Homer1a disruption increases vulnerability to predictable subtle stress normally sub-threshold for behavioral changes

brain research 1605 (2015) 70–75

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Research Report

Homer1a disruption increases vulnerability to predictable subtle stress normally sub-threshold for behavioral changes Yuan Shuia,b, Li Wanga,b, Xianwen Luoa,c, Osamu Uchiumia, Ryo Yamamotoa, Tokio Sugaia, Nobuo Katoa,n a

Department of Physiology, Kanazawa Medical University, Ishikawa 920-0293, Japan China–Japan Friendship Hospital, Beijing 100029, China c Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China b

ar t ic l e in f o

abs tra ct

Article history:

Homer1a is implicated in depression in humans and depression-like behavior in mice. To

Accepted 5 February 2015

further understand the role of Homer1a in stress-induced emotional changes, we applied

Available online 13 February 2015

very mild stress to Homer1a knockout (H1a KO) mice. The wild-type (WT) and H1a KO mice

Keywords:

were restrained for 2 h daily for 7 consecutive days at the same time of the day. The

Chronic restraint stress

restraint was so mild that no changes in anxiety- or depression-like behavior were

Homer1a

detected in either type of mice. However, total locomotion in the open field test and

Neuronal excitability

forced swimming test was increased by restraint in H1a KO mice only. After behavior, we

Synaptic efficiency

made brain slices to examine neuronal excitability in cingulate cortex pyramidal cells and synaptic efficiency in hippocampal CA1 synapses. The excitability, assessed on the basis of the frequency of spikes elicited by current injection, was increased by restraint in H1a KO mice. The synaptic efficiency was evaluated by comparing the input–output relationship between the size of fiber volley and the slope of field excitatory postsynaptic potentials, and was shown to be increased by restraint in H1a KO mice only. Thus, predictable subtle stress, which failed to induce behavioral or electrophysiological changes in WT mice, resulted in a minor behavioral change that accompany upregulation of neuronal excitability and synaptic efficiency in H1a KO mice, suggesting that Homer1a may play a critical role in resilience to subtle stress. & 2015 Elsevier B.V. All rights reserved.

1.

Introduction

Homer1, a member of the scaffold protein family Homer, consists of the longer and shorter splice variants, Homer1b/c and Homer1a (Brakeman et al., 1997; Kato et al., 1997). Both

n

Corresponding author. Fax: þ81 76 286 3523. E-mail address: [email protected] (N. Kato).

http://dx.doi.org/10.1016/j.brainres.2015.02.008 0006-8993/& 2015 Elsevier B.V. All rights reserved.

variants bind to diverse receptors including metabotropic glutamate receptors (mGluRs). These receptors are crosslinked by self-multimerization of Homer1b/c, the longer constitutively-expressed variant. Homer1a, which lacks the multimerizing motif, is activity-dependently induced and prevents the cross-linkage by Homer1b/c multimerization.

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2.

Results

2.1.

Behavior

In the open field test, there was an across-group difference in the total distance traveled among the 4 groups (Fig. 1a): wild-type (WT) and Homer1a-knockout (H1a KO) mice with or without restraint (WT-control, WT-restraint, H-control, H-restraint; one-way ANOVA, F(3,95)¼29.193, P ¼0.001). The H-restraint group exhibited a reduced locomotion (9.6270.37 m; N¼ 26) than WT-control group (13.2970.38 m, N¼ 25; post hoc Tukey HSD test, P¼ 0.001). Restraint increased the mobility in H1a KO mice (H-restraint; 11.1770.35 m, N¼ 24, P¼ 0.031), but not in WT mice (WT-restraint; 14.2970.45 m, N¼ 24, P ¼0.277). However, among the 4 groups, there was no across-group difference in the time spent in the inner zone (F(3,95)¼1.530, P ¼0.212), indicating that neither Homer1a disruption nor restraint induced anxiety-like tendency (Fig. 1b). This was confirmed by the light/dark test (Fig. 1c), in which no across-group difference was detected in the time spent in the light compartment

15.0

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30.0

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Fig. 1 – Test results evaluating anxiety-like behavior of WT and H1a KO mice. (a,b) Open field test results. The total distance traveled was increased by restraint in H1a KO mice only, but not WT mice (a). n, Po0.05. The time spent in the inner zone was not altered by restraint in H1a KO or WT mice (b). (c) The light/dark test results. Restraint did not alter the time spent in the light compartment in H1a KO or WT mice.

