mPFC circuits playing central roles in regulating social behavior, social impairments have been rescued in several contexts by directly manipulating mPFC activity while animals are executing behavioral tests^4,51,52,75. Here in this study, we were able to developmentally ameliorate the sociability deficits in our ASD model animals. Thus, our findings on the putative ASD critical period pinpoints a developmental stage in GABAergic circuits that offers a possible route for therapeutic intervention.

Methods

FoxG1 perturbation model animals

All animal handling and experiments were performed in accordance with protocols approved by the respective Institutional Animal Care and Use Committees of the NYU School of Medicine, Tokyo Metropolitan Institute of Medical Science and Tokyo Women’s Medical University. Animal cages are maintained at 22 °C ± 1 °C, 50 ± 15% humidity with a 12 h light/dark cycle. To generate the FoxG1 Heterozygous model, we combined a FoxG1 LacZ knock-in null allele¹³ with the wild type FoxG1 allele. FoxG1 Low models were generated by crossing male Nex-Cre⁴³; Dlx-Cre animals⁴⁴ with female FoxG1-C/C; R26-CAG-FRTed stop-EGFP homozygous animals⁵⁹ (RCE:FRT, Jackson Laboratories stock #10812). Upon Cre-mediated recombination, the conditional floxed-FoxG1 allele is converted into a Flpe knock-in allele²⁰, and thus the recombination efficiency in each animal can be evaluated by EGFP expression from the Flp-dependent EGFP reporter RCE:FRT. FoxG1 High models were generated by crossing male Nex-Cre; Dlx-Cre animals to female R26-stop-tTA homozygous⁷⁸ (Jackson Laboratories stock #008600); TRE-FoxG1 homozygous animals⁷⁹. In order to delay FoxG1 augmentation until after postnatal 1 week, a doxycycline-containing diet (200 PPM, #1810413 Test Diet) was provided to the pregnant females from the date of the observed plug until postnatal day 7. 3 week High and 1–3 week High models were generated by replacing the doxycycline diet with a regular one at P21 and during P7 to P21, respectively. Other variations of 1–2 and 2–3 week High animals were generated in a similar manner. The TRE-FoxG1 allele contains an IRES-LacZ cassette downstream of the FoxG1 coding sequence and thus, the corresponding FoxG1 GOF population can be visualized based on X-gal staining or by using anti-beta-galactosidase immunohistochemistry. In order to carry out littermate studies for FoxG1;Gad2 compound mutants, double-heterozygous male FoxG1-LacZ; Gad2 animals were crossed to female Gad2 heterozygotes^53,54.

Tissue preparation and immunohistochemistry

Embryos were dissected out, and transcardiac perfusion was performed with cold 4% formaldehyde/PBS solution (w/v). Brain dissection was carried out in PBS in a petri dish, and brains were subsequently placed into 4% formaldehyde/PBS solution on ice. The P7 to P21 as well as adult brains were also perfused with the same fixative. Brains were postfixed according to developmental stages (E14.5: 20 min, E16.5: 40 min, E18.5 onwards: 60 min) and, following a brief rinse in PBS, were placed into cold 25% sucrose/PBS (w/v) solution for cryoprotection until they sunk to the bottom. Subsequently, brains were mounted in OCT compound (Sakura Finetek) and stored at −80 °C. Cryosections were prepared at 12 μm thickness and collected on glass slides (Fisher or Matsunami), and, after over 1 h of drying, the sections were stored at −80 °C. Immunohistochemistry was performed similarly to previously described^20,59,60. All reactions were carried out in PBS containing 1.5% normal donkey serum (Jackson Immunoresearch) (v/v) and 0.1% Triton X-100 (v/v) (DST solution), which we generally keep at 4 °C up to two weeks. By using a liquid blocker pen (#Z377821, Sigma), two lines are placed in between the section nearest to the frost of the slide and the frost to restrict the movement of liquid on the slide glass. Sections were rinsed 3 times in PBS to remove residual OCT, followed by non-specific antibody blocking, which was carried out in DST solution at room temperature for 30–60 min. Primary antibody incubation was subsequently performed overnight at 4 °C. Primary antibody was included in the DST solution at the following concentrations: Rabbit anti-GFP (1:2000; Molecular Probes, #A11122), Rat anti-GFP #GF090R (1:2000; Nacalai Tesque, #04404-84), Rabbit anti-RFP Living Colors DsRed Polyclonal Antibody (1:2000; Takara Bio Clontech, #632496), Rat anti-RFP #5F8 (1: 2000; Allele Biotechnology, #ACT-CM-MRRFP10). Secondary antibodies conjugated with Alexa Fluor dyes 488 or 594 for anti-Rabbit Alexa 488 (Invitrogen, #A-21206), anti-Rat Alexa 488 (Invitrogen, #A-21208), anti-Rabbit Alexa 594 (Invitrogen, #A-21207), anti-Rat Alexa 594 (Invitrogen, #A-21209) raised in donkey and cross-adsorbed against other species were used in 1:2000 dilution for fluorescent immunohistochemistry. In order to visualize the cell nuclei and laminar structure of the developing neocortex, DAPI (Sigma) (1 ng/µl in PBS and filtrated) was added on slides for 20–30 min following the immunohistochemistry staining procedure described above. Slides were rinsed a couple of times in PBS and then coverslipped using Fluoromount G (Southern Biotech). Fluorescent images were captured with a Zeiss AxioScope.A1 (Carl Zeiss) by using a cooled-CCD camera (Princeton Scientific Instruments, NJ) with Metamorph software (Universal Imaging, Downingtown, Pennsylvania) or by using a QSI RS 6.1s cooled-CCD camera (2758×2208 pixels, QSI) with micro-manager (open source software). Fluorescent in situ hybridization was carried out by using a Gad1 anti-sense probe and by visualizing signals with Alexa Fluor 594 Tyramide Reagent (#B40957, Thermo Fisher Scientific) following a similar protocol similarly to previously described⁸⁰. In order to facilitate the visualization of gene expression, acquired images were inverted in Photoshop (Adobe) and subsequently combined to generate the figures. For example, tdTomato fluorescent images were placed into both the green and blue channels of the RGB format file and then all color channels were simultaneously inverted to generate a figure with red signals in the white background. For a multicolor presentation, layers including different color signals were assembled by using the function of multiply layers.

