<em>Aplysia</em> Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

The Scripps Research Institute, Florida

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (approximately 7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is approximately 8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy.

&#x6D77;&#x5154;&#x3001; &#x79FB;&#x52A8;&#x7535;&#x8BDD;&#x548C;&#x7CFB;&#x7EDF;&#x7684;&#x795E;&#x7ECF;&#x79D1;&#x5B66;&#x3001; &#x5B8C;&#x5584;&#x7684;&#x6A21;&#x5F0F;&#x751F;&#x7269;&#x5206;&#x5B50;&#x5206;&#x6790;&#x4F1A;&#x53D7;&#x5230;&#x4E25;&#x91CD;&#x963B;&#x788D;&#xFF0C;&#x7531;&#x4E8E;&#x7F3A;&#x4E4F;&#x8DB3;&#x591F;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x4FE1;&#x606F;&#x3002;&#x901A;&#x8FC7;&#x6D4B;&#x5E8F;&#x4ECE;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF; (CNS) &#x7684;&#x57FA;&#x56E0;&#x5E93;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x6709;&#x8D85;&#x8FC7; 175,000 &#x8868;&#x8FBE;&#x7684;&#x5E8F;&#x5217;&#x6807;&#x7B7E; (&#x65E0;&#x5BB3;&#x73AF;&#x5883;&#x6280;&#x672F;&#xFF09;&#xFF0C;&#x5176;&#x4E2D; 19,814 &#x662F;&#x552F;&#x4E00;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x57FA;&#x56E0;&#x4EA7;&#x54C1;&#xFF0C;&#x5360; 50%-70%&#x7684;&#x603B;&#x7684;&#x6D77;&#x5154;&#x795E;&#x7ECF;&#x5143;&#x8F6C;&#x5F55;&#x8FC7;&#x7A0B;&#x3002;&#x6211;&#x4EEC;&#x6709;&#x7279;&#x70B9;&#x4E09;&#x4E2A;&#x5C42;&#x9762;&#x8F6C;&#x5F55;&#x8FC7;&#x7A0B;&#xFF1A; (1) &#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x3001; &#x7B80;&#x5355;&#x7684;&#x884C;&#x4E3A; (2) &#x57FA;&#x672C;&#x6210;&#x5206;&#xFF1A; &#x9CC3;&#x64A4;&#x51FA;&#x53CD;&#x5C04;-&#x901A;&#x8FC7;&#x5206;&#x6790;&#x611F;&#x5B98;&#x3001; &#x7535;&#x673A;&#x3001; &#x548C; serotonergic &#x7684;&#x8C03;&#x8282;&#x795E;&#x7ECF;&#x5143;&#x548C; (3) &#x5355;&#x4E2A;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x5DE5;&#x4F5C;&#x6D41;&#x7A0B;&#x3002;&#x9664;&#x4E86;&#x589E;&#x52A0;&#x53EF;&#x7528;&#x57FA;&#x56E0;&#x5E8F;&#x5217;&#x7684;&#x6D77;&#x5154;&#x7684;&#x4E24;&#x4E2A;&#x6570;&#x91CF;&#x7EA7;&#x7684;&#x91CF;&#xFF0C;&#x6B64;&#x96C6;&#x5408;&#x8868;&#x793A;&#x4EFB;&#x4F55;&#x6210;&#x5458;&#x7684; Lophotrochozoa &#x7684;&#x6700;&#x5927;&#x53EF;&#x7528;&#x7684;&#x6570;&#x636E;&#x5E93;&#xFF0C;&#x56E0;&#x6B64;&#x63D0;&#x4F9B;&#x4E86;&#x66F4;&#x591A;&#x6DF1;&#x5165;&#x4E86;&#x89E3;&#x6B64;&#x9AD8;&#x5EA6;&#x6210;&#x529F;&#x7684;&#x591A;&#x5143;&#x5316;&#x6CBF;&#x88AD;&#xFF0C;&#x53CC;&#x8FB9;&#x52A8;&#x7269;&#x7684;&#x4E09;&#x4E2A;&#x5EFA;&#x8BAE; superclades &#x4E4B;&#x4E00;&#x6240;&#x4F7F;&#x7528;&#x7684;&#x8FDB;&#x5316;&#x7B56;&#x7565;&#x3002;

&#x6D77;&#x5154;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x8F6C;&#x5F55;&#x8FC7;&#x7A0B;&#xFF1A; &#x795E;&#x7ECF;&#x5143;&#x8F66;&#x53A2;&#x548C;&#x7535;&#x8DEF;&#x3002;

Molecular analyses of Aplysia, a well-established model organism for cellular and systems neural science, have been seriously handicapped by a lack of adequate genomic information. By sequencing cDNA libraries from the central nervous system (CNS), we have identified over 175,000 expressed sequence tags (ESTs), of which 19,814 are unique neuronal gene products and represent 50%-70% of the total Aplysia neuronal transcriptome. We have characterized the transcriptome at three levels: (1) the central nervous system, (2) the elementary components of a simple behavior: the gill-withdrawal reflex-by analyzing sensory, motor, and serotonergic modulatory neurons, and (3) processes of individual neurons. In addition to increasing the amount of available gene sequences of Aplysia by two orders of magnitude, this collection represents the largest database available for any member of the Lophotrochozoa and therefore provides additional insights into evolutionary strategies used by this highly successful diversified lineage, one of the three proposed superclades of bilateral animals.

Neuronal transcriptome of Aplysia: neuronal compartments and circuitry.

