A neuron type-specific microexon in Ank3/ankyrin-G modulates calcium activity and neuronal excitability

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: major_revision

Abstract
Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

Introduction
Proper function of the mammalian brain relies on a complex assembly of hundreds of neuronal cell types with distinct morphological and electrophysiological properties that are building blocks of intricate neural circuits. This remarkable cellular diversity is specified by precise neuron type-specific gene-regulatory programs1,2. In addition to transcriptional regulation, alternative splicing of precursor messenger RNA (pre-mRNA) generates multiple mRNA and protein isoforms from a single gene by removing introns from pre-mRNAs and connecting exons in different combinations, thus dramatically amplifying the genetic information encoded in the genomic DNA3. Regulation of alternative splicing is particularly extensive in the brain. In recent years, global transcriptome profiling using bulk or single-cell RNA-sequencing (RNA-seq) has enabled the discovery of distinct splicing patterns in the brain compared with non-neuronal organs4–6, in neurons compared with non-neuronal cells in the brain7,8, in central nervous system (CNS) neurons compared with peripheral sensory neurons9, as well as across different developmental stages in the brain9. More recently, we and others have also identified hundreds of alternative exons differentially spliced in different neuronal types using RNA-seq data derived from purified neuronal populations or large-scale single-cell RNA-seq (scRNA-seq)10–12. These include microexons with lengths ≤30 nt, which are frequently neuron-specific and can be dysregulated in neurodevelopmental disorders such as autism13,14. The functional roles of a few such exons in the nervous system have been extensively studied. One example is Neurexin 1 (Nrxn1) exon 20 (also known as alternatively spliced site 4, or SS4), which is specifically included in GABAergic interneurons but skipped in excitatory neurons in a manner that is dependent on neuronal activity15. This alternative exon modulates excitatory synapse formation during brain development by altering the postsynaptic binding partners of Nrxn115–17. Multiple neuron type-specific exons and their regulatory mechanisms have also been studied in C. elegans. Two RNA-binding protein (RBP) splicing factors UNC-75 and EXC-7, whose mammalian homologs are CELF and ELAVL proteins, respectively, regulate a differential splicing program between GABAergic and cholinergic neurons18. Among these, depletion of a neuron type-specific isoform of unc-64/Syntaxin, a critical component in the SNARE complex that mediates synaptic vesicle fusion, led to movement coordination deficits. Despite these salient examples, it remains unknown how most neuron type-specific alternative exons contribute to cellular and organismal phenotypes.

The family of ankyrins, composed of ankyrin-R (ANK1), ankyrin-B (ANK2), and ankyrin-G (ANK3), encoded by homologous genes, are adaptor proteins with shared domain architecture that link membrane proteins to the spectrin/actin cytoskeleton19,20. Each family member is composed of 24 ankyrin repeats as a membrane-binding domain (MBD), two ZU5 domains and a UPA domain as a spectrin-binding domain (SBD), and a death domain (DD) followed by the C-terminal regulatory region. In this study, we focus on ankyrin-G (AnkG), encoded by ANK3, a leading risk gene for bipolar disorder in humans that has additional associations with schizophrenia (SCZ) and autism spectrum disorder (ASD)21–24. AnkG is best known as the key organizer of the axon initial segment (AIS) and nodes of Ranvier, where it clusters ion channels, transporters, and cell adhesion molecules to physically separate axonal and somatodendritic compartments and enable the initiation and propagation of action potentials25–30. Alternative splicing in ANK3 is well known to contribute to AnkG isoform diversity31 (Supplementary Fig. 1). The best known alternative exon is a giant exon (exon 37), which is specifically included in neurons to produce a 480 kDa isoform localized to the AIS and required for AIS establishment32. In a previous study, we demonstrated that exon 27a (Supplementary Fig. 1), a developmentally regulated exon that is skipped in adult neurons, is also critical for AnkG to accumulate in the AIS by modulating AnkG-spectrin interactions. Forced inclusion of this exon results in ectopic expression of the embryonic isoform in maturing neurons, causing impaired AIS formation and dramatic reduction in neuronal excitability33. This exon also contributes to AIS plasticity through neuronal activity-dependent splicing34. Regulation of most of these alternative exons appears to be pan-neuronal, which is consistent with the notion that the AIS is a characteristic subcellular structure shared by most neuron types.

Here, we investigate a highly conserved, neuron-type-specific microexon, Ank3 exon 35a or E35a, of 27 nucleotides (nt) long, which is highly included in GABAergic interneurons, but is predominantly skipped in glutamatergic excitatory neurons in the adult mouse cortex. We determine the major splicing factors that regulate cell-type-specific splicing of this microexon. Furthermore, using an E35a-deletion mouse model, we demonstrate that AnkG has acquired neuron type-specific functions outside the AIS through alternative splicing, allowing it to fine-tune the electrophysiological properties of different neuron types.

Results
A deeply conserved, highly regulated neuron type-specific microexon in Ank3
We recently performed a systematic analysis of neuron type-specific alternative splicing in adult mouse neocortex using scRNA-seq data with deep, full-transcript read coverage12,35,36. In this analysis, we found differential splicing among different neuron types defined at multiple hierarchical levels, including 469 cassette exons that showed differential splicing between glutamatergic and GABAergic neurons, the two major neuronal classes in the neocortex (Fig. 1a). Among them is a 27-nt microexon in Ank3 (denoted E35a hereafter) located between constitutive exons E35 and E36 and upstream of the giant exon E37 (Supplementary Fig. 1). Ank3 E35a is highly included in GABAergic interneurons, but is predominantly skipped in glutamatergic excitatory neurons (percent spliced-in, PSI or Ψ = 0.65 vs. 0.14) (Fig. 1b and Supplementary Fig. 2a, b). Splicing of the exon is neuron-specific, as it is skipped in non-neuronal brain cell types (Fig. 1b) and in non-brain tissues of both mice and humans (Supplementary Fig. 3a, b). Examination of additional neuron types in different brain regions using published RNA-seq data also revealed a striking degree of neuron type-specific splicing, with low exon inclusion in hippocampal glutamatergic neurons and medium spiny neurons of the dorsal striatum, similar to cortical glutamatergic neurons, but high inclusion in hippocampal GABAergic neurons, dopaminergic neurons, cerebellar Purkinje and granule cells, and olfactory sensory neurons, similar to cortical GABAergic neurons (Fig. 1c and Supplementary Fig. 2c; see further details in Supplementary Table 1 and Methods). During neurodevelopment, the exon inclusion is initially high in young glutamatergic neurons, as observed during neuronal differentiation in vitro, and is then reduced to low levels in mature neurons, a trajectory consistent with the overall decrease in exon inclusion observed in bulk cortex tissue in both the mouse and human brains; in contrast, E35a inclusion is maintained at a high level in GABAergic neurons (Supplementary Fig. 4a–c).

Multiple observations led us to focus on this exon as a model for detailed mechanistic and functional dissection of neuron type-specific alternative splicing. First, the peptide encoded by E35a is located in an intrinsically disordered region between the UPA domain and DD domain, based on the crystal structure of AnkB37 and the AlphaFold3-predicted structure of AnkG38 (Fig. 1d, e, and Supplementary Fig. 5a). This region is important for protein-protein interactions and is enriched in disease-related mutations37. Interestingly, the inclusion of the nine amino acids encoded by E35a appears to convert a loop into part of an extended alpha helix upstream of the DD domain, based on AlphaFold3 prediction (Supplementary Fig. 5b). Second, E35a and its flanking intronic regions are highly conserved across vertebrate species, including humans and fish, which implies considerable functional significance and strong purifying selection to maintain the highly regulated splicing pattern over ~450 million years of evolution (Fig. 1f). Third, to the best of our knowledge, the function of this microexon has not been characterized in the literature. However, when we searched for additional homologous sequences in the genome, we found a paralogous exon in the same region in Ank2, but not in Ank1. In contrast to the highly regulated alternative splicing for the Ank3 microexon, the Ank2 exon has six extra nucleotides, so that it encodes a total of 11 amino acids, and appears to be constitutively included in mouse tissues and cell types we examined (Fig. 1g). The homology between the Ank2 and Ank3 exons is also clear from amino acid sequence alignments (Fig. 1g). Interestingly, previous studies found that a missense E1425G mutation in the homologous region in AnkB, which disrupts its interaction with inositol trisphosphate receptors (InsP3Rs), is associated with type 4 long QT syndrome and sudden cardiac death in humans39,40 (Fig. 1g). The deep conservation of AnkG E35a and the health relevance of the homologous region in AnkB suggest that it may play a role in delineating neuron type-specific physiological properties through highly regulated alternative splicing.

Regulatory mechanisms of Ank3 E35a alternative splicing
To study Ank3 E35a, we first investigated which splicing factors contribute to the specific inclusion of the microexon in certain neuron types including GABAergic neurons. Our recent work identified multiple RBPs contributing to the differential splicing between glutamatergic and GABAergic neurons in the cortex, with Mbnl2, Celf2 and Khdrbs3 (Slm2) more highly expressed in glutamatergic neurons and facilitating glutamatergic neuron-specific splicing, while Elavl2 and Qk are more highly expressed in GABAergic neurons, facilitating GABAergic neuron-specific splicing12 (Fig. 2a). By analyzing differential splicing upon genetic depletion of RBP splicing factors using RNA-seq datasets9,41, we identified at least four candidate RBPs regulating microexon E35a (Fig. 2b). In the embryonic brain, this exon is suppressed by Ptbp2. In contrast, Celf2 and nSR100 (Srrm4), which are more highly expressed in glutamatergic neurons and GABAergic neurons, respectively, activate inclusion of the exon. In the adult brain, a major splicing suppressor is Mbnl2, which is more highly expressed in glutamatergic neurons. E35a has a weak 3ʹ splice site (SS) immediately flanked by multiple purines (A/G) in the polypyrimidine tract (3ʹ SS: −0.98, 5ʹ SS: 7.96; based on the human homolog in VastDB42). Accordingly, the upstream intron appears to be the main site of splicing regulation, which is also reflected in a high level of sequence conservation across vertebrate species. Indeed, the upstream intron contains binding sites of candidate RBP regulators we identified, based on CLIP data and bioinformatic motif site predictions9 (Fig. 2c), suggesting that the regulation of E35a by these RBPs is direct and likely conserved during vertebrate evolution. Interestingly, we also found that splicing of E35a is neuronal activity-dependent through our reanalysis of published RNA-seq datasets43,44. Specifically, E35a inclusion is reduced in cultured cortical cells after depolarization induced by KCl treatment. In parallel, there is also an increase in Mbnl2 expression and a decrease in nSR100 activity (Supplementary Fig. 4d, e and ref. 43), which are consistent with, and presumably contribute to, the observed activity-dependent splicing change. Together, these data suggest that combinatorial regulation by multiple RBPs dictates the differential splicing of E35a between cortical glutamatergic and GABAergic neurons, which can be further modulated by neuronal activity.

To further validate our results obtained from RNA-seq analysis, we focused on nSR100 and Mbnl2 as likely major regulators in mature neurons. We generated a mini-gene splicing reporter using sequences encompassing E35, E35a, E36, and intronic sequences in between. When the mini-gene was transiently transfected into differentiated mouse neuroblastoma N2a cells, E35a was completely skipped, similar to the splicing pattern of the endogenous Ank3 in non-neuronal cells and tissues. Overexpression of nSR100 with the mini-gene led to an increase in exon inclusion to 20%, suggesting that nSR100 can effectively activate exon inclusion (Fig. 2d). Moreover, simultaneous overexpression of Mbnl2 together with nSR100 reduced exon inclusion to 12%, as compared to nSR100 expression alone, and the magnitude of Mbnl2-regulated splicing repression was dose-dependent (Fig. 2d). We also repeated this experiment in HEK293T cells and obtained similar results (Fig. 2e). The magnitude of Mbnl2-dependent skipping in HEK293T cells is less than that observed in N2a cells, probably because N2a cells more closely recapitulate the neuronal cellular context. Together, these cell-based experiments validated our results obtained from global transcriptome analysis using in vivo data derived from various mouse models.

Generation of Ank3 E35a deletion (E35a−/−) mice
To investigate the functional impact of E35a’s neuron type-specific splicing, we generated a mouse line lacking this exon using CRISPR-Cas9. Two single guide RNAs (sgRNAs) were designed to target the intronic sequences flanking E35a, resulting in precise deletion of the exon (Supplementary Fig. 6a, b). Homozygous mutants, denoted Ank3 E35a−/−, were successfully obtained using this approach (Supplementary Fig. 6c). RT-PCR analysis of RNA from the cortex and cerebellum confirmed that these mice expressed only the Ank3 E35a-skipping isoform, while both isoforms were detected in wild-type (WT) controls; a higher exon inclusion level was observed in the cerebellum than the cortex, as expected from the major neuron types populating these brain regions (Supplementary Fig. 6d). Ank3 E35a−/− mice were born according to the expected Mendelian ratios and showed superficially normal development. Body weight assessment of adult Ank3 E35a−/− mice at 3 months of age did not exhibit any notable changes compared with WT controls (Supplementary Fig. 6e).

Ank3 E35a−/− GABAergic neurons show increased neuronal excitability without AIS disruption
With the establishment of E35a−/− mice, we first investigated the impact of E35a deletion on GABAergic neurons at the cellular level, with an initial hypothesis that the mutant mice might exhibit impaired AIS function and defects in neuronal excitability. For this analysis, given the associations of AnkG with epilepsy and bipolar disorders45, we focused on the orbitofrontal cortex (OFC), a prefrontal region implicated in decision making and social behavior46,47.

To selectively label GABAergic interneurons with tdTomato, we crossed Ank3 E35a−/− mice with Dlx6a-Cre;Ai9 mice expressing tdTomato. Whole-cell patch clamp recordings were performed in tdTomato+ OFC interneurons using brain slices from Ank3 E35a−/− and WT control mice (Fig. 3a). Current-clamp measurements of passive and active membrane properties from resting membrane potential (RMP) in each neuron suggested that there was an increase in input resistance for Ank3 E35a−/− interneurons, while values of RMP and capacitance were similar to those of WT interneurons (Fig. 3b–d). Action potentials (APs) were elicited at RMP using depolarizing current commands, and the excitability of interneurons in both groups was compared using rheobase values and the number of APs evoked by each current step. We found that interneurons from Ank3 E35a−/− mice were more excitable than WT interneurons, as reflected in reduced rheobase (Mann–Whitney U test, p = 0.0391) and increased firing rate (two-way ANOVA, p = 0.0129, n = 46 neurons for E35a−/− and 48 neurons for WT) (Fig. 3e).

Interneurons in the cortex consist of diverse neuronal subtypes with distinct firing properties2,48–50. To refine our analysis and reduce the variability in electrophysiological parameters due to interneuron diversity, we divided tdTomato+ interneurons into two groups: fast-spiking (FS; n = 42) interneurons and non-fast-spiking (non-FS; n = 52) interneurons. This categorization was based on the characteristic fast membrane time constant and higher firing rate of FS interneurons (Supplementary Fig. 7a)51–53. We also confirmed that the age distribution of recorded neurons did not differ significantly between WT and Ank3 E35a-/- mice (Supplementary Fig. 7b), and that FS interneuron excitability was comparable between late adolescent and adult mice in both groups (Supplementary Fig. 7c).

We found that FS interneurons (n = 21 for E35a−/− and 21 for WT) exhibited a higher firing frequency in Ank3 E35a-/- than WT (500 ms at 400 pA; 79 ± 5 vs. 55 ± 6 APs) (Fig. 3f, g), although the rheobase was not significantly lower in Ank3 E35a−/− neurons (Fig. 3h, top panel). RMP was similar between both genotypes (Fig. 3h, bottom panel), but FS interneurons from Ank3 E35a−/− mice displayed higher input resistance and lower capacitance, consistent with increased intrinsic excitability (Fig. 3i). APs evoked by rheobase + 100 pA also showed shorter latency to the first AP and a decrease in the first inter-spike interval (ISI) in Ank3 E35a−/− (Fig. 3j, k), while the fast afterhyperpolarization (fAHP) was unaltered (Supplementary Fig. 7d). By contrast, non-FS interneurons showed no significant differences in firing properties between Ank3 E35a−/− and WT (Supplementary Fig. 7e, f), although the rheobase was lower in Ank3 E35a−/− cells (Supplementary Fig. 7g). The passive membrane properties of non-FS interneurons in Ank3 E35a−/− did not differ between genotypes (Supplementary Fig. 7h-j).

To further investigate the potential effect of the AnkG microexon on AP initiation related to the AIS, we analyzed the properties of the first AP evoked by rheobase stimulation50,54,55. For FS interneurons, no differences were found in single AP properties elicited by either 5 ms current injections at a standardized membrane potential of −70 mV (Fig. 3l) or by 500 ms at RMP, except for the lower AP amplitude in Ank3 E35a−/− than in WT controls (Supplementary Fig. 7k). For non-FS interneurons, the properties of single APs were also similar between genotypes, whether evoked by long duration current steps at RMP (Supplementary Fig. 7l) or by short current pulses when neurons were held at −70 mV (Supplementary Fig. 7m). Taken together, these data show that Ank3 E35a−/− is associated with increased excitability in interneurons, specifically in presumed FS interneurons.

Given the altered neuronal excitability of FS cells, we examined AIS morphology to detect any potential structural changes56. To our surprise, the comparison between Ank3 E35a−/− and WT mice did not show any apparent alterations in AIS length and distance of the AIS to the soma in cortical vGAT+ interneurons within the OFC (Fig. 3m, n). To further confirm this result, we transfected 480kDa giant AnkG isoforms with or without E35a into dissociated hippocampal neurons from genetic Ank3 knockout mice (Ank3−/−) cultured in vitro (Supplementary Fig. 8a). Quantification of AIS length by immunostaining for AnkG or another AIS marker, β4-spectrin, at in vitro day 7 (DIV7) did not reveal any notable differences (Supplementary Fig. 8b–d). Together, these results demonstrate that deletion of E35a increases interneuron excitability, particularly in FS subtypes, without detectable disruption of the AIS structure.

