総説北里医学 2013; 43: 95-104

パーキンソン病に対する外科治療
佐藤

澄人，隈部

俊宏

北里大学医学部脳神経外科学
パーキンソン病に対する外科治療は長い歴史を持ち，薬物療法と相補的関係にある。外科的治療のよい適応は，長期間のL-ドパ療法によるwearing offやドパ誘発性ジスキネジアなどの運動合併症，薬物抵抗性の振戦である。主として淡蒼球や視床下核の脳深部刺激療法 (deep brain stimulation:
DBS) が行われ，術前にL-ドパ療法の効果が保たれていることが重要である。術後に運動機能の著明な改善が認められる一方，認知・精神機能に対する影響も考慮する必要がある。長期経過において振戦に対する効果は維持されるが，体軸症状の悪化が問題となる。DBSの作用機序は現在なお明らかでなく，病態生理の解明とともに今後の研究が待たれる。
Key words: パーキンソン病，視床凝固術，脳深部刺激療法，大脳基底核

どについて概説する。

はじめに
パーキンソン病 (Parkinson's disease) は，1918年に英国のJames Parkinsonによって報告1されて以来，病因の解明や治療法の進歩において他の神経変性疾患と比べ際立っている。黒質緻密部のドパミンニューロンの変性・脱落 (Trétiakoff，1921年2) に始まり，Lewy小体の発見 (Lewy，1912年3) やその進展様式 (Braak，2003年4)が明らかにされた。また，MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine) を用いた実験的パーキンソニ
ズムにより病態生理の研究が進み，原因遺伝子も次々発見されてきている。治療法においても1960年代にLドパ療法が開発され，予後は著しく改善された。さらにドパミンアゴニスト，末梢性カテコラミン分解酵素阻害薬，モノアミン酸化酵素B阻害薬，アデノシン
A2A受容体阻害薬などさまざまな治療薬が開発されつつある。今後はiPS細胞などの再生医療の分野に期待が持たれている。
一方，外科的治療に目を転じると，L-ドパ療法に先立つ1950年代より定位脳手術が行わるようになり，有効な治療法がなかった暗黒の時代に光明が差し込んだ5,6。その後，微小電極記録による神経生理学的手法の導入 (Albe-Fessardら，1963年7) ，MRI誘導によるターゲットの設定，神経科学的知見の蓄積による神経回路や病態生理の解明，脳深部刺激療法 (deep brain
stimulation: DBS) などのエレクトロニクスの進歩により，さらに精度の高い理論的な手術が可能となった。
本稿ではパーキンソン病の外科治療における歴史，適応，手術法，現在考えられているDBSのメカニズムな外科的治療の歴史的概観8-11
振戦や筋固縮に対する外科治療の創始期は，まさに試行錯誤の連続であったと思われる。錐体路の遮断を目的とした一次運動野の切除 (Bucy，1939年12)，固有
感覚入力の遮断のための後外側コルドトミー(Putnam，1933年13)，錐体外路系に対する侵襲として大脳脚切截術 (Walker，1949年14) などが試みられたが，必然的に重度の運動麻痺を生じる結果となった。
Meyersは尾状核頭部や淡蒼球などの皮質下構造物に目を向け，レンズ核ワナの遮断が振戦や筋固縮に効果があることを1942年に報告15した。しかし，定位的方法
ではなく半球間裂および側脳室を経由するアプローチのため，侵襲の大きな手術法であった。Cooper 11 は
Walkerの大脳脚切截術を行う際，意図せず前脈絡叢動
脈を閉塞させてしまった。ところが，術後に筋固縮や振戦が改善しているのに気づき，前脈絡叢動脈の結紮術を試みるようになった。その後，ターゲットを淡蒼球に修正し，1955年にフリーハンドでアルコールを注入する淡蒼球破壊術 (chemopallidectomy) を報告した。
定位脳手術 (stereotactic surgery) の歴史は，1908年にHorsleyとClarke16によって動物実験用の装置が考案され，1947年にSpiegelとWycis17によって初めて臨床応用された。定位脳手術とは，皮質下の神経核や神経路に限局した破壊巣を作成することを目的とし，立体的な位置と方向を定め，機械的な誘導によって正確にアプ

Received 29 November 2013, accepted 3 December 2013
連絡先: 佐藤澄人 (北里大学医学部脳神経外科学)〒252-0374 神奈川県相模原市南区北里1-15-1
E-mail: sumito@med.kitasato-u.ac.jp

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ローチする手法である。最小限の侵襲で最大限の効果を図る発想で，現在の低侵襲外科 (minimally invasive
surgery) に通じるものと言える。この定位的方法により脳深部の手術は安全なものとなり，レンズ核ワナの凝固術 (ansotomy) が施行された (SpiegelとWycis，1954
年18)。時を同じくして，本邦でも楢林らが独自の定位
脳手術装置を開発し，1952年にパーキンソン病に対して淡蒼球手術を行った19。最初は効果を確かめるために塩酸プロカイン水溶液を注入し，次いで効果を持続させるために塩酸プロカイン油性懸濁質を注入した。
振戦及び筋固縮は消失し，運動麻痺や感覚障害などは出現せず，この効果は約40日間持続した。その後振戦は再燃してきたが，筋固縮の軽減は持続した20。HasslerとRiechertは淡蒼球内節の投射先である視床の破壊も有効であろうと考え，1952年にventrolateral thalamotomyを行ったところ，筋固縮のみならず振戦に対しても有効であった。
1960年代より，L-ドパ療法がパーキンソン病治療の主役となるが，L-ドパを長期間続けることにより様々な副作用が生じることが次第に明らかとなった。1992
年，LaitinenらはLeksellによる淡蒼球後腹側部の破壊がパーキンソン症状に効果があったとの報告 (Svennilson
ら，1960年21) から後腹側淡蒼球破壊術 (posteroventral
pallidotomy) の有効性を報告した22。さらに同じ頃，
Benabidらが破壊術に代わる安全で効果的な方法として，視床23,24や視床下核 (subthalamic nucleus: STN)25の高頻度刺激療法，すなわちDBSを導入した。これら外科治療が薬物治療を補う治療戦略として再び注目されるようになった。

い。オフ症状としてのすくみは改善が期待できるが，オン状態のときにみられるすくみはDBSでも治療が困難である27。オフ状態L-ドパ治療に反応が乏しい場合には手術の効果は期待できず，多系統萎縮症や進行性
核上性麻痺などのパーキンソン症候群においても手術適応はない。手術は十分な薬物療法の後に考慮されるべきものであるが，年齢としては70歳以下で行うことが望ましい。手術時の年齢が若い方が良好な経過 2 8で，後述する認知・精神機能への影響も少ないと考えられる。また，比較的若年で社会的・家庭的に活動度が高いほど，高い満足度と社会への還元効果が得ら
れ，医療経済的にも有利29といえる。最近，比較的早期のパーキンソン病患者に対するDBSの有用性を検証するための多施設共同無作為試験 (EARLYSTIM試験)が行われた30。対象は罹患期間4年以上，早期運動合併症がみられる症例で，STN-DBS + 内科的治療群124例(平均年齢52.9歳，平均罹病期間7.3年)，内科的治療単独群127例 (52.2歳，7.7年) が割り付けられた。2年間の評価において，前者が運動症状，ADL，L-ドパ誘発性
運動合併症の改善効果が優れていた。今後はDBSが比較的早期に治療の選択枝に挙げる必要があるかもしれない。
その他，副作用によって十分な薬物療法が行えない
場合31や，薬物療法に抵抗性の振戦症状に対しては，時期を問わず外科的治療の適応となる。薬物の副作用によって生じている幻覚はDBS後に減薬することで改善され得るが，もともと幻覚などの精神疾患を合併している場合は悪化する恐れがあるため除外する。Table
1に外科治療の適応32を示す。

手術適応および時期

PDの病態生理とDBSのメカニズム

L-ドパ療法を長期間継続すると，薬効が一定せず，症状の日内変動 (motor fluctuation) や，ドパ誘発性ジス
キネジアが生じるようになる。これらを運動合併症(motor complication) という。運動症状の日内変動には，L-ドパの薬効時間の短縮するwearing-off現象，突然効果が切れて動けなり，また再び動けるようになる
オン・オフ現象などがある。ドパ誘発性ジスキネジア
はオン時，最もL-ドパ濃度が高まっているときに生じるpeak-doseジスキネジアと，オフからオンあるいはオンからオフに切り替わるタイミングで生じるdiphasicジ
スキネジアとに大別される。Wearing-offの頻度はさま
ざま報告されているが，L-ドパ治療開始後5年で50%程
度26と考えられる。
DBSはこのようなwearing-offとジスキネジアに対して有効な治療法である。オフ状態がHoehn-Yahr重症度
で3度以上が手術を勧める時期であるが，手術による改善効果は薬物療法における最良のオン状態を越えるものではなく，オン状態が3度以下であることが望まし

1989年から1990年にかけて，Albinら33やDeLongら34,35のグループによって大脳基底核の直接路・間接路モデルが提唱され，大脳基底核の神経回路やパーキンソン病をはじめとする大脳基底核疾患の病態生理の理解は大きく進歩した36 (Figure 1A，1B)。大脳基底核の出力部である淡蒼球内節 (internal segment of the globus pallidus:
GPi) および黒質網様部 (substantia nigra pars reticulate:
Table 1
・長期L-ドパ療法による運動合併症 (wearing-off現象、ドパ
誘発性ジスキネジアなど)
・薬物抵抗性の重度の振戦・副作用 (嘔気，アレルギー，幻覚などの精神症状) により薬物治療が困難・L-ドパ療法の反応性が保たれている (オン状態でHoehnYahr重症度3度以下)
・一般的には70歳以下で重度の合併症がない
・既存の精神疾患がない
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SNr) はGABA作動性の抑制性ニューロンで，高頻度で連続して発射しているため，投射先の視床はふだん抑制された状態にある。大脳皮質からの入力によって線
条体 (striatum) ニューロンが興奮すると，線条体の投射ニューロンはGABA作動性の抑制性ニューロンなので，GPi/SNrニューロンが脱抑制され (直接路)，投射
先のニューロンが興奮することにより必要な運動が起こる。一方，淡蒼球外節 (external segment of the globus
pallidus: GPe)，STNを経由してGPi/SNrニューロンを興奮させ (間接路)，直接路と逆の作用をもたらす。直接路は意図した運動を脱抑制によって発現させ，間接路は不要な運動を抑制することにより，運動のSN比を上げていると考えられている37 (focused selection)。
DBSの臨床応用は，1950年代にHeathら38が精神疾患の患者に対して実験的に始められた。報酬系について調べられ，相反する疼痛に対する治療の道を開いた39。
運動障害に対するDBSは，1987年にBenabidら23,24がthalamotomyに代わる治療法としてVim-DBSを試みたのが始まりである。1990年にBergmanら40はMPTPによる
パーキンソン病モデルのサルが，一側の視床下核の破壊によって対側肢の無動，筋固縮，振戦などの運動症状が改善することを見出した。この知見をもとにBenabidらのグループ25が1995年，パーキンソン病患者に両側STN-DBSを開始したところ，著明な効果が得ら
れ広く普及するに至った。MPTPは自家製麻薬の副産物として生成され，それを含む麻薬を注射するとパーキンソニズムが発生する (Davisら，1979年41) ことから，実験的パーキンソニズムに利用されるようになった。MPTPは脳内でモノアミン酸化酵素によって毒性の強いMPP+に変換され，黒質線条体ニューロンに選択的に取り込まれる。MPTPを投与されたサルは，
パーキンソン病と似た無動や寡動，前屈姿勢，筋固縮，振戦 (一部の種) が出現する35。
DBSの作用機序については，現在のところまだ明らかでない。DBSが破壊術と同様の効果を示すことか
ら，高頻度連続刺激が局所のニューロン活動をブロッA

B

Figure 1. Schematic diagram of the basal ganglia-thalamocortical circuitry under (A) normal and (B) parkinsonian conditions (modified
from references 25, 53)
Inhibitory connections are shown as black arrows, excitatory connections are gray arrows. Parkinsonism leads to differential changes inthe two striatopallidal projetions, which are indicated by the thickness of the connecting arrows. Basal ganglia output to the thalamus isincreased.
CM, centromedian nucleus; GPe, external segment of the globus pallidus; GPi, internal segment of the globus pallidus; PPN,
pedunculopontine nucleus; SNr, substantia nigra, pars reticulate; SNc, substantia nigra, pars compacta; STN, subthalamic nucleus; VA,
motor thalamus ventralis anterior; VL, ventrolateral thalamus
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振戦に対しては視床腹中間核 (ventrointermedius: Vim)が著効を示す (Table 2)。STNとGPiを比較した多数例の無作為比較試験48-50では，総じて運動機能の改善はほぼ互角ないしSTNの方が薬剤の減量やDBSの刺激強度が小さくて済む分優位ととらえられている。しかし，
STN-DBS後の減薬はうつやアパシー (apathy) を悪化させる51ことが指摘されている。ドパミン補充療法は運動障害改善に必要な黒質線条体系に作用するだけでな
く，中脳辺縁系や中脳皮質系にも作用するため，減薬によりこれら非運動症状が悪化する可能性がある52。
そのため，われわれはSTN-DBS後の減薬は控えるようにしている。
STN-DBSによる認知機能の問題は41%に生じたとの報告53もあり，遂行機能障害 (executive dysfunction)54，
言語流暢性 (verbal fluency) の低下55が指摘されている。
STN-DBSがGPi-DBSと比較して認知・精神機能に影響を与えやすい原因としては，感覚運動系 (sensorimotor)・連合系 (associative)・辺縁系 (limbic) の皮質−大脳基底核連関において，STNが最もコンパクトに情報が集まっている56ためと考えられる。体積で比較すると，クするという説 (抑制仮説) がある。これには，①脱分
極ブロックによるもの，②抑制性入力線維や抑制性介在ニューロンが刺激されることによるもの，③入力線
維が逆行性に刺激され，標的ニューロンがブロックされたと同じ効果による，などの可能性が挙げられる42。
抑制仮説は，パーキンソン病が黒質緻密部のドパミン
ニューロンが変性・脱落することにより，線条体の直接路のニューロンの活動が減弱，間接路のニューロンの活動が亢進し，結果的にSTNおよびGPiのニューロン
活動の亢進，すなわち平均発射頻度の上昇すること (発射頻度仮説) と論理が一致している (Figure 1B)。しかし，振戦やドパ誘発性ジスキネジアに対する効果は説明できない。
一方，高頻度連続刺激は局所のニューロンを興奮させる考え方 (興奮仮説) もある。STNからGPiに投射するニューロンは興奮性 (グルタミン作動性) であり，
MPTPによるサルのパーキンソン病モデルにおいて，STNの高頻度刺激によりGPiの発射頻度が増加し，パーキンソン症状が回復したとの報告43がある。興奮仮説
はパーキンソン病の発射頻度仮説では説明できないた
め，発射パターンを規則的に変化させることにより病態を改善させていると考えられている (発射パターン仮説) 。すなわち，パーキンソン病では大脳基底核のニューロン活動が発振・共振するために情報処理が阻害され，そのような発振が視床を介して大脳皮質に伝達されるため，諸症状が起きると考えられる44-47。これは，正常では大脳基底核内の情報処理が独立して行われているが，ドパミンが不足すると互いに独立性が失われ情報の混信が起きることを示唆している42,44。高頻度連続刺激は局所のニューロンを刺激に同期して興奮させることにより，このような発振や共振を強制的に脱同期させ，症状を改善すると考えられる42,43。

ターゲットの選択
パーキンソン病に対する定位脳手術のターゲット
は，STN，GPi，視床 (特に腹中間核)，脚橋被蓋核(pedunculopontine nucleus: PPN) である (Figure 2)。現在のところ，主として選ばれるのはSTNとGPiで，いずれもパーキンソン病の主要運動症状に広く有効である。

Figure 2. Stereotactic targets for Parkinson's diseaseThal Vim, thalamic ventrointermedius nucleus; STN,
subthalamic nucleus; GPi, internal segment of the globus
pallidus; PPN, pedunclopontine nucleus

Table 2. Efficacy of each target

ThalamusGPi
STN

Tremor

Rigiity

Akinesia

Gait posturedisturbance

Wearing off

Dyskinesia

Drug reduction

○
○
○

△
○
○

×
○
○

×
○
○

×
○
○

×
○
○

×
○
×

○: effective, △: partially effective, ×: not effective
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大脳基底核の入力部である尾状核は4,316 mm3，被殻は5,625 mm3，介在部である淡蒼球外節は808 mm3，視床下核は158 mm3，出力部である淡蒼球内節は478 mm3，
黒質網様部は412 mm3である57。すなわち，視床下核は体積が淡蒼球内節の約1/3で細胞密度も高く，感覚運動系ループのみならず，連合系や辺縁系ループも近接しており，DBSによる刺激がそれらに波及しやすいと推測される。したがって，運動機能への効果と同時に認知・精神機能への作用も大きいと考えられる。
PPNは橋中脳被蓋尾側に位置しており，大脳基底核や運動調節に関連する他の脳領域と密接に関連している 5 8 。除脳ネコやラットで電気刺激すると歩行動作(locomotion) が誘発される59,60ため，PPNは中脳歩行動
作領域 (mesencephalon locomotor region，MLR) の一部と考えられ，S T N やG P i では改善できないすくみ(freezing) や歩行障害などの体軸症状に対しての効果が期待されるようになった。2005年よりPPN-DBSがPDの患者に試みられ始め61，年々報告例が増加しているものの，S T N - D B S との併用効果を述べた論文 6 2 や，
UPDRSスコアでは有意な改善が得られなかったとの報
告63など，まだ一定した評価が得られていないのが現状である64。その背景には，PPNは脳幹網様体に属する細胞集団で境界が不鮮明であること58,65，解剖学的用語の混乱66，種による違い67などが考えられる。除脳ネコ
やラットのPPNで確認されている歩行制御などの証拠
はサルではまだ確認されておらず65，また四足歩行の動物と二足歩行であるヒトでは歩行のメカニズムが異なることが推測される67。現在までのところ，本邦でのPPN-DBSの報告例はみられていない。
まとめると，比較的若年で認知・精神機能に問題がなく，薬物の減量を期待したいときはSTN-DBSを，反対に高齢で術後の認知機能低下が危惧されるような症

Figure 3. Target selection

Figure 4. Number of each surgical procedure in our institute
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い。そのため，微小電極記録による神経生理学的な検証が必要となる。先端が極めて細く，電極のインピー
ダンスが数メグオームの微小電極 (microelectrode) と，それに比べて先端がやや太くインピーダンスが数十〜
百キロオーム程の準微小電極 (semi-microelectrode) とがあり，前者は単一ニューロン活動の記録 (single-unit
recording)，後者は複数のニューロン発射や集合電位の記録 (multi-unit recording) に用いる。
DBS電極の留置は原則的に局所麻酔下で行う。覚醒下での脳深部神経活動の記録と臨床所見の観察が可能
で，術中に試験刺激による効果や副作用を確認できる利点がある。しかし，両側の電極を同じ日に留置する
場合は手術時間が長くなり，患者の身体的・精神的苦痛が少なくない。不穏をきたした際には，頭部にフレームを装着している状態での過鎮静は気道確保の点で危険を伴う。最近，全身麻酔下でも微小電極もしく
は準微小電極記録による神経核の同定は可能で，良好な長期成績が得られたという報告69,70もあり，当施設でも患者の希望に応じて全身麻酔下でのDBS電極留置を行っている。
DBSシステムは，電極リードと完全埋設型刺激発生装置 (implantable pulse generator: IPG)，両者を接続するケーブルで構成される (Figure 5A，5B)。電極リード
は直径1.27 mmで長さ1.5 mmの電極が1.5 mmもしくは0.5 mm間隔で4個連なっている。IPGは心臓ペースメー
カーと似た装置で，リチウム電池を内蔵し電池の寿命は通常4〜5年である。一般的には低電圧 (数V以下)，
持続時間の短い (数十μs)，高頻度 (100 Hz以上) の刺激が用いられ，1 mA程度の電流が流れるが，電流密度

例にはGPi-DBSの方が安全といえる。ジスキネジアやジストニアが目立つ場合はGPi-DBSを，振戦が主症状であればVimをターゲットに選択する。当施設での術式の選択基準 (Figure 3) と，これまで行った手術術式(Figure 4) を示す。2011年よりGPi-DBSがやや増加しているのが分かるが，DBS療法を開始した当初はSTNDBSのみ行っており，術後に認知機能の低下をきたした症例を経験した。そこで，STN-DBSを行った場合に術後の認知機能に悪影響を及ぼすことが懸念される症例に対し，GPi-DBSを行ったところ良好な結果が得られたため，以後積極的にGPi-DBSを取り入れるようになった。Vimをターゲットにする際はDBSではなく凝固術 (thalamotomy) を行っているが，Vim-DBSとVimthalamotomyは同等に有効であり68，インフォームドコ
ンセントにおいて患者がデバイスを留置しない凝固術を選択したためである。

手術の方法
定位脳手術用フレームを患者の頭部に装着し，フレームを基準にした位置情報を与えるインジケータを取り付けてMRIを撮影する。得られた画像データをプ
ランニング・ソフトウェアに取り込み，仮標的(tentative target) を設定する。仮標的とはアトラスや画像所見から計測する位置で，標準アトラスから求められる座標と画像所見に基づく解剖学的な個人差とをすり合わせて決定される。実際の手術では，MRI画
像の歪みや様々な過程で生じる僅かな誤差の積み重ねにより，必ずしも意図した結果が得られるとは限らな

A

B

Figure 5. System for deep brain stimulation composed of (A) electrode leads and (B) implanted stimulators.
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3. Vim-DBS or thalamotomy
ヒトの視床の亜核分類法はHassler75やHiraiとJones79によるものがあるが，定位脳手術で広く用いられている
Schaltenbrandらのアトラス80では前者による用語が記載されている。Vimは視床腹外側核群で淡蒼球内節から入力を受ける腹吻側核 (Vo，ventrooralis) の後方，表在感覚の入力を受ける腹尾側核 (Vc，ventrocaudalis) の前方に位置し，小脳核からの入力と深部感覚の入力を受けている81-83。深部感覚の入力を受けるニューロンはVim核の最外側に存在し，微小電極記録を行うと，四肢関節の受動運動に対する反応 (kinesthetic response)84
や振戦リズムに一致した群過放電 (tremor-frequency
rhythmic discharge) が観察される。それらの位置の凝固もしくはDBSにより振戦は直ちに抑制される。

は低い。持続時間が短く電流密度が低いため，ニューロンの細胞体よりは軸索，しかも太い軸索が刺激されやすい傾向にあるとされる議論もある42,71。刺激のパラ
メータはプログラマーを用いて体外から調整を行うことができる。この可逆性，調節性がDBS療法の最大の利点である。
1. GPi-DBS
Laitinenら22がパーキンソン症状に対する有効性を指摘したGPi後腹側部は，線条体の運動領域から投射を受ける淡蒼球尾側の腹側2/3の部位と一致しており，これらは淡蒼球の運動領域で体部位局在をもっている。
すなわち，腹側から背側に向かって，口腔顔面，上肢，下肢の領域が存在する72。これら運動系ループに属する運動淡蒼球 (motor pallidum) が治療のターゲットとなる。サルを使った研究73では，運動淡蒼球の腹外側部は一次運動野および固有補足運動野の関連領域
で，視床への投射線維はレンズ束 (Forel H2野) となり，
STNの背側を内側に向かいForel H野に到達し，視床束
Forel H1野) となり，視床VLo核 (Olszewski74によるサルの亜核分類で，Hassler75によるヒトのVoa核に相当)に終止する。一方，運動淡蒼球の背内側部は運動前野
および前補足運動野に関連しており，視床投射線維は腹側に向かいレンズ核ワナを形成し，視床VApc核(同74,75，ヒトのLpo核) に終止する。
GPi-DBSにおいて，刺激電極の位置によって臨床効
果が異なることが報告されている。すなわち，GPiの腹側部刺激では，筋固縮やドパ誘発性ジスキネジアが改善するが，逆に歩行や無動が増悪し，背側部刺激ではオフ時の無動，歩行障害，筋固縮が改善するが，刺激強度を上げるにつれてドパ誘発性ジスキネジアが誘発された76。上記のようなGPiの腹側と背側とで異なった投射先であることとの関連が示唆される。

合併症85
定位脳手術における脳内出血合併症の発生率は，諸報告を総合して0〜5.0%であり，症候性の出血は0〜2.0%である。脳内出血を起こす危険因子として，高血圧の既往，高齢であることが指摘されている。また，
微小電極記録の穿刺回数は必要最小限にすべきであろう。DBSシステムの機器に関連する合併症は，2006年
以降の報告では，4.0〜9.7%で，頭蓋内電極の迷入・脱落，電極・延長コードの断線，コード・刺激装置埋没部の皮膚潰瘍，感染，アレルギー性皮膚反応，刺激装置の故障などが報告されている。

長期効果
STN-DBS，GPi-DBSともに3〜6年にわたり有効性が維持されていることが報告されている86,87。効果の持続は症状によって異なり，STN-DBSの8〜10年の経過では良好な効果が持続されるのは振戦で，すくみや姿勢
反射障害といった体軸症状は効果が低下しやすい傾向にあった88,89。しかし，これは効果が減弱するという意
味だけではなく，病期の進行に追いつかなくなったともいえる。DBS療法はパーキンソン病の進行を抑える
効果はないが，DBSをオフにすると症状は増悪するのでDBSの効果自体は持続していると考えられる。ちな
みに，淡蒼球内節破壊術 (pallidotomy) はパーキンソン病の運動症状全般に有効であるが，その効果の持続時
間は2年程度90である。