Western blotting of mPFC tissue

Brain slices were prepared in a similar manner to the in vitro electrophysiological recordings (300 µm thickness) or by generating a coronal slice with a cutting blade (typically ~400 µm). In the petri dish, mPFC was bilaterally dissected out from the slice with forceps, placed into a 1.5 ml tube and immediately stored in −80 °C freezer. After obtaining tissue samples from the Het, Low and High models at various postnatal stages, and in addition, E16 embryonic cortical Het and Null tissue as positive controls, protein lysate was prepared. For each sample, 200 µl of SDS lysis buffer (1% SDS, 10 mM Tris pH7.5, 5 mM EDTA, 1tablet/10 ml Protease inhibitor cocktail (Roche: 11-836-153-001), 1tablet/10 ml PhosSTOP (Roche: 04-906-837-001)) was added and homogenized with pipetting. After vortex mixing the samples, tubes were centrifuged for 15 min with 20,630 × g/15,000 rpm and subsequently, the supernatant protein concentration was analyzed by using the BCA Protein Assay Kit (Thermo Scientific #23227). Total 4 µg of protein lysate from each sample was applied to the well of Mini-Protean TGX 4–15% Gel (BIORAD #456-1086) for western-blotting analysis. We initially compared three FoxG1 antibodies of Rabbit anti-FoxG1 (Neuracell, #NC-FAB), Rabbit anti-FoxG1 (Abcam, #ab18259) and Rabbit anti-FoxG1 monoclonal # EPR18987 (Abcam, #ab196868) in 1:10,000 concentration for E16 wild type and FoxG1 null cortical tissues. We decided to use the rabbit monoclonal # EPR18987 (1:10,000 concentration) because we detected almost no background bands in Null tissues, as well as the positive band was clear (Supplementary Fig. 5a). Gel blotting was performed with a regular procedure by using a 0.45 µm Nitrocellulose membrane (Biorad #1620116) or Immobilon PVDF membrane (Millipore#IPVH07850) and after using 5% ECL blocking reagent (GE Healthcare # RPN2125V) in TBST, antibody reactions were carried out for rabbit mono FoxG1 or mouse monoclonal βactin antibody #AC-15 (1:50,000; SIGMA Aldrich #A5441-2ml) or mouse monoclonal Tuj1 antibody #TU-20 (1:5000; Millipore # MAB1637). After 1 h of secondary reaction with 1:5000 HRP-conjugated anti Rabbit or Mouse antibodies (Jackson ImmunoResearch #115-035-003 or 111-035-003) and subsequent washing of the membrane, signals were recorded by Clarity Western ECL Substrate (Biorad #1705061) and Image Quant LAS 4000 (GE Healthcare). When required, antibody stripping from the membrane was performed by 30 min incubation at 55 °C with a stripping solution containing 2% SDS, 100 mM mercaptoethanol, and 62.5 mM Tris-HCl (pH 6.8). Obtained signals were analyzed by using the Image J software.

GABAergic cell precursor transplantation

In order to prepare donor EGFP-labeled GABAergic cell precursors, we crossed males double positive for Dlx5a-Cre⁴⁴ (Jackson Laboratories stock #008199) and R26-CAG-loxPstop-EGFP reporter homozygous^59,81 (RCE:loxP; Jackson Laboratories stock #10701) to wild type female animals. At E13.5, embryos were dissected out and the ones harboring green brains were confirmed by using a fluorescent dissection microscope (Olympus, SZX 12). From each embryo, two MGEs (medial ganglionic eminence)⁶² from both hemispheres were collected. We typically used total 8 embryos for donor but in case we do not have 8 EGFP-positives, we combined EGFP-negative embryos as well. For cell dissociation, brains were dissected out in HBSS solution (Gibco #14175-095) one by one, each pair of MGEs were dissected out and stored in a 1.5 ml flat-bottom micro tube (Watson #131-815CS) with 500 µl of HBSS solution and kept on ice until the dissection was complete for maximum 8 embryos. Later, tissues were rinsed twice with HBSS and 100 μl of DNAse I (20,000 ku/ml in HBSS, Worthington) and 400 μl of Papain (20 u/ml in HBSS, Worthington #LK003176) solution were immediately added. By gentle pipetting (typically around 5 times) with a 200 μl filter tip pipette, tissues were broken up into small pieces. After a 20 min of incubation at 37 °C with shaking, 750 μl of 1% (v/v) heat-inactivated horse serum (Gibco #26050-070) in ice-cold HBSS and 75 μl of DNAse I (2000u/ml in HBSS) were added. After gently inverting the tube a couple of times, the tube was centrifuged for 5 min at 200 g/1480 rpm at 4 °C, the supernatant was discarded, 500 μl of ice-cold DMEM/GlutaMax (containing 5 μl of DNAse I) was added, and the cell pellet was resuspended by gentle pipetting using a 200 μl filter tip pipette. Cells are then collected into a 1.5 ml flat-bottom tube through 40 µm mini cell-strainer II (Hi-tech-inc# HT-AMS-04002, blue). The cell-strainer is rinsed with another 500 μl of ice-cold DMEM/GlutaMax containing 5 μl of DNAse I to make total 1 ml cell solution. Cell numbers were counted on OneCell counter (Fine Plus International Ltd). MGEs from 8 embryos typically ended up with 6000–8000 cells/µl in 1 ml cell solution. 1 ml of cell solution was centrifuged for 5 min at 4 °C, 200 g/1480 rpm and supernatant was removed to leave 15-20 µl with the cell pellet. After an addition of 0.5 µl of DNAse I (2000 u/ml in HBSS), the tube was vortex mixed and kept on ice for cell transplantation surgery.

