Serotonin induces Arcadlin in hippocampal neurons

Journal Pre-proof Serotonin induces Arcadlin in hippocampal neurons Hidekazu Tanaka, Toshinori Sawano, Naoko Konishi, Risako Harada, Chiaki Takeuchi, Yuki Shin, Hiroko Sugiura, Jin Nakatani, Takahiro Fujimoto, Kanato Yamagata

PII:

S0304-3940(20)30053-7

DOI:

https://doi.org/10.1016/j.neulet.2020.134783

Reference:

NSL 134783

To appear in:

Neuroscience Letters

Received Date:

4 September 2019

Revised Date:

16 December 2019

Accepted Date:

21 January 2020

Please cite this article as: Tanaka H, Sawano T, Konishi N, Harada R, Takeuchi C, Shin Y, Sugiura H, Nakatani J, Fujimoto T, Yamagata K, Serotonin induces Arcadlin in hippocampal neurons, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134783

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Serotonin induces Arcadlin in hippocampal neurons Hidekazu Tanaka1, Toshinori Sawano1, Naoko Konishi1, Risako Harada1, Chiaki Takeuchi1, Yuki Shin1, Hiroko Sugiura2, Jin Nakatani1, Takahiro Fujimoto3, Kanato Yamagata2 1 Department

2 Synaptic

3

of Biomedical Sciences, College of Life Sciences, Ritsumeikan University

Plasticity Project, Tokyo Metropolitan Institute of Medical Science

Department of Pathology and Applied Neurobiology, Graduate School of Medical Science,

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Kyoto Prefectural University of Medicine Correspondence to: Hidekazu Tanaka, MD, PhD

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Department of Biomedical Sciences College of Life Sciences

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Ritsumeikan University 1-1-1 Noji-Higashi, [email protected]

Highlights

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+81-77-599-4326 (phone, fax)

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Kusatsu, Shiga 525-8577, Japan

Neural activity induces Arcadlin, which mediates the activation of p38 MAP kinase

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Dual inputs by serotonin and glutamate induce Arcadlin in hippocampal neurons

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Dual inputs by serotonin and glutamate activate p38 MAP kinase Chronic administration of fluoxetine induces Arcadlin in the hippocampus

Abstract The monoamine hypothesis does not fully explain the delayed onset of recovery after

antidepressant treatment or the mechanisms of recovery after electroconvulsive therapy (ECT). The common mechanism that operates both in ECT and monoaminergic treatment presumably involves molecules induced in both of these conditions. A spine density modulator, Arcadlin (Acad), the rat orthologue of human Protocadherin-8 (PCDH8) and of Xenopus and zebrafish Paraxial protocadherin (PAPC), is induced by both electroconvulsive seizure (ECS) and antidepressants; however, its cellular mechanism remains elusive. Here we confirm induction

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of Arcadlin upon stimulation of an N-methyl-D-aspartate (NMDA) receptor in cultured hippocampal neurons. Stimulation of an NMDA receptor also induced acute (20 min) and delayed (2 h) phosphorylation of the p38 mitogen-activated protein (MAP) kinase; the delayed

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phosphorylation was not obvious in Acad–/– neurons, suggesting that it depends on Arcadlin induction. Exposure of highly mature cultured hippocampal neurons to 1–10 M serotonin for 4

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hours resulted in Arcadlin induction and p38 MAP kinase phosphorylation. Co-application of

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the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (APV) completely blocked Arcadlin induction and p38 MAP kinase phosphorylation. Finally, administration of

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antidepressant fluoxetine in mice for 16 days induced Arcadlin expression in the hippocampus. Our data indicate that the Arcadlin-p38 MAP kinase pathway is a candidate neural network modulator that is activated in hippocampal neurons under the dual regulation of serotonin and

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glutamate and, hence, may play a role in antidepressant therapies.