2. STN-DBS
STNはその名の通り視床の下に位置し，外側に内包，背側にレンズ束 (Forrel H2野)，腹側には黒質に囲まれた158 mm3程の小さな核である。STNの背側部が運動系ループに属している感覚運動領域で，DBS電極の留置に適した部位である。このうち，外側部は一次運動野から体部位局在的な入力を受けており，外側から内側にかけて口腔顔面，上肢，下肢の領域である。感覚運動領域の内側部は高次運動野から入力を受けており，体部位局在は一次運動野からのものと鏡像関係になっている72。感覚運動領域の腹側には眼球運動に関連した領域，さらに腹側には前頭前野系ループに属する領域，腹内側部には辺縁系ループに属する領域が存在する56,77,78。

今後の展望エレクトロニクスの進歩により，デバイスの性能は向上してきている。刺激条件の設定はこれまで定電圧式であったが，定電流式も可能になり有効性が示されている 9 1 。パーキンソン病患者の通常の使用条件で
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8. Gildenberg PL, Krauss JK. History of Stereotactic Surgery. In:
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nucleus. Lancet 1991; 337: 403-6.
25. Limousin P, Pollak P, Benazzouz A, et al. Effect of parkinsoniansigns and symptoms of bilateral subthalamic nucleus stimulation.
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は，バッテリーの寿命は4〜5年程度である。その度にIPGの交換が必要となるが，最近は充電式のもの92も利用できるようになった。現在のところ頻回の充電が必要であるが，今後の改良が期待される。MRIは原則禁忌であるが，進行期パーキンソン病の患者は腰痛など
整形外科疾患が併存することも多く，MRI撮影ができないことは診療上不利である。この分野で先行する心臓ペースメーカーではMRI耐性のデバイスが既に開発されているので，近い将来解決されるものと思われる。現在のDBSシステムは，設定したパラメータの電気刺激を脳の局所に一方的に与えるだけであるが，センシング機能を持った心臓ペースメーカーのように，脳の神経活動を記録しながらそれに合わせて刺激を調整するclosed-loop DBSシステム93が開発されつつある。

おわりに不随意運動疾患に対する定位脳手術などは機能的脳神経外科 (functional neurosurgery) と呼ばれる，神経組
織自体に手を加えて，その機能を変化させることにより，目的とする臨床効果を引き出す治療概念である。
最近は，ニューロモデュレーション (neuromodulation)という，神経組織に微弱な電気や薬剤を流すことにより神経活動を調節する治療法としてとらえられ，医学と工学技術が融合した最先端治療としてさらなる進歩が期待されている。また，神経科学の基礎的な研究とも密接に関連しており，基礎医学と臨床医学が連携して発展させていく必要がある。

文

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Surgical treatment for Parkinson's disease
Sumito Sato, Toshihiro Kumabe
Department of Neurosurgery, Kitasato University School of Medicine
Surgery is complementary to medical treatment in patients with Parkinson's disease (PD). The chief surgicalindications are drug-resistant severe tremor and motor complications such as wearing-off of drug effectivenessand dyskinesia due to the prolonged administration of levodopa. Whether the subthalamic nucleus (STN) or
the pallidum should be the main target of deep brain stimulation (DBS) remains controversial. While inpatients treated by STN-DBS the administrated dose of antiparkinsonian medications can be reduced, it tends
to cause a cognitive decline and/or emotional changes. Patients with tremor-dominant PD may be goodcandidates for ventrointermedius thalamotomy or DBS. The pedunculopontine nucleus has become a newtarget for axial symptoms of PD. Although surgical treatment cannot stop the progression of PD, DBS iseffective for longer than 6 years. Animal studies have contributed to an understanding of basal ganglia
function and the pathophysiology of PD; however, more studies are warranted to clarify the precise mechanismof DBS.
Key words: Parkinson's disease, thalamotomy, deep brain stimulation, basal ganglia

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Contents lists available at ScienceDirect

Neuroscience Research
journal homepage: www.elsevier.com/locate/neures

The NR2B antagonist, ifenprodil, corrects the l-DOPA-induced deﬁcit
of bilateral movement and reduces c-Fos expression in thesubthalamic nucleus of hemiparkinsonian rats
Masakazu Igarashi a,∗ , Toshiya Habata a,b , Hisanao Akita a,c , Kazuko Noda c ,Masanori Ogata c , Makoto Saji a,caDivision of Brain Science, Graduate School of Medical Sciences, Kitasato University, Sagamihara, Japan
Department of Occupational Therapy, School of Allied Health Sciences, Kitasato University, Sagamihara, Japanc
Department of Physiology, School of Allied Health Sciences, Kitasato University, Sagamihara, Japanba r t i c l e

i n f oArticle history:
Received 20 September 2014
Received in revised form 26 January 2015
Accepted 6 February 2015
Available online 16 February 2015
Keywords:
Parkinson’s disease
Cylinder test
l-DOPA
Striatum
Subthalamic nucleus
Bilateral movementa b s t r a c t
The use of NR2B antagonists in Parkinsonism is still controversial. To examine their anti-parkinsonian
effects, the NR2B antagonist, ifenprodil, and l-DOPA were administered together and separately in hemiparkinsonian rats (hemi-PD) that were subjected to a cylinder test. Recovery from hypoactivity wasachieved by single administration of 3–7 mg/kg of l-DOPA; however, improvement in the deﬁcit ofbilateral forelimb use was not observed. When administered alone, ifenprodil had no anti-parkinsonian
effects; however, combined administration of ifenprodil and 7 mg/kg of l-DOPA signiﬁcantly reversed thedeﬁcit of bilateral forelimb use without adversely affecting the l-DOPA-induced improvement in motor
activity. Next, in order to identify the brain area inﬂuenced by l-DOPA and ifenprodil, quantitative analysis
of l-DOPA-induced c-Fos immunoreactivity was performed in various brain areas of hemi-PD following
administration of l-DOPA with and without ifenprodil. Among brain areas with robust c-Fos expression
within the motor loop circuit in dopamine-depleted hemispheres, co-administered ifenprodil markedly
attenuated l-DOPA-induced c-Fos expression in only the subthalamic nucleus (STN), suggesting that the
STN is the primary target for the anti-parkinsonian action of NR2B antagonists.
© 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction
l-Dihydroxyphenylalanine (l-DOPA), the precursor of
dopamine, is the most effective drug for the treatment of Parkinson’s disease (PD). Dopamine-replacement therapy with l-DOPA
produces remarkable recovery from motor deﬁcits in patientswith PD during its initial period; however, in most PD patientsand in animal models of PD, chronic treatment with l-DOPA
leads to the development of uncontrollable movement, known as
l-DOPA-induced dyskinesia (LID). Therefore, non-dopaminergic
treatments of PD have been investigated in order to avoid development of LID. It has been reported that N-methyl-d-aspartate∗ Corresponding author at: Division of Brain Science, Graduate School of Medical
Sciences, Kitasato University, 1-15-1 Kitasato Minami-ku, Sagamihara, Kanagawa,Japan. Tel.: +81 42 778 8153.
E-mail addresses: mi07511r@gmail.com (M. Igarashi),habata@kitasato-u.ac.jp (T. Habata), akita310@ahs.kitasato-u.ac.jp (H. Akita),
noda1219@ahs.kitasato-u.ac.jp (K. Noda), masa422@ahs.kitasato-u.ac.jp (M. Ogata),sajim@ahs.kitasato-u.ac.jp (M. Saji).

(NMDA) receptor antagonists improve motor symptoms of PD(Merello et al., 1999; Chase et al., 2000; Putzki et al., 2002).
However, NMDA receptor antagonists could potentially cause
problems such as mental illness, because NMDA antagonists block
all subtypes of NMDA receptors distributed throughout almost
the entire brain. NMDA receptors are heterotetramers consistingof two NR1 subunits and two NR2 subunits. NR2 subunits are
categorized into A–D subtypes (Erreger et al., 2007; Yuan et al.,2009). In rodents, the striatum is enriched with NR2B (Chen andReiner, 1996; Standaert et al., 1999), and NR2B-bearing NMDA
receptors are also distributed in the subthalamic nucleus (STN), theentopeduncular nucleus (EP), and the cerebral cortex (Standaert
et al., 1994; Sgambato-Faure and Cenci, 2012). Consequently,
it is probable that NR2B-selective antagonists are candidates
for treatment of PD minimizing undesirable side effects. In fact,
anti-parkinsonian effects of NR2B antagonists have been studied
for the last couple of decades. Monotherapy with NR2B antagonists
or combined administration of NR2B antagonists and l-DOPA
have been somewhat encouraging (Steece-Collier et al., 2000;
Nash et al., 2000, 2004; Nash and Brotchie, 2002; Löschmann

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0168-0102/© 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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M. Igarashi et al. / Neuroscience Research 96 (2015) 45–53

et al., 2004), whereas no anti-parkinsonian effect was observedin clinical studies with administration of single NR2B antagonists(Addy et al., 2009). Thus, ameliorative effects of NR2B antagonists
on motor symptoms of PD are still controversial. It is also unknown
whether systemically administered NR2B antagonists speciﬁcallyaffect neurons in restricted regions within motor circuits, such as
the striatum, the STN, or the motor cortex, in order to improve themotor symptoms of PD.
Since there are various functional differences between the glutamate system and the dopamine system, it is unlikely that NR2B
antagonists have anti-parkinsonian effects equivalent to those ofl-DOPA. We hypothesized that NR2B antagonists potentiate antiparkinsonian effects of l-DOPA or affect particular symptoms of
PD. In the present study, we ﬁrst compared the monotherapeutic
effect of l-DOPA on motor deﬁcits of hemiparkinsonian rats (hemiPD) with that of the NR2B antagonist, ifenprodil, using a cylinder
test. Secondly, we used the cylinder test to evaluate how ifenprodil inﬂuences l-DOPA effects when l-DOPA and ifenprodil wereco-administered. Following behavioral experiments, quantitative
analysis of neurons showing c-Fos expression induced by l-DOPA,
with or without co-administered ifenprodil, was performed in various brain areas, in order to identify the area that is responsible
for the anti-parkinsonian effect of ifenprodil in the basal gangliathalamo-cortical loop for motor regulation.
2. Materials and methods
2.1. Animals
Male Hannover Wistar rats (CLEA Japan Inc., Japan) weighing
220–250 g were used in this study. Rats were housed in clear plastic
cages in groups of 2–3 under a 12 h light/dark cycle with constant temperature (25 ◦ C) and humidity (50%), and were allowed
free access to food and water throughout the experiments. All
experiments were approved by the Kitasato University Institutional
Animal Care and Use Committee and were conducted in accordancewith the rules of the National Research Council for the Care and Useof Laboratory Animals.
2.2. Preparation of hemiparkinsonian rats
Under anesthesia with pentobarbital sodium (40 mg/kg, i.p.,
Abbott Lab., USA), rats received a unilateral, stereotaxic injection of
8 g of 6-OHDA (2 g/L, Sigma, USA) dissolved in 0.1% l-ascorbic
acid/saline into the lateral sector of the medial forebrain bundle(MFB). A glass micropipette (tip size: 30 m), made from a disposable 20 L micropipette (Drummond, USA) and connected to an
air pressure system with polyethylene tubing, was used for intraMFB injection. Stereotaxic coordinates of the MFB were as follows:AP, 2.1 mm caudal to the bregma; L, 2.0 mm right of the midline;
V, 8.0 mm ventral to the dural surface, according to Akita et al.
(2010). Thirty minutes prior to surgery, all rats were pretreatedwith desipramine HCl (15 mg/kg, i.p., Sigma, USA) to preserve noradrenergic neurons.
2.3. Rotation test
Three weeks after 6-OHDA injection into the MFB, anapomorphine-induced rotation test was performed to evaluate theseverity of dopamine depletion in hemi-PD. The behavior of rats
placed in a white square box (50 cm × 50 cm × 50 cm), was recorded
using a CCD camera. Prior to the i.p. injection of apomorphine(1 mg/kg, i.p., Sigma, USA), rats were habituated to the square box
for 15 min. Thirty minutes after drug administration, rat behavior was recorded for 15 min test periods. The number of turning
behaviors, a rotation of more than 180◦ (a half turn), was used as

an index of the severity of apomorphine-induced rotation behavior.
6-OHDA-injected rats demonstrating more than 100 rotations per
15 min test period were used for behavioral investigations (Ogura
et al., 2005).
2.4. Cylinder test
Two weeks after the rotation test, the cylinder test was performed to evaluate the ameliorative effect of l-DOPA and/or
ifenprodil on motor deﬁcits of hemi-PD. Rats were placed in atransparent cylinder (20 cm diameter; 30 cm height), and their
exploratory behavior in the cylinder was recorded from beneathfor 15 min using a CCD camera. The test was performed in lateafternoon in order to induce rats to explore. Rats normally movealong the wall of the cylinder, rearing and touching the wall. In
the wall-touching behavior, rats show various types of forelimb
movement: single, consecutive, and simultaneous touches. Walltouches with the forepaws can be classiﬁed into three behaviors:(1) ipsilateral forelimb use, where rats touch the wall with only
the forepaw on the same side as the injection, (2) contralateralforelimb use, in which rats touch the wall with the forepaw on
the opposite side from the injection (parkinsonian paw) independently, and (3) ambidextrous forelimb use, deﬁned as consecutive
touches in which an ipsilateral touch followed a contralateral touchor vice versa, or simultaneous touches, with both forepaws. If
rats returned to a tetrapodal stance between wall contacts, thosetouches were not scored as “consecutive touches” and did not countas ambidextrous forelimb use. The total number of wall touches(TWT) per 15 min was estimated by summing ipsilateral, contralateral, and ambidextrous touches. The rate of forelimb use (RFU) was
expressed by three parameters: the ratio of ipsilateral touches to
TWT; the ratio of contralateral touches to TWT; and the percent of
ambidextrous forelimb use.
2.5. Drug administration and treatment groups
Thirty minutes prior to the cylinder test, l-DOPA was administered i.p. to hemi-PD rats, with or without ifenprodil. Hemi-PD
rats were divided into four treatment groups: 1. l-DOPA, hemiPD rats administered various doses of l-DOPA (1 mg/kg, 3 mg/kg,
5 mg/kg, or 7 mg/kg, Sigma, USA); 2. ifenprodil, hemi-PD rats,
administered various doses of ifenprodil (0.01 mg/kg, 0.1 mg/kg, or
1 mg/kg, Sigma, USA); 3. l-DOPA + ifenprodil, hemi-PD rats administered 3 mg/kg or 7 mg/kg l-DOPA with various doses of ifenprodil(0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg); 4. vehicle, hemi-PD rats administered a mixture of l-DOPA and ifenprodil solvents only. All
animals in treatment groups 1 and 3 were given a single dose ofl-DOPA because repeated administration induces behavioral sanitization, called “priming” (Di Chiara et al., 1992; Nadjar et al., 2009),
where the brain becomes sensitized to subsequent administrationsof l-DOPA. The solvent for l-DOPA was 0.1 N HCl diluted with 0.1 M
phosphate buffer (pH 6.4), adjusted with 1 N NaOH, while thatfor ifenprodil was 0.1% L-ascorbic acid/saline. To prevent decarboxylation of l-DOPA, various doses of l-DOPA (1 mg/kg, 3 mg/kg,
5 mg/kg or 7 mg/kg) were mixed with benserazide (2.5 mg/kg,
Wako, Japan), dissolved in 0.1% l-ascorbic acid/saline just before
administration.
2.6. c-Fos immunohistochemistry
Two h after administration of the drug (70 min after the cylinder test), rats were deeply anesthetized with ethyl carbamate(1.4 g/kg, i.p.) and perfused transcardially with 200 mL of heparinsaline followed by 200 mL of 4% paraformaldehyde buffered witha 0.1 M phosphate buffer (pH 7.4). Removed brains were postﬁxed
for 1–2 h in perfusate and saturated overnight in 30% sucrose145

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for cryoprotection. Coronal 40 m sections were cut on a sliding
microtome and sampled every 80 m throughout the striatum, themotor cortex, and the subthalamic nucleus, and were stored in a
cryoprotectant solution (20 mM phosphate buffer containing 30%
ethylene glycol and 30% glycerin) at −20 ◦ C. Immunohistochemical
staining with anti-c-Fos rabbit polyclonal antibody (sc-52, Santa
Cruz, USA) was performed using the avidin-biotin peroxidase
technique (ABC system). Sections were ﬁrst incubated in 50 mM
phosphate-buffered saline (PBS) containing 0.3% H2 O2 to preventthe reaction with endogenous peroxidase, then in a blocking
solution of PBS containing 3% goat serum and 0.25% Triton X-100,
and ﬁnally in 50 mM PBS containing 1:2000 anti-c-Fos antibody
and 0.25% Triton X-100 overnight at 4 ◦ C. After washing with
PBS, sections were incubated in 50 mM PBS containing 1:400
biotinylated anti-rabbit-IgG antibody and 0.25% Triton X-100 and
subsequently exposed to the 1:100 ABC reagent (ABC Kit, Vector
Lab., USA) diluted in 50 mM PBS containing 0.5% Triton X-100. To
visualize antibody-positive neurons, sections were incubated with
0.05% diaminobenzidine and 0.015% H2 O2 for 5 min, and then were
mounted on gelatin coated slides.
2.7. Quantitative analysis of c-Fos expression
Coronal sections immunostained with c-Fos antibody were chosen from the M1 and M2 motor cortex (3.70 mm rostral to thebregma), the striatum (0.0 mm to the bregma), and the subthalamic
nucleus (STN) (3.60 mm caudal to the bregma). Digitized photomicrographs of the coronal section, showing robust c-Fos expression,
were analyzed with automated imaging software (cellSens, Olympus, Tokyo). Robust c-Fos expression in the three regions in thebasal ganglia-thalamo-cortical loop for motor regulation was analyzed quantitatively using imaging software as follows: the motor
cortex, the striatum, or the STN where the area densely packed
with neurons containing c-Fos-positive nucleus is outlined, andimmunoreactive particles as c-Fos-positive nuclei within the outlined area were automatically counted with the software if it
showed stain density beyond a threshold level. Subsequently, thedensity (cells/mm2 ) of c-Fos-positive neurons in each region was
estimated.
2.8. Statistical analysis
All values indicated in the ﬁgures were expressed asmeans ± S.E.M. RFU scores were compared with one-way ANOVA,
followed by Dunnett’s multiple comparisons test for more than
two groups, or Student’s t-test for two groups, if signiﬁcant. TWT
scores and c-Fos quantitative scores were compared with the
Steel multiple-comparisons test for more than two groups or theMann–Whitney U test for two groups. Statistical signiﬁcance wasset at P < 0.05 for all statistical analyses.
3. Results
3.1. Abnormalities of forelimb use in hemi-PD during the cylinder
test
In order to deﬁne motor deﬁcits of hemi-PD, abnormalities offorelimb use were evaluated against normal forelimb use of control
rats (Fig. 1). Controls actively moved along the wall of the cylinder,
touching it with forepaws, alternately or simultaneously, with
TWT of 145.6 ± 33.0 per 20 min. Forelimb use was predominantly
ambidextrous (74.3 ± 1.8%), with some ipsilateral (12.3 ± 1.1%)
and contralateral forelimb use (13.5 ± 2.0%). In contrast, hemi-PD
rats moved less actively along the wall, with TWT of 21.8 ± 9.4 per
20 min. Meanwhile, the RFU consisted of decreased ambidextrous
forelimb use (20.1 ± 10.3%), dominant ipsilateral forelimb use

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Fig. 1. Hemi-parkinsonian (hemi-PD) rats display a marked deﬁcit of ambidextrous
and contralateral forelimb use. Hemi-parkinsonian (n = 10) and control rats (n = 8)
were subjected to a cylinder test. (A) The bar indicates the average total wall touches(TWT) per 20 min. A marked reduction of TWT was observed in hemi-PD. (B) Bars
indicate the mean RFU, the ratio of ipsilateral and contralateral forelimb use. HemiPD rats showed a signiﬁcant increase in ipsilateral forelimb use and a signiﬁcantdecrease in contralateral forelimb use and ambidextrous forelimb use, comparedwith control animals. **P < 0.01.

(79.2 ± 5.2%), and a few contralateral forepaw touches (0.6 ± 0.5%).
RFU data indicate that hemi-PD rats have a serious disability inregard to contralateral forelimb use (parkinsonian paw); therefore
they tend to use their ipsilateral forelimb instead of their parkinsonian paw. Furthermore, signiﬁcant loss of ambidexterity wasobserved.
3.2. Effect of various doses of l-DOPA on abnormal forelimb use
in hemi-PD
Single l-DOPA doses of 3 mg/kg, 5 mg/kg or 7 mg/kg signiﬁcantly increased TWT of hemi-PD rats in a dose-dependent manner(Fig. 2A). In particular, complete reversal of hypoactivity wasobserved when 7 mg/kg l-DOPA (151.9 ± 45.6) were administered. In contrast to the TWT results, no signiﬁcant increase ofambidextrous forelimb use was observed with any l-DOPA dose,compared with PD/veh. While reduced contralateral forelimb use in
PD/veh (0.2 ± 0.2%) was signiﬁcantly increased in a dose-dependent
manner, predominant use of the ipsilateral forelimb in PD/veh(84.9 ± 9.8%) was reversed. Thus, the imbalance of forelimb use due
to predominant use of the ipsilateral forelimb in PD/veh was converted to a different imbalance by dominant use of the contralateralforelimb, while ambidextrous forelimb use remained unchanged.
3.3. No amelioration by various doses of ifenprodil of motor
deﬁcits in hemi-PD
No signiﬁcant recovery from reduced TWT of PD/veh wasobserved when various single doses of ifenprodil were administered alone (Fig. 3A). In addition, there was no signiﬁcant change
in the ipsilateral dominance of RFU for any dose of ifenprodil,
compared with PD/veh (Fig. 3B). Consequently, single doses of ifenprodil demonstrated no ameliorative effect on either reduced TWT
or imbalanced RFU in hemi-PD.
3.4. Effect of combined administration of 3 mg/kg l-DOPA with
ifenprodil
To investigate whether ifenprodil potentiates the effect of
3 mg/kg of l-DOPA, a dose that by itself did not have signiﬁcanteffects on either TWT or RFU (Fig. 2), co-administration of ifenprodil and 3 mg/kg l-DOPA was performed. Co-administration of
3 mg/kg l-DOPA and ifenprodil slightly reduced the ameliorative
effect of 3 mg/kg of l-DOPA on TWT (Fig. 4A). When combined with
3 mg/kg l-DOPA, neither 0.01 mg nor 1 mg of ifenprodil changed
the effect of l-DOPA on RFU, while 0.1 mg of ifenprodil shifted the48

M. Igarashi et al. / Neuroscience Research 96 (2015) 45–53

Fig. 2. Various doses of l-DOPA fail to completely reverse deﬁcits of ambidextrous forelimb use in hemi-PD rats. Various i.p. doses of l-DOPA were tested. Treatment groups
were control, untreated controls (n = 8); PD/veh (n = 7), hemi-PD rats treated with vehicle; and PD/l-DOPA, hemi-PD rats treated with various doses of l-DOPA (1 mg/kg,
n = 6; 3 mg/kg, n = 7; 5 mg/kg, n = 7; or 7 mg/kg, n = 12). (A) Effect of various doses of l-DOPA on TWT. TWT in hemi-PD was signiﬁcantly increased when treated with 3 mg/kg,
5 mg/kg or 7 mg/kg of l-DOPA. (B) Effect of various doses of l-DOPA on RFU. There was no signiﬁcant difference in the ratio of forelimb use between PD/veh and PD/l-DOPA.
The increased use of the ipsilateral forelimb in hemi-PD was reversed with l-DOPA in a dose-dependent manner. l-DOPA signiﬁcantly increased the reduced use of thecontralateral forelimb in hemi-PD in a dose-dependent manner. *P < 0.05, **P < 0.01 relative to PD/veh. ns, no signiﬁcant difference with PD/veh.

Fig. 3. Various doses of ifenprodil have no effect on deﬁcits of TWT and RFU in hemi-PD rats. Hemi-PD rats were injected i.p. with various doses of ifenprodil 30 min prior tothe cylinder test. Treatment groups were: controls (n = 8), PD/veh (n = 7), and PD + ifenprodil treated with various doses of ifenprodil (0.01 mg/kg, n = 4; 0.1 mg/kg, n = 5; or
1 mg/kg, n = 7). (A) No signiﬁcant change in reduced TWT was observed in hemi-PD. (B) PD/veh and PD + ifenprodil did not differ signiﬁcantly in RFU.

ipsilateral dominance to contralateral dominance (Fig. 4B). Taken
together, ifenprodil slightly modulated the effect of 3 mg/kg lDOPA on reduced TWT and imbalanced RFU in hemi-PD; however,the direction of modulation was inconsistent.
3.5. Effect of combined administration of 7 mg/kg l-DOPA and
ifenprodil
Combined administration of ifenprodil with 7 mg/kg of l-DOPA
was tested to see whether ifenprodil affects the inefﬁcacy of asingle dose of 7 mg/kg l-DOPA on forelimb use, and/or reverses
the excessive contralateral forelimb use induced by l-DOPA. In
contrast to results with 3 mg/kg l-DOPA, signiﬁcant recovery fromreduced TWT was observed when 7 mg/kg l-DOPA was combinedwith various doses of ifenprodil (Fig. 5A). With co-administration of
7 mg/kg l-DOPA, predominant use of the contralateral forelimb wasslightly reduced, while reduced use of the ipsilateral forelimb was
unaffected. Consequently, reduced ambidextrous forelimb use with
7 mg/kg l-DOPA was signiﬁcantly reversed by co-administrationof all doses of ifenprodil (0.01, 0.1, and 1 mg/kg). Thus, when

Fig. 4. Low doses of l-DOPA have no signiﬁcant synergistic effects on motor deﬁcits in hemi-PD rats when combined with various doses of ifenprodil. Hemi-PD rats were
injected i.p. with a combination of 3 mg/kg l-DOPA and ifenprodil 30 min prior to the cylinder test. Treatment groups were: controls (n = 8), PD/veh (n = 7), PD/3 mg/kg
l-DOPA + (−) (n = 7), hemi-PD treated with 3 mg/kg l-DOPA without ifenprodil, PD/3 mg/kg l-DOPA + ifenprodil, treated with 3 mg/kg l-DOPA and various doses of ifenprodil(0.01 mg/kg: n = 6, 0.1 mg/kg: n = 7 or 1 mg/kg: n = 7). (A) The ameliorative effect of 3 mg/kg l-DOPA on reduced TWT in hemi-PD rats was blocked by ifenprodil. (B) No
ameliorative effect of ifenprodil on the ratio of ambidextrous forelimb use was observed. Increased use of ipsilateral forelimb and reduced use of contralateral forelimb inhemi-PD were sustained or slightly changed by ifenprodil. *P < 0.05, **P < 0.01 relative to PD/veh.