In the afternoon, P7 pup was placed in a bag and buried under crushed ice for 15 min to have sufficient anesthetization. For 3 week old experiments, animals were first anesthetized with 2% isoflurane and then intraperitoneal injection of ketamine (100 mg/kg) and xylazine (16 mg/kg) mixture was carried out. 3 µl of cell solution was placed on a parafilm (typically for 3–4 pups) and was adsorbed into a 36 g beveled NanoFil injection needle (World Precision Instruments, #NF35BV-2) at 300 nl/min ratio. Surgical tape-protected ear-bars are placed bilaterally to fix the pup head. The skin of the head above the olfactory bulbs was cut in the center for about 3–5 mm to visualize the skull. 1.7 mm posterior and 0.2 mm lateral from the blood vessel crossing, which is located above the border of nasal and frontal skull was aimed. Prior to needle injection, cells are released from the needle to confirm the clogging. The needle tip is placed 1.5 mm deep from the surface, and 200 nl of cells are injected at 200 nl/min speed. Upon completing the cell injection, the needle was kept still for another minute and then removed from the skull slowly. Upon completing the cell injection into the right hemisphere, pup status was confirmed, and if necessary, a small ice pack was placed below the belly of the pup before proceeding to the left hemisphere injection. When done with the injections into both hemispheres, the head was thoroughly wiped, the skins were put together with instant glue and the pup was placed on the warm pad for a while before sending back to the littermate cohort. The entire surgery takes approximately 25 min for one pup.

Behavioral assays

The date of the observed plug was designated as E0 and pups were usually delivered on E19. At postnatal day 21, the whole litter was weaned and placed into a larger cage (W270 × L440 × H187 mm, KN-601, Natsume Seisakusyo) from the original cage (W220 × L320 × H135 mm). Genotyping of the animals by tail PCR was carried out by postnatal 2 weeks. Upon completing battery of behavioral analyses, we again carried out tail PCR to assure the genotypes of the animals. Open field and elevated plus maze behavioral assays were carried out on cohorts of animals at 5 weeks of age. The following week, the three-chamber assay and 8-arm radial maze working memory task were performed. Mice of both genders were included in our analysis. All of the assays were carried out in a sound-proof room (S-1220 soft DX, STAR LITE) and mouse behavior in each specific arena was recorded by a CCD camera (Chameleon3 1.3 MP Mono USB3 Vision, CM3-U3-13S2M-CS, FLIR) at 15 frames/sec and saved as M-JPEG file with 75% compression by using FlyCapture2 camera software 2.9.3.11 (Photonics). Prior to initiating the recording, the information of the test animal, such as date of filming, animal number, and presence or absence of a social animal, were written on a small white board (22 × 33 cm) and placed on the behavior testing set. After video recording had been initiated, the white board was removed and the test animal was placed in the arena or the starting dome was removed to release the test animal. Once testing had begun, the experimenter would quickly leave the sound-proof chamber and quietly close the door in order to provide a non-disturbing environment to the test animal during the assay. The experimenter was always blind to the genotype of the test animals. Video files were later analyzed by using the ANY-maze video tracking software 5.1 (Stoelting, USA), with the video analysis typically initiated after the door had been closed. All behavioral analysis data are shown as mean ± SEM and p values are from two-tailed t-test.

Open field assay

The open field apparatus consists of a gray acrylic-modified polyvinyl-chloride floor with a 50 × 50 cm square field and gray vinyl chloride 40 cm height walls (OF-36MSQ, Muromachi). Animals were placed in the center of the arena and the recording was performed for 10 min and 30 s. Data were analyzed over the 10 min session for the distance traveled and time spent in the center (30 × 30 cm square center) and margin of the chamber.

Elevated plus maze

The apparatus material is similar to that of the open field chamber, and the length of each of the open and closed arms is 30 cm, with a corridor width of 6 cm (EPM-04M, Muromachi). The walls of the closed arm are 15 cm in height. At the beginning of the assay, animals are placed on one end of the open arms and allowed to explore freely during the 10 min and 30 s recording session. Distance and time each animal spent on the open and closed arms were analyzed for the first and second 5 min of the assay.

Three-chamber social interaction assay

The apparatus consists of a gray acrylic-modified polyvinyl-chloride floor and three chambers (each 20 × 40 cm) connected by 5 cm width × 7 cm height windows in transparent acryl 22 cm height walls (SC-03M, Muromachi). Wired cages are 18.5 cm height with 9 cm diameter circle bottom and top, both of which are connected by 3 mm diameter wires (total 16) placed in a circle with 7 mm distance between wires. Out of the four wired cages, two were used to place the stranger mouse but the other two did not receive animals and were used for the Habituation (1st 10 min) and non-social side of the Sociability session (2nd 10 min). The chambers and wired cages were cleaned and dried with paper towels between each test animal trial. The test mouse is first placed in a start dome (20 cm diameter circle tube, transparent acryl) in the middle chamber. Upon removal of the start dome, the experimenter quickly left the sound-proof room and closed the door quietly. In the first session (Habituation), the animal was allowed to freely move inside the three-chambers with two empty wired cages placed in the centers of the lateral chambers. After 10 min and 30 s video recording was obtained, the test animal was allowed to move into the middle chamber and was trapped in the start dome. At the beginning of second session (Sociability), one of the empty wired cage was replaced with another wired cage holding an age- and gender-matched stranger mouse. After 10 min and 30 s of video recording, the test animal was again trapped inside the starting dome in the middle chamber. Then, the remaining empty wired cage was replaced with a stranger animal-containing wired cage for the final session (Social Novelty). After the video recording was complete, the test animal was returned to its littermate home cage. For each 10 minutes of assay, time spent in each of the three chambers was measured and analyzed.