Keywords:

serotonin,

protocadherin,

mitogen-activated protein kinase

Abbreviations Acad, Arcadlin

adhesion,

synaptic

plasticity,

antidepressant,

APV, D-(-)-2-amino-5-phosphonopentanoic acid DIV, days in vitro DMSO, dimethyl sulfoxide DUSP-1, dual-specificity phosphatase-1 E18, embryonic day 18 ECS, electroconvulsive seizure

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ECT, electroconvulsive therapy GABA, gamma aminobutyric acid GI, glycine/bicuculline/strychnine

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HEPES, 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic Acid

IBMX, 3-isobutyl-1-methylxanthine

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LTP, long-term potentiation

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5-HT, 5-hydroxytryptamine, or serotonin

MAP kinase, mitogen-activated protein kinase;

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mPFC, medial prefrontal cortex MEK3, MAP kinase kinase 3

MKP-1, mitogen-activated protein kinase phosphatase-1 NMDA, N-methyl-D-aspartate

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P0, postnatal day 0

PAPC, Paraxial protocadherin PBS, phosphate buffered saline PFA, paraformaldehyde SSRI, selective serotonin reuptake inhibitor TAO2, thousand-and-one kinase 2

1. Introduction The hypofunction of monoamine neurons has long been the hypothetical cause of depression, known as the monoamine hypothesis. Prolongation of monoamine half-life with selective serotonin reuptake inhibitors (SSRI) or other antidepressants ameliorates depression symptoms. However, the monoamine hypothesis does not fully explain the delayed onset of recovery after antidepressant treatments or the mechanisms of recovery after electroconvulsive

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therapy (ECT), ketamine, and scopolamine [2, 12]. Serotonin and noradrenaline neurons abundantly project their axons to the limbic system, including the hippocampus and the medial

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prefrontal cortex (mPFC). Recent studies indicate that depression and its treatment modifies neuronal dendritic morphology and spine density in the mPFC, the hippocampus, and the

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amygdala, and the volume of these brain regions [7, 8, 13, 15, 21, 25].

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The common mechanism that operates both in ECT and monoaminergic treatment presumably involves molecules induced under these conditions. Electroconvulsive seizure

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(ECS) mimics ECT in rodents and is widely used to investigate the underlying mechanism of ECT. Several screening studies have identified the array of molecules that are induced in the hippocampus by ECS [5, 14, 17, 20, 23, 26-30]. Among these ECS-induced molecules, Arcadlin/Paraxial Protocadherin (PAPC)/Protocadherin-8, a non-clustered protocadherin cell

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adhesion protein, is also listed in the array of chronic fluoxetine (an antidepressant)-induced molecules (GEO profile GDS2803/1417051_at). Arcadlin has been identified as one of the effector immediate-early genes induced in

the hippocampus by ECS or tetanic stimulation [28]. Arcadlin is a shorter version of the human orthologue Protocadherin-8 (PCDH8), lacking a 97 amino acid stretch found in the cytoplasmic region of the longer version. The cytoplasmic domain of Arcadlin binds to a splice variant of

thousand-and-one kinase 2 (TAO2), known as a mitogen-activated protein kinase kinase kinase (MAPKKK) [32]. Homophilic dimerization of Arcadlin at its extracellular domain triggers the phosphorylation of p38 MAP kinase through TAO2 and MAPKK MEK3 [32]. The phosphorylated p38 MAP kinase reciprocally phosphorylates the regulatory domain of TAO2, resulting in endocytosis of Arcadlin itself [32]. Arcadlin binds laterally to N-cadherin, an abundant synaptic cell adhesion protein, and enhances its co-endocytosis. Overexpression and

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loss of function of Arcadlin leads to a reduction and an increase, respectively in dendritic spine density of cultured hippocampal neurons [32].

Arcadlin is induced in cultured hippocampal neurons in response to bath-applied

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glutamate and the mixture of forskolin and 3-isobutyl-1-methylxanthine (IBMX) [32]. These

protocol in hippocampal slices [16].

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conditions are similar to the glycine-induced (GI) chemical long-term potentiation (cLTP)

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Although Arcadlin is induced by both ECS and antidepressants, its cellular mechanism remains elusive. Here, we first confirm induction of Arcadlin and phosphorylation

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of p38 MAP kinase upon the stimulation of an N-methyl-D-aspartate (NMDA) receptor in cultured hippocampal neurons. We then examined whether stimulation of serotonin receptors on hippocampal neurons influences the induction of Arcadlin and the phosphorylation of p38 MAP kinase. Finally, we describe the profile of Arcadlin expression in response to chronic

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treatments using the antidepressant fluoxetine. Our findings indicate an involvement of the Arcadlin-p38 MAP kinase pathway in recovery from depression through both antidepressant and ECT.

