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. 2021 Aug 3;7(3-4):71–79. doi: 10.1159/000518819

Ankyrin-G Heterozygous Knockout Mice Display Increased Sensitivity to Social Defeat Stress

Zachary A Cordner a, Seva G Khambadkone a,b, Shanshan Zhu c, Justin Bai c, R Rasadokht Forti c, Ethan Goodman a, Kellie LK Tamashiro a,*, Christopher A Ross a,c,d,*
PMCID: PMC8740233  PMID: 35928299

Abstract

The ANK3 locus has been repeatedly found to confer an increased risk for bipolar disorder. ANK3 codes for Ankyrin-G (Ank-G), a scaffold protein concentrated at axon initial segments, nodes of Ranvier, and dendritic spines, where it organizes voltage-gated sodium and potassium channels and cytoskeletal proteins. Mice with homozygous conditional knockout of Ank-G in the adult forebrain display hyperactivity and reduced anxiety-like behaviors, responsive to mood stabilizers. Their behavior switches to a depression-like phenotype when exposed to chronic social defeat stress (SDS), and then spontaneously reverts to baseline hyperactivity. Ank-G heterozygous conditional knockouts (Ank-G Het cKO) have not previously been characterized. Here, we describe the behavior of Ank-G Het cKO mice compared to littermate controls in the open field, elevated plus maze, and forced swim test, under both unstressed and stressed conditions. We found that Ank-G Het cKO is not significantly different from controls at baseline or after chronic SDS. The chronic stress-induced “depression-like” behavioral phenotype is persistent for at least 28 days and is responsive to fluoxetine. Strikingly, Ank-G Het cKO mice display increased sensitivity to a short duration SDS, which does not affect controls. The heterozygous Ank-G genetic model may provide novel insights into the role of Ank-G in the pathophysiology of stress sensitivity and “depression-like” phenotypes and could be useful for studying Ank-G-related gene-environment interactions.

Keywords: Depression, Gene-environment interaction, Antidepressant, Selective serotonin reuptake inhibitor

Introduction

Affective disorders, including both bipolar disorder and major depression, have a high prevalence and significant morbidity [1, 2]. Symptoms and signs of mania include elevated, expansive, or irritable mood, grandiosity, increased activity, distractibility, and decreased anxiety [3, 4]. By contrast, symptoms and signs of depressive episodes include low mood, anhedonia, a sense of worthlessness or guilt, decreased activity, and impaired concentration. While the neurobiological mechanisms underlying affective disorders and contributing to transitions between depressive, manic, and mixed mood states are only partially understood [5, 6], genetic factors clearly contribute to risk [3, 7, 8, 9]. Environmental factors are also important, with stress recognized as a trigger for both depression [6, 10] and mania [11, 12]. Additionally, a number of gene-environment interactions have now been found to contribute to major depression and bipolar disorder [7, 13, 14, 15, 16, 17].

Early genetic association findings for bipolar disorder implicated loci within or very near the ANK3 gene, and these loci were again implicated in subsequent studies [8, 9, 18, 19, 20, 21, 22, 23]. ANK3 codes for Ankyrin-G (Ank-G), a highly expressed scaffold protein, with a 480 kDa isoform crucial for the organization of the axon initial segment and Nodes of Ranvier. Among its functions, Ank-G clusters sodium and potassium channels at the axon initial segment, thereby playing an important role in regulation of neuronal action potential firing. Analysis of the bipolar disorder-associated loci within ANK3 suggests that some may be associated with decreased expression of major isoforms [19, 24, 25] and lead to loss of Ank-G function [21]. Data from a postmortem study of patients with schizophrenia, a disorder for which ANK3 loci have also been implicated, supported this loss of function hypothesis. Ank-G protein expression was reduced in the AIS of pyramidal neurons in the cortical superficial layer of schizophrenia postmortem brains [26].