Des analyses mol&eacute;culaires d&#39;Aplysia, un organisme mod&egrave;le bien &eacute;tabli pour la science neurale cellulaires et syst&egrave;mes, ont &eacute;t&eacute; s&eacute;rieusement handicap&eacute;s par un manque de renseignements g&eacute;nomiques. En s&eacute;quen&ccedil;ant les banques d&#39;ADNc du syst&egrave;me nerveux central (CNS), nous avons identifi&eacute; plus de 175 000 &eacute;tiquettes de s&eacute;quences exprim&eacute;es (est), dont 19 814 produits g&eacute;niques neuronale uniques et repr&eacute;sentent 50 % - 70 % de la total transcriptome neuronal Aplysia. Nous avons caract&eacute;ris&eacute; le transcriptome &agrave; trois niveaux: (1) le syst&egrave;me nerveux central, (2) les composants &eacute;l&eacute;mentaires d&#39;un comportement simple : le r&eacute;flexe gill-retrait en analysant les neurones modulateurs s&eacute;rotoninergiques, moteurs et sensoriels et (3) le processus de neurones individuels. En plus d&#39;augmenter la quantit&eacute; de s&eacute;quences de g&egrave;nes disponibles d&#39;Aplysia par deux ordres de grandeur, cette collection repr&eacute;sente la plus grande base de donn&eacute;es disponible pour n&#39;importe quel membre de la Lophotrochozoa et donc donne une id&eacute;e des strat&eacute;gies &eacute;volutives utilis&eacute;s par cette lign&eacute;e diversifi&eacute;e tr&egrave;s r&eacute;ussie, l&#39;un de le superclades propos&eacute;es trois animaux bilat&eacute;raux suppl&eacute;mentaires.

Transcriptome neuronale d&#39;Aplysia : compartiments neurones et circuits.

Molekulare Analysen der Aplysia, etablierten Modellorganismus f&uuml;r Mobiltelefone, Systeme neuralen Wissenschaft, haben durch einen Mangel an ausreichenden genomische Informationen schwer behinderter wurde. Durch Sequenzierung cDNA-Bibliotheken aus dem zentralen Nervensystem (ZNS), haben wir mehr als 175.000 exprimierten Sequenz Markierungen (ESTs), identifiziert, die darunter 19.814 einzigartige neuronale Genprodukte und 50 % - 70 % von der gesamten Aplysia neuronale Transkriptom darstellen. Wir haben das Transkriptom auf drei Ebenen gepr&auml;gt: (1) des zentralen Nervensystems, (2) die elementaren Bestandteile eines einfachen Verhaltens: die Gill-R&uuml;ckzug-Reflex-von Analyse, sensorische, motorische und serotonergen modulierende Neuronen und (3) die Prozesse der einzelnen Neuronen. Neben der Erh&ouml;hung der Menge an verf&uuml;gbaren Gensequenzen von Aplysia um zwei Gr&ouml;&szlig;enordnungen, diese Sammlung stellt die gr&ouml;&szlig;te Datenbank verf&uuml;gbar f&uuml;r jedes Mitglied der Lophotrochozoen und bietet daher zus&auml;tzliche Einblicke in evolution&auml;ren Strategien, die von dieser sehr erfolgreichen diversifizierten Linie, eines der drei vorgeschlagenen Superclades von bilateralen Tieren verwendet.

Neuronale Transkriptom von Aplysia: Neuronale F&auml;cher und Schaltungen.

Analisi molecolari di Aplysia, un organismo modello consolidato per cellulari e sistemi neural science, hanno stato gravemente handicappati dalla mancanza di adeguate informazioni genomiche. Dal sequenziamento del cDNA librerie dal sistema nervoso centrale (SNC), abbiamo identificato oltre 175.000 espresso sequence tags (ESTs), di cui 19.814 prodotti unico gene neuronale e rappresentano il 50-70% del totale Aplysia transcriptome neuronale. Noi abbiamo caratterizzato il trascrittoma a tre livelli: (1) il sistema nervoso centrale, (2) i componenti elementari di un semplice comportamento: il riflesso-da gill-ritiro analisi sensoriali, motori e serotoninergici modulatrice neuroni e (3) i processi di singoli neuroni. Oltre ad aumentare la quantit&agrave; di sequenze di geni disponibili di Aplysia da due ordini di grandezza, questa collezione rappresenta il pi&ugrave; grande database disponibile per qualsiasi membro i Lophotrochozoa e pertanto fornisce ulteriori approfondimenti strategie evolutive utilizzati da questo lignaggio diversificato altamente riuscito, uno dei tre superclades proposto di animali bilaterali.

Neuronale transcriptome di Aplysia: compartimenti neuronali e circuiti.

