Genetic background determines synaptic phenotypes in Arid1b-mutant mice

ARID1B, a chromatin remodeler, is strongly implicated in autism spectrum disorders (ASD). Two previous studies on Arid1b-mutant mice with the same exon 5 deletion in different genetic backgrounds revealed distinct synaptic phenotypes underlying the behavioral abnormalities: The first paper reported decreased inhibitory synaptic transmission in layer 5 pyramidal neurons in the medial prefrontal cortex (mPFC) region of the heterozygous Arid1b-mutant (Arid1b+/−) brain without changes in excitatory synaptic transmission. In the second paper, in contrast, we did not observe any inhibitory synaptic change in layer 5 mPFC pyramidal neurons, but instead saw decreased excitatory synaptic transmission in layer 2/3 mPFC pyramidal neurons without any inhibitory synaptic change. In the present report, we show that when we changed the genetic background of Arid1b+/− mice from C57BL/6 N to C57BL/6 J, to mimic the mutant mice of the first paper, we observed both the decreased inhibitory synaptic transmission in layer 5 mPFC pyramidal neurons reported in the first paper, and the decreased excitatory synaptic transmission in mPFC layer 2/3 pyramidal neurons reported in the second paper. These results suggest that genetic background can be a key determinant of the inhibitory synaptic phenotype in Arid1b-mutant mice while having minimal effects on the excitatory synaptic phenotype.

Previous studies on Arid1b-mutant mice reported various ARID1B deficiency-related phenotypes and underlying mechanisms (7)(8)(9)(10), including suppressed insulin-like growth factor signaling (8), impaired inhibitory synaptic transmission (9), and impaired excitatory synaptic transmission (10).The impaired inhibitory synaptic transmission was observed among layer 5 pyramidal neurons in the medial prefrontal cortex (mPFC) of heterozygous Arid1b-mutant (Arid1b +/− ) mice that lacked exon 5 of the Arid1b gene (9).In a similar and more recent study on Arid1b +/− mice also lacking exon 5, our group did not find inhibitory synaptic changes in layer 5 pyramidal mPFC neurons, but rather observed impaired excitatory synaptic transmission in layer 2/3 mPFC pyramidal neurons (10).This discrepancy in the synaptic phenotypes of two Arid1b +/− mouse lines with the same exon 5 deletion may reflect a difference in the genetic background of the mutant mouse lines: The first study used C67BL6/J mice, while we used C67BL/N mice.These genetic backgrounds have been reported to exhibit multiple phenotypic differences, including differences in synaptic transmission and metabolism (11)(12)(13)(14).
In the present study, we attempted to change the genetic background of our Arid1b +/− mice from C57BL/6 N to C57BL/6 J by backcrossing with wild-type C57BL/6 J mice for more than seven generations.We found that our new Arid1b +/− mice with the C57BL/6 J background show impaired inhibitory synaptic transmission in layer 5 mPFC pyramidal neurons, similar to the previous results published by the other group (9).However, our new Arid1b +/− mice with the C57BL/6 J background continued to show impaired excitatory synaptic transmission in layer 2/3 mPFC pyramidal neurons, similar to our previous results (10).These findings suggest that genetic background is a key determinant of the inhibitory synaptic phenotype in Arid1b +/− mice carrying exon 5 deletion, whereas the excitatory synaptic phenotypes are minimally affected by the genetic background.

Synaptic transmission differs between two exon 5-deleted Arid1b +/− mouse lines
Two previous studies on Arid1b +/− mice (exon 5 deletion) reported distinct changes among excitatory and inhibitory synaptic transmissions in layer 2/3 or 5 pyramidal neurons in the mPFC (Figure 1A).Specifically, a previous study on Arid1b +/− mice in the genetic background of C57BL/6 J (EUCOMM; MGI:4435087) reported that the frequency but not amplitude of miniature inhibitory postsynaptic currents (mIPSCs) was decreased in layer 5 pyramidal neurons in the mPFC while excitatory miniature excitatory postsynaptic currents (mEPSCs) were not altered (9).This functional change was observed in adult brains and further supported by a decrease in the excitatory synapse number, as revealed by electron microscopy (EM) (9).
In contrast, our more recent study on Arid1b +/− mice with the same exon 5 deletion in the genetic background of C57BL/6 N (NorCOMM2; MGI:6156423) revealed normal mIPSCs and mEPSCs in layer 5 mPFC pyramidal neurons but a decrease in the frequency (not amplitude) of mEPSCs in layer 2/3 pyramidal neurons without changes in mIPSCs (10).These excitatory synaptic changes were supported by EM results and observed at both juvenile and adult stages (10).
We hypothesized that the difference in the genetic backgrounds of the Arid1b +/− mouse lines (hereafter termed "Aridb1-Jung-J" and "Arid1b-Kim-N" mice) might contribute to the discrepancy in the observed synaptic phenotypes.To test this hypothesis, we attempted to change the genetic background of our Arid1b +/− mice from C57BL/6 N to C57BL/6 J (i.e., Arid1b-Kim-N mice to Arid1b-Kim-J mice) by backcrossing Arid1b-Kim-N mice with wild-type (WT) C57BL/6 J mice for more than seven generations (Figure 1B).This change in the genetic background was confirmed by PCR genotyping for exon 9 of the Nnt (nicotinamide nucleotide transhydrogenase) gene (14), which was present in Arid1b-Kim-N mice but absent from Arid1b-Kim-J mice.