We recently developed a mouse model with homozygous conditional knockout of all major isoforms of Ank-G in forebrain pyramidal neurons via the CaMKII promoter (Ank-G cKO). The CaMKII promoter drives Cre expression in the mouse forebrain, a brain region relevant to human psychiatric disease, starting at 2–3 weeks of age postnatal. Previous Ank-G knockout mouse models, which delete Ank3 gene expression during embryonic stages have developmental defects and do not survive long. The Ank-G cKO mice do not have any obvious developmental defects and have home cage behavior similar to control. However, they displayed a robust behavioral phenotype, including hyperactivity, increased exploratory behavior, and decreased anxiety that was substantially normalized by either lithium or valproic acid at therapeutic levels. When exposed to chronic social defeat stress (SDS), a paradigm that is well studied in mice [27, 28, 29, 30, 31], the behavior of Ank-G homozygous cKO mice switched to a “depression-like” phenotype, before spontaneously returning to their baseline level of hyperactivity within 1–2 weeks [32], which may be relevant as a model for some aspects of the neurobiology related to mania.

Human genetic variation at the ANK3 locus is unlikely to involve complete loss of function but may involve partial loss of expression or other partial loss of function. Clinical and translational studies have shown that heterozygous Ank-G loss of function variants and variants with diminished expression of Ank-G are associated with increased disease risk and disrupted neuronal function [21, 24, 25]. Our previous study was limited to homozygous conditional knockouts. We have, therefore, turned attention in this study to the heterozygous conditional knockout mouse (Ank-G Het cKO).

In this study, we show that several aspects of the baseline behavior of Ank-G Het cKO mice are not significantly different from wild-type (WT) control littermates. When exposed to 14 days of SDS, Ank-G Het cKO mice respond comparably to WT controls, and the stress-induced phenotype persists for at least 28 days in both groups. When exposed to 14 days of SDS, then treated with a low dose of the selective serotonin reuptake inhibitor (SSRI) fluoxetine, the stress-induced behavioral phenotype improves in both WT and Ank-G Het cKO mice.

Finally, when exposed to a short duration of SDS, Ank-G Hets, but not WT controls, developed a “depression-like” stress-induced behavioral phenotype. The data presented here indicate that the Ank-G Het cKO mouse model may be useful for studies of gene-environment interactions between Ank-G and stress, as well as for investigations of the role of Ank-G in stress sensitivity.

Methods

Animals

As previously described, we generated mice on a C57BL6/J background with homozygous disruption of Ank-G in adult forebrain pyramidal neurons using the Camk2a promoter to drive Cre recombinase expression (Camk2a-Cre; Ank3flox/flox) [32]. Ank-G Het cKO mice were then generated by mating male Ank3flox/flox mice with female heterozygous Camk2a-Cre mice on a C57BL6/J background. Male littermates that express Ank3flox/flox were used as controls. Studies were conducted using male mice 3–6 months old. Mice were housed in standard cages (52 × 32 × 24 cm) on a 12 h:12 h light-dark cycle with ad libitum access to water and standard chow (Envigo 2018 rodent diet). All mice were handled daily and housed in the same room except during stress procedures and behavioral testing. All protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.

In this study, there were 5 cohorts of mice. The first was used only to assess baseline behavior. The next 4 cohorts were used in stress studies.

Social Defeat Stress

SDS was applied using a standard protocol described previously [30]. Briefly, WT or Ank-G Het cKO mice were paired with a different male aggressor (CD-1 retired breeders, Charles River) each day. The SDS mouse was placed into the aggressor's home cage, and mice were allowed to interact for 10 min. After 10 min, mice were separated by a clear, ventilated barrier, and they remained co-housed for the next 24 h before SDS mice were moved to a different resident aggressor's home cage. Unstressed controls were pair housed in cages divided by a clear, ventilated barrier.

In the first stress cohort, mice were exposed to standard conditions or 14 days of SDS followed by behavioral testing. In a separate cohort, mice were exposed to 14 days of SDS, tested, and then divided into 2 groups. To determine the persistence of the SDS phenotype, half of the mice were returned to standard housing conditions for 28 days then retested. To determine the effects of SSRI treatment, which has previously been shown to normalize the SDS phenotype [33, 34], half of the mice were treated with low-dose fluoxetine then retested.