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* 100

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es t

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Homers are involved in a wide variety of neuropsychiatric abnormalities (Szumlinski et al., 2006). In particular, a genome-wide association study implicated Homer1 in pathogenesis of major depression (Rietschel et al., 2010). By using a modified version of the original forced swimming model of depression in mice, we have recently revealed that expression of Homer1a in the neocortex is decreased in depression model mice and recovered by imipramine application or repetitive transcranial magnetic stimulation (rTMS; Sun et al., 2011). Such Homer1a upregulation induced by rTMS is considered to constitute a part of negative feedback regulation of neuronal excitability, in which neural activity-induced Homer1a facilitates the large conductance calcium-activated potassium (big-K; BK) channel and thereby decreases excitability (Sakagami et al., 2005). Consistently, anti-epileptigenic effects of Homer 1a has been more directly demonstrated (Potschka et al., 2002; Klugmann et al., 2005). To further elucidate the role played by Homer1a in depression, the present study relied upon two different approaches. First, chronic mild stress was adopted as the stressor, instead of forced swimming. The suitability of forced swimming as a stressor inducing depression-like behavior in rodents has been frequently in question (Holmes, 2003; Nestler and Hyman, 2010; Veenema et al., 2003). It has been considered that changes in animal behavior induced by acute intensive insults are less likely to represent human depression than those induced by chronic accumulation of mild stress (Holmes, 2003; Nestler and Hyman, 2010; Willner, 1997; Willner, 2005). In the present experiments, therefore, forced swimming was used only as a test paradigm detecting depressive states. Second, we used Homer1a knockout (KO) mouse (Inoue et al., 2009), in which constitutive expression of Homer1b/c is normal but activity-dependent expression of Homer1a is disrupted. By introducing these two modifications, we attempted to demonstrate an essential involvement of Homer1a in resilience to stressors.

Fig. 2 – Forced swimming test results. In all the 4 experimental groups, the distance traveled (a) was shortened and the immobility time (b) elongated on Day 2 as compared with those on Day 1, confirming that depressionlike behavior was similarly detected in all the groups, irrespective of whether restraint was imposed. Multiple comparisons revealed that the locomotor activity on Day 1, assessed by both measures (a,b), was higher in the Hrestraint group than in any other groups. n, always Po0.05 as compared with the other 3 groups. among the 4 groups (WT-control, 24.6772.44 s, N¼ 20; WTrestraint, 28.4271.83 s, N ¼19; H-control, 22.573.17 s, N¼ 26; H-restraint, 22.4773.18 s, N¼ 24; one-way ANOVA, F(3,85)¼ 0.893, P¼ 0.448). Depression-like behavior was examined by the forced swimming test (Fig. 2). The distance swum on Day 2, as expressed by percent of that on Day 1, did not differ among the 4 groups (Fig. 2a; one-way ANOVA, F(3,55)¼ 0.369, P¼ 0.776). Neither did the immobility time on Day 2 as compared with Day1 (Fig. 2b; one-way ANOVA, F(3,55)¼ 1.206, P¼ 0.312), suggesting that restraint failed to induce

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2.2.