Ank3 E35a−/− GABAergic neurons show increased somatic calcium activity
To investigate whether E35a contributes to neuron type-specific physiological properties in vivo, we performed two-photon Ca2+ imaging to examine the somatic activity of cortical neurons expressing the genetically encoded Ca2+ indicator GCaMP in awake, head-restrained mice57,58. This experiment was motivated by prior findings that a loss-of-function mutation in the homologous AnkB region causes aberrant Ca2+ handling in cardiac cells39,40. Ank3 E35a-/- or WT control mice crossed with Dlx6a-Cre;Ai9 mice were injected with an adeno-associated virus (AAV) encoding GCaMP6s under the control of the synapsin promoter to drive indicator expression in both tdTomato-pyramidal neurons and tdTomato+ interneurons (Fig. 4a). Three weeks post-injection, GCaMP6s-expressing neurons in the prelimbic (PL) area of the medial prefrontal cortex (mPFC) were imaged through a transcranial glass window (Fig. 4b). The PL region was chosen for accessibility for two-photon imaging; OFC and mPFC are reciprocally connected59 and are both implicated in decision-making and social behavior46,47.

We found that interneurons co-expressing GCaMP6s and tdTomato in Ank3 E35a−/− mice exhibited ~2-fold higher somatic Ca2+ activity compared with WT controls during quiet resting states (Fig. 4c–h). Quantitative analysis revealed significant increases in area under the curve (AUC; 8.05 ± 0.31 vs. 4.75 ± 0.23, p < 0.0001), peak amplitude (0.42 ± 0.02 vs. 0.23 ± 0.01, p < 0.0001), duration (0.88 ± 0.06 vs. 0.53 ± 0.02, p < 0.0001), and frequency (1.08 ± 0.10 vs. 0.52 ± 0.01, p < 0.0001) of Ca2+ transients (n = 110 neurons from three mice for WT and 164 cells from four mice for E35a-/-; Mann-Whitney U test) (Fig. 4c–h). By contrast, pyramidal neurons (GCaMP6s+, tdTomato–) from Ank3 E35a-/- mice showed little change relative to WT (Fig. 4i). Minor differences were observed in AUC (Fig. 4j, k) and peak amplitude (Fig. 4l), whereas Ca2+ transient duration (Fig. 4m) and frequency (Fig. 4n) were unchanged (n = 129 neurons from three WT mice and 170 neurons from four Ank3 E35a-/- mice; Mann–Whitney U test) (Fig. 4c–h). Together, these findings demonstrate that E35a deletion primarily increases somatic Ca2+ activity in cortical interneurons. This is consistent with the neuron type-specific splicing pattern of the microexon and with our in vitro electrophysiological results.

E35a modulates protein–protein interactions with the NCX/NKA/InsP3R complex
It was previously shown in cardiac cells that AnkB can tether Na+/K+ ATPase (NKA) and Na+/Ca2+ exchanger 1 (NCX1) localized to T-tubules, invaginations of the plasma membrane (PM), and InsP3Rs in sarcoplasmic reticulum (SR) membrane to form a complex clustered into microdomains at SR-PM junctions. This complex plays an essential role in modulating cytoplasmic Ca2+ levels that are required for normal rhythmic contraction40. Interestingly, the E1425G mutation in AnkB which causes type 4 long QT syndrome disrupts the NCX/NKA/InsP3R complex and the localization of its components at Z-line/T-tubules, resulting in altered Ca2+ signaling in cardiomyocytes39,40. We therefore tested whether alternative splicing of Ank3 E35a similarly regulates Ca2+ activity in neurons by modulating protein-protein interactions.

We performed co-immunoprecipitation (co-IP) of type 1 InsP3R (InsP3R1) from mouse cerebellum extracts using an anti-AnkG antibody. The cerebellum was chosen over cortex because of the high E35a inclusion at the bulk tissue level in the former but not the latter (Supplementary Fig. 6d), reflecting major neuron types constituting these different brain regions (i.e., both granule cells and Purkinje cells in the cerebellum have high E35a inclusion, whereas the GABAergic neurons with high E35a inclusion represent a minority population in the cortex; Fig. 1b, c). Among the three InsP3R isoforms, InsP3R1 is the predominant brain subtype, forming micro-domains at the junctional region of the endoplasmic reticulum (ER; the equivalent in non-muscle cells of SR) and PM (ER-PM junctions; the equivalent of SR-PM junctions in non-muscle cells)60,61. Western blotting of the precipitated proteins using anti-AnkG antibodies identified three bands at approximately 190 kDa, 270 kDa, and, at a lower abundance, ~480 kDa; the expression of the AnkG 190 kDa isoform was used for quantification in our analysis. An anti-InsP3R1 antibody detected a band corresponding to InsP3R1 with a molecular weight of 268 kDa. In input samples, the abundance of InsP3R1 and AnkG is comparable between Ank3 E35a−/− and WT mice, suggesting that E35a deletion does not affect their expression levels. Importantly, in the co-IP samples, the amount of InsP3R1 pulled down from the Ank3 E35a−/− cerebellum showed a 77% reduction compared with that from WT (p = 0.003; n = 4 per group; paired t-test), confirming a reduced interaction between AnkG and InsP3R1 in the absence of the E35a microexon (Fig. 5a). We also probed NKAα1 and observed a ~66% reduction in interaction with the complex in the absence of the E35a microexon (p = 0.048; n = 3 per group; paired t-test; Fig. 5b).

To test whether the E35a-encoded peptide directly mediates these interactions, we generated FLAG-tagged mini-gene constructs encoding AnkG peptides encompassing UPA and DD domains, with or without the peptide encoded by the E35a microexon (Fig. 5c). Of note, since the full-length AnkG has a plethora of interacting proteins, the use of these constructs helps assess the specific impact of the microexon in mediating protein-protein interactions. The UPA-DD mini-gene, with or without E35a, was transfected into N2a cells, and co-IP of InsP3R1 was performed using the anti-FLAG antibody. While the levels of the mini-gene and InsP3R1 expression were comparable in the input samples, the UPA-DD mini-gene without E35a pulled down only ~30% of InsP3R1 relative to its counterpart containing E35a (p = 0.0046; n = 3 per group; paired t-test; Fig. 5d). This experiment corroborates our results in vivo indicating that the E35a-encoded peptide and its flanking protein domains can directly mediate protein-protein interactions between AnkG and the NCX1/NKA/InsP3R complex at ER-PM junctions involved in intracellular Ca2+ transport.

Behavioral analyses in Ank3 E35a-/- mice
Given the altered AnkG isoform ratios, disrupted protein-protein interactions, and changes in neuronal excitability that we observed in Ank3 E35a−/− mice, we next tested whether these molecular and cellular phenotypes manifest at the behavioral level (Supplementary Fig. 9a). Because E35a is highly included in cerebellar neurons, we first assessed motor function. Gait analysis revealed that Ank3 E35a−/− mice exhibited an expanded step length (p = 0.0004) and stance width (p = 0.0023), implying a broader base of support during locomotion (Supplementary Fig. 9b; n = 15 mice for WT and 14 mice for Ank3 E35a−/− group; Mann–Whitney U test; the same for other behavioral tests below). Front paw (FP)—hind paw (HP) distance was unchanged, but the alternation coefficient was decreased, indicating highly coordinated stepping with consistent left-right alternation in Ank3 E35a−/− mice. Mutant mice also crossed the testing platform faster than WT littermates (p = 0.0075) (Supplementary Fig. 9b). By contrast, beam walk performance was unaffected (Supplementary Fig. 9c). Collectively, these data suggest that Ank3 E35a−/− mice do not have gross coordination deficits but instead display a pattern of fast, frenetic locomotor activity.

Because ANK3 variants are genetically linked to bipolar disorder21, we also assessed the mice with assays of anxiety-related responses (open field and elevated plus maze; Supplementary Fig. 9d, e), memory (Y-maze, Supplementary Fig. 9f), and social behavior (3-chamber social, Supplementary Fig. 9g). No significant differences were observed between the WT and Ank3 E35a-/- mice in these assays. However, mutants showed a trend toward increased velocity in the 3-chamber test and more vertical counts in the open field, consistent with the hyperactive locomotor phenotype detected by gait analysis. Overall, the behavioral phenotypes of Ank3 E35a−/− mice are relatively mild.

Methods
Mice
All experimental procedures followed the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals (National Research Council, 2011). All animal procedures were approved and performed following the institutional animal care and use committee’s policies at Columbia University. Male and female mice were group-housed in polycarbonate cages with corncob bedding; they were maintained in a humidity- and temperature-controlled vivarium (20-22°C) on a 12/12 h light/dark schedule (light on at 07:00 am and off at 07:00 pm). Animals had access ad libitum to food and water except during behavioral testing. Ank3 E35a-/- mice were generated in this study.

Generation of Ank3 microexon E35a deletion mice
To generate Ank3 E35a deletion (E35a−/−) mice using CRISPR-Cas9, we designed two single guide RNAs (sgRNAs) flanking the microexon. The sgRNAs (IDT) were assembled with the Alt-R™ S.p. Cas9 Nuclease V3 (IDT) in an injection buffer containing 5 mM Tris-HCl (pH 8) and 0.2 mM EDTA. This assembly process aimed to form the CRISPR RNP (ribonucleoproteins) with a final concentration of 0.1 µM. The assembly was conducted at room temperature for 10 min. Subsequently, the assembled RNP was microinjected into the pronuclei of fertilized B6CBAF1 eggs. Eggs that survived the injection were then transferred into the oviducts of E0.5 pseudo-pregnant surrogates. Pups resulting from this injection were genotyped at the age of 15 days old (P15) using PCR. Heterozygous Ank3 E35a+/− mice were generated in a B6CBAF1 mixed background (Jackson Laboratory Stocks #100011) and bred to Dlx6a-Cre; Ai9fl/fl mice (Jackson Laboratory Stocks #008199 and #007909). The mouse strain Ank3 E35a−/− generated in this study is available from The Jackson Laboratory (B6.Cg-Ank3<em1Czhng>/J; Jackson Laboratory Stock #040687).

RNA-seq analysis
RNA-seq data used for Ank3 E35a inclusion quantification in different neuron types were obtained from previous studies: cortical glutamatergic and GABAergic neurons, and non-neuronal cells from single-cell RNA-seq data35,36, hippocampal glutamatergic and GABAergic neurons10, dopaminergic neurons85, dorsal medium spiny neurons86, cerebellar Purkinje cells and granule cells87, olfactory sensory neurons88, dorsal root ganglion (DRG) neurons89–91, rod and cone cells92,93, and motor neuron94. We also examined exon 35a splicing in mouse95 and human tissues96, during cortex development of mouse9 and human brain97, and differentiation of mouse embryonic stem cells (mESCs) to excitatory glutamatergic neurons98. Most of these datasets were analyzed using Quantas7 in a previous study9 or otherwise analyzed using the same pipeline. Splicing across human tissues was calculated using exon junction read counts generated by the GTEx consortium96. To identify potential regulators of exon 35a, we used RNA-seq data from mouse brains with genetic depletion of the following splicing factors, Ptbp2 (ref. 99), nSR100 (ref. 41), and Mbnl1/2 (ref. 9). These datasets were also analyzed previously or in this study using the same pipeline9. Conditional Celf2 knockout in cortical excitatory neurons were generated by breeding Celf2fl/fl mice with CamKII-Cre, which was used to derive RNA-seq data of Celf2 KO and WT control. The RNA-seq data was analyzed as described previously9; only Ank3 E35a inclusion was examined in this study, and additional analysis of the dataset will be described elsewhere. Neuron-activity dependent splicing was analyzed with the same pipeline9 using RNA-seq data derived from primary cortical44 and hippocampal43 neurons depolarized by KCl. Public RNA-seq data analyzed in this study were summarized in Supplementary Table 1.

Plasmid cloning
Ank3 E35a minigene (genomic sequence extending from exon E35-E36 including E35a and flanking intronic sequences; 1,926 bp) was cloned from P7 mouse cortex genomic DNA in a TOPO TA vector (Thermo Scientific #450030) according to kit instructions (see primer sequences in Supplementary Table 2). Next, this sequence was subcloned to the pcDNA3.1 (+) vector, with EcoRI digest to both TOPO TA vector (+minigene) and pcDNA3.1 (+), followed by ligation and transformation.

The giant AnkG 480kDa isoform construct without E35a (AnkG480-GFP) was described in our previous studies32,100. Due to the relatively large coding sequence (approximately 14 K) of giant AnkG, we generated AnkG480-E35a-GFP, including E35a, using a cloning-based strategy. In brief, the AnkG480-GFP plasmid was digested with different restriction enzymes, NheI and Bstz17I (Supplementary Table 2) to remove a fragment that contains the target region to insert E35a, and the cut vector fragment was purified. Fragment 1 was created by a forward primer that contains a 15 bp overlap with the digested vector and a reverse primer that contains the mutated site and 15 bp overlap with fragment 2. Fragment 2 was created using a forward primer that contains the mutated site and 15 bp overlap with fragment 1 and a reverse primer that contains 15 bp overlap with the digested vector. Finally, the digested vector, fragment 1 and fragment 2 were assembled using In-Fusion® clone kit (Takara Bio USA, Inc.; #638920). Primers used to generate giant AnkG mutations are listed in Supplementary Table 2.

We generated plasmids expressing FLAG-tagged AnkG peptides containing specific UPA-DD domains with and without the E35a microexon under the control of the pCAGGs promoter. These sequences were cloned into the TOPO TA vector from cDNA derived from P7 mouse cortex with specific primers (Supplementary Table 2). These sequences were then subcloned into a pCAGGs plasmid together with an ATG start codon, 3X-FLAG, and stop codon sequences to allow for translation of these peptides. The plasmid constructs generated in this study have been deposited in the Addgene repository under accession codes 238352, 238353, 241435, and 241434.

Cell cultures
HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco™ #12491015) supplemented with 10% FBS (Thermo Fisher Scientific #A5256801), and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific #15140122). N2a cells (ATCC #CCL-131) were cultured in DMEM, supplemented with 10% bovine serum (Thermo Fisher Scientific #A5256801), 1x non-essential amino acids (Thermo Fisher Scientific #11140050), and 100 U/ml penicillin-streptomycin. For N2a differentiation, cells were kept in the presence of 0.2% FBS and 2% DMSO (Sigma Aldrich # 34869) for 7 days.

Ank3 E35a minigene splicing reporter assay
HEK293T cells were plated at a density of 400,000 cells per cell in 6-well plates. Cells were co-transfected using calcium phosphate (CalPhos™ Mammalian Transfection Kit, #631312, Takara) with 125 ng Ank3 minigene, 1 μg nSR100 (pLDPuro-MmnSR100N, Addgene #35171) and increasing amounts of MBNL2 (Addgene #217032; ref. 9) (1 μg, 1.5 μg, and 2 μg). N2a cells were plated at a density of 300,000 cells per cell in 6-well plates and differentiated for 7 days in the presence of 0.2% FBS and 2% DMSO. Cells were then co-transfected using lipofectamine 3000 (Thermo Fisher Scientific #L3000001) with 125 ng Ank3 minigene, 0.5 μg nSR100, and increasing amounts of MBNL2 (0.5 μg, 1 μg, and 1.5 μg). Expression of transfected splicing factors was validated using western blots of RIPA cell lysates (Thermo Fisher Scientific #89901).

RT-PCR validation of splicing quantification
To measure exon inclusion, RNA was extracted using TRIzol reagent (Invitrogen #15596026) or Direct-zol RNA kits (Zymo Research #R2061) from cultured cells or brain tissues. Total RNA was reverse transcribed using SuperScript III RT (Thermo Fisher Scientific #12574026,) in 20 μL reaction with random hexamer primers (f.c. 0.5 ng/μL). The cDNA was PCR amplified using gene-specific primers (Supplementary Table 2) and analyzed by the Qiaxcel system (Qiagen, #9001421) or agarose gel electrophoresis.

AIS analysis using brain tissues
To analyze AIS of GABAergic neurons in Ank3 E35a-/- and WT adult mice (3-4 months old), we first labeled GABAergic neurons by in situ hybridization via the vesicular GABA transporter (vGAT) probe (SLC32A1, Advanced Cell Diagnostics #319191). Immunohistochemistry (IHC) was then performed to stain the AIS structure using anti-AnkG (Synaptic Systems #386003). Fluorescence in situ hybridization (FISH) was conducted using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics, #323110) following the manufacturer’s protocol. 100 μm coronal brain sections were cut using a vibratome (Leica VT1000s) from bregma 2.5 to 2.0 mm from 4% paraformaldehyde (Fisher Scientific #AAJ19943K2) perfusion-treated mice. The brain sections were then post-fixed with cold acetone for 2 min, followed by treatment with hydrogen peroxide, target retrieval reagent (Advanced Cell Diagnostics, #322000), and digestion with Protease III (Advanced Cell Diagnostics, #322337). The vGAT RNA probe (SLC32A1, Advanced Cell Diagnostics, #319191) was hybridized for 2 h. Signal amplification was performed with HRP, followed by the fluorescent labeling with Opal 690 reagent (Akoya Biosciences, #FP1497001KT), which was diluted 1:1000 in TSA buffer (Advanced Cell Diagnostics, #322810). IHC was processed after the step of HRP blocker treatment in the FISH. The brain sections were blocked in blocking solution (5% donkey serum (Jackson ImmunoResearch, #017000121) with 0.3% Tween-20 in PBS solution) at room temperature for 1 h, followed by incubation with anti-AnkG antibody (Synaptic Systems, #386003, 1:500 dilution in blocking solution) at 4°C overnight. Alexa Fluor 488 donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, #711545152), diluted 1:500 in 3% BSA (Millipore Sigma, #12660) with 0.1% Tween-20 in PBS solution, was used to incubate the brain sections at room temperature for 1 h. Fluoro-Gel II (Electron Microscopy Sciences, #1798551), which contains DAPI, was used to mount the brain sections. Images were acquired using a Zeiss 810 confocal microscope with a 40x objective and Z-stack processing for the orbitofrontal cortical regions, the same brain region where our patch clamp analysis was performed. The positions of vGAT-labeled GABAergic neurons and their associated AIS structures were identified by observing different focal planes and 3D images from the Z-stack in ZEISS ZEN 3.8. The Extended Depth of Field (EDF) plugin (Biomedical Imaging Group) in ImageJ (FIJI)101 was used to combine Z-stack images into a single, in-focus image102. AIS length and AIS-to-Soma distance (from the root of the AIS to the soma boundaries) measurements were done by manual identification of the AIS region based on AnkG staining on EDF-treated images using ImageJ.