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49

Fig. 5. High doses of l-DOPA combined with ifenprodil cause a signiﬁcant recovery of the deﬁcit of ambidextrous forelimb use in hemi-PD rats. Hemi-PD rats were injected
i.p. with 7 mg/kg l-DOPA and various doses of ifenprodil. Treatment groups were: controls (n = 8), PD/veh (n = 7), treated with vehicle (veh), PD/7 mg/kg l-DOPA (n = 12),treated with 7 mg/kg l-DOPA without ifenprodil; PD/7 mg/kg l-DOPA + ifenprodil, treated with 7 mg/kg l-DOPA (n = 7) and various doses of ifenprodil (0.01 mg/kg: n = 10,
0.1 mg/kg: n = 9 or 1 mg/kg: n = 8). (A) Reduced TWT in hemi-PD was completely reversed by 7 mg/kg l-DOPA with or without various doses of ifenprodil. (B) The ameliorative
effect of 7 mg/kg l-DOPA on RFU was potentiated by concomitant administration of various doses of ifenprodil. *P < 0.05, **P < 0.01 relative to PD/veh. #P < 0.05, ##P < 0.01
relative to PD/7 mg/kg l-DOPA without ifenprodil.

administered with l-DOPA (7 mg/kg), ifenprodil markedly reversed
abnormal forelimb use without adversely affecting the l-DOPAinduced improvement in TWT.
3.6. Attenuation of l-DOPA-induced c-Fos expression in the STN
by ifenprodil
Two hours after administration of 7 mg/kg l-DOPA, inductionof c-Fos expression was assessed in the dorsal striatum, the STN,
and the motor cortices, M1 and M2. l-DOPA induced c-Fos expression is mainly observed in ipsilateral hemisphere (6-OHDA lesionedside) (Fig. 6). Conversely, c-Fos expression was scarcely observedin the same four regions in the hemisphere that was contralateral to the lesion (intact side). Neurons with c-Fos-positive nuclei
were abundant in the dorsal striatum and the STN, ipsilateral to thelesion (Figs. 6C and G). On the other hand, induction of robust c-Fos
expression was attenuated in the STN following co-administrationof ifenprodil and 7 mg/kg l-DOPA (Fig. 6H). A single dose of 7 mg/kg
l-DOPA induced robust c-Fos expression in the dorsal striatum andthe STN, and moderate c-Fos expression in the M1 and M2 cortexeson the DA depleted side, while l-DOPA treatment induced weak cFos expression in the four regions on the intact side (Fig. 7). Whenvarious doses of ifenprodil (0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg) wereco-administered with 7 mg/kg l-DOPA, a relatively high dose of
ifenprodil signiﬁcantly attenuated robust c-Fos expression in the
STN (Fig. 7D and H). In the dorsal striatum and in M1 and M2 ipsilateral to the lesion, l-DOPA-induced c-Fos expression was slightly
decreased by ifenprodil, but the reduction was not statistically signiﬁcant (Fig. 7A–C).
4. Discussion
During the cylinder test, the motor abnormality of hemi-PD wascharacterized by reduced TWT and imbalanced RFU, in agreementwith a previous report (Shi et al., 2004). Reduced TWT in PD isbelieved to correspond to hypoactivity, like akinesia. On the other
hand, it is likely that imbalanced RFU, due to the predominant useof the ipsilateral forelimb and reduced ambidextrous forelimb use
is associated with the deﬁcit of bilaterally coordinated forelimb
movement (ambidextrous, alternate, or simultaneous use of bothforelimbs). Using the cylinder test, it appears that in addition to
TWT, the deﬁcit of bilaterally coordinated movement (RFU) can beused to quantify therapeutic drug-induced recovery, as a measureof akinesia. In fact, Shi et al. (2004) reported the complete reversalof imbalanced use of both forelimbs in hemi-PD during the cylinder
test was achieved with deep brain stimulation in the STN.

148

Reduced TWT in hemi-PD (akinesia) was apparently improvedby l-DOPA in a dose-dependent manner. On the other hand, imbalanced RFU (deﬁcit of bilaterally coordinated movement) was notreversed by l-DOPA. In particular, the motor abnormality deteriorated further with 5–7 mg/kg l-DOPA due to predominant use of
the contralateral forelimb (parkinsonian paw). This implies thatthe improvement of motor deﬁcits of hemi-PD with l-DOPA is
incomplete. Why does l-DOPA cause excessive use of the contralateral forelimb (parkinsonian paw) in hemi-PD? In the basal ganglia
circuit, two functionally distinct micro circuits, “direct” and “indirect” pathways, have been described (Albin et al., 1989; DeLong,
1990; Smith et al., 1998). In the classical model, the primate direct
pathway involves projection of GABAergic medium spiny neurons(MSN) in the striatum to the internal globus pallidus (GPi, entopeduncular nucleus in rats) and to the substantia nigra pars reticulata(SNR). The indirect pathway originates from GABAergic MSNs, andprojects to the external globus pallidus (GPe, Globus pallidus inrats). GABAergic neurons, receiving afferents from the MSNs, inthe GPe project to the subthalamic nucleus (STN). Excitatory neurons in the STN ﬁnally project to the GPi and the SNR. In fact,direct activation of direct or indirect pathway MSN causes increased
locomotion or decreased locomotor initiation respectively (Kravitz
et al., 2010). Dopamine D1 receptors are dominantly expressed indirect pathway MSNs, whereas dopamine D2 receptors are mainly
expressed in indirect pathway MSNs in the striatum (Gong et al.,
2003; Bertran-Gonzalez et al., 2008, 2010; Matamales et al., 2009;
Valjent et al., 2009). In addition, D1 receptors are sensitized in
the dopamine-depleted striatum of hemi-PD (Corvol et al., 2004).
Therefore, in the present study, it is likely that administration of
5–7 mg/kg l-DOPA activated sensitized D1-like receptors in the
dopamine-depleted striatum and overly triggered movement initiation of the contralateral forelimb via activation of direct pathway
MSNs. Actually, D1 agonists reportedly improve reduced locomotor
activity and produce abnormal involuntary movement in hemi-PD
simultaneously (Darmopil et al., 2009; Li and Zhou, 2013).
Several studies have reported that single administration of NR2B
receptor antagonists improve motor deﬁcits in an animal modelof PD (Nash et al., 2000; Nash and Brotchie, 2002; Löschmann
et al., 2004). In particular, Nash and Brotchie (2002) have reported
that intrastriatal injection of ifenprodil induces rotation behaviorof hemi-PD rats, so we had anticipated that systemic administration of ifenprodil would also enhance either total forelimb touches
or the ratio of contralateral forelimb use. However, in the present
study, single administration of ifenprodil did not improve deﬁcits
of forelimb movement of hemi-PD. One possible interpretation isthat this discrepancy resulted from a difference in the expression

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M. Igarashi et al. / Neuroscience Research 96 (2015) 45–53

Fig. 6. c-Fos-immunostained sections of striatum and STN ipsilateral to the 6-OHDA lesion, showing neurons with drug-induced c-Fos expression. (A and E) Schematic
drawings of coronal sections of the striatum (0.20 mm rostral to the bregma) and the STN (3.80 mm caudal to the bregma). The black frame indicates where photomicrographs
were taken. (B and F) Sections of the striatum and STN of a hemi-PD rat treated with vehicle (veh) demonstrating several c-Fos positive neurons. (C and G) Robust c-Fospositive neurons observed in a hemi-PD rat treated with 7 mg/kg of l-DOPA. (D and H) Sections in a hemi-PD rat treated with 7 mg/kg of l-DOPA and ifenprodil (0.1 mg/kg).
Scale bars represent 250 m.

level of NR2B receptors in the striatum among the dopaminedepleted PD models. It has been reported that the expression
level of NR2B receptors in the striatum of a PD model with full
dopamine-lesions was lower than that with a partial dopaminelesions (Gardoni et al., 2006, 2009; Paillé et al., 2010). Since hemi-PD
are considered to have full dopamine-lesions, equivalent to theloss of more than 97% of nigrostriatal dopaminergic cells (Ogura
et al., 2005), the low expression of striatal NR2B receptors in ourhemi-PD might have weakened the effect of NR2B antagonists
in the striatum. Another possible explanation is a route of drug
administration. In the present study, ifenprodil was administeredintraperitoneally; therefore, ifenprodil could affect entire regionsof the CNS, which might inhibit the effect of ifenprodil on motor
initiation. Possible sources of interference include the motor cortex
and the ventromedial (VM) thalamic nucleus. This is because NR2Bcontaining NMDA receptors are also expressed in the motor cortex

and the VM thalamic nucleus (Khan et al., 2000; Zheng and Fan,
2011; Chen et al., 2013). Layer 5 of the motor cortex is thought tobe a major output of motor execution via pyramidal tract neurons,
and the VM thalamic nucleus is the output of SNr/EP in rodents;
thus, it is possible that motor initiation induced by blockade of striatal NR2B receptors was not sufﬁcient to override inhibition by the
motor cortex and/or the VM thalamic nucleus.
The ameliorative effect of a single dose of l-DOPA on motor
abnormalities of hemi-PD was incomplete, because of an apparent lack of recovery from imbalanced RFU. Therefore, we examined
whether the NR2B antagonist, ifenprodil, boosts the incomplete
recovery from deﬁcits of forelimb movement by l-DOPA. As aresult, combined treatment with 7 mg/kg l-DOPA and ifenprodil
demonstrated signiﬁcant recovery of TWT, involving a reversal of
reduced ambidextrous forelimb use (Fig. 5), while combined treatment with 3 mg/kg l-DOPA and 0.1 mg/kg ifenprodil prevented149

M. Igarashi et al. / Neuroscience Research 96 (2015) 45–53

51

Fig. 7. Ifenprodil suppresses l-DOPA-induced c-Fos expression in the STN. (A–H) The density of c-Fos positive cells (number/mm2 ) was calculated on both sides of the dorsal
striatum (A and E), the M1 cortex (B and F), the M2 cortex (C and G) and the STN (D and H). Treatment groups were: PD/veh (n = 4), PD/7 mg/kg l-DOPA + (−) (n = 7), hemi-PD
rats treated with 7 mg/kg l-DOPA without ifenprodil; PD/7 mg/kg l-DOPA + ifenprodil, hemi-PD rats treated with 7 mg/kg l-DOPA and various doses of ifenprodil (0.01 mg/kg:
n = 5, 0.1 mg/kg: n = 5 or 1 mg/kg: n = 5). #P < 0.05, ##P < 0.01 relative to PD/7 mg/kg l-DOPA without ifenprodil.

the ameliorative effect of l-DOPA on abnormalities of forelimb
movement. What is the pathophysiological mechanism of ifenprodil’s action on motor abnormalities? It is thought that l-DOPA
facilitates motor activity mediated by activation of striatal D1
receptors. The predominant use of the contralateral (Parkinsonian)paw observed in these experiments is most likely due to hypersensitivity of D1 receptors. Since improvement of bilateral forelimb
use by NR2B antagonists necessarily involves a signiﬁcant improvement in motor activity, it is likely that ifenprodil’s enhancement of
l-DOPA’s beneﬁcial effects is also mediated by D1 receptor activation. It may be that ifenprodil normalizes the hypersensitivity of150

D1 receptors in the dopamine-depleted brains of hemi-PD rats byacting upon NMDA receptors in the striatum or other nuclei of thebasal ganglia to reduce the l-DOPA-induced contralateral (Parkinsonian) paw use, and to recover bilaterally coordinated forelimb
movement.
In the present study, it is noteworthy that the four regions
demonstrating l-DOPA-induced c-Fos expression are involved in
the basal ganglia-thalamo-cortical loop circuit for motor regulation. However, robust c-Fos expression was not observed in the
SNr or the GPe, which receive GABAergic input from the striatum, which also manifests signiﬁcant c-Fos expression. It has

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M. Igarashi et al. / Neuroscience Research 96 (2015) 45–53

been thought that expression of immediate early genes, such as
c-fos, is a biological marker of hyperactivity or over-excitationof neurons in the central nervous system (Sheng and Greenberg,
1990; Herrera and Robertson, 1996; Andersson et al., 1999; Ebihara
et al., 2011). Regionally speciﬁc c-Fos expression in the striatum
is probably due to hypersensitive D1 receptors on striatal neurons caused by dopamine-depletion in PD models (Darmopil et al.,2009; Li and Zhou, 2013). It is well known that developmentof D1 receptor-mediated long-term potentiation (LTP) in striatal
MSN results from treatment with D1 receptor agonists (Kerr andWickens, 2001). Since D1 receptors are dominantly expressed indirect pathway MSNs, l-DOPA-induced c-Fos expression in the striatum probably indicates facilitation of direct pathway MSNs causedby the D1 receptor-mediated LTP underlying the mechanism of the
improvement of TWT (Fig. 3). As for c-Fos expression in the STN, apossible interpretation is that prolonged disinhibition via blockade of inhibitory afferents from the GP caused signiﬁcant c-Fos
expression in the STN (Saji et al., 1995). However, in the present
study, it is difﬁcult to conclude that l-DOPA-induced c-Fos expression in the STN is mediated by suppression of the GPe. First, it has
been reported that l-DOPA administration only leads to predominance of LTD in indirect pathway MSNs, but not vice versa (Thiele
et al., 2014); consequently, it is less likely that l-DOPA potentiates GABAergic indirect pathway MSNs and suppresses neuronal
activity of the GPe. Second, it has been reported that l-DOPA treatment does not change neuronal activity of the GPe in animal modelsof PD (Boraud et al., 1998). Those reports contradict the conclusion that c-Fos expression in the STN results from facilitation ofindirect pathway MSNs or inhibition of GABAergic neurons in the
GP. Another possible interpretation is that l-DOPA administration
directly stimulates D1/D5 receptor on STN neurons, and this idea
seems supported by more evidence, as when expression of the cfos gene is followed by systemic administration of SKF 82958, a
D1/D5 dopamine receptor agonist, in rats (Svenningsson and Le
Moine, 2002). Since D1 receptors are expressed on direct pathway
MSNs, D1 agonists necessarily altered activity of the STN without affecting the indirect pathway, supporting the idea of direct
stimulation of STN by l-DOPA. Further investigation is required tounderstand the causality of c-Fos expression in the STN. Althoughc-Fos expression in the STN indicates strong excitatory inputs to
the SNr, c-Fos expression was not observed in the SNR. No c-Fos
expression in the SNR suggests that the striatal direct pathway was
dominantly activated by l-DOPA, compared with excitatory input
from the STN. Predominance of the direct pathway reinforces theidea that l-DOPA-induced excessive contralateral use of forepaw
in the present study is mediated by direct pathway MSNs in the
striatum.
In the present study, co-administration of ifenprodil with lDOPA did not attenuate striatal c-Fos expression signiﬁcantly(Fig. 7), and this result agrees with a previous report using another
NR2B antagonist (Rylander et al., 2009). In contrast, we foundthat co-administration of ifenprodil signiﬁcantly reduced l-DOPA
induced c-Fos expression in the STN. Since both the improvementof bilateral forelimb use and the attenuation of robust c-Fos expression in the STN occurred with co-administration of ifenprodil and
l-DOPA, it is quite likely that the two l-DOPA-induced phenomena
inﬂuenced by ifenprodil-treatment are interrelated. Spontaneous
tonic activity of excitatory projections from the STN to the SNr/GPi(or the EP in rodents) reportedly suppress unnecessary movement
via motor thalamus inhibition by GABAergic projections from the
SNR/GPi (Mink, 1996; Nambu et al., 2002; Leblois et al., 2006).
Thus, unilateral chemical lesioning of the STN with microinjectionof the GABAA agonist, muscimol, causes uncontrollable hyperactivity of limbs contralateral to the lesion (Dybdal and Gale, 2000). In
addition, the STN is considered to have another role in motor regulation. That is, phasic activity of the STN elicited via an indirect

pathway from the stimulated striatum inhibits the motor thalamus via GABAergic efferents from the SNr. This terminates motor
activity previously driven directly by the stimulated striatum
and thereby reverses one-sided ipsilateral motion to contralateral movement. It is possible that this role of the STN underlies
the bilaterally coordinated movement of forelimbs. However, thishypothesis should be investigated further.
Acknowledgements
We thank Dr. Steven Douglas Aird for English proofreading andediting, and also thank for Mr. Yousuke Kitahara for assistance. This
work was supported by JSPS KAKENHI Grant Number 22659264.
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A Single Amino Acid Mutation in SNAP-25 Induces
Anxiety-Related Behavior in Mouse
Masakazu Kataoka1., Saori Yamamori2., Eiji Suzuki3, Shigeru Watanabe2, Taku Sato2, Hitoshi Miyaoka2,
Sadahiro Azuma2, Shiro Ikegami4¤a, Reiko Kuwahara4, Rika Suzuki-Migishima4, Yohko Nakahara4, Itsuko
Nihonmatsu4, Kaoru Inokuchi4¤b, Yuko Katoh-Fukui4¤c, Minesuke Yokoyama4¤d, Masami Takahashi2*
1 Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Nagano-shi, Nagano, Japan, 2 Kitasato University School of Medicine,
Sagamihara-shi, Kanagawa, Japan, 3 Department of Psychiatry, International University of Health and Welfare Atami Hospital, Atami-shi, Shizuoka, Japan, 4 Mitsubishi
Kagaku Institute of Life Sciences, Machida-shi, Tokyo, Japan

Abstract
Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release.
Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter
release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 isessential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We
therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using ‘‘knock-in’’ technology. The moststriking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response toenvironmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests.
In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis
measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly
indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as
the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible
mechanisms underlying these defects.
Citation: Kataoka M, Yamamori S, Suzuki E, Watanabe S, Sato T, et al. (2011) A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in
Mouse. PLoS ONE 6(9): e25158. doi:10.1371/journal.pone.0025158
Editor: Thomas Claudepierre, Faculty of Medicine University of Leipzig, Germany
Received April 13, 2011; Accepted August 27, 2011; Published September 20, 2011
Copyright:  2011 Kataoka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (B) (#18300127, 21300141) from the Japan Society for the Promotionof Science (JSPS), Grant-in-Aid 15082206 for Scientific Research on Priority Areas on ‘‘Elucidation of Glia–Neuron Network-Mediated Information Processing
Systems’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for Core Research for Evolutional Science andTechnology (CREST) from the Japan Science and Technology Agency (JST). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: masami@med.kitasato-u.ac.jp. These authors contributed equally to this work.
¤a Current address: Department of Psychology, Saitama Institute of Technology, Fukaya-shi, Saitama, Japan¤b Current address: Department of Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama-shi, Toyama, Japan¤c Current address: National Center for Geriatrics and Gerontology, National Institute for Longevity Science, Morioka-machi, Obu, Aichi, Japan¤d Current address: Animal Resources Branch, Center for Bioresource-based Researches, Brain Research Institute, Niigata University, Niigata-shi, Niigata, Japan

Introductionof secretory vesicles containing DA and ACh was enhanced by theactivation of PKC [12]. In adrenal chromaffin cells and insulin
secreting cells, PKC activation enhanced exocytotic release of
these hormones by increasing the size of the readily releasable
vesicle pool and the highly Ca2+-sensitive vesicle pool (HCSP), andphosphorylation of SNAP-25 at Ser187 was essential for theseeffects [13–16]. Immunoblotting analysis using phosphorylationspecific antibodies revealed that the phosphorylation of SNAP-25
at Ser187 also occurred in brain [17–21] and interestingly thephosphorylation of SNAP-25 was dramatically changed in epilepsy[20,21]. However, the physiological roles of phosphorylation at
this site are still obscure [18,22].
To address the issue, we generated a knock-in mouse with a singleamino acid substitution of Ala at Ser187. We found that the mutant
mouse displayed a variety of interesting behavioral phenotypes
consistent with the conclusion that the phosphorylation of SNAP-25
plays an important role in synaptic function after birth.

Synaptic transmission requires neurotransmitter release frompresynaptic nerve terminals. Three SNARE proteins, VAMP-2/
synaptobrevin 2 in the synaptic vesicle membrane, and SNAP-25
and syntaxin 1 in synaptic plasma membrane, play crucial roles inthe exocytotic release of neurotransmitters [1–4]. Neurotransmitter release is regulated both positively and negatively by various
kinds of protein kinases [5–7], and these regulations are some of
the important mechanisms of synaptic plasticity underlying
learning and memory.
In many neuronal preparations, neurotransmitter release isenhanced by the activation of protein kinase C (PKC) [8,9].
Previously, we showed that PKC activation induced enhancementof Ca2+-dependent release of dopamine (DA) and acetylcholine(ACh) from PC12 cells, and Ser187 was specifically phosphorylated
in these conditions [10,11]. We also showed that the recruitment
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Anxiety-Related Behavior of SNAP-25 Mutant Mouse

Results

mutant mice were obtained using the blastocyst injection method.
Mice heterozygous for the Snap25S187A mutation (Snap25+/S187A) wererobust, fertile and phenotypically indistinguishable from wild-type(WT) littermates. The Snap25+/S187A mice were bred with C57BL/
6N, and the N2 offspring were crossed to obtain mice homozygous
for the Snap25S187A mutation (Snap25S187A/S187A). Figure 1B shows
the results of PCR genotyping using a primer set just outside exon 7.
0.3 kbp and 2.2 kbp PCR products originating from WT and
Snap25S187A mutant alleles, respectively, were obtained, and both

Generation of mutant mice
The Snap25 gene encodes two isoforms, SNAP-25a and SNAP25b, derived from alternative splicing of exon 5 [23,24]. To avoid aneffect of gene targeting on alternative splicing, exon 7 was replacedwith a mutated minigene in which the Ala codon was substituted for
Ser187 in the targeting vector (Fig. 1A). ES cells with the heterozygous Snap25S187A mutant allele were generated and heterozygous

Figure 1. Generation of Snap-25 ‘‘knock-in’’ mouse. (A) The targeting vector (middle) was constructed by insertion of the DNA fragment(lower), consisting of part of a mutated SNAP-25 minigene, the PGK 39 end poly-A signal, the PGK promoter and the neo gene, into the SmaI site atexon 7 of the region between exon 6 and exon 8 of the wild-type allele (upper). The black box in the lower panel indicates the mutation point of thecodon for Ser187 to Ala187. Arrows indicate the primers used in ES cell screening. (B) The genotyping of the mice was conducted by PCR using primers
overlapping the 39 and 59 extremities of exon 7 (arrowheads in upper panel in (A)). The DNA fragments from wild-type and mutant alleles areindicated with ‘‘W’’ and ‘‘M’’, respectively. Wt, wild-type mouse; He, Snap25+/S187A mouse; Hm, Snap25S187A/S187A mouse. Mw, molecular weight marker.
(C) Northern blotting of total forebrain RNAs from each mouse genotype (P25) was conducted using SNAP-25b cDNA as a probe. The signals oftranscripts from each allele are indicated as in (B). 18S, 18S rRNA; 28S, 28S rRNA. (D) Immunoblot analysis of the phosphorylation level of SNAP-25 andthe expression levels of major synaptic proteins in mouse brain.
doi:10.1371/journal.pone.0025158.g001

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Anxiety-Related Behavior of SNAP-25 Mutant Mousebands were detected in Snap25+/S187A mice. Northern blot analysis
using SNAP-25b ORF as a probe revealed transcripts from the WT
allele of 2.1 kb and the shortened transcript from the Snap25S187A
allele of 1.3 kb (Fig. 1C). Thus the Snap25S187A/S187A mice are viable.
Figure 1D illustrates immunoblots of forebrain homogenates from
WT, Snap25+/S187A and Snap25S187A/S187A of 25-day-old littermate
mice. Immunoblots probed with an antibody directed against
SNAP-25 phosphorylated at Ser187 showed dose-dependent loss of
signal and no significant bands were detected in the brain
homogenates of Snap25S187A/S187A mice, indicating successful
generation of mutant mice in which Ser187 of SNAP-25 cannot be
phosphorylated. Immunoblots with anti-SNAP-25 antibody indicated that SNAP-25 expression in Snap25S187A/S187A mice was also
decreased to 50% of that in WT mice. Immunoblotting for various
other synaptic proteins including syntaxin-1 (Stx-1), VAMP-2, a/bSNAP, synaptotagmin I (Stg-1), rab3A, cystein string protein (csp),
N-ethylmaleimide-sensitive factor (NSF), complexin I (CPLX), Na/
K-ATPase, NMDA receptor (NR2A), AMPA receptor (GluR2), anda1 subunit of L-type Ca channel (L-VACC), showed no significant
difference in their expression between Snap25S187A/S187A and WT
mice (Fig. 1D).

Possible early postnatal role of SNAP-25 phosphorylation
No embryonic lethal phenotype was apparent, sinceSnap25S187A/S187A mice were recovered at the expected Mendelian
ratio following crossing of heterozygous mice. Nissl stained brainslices from WT and Snap25S187A/S187A mice at 2.5 weeks of age
seemed to show no significant differences (see Fig. S1), suggestingthat the phosphorylation of SNAP-25 at Ser187 was not importantin embryogenesis. The mutant mice were not distinguishable bytheir morphology at birth, however, some Snap25S187A/S187A micedied between postnatal week 2 and 3. The mortality ofSnap25S187A/S187A mice in this period was 12.5% (28/224), whereas
those of WT and Snap25+/S187A mice were 0.5% (1/196) and 1.2%
(5/430), respectively. Surprisingly, the fragility of Snap25S187A/S187A
mice was characteristic of this particular period, and surviving micegrew well thereafter for almost two years. This phenotype was stillobserved after backcrossing to the C57BL/6N background 13
times. Snap25S187A/S187A mice sometimes exhibited spontaneously
occurring convulsive seizures after postnatal day 21–24.