&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x3001;&#x5B9A;&#x8A55;&#x306E;&#x3042;&#x308B;&#x30E2;&#x30C7;&#x30EB;&#x751F;&#x7269;&#x7D30;&#x80DE;&#x304A;&#x3088;&#x3073;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x795E;&#x7D4C;&#x79D1;&#x5B66;&#x3001;&#x5206;&#x5B50;&#x89E3;&#x6790;&#x771F;&#x5263;&#x306B;&#x5341;&#x5206;&#x306A;&#x30B2;&#x30CE;&#x30E0;&#x60C5;&#x5831;&#x306E;&#x6B20;&#x5982;&#x306B;&#x3088;&#x3063;&#x3066;&#x969C;&#x5BB3;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB; &#xFF08;CNS&#xFF09; &#x304B;&#x3089;&#x306E; cDNA &#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x3092;&#x914D;&#x5217;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005; &#x306F; 19,814 &#x306F;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x795E;&#x7D4C;&#x907A;&#x4F1D;&#x5B50;&#x88FD;&#x54C1;&#x3067;&#x3042;&#x308A;&#x3001;&#x5408;&#x8A08;&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30FC;&#x30E0;&#x306E; 50%-70% &#x3092;&#x8868;&#x3059;&#x4EE5;&#x4E0A; 175,000 &#x767A;&#x73FE;&#x914D;&#x5217;&#x30BF;&#x30B0; &#xFF08;Est&#xFF09; &#x3092;&#x8B58;&#x5225;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F; 3 &#x3064;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30FC;&#x30E0;&#x7279;&#x5FB4;&#x304C;&#x3042;&#x308B;: (1) &#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;, (2) &#x5C0F;&#x5B66;&#x6821;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3092;&#x5358;&#x7D14;&#x306A;&#x52D5;&#x4F5C;&#x306E;: &#x611F;&#x899A;, &#x30E2;&#x30FC;&#x30BF;, &#x3068;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;&#x4F5C;&#x52D5;&#x97F3;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3068; (3) &#x500B;&#x3005; &#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x5206;&#x6790;&#x30FB; &#x30AE;&#x30EB;&#x64A4;&#x9000;&#x53CD;&#x5C04;&#x306B;&#x3088;&#x3063;&#x3066;&#x3002;&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x306E;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5869;&#x57FA;&#x914D;&#x5217;&#x306B;&#x3088;&#x308B; 2 &#x6841;&#x306E;&#x5897;&#x52A0;&#x3001;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3053;&#x306E;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x6700;&#x5927;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x30D9;&#x30FC;&#x30B9;&#x3092;&#x5229;&#x7528;&#x3067;&#x304D;&#x308B;&#x3001;Lophotrochozoa &#x306E;&#x4EFB;&#x610F;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x9032;&#x5316;&#x6226;&#x7565;&#x306E;&#x3053;&#x306E;&#x975E;&#x5E38;&#x306B;&#x6210;&#x529F;&#x3057;&#x305F;&#x591A;&#x69D8;&#x306A;&#x7CFB;&#x7D71;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x4E8C;&#x56FD;&#x9593;&#x306E;&#x52D5;&#x7269;&#x306E; 3 &#x3064;&#x306E;&#x63D0;&#x6848; superclades &#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x3092;&#x4F7F;&#x7528;&#x306B;&#x8FFD;&#x52A0;&#x306E;&#x6D1E;&#x5BDF;&#x529B;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x306E;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30FC;&#x30E0;: &#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8;&#x304A;&#x3088;&#x3073;&#x56DE;&#x8DEF;&#x3002;

Aplysia, &#xD734;&#xB300; &#xBC0F; &#xC2DC;&#xC2A4;&#xD15C; &#xC2E0;&#xACBD; &#xACFC;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xAE30;&#xCD08;&#xAC00; &#xD2BC;&#xD2BC;&#xD55C; &#xBAA8;&#xB378; &#xC720;&#xAE30; &#xCCB4;&#xC758; &#xBD84;&#xC790; &#xBD84;&#xC11D; &#xC801;&#xC808; &#xD55C; &#xAC8C;&#xB188; &#xC815;&#xBCF4;&#xAC00; &#xBD80;&#xC871; &#xD558; &#xC5EC; &#xC7A5;&#xC560;&#xC778;&#xC744; &#xC2EC;&#xAC01; &#xD558; &#xAC8C; &#xB418;&#xC5B4; &#xC788;&#xB2E4;. &#xC911;&#xC559; &#xC2E0;&#xACBD; &#xC2DC;&#xC2A4;&#xD15C; (CNS)&#xC5D0;&#xC11C; cDNA &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xB97C; &#xC2DC;&#xD000;&#xC2F1; &#xD558;&#xB294; &#xBC29;&#xBC95; &#xC6B0;&#xB9AC;&#xAC00; &#xC774;&#xC0C1; 175000 &#xD45C;&#xD604;&#xB41C; &#xC2DC;&#xD000;&#xC2A4; &#xD0DC;&#xADF8; (ESTs)&#xB294; 19,814 &#xACE0;&#xC720; &#xC2E0;&#xACBD; &#xC9C4; &#xC81C;&#xD488; &#xBC0F; &#xCD1D; Aplysia &#xC2E0;&#xACBD; transcriptome&#xC758; 50%-70%&#xB97C; &#xB300;&#xD45C; &#xD30C;&#xC545; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC138; &#xAC00;&#xC9C0; &#xC218;&#xC900;&#xC5D0;&#xC11C; transcriptome &#xD2B9;&#xC9D5; &#xC788;&#xB2E4;: (1) &#xC911;&#xC559; &#xC2E0; &#xACBD;&#xACC4;, &#xB2E8;&#xC21C;&#xD55C; &#xB3D9;&#xC791;&#xC758; &#xAE30;&#xBCF8; &#xAD6C;&#xC131; &#xC694;&#xC18C; (2):&#xB294; &#xAE38; &#xCCA0;&#xC218; &#xBC18;&#xC0AC;-&#xBD84;&#xC11D; &#xD558; &#xC5EC;, &#xBAA8;&#xD130;, &#xAC10;&#xAC01; &#xBC0F; serotonergic modulatory &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xBC0F; (3) &#xAC1C;&#xBCC4; &#xB274;&#xB7F0;&#xC758; &#xD504;&#xB85C;&#xC138;&#xC2A4;. 2 &#xC99D;&#xAC00;&#xD560; Aplysia&#xC758; &#xC0AC;&#xC6A9; &#xAC00;&#xB2A5;&#xD55C; &#xC720;&#xC804;&#xC790; &#xC2DC;&#xD000;&#xC2A4;&#xC758; &#xC591;&#xC744; &#xC99D;&#xAC00; &#xC774;&#xC678;&#xC5D0;&#xC774; &#xCEEC;&#xB809;&#xC158;&#xC740; Lophotrochozoa&#xC758; &#xBAA8;&#xB4E0; &#xAD6C;&#xC131;&#xC6D0;&#xC5D0; &#xB300;&#xD574; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xCD5C;&#xB300; &#xB370;&#xC774;&#xD130;&#xBCA0;&#xC774;&#xC2A4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4; &#xD558; &#xACE0; &#xB530;&#xB77C;&#xC11C; &#xC591;&#xC790; &#xB3D9;&#xBB3C;&#xC758; &#xC138; &#xAC00;&#xC9C0; &#xC81C;&#xC548; &#xB41C; superclades&#xC758; &#xB9E4;&#xC6B0; &#xC131;&#xACF5;&#xC801;&#xC778; &#xB2E4;&#xC591; &#xD55C; &#xD608;&#xD1B5;&#xC774; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xC9C4;&#xD654; &#xC804;&#xB7B5;&#xC5D0; &#xCD94;&#xAC00;&#xC801;&#xC778; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC81C;&#xACF5;.