AIS analysis using cultured hippocampal neurons
The method for culturing primary hippocampal neurons has been previously described in detail103,104. Hippocampi were harvested from genotyped postnatal day 0–1 (P0-P1) mice (AGE22.23fl/fl), then treated with trypsin, dissociated, and plated on poly-L-lysine-coated 18-mm glass coverslips at a density of approximately 600,000 neurons per 60-mm culture dish. The initial plating medium consisted of MEM supplemented with 0.6% (w/v) glucose and 10% (v/v) horse serum. After allowing the neurons to adhere for 2–3 h, the coverslips were carefully transferred into preconditioned growth medium, which included Neurobasal-A medium with 2% (v/v) B27 supplement and 1× GlutMAX® (Thermo Fisher #21103049, #17504044, #35050061). Cultures were maintained in a 37°C incubator with 5% CO2.

Neuron transfection. In 3 DIV, 0.25 μg total AnkG plasmids (AnkG480-GFP and AnkG480-E35a-GFP) for each coverslip were mixed with 25 μL opti-MEM (Thermo Fisher, #31985062) at room temperature for 5 min. Then the DNA mixture was combined with 0.75 μL Lipofectamine 2000® (Thermo Fisher, #11668019) and 25 μL Opti-MEM for 10 min at room temperature. Each coverslip was added 50 μL of the DNA/lipofectamine mix and incubated for 30 min in the 37°C incubator. Then the coverslips were flipped back to the previous neuron growth media. Transfected hippocampal neurons were maintained in the incubator until fixation.

Immunocytochemical staining. In the fixation step, we used incubate-warmed 4% wt/vol PFA with corresponding 4% wt/vol sucrose in 1x PBS to add on each coverslip and incubated for 10 min at 37°C, and then washed with PBS three times for 5 min. For immunostaining, fixed neurons were permeabilized with 0.25% Triton X-100 for 10 min, washed with PBS three times for 5 min and then blocked with 5% BSA in PBS for 1 h at room temperature. The primary antibodies (anti-AnkG or anti-beta4 spectrin, custom-made rabbit polyclonal; ref. 32) were diluted (1:1000) in 5% BSA and incubated at 4°C overnight. On the next day, neurons were washed with PBS for 5 min and incubated with the appropriate fluorescent secondary antibodies in blocking buffer for 1 h at room temperature. Finally, neurons were mounted with Mowiol mounting medium (Mowiol 4-88, Glycerol, Tris-HCl pH 8.5 and DABCO (1,4-diazabicyclo-[2,2,2]-octane) onto glass slides and allowed to cure for 24 h before imaging.

Imaging and data analysis. Immunofluorescent-stained neurons or paraffin-embedded tissue sections were imaged with confocal microscopy (Olympus FV3000). For AIS intensity quantification, images were captured using a 100x NA/1.4 oil objective. Each experiment was repeated independently three times, and the final quantification was derived from 8-10 neurons in vitro. To ensure the reproducibility of intensity measurements, all neurons from the same experiment were imaged under identical microscope settings. A standard AIS image was obtained as a three-step Z-series with a 0.30 μm interval between each step. These Z-series images were processed using the maximum intensity projection function in Fiji software101. The resulting maximum intensity projections were used for AIS intensity quantification, following the method adapted from Berger et al. study105. AIS length was quantified using a custom MATLAB script, similar as previously described100.

Electrophysiology
Brain slices were obtained from Ank3 E35a−/− (8) and WT control (8) mice (6 to 12 weeks old, except for one 15-week-old mouse in each group) of both sexes. Mice were fully anesthetized with isoflurane and decapitated. Brains were dissected and sectioned in ice-cold (4 °C) artificial cerebrospinal fluid (aCSF) composed of (in mM): 124 NaCl, 2.5 KCl, 2 MgSO4, 1.25 NaH2PO4, 2 CaCl2, 26 NaHCO3, 10 glucose, and 2.5 sucrose, to yield coronal slices (300 μm thick) containing the orbitofrontal cortex (OFC) using a vibrating blade (VT1000S; Leica Biosystems). Slices were placed in a holding chamber containing oxygenated (95% O2–5% CO2) aCSF at 34°C for 30 min and were subsequently transferred to room temperature for at least another 30 min before recordings were initiated. For recordings, slices were submerged in a chamber continuously perfused with oxygenated aCSF at 32°C at a flow rate of 1–2 ml/min. Brain slices were visualized under an upright light microscope (BX51WI, Olympus) coupled to a camera (Hamamatsu #C8484), and the OFC area was identified according to cytoarchitectural criteria as previously defined in the Paxinos and Watson mouse brain atlas106. Dlx6a-Cre interneurons were identified by the Ai9-tdTomato fluorescence (mercury arc lamp or LED 525 nm).

Whole-cell patch-clamp electrophysiology was performed as previously described107. Intracellular recordings were made using pipettes pulled from borosilicate glass (World Precision Instruments, TW150F-4) with a resistance of 3–6 MΩ were filled with a potassium gluconate intracellular solution containing (in mM): 130 potassium gluconate, 4 NaCl, 10 HEPES, 0.3 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine (300-305 mOsm). The pH was adjusted to 7.2-7.3 using KOH. 0.4% biocytin (Sigma #B4261) was added to the potassium gluconate intracellular solution for later morphological identification of a subset of recorded neurons. After recordings, slices were fixed for 24–48 h at 4°C in 4% formaldehyde in PBS. After washing out the PFA, the slices containing biocytin-filled neurons were incubated with Streptavidin Alexa Fluor-488 conjugate (1:300 v/v dilution, ThermoFisher #S11223) in PBS and Triton-X 0.3% for 3-5 h at room temperature. The slices were rinsed with PBS and mounted on glass slides to acquire images with a confocal microscope (Nikon Eclipse Ti) and then processed with ImageJ.

Recordings of the electrophysiological membrane properties and intrinsic excitability were performed in the absence of synaptic blockers. Electrophysiological recordings were performed with a MultiClamp 700B amplifier (Molecular Devices), and 1440 Digitizer (Molecular Devices), interfaced with Clampex 10.7 software (Molecular Devices). Data were collected at either 10 or 20 kHz and low-pass filtered at 2 or 10 kHz. Series resistance was compensated in current clamp using the bridge balance. Patch-clamp data was acquired only if the resting membrane potential was below −50 mV and initial access resistance < 25 MΩ.

Following equilibration (~2–3 min) after break-in, a current protocol of 10 pA command increments (6 steps starting at −30 pA, 500 ms) was used to measure passive membrane properties, including the resting membrane potential (RMP), the membrane time constant and capacitance. Input resistance (in MΩ) was calculated from the slope of a linear regression fit to the voltage-current relation in the 10 pA command increments. Using the same protocol, the time constant (tau) was estimated by the exponential fit between the beginning of the decay and the resting membrane potential. Then, capacitance was calculated from input resistance and time constant. Firing properties were characterized using 20 pA command increments from 0 to 400 pA, 500 ms duration, and APs were identified as events exceeding a threshold of −10 mV or with an amplitude of at least 70 mV from baseline voltage. The input-output curves (the number of spikes in response to each current step) and rheobase (minimum current to elicit an AP) were determined using this protocol and the following parameters were also calculated for the first evoked AP: voltage threshold (voltage where dV/dt > 5% of the maximal increased dV/dt), amplitude (the difference between the AP voltage peak and the nadir of the fast afterhyperpolarization), duration (“width” at half-height), maximum rise slope (maximum dV/dt during depolarization phase), and maximum decay slope (maximum rate of fall of membrane potential during repolarization phase). Latency to the first AP (time between the start of the stimulus until the time of the first AP), first ISI (the ISI between the first two APs), and fAHP (voltage difference between voltage threshold and the minimum voltage after the AP peak), were measured at rheobase + 100 pA. The 10 pA and 20 pA step protocols were repeated at RMP and from a holding potential of −70 mV. Additionally, single APs were evoked by short current pulses (5 ms) in 10 pA increments with the membrane potential held at −70 mV. AP properties using this protocol were analyzed as described above.

Offline analysis of in vitro electrophysiological data was performed using Clampfit 10.7 and Python scripts adapted from the IPFX package (https://github.com/alleninstitute/ipfx). To include neurons in the analysis, the following criteria were applied: cell with a stable resting membrane more negative than −55 mV, access resistance lower than 25 MΩ or variation < 30% throughout the recording, input resistance lower than 1000 MOhm, and more than one AP evoked at RMP by the range of current stimuli (0 to 400 pA). Following these criteria, 5 WT and 3 Ank3 E35a-/- interneurons were excluded from the analysis. Graphing and statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Data are shown as scatter plots and as mean ± SEM in the input-output curves. Data was reported uncorrected for the liquid junction potential. Comparisons were considered significantly different when p < 0.05 and exact p-values were reported in the figures.

In vivo calcium imaging
Surgical preparation. In vivo Ca2+ imaging was performed in the prelimbic (PL) area of the medial prefrontal cortex of awake, head-restrained mice, as previously described108,109. Mice were deeply anesthetized with an i.p. injection of 100 mg/kg ketamine and 15 mg/kg xylazine. After shaving the fur, the skull surface was exposed with a midline scalp incision, and the periosteum was removed. A head bar was glued to the skull with adhesive to minimize motion artifacts during imaging. A small skull region (~1 mm in diameter) over the PL (anterior-posterior (AP) +2.68 mm, medial-lateral (ML) 0.5 mm) was carefully removed without damaging the dura mater. A round glass coverslip, matching the size of the removed bone, was glued in place, and dental acrylic cement was applied to secure the glass window. Throughout the procedure and recovery, the animal’s body temperature was maintained at ~37°C. After recovery, mice were habituated to head restraint on the imaging platform in three 10-min sessions to minimize stress. Imaging experiments were performed 24 h after surgery to ensure no lingering effects from anesthesia.

In vivo Ca2+ imaging and data analysis. GCaMP6s was expressed in PL neurons using recombinant adeno-associated virus (AAV) under the synapsin promoter (AAV1-Syn-GCaMP6s; Addgene #100843). 0.1 μl of diluted AAV (1:10) was injected into the left PL (AP +2.68 mm, ML 0.5 mm, subpial (SP) 0.85 mm) of Dlx6a-Cre;Ai9+; Ank3 E35a−/− or Dlx6a-Cre;Ai9+; Ank3 WT mice. The injection was performed over 10–15 min using a Picospritzer III (15 p.s.i., 10 ms, 0.5 Hz) and a glass microelectrode. Imaging was performed approximately 3 weeks post-injection. In vivo Ca2+ imaging was conducted using a Scientifica two-photon system equipped with a Ti:sapphire laser (Chameleon Vision-S, Coherent) tuned to 920 nm. Imaging was performed using a 25× objective (1.05 N.A.) immersed in artificial cerebrospinal fluid, with a digital zoom of 1.5×. Images were acquired at ~1.7 Hz (2-μs pixel dwell time) with a resolution of 512 × 512 pixels. Data acquisition was performed using ScanImage software, and subsequent analyses were conducted using NIH ImageJ software. Regions of interest (ROIs) corresponding to visually identifiable somas labeled with GCaMP6, with or without tdTomato, were selected for quantification. The fluorescence time course for each ROI was determined by averaging pixel intensities within the ROI. Background fluorescence was subtracted from the GCaMP6s signal, and Ca2+ transients were calculated as ΔF/F0, where ΔF/F0 = (F–F0)/F0, with F0 representing the baseline fluorescence averaged over a 3.5-s period of minimal fluorescence during the recording. Motion artifacts due to respiration and heartbeat were generally <2 μm and were minimized by habituation and head bar fixation. Vertical movements were rare, and segments with struggling behavior were excluded from analyses.

Quantification and statistical analysis. The average integrated Ca2+ activity was quantified as the area under the curve (AUC) of ΔF/F0 over 35 s. Ca2+ events were defined as fluorescence signals > 3× the standard deviation (s.d.) of baseline fluorescence. The peak amplitude of Ca2+ transients was defined as the average of peak ΔF/F0 during the 35 s recording. Frequency was calculated as the number of Ca2+ transients per min, and duration was measured as the average full width of Ca2+ transients during the recording period.

Summary data are presented as means ± SEM. Sample sizes were chosen to ensure statistical power while minimizing animal usage. No successfully imaged animals were excluded from analysis. Data normality was assessed using the Shapiro-Wilk test. Nonparametric statistical comparisons were made using the Mann-Whitney U test, with significance set at p < 0.05. All statistical analyses were performed using GraphPad Prism software.

Co-Immunoprecipitation
Brain tissue (cerebellum). Cerebellum lysates from WT and Ank3 E35a-/- mice brains were prepared by homogenizing the tissue using a hand Dounce in lysis buffer (30 mM Tris, 150 mM NaCl, 1% IGEPAL, 0.5% Na Deoxycholate, 0.1% SDS, 2 mM EDTA, pH 6.8) with freshly added protease and phosphatase inhibitors. The homogenates were ultracentrifuged at 435,400g for 30 min at 4°C to collect clear whole tissue lysates.

For the co-immunoprecipitation (co-IP) process, 50 μL of magnetic beads (Protein G, Invitrogen, #10003D) were prepared by resuspending them in a tube, separating them from the solution using a magnet, and washing them with NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40). Subsequently, 5 μg of the anti-AnkG antibody (Santa Cruz #sc-12719) was added to the magnetic beads and incubated for 4 h at room temperature. The supernatant was removed using a magnet, and the magnetic bead-antibody-antigen complex was resuspended in whole tissue lysates (500 μg total protein) and rotated overnight at at 4°C. The following day, the bead-antibody-antigen complex was washed three times with NT2 buffer and once with wash buffer (NT2 buffer without NP-40), then resuspended in a clean tube. The target antigen was eluted from the complex using 50 μL elution buffer (50 mM glycine, pH 2.8) by resuspending the magnetic bead-antibody-antigen complex and placing it on a thermomixer at 23°C and rotated for 15–20 min. The tubes were then placed on the magnet, and the supernatant was transferred to new tubes. To adjust the pH, 2 μL of Tris-HCl pH 7.5 was added along with 12.5 μL of premixed NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific #NP0007), and the samples were stored at −20°C. When ready for Western blotting, NuPAGE™ Sample Reducing Agent (Thermo Fisher Scientific #NP0009) was added to the samples, which were heated for 10 min at 68°C.

N2a cells. N2a cells were cultured in DMEM GlutaMAX (Invitrogen #10566-016) supplemented with 10% fetal bovine serum. For the co-IP experiments, cells were plated on a 6-well plate, with three wells designated for each condition: with and without E35a. After 48 h of transfection with minigene plasmids, cells from three wells of each condition were pooled together. The cells were lysed in 400 μL of IP lysis buffer (Pierce™ IP Lysis Buffer, Thermo Fisher Scientific #87787) with protease and phosphatase inhibitors and ultracentrifuged at 435,400g for 30 min at 4°C. Total protein isolated from all three wells for each condition was used for IP using Anti-Flag M2 antibody (Sigma-Aldrich F3165), following the same protocol as described above for cerebellum samples.

Western immunoblotting. Protein lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane using the iBlot Dry Blotting System (Thermo Fisher Scientific #IB21001). The membranes were blocked with 5% bovine serum albumin in TBS-T (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20) for 30 min at room temperature. They were then incubated overnight at 4°C with the primary antibodies IP3R1 (Millipore Sigma #ABS55) and/or Na-K ATPase (Thermo Fisher Scientific # MA5-36259) (1:1000). Following incubation, the membranes were washed three times with TBS-T buffer (10 min each wash) and then incubated for 1 h at room temperature on a rotator with an HRP-conjugated secondary antibody. The membranes were washed again three times (10 min each wash) to remove excess secondary antibody. Next, the membranes were incubated with infrared Dye 800-conjugated secondary antibodies (1:10,000 dilution, LI-COR Biosciences, #926-32210 and #926-32213) for 1 h at room temperature. Images were captured using the Odyssey infrared imaging system (LI-COR Biosciences). Quantification was performed using ImageJ and Prism GraphPad software 9.0.

Behavioral assays
All behavior analyses were performed using ~3-month-old mice during the light phase of the light/dark cycle. All behavior tests, except for the 3-chamber test, were performed on 15 WT (9 males and 6 females) and 14 Ank3 E35a-/- (6 males and 8 females). Three-chamber test included 21 WT (11 males and 10 females) and 19 Ank3 E35a-/- (9 males and 10 females).

Gait analysis. Gait analysis was performed as previously described110. In brief, a transparent Plexiglass walkway (66 cm long, 6 cm wide) connected to a black box at its end was used to entice the animals in. The walkway has a mirror underneath with an inclination of 45° to allow a clear vision of the paws disposition from the front. Mice were filmed with a digital camera 12MP and 60 fps placed horizontally, 35 cm from the walkway. Ank3 E35a-/- and WT littermates were encouraged to walk at least three consecutive steps per trial, for a total of three trials. Still-frames from the recordings were extracted and analyzed offline using the Manual Tracking plugin of Fiji software101 in order to obtain data concerning stride length (the distance of the same paw in a consecutive step), width (the distance between the center of the two hind or fore paws), the distance between ipsilateral forepaw and hind paw placements and the fore and hind stance (alternation coefficient). The walking speed was also measured in order to exclude differences, which might affect gait parameters.

Beam walk test. The beam walk test was performed using a beam (55 cm long section, 5 mm wide) connected to a black box at its end to entice the animals in. Mice were trained in the beam one day prior to testing by allowing them to cross the beam for three consecutive runs. The test result was recorded using Noldus’ Ethovision software, which uses mouse body point tracking features while crossing the beam. Parameters such as Mean Velocity (cm/s), Mean Meander (deg/cm) and % Mean Mobility were analyzed. A camera was also used in parallel to assess manually the number or toe slips of each mouse.