Characteristic behavior of homozygous mutant mice inthe open field
The most striking phenotype of Snap25S187A/S187A mice wasabnormal behaviour possibly attributed to increased anxiety.
Snap25S187A/S187A mice froze very readily in response to environmental change (see Videos S1 and S2). Since freezing behavior isoften observed in conditions of increased anxiety, we examined
locomotor activity in an open-field box, a classical test of anxiety.
As shown in Figure 2A, WT (Wt) and the heterozygous mutant
mice (He) showed active exploratory behavior when theyencountered a novel environment and visited the whole area
during the test period. Two different phenotypes were observed in
Snap25S187A/S187A mice. The first type spent most of the time near
the wall throughout the test period (Fig. 2A, Hm-1). The second
type exhibited hyperlocomotor activity and moved continuously
along the wall throughout the test period (Fig. 2A, Hm-2). As
shown in Fig. 2B, initial delay of moving was significantly longer in
Snap25S187A/S187A mice (4.5764.31 s, n = 11) than in WT mice
(0.3860.31 s, n = 6) and in heterozygous mice (0.7260.59 s,
n = 11). The percentage of time spent near the wall (i.e. within
5.6 cm) was much higher in Snap25S187A/S187A mice (92.9167.8%,
n = 11) than in WT mice (62.30613.07%, n = 6) and in
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Figure 2. General locomotor activity in open field. (A) Typical
traces of WT (Wt), Snap25+/S187A (He), and Snap25S187A/S187A mice (Hm-1and Hm-2) during 30 min. (B) Initial delay time before moving. (C)Percentage of time spent near the wall of the open-field box. (D) Total
moving time in 30 min of test period. (E) Average velocity. (F) Timecourse of rearing frequency during the 30 min. Values represent mean
6 SD. **, p,0.01 and *, p,0.05 by paired Student’s t test between Wt
mice and Snap25S187A/S187A mice.
doi:10.1371/journal.pone.0025158.g002

heterozygous mice (61.03611.66%, n = 11) (Fig. 2C). The averagevalue of the total time in movement of Snap25S187A/S187A mice wasvery similar to those of WT and heterozygous mice, however,variation of the individual values was much larger compared to
WT and heterozygous mice (Fig. 2D). The average velocity wassignificantly larger in Snap25S187A/S187A mice (26.5167.19 cm/s,
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tterns including increased anxiety, we determined the content
of 5-HT and its major metabolite, 5-hydroxyindole acetic acid
(5-HIAA), in various brain regions. We also measured the content of
DA and homovanillic acid (HVA), a major metabolite of DA.
Except for slight increases in HVA in the hypothalamus and brain
stem of Snap25S187A/S187A mice, no significant change was observed(Fig. 5A). Next, we examined the release of DA and 5-HT in
amygdala by microdialysis. As shown in Fig. 5B, spontaneous
release of both DA and 5-HT were reduced by approximately
58% and 40% for DA and 5-HT in the Snap25S187A/S187A micecompared to WT mice, respectively. High-K+-evoked releaseover 40 min was also decreased in Snap25S187A/S187A mice byapproximately 60% and 38% for DA and 5-HT, respectively(Fig. 5C).

n = 11) than in WT mice (19.2261.00 cm/s, n = 6) and inheterozygous mice (19.9663.23 cm/s, n = 11) (Fig. 2E). Althoughrearing frequencies were not significantly changed in WT andheterozygous mice throughout the test period of 30 min, it
gradually decreased with time in Snap25S187A/S187A mice
(Fig. 2F).
To know the cause of this increased variability, we repeated theopen-field test three times with one week intervals in thirteen WT
mice and thirteen Snap25S187A/S187A mice. There was no significant
difference in the behavior of WT mice (Fig. 3A, W1 to W13). In
contrast, Snap25S187A/S187A mice showed different behaviour
pattern even in the same mouse in these three trials (Fig. 3A,
H1 to H13). More interestingly, the inconstancy of the behaviour was even observed in each test period of 30 min in
Snap25S187A/S187A mice. As shown in Fig. 3B, WT always
continued to move at almost constant velocities throughout thetest period (W1 to W13), whereas Snap25S187A/S187A mice showedstriking variability (H1 to H13). Sometimes, they start to movesuddenly after a long stationary period (arrowhead), but at othertimes, they suddenly stopped after active movement (arrow). In
order to know the origin of variability in behavior, we counted anumber of trials having total stopping period longer than 200 s.
Except for one case (212 s), the total stopping period was less than
200 s in 38 trials of WT mice. In a striking contrast, the stopping
was varied even in each three trials of Snap25S187A/S187A mice
(Fig. 3C). The value of standard deviation of moving distance ineach three trials divided by the average of moving distance wasmuch larger in Snap25S187A/S187A mice than that in WT mice
(Fig. 3D). These results clearly indicated that the variability of
behavior was not derived by heterogeneity of genetic backgroundbut by characteristic feature of Snap25S187A/S187A mice.

Discussion
In the present study, we generated SNAP-25 mutant mice witha single amino acid substitution at the PKC-dependent phosphorylation site and found that the homozygous mice show various
striking phenotypes with abnormal emotional behavior. We also
found that DA and 5-HT release in the amygdala were markedly
reduced despite the fact that neurotransmitter content were notdecreased in the mutant mice.
The expression level of SNAP-25 in Snap25S187A/S187A micedecreased to about 50% of that in WT mice and thus certain
aspects of the phenotypes might be uniquely derived from reduced
expression. However, this possibility seems unlikely since SNAP-25
expression in the SNAP-25+/2 mice was decreased to 50% of thatof wild-type, but they were phenotypically indistinguishable fromwild-type litter mates [25].
The most striking phenotype of Snap25S187A/S187A mice was theabnormal behavior possibly attributed to increased anxiety.
Snap25S187A/S187A mice froze very readily in response to environmental change. The phenotype was well quantified by traditional
assays for anxiety, such as general activity test in an open-field box
and the light and dark preference test. There seemed no significant
defect in locomotor activity since the average velocity ofSnap25S187A/S187A mice was even higher than WT mice in open
field (Fig. 2E) and light-dark test (Fig. 4C). There seemed to be nosensory defect since (1) startle response to auditory stimuli (120 dB)
was even bigger in Snap25S187A/S187A mouse than that in
WT mouse (WT, 179.3647.2; Snap25S187A/S187A, 268.1697.4;
p,0.01), (2) strength of electric shock to induce jumping was
almost same between WT and Snap25S187A/S187A mouse (data notshown), and (3) Snap25S187A/S187A mouse recognized the dimly lit
area near to the entrance in light-dark test (Fig. 4A, right). Thus, it
is reasonable to conclude that the abnormal behaviour of theSnap25S187A/S187A mice was attributed to the point mutation at the
PKC-dependent phosphorylation site in SNAP-25.
Monoamines play crucial roles in the expression and regulationof emotional behavior, and a decrease in extrasynaptic 5-HT levels
results in increased anxiety [26]. In PC12 cells and adrenal
chromaffin cells, phosphorylation of SNAP-25 at Ser187 is essential
for the PKC-dependent enhancement of catecholamine release byincreasing the size of the readily releasable vesicle pool and thehighly Ca2+-sensitive vesicle pool (HCSP) [10–15]. In the present
study, we showed that DA and 5-HT release in amygdala wasmarkedly decreased in Snap25S187A/S187A mice, possibly due to alack of phosphorylation-dependent enhancement of monoamine
release. Thus, it is quite likely that some of the emotional defects of
Snap25S187A/S187A mice derive from the absence of PKC-dependent enhancement of monoamine release mediated by aphosphorylation of SNAP-25 at Ser187.

Light-dark test
Next we performed another classical test for anxiety-related
behavior, the light and dark preference test. WT mice tended tostay a little bit longer in the dark compartment, however, they also
exhibited an active exploratory behavior in the light compartment(Fig. 4A, left). In striking contrast, all of Snap25S187A/S187A mice
showed a very strong preference for the dark component (Fig. 4A,
right and Fig. 4B). The activity of Snap25S187A/S187A mouse was very
high in the dark compartment, and the average velocity was larger
than that of WT mouse (Fig. 4C), while active rearing behavior
was similar to WT mouse (Fig. 4E). Latency to enter the dark box
in L-D test was significantly larger in Snap25S187A/S187A mice thanthat in Wt mice (Fig. 4D). Interestingly, all of the Snap25S187A/S187A
mice avoided the dimly lit area near to the entrance (Fig. 5A, right).

Behavior on the elevated platform
Since the open field test and the light-dark test suggested thatanxiety was increased in Snap25S187A/S187A mice, we attempted toperform another test for anxiety-related behaviour, the elevatedplus-maze test, but without success. Generally, mice are scared ofheights and will not jump off an elevated platform. Surprisingly,
most of Snap25S187A/S187A mice put on an elevated platform (50 cmhigh) jumped off without any hesitation, while all the WT mice
stayed on the platform throughout the test period (15 min) (seeVideo S3). Snap25S187A/S187A mice sometimes froze on the platform
without jumping.

Serotonin (5-HT) and DA release from amygdala
It has been proposed that perturbation of 5-HT levels in the brain
contributes to anxiety and depression. Since Snap25S187A/S187A
mice showed a variety of abnormal emotional behavior paPLoS ONE | www.plosone.org

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Figure 3. (A) Traces of thirteen Wt (W1 to W13) and thirteen Snap25S187A/S187A mice (H1 to H13) in open field performed three times with one week
intervals. (B) Time courses of locomotor activities in open field shown in A. Total moving distances are plotted against time. Red, purple and yellow
lines represent 1st, 2nd and 3rd trial, respectively. Sometimes, they start to move suddenly after a long stationary period (arrowhead), but at othertimes, they suddenly stopped after active movement (arrow). (C) Number of mice showing stopping period longer than 200 s during 30 min test
period either 0 time (0/3), once (1/3), twice (2/3), or three times (3/3) in each three trials. (D) The value of standard deviation of moving distance ineach three trials divided by the average of moving distance in Wt and Snap25S187A/S187A mice (Hm). Values represent mean 6 SD. ***, p,0.001 bypaired Student’s t test between Wt mice and Snap25S187A/S187A mice.
doi:10.1371/journal.pone.0025158.g003

Expression and phosphorylation of SNAP-25 were quite low
during the embryonic stage [20,27,28], and SNAP-25 knock-out
mice survived normally until just after the birth [25]. No

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embryonic lethal phenotype and no significant abnormality inthe brain structure of 2.5 week-old mice were observed in
Snap25S187A/S187A mice. Taken together, it is thus likely that

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velocity in the open field were much larger in Snap25S187A/S187A
mice than WT mice (Figs. 2D and 2E). Snap25S187A/S187A micesuddenly stopped or started moving without any noticeable stimuli(Figs. 3B and 3C). The homozygous mice often showed impulsivity
on the elevated platform (Movie S3). It may be more likely thatthese phenotypes derive not from an emotional abnormality but
from a defect in decision making. An interesting hypothesis has
been proposed that 5-HT plays a critical role in changing the risk
factor for decision making [32]. It is possible that the variability of
behavior in Snap25S187A/S187A mice can be attributed to depressed
5-HT release in basal nuclei.
Although the phosphorylation of SNAP-25 at Ser187 has beenshown to be essential for PKC-dependent enhancement ofhormone release in secretory cells, the involvement of SNAP-25
phosphorylation in the regulation of neurotransmitter release in
the brain is still debated [18,22]. Furthermore, some reportssuggest that at least a fraction of GABAergic neurons do not
express SNAP-25 [33,34]. Thus, it is likely that phosphorylation of
SNAP-25 is not ubiquitously involved in the regulation ofneurotransmitter release but only in some particular types ofneurons including monoaminergic neurons.

Materials and Methods
Generation of mutant mice
All procedures involving animals complied with the guidelinesof the National Institutes of Health, and were approved by the
Animal Experimentation and Ethics Committees of the Kitasato
University School of Medicine (permit number 2010109) and
Mitsubishi Kagaku Institute of Life Sciences. All efforts were madeto minimize animal suffering and to reduce the number of animals
used. The 7.5 kbp DNA region between exon 6 and exon 8 of theSnap25 gene was cloned, and used for gene targeting. In brief, themutated mini cDNA for SNAP-25S187A, followed by the PGK poly
A signal, the PGK promoter and the Neor gene, was inserted into
the SmaI site within exon 7 using the corresponding site in themutated cDNA. Two transcripts, for SNAP-25S187A and forneomycin phosphotransferase, must therefore be generated fromthe mutated Snap25 locus. The targeting vector was linearized andelectroporated into E14TG2a mouse embryonic stem cells (akind gift from Dr. Augustin G. Smith). Cells were selected forhomologous recombination with G418 followed by clonalpassaging. Clones were screened for homologous recombination
by PCR using primers for the region within the Neor gene and thatwithin the exon 8, not included in the targeting vector. Twocorrectly targeted clones were expanded and microinjected into
C57BL/6N blastocysts. Chimeric males were crossed with
C57BL/6N female mice, and heterozygous agouti offspring wereobtained. Genotyping of the mice was performed by PCR. Micewith heterozygous Snap25S187A locus were bred with C57BL/6N
and maintained using standard husbandry procedure. After
finishing back-cross into C57BL/6N genetic background 13 times,
homozygous mice were routinely obtained by in vitro fertilization
using ICR mice as foster mothers. When the mice were obtainedby in vitro fertilization, the littermate WT mice were used ascontrols for the behavior tests.

Figure 4. Orbital of locomotor activity in light and dark boxes.
(A) Typical traces of WT (left five, Wt) and Snap25S187A/S187A mice (rightfive, Hm) during 30 min. (B) % time in dark room. (C) Average moving
velocity. (D) Latency to enter dark box. (E) Total number of rearing
behaviors. Values represent mean 6 SD. ***, p,0.001, **, p,0.01
and *, p,0.05 by paired Student’s t test between Wt mice andSnap25S187A/S187A mice.
doi:10.1371/journal.pone.0025158.g004

neither phosphorylation of SNAP-25 nor SNAP-25 expression
play an important role during embryonic stages.
The phosphorylation of SNAP-25 increased remarkably during
two to three weeks after birth [26], and spontaneously occurringconvulsive seizures appeared after this period. Defects in 5-HT
receptor-mediated signaling in the early postnatal period havesevere consequences including increased anxiety and stressvulnerability in adulthood [29,30]. In recent years, there has been
increasing evidence that serotonergic neurotransmission modulates a wide variety of experimentally induced seizures [31]. Thus,
it is likely that some of the phenotypes that appeared in
Snap25S187A/S187A mice may be derived from the reduction in 5HT release during the early postnatal period. Further studies arenecessary to evaluate this possibility.
Another characteristic phenotype of Snap25S187A/S187A mice isinconstancy of behavior. Variations in the moving period and
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Northern and Western blotting
Mouse brains were removed after cervical dislocation for RNA
and protein samples. For Northern blot analysis, total RNAs wereisolated from P15 mouse forebrain using an RNAeasy RNA
isolation kit (QIAgene). Ten micrograms of total RNA was loaded
for Northern blotting using the cDNA for SNAP-25b as a probe.
For Western blotting, brains from P25 mice were removed and
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Anxiety-Related Behavior of SNAP-25 Mutant Mouse

Figure 5. Monoamine metabolisms in brain. (A) Contents of dopamine (DA), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole
acetic acid (5-HIAA) in various brain regions of WT (red columns) and Snap25S187A/S187A mice (blue columns). Fcx, frontal cortex; CP, striatum; Amy,
amygdala; Hip, hippocampus; Th, thalamus; Hypo, hypothalamus; BS, brain stem; Ce, Cerebellum. *, p,0.05. (B) Spontaneous release of DA and 5-HTin amygdala of WT (red column) and Snap25S187A/S187A mouse (blue column). **, p,0.01. (C) Time-dependent changes of high-K+ (100 mM)-evoked
DA and 5-HT release in amygdala of WT (red circles) and Snap25S187A/S187A mouse (blue circles). KCl concentration was elevated at a time indicated byarrows. *, p,0.05 compared with WT and Snap25S187A/S187A mouse in each period.
doi:10.1371/journal.pone.0025158.g005

homogenized in 5 ml SDS sample buffer with an ultrasonicator.
The samples were boiled and their protein concentration was
estimated using the BCA protein assay kit (Pierce). Western
blotting was performed with enhanced chemiluminescence(Amersham) as described previously [11].

from 6:00 a.m. under constant temperature (2361uC) in a roomwith a clean air conditioning system. One week before theexperiment, they were handled once daily for 3 days.
General activity was measured in an open-field box(50650640 cm) constructed from grey vinylchloride plates. The
apparatus was placed in a sound-attenuating room in which
external noise was greatly reduced. Two pairs of 24624 array
infrared photosensors were attached to the outer wall, equally
spaced in rows 2.5 cm and 6.5 cm from the floor. The lower row

Behavior experiment
Animals were housed at one per cage with free access to foodand water. They were maintained in a 14:10 h light-dark cycle
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7

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Anxiety-Related Behavior of SNAP-25 Mutant Mouseof photocells was used to measure locomotor activity and theupper row to detect rearing behavior. The sensor state was
sampled every 0.1 sec. A computer recorded the number ofhorizontal photobeam interruptions caused by animal movement.
Each mouse remained in the apparatus for 30 min.
For the light and dark preference test, the apparatus consisted of
two compartments of grey vinylchloride plates, and was placed ina darkened and sound-attenuating room. One compartment was abright (250 lux) chamber (25650640 cm) illuminated by afluorescent lamp, and the other was a dark (0.5 lux) chamber(25650640 cm). The two compartments were separated by a wallwith a small opening (8616 cm). A mouse was placed in the centerof the light chamber facing the opening, and its behavior wasrecorded for 30 min by the computer. The mouse was considered
to have entered a new area when all four feet were in this area.
The behavior on the elevated platform (50 cm high) was recorded
and analyzed by video. Student’s t test was used for statisticalanalysis.

probe was inserted into the left amygdala. To measure DA and 5HT, we modified the previously described methods [36]. To
determine levels of extracellular DA and 5-HT, the microdialysis
probes (length: 1.0 mm, diameter: 0.22 mm, MW cutoff: 50,000
daltons, Eicom, Kyoto, Japan) were perfused with Ringer’s
solution (147 mM Na+, 4 mM K+, 2.3 mM Ca2+, 155.6 mMCl2) at a flow rate of 2.0 ml/min. To investigate DA and 5-HTrelease in the amygdala, we changed the perfusion solution from
Ringer’s to a high-K+ solution (147 mM Na+, 100 mM K+,
2.3 mM Ca2+, 155.6 mM Cl2). These dialysate samples werecollected with an Auto Injector, and to measure DA and 5-HT ona real-time basis, put in the HPLC-ECD system described above
every 20 min. Following completion of the experiment, mice were
given an overdose of sodium pentobarbital (150 mg/kg) andtranscardially perfused with physiological saline, followed by 10%
buffered formalin. Brains were post-fixed in 10% bufferedformalin for 1 day to 1 week, preserved in 20% sucrose for 1day, frozen, and cut on a sliding microtome into 50 mm sections.
Every fourth section was collected in distilled water, mounted on asilane-coated slide, air-dried, and stained with thionine. The
accuracy of placements was then confirmed. This procedure wasdescribed previously [37]. Data are presented as means 6
standard error of the mean. Data were analyzed using one-way
analysis of variance (ANOVA). The post-hoc Tukey-Kramer test
was employed.

Monoamine content
Mice were decapitated under the sodium pentobarbital (50 mg/
kg i.p.) anesthesia and the brains were immediately removed. Eachbrain was rapidly frozen on dry ice. For assay of dopamine andserotonin, the frozen brains were divided into the following
regions: frontal cortex; striatum; amygdala; hippocampus; thalamus; hypothalamus; cerebellum; and brainstem. All samples werestored at 280uC until assay. Levels of the following monoamines
and metabolites were measured as described previously [35].
Briefly, each tissue sample was homogenized in 500 ml of 0.05 M
perchloric acid, containing isoproterenol (Sigma) as an internal
standard, then centrifuged for 5 min at 15,000 rpm at 4uC. Aftercentrifugation, supernatants were filtered through a 0.45 mm
membrane filter. The 100-ml aliquots thus obtained were injected
into a high performance liquid chromatography-electrochemical
detection (HPLC-ECD) system, comprised of an EP-300 liquid
chromatography pump (Eicom, Kyoto, Japan), a CA-5ODS
reversed-phase octadecylsilyl column (2.16150 mm; Eicom) witha mobile phase consisting of 80% sodium phosphate buffer, 20%
methanol, 700 mg/l sodium octanesulphonate, and 50 mg/l
EDTA (2 Na). This system’s detector (ECD-300 electrochemical
detector; Eicom) had a graphite working electrode set at +0.45 V
relative to an Ag/AgCl reference electrode. Use of the Auto
Injector (ESA-20: EICOM) enabled dopamine and serotonin to bemeasured without any sample decomposition or loss caused byoxidation.

Supporting Information
Figure S1 Nissl staining of sagittal brain sections ofwild-type (Wt) and Snap25S187A/S187A (Hm) mouse.
(TIF)
Video S1

Behavior of WT mouse in a cage box.

(WMV)Video S2 Behavior of Snap25S187A/S187A mouse in a cagebox. The homozygous mouse freeze very readily in response toenvironmental change.
(WMV)Video S3 Behavior of one WT and four Snap25S187A/S187A

mice (H-1 to H-4) on an elevated platform. All of these mice
were littermates having same experience of the behavior tests.
(WMV)

Acknowledgments
We thank Michael J. Seagar for critical reading of the manuscript.

In vivo microdialysis

Author Contributions

Mice were anesthetized with sodium pentobarbital (50 mg/kg
i.p.) for stereotaxic surgery. A guide cannula was inserted(1.34 mm posterior and 2.9 mm lateral to the bregma, at a depthof 5.8 mm from the bone surface). One week later, a microdialysis

Conceived and designed the experiments: MT. Performed the experiments: MK SY ES SW TS HM SA SI RK RS-M YN IN KI YK-F MY.
Analyzed the data: MK SY SW TS SI MY MT. Wrote the paper: MT.

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Neuroscience Letters 561 (2014) 182–187

Contents lists available at ScienceDirect

Neuroscience Lettersjournal homepage: www.elsevier.com/locate/neulet

Stress-induced phosphorylation of SNAP-25
Saori Yamamori a,∗ , Daichi Sugaya a , Yuuki Iida b , Hirotoshi Kokubo a , Makoto Itakura a ,Eiji Suzuki c , Masakazu Kataoka d , Hitoshi Miyaoka b , Masami Takahashi aaDepartment of Biochemistry, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan
Department of Psychiatry, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0380, Japanc
International University of Health and Welfare Atami Hospital, Atami, Shizuoka 413-0012, Japand
Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Nagano-shi, Nagano 380-8553, Japanbh i g h l i g h t s

g r a p h i c a l

a b s t r a c t• Cold-water restraint stress induces

SNAP-25 phosphorylation in themouse brain.
• SNAP-25 phosphorylation was highin stress-related brain regions.
• The extent of phosphorylation
increased with increasing amountsof stress.
• SNAP-25 phosphorylation is regulated through both central andperipheral mechanisms.

a r t i c l e

i n f oArticle history:
Received 24 September 2013
Received in revised form
25 November 2013
Accepted 20 December 2013
Keywords:
Protein kinase C
SNARE protein
Exocytosis
Presynaptic regulationa b s t r a c t
Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser187 , however thephysiological signiﬁcance of this phosphorylation event in brain function remains unclear. In the present
study, we found that SNAP-25 phosphorylation increased rapidly in the mouse brain following cold-water
restraint stress. Both basal and stress-induced phosphorylation of SNAP-25 were high in stress-related
brain regions, including the cerebral cortex, hippocampus, and amygdala, and the extent of phosphorylation increased with increasing amounts of stress. Intravenous administration of adrenaline increased
SNAP-25 phosphorylation, although stress-induced phosphorylation was still observed in adrenalectomized mice. These results indicate that SNAP-25 phosphorylation is regulated in a stress-dependent
manner through both central and peripheral mechanisms.
© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Synaptic transmission involves the release of neurotransmitters from presynaptic terminals by exocytosis. Protein kinases
both activate and inhibit neurotransmitter release in various
contexts [25,26] and are important regulators of synaptic plasticity underlying higher brain functions, such as learning and

Abbreviations:
HPA-axis, hypothalamic-pituitary-adrenal-axis; SNAP-25,
synaptosomal-associated protein of 25 kDa.
∗ Corresponding author. Tel.: +81 42 778 8626; fax: +81 42 778 8441.
E-mail address: yamamori@med.kitasato-u.ac.jp (S. Yamamori).

memory. Synaptosomal-associated protein of 25 kDa (SNAP-25),
a t-SNARE protein, plays an essential role in neurotransmitter
release [18]. In several secretory cell types, Ser187 in the Cterminus of SNAP-25 is phosphorylated by protein kinase C (PKC)[5,12,22,24]. Moreover, PKC activation enhances the exocytotic
release of hormones in endocrine cells by recruiting secretory
vesicles to the plasma membrane [13], or by increasing the sizeof the readily releasable vesicle pool [12,24]. Notably, SNAP25 phosphorylation at Ser187 is essential for these effects [12].
SNAP-25 is phosphorylated at Ser187 in brain tissue as well
[3,5,7,27], although the involvement of this phosphorylation eventin the regulation of neurotransmitter release is controversial[1,17,23].