Electrophysiology

We previously reported that a forced swimming paradigm, in which mice swim for 10 min daily for 5 consecutive days, result in reduced mobility in behavior and increased cingulate cortex pyramidal cell excitability (Sun et al., 2011). Here, we examined whether excitability of cingulate cortex neurons was changed by restraint. Spikes were evoked by injecting depolarizing currents of various current intensities, and their frequencies were compared among the 4 experimental groups (Fig. 3a). No interaction was detected between the effects of the group and current intensity on the spike frequency (repeated measure ANOVA, F(27)¼ 1.275, P¼0.157). The group effect was significant (F(3)¼ 11.586, P¼0.001), indicating that neurons in the H-control group (N¼74 cells) were more excitable than those from the WTcontrol group (N¼ 49 cells; Tukey HSD test, P¼ 0.002) and that restraint increased pyramidal cell excitability in H1a KO mice (Hcontrol vs. H-restraint groups; N¼ 74 vs 20 cells, P¼0.001) but not in WT mice (WT-control vs. WT-restraint groups; N¼ 49 vs. 25 cells, P¼ 0.724). It was thus demonstrated that H1a KO mice are more vulnerable to restraint stress, which is manifested in terms of increase in cingulate cortical excitability. To examine synaptic efficiency, hippocampal slices were used. This was because the clear elicitation of fiber volley and field EPSP at the Schaffer collateral-CA1 synapse allows us to analyze the input–output relationship at the synapse in a relatively quantitative manner. The inclination of the input– output curve is represented by the regression line between the fiber volley amplitude and the slope of field excitatory postsynaptic potential (fEPSP) (Fig. 3b). The inclination was steeper in the H-restraint group (3.6470.19 ms  1, N¼94 fEPSP recordings) than in any other groups (WT-control, 2.2870.15 ms  1, N¼ 125 fEPSPs, t¼ 4.663, Po0.01, t-test with Bonferroni correction; WTrestraint, 2.1670.26 ms  1, N¼151 fEPSPs, t¼ 5.865, Po0.01; Hcontrol, 2.2870.08 ms  1, N¼ 67 fEPSPs, t¼ 4.258, Po0.01). It was thus shown that restraint increased the inclination in H1a KO mice, but not in WT mice. The relationship between stimulation strengths and fiber volley amplitudes was also examined (Fig. 4). The inclination of the regression line was compared in the same manner as

Spike frequency (Hz)

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fEPSP slope (mV/ms)

depression-like behavior in both WT and H1a KO mice. However, in absolute values, the distance traveled was longer in the H restraint group than in any of the 3 other groups (one-way ANOVA, F(3,55)¼ 7.782, P¼ 0.001; post hoc Dunnett test, P¼ 0.001 with the WT control group, P¼ 0.001 the WT restraint group, P ¼0.028 with the H control group), but was not different among the other 3 groups. Likewise, the immobility time was shorter in the H restraint group (one-way ANOVA, F(3,55)¼3.254, P¼ 0.028; post hoc Dunnett test, P¼ 0.047 with the WT control group, P¼ 0.019 with the WT restraint group, P ¼0.031 with the H control group), but was not different among the other 3 groups. The distance traveled and immobility time on Day 1, unlike those on Day 2, are considered to reflect spontaneous activity rather than depression-like tendency. It is thus shown that, in agreement with the open field result, restraint increased locomotion in H1a KO mice but not in WT mice. By contrast, locomotion in the open field, but not that in the water, was reduced by Homer1a disruption per se.

WT-Restraint H-Resraint WT-Restraint H-Restraint

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Fig. 3 – Electrophysiological changes induced by restraint. (a) Frequencies of action potentials evoked by current injection in cingulate cortex pyramidal cells. Many error bars are too short to be seen. Inset, specimen recording of an action potential train in a neuron from a WT mouse. Scale bars; 20 mV, 50 ms. (b) The input–output curve of the synaptic response. In each slice, fEPSPs were evoked by different intensities of stimulation (2.0, 2.4, 2.8, 3.2, 3.6 and 4.0 V). From each fEPSP recording, both the fiber volley amplitude and fEPSP slope were measured, and then the slope was plotted against the fiber volley amplitude. Inset, superimposition of responses evoked by 4 different intensities of stimulation in a slice obtained from a WT mouse. Note that both the fiber volley and fEPSP increase stepwise. Scale bars; 0.5 mV, 2 ms.