Y-maze (spontaneous activity). Spontaneous alteration is assessed in a Y-shaped maze with three white, opaque plastic arms at a 120° angle from each other. After introduction to the center of the maze, the animal is allowed to freely explore the three arms for a total of 8 min. Over the course of multiple arm entries, the subject should show a tendency to enter a less recently visited arm. The number of arm entries and the number of triads are recorded in order to calculate the percentage of alternation. An entry occurs when all four limbs are within the arm. This test is used to quantify cognitive deficits in transgenic strains of mice. Percent alternation can be analyzed as a measure of working memory. Alternation % = (Number of spontaneous alternations) / (total number of arm entries - 2) × 100, where one alternation refers to a sequence of three different arms. The theoretical chance alternation % = 1 × 2/3 × 1/3 ≈ 22%.

Three-chamber test. Social approach was assayed using an automated three-chambered apparatus (NIMH Research Services Branch, Bethesda, MD) as previously described111,112. Novel target mice were 3-month-old FVB/NJ mice of the same sex as the subjects. The apparatus was a rectangular, three-chambered box made of clear polycarbonate. Retractable doorways built into the two dividing walls controlled access to the side chambers. The number of entries and time spent in each chamber were automatically detected by photocells embedded in the doorways and tallied by the software. The test session began with a 10-min habituation session in the center chamber only, followed by a 10-min habituation to all three empty chambers. Lack of innate side preference was confirmed during the second 10-min habituation. The subject was then briefly confined to the center chamber while the clean novel object (an inverted stainless-steel wire pencil cup; Galaxy; Kitchen-Plus; http://www.kitchen-plus.com) was placed in one of the side chambers. A novel mouse previously habituated to the enclosure was placed in an identical wire cup located in the other side chamber (a disposable plastic drinking cup containing a lead weight was placed on the top of each inverted wire pencil cup to prevent the subject from climbing on top). After both stimuli were positioned, the two side doors were simultaneously lifted and the subject was allowed access to all three chambers for 10 min. Time spent in each chamber and entries into each chamber were automatically tallied. Time (frequency and duration) spent sniffing and rearing towards the novel object and time spent sniffing and rearing toward the novel mouse during the 10-min test session were later scored from video recording, by an observer using two stopwatches. The apparatus was cleaned with 70% ethanol and water between subjects. FVB/NJ mice were used as target novel mice because this strain is generally inactive, passive, and does not exhibit aggressive behaviors toward subject mice. Using a minimally active partner is a strategy that allows all approaches to be initiated by the subject mouse only.

Open field. Open field test was performed according to published protocols113,114. Each mouse was gently placed in the center of a clear Plexiglas arena (27.31 × 27.31 × 20.32-cm, Med Associates ENV-510) lit with dim light (~5 lux), and was allowed to ambulate freely. Infrared (IR) beams embedded along the x, y, z axes of the arena automatically track distance moved, horizontal movement, vertical movement, stereotypies, and time spent in the center zone. Exploration was monitored during a 30-min session with Activity Monitor Version 7 tracking software (Med Associates Inc.). Parameters such as ambulatory distance (cm), center time (sec), and vertical counts were recorded. Data were analyzed in six, 10-min time bins. Areas were cleaned with 70% ethanol and thoroughly dried between trials.

Elevated plus maze (EPM). The elevated plus maze (EPM) apparatus consists of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm) extending from a central junction (5 cm × 5 cm) area. Each mouse completes one trial with a duration of 5 min. Each mouse is gently placed in the junction area to start the trial and is allowed to ambulate freely in the maze for the duration of the trial. Exploration of the closed and open arms as well as total entries were monitored via the Ethovision XT software. Percent (%) time spent in closed/open arm was calculated by dividing the time spent in closed/open arm by 300 s, and multiply by 100). Total entries are the sum of the open entrance and closed entrance. Areas were cleaned with 70% ethanol and thoroughly dried between trials.