8-arm radial maze working memory task

The maze is made from a material which is similar to the open field, and each arm is 35 cm length with 6 cm wall heights (RM-08M, Muromachi). The center part of the arms (10 cm) have higher walls (12 cm) and the distance between the opposing two arms spanning the center area is 20 cm. At the end of each arm, there is a circle hole (2 cm diameter, 1 cm depth) with a small dip (0.5 cm diameter, reverse cone shape) in the center. Prior to initiating the task, 50 µl of water droplets are carefully placed into all of the eight dips by using a pipette. The test animal is placed in a start dome (20 cm diameter circle tube, transparent acryl), which is placed in the center of the arena. The assay starts by removing the start dome and the experimenter leaving the sound-proof chamber and closing the door. In terms of the test model animals, water bottles were removed from the cage in the morning of one day prior to the first experiment. On the first day, assays are carried out for two rounds (R1 and R2) typically in the morning and the afternoon. On the second day, another two rounds of experiments (R3 and R4) are carried out. In all of the tests, video recording was carried out for 5 min and 30 s. With the ANY-maze software, the analysis of video file is initiated when the test animal reaches the first end of the arm by its body trace dot crossing a line located 10 cm away from the end of arm. When the test animal has entered the eighth end of the arm, the trial is terminated; otherwise it ends after 5 min. The duration to complete the task, total numbers of the end of the arms visited and the mean speed was measured for each animal in the R3 and R4 trials for analysis. Note that when a test animal does not complete the task within 5 min, the total numbers of visits, as well as duration, are both underestimated.

Electroencephalogram recording

EEG and EMG recordings of freely-moving animals

Stereotaxic surgery was performed under anesthesia with a ketamine-xylazine mixture (100 and 10 mg/kg, respectively, i.p.). Electrodes for EEG were implanted on the skull over the left and right prefrontal cortex (+3.0 mm anteroposterior, +1.5 mm mediolateral from bregma; +1.1 mm anteroposterior, +1.5 mm mediolateral from bregma) according to the mouse brain atlas⁸². Electrodes for EMG were inserted into the neck muscles. Continuous EEG and EMG recordings were performed through a slip ring (SPM-12, Hikari Denshi Kogyo) designed so that the movement of mouse was unrestricted. EEG and EMG signals were amplified (BA1008, Miyuki Giken), low-pass-filtered at 100 Hz, digitized at a sampling rate of 400 Hz, and recorded using LabChart v7.3.8 software (AD instruments). For the vigilance state determination, EEG/EMG recordings were scored by visual inspection as wakefulness, NREM sleep, or REM sleep in 8-sec epochs following the criteria⁸³. The same individual, blinded to experimental conditions, scored all EEG/EMG recordings. Spectral analysis of the EEG was performed by fast Fourier transform (SleepSign software version 3.0, Kissei Comtec). The EEG signal were filtered by 0.5 Hz high-pass filter and 50 Hz notch filter. This analysis yielded a power spectral profile over a 0.5–80 Hz window with a 0.2 Hz resolution divided into delta (0.75–4 Hz), theta (4–12 Hz), alpha/beta (12-30 Hz), low-gamma (30–45 Hz), and high-gamma (55–80 Hz) waves. Whereas the recordings were obtained for more than 24 hr, the EEG data in wakefulness state early in the dark period (from 22:00 to 0:00) was used for the spectral analyses. The absolute power was divided by the total power (0.5–80 Hz) in 24 hr. The mean normalized power in each frequency band was then calculated for each subject.

Sensory-evoked EEG recordings in awake head-fixed animals

In order to prepare for EEG recording, a custom-made head plate was implanted onto the skull of 7–8 week-old adult FoxG1 model animals using acrylic dental resin (Super-Bond, Sun Medical, Shiga, Japan). Prior to this surgery, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (16 mg/kg) mixture and held in a stereotaxic apparatus. The head plate-implanted animals were placed back in the home cage and allowed to recover from the surgery for 2 or 3 days. For EEG recordings, the animals were first anesthetized with 2% isoflurane to insert screws. A reference screw was inserted into the skull on top of the right cerebellum and recordings were from a screw on top of the right cortex (1 mm anterior and 0.5 mm lateral to bregma). EEG recordings were obtained from head-fixed mice during the awake state. The EEG signal was amplified and digitized at 4 kH by Plexon OmniPlexD system Ver. 1.12.0.0 (Plexon Inc., Dallas, TX, USA). EEG data were analyzed using a custom-written MATLAB program (MathWorks, Natick, MA, USA). Whiskers were stimulated by using a piezoelectric device controlled by a custom-written MATLAB program. The codes are available from the corresponding author upon reasonable request. All whiskers were deflected toward forward and backward directions for 30 ms with a 170 ms steady state interval after each deflection. This stimulation protocol was carried out four times in one session, which was repeated 20 times at 3.6 s intervals. Recordings with and without stimulation were carried out for 1.6 s after and before the first whisker deflection, respectively.

In vitro electrophysiological recordings

Juvenile (P12-14) and adult (6–8 weeks) male or female mice were deeply anesthetized with pentobarbital (100 mg/kg, intraperitoneal) and the brains were quickly removed and submerged in an ice-cold cutting solution (234 mM sucrose, 2.5 mM KCl, 10 mM MgCl₂, 0.5 mM CaCl₂, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 0.5 mM myo-inositol, and 11 mM glucose). Coronal brain slices were cut using a vibratome (300 µm, VT-1200S, Leica) and immersed in a physiological solution (125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 20 mM glucose, 5 mM Na-ascorbate, 3 mM Na-pyruvate, and 2 mM thiourea) that was continuously bubbled with a mixture of 95% O₂ and 5% CO₂. Na-ascorbate, Na-pyruvate, and thiourea were omitted during recordings. Whole-cell patch clamping was performed at 31–32 °C. Voltage-clamp recordings were made from visually identified layer 2/3 pyramidal neurons in the prelimbic cortex (PFC, prefrontal cortex) or barrel fields of the primary somatosensory cortex (S1).