2. Material and methods 2.1.

Animals and antidepressant treatments

Seven-week-old C57BL/6J male mice were purchased from SLC (Hamamatsu, Japan) and allowed to habituate to the animal facility under controlled conditions (21–23 ℃, 12 h light/dark cycle) for 1 week. Fluoxetine and its vehicle control dimethyl sulfoxide (DMSO) were diluted with saline and administered through the intraperitoneal injection (solution corresponding to the 1% of body weight [v/w]). All animal experiments were conducted in accordance with the guidelines and laws of the Japanese government and approved by the

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Animal Care Committee of Ritsumeikan University, Biwako-Kusatsu Campus.

Neuronal cultures

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Neuronal cultures were performed as previously described [19]. Neurons dissected from E18 Sprague-Dawley rats (SLC, Hamamatsu, Japan) or from wild type and Acad–/–- P0

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C57BL/6J mice [31] hippocampi were plated at a density of 2.1 x 104 cells/cm2 on poly-L-lysine

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coated 6-well plates (well diameter: 35 mm) and cultured in Neurobasal Medium supplemented with B-27 (Life Technologies, Carlsbad, CA, USA). After being maintained in culture for 19-60

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days, neurons were treated by bath applications of agonists/antagonists for 20 min–7 hours. Bath applications of reagents were performed by the addition of the 100–10,000 times stock solutions dissolved in H2O or DMSO. Final concentrations were as follows: serotonin-HCl (1–10 M), -Me-5HT (20 M), 8-OH-DPAT (20 M), WAY-100635 (100 M), ketanserin (20 M),

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D-(-)-2-amino-5-phosphonopentanoic acid (APV, 100 M). GI was applied to neurons as previously described [18]. Neurons were briefly washed with 2 mL of GI-wash solution (5 mM HEPES-NaOH [pH 7.4], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 33 mM glucose) and then stimulated with 1 ml of the GI solution (0.2 mM glycine, 0.02 mM bicuculline, 0.003 mM strychnine added to GI-wash) for 15 min. Stimulated neurons were further cultured in the conditioned medium supplemented with 100 M APV.

2.3.

Immunostaining Mice were transcardially perfused with phosphate buffered saline (PBS) followed by

4% paraformaldehyde (PFA). Brains were then isolated and post-fixed in PFA overnight followed by sectioning with a vibratome to obtain 25 m-thick coronal slices. After washing with PBS, slices were treated with microwave heating (200 W x 10 sec, repeated five times

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between cooling periods of 20 sec) in a 10 mM citrate buffer (pH 6.0) for antigen retrieval. Slices were incubated in a Blocking Solution (BL; 0.1% Triton X-100, 5% normal goat serum in PBS) for 5 min and then incubated with primary antibodies diluted in BL overnight (4 ℃), followed by

Western blot

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2.4.

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secondary antibodies for 1 h (room temperature).

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Hippocampi were dissected immediately after euthanasia and homogenized in a RIPA buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA-NaOH, 1% Nonidet-P40, 0.5%

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sodium deoxy cholate, 0.1% sodium dodecyl sulfate, 0.2 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin A, 20 mM NaF, 20 mM -glycerophosphate, 1 mM Na3VO4) (five times their volume) using a Teflon-glass homogenizer (15 strokes). Samples were centrifuged for 15 min at 15,000 x g at 4 ℃. After checking the protein

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concentrations of the resultant supernatants by BCA assay, 20 g of each sample were loaded in a 7% SDS-PAGE gel followed by transfer into nitrocellulose membranes for detection with antibodies.

2.5.

Antibodies The following antibodies were used in this study: anti-Arcadlin/Protocadherin-8

antibody (Abnova, H00005100-M01, Taipei, Taiwan; 1:400 dilution), anti-phospho-p38 MAP kinase, and anti-p38 MAP kinase antibodies (Cell Signaling Technology Japan, #9211/#8690, Tokyo, Japan; 1:1000 dilution), anti--Actin antibodies (Medical & Biological Laboratories, PM053, Aichi, Japan; 1:1000 dilution; Cell Signaling Technology, #4970; 1:5000 dilution), HRP-conjugated

secondary

antibodies

(MP

Biomedicals

Japan,

Tokyo,

Japan),

and

Alexa-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific, Tokyo,

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Japan). Productions and purifications of anti-Arcadlin and anti-Arc antibodies were previously described [11, 28].

