Using Microfluidics Chips for Live Imaging and Study of Injury Responses in <em>Drosophila</em> Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

University of Michigan

The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior.

Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans.

&#x867D;&#x7136;&#x5728;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x7684;&#x5F88;&#x591A;&#x5C5E;&#x6027;&#x52A8;&#x7269;&#x548C;&#x7CFB;&#x7EDF;&#x4E4B;&#x95F4;&#x5171;&#x4EAB;&#x7684;&#x5B83;&#x4E0D;&#x662F;&#x77E5;&#x9053;&#x4E0D;&#x540C;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x7535;&#x8DEF;&#x662F;&#x5426;&#x4F7F;&#x7528;&#x5171;&#x540C;&#x7684;&#x6218;&#x7565;&#x6765;&#x6307;&#x5BFC;&#x884C;&#x4E3A;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x8FD9;&#x91CC;&#x63CF;&#x8FF0;&#x4FE1;&#x606F;&#x5904;&#x7406;&#x7684;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x55C5;&#x89C9;&#x795E;&#x7ECF;&#x5143; (AWC&#xFF09; &#x548C; interneurons &#xFF08;AIB &#x548C; AIY&#xFF09; &#x63A7;&#x5236;&#x98DF;&#x54C1;&#x548C;&#x6C14;&#x5473;&#x8BF1;&#x53D1;&#x7684;&#x884C;&#x4E3A;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x9499;&#x6210;&#x50CF;&#x548C;&#x7A81;&#x53D8;&#xFF0C;&#x5C31;&#x4F1A;&#x5F71;&#x54CD;&#x7279;&#x5B9A;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x8FDE;&#x63A5;&#xFF0C;&#x663E;&#x793A; AWC &#x795E;&#x7ECF;&#x5143;&#x88AB;&#x6FC0;&#x6D3B;&#x7684;&#x6C14;&#x5473;&#x53BB;&#x9664;&#x548C;&#x6FC0;&#x6D3B; AMPA &#x578B;&#x8C37;&#x6C28;&#x9178;&#x53D7;&#x4F53;&#x901A;&#x8FC7; AIB interneurons&#x3002;&#x51E0;&#x5206;&#x949F;&#x540E;&#x53BB;&#x9664;&#x6C14;&#x5473;&#xFF0C;&#x6C14;&#x5473;&#x64A4;&#x51FA;&#x957F;&#x671F;&#x884C;&#x4E3A;&#x56DE;&#x5E94;&#x795E;&#x7ECF;&#x5143;&#x76F8;&#x5173;&#x9AD8;&#x67B6;&#x662F; AIB interneurons &#x4E2D;&#x9499;&#x7684;&#x6C34;&#x5E73;&#x3002;AWC &#x795E;&#x7ECF;&#x5143;&#x6291;&#x5236;&#x8C37;&#x6C28;&#x9178;&#x95E8;&#x63A7;&#x6C2F;&#x901A;&#x9053; &#xFF1B; &#x901A;&#x8FC7;&#x827E;&#x4F0A; interneurons&#x6C14;&#x5473;&#x6F14;&#x793A;&#x6587;&#x7A3F;&#x53EF;&#x4EE5;&#x51CF;&#x8F7B;&#x6B64;&#x6291;&#x5236;&#x548C;&#x6FC0;&#x6D3B;&#x7684;&#x827E;&#x4F0A; interneurons &#x7ED3;&#x679C;&#x3002;AIY &#x548C; AIB interneurons &#x5BF9;&#x9762;&#x89C4;&#x7BA1;&#x751F;&#x6210;&#x4E00;&#x4E2A;&#x534F;&#x8C03;&#x4E00;&#x81F4;&#x7684;&#x884C;&#x4E3A;&#x54CD;&#x5E94;&#x3002;&#x4FE1;&#x606F;&#x5904;&#x7406;&#x6B64;&#x7535;&#x8DEF;&#x7684;&#x7C7B;&#x4F3C;&#x4E8E;&#x4E3A; &#39;OFF&#39; &#x4E24;&#x6781;&#x548C; &#39;&#x5BF9;&#39; &#x53CC;&#x6781;&#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x6307;&#x793A;&#x611F;&#x5B98;&#x4FE1;&#x606F;&#x52A0;&#x5DE5;&#x5B88;&#x6052;&#x6216;&#x6536;&#x655B;&#x7B56;&#x7565;&#x4ECE;&#x810A;&#x690E;&#x52A8;&#x7269;&#x611F;&#x53D7;&#x5668;&#x7684;&#x4FE1;&#x606F;&#x6D41;&#x52A8;&#x3002;