Statistical analysis
Statistical analysis was performed using GraphPad Prism unless indicated otherwise. Specific tests used are provided in the Results section or figure legends. A summary of all statistical tests and parameters is provided in the Supplementary Table 3.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study characterizes a neural-specific microexon (E35a) in the Ank3 gene with several remarkable findings that provide the best rationale so far for the link between ANK3 and human bipolar disorder first reported in 2008. The authors demonstrate for the first time that E35a is preferentially expressed in adult GABAergic interneurons. They find that E35a is paralogous to a constitutively expressed sequence in Ank2 that is the target for mutation causing long QT cardiac arrythmia. The E35a equivalent exon in Ank2 was demonstrated previously to promote interaction of between ankyrin-B with IP3 receptors in the ER and Na/K ATPase, and the Na/Ca exchanger in the plasma membrane. In cardiomyocytes this ankyrin-B/IP3 receptor, NKA, NCX complex is responsible for electroneutral export of calcium ions, and its loss results in abnormal calcium dynamics. The current study demonstrates that retention of E35a in GABAergic neurons confers a gain of function of linking ankyrin-G to the IP3 receptor and Na/K ATPase. Furthermore, mice lacking E35a exhibit increased calcium transients in prefrontal GABAergic neurons imaged in awake animals using 2-photon microscopy. Consistent with these findings E35a-null mice exhibit increased excitability in behavioral assays.\nFor the first time in my recent memory, I am recommending acceptance of a paper with minimal revision. The data are clean, the experiments well designed and cutting edge in cellular neuroscience as well as RNA biology, and the results are clear and well presented. The findings are original and of high impact with the first evidence of how ANK3 mutation could provide a substrate for bipolar disease. I have a few minor points that could further improve the presentation:\n1. Lines 196-197. Interestingly, we also found that splicing of E35a is neuron activity-dependent. E35a inclusion is reduced in cultured cortical cells after depolarization induced by KCl treatment 41,42\nAs written, a reader could infer that activity-dependent inclusion of E35a was discovered by the authors of the current paper. Please clarify.\n2. Please comment on implications of the current findings for a potential functional link between ANK3 and CACNA1C, which are both implicated in bipolar disorder in genome-wide association studies.", "reviewer_2": "The manuscript by Alam and colleagues describes a neuron type-specific mechanism of inclusion of a microexon in Ank3/ankyrin-G in GABAergic neurons, which modulates neuronal excitability and calcium activity in the prefrontal cortex. Based on previously acquired transcriptomic datasets, the authors have identified microexon E35a in Ank3 as being predominantly included in GABAergic and cerebellum neurons but excluded from cortical glutamatergic cells. This exon encodes an amino acid stretch which is also present in ankyrin-B, and a mutation in the homologous region in ankyrin-B can cause type 4 long QT syndrome and sudden cardiac death in humans. In the present study, authors explore regulatory mechanisms of Ank3 E35a alternative splicing, and use a mouse model in which E35a is deleted to investigate consequences for animal behavior, excitability and calcium activity of GABAergic neurons, as well as ankyrin-G binding to InsP3R.\nThe neuron type-specific pattern of E35a splicing is very curious and potentially relevant, and the approaches used in this study to investigate its functional importance are appropriate and state-of-the-art. Main findings are increased neuronal excitability in GABAergic cells in the orbitofrontal cortex, in particular in fast spiking interneurons, as well as increased somatic calcium activity in interneurons in the prelimbic area of the mPFC, in the absence of E35a. The E35a-encoded amino acid stretch binds to the InsP3R and to NKA/NCX1 in the cerebellum. Authors also explored the behavioral impact of the loss of E35a, but results are not very compelling at this point.\nDespite the clear interneuronal phenotypes triggered by E35a loss, which is very interesting and relevant, there are aspects of the study that need to be improved for publication to be considered:\n1) Behavior analyses do not show a clear phenotype for mice lacking E35a in Ank3. A battery of tests was performed, and the authors claim that Ank E35a-/-mice displayed altered motor and social behaviors (Fig. 3). However, behavioral data are not very convincing. The number of animals per sex appears to be small and not even, which could result in bias, given sex differences. There is no mention to which phase of the light/dark cycle the behaviour tests were performed in. Major issues are the following:\na. No coordination deficits were found in these animals, and their locomotor activity was in fact faster than that of wild-type animals when gait analysis was performed. Nevertheless, in the open field test the ambulatory distance was not affected; it would be important to report the average instant speed in the open field.\nb. The three-chamber test was performed, which is a test for sociability, but authors do not compare contact with the empty cage with contact with the stranger mouse. It is essential to make this comparison, to determine whether sociability is impaired in E35a-/- mice (which does not seem to be the case). Authors report a significant decrease in rearing duration in the contact with the target mouse for E35a-/- mice, an effect which is driven by female mice only. It is unclear what the significance of this finding is, in particular because the three-chamber test does not seem to be working as expected for female mice (no clear differences between exploration of empty cage and stranger mouse cage seem to be found). Based on a marginal difference in rearing duration in the exploration of the stranger mouse, driven by female animals (for which no explicit sociability is found in wild-type animals), it is not convincing that these animals have social behavior deficits.\nc. Are there sex-differences in other behavioral tests?\nd. In the prepulse inhibition test, do WT and E35a(-/-) animals respond similarly to the 110dB pulse? Do the prepulses induce a startle response on their own? How was the %PPI calculated? What is the \"baseline\" %PPI? Isn't the %PPI a function of the startle response to the pulse alone and the different prepulse-pulse combinations? What is the physiological meaning of a negative value of prepulse inhibition? The authors should show the absolute startle responses for the 110dB pulse alone, for the different intensity prepulses alone and for the different prepulse-pulse combinations.\ne. It would be important to test E35a-/- animals for memory, and prefrontal cortex-dependent behavior, given that functional analysis was performed in the prefrontal cortex. A spatial or recognition memory test, fear conditioning and extinction tests, as well as impulsivity tests could be informative here.\nOverall, complementing and improving behavioral analyses is essential to determine the relevance of E35a cell type-specific splicing.\n2) Electrophysiology experiments were performed in the orbitofrontal cortex (OFC) using animals between 6 and 12 weeks old. Previous studies have shown that the rodent PFC has a late developmental stage that extends up to the 8th postnatal week. Pöpplau et al have recently demonstrated that adolescence in mice (PND28 to PND52, approximately 4-7.5 postnatal weeks) is a critical period for medial PFC circuitry reorganization, accompanied by characteristic fluctuations in basal activity levels (Pöpplau et al Neuron 2024). Did the authors consider the developmental timeframe of the PFC when selecting the age range for this experiment? By pooling data from adolescent and adult animals, could the results be skewed?\n3) Did the authors analyse the fast afterhyperpolarization (fAHP) in action potential curves? In representative tracings, the fAHP seems to have a higher amplitude in WT animals. Could this be tied to the proposed mechanism?\n4) Electrophysiological recordings were conducted in the orbitofrontal cortex, while axon initial segment (AIS) analysis was performed in the primary motor and somatosensory cortices. This raises the question: is interneuronal excitability altered in E35a⁻/⁻ animals in these regions? In contrast, calcium imaging was carried out in the prelimbic area of the prefrontal cortex, and biochemical studies examining ankyrin-G interactions with InsP₃R and NKA/NCX were conducted using cerebellar tissue. The use of distinct brain regions for different experimental approaches complicates data interpretation and integration.\n5) Cerebellar granule cells are glutamatergic, but show high levels of E35a inclusion in ankyrin-G. How is this compatible with the regulatory mechanisms of E35a alternative splicing that the authors propose?\n6) Biochemical analysis of ankyrin-G interactome was performed in the cerebellum, contrarily to functional analyses, that were done in the PFC. Cerebellar neurons, and Purkinje cells specifically, have a very distinct activity pattern when compared to other inhibitory neuronal populations. From the extensive published body of evidence, we know that PCs have high levels of intrinsic activity, with some PCs firing tonically and others in a TBS (Tonic-Burst-Silence) pattern. This may lead us to infer that the mechanisms regulating calcium dynamics in PCs are different from other neurons. Therefore, it is difficult to compare the biochemical data obtained in the cerebellum with functional data from the PFC.\n7) The final dataset in the manuscript is a comparative RNA-seq analysis in the cerebellum and olfactory epithelium of WT and E35-/- mice. The relevance of this analysis is unclear, and the obtained data is very poorly discussed.", "reviewer_3": "This manuscript investigates a neuron-type-specific microexon (E35a) in Ank3, demonstrating its preferential inclusion in GABAergic neurons and proposing that it modulates neuronal excitability and intracellular Ca2+ via interaction with InsP3R/NCX/NKA complexes. The Authors investigate the molecular bases for the selective inclusion of the microexon in certain neuron types, and suggest that combinatorial regulation by multiple RBPs dictates the differential splicing of E35a between glutamatergic and GABAergic neurons, which can be further modulated by neuronal activity. Using E35a-deletion mice, the authors report changes in electrophysiological properties, Ca2+dynamics, and behavior.\nThe study is original and addresses a timely topic in neuroscience. It uses a variety of state-of-the-art approaches, which are not always adequately described in the Methods section. Specifically, the conceptual framework, statistical rigor, and integration of behavioral data must be significantly improved. Major concerns that need to be addressed are outlined below.Major Comments\n1. The link between E35a deletion and increased Ca2+ activity is merely correlative\nThe increase in Ca2+ transients in GABAergic neurons of E35a⁻/⁻ mice is striking but not mechanistically demonstrated to depend on the postulated alterations induced by the lack of the microexon. The claim that this reflects disruption of the AnkG–InsP3R/NCX/NKA complex is not directly tested.\no The Authors might consider, if possible, to add a pharmacological inhibition of NCX (e.g., SEA0400) or InsP3R (e.g., xestospongin C) in E35a⁻/⁻ vs WT mice, or rescue of E35a+ isoform in interneurons.\no Alternatively, further colocalization studies or proximity ligation assays could bolster the structural claim.\n2. The interpretation of increased excitability in FS interneurons is not fully explored.\nThe Authors report that FS interneurons in E35a⁻/⁻ mice are more excitable (reduced rheobase, higher firing rate), but they do not investigate at the ionic level why this happens.\n• Could these changes be due to altered Ca2+ signaling? For example, using intracellular BAPTA could help determining whether intracellular [Ca2+] plays a role.\n• Additional analyses (if possible) of action potential threshold, afterhyperpolarization (AHP), or waveform shape could clarify which specific ion conductances are affected\n3. Lack of clear rationale for behavioral testing in the Introduction, and poor integration in the Discussion\nThe manuscript introduces a behavioral battery (gait analysis, 3-chamber test, open field, EPM, Y-maze) without any stated hypothesis or rationale in the Introduction. There is no explanation of why these specific tests were selected or what phenotypes were expected.\no The Introduction should be revised to clearly justify the inclusion of behavioral testing — e.g., in light of ANK3’s implication in bipolar disorder or the known role of GABAergic interneurons in behavior.\no In the Discussion, the behavioral findings (hyperactivity, altered gait, subtle social differences) are disconnected from the molecular and cellular data, and the reference to bipolar disorder is speculative.\no If the behavioral work is exploratory (which must not be seen as a negative aspect per se), the Authors should state this explicitly and moderate their interpretation accordingly.\n.\n4. Statistical methods are insufficiently reported\nThe manuscript frequently mentions two-way ANOVA but fails to specify the factors or interaction terms (e.g., genotype × current, genotype × sex). This is a significant omission that limits interpretability.\no Please clearly define all factors used in each ANOVA and report F-values, degrees of freedom, and exact p-values.\no Consider including a statistical summary table in the supplementary material for transparency.\n5. Timeline and procedural details for behavioral tests are unclear\nThe order, timing, and conditions of behavioral testing are not provided. These are critical variables that can influence results.\no What was the sequence of tests? What time of day were they performed (light/dark phase)? Were animals habituated?\no The addition of a timeline schematic and consolidated “Behavioral Methods” section would help.\n6. Overstated psychiatric relevance\nThe reference to bipolar disorder based on subtle social deficits and hyperactivity is premature and speculative.\no The authors should moderate their language or include further data (e.g., affective tests, pharmacological rescue) if they wish to support such a claim.\nMinor Comments\n• The Discussion should be reorganized to improve clarity — e.g., separating behavioral, physiological, and molecular interpretation.\n• Sex differences are briefly mentioned (e.g., Extended Fig. 7) but not consistently addressed across experiments. Please clarify whether sex was included as a factor in all analyses.\n• Figure legends should explicitly state: sample sizes, statistical tests used, and whether data meet assumptions for parametric tests.\n• The hypothetical model shown in Fig. 7b is not adequately described in the Discussion\n• “calcium” should be substituted with Ca2+, since the referral is to the concentration of the ion, and not of total calcium.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point responses to the Reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThis study characterizes a neural-specific microexon (E35a) in the Ank3 gene with several remarkable\nfindings that provide the best rationale so far for the link between ANK3 and human bipolar disorder\nfirst reported in 2008. The authors demonstrate for the first time that E35a is preferentially expressed in\nadult GABAergic interneurons. They find that E35a is paralogous to a constitutively expressed sequence\nin Ank2 that is the target for mutation causing long QT cardiac arrythmia. The E35a equivalent exon in\nAnk2 was demonstrated previously to promote interaction of between ankyrin-B with IP3 receptors in\nthe ER and Na/K ATPase, and the Na/Ca exchanger in the plasma membrane. In cardiomyocytes this\nankyrin-B/IP3 receptor, NKA, NCX complex is responsible for electroneutral export of calcium ions, and\nits loss results in abnormal calcium dynamics. The current study demonstrates that retention of E35a in\nGABAergic neurons confers a gain of function of linking ankyrin-G to the IP3 receptor and Na/K ATPase.\nFurthermore, mice lacking E35a exhibit increased calcium transients in prefrontal GABAergic neurons\nimaged in awake animals using 2-photon microscopy. Consistent with these findings E35a-null mice\nexhibit increased excitability in behavioral assays.\nFor the first time in my recent memory, I am recommending acceptance of a paper with minimal\nrevision. The data are clean, the experiments well designed and cutting edge in cellular neuroscience as\nwell as RNA biology, and the results are clear and well presented. The findings are original and of high\nimpact with the first evidence of how ANK3 mutation could provide a substrate for bipolar disease. I\nhave a few minor points that could further improve the presentation:\nWe thank the reviewer for their excellent summary of this study. We are grateful that this reviewer\nagrees with the significance, novelty and potential impact of our findings and has only minor points we\nneed to address.\n1. Lines 196-197. Interestingly, we also found that splicing of E35a is neuron activity-dependent. E35a\ninclusion is reduced in cultured cortical cells after depolarization induced by KCl treatment 41,42\nAs written, a reader could infer that activity-dependent inclusion of E35a was discovered by the authors\nof the current paper. Please clarify.\nActivity-dependent splicing of the microexon is indeed discovered by our study through a re-analysis of\npublished RNA-seq datasets. We have rephrased the relevant sentences to avoid potential confusion\n(p.7):\n“Interestingly, we also found that splicing of E35a is neuron activity-dependent through our\nreanalysis of published RNA-seq datasets 42, 43. Specifically, E35a inclusion is reduced in cultured\ncortical cells after depolarization induced by KCl treatment. In parallel, there is also an increase\nin Mbnl2 expression and a decrease in nSR100 activity (Extended Data Fig. 4d,e and ref. 42),\nwhich are consistent with and presumably contribute to the observed activity-dependent\nsplicing change.”\n2. Please comment on implications of the current findings for a potential functional link between ANK3\nand CACNA1C, which are both implicated in bipolar disorder in genome-wide association studies.\nbetween ANK3 and CACNA1C in the Discussion (p. 16):\n“…Notably, another gene implicated in bipolar disorder is CACNA1C, encoding the alpha 1C\nsubunit of the L-type voltage-gated calcium channel 21, suggesting that convergent mechanisms\nmight alter calcium signaling pathway in the disease (Fig. 6b). Whether AnkG directly or\nindirectly interacts with CACNA1C awaits further investigation.”\nReviewer #2 (Remarks to the Author):\nThe manuscript by Alam and colleagues describes a neuron type-specific mechanism of inclusion of a\nmicroexon in Ank3/ankyrin-G in GABAergic neurons, which modulates neuronal excitability and calcium\nactivity in the prefrontal cortex. Based on previously acquired transcriptomic datasets, the authors have\nidentified microexon E35a in Ank3 as being predominantly included in GABAergic and cerebellum\nneurons but excluded from cortical glutamatergic cells. This exon encodes an amino acid stretch which\nis also present in ankyrin-B, and a mutation in the homologous region in ankyrin-B can cause type 4\nlong QT syndrome and sudden cardiac death in humans. In the present study, authors explore\nregulatory mechanisms of Ank3 E35a alternative splicing, and use a mouse model in which E35a is\ndeleted to investigate consequences for animal behavior, excitability and calcium activity of GABAergic\nneurons, as well as ankyrin-G binding to InsP3R.\nThe neuron type-specific pattern of E35a splicing is very curious and potentially relevant, and the\napproaches used in this study to investigate its functional importance are appropriate and state-of-the-\nart. Main findings are increased neuronal excitability in GABAergic cells in the orbitofrontal cortex, in\nparticular in fast spiking interneurons, as well as increased somatic calcium activity in interneurons in\nthe prelimbic area of the mPFC, in the absence of E35a. The E35a-encoded amino acid stretch binds to\nthe InsP3R and to NKA/NCX1 in the cerebellum. Authors also explored the behavioral impact of the loss\nof E35a, but results are not very compelling at this point.\nDespite the clear interneuronal phenotypes triggered by E35a loss, which is very interesting and\nrelevant, there are aspects of the study that need to be improved for publication to be considered:\nWe thank this reviewer for their thorough review and excellent summary of our study. We are pleased\nthat the reviewer concurred on the novelty and functional relevance of neuron-cell type-specific\nsplicing of Ank3 E35a and praised the approach we used for our analysis at the molecular and cellular\nlevels.\nWe also thank the reviewer for their detailed critique and constructive suggestions on our behavior and\nelectrophysiological studies. We have performed additional experiments and data analyses to address\ntheir concerns. We confirmed the robustness of our electrophysiological findings. On the other hand,\nthe behavior phenotypes of the E35a-/- mice are less than clear. Given the moderate behavioral\nphenotypes observed in the E35a-/- mice and some of the experimental caveats for this part of the\nstudy, we have decided to move all the behavior data to Extended Data Fig. 9 and we now describe\nthese data in the final section of the results. We have amended the interpretation of the behavioral\ndata accordingly throughout the text (see more detailed response below).\nexons that has been characterized at the molecular and cellular levels in detail (see also the\nintroduction). Using a combination of in vivo and in vitro systems, our study provides novel and\nintriguing insights into alternative splicing regulation and function, expanding our understanding of a\nclassic neuronal gene. We are confident that this study is of significant interest to the audience in both\nthe RNA and the broader molecular neurobiology communities.\n1) Behavior analyses do not show a clear phenotype for mice lacking E35a in Ank3. A battery of tests\nwas performed, and the authors claim that Ank E35a-/-mice displayed altered motor and social\nbehaviors (Fig. 3). However, behavioral data are not very convincing. The number of animals per sex\nappears to be small and not even, which could result in bias, given sex differences. There is no mention\nto which phase of the light/dark cycle the behaviour tests were performed in. Major issues are the\nfollowing:\nIn response to the reviewer’s suggestion, we performed additional experiments with mice in\nthe 3-chamber assay to increase the sample size and to make sexes more even (WT: N=11\nmales, N=10 females; Mut: N=9 males, N=10 females; updated mouse number).\nUnfortunately, we did not observe significant differences between E35a-/- and control mice with\nthis expanded dataset. As we responded above, one limitation of our behavior analyses is the\nuse of mice with mixed C57BL/6J and CBA/J mixed background (B6CBAF1) genetic background,\nwhich might have affected our behavior findings. We have decided to move the behavioral data\ninto Extended Data Fig. 9 and to amend the discussion related to behavior phenotypes.\nWe also added information in Methods about light/dark cycle and clarified that all behavioral\ntests were performed during the light phase.\na. No coordination deficits were found in these animals, and their locomotor activity was in fact faster\nthan that of wild-type animals when gait analysis was performed. Nevertheless, in the open field test\nthe ambulatory distance was not affected; it would be important to report the average instant speed in\nthe open field.\nUnfortunately, we did not have instant speed measurements available from our open field test.\nHowever, in the 3-chamber assay, the software tracked the distance and speed. As shown in\nthe revised Fig. 9e (also provided as Fig. R1a below for your convenience), the mutant mice\ndid display a slight increase in speed although the difference did not reach the threshold of\nstatistical significance (p =0.174). Also see our response above.\n9e) Quantification in WT and Ank3 E35a-/- mice (mean ±SEM) is shown. b, Quantification of time in\nchamber with males and females separated.\nb. The three-chamber test was performed, which is a test for sociability, but authors do not compare\ncontact with the empty cage with contact with the stranger mouse. It is essential to make this\ncomparison, to determine whether sociability is impaired in E35a-/- mice (which does not seem to be\nthe case). Authors report a significant decrease in rearing duration in the contact with the target mouse\nfor E35a-/- mice, an effect which is driven by female mice only. It is unclear what the significance of this\nfinding is, in particular because the three-chamber test does not seem to be working as expected for\nfemale mice (no clear differences between exploration of empty cage and stranger mouse cage seem to\nbe found). Based on a marginal difference in rearing duration in the exploration of the stranger mouse,\ndriven by female animals (for which no explicit sociability is found in wild-type animals), it is not\nconvincing that these animals have social behavior deficits.