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3. Results

3.1. Delayed phosphorylation of p38 MAP kinase depends on Arcadlin induction

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Arcadlin has been described as an immediate early gene induced in hippocampus by

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ECS [28]. The induction has been reproduced in rat cultured hippocampal neurons treated with glutamate, depolarization, or the mixture of forskolin and IBMX [32], suggesting that the

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induction is mediated by calcium influx and the production of cAMP. Here, GI [16] was found to induce Arcadlin (Fig. 1A). GI allow the NMDA receptors to open upon spontaneous neural activity by lowering the [Mg2+]o and blocking GABA receptors. We adopted GI as a mild NMDA receptor-stimulating protocol, because direct bath-application of NMDA was

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sometimes excitotoxic to our neurons. Incubation for 225 min after GI-stimulation (15 min) significantly induced Arcadlin. This induction was not observed in the presence of an NMDA receptor antagonist APV (Fig. 1A). The intracellular domain of Arcadlin binds to TAO2. Homophilic interaction of Arcadlin extracellular domains triggers the phosphorylation of p38 MAP kinase, a downstream target of TAO2 [32]. Although there may be a relationship between the overexpressed

Arcadlin and the phosphorylated p38 MAP kinase, other signals should also trigger phosphorylation. In fact, simple NMDA receptor activation by GI led to strong phosphorylation at 20 min when Arcadlin still remained at the basal level (Fig. 1B). This initial phase of p38 MAP kinase activation is supposed to be triggered by the preexisting signaling pathways downstream of the NMDA receptor. The initial phase of phosphorylation was ceased 1 hour later, and then upregulated again after 2 hours when Arcadlin was induced (Fig. 1B).

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To investigate whether the induced Arcadlin influences the latter phase (2–4 hours after the stimulation) of p38 MAP kinase phosphorylation, we compared the phosphorylation levels after GI administration between wild-type and Acad–/– neurons (Figs. 1C and 1D).

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Phosphorylation at 20 min after GI administration was observed both in wild-type and Acad–/– neurons to the same extent. By contrast, phosphorylation at 4 hours after GI administration was

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clearly observed in wild-type neurons, but was not obvious in Acad–/– neurons. The data suggest

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that the latter phase of p38 MAP kinase phosphorylation depends on the induction of Arcadlin. A typical neural activity marker Arc [14] was induced 4 hours (but not in the first 20 min) after

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GI administration in both WT and Acad–/– neurons indicating that the neurons were stimulated to the same extent (Fig. 1C).

3.2. Serotonin induces Arcadlin in cultured hippocampal neurons

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In addition to the glutamatergic input, the hippocampus also receives monoaminergic

projections including serotonin neurons. These inputs, through various types of 5-HT receptors, modify the activity of hippocampal neurons in combination with inputs through other receptors such as glutamate receptors. An initial administration of serotonin onto cultured hippocampal neurons (21 days in vitro [div]) failed to induce Arcadlin significantly (data not provided). However, the exposure of highly mature neurons (40–60 div) to 1–10 M serotonin

for 4 hours resulted in the induction of Arcadlin (Fig. 2A). Arc was also induced by these treatments indicating that neural activity was enhanced by serotonin (Fig. 2A). Sustained induction of Arcadlin was observed 4–7 hours after the stimulation (data not presented).

3.3. Serotonin-induced Arcadlin expression requires coactivation of an NMDA receptor To identify the type of serotonin receptors responsible for the induction of Arcadlin,

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we utilized several agonists and antagonists of serotonin receptors. The 5-HT2A/2C receptor agonist -methyl 5-hydroxytryptamine (-Me-5HT) or the 5-HT1A receptor agonist 8-OH-DPAT seemed to induce Arcadlin, but the extent of the induction was not significant (Fig. 2B). In

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addition, the 5-HT1A receptor antagonist WAY-100635, or the 5-HT2 receptor antagonist ketanserin did not block the induction completely (data not presented). It is, therefore, possible

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is required for Arcadlin induction.

