imaging-subcellular-structures-in-the-living-zebrafish-embryo

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Technische Universität München

Utrecht University

CCL3 and CCL4 are proinflammatory chemokines belonging to the CC family. Increase in expression of mRNA coding for various chemokines including CCL3 and CCL4 has been often detected with global transcriptome profiling of brain tissue following epileptogenic stimuli as well as in epilepsy in experimental models and in human patients. Despite this, little is known about the expression of these proteins in epileptogenesis or epilepsy. In the present work CCL3 and CCL4 mRNA and protein expression were studied in the amygdala stimulation model of temporal lobe epilepsy using quantitiative PCR and immunohistochemistry. Expression of CCL3 and CCL4 mRNA in the block of tissue containing enthorinal and piriform cortices, amygdala and piriform nucleus was markedly up-regulated at 1, 4, 14 and 30 days following stimulation and in hippocampal CA1 was significantly increased at 1 and 4 days following stimulation. Expression of CCL3 and CCL4 proteins was elevated in astrocytes in the enthorinal and piriform cortices, amygdala, and hippocampus showing the largest increase at 4D after status epilepticus. Increase in mRNA and protein levels of CCL3 and CCL4 in the animal model of temporal lobe epilepsy suggests their role in disease development or recovery form epileptogenic insult. Existence of multiple targets for these chemokines in the damaged brain allows several possibilities of influencing neuronal and glial functions.

Status epilepticus evokes prolonged increase in the expression of CCL3 and CCL4 mRNA and protein in the rat brain.

Approximately 30% of all epilepsy cases are acquired. At present there is no effective strategy to stop epilepsy development after the precipitating insult. Recent data from experimental models pointed to the mTOR pathway, which can be potently inhibited by rapamycin. However, data on the antiepileptic and antiepileptogenic properties of rapamycin are conflicting. Therefore, we tested whether rapamycin post-treatment influences epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy in rats. Animals were treated with rapamycin (6mg/kg) or vehicle daily for 2 wks, beginning 24h after stimulation. Sham-operated animals were treated with rapamycin or vehicle but were not stimulated. Animals were video-EEG monitored to detect spontaneous seizures. Animals were sacrificed 4 wks later and brains were collected for Timm staining. There were no significant differences in the number of stimulated rats developing epilepsy; latency to first spontaneous seizure; number of seizures, or seizure frequency in epileptic animals. The area occupied by mossy fibers was significantly increased in stimulated vs. sham-operated animals but was not different in animals treated with rapamycin vs. vehicle. Collectively, our data suggest that the antiepileptic or antiepileptogenic action of rapamycin is not a universal phenomenon and might be limited to certain experimental models or experimental conditions.

Post-treatment with rapamycin does not prevent epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy.