for the analysis of the relationship between fiber volley amplitudes and fEPSP slopes. There were no statistical differences among the 4 experimental groups (WT-control, 0.10270.013 mV/V, N¼ 125 fiber volleys; WT-restraint, 0.12570.011 mV/V, N ¼151 fiber volleys; H-control, N ¼67 fiber volleys; H-restraint, 0.09970.015 ms  1, 0.10570.013 mV/V, N¼ 94 fiber volleys; always P40.1, t-test). It was thus suggested that axonal excitability was not altered by H1a knockout and/or restraint. Finally, to exclude the possibility that behavioral tests critically affected the electrophysiological findings, we

brain research 1605 (2015) 70–75

WT-Control WT-Restraint

Fiber volley (mV)

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Stimulation intensity (V)

Fig. 4 – The input–output curve of the fiber volley activation. Fiber volley amplitudes were plotted against the intensities of stimulation (2.0, 2.4, 2.8, 3.2, 3.6 and 4.0 V). The inclination of regression lines did not statistically differ. examined the relationship between fiber volley amplitude and fEPSP slopes in separate sets of H1a-KO mice that were not subjected to the present series of behavioral tests. There were no statistical differences in the inclination of the regression line between the H-control mouse group that was subjected to the behavioral tests and that which was not (2.2070.24 ms  1, N¼19 fEPSPs, P40.1), or between the two H-restraint mouse groups with and without the behavioral tests (3.7170.39 ms  1, N¼ 15 fEPSPs, P40.1). Thus, the effects of the present behavioral tests on the electrophysiological findings, if any exists, were likely to be negligible.

3.

Discussion

The chronic imposition of stress affects behavioral, neuroendcrinological, neurophysiological and cognitive states in animals and humans (McEwen and Stellar, 1993). One of the major factors that determine the susceptibility of animals to stress is the genotype. Among the many genes that could influence susceptibility and resilience to chronic stress, we chose to analyze Homer1a, since a human genomic study has revealed that Homer1 is associated with major depression (Rietschel et al., 2010). In addition, findings from animal studies show that targeted disruption of Homer1 gene results in behavioral anomalies that have relevance to neurological and psychiatric disorders. Inoue et al. (2009) demonstrated an essential involvement of Homer 1a in fear memory formation. It was shown that mice with pan-Homer1 KO, including Homer1a and Homer1b/c, exhibit a prolonged floating behavior after repeated swimming in the forced swimming paradigm (Lominac et al., 2005). In agreement, Sun et al. (2011) showed that recovery of depression-like behavior in the forced swimming is accompanied by upregulation of Homer 1a expression. Lominac et al. (2005) further demonstrated that the virus-induced overexpression of Homer 1a, but not Homer 1c, in the prefrontal cortex (PFC) reversed the panHomer1-KO-induced floating behavior, revealing a critical role of Homer1a in adaptive responses to repeated stress exposure. The present experiments showed that Homer1arestricted KO causes hypoactivity as revealed by the open field test, which is basically consistent with the report that Homer1a expression suppresses anxiety-like behaviors