0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neulet.2013.12.044

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Although SNAP-25 phosphorylation is dramatically altered inepileptic brains, its status in healthy animals is unclear. In thepresent study, we found that SNAP-25 phosphorylation is dynamically regulated by stress in stress-related regions of the mouse brain
by at least two different mechanisms.
2. Materials and methods
2.1. Antibodies
Anti-SNAP-25 C-terminus [13], anti-phospho-Ser187 -SNAP-25[5], and anti-phospho-Ser120 -SNAP-23 [28] antibodies were prepared as previously described.
2.2. Animals
All experiments, were performed with 9–13 week-old male
C57BL/6N (CLEA, Japan) mice. For further details, see Supplementary methods.
2.3. Stress treatments
Cold-water restraint stress. Mice were placed into 50-ml plastic
centrifuge tubes with 8 small holes drilled at the top and bottom of
the tubes, and were submerged horizontally in water at 18 ± 1 ◦ C
up to the level of the clavicle for the indicated durations. To observe
recovery from stress, some mice were returned to their home cages.
Restraint stress. Mice were placed into the same plastic tubes ofcold-water restraint stress, and were kept at room temperature for
30 min.
Tape-immobilization stress. Mice were ﬁxed on a table with adhesive tape in the supine position for 30 min.
Cold-water forced swim stress. Mice were forced to swim in apool (45 cm W × 26 cm D × 30 cm H) with water added to a heightof 15 cm at indicated temperatures for 30 min.
Unstressed control. All control mice were placed in home cages.

183

at about 30 min after exposure to stress, conﬁrming the potencyof the stress (Fig. 1A). We then examined the extent of Ser187
phosphorylation and total protein levels of SNAP-25 in the hippocampus before and after the stress. Even in stress-free control
mice, SNAP-25 phosphorylated at Ser187 was detected in hippocampal homogenates, indicating that a fraction of SNAP-25
is phosphorylated in vivo. SNAP-25 phosphorylation increased
rapidly in response to stress, with a time-course similar to thatof serum corticosterone. However, the amount of total SNAP-25
protein remained constant during the test period. When stressed
mice were returned to their home cages, SNAP-25 phosphorylation rapidly decreased and returned to basal levels within 60 min(Fig. 1B). Serum corticosterone levels also decreased, but to a lesser
extent than was observed with SNAP-25 phosphorylation. These
results indicate that SNAP-25 phosphorylation is dynamically regulated in vivo.
SNAP-23 is a homolog of SNAP-25 that is expressed ubiquitously
in various tissues. PKC phosphorylates SNAP-23 at several serine
residues, including Ser120 [28]. No signiﬁcant change was observedin SNAP-23 phosphorylation at Ser120 in response to cold-water
restraint stress (data not shown).
3.2. Regional differences in stress-induced SNAP-25
phosphorylation
Next, we compared levels of total SNAP-25 protein and SNAP-25
phosphorylation in various brain regions with or without coldwater restraint stress (Fig. 2). Although SNAP-25 protein levels
remained equal in all brain regions tested, SNAP-25 phosphorylation was high in brain regions involved in stress response, including
the cerebral cortex, hippocampus, and amygdala. Furthermore,while cold-water restraint stress further induced SNAP-25 phosphorylation in the cerebral cortex, hippocampus, and amygdala, nofurther elevation was observed in the hypothalamus or cerebellum.
3.3. Extent of SNAP-25 phosphorylation depends on degree ofstress

2.4. Sample preparation
Mice were decapitated and blood was collected, as needed.
Brains were quickly removed from the skull and placed in icecold phosphate buffered saline, separated into each region, andhomogenized in homogenization buffer (0.32 M sucrose, 5 mM
Hepes-NaOH, pH 7.4) supplemented with phosphatase inhibitors(Phos-stop, Roch). Homogenates were mixed with SDS sample
buffer, boiled at 100 ◦ C for 10 min, and stored at −80 ◦ C until use
for immunoblotting. Sera were stored at −80 ◦ C until use.
2.5. Immunoblotting
They were performed as previously described [3] (see Supplementary methods).

If SNAP-25 phosphorylation occurs as a step in the stressresponse, a correlation should exist between the degree of stressand extent of SNAP-25 phosphorylation. Tape-immobilizationstress is a stronger stress stimulus than restraint stress [20].
SNAP-25 phosphorylation was not signiﬁcantly increased by acuterestraint stress in a small tube, whereas a marked increase
was observed after tape-immobilization stress (Fig. 3A). In the
cold-water forced swim test, the strength of stress increases
with decreasing water temperature. SNAP-25 phosphorylation
increased in mice forced to swim as the water temperature wasdecreased (Fig. 3B). These results collectively show a correlation
between the extent of SNAP-25 phosphorylation and degree ofstress.
3.4. Central and peripheral regulation of SNAP-25
phosphorylation in the brain

2.6. Statistics
Multiple comparisons were performed using one-way or twoway analysis of variance (ANOVA) with either Tukey–Kramer’s orDunnett’s post hoc correction. P < 0.05 was considered signiﬁcant.
3. Results
3.1. Cold-water restraint stress induces SNAP-25 phosphorylationin the mouse brain
Mice were subjected to cold-water restraint stress. Serum corticosterone levels increased rapidly and reached a maximal level

163

Adrenaline is released from the adrenal medulla in response tovarious stress stimuli. Intraperitoneal administration of adrenaline
markedly induced SNAP-25 phosphorylation at Ser187 in various brain regions, indicating that SNAP-25 phosphorylation could
be regulated, at least in part, through feedback regulation byadrenaline in peripheral tissues (Fig. 4). Although peripherallyadministrated adrenaline induced SNAP-25 phosphorylation in
the brain (Fig. 4), it was possible that cold-water restraint stressinduced SNAP-25 phosphorylation via a mechanism that did not
involve peripheral feedback. To test this possibility, we examined stress-induced SNAP-25 phosphorylation in adrenalectomized

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S. Yamamori et al. / Neuroscience Letters 561 (2014) 182–187

Fig. 1. Stress-induced phosphorylation of SNAP-25 in the hippocampus and changes in serum corticosterone levels after subjecting mice to cold-water restraint stress. (A)Mice were subjected to cold-water restraint stress for the indicated durations. Protein amounts and phosphorylation levels of SNAP-25 in the hippocampus were determinedby immunoblotting with anti-SNAP-25 antibody and anti-phospho-Ser187 -SNAP-25 antibody, respectively. Serum corticosterone levels were determined by ELISA. (Upper)
Typical immunoblots. (Lower) Quantiﬁcation of SNAP-25 phosphorylation (circles) and serum corticosterone levels (triangles) before (open symbols) and after (closed
symbols) stress. SNAP-25 phosphorylation was quantiﬁed densitometrically by dividing the band intensity value for SNAP-25 phosphorylation by the value for total SNAP-25
protein. Values are expressed relative to that of control mice. (B) Mice were subjected to cold-water restraint stress for 30 min and then returned to their home cages.
Protein amounts, phosphorylation levels of SNAP-25 in the hippocampus, and serum corticosterone levels were determined and were represented as in (A). Values represent
mean ± SE (A, control, n = 4; others, n = 3; B, 90 min, n = 4; others, n = 3). **, p < 0.01 and *, p < 0.05 comparing with and without stress, ## , p < 0.01 and # , p < 0.05 comparing
before and after returning mice to their home cages, by one-way ANOVA with Tukey–Kramer’s post hoc correction.

mice. Cold-water restraint stress signiﬁcantly increased serum
corticosterone levels in sham-operated mice, but not in adrenalectomized mice (data not shown). In contrast, cold-water restraint
stress induced SNAP-25 phosphorylation in both sham-operated
mice and adrenalectomized mice (Fig. 5). These results indicatethat a direct pathway in the brain, which is not downstream of theperipheral feedback system, links stress to SNAP-25 phosphorylation. We also found that stress-induced SNAP-25 phosphorylation
was signiﬁcantly higher in adrenalectomized mice compared to
sham-operated mice only in the hippocampus (two-way repeatedmeasures ANOVA interaction effect between stress and adrenalectomy, p = 0.0044; main effect of stress, p < 0.0001; main effectof adrenalectomy, p = 0.015). A small but statistically signiﬁcanceincrease of SNAP-25 phosphorylation was observed in cerebellumof adrenalectomized mice. The reason is not clear at the present,
however, prolonged effects of sham-operation may affect on the
response in cerebellum.
4. Discussion
Fig. 2. Basal and stress-induced SNAP-25 phosphorylation in various brain regions.
Mice were subjected to cold-water restraint stress for 30 min. Various brain regions
were isolated from either controls (−) or mice subjected to cold-water restraint
stress for 30 min (+). Protein amounts and phosphorylation levels of SNAP-25 were
determined by immunoblotting with anti-SNAP-25 antibody and anti-phosphoSer187 -SNAP-25 antibody, respectively. (Upper) Typical immunoblots. Cx, cerebral
cortex; HC, hippocampus; Amy, amygdala; HT, hypothalamus; CB, cerebellum.
(Lower) Quantiﬁed results of SNAP-25 phosphorylation with (closed columns) orwithout (open columns) stress. The values were normalized to those of SNAP25 amounts in each region, and were expressed as relative values to that in the
hippocampus of control mice. The result represents a typical example of three independent experiments. Values represent mean ± SE (n = 3). **, p < 0.01 comparing withand without stress, by one-way ANOVA with Tukey–Kramer’s post hoc correction.

In previous studies, SNAP-25 phosphorylation was induced
using artiﬁcial techniques including PKC activator-like phorbol
esters in cultured cells and electrical stimulation in hippocampal
slice cultures. In mice, however, only epileptic seizures have successfully induced SNAP-25 phosphorylation. In the present study,
we demonstrate for the ﬁrst time that SNAP-25 phosphorylation
is increased in healthy animals subjected to physical stress. This
phosphorylation event was speciﬁc to SNAP-25, as SNAP-23, aubiquitously expressed SNAP-25 homolog, was not phosphorylated under the same conditions. Basal and stress-induced SNAP-25
phosphorylation was high in brain regions suggested to be involvedin stress response, and a correlation was found between the164

S. Yamamori et al. / Neuroscience Letters 561 (2014) 182–187

185

Fig. 3. Extent of SNAP-25 phosphorylation by degree of stress. (A) Mice were restrained either in a plastic tube or with adhesive tape for 30 min at room temperature. Protein
amounts and phosphorylation levels of SNAP-25 in the hippocampus of either control or stressed mice were determined by immunoblotting with anti-SNAP-25 antibody
and anti-phospho-Ser187 -SNAP-25 antibody, respectively. Values were normalized to those of SNAP-25 amounts and were represented relative to control mice. (B) Mice
were subjected to the forced swim test for 30 min at 18, 30 or 35 ± 1 ◦ C, and protein amounts and phosphorylation levels of SNAP-25 in the hippocampus were determined
and represented as in (A). Values represent mean ± SE (n = 3). **, p < 0.01 and *, p < 0.05 compared to control unstressed mice, by one-way ANOVA with Dunnett’s post hoc
correction.

extent of SNAP-25 phosphorylation and degree of stress. Collectively, these results suggest that SNAP-25 phosphorylation plays
an important role in stress response in the brain.
When the brain senses stress, the HPA-axis and sympathetic nervous system are activated, leading to glucocorticoid andFig. 4. Intraperitoneal injection of adrenaline increases SNAP-25 phosphorylationin the mouse brain. Adrenaline (0.5 mg/kg; ip) was intraperitoneally administered
to mice. After 30 min, brain regions were isolated from mice receiving either saline(−) or adrenaline (+). Protein amounts and phosphorylation levels of SNAP-25 were
determined by immunoblotting with anti-SNAP-25 antibody and anti-phosphoSer187 -SNAP-25 antibody, respectively. (Upper) Typical immunoblots. Cx, cerebral
cortex; HC, hippocampus; Amy, amygdala; HT, hypothalamus; CB, cerebellum.
(Lower) Quantiﬁed results of SNAP-25 phosphorylation with (closed columns) orwithout (open columns) administration of adrenaline. Values were normalized tothose of SNAP-25 amounts in each region, and were expressed as relative values tosaline-injected control mice. Values represent mean ± SE (n = 3). **, p < 0.01 and *,p < 0.05 compared to control mouse, by one-way ANOVA with Tukey–Kramer’s post
hoc correction.

165

adrenaline release from the adrenal glands into the blood. The
released glucocorticoid and adrenaline affect not only peripheral
cells, but also the brain by feedback mechanisms. This suggeststhat there are two possible pathways for regulating stress-induced
SNAP-25 phosphorylation in the brain—one is direct (i.e., without
participation of peripheral mechanisms) and the other is a feedback
mechanism from the peripheral system. Our results support bothmechanisms. For example, peripherally-administered adrenaline
increased SNAP-25 phosphorylation in the brain. Since adrenaline
does not cross the blood-brain barrier, adrenaline likely exerted
its effect on the brain by activating the vagus nerve [11]. On theother hand, adrenalectomized mice also exhibited stress-induced
SNAP-25 phosphorylation, which would suggest that peripheral
feedback is not essential for SNAP-25 phosphorylation. While wefound an interaction effect between stress and adrenalectomyin the hippocampus, no such effect was found in the amygdala.
The hippocampus and amygdala play reciprocal roles in stressresponse; the former plays an inhibitory role, while the latter plays
an excitatory role. Thus, the interaction effect between stress andadrenalectomy in the hippocampus might be attributable to thecharacteristic properties of the hippocampus in stress response.
However, further studies will be needed to elucidate the functionalroles of SNAP-25 phosphorylation in stress response in the brain.
We previously showed that serotonin and dopamine release aremarkedly reduced in the amygdala of Snap-25S187A/S187A mutant
mice [8]. In secretory cells, the activation of PKC by phorbol
ester treatment induces SNAP-25 phosphorylation at Ser187 andenhances catecholamine and peptide hormone release fromthese cells [5,12,22,24]. However, in hippocampal glutamatergic
neurons, SNAP-25 phosphorylation was dispensable for phorbol
ester-induced enhancement of synaptic transmission [1,17]. Taken
together, these results suggest that SNAP-25 phosphorylation is
selectively involved in the regulation of monoamine release in
the mouse brain. Various stressors induce monoamine release in
speciﬁc brain regions [4,16,29]. Thus, it is possible that SNAP-25
phosphorylation participates in the stress response by regulating
monoamine release in the brain. SNAP-25 is a multifunctional
protein that plays a role in several processes unrelated to186

S. Yamamori et al. / Neuroscience Letters 561 (2014) 182–187

Fig. 5. Basal and stress-induced SNAP-25 phosphorylation in the brain of adrenalectomized mice. Adrenalectomized (ADX) and sham-operated (Sham) mice were restrainedin cold water for 30 min. Various brain regions were isolated from either control mice (−) or mice subjected to cold-water restraint stress for 30 min (+). Protein amounts
and phosphorylation levels of SNAP-25 were determined by immunoblotting with anti-SNAP-25 antibody and anti-phospho-Ser187 -SNAP-25 antibody, respectively. (Upper)
Typical immunoblots. Cx, cerebral cortex; HC, hippocampus; Amy, amygdala; HT, hypothalamus; CB, cerebellum. (Lower) Quantiﬁed results of SNAP-25 phosphorylation with(closed columns) or without (open columns) stress. Values were normalized to SNAP-25 levels in each region, and were expressed as relative values to that in sham-operated
control mice. Values represent mean ± SE (n = 3 per group). **, p < 0.01 and *, p < 0.05 compared to control mice, by two-way ANOVA with Tukey–Kramer’s post hoc correction.
A signiﬁcant interaction effect was observed in the hippocampus between stress and adrenalectomy (p = 0.0044).

neurotransmitter release, such as neurite extension [14], incorporation of ion channels and receptors into the plasma membraneby exocytosis [9,10,15,21], and regulation of ion channel function[2,6,19]. At least some of these processes are considered to be
regulated by SNAP-25 phosphorylation at Ser187 [9,10,15,21]. This
raises the possibility that SNAP-25 phosphorylation may modulate
the function of stress-related neurons via mechanisms independent of monoamine release. Further studies will be necessary toaddress these possibilities.
Acknowledgements
This work was supported by: Grants-in-Aid 18300127 and
21300141 for Scientiﬁc Research (B), 24700385 for Young Scientists (B), and 21650091 for Challenging Exploratory Research fromthe Japan Society for the Promotion of Science, and a Grant-in-Aid
for Core Research for Evolutional Science and Technology from theJapan Science and Technology Agency.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.neulet.2013.
12.044.
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Mol Neurobiol (2015) 52:1210–1222
DOI 10.1007/s12035-014-8923-2

Stimulation of Sigma-1 Receptor Ameliorates Depressive-like
Behaviors in CaMKIV Null Mice
Shigeki Moriguchi & Hiroyuki Sakagami & Yasushi Yabuki &Yuzuru Sasaki & Hisanao Izumi & Chen Zhang & Feng Han &Kohji Fukunaga

Received: 4 August 2014 / Accepted: 6 October 2014 / Published online: 15 October 2014
# Springer Science+Business Media New York 2014

Abstract Sigma-1 receptor (Sig-1R) is a molecular chaperone regulating calcium efflux from the neuronal endoplasmic
reticulum to the mitochondria. Calcium/calmodulindependent protein kinase IV (CaMKIV) null mice exhibit
depressive-like behaviors and impaired neurogenesis asassessed by bromodeoxyuridine (BrdU) incorporation into
newborn cells of the hippocampal dentate gyrus (DG). Here,
we demonstrate that chronic stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for
14 days improves depressive-like behaviors in CaMKIV nullmice. By contrast, treatment with paroxetine, which lacks
affinity for Sig-1R, did not alter these behaviors. Reduced
numbers of BrdU-positive cells and decreased brain-derived
neurotrophic factor (BDNF) mRNA expression and protein
kinase B (Akt; Ser-473) phosphorylation seen in the DG of
CaMKIV null mice were significantly rescued by chronic Sig1R stimulation. Interestingly, reduced ATP production observed in the DG of CaMKIV null mice was improved bychronic Sig-1R stimulation. Such stimulation also improved
hippocampal long-term potentiation (LTP) induction andmaintenance, which are impaired in the DG of CaMKIV nullmice. LTP rescue was closely associated with both increases

in calcium/calmodulin-dependent protein kinase II (CaMKII)
autophosphorylation and GluA1 (Ser-831) phosphorylation.
Taken together, Sig-1R stimulation by SA4503 or
fluvoxamine treatment increased hippocampal neurogenesis,
which is closely associated with amelioration of depressivelike behaviors in CaMKIV null mice.
Keywords Sigma-1R . CaMKIV null mice . Depressive-likebehaviors . Hippocampal neurogenesis
Abbreviations
AktProtein kinase B
BDNF
Brain-derived neurotrophic factor
BrdU
Bromodeoxyuridine
CaMKII Calcium/calmodulin-dependent protein kinase II
CaMKIV Calcium/calmodulin-dependent protein kinase IV
CREB
cAMP-responsive element binding protein
DG
Dentate gyrus
DHEA
Dehydroepiandrosterone
ERK
Extracellular signal-regulated kinase
ER/SR
Endoplasmic/sacroplasmic reticulum
fEPSPsField excitatory postsynaptic potentials
HFS
High-frequency stimulation
LTP
Long-term potentiation
NMDAR N-methyl-D-aspartate receptor
Sig-1R
Sigma-1 receptor
SSRIs
Selective serotonin reuptake inhibitors

S. Moriguchi (*) : Y. Yabuki : Y. Sasaki : H. Izumi :K. Fukunaga (*)
Department of Pharmacology, Graduate School of Pharmaceutical
Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai,
Miyagi 980-8578, Japane-mail: shigeki@m.tohoku.ac.jp
e-mail: kfukunaga@m.tohoku.ac.jp

Introduction

H. Sakagami
Department of Anatomy, Kitasato University School of Medicine,
Sagamihara, Japan

Depressive-like behavior is believed to be associated with
impaired adult neurogenesis in the subgranular zone of the
hippocampal dentate gyrus (DG) [1]. Restricted exposure of
the hippocampus to X-irradiation blocks DG neurogenesis

C. Zhang : F. Han
Department of Pharmacy, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, China

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Materials and Methods

and compromises the ability of anti-depressants to improve
depressive behaviors [2]. Human postmortem brain studies
reveal that chronic treatment with tricyclic anti-depressantssuch as imipramine increases the number of neural progenitor
cells in the DG in humans with major depression disorders [3].
Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine or fluvoxamine also improve impaired adult hippocampal neurogenesis in the rodent DG [2, 4]. Notably, SSRIs,
including fluoxetine and fluvoxamine, and imipramine
show an affinity for the sigma-1 receptor (Sig-1R) [5].
Knockout mice lacking Sig-1R exhibit depressive-likebehaviors [6]. We also recently reported that impaired
cognitive-related and depressive-like behaviors in olfactorybulbectomized mice are improved by chronic oral administration of dehydroepiandrosterone (DHEA), an endogenous
Sig-1R ligand [7, 8].
Sig-1R has been cloned in humans and several species[9–13]. In the brain, Sig-1R is widely distributed in bothneurons and in glial cells such as oligodendrocytes, and is
especially enriched in the prefrontal cortex, hippocampus, andstriatum [14, 15]. Sig-1R mainly localizes in membranes ofthe endoplasmic/sarcoplasmic reticulum (ER/SR) where it
regulates Ca2+ signaling through the inositol 1,4,5-triphosphate receptor in ER membranes closely associated withmitochondria [16, 17]. Sig-1R stimulation increases releaseof the neurotransmitters dopamine and glutamate [18, 19].
However, the mechanism underlying this activity remains
unclear.
Calcium/calmodulin-dependent protein kinase IV(CaMKIV) is a serine-threonine protein kinase activated by
nuclear Ca2+ elevation and which mediates phosphorylationof cyclic AMP-responsive element binding protein (CREB) at
residue Ser-133 [20, 21]. CREB phosphorylation by CaMKIV
regulates expression of several genes (including BDNF) thatfunction in synaptic plasticity [22], learning and memory[23–25], and emotional behaviors in rodents [26–28].
CaMKIV is widely distributed in the anterior cingulate cortex,
somatosensory cortex, insular cortex, cerebellum, hippocampus, and amygdala, where the kinase is primarily localized toneuronal nuclei [29]. Accumulating evidence demonstrates
that CaMKIV null mice display deficits in contextual andcued fear conditioning memory [30], decreased anxiety-likebehaviors [30, 31], and impaired memory of eyeblink conditioning [32]. More importantly, treatment with the typical
SSRI fluoxetine fails to induce DG neurogenesis and lacksof its anti-depressive activity in CaMKIV null mice [33].
Based on these findings, we concluded that CaMKIV nullmice showing resistance to SSRI treatment constitute a usefulmodel of refractory depression in which to test therapeuticstrategies to improve these behaviors. Using novel therapeutics of SA4503 or fluvoxamine, we found that Sig-1R stimulation rescues impaired neurogenesis and improvesdepressive-like behaviors in these mice.