The pipette solution for current-clamp recordings consisted of 130 mM potassium gluconate, 0.2 mM EGTA, 2 mM MgCl₂, 2 mM Na₂ATP, 0.3 mM NaGTP, and 10 mM HEPES. The pipette solution for voltage-clamp recording consisted of 108 mM cesium gluconate, 0.4 mM EGTA, 2.8 mM NaCl, 5 mM TEA-Cl, 4 mM MgATP, 0.3 mM NaGTP, 10 mM Na₂-phosphocreatine and 20 mM HEPES. In some recordings, 0.5% biocytin was included in the pipette solution to visualize neuron morphology. The pH of the solution was adjusted to 7.2 and the osmolarity was 290 mOsm. The membrane potentials were not corrected for liquid junction potentials. The series resistance of the recording cells was <25 MΩ. For current-clamp recordings, the resting membrane potentials and firing responses to depolarizing current pulses (1-s duration, 0–600 pA with 50 pA increments from the resting membrane potential) were recorded within 5 min from whole-cell break-in. The input resistance and time constant were determined from the transient voltage response to a hyperpolarizing current injection (−50 pA). The voltage sag amplitude was determined by subtracting the steady state amplitude from the maximum amplitude in response to a hyperpolarizing current pulse (−200 pA, 1-s duration). For voltage-clamp recordings, miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded as inward currents at −60 mV and outward currents at 0 mV, respectively, from the same neuron in the presence of physiological solution containing 0.5 µM tetrodotoxin (Alomone Labs, Jerusalem, Israel). After recordings, slices containing biocytin-loaded cells were fixed overnight at 4 °C in a fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M PB) and incubated with Alexa Fluor 488-conjugated streptavidin (1:500, Thermo Fisher Scientific, Waltham, MA, USA).

Recordings were amplified with a Multiclamp 700A amplifier (Molecular Devices, LLC, Sunnyvale, CA, USA), digitized at 10 kHz using a Digidata 1322A apparatus (Molecular Devices, LLC), and collected with pClamp 8.2 software (Molecular Devices, LLC). mEPSC/mIPSC events were detected with a scaled-template algorithm and analyzed using IGOR Pro 6.36 software (WaveMetrics, Inc., Lake Oswego, OR, USA) and Clampfit 8.2 software (Molecular Devices, LLC). The amplitude and the frequency of mEPSC/mIPSC events were determined for each neuron over a 5-min period of recording. Statistical analyses were performed as follows using Graphpad Prism 7.05 software (GraphPad Software, San Diego, CA, USA): two-way ANOVA and post-hoc Tukey’s test were used for comparisons of current-frequency relationships; Welch’s t-test was used for two group comparisons; and the chi-square test was used for comparison of firing type composition.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are provided within the paper and its Supplementary information. A Source data file is provided with this paper. We confirm that all unique materials used here are readily available from the authors (FoxG1-LacZ, floxed-FoxG1, TRE-FoxG1, Nex-Cre, Gad2 null) or from Jackson labs (Dlx5a-Cre, RCE:FRT, RCE:loxP, R26-stop-tTA, Ai9). Further information on research design is available in the Nature Research Reporting Summary linked to this article. Any data are available from the authors upon request.

Code availability

A custom code was used in the EEG analysis of head-fixed animals (Supplementary Fig. 4). All scripts used to analyze or display the data are available upon request.