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that the combination of different classes of 5-HT receptors, including other unknown subtypes,

In the present study, the hippocampal neurons are consisted largely of glutamatergic

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pyramidal neurons and approximately 6% of GABAergic interneurons [1]. In this culture system, neurons are spontaneously active and excrete certain amounts of glutamate [22]. The bath application of serotonin in these cultures, therefore, results in the co-stimulation of serotonin and glutamate receptors. Application of the NMDA receptor antagonist APV

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completely blocked induction of Arcadlin by serotonin (Figs. 2A and 2B). Activation of an NMDA receptor is, therefore, the prerequisite for Arcadlin-induction, and activation of serotonin receptors may lower the gating threshold of the NMDA receptor. Induction of Arc was similarly blocked with the co-application of APV (Figs. 2A and 2D). Serotonin 5-HT2A/2C and 5-HT1A receptor agonists, -Me-5HTand 8-OH-DPAT, respectively, induced Arc significantly (Fig. 2D). These agonists had similar trends in the induction of Arcadlin, although

they were not significant (Fig. 2B). Arcadlin and Arc, therefore, seem to share the overlapping gene induction pathway through the dual input through serotonin and NMDA receptors.

3.4. Serotonin induces the phosphorylation of p38 MAP kinase To understand whether the serotonin-induced Arcadlin has any influence on the downstream signal of Arcadlin, we investigated the phosphorylation level of p38 MAP kinase.

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The conditions that led to Arcadlin induction resulted in an enhancement of p38 MAP kinase phosphorylation (Fig. 2C). Phosphorylation of p38 MAP kinase 4 hours after the application of serotonin and -Me-5HT was significantly higher than in the control (Fig. 2C); this was

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completely blocked under the presence of APV (Fig. 2C). Similar to the case of Arcadlin induction, activation of an NMDA receptor may also be the prerequisite for the signal upstream

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of the p38 MAP kinase. The involvement of the induced Arcadlin in the phosphorylation of p38

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MAP kinase in response to serotonin could not be determined. Cultured hippocampal neurons from wild-type and Acad–/– mice at 40–60 div were not healthy enough to evaluate

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serotonin-induced Arcadlin expression, which, however, were detectable using rat neurons.

3.5. Chronic fluoxetine induces Arcadlin in mouse hippocampus The above observations indicate that the sustained elevation of extracellular serotonin

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leads to the induction of Arcadlin in hippocampal neurons. The in vivo administration of fluoxetine, a SSRI, is supposed to mimic this extracellular condition. To test this hypothesis, mice were intraperitoneally administered with 25 mg/kg (body weight) of fluoxetine daily for up to 21 days until they were euthanized to obtain bilateral hippocampi, which were subjected to the western blot. As a positive control, mice were subjected to ECS administered transcranially. Chronic administration of fluoxetine for 16 days induced Arcadlin in the

hippocampus (Fig. 3A). This induction was not observed when a shorter period of treatment, such as 14 days, was applied (Fig. 3A). However, a higher dose of daily fluoxetine (35−40 mg/kg) was sufficient to induce Arcadlin during a 14-day treatment (Fig. 3B). Conversely, the smaller dose (20 mg/kg) was sufficient to induce Arcadlin under the longer treatment protocol for 21 days (Fig. 3C). The chronically induced Arcadlin during an 18-day fluoxetine treatment (25 mg/kg)

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remained at a high level for more than 4 days, whereas the electrically induced Arcadlin reverted to background levels within 1 day (Figs. 3D and 3E). The acutely induced Arcadlin largely localized to the soma of dentate granule cells and CA1-3 pyramidal cells. The Arcadlin

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also showed weak expression in apical dendrites of CA1 pyramidal cells. By contrast, chronically induced Arcadlin was distributed in granule cell dendrites within the molecular

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layer of the dentate gyrus, in puncta of CA3 stratum lucidum, and in apical dendrites of CA1

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4. Discussion

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pyramidal cells within the stratum radiatum (Fig. 3F).