Generation of CaMKIV Null Mice CaMKIV null mice were
established by Takao et al. [30]. To disrupt the CaMKIV gene,
the exon containing the CaMKIVα initiation codon was replaced with a neomycin resistance cassette. Southern blot
analysis with a 3′ external probe confirmed homologous recombination in CCE embryonic stem (ES) cells derived fromthe 129/SvJ mouse strain. A more detailed protocol used toestablish these mice is provided in our previous study [30].
Adult (8–9 weeks old) male CaMKIV null mice were housedin cages with free access to food and water at a constanttemperature (23±1 °C) and humidity (55±5 %) with a 12-h
light/dark cycle (09:00–21:00 hours). All experimental animal
procedures were approved by the Committee on Animal Experiments at Tohoku University and Kitasato University.
Depressive-Like Behavioral Tasks (1) Tail suspension task: A
detailed protocol of this task is described [8]. The test is basedon the fact that normally, animals exhibit immobile posturewhen subjected to short-term, inescapable stress by tail suspension. (2) Forced swim task: The forced swim task is
described elsewhere [8]. Mice are placed individually in glass
cylinders (height, 20 cm; diameter, 15 cm) filled with 25 °C
water and the duration of immobile periods within a 5-min test
is scored.
Memory-Related Behavioral Tasks (1) Y-maze task: The Ymaze task is described elsewhere [7]. Spontaneous alternation
behavior in a Y-maze is assessed as an indicator of spatialreference memory. The apparatus consists of three identicalarms (50×16×32 cm) made of black Plexiglas. A mouse isplaced at the end of one arm and allowed to move freely
through the maze during an 8-min session to score the alteration behavior. (2) Novel object recognition task: A detailed
protocol of this task has been reported [7]. The task is based on
the tendency of normal rodents to discriminate a familiar froma new object. Mice are individually habituated to an openfield box (35×25×35 cm) for two consecutive days. The
experimenter scoring behavior is blinded to the treatment.
During the acquisition phases, two objects of the same material are placed in a symmetric position in the center of thechamber for 5 min. One hour later, one object is replaced by anovel object, and exploratory behavior is again analyzed for
5 min to score the number of approaching to two objects. (3)Step-through passive avoidance task: This task is described
elsewhere [7]. Briefly, training and retention trials are conducted in box that consists of dark (25×25×25 cm) and light(14×10×25 cm) compartments. The floor is constructed ofstainless steel rods; those in the dark compartment are connected to an electronic stimulator (Nihon Kohden, Tokyo,
Japan). Mice are habituated to the apparatus the day before
avoidance acquisition. On training trials, a mouse is placed in

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Mol Neurobiol (2015) 52:1210–1222

vibratome (Dosaka EM Co. Ltd., Kyoto, Japan, or Leica
VT1000S, Nussloch, Germany). Coronal brain sections werepermeabilized with 0.3 % Triton X-100 in PBS and blockedwith 5 % normal goat or donkey serum for 3 h and thenincubated overnight with mouse anti-NeuN monoclonal antibody (1: 500) (Millipore), anti-Sigma-1R polyclonal antibody(1:500) (Abcam), a rat anti-bromodeoxyuridine (BrdU) polyclonal antibody (1:500) (Accurate Chemical and Scientific,
Oxford Biotechnology, Oxfordshire, UK), anti-PSD95 monoclonal antibody (1:500) (Abcam), anti-Synaptophysin monoclonal antibody (1:500) (Sigma-Aldrich), anti-CaMKIV polyclonal antibody (1:500 [30]), anti-BLBP monoclonal antibody(1:500, BLBP-GF-Af291, Frontier Institute Co., Ltd., Hokkaido, Japan), anti-PSA-NCAM monoclonal antibody (1:500[37]), or anti-calbindin polyclonal antibody (1:500, Santa
Cruz Biotech, Santa Cruz, CA) in blocking solution at 4 °C.
After thorough washing in PBS, sections were incubated 3-hin Alexa 488-labeled anti-rat IgG (anti-BrdU) or Alexa 594labeled anti-mouse IgG (anti-NeuN). After several PBS
washes, sections were mounted on slides with Vectashield(Vector Laboratories, Burlingame, CA, USA). Immunofluorescent images were analyzed using a confocal laser-scanningmicroscope (Nikon EZ-C1, Nikon, Tokyo, Japan or LSM 710,
Zeiss, Oberkochen, Germany). To count BrdU and NeuN
double-positive cells after immunohistochemistry, six hippocampal sections were cut every 50 μm beginning at 1.7 to
2.2 mm caudal to the bregma. The number of BrdU/NeuN or
BrdU/β-catenin double-positive cells in the DG region wasdetermined in a 300×300-μm area in a section. In the DG, theGCL (approximately 50 μm wide) and the SGZ (defined as azone two cell bodies wide (5 μm) along the border of the GCL
and hilus) were quantified together. The number of
BrdU/NeuN double-positive cells counted per mouse wasexpressed as the number of the double-positive cells per a
300×300-μm area. Six sections per mouse and six mice percondition were used. The person responsible for cell counts
was blind to experimental conditions.

the lighted compartment of the box. When the mouse enters
the dark compartment, the door is closed and the animal
receives an inescapable electric shock (1 mA for 500 ms) fromthe floor. The mouse is removed from the apparatus 30 s later.
The same procedure without footshock is repeated 24 h laterto assess retention level.
Electrophysiology Hippocampal slices were prepared as described [34]. Transverse hippocampal slices (400 μm thick)cut using a vibratome (Microslicer DTK-1000) were incubated for 2-h in continuously oxygenized (95 % O2, 5 % CO2)
artificial cerebrospinal fluid at room temperature. Then, slices
were transferred to an interface recording chamber and perfused at a flow rate of 2 ml/min with artificial cerebrospinal
fluid warmed to 34 °C. Field excitatory postsynaptic potentials were evoked by a 0.05-Hz test stimulus through a bipolarstimulating electrode placed on the perforant path from theentorhinal cortex and recorded from the inside granule cell
layer using a glass electrode filled with 3 M NaCl. Highfrequency stimulation (HFS) of 100 Hz with a 1-s duration
was applied twice with a 10-s interval, and test stimulation
was continued for the indicated periods. After recording,slices were transferred to a plastic plate cooled on ice to dissect
out the DG, which was immediately frozen in liquid nitrogenand stored at −80 °C until biochemical analysis was
performed.
Biochemical Analysis Analysis was performed as described[34] using the following antibodies: anti-phospho CaMKII(1:5000 [35]), anti-CaMKII (1:5000 [36]), anti-phosphoGluA1 (Ser-831) (1:1000, Millipore, Billerica, MA, USA),anti-GluA1 (1:1000, Millipore), anti-phospho-Akt (Ser-473)(1:1000, Millipore), anti-Akt (1:1000, Millipore), antiphospho-MAP kinase (diphosphorylated extracellular signalregulated kinase (ERK) 1/2) (1:2000, Sigma-Aldrich, St. Louis, MO, USA), anti-MAP kinase (1:1000, Millipore), antiCREB (Ser-133) (1:1000, Millipore), anti-CREB (1:1000,
Millipore), anti-sigma-1R (1:1000, Abcam, Cambridge, MA,
USA), and anti-β-tubulin (1:5000, Sigma-Aldrich). Bound
antibodies were visualized using the enhanced chemiluminescence detection system (Amersham Life Science, Buckinghamshire, UK) and analyzed semiquantitatively using the
National Institutes of Health Image program.

ATP Assay DG samples were dissected, rapidly frozen inliquid nitrogen and stored at −80 °C before use. ATP measurement was performed using an ATP assay kit (Toyo Ink,
Tokyo, Japan), according to the manufacturer’s protocol.
Briefly, frozen samples were homogenized in homogenate
buffer (0.25 M sucrose, 10 mM HEPES-NaOH, pH 7.4),
and lysates were cleared by centrifugation at 1000g for
10 min at 4 °C. The supernatant was collected, and supernatant proteins were solubilized in extraction buffer. After
30 min, buffer containing luciferin was added to each sample
and oxyluciferin was detected using a luminometer (GeneLight 55, Microtec, Funabashi, Japan).

Immunohistochemistry Immunohistochemistry was performed as described [8, 30]. Mice at 10–12 weeks of age wereanesthetized with sevoflurane and perfused via the ascending
aorta with phosphate-buffered saline (PBS; pH 7.4) until theoutflow became clear. The perfusate was then switched tophosphate buffer (pH 7.4) containing 4 % paraformaldehyde
in 0.1 M phosphate buffer (pH 7.4) for 15 min. The brain wasremoved, postfixed in the same solution for 2 h at 4 °C,
embedded in 2 % agarose and sliced at 50 μm using a

Real-Time PCR Quantification of BDNF mRNA Analysis was
performed in 48-well plates (Mini Opticon Real Time PCR

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1R antagonist NE100 (1 mg/kg, i.p.) eliminated the effects ofeither fluvoxamine or SA4503 in CaMKIV null mice (NE100
plus fluvoxamine-treated CaMKIV null mice, 165.1 ±11.1 s, n=5; NE100 plus SA4503-treated CaMKIV null
mice, 162.3±9.9 s, n=5) (Fig. 1a).
CaMKIV null mice also showed significantly increased immobility time relative to wild-type mice in aforced swimming task (wild-type mice, 44.7±2.5 s, n=5;
CaMKIV null mice, 86.7 ± 8.5 s, n = 5). Chronic
fluvoxamine or SA4503 treatment, but not treatment withparoxetine, significantly ameliorated decreased immobility time (fluvoxamine-treated CaMKIV null mice, 62.0±
4.9 s, n=5; SA4503-treated CaMKIV null mice, 64.8±
4.6 s, n = 5) (Fig. 1b). Pretreatment with NE100(1.0 mg/kg, i.p.) significantly canceled the effects of
fluvoxamine or SA4503 treatments in CaMKIV nullmice (NE100 plus fluvoxamine-treated CaMKIV null
mice, 92.5 ± 10.2 s, n = 5; NE100 plus SA4503-treated
CaMKIV null mice, 96.1±7.3 s, n=5) (Fig. 1b).
Notably, CaMKIV null mice showed normal memoryrelated behaviors, as assessed by Y-maze (n=6) (Fig. 1c, d),novel object recognition (n=6) (Fig. 1e) or passive avoidancetasks (n=6) (Fig. 1f, g).

System, Bio-Rad) using iQ SYBR Green Supermix 2× (BioRad). Primer sequences used were: BDNF-IF (CCTGCATC
TGTTGGGGAGAC), BDNF-IR (GCCTTGTCCGTGGACGTTTA), BDNF-IVF (CAGAGCAGCTGCCTTGATGTT), BDNF-IVR (GCCTTGTCCGTGGACGTTTA),GAPDH-F (TGTGTCCGTCGTGGATCTGA), and
GADPH-R (CACCACCTTCTTGATGTCATCATAC). Relative quantities of target transcripts were determined by thecomparative threshold cycle (ΔCT) method and normalized
to GAPDH. Product purity and specificity were confirmed byomitting the template and performing a standard melting curve
analysis.
Other Chemicals Paroxetine, fluvoxamine, DL-2-amino-5phosphonovaleric acid (APV), nifedipine, and BrdU
were purchased from Sigma-Aldrich (Tokyo, Japan).
NE-100 was purchased from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA, USA). SA4503 was synthesizedin the Laboratory of Medical Chemistry, Zhejiang University(Hangzhou, China).
Data Analysis Comparison between two experimentalgroups was made using the unpaired Student’s t test.
Statistical significance for differences among groups wastested by one-way analysis of variance (ANOVA),
followed by multiple comparisons between control andother groups using Scheffe’s test by StatView (Hulinks, Inc).
P<0.05 was considered significant.

Fluvoxamine or SA4503 Treatment Ameliorates Decreased
Hippocampal Neurogenesis in CaMKIV Null MiceTo address mechanisms underlying anti-depressive effects of fluvoxamine and SA4503, we evaluated hippocampal neurogenesis in CaMKIV null mice. Mice were
injected with BrdU on the first day of drug treatment forfive consecutive days during 2 weeks treatment withdrugs. To identify BrdU-positive neuronal cells, hippocampal slices were double-stained with antibodies to
BrdU and the neuronal marker NeuN. As compared towild-type mice (Fig. 2a), the number of BrdU-positive
neuronal cells significantly decreased in CaMKIV nullmice (wild-type mice, 104.8±2.4 cells, n=8; CaMKIV
null mice, 71.8± 3.1 cells, n=8) (Fig. 2a, b). Chronic
treatment with fluvoxamine (2.5 mg/kg) or SA4503(0.3 mg/kg), but not paroxetine at 1.0 mg/kg, for 14 days
significantly increased the number of BrdU/NeuN
double-positive cells in CaMKIV null relative to wildtype mice (fluvoxamine-treated CaMKIV null mice,
111.1 ± 2.5 cells, n = 8; SA4503-treated CaMKIV null
mice, 92.4±2.3 cells, n=8) (Fig. 2a. b). Pretreatment of
CaMKIV null mice with NE100 (1.0 mg/kg i.p.) eliminated the effects of fluvoxamine or SA4503 in CaMKIV
null mice (NE100 plus fluvoxamine-treated CaMKIV
null mice, 66.2±3.8 cells, n=8; NE100 plus SA4503treated CaMKIV null mice, 59.8 ± 6.3 cells, n = 8)
(Fig. 2a, b).

Results
Fluvoxamine or SA4503 Treatment Ameliorates
Depressive-like Behaviors in CaMKIV Null MiceTo determine whether Sig-1R stimulation improvesdepressive-like behaviors in CaMKIV null mice, we tested
the effects of the SSRI fluvoxamine, which has a high affinity
for Sig-1R, the Sig-1R agonist SA4503, and paroxetine, an
SSRI that lacks Sig-1R affinity, in these mice. Mice weretreated chronically with paroxetine at 1.0 mg/kg, p.o.,
fluvoxamine at 2.5 mg/kg, p.o., or SA4503 at 0.3 mg/kg,
p.o. for 14 days and then subjected to a tail suspension task.
Immobility time was significantly increased in CaMKIV nullcompared to wild-type mice (wild-type mice, 127.8±5.1 s, n=5; CaMKIV null mice, 172.8±7.9 s, n=5), indicative ofdepressive-like behavior in the null group. Interestingly,
chronic treatment with fluvoxamine or SA4503, but not withparoxetine, significantly decreased immobility time
(fluvoxamine-treated CaMKIV null mice, 130.4±7.3 s, n=5;SA4503-treated CaMKIV null mice, 122.4±8.8 s, n=5)
(Fig. 1a). Pretreatment of CaMKIV null mice with the Sig-

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Fig. 1 Fluvoxamine or SA4503 but not paroxetine improves depressivelike behaviors in CaMKIV null mice. a–b Immobility time as determinedby tail suspension task (a) or forced swim task (b) were measured
following chronic treatment with paroxetine at 1.0 mg/kg, p.o.,
fluvoxamine at 2.5 mg/kg, p.o., or SA4503 at 0.3 mg/kg, p.o. for 14 days.
Fluvoxamine or SA4503 significantly rescued increased immobility time
seen in CaMKIV null mice, an effect blocked by pretreatment ofNE100 at 1.0 mg/kg, i.p. (Mann-Whitney U test, n=5 per group). c–dLocomotor activity as assessed by total arm entries and alternation in a Ymaze task were measured in CaMKIV null and wild-type (WT) mice. No

differences in either were seen in CaMKIV null versus wild-type mice. eShown are results from a novel object recognition task. There were nodifferences in discrimination of a novel object seen between CaMKIV
null and wild-type mice. f–g Shown is latency time in a passive avoidancetask. CaMKIV null and wild-type mice showed no significant differences.
Vertical lines show the S.E.M. **p<0.01 versus the wild-type mice.
++
p<0.01 versus CaMKIV null mice. #p<0.05 versus fluvoxamine-treated CaMKIV null mice. †p<0.05 versus SA4503-treated CaMKIV nullmice

Fluvoxamine or SA4503 Treatment Improves Decreased
CREB and Akt Phosphorylation, CaMKIIαAutophosphorylation and BDNF Expression in the DG
of CaMKIV Null Mice

increased phosphorylation of Akt (Ser-473) and CREB (Ser133) in the DG (NE100 plus fluvoxamine-treated CaMKIV
null mice: Akt (Ser-473), 51.2±2.8 % of control, n=4; CREB
(Ser-133), 32.1±6.0 % of control, n=4; NE100 plus SA4503treated CaMKIV null mice: Akt (Ser-473), 48.6±3.4 % ofcontrol, n=4; CREB (Ser-133), 31.8±6.4 % of control, n=4)
(Fig. 3a, b).
By contrast, phosphorylation of ERK (Thr-202/Tyr-204) inthe DG of CaMKIV null mice was unchanged compared towild-type mice. However, SA4503 treatment (0.3 mg/kg) significantly increased ERK phosphorylation (ERK, 160.4±
9.3 % of control, n=4) and increased ERK phosphorylation
was inhibited by NE100 (1.0 mg/kg) treatment (ERK, 94.9±
3.1 % of control, n=4) (Fig. 3c, d). Interestingly, CaMKIIα(Thr-286) autophosphorylation and GluA1 (Ser-831) phosphorylation were significantly decreased in the DG of
CaMKIV null mice (CaMKIIα (Thr-286), 57.4±3.5 % ofcontrol, n=4; GluA1 (Ser-831), 54.86±2.5 % of control,n=4) (Fig. 3c, d). Chronic treatment with fluvoxamine(2.5 mg/kg) or SA4503 (0.3 mg/kg), but not paroxetine(1.0 mg/kg), significantly rescued decreased CaMKIIα(Thr-286) autophosphorylation and GluA1 (Ser-831)
phosphorylation (fluvoxamine-treated CaMKIV null

Since activities of the phosphatidylinositol 3-kinase (PI3K)/
Akt pathway and ERK signaling are required for proliferationand maturation of neural progenitor cells [38], we assessed
those activities following fluvoxamine or SA4503-induced
hippocampal neurogenesis in the DG of CaMKIV null mice.
In those mice, phosphorylation of Akt (Ser-473) and CREB
(Ser-133) in the DG was markedly decreased compared towild-type mice (Akt (Ser-473), 60.0±7.2 % of control, n=4;
CREB (Ser-133), 31.5±4.9 % of control, n=4) (Fig. 3a, b).
Chronic treatment with fluvoxamine (2.5 mg/kg) or SA4503(0.3 mg/kg), but not with paroxetine (1.0 mg/kg), significantly
restored phosphorylation of Akt (Ser-473) and CREB (Ser133) in the DG (fluvoxamine-treated CaMKIV null mice: Akt(Ser-473), 86.7±7.9 % of control, n=4; CREB (Ser-133),52.9±2.6 % of control, n=4; SA4503-treated CaMKIV null
mice: Akt (Ser-473), 95.0±9.1 % of control, n=4; CREB
(Ser-133): 61.4±1.7 % of control, n=4) (Fig. 3a, b). Pretreatment with NE100 (1.0 mg/kg) completely eliminated172

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Fig. 2 Fluvoxamine or SA4503 but not paroxetine enhances hippocampal neurogenesis in CaMKIV null mice. a Confocal microscopy images
showing double staining for BrdU (green), NeuN (red) and merged
images in hippocampal slices from wild-type mice, CaMKIV null mice,paroxetine-treated CaMKIV null mice, fluvoxamine-treated CaMKIV
null mice, SA4503-treated CaMKIV null mice, NE100 plus
fluvoxamine-treated CaMKIV null mice and NE100 plus SA4503-treated
CaMKIV null mice. Mice were injected with BrdU on the first day ofdrug treatment for consecutive 5 days during 2 weeks treatment withdrugs. Mice were treated with paroxetine, fluvoxamine, or SA4503
treatments for 2 weeks (n=8). b Quantitative analyses of the number of
BrdU/NeuN double-positive cells in the DG (n=8). Vertical lines show
SEM. **p<0.01 versus wild-type mice. ++p<0.01 versus CaMKIV nullmice. ##p < 0.01 versus fluvoxamine-treated CaMKIV null mice.
††
p<0.01 versus SA4503-treated CaMKIV null mice

mice: CaMKIIα (Thr-286), 93.0±6.9 % of control, n=4;
GluA1 (Ser-831), 90.7±9.7 % of control, n=4; SA4503treated CaMKIV null mice, CaMKIIα (Thr-286), 93.9±
8.6 % of control, n=4; GluA1 (Ser-831), 99.3±4.8 % ofcontrol, n = 4) (Fig. 3c, d). Pretreatment with NE100(1.0 mg/kg) eliminated CaMKIIα (Thr-286) autophosphorylation and GluA1 (Ser-831) phosphorylation increased by fluvoxamine or SA4503 treatment (NE100
plus fluvoxamine-treated CaMKIV null mice: CaMKIIα(Thr-286), 52.5±1.7 % of control, n=4; GluA1 (Ser831), 57.7 ± 4.2 % of control, n = 4; NE100 plus
SA4503-treated CaMKIV null mice: CaMKIIα (Thr286), 50.8±2.5 % of control, n=4; GluA1 (Ser-831),54.1±3.1 % of control, n=4) (Fig. 3c, d).
We next examined levels of transcripts encoding brainderived neurotrophic factor (BDNF) in the DG. Since BDNF
exons I and IV required Ca2+ signal [39, 40], we assessed
levels of BDNF transcripts containing exons I and IV. In
CaMKIV null versus wild-type mice, expression levels of
BDNF mRNA containing those exons significantly decreasedin the DG (Exon I, 49.1±5.5 % of control, n=10; Exon IV,
65.5 ± 5.6 % of control, n = 10). Chronic treatment withfluvoxamine or SA4503, but not with paroxetine, restoredlevels of transcripts containing both exons in the DG(fluvoxamine-treated CaMKIV null mice: Exon I, 83.7±
6.1 % of control, n=10; Exon IV, 101.6±22.4 % of control,
n=10; SA4503-treated CaMKIV null mice: Exon I, 91.9±
7.3 % of control, n=10; Exon IV, 101.6±22.4 % of control,n=10) (Fig. 3e, f). Pretreatment with NE100 (1.0 mg/kg)
completely eliminated these effects (NE100 plus
fluvoxamine-treated CaMKIV null mice: Exon I, 51.0±
13.8 % of control, n = 10; Exon IV, 46.4 ± 5.3 % ofcontrol, n=10; NE100 plus SA4503-treated CaMKIV null
mice: Exon I, 50.4±23.8 % of control, n=10; Exon IV, 43.2±
5.0 % of control, n=10) (Fig. 3e, f).
Fluvoxamine or SA4503 Treatment Restores Decreased
Expression of Sig-1R and ATP Production in the DG
of CaMKIV Null Mice
Since we hypothesized that fluvoxamine and SA4503 elicit
anti-depressive effects through Sig-1R stimulation, as reported previously [8], we next assessed localization of Sig-1R
protein in the wild-type hippocampus. Hippocampal slices
were double-stained with antibodies against Sig-1R and
PSD95 or synaptophysin as postsynaptic or presynaptic

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Fig. 3 Fluvoxamine or SA4503
treatment but not paroxetine
rescues decreased
phosphorylation of CREB
(Ser-133), Akt (Ser-473) and
CaMKIIα (Thr-286)
autophosphorylation and reduced
BDNF mRNA expression in the
DG of CaMKIV null mice. a or c
Representative images of
immunoblots using antibodies
against phosphorylated CREB
(Ser-133), CREB, phosphorylated
Akt (Ser-473), Akt,
autophosphorylated CaMKII,CaMKII, phosphorylated GluA1(Ser-831), GluA1,
phosphorylated ERK (Thr-202/
Tyr-204), ERK and β-tubulin. bor d Quantitative analyses ofphosphorylated CREB (Ser-133),
phosphorylated Akt (Ser-473),
autophosphorylated CaMKIIα(Thr-286), phosphorylated GluA1(Ser-831) and phosphorylated
ERK (Thr-202/Tyr-204). e–f
Quantitative analyses of BDNF
mRNA containing exon I (e) orexon IV (f). Vertical lines show
SEM. *p<0.05, **p<0.01 versus
wild-type mice. +p<0.05,++
p<0.01 versus CaMKIV nullmice. #p<0.05, ##p<0.01 versus
fluvoxamine-treated CaMKIV
null mice. †p<0.0, ††p<0.01
versus SA4503-treated CaMKIV
null micemarkers, respectively. Although Sig-1R was reported to behighly expressed in astrocytes [41], Sig-1R was moderately
expressed in cell bodies of pyramidal neurons in hippocampal
regions CA3 and CA1 and faintly in DG granule cells. In all ofthese regions, Sig-1R colocalized with PSD95 immunoreactivity in postsynaptic regions and was expressed in somepresynaptic terminal co-expressing synaptophysin in the DG(Fig. 4a, b).
We next assessed Sig-1R protein levels in wild-type and
CaMKIV null mice by immunoblot. Sig-1R levels significantly decreased in the DG of CaMKIV null mice (61.5±10.1 %of control, n=4). Chronic treatment with fluvoxamine orSA4503, but not with paroxetine, significantly rescued decreased Sig-1R expression in the DG (fluvoxamine-treated

CaMKIV null mice, 99.9±9.7 % of control, n=4; SA4503treated CaMKIV null mice, 91.8±15.4 % of control, n=4).
Enhancement of Sig-1R expression by fluvoxamine orSA4503 was eliminated by NE100 treatment (1.0 mg/kg)(NE100 plus fluvoxamine-treated CaMKIV null mice,
53.9±6.9 % of control, n=4; NE100 plus SA4503-treated
CaMKIV null mice, 50.9±3.9 % of control, n=4) (Fig. 4c).
We previously reported that Sig-1R stimulation elicits
mitochondria-dependent ATP production [17]. Therefore, we
assessed ATP content in the DG of CaMKIV null mice. ATP
production significantly decreased in the DG of CaMKIV null
relative to wild-type mice, similar to Sig-1R expression levels(80.3 ± 1.2 % of control, n = 4). Chronic treatment withfluvoxamine or SA4503, but not with paroxetine, significantly

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Fig. 4 Fluvoxamine or SA4503
treatment but not paroxetine
restores decrease in Sig-1R and
ATP production in the DG of
CaMKIV null mice. a–b
Confocal microscopy images
showing double staining for
Sig-1R (green), PSD95 (a) orsynaptophysin (b) (red) andmerged images in hippocampal
slices. Far right columns showhigh magnification images of
boxed regions in adjacent image.
Scale bars, a and b, 50 μm in low
magnification and 20 μm in highmagnification images. c
Representative images of
immunoblots using antibodies
against Sig-1R and quantitative
analyses. d Quantitative analysesof ATP production. Vertical lines
show SEM. **p<0.01 versus
wild-type mice. ++p<0.01 versus
CaMKIV null mice. ##p<0.01
versus fluvoxamine-treated
CaMKIV null mice.††p<0.01
versus SA4503-treated CaMKIV
null mice

LTP in the DG, which lasted over 60 min (146.3±6.2 % ofbaseline at 60 min, n=5) (Fig. 5a–c). However, relative towild-type mice, markedly reduced LTP was observed in the
DG of CaMKIV null mice (122.0±5.8 % of baseline at
60 min, n=5) (Fig. 5a–c). SA4503 treatment significantly
improved LTP in the DG of CaMKIV null mice (150.2±
8.4 % of baseline at 60 min, n=5) (Fig. 5a–c). Enhancementof LTP by SA4503 was eliminated by NE100 treatment(1.0 mg/kg) (121.2± 7.1 % of baseline at 60 min, n=4)
(Fig. 5a–c).

restored decreased ATP production in the DG of CaMKIV
null mice (fluvoxamine-treated CaMKIV null mice, 104.7±
3.3 % of control, n=4; SA4503-treated CaMKIV null mice,
102.7±2.6 % of control, n=4) (Fig. 4d), and ATP production
enhanced by fluvoxamine or SA4503 was inhibited by NE100(1.0 mg/kg) (NE100 plus fluvoxamine-treated CaMKIV null
mice, 79.6±3.2 % of control, n=4; NE100 plus SA4503treated CaMKIV null mice, 69.7±1.1 % of control, n=4)
(Fig. 4d).
SA4503 Treatment Rescues Decreased Long-TermPotentiation in the DG of CaMKIV Null Mice

Elevation of CaMKIIα Autophosphorylation by SA4503
Treatment is Inhibited by APV in the DG of CaMKIV NullMice

We previously reported that stimulation of Sig-1R by chronic
DHEA treatment ameliorates impaired long-term potentiation(LTP) in the hippocampal DG of OBX mice [8]. Therefore, we
analyzed LTP in the DG of wild-type mice, CaMKIV null
mice, and CaMKIV null mice treated every day for 14 days
with SA4503. In control slices from wild-type mice, HFS(100 Hz, 2 trains) of the inside granule cell layer induced

We also examined that mechanism of elevation of CaMKII
activity by Sig-1R stimulation in the DG of CaMKIV nullmice. In hippocampal DG slices of CaMKIV null mice, acutetreatment with SA4503 (1 μM) significantly restored downregulated CaMKIIα (Thr-286) autophosphorylation relative

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Fig. 5 SA4503 ameliorates impaired LTP seen in the DG of CaMKIV
null mice. a Representative field excitatory postsynaptic potentials(fEPSPs) were recorded from the DG of wild-type mice, CaMKIV null
mice, SA4503-treated CaMKIV null mice, and SA4503 plus NE100treated CaMKIV null mice. SA4503 treatment significantly improved
impaired LTP in CaMKI null mice. b–c Changes in fEPSP slope following HFS are shown in wild-type mice, CaMKIV null mice, SA4503-

treated CaMKIV null mice, and SA4503 plus NE100-treated CaMKIV
null mice. d Representative images of immunoblots using antibodies
against autophosphorylated CaMKII and CaMKII. e Quantitative analyses of autophosphorylated CaMKIIα (Thr-286). Vertical lines show
SEM. *p<0.05; **p<0.01 versus wild-type mice. +p<0.05; + +p<0.01
versus CaMKIV null mice. #p<0.05 versus SA4503-treated CaMKIV
null mice

to wild-type mice (CaMKIV null mice, 62.8±4.3 % of baseline, n=4; SA4503-treated CaMKIV null mice, 82.0±3.8 % ofbaseline, n=4). The elevation of CaMKIIα (Thr-286) autophosphorylation by SA4503 was significantly inhibited by
APV treatment at 50 μM (69.7±3.1 % of control, n=4) but
not by nifedipine treatment at 10 μM (Fig. 5d, e).

Discussion
Phenotypes of Sig-1R null mice are associated with cognitive
deficits, and anxiety- and depressive-like behaviors [6]. Since
impaired adult hippocampal neurogenesis in DG correlateswith depressive-like behaviors in OBX mice [7, 8] and Sig1R null mice showed impaired adult hippocampal
neurogenesis in DG [44], we hypothesized that depressivelike behaviors in CaMKIV null mice are associated with lostor impaired Sig-1R function in hippocampal DG and thatparoxetine-resistant depression may be improved by treatment
with Sig-1R agonists.
Consistent with previous studies showing that fluoxetineand fluvoxamine enhance adult hippocampal neurogenesis in
the rodent DG [2, 4], we observed that fluvoxamine treatment
ameliorated decreased neurogenesis seen in CaMKIV nullmice. Surprisingly, the potent SSRI paroxetine failed to stimulate neurogenesis with concomitant lack of anti-depressive
effects. The order of inhibitory constants (Ki) for inhibition ofserotonin uptake into rat brain is: paroxetine (Ki =0.7 nM)>

CaMKIV Colocalizes with PSA-NCAM and Calbindin
but not with a Glial Marker in the DG
Finally, we assessed whether CaMKIV is expressed inimmature neurons in addition to mature granule cells in
the DG. To do so, we double-stained hippocampal slices
with antibodies to CaMKIV and anti-BLBP (a marker ofradial glia and astrocytes), anti-PSA-NCAM (a markerof newly generated immature granule cells), and anticalbindin (a marker of mature granule cells) [37, 42,43]. CaMKIV was expressed in both mature and immature granule cells in the DG not in either neural stem
cells or astrocytes in the DG (Fig. 6).

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Fig. 6 CaMKIV colocalizes with
PSA-NCAM and calbindin but
not with the glial marker BLBP in
the DG. Confocal microscopy
images showing double
immunofluorescence staining of
the adult DG for CaMKIV (green)and BLBP, PSA-NCAM, or
calbindin (red). Merged images
show nuclear staining with 4′,
6-diamidino-2-phenylindole
dihydrochloride (DAPI) (blue).
Scale bars, 500 μm in low
magnification and 10 μm in highmagnification images[44] cannot be explained due to a low affinity for Sig-1R. Our
hypothesis that fluvoxamine-induced anti-depressive action in
CaMKIV null mice is mediated by Sig-1R stimulation was
confirmed by the observation that the Sig-1R-specific agonist
SA4503 ameliorates impaired adult hippocampal
neurogenesis in DG and depressive behaviors in CaMKIV
null mice.
Enhanced adult hippocampal neurogenesis is associatedwith activation of both the PI3K/Akt [8, 47, 48] andCREB/BDNF pathways [8, 47]. Activation of both pathways

citalopram (K i = 2.6 nM) > sertraline (K i = 3.4 nM) >fluvoxamine (Ki =6.2 nM)>fluoxetine (Ki =14 nM) [45]. On
the other hand, the order of affinity of SSRIs for the sigma-1
receptor is: fluvoxamine (Ki =36 nM)>sertraline (Ki =57 nM)>fluoxetine (Ki =120 nM)>citalopram (Ki =292 nM)>paroxetine (Ki =1893 nM) [46]. Thus, the lack of an anti-depressive
effect by paroxetine in CaMKIV null mice suggests that Sig1R stimulatory action rather than serotonin reuptake inhibition
accounts for fluvaxamine’s anti-depressive action. However,lack of amelioration by fluoxetine as reported by Sha et al.

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may alter their proliferation. However, the total number of
granule cells in the DG was comparable in wild-type and
CaMKIV null mice. We previously documented that reduced
CaMKII activity in CaMKIIα heterozygous knockout miceincreased the number of immature granule cells in the hippocampal DG but decreased the number of mature granule cells[51]. Moreover, the number of BrdU-positive cells slightly
increased CaMKIIα heterozygous knockout mice [51]. Thus,
CaMKIV but not CaMKIIα likely functions in proliferation or
maturation of granule cells in the mouse DG. However, further studies are required to define the mechanism underlying
decrease in Akt activity and neurogenesis in CaMKIV null
mice.
Interestingly, Sig-1R stimulation enhances N-methyl-D-aspartate receptor (NMDAR)-evoked intracellular calcium mobilization [18, 52, 53]. In fact, the elevation of CaMKII
activity by SA4503 was eliminated by NMDAR antagonist.
The regulation of NMDAR activity by Sig-1R was also reported in Sig-1R null mice in which adult hippocampal
neurogenesis is impaired via downregulation of NMDAR
subunit NR2B [44]. Martina et al. [54] reported that a Sig1R agonist inhibits small-conductance calcium-activated potassium channel in the hippocampus and consequently increased Ca2+ mobilization by an NMDAR-dependent mechanism. We here confirmed that SA4503 activation of Sig-1R
increases CaMKII activity through NMDAR stimulation,
thereby ameliorating the impaired hippocampal LTP seen in
the DG of CaMKIV null mice. Akt activation by fluvoxamineand SA4503 is likely mediated by tyrosine kinase signaling
associated with NMDAR activation [55] or NMDARdependent BDNF expression through CaMKII signalingin neuronal progenitors plays an essential role in cell proliferation and maturation [38]. In the present study, stimulation of
Sig-1R by fluvoxamine or SA4503 markedly activated both
PI3K/Akt and CREB/BDNF signaling in DG of CaMKIV
null mice. Although Akt and CaMKII phosphorylation wasclosely associated with CREB (Ser-133) phosphorylation,
CaMKII likely accounts for the CREB phosphorylation and
BDNF expression. This idea is also confirmed by the observation that expression of BDNF mRNA containing exons I or
IV in the DG of CaMKIV null mice is upregulated by Sig-1R
stimulation. Indeed, Sun et al. [49] reported that unlike
CaMKIV, CaMKII regulates CREB activity through additional phosphorylation of CREB at Ser-142 residue (in addition toSer-133). We also confirmed that CaMKII overexpression
increases levels of BDNF transcripts containing exon IV in
NG108-15 cells [50]. However, mechanisms underlying
CaMKII activation by Sig-1R stimulation remain unclear.
Stimulation of Sig-1R increases calcium transport from ER/
SR to mitochondria, which in turn enhances ATP production[17]. Rescued ATP production may improve Ca2+ storage in
ER by enhancing activity of the sarcoplasmic/endoplasmic
Ca2+-ATPase pump, which could promote Ca2+-induced
Ca2+-release from the ER and in turn activate CaMKII
activity in neurons.
Mechanisms underlying decreased neurogenesis seen in
CaMKIV null mice are also largely unknown. As shown
Fig. 6, CaMKIV is expressed ubiquitously in pyramidal neurons in both CA1 and CA3 regions and in DG granule cells.
Interestingly, CaMKIV is expressed both in immature neurons
stained with PSA-NCAM and in mature neurons positive forcalbindin in the DG. Loss of CaMKIV in neural progenitors

Fig. 7 Schematic representationof altered adult hippocampal
neurogenesis in the DG.
Stimulation of Sig-1R increases
intracellular calcium mobilization
through NMDARs in the plasma
membrane or through the ER/SR
via mitochondrial ATP
production. Increased
intracellular calcium increases
CaMKII autophosphorylation andpromotes CREB (Ser-133)
phosphorylation and BDNF
expression, which in turnincreases Akt phosphorylationand promotes adult hippocampal
neurogenesis

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References[56]. In addition, an essential role of link between Akt and
CREB activities has been demonstrated in neural progenitor
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NMDAR and Akt signaling likely promote BDNF transcription, which would increase Akt phosphorylation via TrkB
(Fig. 7). CaMKII and Akt signaling possibly ameliorate
depressive-like behaviors through enhanced neurogenesis in
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Acknowledgments This work was supported in part by grants from the
Ministry of Education, Culture, Sports, Science and Technology, and theMinistry of Health and Welfare of Japan (22390109 to K.F.; 20790398 toS.M.), the Smoking Research Foundation (to K.F.), the Takeda Science
Foundation (to S.M.), the Suzuken Memorial Foundation (to S.M.), theYokoyama Foundation for Clinical Pharmacology (to S.M.) and theComprehensive Brain Science Network (CBSN).
Authors Contributions S.M., H.S., Y.S., N.I., and Y.Y. performed theexperiments. H.S. provided knockout mice. C.Z. and F.H. provided
materials. S.M. and K.F. wrote the manuscript and designed the study.
Conflicts of Interest
interests.

The authors declare no competing financial

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Roles of receptor activity-modifying protein 1 inangiogenesis and lymphangiogenesis during skinwound healing in mice
Chie Kurashige,*,† Kanako Hosono,* Hiromi Matsuda,*,† Kazutake Tsujikawa,‡
Hirotsugu Okamoto,† and Masataka Majima*,1*Department of Pharmacology and †Department of Anesthesiology, Kitasato University School of
Medicine, Sagamihara, Japan; and ‡Department of Immunology, Graduate School of Pharmaceutical
Sciences, Osaka University, Suita, Japan
ABSTRACT
Receptor activity-modifying protein 1(RAMP1) forms a complex with calcitonin receptor-likereceptor (CLR) to produce the receptor for calcitonin
gene-related peptide (CGRP). CGRP, a 37-aa neuropeptide, is widely distributed in neuronal tissues andexerts its biological effects via CLR/RAMP1; however,the pathophysiological roles of CLR/RAMP1 remain to
be clarified. To study the functions of CLR/RAMP1,
we generated RAMP1-knockout (RAMP1/) mice.
Compared with those of wild-type (WT) mice, woundhealing and wound-induced angiogenesis were significantly suppressed in RAMP1/ mice, with reduced
expression of vascular endothelial growth factor(VEGF)-A. Formation of the lymphatic vessels thatdrain interstitial fluids was also suppressed in
RAMP1/ mice, with reduced expression of VEGF-C
and VEGFR-3 in wound granulation tissues. RAMP1
was expressed in endothelial cells (ECs) in the preexisting skin blood vessels, but was not observed in ECs innewly formed blood or lymphatic vessels. Macrophages
in the wound granulation tissues expressed RAMP1 andproduced substantial amounts of VEGF-C in response
to CGRP in vitro. RAMP1/ bone marrow chimericmice showed delayed wound healing with reduced
angiogenesis/lymphangiogenesis in wound granulation
tissues. These findings suggest that RAMP1 plays acrucial role in wound healing and wound-induced angiogenesis and lymphangiogenesis and that it is a promising target for controlling angiogenesis and lymphangiogenesis.—Kurashige, C., Hosono, K., Matsuda, H.,
Tsujikawa, K., Okamoto, H., Majima, M. Roles ofreceptor activity-modifying protein 1 in angiogenesis
and lymphangiogenesis during skin wound healing in
mice. FASEB J. 28, 1237–1247 (2014). www.fasebj.org
Key Words: calcitonin gene-related peptide  CD31  VEGFR-3
 macrophages  vascular endothelial growth factor
Calcitonin gene-related peptide (CGRP) is a 37-aa
neuropeptide that is produced in the neural body of
dorsal root ganglion (DRG) cells and released fromsensory nerve endings. Receptor activity-modifying proteins (RAMPs) are a family of proteins that interact withand modulate the activities of several class B G-proteincoupled receptors (GPCRs). The RAMP protein family
consists of 3 members, RAMP1, RAMP2, and RAMP3,
each of which is encoded by a separate gene and has
diverse spatiotemporal expression patterns (1). CGRP
binds to CGRP receptors composed of RAMP1 andcalcitonin receptor-like receptor (CLR) to modulate
various functions, such as vasodilation and pain transmission (2, 3). CGRP couples to the activation of
adenylate cyclase (AC) to elevate cAMP levels; however,biochemical and pharmacologic studies in cultured
cells show that RAMPs can modulate additional aspectsof CGRP receptor signaling, including receptor trafficking, ligand binding affinity, second-messenger signaling, and receptor desensitization. Several gene-targeted
knockout and transgenic models have been generated
and characterized in recent years (4).
CLR is a common receptor subunit for both CGRP
and adrenomedullin (AM), a 52-aa peptide that sharessome degree of homology with CGRP (5). The specificity of the CGRP receptor is believed to be decided by
RAMP1, in light of the finding that the N terminus of
RAMP1 is essential for CGRP binding (6). To elucidate
the physiological functions of CGRP mediated throughAbbreviations: AC, adenylate cyclase; AM: adrenomedullin;
ANOVA, analysis of variance; bFGF, basic fibroblast growth
factor; BM, bone marrow; BMT, bone marrow transplantation; CGRP, calcitonin gene-related peptide; CLR, calcitoninreceptor-like receptor; CTGF, connective tissue growth factor;
DRG, dorsal root ganglion; EC, endothelial cell; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G-protein-coupled receptor; LYVE-1,
lymphatic vessel endothelial hyaluronan receptor-1; PBS,
phosphate-buffered saline; RAMP, receptor activity-modifying
protein; TGF, transforming growth factor; VEGF, vascularendothelial growth factor, WT, wild type1
Correspondence: Kitasato University School of Medicine,
Sagamihara, Kanagawa 252-0374, Japan. E-mail: mmajima@med.
kitasato-u.ac.jp
doi: 10.1096/fj.13-238998
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.

0892-6638/14/0028-1237 © FASEB

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Determination of expressions of Pro-CGRP, CGRP,
and RAMP1 in DRGs

CLR/RAMP1 receptors in vivo, we generated RAMP1deficient (RAMP1/) mice by using a conditional
gene-targeting technique. We discovered that thesemice exhibit potent hypertension, but their heart rate isunaffected (7). In addition, the RAMP1/ mice exhibit a significant increase in serum CGRP levels andmarkedly higher serum levels of proinflammatory cytokines after LPS administration, compared with theirwild-type (WT) counterparts. Dysregulation of proinflammatory cytokine levels in RAMP1/ mice is attributed to deficient signal transduction by CGRP in dendritic cells, suggesting that CLR/RAMP1 receptors
regulate not only blood pressure but also inflammatory
responses.
We have reported (8 –10) that gastrointestinal tracts
contain a large amount of CGRP and experience theprotective actions from the effects of ethanol, a harmful
necrotizing agent. Recently, we found that CGRP also
exhibits proangiogenic activity in models of cancer andhindlimb ischemia (11, 12). These studies suggested
that CGRP functions as an intermediate between vascular and neuronal systems under pathologic conditions.
Further, we identified increased levels of CGRP during
gastric ulcer healing that facilitate angiogenesis (13).
The establishment of a vascular supply is essential,
not only for organ development and differentiation
during embryogenesis, but also for the process ofwound healing (14). The formation of lymphatic vessels, or lymphangiogenesis, occurs during the development of the corpus luteum and during wound healing
and may be increased in certain pathologic processes(15). It typically occurs at sites of inflammation inducedby factors produced by macrophages. The process oflymphangiogenesis contributes to the reduction of tissue edema and activation of immune responses byfacilitating fluid drainage and the transport of dendritic cells and macrophages (16 –18). In the present
study, we used RAMP1/ mice to investigate the roleof RAMP1 in lymphangiogenesis during the healing ofskin wounds. The results showed that CLR/RAMP1
signaling plays a crucial role in wound healing andwound-induced angiogenesis and ymphangiogenesis
and that it presents a promising target for controllingthese processes in vivo.

The medulla spinalis was removed, and the DRGs of the L1–5
vertebrae were isolated. DRG tissues were homogenized in 1
ml of TRIzol reagent (Gibco-BRL; Life Technologies, Rockville, MD, USA). A sample of RNA was extracted from thetissue according to the manufacturer’s instructions. Real-time
PCR was performed as described below. The oligonucleotide
primers were as follows: for pro-CGRP, 5=-CCCCAGAATGAAGGTTACACA-3= (sense) and 5=-TGTCAAAGGGAGAAGGGTTTT-3=(antisense); for CGRP, 5=-CCCCAGAATGAAGGTTACACA-3=(sense) and 5=-TGTCAAAGGGAGAAGGGTTTT-3= (antisense);
and for GAPDH, 5=-CCCTTCATTGACCTCAACTACAATGGT-3=(sense) and 5=-GAGGGGCCATCCACAGTCTTCTG-3= (antisense).
Skin wound model and evaluation of the wound-healing
process
Surgical wounds were made on the backs of the mice, as
described elsewhere (19). In brief, after the mice wereanesthetized with pentobarbital sodium (50 mg/kg, i.p.), thedorsal hair was shaved, the exposed skin was cleaned with
70% ethanol, and full-thickness skin wounds of 6-mm diameter were made aseptically on either side of the dorsal
midline. The day of surgical treatment was defined as d 0.
Each wound region was digitally photographed at a fixed
time, and the wound areas were calculated by ImageJ image
analysis software (U.S. National Institutes of Health,Bethesda, MD, USA). Changes in wound area were expressed
as a percentage of the wound area at d 0.
Immunohistochemistry

MATERIALS AND METHODS

For immunohistochemistry, wound tissues were immediately
fixed with 4% paraformaldehyde and stained with anti-CD31
antibody (1:500 dilution) and anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1; 1:500 dilution), to identify blood and lymphatic vessels, respectively. Immune complexes were detected with an LSABSystem-HRP kit(DakoCytomation, Carpinteria, CA, USA). Wound-induced
angiogenesis and lymphangiogenesis in granulation tissues
were estimated by micro blood vessel density and lymphatic
vessel density and expressed as the number of vessels per
square millimeter.
For immunofluorescence, the primary antibodies usedwere anti-RAMP1 antibody (1:100 dilution), CD31 (1:500
dilution), CD11b (1:200 dilution), and S100A4 (1:200 dilution). Images were captured with a confocal scanning laser
microscope (LSM700; Zeiss, Jena, Germany), and computerassisted morphometric analyses were performed with ZEN
2008 software (Zeiss).

Animals

Collection and culture of peritoneal macrophages

RAMP1/ mice were developed as described elsewhere (7).
Male 8- to 10-wk-old C57BL/6 WT mice and RAMP1/ mice
were used in this study. RAMP1/ mice exhibit good health,show normal behavior, and have no visible phenotypes different from normal mice. All animals were housed individually
under a 12-h light-dark cycle at a constant temperature(251°C) and humidity (605%). All mice had free access to
tap water and rodent chow. All experiments were performedin accordance with the guidelines for animal experiments
established by the Kitasato University School of Medicine.

Peritoneal macrophages were obtained from 8- to 10-wk-old
WT and RAMP1/ mice that had received 2 ml of thioglycolate i.p. 3 d earlier. The macrophages were washed, resuspended, placed in 12-well culture plates (1106/well), andtreated for 24 h in RPMI 1640 medium (Cambrex, Baltimore,
MD, USA), containing 10% FBS at 37°C in 5% humidified
CO2. After 24 h, the plates were washed with phosphatebuffered saline (PBS) to remove nonadherent cells. Approximately 60% of the cells remained adherent and were used inall subsequent experiments.

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KURASHIGE ET AL.

Real-time RT-PCR

ton, DE, USA) was used to quantitate the intensity of thefluorescence of the extracted dye.

Transcripts encoding CD31, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGFR-3, epidermal growth factor(EGF), transforming growth factor (TGF)-, basic fibroblast
growth factor (bFGF), connective tissue growth factor(CTGF), and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were quantified by real-time RT-PCR analysis. Total
RNA was extracted from mouse tissues with TRIzol (GibcoBRL), and single-stranded cDNA was generated from 1 g of
total RNA via reverse transcription with ReverTra Ace-
(Toyobo, New York, NY, USA). Quantitative PCR amplification was performed with SYBR Premix Ex Taq (Takara
Bio, Shiga, Japan) and the following gene-specific primers: 5=-ACTTCTGAACTCCAACAGCGA-3= (sense) and
5=-CCATGTTCTGGGGGTCTTTAT-3= (antisense) for CD31;
5=-GAGAGAGGCCGAAGTCCTTT-3= (sense) and 5=-TTGGAACCGGCATCTTTATC-3= (antisense) for VEGF-A; 5=-TCTGTGTCCAGCGTAGATGAG-3= (sense) and 5=-GTCCCCTGTCCTGGTATTGAG-3= (antisense) for VEGF-C; 5=-CCTATTGACATGCTGTGGGAT-3= (sense) and 5=-GTGGGTTCCTGGAGGTAAGAG-3=(antisense) for VEGF-D; 5=-TTTATGTCCCACCCCCACTAC-3=(sense) and 5=-GGCTGAGCTACAAGGCAATCG-3= (antisense) for
VEGFR-3; 5=-ATGGGAAACAATGTCACGAAC-3= (sense) and 5=TGTATTCCGTCTCCTTGGTTC-3= (antisense) for EGF; 5=-AACAATTCCTGGCGTTACCTT-3= (sense) and 5=-TGTATTCCGTCTCCTTGGTTC-3= (antisense) for TGF-; 5=-GGCTGCTGGCTTCTAAGTGTG-3= (sense) and 5=-TTCCGTGACCGGTAAGTATTG-3= (antisense) for bFGF; 5=-AACCGGGGAGGGAAATTATAG-3= (sense) and 5=-TGGAATCAGAATGGTCAGAGG-3=(antisense) for CTGF; and 5=-ACATCAAGAAGGTGGTGAAGC-3=(sense) and 5=-AAGGTGGAAGAGTGGGAGTTG-3= (antisense)for GAPDH. The obtained threshold cycle (Ct) values were processed for further calculations according to the comparative Ct
method. Expression levels of target genes were normalized to thehousekeeping gene GAPDH, giving the Ct value. Finally, the gene
expression level was calculated as 2Ct, giving the final value thatwas normalized to that of the housekeeping gene.

Murine bone marrow transplantation (BMT) model
Bone marrow (BM) cells were obtained by flushing thecavities of freshly dissected femurs and tibiae from donor
male RAMP1/ mice with PBS. Donor BM cells were harvested from their WT counterparts (8-wk-old male C57BL/6
mice) by the same method. The flushed BM cells of eachdonor were dispersed by pipetting and resuspended in PBS ata density of 1  107 cells/ml. WT mice were lethally irradiatedat 9.0 Gy, with an MBR-1505R X-ray irradiator (Hitachi
Medico Co., Tokyo, Japan) with a filter (copper, 0.5 mm;aluminum, 2 mm) monitoring the cumulative radiation dose.
Donor BM mononuclear cells (2106 cells) in 200 l of PBS
were transplanted via the tail vein of irradiated WT mice.
After 12 wk, peripheral blood was collected, and RAMP1
expression was tested to confirm chimerism.
GFP transgenic mice (a gift from Dr. M. Okabe, Genome
Information Research Center, Osaka University, Osaka, Japan) with a C57BL/6 background were used as WT mice toconfirm the chimerism. The mice, in which 95% of theperipheral leukocytes exhibited GFP positivity in fluorescence-activated cell-sorting analysis, were used in this experiment as the control group.
Statistical analysis
Data are expressed as means  sem. Comparisons among
multiple groups were performed via factorial analysis ofvariance (ANOVA), followed by Scheffé’s test. Comparisons
between 2 groups were performed with the Mann-Whitney U
test. Values of P  0.05 were considered statistically significant.

RESULTS

RT-PCR analysis in macrophages

Suppression of wound-induced angiogenesis in
RAMP1/ miceThioglycolate-induced macrophages were incubated for 4 hwith CGRP (3 and 30 nM) and RPMI 1640 medium. Next, themacrophages were collected, and mRNA was isolated with
Trizol reagent (Life Technologies-Invitrogen, Carlsbad, CA,
USA). Expression levels of VEGF-A and VEGF-C mRNA inmacrophages were measured by real-time RT-PCR.

The RAMP1/ mice appeared healthy, and their small
size was the same as that of their WT counterparts. The
basal levels of mRNAs of pro-CGRP and CGRP in the
DRG tissues in the nontreated RAMP1/ mice were
not different from those in the WT (Fig. 1A, B),suggesting that the CGRP levels in the neuronal systemwere not reduced in RAMP1/. By contrast, theRAMP1 expressed in the DRGs was much lower in the
RAMP1/ mice in comparison with the WT (Fig. 1C).
These results suggest that RAMP1/ mice are goodtools for evaluating CGRP signaling in pathologic conditions.
Full-thickness skin wounds were surgically created on
the backs of WT mice, and the closure of the wounds
was monitored. The healing process observed in thismouse model occurred in a manner essentially thesame as the process reported previously. At 24 h after
wounding, the wound areas were reduced to 50% in thecontrol WT mice, and the wounds were completely
closed after 13 d. Invasion of granulation tissue fromthe wound margins into the wound bed was observed 2
d after injury. At d 3, the wound bed was filled

RT-PCR analysis in fibroblasts
Murine fibroblasts (L929) were obtained from the Cell Bank,RIKEN Bioresource Center (Ibaraki, Japan). The cells were
cultured in DMEM supplemented with 10% (v/v) FBS and
100 U/500 ml penicillin (Gibco-BRL) at 37°C in 5% humidified CO2. L929 fibroblasts (3105cells/well) were incubatedfor 6 h with CGRP (3 and 30 nM) and PBS, and the mRNA
expression levels of VEGF-A, VEGF-C, and VEGF-D in fibroblasts were determined by real-time RT-PCR.
Drainage of fluorescein isothiocyanate-dextran from woundareas
At 7 d after wounding, 200 l of 3% fluorescein isothiocyanate-dextran was injected s.c. into the wound granulation
tissue. At 2 h after the injections, axillary lymph nodes were
excised to measure the intensity of fluorescence drained fromthe wound area via newly formed lymphatic vessels. A NanoDrop 3300 fluorospectrometer (ThermoScientific, WilmingRAMP1 AND ANGIOGENESIS/LYMPHANGIOGENESIS

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pro-CGRP

CGRP

Figure 1. Reduced RAMP1 mRNA levels and delayed woundhealing in RAMP1/ mice. A–C) mRNA levels of pro-CGRP(A), CGRP (B), and RAMP1 (C) in DRGs. Data are expressed asmeans  sem of 4 mice/group. There was no difference
between 2 groups before wounding (A, B), whereas a marked
reduction in RAMP1 mRNA levels was seen in RAMP1/ mice(C). D) Time course of wound closure in RAMP1/ and WT
mice. Surgical wounds were made on the backs of RAMP1/
mice and their WT counterparts, and wound closure wasmonitored as described in Materials and Methods. Data areexpressed as means  sem of 8 mice/group. *P  0.05, **P 
0.01 vs. WT mice. E) Typical appearance of wounds in
RAMP1/ and WT mice at d 3, 5, and 7. Original diameter ofthe wounds was 6 mm.

completely with granulation tissue, and at this stage,
marked neovascularization was evident.
To evaluate the roles of endogenous CGRP-RAMP1
signaling in wound-dependent angiogenesis andwound closure, we created skin wounds in both
RAMP1/ mice and WT mice, and monitored thehealing process for 14 d. As shown in Fig. 1D, E, woundclosure in the RAMP1/ mice was significantly delayed compared with that in the WT mice, especiallyduring the early phase of the healing process.
At d 2, 3, 4, 5, 6, and 7, we compared the histologic
appearance of wounds in the WT and RAMP1/ mice.
Although immunostaining of CD31-positive microvessels indicated the presence of abundant microvessel
assemblies in the wound granulation tissues (Fig. 2B) of
the WT mice, few were observed in the RAMP1/
mice (Fig. 2B, C). Moreover, the RAMP1/ micedemonstrated significantly lower microvessel densities
throughout the observation periods than did the WT
mice (Fig. 2D). Real-time RT-PCR analysis also showedsignificantly lower CD31 and VEGF-A mRNA levels in
the RAMP1/ mice than in the WT mice during theearly phase of the healing process (d 2, 5, and 7 for
CD31; d 2 and 3 for VEGF-A, Fig. 2E, F). When theexpressions of other cytokines were determined in thewound granulation tissues, TGF-, bFGF, and CTGF
were also reduced in the early stage of wound healing
in the RAMP1/ mice, whereas there was no marked
reduction in EGF (Supplemental Fig. S1). These results
suggest that endogenous CGRP-RAMP1 signaling upregulates angiogenesis in the wound granulation tissues
by inducing the growth factors, including VEGF-A, andthereby facilitates wound healing.
To examine the localization of RAMP1 in the endothelial cells (ECs), we performed double immunostain1240

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ing of RAMP1 and CD31 (Fig. 2A). No RAMP1/CD31
double-positive vessels were observed in granulation
tissues, although they were present in uninjured regions around the wounds. This observation indicatesthat newly formed vessels in granulation tissues lack
RAMP1 expression. These results suggest that CGRPRAMP1 signaling does not play a direct role in forming
new vessels.
Cellular components relevant to CGRP-RAMP1
signaling-dependent angiogenesis
To identify the cells responsible for enhancing woundinduced angiogenesis via CGRP-RAMP1 signaling, we
evaluated macrophages and fibroblasts, the 2 major
components of wound granulation tissues. We performed double immunostaining of RAMP1 and CD11b,a macrophage marker (Fig. 3A), and of RAMP1 and
S100A4, an activated fibroblast marker (Fig. 3B) in
granulation tissues 7 d after wounding. A large numberof CD11b- and S100A4-positive cells were found to
express RAMP1. We then tested the in vitro effects of
CGRP on macrophages and fibroblasts. When mouse
fibroblasts (L929 cells) were treated with CGRP agonists, no increase in VEGF-A expression was observed(Fig. 3F). By contrast, VEGF-A expression increased inmacrophages isolated from the peritoneal cavities of
WT mice in a dose-dependent manner when treatedwith CGRP agonists (Fig. 3C). These results suggest that
CGRP-RAMP1 signaling up-regulates angiogenesis in
granulation tissues by inducing VEGF-A expression in
macrophages.
Furthermore, we tested whether lymphangiogenic
factors increase in response to CGRP agonists. Neither
VEGF-C nor VEGF-D production was significantly en-

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Figure 2. Inhibition of wound-induced angiogenesis in RAMP1/ mice. Surgical wounds were made on the backs of
RAMP1/ mice and their WT counterparts, and angiogenesis in the wound granulation tissues was monitored as described inMaterials and Methods.A) Double immunostaining of normal tissue in an uninjured region (left) and granulation tissue (right)
from WT mice at d 5 with antibodies against CD31 (red) and RAMP1 (green). Scale bars  100 m.B, C) Typical results ofhematoxylin and eosin staining (d 6; B) and CD31 immunostaining (d 2–7; C) of wound granulation tissues in RAMP1/ andWT mice. A reduced number of CD31-positive microvessels (brown stains indicated by red arrows) was observed in the
granulation tissues of RAMP1/ mice compared with that in WT mice. Scale bars  100 m.D) Changes in microvessel density
in granulation tissues at d 2–7. Tissues were stained for CD31 and photographed, and microvessels in the wound granulation
tissues were counted. Microvessel density is expressed as microvessels per square millimeter. Data are expressed as means  semfrom 6 mice/group; significantly different from WT mice at d 2–7. *P  0.05 vs. WT mice. E, F) mRNA expression levels of CD31
(E) and VEGF-A (F) in the wound granulation tissues of RAMP1/ and WT mice determined by real-time RT-PCR at d 0, 2,
3, 5, 7, 10, and 14. Data are expressed as means  sem of 6 mice/group. *P  0.05, **P  0.01 vs. WT mice.

to the lymphatic-specific marker LYVE-1 revealed fewer
LYVE-1-positive lymphatic structures in the RAMP1/
mice than in the WT mice (Fig. 4B, C). Moreover,quantitative analysis of lymphatic vessel density confirmed that wound-induced lymphangiogenesis in the
RAMP1/ mice was significantly reduced at d 6, 7, and10 compared with that in the WT mice (Fig. 4D).
Real-time RT-PCR analysis also showed significantly
lower VEGF-C and VEGFR-3 mRNA levels in the
RAMP1/ mice than in the WT mice from earlyphases of the healing process (Fig. 4E, F). These results
suggest that lymphangiogenesis in wound granulation
tissues is accelerated by VEGF-C and VEGFR-3 signalingfrom early stages and is dependent on endogenous

hanced in L929 cells (Fig. 3G, H). In macrophages,
VEGF-C expression increased, whereas VEGF-D expression did not (Fig. 3D, E). These results suggest that
CGRP-RAMP1 signaling up-regulates lymphangiogenesis in granulation tissues by inducing VEGF-C inmacrophages, but not in fibroblasts.
Suppression of wound-induced lymphangiogenesis in
RAMP1/ mice
On d 3, 5, 6, 7, and 10, we compared the histologic
appearance of the wounds in the WT and RAMP1/
mice, to evaluate lymphangiogenesis in granulation
tissues. Immunohistochemical analysis with an antibody
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Figure 3. Effects of CGRP on the expression of VEGF-A, VEGF-C, and VEGF-D in peritoneal thioglycolate-elicited macrophages
and mouse fibroblast L929 cells.A) Double immunostaining of granulation tissue from WT mice at d 7 with antibodies against
CD11b (red) and RAMP1 (green). Scale bar  10 m.B) Double immunostaining of granulation tissue from WT mice at d 7
with antibodies against S100A4 (red) and RAMP1 (green). Scale bar  20 m.C–E) Macrophages isolated from WT and
RAMP1/ mice were treated with CGRP agonists, and the mRNA levels of VEGF-A (C), VEGF-C (D), and VEGF-D (E) were
determined 4 h after incubation. Real-time quantitative RT-PCR assays were used to assess mRNA expression. Data are expressed
as means  sem of 3 independent experiments. *P  0.05 vs. vehicle. F–H) Mouse fibroblasts, L292 cells, were treated withCGRP agonists for 4 h. mRNA levels of VEGF-A (F), VEGF-C (G), and VEGF-D (H) were determined by real-time quantitative
RT-PCR assays. Data are expressed as means  sem of 3 independent experiments. CGRP agonists did not significantly increase
VEGF-A, VEGF-C, or VEGF-D production in L929 cells. *P  0.05 vs. vehicle.

CGRP-RAMP1 signaling. In addition, as shown in Fig.
4G, H, the drainage of fluorescent dye injected into thewound area into the regional lymph nodes (axillary
lymph nodes) was reduced in the RAMP1/ mice at d7, when lymphangiogenesis was suppressed in the mice.
Therefore, lymphangiogenesis appears to modulate
lymphatic flow in the granulation tissues with newly
formed lymphatic vessels via a CGRP-dependent mechanism.
To examine the localization of RAMP1 in lymphatic
ECs, we performed double immunostaining for RAMP1and LYVE-1 (Fig. 4A). No RAMP1 and LYVE-1 doublepositive vessels were observed in granulation tissues,
indicating that newly formed lymphatic vessels in granulation tissues do not express RAMP1. These results
suggest that CGRP-RAMP1 signaling does not directly
affect newly formed lymphatic vessels, rather, it upregulates lymphangiogenesis by inducing VEGF-C expression in macrophages.

sis and lymphangiogenesis. In comparison with micewith transplanted WT BM cells, mice with BM cells
transplanted from RAMP1/ mice demonstrated significantly delayed wound healing, especially during theearly phase of the healing process (Fig. 5A). A reduced
accumulation of RAMP1-postive cells was observed at d
7 after wounding in the wound granulation tissues ofmice with BM cells transplanted from RAMP1/ mice
(Supplemental Fig. S2A, B). Immunohistochemical
analysis with a CD31 antibody showed that microvessel
density at d 3 after wounding in mice with BM cells
transplanted from RAMP1/ mice was significantly
suppressed compared with mice receiving WT BM cells(Fig. 5B, C). Immunohistochemical analysis using an
LYVE-1 antibody revealed significantly decreased lymphatic vessel density at d 7 after wounding in mice with
BM cells transplanted from RAMP1/ mice, compared with mice receiving WT BM cells (Fig. 5D, E).
Histologically, GFP-positive cells accumulated in thewound granulation tissues in the WT mice with BM cells
transplanted from GFP-transgenic mice with a
C57BL/6 background, suggesting that transplanted BM
cells were recruited into the wound granulation tissues.
When we performed immunostaining of RAMP1,
CD11b, and S100A4 in the granulation tissues of micewith BM cells transplanted from GFP transgenic mice 7

Role of recruited BM cells expressing RAMP1 inwound healing
Recent results suggest that recruitment of hematopoietic cells from the BM plays a crucial role in woundhealing. We next examined the role of recruited BM
cells expressing RAMP1 in wound-induced angiogene1242

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Figure 4. Inhibition of wound-induced lymphangiogenesis in RAMP1/ mice. Surgical wounds were made on the backs of
RAMP1/ mice and their WT counterparts, and lymphangiogenesis in the wound granulation tissues was assessed, as describedin Materials and Methods.A) Double immunostaining of granulation tissues from WT mice at d 7 with antibodies against LYVE-1(red) and RAMP1 (green). Scale bar  50 m. B, C) Typical results of hematoxylin and eosin staining (d 7; B) and LYVE-1
immunostaining (d 3, 5, 6, 7, and 10; C) in the wound granulation tissues in RAMP1/ and WT mice. A lesser number of
LYVE-1-positive microvessels (brown stains indicated by red arrows) was observed in the granulation tissues of RAMP1/ micethan in WT mice. Scale bars  100 m.D) Changes in lymphatic vessel density in the granulation tissues at d 3, 5, 6, 7, and 10.
Tissues were stained for LYVE-1 and photographed, and lymphatic vessels in the wound granulation tissues were counted.
Lymphatic vessel density is expressed per square millimeter. Data are expressed as means  sem of 6 mice/group, significantly
different from WT mice at d 6, 7, and 10. *P  0.05 vs. WT mice.E, F) mRNA expression levels of VEGF-C (E) and VEGFR-3 (F)in the wound granulation tissues of RAMP1/ and WT mice, determined by real-time RT-PCR at d 0, 2, 3, 5, 7, 10, and 14. Data
are expressed as means  sem of 6 mice/group. *P  0.05, **P  0.01 vs. WT mice. G) Fluorescence images of axillary lymph
nodes 2 h after injection of 200 l of 3% fluorescein isothiocyanate– dextran into the wounds on d 7 with a microsyringe. Scalebars  5 mm. H) Quantification of the fluorescence of the extracted dye indicated that drainage from the wound area to theregional lymph nodes was reduced in RAMP1/ mice (n5 for RAMP1/; n10 for WT).

d after wounding, a large number of GFP and RAMP1
double-positive cells and GFP and CD11b doublepositive cells were observed in the tissues (Supplemental Fig. S2C, D). These results suggest that macrophages
from the BM are recruited into inflammatory lesions,where they appear to secrete VEGF-A and VEGF-C inresponse to CGRP stimulation, thereby contributing toangiogenesis and lymphangiogenesis.
In separate experiments, we confirmed that neuronalsystem-derived CGRP was relevant to RAMP1 stimulation during the healing process of this model. The
wound-healing processes and angiogenesis/
lymphangiogenesis in WT mice denervated either sur-gically or chemically, in which CGRP release to theperipheral tissues may be reduced, were suppressed incomparison with those in sham-surgery mice and vehicle-treated mice (Supplemental Fig. S3A–E). These
strengthen the significance of RAMP1 signaling in thewound-healing processes.
DISCUSSIONCGRP is a 37-aa neuropeptide released in response to
sensory stimuli that contributes to various biologicalprocesses, such as cardiovascular regulation and vaso-

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Figure 5. Time course of wound healing, angiogenesis, and lymphangiogenesis in mice with
BM cells transplanted from RAMP1/ mice. A
lethal dose of radiation was administered to WT
mice and BM cells from either WT mice or
RAMP1/ mice were injected into the tail vein
(WT¡WT or RAMP1/¡WT). Surgicalwounds were made on the backs of the mice,
and wound closure was assessed as described inMaterials and Methods.A) Time course ofwound closure in the WT mice transplantedwith BM cells from RAMP1/ or WT mice.
Data are expressed as means  sem from 8 mice/group. *P  0.05, **P  0.01 vs. WT mice. B) Typical results of CD31
immunostaining of the wound granulation tissues in the WT mice with BM cells transplanted from RAMP1/ or WT mice at
d 3. A lesser number of CD31-positive microvessels (brown staining indicated by red arrows) was observed in the granulation
tissues of RAMP1/ ¡ WT mice than in the WT ¡ WT mice. Scale bars  100 m.C) Microvessel density in granulation tissues
on d 3. Data are expressed as means  sem of 6 mice/group, significantly different from WT ¡ WT mice. **P  0.01 vs. WT¡ WT mice.D) Typical results of LYVE-1 immunostaining of the wound granulation tissues of WT mice with BM cells
transplanted from either RAMP1/ or WT mice at d 7. A lower number of LYVE-1-positive microvessels (brown staining
indicated by red arrows) was observed in the granulation tissues of RAMP1/ ¡ WT mice than in WT ¡ WT mice. Scale bars
 100 m.E) Lymphatic vessel density in granulation tissues on d 7. Data are expressed as means  sem of 6 mice/group,
significantly different from WT ¡ WT mice. **P  0.01 vs. WT ¡ WT mice.

dilation (20). Using the peptidic antagonist CGRP
(8 –37) and the antagonist BIBN4096BS, pharmacologic researchers have classified CGRP receptors into 2
subtypes: CGRP-1 and CGRP-2 (21, 22). Although more
CGRP receptor subtypes have been suggested, only one
receptor, CLR, which belongs to a large family of
GPCRs, has been identified molecularly (23). This
receptor of the B-family of GPCRs forms a heterodimer
with a single transmembrane domain protein, RAMP.
Three distinct RAMPs have been cloned and beenshown to determine the selectivity of the CRLRreceptor for the different members of the calcitonin/
CGRP family of peptides (24). Coexpression of CLRwith RAMP1 results in formation of the CGRP receptor,
whereas coexpression of CLR with RAMP2 or RAMP3
corresponds to the formation of AM1 or AM2 receptors, respectively (24). CGRP binds to the AM2 receptor
with 50-fold lower affinity than does AM and has aneven lower affinity for AM1 receptors (24). This suggests that RAMP1 is the key receptor for supporting
CGRP activity and that a RAMP1-null mouse model is agood tool for clarifying the roles of CGRP and RAMP1
in vivo.
In the present experiment, we used RAMP1/ mice
to define the roles of RAMP1 in angiogenesis andlymphangiogenesis during wound healing. As shown in
Fig. 2C–E, angiogenesis in the granulation tissues in thebase of the wounds was suppressed from the early stages
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WT counterparts. Expression of RAMP1 in vascular ECs
in granulation tissues in the WT mice was faint compared with the expression observed in establishedblood vessels in normal mouse skin. The potent hypotensive action of CGRP is explained by the stimulationof CGRP receptor in which RAMP1 commits as a
cofactor for the intracellular signaling.
The low level of RAMP1 expression in neovascularized blood vessels was a surprising observation. It has
been frequently reported that microvascular ECs display a large degree of functional heterogeneity, depending on their location in the vascular tree (25). The
existence of organ-specific, microvascular-bed-specific,
and even intravascular variations in endothelial cellgene expression emphasizes their high cell-to-cell variability, which allows them to adapt to changing conditions. Further, the ability of microvascular ECs torespond dynamically to pathology-related microenvironmental changes is particularly apparent with respect
to angiogenesis. The present study demonstrates thatthe site of CGRP activity and the localization of RAMP1
in neovascularized areas are different than in vascularECs. Newly formed blood vessels in a pathologic setting
may be quite different from the mature blood vessels
formed during development and tissue maintenanceand homeostasis.
The major cellular components of wound granulation tissues relevant to healing and angiogenesis are
fibroblasts and macrophages (19). Both fibroblasts and

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angiogenesis and is responsible for increasing VEGF
expression in hematopoietic cells such as macrophages(Fig. 3C, D), consistent with a previous in vitro report (34).
In the present study, we did not measure the amountof CGRP released. However, we have characterized therelease of CGRP in angiogenic responses under pathologic conditions in previous reports (8, 11–13). Healingof skin wounds in neonatal mice was suppressed bycapsaicin treatment, which depletes CGRP from sensory nerve endings (35). We had confirmed the effects
of chemical denervation in the WT mice used in thisstudy (Supplemental Fig. S3E). C fibers, which contain
CGRP in their nerve endings, may be relevant to
RAMP1-mediated neovascularization. In fact, surgicaldenervation of primary sensory neurons resulted in
reduced angiogenesis and lymphangiogenesis (Supplemental Fig. S3A–D). Further, in our previous reports,
CGRP enhanced the healing of gastric ulcers, with
substantial induction of angiogenesis in the ulcer basetissues (13), and recovery from hindlimb ischemia
induced by the ligation of femoral arteries was augmented by the release of CGRP from the sensory nerves(12). In these instances, it is plausible to hypothesizethat C fibers were stimulated to release CGRP by protonions secreted from the gastric mucosa or by ischemia,
suggesting that the release of CGRP in such pathologic
settings plays a significant role in enhancing angiogenesis. RAMP1 may be an element that is responsible for
CGRP released during pathologic processes.
The functional and morphologic relationships between primary afferent neurons and blood vessels inpathologic conditions, such as ischemia and cancer
development, are poorly understood. However, recent
evidence suggests that the physical and biochemicalinteractions between these peripheral components areimportant in tumor biology and cancer-associated pain(36). Angiogenesis is a critical determinant for tumor
growth, and an angiogenic switch, which allows thetumor to receive an adequate blood supply, is activatedin the quiescent vasculature around tumors, contributing to tumor growth and metastasis, and ultimately
decides a patient’s prognosis. The neuronal system
plays a fundamental role in the maturation of primitiveembryonic vasculature. Mutations that disrupt peripheral sensory nerves or Schwann cells prevent proper
arteriogenesis, whereas those that disorganize thenerves result in arteries with misrouted axons (37). It isalso reported that sensory neurons modulate the expression of arterial markers on ECs via the secretion of
VEGF 164/120. These data suggest that during development, peripheral nerves play a role in determining
organ-specific patterns of blood vessel branching andarterial maturation (38).
Some neuronal factors, such as Notch, are also
reported to contribute to pathologic angiogenesis. Specialized ECs called tip cells, which lead and guidesprouting, share many features with another guidance
structure, the axonal growth cone (39). Axonal growthcones and endothelial tip cells both respond to signals
originating from the same molecular families, such as

macrophages express RAMP1, but when cultured fibroblasts were incubated with CGRP, no increase in theexpression of VEGF isoforms was observed (Fig. 3F–H).
By contrast, when exposed to CGRP, macrophages
isolated from the mouse peritoneal cavity exhibited
increased expression of VEGF-A and VEGF-C, but not
VEGF-D, in a dose-dependent manner (Fig. 3C–E).
VEGF-A is active in this model, since the addition ofa VEGF neutralizing antibody or VEGF receptor tyrosine
kinase inhibitor strongly inhibits proangiogenic processes (26). Furthermore, VEGF-C is a potent inducerof lymphangiogenesis (27). LYVE-1-positive cells did
not express RAMP1, indicating that lymphatic ECs do
not express RAMP1 in the same manner as neovascularized vascular ECs. Newly formed lymphatics also
failed to exhibit RAMP1 expression, suggesting that theimportant cellular components are non-ECs, such as
macrophages. VEGF-C reduction was seen in the
RAMP1/ mice from the early stages of the healing.
Thus, it appears that lymphangiogenesis, as seen in thepresent study, may be regulated by VEGF-C released
from macrophages in the early stages.
The established lymphatic vessels observed in thisstudy effectively drained the interstitial fluids, as demonstrated by the movement of dye injected subcutaneously (Fig. 4G, H). AM induces lymphangiogenesis(28), and the presence of the AM receptor on lymphatic ECs is reported, suggesting that the cellularcomponents necessary for RAMP1 signaling are different from those needed for AM activity. Immunohistochemical staining revealed that RAMP1 expression colocalized with CD11b expression, suggesting thatmacrophages actively induce lymphangiogenesis in
granulation tissues in the wound base. This is the firstreport describing the involvement of RAMP1 expressedby macrophages in enhancing angiogenesis and lymphangiogenesis in vivo. Controlling RAMP1 signaling is agood option for the treatment of angiogenesis/
lymphangiogenesis-related pathologic conditions. In
addition, recruited macrophages were shown to be
crucial for angiogenesis/lymphangiogenesis, since
RAMP1/ BM chimeric mice exhibited decreased
angiogenesis and lymphangiogenesis in wound granulation tissues and showed delayed wound healing (Fig. 5).
Activation of the CGRP receptor results in Gsmediated activation of AC with a subsequent increase incAMP and activation of nitric oxide synthase (NOS),
both of which are closely related to vasodilatation(29 –32). We previously reported that cAMP elevation
up-regulated the expression of VEGF and enhanced
angiogenesis in a sponge implantation model (33).
Forskolin, which activates AC, enhanced angiogenesis,
and the phosphodiesterase inhibitor amrinone, which
induces the accumulation of intracellular cAMP, increased neovascularization in sponge granulation tissues (33). These intracellular signaling cascades havebeen shown to couple CGRP receptors related tophysiological responses, such as vasodilatation of bloodvessels. This study confirmed that CGRP receptor signaling relevant to cAMP elevation increased pathologic
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Slits and Roundabouts; Netrins and UNC5 receptors;
Semaphorins, Plexins and Neuropilins; and Eph receptors and ephrin ligands. Activation of the Notch pathway and VEGF signaling induces angiogenic growth of
ECs and the selection of tip cells, and Notch signaling
is a key pathway that is activated during ischemiainduced neovascularization (40). Indeed, Notch signaling regulates several steps of the reparative process inischemic tissues, including sprouting angiogenesis, vessel maturation, interaction of vascular cells with leukocytes, and skeletal myocyte regeneration. Together withfindings in our previous studies, the present results
suggest that CGRP has a major role in pathologic
angiogenesis (11–13). Thus, RAMP1 is also a major
player in these interactions. Thus, renewed focus
should be placed on the interactions that occur between the neuronal systems and the lymphatic systems
in pathologic conditions.
As discussed above, the findings in the present study
indicate that both angiogenesis and lymphangiogenesis
are enhanced by RAMP1 signaling in wound granulation tissues. RAMP1 was not expressed on blood vessel
ECs or lymphatic ECs during neovascularization.
RAMP1 signaling was relevant to the expression of
VEGF isoforms in macrophages derived from the BM,
as tested in RAMP1/ BM chimeric mice. Unlike
AM-null mice, which exhibit edema during development, RAMP1-null mice are healthy, with no evidenceof edema formation. Pathologic lymphangiogenesis washampered in RAMP1/ mice, as was drainage ofinterstitial fluids. The findings presented in this study
indicate that RAMP1 signaling holds therapeutic potential for the treatment of pathologic angiogenesis/
lymphangiogenesis-related conditions.

8.

9.

10.

11.

12.

13.

14.
15.
16.

The authors thank Michiko Ogino, Kyoko Yoshikawa,
Mieko Hamano, and Yoshihiro Nara for technical assistance.
This study was supported by the Integrative Research Program of the Graduate School of Medical Science at Kitasato
University. The authors declare no conflicts of interest.

17.

18.

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Received for publication July 30, 2013.
Accepted for publication November 18, 2013.

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