References

Sztainberg, Y. & Zoghbi, H. Y. Lessons learned from studying syndromic autism spectrum disorders. Nat. Neurosci. 19, 1408–1417 (2016).
Article CAS PubMed Google Scholar
Rubenstein, J. L. & Merzenich, M. M. Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav. 2, 255–267 (2003).
Article CAS PubMed PubMed Central Google Scholar
Nelson, S. B. & Valakh, V. Excitatory/inhibitory balance and circuit homeostasis in autism spectrum disorders. Neuron 87, 684–698 (2015).
Article CAS PubMed PubMed Central Google Scholar
Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).
Article CAS PubMed PubMed Central ADS Google Scholar
Marin, O. Interneuron dysfunction in psychiatric disorders. Nat. Rev. Neurosci. 13, 107–120 (2012).
Article CAS PubMed Google Scholar
Lewis, D. A., Hashimoto, T. & Volk, D. W. Cortical inhibitory neurons and schizophrenia. Nat. Rev. Neurosci. 6, 312–324 (2005).
Article CAS PubMed Google Scholar
Hashemi, E., Ariza, J., Rogers, H., Noctor, S. C. & Martinez-Cerdeno, V. The number of parvalbumin-expressing interneurons is decreased in the prefrontal cortex in autism. Cereb. Cortex 1, 1931–1943 (2017).
Chao, H. T. et al. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269 (2010).
Article CAS PubMed PubMed Central ADS Google Scholar
Judson, M. C. et al. GABAergic neuron-specific loss of Ube3a causes angelman syndrome-Like EEG abnormalities and enhances seizure susceptibility. Neuron 90, 56–69 (2016).
Article CAS PubMed PubMed Central Google Scholar
Hensch, T. K. Critical period plasticity in local cortical circuits. Nat. Rev. Neurosci. 6, 877–888 (2005).
Article CAS PubMed Google Scholar
Southwell, D. G., Froemke, R. C., Alvarez-Buylla, A., Stryker, M. P. & Gandhi, S. P. Cortical plasticity induced by inhibitory neuron transplantation. Science 327, 1145–1148 (2010).
Article CAS PubMed PubMed Central ADS Google Scholar
Antoine, M. W., Langberg, T., Schnepel, P. & Feldman, D. E. Increased excitation-inhibition ratio stabilizes synapse and circuit excitability in four autism mouse models. Neuron 101, 648–661 (2019). e644.
Article CAS PubMed PubMed Central Google Scholar
Xuan, S. et al. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 14, 1141–1152 (1995).
Article CAS PubMed Google Scholar
Hanashima, C., Li, S. C., Shen, L., Lai, E. & Fishell, G. Foxg1 suppresses early cortical cell fate. Science 303, 56–59 (2004).
Article CAS PubMed ADS Google Scholar
Muzio, L. & Mallamaci, A. Foxg1 confines Cajal-Retzius neuronogenesis and hippocampal morphogenesis to the dorsomedial pallium. J. Neurosci. 25, 4435–4441 (2005).
Article CAS PubMed PubMed Central Google Scholar
Shen, Q. et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9, 743–751 (2006).
Article CAS PubMed Google Scholar
Godbole, G. et al. Hierarchical genetic interactions between FOXG1 and LHX2 regulate the formation of the cortical hem in the developing telencephalon. Development 145, dev154583 (2018).
Martynoga, B., Morrison, H., Price, D. J. & Mason, J. O. Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis. Dev. Biol. 283, 113–127 (2005).
Article CAS PubMed Google Scholar
Manuel, M. et al. The transcription factor Foxg1 regulates the competence of telencephalic cells to adopt subpallial fates in mice. Development 137, 487–497 (2010).
Article CAS PubMed PubMed Central Google Scholar
Miyoshi, G. & Fishell, G. Dynamic FoxG1 expression coordinates the integration of multipolar pyramidal neuron precursors into the cortical plate. Neuron 74, 1045–1058 (2012).
Article CAS PubMed PubMed Central Google Scholar
Tian, C. et al. Foxg1 has an essential role in postnatal development of the dentate gyrus. J. Neurosci. 32, 2931–2949 (2012).
Article CAS PubMed PubMed Central Google Scholar
Won, H. et al. Chromosome conformation elucidates regulatory relationships in developing human brain. Nature 538, 523–527 (2016).
Article PubMed PubMed Central ADS CAS Google Scholar
Mariani, J. et al. FOXG1-Dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).
Article CAS PubMed PubMed Central Google Scholar
Brunetti-Pierri, N. et al. Duplications of FOXG1 in 14q12 are associated with developmental epilepsy, mental retardation, and severe speech impairment. Eur. J. Hum. Genet. 19, 102–107 (2010).
Yeung, A. et al. 4.45 Mb microduplication in chromosome band 14q12 including FOXG1 in a girl with refractory epilepsy and intellectual impairment. Eur. J. Med. Genet. 52, 440–442 (2009).
Article PubMed Google Scholar
Ariani, F. et al. FOXG1 is responsible for the congenital variant of Rett syndrome. Am. J. Hum. Genet. 83, 89–93 (2008).
Article CAS PubMed PubMed Central Google Scholar
Bahi-Buisson, N. et al. Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant. Neurogenetics 11, 241–249 (2010).
Article CAS PubMed Google Scholar
Kortum, F. et al. The core FOXG1 syndrome phenotype consists of postnatal microcephaly, severe mental retardation, absent language, dyskinesia, and corpus callosum hypogenesis. J. Med. Genet 48, 396–406 (2011).
Article PubMed CAS Google Scholar
Takahashi, S. et al. FOXG1 mutations in Japanese patients with the congenital variant of Rett syndrome. Clin. Genet. 82, 569–573 (2012).
Article CAS PubMed Google Scholar
Florian, C., Bahi-Buisson, N. & Bienvenu, T. FOXG1-Related Disorders: From Clinical Description to Molecular Genetics. Mol. Syndromol. 2, 153–163 (2012).
CAS PubMed Google Scholar
Seltzer, L. E. et al. Epilepsy and outcome in FOXG1-related disorders. Epilepsia 55, 1292–1300 (2014).
Article CAS PubMed PubMed Central Google Scholar
Wong, L. C. et al. FOXG1-related syndrome: from clinical to molecular genetics and pathogenic mechanisms. Int. J. Mol. Sci. 20, 4176 (2019).
Silverman, J. L., Yang, M., Lord, C. & Crawley, J. N. Behavioural phenotyping assays for mouse models of autism. Nat. Rev. Neurosci. 11, 490–502 (2010).
Article CAS PubMed PubMed Central Google Scholar
Peca, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442 (2011).
Article CAS PubMed PubMed Central ADS Google Scholar
Orefice, L. L. et al. Peripheral mechanosensory neuron dysfunction underlies tactile and behavioral deficits in mouse models of ASDs. Cell 166, 299–313 (2016).
Article CAS PubMed PubMed Central Google Scholar
Han, S. et al. Autistic-like behaviour in Scn1a+/- mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489, 385–390 (2012).
Article CAS PubMed PubMed Central ADS Google Scholar
Schmeisser, M. J. et al. Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2. Nature 486, 256–260 (2012).
Article CAS PubMed ADS Google Scholar
Shen, L., Nam, H. S., Song, P., Moore, H. & Anderson, S. A. FoxG1 haploinsufficiency results in impaired neurogenesis in the postnatal hippocampus and contextual memory deficits. Hippocampus 16, 875–890 (2006).
Article CAS PubMed Google Scholar
Eagleson, K. L. et al. Disruption of Foxg1 expression by knock-in of cre recombinase: effects on the development of the mouse telencephalon. Neuroscience 148, 385–399 (2007).
Article CAS PubMed Google Scholar
Cargnin, F. et al. FOXG1 Orchestrates neocortical organization and cortico-cortical connections. Neuron 100, 1083–1096.e5. (2018).
Dastidar, S. G., Landrieu, P. M. & D’Mello, S. R. FoxG1 promotes the survival of postmitotic neurons. J. Neurosci. 31, 402–413 (2011).
Article CAS PubMed PubMed Central Google Scholar
Siegenthaler, J. A., Tremper-Wells, B. A. & Miller, M. W. Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression. Cereb. Cortex 18, 1865–1875 (2008).
Article PubMed Google Scholar
Goebbels, S. et al. Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice. Genesis 44, 611–621 (2006).
Article CAS PubMed Google Scholar
Monory, K. et al. The endocannabinoid system controls key epileptogenic circuits in the hippocampus. Neuron 51, 455–466 (2006).
Article CAS PubMed PubMed Central Google Scholar
Verginelli, F. et al. Transcription factors FOXG1 and Groucho/TLE promote glioblastoma growth. Nat. Commun. 4, 2956 (2013).
Article PubMed ADS CAS Google Scholar
Weise, S. C. et al. TGFbeta-signaling and FOXG1-expression are a hallmark of astrocyte lineage diversity in the murine ventral and dorsal forebrain. Front. Cell. Neurosci. 12, 448 (2018).
Article CAS PubMed PubMed Central Google Scholar