Synaptic transmission in Arid1b-Kim-J mice
We then measured mIPSCs in Arid1b-Kim-J mice to see if the shift of genetic background from C57BL/6 N to C57BL/6 J had any effect on inhibitory synaptic transmission in layer 5 mPFC pyramidal neurons at postnatal day 90 (P90), when mIPSCs were measured in the first study (9).Similar to the previous results (9), we observed a decrease in the frequency but not amplitude of mIPSCs in layer 5 mPFC neurons of Arid1b-Kim-J mice (Figure 2A).This suggests that Arid1b haploinsufficiency in mice leads to inhibitory synaptic deficits in the C57BL/6 J background but not the C57BL/6 N background.
We next tested if the mEPSCs found to be suppressed in the Arid1b-Kim-N mice (10) were affected by the change of genetic background from C57BL6/N to C57BL6/J.Measurements of mEPSCs in layer 2/3 mPFC pyramidal neurons in Arid1b-Kim-J mice at P21 revealed a decrease in the mEPSC frequency but not the amplitude (Figure 2B), which was similar to the results we previously obtained in P21 Arid1b-Kim-N mice (10).This suggests that excitatory synaptic transmission deficits are consistently observed in Arid1b +/− mouse lines of the two different genetic backgrounds (Arid1b-Kim-N and Arid1b-Kim-J mice).

Discussion
In this study, we demonstrate that the genetic background has strong effects on inhibitory synaptic transmission but not excitatory synaptic transmission in Arid1b +/− mice.This explains the distinct synaptic phenotypes observed in two previously reported Arid1b +/− mice having the same exon 5 deletion in different genetic backgrounds: Decreased mIPSC frequency was reported in Arid1b-Jung-J mice but decreased mEPSC frequency was found in Arid1b-Kim-N mice.The results from the present study suggest that genetic background of Arid1b +/− mice can modulate an excitation/inhibition imbalance, a mechanism implicated in ASD (15)(16)(17)(18)(19), through the regulation of inhibitory synaptic transmission.
It remains unclear how altering the genetic background of Arid1b +/− mice from C57BL/6 N to C57BL/6 J can decrease inhibitory synaptic transmission.We do know that mice of C57BL/6 N and C57BL/6 J backgrounds differ in their genetic and phenotypic profiles (12-14).Genetic differences between C57BL/6 N and C57BL/6 J mice include 34 single-nucleotide polymorphisms, 2 indels, and 15 structural variants.Phenotypic differences were observed in various domains, including dysmorphology, ophthalmology, cardiovascular, metabolism, neurological, behavioral, sensory, clinical chemistry, hematology, and immune/allergy.
Previous studies on Arid1b-Jung-J mice showed that Arid1b haploinsufficiency dramatically decreases the number of parvalbumin-positive GABAergic interneurons through various mechanisms, including altered Wnt/β-catenin signaling and impaired modulation of histone acetyltransferase activity in the promotor region of the parvalbumin/Pvalb gene (9,20).We speculate that the genetic background switch from Arid1b-Kim-N to Arid1b-Kim-J mice might have altered these mechanisms involved in parvalbumin-positive GABA neuronal development.With regard to a candidate mechanism, wild-type mice of C57BL/6 N background carry a mutation (S968F) in the CYFIP2 (cytoplasmic FMR1-interacting protein 2) protein (11).CYFIP2 is a component of the actin regulatory WAVE complex, which binds the fragile X messenger ribonucleoprotein (FMRP) (21-23) and has been implicated in developmental delay, epilepsy, West syndrome (a developmental and epileptic encephalopathy), and Alzheimer's disease (23-25).The CYFIP2-S968F mutation was originally reported to destabilize the protein (11), but later shown to also induce excessive Rac1-mediated WAVE complex activation (26).In addition, in vitro and in vivo studies have shown that CYFIP2 regulates various neuronal and synaptic functions, including neuronal excitability, excitatory and inhibitory synaptic structure/ function, presynaptic/mitochondrial function, neuronal excitability, and local translation (11,23,(27)(28)(29)(30)(31)(32)(33).In the context of the present work, it is notable that CYFIP2 is enriched at inhibitory synapses, and its neuronal overexpression suppresses inhibitory synaptic structure (30).Thus, we speculate that the loss of the CYFIP2-S968F mutation in Arid1b-Kim-J mice during the genetic background switch might have contributed to inducing inhibitory synaptic deficits.Future studies will be needed to explore this possibility in detail.Secondly, Arid1b haploinsufficiency in mice has been shown to cause growth impairment and deficiency of insulin-like growth factor (IGF) (8); this key regulator of brain/neuronal/synaptic development and metabolism has been implicated in various brain disorders, including ASD and neurodegenerative disorders (34)(35)(36)(37)(38). IGF-1, in particular, has been shown to induce long-lasting changes of inhibitory synaptic transmission in the neocortex, cerebellum, and olfactory bulb (39-42).WT C57BL/6 J mice reportedly show distinct metabolic states (body weight, tissue weight, and plasma insulin levels) compared to those of WT C57BL6/N mice (43).This is thought to involve differential gene expression and spontaneous genetic mutations.For example, deletion of exons 7-11 from the Nnt gene in C57BL/6 J mice is known to induce metabolic changes, such as glucose intolerance and body weight changes (44)(45)(46).We therefore suggest that In summary, we herein show that the genetic background is a key determinant of inhibitory synaptic phenotype in cortical neurons from Arid1b-mutant mice of C57BL/6 N and C57BL/6 J genetic backgrounds, whereas the excitatory synaptic phenotype is minimally affected by these genetic backgrounds.