ECT/ECS and recently nominated antidepressants beyond the monoamine hypothesis, such as ketamine and scopolamine, provide novel insight into the pathophysiology of depression. The common target of different classes of treatments includes the limbic system and

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the mPFC. In fact, volumes and neural cell structures in these regions are modified during depression and after its recovery [7, 8, 13, 15, 21, 25]. On dendrites of hippocampal neurons, in particular, spine density is suppressed by stress and recovered by antidepressants [8]. Here, Arcadlin was induced in the hippocampus by both ECS and SSRI (Fig. 3). In addition, the induction of Arcadlin in cultured hippocampal neurons required dual input through serotonin and NMDA receptors (Fig. 2). In this case, the stimulation of an NMDA

receptor was essential for the induction, and the serotonin signal potentiated it. It seems that the monoaminergic antidepressants potentiate the glutamate-induced activity of limbic neurons resulting in an effect similar to that caused by ECS. Thus, Arcadlin may be involved in the antidepressant mechanism that operates both in monoaminergic and non-monoaminergic pathways. As Arcadlin is involved in the remodeling of dendritic spines, the induced Arcadlin

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may yield the dendritic remodeling in the antidepressant-treated brain. In cultured hippocampal neurons, Arcadlin has been demonstrated to downregulate spine density [32]. Theoretically, therefore, the serotonin-induced Arcadlin may not mediate the upregulation of

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spine density in response to antidepressants, but rather suppress spine density, thus playing a homeostatic role.

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The cytoplasmic domain of Arcadlin binds to TAO2, a splice variant of TAO2 () [32].

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TAO2 is a MAPKKK located upstream of p38 MAP kinase [3, 4]. The homophilic dimerization of Arcadlin at its extracellular domain in fact triggers phosphorylation of p38 MAP kinase

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through TAO2 and MEK3 [32]. Although it is obvious that variety of cell signals mediate the phosphorylation of p38 MAP kinase, the overexpressed Arcadlin may also potentiate this phosphorylation, especially in the delayed phase after neural stimulation when Arcadlin is upregulated (Fig. 1). In addition to the enhanced endocytosis [32], a variety of cellular

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responses triggered by the phosphorylated p38 MAP kinase should be responsible for the mechanisms underlying antidepressant. In fact, a negative regulator of MAP kinases, mitogen-activated protein kinase phosphatase-1 (MKP-1, or dual-specificity phosphatase-1, DUSP-1) is increased in the hippocampus of depressive patients [9]. It is, therefore, possible that enhancement of the p38 MAP kinase pathway due to the sustained overexpression of Arcadlin could counteract the MKP-1/DUSP-1 pathway and thus exert an alleviating activity.

The discrepancy between the latencies of Arcadlin induction in the brain (16 days) and cultured neurons (2-4 hours) remains an open question. Daily injections of fluoxetine induce repeated activation of limbic neurons, probably resulting in the remodeling of the neural network such as spinogenesis, which, in turn, may elevate the network activity and drive the biogenesis of the Arcadlin gene product. Many lines of evidence indicate that neural activity increases the complexity of neural networks. For example, seizures caused by abnormal activity

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result in axon sprouting [6, 24]. LTP-inducing stimuli trigger the protrusion of novel spines [10]. Chronic antidepressants may induce long-lasting remodeling of the architecture and function of neural networks, which may elevate the basal activity resulting in a delayed overexpression of

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Arcadlin and enhanced signaling of the antidepressant MAP kinase pathway.

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5. Conclusion

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The Arcadlin-p38 MAP kinase system is a candidate neural network modulator activated in hippocampal neurons under the dual regulation of serotonin and glutamate

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signals; therefore, it may play a role in a common antidepressant mechanism. On one hand, its morphological outcome, demonstrated by sparse spines may play a homeostatic role in normalizing the excess complexity of neural networks due to the hyperactivity of the treated brain. On the other hand, the enhanced p38 MAP kinase signal may alleviate the hyperactivity

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of MKP-1/DUSP-1 resulting in an antidepressant activity.

Author contribution H.T. and K.Y. designed experiments and wrote the paper. H.T. and T.S. performed western blots.

R.H.,

J.N.,

Y.S.,

and

C.T.

performed

drug

treatment

of

animals

and

immunohistochemistry. H.T. and N.K. performed neuronal culture. H.S., K.Y., and T.F.

produced antisera.

Acknowledgements We thank Eddy M. De Robertis for the PAPC/Acad–/– mice. This work was supported by the MEXT-supported program for the strategic research foundation at private universities,

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AMED (grant number JP18ek0109311 (K.Y.)) and JSPS KAKENHIs (grant numbers JP 23590300 (H.T.), JP 24659093 (K.Y.), and JP 25293239 (K.Y.)). The authors declare no competing financial interests. H.T. and K.Y. designed experiments and wrote the paper. H.T. and T.S. performed

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western blots. R.H., J.N., Y.S., and C.T. performed drug treatment of animals and immunohistochemistry. H.T. and N.K. performed neuronal culture. H.S., K.Y., and T.F.