&#x89E3;&#x5256;&#x4E00;&#x6746;&#x7EBF;&#x866B;&#x7684;&#x55C5;&#x89C9;&#x884C;&#x4E3A;&#x7684;&#x7535;&#x8DEF;&#x3002;

Although many properties of the nervous system are shared among animals and systems, it is not known whether different neuronal circuits use common strategies to guide behaviour. Here we characterize information processing by Caenorhabditis elegans olfactory neurons (AWC) and interneurons (AIB and AIY) that control food- and odour-evoked behaviours. Using calcium imaging and mutations that affect specific neuronal connections, we show that AWC neurons are activated by odour removal and activate the AIB interneurons through AMPA-type glutamate receptors. The level of calcium in AIB interneurons is elevated for several minutes after odour removal, a neuronal correlate to the prolonged behavioural response to odour withdrawal. The AWC neuron inhibits AIY interneurons through glutamate-gated chloride channels; odour presentation relieves this inhibition and results in activation of AIY interneurons. The opposite regulation of AIY and AIB interneurons generates a coordinated behavioural response. Information processing by this circuit resembles information flow from vertebrate photoreceptors to 'OFF' bipolar and 'ON' bipolar neurons, indicating a conserved or convergent strategy for sensory information processing.

Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.

Bien que de nombreuses propri&#xE9;t&#xE9;s du syst&#xE8;me nerveux sont partag&#xE9;s entre les animaux et des syst&#xE8;mes, on ne sait pas si les diff&#xE9;rents circuits neuronaux utiliser des strat&#xE9;gies communes pour guider le comportement. Ici nous caract&#xE9;risons traitement de l&#x26;#39;information par les neurones olfactifs Caenorhabditis elegans (AWC) et des interneurones (AIB et AIY) qui contr&#xF4;lent les comportements alimentaires et les odeurs &#xE9;voqu&#xE9;es. En utilisant l&#x26;#39;imagerie de calcium et de mutations qui affectent les connexions neuronales sp&#xE9;cifiques, nous montrons que les neurones sont activ&#xE9;s par AWC &#xE9;limination des odeurs et d&#x26;#39;activer les interneurones AIB &#xE0; travers des r&#xE9;cepteurs du glutamate de type AMPA. Le niveau de calcium dans les interneurones AIB est &#xE9;lev&#xE9; pendant plusieurs minutes apr&#xE8;s l&#x26;#39;&#xE9;limination des odeurs, un neuronale corr&#xE9;l&#xE9;e &#xE0; la r&#xE9;ponse comportementale prolong&#xE9;e aux odeurs de retrait. Le neurone AWC inhibe interneurones AIY travers les canaux chlorure glutamate-d&#xE9;pendants, la pr&#xE9;sentation d&#x26;#39;odeur soulage cette inhibition et les r&#xE9;sultats dans l&#x26;#39;activation des interneurones AIY. Le r&#xE8;glement en face des interneurones AIY et AIB g&#xE9;n&#xE8;re une r&#xE9;ponse coordonn&#xE9;e de comportement. Traitement de l&#x26;#39;information par ce circuit ressemble le flux d&#x26;#39;informations &#xE0; partir de photor&#xE9;cepteurs vert&#xE9;br&#xE9;s &#xE0; &#x26;quot;OFF&#x26;quot; bipolaire et &#x26;#39;ON&#x26;#39; neurones bipolaires, indiquant une strat&#xE9;gie conserv&#xE9;e ou convergentes pour le traitement de l&#x26;#39;information sensorielle.

Dissection d&#x26;#39;un circuit de comportement olfactif chez Caenorhabditis elegans

Obwohl viele Eigenschaften des Nervensystems Tiere und Systeme gemeinsam sind, ist nicht bekannt, ob unterschiedliche neuronale Schaltkreise gemeinsame Strategien verwenden, um Verhalten zu f&uuml;hren. Hier wir charakterisieren die Informationsverarbeitung von Caenorhabditis Elegans olfaktorischen Neuronen (AWC) und Interneuronen (AIB und AIY), die Nahrung und Geruch hervorgerufen Verhaltensweisen zu steuern. Mit Kalzium Bildgebung und Mutationen, die bestimmte neuronale Verbindungen beeinflussen, zeigen wir, dass die AWC Neuronen durch Geruchsbeseitigung aktiviert und die AIB Interneuronen durch AMPA-Typ Glutamat-Rezeptoren zu aktivieren. Die H&ouml;he der Kalzium in AIB Interneuronen ist f&uuml;r einige Minuten nach Geruchsbeseitigung, einer neuronalen Korrelat der anhaltenden Verhaltensst&ouml;rungen Antwort auf Geruch R&uuml;ckzug erh&ouml;ht. Das AWC-Neuron hemmt AIY Interneuronen durch Glutamat-Gating-Chlorid-Kan&auml;le; Geruch Pr&auml;sentation entlastet diese Hemmung und f&uuml;hrt zu einer Aktivierung von AIY Interneuronen. Die entgegengesetzte Regulierung von AIY und AIB Interneuronen generiert ein abgestimmtes Verhalten. Informationsverarbeitung durch diese Schaltung &auml;hnelt Informationsflu&szlig; von vertebrate Photorezeptoren auf &#39;OFF&#39; bipolar und &#39;ON&#39; bipolare Nervenzellen, die angibt, eine konservierte oder konvergente Strategie f&uuml;r sensorische Informationsverarbeitung.

Sezieren einer Schaltung f&uuml;r olfaktorische Verhalten in Caenorhabditis Elegans.

Anche se molte propriet&agrave; del sistema nervoso sono condivisi tra i sistemi e gli animali, non &egrave; noto se diversi circuiti neuronali utilizzano strategie comuni per guidare il comportamento. Qui ci caratterizzano information processing di Caenorhabditis elegans neuroni olfattivi (AWC) e interneurons (AIB e AIY) che controllano i comportamenti alimentari e odori evocati. Utilizzando calcio imaging e mutazioni che colpiscono specifiche connessioni neuronali, mostriamo che i neuroni AWC sono attivati dalla rimozione degli odori e attivare il interneurons AIB attraverso i recettori del glutammato di tipo AMPA. Il livello di calcio nel interneurons AIB &egrave; elevato per diversi minuti dopo la rimozione degli odori, un correlato neuronale alla prolungata risposta comportamentale al ritiro degli odori. Il neurone AWC inibisce AIY interneurons attraverso canali cloruro glutammato-gated; presentazione di odore allevia Questa inibizione e risultati nell&#39;attivazione dei interneurons AIY. Il regolamento opposto di interneurons AIY e AIB genera una risposta coordinata e comportamento. Informazioni trattamento da parte di questo circuito assomiglia a flusso di informazioni da fotorecettori vertebrati su &#39;OFF&#39; bipolare e &#39;Su&#39; neuroni bipolari, che indica una strategia convergente o conservata per l&#39;elaborazione di informazioni sensoriali.

Dissezione di un circuito per comportamento olfattiva in Caenorhabditis elegans.

&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x591A;&#x304F;&#x306E;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x306F;&#x3001;&#x52D5;&#x7269;&#x3084;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x9593;&#x3067;&#x5171;&#x6709;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x304C;&#x3001;&#x305D;&#x308C;&#x306F;&#x7570;&#x306A;&#x3063;&#x305F;&#x795E;&#x7D4C;&#x56DE;&#x8DEF;&#x306E;&#x52D5;&#x4F5C;&#x3092;&#x30AC;&#x30A4;&#x30C9;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5171;&#x901A;&#x306E;&#x6226;&#x7565;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x306F;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x7DDA;&#x866B;Caenorhabditis elegans&#x306E;&#x55C5;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#xFF08;AWC&#xFF09;&#x3068;&#x4ECB;&#x5728;&#xFF08;AIB&#x3068;AIY&#xFF09;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x98DF;&#x54C1;&#x3068;&#x81ED;&#x6C17;&#x8A98;&#x767A;&#x884C;&#x52D5;&#x306B;&#x3088;&#x308B;&#x60C5;&#x5831;&#x51E6;&#x7406;&#x3092;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x308B;&#x3002;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x795E;&#x7D4C;&#x63A5;&#x7D9A;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x5909;&#x7570;&#x3092;&#x7528;&#x3044;&#x3066;&#x3001;AWC&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;&#x81ED;&#x6C17;&#x3092;&#x9664;&#x53BB;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x3001;AMPA&#x578B;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x4ECB;&#x3057;&#x3066;AIB&#x4ECB;&#x5728;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; AIB&#x4ECB;&#x5728;&#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x81ED;&#x6C17;&#x3092;&#x9664;&#x53BB;&#x3057;&#x305F;&#x5F8C;&#x306E;&#x6570;&#x5206;&#x9593;&#x4E0A;&#x6607;&#x3055;&#x305B;&#x3001;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306F;&#x3001;&#x64A4;&#x9000;&#x3092;&#x81ED;&#x6C17;&#x306B;&#x9577;&#x6642;&#x9593;&#x884C;&#x52D5;&#x53CD;&#x5FDC;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; AWC&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x4F9D;&#x5B58;&#x6027;&#x30AF;&#x30ED;&#x30E9;&#x30A4;&#x30C9;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3092;&#x4ECB;&#x3057;&#x3066;AIY&#x4ECB;&#x5728;&#x3092;&#x963B;&#x5BB3;&#x3057;&#x3001;&#x81ED;&#x6C17;&#x306E;&#x30D7;&#x30EC;&#x30BC;&#x30F3;&#x30C6;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;AIY&#x4ECB;&#x5728;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x963B;&#x5BB3;&#x3057;&#x3001;&#x7D50;&#x679C;&#x3092;&#x7DE9;&#x548C;&#x3057;&#x307E;&#x3059;&#x3002; AIY&#x3068;AIB&#x4ECB;&#x5728;&#x306E;&#x53CD;&#x5BFE;&#x5074;&#x306E;&#x898F;&#x5236;&#x306F;&#x3001;&#x5354;&#x8ABF;&#x884C;&#x52D5;&#x306E;&#x5FDC;&#x7B54;&#x3092;&#x751F;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x56DE;&#x8DEF;&#x306B;&#x3088;&#x308B;&#x60C5;&#x5831;&#x51E6;&#x7406;&#x3067;&#x306F;&#x3001;&#x611F;&#x899A;&#x60C5;&#x5831;&#x306E;&#x51E6;&#x7406;&#x306E;&#x305F;&#x3081;&#x306B;&#x4FDD;&#x5B58;&#x307E;&#x305F;&#x306F;&#x53CE;&#x675F;&#x6226;&#x7565;&#x3092;&#x793A;&#x3059;&#x3001;&#x53CC;&#x6975;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3; &#x26;quot;ON&#x26;quot; &#x26;quot;OFF&#x26;quot;&#x30D0;&#x30A4;&#x30DD;&#x30FC;&#x30E9;&#x3068;&#x306E;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;&#x5149;&#x53D7;&#x5BB9;&#x4F53;&#x304B;&#x3089;&#x306E;&#x60C5;&#x5831;&#x306E;&#x6D41;&#x308C;&#x306B;&#x4F3C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x7DDA;&#x866B;Caenorhabditis elegans&#x306E;&#x55C5;&#x899A;&#x884C;&#x52D5;&#x306E;&#x305F;&#x3081;&#x306E;&#x56DE;&#x8DEF;&#x3092;&#x89E3;&#x5256;

&#xC2E0;&#xACBD; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xB9CE;&#xC740; &#xC18D;&#xC131; &#xB3D9;&#xBB3C;&#xACFC; &#xC2DC;&#xC2A4;&#xD15C; &#xACF5;&#xC720; &#xD558;&#xB294;, &#xC788;&#xC9C0;&#xB9CC; &#xB2E4;&#xB978; &#xC2E0;&#xACBD; &#xD68C;&#xB85C; &#xD589;&#xB3D9;&#xC744; &#xC548;&#xB0B4; &#xD558; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xC804;&#xB7B5;&#xC744; &#xC0AC;&#xC6A9; &#xD558;&#xB294;&#xC9C0; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC9C0; &#xC54A;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC; &#xC815;&#xBCF4; Caenorhabditis elegans &#xD6C4; &#xAC01; &#xC2E0;&#xACBD; (AWC) &#xC758;&#xD574; &#xCC98;&#xB9AC; &#xD2B9;&#xC9D5; &#xBC0F; &#xC74C;&#xC2DD; &#xB0C4;&#xC0C8; &#xAC16;&#xB294; &#xD589;&#xB3D9;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xC218; (AIB &#xBC0F; AIY). &#xCE7C;&#xC298; &#xC774;&#xBBF8;&#xC9D5; &#xBC0F; &#xD2B9;&#xC815; &#xC2E0;&#xACBD; &#xC5F0;&#xACB0;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xB294; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC; AWC &#xB274;&#xB7F0; &#xB0C4;&#xC0C8; &#xC81C;&#xAC70;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; &#xB418; &#xACE0; &#xD65C;&#xC131;&#xD654; AMPA &#xD615; glutamate &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xD1B5;&#xD574; AIB &#xC218; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. AIB &#xC218;&#xC5D0; &#xCE7C;&#xC298; &#xC218;&#xC900;&#xC740; &#xB0C4;&#xC0C8; &#xC81C;&#xAC70;, &#xB0C4;&#xC0C8; &#xCCA0;&#xC218;&#xC5D0; &#xC7A5;&#xAE30;&#xAC04;&#xB41C; &#xD589;&#xB3D9; &#xBC18;&#xC751;&#xC5D0; &#xC2E0;&#xACBD; &#xC5F0;&#xACB0; &#xD6C4; &#xBA87; &#xBD84; &#xB3D9;&#xC548; &#xC0C1;&#xC2B9; &#xD569;&#xB2C8;&#xB2E4;. AWC &#xC2E0;&#xACBD; &#xC5B5;&#xC81C; AIY &#xC218; &#xC0B0; &#xC5FC; &#xBB38;&#xC774; &#xC5FC;&#xD654; &#xCC44;&#xB110;;&#xC744; &#xD1B5;&#xD574; &#xB0C4;&#xC0C8; &#xD504;&#xB808; &#xC820; &#xD14C;&#xC774; &#xC158;&#xC774;&#xC774; &#xC800;&#xD574;&#xB97C; &#xD574;&#xC18C; &#xD558; &#xACE0; AIY &#xC218; &#xD65C;&#xC131;&#xD654; &#xACB0;&#xACFC;. AIY &#xBC0F; AIB &#xC218;&#xC758; &#xBC18;&#xB300; &#xADDC;&#xC81C; &#xC870;&#xC728; &#xB41C; &#xD589;&#xB3D9; &#xC751;&#xB2F5;&#xC744; &#xC0DD;&#xC131;&#xD569;&#xB2C8;&#xB2E4;. &#xC815;&#xBCF4; &#xCC98;&#xB9AC;&#xC774; &#xD68C;&#xB85C; &#xC758;&#xD574; &#xCC99;&#xCD94; &#xB3D9;&#xBB3C; &#xB300;&#xB1CC;&#xC5D0;&#xC11C; &#xC815;&#xBCF4;&#xC758; &#xD750;&#xB984;&#xC744; &#39;OFF&#39; &#xBC14;&#xC774; &#xD3F4;&#xB77C; &#xBC0F; &#39;&#xBC14;&#xC774; &#xD3F4;&#xB77C; &#xB274;&#xB7F0;, &#xAC10;&#xAC01; &#xC815;&#xBCF4; &#xCC98;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xC874; &#xB610;&#xB294; &#xC9D1;&#xC911; &#xC804;&#xB7B5;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xC5D0;&#39;&#xC640; &#xBE44;&#xC2B7;&#xD569;&#xB2C8;&#xB2E4;.