\nWe thank reviewer for the suggestion. We have added a plot showing the time in each\nchamber and compared them between object and stranger (revised Extended Fig. 9e; also shown\nhere in Fig. R1a). Our analysis demonstrated that E35a-/- mice displayed normal preference for\nthe stranger mouse, suggesting their normal sociability. There is also no difference when we\nseparated male and female mice in our analysis (shown in Fig. R1b for reviewers). Given this\nresult, we agree with the reviewer’s assessment and have decided to move all behavioral data\ninto Extended Data Fig. 9.\nc. Are there sex-differences in other behavioral tests?\nWe did not observe any sex-differences in other behavioral tests. Therefore, in the revised\nversion, male and female mice were pooled together in our statistical analyses. However, for\ncertain tests, statistical analyses with male and female mice separated are presented to the\nreviewers here in the response letter to provide additional information.\n4\nd. In the prepulse inhibition test, do WT and E35a(-/-) animals respond similarly to the 110dB pulse? Do\nthe prepulses induce a startle response on their own? How was the %PPI calculated? What is the\n\"baseline\" %PPI? Isn't the %PPI a function of the startle response to the pulse alone and the different\nprepulse-pulse combinations? What is the physiological meaning of a negative value of prepulse\ninhibition? The authors should show the absolute startle responses for the 110dB pulse alone, for the\ndifferent intensity prepulses alone and for the different prepulse-pulse combinations.\nWe thank the reviewer for these legitimate questions and apologize for the oversight. We have\nnow added additional descriptions about the PPI calculation in Methods. Briefly, PPI = (110db\naveraged mV -averaged stimulus mV )/110db averaged mV , where mV is the\nMax Max Max Max\nresponse of the mouse to a given stimulus. Also, as the reviewer suggested, we have added a\nplot showing startle responses to 110db pulse alone and different prepulse-pulse combinations\n(revised Extended Fig. 9h, also shown as Fig. R2 below). Unfortunately, we did not examine\nthe startle responses to different intensities of prepulses alone.\nFig. R2 (revised Extended Fig. 9h): Pre-pulse inhibition test. Left, quantification of startle\nresponse to different prepulse-pulse combinations and 110db pulse alone (most right barplots).\nRight, quantification of % Prepulse inhibition following a pulse (ranging from 74 to 92 dB) in\nWT and Ank3 E35a-/- mice.\ne. It would be important to test E35a-/- animals for memory, and prefrontal cortex-dependent behavior,\ngiven that functional analysis was performed in the prefrontal cortex. A spatial or recognition memory\ntest, fear conditioning and extinction tests, as well as impulsivity tests could be informative here.\nWe agree that memory tests could be informative. However, given the limited availability of\nmutant mice, it is difficult for us to perform the suggested tests for this revision. That said, we\ndid include a Y-maze task (Extended Data Fig 9d), which provides information on spatial\nworking memory. We did not see any significant differences in performance in this task.\nOverall, complementing and improving behavioral analyses is essential to determine the relevance of\nE35a cell type-specific splicing.\nOverall, we agree with the reviewer regarding some of the limitations in the behavioral phenotypes\nobserved from E35a-/- mice. However, it is not totally unexpected this phenotype is more subtle\ncompared to previous studies where AnkG knockout resulted in a significant loss of AnkG in forebrain\npyramidal neurons (Zhu et al., 2017; PMID 28894008) or PV interneurons (Lopez et al., 2017; PMID\n5\n27956739). Our findings showed that there is no significant disruption of AIS in prefrontal cortical\nneurons, nor changes in action potential parameters associated with the AIS in E35a-/-. Moreover,\ncalcium imaging showed increased interneuron activity along with a modest elevation in pyramidal\nneuron activity in the prefrontal cortex of E35a⁻/⁻ mice, suggesting a nuanced shift in cortical network\ndynamics. These results may reflect compensatory adjustments in excitatory-inhibitory balance which\nmay be related with precise timing responses in social behaviors and memory tests.\nAlternatively, we would like to point out that our behavior analyses described in the paper were\nperformed using mice with mixed C57BL/6J and CBA/J background (B6CBAF1), which might have\naffected our behavioral findings. We have been backcrossing the mice to the inbred C57BL/6J\nbackground, but we respectfully suggest that the behavioral analysis of these mice is beyond the scope\nof the current study but more appropriate for future work. This is especially true given the extensive\nmolecular and cellular data that have already been presented in this manuscript.\nThese points are now elaborated more clearly in Discussion (p.17):\n“Based on the pronounced changes in neuronal electrophysiological properties associated with\nE35a splicing, it was somewhat unexpected that the Ank3 E35a-/- mice did not show overt\ndevelopmental abnormalities or major behavioral deficits. Across a battery behavioral assays\ntesting motor function, memory, anxiety- and depression-related behaviors, and social\ninteraction, the only significant differences were increased step length and locomotion in gait\nanalysis, consistent with a hyperactive phenotype. No major anxiety- or social-related deficits\nwere observed. The divergence between the cellular and behavior phenotypes may reflect\ncompensatory adjustments in excitatory-inhibitory balance, particularly in behaviors requiring\nprecise timing such as social interaction and memory. Alternatively, the mixed C57BL/6J and\nCBA/J (B6CBAF1) genetic background of the mice used here may masked subtle deficits or\nintroduced variability in behavior outcomes. Further studies using inbred C57BL/6J mice and\nprefrontal cortex-dependent cognitive tasks, such as fear conditioning and extinction, will be\nimportant to fully assess the organismal impact of this microexon.”\n2) Electrophysiology experiments were performed in the orbitofrontal cortex (OFC) using animals\nbetween 6 and 12 weeks old. Previous studies have shown that the rodent PFC has a late developmental\nstage that extends up to the 8th postnatal week. Pöpplau et al have recently demonstrated that\nadolescence in mice (PND28 to PND52, approximately 4-7.5 postnatal weeks) is a critical period for\nmedial PFC circuitry reorganization, accompanied by characteristic fluctuations in basal activity levels\n(Pöpplau et al Neuron 2024). Did the authors consider the developmental timeframe of the PFC when\nselecting the age range for this experiment? By pooling data from adolescent and adult animals, could\nthe results be skewed?\nWe thank the reviewer for highlighting the importance of developmental timing in a late-maturing\nregion such as the prefrontal cortex. We have now added new data showing that the age distribution of\nmice used for our recordings did not differ significantly between WT and Ank3 E35a-/- groups\n(Extended Data Fig. 7b), and the firing properties of FS interneurons were similar between groups\n(Extended Data Fig. 7c) (also shown as Fig. R3b,c below).\nproperties. b, Age distribution of patch clamp recordings for WT (8 mice, 48 cells) and Ank3 E35a-/- (8\nmice, 46 cells) genotypes. c, Average firing frequency during 500-ms current steps in FS interneurons,\ngrouped by age into late-adolescent (P < 60) and adult (P > 60) mice. No age-related differences were\ndetected in WT (left; F = 0.1577, p = 0.6957) or Ank3 E35a⁻/⁻ mice (right; F = 0.0315, p =\n(1,19) (1,18)\n0.8614).\nA brief description about the new analysis was added in p. 9:\n“…We also confirmed that the age distribution of recorded neurons did not differ significantly\nbetween WT and Ank3 E35a-/- mice (Extended Data Fig. 7b), and that FS interneuron excitability\nwas comparable between late adolescent and adult mice in both groups (Extended Data Fig.\n7c).”\nThese results align with previous studies showing that even FS interneurons, which emerge relatively\nlate in postnatal development (Okaty et al., 2009; PMID 19474331), show mature intrinsic\nelectrophysiological properties already by the 6th postnatal week (Miyamae et al., 2017; PMID\n28408413, Pan et al., 2016; PMID 27319800). Together, these findings support the conclusion that\ndevelopmental age is unlikely to confound our electrophysiological results.\n3) Did the authors analyse the fast afterhyperpolarization (fAHP) in action potential curves? In\nrepresentative tracings, the fAHP seems to have a higher amplitude in WT animals. Could this be tied to\nthe proposed mechanism?\nWe appreciate the reviewer’s suggestion. We have now incorporated fAHP analysis in Extended Data\nFig. 7d (also shown as Fig. R4 below) and briefly discussed the new data in the main text (p. 9):\n“APs evoked by rheobase +100 pA also showed a shorter latency to the first AP and a decrease\nin the first interspike interval (ISI) in Ank3 E35a-/- (Fig. 3j,k), while fast AHP (fAHP) was unaltered\n(Extended Data Fig. 7d).”\nfAHP was measured from the first 5 action potentials evoked by 500-ms current step at rheobase + 100\npA from RMP. No differences were observed between genotypes in either non-FS (left) or FS (right)\ninterneurons.\nIn summary, we found that the fAHP amplitude in putative non-FS interneurons is comparable between\ngenotypes, while the slightly smaller fAHP in putative FS interneurons in Ank35a-/- did not reach\nstatistical significance. Although increases in intracellular calcium, via RyR/IP3R, or other calcium\nchannels, have been reported to activate BK and SK channels associated with fAHP and sAHP in various\nneuronal types (Storm et al., 1987; PMID 2443676, Maylie et al., 2003; PMID 14500775, Gu et al., 2007;\nMPID 17303637), the fAHP in FS interneurons is mainly mediated by the voltage-gated Kv3.1-Kv3.2\nchannels (Erisir et al., 1999; PMID 10561420, Lien and Jonas, 2003; PMID 12657664) and is likely\nindependent of BK/SK channels gated by calcium. We have integrated the interpretation of these\nresults into the discussion (p. 15-16):\n“…Intracellular Ca2+ can also activate SK and BK channels that contribute to the slow and fast\nAHP in several neuronal types 70-72. However, because no differences in fAHP were observed\nbetween genotypes, and fAHP in FS cells is primarily mediated by voltage-gated Kv3.1–Kv3.2\nchannels 73-75, the increased excitability of Ank3 E35a-/- FS cells is likely independent of Ca2+-\ngated BK/SK channels…”\n4) Electrophysiological recordings were conducted in the orbitofrontal cortex, while axon initial segment\n(AIS) analysis was performed in the primary motor and somatosensory cortices. This raises the question:\nis interneuronal excitability altered in E35a⁻/⁻ animals in these regions? In contrast, calcium imaging\nwas carried out in the prelimbic area of the prefrontal cortex, and biochemical studies examining\nankyrin-G interactions with InsP₃R and NKA/NCX were conducted using cerebellar tissue. The use of\ndistinct brain regions for different experimental approaches complicates data interpretation and\nintegration.\nAlthough the use of different cortical regions highlights the pancortical effect of E35a-/-, which is\nconsistent with the splicing pattern of the microexon across cell types and brain regions, we agree with\nthe reviewer that it may complicate data integration and intepretation. To address the reviewer’s\nconcern, we have quantified AIS morphology in the prefrontal cortex to align the analysis with the\nneurophysiological data. Our data demonstrated that the AIS morphology in GABAergic neurons of the\nOFC, including its length and the distance from the soma, did not differ significantly between WT and\nE35a-/- mice. These results are consistent with those observed in the primary motor and somatosensory\ncortices, as well as cultured hippocampal neurons in vitro, as described in our original manuscript. The\noriginal AIS morphology data have been replaced with the updated data in Figure 3m,n. We also did not\nobserve any differences when we separated male and female mice in our analysis (Fig. R5).\nFig. R5 (results with males and females pooled together are provided in revised Fig. 3m,n). AIS\nmorphology analysis in WT and Ank3 E35a-/- OFC. m, Representative double-staining (FISH and IHC)\nimage of AIS structures in vGAT+ GABAergic interneurons of WT and Ank3 E35a-/- mouse cortex for each\nsex. Arrowheads indicate AIS that can be clearly associated with vGAT+ cells selected for quantification.\nScale bar: 20 µm. n, Quantification of AIS lengths (up) and AIS-to-Soma distances (down) in OFC vGAT+\nGABAergic neurons. The numbers of AIS related measurements (from 3 male and 3 female WT, and 3\nmale and 3 female Ank3 E35a-/- mice) are indicated in the graph. No significant differences were\nidentified in the t-test results of AIS length and AIS-to-Soma distance in GABAergic neurons between\nAnk3 E35a-/- and WT mice for each sex. All data are presented as mean ± SEM. By Mann-Whitney U\ntest. ns indicates not significant (p>0.05).\nFor in vivo two-photon calcium imaging, the PL area of medial prefrontal cortex rather than OFC was\nselected due to its improved accessibility for imaging using a cranial window. OFC and mPFC share\nreciprocal connectivity (Hoover and Vertes, 2011; PMID: 21800317) and are both implicated in\ndecision-making and social behavior (Klein-Flugge et al., 2022; PMID: 35705077). The cellular\nphysiology of interneuron subtypes in OFC and mPFC is very similar (Kim et al., 2017; PMID: 28985566).\nRegarding the co-IP experiments, we do not have a lot of options other than identifying a region with\nhigh E35a inclusion at the bulk tissue level in the wild type mice, which can then be contrasted with\nE35a-/- mice. This indeed potentially complicates interpretation of the data, but reflects the impact of\nthis highly regulated microexon in a broader cellular context (see also responses to points #5 and #6\nbelow).\n5) Cerebellar granule cells are glutamatergic, but show high levels of E35a inclusion in ankyrin-G. How is\nthis compatible with the regulatory mechanisms of E35a alternative splicing that the authors propose?\ncerebellar granule cells, we also noticed that striatal medium spiny neurons are GABAergic (although\nquite different from FS interneurons in many ways) but show low inclusion of the exon. Therefore, the\nsplicing pattern of E35a is not likely driven by the neurotransmitter phenotypes alone, but by other\nmechanisms (e.g., developmental lineages of different neuronal types) that are imperfectly correlated\nwith neurotransmitter types. When we state that the microexon has differential splicing between\nglutamatergic and GABAergic neurons, we always carefully limit the context of this statement to cortex\nand hippocampus, where this difference is consistently observed. We have briefly elaborated this point\nin Discussion (p. 14):\n“…However, it is important to note that while there is a good correlation between splicing\npattern of the exon and neurotransmitter types in the cortex and hippocampus, this is not\nalways true in the other brain regions. For example, cerebellar granule cells are glutamatergic\nbut have a high level of E35a inclusion, whereas striatal medium spiny neurons are GABAergic\nbut have a low level of exon inclusion. Therefore, the neuron cell type-specific splicing of the\nexon is unlikely to be dictated simply by neurotransmitter phenotype, but by other mechanisms\nsuch as developmental lineages of neuron types that are imperfectly correlated with\nneurotransmitter phenotypes.”\n6) Biochemical analysis of ankyrin-G interactome was performed in the cerebellum, contrary to\nfunctional analyses, that were done in the PFC. Cerebellar neurons, and Purkinje cells specifically, have a\nvery distinct activity pattern when compared to other inhibitory neuronal populations. From the\nextensive published body of evidence, we know that PCs have high levels of intrinsic activity, with some\nPCs firing tonically and others in a TBS (Tonic-Burst-Silence) pattern. This may lead us to infer that the\nmechanisms regulating calcium dynamics in PCs are different from other neurons. Therefore, it is\ndifficult to compare the biochemical data obtained in the cerebellum with functional data from the PFC.\nFor this biochemical analysis, we had to identify a brain region with high E35a inclusion at the bulk\ntissue level in the wild type mice, which can then be contrasted with E35a-/- mice. Cerebellum is our\nbest candidate for this purpose. Our rationale is that since InsP3R is ubiquitously expressed in different\nneuron types, its preferential interaction with AnkG isoforms should be similar across the cell types,\nalthough the downstream impact of such interaction might vary, as this reviewer suggested. The\nbottom line is that cerebellum provided a complementary and more physiologically relevant system\nthan the HEK293 cells we used for our over-expression experiment. Nevertheless, in response to the\nreviewer’s critique, we performed Co-IP using cortex tissues, but were unable to successfully pull down\na notable amount of InsP3R1, presumably due to lower E35a inclusion at the bulk tissue level in the\ncortex (Fig. R6).\n(IP3R1) from cortical lysate was co-immunoprecipitated using anti-AnkG and analyzed by\nimmunoblotting using anti-IP3R1.\nWe have added additional justifications on the use of cerebellar tissues for the Co-IP experiments (p.\n11-12:\n“...Cerebellum was chosen instead of cortex because E35a inclusion is high in Purkinje cells and\ngranule cells, the dominant cerebellar neuronal types (Fig. 1c and Fig. 3b), whereas cortical\nGABAergic neurons are a minority population…”\n7) The final dataset in the manuscript is a comparative RNA-seq analysis in the cerebellum and olfactory\nepithelium of WT and E35-/- mice. The relevance of this analysis is unclear, and the obtained data is\nvery poorly discussed.\nOur original intention is to use the RNA-seq data to determine downstream genes and pathways\nregulated by E35a inclusion. While we did identify a short list of genes consistently affected by E35a\ndeletion, we agree that our analysis is very limited and the data is not as informative as we had hoped.\nWe thus removed this part in the revised manuscript, as we agree that its relevance is questionable\nhere.\nReviewer #3 (Remarks to the Author):\nThis manuscript investigates a neuron-type-specific microexon (E35a) in Ank3, demonstrating its\npreferential inclusion in GABAergic neurons and proposing that it modulates neuronal excitability and\nintracellular Ca2+ via interaction with InsP3R/NCX/NKA complexes. The Authors investigate the\nmolecular bases for the selective inclusion of the microexon in certain neuron types, and suggest that\ncombinatorial regulation by multiple RBPs dictates the differential splicing of E35a between\nglutamatergic and GABAergic neurons, which can be further modulated by neuronal activity. Using\nE35a-deletion mice, the authors report changes in electrophysiological properties, Ca2+dynamics, and\nbehavior.\nThe study is original and addresses a timely topic in neuroscience. It uses a variety of state-of-the-art\napproaches, which are not always adequately described in the Methods section. Specifically, the\nconceptual framework, statistical rigor, and integration of behavioral data must be significantly\nimproved. Major concerns that need to be addressed are outlined below.\nWe thank this reviewer for their excellent summary of our study and their endorsement on the\nsignificance and novelty of our work. We also thank the reviewer for their insightful and constructive\nsuggestions.\nMajor Comments\n1. The link between E35a deletion and increased Ca2+ activity is merely correlative\nThe increase in Ca2+ transients in GABAergic neurons of E35a⁻/⁻ mice is striking but not mechanistically\ndemonstrated to depend on the postulated alterations induced by the lack of the microexon. The claim\n11\nthat this reflects disruption of the AnkG–InsP3R/NCX/NKA complex is not directly tested.\no The Authors might consider, if possible, to add a pharmacological inhibition of NCX (e.g., SEA0400) or\nInsP3R (e.g., xestospongin C) in E35a⁻/⁻ vs WT mice, or rescue of E35a+ isoform in interneurons.\no Alternatively, further colocalization studies or proximity ligation assays could bolster the structural\nclaim.\nWe completely agree with the reviewer on the importance of understanding the underlying\nmechanism. We thank the reviewer for the suggestion to examine a direct interaction between\nankyrin-G (AnkG) and inositol 1,4,5-trisphosphate receptor (InsP3R) using an orthogonal approach to\nstrengthen our co-IP data. To investigate this, we performed colocalization experiments in HEK293T\ncells. Fig. R7 below shows the recruitment of AnkG-GFP, with or without E35a, to membrane-bound\nInsP3R. We did not include these results in the main manuscript due to several experimental caveats,\nwhich we outlined below.\nFig. R7: AnkG-InsP3R colocalization through HEK cell recruitment assay. a, Representative images\nshowing detection of Neurofascin (NF)-InsP3R chimera without (left) or with (right) ankyrin-binding\ndomain (ABD). NF is labeled in far red and AnkG-GFP in green. DAPI (blue) stains the nucleus. b,\nRepresentative plot profiles of NF chimera and AnkG-GFP fluorescence intensity (a.u.) in control (top)\nand NF-InsP3R (bottom) determined by yellow line in panel (a) c, Quantification of membrane-to-\ncytoplasm GFP ratio with and without E35a. Statistical analysis of membrane / cytoplasm GFP ratio\nusing two-way ANOVA revealed no interaction between chimera type and presence of E35a in AnkG; P\nvalue = 0.94. Simple main effects analysis showed that IP3R-ABD did have a statistically significant\neffect on AnkG recruitment; P value < 0.00).\nneurofascin chimera in which the native cytoplasmic domain was replaced with the InsP3R ankyrin-\nbinding domain (NF-InsP3R-ABD), thereby targeting the InsP3R peptide to the plasma membrane. Co-\ntransfection of GFP-tagged AnkG with NF-InsP3R-ABD resulted in a significantly higher membrane-to-\ncytoplasm ratio of GFP compared with cells co-transfected with AnkG-GFP and an NF construct lacking\nthe C-terminus. However, deletion of E35a from AnkG did not alter this recruitment. Here, although\nrecruitment was statistically significant compared to controls, the increase was only ~2-fold in the\nmembrane-to-cytoplasm ratio. For strong interactions, an average ~10-fold enrichment is typically\nexpected. To further assess the performance of the chimera, we co-transfected NF–InsP3R-ABD with\nankyrin-B, a well-established InsP3R binding partner, as we described in our manuscript. The results\nmirrored those observed with AnkG, suggesting that the construct is limited in its ability to reveal\nstrong recruitment. This limitation may stem from the presence of cytoplasmic chimera that remains\nundetected, as visualization of the chimera relies on selective labeling of the extracellular domain of\nneurofascin. The truncated form of InsP3R in the chimera may also be insufficient for full recruitment\nof ankyrin, which could explain the lack of robust recruitment.\nAdditionally, HEK293T cells lack certain neuronal mechanisms; for example, the absence of specific\npost-translational modifications may explain why strong recruitment was not observed in this assay, in\ncontrast to the Co-IP results from mouse cerebellum and N2a cells presented in the manuscript.\nMoreover, since all three major isoforms (190 kDa, 270 kDa, and 480 kDa) were expressed in the brain\ntissue, it is possible that E35a-dependent interaction with InsP3R is more specific to a particular AnkG\nisoform.\nWhile this recruitment assay provided supportive but modest evidence for AnkG-InsP3R interaction,\nwe believe that further optimization is required to fully capture the role of E35a in this assay. Future\nstudies investigating the contribution of different ankyrin isoforms and post-translational modifications\nin this novel interaction will be important, but are beyond the scope of the current manuscript.\nLikewise, we consider that pharmacological inhibition of components in the pathway related to\nintracellular Ca²⁺ storage/release, as suggested by the reviewer, as well as E35a+ isoform rescue, may\nrequire substantial in vivo and in vitro experiments aimed at elucidating the potentially complex\nmechanism that we describe here for the first time.\n2. The interpretation of increased excitability in FS interneurons is not fully explored.\nThe Authors report that FS interneurons in E35a⁻/⁻ mice are more excitable (reduced rheobase, higher\nfiring rate), but they do not investigate at the ionic level why this happens.\n• Could these changes be due to altered Ca2+ signaling? For example, using intracellular BAPTA could\nhelp determining whether intracellular [Ca2+] plays a role.\n• Additional analyses (if possible) of action potential threshold, afterhyperpolarization (AHP), or\nwaveform shape could clarify which specific ion conductances are affected\nSee also our response to related comments (point #2) from reviewer 2 above.