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produced antisera.

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Figure Legends Figure 1. Delayed phosphorylation of p38 MAP kinase depends on the induction of Arcadlin. (A) Rat hippocampal neurons (21 div) were not treated (Control), treated with 100 M APV for 240 min (APV), treated with GI for 15 min and further cultured for 225 min in the presence of APV (GI), or treated with GI + APV for 15 min and further cultured for 225 min in the presence of APV (GI + APV), followed by a western blot. Relative Acad band intensities are shown

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(Mean ± S.E.M.). (B-D) Hippocampal neurons (19–20 div) from wild-type and Acad–/– mice were treated with GI for 15 min followed by further incubation for 5–225 min in the presence of APV before a western blot analysis. (B) A representative blot of phosphorylated and total p38 MAP

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kinase in neurons prepared from wild-type mice. Relative pp38 band intensities are shown (Mean ± S.E.M.). (C) A representative blot of phosphorylated and total p38 MAP kinase and Arc

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in neurons prepared from wild-type (left) and Acad–/– (right) mice. (D) Relative pp38 band

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intensities of wild-type and Acad–/–- mouse neurons 225 min after a 15 min period of GI-treatment. Mean ± S.E.M. The numbers of culture dishes are shown above/in each bar (n). *

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p < 0.05; ** p < 0.01; *** p < 0.005; one-way ANOVA followed by Bonferroni’s multiple

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comparison test or Games-Howell posthoc test.

ro of -p re lP ur na Jo Figure 2. Stimulation of serotonin receptors induces Arcadlin in cultured hippocampal

neurons. (A) Rat hippocampal neurons (43 div) were bath-applied with serotonin (5HT) with or without APV and cultured for 4 hours followed by a western blot. Acad, Arcadlin; pp38, phosphorylated p38 MAP kinase; p38, total p38 MAP kinase; Ncad, N-cadherin. (B-D) Rat hippocampal neurons (40–60 div) were bath-applied with vehicle (C, control), APV, 10 M serotonin (5HT), 10 M -Me-5HT (aMe), 20 M 8-OH-DPAT (dpat), and the mixture of

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serotonin and APV (5HT-APV). Neurons were then cultured for 4 hours before the western blot analysis. The relative intensities of desired bands were quantified compared to the control band in each gel. (B) Arcadlin, (C) phosphorylated p38 MAP kinase (gray column) and total p38 MAP

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kinase (open column), (D) Arc. Mean ± S.E.M. The numbers of culture dishes are shown in/above each bar (n). * p < 0.05; ** p < 0.01; one-way ANOVA followed by Games-Howell

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posthoc test.

ro of -p re lP ur na Jo Figure 3. Chronic administration of fluoxetine induces Arcadlin in mouse hippocampus.

(A) Eight-week-old male C57BL/6J mice (WT or Acad–/–) were injected (i.p.) daily with 25 mg/kg fluoxetine during the indicated days followed by a western blot of hippocampal extracts. A band below the full length Arcadlin in ECS lane is a degradation product of Arcadlin. (B) Mice were injected with 10–40 mg/kg fluoxetine for 14 days before being subjected to a western blot analysis. (C) Mice were injected with 20–30 mg/kg of fluoxetine for 21 days and then subjected to a western blot analysis. (D) Mice were injected with 25 mg/kg of fluoxetine for 18

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days, and then maintained without drug administration for the indicated days before the hippocampal sampling for the western blot. (E) Mice were ECS-treated and sacrificed after the indicated hours when the hippocampal sampling for western blot analysis took place. (F) Mice

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were perfused 4 hours after ECS (top), daily injection of fluoxetine for 18 days (middle), or without pretreatment (bottom), and their brains were isolated for sectioning and

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immunostaining using an anti-Arcadlin antibody. GC, granule cells; PC, pyramidal cells.

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Arrows indicate projection or punctate staining of dendrites. Scale bar = 30 m. (A-F) Representative results of at least three experiments (using more than three mice each) are

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shown.

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