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(Lominac et al., 2005). Although Tappe and Kuner (2006) used the light–dark choice test to find that Homer1a overexpression in the striatum induces higher levels of anxiety, the forebrain including the hippocampus was devoid of Homer1a expression in the mice they used. It thus appears that the failure of Homer1a expression in the forebrain predisposes to maladaptation to repeated stress (Szumlinski et al., 2006). In this line, Ary et al. (2007) reported that a sustained expression of Homer1a is induced by chronic stress even after elimination of the stressor, suggesting that stress-induced Homer1a may have a defensive role in coping with a stressful environment. It would then be reasonable that the present Homer1aKO mice are vulnerable to very mild stress that fails to affect behavior in wild-type mice. The present H1a KO mice exhibited stress-induced increase in locomotion. Different research groups documented increase (Gronli et al., 2005; Mineur et al., 2006), decrease (Grippo et al., 2003) and no change (Harris et al., 1997) in locomotion after chronic stress. This difference is most likely attributable to the variety of the stress procedures used. The present stress paradigm, which consists of daily physical restraint only and is fairly predictable, appears to be much milder than in those other studies, and may well leave the WT mouse unaffected. In fact, D'Aquila et al. (2000) demonstrated that exploratory activity in the open field was reduced by chronic mild stress but not by milder restraint stress in WT mice. However, in the present H1a KO mice, even such an extremely mild stress induced hyper-locomotion in behavior, and cingulate pyramidal cell hyper-excitability and elevated hippocampal synaptic efficiency. These findings raise the possibility that Homer1a disruption enhances the vulnerability of mice to extremely mild chronic stress. In other words, Homer1a may stabilize the animals' reaction to chronically imposed mild stressors and, by this way, play a role in resilience to mild stress. The present experiments revealed that Homer1a disruption reduced cingulate cortex pyramidal cell excitability, which is reversed partially by chronic restraint, and that hippocampal synaptic transmission is increased by chronic restraint in H1a KO mice but not WT mice. Combined with the behavioral data, the reduced cingulate cortex excitability may be related to hypo-locomotion found in H1a KO mice. Conversely, chronic restraint stress is reported to induce disinhibition of synaptic transmission in the cingulate cortex, which accompanies neuronal hyper-excitability and behavioral hyper-locomotion (Ito et al., 2010). Generally, the cingulate cortex is implicated in locomotion in the context of attention-deficit hyperactivity disorder (Bush et al., 2005; Seidman et al., 2006; Bledsoe et al., 2013). It is thus considered that dysfunction of the cingulate cortex has intimate relevance to hyperactivity. Finally, there is a possibility that an altered hypothalamopituitary-axis (HPA) activity may relate to the hypoactivity in H1a KO mice. Seizure increases HPA axis activities, and HPA axis activity contributes to reduce seizure thresholds (Maguire and Salpekar, 2013; O'Toole et al., 2014), thus forming a positive feedback exacerbation of neuronal excitability. By contrast, we have reported that neocortical neuronal activities induce expression of Homer1a, which facilitates the BK-type potassium channels. By this way, a negative

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feedback regulation of cortical excitability is constituted (Sakagami et al., 2005). In H1a KO mice, since this Homer1amediated negative feedback regulation should be negated, the HPA axis-led positive feedback exacerbation may dominate. It is therefore expected that H1a disruption may upregulate HPA axis activity. Moreover, it is reported that sleep loss increases Homer1a expression and HPA axis activity (Maret et al., 2007). In this case also, Homer1a is considered to function as a negative regulator of neuronal hyperactivity. Overall, once Homer1a expression is disrupted, activities of neural structures including the HPA axis may well be upregulated. In summary, the present restraint paradigm induced no clear changes in anxiety- or depression-related behavior in WT mice, suggesting that the restraint stressor used here was subthreshold for inducing behavioral changes in WT mice. In H1a KO mice, however, the same restraint induced an increased ambulation in the open field test and, concomitantly, increases in both cingulate cortical excitability and hippocampal synaptic transmission, pointing to a role of Homer1a in resilience to subtle stress.

4.

Experimetnal procedures

4.1.

Animals

All experiments were performed in accordance with the guiding principles of the Physiological Society of Japan and with the approval of the Animal Care Committee of Kanazawa Medical University. C57BL/6 (wild-type; WT) and Homer1a ( / ) male mice (12–20 weeks old; 23–36 g-weight; H1a KO) were kept under an automatic day–night control (12:12 h) and allowed ad libitum access to food and water. Homer1a (  / ) mice (Inoue et al., 2009) were obtained from Dr. Inokuchi (Toyama University, Toyama, Japan). Mice of each genotype were randomly assigned to two groups: one is the control group, and the other was subjected to restraint stress as chronic mild stress (restraint group). For restraint, each mouse was immobilized for 2 h/day on 7 consecutive days in the late morning, by using a 50 ml plastic centrifuge tube with a small hole at the tip, from which ventilation was possible. The four experimental groups are referred to as the WT-control, WT-restraint, H-control, and H-restraint groups.