Aplysia &#xC2E0;&#xACBD; transcriptome: &#xC2E0;&#xACBD; &#xAD6C;&#xD68D; &#xBC0F; &#xD68C;&#xB85C;.

An&aacute;lisis moleculares de Aplysia, un organismo modelo establecido para celulares y sistemas neuronal ciencia, han sido seriamente perjudicados por la falta de informaci&oacute;n adecuada sobre el genoma. Mediante la secuenciaci&oacute;n de las bibliotecas de cDNA del sistema nervioso central (CNS), hemos identificado m&aacute;s de 175.000 etiquetas expressed sequence (ESTs), de ellas 19.814 productos g&eacute;nicos neuronal &uacute;nica y representan el 50% - 70% de la total transcriptoma neuronal de Aplysia. Hemos caracterizado el transcriptoma en tres niveles: (1) el sistema nervioso central, (2) los componentes elementales de un comportamiento simple: el gill-retiro reflejo-mediante el an&aacute;lisis sensoriales, motores y serotonin&eacute;rgicas neuronas moduladores y (3) procesos de neuronas individuales. Adem&aacute;s de aumentar la cantidad de secuencias de genes disponibles de Aplysia por dos &oacute;rdenes de magnitud, esta colecci&oacute;n representa la mayor base de datos disponible para cualquier miembro de la Lophotrochozoa y por lo tanto proporciona perspectivas adicionales en estrategias evolutivas utilizados por este linaje diversificada exitoso, uno de los tres superclades propuestas de animales bilaterales.

Transcriptoma neuronal de Aplysia: compartimentos neuronales y circuitos.

The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that, following repeated pulses of serotonin (5-HT), the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF.

Sustained CPEB-dependent local protein synthesis is required to stabilize synaptic growth for persistence of long-term facilitation in Aplysia.

To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.

A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex.

&#x5185;&#x5B58;&#x5B58;&#x50A8;&#x4E0E;&#x5185;&#x5B58;&#x76F8;&#x5173;&#x7684;&#x7A81;&#x89E6;&#x53EF;&#x5851;&#x6027;&#x4F9D;&#x9760;&#x7CBE;&#x786E;&#x65F6;&#x7A7A;&#x8C03;&#x63A7;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x5728;&#x5B66;&#x4E60;&#x6709;&#x5173;&#x7A81;&#x89E6;&#x53EF;&#x5851;&#x6027;&#x63A2;&#x7D22;&#x76D1;&#x7BA1;&#x5C0F; RNAs &#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x6211;&#x4EEC;&#x8FDB;&#x884C;&#x4E86;&#x5927;&#x89C4;&#x6A21;&#x5E76;&#x884C;&#x7684;&#x5148;&#x540E;&#x987A;&#x5E8F;&#x6765;&#x5206;&#x6790;&#x5C0F; RNAs &#x7684;&#x6D77;&#x5154;&#x591C;&#x86FE;&#x3002;&#x6211;&#x4EEC;&#x627E;&#x51FA; 170 &#x4E0D;&#x540C; Mirna&#xFF0C;&#x5176;&#x4E2D; 13 &#x4E2A;&#x662F;&#x65B0;&#x9896;&#x7684;&#x3001; &#x7279;&#x5B9A;&#x4E8E;&#x6D77;&#x5154;&#x3002;&#x4E5D;&#x4E2A; Mirna &#x4E30;&#x5BCC;&#xFF0C;&#x5927;&#x8111;&#xFF0C;&#x5176;&#x4E2D;&#x7684;&#x51E0;&#x4E2A;&#x8FC5;&#x901F;&#x7531;&#x77AC;&#x6001;&#x66B4;&#x9732;&#x4E8E; 5-&#x7F9F;&#x8272;&#x80FA;&#xFF0C;&#x8C03;&#x8282;&#x795E;&#x7ECF;&#x9012;&#x8D28;&#xFF0C;&#x5728;&#x5B66;&#x4E60;&#x671F;&#x95F4;&#x91CA;&#x653E;&#x7684;&#x7F16;&#x7801;&#x3002;&#x5BCC;&#x8111; Mirna &#x8FDB;&#x4E00;&#x6B65;&#x9274;&#x5B9A;&#x8868;&#x660E;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9;-124&#xFF0C;&#x6700;&#x4E3A;&#x4E30;&#x5BCC;&#x3001; &#x6700;&#x4E3A; well-conserved &#x5927;&#x8111;&#x7279;&#x5B9A;&#x7C73;&#x5C14;&#xFF0C;&#x4E13;&#x672C; presynaptically &#x5728;&#x611F;&#x5B98;&#x7535;&#x673A;&#x7A81;&#x89E6;&#x90A3;&#x91CC;&#x5B83;&#x7EA6;&#x675F;&#x901A;&#x8FC7;&#x8F6C;&#x5F55;&#x56E0;&#x5B50; CREB &#x8C03;&#x63A7;&#x81F4; 5-&#x7F9F;&#x8272;&#x80FA;&#x7A81;&#x89E6;&#x4FBF;&#x5229;&#x5316;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x76EE;&#x524D;&#x76F4;&#x63A5;&#x8BC1;&#x636E;&#x8C03;&#x8282;&#x795E;&#x7ECF;&#x9012;&#x8D28;&#x91CD;&#x8981;&#x7684;&#x5B66;&#x4E60;&#x53EF;&#x4EE5;&#x53D1;&#x6325;&#x4F5C;&#x7528;&#xFF0C;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 124 &#x5883;&#x5185;&#x957F;&#x671F;&#x6210;&#x719F;&#x7684;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x7A81;&#x89E6;&#x7684;&#x53EF;&#x5851;&#x6027;&#x548C;&#x89C4;&#x7BA1;&#x5C0F; RNAs &#x7684;&#x6C34;&#x5E73;&#x3002;