Animals
Experimental procedures using mice (Mus musculus) were approved by the Committee on Animal Research at KAIST (KA2020-51 and KA2023-092-v1) and performed in compliance with all relevant ethical regulations.Mice were maintained according to the Requirements of Animal Research at KAIST, fed ad libitum, and housed under a 13:00-01:00 dark/light cycle.
As part of the NorCOMM2 project (TCPC317) funded by Genome Canada and the Ontario Genomics Institute (OGI-051) at the Toronto Centre for Phenogenomics, Arid1b +/− mice were generated by targeting exon 5 of Arid1b using CRISPR/Cas9 (C57BL/6 N-Arid1b em1Tcp ).This involved the use of Cas9 nickase (D10A) and single-guide RNAs with spacer sequences CTGCTTAGCAAGTTACCACT and GCCTGATACAGCACTTACAT for targeting the 5′ side of exon OTTMUSE00000314956 (exon 5), and sequences ACACTAAAGGGGTTGCTTTC and CTTGTAATCCCCC TGTAGTA for targeting the 3′ side.This resulted in deletion of Chr17 from 5242523 to 5243410 with insertion of "TT." For the present work, these mice were obtained from the Canadian Mouse Mutant Repository.
The originally published Arid1b-mutant mice were maintained in the C57BL/6 N background (10).To change the genetic background from C57BL/6 N to C57BL/6 J, male Arid1b +/− mice of C57BL/6 N background were backcrossed with female wild-type C57BL/6 J mice for more than seven generations.All mice used in the present study were generated by crossing backcrossed male Arid1b +/− mice of the C57BL/6 J genetic background with female wild-type C57BL/6 J mice.Pups were weaned at postnatal day (P) 21.After weaning, three to six mice of the same genetic background were co-housed in a single cage.The following primers were used for PCR genotyping: Arid1b HT/ heterozygotes, F/forward CATTACAGTGTCCTCTCCCATCTTG and WT-R/reverse GAAAGAGAAAGCGGGTGTTCATAC plus knockout (KO)-R CGGTGTGTGACTGTGATCATAGATG; for Nnt, primer ex6 F GGGTTTCGATTGCTGTCATT and ex6 R AGTCAGCAGCACTCCTCCAT; and ex9 F CCAGCATGCACT CTCTTCTG and ex9 R TGGTCTCCAAGTGCACAGAG (47).

Statistical analysis
Data are presented as means with standard error of mean (SEM).Statistical analyses were performed using GraphPad Prism software.Details on mice and statistical details are summarized in Supplementary Table S1.

FIGURE 1
FIGURE 1 Genetic backgrounds of Arid1b +/− mice and their changes.(A) Summary of the electrophysiological properties of layer 2/3 or layer 5 pyramidal neurons in mPFC regions representing two Arid1b +/− mouse lines of different genetic backgrounds (C57BL/6 J and C57BL6/N) having the same exon 5 deletion, as reported in two different studies (termed Arid1b-Jung-J and Arid1b-Kim-N mice, respectively).mEPSC, miniature excitatory postsynaptic currents; mIPSC, miniature inhibitory postsynaptic currents.(B) (Upper) A schematic diagram showing strategy of backcrossing Arid1b-Kim-N mice with wildtype (WT) C57BL/6 J mice for more than seven generations to generate Arid1b-Kim-J mice.(Lower left) Schematic diagrams of the WT and mutant Arid1b alleles and the Nnt alleles of the C57BL/6 N and C57BL/6 J backgrounds, along with the primers used to genotype them.(Lower right) PCR genotyping results indicating that the genetic background was successfully switched from C57BL/6 N to C57BL/6 J, as tracked by the lack of Nnt exon 9 in the C57BL/6 J background.