4.2.

Behavior

The open field test was performed in a plastic cylindrical arena (80 cm ∅) with a wall of 45 cm high. Room illumination was adjusted to about 10l  . The mice were allowed to walk around for 5 min, and the walking trajectory was analyzed off-line (SMART, Panlab s.l.u., Cornella, Spain). The bottom of the arena was virtually divided into the two concentric regions with the equal floor areas, which were called the inner and outer zones. Preference to the outer and inner zones indicates more and less anxiety-like tendency, respectively. The arena used for the light/dark test consists of two connected plastic boxes (25 wide  16 long  24 high cm3, blackcolored; 25 w  25 l  24 h cm3, white-colored; purchased from

Panlab), with an opening (7  7 cm2) that allows mice to shuttle between the two compartments. The mouse was initially located in the center of the white box. Crossing between the two boxes were checked for 5 min. Which box the mouse was located in was monitored by a gravity censor incorporated in the arena base. By this way, the time spent in the light box during the 5-min test period was reported as an index for anti-anxiety-like behavior by using software (PPCWIN, Panlab). For forced swimming, we used the test paradigm originally meant for testing rats' behavior (Porsolt et al., 1978), but not the 5-day paradigm that we previously used to induce depression-like behavior in mice (Sun et al., 2011). The mice were forced to swim for 10 min daily for 2 consecutive days (on days 1 and 2) in a transparent cylinder of acrylate (24 cm ∅, 60 cm high) filled with 25 1C water (25 cm deep). The data were analyzed by ANY-Maze (Stoelting, Wood Dale, IL, USA).

4.3.

Electrophysiology

After behavior, the mice were subjected to electrophysiological studies performed as described (Isomura et al., 1999; Sun et al., 2011). Briefly, slices of the cingulate cortex and hippocampus were made in a medium (pH 7.4; 2–5 1C) containing (in mM): NaCl 124, KCl 3.3, KH2PO4 1.3, NaHCO3 26, CaCl2 2.5, MgSO4 2.0, and glucose 10. Slices were placed in a recording chamber on the stage of an upright microscope (Eclipse E600FN, Nikon) with a 40  water immersion objective (Fluor 40  0.80 W, Nikon). For patch recording, we used patch pipettes (resistance, 4–10 MΩ) filled with a solution (pH 7.3) containing (in mM) KCl 7, K-gluconate 144, KOH 10, HEPES 10. Whole-cell recordings were made from cingulate cortex pyramidal cells that had sufficiently negative resting membrane potentials ( r 55 mV) without spontaneous action potentials. To assess membrane excitability of recorded neurons, depolarizing currents (0.05–0.5 nA for 500 ms) were injected through the patch pipette. For field potential recordings at Schaffer collateral-CA1 synapses, recording electrodes (2–5 MΩ) filled with 2.5 M NaCl were placed in the stratum radiatum and a set of bipolar tungsten electrodes was inserted nearby.

4.4.

Statistics

Data are expressed as averages7SEM. For statistics, unpaired t tests and repeated-measures or one-way ANOVA followed by t tests with Bonferroni correction, Dunnette test or Tukey's HSD (Honestly Significant Difference) test were used (SPSS, version 18; Japan IBM). The significance level was set at po0.05.

Acknowledgments We express our gratitude to Dr. K. Inokuchi (Toyama University, Toyama, Japan) for the Homer1a-KO mouse. Thanks are also due to Dr. T. Masuoka (Kanazawa Medical University, Ishikawa, Japan) for advice, and to Mr. H. Adachi, Mr S. Muramoto, Ms. Y. Hori, and Ms. K. Yamada for assistance. This work was supported by the High-Tech Research Project

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(H2010-14, H2012-15 and H2013-16) of Kanazawa Medical University, and Grants from the Japan-China Medical Association and the National Natural Science Foundation of China (81300942). Y.S. was a Japan-China Sasakawa Medical Fellow.

r e f e r e n c e s

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