&#x6D77;&#x5154;&#x5C0F; RNAs &#x7684;&#x8868;&#x5F81; CREB &#x901A;&#x8FC7;&#x5236;&#x7EA6;&#x7A81;&#x89E6;&#x53EF;&#x5851;&#x6027;&#x4E2D;&#x63ED;&#x793A;&#x4E86;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 124 &#x65B9;&#x9762;&#x53D1;&#x6325;&#x7684;&#x4F5C;&#x7528;&#x3002;

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a role for miR-124 in long-term plasticity of synapses in the mature nervous system.

Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.

M&#xE9;moire de stockage et de la m&#xE9;moire li&#xE9;e &#xE0; la plasticit&#xE9; synaptique repose sur une r&#xE9;glementation pr&#xE9;cise spatio-temporelle de l&#x26;#39;expression g&#xE9;nique. Pour explorer le r&#xF4;le des petits ARN r&#xE9;gulateurs dans l&#x26;#39;apprentissage li&#xE9; &#xE0; la plasticit&#xE9; synaptique, nous avons r&#xE9;alis&#xE9; le s&#xE9;quen&#xE7;age massivement parall&#xE8;le pour &#xE9;tablir le profil des petits ARN de Aplysia californica. Nous avons identifi&#xE9; 170 miARN distinctes, dont 13 &#xE9;taient nouvelles et sp&#xE9;cifiques &#xE0; l&#x26;#39;aplysie. Neuf miARN ont &#xE9;t&#xE9; enrichi cerveau, et plusieurs d&#x26;#39;entre eux ont &#xE9;t&#xE9; rapidement r&#xE9;prim&#xE9;e par l&#x26;#39;exposition temporaire &#xE0; la s&#xE9;rotonine, un neurotransmetteur modulateur lib&#xE9;r&#xE9;s lors de l&#x26;#39;apprentissage. Une caract&#xE9;risation plus pouss&#xE9;e des cerveau-enrichis miARN miR-a r&#xE9;v&#xE9;l&#xE9; que 124, le plus abondant et bien conserv&#xE9; sp&#xE9;cifique du cerveau miRNA, a &#xE9;t&#xE9; exclusivement pr&#xE9;senter pr&#xE9;synaptique dans une synapse sensori-moteur, o&#xF9; elle contraint la s&#xE9;rotonine induite par la facilitation synaptique par la r&#xE9;glementation du facteur de transcription CREB. Nous pr&#xE9;sentons donc une preuve directe que un neurotransmetteur modulateur important pour l&#x26;#39;apprentissage peut r&#xE9;gler les niveaux de petits ARN et de pr&#xE9;senter un r&#xF4;le &#xE0; jouer miR-124 &#xE0; long terme la plasticit&#xE9; des synapses dans le syst&#xE8;me nerveux mature.

Caract&#xE9;risation des petits ARN chez l&#x26;#39;aplysie r&#xE9;v&#xE8;le un r&#xF4;le pour Mir-124 en Contraindre Plasticit&#xE9; synaptique Gr&#xE2;ce CREB

Speicher und speicherbezogene synaptische Plastizit&auml;t beruhen auf exakte raumzeitliche Regulierung der Genexpression. Um die Rolle der kleine regulatorische RNAs in synaptische Plastizit&auml;t im Zusammenhang mit lernen zu untersuchen, haben wir durchgef&uuml;hrt massive parallele Sequenzierung, die kleine RNAs von Aplysia Californica profilieren. Wir haben 170 unterschiedliche MiRNAs, die 13 die neuartige und spezifisch f&uuml;r Aplysia wurden identifiziert. Neun MiRNAs waren Gehirn bereichert, und mehrere von diesen waren schnell Downregulated durch vor&uuml;bergehende Aussetzung zu Serotonin, ein modulierende Neurotransmitter w&auml;hrend lernen ver&ouml;ffentlicht. Weitere Charakterisierung von dem Gehirn angereicherten MiRNAs ergab, dass MiR-124, die meisten reichlich vorhanden und Stadtgrundriss Gehirn-spezifische-MiRNA, ausschlie&szlig;lich presynaptically in eine sensorisch-motorischen Synapse vorhanden wo es schr&auml;nkt Serotonin-induzierte synaptische Erleichterung durch Regulierung des Faktors transkriptionelle CREB. Wir pr&auml;sentieren daher direkte Beweise, dass eine modulierende Neurotransmitter, die wichtig f&uuml;r das Lernen die Ebenen der kleine RNAs regulieren und eine Rolle f&uuml;r die MiR-124 in langfristige Plastizit&auml;t der Synapsen im Nervensystem ausgereift pr&auml;sentieren kann.