Falcone, C. et al. Foxg1 antagonizes neocortical stem cell progression to astrogenesis. Cereb. Cortex 29, 4903–4918 (2019).
Article PubMed Google Scholar
Inamura, N. et al. Intrinsic and extrinsic mechanisms control the termination of cortical interneuron migration. J. Neurosci. 32, 6032–6042 (2012).
Article CAS PubMed PubMed Central Google Scholar
Orekhova, E. V. et al. Excess of high frequency electroencephalogram oscillations in boys with autism. Biol. psychiatry 62, 1022–1029 (2007).
Article PubMed Google Scholar
Rojas, D. C., Maharajh, K., Teale, P. & Rogers, S. J. Reduced neural synchronization of gamma-band MEG oscillations in first-degree relatives of children with autism. BMC Psychiatry 8, 66 (2008).
Article PubMed PubMed Central Google Scholar
Cao, W. et al. Gamma oscillation dysfunction in mPFC leads to social deficits in neuroligin 3 R451C knockin mice. Neuron 98, 670 (2018).
Article CAS PubMed Google Scholar
Selimbeyoglu, A. et al. Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice. Sci. Transl. Med. 9, eaah6733 (2017).
Yamamoto, T. et al. Development of autoimmune diabetes in glutamic acid decarboxylase 65 (GAD65) knockout NOD mice. Diabetologia 47, 221–224 (2004).
Article CAS PubMed Google Scholar
Yanagawa, Y. et al. Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res. 8, 215–221 (1999).
Article CAS PubMed Google Scholar
Fagiolini, M. & Hensch, T. K. Inhibitory threshold for critical-period activation in primary visual cortex. Nature 404, 183–186 (2000).
Article CAS PubMed ADS Google Scholar
Southwell, D. G. et al. Interneurons from embryonic development to cell-based therapy. Science 344, 1240622 (2014).
Article PubMed PubMed Central CAS Google Scholar
Martinez-Cerdeno, V. et al. Embryonic MGE precursor cells grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-lesioned rats. Cell Stem Cell 6, 238–250 (2010).
Article CAS PubMed PubMed Central Google Scholar
Southwell, D. G. et al. Interneuron transplantation rescues social behavior deficits without restoring wild-type physiology in a mouse model of autism with excessive synaptic inhibition. J. Neurosci. 40, 2215–2227 (2020).
Article CAS PubMed PubMed Central Google Scholar
Miyoshi, G. et al. Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons. J. Neurosci. 30, 1582–1594 (2010).
Article CAS PubMed PubMed Central Google Scholar
Miyoshi, G. et al. Prox1 Regulates the subtype-specific development of caudal ganglionic eminence-derived GABAergic cortical interneurons. J. Neurosci. 35, 12869–12889 (2015).
Article CAS PubMed PubMed Central Google Scholar
Bourgeron, T. From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat. Rev. Neurosci. 16, 551–563 (2015).
Article CAS PubMed Google Scholar
Miyoshi, G., Butt, S. J., Takebayashi, H. & Fishell, G. Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors. J. Neurosci. 27, 7786–7798 (2007).
Article CAS PubMed PubMed Central Google Scholar
Peixoto, R. T., Wang, W., Croney, D. M., Kozorovitskiy, Y. & Sabatini, B. L. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(-/-) mice. Nat. Neurosci. 19, 716–724 (2016).
Article CAS PubMed PubMed Central Google Scholar
Miyoshi, G. Elucidating the developmental trajectories of GABAergic cortical interneuron subtypes. Neurosci. Res. 138, 26–32 (2019).
Article CAS PubMed Google Scholar
Pancrazi, L. et al. Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics. Proc. Natl Acad. Sci. USA 112, 13910–13915 (2015).
Article CAS PubMed ADS PubMed Central Google Scholar
Hollis, F., Kanellopoulos, A. K. & Bagni, C. Mitochondrial dysfunction in autism spectrum disorder: clinical features and perspectives. Curr. Opin. Neurobiol. 45, 178–187 (2017).
Article CAS PubMed Google Scholar
Siddiqui, M. F., Elwell, C. & Johnson, M. H. Mitochondrial dysfunction in autism spectrum disorders. Autism Open Access 6, 1000190 (2016).
Chen, L. et al. MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome. Proc. Natl Acad. Sci. USA 112, 5509–5514 (2015).
Article CAS PubMed ADS PubMed Central Google Scholar
Gabel, H. W. et al. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 522, 89–93 (2015).
Article CAS PubMed PubMed Central ADS Google Scholar
Katayama, Y. et al. CHD8 haploinsufficiency results in autistic-like phenotypes in mice. Nature 537, 675–679 (2016).
Article CAS PubMed ADS Google Scholar
Wade, A. A., Lim, K., Catta-Preta, R. & Nord, A. S. Common CHD8 genomic targets contrast with model-specific transcriptional impacts of CHD8 haploinsufficiency. Fron. Mol. Neurosci. 11, 481 (2019).
Zoghbi, H. Y. Rett syndrome and the ongoing legacy of close clinical observation. Cell 167, 293–297 (2016).
Article CAS PubMed Google Scholar
Dastidar, S. G. et al. Isoform-specific toxicity of Mecp2 in postmitotic neurons: suppression of neurotoxicity by FoxG1. J. Neurosci. 32, 2846–2855 (2012).
Article CAS PubMed PubMed Central Google Scholar
Ma, M., Adams, H. R., Seltzer, L. E., Dobyns, W. B. & Paciorkowski, A. R. Phenotype differentiation of FOXG1 and MECP2 disorders: a new method for characterization of developmental encephalopathies. J. Pediatr. 178, 233–240 (2016). e210.
Article CAS PubMed PubMed Central Google Scholar
Bicks, L. K. et al. Prefrontal parvalbumin interneurons require juvenile social experience to establish adult social behavior. Nat. Commun. 11, 1003 (2020).
Article CAS PubMed PubMed Central ADS Google Scholar
Miyamae, T., Chen, K., Lewis, D. A. & Gonzalez-Burgos, G. Distinct physiological maturation of parvalbumin-positive neuron subtypes in mouse prefrontal cortex. J. Neurosci. 37, 4883–4902 (2017).
Article CAS PubMed PubMed Central Google Scholar
Del Pino, I., Rico, B. & Marin, O. Neural circuit dysfunction in mouse models of neurodevelopmental disorders. Curr. Opin. Neurobiol. 48, 174–182 (2018).
Article PubMed CAS Google Scholar
Wang, L. et al. Restricted expression of mutant SOD1 in spinal motor neurons and interneurons induces motor neuron pathology. Neurobiol. Dis. 29, 400–408 (2008).
Article CAS PubMed Google Scholar
Hanashima, C., Shen, L., Li, S. C. & Lai, E. Brain factor-1 controls the proliferation and differentiation of neocortical progenitor cells through independent mechanisms. J. Neurosci. 22, 6526–6536 (2002).
Article CAS PubMed PubMed Central Google Scholar
Mayer, C. et al. Developmental diversification of cortical inhibitory interneurons. Nature 555, 457–462 (2018).
Article CAS PubMed PubMed Central ADS Google Scholar
Sousa, V. H., Miyoshi, G., Hjerling-Leffler, J., Karayannis, T. & Fishell, G. Characterization of Nkx6-2-derived neocortical interneuron lineages. Cereb. Cortex 19, i1–i10 (2009). Suppl 1.
Article PubMed PubMed Central Google Scholar
Franklin, K. B. & Paxinos, G. The mouse brain in stereotaxic coordinates (Academic press, 2008).
Tobler, I., Deboer, T. & Fischer, M. Sleep and sleep regulation in normal and prion protein-deficient mice. J. Neurosci. 17, 1869–1879 (1997).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank the following Drs. for kindly sharing their reagents with us: Eseng Lai (FoxG1-LacZ knock-in mutant), Carina Hanashima (TRE-FoxG1 transgenic), Marc Ekker (Dlx5a-Cre transgenic), and Klaus-Armin Nave (Nex-Cre knock-in). We also wish to thank Drs. Kazutaka Ikeda and Hiroko Kotajima for helpful inputs regarding mouse behavioral assays and Dr. Mitsuharu Midorikawa for help with photo acquisition. We thank Mai Hatakenaka, Emiko Naraba, Tomoya Tsuchida, Sachie Sekino, Fumiya Urata and Yumi Tani for technical help. We greatly appreciate Takanori Maeda and Junnosuke Ohnuma for the effort in mouse behavioral analysis. We also thank Dr. Tomomi Shimogori for critically reading this manuscript prior to submission. We are grateful for the communications with Japan and international FOXG1 family communities. This work was supported by Grants-in-Aid for Scientific Research JP17H05775, JP17K07102, JP19H04789, JP19H05228, and JP20K07362 (G.M.), JP15H01667, JP19H03343, JP20H05481 and JP20H05916 (M.M.), and NIH grant R01MH095147 (G.F.). G.M. is supported by the Believe in a Cure, Inc., Mochida Memorial Foundation for Medical and Pharmaceutical Research, Cell Science Research Foundation, Takeda Science Foundation, and Brain Science Foundation.

Author information

Authors and Affiliations

Department of Neurophysiology, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan
Goichi Miyoshi, Yoshifumi Ueta, Kou Hiraga, Hironobu Osaki, Yuki Yagasaki & Mariko Miyata
Sleep Disorders Project, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo, Japan
Akiyo Natsubori
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
Yusuke Kishi
Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan
Yuchio Yanagawa
NYU Neuroscience Institute, Smilow Research Center, New York University School of Medicine, New York, NY, USA
Gord Fishell & Robert P. Machold
Department of Neurobiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
Gord Fishell
Stanley Center at the Broad Institute, Cambridge, MA, USA
Gord Fishell

Authors

Goichi Miyoshi
View author publications
You can also search for this author in PubMed Google Scholar
Yoshifumi Ueta
View author publications
You can also search for this author in PubMed Google Scholar
Akiyo Natsubori
View author publications
You can also search for this author in PubMed Google Scholar
Kou Hiraga
View author publications
You can also search for this author in PubMed Google Scholar
Hironobu Osaki
View author publications
You can also search for this author in PubMed Google Scholar
Yuki Yagasaki
View author publications
You can also search for this author in PubMed Google Scholar
Yusuke Kishi
View author publications
You can also search for this author in PubMed Google Scholar
Yuchio Yanagawa
View author publications
You can also search for this author in PubMed Google Scholar
Gord Fishell
View author publications
You can also search for this author in PubMed Google Scholar
Robert P. Machold
View author publications
You can also search for this author in PubMed Google Scholar
Mariko Miyata
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

conceptualization (G.M. and R.M.), project administration and supervision (G.M. and M.M.), data curation, formal analysis, investigation and methodology (G.M., Y.U., A.N., K.H., H.O., Y. Yagasaki, and Y.K.), funding acquisition and resources (G.M., Y. Yanagawa, G.F., and M.M.), software (G.M., Y.U., A.N., and H.O.), validation and visualization (G.M., Y.U., A.N., K.H., and R.M.), writing original draft (G.M. and R.M.) and editing (G.M., G.F., R.M., and M.M.).

Corresponding author

Correspondence to Goichi Miyoshi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Communications thanks Takanobu Nakazawa, Zhengping Jia and the other anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Miyoshi, G., Ueta, Y., Natsubori, A. et al. FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes. Nat Commun 12, 3773 (2021). https://doi.org/10.1038/s41467-021-23987-z

Download citation

Received: 13 September 2020
Accepted: 27 May 2021
Published: 18 June 2021
DOI: https://doi.org/10.1038/s41467-021-23987-z

This article is cited by

iPSC-derived models of PACS1 syndrome reveal transcriptional and functional deficits in neuron activity
- Lauren Rylaarsdam
- Jennifer Rakotomamonjy
- Alicia Guemez-Gamboa
Nature Communications (2024)
FoxG1 as a Potential Therapeutic Target for Alzheimer’s Disease: Modulating NLRP3 Inflammasome via AMPK/mTOR Autophagy Pathway
- Qi Yun
- Si-Fei Ma
- Jia Wang
Cellular and Molecular Neurobiology (2024)
Differential vulnerability of adult neurogenic niches to dosage of the neurodevelopmental-disorder linked gene Foxg1
- Iris Schäffner
- Marie-Theres Wittmann
- D. Chichung Lie
Molecular Psychiatry (2023)
Epigenetic genes and epilepsy — emerging mechanisms and clinical applications
- Karen M. J. Van Loo
- Gemma L. Carvill
- David C. Henshall
Nature Reviews Neurology (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.