In another cohort intended to determine sensitivity to a short duration of stress, mice were exposed to standard conditions or 4 days of SDS followed by behavioral testing. To confirm results from these studies, a final cohort was tested at baseline and then retested after 4 and 10 days of SDS.

Fluoxetine Treatment

Fluoxetine (Sigma F132), an SSRI commonly used in clinical settings and rodent studies, was dissolved in drinking water to a final concentration of 50 mg/L. Throughout treatment, mice continued to have ad libitum access to water and standard chow. For all mice, water intake was monitored every 2 days, and water was changed every 2 days. Mice were treated with fluoxetine for a total of 14 days. An average daily fluoxetine dose (mg/kg) was calculated for the entire 14 days period.

Behavior

Behavioral tests were performed during the middle of the light phase. The interval between behavioral tests was 1 day.

Open Field Test

The open field consists of a plastic box (40 cm × 40 cm × 30 cm). Each mouse was placed into the center of the field, and locomotor activity of was measured for 30 min. Distance traveled was calculated by a computerized detection system (Omnitech Electronics, Columbus, OH).

Elevated Plus Maze

The elevated plus maze consists of 2 open arms (6 cm × 30 cm) and 2 closed arms (6 cm × 30 cm × 40 cm) connected by a center platform (6 cm × 6 cm). Each mouse was placed on the center platform facing an open arm and allowed to explore the plus maze for 5 min. Behavior was video recorded and coded by a blinded observer for time spent in the open arms, closed arms, and center platform.

Forced Swim Test

The swim chamber is a clear glass cylinder (26 cm high and 22 cm diameter) filled with water (21–23°C, 17 cm water height). Each mouse was started at the center of the cylinder and allowed to explore for 6 min. Behavior during the final 4 min was video recorded and coded by a blinded observer for time spent immobile. Because the forced swim test is itself particularly stressful [35] and prone to effects of habituation [36], here only the open field and elevated plus maze were used in cohorts where mice were repeatedly tested.

Statistical Analyses

Statistical analyses were completed using Statistica 7 (StatSoft, Inc., Tulsa, OK, USA) or Prism 8 (GraphPad Software, San Diego, CA, USA). Differences between groups were assessed by Student's t test, factorial ANOVA, or repeated measures ANOVA followed by Tukey post hoc analysis. For all tests, p < 0.05 was considered significant. Data are expressed as averages ±standard error of the mean.

Results

As adults, male Ank-G Het cKO and WT littermates had similar locomotor activity in the open field and similar exploratory behavior in the elevated plus maze. Likewise, they behaved similarly in the forced swim test (Fig. 1).

Fig. 1.

Fig. 1

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Behavior at baseline. a–c at baseline, Ank-G Hets behaved similarly to WT controls in the open field, elevated plus maze, and forced swim test. Graphs indicate mean ± standard error of the mean. Ank-G, Ankyrin-G; Ank-G Hets, Ank-G cKO Heterozygotes; WT, wild type.

We next assessed the phenotype of Ank-G Het cKO in response to 14 days of SDS, which has been used in prior studies of chronic stress in mice [30]. Here, there were main effects of stress exposure such that both Ank-G Het cKO and WT controls displayed significantly reduced locomotor activity in the open field, reduced time on the open arm of the elevated plus maze, and increased immobility in the forced swim test in response to SDS (Fig. 2).

Fig. 2.

Fig. 2

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Behavior after 14 days of SDS (chronic stress). In a separate cohort, 14 days of SDS resulted in reduced locomotor activity in the open field (a), reduced time on the open arm of the elevated plus maze, (b) and increased immobility in the forced swim test among both WT controls and Ank-G Hets (c). *p < 0.05. Graphs indicate mean ± standard error of the mean. Ank-G, Ankyrin-G; SDS, social defeat stress; Ank-G Hets, Ank-G cKO Heterozygotes; WT, wild type.