\nWe thank the reviewer for these thoughtful and constructive comments. We have now included an\nanalysis of fast AHP (Extended Data Fig. 7d, also shown as Fig. R3 above). However, as we did not\nobserve clear differences between WT and E35a-/- mice and given that fAHP in FS interneurons is\n13\nprimarily mediated by Kv3 channels (Erisir et al., 1999; PMID 10561420, Lien and Jonas, 2003; PMID\n12657664). Thus, we have not further elaborated on a potential link between Ca²⁺ signaling and fAHP,\nbut we have added this part to the discussion (p. 15-16):\n“…Intracellular Ca2+ can also activate SK and BK channels that contribute to the slow and fast\nAHP in several neuronal types 70-72. However, because no differences in fAHP were observed\nbetween genotypes, and fAHP in FS cells is primarily mediated by voltage-gated Kv3.1–Kv3.2\nchannels 73-75, the increased excitability of Ank3 E35a-/- FS cells is likely independent of Ca2+-\ngated BK/SK channels…”\nWe would also like to note that action potential parameters, such as voltage threshold, rise and fall\nslope, and width, were already reported in the original manuscript (Fig. 3l, and Extended Data Fig. 7k-\nm). The analysis of action potential properties at rheobase minimizes the contribution of intracellular\ncalcium loading, particularly when using short current pulses. Our findings show that action potential\nshape is not significantly altered in E35a-/- mice, and together with preserved AIS morphology (Fig.\n3m,n), supports the conclusion that the AnkG microexon mutation does not critically impair action\npotential generation at the AIS.\nThe larger and prolonged Ca2+ transients along with our biochemical co-IP data, point to a potential role\nfor altered calcium signaling underlying increased excitability observed in patch-clamp recordings. In\naddition to the previously discussed mechanisms consistent or compatible with calcium-induced\ncalcium release, we have also considered potential inhibition of leak potassium currents that may\ncontribute to increase input resistance and excitability (Goldberg et al., 2008; PMID 20705896).\nOverall, we provide strong evidence for a role for the AnkG microexon in regulating interneuron\nexcitability through mechanisms extending beyond its classic function in the AIS. We respectfully\nsuggest that a comprehensive dissection of the calcium-related pathways would require a dedicated\nstudy beyond this work, given the complexity of intracellular calcium dynamics during neuronal activity\n(Grienberger and Konnerth, 2012; PMID: 22405199), particularly in interneurons that are highly\nenriched in Ca2+-buffering proteins such as parvalbumin (Aponte et al., 2008; PMID: 18276734).\n3. Lack of clear rationale for behavioral testing in the Introduction, and poor integration in the\nDiscussion\nThe manuscript introduces a behavioral battery (gait analysis, 3-chamber test, open field, EPM, Y-maze)\nwithout any stated hypothesis or rationale in the Introduction. There is no explanation of why these\nspecific tests were selected or what phenotypes were expected.\no The Introduction should be revised to clearly justify the inclusion of behavioral testing — e.g., in light\nof ANK3’s implication in bipolar disorder or the known role of GABAergic interneurons in behavior.\no In the Discussion, the behavioral findings (hyperactivity, altered gait, subtle social differences)\nare disconnected from the molecular and cellular data, and the reference to bipolar disorder is\nspeculative.\no If the behavioral work is exploratory (which must not be seen as a negative aspect per se), the\nAuthors should state this explicitly and moderate their interpretation accordingly.\nWe have now provided a more clear rationale for the specific behavior assays we tested in our study (p.\n13):\n14\n“…Because E35a is highly included in cerebellar neurons, we first assessed motor function…\nBecause ANK3 variants are genetically linked to bipolar disorder 21, we also tested memory (Y-\nmaze, Extended Data Fig. 9d), social behavior (3-chamber social, Extended Data Fig. 9e) and\nanxiety-related responses (open field, elevated plus maze (EPM), prepulse inhibition; Extended\nData Fig. 9f-h)…”\nNevertheless, we agree with the reviewer that our behavioral analysis is exploratory and has limitations\n(e.g., the use of mice with mixed genetic background), which needs to be strengthened in a future\nstudy. We have moved all behavioral data to Extended Data Fig. 9 and tuned down the conclusion.\nThese caveats are now elaborated in Discussion (p. 17-18). See also our response to related comments\n(point #1) from reviewer 2 above.\n4. Statistical methods are insufficiently reported\nThe manuscript frequently mentions two-way ANOVA but fails to specify the factors or interaction terms\n(e.g., genotype × current, genotype × sex). This is a significant omission that limits interpretability.\no Please clearly define all factors used in each ANOVA and report F-values, degrees of freedom, and\nexact p-values.\no Consider including a statistical summary table in the supplementary material for transparency.\nWe have added additional details on statistical tests to the figure legends. We have also added a\n“Statistical analysis” section in Methods to ensure transparency.\n5. Timeline and procedural details for behavioral tests are unclear\nThe order, timing, and conditions of behavioral testing are not provided. These are critical variables that\ncan influence results.\no What was the sequence of tests? What time of day were they performed (light/dark phase)? Were\nanimals habituated?\no The addition of a timeline schematic and consolidated “Behavioral Methods” section would help.\nAs suggested, we have added the schematic in the revised Extended Data Fig. 9a showing the timeline\nand sequence of the behavior tests. Additional details of behavior tests have also been included in the\nMethods.\n6. Overstated psychiatric relevance\nThe reference to bipolar disorder based on subtle social deficits and hyperactivity is premature and\nspeculative.\no The authors should moderate their language or include further data (e.g., affective tests,\npharmacological rescue) if they wish to support such a claim.\nWhile the genetic link between AnkG and bipolar disorder described in the literature is strong, we\nagree that our behavior data is more explorative, and we have toned down our conclusion in this\nregard. Please also see our responses above.\nMinor Comments\n15\n• The Discussion should be reorganized to improve clarity — e.g., separating behavioral,\nphysiological, and molecular interpretation.\nWe have revised the discussion to streamline the logic flow and make the focus of each paragraph\nmore clear.\n• Sex differences are briefly mentioned (e.g., Extended Fig. 7) but not consistently addressed across\nexperiments. Please clarify whether sex was included as a factor in all analyses.\nWe have added sex information. Since we did not observe sex-specific differences, we have now\npooled both sexes for our statistical analyses.\n• Figure legends should explicitly state: sample sizes, statistical tests used, and whether data meet\nassumptions for parametric tests.\nWe have added relevant information in figure legends.\n• The hypothetical model shown in Fig. 7b is not adequately described in the Discussion\nThe discussion has been revised, so we hope the section relevant for the new Fig. 6b is more clear.\n• “calcium” should be substituted with Ca2+, since the referral is to the concentration of the ion, and\nnot of total calcium.\nAs suggested, we have replaced calcium with Ca2+ where appropriate.\nAs noted in my initial review, this report provides the best rationale so far for the link between\nANK3 and human bipolar disorder first reported in 2008. The data are clean, the experiments well\ndesigned and cutting edge in cellular neuroscience as well as RNA biology, and the results are\nclear and well presented. The findings are original and of high impact with the first evidence of\nhow ANK3 mutation could provide a substrate for bipolar disease. I have two minor points that\ncould further improve the presentation:\n1. Ref 26 is an abstract, later published as a paper, which should be cited instead: Zhou D et\nal J Cell Biol. 1998 Nov 30;143(5):1295-304.\n2. Consider citing the first paper documenting the role of ankyrin-G in coordinating spectrin,\nL1CAM cell adhesion molecules and voltage-gated sodium channels at axon initial segments:\nAnkyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated\nsodium channels, and L1 CAMs at Purkinje neuron initial segments. Jenkins SM, Bennett V. J\nCell Biol. 2001 Nov 26;155(5):739-46.\nWe thank the reviewer for their constructive and positive feedback on our manuscript and for\nrecognizing the originality and impact of our findings potentially linking ANK3 to bipolar disorder.\nWe also appreciate the reviewer’s suggestions to strengthen the citation accuracy. These citations\nhave been incorporated into the revised manuscript in the relevant sections.\nReviewer #2 (Remarks to the Author):\nAuthors have appropriately addressed some of the concerns that were raised, and improved the\nmanuscript, but behavioral data presented in this study are still not conclusive:\nRegarding social behavior, in figure R1 and extended data figure 9e, the authors have either not\nincluded an intra-genotype comparison between time spent in social chamber versus time spent\nin non-social chamber, or that comparison was made but differences are not statistically\nsignificant. This means that WT animals are not displaying a preference for social behaviour,\nwhich makes the results for the mutant animals inconclusive. These results should be removed\nfrom the data set.\nWe apologize for the omission of some relevant information from our behavioral results in the first\nround of revision, which may have caused confusion. In the 3-chamber test, we did actually\nperform the intra-genotype comparisons. As expected, the WT animals did show a preference for\nsocial interactions, as indicated by the significantly increased time (P=0.02, paired t-test) and\nsniffing duration (P=0.002, paired t-test) in the stranger chamber, as compared to the object\nchamber (Fig. R1). The 3-chamber data we obtained is therefore reliable and we were able to\nconclude that Ank3 E35a-/- mice did not show significant social deficits in this test.\nFig. R1. Intra-genotype comparisons in the 3-chamber test (paired t-test).\nIn the PPI test, measuring the startle responses to the different intensity prepulses is a necessary\ncontrol, as it excludes the potential contribution of these prepulses to the observed startle\nresponse in each prepulse-pulse combination. This is even more relevant when we take into\nconsideration that in this particular data set the startle response of WT animals to the presentation\nof 74dB or 78dB prepulse together with the 110dB pulse elicit higher startle responses than when\npresenting the pulse alone. On a related subject, the authors did not provide any hypothesis on\nthe physiological meaning of negative values of prepulse inhibition.\nThe reviewer makes some good points here and in response to the reviewer’s concern, we\ndecided to remove the PPI data from the manuscript. We agree that it would be ideal to include\nthe startle responses to prepupses alone for better data interpretation. However, given the overall\nnegative result (i.e., no difference between WT and mutant) and the challenge of obtaining enough\nmutant mice for behavior tests, we consider that performing this additional control experiment is\nnot practical and will not contribute to any positive results. With regard to the negative values of\nprepulse inhibition, we do not think they convey significant physiological meaning, rather, this is\nmore likely caused by variations of behavioral responses in those two conditions (e.g., response\nto 74dB-110dB combination is slightly larger than response to 110dB alone). The large error bars\nalso support this interpretation.\nIn the Y-maze, values for spontaneous alternation in C57BL/6 WT mice are typically significantly\nabove 50%, indicating a non-random pattern of spatial exploration, and thus an intact short-time\nspatial working memory. This is not the case in extended data figure 9d, in which the average %\nof spontaneous alternation in the Y-maze for WT animals appears to be below 50%. As a result,\nthis data set cannot be conclusively interpreted and should be removed from the paper.\nWe apologize for the confusion here, which resulted from a lack of clarity on our part regarding\nthe three-arm maze used for these experiments. Based on our calculation of alternation%=\n(number of spontaneous alternations)/(total number of arm entries-2) x100, the alternation% for\na totally random pattern in a three-arm maze would be expected to be ~22% not 50% (1 X 2/3 X\n1/3 = 2/9 = ~22%). This issue has now been clarified in Methods.\nOverall, behavioral characterization in this study is not as mature as the other datasets. Only\nrobust behavior datasets should be included, and behavior paradigms that do not work for wild-\ntype mice, or which lack appropriate controls (as highlighted above) should be removed.\nWe thank the reviewer for the careful reassessment of our manuscript and for their valuable\ninsights regarding the entire set of behavioral data. We appreciate these points greatly and we\nhave revised the manuscript extensively, as described above.\nReviewer #3 (Remarks to the Author):\nThe authors have made several improvements to the manuscript in response to the Referees’\ncomments. Several additional experiments have been performed following ours and the other\nReferees’ suggestions, with variable results.\nElectrophysiological data have been added, and the behavioral testing procedures are now better\ndescribed. Unfortunately, the additional experiments do not fully support the hypothesis of the role\nof E35A in modulating behavior, thus posing a question on the link of the observed phenotype\nwith human bipolar disorder. Given that the results of the behavioral data are not compelling, they\nhave been moved to Supplementary Data. This is not a proper strategy: the Supplementary Data\nsection should include data which are less important, but equally rigorous, than those presented\nin the main sections. In addition, if behavioral data are not convincing, they should not be a central\npart of the Discussion.\nWe thank the reviewer for their thoughtful feedback and recognition of our previous revisions. We\nagree with the reviewer that only rigorous data should be included in the manuscript. Following\nthe Reviewers’ critiques, we carefully re-examined all behavior datasets and made the following\nchanges:\n1. Prepulse inhibition (PPI): We identified a missing control (the startle responses to\nprepulses alone), rendering the dataset inconclusive. We have removed the PPI dataset\nfrom the revised manuscript.\n2. Three-chamber sociability: We added methodological clarifications and analysis details to\nsupport the reliability of the data.\n3. Given the limitations imposed by mixed genetic background and the lack of convergent\npositive findings across assays (as already discussed), we now present the remaining\nbehavioral datasets in the Supplementary information because they are rigorous but\nsecondary to the paper’s primary molecular and cellular focus. In the Discussion, we have\nnow substantially de-emphasize behavioral interpretations and instead briefly note the\nmost robust observations, -gait alterations in the Ank3 E35a-/- mice, as a phenotype\nwarranting future study.\nWe believe these changes resolve the concern by ensuring that (1) only rigorous behavioral\nresults are retained, (2) their placement reflects their weight relative to the mechanistic data,\nand (3) the Discussion no longer treats behavior as a central focus of the paper.\nIn addition, the core mechanistic question remains insufficiently addressed, and this continues to\nundermine the central claims of the manuscript. Indeed, the central hypothesis that E35a deletion\nalters Ca²⁺ dynamics through disruption of the AnkG/InsP3R/NCX/NKA complex is still not\nsupported by direct experimental evidence.\nWe recognize the limitations of our data, and we have followed the reviewer’s suggestion to tone\ndown our model relating E35a and Ca²⁺ activity. We have therefore substantially revised our\ndiscussion and we have removed the schematics of the model (original Fig. 6), which might be\nperceived to be over-reaching. The limitations of our study are also elaborated in Discussion\n(also see our response to the last point below).\nSpecifically, we recognize the reviewer's concerns and agree that our proposed mechanism\nregarding the AnkG/InsP3R/NCX/NKA complex can benefit from additional future experiments on\nCa²⁺ dynamics. These experiments are obviously of great interest but are not possible at this time.\nWithout going into minute detail, the events of this year at our university have severely impacted\nour ability to work. New techniques that involve significant resource and time expenditures have\nproved impossible for the time being.\nWe would like to emphasize that\n1. Our co-IP/western blot experiments performed in both cerebellar tissue and HEK293T\ncells consistently confirmed that the inclusion of E35a facilitates the interaction of AnkG\nwith InsP3R and NKA, providing direct evidence of the proposed model. These two\nexperiments were designed to be complementary to each other: the N2a experiments\ntested the AnkG fragment encompassing the E35a microexon, which helps to determine\nthe specific domains directly modulating protein-protein interactions, while the cerebellum\nexperiment tested the full-length AnkG in a physiologically relevant context. In the\nprevious round of revision, we explained why cerebellum, instead of cortex, was chosen\nfor this experiment. This is because bulk cerebellar tissue, but not cortical tissue, has a\nhigh level of E35a inclusion (reflecting the major neuronal types in these brain regions),\nand we need to use bulk tissues for the co-IP/western blot experiment. While this choice\nis not ideal, cerebellar tissue does represent a more physiologically relevant context than\ncell lines like N2a. The narrative is rephrased to make this point more clear (pp. 12):\n“Cerebellum was chosen here instead of cortex, because of the high E35a inclusion\nat the bulk tissue level in the former but not the latter (Extended Data Fig. 6d),\nreflecting major neuron types constituting these different brain regions (i.e., both\ngranule cells and Purkinje cells in the cerebellum have high E35a inclusion,\nwhereas the GABAergic neurons with high E35a inclusion represent a minority\npopulation in the cortex; Fig. 1b,c).”\n2. As we cited in the Discussion, it was previously shown that the spectrin-binding domain of\nAnkG interacts directly with NKA, corroborating our proposed model.\nThey introduced an assay in HEK293T cells using an InsP3R-derived chimera, but this system\nlacks neuronal context (as well as several caveats, as the authors themselves indicate), and their\nresults show no effect of E35a deletion on AnkG recruitment.\nAs explained in the first round of revision, despite substantial efforts we made to optimize the\nrecruitment assay, our positive control (using the NF–InsP3R-ABD construct to recruit AnkB, an\nestablished interactor of InsP3R), did not show sufficient efficiency, so that the results from this\nexperiment was inconclusive. However, we refer the reviewers to our co-IP/western blot\nexperiments that validated a difference between the interaction of the two AnkG isoforms with the\nInsP3R/NCX/NKA complex.\nImportantly, the Authors did not follow and execute any of the suggested strategies, such as i)\npharmacological inhibition of NCX or InsP3R in E35a⁻/⁻ vs WT neurons; ii) rescue of the E35a+\nisoform in interneurons; iii) In situ PLA or co-IP in neuronal tissue...\n…The justification that the demonstration of a mechanistic link is “beyond the scope of the\nmanuscript” is not properly compelling, given the weight of the mechanistic claims being made. If\nthese experiments cannot be performed, the Authors should substantially tune down the\ninterpretation of their Ca²⁺ findings throughout the text.\nWe appreciate these constructive suggestions and agree they would strengthen the mechanistic\nlink. However, several factors limit feasibility at this stage. All these experiments require\nsubstantial efforts to optimize the assay conditions before we can obtain trustworthy results.\nLogistically, our labs at Columbia faced unprecedented financial uncertainty in the past year,\nwhich also severely affected our ability to perform these experiments that require substantial\ninvestment in resource and time.\n1. Pharmacological inhibition. The reviewer makes some interesting suggestions for some\nproposed pharmacology experiments to probe the role of the Na/Ca exchanger (NCX) and\nthe IP3 receptor (InsP3R). We were also enthusiastic about this initially, but on reading\nfurther we realized that there is some potential complexity to these experiments that is not\nimmediately apparent. Even the interpretation of the results can be difficult, given the\nheterogeneity of Ca2+ dynamics between different neuronal microdomains, as reported\nby the Yuste lab (Goldberg et al., 2003: PMID 14622584). The NCX exists as three\nsubtypes, NCX1-3, which are differentially expressed across cell types (Khananshvili,\n2023: PMID 37918135), and NCX2 seems to be highly expressed in interneurons (Minelli\net al., 2007; PMID 16914199). Most of the NCX inhibitors are less potent against NCX2\nthan other isoforms, and at higher concentrations can have off-target effects on VDCC. So\nestablishing an effective and specific concentration for inhibitors like SEA0400 or YM-\n244769, which preferentially affect NCX1 and NCX3 (PMID: 14660663, 16973719) is not\na trivial matter and would likely involve a systematic dose-response approach. InsP3R\ninhibition presents further complications. Although there are inhibitors available, such as\nxestospongin C, these often inhibit the ER calcium pump as well (De Smet et al., 1999:\nPMID 10892566), complicating interpretation of the data. Another example of target\noverlap is caffeine, which interacts with ryanodine-sensitive calcium stores as well as\nInsP3Rs. Clearly, this is a tricky area for pharmacological study.\n2. In situ PLA is an attractive next stepbecause of its capability to provide spatial information\nof protein-protein interactions. However, its success depends on the epitopes of\nantibodies of the interacting proteins to be in close proximity (which may not hold given\nthe large size of AnkG and its interactor proteins) and the assay could be susceptible to\ntechnical artifacts. Similar to our co-IP experiments, it might also be challenging to perform\nquantitative analysis of protein-protein interactions in the cortex, where E35a inclusion is\nlow in the WT brain. The information from this assay is also more confirmative given the\nco-IP experiments we have already performed.\n3. Rescuing the E35a+ isoform in interneurons would require generating AAV vectors\ncapable of expressing the full-length Ankyrin-G (AnkG) cDNA with the E35a exon, which\nis challenging because this isoform is over 18 kb, far exceeding the optimal AAV genome\npackaging capacity of ~4.7–5 kb.\nWe have now presented additional studies, including these suggested experiments, as future\ndirections in the Discussion, and have revised claims throughout so that the manuscript does not\ndepend on these yet-to-be performed experiments.\nConcerning the statistical analysis, the revised manuscript includes a dedicated section on\nstatistical methods and improved figure legends. However, F-values and degrees of freedom are\nnot consistently reported, and there is no statistical summary table in the supplement as\npreviously recommended.\nWe thank the reviewer for highlighting the need for greater statistical transparency. We have now\nincluded consistent reporting of F-values and degrees of freedom in all relevant figure legends\nand Results sections. In addition, as suggested by the reviewer, a comprehensive statistical\nsummary table (Extended Data Table 3) has been added, detailing the statistical tests used,\nsample sizes, F-values, degrees of freedom, p-values, and post-hoc comparisons for all datasets\nin the manuscript.\nFinally, the psychiatric relevance is now framed more cautiously, which is appreciated. However,\nthe discussion of calcium signaling and the proposed molecular complex. Overall, while\nimprovements have been made, the intrerpretation of the data still relies heavily on a mechanistic\nmodel that is not fully supported by experimental evidence. Without functional validation or at\nleast a stronger anatomical and physiological demonstration of the proposed complex, the claims\nshould be significantly revised.\nSee also our response to the critiques above. We appreciate the reviewer's guidance in\nstrengthening the scientific rigor of this manuscript. In the revised Discussion, we have toned\ndown all discussion of the calcium-related findings and the limitations of our studies are now\nexplicitly acknowledged. We believe these revisions appropriately align our interpretations with\nthe experimental evidence provided and address the reviewer's core concerns regarding the\nmechanistic claims.