Caenorhabditis elegans&#xC5D0; &#xD6C4; &#xAC01; &#xD589;&#xB3D9;&#xC5D0; &#xB300; &#xD55C; &#xD68C;&#xB85C; &#xBD80;.

Aunque muchas de las propiedades del sistema nervioso son compartidas entre los animales y de los sistemas, no se sabe si los diferentes circuitos neuronales utilizan estrategias comunes para guiar la conducta. Aqu&#xED; se caracterizan por el procesamiento de la informaci&#xF3;n Caenorhabditis elegans neuronas olfativas (AWC) y las interneuronas (AIB y Aiy) que controlan las conductas alimentarias y el olor evoca-. Utilizando im&#xE1;genes de calcio y las mutaciones espec&#xED;ficas que afectan a las conexiones neuronales, se muestra que las neuronas de AWC son activados por la eliminaci&#xF3;n de olores y activar las interneuronas de AIB a trav&#xE9;s de receptores de glutamato AMPA-tipo. El nivel de calcio en las interneuronas de AIB se eleva durante varios minutos despu&#xE9;s de la eliminaci&#xF3;n de olores, una neuronal se correlaciona con la respuesta al olor de comportamiento prolongada abstinencia. La neurona AWC inhibe interneuronas Aiy trav&#xE9;s de los canales de cloruro glutamato, presentaci&#xF3;n olor alivia esta inhibici&#xF3;n y los resultados en la activaci&#xF3;n de las interneuronas Aiy. La regulaci&#xF3;n contrario de las interneuronas Aiy y AIB genera una respuesta de comportamiento coordinado. Procesamiento de la informaci&#xF3;n por este circuito se parece a un flujo de informaci&#xF3;n de los fotorreceptores de vertebrados en &#x26;#39;OFF&#x26;#39; bipolar y &#x26;#39;en&#x26;#39; las neuronas bipolares, lo que indica una estrategia conservada o convergentes para el procesamiento de informaci&#xF3;n sensorial.

Disecci&#xF3;n de un circuito de comportamiento olfativo en Caenorhabditis elegans

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies.

We present two microfluidic approaches for immobilizing the roundworm C. elegans on-chip. The first approach creates a CO(2) micro-environment while the second one utilizes a deformable PDMS membrane to mechanically restrict the worm's movement. An on-chip 'behavior' module was used to characterize the effect of these methods on the worm's locomotion pattern. Our results indicate that both methods are appropriate for the short-term (minutes) worm immobilization. The CO(2) method offers the additional advantages of long-term immobilization (1-2 hours) and reduced photobleaching, if fluorescent imaging during immobilization is required. We envision the use of these methods in a wide variety of biological studies in C. elegans, including cell developmental and neuronal regeneration studies.

CO2 and compressive immobilization of C. elegans on-chip.

Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2-dependent behaviors.

Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases.

The nematode Caenorhabditis elegans is a widely adopted model organism for studying various neurobiological processes at the molecular and cellular level in vivo. With a small, flexible, and continuously moving body, the manipulation of C. elegans becomes a challenging task. In this review, we highlight recent advances in microfluidic technologies for the manipulation of C. elegans. These new family of microfluidic chips are capable of handling single or populations of worms in a high-throughput fashion and accurately controlling their microenvironment. So far, they have been successfully used to study neural circuits and behavior, to perform large-scale phetotyping and morphology-based screens as well as to understand axon regeneration after injury. We envision that microfluidic chips can further be used to study different aspects of the C. elegans nervous system, extending from fundamental understanding of behavioral dynamics to more complicated biological processes such as neural aging and learning and memory.

Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans.

We present a novel microfabrication approach for obtaining arrays of planar, polymer-based microlenses of high numerical aperture. The proposed microlenses arrays consist of deformable, elastomeric membranes that are supported by polymer-filled microchambers. Each membrane/microchamber assembly is converted into a solid microlens when the supporting UV-curable polymer is pressurized and cured. By modifying the microlens diameter (40-60 microm) and curing pressure (7.5-30 psi), we demonstrated that it is possible to fabricate microlenses with a wide range of effective focal lengths (100-400 microm) and numerical apertures (0.05-0.3). We obtained a maximum numerical aperture of 0.3 and transverse resolution of 2.8 microm for 60 microm diameter microlenses cured at 30 psi. These values were found to be in agreement with values obtained from opto-mechanical simulations. We envision the use of these high numerical microlenses arrays in optical applications where light collection efficiency is important.

A high numerical aperture, polymer-based, planar microlens array.

The study of small-size animal models, such as the roundworm C. elegans, has provided great insight into several in vivo biological processes, extending from cell apoptosis to neural network computing. The physical manipulation of this micron-sized worm has always been a challenging task. Here, we discuss the applications, capabilities and future directions of a new family of worm manipulation tools, the 'worm chips'. Worm chips are microfabricated devices capable of precisely manipulating single worms or a population of worms and their environment. Worm chips pose a paradigm shift in current methodologies as they are capable of handling live worms in an automated fashion, opening up a new direction in in vivo small-size organism studies.

Worm chips: microtools for C. elegans biology.

Functional fluorescence imaging methods are widely used to study cellular physiology. When applied to small organisms, these methods suffer from low-throughput due to the laborious immobilization/stimulus delivery procedure that is typically involved during imaging. Here, we describe the development of an automated microfluidic-based platform for performing automated neuronal functional (calcium) imaging in the roundworm Caenorhabditis elegans. The platform, capable of processing tens to hundreds of worms per hour, immobilizes individual worms, delivers a chemical odor to their nose and collects calcium imaging data from single neurons without any manual intervention. We used the developed platform to obtain a large number of calcium responses from worms of different ages (212 worms were imaged in total). The calcium imaging data revealed significant difference in the responses from young and old worms, indicating that neural functionality is age-dependent. We believe that such a technology will be an essential tool for obtaining repeatable and accurate functional imaging data from a large population of worms, in order to minimize stochastic biological noise and identify statistically significant trends.

An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans.

Electrical stimulation has been widely used to modulate and study the in vitro and in vivo functionality of the nervous system. Here, we characterized the effect of electrical stimulation on ASH neuron in Caenorhabditis elegans and employed it to probe the neuron's age dependent properties. We utilized an automated microfluidic-based platform and characterized the ASH neuronal activity in response to an electric current applied to the worm's body. The electrically induced ASH neuronal response was observed to be dependent on the magnitude, polarity, and spatial location of the electrical stimulus as well as on the age of the worm.

Probing the physiology of ASH neuron in Caenorhabditis elegans using electric current stimulation.

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.

Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae.

We present a microfluidic biochip for trapping single white blood cells (WBCs). The novel biochip, microfabricated using standard surface micromachining processes, consists of an array of precisely engineered microholes that confine single cells in a tight, three dimensional space and mechanically immobilize them. A high (> 87%) trapping efficiency was achieved when WBC-containing samples were delivered to the biochip at the optimal pressure of 3 psi. The biochip can efficiently trap up to 7,500 cells, maintaining a high trapping efficiency even when the number of cells is extremely low (~200 cells). We believe that the developed biochip can be used as a standalone unit in a biology/clinical lab for trapping WBCs as well as other cell types and imaging them using a standard fluorescent microscope at the single cell level. Furthermore, it can be integrated with other miniaturized optical modules to construct a portable platform for counting a wide variety of cells and therefore it can be an excellent tool for monitoring human diseases at the point-of-care.

Nikos Chronis

Department of Biomedical Engineering,
Department of Mechanical Engineering

University of Michigan

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Nikos Chronis

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

University of Michigan

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Department of Biomedical Engineering,
Department of Mechanical Engineering