In a separate cohort, mice were exposed to 14 days of SDS then either returned to standard conditions for 28 days to determine the persistence of the stress-induced phenotype, or treated with low-dose fluoxetine. Throughout exposure to fluoxetine dissolved in drinking water, mice received an average daily dose of 9.62 ± 0.43 mg/kg based on water intake, consistent with low doses used in prior studies [37]. There were no differences in average daily dose between fluoxetine treated groups (WT control 9.74 ± 0.63 mg/kg; Ank-G Het cKO 9.50 ± 0.60 mg/kg; p = 0.8). When behavior was assessed, there were main effects of fluoxetine exposure in the open field and elevated plus maze. That is, after 14 days of SDS, Ank-G Het cKO and WT controls behaved similarly. Following SDS and 28 days of standard housing conditions, the behavior of Ank-G Het cKO and WT controls was not different from their behavior immediately after SDS, suggesting persistence of the stress-induced phenotype. Following SDS and treatment with low-dose fluoxetine, Ank-G Het cKO and controls showed similar increases in locomotor activity in the open field and exploratory behavior in the elevated plus maze. There was a nonsignificant trend for Ank-G Het cKO mice having a larger response to fluoxetine than WT controls in the elevated plus maze (p = 0.07) (Fig. 3).

Fig. 3.

Fig. 3

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Effect of recovery and fluoxetine on the social defeat phenotype. a, b In the open field and elevated plus maze, WT controls and Ank-G Hets behaved similarly after 14 days of SDS. The SDS-induced phenotype persisted for 28 days post-stress. Treatment with fluoxetine post-stress resulted in increased exploratory behavior in the open field and elevated plus maze. *p < 0.05. Graphs indicate mean ± standard error of the mean. Ank-G, Ankyrin-G; Ank-G Hets, Ank-G cKO Heterozygotes; SDS, social defeat stress; WT, wild type.

In order to determine whether there might be an interaction between the Ank-G genetic alteration and sensitivity to stress, we exposed mice to 4 days of SDS, a duration not expected to produce prominent behavioral changes among WT mice. Again, unstressed Ank-G Het cKO and controls behaved similarly. In the open field, 4 days of SDS had no effect on either group. In the elevated plus maze and forced swim test, there were genotype-by-stress interactions. That is, after 4 days of SDS, stressed WT controls were not significantly different from unstressed WT controls while stressed Ank-G Het cKO mice displayed significantly reduced time on the open arm of the elevated plus maze and increased immobility in the forced swim test (Fig. 4a–d).

Fig. 4.

Fig. 4

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Behavior after 4 and 10 days of SDS (subthreshold stress). Among Ank-G Hets, 4 days of SDS resulted in no change in locomotor activity in the open field, (a, b) reduced time on the open arm of the elevated plus maze, (c) and increased immobility in the forced swim test (d). By contrast, 4 days of SDS had no significant effect on the behavior of WT controls. e, f In a separate cohort tested at baseline then again after 4 and 10 days of SDS, Ank-G Hets were again found to behave similarly to WT controls in the open field and elevated plus maze at baseline. However, when exposed to 4 days of SDS, Ank-G Hets displayed evidence of reduced exploratory behavior, while controls did not. After 10 days of SDS, WT controls and Ank-G Hets displayed similarly reduced exploratory behavior in the open field and elevated plus maze. *p < 0.05. Graphs indicate mean ± standard error of the mean. Ank-G, Ankyrin-G; Ank-G Hets, Ank-G cKO Heterozygotes; SDS, social defeat stress; WT, wild type.

The sensitivity of Ank-G Het cKO mice to a short duration of SDS was then replicated in a separate cohort in which mice were repeatedly tested in the open field and elevated plus maze at baseline, after 4 days of SDS, and after 10 days of SDS. Repeated measures ANOVA revealed a genotype-by-time interaction. Post hoc analysis showed that Ank-G Het cKO and WT controls again behaved similarly at baseline. Following 4 days of SDS, Ank-G Het cKO mice displayed evidence of decreased exploratory behavior compared to WT controls. Following 10 days of SDS, WT controls and Ank-G Het cKO mice displayed a similar stress-induced phenotype (Fig. 4e, f).

Discussion

Genetic loci in or very near the ANK3 gene have been associated with several psychiatric disorders, especially bipolar disorder. Ank-G biology has a crucial role in neuronal organization, especially at the AIS [38], and genetic loss of function has been implicated as a mechanism. The neurobiology of Ank-G loss of function and relation to behavioral phenotypes, and the possibility that Ank-G-related pathways may be treatment targets remain active areas of investigation. While bipolar disorder is a uniquely human disease, translational animal studies remain useful for both understanding-related neurobiology, and screening for potential novel treatment approaches [39, 40, 41].