\n“At this point, we are uncertain about the origin of the changes in Ca2+ dynamics in FS\ncells of the Ank3 E35a-/- mice. Despite the complexity of the calcium dynamics in neurons,\nbroadly speaking, Ca2+ may enter the cells via receptor-operated channels (ROCs) such\nas NMDA receptors or through voltage-dependent calcium channels (Ca s). In addition,\nv\nCa2+ can be released from intracellular stores, which can control cellular Ca2+ dynamics\n67, 70. An increase in the frequency of calcium transients in some GABAergic neurons of\nthe PFC is certainly consistent with the increase in intrinsic excitability of FS cells recorded\nin brain slices, but could also be explained by alterations in one or more other\nmechanisms…\nWe investigated the possible biochemical basis underlying the observed changes in\nneuronal excitability and calcium activity in cortical GABAergic neurons of E35a-/- mice…\n…The disruption of the NCX/NKA/InsP3R complex in Ank3 E35a-/- in GABAergic neurons\nof Ank3 E35a-/- mice might result in reduced export of Ca2+ released from the ER storage,\nwhich would be consistent with the observed increased somatic Ca2+ activity. However,\nthis model needs to be corroborated by direct observation of the complex at ER-PM\njunctions and its dependence on E35a inclusion. In addition, functional evidence for this\nproposed mechanism is currently lacking and awaits further investigation. Therefore, it\nremains unclear for now how, and to what extent, the ER storage mechanisms contribute\nto the observed Ca2+ activity changes, as compared to alternative mechanisms such as\nincreased Ca2+ influx through ROCs or Ca s upon neuronal depolarization.\nv\nDespite all of the above comments on the limitations of the study, given the substantial amount of\nmolecular, cellular and physiological data we have already presented in the manuscript (including\nthose from a new mouse model), we believe this study already stands as a significant contribution\nto the RNA splicing and molecular neuroscience field as it presents one of few examples of neuron\ntype-specific exons whose regulation and function have been characterized in detail at both\nmolecular and cellular levels.\nReviewer #2 (Remarks to the Author):\nThe authors have appropriately addressed the issues that were raised, and the manuscript is\nnow appropriate for publication.\nWe thank the reviewer’s support for publication of our work.\nReviewer #3 (Remarks to the Author):\nWe thank the authors for their thoughtful revision of the manuscript. The authors have made\nsignificant efforts to address our previous concerns. The revised version is notably improved in\nclarity and overstatements have been removed.\nMajor strengths of the revised version:\n• The authors have appropriately toned-down mechanistic interpretations regarding the AnkG–\nInsP3R/NCX/NKA complex and removed the speculative cartoon figure.\n• They now explicitly acknowledge the correlative nature of the calcium findings and the\nabsence of direct causal evidence.\n• The statistical reporting has been considerably improved with the inclusion of a dedicated table\n(Extended Data Table 3), including F-values, degrees of freedom, and p-values.\n• The behavioral data have been restructured appropriately: weaker or inconclusive datasets\nwere removed, and the remaining data are now placed in the Supplement and discussed more\ncautiously.\nWe understand that additional mechanistic experiments could not be performed due to technical\nand funding constraints, which the authors have explained transparently. While the manuscript\nremains descriptive in nature, it provides robust and novel insights into neuron-specific\nalternative splicing and its physiological consequences, particularly in fast-spiking (FS)\ninterneurons.\nWe find the revised manuscript acceptable for publication, provided the authors retain the\ncurrent cautious interpretation of the mechanistic findings and maintain the focus on cell-type-\nspecific regulation and electrophysiological phenotypes.\nWe thank the reviewer’s understanding and support for publication of our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "As noted in my initial review, this report provides the best rationale so far for the link between ANK3 and human bipolar disorder first reported in 2008. The data are clean, the experiments well designed and cutting edge in cellular neuroscience as well as RNA biology, and the results are clear and well presented. The findings are original and of high impact with the first evidence of how ANK3 mutation could provide a substrate for bipolar disease. I have two minor points that could further improve the presentation:\n1. Ref 26 is an abstract, later published as a paper, which should be cited instead: Zhou D et al J Cell Biol. 1998 Nov 30;143(5):1295-304.\n2. Consider citing the first paper documenting the role of ankyrin-G in coordinating spectrin, L1CAM cell adhesion molecules and voltage-gated sodium channels at axon initial segments: Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments. Jenkins SM, Bennett V. J Cell Biol. 2001 Nov 26;155(5):739-46.", "reviewer_2": "Authors have appropriately addressed some of the concerns that were raised, and improved the manuscript, but behavioral data presented in this study are still not conclusive:\nRegarding social behavior, in figure R1 and extended data figure 9e, the authors have either not included an intra-genotype comparison between time spent in social chamber versus time spent in non-social chamber, or that comparison was made but differences are not statistically significant. This means that WT animals are not displaying a preference for social behaviour, which makes the results for the mutant animals inconclusive. These results should be removed from the data set.\nIn the PPI test, measuring the startle responses to the different intensity prepulses is a necessary control, as it excludes the potential contribution of these prepulses to the observed startle response in each prepulse-pulse combination. This is even more relevant when we take into consideration that in this particular data set the startle response of WT animals to the presentation of 74dB or 78dB prepulse together with the 110dB pulse elicit higher startle responses than when presenting the pulse alone. On a related subject, the authors did not provide any hypothesis on the physiological meaning of negative values of prepulse inhibition.\nIn the Y-maze, values for spontaneous alternation in C57BL/6 WT mice are typically significantly above 50%, indicating a non-random pattern of spatial exploration, and thus an intact short-time spatial working memory. This is not the case in extended data figure 9d, in which the average % of spontaneous alternation in the Y-maze for WT animals appears to be below 50%. As a result, this data set cannot be conclusively interpreted and should be removed from the paper.\nOverall, behavioral characterization in this study is not as mature as the other datasets. Only robust behavior datasets should be included, and behavior paradigms that do not work for wild-type mice, or which lack appropriate controls (as highlighted above) should be removed.", "reviewer_3": "The authors have made several improvements to the manuscript in response to the Referees’ comments. Several additional experiments have been performed following ours and the other Referees’ suggestions, with variable results.\nElectrophysiological data have been added, and the behavioral testing procedures are now better described. Unfortunately, the additional experiments do not fully support the hypothesis of a role of E35A in modulating behavior, thus posing a question on the link of the observed phenotype with human bipolar disorder.\nGiven that the results of the behavioral data are not compelling, they have been moved to the Supplementary Data. This is not a proper strategy: the Supplementary Data section should include data which are less important, but equally rigorous, than those presented in the main sections. In addition, if behavioral data are not convincing, they should not be a central part of the Discussion.\nIn addition, the core mechanistic question remains insufficiently addressed, and this continues to undermine the central claims of the manuscript. Indeed, the central hypothesis that E35a deletion alters Ca²⁺ dynamics through disruption of the AnkG/InsP3R/NCX/NKA complex is still not supported by direct experimental evidence. Importantly, the Authors did not follow and execute any of the suggested strategies, such as: i) pharmacological inhibition of NCX or InsP3R in E35a⁻/⁻ vs WT neurons; ii) rescue of the E35a+ isoform in interneurons; iii) In situ PLA or co-IP in neuronal tissue. They introduced an assay in HEK293T cells using an InsP3R-derived chimera, but this system lacks neuronal context (as well as several caveats, as the authors themselves indicate), and their results show no effect of E35a deletion on AnkG recruitment.\nThe justification that the demonstration of a mechanistic link is “beyond the scope of the manuscript” is not properly compelling, given the weight of the mechanistic claims being made. If these experiments cannot be performed, the Authors should substantially tune down the interpretation of their Ca²⁺ findings throughout the text.\nConcerning the statistical analysis, the revised manuscript includes a dedicated section on statistical methods and improved figure legends. However, F-values and degrees of freedom are not consistently reported, and there is no statistical summary table in the supplement as previously recommended.\nFinally, the psychiatric relevance is now framed more cautiously, which is appreciated. However, the discussion of calcium signaling and the proposed molecular complex still goes beyond what the data allow. The conclusions should be moderated unless further experimental support is added.\nOverall, while improvements have been made, the intrerpretation of the data still relies heavily on a mechanistic model that is not fully supported by experimental evidence. Without functional validation or at least a stronger anatomical and physiological demonstration of the proposed complex, the claims should be significantly revised.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for their constructive and positive feedback on our manuscript and for recognizing the originality and impact of our findings potentially linking ANK3 to bipolar disorder. We also appreciate the reviewer’s suggestions to strengthen the citation accuracy. These citations have been incorporated into the revised manuscript in the relevant sections.\nWe apologize for the omission of some relevant information from our behavioral results in the first round of revision, which may have caused confusion. In the 3-chamber test, we did actually perform the intra-genotype comparisons. As expected, the WT animals did show a preference for social interactions, as indicated by the significantly increased time (P=0.02, paired t-test) and sniffing duration (P=0.002, paired t-test) in the stranger chamber, as compared to the object chamber (Fig. R1). The 3-chamber data we obtained is therefore reliable and we were able to conclude that Ank3 E35a-/- mice did not show significant social deficits in this test.\nFig. R1. Intra-genotype comparisons in the 3-chamber test (paired t-test).\nThe reviewer makes some good points here and in response to the reviewer’s concern, we decided to remove the PPI data from the manuscript. We agree that it would be ideal to include the startle responses to prepupses alone for better data interpretation. However, given the overall negative result (i.e., no difference between WT and mutant) and the challenge of obtaining enough mutant mice for behavior tests, we consider that performing this additional control experiment is not practical and will not contribute to any positive results. With regard to the negative values of prepulse inhibition, we do not think they convey significant physiological meaning, rather, this is more likely caused by variations of behavioral responses in those two conditions (e.g., response to 74dB-110dB combination is slightly larger than response to 110dB alone). The large error bars also support this interpretation.\nWe apologize for the confusion here, which resulted from a lack of clarity on our part regarding the three-arm maze used for these experiments. Based on our calculation of alternation%=(number of spontaneous alternations)/(total number of arm entries-2) x100, the alternation% for a totally random pattern in a three-arm maze would be expected to be ~22% not 50% (1 X 2/3 X 1/3 = 2/9 = ~22%). This issue has now been clarified in Methods.\nWe thank the reviewer for the careful reassessment of our manuscript and for their valuable insights regarding the entire set of behavioral data. We appreciate these points greatly and we have revised the manuscript extensively, as described above.\nWe thank the reviewer for their thoughtful feedback and recognition of our previous revisions. We agree with the reviewer that only rigorous data should be included in the manuscript. Following the Reviewers’ critiques, we carefully re-examined all behavior datasets and made the following changes:\n1. Prepulse inhibition (PPI): We identified a missing control (the startle responses to prepulses alone), rendering the dataset inconclusive. We have removed the PPI dataset from the revised manuscript.\n2. Three-chamber sociability: We added methodological clarifications and analysis details to support the reliability of the data.\n3. Given the limitations imposed by mixed genetic background and the lack of convergent positive findings across assays (as already discussed), we now present the remaining behavioral datasets in the Supplementary information because they are rigorous but secondary to the paper’s primary molecular and cellular focus. In the Discussion, we have now substantially de-emphasize behavioral interpretations and instead briefly note the most robust observations, -gait alterations in the Ank3 E35a-/- mice, as a phenotype warranting future study.\nWe believe these changes resolve the concern by ensuring that (1) only rigorous behavioral results are retained, (2) their placement reflects their weight relative to the mechanistic data, and (3) the Discussion no longer treats behavior as a central focus of the paper.\nWe recognize the limitations of our data, and we have followed the reviewer’s suggestion to tone down our model relating E35a and Ca²⁺ activity. We have therefore substantially revised our discussion and we have removed the schematics of the model (original Fig. 6), which might be perceived to be over-reaching. The limitations of our study are also elaborated in Discussion (also see our response to the last point below).\nSpecifically, we recognize the reviewer's concerns and agree that our proposed mechanism regarding the AnkG/InsP3R/NCX/NKA complex can benefit from additional future experiments on Ca²⁺ dynamics. These experiments are obviously of great interest but are not possible at this time. Without going into minute detail, the events of this year at our university have severely impacted our ability to work. New techniques that involve significant resource and time expenditures have proved impossible for the time being.\nWe would like to emphasize that\n1. Our co-IP/western blot experiments performed in both cerebellar tissue and HEK293T cells consistently confirmed that the inclusion of E35a facilitates the interaction of AnkG with InsP3R and NKA, providing direct evidence of the proposed model. These two experiments were designed to be complementary to each other: the N2a experiments tested the AnkG fragment encompassing the E35a microexon, which helps to determine the specific domains directly modulating protein-protein interactions, while the cerebellum experiment tested the full-length AnkG in a physiologically relevant context. In the previous round of revision, we explained why cerebellum, instead of cortex, was chosen for this experiment. This is because bulk cerebellar tissue, but not cortical tissue, has a high level of E35a inclusion (reflecting the major neuronal types in these brain regions), and we need to use bulk tissues for the co-IP/western blot experiment. While this choice is not ideal, cerebellar tissue does represent a more physiologically relevant context than cell lines like N2a. The narrative is rephrased to make this point more clear (pp. 12):\n“Cerebellum was chosen here instead of cortex, because of the high E35a inclusion at the bulk tissue level in the former but not the latter (Extended Data Fig. 6d), reflecting major neuron types constituting these different brain regions (i.e., both granule cells and Purkinje cells in the cerebellum have high E35a inclusion, whereas the GABAergic neurons with high E35a inclusion represent a minority population in the cortex; Fig. 1b,c).”\n2. As we cited in the Discussion, it was previously shown that the spectrin-binding domain of AnkG interacts directly with NKA, corroborating our proposed model.\nAs explained in the first round of revision, despite substantial efforts we made to optimize the recruitment assay, our positive control (using the NF–InsP3R-ABD construct to recruit AnkB, an established interactor of InsP3R), did not show sufficient efficiency, so that the results from this experiment was inconclusive. However, we refer the reviewers to our co-IP/western blot experiments that validated a difference between the interaction of the two AnkG isoforms with the InsP3R/NCX/NKA complex.\nWe appreciate these constructive suggestions and agree they would strengthen the mechanistic link. However, several factors limit feasibility at this stage. All these experiments require substantial efforts to optimize the assay conditions before we can obtain trustworthy results. Logistically, our labs at Columbia faced unprecedented financial uncertainty in the past year, which also severely affected our ability to perform these experiments that require substantial investment in resource and time.\n1. Pharmacological inhibition. The reviewer makes some interesting suggestions for some proposed pharmacology experiments to probe the role of the Na/Ca exchanger (NCX) and the IP3 receptor (InsP3R). We were also enthusiastic about this initially, but on reading further we realized that there is some potential complexity to these experiments that is not immediately apparent. Even the interpretation of the results can be difficult, given the heterogeneity of Ca2+ dynamics between different neuronal microdomains, as reported by the Yuste lab (Goldberg et al., 2003: PMID 14622584). The NCX exists as three subtypes, NCX1-3, which are differentially expressed across cell types (Khananshvili, 2023: PMID 37918135), and NCX2 seems to be highly expressed in interneurons (Minelli et al., 2007; PMID 16914199). Most of the NCX inhibitors are less potent against NCX2 than other isoforms, and at higher concentrations can have off-target effects on VDCC. So establishing an effective and specific concentration for inhibitors like SEA0400 or YM-244769, which preferentially affect NCX1 and NCX3 (PMID: 14660663, 16973719) is not a trivial matter and would likely involve a systematic dose-response approach. InsP3R inhibition presents further complications. Although there are inhibitors available, such as xestospongin C, these often inhibit the ER calcium pump as well (De Smet et al., 1999: PMID 10892566), complicating interpretation of the data. Another example of target overlap is caffeine, which interacts with ryanodine-sensitive calcium stores as well as InsP3Rs. Clearly, this is a tricky area for pharmacological study.\n2. In situ PLA is an attractive next stepbecause of its capability to provide spatial information of protein-protein interactions. However, its success depends on the epitopes of antibodies of the interacting proteins to be in close proximity (which may not hold given the large size of AnkG and its interactor proteins) and the assay could be susceptible to technical artifacts. Similar to our co-IP experiments, it might also be challenging to perform quantitative analysis of protein-protein interactions in the cortex, where E35a inclusion is low in the WT brain. The information from this assay is also more confirmative given the co-IP experiments we have already performed.\n3. Rescuing the E35a+ isoform in interneurons would require generating AAV vectors capable of expressing the full-length Ankyrin-G (AnkG) cDNA with the E35a exon, which is challenging because this isoform is over 18 kb, far exceeding the optimal AAV genome packaging capacity of ~4.7–5 kb.\nWe have now presented additional studies, including these suggested experiments, as future directions in the Discussion, and have revised claims throughout so that the manuscript does not depend on these yet-to-be performed experiments.\nWe thank the reviewer for highlighting the need for greater statistical transparency. We have now included consistent reporting of F-values and degrees of freedom in all relevant figure legends and Results sections. In addition, as suggested by the reviewer, a comprehensive statistical summary table (Extended Data Table 3) has been added, detailing the statistical tests used, sample sizes, F-values, degrees of freedom, p-values, and post-hoc comparisons for all datasets in the manuscript.\nSee also our response to the critiques above. We appreciate the reviewer's guidance in strengthening the scientific rigor of this manuscript. In the revised Discussion, we have toned down all discussion of the calcium-related findings and the limitations of our studies are now explicitly acknowledged. We believe these revisions appropriately align our interpretations with the experimental evidence provided and address the reviewer's core concerns regarding the mechanistic claims.\n“At this point, we are uncertain about the origin of the changes in Ca2+ dynamics in FS cells of the Ank3 E35a-/- mice. Despite the complexity of the calcium dynamics in neurons, broadly speaking, Ca2+ may enter the cells via receptor-operated channels (ROCs) such as NMDA receptors or through voltage-dependent calcium channels (Ca s). In addition, Ca2+ can be released from intracellular stores, which can control cellular Ca2+ dynamics 67, 70. An increase in the frequency of calcium transients in some GABAergic neurons of the PFC is certainly consistent with the increase in intrinsic excitability of FS cells recorded in brain slices, but could also be explained by alterations in one or more other mechanisms…\nWe investigated the possible biochemical basis underlying the observed changes in neuronal excitability and calcium activity in cortical GABAergic neurons of E35a-/- mice…\n…The disruption of the NCX/NKA/InsP3R complex in Ank3 E35a-/- in GABAergic neurons of Ank3 E35a-/- mice might result in reduced export of Ca2+ released from the ER storage, which would be consistent with the observed increased somatic Ca2+ activity. However, this model needs to be corroborated by direct observation of the complex at ER-PM junctions and its dependence on E35a inclusion. In addition, functional evidence for this proposed mechanism is currently lacking and awaits further investigation. Therefore, it remains unclear for now how, and to what extent, the ER storage mechanisms contribute to the observed Ca2+ activity changes, as compared to alternative mechanisms such as increased Ca2+ influx through ROCs or Ca s upon neuronal depolarization.\nv\nDespite all of the above comments on the limitations of the study, given the substantial amount of molecular, cellular and physiological data we have already presented in the manuscript (including those from a new mouse model), we believe this study already stands as a significant contribution to the RNA splicing and molecular neuroscience field as it presents one of few examples of neuron type-specific exons whose regulation and function have been characterized in detail at both molecular and cellular levels.\nWe thank the reviewer’s support for publication of our work.\nWe thank the reviewer’s understanding and support for publication of our work."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have appropriately addressed the issues that were raised, and the manuscript is now appropriate for publication.", "reviewer_3": "We thank the authors for their thoughtful revision of the manuscript. The authors have made significant efforts to address our previous concerns. The revised version is notably improved in clarity and overstatements have been removed.\nMajor strengths of the revised version:\n• The authors have appropriately toned-down mechanistic interpretations regarding the AnkG–InsP3R/NCX/NKA complex and removed the speculative cartoon figure.\n• They now explicitly acknowledge the correlative nature of the calcium findings and the absence of direct causal evidence.\n• The statistical reporting has been considerably improved with the inclusion of a dedicated table (Extended Data Table 3), including F-values, degrees of freedom, and p-values.\n• The behavioral data have been restructured appropriately: weaker or inconclusive datasets were removed, and the remaining data are now placed in the Supplement and discussed more cautiously.\nWe understand that additional mechanistic experiments could not be performed due to technical and funding constraints, which the authors have explained transparently. While the manuscript remains descriptive in nature, it provides robust and novel insights into neuron-specific alternative splicing and its physiological consequences, particularly in fast-spiking (FS) interneurons.\nWe find the revised manuscript acceptable for publication, provided the authors retain the current cautious interpretation of the mechanistic findings and maintain the focus on cell-type-specific regulation and electrophysiological phenotypes.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter