モノアラガイでの味覚嫌悪学習のキー・ニューロン内でのCREB1タンパク質の定量を試みるのが本研究の目的である。まずはその検出方法であるが、当該のタンパク質の抗体を用いたELISA法に、チオNAD酵素サイクリング法を組み合わせたものを適用し、極微量のタンパク質でも定量できるようにその測定方法の開発に努めた。サイクリング法での脱水素酵素として3α-hydroxysteroid dehydrogenaseを用い、かつ基質としては改変したandrosteroneを用いて、300回転/分以上のサイクリングを実現した。これは、androsteroneの17位を修飾することによって成功した。また酵素反応の至適条件も見直し、バッファー組成のリン酸基濃度を増やした。さらには、標識酵素としてアルカリホスファターゼまたはβガラクシダーゼのどちらを適用し、市販のプレートリーダーを用いた測定系で、10^<-20>moles/well以上の検出限界を達成できた。さて次に、CREB1タンパク質を、上述のアルカリホスファターゼを標識酵素として用いて定量してみた。しかしながら、現時点では高感度は得られておらず、行動変容に特に関与するニューロン1個内のCREB1タンパク質を定量できるまでには至っていない。問題点は、抗体の精度にあることは明らかとなったので、現在は、その抗体の精度を上げるように努めている。

Although the aim of this study was to investigate the molecular mechanisms of translational coactivators in honeybees, we could not isolate these functional genes. Then, we changed the experimental targets with the RNA polymerases of influenza A virus, which has histone-acetylating activity as well as those of honeybee genes. We succeeded in cloning the target genes and expressing its recombinant proteins, which will be used for analyzing tertiary structure and functions.

花から巣に戻ったミツバチは訪れた花の位置(情報)を、「8の字ダンス」という特異的な行動によって巣内のミツバチに伝える。このようなミツバチの社会性の研究は生物学だけからのアプローチでは解明が難しく、工学系研究者との共同研究によって8の字ダンスに挑戦すれば、必ずや世界に先駆けてそのメカニズムを明らかにできると考え、本研究を進めてきた。われわれはミツバチの社会性適応行動を例に取り、特にその中でも他者とのコミュニケーション手段である「8の字ダンス」の基本機序をシステムとして理解を深めながら、社会性適応のための行動変容機構を明らかにすることが本研究課題の目的である。これまで生物データの取得のためにミツバチのダンス行動をビデオ撮影してきた。解析の結果、巣内ミツバチの歩行パターンと1秒あたりの最大移動距離、ダンスの発生場所の分布、ダンス情報の性質、追従バチの追従パターンなど、モデル構築に必要な多くのパラメータを取得した。そこで今年度はこれらのパラメータを利用して、ミツバチの採餌行動のマルコフモデルを構築後、シミュレーション実験を行った。その結果、われわれのモデルはミツバチの採餌行動をよく表現できていること、ダンスによる情報伝達は採餌の効率をあげていることなどがわかった。これらと平行して、ミツバチの行動は巣内外の環境に柔軟に適応して調節されるので巣内外の環境を自動で経時的に計測するためのシステムも同時に構築した。また、効率よく行動パラメータを取得するために、ベクトル量子化法を用いた、ミツバチの自動追跡システムを開発した。これらのことから得られる結果を組み合わせることで、ダンス行動によるコロニー維持の効果や情報の伝搬様式などのより深い理解につながると期待される。

The aims of the present study were (1) to clarify a series of mechanisms from gene expression to behavioral changes in a snail as well as (2) to develop the experimental methods to achieve the first purpose. We here noticed the conditioned taste aversion in the pond snail Lymnaea stagnalis. We have first developed the determination method for mRNA and protein of CREB in a single neuron and demonstrated the quantitative changes in these molecules during the long-term memory. Then we have also attempted to establish the bottom-up studies from a single neron to behavioral changes. Our present findings have paved the new way for studies of neurobiology.

生物学の定義では,ミツバチはヒト以上に社会性適応行動を示す.そこでミツバチの社会性適応行動を例に取り,特にその中でも他者とのコミュニケーション手段である「8の字ダンス」の基本機序をシステムとして理解することによって,社会性適応のための脳システム変容機構を明らかにすることが目的である.さらには,工学系研究者との共同研究を進め,そのモデル化にも挑戦している.これまでの研究によって,脳内の特定単一ニューロンの転写・翻訳の機構が,脳システムの機能変化を生じさせ,さらには動物行動の適応性を生み出してくることを明らかにしてきた.またミツバチをはじめとする社会性昆虫のさまざまな行動形態やそのメカニズムについての理解を深めてきた.
ミツバチは訪れた花の位置(情報)を8の字ダンスによって巣内の他のミツバチに伝える.われわれはこの行動を社会性維持のための「情報の伝搬と共有」のモデルとしてとらえ,8の字ダンスのコロニーへの効果を,行動観察の結果を盛り込んだ数理モデルによるコンピュータシミュレーションを用いて調べた.とくに採餌行動の数理モデルの構築を行い,その基礎となる巣内状態や個体行動データの取得を進めた.行動観察から,8の字ダンスは巣板上の特定の場所で数回踊った後,別の場所に移動して再び踊ることを繰り返す傾向があり,また,伝達する餌場情報にはかなり誤差が含まれていることがわかった.さらには,情報伝達の基本信号と考えられるダンス音の録音,巣内活動度の計測,評価方法についても検討を行った.また,8の字ダンスがコロニー維持に及ぼす効果については,現在さらなる検討中である.

本研究では、高いターンオーバー数をもつ標識酵素のELISA法と、その高感度活性測定法とを組み合わせて、タンパク質の超高感度測定法を作り上げようと試みた。そこで、まずは標識酵素の高感度活性測定法を開発した結果、従来法で最も感度の高い方法よりもさらに20倍高感度な方法に仕上がった。また本研究では、用いている抗体標識酵素が普遍的に存在するものであるため、酵素サイクリング法内にもその酵素が混在していることが明らかとなった。この混在が検出シグナルの大きなバックグランドの原因となり、検出感度(S/N)の低下をもたらすことがわかった。そのため、酵素サイクリング法に利用する酵素に含まれる標識酵素活性の除去法の開発を行った。標識酵素の自殺基質の適用により、混在酵素を除去することができた。さらには、その酵素の抗体カラムを作製し、混入酵素を除去することができた。両者とも程度の差はあるが、混入酵素の除去に役立つことがわかり、酵素サイクリング応用酵素免疫測定法が汎用に耐えるものであることが実証された。もう一つの課題である、単一細胞の単離のために、マイクロダイセクションに関する最も効率の良い手法を解析し、半自動化のマイクロサンプリングシステムを完成させた。開発した半自動化サンプリングを用いて細胞単離を行った場合、従来のマニピュレーターを用いた手動サンプリングに比べて飛躍的に短時間かつ容易に行えることが示された。

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. We therefore developed a procedure for determining the exact copy numbers of mRNAs within a single cell. We first isolated the cerebral giant cell of Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior of feeding. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs) by quantitative real-time PCR. Next, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time PCR to CREB in the cerebral giant cells. The cerebral giant cells employ a CREB cascade for the synaptic enhancement to neurons such as the B1 motoneurons. We injected the siRNA of the activator type of CREB (CREB1) or the repressor type (CREB2) into the cerebral giant cells and examined the changes in amplitude of EPSP recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation.

本研究は,行動生物学の気鋭研究者を関連各分野から結集し,動物行動の強力な研究体制を確立して,「統合行動生物学」を構築することを最終目的とする活動の一環である.平成16年度は,統合行動生物学の構築を目指した特定領域研究をスタートさせるための準備を行った.実際に,平成17年度発足予定の特定領域研究として,『昆虫における高次社会行動の発現機構の解明』(東北大学・水波誠代表)を申請することができた.
統合行動生物学の構築を成功させるためには,分子から個体,さらには社会レベルまでの関連分野の研究者が一堂に会し,共通の言葉を使って情報を提供しあうことが重要であり,この企画調査では,普段同じ学会で活動している研究者は半数以下に抑え,他分野の研究者を加えて,いわばヘテロな集団を構築した.この体制で2回の研究会研究を開催し,研究の現状について忌憚無い意見を交わした.また,問題点を分析・整理し,行動生物学を新しい視点でとらえなおすことができた.さらには,当該分野の著名外国人研究者を複数招聘し,彼らと意見交換し,われわれの研究の方向性や活動方針ならびに研究内容について議論していただいた.
この研究を通じて,実験室での研究者のみならず,フィールドの研究者やロボティクスの研究者にも,動物行動に強い興味をお持ちになられている方が多いこともわかった.

アゴニストの結合によって引き起こされるグルタミン酸受容体タンパク質での振動励起エネルギーが,再配分して行く過程が存在することは既にわかっている.そこで,リガンド結合部位のArg485側鎖から,振動エネルギーの移動経路を主成分分析法によって明らかにすることを試みた.この解析法を用いることにより,受容体が最も大きくゆらぐ方向を明らかにすることが示され始めた.
受容体のある特定の振動モード(GluR2ではArg485側鎖の変角振動)にエネルギーを与えると,受容体はそのエネルギーを機能部位に移動させて構造変化を起こすことがわかった.それ以外の場所にエネルギーを与えても,散逸するだけで生物学的な意義はないようであった.
次に,グルタミン酸受容体のリガンド結合部位であるArg485を振動励起させた場合と振動励起させない場合のシミュレーション結果を比較した.振動エネルギーによるリガンド結合領域の構造変化が現在明らかになりつつある.
さらには,Arg485の振動励起エネルギーの移動による膜貫通領域の構造変化の考察を試みた.リガンド結合領域は,膜貫通領域と3本のリンカーペプチドで連結されている.GluR2リガンド結合領域の結晶構造は,リンカーペプチドのアミノ酸残基を含んでいる.そこでシミュレーション中のリンカーペプチドの動きに着目し,リンカーペプチドの動きが膜貫通領域をどれくらい変位させる力を生むのかを,膜貫通領域をシリンダーでモデル化した.特に,Kチャネルの膜内構造との比較を重視し,これとの相同点・相違点について現在考察しているところである.

本研究ではイオンチャネル型グルタミン酸受容体の結晶構造を用い,この受容体のリガンド認識および活性化のダイナミクスを理論的に解析した.まずは,グルタミン酸受容体のアゴニストおよびアンタゴニストの静電ポテンシャルおよび受容体リガンド結合ドメインの静電ポテンシャルを,量子化学計算によって調べた.全てのリガンドは,分子の両端に負の静電ポテンシャルを形成している一方,受容体リガンド結合部位は,Arg485を中心に正の静電ポテンシャルを形成していた.これらのリガンド・受容体間の静電ポテンシャルの相補性から,グルタミン酸受容体のリガンド認識には,静電気力が中心的な働きをしていることが明らかにされた.
また,リガンドの結合は,まずArg485への結合から始まることも示唆された.受容体活性化メカニズムの解明のために,リガンドおよび受容体の基準振動を調べ,アゴニストとアンタゴニストの結果を比較したところ,アゴニストが共通に持っているもののアンタゴニストは持っていない振動モードが,受容体と水素結合を介して振動相互作用することをつきとめた.そこで,この振動モードがアゴニストとArg485との間の衝突によって振動励起する確率を量子力学的に計算した.その結果,サブピコ秒の時間オーダーでこれら2つの振動モードは励起することがわかった.この振動励起のエネルギー源は,静電相互作用によって加速されたアゴニストの運動エネルギーであった.最後に,受容体リガンド結合ドメインにおけるArg485の振動励起エネルギーの移動過程を,分子動力学シミュレーションによって調べたところ,約3ピコ秒でJヘリックスの原子にサブÅの変位が起こることがわかった.Jヘリックスは,サブユニット間の結合部位に位置している.サブユニット間の結合部位に起こる原子変位は,サブユニット間の会合に摂動を与え,イオンチャネルを開口へと導くのであろう.

When an appetitive stimulus (sucrose) is presented with an aversive stimulus (potassium chloride) to the pond snails Lymnaea stagnahs, associative learning, which is referred to as conditioned taste aversion (CTA), is quickly acquired and persisted for up to a month in snails. Previous results obtained by our cellular analyses showed that the cerebral giant cell is the key neuron for the formation of learning and the consolidation of long-term memory for CTA in L.stagnalis. In the present study, the molecular mechanism of CTA was analyzed with paying attention to a transcription factor. The transcription factor, cyclic AMP responsive element binding proteins (CREB), is considered to play major roles in the consolidation of long-term memory in various animals. We therefore cloned the cDNAs in L.stagnalis and analyzed their mRNA localization in identifiable neurons in the central nervous system. We found that the cerebral giant cells contain these mRNAs. Further, the splicing variants of these genes were studied, and 7 isoforms were found for CREB1 (so called "activator"). However, three of these CREB1 isoforms do not have any potential sites of phosphorylation by protein kinase A in their predicted amino acid sequences. This result suggests that the functions of these 3 isoforms as transcription factors differ from the other 4 isoforms and that all the isoforms affect to each other, resulting in the change in their DNA binding ability. Finally, we succeeded in measuring the changes in copy numbers of the CREB mRNAs in the cerebral giant cells during CTA by single-cell real-time quantitative RT-PCR. The reduction of CREB2 (represser) was found to be essential for the consolidation of long-term memory.

Gonadotropin-releasing hormone (GnRH) regulates synthesis and release of gonadotropins (GTHs) and other pituitary hormones, such as growth hormone (GH), prolactin (PRL), and somatolactin (SL) in a seasonally different manner. In masu salmon (Oncorhynchus masou), expression of two genes encoding salmon GnRH (sGnRH) precursors increases at prepubertal stages and changes with seasons during sexual maturation. Recently, we demonstrated in masu salmon that five types of GnRH receptor (GnRH-R) genes are expressed in the brain, pituitary and other peripheral tissues in different patterns. In the present study, we examined seasonal expression patterns of sGnRH and GnRH-R genes, in the brain and pituitary during growth and sexual maturation, to elucidate roles of the multiple GnRH-Rs in GnRH action. Moreover, seasonal changes in response to GnRH of GnRH-R genes _were examined by a GnRH analog (GnRHa) implantation experiment. The fish were treated with GnRHa for one week and the brains and pituitaries were taken out. This implantation experiment was repeated every month from immature to spawning periods. The absolute amounts of mRNAs for two sGnRHs and five GnRH-Rs were determined by a quantitative real-time PCR method. In the brain, the amounts of two sGnRH mRNAs tended to be high in winter and spring, and decreased toward summer, and again increased from late summer to spawning. The changes in the amounts of five types of GnRH-R mRNA were similar to that of sGnRHs. In the pituitary, the amounts of five GnRH-R mRNAs showed different changes during immature and maturing stages but increased in pre-spawning periods, in which GnRHa treatment increased the amounts in the females, whereas decreased in the males. The coincidental elevation of expression of sGnRH, GnRH-R and GTH subunit genes in the pre-spawning stage may be important in the regulation of GTH subunit gene expression by GnRH, and further control of upstream migration.

高次脳機能の発現機構を解明するためには,ミエリン鞘形成を伴う神経回路の発達機構を明らかにすることが必須である.本研究では,オリゴデンドロサイトによるミエリン鞘形成機構に着目し,その際に利用されている神経情報伝達系,特にセカンドメッセンジャー系(cAMPとカルシウム)がどのように使い分けられているのかを,転写調節因子であるcAMP response element binding protein (CREB)のリン酸化という点に的を絞って検討した.培養は分化に合わせて各種の成長因子などを加えた培養液を用いて行なった.分化段階は特異的に発現してくる抗原の抗体(A2B5,O4,O1,MBP)で区別した.オリゴデンドロサイトにおけるCREBの発現を,「リン酸化されている場合」と「されていない場合」との局在の違いに注意して,分化段階別に免疫細胞化学法によって調べた.その結果,分化段階の進行に伴って,CREBおよびリン酸化したCREBの発現量が増加することがわかった.また,発現しているCREBのほとんどがリン酸化されていることも明らかとなった.次に,細胞分化にはcAMPが重要であることから,オリゴデンドロサイトのCREBリン酸化の経路としてcAMP-タンパク質キナーゼAの系について検討した。その結果,未成熟なオリゴデンドロサイトではCREBのリン酸化はタンパク質キナーゼAに依存しているが,成熟したオリゴデンドロサイトでは,タンパク質キナーゼAには依存しないことも示された.従って,未成熟のオリゴデンドロサイトと成熟のものとでは,リン酸化に使われているセカンド・メッセンジャー系が異なることが明らかとなった.

本基盤研究(C)(1)企画調査では「動物の学習の生物学的理解」を中心課題に設定して、動物種による違いを生みだした進化の過程を再構築しながら、なおその中で保存されている共通性と普遍性を発見することを目的とした。そのために、動物行動学・行動生理学・認知心理学・神経科学の広い分野にわたって関連する研究者が最新の成果を持ち寄り二つのシンポジウム(神経科学会大会と動物学会大会)を実施すると共に研究会を開催して研究者間の緊密なネットワークを形成した。これらの一連の活動は動物学の新しい側面を拓く活動である。
(1)第23回日本神経科学会大会シンポジウム「学習する本能」
平成12年9月5日、横浜市「横浜みなとみらい21・パシフィコ横浜」にて、シンポジウムを開催した。
サケの母川回帰と嗅覚記憶形成:佐藤真彦(横浜市立大学・大学院総合理学研究科)
雛鳥の視覚性刷込みによる遺伝子発現の増大:木村尚博(創価大学・工学部)
小脳の新しい機能:プルキンエ細胞とニューロステロイド:筒井和義(広島大学・総合科学部)
鳥の歌文法の神経基盤:岡ノ谷一夫(千葉大学・文学部、科学技術振興事業団さきがけ研究21)
The Neural Basis of Vocal Learning in Songbirds:A.J.Doupe(University of California,San Francisco)
総合討論「学習する本能」松本元、松島俊也、佐藤真彦、木村尚博、筒井和義、岡ノ谷一夫、Allison J.Doupe
本シンポジウムを実施することにより、学習の生物学的基盤に関する最新の知見を得ることができた。様々な研究対象における記憶研究の重要性とその展開を強く印象付けるものであった。神経科学の新しい側面を拓くものとして高く評価された。
(2)第71回日本動物学会大会シンポジウム「学習の生物学的基盤に関する総合的研究」
平成12年9月21日(木)午後3時から6時まで、東京都目黒区「東京大学駒場キャンパス」にて、日本動物学会第71回大会の[国際ブレインサイエンスシンポジウム」として開催した。
トリの歌学習システム:H.-J.Bischof(University of Bielefeld,Germany)
指定討論者:岡ノ谷一夫(千葉大・文学部)、竹内浩昭(静岡大・理学部)
サケの母川回帰と嗅覚記憶システム:Thomas Quinn(University of Washington,USA)
指定討論者上田宏(北海道大・水産学部)、松島俊也(名古屋大・大学院生命農学研究科)
記憶学習の研究がつねに動物の進化的背景と行動生態学的背景を見失ってはならないこと、様々な研究手段を糾合し様々な現象を広く深く俯瞰する研究戦略が、今後の学習研究の主流となる事を強く印象付けるものとなった。翌日開催された大会本部企画シンポジウム「20紀の動物学と21世紀の展望」においても、講演者の一人(日高敏隆、滋賀県立大学学長)により動物学の新しい側面を拓くものとして高く評価された。
(3)「学習の生物学的基盤に関する総合研究」研究会
平成12年9月24日25日に、水産庁養殖研究所日光支所において、本企画調査メンバーと「微小脳システムの適応的設計:学習・認知およびホルモンによる行動変容研究会」(文部省科学研究費特的領域研究「微小脳システムの適応的設計」代表:高畑雅一)の協力を得て、水産庁養殖研究所日光支所(栃木県日光市)において合同研究会を実施し、研究者間の緊密なネットワークを形成した。

Spawning migration of salmon is a typical stereotyped or instinctive behavior which is genetically programmed. However, molecular approaches are rare to reveal what is a genetic program of salmon migration. Aiming clarification of such genetic program as the final goal, we examined molecular bases of neuroendocrine mechanisms for salmon spawning migration. Since spawning migration is reproductive behavior, we hypothesized that preoptic neurons producing gonadotropin-releasing hormone (GnRH) and vasotocin (VT) have a crucial role in coordination of the sensory, motor and motivational function of the central nervous system and the neuroendocrine function of the brain-pituitary-gonadal axis. The coordinated activity of the central nervous system and the neuroendocrine system should be crucial for the success of reproduction, that is, the neural controls of migratory and spawning behavior must be temporally and spacially coincident. Herewith. we determined absolute changes in the amounts of mRNAs encoding neurohormones and pituitary hormones along migratory pathways to elucidate changes in expression of hormonal genes during migration. We further analyzed regulatory mechanisms of expression of hormonal genes in salmonids. The results obtained through the present project actually support our hypothesis mentioned above.

1 ヨーロッパモノアラガイにおけるFos,Jun,CREBの免疫組織化学
市販されているFos,Jun,CREBの抗体は哺乳類由来のものであり,無脊椎動物で認識されるか否かは不明であった。そこでまず,ヨーロッパモノアラガイから中枢神経系単離し,複数種類の抗体を用いてウエスタン・ブロッティング法により認識蛋白を確認した。その結果、哺乳類で報告されている大きさの蛋白質の前後に数本バンドが確認され、哺乳類の抗体が,少なくともモノアラガイでは不適切なことが分かった。このような結果は残念ながら,単発的もしくは必要最低限のデータのみを報告している無脊椎動物におけるこれらの蛋白質に対する免疫組織化学の論文に対して,その正当性に疑問を投げかけるものであった。従って、本研究者は実験のストラテジーを変更し,ヨ-リッパモノアラガイのfos,jun,CREBのクローニングを行って塩基配列を決定して,in situハイブリダイゼーション法でmRNAレベルの解析を,先に行うことにした。
2 cDNAライブラリーの作製とfos,jun,CREBのクローニング
モノアラガイの中枢神経系から抽出したmRNAを逆転写酵素でDNAに直し,それをvectorでファージに挿入して,cDNAライブラリーを作製した。次にそのファージ液を大腸菌に感染させて,組み込んだcDNAを発現させたものを1で使った抗体でスクリーニングした。このスクリーニングを何回か行い,ポジティブプラークだけが取れる段階まで来た。現在は,これらをPCRで増幅させ,シークエンスを調べている最中である。もっとも1の結果から分かるように,用いている抗体は目的の蛋白質以外も認識しているので,このプロセスも何回か繰り返し最適なものを見つける必要がある。しかし,これは数を重ねれば必ず正解に行き当たるため,次年度早々には,次のステップに入れると確信している。

1 培養神経細胞系でのin vitro学習系の確立
本年度は,ヨーロッパモノアラガイ中枢神経系から単離した神経細胞,ラット大脳皮質から単離した神経細胞,およびニューロブラストーマ・グリオーマの雑種細胞であるNG108-15細胞を各々用いて,神経ネットワークを作製し,それぞれの系の中の2つの神経細胞に着目して,刺激・記録を行う実験方法を確立した。そして上記の培養方法のうち大脳皮質を用いた場合にのみ,促通現象が得られることが分った。この結果は,大脳皮質の初代培養系がin vitro系での学習モデルとして大いに利用できることを示している。現在は,基板電極上で細胞を培養し,ガラス電極などを挿入することなしに刺激・記録できる方法を模索している。
2 培養神経細胞の3次元極微細構造観察
上記1での培養系のうち,モノアラガイ神経細胞の終末ならびにシナプス部位での無刺激下における自発的な極微細構造の経時変化を,原子間力顕微鏡によって観察することに成功した(研究発表の項を参照)。1で報告したように,次年度に基板培養法が確立すれば,原子間力顕微鏡観察と同時にin vitro学習実験が行えるようになり,まさに学習時の極微細形態変化をリアルタイムで観察出来るようになる。
3 フォトダイオードアレイを用いた単一神経細胞の膜電位光学測定
上記1で示した培養系を,膜電位感受性色素で染色して,単一神経細胞の膜電位測定を試みた。しかし残念ながら今年度はこの点を成功できないで終わった。しかし,染色や顕微鏡回りの各種の最適条件がかなり分かり始めて来ているので,次年度は成功できると確信している。

The spawning migration is a kind of innate behavior which is essential for successful reproduction in salmon. To realize a genetic program of the behavior. we have examined molecular basis of neuro-endocrine mechanisms of salmon migration. Our research has been focused mainly on the following three points ; 1)Changes in expression levels of hypothalamic and pituitary hormone genes during spawning migration, 2)Structural analysis of the neurohormone genes. 3)Roles of gonadotropin-releasing hormone (GnRH) in the control of spawning behavior. The results of this project are summarized as follows.
1. The levels in neurohypophysial hormone (NH) mRNAs decrease in the pre-spawning females. The sexual dimorphic expression of the NH genes suggests that the NH may be involved in reproductive behavior as well as osmotic regulation in the homing salmon. The gonadal and adrenocortical stroid hormones may be concerned in the control of the NH gene expression.
2. The expression of salmon GnRH (sGnRH) gene in the preoptic area increases during final maturation. The 5' upstream regions of the two sGnRH genes (sGnRH-I and-II) are distinctively divergent. The expression of gonadotropin alpha and IIbeta genes also. increases temporally in the final maturation, and is stimulated by the administration of genre.
3. The adminiatration of GnRH shortens duration of the homing migration. The GnRH may play roles in the stimulation of not only gonadal maturation but also migratory behavior.
The molecular neuroendocrine basis of spawning migration is considered to be mostly established at present. In the new project ''Moleclular neuroendocrine regulation of spawning migration in salmon", which is supported by the Grant-in-Aid for Specially Promoted Research, further investigations on the molecular mechanisms of neurohormone action in the control of salmon spawning migration will be progressively done.

An optical recording system that can be used in electrophysiological experiments using the whole animal preparation of invertebrates has been developed to visualize the change in intracellular calcium concentration associated with the synaptic activity. The system consists of an epifluorescence device for exciting Fura-2 that is iontophoretically injected into a cell, a dissecting microscope with the working distance of 187mm, a Peltier-cooled CCD video camera, and a personal computer with newly developed programs for image analysis. To test the performance of this recording system, nerve cells in the terminal abdominal ganglion of crayfish were loaded with the dye and observed from the dorsal side with the CCD exposure time of 16sec. The whole cell structure having the soma, dendrites and axon could be visualized in situ together with the electrode tip under the dissecting microscope by either 380nm or 340nm excitation. When the sensory nerve bundle was stimulated repeatedly at 1Hz during the exposure time, a decrease in the fluorescence of the dye to excitation at 380nm was detected in a specific dendritic region of the cell. However, any significant change in the ratio of fluorescence by 380nm to that by 340nm excitation during synaptic activation could not be detected probably due to the relatively weak strength of the latter. It is concluded that although the sensitivity and time resolution are not enough to quantify the dynamics of intracellular calcium ions, the presently developed system can be used to localize the functional synaptic region within a cell by visualizing the change in intracellular calcium concentration due to synaptic activity together with the whole cell structure.

淡水棲カタツムリ(モノアラガイ)に味覚嫌悪学習を施し,その学習効果に対して行動学と生理学とを同時に組み合わせた神経行動学的解析を行った。そして,我々が既に提出している味覚嫌悪学習に関するニューロモジェレーションモデルの証明を行い,その学習・記憶機構にとっての必要十分条件を見出すことに努めた。まずは,学習を施された実験動物と施されていない実験動物の両者の中枢神経系を摘出して、神経生理学実験を行った。そして,咀嚼リズムを司っているcentral pattern generatorの介在神経細胞において,その性質の違いを抑制性シナプス後電位の延長という形で見出し,味覚嫌悪学習に対する我々のニューロモジュレーションモデルを部分的に検証した。一方,central pattern generatorでの抑制性シナプス後電位の延長を人工的に起した後に,実際に固体の条件刺激に対する嗜好性応答(咀嚼運動)を行動実験で観察し,それが減少しているか(味覚嫌悪)を調べた。そのためには,中枢神経系を摘出するのではなく,動物固体を生きたまま不動化し,その我意膜を切り開いて中枢神経系を露出させた標本を作った。そして着目している細胞の膜電位を過分極状態にした上で,条件刺激に対する咀嚼運動の減少を確認することができた。このように部分的ではあるが,咀嚼リズムを司っているcentral pattern generatorの介在神経細胞における抑制性シナプス後電位の延長が,味覚嫌悪学習における必要十分条件であることが明らかになった。

ヨーロッパモノアラガイに味覚嫌悪学習を施すと,条件づけられる食物に対する嗜好性が低下する。我々はすでに,この学習成立の一因が咀嚼リズムを司るcentral pattern generatorへの調節細胞からの抑制の増強であることを示した。本研究では発生過程を追ってこの学習の成立を調べたところ,veligerの最終期で咀嚼運動の開始が確認され,孵化寸前に味覚嫌悪学習が成立することが分かった。しかし,それが長期記憶として保持されるには性成熟の直前まで待たねばならなかった。この学習行動に対する神経細胞の変化を組織学的に解析したところ,学習を獲得できる発生段階では,神経繊維の異常な収縮が起こっており,シナプスの再編成があると予想できた。また、学習を長期記憶化できる発生段階では,神経細胞数の莫大な増加が認められた。次に,central pattern generatorの調節細胞を蛍光染色して,長期記憶化時の形態を調べてみた結果,この細胞の形態はadultのものと全く同じであった。すなわち,長期記憶を得るためには重要な神経細胞の形態は整備される必要であった。一方,モノアラガイの神経細胞を初代培養しネットワークを作らせ,in vitro系での学習法を確立することも目指した。神経細胞の機能変化に伴って微細形態変化が起こっていると予想されるので,まず手始めに,初代培養した神経細胞の超微細構造を原子間力顕微鏡で観察することを努めた。細胞膜上の蛋白質や糖鎖のゆらぎを考慮すると,高さ方向は最低限40nmの分解能があることが分かった。また時間経過に伴って,シナプス領域での突起どうしの接近・反発,また成長円錐の伸長過程などが観察できた。現在,神経伝達物質など薬物投与時の超微細形態変化の観察を試みており,学習とシナプス形態変化との関連を明らかにされると考えられる。

本研究では、淡水棲カタツムリであるヨーロッパモノアラガイを使って,嗜好性学習と忌避性学習との成立・保持機構での相異点について調べた。古典的条件づけの手法を用いて実験動物に学習を施した場合,忌避性学習は成立が早くその保持も長期に渡った。しかし嗜好性学習はその成立に多くの訓練を要し,忘却も早く起きた。この相異点を神経細胞レベルで明らかにするための作業仮説として,我々は各々に対するニューロモジュレーションモデルを提出した。例えば,忌避性学習においては,無条件刺激の伝達経路側から条件刺激の経路側への抑制性シナプスが,長期に渡って増強されることを予想した。そこでこのモデルを証明するために,味覚嫌悪学習が成立した動物個体の中枢神経細胞を電気生理学的に調べたところ,味覚嗜好性の指標である咀嚼リズムを形成するcentral pattern generatorの1つの介在神経細胞(N1M細胞)において,調節介在神経細胞(CGC)由来のIPSPが長期に渡って増強されていることを見出した。一方,嗜好性学習については,条件刺激の伝達経路側から無条件刺激の経路側への興奮性シナプスの増強を予想した。しかし,嗜好性学習においては条件刺激として中性の刺激を用いる必要があるため,その中性刺激と嗜好性刺激との結び付きを神経細胞レベルで確認することが非常に困難であった。従ってこの点については,まだ実験を遂行している最中である。

本研究では,ヨーロッパモノアラガイを使って,味覚嫌悪学習の成立と神経回路発達との関連を調べた。咀嚼運動自体はベリコンジャー幼生から始めたものの,味覚嫌悪学習は孵化直前の胚で始めて成立することが分かった。胚の組織切片をアザン染色し,光学顕微鏡観察によって神経節や神経細胞の配置を確認した結果,ベリジャー幼生からベリコンジャー幼生に移る際に,脳神経節から口球神経節へのコネクティブが太く明確になってくることを見出した。また,ベリコンジャー幼生と孵化直前の胚では神経節の配置が成体とほぼ同様になっているものの,神経節全体における神経突起の体積は両者の間で1.5倍に増えていることが確認された。これによって味覚嫌悪学習の成立には成体と同じ神経節の配置を必要とし,また神経節または細胞間の結び付きが強固になっている必要があることが示唆された。しかし長期記憶の保持は孵化後10mm程のjuvenileに成長しないと成立せず,この結果の解釈は今後の分子レベルでの解析を待つ必要があった。
この味覚嫌悪学習を神経細胞レベルで明らかにするために,味覚嫌悪学習が成立した動物個体の中枢神経細胞を電気生理学的に調べたところ,咀嚼リズムを形成するcentral pattern generatorの1つの介在神経細胞において,調節介在神経細胞由来のIPSPが長期に渡って増強されていることを見出した。central pattern generatorは口球神経節に,調節介在神経細胞は脳神経節に存在することから,やはり味覚嫌悪学習にとっては,脳神経節から口球神経節への情報伝達経路が重要であることが裏づけられた。

軟体動物腹足類(カタツムリの仲間)の連合学習の一つである古典的条件付けを施した今までの神経科学的研究は、すべて電気ショック等(忌避性刺激)を無条件刺激として使っており,犬に餌(嗜好性刺激)を与えるようなバヴロフの条件付けとは大きく異なっている。我々は、忌避性学習と嗜好性学習とは分けて神経科学的研究を進めるべきと考え,それらの成立・維持・消去・想起の類似点・相異点をシナプスレベルで明らかにする実験を開始した。本年度はその第一歩として,行動学実験からスタートし,その結果として推測される2つの相異なるシナプス・モジュレーション・モデルを提出することに成功した。
1.行動学実験:ヨーロッパモノアラガイに忌避性学習(条件刺激(CS):ショ糖,無条件刺激(UCS):KCI)と嗜好性学習(CS:光または振動,US:ショ糖)の訓練を施した。その結果,忌避性学習は成立が早く,かつ記憶の保持も長いことが示され,嗜好性学習の方は全く逆に,成立に多くの訓練回数を要するにもかかわらず,記憶の消去は早いことが分かった。更には,感性予備条件付け及びオペラント条件付けも忌避性学習では成立しやすいものの,嗜好性学習では困難であることが示された。
2.シナプス・モジュレーション・モデル:忌避性学習は,UCS側の介在神経細胞からCS側のそれへ,強い抑制が長期に渡って不可逆的にかかると考えられた。一方,嗜好性学習は,CSに関与する介在神経細胞からUCSのそれへの興奮性のシナプスが強化されることであると予想された。しかし,これは可逆性が強いため,記憶の消去も早いものと考えられた。現在は,これらのモデルの証明実験を行なっており,着目している細胞の長期興奮によるカルシウム濃度上昇や、それに伴う蛋白質リン酸化の関与が明らかになりつつある。

Since a scanning tunneling microscope (STM) was developed, an observation of living cell surface has been one of the final aims in the biological application of STM. We first attempted to apply a STM for solid state physics to observe the surfaces of platinum-carbon replicas of mouse-bladder membrane and liposome membrane which were prepared for a transmission electron microscope. These results taught us that the STM for cell-membrane observation had to be equipped with the following two abilities : (1) a maximum scanning area is set at more than 10um X 10um, (2) a STM-observation region is identified with an optical microscope (OM). Accordingly, we have developed a new style of STM which was combined with an OM and with a novel system for the centering of both images. By using this STM, we successfully got the STM images of living cell surface of T24 cells (human bladder cancer cell lin) and CHO cells (Chinese hamster ovary fibroblast) cultured on highly oriented pyrolytic graphite under the appropriate condition (V > 8.OV, I < 0.2nA). Unexpectedly, the living T24 cell showed a slightly uneven surface with a steep foot slope. The CHO cell showed more rough surface with steeper slope of cell foot. Although the STM system had a fine spatial resolution less than 3nm, the profile of living cell surface covered with electrolyte was clear only the scanning area was more than 10um x 10um.

In the olfactory center of terrestrial animals, changes in the oscillatory frequency of the local field potential (LFP) are thought to be involved in olfaction-based behavior and olfactory memory. The terrestrial slug Limax has a highly developed olfactory center, the procerebrum, in which the LFP spontaneously oscillates. Although changes in the oscillatory frequency are thought to correspond to the preference for specific odors, our knowledge about the mechanism of this frequency regulation is limited. To clarify the mechanism of the bidirectional frequency changes in the procerebrum, we focused on the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), which is known to have neuromodulatory functions in invertebrate nervous systems. Application of FMRFamide decreased the oscillatory frequency via G-protein-mediated cascades. Immunohistochemistry showed that FMRFamide-like-immunoreactive neuronal cell bodies are located in the cell mass layer of the procerebrum, projecting their neurites to the neuropile layers. The procerebrum was shown to also receive innervation from other regions of the cerebral ganglion. Furthermore, according to their morphological and projection characteristics, FMRFamide-containing neurons belong to the subpopulations of both bursting and nonbursting neurons in the procerebrum. The mRNA splice variant encoding multiple copies of canonical FMRFamide was specifically expressed in the procerebrum. Taking into account previous results showing that serotonin increases the oscillatory frequency, our results indicate that FMRFamide and serotonin both regulate the LFP frequency but in exactly the opposite direction in the olfactory center of the terrestrial slug.

The tentacles of pulmonates regenerate spontaneously following amputation. The regenerated tentacle is equipped with all the elements necessary for normal olfactory functioning, and the slugs can behave as well as they did before the tentacle amputation. However, it is not known what changes occur to the olfactory center procerebrum in the brain at the morphological and physiological levels. Here, we investigated the innervation of tentacular nerves into the procerebrum by examining the size of the terminal mass (input layer from tentacular nerves) of the procerebrum and also by staining afferent nerves immunohistochemically at 15, 58 and 75 days following unilateral amputation of the superior and inferior tentacles. The size of the terminal mass was significantly decreased, and the Phe-Met-Arg-Phe-NH(2)ergic (FMRFamidergic) afferent nerves disappeared by 15 days following the tentacle amputation. However, the size of the terminal mass had recovered substantially by 58 days, as the tentacle regenerated. The FMRFamidergic innervation into the cerebral ganglion was also restored by this time. An extended recovery (75 days), however, did not result in any further increase in the size of the terminal mass. We also recorded the local field potential (LFP) oscillation in the procerebrum. We found that the oscillatory frequency of the LFP had decreased at 15 days following the tentacle amputation but had recovered at 58 and 75 days. These results suggest that the amputation and regrowth of the tentacle are accompanied by the respective degeneration and re-innervation of olfactory nerves, and these changes in the innervation status affect the basal state of LFP oscillation.

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm. (C) 2009 Wiley-Liss, Inc.

In most sensory modalities, neuronal inputs are bilaterally processed in a higher center. In some animal species, however, functional lateralization is sometimes observed in the sensory processing at the higher level. For the terrestrial slug Limax, olfaction is the most important sensory modality and this slug can acquire odor-aversion memories. Previously, it has been demonstrated in bilateral PC ablation experiments that the procerebrum (PC) is necessary for odor-aversion memory, and that the PC is the memory storage site. On the other hand, it has been hypothesized that only the unilateral PC is used for odor-aversion learning. Here we demonstrated that the number of the slugs with intact memory performance was reduced by approximately 50% when the PC was surgically ablated only unilaterally before or after conditioning. There was no difference in the memory performance of the right vs. the left PC-ablated slugs. However, memory deficit from unilateral PC ablation was not observed when the ipsilateral tentacles were also amputated at the same time. We also showed that there was no lateral memory transfer from one PC to the other, after up to 7 days post-conditioning. Our results demonstrated clearly that either the left or right PC is randomly used for olfactory learning, and that the side of use is determined at the level of the olfactory ascending pathway to the PC. (c) 2009 Elsevier Inc. All rights reserved.

The pond snail Lymnaea stagnalis moves along the sides and bottom of an aquarium, but it can also glide upside down on its back below the water&apos;s surface. We have termed these two forms of locomotion "standard locomotion" and "upside-down gliding," respectively. Previous studies showed that standard locomotion is produced by both cilia activity on the foot and peristaltic contraction of the foot muscles. whereas upside-down gliding is mainly caused by cilia activity. The pedal A neurons are thought to receive excitatory octopaminergic input, Which ultimately results in increased cilia beating. However, the relationship between locomotory speed and the responses of these neurons to octopamine is not known. We thus examined the effects of both an agonist and an antagonist of octopamine receptors on locomotory speed and the firing rate of the pedal A neurons. We also examined. at the electron and light-microscopic levels, whether structural changes occur in cilia following the application of either an agonist or an antagonist of octopamine receptors to the central nervous system (CNS). We found that the application of an octopamine antagonist to the CNS increased the speed of both forms of locomotion, whereas application of octopamine increased only the firing rate of the pedal A neurons. Microscopic examination of the cilia proved that there were no chan-e in their morphology after application of octopamine me ligands. These data suggest that there is an unidentified octopaminergic neuronal network in the CNS whose activation reduces cilia movement and thus locomotory speed.

Background: Of all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug&apos;s cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.
Principal Findings: In the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.
Significance: Our findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.

Cyclic AMP-responsive element binding protein1 (CREB1) has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1) mRNA isoforms of spliced variants in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA) learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real-time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum. (C) 2009 wiley-Liss, Inc.

Nitric oxide (NO) plays important roles in the olfactory center of various animals. In the terrestrial slug, NO is indispensable for field potential oscillation in the higher olfactory center, the procerebrum (PC), and also for odor learning. Here we identify a novel NO synthase (NOS) gene, limNOS2, in the terrestrial slug. The mRNA (similar to 10 kb) of limNOS2 encodes a protein consisting of 1616 amino acids, including a PDZ domain. The protein has 70.0% sequence identity with the previously identified limNOS1 gene. In contrast to limNOS1, however, limNOS2 is expressed specifically in the PC. Moreover, most of the cells in the PC contain limNOS2 mRNA, indicating that the nonbursting neurons, the major constituent of the PC, have this mRNA. The expression pattern of limNOS2 conforms well to the pattern of NOS enzymatic histochemical staining. Our present findings indicate that limNOS2 is responsible for most of the NO generation in the PC. (C) 2009 Elsevier Inc. All rights reserved.

The pond snail Lymnaea stagnalis can often be observed moving upside down on its back just below the surface of the water. We have termed this form of movement "upside-down gliding." To elucidate the mechanism of this locomotion, we performed a series of experiments involving behavioral analyses and microscopic observations. These experiments were designed (1) to measure the speed of this locomotion; (2) to determine whether the mucus secreted from the foot of Lymnaea repels water, thereby allowing the snail to exploit the surface tension of the water for upside-down gliding; and (3) to observe the beating of foot cilia in this behavior. The beating of these cilia is thought to be the primary driving force for upside-down gliding. Our results demonstrate that upside-down gliding is an efficient active process involving the secretion of mucus that floats up to the water surface to serve as a substrate upon which cilia beat to cause locomotion at the underside of the water surface.

Atomic force microscopy (AFM) creates a topographic map of a sample surface and has enabled the imaging of biological surfaces. As an initial step in elucidating gene expression mechanisms from the perspective of structural biology, we have started to characterize the structure of nucleosomes that consist of core histones and DNA promoter regions, including specific transcription factor-related binding sites. We formed nucleosomes using core histones and tyrosine hydroxylase promoter regions that include cAMP- and TPA-responsive elements, and then we observed these nucleosomes using the dynamic-force (tapping) mode in AFM. Good correspondence was seen between the lengths of the DNA molecules observed by AFM and the theoretical values estimated from the base pairs of DNA molecules, thus confirming that the present AFM study acquired images of nucleosomes, including promoter regions.

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of beta-actin, gamma-actin, and beta-tubulin mRNAs in C6 cells, but not alpha-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except alpha-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.

Terrestrial slugs have the ability to learn and remember a food odor paired with an aversive stimulus. Olfaction in slugs involves the tips of two pairs of tentacles, the superior and the inferior tentacles. Sensory nerves in both pairs of the tentacles transmit olfactory information to the structure in the CNS, the procerebrum where learning and memory formation occur. We investigated the role of each pair of tentacles in odor-aversion learning, and examined the ability of slugs to recall memory after selective surgical amputation. Our results show that memory formation was not altered by the amputation of either one of the pairs before or after odor-aversion learning, while the odor sensibility of the slugs was maintained. These data suggest that either pair of tentacles is sufficient for the acquisition and retrieval of aversive olfactory memory.

The analysis of biological function produced by both of individual and assemblage activity of animals is a challenging topic of behavioral ecology. Social insects are possible tool for the analysis because numerous studies have been already done to clarify the behavioral factors of individuals. Especially, honeybee is an ideal animal for the experiments because of the development of breeding techniques, existence of clear caste according to their age, efficient forage behavior adaptive for variable feeding places and high ability of honey storage in their colony. Seeley et al. conducted many behavioral experiments in order to scrutinize honeybee behavior inside and outside of the nests. However, these experiments normally have to put numbers on each body of thousands of honeybees to identify individuals and follow their behavior by hand, which require huge time and labors. In this study, we developed a system to identify honeybee in a nest image and to follow plural honeybees simultaneously. The system is composed by three parts, determination of the region of honeybee body from the other parts with vector quantization, extraction of the region of single honeybee bodies, and reorganization of single honeybees. Our system succeeded to identify nearly 70% of honeybees in a hive automatically.

A honeybee informs her nestmates of flower locations by a unique behavior called a &apos;waggle dance&apos;. We regard this behavior as a good model of the &apos;propagation and sharing of knowledge&apos; to maintain a society. We have attempted to reveal how this dance benefits the colony using mathematical models and computer simulation based on parameters obtained from observations of bee behavior. Our simulation indicated that the most successful forages were made by a putative bee colony that used the dance to communicate. Video analysis of worker honeybee behavior in the field showed that a bee does not dance in a single, random place in the hive, but waggles several times in one place and several times in another. The orientation and duration of waggle runs varied from run to run, within ranges of +/- 15 degrees and +/- 15%, respectively. We also found that most of the bees that listened to the waggle dance turned away from the dancer after listening to one or two runs. These data suggest that honeybees use the waggle dance as a method of communication, but that they must base their forages oil ambiguous information about the location of a food source. (C) Koninklijke Brill NV, Leiden and The Robotics Society of Japan, 2008

A honeybee informs her nestmates of the location of a flower she has visited by a unique behavior called a "waggle dance." On a vertical comb, the direction of the waggle run relative to gravity indicates the direction to the food source relative to the sun in the field, and the duration of the waggle run indicates the distance to the food source. To determine the detailed biological features of the waggle dance, we observed worker honeybee behavior in the field. Video analysis showed that the bee does not dance in a single or random place in the hive but waggled several times in one place and then several times in another. It also showed that the information of the waggle dance contains a substantial margin of error. Angle and duration of waggle runs varied from run to run, with the range of +/- 15 degrees and +/- 15%, respectively, even in a series of waggle dances of a single individual. We also found that most dance followers that listen to the waggle (lance left the dancer after one or two sessions of listening.

The pond snail, Lymnaea stagnalis, can locomote on its back utilizing the surface tension of the water. We have called this form of movement 'back-swimming'. In order to perform this behavior, the snail must flip itself over on its back so that its foot is visible from above. Little is known about the mechanism of this back-swimming. As a first step for the elucidation of this mechanism, we measured the speed of back-swimming of Lymnaea at the different times of the day. They back-swam significantly faster in the morning than just before dark. These data are consistent with Our earlier findings on circadian-timed activity pattern in Lymnaea.. Lymnaea appear to secrete a thin membrane-like substance from their foot that may allow them to back-swim. To confirm the existence of this substance and to examine whether this substance is hydrophobic or hydrophilic, we applied a detergent onto the foot during back-swimming. A single drop of 1% Tween 20 drifted Lymnaea away that were still kept at the water surface. These results suggest that Lymnaea secrete a hydrophobic substance from their foot that floats to the water surface allowing Lymnaea to back-swim.

Gamma-aminobutyric acid (GABA)-containing neurons are capable of controlling network oscillations and their patterns in the widely divergent species. In the terrestrial slug, Limax valentianus, oscillatory activity of the procerebrum neurons is considered to encode the odor information. Previous studies showed that GABA is present in the central nervous system and may be involved in the control of neuronal activity of Limax. In the present study, we examined the effects of GABA on the generation of oscillatory activity in the procerebrum. The results suggest that the GABAergic synaptic transmission may be involved in the oscillatory neural network of the procerebrum.

The procerebrum (PC) is indispensable for odor-aversion learning in Limax. On the other hand, the central nervous system (CNS) of some Pulmonata shows robustness against injury, recovering from nerve injury both at the histological and functional levels. To investigate whether the PC of Limax also shows robustness against nerve injury, we tested whether or not the slugs can acquire and retrieve odor-aversion memory after a long recovery period from PC ablation. When the recovery period is short (7 days), the PC-ablated slugs failed to avoid the conditioned odor. But when the recovery period is long (I month), the PC-ablated slugs successfully avoided the conditioned odor. These results indicate that the CNS including the PC can recover from injury at least at the functional level.

The serotonin transporter, SERT, is reported as a key molecule that regulates serotonergic neurotransmission. In the present study, we analyzed the localization of Lymnaea SERT (LymSERT) mRNA-containing neurons by in situ hybridization using frozen sections of the central nervous system (CNS) of Lymnaea. To precisely demonstrate the distribution of LymSERT mRNA-containing neurons, colocalization with serotonin immunoreactivity was also examined. The results showed that LymSERT mRNA was constitutively expressed and localized in the serotonin-containing neurons in the CNS.

We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50-60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.

Repeated spaced training sessions of contingent tactile stimulation to the pneumostome as it opens are required to cause long-term memory (LTM) formation of aerial respiratory behaviour making if difficult to determine exactly when memory forms. We have devised a single-trial aversive operant conditioning training procedure in Lymnaea to be better able to elucidate the causal mechanisms of LTM formation. Observations of baseline breathing behaviour in hypoxia were first made. Twenty-four hours later the snails were trained using the single trial procedure, by placing them in a small Petri dish containing 4 ml of 25 mM KCl for 30-35 s as soon as the first pneumostome opening in hypoxia was attempted. LTM was present if (1) breathing behaviour following training was significantly less than before; and (2) breathing behaviour post-training was significantly less in experimental groups than in yoked control groups. LTM persisted for 24 It but not 48 h. Yoked controls that received an aversive stimulus not contingent with pneumostome opening had no evidence of memory. Cooling directly after, but not at any other time, blocks LTM formation. LTM formation was also prevented by removal of the cell body of the neuron RPeD1 before training. (C) 2007 Elsevier Inc. All rights reserved.

In the majority of studies designed to elucidate the causal mechanisms of memory formation, certain members of the experimental cohort, even though subjected to exactly the same conditioning procedures, remember significantly better than others, whereas others show little or no long-term memory (LTM) formation. To begin to address the question of why this phenomenon occurs and thereby help clarify the causal mechanism of LTM formation, we used a conditioned taste aversion (CTA) procedure on individuals of the pond snail Lymnaea stagnalis and analyzed their subsequent behavior. Using sucrose as an appetitive stimulus and KCI as an aversive stimulus, we obtained a constant ratio of 'poor' to 'good' performers for CTA-LTM. We found that approximately 40% of trained snails possessed LTM following a one-trial conditioning procedure. When we examined the time-window necessary for the memory consolidation, we found that if we cooled snails to 4 degrees C for 30 min within 10 min after the one-trial conditioning, LTM was blocked. However, with delayed cooling (i.e. longer than 10 min), LTM was present. We could further interfere with LTM formation by inducing inhibitory learning (i.e. backward conditioning) after the one-trial conditioning. Finally, we examined whether we could motivate snails to acquire LTM by depriving them of food for 5 days before the one-trial conditioning. Food-deprived snails, however, failed to exhibit LTM following the one-trial conditioning. These results will help us begin to clarify why some individuals are better at learning and forming memory for specific tasks at the neuronal level.

Nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO) are thought to act as gaseous neuromodulators in the brain across species. For example, in the brain of honeybee Apis mellifera, NO plays important roles in olfactory learning and discrimination, but the existence of H2S- and CO-mediated signaling pathways remains unknown. In the present study, we identified the genes of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), cystathionine beta-synthase (CBS), and heme oxygenase (HO) from the honeybee brain. The honeybee brain contains at least one gene for each of NOS, CBS, and HO. The deduced proteins for NOS, CBS, and HO are thought to contain domains to generate NO, H2S, and CO, respectively, and to contain putative Ca2+/calmodulinbinding domains. On the other hand, the honeybee brain contains three subunits of sGC: sGC alpha 1, sGC beta 1, and sGC beta 3. Phylogenetic analysis of sGC revealed that Apis sGC alpha 1 and sGC beta 1 are closely related to NO- and CO-sensitive sGC subunits, whereas Apis sGC beta 3 is closely related to insect O-2-sensitive sGC subunits. In addition, we performed in situ hybridization for Apis NOS mRNA and NADPH-diaphorase histochemistry in the honeybee brain. The NOS gene was strongly expressed in the optic lobes and in the Kenyon cells of the mushroom bodies. NOS activity was detected in the optic lobes, the mushroom bodies, the central body complex, the lateral protocerebral lobes, and the antennal lobes. These findings suggest that NO is involved in various brain functions and that H2S and CO can be endogenously produced in the honeybee brain. (c) 2007 Wiley Periodicals, Inc.

Gonadotropin-releasing hormone (GnRH) is a hypophysiotropic decapeptide that stimulates the release of gonadotropins from the pituitary. In addition, there are extra-hypothalamic GnRH neurons that project to all regions of the brain and whose function remains unknown. Here, we investigated the effects of GnRH on retinotectal synaptic transmission, the synapses of which are formed between retinal fibers and tectal periventricular neurons that express GnRH receptor mRNA. We used rainbow trout as our study model. The excitatory postsynaptic currents (EPSCs), which were evoked by electrical stimulation of the retinal fibers and recorded in periventricular neurons, were suppressed by antagonists of ionotropic glutamate receptors. EPSCs were increased by application of each of two types of GnRH (GnRH2 and GnRH3) in the trout tectum. Such facilitation lasted for at least 10 min after application of the GnRH. To our knowledge, this is the first report of GnRH modulating conventional synaptic transmission in the brain, suggesting that tectal GnRH enhances tectal sensitivity for retinal inputs. Furthermore, such long-lasting facilitation might occur across all the brain regions innervated by GnRH neurons, and GnRH might simultaneously switch neuronal activities in the brain regions relevant to reproductive behaviors.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Hybrid quantum mechanics/molecular mechanics simulations, coupled to the recently introduced metadynamics method, performed on the adenosine triphosphate (ATP) of the bovine Hsc70 ATPase protein, show which specific water molecule of the solvation shell of the Mg2+ metal cation acts as a trigger in the initial phase of the ATP hydrolysis reaction in ATP synthase. Furthermore, we provide a detailed picture of the reaction mechanism, not accessible to experimental probes, that allows us to address two important issues not yet unraveled: (i) the pathway followed by a proton and a hydroxyl anion, produced upon dissociation of a putative catalytic H2O molecule, that is crucial in the selection of the reaction channel leading to the hydrolysis; (ii) the unique and cooperative role of K+ and Mg2+ metal ions in the reaction, acting as cocatalysts and promoting the release of the inorganic phosphate via an exchange of the OH- hydroxyl anion between their respective solvation shells. This is deeply different from the proton wire mechanism evidenced, for instance, in actin and lowers significantly the free energy barrier of the reaction.

The efferent connections and axonal and dendritic morphologies of periventricular neurons were examined in the optic tectum of rainbow trout to classify periventricular efferent neurons in salmonids. Among the target nuclei of tectal efferents, tracer injections to the following four structures labeled periventricular neurons: the area pretectalis pars dorsalis (APd), nucleus pretectalis superficialis pars magnocellularis (PSm), nucleus ventrolateralis of torus semicircularis (TS), and nucleus isthmi (NI). Two types of periventricular neurons were labeled by injections to the APd. One of them had an apical dendrite ramifying at the stratum fibrosum. et griseum. superficiale (SFGS), with an axon that bifurcated into two branches at the stratum griseum. centrale (SGC), and the other had an apical dendrite ramifying at the SGC. Two types of periventricular neurons were labeled after injections to the TS. One of them had an apical dendrite ramifying at the boundary between the stratum opticum (SO) and the SFGS, and the other had dendritic branches restricted to the stratum album centrale or stratum periventriculare. Injections to the PSm and NI labeled periventricular neurons of the same type with an apical dendrite ramifying at the SO and a characteristic axon that split into superficial and deep branches projecting to the PSm and NI, respectively. This cell type also possessed axonal branches that terminated within the tectum. These results indicate that periventricular efferent neurons can be classified into at least five types that possess type-specific axonal and dendritic morphologies. We also describe other tectal neurons labeled by the present injections.

The pond snail Lymnaea stagnalis is capable of learning conditioned taste aversion (CTA) and then consolidating that learning into long-term memory (LTM) that persists for at least 1 month. LTM requires cle novo protein synthesis and altered gene activity. Changes in gene activity in Lymnaea that are correlated with, much less causative, memory formation have not yet been identified. As a first step toward rectifying this situation, we constructed a cDNA microarray with mRNAs extracted from the central nervous system (CNS) of Lymnaea. We then, using this microarray assay, identified genes whose activity either increased or decreased following CTA memory consolidation. We also identified genes whose expression levels were altered after inhibition of the cyclic AMP response element-binding protein (CREB) that is hypothesized to be a key transcription factor for CTA memory. We found that the molluscan insulin-related peptide II (MIP II) was up-regulated during CTA-LTM, whereas the gene encoding pedal peptide preprohormone (Pep) was down-regulated by CRE132 RNA interference. We next examined mRNAs, of MIP II and Pep using real-time RT-PCR with SYBR Green. The MIP II mRNA level in the CNS of snails exhibiting "good" memory for CTA was confirmed to be significantly higher than that from the CNS of snails exhibiting.,poor" memory. In contrast, there was no significant difference in expression levels of the Pep mRNA between "good" and "poor" performers. These data suggest that in Lymnaea MIP II may play a role in the consolidation process that forms LTM following CTA training. (c) 2006 Wiley-Liss, Inc.

Interaction between the activator type of cyclic AMP response element binding protein (CREB1) and the repressor type (CREB2) results in determining the emergence of long-lasting synaptic enhancement involved in memory consolidation. However, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time polymerase chain reaction (PCR) to CREB in the cerebral giant cells of Lymnaea. The cerebral giant cells play an important role in associative learning and employ a CREB cascade for the synaptic enhancement to neurons such as the B1 moto-neurons. We injected the small interfering RNA (siRNA) of CREB1 or CREB2 into the cerebral giant cells and examined the changes in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation. (c) 2006 Wiley-Liss, Inc.

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP-treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP-treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes. (c) 2006 Wiley-Liss, Inc.

Super water repellency of solid surfaces can be realized by formation of fractal structures, which were verified and explained by various materials, such as alkylketene dimer (AKD), platinum-palladium alloy-covered AKD, and poly(alkylpyrrole), and also by comparison of fractal surfaces with smooth ones. Novel potential applications of the fractal solid surfaces to unique biological studies are suggested based on our previous and the present biological studies on fractal AKD surfaces. (c) 2005 Elsevier B.V. All rights reserved.

The midbrain roof is a retinorecipient region referred to as the optic tectum in lower vertebrates, and the superior colliculus in mammals. The retinal fibers projecting to the tectum transmit visual information to tectal retinorecipient neurons. Periventricular neurons are a subtype of these neurons that have their somata in the deepest layer of the teleostean tectum and apical dendrites ramifying at more superficial layers consisting of retinal fibers. The retinotectal synapses between the retinal fibers and periventricular neurons are glutamatergic, and ionotropic glutamate receptors mediate the transmission in these synapses. This transmission involves long-term potentiation, and is modulated by hormone action. Visual information processed in the periventricular neurons is transmitted to adjacent tectal cells and target nuclei of periventricular neuron axonal branches, some of which relay the visual information to other brain areas controlling behavior. We demonstrated that periventricular neurons play a principal role in visual information processing in the teleostean optic tectum; the effects of tectal output on behavior is discussed also in the present review. (c) 2006 Elsevier Ltd. All rights reserved.

Conditioned taste aversion (CTA) in the pond snail Lymnaea stagnalis has been widely used as a model for gaining an understanding of the molecular and behavioral mechanisms underlying learning and memory. At the behavioral level, however, it is still unclear how taste discrimination and CTA interact. We thus examined how CTA to one taste affected the feeding response induced by another appetitive food stimulus. We first demonstrated that snails have the capacity to recognize sucrose and carrot juice as distinct appetitive stimuli. We then found that snails can become conditioned (i.e. CTA) to avoid one of the stimuli and not the other. These results show that snails can distinguish between appetitive stimuli during CTA, suggesting that taste discrimination is processed upstream of the site where memory consolidation in the snail brain occurs. Moreover, we examined second-order conditioning with two appetitive stimuli and one aversive stimulus. Snails acquired second-order conditioning and were still able to distinguish between the different stimuli. Finally, we repeatedly presented the conditional stimulus alone to the conditioned snails, but this procedure did not extinguish the long-term memory of CTA in the snails. Taken together, our data suggest that CTA causes specific, irreversible and rigid changes from appetitive stimuli to aversive ones in the conditioning procedure.

Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L. stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation. (c) 2005 Elsevier Ltd. All rights reserved.

The fractal alkylketene dimer (AKD) surface is an artificial super water-repellent one with a high contact angle of 174 degrees, therefore, may provide special surface circumstances for studies of biological cells such as cell cultures. The experimental results indicated that the distribution of F-actin in the astrocytes cultured on the fractal AKD-coated dishes showed the stellate shape, while that in the astrocytes cultured on the poly-L-lysine-coated coverslips showed the formation of long alignment. The morphological change of astrocytes is induced by the fractal AKD surface, and the result suggests that astrocyte differentiation is stimulated by the fractal AKD surface.

A transcription factor, cyclic AMP-response element binding protein (CREB), which is phosphorylated by protein kinases (PKA and PKC), is known to be involved in the regulation of oligodendrocyte differentiation. However, it is still unclear whether protein kinase A (PKA) and protein kinase C (PKC) are used simultaneously or at different time points to phosphorylate CREB in oligodendrocytes and whether CREB phosphorylation advances oligodendrocyte differentiation or vise versa. Our previous experiments have shown that in the differentiation process from immature to mature cells, CREB phosphorylation depends on PKC activity and leads to the progression of differentiation. In order to gain a better understanding of the process of differentiation from progenitor to immature cells, we identified which protein kinase, i.e., PKA or PKC, regulates CREB phosphorylation and we determined whether CREB phosphorylation advances differentiation or the reverse. Our results showed that CREB phosphorylation is principally regulated by PKA activity in progenitor cells but not by PKC activity, and that this phosphorylation advances the differentiation of progenitor cells to immature cells in oligodendrocytes. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 10(5) copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (&lt; 25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.

Previous experiments showed that the expression and phosphorylation levels of cyclic AMP-response element binding protein (CREB) are important factors that regulate oligodendrocyte differentiation. The present study was designed to determine whether CREB phosphorylation advances oligodendrocyte differentiation or vice versa and to identify the protein kinase that primarily regulates CREB phosphorylation. We examined the expression and phosphorylation levels of CREB in developing oligodendrocytes at a specific differentiation stage by double-immunocytochemical staining with specific differentiation markers and antibody for phosphorylated CREB. We found that the CREB expression level increased along oligodendrocyte differentiation, and that its phosphorylated level was highest in immature oligodendrocytes. We also showed that CREB phosphorylation was regulated principally by protein kinase C (PKC) activity in immature oligodendrocytes. Our findings suggest that CREB phosphorylation is dependent on a PKC signaling cascade, and this phosphorylation activates CREB-mediated transcription and advances the differentiation of immature to mature oligodendrocytes. (c) 2005 Wiley-Liss, Inc.

Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. (C) 2005 Wiley-Liss, Inc.

We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in Slugs. (C) 2004 Wiley Periodicals, Inc.

The transcription factor, CCAAT/enhancer-binding protein (C/EBP), is involved in important physiological processes, such as cellular proliferation and differentiation, homeostasis, and higher-order functions of the brain. In the present study, we investigated the distribution of mRNA and protein of C/EBP in the central nervous system of the pond snail, Lymnaea stagnalis, by in situ hybridization and immunohistochemistry. Specificity of the anti-mammalian C/EBP antibody against Lymnaea C/EBP (LymC/EBP) was confirmed by combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing and immunoblotting. Cells positive for in situ hybridization were immunoreactive for LymC/EBP in all 11 ganglia. The motoneurons (B1, B2, B4, and B4 clusters) in the buccal ganglia and interneurons ( cerebral giant cell, CGC) in the cerebral ganglia were positive for in situ hybridization and were immunopositive. In the pedal ganglion, the right pedal dorsal 1 (RPeD1), pedal A, and pedal C clusters exhibited positive signals of in situ hybridization and immunohistochemistry for LymC/EBP. CGC and RPeD1 are key neurons for associative learning. In addition, the neuropeptidergic cells in the cerebral, pleural, parietal, and visceral ganglia were positive for in situ hybridization and immunoreactive. Interestingly, although the cytoplasm of almost all immunopositive cells was stained, some neuropeptidergic cells located in the light parietal and visceral ganglia exhibited immunoreactivity in nuclei. Our results suggest that LymC/EBP is involved in learning and memory and in the expression and/or secretion of neuropeptides in Lymnaea.