&#x7EBF;&#x7C92;&#x4F53;&#x63D0;&#x4F9B; ATP&#x3001; &#x7EF4;&#x6301;&#x9499;&#x7A33;&#x6001;&#x548C;&#x8C03;&#x8282;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x3002;&#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x5B83;&#x4EEC;&#x7684;&#x5927;&#x5C0F;&#x548C;&#x51E0;&#x4F55;&#x5F62;&#x72B6;&#x590D;&#x6742;&#xFF0C;&#x7531;&#x4E8E;&#x662F;&#x7279;&#x522B;&#x4F9D;&#x8D56;&#x7684;&#x6B63;&#x5E38;&#x8FD0;&#x8F6C;&#x548C;&#x5206;&#x5E03;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x3002;&#x56E0;&#x6B64;&#x8FD9;&#x4E9B;&#x7EC6;&#x80DE;&#x5668;&#x548C;&#x5176;&#x8FD0;&#x8F93;&#x4E2D;&#x65AD;&#x5728;&#x8303;&#x56F4;&#x5E7F;&#x6CDB;&#x7684;&#x795E;&#x7ECF;&#x9000;&#x884C;&#x6027;&#x75BE;&#x75C5;&#x4E2D;&#x53D1;&#x6325;&#x4E2D;&#x5FC3;&#x4F5C;&#x7528;&#x3002;&#x867D;&#x7136;&#x4F53;&#x5916;&#x7814;&#x7A76;&#x5927;&#x5927;&#x6269;&#x5C55;&#x4E86;&#x6211;&#x4EEC;&#x5BF9;&#x7EBF;&#x7C92;&#x4F53;&#x52A8;&#x529B;&#x5B66;&#x7684;&#x77E5;&#x8BC6;&#xFF0C;&#x6211;&#x4EEC;&#x4F53;&#x5185;&#x7684;&#x7406;&#x89E3;&#x4ECD;&#x7136;&#x6709;&#x9650;&#x3002;&#x4E3A;&#x4E86;&#x89E3;&#x51B3;&#x8FD9;&#x4E00;&#x7F3A;&#x9677;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x5DE5;&#x5177;&#x7814;&#x7A76;&#x7EBF;&#x7C92;&#x4F53;&#x52A8;&#x6001;&#x5149;&#x5B66;&#x8BBF;&#x95EE;&#x6591;&#x9A6C;&#x9C7C;&#x4F53;&#x5185;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x8FD9;&#x91CC;&#x5C55;&#x793A;&#x6211;&#x4EEC;&#x65B0;&#x751F;&#x6210;&#x7684;&#x5DE5;&#x5177;&#xFF0C;&#x5305;&#x62EC;&#x8F6C;&#x57FA;&#x56E0;&#x7684;&quot;MitoFish&quot;&#xFF0C;&#x53EF;&#x4EE5;&#x7528;&#x6765;&#x7814;&#x7A76;&#x7EBF;&#x7C92;&#x4F53;&#x7684;&#x4F53;&#x5185;&quot;&#x751F;&#x547D;&#x5468;&#x671F;&quot;&#xFF0C;&#x5E76;&#x5141;&#x8BB8;&#x67E5;&#x660E;&#x836F;&#x7406;&#x548C;&#x9057;&#x4F20;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x52A8;&#x529B;&#x5B66;&#x8C03;&#x5236;&#x5668;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x89C2;&#x5BDF;&#x5230;&#x6DF1;&#x523B;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x8FD0;&#x8F93;&#x8D64;&#x5B57;&#x5B9E;&#x65F6;&#x6591;&#x9A6C;&#x9C7C; tauopathy &#x6A21;&#x578B;&#x4E2D;&#x3002;&#x7531;&#x62A2;&#x6551;&#x4F7F;&#x7528; MARK2 &#x6B64;&#x8868;&#x578B; &#xFF08;&#x5FAE;&#x7BA1;&#x4EB2;&#x548C;&#x8C03;&#x8282;&#x6FC0;&#x9176; 2&#xFF09;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x4F9B;&#x6B64;&#x6FC0;&#x9176;&#x8C03;&#x8282;&#x8F74;&#x7A81;&#x8FD0;&#x8F93;&#x9A6C;&#x5934;&#x4F9D;&#x8D56;&#x7684;&#x65B9;&#x5F0F;&#x76F4;&#x63A5;&#x4F53;&#x5185;&#x8BC1;&#x636E;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x65B9;&#x6CD5;&#x5728;&#x81EA;&#x7136;&#x73AF;&#x5883;&#x4E0B;&#x6B63;&#x5E38;&#x548C;&#x75BE;&#x75C5;&#x7684;&#x6761;&#x4EF6;&#x4E0B;&#x5141;&#x8BB8;&#x52A8;&#x6001;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x7684;&#x8BE6;&#x7EC6;&#x7684;&#x7814;&#x7A76;&#x3002;

&#x75BE;&#x75C5;&#x76F8;&#x5173;&#x7EBF;&#x7C92;&#x4F53;&#x4E2D;&#x7684;&#x52A8;&#x529B;&#x5B66;&#x7684;&#x810A;&#x690E;&#x52A8;&#x7269;&#x6A21;&#x578B;&#x7CFB;&#x7EDF;&#x7684;&#x6D3B;&#x4F53;&#x6210;&#x50CF;&#x3002;

Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.

In vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system.

Mitochondries fournissent de l&#39;ATP, maintiennent l&#39;hom&eacute;ostasie du calcium et r&eacute;glementent l&#39;apoptose. Les neurones, en raison de leur taille et la g&eacute;om&eacute;trie complexe, sont particuli&egrave;rement d&eacute;pendantes sur le bon fonctionnement et de la distribution des mitochondries. Ainsi les perturbations de ces organites et leur transport jouent un r&ocirc;le central dans un large &eacute;ventail de maladies neurod&eacute;g&eacute;n&eacute;ratives. Alors que des &eacute;tudes in vitro ont consid&eacute;rablement &eacute;largi notre connaissance de la dynamique mitochondriale, notre compr&eacute;hension in vivo reste limit&eacute;e. Pour combler cette lacune, nous avons d&eacute;velopp&eacute; des outils pour &eacute;tudier la dynamique mitochondriale in vivo chez le poisson z&egrave;bre optiquement accessible. Nous montrons ici que nos outils nouvellement cr&eacute;&eacute;es, y compris transg&eacute;nique &laquo; MitoFish &raquo;, peut &ecirc;tre utilis&eacute; pour &eacute;tudier le &laquo; cycle &raquo; in vivo des mitochondries et permet d&#39;identifier des modulateurs pharmacologiques et g&eacute;n&eacute;tiques de la dynamique mitochondriale. En outre, nous avons observ&eacute; des d&eacute;ficits profonds mitochondriale de transport en temps r&eacute;el dans un mod&egrave;le de tauopathy de poisson-z&egrave;bre. En sauvant ce ph&eacute;notype &agrave; l&#39;aide de MARK2 (kinase r&eacute;gulatrice de microtubule-affinit&eacute; 2), nous fournissons une preuve in vivo directe que cette kinase r&eacute;gule le transport axonal de mani&egrave;re Tau-d&eacute;pendante. Ainsi, notre approche permet des &eacute;tudes d&eacute;taill&eacute;es de la dynamique des mitochondries dans leur milieu naturel dans des conditions normales et la maladie.

Imagerie in vivo de la dynamique mitochondriale li&eacute;s &agrave; la maladie dans un syst&egrave;me mod&egrave;le de vert&eacute;br&eacute;s.

Mitochondrien ATP liefern Kalzium-Hom&ouml;ostase aufrechtzuerhalten und Apoptose zu regulieren. Neuronen, aufgrund ihrer Gr&ouml;&szlig;e und komplexe Geometrie, sind besonders f&uuml;r das ordnungsgem&auml;&szlig;e Funktionieren und die Verteilung der Mitochondrien abh&auml;ngig. Somit Rolle St&ouml;rungen in diesen Organellen und deren Transport eine zentrale in einer Vielzahl von neurodegenerativen Erkrankungen. W&auml;hrend in vitro Studien unseres Wissens der mitochondrialen Dynamik stark erweitert haben, bleibt unser Verst&auml;ndnis in-vivo begrenzt. Um dieses Manko zu beheben, entwickelten wir Tools zur in-vivo in optisch zug&auml;nglichen Zebrafisch mitochondriale Dynamik zu studieren. Hier zeigen wir, dass unsere neu erstellte Tools, darunter transgene &quot;MitoFish,&quot; kann verwendet werden, um den in-vivo &quot;Lebenszyklus&quot; von Mitochondrien zu studieren und erm&ouml;glicht die Identifizierung pharmakologischer und genetischer Modulators des mitochondrialen Dynamik. Des weiteren beobachteten wir profunde mitochondriale Transport Defizite in Echtzeit in einem Zebrafisch Tauopathie Modell. Durch die Rettung dieser Ph&auml;notyp mit MARK2 (Microtubule-Affinit&auml;t regulierende Kinase 2), wir liefern direkt in-vivo Beweis, dass diese Kinase axonale Transport in Tau-abh&auml;ngigen Weise reguliert. Unser Ansatz erm&ouml;glicht so detaillierte Untersuchungen der Dynamik der Mitochondrien in ihrer nat&uuml;rlichen Umgebung unter Normal und Krankheit.

In-vivo Bildgebung Krankheitsbezogene mitochondriale Dynamik in vertebrate Modellsystem.

Mitocondri forniscono ATP, mantengono l&#39;omeostasi del calcio e regolano l&#39;apoptosi. I neuroni, a causa della loro dimensione e geometria complessa, sono particolarmente dipendenti il corretto funzionamento e distribuzione dei mitocondri. Cos&igrave; interruzioni di questi organelli e loro trasporto giocano un ruolo centrale in una vasta gamma di malattie neurodegenerative. Mentre gli studi in vitro hanno notevolmente ampliato la nostra conoscenza delle dinamiche mitocondriali, la nostra comprensione in vivo rimane limitata. Per risolvere questo inconveniente, abbiamo sviluppato strumenti per studiare dinamiche mitocondriali in vivo in zebrafish otticamente accessibile. Qui dimostriamo che i nostri strumenti appena generati, tra cui transgenici &quot;MitoFish&quot;, pu&ograve; essere utilizzato per studiare in vivo &quot;ciclo di vita&quot; dei mitocondri e permette di identificare modulatori farmacologiche e genetiche delle dinamiche mitocondriali. Inoltre abbiamo osservato deficit profondo trasporto mitocondriale in tempo reale in un modello di Danio rerio Taupatie. Di salvataggio di questo fenotipo utilizzando MARK2 (microtubule-affinit&agrave; regolazione chinasi 2), forniamo prove dirette in vivo, che questa chinasi regola il trasporto assonale in modo Tau-dipendente. Cos&igrave;, il nostro approccio permette studi dettagliati della dinamica dei mitocondri nel loro ambiente naturale, in condizioni normali e malattia.

Imaging in vivo riguardanti le malattie mitocondriali dinamiche in un sistema modello vertebrati.