Several studies in translational models have now provided intriguing support for the role of Ank-G in neuronal function related to bipolar disorder. For example, virally mediated knockdown of Ank3 expression in the dentate gyrus of mice led to a phenotype characterized by hyperactivity during the light phase, and reduced anxiety-like behavior, reversed by lithium treatment [42]. Lending further support for the role of Ank-G in aspects of neurobiology relevant to bipolar disorder, a recent report described a small family with bipolar disorder segregating with a heterozygous ANK3 missense variant. A knock-in mouse model carrying the same variant was found to have neuronal hyper excitability due to impaired GABAergic signaling [21]. We also previously reported that homozygous conditional knockout of Ank-G in mouse forebrain neurons recapitulated several aspects of behavior potentially relevant to human bipolar disorder, including baseline hyperactivity and reduced anxiety-like behaviors, responsive to lithium. After chronic SDS, there was a switch to a “depression-like” phenotype, with spontaneous recovery back to the hyperactive behavior [32].

Here, we have turned attention to Ank-G Het cKO mice. We found that Ank-G Het cKO mice are similar in appearance, and in several behaviors as described above, to WT controls at baseline.

When exposed to 4 days of SDS − a brief duration not expected to produce substantial behavior changes in controls − Ank-G Het cKO displayed evidence of reduced exploratory behavior, increased anxiety-like and depression-like behavior, while controls did not. In contrast, after 10 days of SDS − a “full course” of defeat stress used in many other studies to induce depression-like and anxiety-like behaviors among WT mice − controls and Ank-G Hets displayed similarly reduced exploratory behavior in the open field and elevated plus maze. This pattern, we believe, suggests that Ank-G Hets display increased stress sensitivity, but a typical response to more severe/prolonged stressors. The 10-days' of stress-induced phenotype persists for at least 4 weeks without intervention but responds to treatment in the interim with an SSRI.

These findings appear consistent with other studies suggesting that partial disruption of Ank-G expression in mice results in a predisposition to depression-like and anxiety-like behaviors, without robust mania-like behaviors. In one study, mice with a heterozygous disruption of Ank3 exon 1b, which is most highly expressed in the cerebellum, displayed some evidence of reduced novelty-induced anxiety-like behaviors at baseline. However, in response to 6 weeks of social isolation stress, these mice developed an anxiety-like and depression-like phenotype [42]. In another study, Ank3 hemizygous mice were found to have increased depression-like and anxiety-like behavior, as well as cognitive impairments, and alterations of neuromodulator pathways including GABA [43]. Mechanisms explaining the contrasting phenotypes between mice with partial versus more complete disruption of Ank-G expression may be an important area for future study.

Our model involves deleting or reducing Ank3 gene expression postnatally in forebrain regions, therefore, the “mania-like” behavior in homozygous knockout and “increased sensitivity to stress” in heterozygous knockout are the results of the postnatal loss of the Ank3 gene in the forebrain. Future mechanistic studies such as electrophysiological recording or single-cell RNAseq analysis will provide more insight into molecular and cellular mechanisms. Ank-G concentrates at AIS, and anchors most of the AIS components to the site, including voltage-gated sodium (excitatory) and potassium channels (inhibitory). In addition, chandelier inhibitory neurons which innervate AIS of pyramidal neuron show decreased or loss of innervation in heterozygous versus homozygous knockouts. Therefore, the E/I balance (decided by activity and presence of sodium channel, potassium channel, and chandelier neuron at the AIS) may be altered in complex ways in heterozygous versus homozygous knockout mice, leading to distinct behavioral phenotypes. Another possibility relates to the fact that the 190 kDa Ank-G isoform is mainly present at the synapse, while the 270 and 480 kDa Ank-G isoforms are present at the AIS. The quantitative localizations may be altered in heterozygous knockouts, leading to different behavioral phenotypes.