Charakterisierung der kleine RNAs in Aplysia enth&uuml;llt eine Rolle f&uuml;r die MiR-124 in einschr&auml;nkenden synaptische Plastizit&auml;t durch CREB.

Memoria e plasticit&agrave; sinaptica relativi alla memoria si basano su precisi spatiotemporal regolazione dell&#39;espressione genica. Per esplorare il ruolo di piccolo RNAs normativo nella plasticit&agrave; sinaptica apprendimento correlati, abbiamo effettuato sequenziamento massiccio parallelo al profilo del piccolo RNAs di Aplysia californica. Abbiamo identificato 170 miRNAs distinti, 13 dei quali erano nuovi e specifici di Aplysia. Nove miRNAs erano cervello arricchito, e molti di questi sono stati rapidamente downregolata da esposizione transitoria di serotonina, un neurotrasmettitore modulatrice rilasciato durante l&#39;apprendimento. Ulteriore caratterizzazione dei miRNAs arricchita di cervello rivelato che miR-124, il miRNA specifici del cervello pi&ugrave; abbondante e ben conservato, era presente esclusivamente presynaptically in una sinapsi sensoriale-motoria dove essa vincola facilitazione sinaptica indotta da serotonina attraverso la regolazione del fattore di trascrizione CREB. Vi presentiamo quindi una prova diretta che un modulatore neurotrasmettitore importante per l&#39;apprendimento pu&ograve; regolare i livelli di piccolo RNAs e presentare un ruolo per miR-124 nel lungo termine plasticit&agrave; delle sinapsi del sistema nervoso maturo.

Caratterizzazione del piccolo RNAs in Aplysia rivela un ruolo per miR-124 a vincolare plasticit&agrave; sinaptica attraverso CREB.

&#x8A18;&#x61B6;&#x88C5;&#x7F6E;&#x3068;&#x30E1;&#x30E2;&#x30EA;&#x95A2;&#x9023;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x53EF;&#x5851;&#x6027;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306E;&#x6B63;&#x78BA;&#x306A;&#x6642;&#x7A7A;&#x9593;&#x5236;&#x5FA1;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5B66;&#x7FD2;&#x95A2;&#x9023;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x53EF;&#x5851;&#x6027;&#x306E;&#x4F4E;&#x5206;&#x5B50;&#x5236;&#x5FA1;RNA&#x306E;&#x5F79;&#x5272;&#x3092;&#x63A2;&#x6C42;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;californica&#x306E;small RNA&#x3092;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EA;&#x30F3;&#x30B0;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x5927;&#x898F;&#x6A21;&#x306A;&#x4E26;&#x5217;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x5C0F;&#x8AAC;&#x3084;&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x306B;&#x7279;&#x7570;&#x7684;&#x3067;&#x3042;&#x3063;&#x305F;13&#x306F;170&#x306E;&#x7570;&#x306A;&#x308B;miRNA&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x30CA;&#x30A4;&#x30F3;miRNA&#x304C;&#x8133;&#x6FC3;&#x7E2E;&#x3055;&#x308C;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306F;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;&#x3078;&#x306E;&#x4E00;&#x6642;&#x7684;&#x306A;&#x9732;&#x51FA;&#x3001;&#x5B66;&#x7FD2;&#x4E2D;&#x306B;&#x653E;&#x51FA;&#x3055;&#x8ABF;&#x7BC0;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x7269;&#x8CEA;&#x306B;&#x3088;&#x3063;&#x3066;&#x6025;&#x901F;&#x306B;&#x30C0;&#x30A6;&#x30F3;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x305F;&#x3002;&#x8133;&#x6FC3;&#x7E2E;miRNA&#x306E;&#x3055;&#x3089;&#x306A;&#x308B;&#x7279;&#x5FB4;&#x306F;&#x3001;miR-124&#x3001;&#x6700;&#x3082;&#x8C4A;&#x5BCC;&#x3067;&#x3001;&#x3088;&#x304F;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x8133;&#x7279;&#x7570;&#x7684;miRNA&#x304C;&#x3001;&#x6392;&#x4ED6;&#x7684;&#x306B;&#x305D;&#x308C;&#x304C;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x8ABF;&#x7BC0;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x8A98;&#x767A;&#x3055;&#x308C;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x5186;&#x6ED1;&#x5316;&#x3092;&#x5236;&#x7D04;&#x3059;&#x308B;&#x611F;&#x899A;&#x904B;&#x52D5;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x524D;&#x306B;&#x5B58;&#x5728;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;CREB&#x3002;&#x305D;&#x3053;&#x3067;&#x6211;&#x3005;&#x306F;&#x5B66;&#x7FD2;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x8ABF;&#x7BC0;&#x795E;&#x7D4C;&#x4F1D;&#x9054;&#x7269;&#x8CEA;&#x306F;&#x3001;&#x5C0F;&#x3055;&#x306A;RNA&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x3001;&#x6210;&#x719F;&#x3057;&#x305F;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306B;&#x304A;&#x3051;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x9577;&#x671F;&#x53EF;&#x5851;&#x6027;&#x306B;&#x304A;&#x3051;&#x308B;miR-124&#x306E;&#x5F79;&#x5272;&#x3092;&#x63D0;&#x793A;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x76F4;&#x63A5;&#x7684;&#x306A;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30E1;&#x30D5;&#x30E9;&#x30B7;&#x306E;&#x5C0F;&#x3055;&#x306A;RNA&#x306E;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x306F;&#x3001;CREB&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x53EF;&#x5851;&#x6027;&#x306E;&#x5236;&#x7D04;&#x306B;miR-124&#x306E;&#x5F79;&#x5272;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;

&#xBA54;&#xBAA8;&#xB9AC; &#xC2A4;&#xD1A0;&#xB9AC;&#xC9C0; &#xBC0F; &#xBA54;&#xBAA8;&#xB9AC; &#xAD00;&#xB828; &#xB41C; &#xC2DC; &#xB0C5; &#xC2A4;&#xAC00; &#xC18C;&#xC131; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC758; &#xC815;&#xD655;&#xD55C; spatiotemporal &#xADDC;&#xC815;&#xC5D0; &#xC758;&#xC874; &#xD569;&#xB2C8;&#xB2E4;. &#xD559;&#xC2B5; &#xAD00;&#xB828; &#xB41C; &#xC2DC; &#xB0C5; &#xC2A4;&#xAC00; &#xC18C;&#xC131;&#xC5D0; &#xC791;&#xC740; &#xADDC;&#xC81C; RNAs &#xC5ED;&#xD560;&#xC744; &#xD0D0;&#xAD6C;, &#xC6B0;&#xB9AC;&#xB294; &#xC791;&#xC740; Aplysia RNAs californica &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xB9C1; &#xB300;&#xADDC;&#xBAA8; &#xBCD1;&#xB82C; &#xC2DC;&#xD000;&#xC2F1; &#xC2E4;&#xC2DC;. &#xC6B0;&#xB9AC;&#xB294; 13 &#xD588;&#xB2E4; &#xC18C;&#xC124;&#xACFC; &#xAD00;&#xB828; Aplysia 170 &#xAC00;&#xC9C0; miRNAs &#xC2DD;&#xBCC4;. 9 MiRNAs &#xB1CC; &#xB18D;&#xCD95; &#xB418;&#xC5C8;&#xACE0; &#xC774;&#xB4E4; &#xC911; &#xBA87; &#xAC00;&#xC9C0; &#xD588;&#xB2E4; &#xC2E0;&#xC18D; &#xD558; &#xAC8C; &#xD559;&#xC2B5; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xBC1C;&#xD45C; modulatory &#xC2E0;&#xACBD; &#xC804;&#xB2EC; &#xBB3C;&#xC9C8; &#xC138;&#xB85C;&#xD1A0;&#xB2CC;&#xC5D0; &#xACFC;&#xB3C4; &#xB178;&#xCD9C;&#xC5D0; &#xC758;&#xD574; downregulated. &#xB450;&#xB1CC; &#xB18D;&#xCD95; Mirnas&#xC758; &#xC790;&#xC138;&#xD55C; &#xBB18;&#xC0AC;&#xB294; &#xBBF8;&#xB974;-124, &#xAC00;&#xC7A5; &#xD48D;&#xBD80; &#xD558; &#xACE0; &#xC6B0;&#xC218; &#xB450;&#xB1CC; &#xAD00;&#xB828; &#xBBF8;&#xB974; &#xB3C5;&#xC810;&#xC801;&#xC73C;&#xB85C;&#xC5D0; &#xC874;&#xC7AC; &#xD588;&#xB2E4; presynaptically &#xAC10;&#xAC01; &#xBAA8;&#xD130; &#xB0C5; &#xADF8;&#xAC83; transcriptional &#xC694;&#xC18C; CREB&#xC758; &#xADDC;&#xC81C;&#xB97C; &#xD1B5;&#xD574; &#xC2DC; &#xB0C5; &#xC2A4; &#xCD09;&#xC9C4; &#xC138;&#xB85C;&#xD1A0;&#xB2CC; &#xC720;&#xB3C4; &#xAD6C;&#xC18D; &#xBC1D;&#xD614;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB530;&#xB77C;&#xC11C; modulatory &#xC2E0;&#xACBD; &#xC804;&#xB2EC; &#xBB3C;&#xC9C8; &#xD559;&#xC2B5;&#xC744; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xC218; &#xC791;&#xC740; Rnas&#xC758; &#xC218;&#xCE58;&#xB97C; &#xC870;&#xC808; &#xD558; &#xACE0; &#xC131;&#xC219;&#xD55C; &#xC2E0; &#xACBD;&#xACC4; &#xC2DC; &#xB0C5; &#xC2A4;&#xC758; &#xC7A5;&#xAE30;&#xAC00; &#xC18C;&#xC131;&#xC5D0; &#xBBF8;&#xB974;-124&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC2DC; &#xD558;&#xB294; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xC99D;&#xAC70; &#xC81C;&#xC2DC;.

Aplysia &#xC791;&#xC740; Rnas&#xC758; &#xD2B9;&#xC131;&#xD654; CREB &#xD1B5;&#xD574; &#xC81C;&#xC57D; &#xC2DC; &#xB0C5; &#xC2A4;&#xAC00; &#xC18C;&#xC131;&#xC5D0; &#xBBF8;&#xB974;-124&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xACC4;&#xC2DC; &#xD55C;&#xB2E4;.

Almacenamiento de la memoria y la memoria relacionada con la plasticidad sin&#xE1;ptica depender de la regulaci&#xF3;n precisa espacio-temporal de la expresi&#xF3;n g&#xE9;nica. Para explorar el papel de los peque&#xF1;os RNAs de reglamentaci&#xF3;n en el aprendizaje relacionado con la plasticidad sin&#xE1;ptica, se llev&#xF3; a cabo la secuenciaci&#xF3;n masiva en paralelo al perfil de los peque&#xF1;os RNAs de Aplysia californica. Se identificaron 170 miRNAs distintas, de las cuales 13 eran nuevas y espec&#xED;ficas para Aplysia. Nueve fueron los miRNAs cerebro enriquecido, y varias de ellas fueron r&#xE1;pidamente downregulated por la exposici&#xF3;n transitoria de la serotonina, un neurotransmisor modulador lanzado durante el aprendizaje. Adem&#xE1;s de la caracterizaci&#xF3;n del cerebro enriquecidos con miRNAs revel&#xF3; que el miR-124, el m&#xE1;s abundante y bien conservado-cerebro-espec&#xED;fica miRNA, se presentar&#xE1; exclusivamente en una sinapsis presin&#xE1;ptica sensorio-motor donde se restringe la serotonina inducida por la facilitaci&#xF3;n sin&#xE1;ptica a trav&#xE9;s de la regulaci&#xF3;n del factor de transcripci&#xF3;n CREB. Por lo tanto, presentar evidencia directa de que un neurotransmisor modulador importante para el aprendizaje puede regular los niveles de ARN peque&#xF1;os y presentar un papel para miR-124 en el largo plazo, la plasticidad de las sinapsis en el sistema nervioso maduro.

Caracterizaci&#xF3;n de los RNAs peque&#xF1;os en Aplysia revela un papel para MIR-124 en la limitaci&#xF3;n de plasticidad sin&#xE1;ptica a trav&#xE9;s de CREB

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.

Neurexin-neuroligin transsynaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in aplysia.

The neuronal isoform of cytoplasmic polyadenylation element-binding protein (CPEB) is a regulator of local protein synthesis at synapses and is critical in maintaining learning-related synaptic plasticity in Aplysia. Previous studies indicate that the function of Aplysia CPEB can be modulated by conversion to a stable prion-like state, thus contributing to the stabilization of long-term memory on a molecular level. Here, we used biophysical methods to demonstrate that Aplysia CPEB, like other prions, undergoes a conformational switch from soluble α-helix-rich oligomer to β-sheet-rich fiber in vitro. Solid-state NMR analyses of the fibers indicated a relatively rigid N-terminal prion domain. The fiber form of Aplysia CPEB showed enhanced binding to target mRNAs as compared to the soluble form. Consequently, we propose a model for the Aplysia CPEB fibers that may have relevance for functional prions in general.

Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB.

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.

A strategy to capture and characterize the synaptic transcriptome.

The human brain is extraordinarily complex, composed of billions of neurons and trillions of synaptic connections. Neurons are organized into circuit assemblies that are modulated by specific interneurons and non-neuronal cells, such as glia and astrocytes. Data on human genome sequences predicts that each of these cells in the human brain has the potential of expressing ∼20 000 protein coding genes and tens of thousands of noncoding RNAs. A major challenge in neuroscience is to determine (1) how individual neurons and circuitry utilize this potential during development and maturation of the nervous system, and for higher brain functions such as cognition, and (2) how this potential is altered in neurological and psychiatric disorders. In this review, we will discuss how recent advances in next generation sequencing, proteomics and bioinformatics have transformed our understanding of gene expression and the functions of neural circuitry, memory storage, and disorders of cognition.

Genomics and proteomics in solving brain complexity.

Gene products such as organelles, proteins and RNAs are actively transported to synaptic terminals for the remodeling of pre-existing neuronal connections and formation of new ones. Proteins described as molecular motors mediate this transport and utilize specialized cytoskeletal proteins that function as molecular tracks for the motor based transport of cargos. Molecular motors such as kinesins and dynein's move along microtubule tracks formed by tubulins whereas myosin motors utilize tracks formed by actin. Deficits in active transport of gene products have been implicated in a number of neurological disorders. We describe such disorders collectively as "transportopathies". Here we review current knowledge of critical components of active transport and their relevance to neurodegenerative diseases.

PATHOLOGIES OF AXONAL TRANSPORT IN NEURODEGENERATIVE DISEASES.

Despite the advances in our understanding of aging-associated behavioral decline, relatively little is known about how aging affects neural circuits that regulate specific behaviors, particularly the expression of genes in specific neural circuits during aging. We have addressed this by exploring a peptidergic neuron R15, an identified neuron of the marine snail Aplysia californica. R15 is implicated in reproduction and osmoregulation and responds to neurotransmitters such as acetylcholine, serotonin and glutamate and is characterized by its action potential bursts.

Age-associated bidirectional modulation of gene expression in single identified R15 neuron of Aplysia.

Several studies have shown that synthesis of new proteins at the synapse is a prerequisite for the storage of long-term memories. Relatively little is known about the availability of distinct mRNA populations for translation at specific synapses, the process that determines mRNA localization, and the temporal designations of localized mRNA translation during memory storage. Techniques such as synaptosome preparation and microdissection of distal neuronal processes of cultured neurons and dendritic layers in brain slices are general approaches used to identify localized RNAs. Exploration of the association of RNA-binding proteins to the axonal transport machinery has led to the development of a strategy to identify RNAs that are transported from the cell body to synapses by molecular motor kinesin. In this article, RNA localization at the synapse, as well as its mechanisms and significance in understanding long-term memory storage, are discussed.

RNA transport and long-term memory storage.

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.

Sathyanarayanan V. Puthanveettil

Department of Neuroscience

The Scripps Research Institute, Florida

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Sathyanarayanan V. Puthanveettil

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neuroscience

The Scripps Research Institute, Florida

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Department of Neuroscience