&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306F; ATP &#x3092;&#x63D0;&#x4F9B;&#x3001;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6052;&#x5E38;&#x6027;&#x3092;&#x7DAD;&#x6301;&#x304A;&#x3088;&#x3073;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306E;&#x30B5;&#x30A4;&#x30BA;&#x3068;&#x8907;&#x96D1;&#x306A;&#x30B8;&#x30AA;&#x30E1;&#x30C8;&#x30EA;&#x306E;&#x305F;&#x3081;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306F;&#x3001;&#x9069;&#x5207;&#x306A;&#x6A5F;&#x80FD;&#x3068;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x5206;&#x5E03;&#x306B;&#x7279;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D30;&#x80DE;&#x5C0F;&#x5668;&#x5B98;&#x3068;&#x305D;&#x306E;&#x8F38;&#x9001;&#x306E;&#x4E2D;&#x65AD;&#x306F;&#x3001;&#x795E;&#x7D4C;&#x5909;&#x6027;&#x75BE;&#x60A3;&#x306E;&#x5E83;&#x3044;&#x7BC4;&#x56F2;&#x3067;&#x4E2D;&#x592E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x307E;&#x3059;&#x3002;In vitro &#x8A66;&#x9A13;&#x306F;&#x5927;&#x5E45;&#x306B;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30AF;&#x30B9;&#x306E;&#x79C1;&#x9054;&#x306E;&#x77E5;&#x8B58;&#x3092;&#x62E1;&#x5927;&#x3057;&#x3066;&#x3044;&#x308B;&#x4E00;&#x65B9;&#x3001;in vivo &#x3067;&#x306E;&#x6211;&#x3005; &#x306E;&#x7406;&#x89E3;&#x306F;&#x9650;&#x3089;&#x308C;&#x305F;&#x307E;&#x307E;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x6B20;&#x70B9;&#x3092;&#x89E3;&#x6C7A;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5149;&#x5B66;&#x7684;&#x30A2;&#x30AF;&#x30BB;&#x30B9;&#x306E;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x306E;&#x751F;&#x4F53;&#x5185;&#x306B;&#x304A;&#x3051;&#x308B;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x52D5;&#x614B;&#x3092;&#x7814;&#x7A76;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x30C4;&#x30FC;&#x30EB;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x3053;&#x3053;&#x3067;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF;&quot;MitoFish&#x3001;&quot;&#x3092;&#x542B;&#x3080;&#x3001;&#x79C1;&#x305F;&#x3061;&#x306E;&#x65B0;&#x3057;&#x304F;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x30C4;&#x30FC;&#x30EB;&#x304C;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x3067; in vivo &#x3067;&#x306E;&#x300C;&#x751F;&#x6D3B;&#x306E;&#x30B5;&#x30A4;&#x30AF;&#x30EB;&#x300D;&#x3092;&#x52C9;&#x5F37;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x3001;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x52D5;&#x614B;&#x306E;&#x85AC;&#x7406;&#x5B66;&#x7684;&#x304A;&#x3088;&#x3073;&#x907A;&#x4F1D;&#x7684;&#x5909;&#x8ABF;&#x5668;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x6DF1;&#x9060;&#x306A;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x8F38;&#x9001;&#x8D64;&#x5B57;&#x30BC;&#x30D6;&#x30E9;&#x30D5;&#x30A3;&#x30C3;&#x30B7;&#x30E5;&#x306E; tauopathy &#x30E2;&#x30C7;&#x30EB;&#x306E;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x3067;&#x89B3;&#x5BDF;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x306B;&#x3088;&#x3063;&#x3066; MARK2 &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3053;&#x306E;&#x8868;&#x73FE;&#x578B;&#x3092;&#x6551;&#x51FA; (&#x5FAE;&#x5C0F;&#x7BA1;&#x89AA;&#x548C;&#x6027;&#x8ABF;&#x7BC0;&#x30AD;&#x30CA;&#x30FC;&#x30BC; 2) &#x3053;&#x306E;&#x30AD;&#x30CA;&#x30FC;&#x30BC; &#x30BF;&#x30A6;&#x4F9D;&#x5B58;&#x7684;&#x8EF8;&#x7D22;&#x8F38;&#x9001;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3053;&#x3068; in vivo &#x3067;&#x306E;&#x76F4;&#x63A5;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005; &#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x6B63;&#x5E38;&#x3068;&#x75C5;&#x6C17;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x81EA;&#x7136;&#x306E;&#x74B0;&#x5883;&#x306B;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x52D5;&#x614B;&#x306E;&#x8A73;&#x7D30;&#x306A;&#x7814;&#x7A76;&#x3092;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;&#x30E2;&#x30C7;&#x30EB; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x95A2;&#x9023;&#x306E;&#x75C5;&#x6C17;&#x306B;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x30C0;&#x30A4;&#x30CA;&#x30DF;&#x30AF;&#x30B9;&#x306E; in vivo &#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3002;