We examined synaptic plasticity in the optic tectum of rainbow trout by extracellular recordings. We found that the field-excitatory postsynaptic potential in the retinotectal synapses was potentiated by repetitive stimuli of 1.0 Hz for 20 s to the retinotectal afferents. The long-term potentiation (LTP) developed slowly, and was maintained for at least 2 h. Applications of an antagonist for N-methyl-D-aspartic acid (NMDA) receptors or Mg2+-free saline showed that activation of NMDA receptors was required to form the UP beyond the induction period. The present findings indicate that presynaptic stimulation in the retinotectal synapses causes LTP mediated by NMDA receptors in the optic tectum of rainbow trout. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Ionotropic glutamate receptors (iGluRs) are postsynaptic ion channels involved in excitatory neurotransmission. iGluRs play important roles in development and in forms of synaptic plasticity that underlie higher order processes such as learning and memory. Neurobiological and biochemical studies have long characterized iGluRs in detail. However, the structural basis for the function of iGluRs has not yet been investigated, because there is insufficient information about their three-dimensional structures. In 1998, a crystal structure called S1S2 lobes was first solved for the extracellular bilobed ligand-binding domain of the GluR2 subunit. Since then, the crystal structures for the S1S2 lobes both in the apo and in various liganded states have been reported, and recent biophysical studies have further elucidated the dynamic aspects of the structure of the S1S2 lobes. In this review, the dynamic structures of the S1S2 lobes and their ligands are summarized, and the importance of their structural flexibility and fluctuation is discussed in light of the mechanisms of ligand recognition, activation, and desensitization of the receptor. (C) 2004 Wiley-Liss, Inc.

Calexcitin (CE) is a low molecular weight Ca2+- and guanosine triphosphate- binding protein, which is phosphorylated during associative learning in both vertebrates and invertebrates. The purpose of this study was to determine the presence of CE in the central nervous system (CNS) of the pond snail Lymnaea stagnalis, which can acquire classical and operant conditioning. Immunoblotting of CE showed that the anti-CE antibody prepared from squid can detect Lymnaea CE. In the cerebral ganglia, CE-like immunoreactivity was exhibited in two pairs of cell clusters that receive taste signals from the superior or median lip nerves. In both pedal ganglia, CE-like immunoreactivity was detected in 1-4 cell of the PeA clusters, which are involved in the withdrawal response. Our results therefore showed that CE is involved in the feeding and withdrawal neural networks, suggesting that CE may function in associative learning of feeding and withdrawal behavior in L. stagnalis.

In the present study, we identified the key protein that modulates the initiation of functional glutamatergic synapses in developing cortical neurons. First, we found a day in vitro that marked a critical increase in the number of functional synapses by the application of cyclic AMP, as demonstrated with Ca2+ imaging. We then examined the changes in the expression levels of proteins, which were expected to play a role in glutamatergic synapses, by the application of cyclic AMP. Our findings suggest that the expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors modulates the initiation of functional synapses in developing neurons, and that immature neurons already contain N-methyl D-aspartate (NMDA) receptors and presynaptic proteins such as synaptophysin.

The digit-like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor-processing circuits in the digits and tentacular ganglia, we studied the effects of gamma-aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of gamma-aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs. (C) 2004 Wiley Periodicals, Inc.

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity. (C) 2003 Wiley Periodicals, Inc.

Differentiating neurons must acquire many unique morphological and functional characteristics in creating the precise neural circuits of the mature nervous system. The phenomenon of 'neuronal differentiation' includes a special set of simple, separate processes, that is, neuritogenesis, neurite outgrowth, pathfinding, targeting and synaptogenesis. All of these processes are critically dependent on the reorganization of actin cytoskeleton by many actin-binding proteins that function downstream of Rho-family GTPases. Furthermore, de novo synthesis of key proteins are critically involved in the reorganization of actin cytoskeleton during neuronal differentiation. In this article, we review recent progresses in the general mechanisms that control actin dynamics by various actin-binding proteins in differentiating neurons, including a series of recent studies from our laboratory on de novo synthesis of several key proteins that are essential for actin reorganization induced by second messengers. We demonstrated that dual regulation of cyclic AMP and Ca2+ determines cofilin (an actin-binding protein) phosphorylation states and LIM kinase 1 (a colifin kinase) expression level during neuritogenesis. (C) 2004 Elsevier Ltd. All rights reserved.

Conformational changes of proteins are dominated by the excitation and relaxation processes of their vibrational states. To elucidate the mechanism of receptor activation, the conformation dynamics of receptors must be analyzed in response to agonist-induced vibrational excitation. In this study, we chose the bending vibrational mode,of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous studies, and we investigated picosecond dynamics of the glutamate receptor caused by the vibrational excitation of Arg485 via molecular dynamics simulations. The vibrational excitation energy in Arg485 in the ligand-binding site initially flowed into Lys730, and then into the J-helix at the subunit interface of the ligand-binding domain. Consequently, the atomic displacement in the subunit interface around an intersubunit hydrogen bond was evoked in about 3 ps. This atomic displacement may perturb the subunit packing of the receptor, triggering receptor activation.

To analyze the expression pattern of genes of cAMP responsive element binding protein (CREB), we performed in situ hybridization for the whole central nervous system (CNS) of the pond snail Lymnaea stagnalis. The CREB1 (activator) and CREB2 (repressor) homologues have already been cloned in L. stagnalis, and they are referred to as LymCREB1 and LymCREB2. Using the frozen sections and the whole mount preparations of the CNS, we mapped the distribution of LymCREB1 and LymCREB2 mRNA containing neurons. The present findings showed that the LymCREB1 mRNA containing neurons are a relatively few, whereas LymCREB2 mRNA is contained ubiquitously in the whole CNS of L. stagnalis.

The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantity mRNA level in cells and tissues and is now a crucial tool for basic biological researches and biotechnology. In the present study, on the basis of the real-time quantitative RT-RCR, we detected and quantified mRNA copies of the transcription factor, CCAAT/enhancer binding protein (C/EBP; an immediate-early gene that is involved in synaptic plasticity and teaming and memory) in the central nervous system of the pond snail Lymnaea stagnalis. We designed the primer set and the probe in the specific insert for the detection of Lymnaea C/EBP (LymC/EBP) clone 1. This insert is not contained in LymC/EBP clone 2 by alternative splicing. The copy number of LymC/EBP clone I was linearly decreased relative to the dilution of cDNA, and it was estimated 30 copies/mu1 in test sample. The availability of the present study showed that the real-time quantitative RT-PCR technique is more accurate and more specific for the detection and quantification of the mRNA level of genes in L. stagnalis than the other PCR methods.

The pond snail Lymnaea stagnalis acquires conditioned taste aversion (CTA) and maintains its memory for more than a month. Snails in our laboratory were cultured at 20 degreesC on a 12: 12 light-dark cycle (light from 7 am to 7 pm). To examine the hours during which snails acquire CTA effectively, we trained some snails in the morning and others in the afternoon, and then compared their scores. CTA developed in both cases, but scores were significantly better in the morning than in the afternoon. To elucidate the cause of this difference in scores, we observed the voluntary activity of snails and found the circadian rhythm reflected in the snails' free-movement distances; distances at the circadian time 0-12 (daytime) were significantly longer than those at the circadian time 12-24 (nighttime). This rhythm was kept up for at least 3 days, even in constant darkness. In conclusion, L. stagnalis should be trained in the morning to acquire associative learning, possibly because of its greater propensity to roam about at that time as opposed to the afternoon.

The tentacular ganglion, the primary olfactory system of terrestrial slugs, exhibits spontaneous oscillations with a spatial coherence. The digit-like extensions (digits) of the tentacular ganglion presumably house the cell bodies of the neurons underlying the oscillations. The present study was designed to identify the anatomical and physiological determinants of these oscillations with a special focus on whether the neurons located in the digits contribute to the coherent oscillations. We recorded field potentials from the spatially separated sites in the digits in the terrestrial slug Limax marginatus. We also simultaneously recorded tentacular nerve to monitor the coherent oscillations. The spatially separated regions in the digits oscillated at the same frequency as the tentacular nerve, indicating a single coherent activity. To study the neural networks underlying the coherent oscillations, we examined the distributions of acetylcholinesterase (AChE)-containing and gamma-aminobutyric acid immunoreactive (GABA-ir) neurons. AChE-containing and GABA-ir fibers were found to connect the neurons in a branch of the digits with those in other branches. We also used a vital staining technique with 1,1&apos;-didodecyl-3,3,3&apos;,3&apos;-tetramethylindocarbocyanine perchlorate to examine the projections of neurons in the digits. Large stained cells were detected in many branches of the digits after placing the dye on one of the cell masses located in right and left sides of the tentacular ganglion. They were detected in the cell masses and in many branches of the digits after placing the dye on a branch of the digits. Our results showed that the slug primary olfactory system has highly interconnected neural networks.

Although primary olfactory systems in various animals display spontaneous oscillatory activity, its functional significance in olfactory processing has not been elucidated. The tentacular ganglion, the primary olfactory system of the terrestrial slug Limax marginatus, also displays spontaneous oscillatory activity at 1-2 Hz. In the present study, we examined the relationship between odor-evoked spike activity and spontaneous field potential oscillations in the tentacular nerve, representing the pathway from the primary olfactory system to the olfactory center. Neural activity was recorded from the tentacular nerve before, during and after application of various odors (garlic, carrot, and rat chow) to the sensory epithelium and the changes in firing rate and spontaneous oscillations were analyzed. We detected the baseline amplitude of the oscillations and baseline spike activity before stimulation. Odor stimulations for 20 s or 60 s evoked a transient increase in the firing rate followed by a decrease in the amplitude of spontaneous oscillations. The decrease in the amplitude was larger in the first 8 s of stimulation and subsequently showed recovery during stimulation. The amplitude of the recovered oscillations often fluctuated. Odor-evoked spikes appeared when the amplitude of the recovered oscillations was transiently small. These results suggest that the large oscillations could inhibit spike activity whereas the first transient increase in spike activity was followed by the decrease in the oscillation amplitude. Our results indicate that there is a significant negative correlation between spontaneous oscillations and odor-evoked spike activity, suggesting that the spontaneous oscillations contribute to the olfactory processing in slugs.

Conformational changes of receptors are dominated by the excitation of their collective motions. The most likely energy source of this excitation is considered to be a collision of an agonist with the binding site of a receptor and a consequential excitation of their vibrational modes. In the present study, as an approach to elucidating the mechanism for receptor activation, we chose both the symmetric stretching vibration of the 1C-carboxyl group of glutamate and the bending vibration of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous study, and we quantum-mechanically calculated the vibrational excitation probability of these two vibrations by glutamate collision. The excitation probability of these two vibrations was found to exceed 0.5 about 260 fs after the onset of collision within the first-order perturbation approximation. Taking into account that the period of these vibrations is about 20 fs, we can expect that the vibrational excitation occurs in Arg485 after tens of vibrations during the collision. This vibrational energy may redistribute to the collective motions of the receptor, resulting in global conformational changes in the receptor. We also confirmed that the charge transfer was small between an agonist and the binding site of GluR2. The glutamate receptor may be oscillatory systems that require the energy injection into the specific vibrational modes of the specific amino acid residues to trigger their activation. Such an injection of energy is provided by agonist collision. (C) 2003 Elsevier B.V. All rights reserved.

The present study was designed to elucidate the roles of dendritic voltage-gated K+ channels in Ca2+ influx mechanism of a rat Purkinje cell using a computer simulation program. First, we improved the channel descriptions and the maximum conductance in the Purkinje cell model to mimic both the kinetics of ion channels and the Ca2+ spikes, which had failed in previous studies. Our cell model is, therefore, much more authentic than those in previous studies. Second, synaptic inputs that mimic stimulation of parallel fibers and induce sub-threshold excitability were simultaneously applied to the spiny dendrites. As a result, transient Ca2+ responses were observed in the stimulation points and they decreased with the faster decay rate in the cell model including high-threshold Ca2+-dependent K+ channels than in those excluding these channels. Third, when a single synaptic input was applied into a spiny dendrite, Ca2+-dependent K+ channels suppressed Ca2+ increases at stimulation and recording points. Finally, Ca2+-dependent K+ channels were also found to suppress the time to peak Ca2+ values in the recording points. These results suggest that the opening of Ca2+-dependent K+ channels by Ca2+ influx through voltage-gated Ca2+ channels hyperpolarizes the membrane potentials and deactivates these Ca2+ channels in a negative feedback manner, resulting in local, weak Ca2+ responses in spiny dendrites of Purkinje cells. (C) 2003 Elsevier B.V. All rights reserved.

The present study was designed to elucidate the cellular and molecular mechanisms of neuritogenesis in differentiating neurons. For this purpose. we used pharmacological and immunochemical techniques to determine the intracellular signal transduction pathways that regulate actin dynamics during neuritogenesis. We confirmed that a rise in intracellular cyclic AMP (cAMP) concentration stimulated cells to increase their neurite numbers, and that this increase of neurites was suppressed by activation of calcineurin induced by a Ca2+ influx through voltage-dependent Ca2+ channels. Expression of a specific cofilin kinase (LIM kinase 1) was increased and decreased by cAMP and Ca2+ cascades. respectively. The phosphorylation state, but not the level of expression, of a potent regulator of actin dynamics (cofilin) was strongly correlated with the expression level of LIM kinase 1. Our results suggest that polymerization and depolymerization of actin by cofilin phosphorylation is necessary for neuritogenesis in differentiating neurons. (C) 2003 Elsevier B.V. All rights reserved.

To understand the mechanism of molecular recognition of glutamate receptors, we calculated the dipole moments, net charges, and electrostatic potentials of the agonists and antagonists of the glutamate receptors under aqueous conditions by the ab initio molecular orbital method at the HF/6-311++G(3df,2pd) level. All of the ligands had negative net charges at both termini and, consequently, formed negative electrostatic potentials at these termini. We further calculated the net charges and electrostatic potential of the S1S2 lobes of the glutamate receptor subunit GluR2 under aqueous conditions by the semempirical PM3 molecular orbital method, and found that the ligand-binding cleft of the S1S2 lobes formed a primarily positive electrostatic potential. A strongly positive electrostatic potential was formed particularly around Arg485 in the S1 lobe. A negative electrostatic potential was observed only in a small region around Glu657 in the S2 lobe in the ligand-binding cleft. When a ligand approaches the ligand-binding cleft, it may proceed to Arg485 in the S1 lobe, due to both repulsion by the negative electrostatic potential of Glu657 as well as the attraction of the strongly positive electrostatic potential of Arg485 itself. (C) 2003 Elsevier B.V. All rights reserved.

Even though glutamic acid contains only one more carboxyl group than gamma-aminobutyric acid (GABA), these neurotransmitters are recognized by their own specific receptors. To understand the ligand-recognition mechanism of the receptors, we must determine the geometric and electronic structures of GABA and glutamic acid in aqueous conditions using the ab initio calculation. The results of the present study showed that the stable structure of GABA was the extended form, and it attracted both cations and anions. Glutamic acid only attracted cations and was stabilized in four forms in aqueous conditions: Type 1 (an extended form), Type 2 (a rounded form), and Types 3 and 4 (twisted forms of Type 1). The former two types had low energy and the energy barrier between them was estimated to be small. These results showed that most free glutamic acid is present as Type a, Type 2, and transient forms. The present results therefore suggest that the flexibility of the geometric structures of ligands should be taken into account when we attempt to elucidate the mechanism of recognition between ligands and receptors, in addition to the physicochemical characteristics of ligands and receptors.

To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two-dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage-sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle-like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization-signal propagating paths along a horizontal layer structure and a vertical bundle-like structure and that these synaptic responses occur via glutamatergic transmission.

Based on the results of previous behavioral experiments, researchers believe that sensitivity to light stimuli is not restricted to the eyes in the pond snail Lymnaea stagnalis. To determine the presence of a non-ocular dermal photoreception system and to examine the synaptic connections between this peripheral system and the central nervous system, we electrophysiologically examined the activities of the pedal nerves in L. stagnalis by light stimulation. The results demonstrated that light stimulation evokes non-ocular dermal photosensitive responses in the foot, that these responses exert inhibitory, afferent influences through the inferior pedal nerves to the pedal ganglia, and that these responses were independent of the ocular photoreception system in L. stagnalis. (C) 2002 Elsevier Science B.V. All rights reserved.

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.

Astrocytes are connected by gap junctions, which provide intercellular pathways that allow a direct exchange of ions and small metabolites including second messengers and the propagation of electric currents. The roles of gap junctional communication on whole-cell morphology, cytoskeletal organization, and intercellular communication in astrocytes are not yet clear even in vitro, though there are many studies that have examined the active relation between gap junctions and actin filaments in astrocytes. Here we examined the effects of gap junction inhibitors, which do not interrupt the formation but rather the function of gap junctions, on whole-cell morphology, cytoskeletal organization. and intercellular communication in rat cultured astrocytes. Functional blockade of gap junctions during the formation of an astrocytic monolayer resulted in discordance of actin stress fibers between neighboring cells. even though whole-cell morphology of these cells did not change by such treatment. Mechanical stimulation-induced calcium wave propagation was significantly reduced in these actin-discordance cells even after thorough wash out. Differentiation of astrocytes in the presence of gap junction inhibitors was associated with morphological disarrangement among neighboring cells due to disordered alignment of actin stress fibers between cells.
Our results indicate that gap junctional communication enables cell-to-cell coordination of actin stress fibers in astrocytes, thus enhancing intercellular communication through calcium spread. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.

Multiple site optical recording was used to analyze the neural activity changes caused by conditioned taste aversion (CTA) training in the pond snail Lymnaea stagnalis. In response to electrical stimulation of the median lip nerve, which transmits chemosensory signals of appetitive taste to the central nervous system, we optically detected large numbers of spikes in several parts of the buccal ganglion. The effects of CTA training on the spike responses were examined in two areas of the ganglion where the most active neural responses occurred. In one area (termed Area I) that included the N1 medial (NUM) cells, a class of central pattern generator interneurons involved in feeding behavior, the number of spikes in a period 1500-2000 ms after median lip nerve stimulation was significantly reduced in conditioned animals compared to control animals. In another area (termed Area II) positioned between buccal motoneurons, the B3 and B4CL (cluster) cells, the evoked spike responses were unaffected by CTA training. These results, taken together with our previous results indicating an enhancement of an inhibitory input to the NIM cells during CTA, suggest that an appetitive taste signal transmitted to the NIM cells through the median lip nerves is suppressed during CTA, resulting in a decrease of the feeding response. (C) 2001 John Wiley & Sons, Inc.

Although the mechanisms of Ca2+, wave propagation in astrocytes induced,by mechanical stimulation have been well studied, it is still not known how the [Ca2+](i) increases in the stimulated cells. Here, we have analyzed the mechanisms of [Ca2+](i) increase in single, isolated astrocytes. Our results showed that there was an autocrine mechanism of Ca2+ regulation mediated by ATP in mechanically stimulated astrocytes. This autocrine mechanism induced the activation of phospholipase C via a G-protein, resulting in Ca2+ release from intracellular Ca2+ stores. A second pathway mediating a [Ca2+](i) increase was via a Ca2+ influx from the extracellular space, which, interestingly, suppressed an intracellular Ca2+ oscillation. These two different Ca2+ cascades are involved in signal transduction and may function separately during intercellular communication. NeuroReport 12:2619-2622 (C) 2001 Lippincott Williams & Wilkins.

As a first approach to understanding the mechanism for the recognition of a ligand by its receptor, we first calculated the electronic and structural states of ionized gamma -aminobutyric acid (GABA) and ionized glutamic acid using the ab initio method with the 6311++G (3df, 2pd) basis set. We paid special attention to the physicochemical characteristics of these molecules, such as the electric dipole moment, electrostatic potential, and electrostatic force, Even though GABA and glutamic acid are known to exert completely opposite influences in the mammalian brain by binding their specific receptors, the only difference in their chemical structures is that glutamic acid contains one more carboxyl group than GABA. As a result, we succeeded in showing that a difference of only one carboxyl group induces significant differences in the electronic and structural states between these molecules. These differences have a crucial influence on the electric dipole moments, the electrostatic potentials, and the electrostatic forces. The most remarkable finding of the present research is that the electrostatic potential formed by glutamic acid is composed of only negative parts, while that formed by GABA is separated into positive and negative parts. These results strongly suggest that GABA can approach either positively or negatively charged amino acids by adjusting its own orientation, while glutamic acid can approach only a positively charged binding site.

To understand the mechanism of activation of a receptor by its agonist, the excitation and relaxation processes of the vibrational states of the receptor should be examined. As a first approach to this problem, we calculated the normal vibrational modes of agonists (glutamate and kainate) and an antagonist (6-cyano-7-nitroquinoxaline-2,3-dione: CNQX) of the glutamate receptor, and then investigated the vibrational interactions between kainate and the binding site of glutamate receptor subunit GluR2 by use of a semiempirical molecular orbital method (MOPAC2000-PM3). We found that two local vibrational modes of kainate, which were also observed in glutamate but not in CNQX, interacted through hydrogen bonds with the vibrational modes of GluR2: (i) the bending vibration of the amine group of kainate, interacting with the stretching vibration of the carboxyl group of Glu705 of GluR2, and (ii) the symmetric stretching vibration of the carboxyl group of kainate, interacting with the bending vibration of the guanidinium group of Arg485, We also found collective modes with low frequency at the binding site of GluR2 in the kainate-bound state. The vibrational energy supplied by an agonist may flow from the high-frequency local modes to the low-frequency collective modes in a receptor, resulting in receptor activation.

We studied the neural oscillatory activity in the peripheral olfactory system of the tentacles in the terrestrial slug, Limax marginatus, by extracellular recording, Recordings from the cut-ends of the inferior tentacular nerves connected to the inferior tentacular ganglia and sensory epithelia showed spontaneous oscillatory activity at frequencies of 0.1-30 Hz. This spontaneous activity was dominated by the 0.6-6 Hz band. Ethanol odor stimulation decreased the amplitude in the 0.6-6 Hz band and increased those in the 6-15 and 15-30 Hz bands. Antagonists of the gamma -aminobutyric acid (GABA) receptor, bicuculline and picrotoxin, resulted in suppression of spontaneous activity and modulated the odor response in the 0.6-6 Hz band. Our results indicate the involvement of GABA-mediated oscillatory activity in the tentacular nerves in the olfactory processing in molluscs. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Complement receptor 3 (CR3), one of cell adhesion molecules, plays a crucial role in secretion of mammalian neutrophils. To study whether CR3 is also involved in neurosecretion of gastropod molluscs, we examined the CR3-like immunoreactivity in the central nervous system (CNS) and the superior and inferior tentacles of the terrestrial slug, Limax marginatus. In the CNS of L. marginatus, we did not detect the specific immunoreactivity at all. In contrast, the CR3-like immunoreactivity was observed in the cell bodies and processes of collar cells of the superior and inferior tentacles. In particular, granules contained in the cell bodies of collar cells exhibited the CR3-like immunoreactivity. Retrograde labeling of horseradish peroxidase applied on the sensory epithelia (SE) of superior and inferior tentacles showed that the collar cells of these tentacles project their processes to the SE. in the previous study, homogenate of the superior tentacles injected into the body cavity of slugs stimulated spermatogenesis and simultaneously inhibited egg-laying behavior, suggesting that the tentacular hormone acts as gonadotropic hormone. Taken together, CR3 expressed in the collar cells is considered to be involved in the secretion of tentacular hormone in L. marginatus.

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-LI-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells. (C) 2000 Elsevier Science BN. All rights reserved.

Although there are many lines of evidence for both the presence of nitric oxide synthase (NOS) in the central nervous system (CNS) and the effects of NO on activating and modulating the feeding circuit in Lymnaea stagnalis, there has been no direct evidence that NO generation in the CNS accompanies feeding behavior. In the present study, we used a NO specific electrode to measure the increase in NO concentration around the buccal ganglia when the lips of semi-intact preparations of L. stagnalis were stimulated by sucrose. The NO concentration of the buccal ganglia was significantly increased by an application of sucrose to the lips. A NO scavenger and a NOS inhibitor suppressed this increase in NO concentration. A pair of putative NO-generative neurons in the buccal ganglia, the B2 cells, are active during the inter-feeding phase, and the bursting of the B2 cell elicited by sucrose application starts simultaneously with the feeding response. The rhythmic pulses of NO generation corresponded well with the rhythmic bursting of the B2 cells, which itself corresponds to the 'fictive feeding response'. The present data provide the first direct evidence that NO is generated in the buccal ganglia of L, staganalis and is involved in a specific behavior such as Feeding. (C) 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

A- and D-type K+ channels (KA and KD channels) have unique physiological properties that play important roles in the integration of excitatory post synaptic potentials (EPSPs) in neuronal dendrites. These functions were analyzed using a computer program, NEURON, to simulate high- frequency sequential synaptic inputs, that can induce long-term potentiation (LTP). We paid close attention to the stability of the reduction of sequential EPSPs. When either KA or KD channels were included in models, the EPSP reduction ratios were less stable than containing both KA and KD channels. When both KA and KD channels were present in the model, the variance of EPSP reduction ratios was significantly smaller in comparison with that in the presence of either KA or KD channels alone. We thus concluded that the co-existence of KA and KD channels is necessary to produce stable EPSPs during the high-frequency synaptic stimulation necessary for induction of LTP. (C) 2000 Elsevier Science Ireland Ltd.

In the present study, the 'clockwork' hypothesis proposed by Schrodinger was examined from the viewpoint of thermodynamics. Firstly, noticing a unidirectional transfer of entropy in a heat engine, the logic was briefly explained about a close relation between this entropy transfer and an irreversible cycle performed by a working body. Next, paying attention to two fundamental differences between a heat engine and a biological system, we considered an isolated system A(Sigma) consisting of three one-component systems (A(i), A, A(o)) and noted a case that the same molecules as the component ones flowed quasistatically into A(i) from the outside. Then, the unidirectional flows of the molecules, energy and entropy, which were induced by the above inflow in A(Sigma), were formulated on the basis of the equilibrium thermodynamics for an open system. Furthermore, it was clarified that the fundamental equation for these flows is the Schrodinger inequality and that the necessary-sufficient condition for this inequality is the existence of an irreversible cycle performed by A. Here A corresponds to a working body in a heat engine. It was, thus, concluded that the 'clockwork' hypothesis by Schrodinger is considered to be reasonable for a biological system composed of various irreversible subsystems. (C) 2000 Elsevier Science B.V. All rights reserved.

To identify the types of neurons and to infer the patterns of connectivity in slug tentacles, we stained the neurons in the superior and inferior tentacles in the terrestrial slug, Limax marginatus, by backfilling of the tentacular nerves with Lucifer yellow. Four types of stained neurons, '(1) sensory neurons', '(2) gamma cells', '(3) ganglion cells', '(4) lateral cells', were identified both in the superior and inferior tentacles. Three subtypes of the sensory neurons, '(la) round sensory neurons', '(lb) spindle-shaped sensory neurons', and '(lc) small sensory neurons', were found in the digits. The gamma cells and the ganglion cells were interneurons. Three subtypes of gamma cells, '(2a) round monopolar gamma cells', '(2b) round bipolar gamma cells', and '(2c) large gamma cells', were present in the digits. The ganglion cells were composed of '(3a) monopolar ganglion cells','(3b) bipolar ganglion cells', and '(3c) elongated ganglion cells'. The monopolar and bipolar types were located both in the tentacular ganglia and digits, whereas the elongated type was present only in the tentacular ganglia. The lateral cells, whose function is unknown, were found in the dermo-muscular sheaths of the tentacles. Our study provides the first description of the neuronal map of inferior tentacles in gastropods. The results showed no differences in the morphological features of stained neurons between the superior and inferior tentacles in L. marginatus. (C) 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The differentiated type of neuroblastoma x glioma hybrid cell line, NG108-15, has widely been used in in vitro studies instead of primary-cultured neurons. Here we examined whether NG108-15 cells can be used as a model for studying the neuronal differentiation process. We compared the expression of neuronal proteins (neurofilament 200 (NF200), phosphorylated-NF200 (p-NF200), microtubule associated protein 2, synaptophysin, syntaxin 1, choline acetyltransferase, and acetylcholinesterase (AChE)) and a glial protein (vimentin) between undifferentiated and differentiated NG108-15 cells by immunocytochemistry and immunoblot analysis. The expression of all neuronal proteins, with the exception of NF200 and p-NF200, was positive in differentiated cells, but almost negative in undifferentiated cells. On the other hand, cytoskeletal intermediate filaments (NF200 and p-NF200) for neurons and that (vimentin) for glia were present in both undifferentiated and differentiated cells. Furthermore, a high expression of AChE mRNA was confirmed in differentiated cells by reverse transcription-PCR analysis. Our results showed that even though the expression of cytoskeletal filaments does not change during differentiation of NG108-15 cells, these cells during differentiation can serve as an appropriate tool for investigating and understanding the mechanisms involved in neuronal development and differentiation. (C) 2000 Elsevier Science Ireland Ltd and the Japanese Neuroscience Society. All rights reserved.

In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.

Fictive feeding activity was monitored in the buccal ganglia of semi-intact preparations of the pond snail, Lymnaea stagnalis, to examine the effects of nitric oxide (NO) released from motoneurons innervating the esophagus on the feeding response. The present results suggest that first
even the low concentration of constitutive NO precisely regulates the feeding rhythm by suppressing high frequency feeding responses
second, that the high concentration of NO released after activation of the feeding central pattern generator following appetitive stimulation of the lips suppresses the feeding rate, resulting in recurrent inhibition. This is the first direct evidence that NO can function to suppress rhythmic activity in the brain. Copyright (C) 2000 Elsevier Science Ireland Ltd.

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. Ln the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which an the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, fur example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis. (C) 2000 Elsevier Science B.V. All rights reserved.

We examined three-dimensionally the arrangement of gamma-aminobutyric acid; (GABA)-like immunoreactive neurons in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis, by a combination of immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations. GABA-like immunoreactivity was detected in all ganglia of the adult CNS. The following distribution of immunoreactive cell bodies was noted in the adult snail. Buccal ganglia: one cell body and five pairs of cell bodies, cerebral ganglia: one pair of cell bodies, pedal ganglia: two single cell bodies, two pairs of cell bodies, and three pairs of cell clusters, and pleural ganglia: one pair of cell bodies. In the asymmetrical parietal ganglia, three cell bodies were located in the left parietal ganglion; three cell bodies and three cell clusters were located in the right parietal ganglion. In the single visceral ganglion, a few scattered individual cell bodies and a cell cluster were GABA-like immunoreactive. Our results showed that the occurrence of GABA is widely spread in the CNS of adult L. stagnalis. GABA-like immunoreactivity in the CNS was not detected in the embryo but; was observed after hatching, although the number of stained cells was less than in the adult, with the exception of those in the cerebral ganglia where their number decreased with maturation. Our results provide detailed maps of the central GABA-like immunoreactive neurons in juveniles, immatures, and adults oft. stagnalis. J. Comp. Neurol. 418:310-322, 2000. (C) 2000 Wiley-Liss, Inc.

The pond snail, Lymnaea stagnalis, is a useful preparation for analyzing the commonality between development and learning. To promote this analysis, the anatomical substrate should be provided upon which learning is superposed during development. Because we previously demonstrated that L. stagnalis change their ability of conditioned taste aversion (CTA) as a long-term memory from veliconcha embryos to immatures, we examined in the present study the numbers of cells and the volume of the buccal and cerebral ganglia in the snails at the critical developmental stages. The buccal and cerebral ganglia include the majority of neurons involved in the CTA. We found that the numbers of cells in these ganglia are almost saturated in the immatures, but the volumes of these ganglia still increase from the immatures to the adults. These results suggested that most of the cells indispensable to the CTA emerge at the immature stage, but that individual cells in the ganglia continue to enlarge even in adulthood. Furthermore, the key neuron for the CTA was found to mature at the immature stage. The present study provided the anatomical substrate upon the long-term CTA, by which snails can eat safe food in a wide territory.

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments - actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity. (C) 2000 Elsevier Science B.V. All rights reserved.

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100 nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques.
Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.

To directly monitor the glial activity in the CNS of the pond snail, Lymnaea stagnalis, we optically measured the electrical responses in the cerebral ganglion and median lip nerve to electrical stimulation of the distal end of the median lip nerve. Using a voltage-sensitive dye, RH155, we detected a composite depolarizing response in the cerebral ganglion, which consisted of a fast transient depolarizing response corresponding to a compound action potential and a slow depolarizing response. The slow depolarizing response was observed more clearly in an isolated median lip nerve and also detected by extracellular recording. In the median lip nerve preparation, the slow depolarizing response was suppressed by an L-type Ca2+ channel blocker, nifedipine, and was resistant to tetrodotoxin and Na+-free conditions. Together with the fact that a delay from the compound action potential to the slow depolarizing response was not constant, these results suggested that the slow depolarizing response was not a postsynaptic response. Because the signals of the action potentials appeared on the saturated slow depolarizing responses during repetitive stimulation, the slow depolarizing response was suggested to originate from glial cells. The contribution of the L-type Ca2+ current to the slow depolarizing response was confirmed by optical recording in the presence of Ba2+ and also supported by intracellular Ca2+ measurement.
Our results suggested that electrical stimulation directly triggers glial Ca2+ entry through L-type Ca2+ channels, providing evidence for the generation of glial depolarization independent of neuronal activity in invertebrates. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.

We have focused on effects of local mechanical properties of the cell on cell motion. By using atomic force microscopy, we measured spatial distribution of local elastic modulus on mouse fibroblasts (NIH3T3), which is living in a physiological condition. In order to examine validity of AFM elastic measurements, we measured local elastic modulus of gels as elastic reference materials. The results obtained with AFM were compared with values obtained by tensile creep method. It is veryfied that these values are proportional each other. The AFM experiments on living cells revealed that center area of cell surface is about 10 times softer than the surroundings and looks like a big softer hole in the elasticity image. We fixed the cell just after the AFM measurements and carried out immunofluorescence observation for cytoskeletal filaments of actin filaments, microtubules and intermediate filaments. A comparison between distribution of local elasticity and cytoskeletones indicates that harder area on the cell results mainly from concentration of actin filaments. However, We found that some areas like the big softer hole do not correspond to distribution of actin filaments.

Using the force modulation mode in atomic force microscopy, we have succeeded in capturing time-lapse viscoelastic images of living mouse fibroblasts (NIH3T3) for several hours in a physiological condition without damaging the fibroblasts. Elongation of the lamellipodia and swelling of blabs were observed in time-lapse topographic images, which were taken every 10 min. The corresponding viscoelastic responses at a frequency of 600 Hz were visualized as consecutive images. The stiffer part of the cell body was fairly stable and did not show morphological changes for over 1 h. This is probably due to excess condensation of the actin network, hardening the cell cortex, and lowering the cytoskeletal activity. The nuclear portion of the cell body seems to be slightly less viscous than the peripheral region.

The topography and elasticity of living and fixed astrocytes cultured from the rat cerebra were studied quantitatively by atomic force microscopy (AFM). Ridge-like structures reflecting F-actin beneath the cell membrane were prominent in the contact-mode images of living astrocytes. Many of these ridges became unclear after fixation (2%, glutaraldehyde). In addition, the ridge-like structures were invisible in the topography of living cells observed at zero-loading force in the force mapping mode, which is considered to show the real cell surface not pressed down by an AFM tip. The topography of fixed cells observed both in the contact mode and at zero-loading force in the force mapping mode was similar to that of living cells observed at zero-loading force in the force mapping mode, although some deformed areas were detected in the fixed cells. The elasticity map images of living astrocytes showed that the cell membrane above the nucleus was softer (2-3 kPa) than the surroundings, and that the cell membrane above F-actin was stiffer (10-20 kPa) than the surroundings. In the elasticity map images of fixed astrocytes, on the other hand, the elasticity of the cells was found to be relatively uniform (200-700 kPa) irrespective of the inner structures of cells. These results show that images observed by AFM should be carefully examined in consideration of the force introduced to specimens and the elasticity of specimens to find out the real surface topography.

The pond snails, Lymnaea stagnalis, change their ability of conditioned taste aversion (CTA) during their development, for example, stage 29 embryos can acquire the CTA, whereas immature snails come to use a long-term memory to maintain the conditioned response. We thus examined the relationships between the learning ability and the development of key neurons (cerebral giant cells: CGCs) for this CTA. The immunoreactivity of serotonin, which is a main neurotransmitter employed in the feeding circuitry, was first observed in the CGCs at the stage 29. After hatching, the CGCs developed their neuropile faster than other cells in the buccal and cerebral ganglia, resulting in their early innervation at the immature stage. The present results, therefore, indicate that the development of key neurons for learning stimulates the developmental changes in learning ability. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

As the first step in the study df morphological changes in neurons associated with their functional changes, we applied atomic force microscopy (AFM) for the observation of fine three-dimensional morphological changes in rat cerebellar granule cells stimulated by an agonist of glutamate receptors, N-methyl-D-aspartate (NMDA). The AFM revealed that NMDA changed the cross-sections of cell bodies from a trapezoid-like form to a triangle-like form within a minute. The fine hill-like structures on the top surfaces of the cell bodies became wider during the same period. These results were suggested to be induced by the depolymerization; of filamentous actin triggered by the entry of Ca2+ via cation channels complexed with the activated NMDA receptors.

The cerebral giant cell (CGC) is known to play a crucial role in the regulation of feeding response in the pond snail, Lymnaea stagnalis. However, the mechanisms of signal transduction from the CGC to the following buccal motor neurons are not clear. In the present study, we examined whether cyclic AMP (cAMP)-dependent protein kinase (PKA) contributes to enhancement of a monosynaptic connection between the presynaptic CGC and the postsynaptic buccal motor neuron 1 (B1 cell). Injection of cAMP into the CGC or inhibition of phosphodiesterase by isobutylmethylxanthine in the CGC increased the amplitude of excitatory postsynaptic potential (EPSP) in the B1 cell, whereas no changes were detected in the electrical properties of the CGC. The synaptic enhancement in the B1 cell was completely blocked by inhibition of PKA in the CGC but did not require a de novo protein synthesis due to a PKA phosphorylation. The increase in the EPSP amplitude of B1 cell was associated with the increase in the amount of serotonin release from the CGC. These results hence provided the physiological evidence of the direct regulation of a synaptic enhancement by PKA in the CNS of L. stagnalis, indicating the completely different mechanism from that in the well-studied siphon- and gill-withdrawal reflex in Aplysia.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using electrophysiological techniques. When the activity of those nerves was induced in lip-tentacle preparations, aversive taste signals were transmitted through all the lip and tentacle nerves, but appetitive signals could be recorded only through the superior lip nerve. In the CNS immersed in high Mg2+-high Ca2+ saline, electrical stimuli applied to any of the nerves failed to induce action potentials in one of the regulatory neurons (cerebral giant cell: CCC) involved in feeding responses, implying that the signals are polysynaptically transmitted to the CGC. Intracellular recordings revealed that the CGCs in semi-intact half-body preparations received both appetitive and aversive taste signals not only through the superior lip nerve but also through the median lip nerve. In addition, an osphradium was ruled out as a candidate for appetitive reception. The present results, together with our preceding data arrived at by the histochemical analyses, indicate that the appetitive taste transduction responsible for generating feeding responses is performed through the superior lip nerve with some contribution of the median lip nerve. The data showing that the CGC can receive various taste signals suggests that it may play a crucial role in feeding behavior as demonstrated in the study of conditioned taste-aversion. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABA(A), nACh, 5-HT3, P-2x, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D,L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using histological techniques. Anatomical drawings showed the detailed distributions of the superior lip, median lip, and tentacle nerves in the lip and tentacle; in particular it was found that the mouth is mainly innervated by the superior lip nerve. The tentacle nerve was clarified to form a zigzag structure along the extension direction in a shrinking tentacle. By backfilling of the superior lip nerve and/or the median lip nerve with fluorescent dyes, the neurons in the CNS made some clusters, whereas those stained from the tentacle nerve made other clusters. These stained neurons were not part of the central pattern generator or its regulatory neurons for feeding. The present results, therefore, suggest that the superior lip nerve may be employed as a principal factor in the chemosensory transduction from the mouth, and that no direct inputs occur through the lip and tentacle nerves to the central pattern generator or its regulatory neurons for feeding. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

As the first step to study relationships between development and learning in the molluscan central nervous system,we examined developmental changes in acquisition and retention of a conditioned taste aversion (CTA) in the pond snail, Lymnaea stagnalis. We found that snails developed ability of a CTA as a long-term memory through three critical stages. Embryos in veliconcha started to respond to appetitive sucrose at the first critical stage. This response was in good agreement with morphological observations that embryos at this developmental stage seemed to be physically ready to eat. However, they could not associate this appetitive stimulus (conditioned stimulus: CS) with an aversive stimulus of KCI (unconditioned stimulus: UCS). At the second critical stage, embryos just before hatching acquired the CTA, but the conditioned response did not persist. Through this stage, they may acquire learning ability to safely seek out food in an external environment. At the third critical stage, immature snails with a 10 mm shell could use a long-term memory to maintain the conditioned response. This memory persisted for at least a month, showing that now they are able to maintain a long-term memory so that they can safely eat a variety of food when they cover wide territory to search for a mate. The present findings indicate that the development of learning ability in snails, which secures acquisition of better survival ability, is coincident with the major changes in their life cycle.

The incomplete morphological characterization of type 2 astrocytes is in pari responsible for the slow progress of studies on these cells. To examine and characterize type 2 astrocytes morphologically, three-dimensional fine structures of the surfaces of type 2 astrocytes cultured from rat cerebella were studied by a combination of atomic force microscopic and immunocytochemical techniques. Atomic force microscopy (AFM) revealed irregular ridge-like structures that form a meshwork distributed throughout the cell body surfaces and the thick processes. These ridges were found to be of two heights (31 nm and 82 nm). This finding indicates two possible configurations responsible for shaping the meshwork: (1) two structures of different thickness are beneath the cell membrane; and (2) two structures are located at two different depths from the cell membrane. On the other hand, immunocytochemical studies for tubulin and glial fibrillary acidic protein (GFAP) revealed that these cytoskeletal filaments are similarly distributed within the resolution power of a light microscope. However, no detectable structures were obtained by actin staining. The immunocytochemical findings suggest that the AFM-revealed ridges forming the irregular meshwork on the cell surfaces may reflect very fine bundles of tubulin and/or GFAP. Therefore, AFM study, with the help of immunocytochemical study, is a powerful tool for characterizing cell morphology. The results of the present study reveal the first morphological characterization of type 2 astrocytes.

We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with actin filaments by immunocytochemistry. During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among different cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere
2) the border of cells when astrocytes tightly adhered
and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhesion of astrocytes. These observations afford valuable clues for understanding the glial communication.

To find the primary mechanism for the frequency changes of electrical oscillations in the procerebral (PC) lobe of a slug, we electrically stimulated the tip, middle and basal regions of the digits of the superior and inferior tentacles and recorded the local field potentials from the PC lobe. Stimuli to the middle and basal regions of the digits of the inferior tentacle significantly decreased the frequency of electrical oscillations in the PC lobe, whereas those to the tip region of the digits of the inferior tentacle and all regions of the digits of the superior tentacle increased it. These findings suggest that the change in the frequency of electrical oscillations in the PC lobe depends on the excited region in the digits, providing the first presentation of the physiological difference in the olfactory function between the superior and inferior tentacles. (C) 1999 Elsevier Science B.V. All rights reserved.

We examined the roles of A- and D-type K channels in the integration of EPSPs, particularly EPSP reduction, in a model dendrite using a computer simulation program (NEURON), instead of conventional electrophysiological methods which present technical difficulties. We obtained two important results: (1) KD channels play a crucial role in every synaptic input event, and (2) KA channels affect reduction of the second EPSP only when the second synaptic input is applied with a short interval (less than or equal to 100 ms). These results suggest that KD channels, which have not yet been analyzed experimentally, play a major role in synaptic integration in dendrites by altering their cable properties. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

We demonstrated a sensory preconditioning in the pond snail, Lymnaea stagnalis. An appetitive sucrose solution (a conditioned stimulus: CS1) and weak vibration (another conditioned stimulus: CS2) were first associated, and then the CS2 and an aversive KCl solution (an unconditioned stimulus: UCS) were done. To build the conditioning, two different training procedures, spaced and massed, were examined. After the both training, the sensory preconditioning was built: significantly fewer feeding response to the CS1 became elicited; slower latency to the first bite to the CS1 was induced. No significant differences on the memory retention between these training procedures were found in the sensory preconditioning. (C) 1998 Elsevier Science B.V. All rights reserved.

Operant conditioning that the pond snail, Lymnaea stagnalis, suppressed its naturally occurring behavior of escape from a water tank was examined by using a negative reinforcement (i.e. an aversive stimulus) prepared outside the tank. During the training period, the number of escapes from a tank was strongly suppressed. One of behavioral factors for this suppression was confirmed as the elongation of latency to the first escape after training. The effects on the memory retention were examined in the massed and spaced training procedures. The latter procedure interposes a rest interval between three sets of 20-min training sessions, whereas the former has the same number of training sessions with no rest interval within 60 min. The memory retention by the massed training was observed within 20 min after training. By the spaced training, the learning acquisition was found to be stronger, which was observed as the slower latency to the first escape, than by the massed training, but the longer-lasting memory retention, which had been expected first, was not formed. These results suggest that once Lymnaea recognize the external environment is safe after training, they may extinguish their memory of the past situation quickly, resulting in no or very little difference in the memory retention by two different training procedures in this operant conditioning. Together with the facts that classical conditioning and its neuronal mechanisms in Lymnaea were previously clarified, the present findings may help to address not only the neuronal basis of operant conditioning but also the relation between classical and operant conditioning.

To examine whether nitric oxide (NO)-generative neurons are included in the central circuitry for generation of feeding pattern in the pond snail, Lymnaea stagnalis, two staining techniques for NADPH diaphorase and serotonin (5-HT) were applied for its central nervous system (CNS). The former technique is known to show localization of NO synthase; the latter is well employed as a marker for the feeding circuitry because 5-HT is a main transmitter in it. In the buccal ganglion, B2 motoneuron was found to be a putative NO-generative neuron. This motoneuron is not involved directly in the coordination of feeding pattern but is activated simultaneously with the feeding to control the oesophageal and gut tissues for the digestion. Taking account of the diffusion effects of NO, the NO released from B2 motoneuron, when the feeding is started, is thought to sufficiently modulate the feeding circuitry. In the cerebral ganglion, the superior lip nerve, the median lip nerve and the tentacle nerve included both putative NO-generative fibers and serotonergic fibers. These fibers are not identical, but the NO released in the nerves may activate the serotonergic fibers, resulting in the influence upon the initiation of the feeding. Therefore, our present findings clearly showed that NO is not involved in transmission within the central circuitry for the feeding, but suggested that NO can crucially affect the feeding behavior, such as initiation and modulation of the feeding pattern. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

We first showed a general theory that reception of information from the outside in a receptor system is accompanied both by an inflow of entropy and by a generation of entropy depending on the reliability of the actual reception mechanism. Then, considering a case for the absorption of light by the visual-pigment system of visual cell, we calculated the time(t)-dependent change in the number N-2(t) of excited visual-pigments to obtain the entropy increase. We thus arrived at the following four conclusions: (1) One or two photons can be detected with a reliability of at least 54%; (2) In compensation for this detection, entropy &gt; 1.12 x log2 is generated; (3) An incident photon of frequency nu from a light source of temperature T-s yields an entropy of h nu/T-s; and (4) Depending on the characteristics of the visual-pigment system, another entropy being different from (2) is generated in proportion to N-2(t). (C) 1998 Elsevier Science B.V. All rights reserved.

Using the force modulation mode in atomic force microscopy (AFM), we have succeeded in imaging elastic properties of agar gels immersed in water. The elastic images of ap ar have been captured simultaneously with the topographic images. Stiffer grains of agar whose size is about 200 nm can be clearly seen in the elastic image of 3.0% agar, while they are not so visible in the case of 1.5% agar. These grains probably correspond to aggregation of agar which cannot be observed in the topographic images. We also measured force-versus-distance curves using AFM to confirm that the absolute values of elastic modulus (Young's modulus) of agar coincide with the bulk values measured using the conventional stress-strain method. The estimated values of the elastic moduli with the AFM were 40 and 90 kPa for 1.5% and 3.0% agar gels, respectively. These are in good agreement with the respective bulk values of 30 and 80 kPa obtained using the conventional method.

Although we can routinely obtain fine structural images of cells by atomic force microscopy (AFM), the adequacy and reliability of morphological information acquired from these AFM images remain to be examined. In this report, we compared images of the line structures of nerve cells as observed by both AFM and scanning electron microscopy (SEM). Although AFM revealed the structure of the top views of cells in greater detail than SEM, their side structures were better observed by SEM. The linear structures in the neural processes detected only by AFM were confirmed, by immunofluorescence staining, to be reflections of the cytoskeletal structures located beneath the cell membrane. These differences between the AFM and the SEM images reflected the characteristics of the detection systems and methods used for sample preparation. Therefore, these results revealed that more detailed information on cell morphology can be obtained by using both AFM and SEM to advantage.

The morphology of cultured glial cells was examined using a combination of atomic force microscopy (AFM) and immunofluorescence staining for cytoskeletons. The meshwork of type-1 astrocytes consisted of thick longitudinal and thin lateral lines on the cell surfaces observed by AFM, the former lines were confirmed to be reflections of actin filaments. The astrocytic processes of type-2 astrocytes were observed to be rugged on AFM. These structures were mainly affected by microtubules. Immunofluorescence imaging of microglia revealed that actin filaments and microtubules were arranged radially and wavily along the cell edge, respectively. AFM could detect these radial and wavy structures clearly. These results show that AFM can provide information on the cytoskeletons of glial cells, indicating that AFM is a useful tool for the morphological characterization of cells.

Using the force modulation mode in atomic force microscopy, we measured elastic properties of living mouse fibroblasts (NIH3T3) in a culture medium, The topographic images of the cellular surface and the corresponding elastic images of the cellular surface were able to be captured simultaneously with high spatial resolution, The consecutive images were useful for examining time-dependent changes in the cellular surface, We observed that some cells continued to shrink and change their softness for 2 hours, Then the force modulation mode in atomic force microscopy shows potential use in analyzing time-dependent regional elastic properties of living cells with high spatial resolution.

To study the neuronal mechanism of a conditioned taste-aversion (CTA) learning in the pond snail Lymnaea stagnalis, we examined the synaptic connection between the neuron 1 medial (N1M) cell and the cerebral giant cell (CGC), the former is an interneuron in central pattern generator for the feeding response and the latter is a regulatory neuron to the central pattern generator. Inhibitory postsynaptic potential (IPSP) which was evoked in the N1M cell by activation of the CGC was larger and lasted longer in the conditioned animal than that in the control animal. The electrical properties of the cell body of CGC and the responses of the CGC to the chemosensory inputs were not changed during the CTA learning. These results, together with the previous report indicating the existence of excitatory projection from the N1M cell to the feeding motoneuron, suggest that enhanced IPSP in the N1M cell may underlie the suppression of feeding responses in the Lymnaea CTA learning. (C) 1997 Elsevier Science Ireland Ltd.

We examined the practical scan speed for the observation of live neurons in a physiological solution using atomic force microscopy (AFM) with a desired vertical resolution of the order of 10(-8) mi which was reasonable when taking into account that a flicker of extracellular protein and saccharide on the neurons in the solution occurred during an observation period of a couple of minutes. The practical scan speed was found to be under 40 mu m/s, therefore, if we applied AFM using 100 lines and 100 pixels per line to an observation area of 20 mu m x 20 mu m, the minimum period for acquiring one image was estimated to be about 2 min. This procedure gave us good images that represented the slow three-dimensional dynamics in live neurons, such as the retrograde movement of surface protuberances, but suggested that another approach was required to detect fast structural changes induced by stimulation.

It is necessary to determine whether, in the same species and for the same behavior, aversive and appetitive conditioning yield different strengths and periods of either acquisition or retention. To this end, we first examined the effects of various chemo-sensory and physical stimuli on feeding and avoidance behavioral responses in the pond snail, Lymnaea stagnalis. Then, using these findings, we constructed classical-conditioning paradigms with aversive and appetitive stimuli. In the aversive conditioning paradigm, an appetitive stimulus (sucrose), which increased the feeding response, was paired with an aversive stimulus (KCI, quinidine sulfate or electric shock), which inhibited the feeding behavior. Upon presentation of KCI, the first type of aversive conditioning, which is generally called ''taste-aversion learning with cessation of feeding response'', was acquired quickly and persisted for up to a month. When using a noxious stimulus (quinidine sulfate or electric shock) inducing pain we additionally found the second type of aversive conditioning, in which the previously appetitive stimulus (sucrose) not only failed to increase the feeding response, but came to elicit an avoidance response. This second type conditioning took longer to acquire and persisted for a shorter period of time than the first type. On the other hand, the appetitive conditioning paradigm paired a neutral stimulus (vibratory) with an appetitive stimulus (sucrose). The strength and period for acquisition and retention of this appetitive leamed response were very similar to those of the second type aversive conditioning but not to the first one. On the basis of these behavioral analyses, the neuronal mechanisms of the two types of aversive and appetitive conditioning were discussed.

We reported that malignant cells are inactivated by photo-excited TiO2 particles (Cai, R.-X., et al. (1992) Cancer Res. 52, 2346-2348). In the present study, the process of cell death of a human bladder cell line T24 with TiO2 irradiated TiO2 was investigated by monitoring the time course change in intracellular calcium concentration ([Ca2+](i)) under UV irradiation using calcium fluorescence indicator, fura-2. In the presence of photo-excited TiO2 particles (100 mu g/ml), [Ca2+](i) showed a sapid two-step increase; while in the absence of TiO2, it exhibited only a slight and monotonous increase or maintained a constant level. The rapid elevation of [Ca2+](i) in the presence of photo-excited TiO2 particles was caused by the influx of extracellular Ca2+ through the plasma membrane and cell death occurred only after the second rapid elevation of [Ca2+](i). These results suggested that the cell membrane permeability to Ca2+ was promoted prior to cell death.

Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon the actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Photonics digital imaging system. Agonists used were bombesin, inositol-1,4,5-trisphosphate (IP3), thapsigargin (Tg), neuropeptide Y (NPY) and calcium chloride. Recordings were typically made on seven cells in each case. We found that IP3 caused a 6-8-fold immediate release of (Ca2+)i in rabbit cells, but skate cells showed no response unless permeabilized with saponin, whereupon an increase of about 50% occurred. Tg induced release from internal stores in rabbit cells, but had no effect on skate cells. Bombesin caused a large increase in (Ca2+)i release in both, while NPY had no effect in either. Skate cells incubated in calcium-free EGTA-Ringer&apos;s solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradual fashion. After calcium imaging, the cells were fixed and stained with rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin cytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit cells, but bombesin had no effect. IP3 receptor was located intracellularly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been caused by the DMSO in which fura-2 was dissolved.
We have found an interesting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capacitative calcium entry, which could reflect a difference in lens accommodative mechanisms. This seems relevant in view of the fact that the rabbit lens accommodates through change in shape, whereas the skate lens does so through translation of position.

Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease-namely, the absence of a 113-picosiemen potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that depends on functional 113-picosiemen potassium channels. was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased [Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by 10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function to produce symptoms such as memory loss by a means other than plaque formation.

Previous observations have implicated GABA as a neurotransmitter released by the vestibular sensory neurons (''hair cells'') of the snail Hermissenda onto visual sensory neurons, the type B cells, whose cell bodies are the sites of biophysical and biochemical changes during and following Pavlovian conditioning. Still other observations demonstrated that light-GABA pairings that simulate stimuli presented during Pavlovian conditioning cause prolonged elevation of intracellular Ca2+ and transformation of GABA-induced synaptic inhibition into excitation. Intracellular Ca2+ signals in response to GABA perfused onto the postsynaptic type B terminal branches are shown here to be prolonged on days after conditioning, but not after control paradigms. These and past results demonstrate two separate sites, i.e., the cell body and the terminal branches, for learning-induced changes after Pavlovian conditioning.

Microspectrofluorometry and video imaging techniques were used to study and to compare the changes in intracellular calcium concentrations ([Ca2+](i)) of individual Fura-2 loaded Spisula oocytes treated with serotonin (5-hydroxytryptamine, 5-HT) or tricyclic antidepressants. In the present study, we showed that 5-HT increased [Ca2+](i) in freshly isolated Spisula oocytes suspended in artificial sea water. In the absence of extracellular Ca2+, 5-HT did not influence [Ca2+](i). Stimulation of [Ca2+](i) by 5-HT was blocked by calcium channel blocker, e.g. verapamil, and by tricyclic antidepressants. These observations combined with our previous results on the effects of 5-HT, tricyclic antidepressants and verapamil on calcium uptake suggest that the increase in [Ca2+](i) induced by 5-HT results from an influx of extracellular calcium through calcium channels, which can be blocked by tricyclic antidepressants.
The use of the Fura-P imaging technique allowed single-cell measurements and defined changes induced by 5-HT In [Ca2+](i) which is the net result of calcium uptake and release of intracellular calcium from storage sites in individual Spisula oocytes.

The recent demonstration of Kf channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca2+-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphate-mediated release of intracellular Ca2+.

Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostk index as well. A 113-pS tetraethylammonium (TEA)-sensftive K+ channel was consistendy absent from AD ribroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ channel was present in all three groups. Elevated external potassium raised intracellular Ca2+ in all cases. TEA depolarized and caused intracellular Ca2+ elevation in young and aged control fibroblasts but not AD fibroblasts. The invariable absence of a 113-pS TEA-sensitive K+ channel and TEA-induced Ca2+ signal indicate K+ channel dysfunction in AD fibroblasts. These results suggest the possibility of a laboratory method that would diagnostically distinguish AD patients, with or without a family history of AD, from normal age-matched controls and also from patients with non-AD neurological and psychiatric disorders.

Previous studies of molluscan and mammalian neural networks have implicated potassium channels, calcium-dependent kinases and a small G-protein (Cp20) in associative memory storage. Since Alzheimer's disease is characterized by memory deficits and possible changes in PKC activation, we studied potassium channel function in AD. Patch-clamp experiments revealed the absence of a 113 pS TEA-sensitive K+ channel in fibroblasts from Alzheimer's but not control patients. These results suggest a role for potassium channel dysfunction in Alzheimer's disease.

For a constant membrane potential, a predominantly inhibitory GABAergic synaptic response is shown to undergo long-term transformation into an excitatory response after pairing of exogenous gamma-aminobutyric acid (GABA) with postsynaptic depolarization or pairing of pre- and postsynaptic stimulation. Current- and voltage-clamp experiments suggest that this synaptic transformation is due to a shift from a net increase of conductance to a net decrease of conductance in response to GABA. GABA-induced elevation of intracellular calcium is prolonged after the same stimulus pairing and may, therefore, contribute to this synaptic transformation via Ca2+-activated phosphorylation pathways. This synaptic transformation, which does not follow unpaired stimulus presentations, occurs in a neuronal compartment spatially separated from the soma, which also changes during stimulus pairing.

To investigate the relation between the function of Ca2+-activated K+ channels and phosphoinositide turnover, we have examined the physiological and pharmacological characteristics of ionotropic and metabotropic quisqualate (QA) receptors in rat cerebellar Purkinje cells during development using the slice-patch method combined with Ca2+ imaging. The typical response to QA obtained from a rat on postnatal day (PND) 21 consisted of three components: (1) a fast inactivating inward current, (2) a slow inward current, and (3) a slow outward current. The slow inward current was abolished in Ca2+-free medium. while the fast inactivating inward current and the slow outward current remained unaffected. The slow outward current which appeared to be activated via a metabotropic receptor was not observed in the Purkinje cell of PND 7 rat, in which dendrites were poorly developed but its amplitude increased linearly with PND. QA caused significant increases in [Ca2+]i in the fully developed dendritic region of the Purkinje cells even in Ca2+-free medium, suggesting a dendritic localization of the metabotropic receptors.

Caudal hair cell impulses cause postsynaptic inhibition of ipsilateral type B photoreceptors in the snail Hermissenda. This inhibition is shown to be GABAergic according to a number of criteria. HPLC, mass spectrophotometric, and immunocytochemical techniques demonstrated the presence of GABA in the hair cells and their axons. GABA agonists and antagonists mimic and block the synaptic effect in a manner consistent with endogenous GABAergic transmission. Other properties, including I-V relations, conductance changes and reversal potentials, are comparable for exogenous GABA responses and endogenous effects of the hair cell impulses. This inhibitory synapse has been found to undergo a long-lasting transformation into an excitatory synapse if GABA release is paired with post-synaptic depolarization. GABA, via GABA(A) and GABA(B) receptors in the B cell, causes the opening of calcium sensitive chloride and potassium channels that leads to the post-synaptic hyperpolarization. GABA also induces a long-lasting intracellular calcium elevation at the terminal branches of the B cell that greatly outlasts the voltage responses. Synaptic transformation induced by pairings is caused by a decrease in both GABA induced chloride and potassium conductances in the post-synaptic B cell, as well as a significant prolongation of the intracellular calcium accumulation in the B cell's terminal axonal branches.

The distribution of functional glutamate receptor subtypes was visualized as regional changes in intracellular Ca2+ concentration induced by administration of a specific agonist for each subtype. An image of the fura-2 fluorescence from a whole hippocampal slice was directly excited at 340 or 380 nm through an optical fibre and observed via a low-magnification objective lens (X 4). A heterogeneous distribution of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes was demonstrated by the ratio of fluorescence intensities before and after administration of each agonist. This 'macro' image analysis can reveal distributions of multiple functional receptors in a living brain slice.

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N-methyl-D-aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex.

In order to examine whether a transient appearance of glutamate receptor in developing rat Purkinje cell in coupled to PI turnover, we have studied Ca2+ mobilization induced by quisqualate or NMDA using fura-2 analyzing system in combination with immunohistochemical staining analysis with anti-PIP2 antibody and/or anti-IP3 binding protein antibody.

親の獲得形質は子に遺伝するのか？この問題をモノアラガイのオランダとカナダの系統ならびにそれらのF1を用い、２つの学習を施して研究した。また、セロトニンとドーパミンの脳内濃度を測定した。味覚嫌悪学習ではオランダが他よりも優秀で、脳内セロトニン量も有意な差があった。呼吸オペラント学習ではカナダが他よりも優秀で、かつドーパミン濃度も有意に異なっていた。一方で、成績が良いまたは悪い系統内での子孫について、味覚嫌悪学習での鍵刺激となるショ糖への応答性を調べたところ、どちらの子孫でも有意な差はなかった。すなわち、優秀な系統とそうでない系統は存在するものの、その機構解明は今後の課題となった。

淡水産巻貝であるモノアラガイは、味覚嫌悪学習が習得でき、それが長期記憶として保持される。この機構にはインスリンとセロトニンの両方の反応過程が関わっていることが、我々の先行研究から示唆されてきた。そこで本研究では両者の関係を調べたところ、（１）弱い飢餓時には味覚嫌悪学習の成績が良く、かつ脳内のセロトニンレベルは低くなっていること、（２）強い飢餓時には味覚嫌悪学習は成立せず、かつ脳内のセロトニンレベルが高くなっていること、（３）強い飢餓時の動物の脳内にインスリンを注入すると、セロトニンレベルが下がり、そして味覚嫌悪学習が改善されることが分かった。

我々は超高感度なタンパク測定ELISA法を開発した実績があり、今回は、さらにこの測定系を拡張するために、今までにない核酸検出に取り組んだ。それは「核酸の増幅を必要としない」超高感度核酸測定の実現である。本研究では、結核菌群に特異的に存在するタンパク質MPB64の１本鎖を用い、かつコントロールとして非結核菌群を用いた。結果として、MPB64の１本鎖に対して、3.7 × 10^5 copies/assay（すなわち4.2 × 10^3 copies/μL）の測定に成功し、また非結核菌群は測定に掛らなかった。すなわち予定通りに、 非増幅・迅速・簡便核酸測定法の開発に成功した。

&nbsp; 過度のストレスから解放されると、動物の学習成績が改善されるのか？この問題を神経生物学で永く利用されている軟体動物モノアラガイで調べた。ストレスからの解放はカンナビノイドの投与によって得た。ほ乳類の脳内には内在性カンナビノイドならびにその受容体が存在する。一方、無脊椎動物はストレスや学習の研究に十分利用可能であるが、それらの存在は不明であった。そこで軟体動物モノアラガイを用いて受容体の存在を調べたところ、ほ乳類と相同な受容体が見つかった、さらには学習行動やストレスが及ぼす影響に対して、カンナビノイド受容体のアゴニスト・アンタゴニストがどのように関与するのかを調べる実験も開始することができた。

&nbsp; 脊椎動物でも無脊椎動物でも、その脳内でインスリンがはたらくことが、近年わかってきた。ただし、このインスリンが学習記憶機構に対して「増強」としてはたらくのか、または「減弱」としてはたらくのかは、不明な点が多い。本研究では、学習記憶研究で永年使用されてきている軟体動物モノアラガイを用いて、①インスリンのシグナルカスケードとセロトニンカスケードが長期記憶形成において拮抗的にはたらくことが明らかとなった。また、②転写因子CREBのシグナルカスケード、ならびにそれに続くCRTCのシグナルカスケードの研究を開始することができた。なお本研究は、同一課題にて特定課題Bでも研究が遂行された。

脊椎動物でも無脊椎動物でも、その脳内でインスリンがはたらくことが、近年わかってきた。ただし、このインスリンが学習記憶機構に対して「増強」としてはたらくのか、または「減弱」としてはたらくのかは、不明な点が多い。本研究では、学習記憶研究で永年使用されてきている軟体動物モノアラガイを用いて、①インスリンのシグナルカスケードとセロトニンカスケードが長期記憶形成において拮抗的にはたらくことが明らかとなった。また、②転写因子CREBのシグナルカスケード、ならびにそれに続くCRTCのシグナルカスケードの研究を開始することができた。なお本研究は、同一課題にて特定課題（新任の教員等）でも研究が遂行された。