These studies provide evidence for a gene-environment interaction, such that Ank-G Het cKO mice were susceptible to a short duration (4-days) SDS, which does not normally cause depressive- or anxiety-like behavior in WT controls. As previously noted, the significance of gene-environment interactions in psychiatry, including in bipolar disorder, has been increasingly recognized [7, 13, 14, 15, 16, 17]. The gene-environment interaction demonstrated in our model would seem to support stress-diathesis [44] or allostatic load [45] models that conceptualize psychiatric disease states as interactions between vulnerabilities and stress, and have been previously used as theoretical frameworks for understanding neurobiological functions potentially related to depression and bipolar disorder [46, 47, 48].

Several limitations and opportunities for future investigations are worth noting. This study, seeking a first assessment of Ank-G Het cKO behavior, relied on a small number of tests that are thought to be relevant for studying depression- and anxiety-like behaviors, but all rely heavily on locomotor activity. A number of other behavioral assays could be used in the future to assess cognitive performance, social interaction, and hedonic response. Additionally, the design of some of our experiments included repeated testing. While informative (e.g., in determining sensitivity to stress over time), this approach limits the ability to use behavioral assays like the forced swim test, which are more inherently stressful, or which are likely to result in habituation. Concerning the stress paradigm used here, the standard protocol, which is employed in a vast majority of SDS studies, can only reliably be applied to males. While this represents a significant limitation of the technique, the robust, reproducible phenotype induced by SDS allows for direct comparison of our findings to other studies using SDS. Future work, however, should investigate potential sex differences in baseline behavior and stress responsivity using modified stress protocols.

Ank-G Het cKO mice appear similar to WT controls at baseline but are hypersensitive to SDS. Ank-G Het cKO mice did not display any evidence of hyperactivity or mania-like behavior. Regarding the treatment of the chronic stress-induced behavioral phenotype, we selected fluoxetine given its common use in such studies and considering that SSRIs are used in clinical practice in the treatment of both bipolar and unipolar depression [49, 50, 51]. Fluoxetine treatment caused a reversal of the stress-induced EPM changes back to the pre-stress level. Thus, the Ank-G Het mice had a greater response to fluoxetine than controls. There may be implications for the “manic switch” behaviors in bipolar disorder patients. Future studies should assess for potential induction of hyperactive behavior using environmental manipulations like sleep deprivation or stimulants. Future studies using mood stabilizers and neuroleptics are warranted. The model may be useful for studying mechanisms underlying Ank-G-related gene-environment interactions and in determining the role of Ank-G in stress sensitivity. Furthermore, the model may have utility in tests of potential novel treatments.

In sum, we found that the Ank-G Het cKO mice display increased sensitivity to a short duration SDS. The heterozygous Ank-G genetic model may provide novel insights into the role of Ank-G in molecular and cellular aspects of stress sensitivity, and “depression-like” phenotypes, and could be useful for studying Ank-G-related gene-environment interactions.

Statement of Ethics

All protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.

Conflict of Interest Statement

Z.A.C. has no conflicts of interest to declare. S.G.K. has no conflicts of interest to declare. S.Z. has no conflicts of interest to declare. J.B. has no conflicts of interest to declare. R.F. has no conflicts of interest to declare. E.G. has no conflicts of interest to declare. K.L.K.T. has no conflicts of interest to declare. C.A.R. has no conflicts of interest to declare.

Funding Sources

This work was supported by the Greif Family Scholar Fund (Z.A.C.), Dalio Philanthropies (K.L.K.T.), the National Institutes of Health (NIH) HD093338 (S.G.K.), MH108944 (K.L.K.T.), and T32 MH15330 (C.A.R. and Z.A.C.). These funding sources had no role in the preparation of data or the manuscript.

Author Contributions

Z.A.C., S.G.K., S.Z., J.B., R.F., E.G., K.L.K.T., and C.A.R. made substantial contributions to the conception of this work, acquisition, analysis, or interpretation of data, and drafting or revising the manuscript. All the authors also gave final approval of the version for publication and agree to be accountable for all aspects of the work.

Data Availability Statement

All data generated and analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Data Availability Statement

All data generated and analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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