&#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; ATP&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xCE7C;&#xC298; &#xD56D;&#xC0C1;&#xC131; &#xC720;&#xC9C0; apoptosis&#xB97C; &#xC870;&#xC808; &#xD569;&#xB2C8;&#xB2E4;. &#xB274;&#xB7F0;&#xC758; &#xD06C;&#xAE30;&#xC640; &#xBCF5;&#xC7A1; &#xD55C; &#xD615;&#xC0C1;&#xC73C;&#xB85C; &#xC778;&#xD574; &#xD2B9;&#xD788; &#xC801;&#xC808; &#xD55C; &#xC791;&#xB3D9; &#xBC0F; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC758; &#xBD84;&#xD3EC;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xB294;. &#xB530;&#xB77C;&#xC11C; &#xC774;&#xB7EC;&#xD55C; organelles &#xBC0F; &#xADF8;&#xB4E4;&#xC758; &#xC804;&#xC1A1;&#xC758; &#xC911;&#xB2E8; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC2E0;&#xACBD; &#xD1F4;&#xD589; &#xC131; &#xC9C8;&#xD658; &#xC911;&#xC2EC; &#xC5ED;&#xD560;&#xC744; &#xB2F4;&#xB2F9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCCB4; &#xC678; &#xC5F0;&#xAD6C; &#xB300;&#xB2E8;&#xD788; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC5ED;&#xD559;&#xC758; &#xC6B0;&#xB9AC;&#xC758; &#xC9C0;&#xC2DD;&#xC744; &#xD655;&#xB300;, &#xC6B0;&#xB9AC;&#xC758; &#xC774;&#xD574;&#xB97C; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xC81C;&#xD55C; &#xB0A8;&#xC544; &#xC788;&#xB2E4;. &#xC774; &#xB2E8;&#xC810;&#xC744; &#xD574;&#xACB0; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC0DD;&#xCCB4; &#xAD11;&#xD559; &#xC561;&#xC138;&#xC2A4;&#xD560; &#xC218; zebrafish&#xC758; &#xC5ED;&#xD559;&#xC744; &#xACF5;&#xBD80; &#xD558;&#xB294; &#xB3C4;&#xAD6C;&#xB97C; &#xAC1C;&#xBC1C; &#xD588;&#xB2E4;. &#xC5EC;&#xAE30; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &quot;MitoFish&quot;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC;&#xC758; &#xC0C8;&#xB85C; &#xC0DD;&#xC131; &#xB41C; &#xB3C4;&#xAD6C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC758; in vivo &quot;&#xB77C;&#xC774;&#xD504; &#xC0AC;&#xC774;&#xD074;&quot; &#xACF5;&#xBD80;&#xB97C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC73C;&#xBA70; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC5ED;&#xD559;&#xC758; &#xC57D;&#xB9AC;&#xD559; &#xBC0F; &#xC720;&#xC804; &#xBCC0;&#xC870;&#xAE30;&#xB97C; &#xC2DD;&#xBCC4; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C; &#xAE4A;&#xC740; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC804;&#xC1A1; &#xC801;&#xC790; zebrafish tauopathy &#xBAA8;&#xB378;&#xC5D0; &#xC2E4;&#xC2DC;&#xAC04;&#xC73C;&#xB85C;&#xC5D0;&#xC11C; &#xAD00;&#xCC30;&#xD569;&#xB2C8;&#xB2E4;. MARK2 &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;&#xC774; &#xD615; &#xAD6C;&#xC870;&#xC5D0; &#xC758;&#xD574; (microtubule &#xC120;&#xD638;&#xB3C4; &#xC870;&#xC808; &#xD0A4; 2), &#xC6B0;&#xB9AC;&#xC774; &#xB2C8; &#xD0C0;&#xC6B0; &#xC885;&#xC18D; &#xBC29;&#xC2DD;&#xC5D0;&#xC11C; axonal &#xC804;&#xC1A1; &#xADDC;&#xC81C; &#xC9C1;&#xC811; vivo&#xC5D0;&#xC11C; &#xC99D;&#xBA85; &#xC815;&#xBCF4;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xC6B0;&#xB9AC;&#xC758; &#xC811;&#xADFC; &#xC815;&#xC0C1;&#xACFC; &#xC9C8;&#xBCD1; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; &#xC790;&#xC5F0; &#xD658;&#xACBD;&#xC5D0;&#xC11C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC758; &#xC5ED;&#xD559;&#xC758; &#xC0C1;&#xC138;&#xD55C; &#xC5F0;&#xAD6C;&#xB97C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xCC99;&#xCD94; &#xB3D9;&#xBB3C; &#xBAA8;&#xB378; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0;&#xC11C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC5ED;&#xD559; &#xC9C8;&#xBCD1; &#xAD00;&#xB828;&#xC758; in vivo &#xC601;&#xC0C1;.

Las mitocondrias proporcionan ATP, mantienen la homeostasis del calcio y regulan la apoptosis. Las neuronas, por su tama&ntilde;o y geometr&iacute;a compleja, son particularmente dependientes sobre el correcto funcionamiento y distribuci&oacute;n de las mitocondrias. As&iacute; interrupciones de estos org&aacute;nulos y su transporte juegan un papel central en una amplia gama de enfermedades neurodegenerativas. Mientras que los estudios in vitro han ampliado enormemente nuestro conocimiento de la din&aacute;mica mitocondrial, nuestro entendimiento en vivo sigue siendo limitada. Para hacer frente a esta carencia, hemos desarrollado herramientas para estudiar la din&aacute;mica mitocondrial en vivo en el pez cebra &oacute;pticamente accesible. Aqu&iacute; demostramos que nuestras herramientas reci&eacute;n generados, incluyendo transg&eacute;nico &quot;MitoFish&quot;, se puede utilizar para estudiar el &quot;ciclo in vivo de la vida&quot; de mitocondrias y permite identificar moduladores farmacol&oacute;gicos y gen&eacute;ticos de la din&aacute;mica mitocondrial. Adem&aacute;s observamos d&eacute;ficit profundo transporte mitocondrial en tiempo real en un modelo de tauopathy de pez cebra. Por rescatar este fenotipo usando MARK2 (regulaci&oacute;n quinasa 2 de microt&uacute;bulos afinidad), Proporcionamos evidencia in vivo directa que esta quinasa regula el transporte axonal en una forma dependiente de Tau. Por lo tanto, nuestro enfoque permite estudios detallados de la din&aacute;mica de las mitocondrias en su ambiente natural en condiciones normales y enfermedad.

Imagen in vivo de la relacionada con la enfermedad mitocondrial din&aacute;mica en un sistema modelo de vertebrados.

Visualizing neuronal mitochondria in a living, intact mammalian organism is a challenge that can be overcome in zebrafish larvae, which are highly accessible for optical imaging and genetic manipulation. Here, we detail an approach to visualize neuronal mitochondria in sensory Rohon-Beard axons, which allows quantitatively measuring mitochondrial shape, dynamics, and transport in vivo. This provides a useful assay for basic studies exploring the behavior of neuronal mitochondria in their natural habitat, for revealing the influence that disease-related alterations have on this behavior and for testing pharmacological compounds and genetic manipulations that might ameliorate disease-related mitochondrial phenotypes in neurons.

In vivo imaging of mitochondria in intact zebrafish larvae.

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2-10 days) in wt mice and 8 days (range 2-16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1-3.4; cD2 KO: 0.57, range 0.1-2.0 seizures/day) or median seizure duration (wt: 51 s, range 23-103; cD2 KO: 51 s, range 23-103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state.

Article	Year
Status epilepticus evokes prolonged increase in the expression of CCL3 and CCL4 mRNA and protein in the rat brain. Acta neurobiologiae experimentalis\| PubMed ID: 21731074	2011
Post-treatment with rapamycin does not prevent epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy. Neuroscience letters\| PubMed ID: 22227620	2012
In vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23152604	2012
In vivo imaging of mitochondria in intact zebrafish larvae. Methods in enzymology\| PubMed ID: 25416357	2014
Epileptogenesis following Kainic Acid-Induced Status Epilepticus in Cyclin D2 Knock-Out Mice with Diminished Adult Neurogenesis. PloS one\| PubMed ID: 26020770	2015

Gabriela Plucinska

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