local-application-drugs-to-study-nicotinic-acetylcholine-receptor

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

artificial-intelligence-approaches-to-assessing-primary-cilia

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Purdue University

Indiana University-Purdue University Indianapolis

&#x70DF;&#x78B1;&#x578B;&#x4E59;&#x9170;&#x80C6;&#x78B1;&#x53D7;&#x4F53; (nAChRs) &#x5305;&#x542B; &alpha;6 &#x4E9A;&#x57FA;&#x8868;&#x793A;&#x53EA;&#x6709;&#x51E0;&#x4E2A;&#x8111;&#x533A;&#xFF0C;&#x5305;&#x62EC;&#x4E2D;&#x8111;&#x591A;&#x5DF4;&#x80FA; (DA) &#x795E;&#x7ECF;&#x5143;&#x3001; &#x84DD;&#x6591;&#x548C;&#x89C6;&#x7F51;&#x819C;&#x795E;&#x7ECF;&#x8282;&#x7EC6;&#x80DE;&#x7684;&#x53BB;&#x7532;&#x80BE;&#x4E0A;&#x817A;&#x7D20;&#x80FD;&#x795E;&#x7ECF;&#x5143;&#x3002;&#x82E5;&#x8981;&#x66F4;&#x597D;&#x5730;&#x4E86;&#x89E3;&#x533A;&#x57DF;&#x548C;&#x4E9A;&#x7EC6;&#x80DE;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x7684; &alpha;6 &#x5305;&#x542B; nAChRs&#xFF0C;&#x6211;&#x4EEC;&#x521B;&#x5EFA;&#x548C;&#x7814;&#x7A76;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x53D8;&#x578B; &alpha;6 &#x4E9A;&#x57FA;&#x4E0E;&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D; (GFP) &#x878D;&#x5408;&#x5728;&#x5E27; M3 M4 &#x80DE;&#x5185;&#x5FAA;&#x73AF;&#x4E2D;&#x3002;&Alpha;6-&#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#xFF0C;&#x4F9D;&#x8D56;&#x4E8E; &alpha;6 &#x7684; synaptosomal DA &#x91CA;&#x653E;&#x548C;&#x653E;&#x5C04;&#x914D;&#x4F53;&#x7ED3;&#x5408;&#x5B9E;&#x9A8C;&#x8BC1;&#x5B9E;&#x6B63;&#x786E;&#x7684;&#x8868;&#x8FBE;&#x5F0F;&#x548C;&#x51FD;&#x6570;&#x4F53;&#x5185;&#x3002;&#x9664;&#x4E86;&#x5F3A; &alpha;6 * nAChR &#x8868;&#x8FBE;&#x5F0F;&#x4E2D;&#x8C37;&#x6C28;&#x9178;&#x80FD;&#x89C6;&#x7F51;&#x819C;&#x795E;&#x7ECF;&#x8F74;&#x7A81;&#x8868;&#x9762;&#x4E0A;&#x4E18; (sSC) &#x7EC8;&#x6B62;&#xFF0C;&#x6211;&#x4EEC;&#x8FD8;&#x53D1;&#x73B0; &alpha;6 &#x4E9A;&#x57FA;&#x8868;&#x8FBE;&#x5B89;&#x57FA;&#x7EC6;&#x80DE;&#x673A;&#x6784;&#x7684;&#x4E00;&#x4E2A;&#x5B50;&#x96C6;&#x5728;&#x8FD9;&#x4E2A;&#x8111;&#x533A;&#x3002;&#x4ECE; sSC &#x795E;&#x7ECF;&#x5143;&#x4ECE;&#x8868;&#x8FBE;&#x8D85;&#x654F; &alpha;6 * nAChRs &#x5C0F;&#x9F20;&#x8111;&#x7247;&#x819C;&#x7247;&#x94B3;&#x5F55;&#x5236;&#xFF0C;&#x5728;&#x6211;&#x4EEC;&#x786E;&#x8BA4;&#x529F;&#x80FD;&#x3001; &#x7A81;&#x89E6;&#x540E; &alpha;6 * nAChR &#x8868;&#x8FBE;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#xFF0C;sSC &#x5B89;&#x57FA;&#x795E;&#x7ECF;&#x5143;&#x8868;&#x8FBE; &alpha;6 * nAChRs &#x8868;&#x73B0;&#x51FA;&#x8FC7;&#x654F; &alpha;6 * &#x7531; ACh nAChRs &#x5E38;&#x8BBE;&#x6FC0;&#x6D3B;&#x4ECB;&#x5BFC;&#x7684;&#x8865;&#x54C1;&#x7535;&#x5BFC;&#x3002;&Alpha;6 * nAChRs &#x4E5F;&#x51FA;&#x73B0;&#x5728;&#x4E9A;&#x7FA4;&#x7684; SC &#x795E;&#x7ECF;&#x5143;&#x7684;&#x8F93;&#x51FA;&#x56FE;&#x5C42;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x9009;&#x62E9;&#x6027;&#x6FC0;&#x6D3B;&#x7684; &alpha;6 * nAChRs &#x4F53;&#x5185;&#x8BF1;&#x5BFC; sSC &#x795E;&#x7ECF;&#x5143;&#x6FC0;&#x6D3B;&#x6240;&#x6D4B;&#x91CF; c Fos &#x7684;&#x8868;&#x8FBE;&#x3002;&#x5171;&#x540C;&#xFF0C;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E; &alpha;6 * nAChRs &#x552F;&#x4E00;&#x4F4D;&#x4E8E;&#x6765;&#x8C03;&#x89E3;&#x7684;&#x8C37;&#x6C28;&#x9178;&#x548C; GABA &#x91CA;&#x653E;&#x7279;&#x6B8A;&#x7684;&#x80C6;&#x78B1;&#x80FD;&#x8C03;&#x5236;&#x4F9B;&#x5E94;&#x94FE;&#x5DF2;&#x6210;&#x4E3A;&#x6F5C;&#x5728;&#x7684;&#x5927;&#x8111;&#x5173;&#x952E;&#x9886;&#x57DF;&#x8D1F;&#x8D23;&#x8F6C;&#x9012;&#x77ED;&#x6F5C;&#x4F0F;&#x671F;&#x7A81;&#x51FA;&#x4FE1;&#x53F7;&#x5411;&#x4E18;&#x8111;&#x548C;&#x4E2D;&#x8111; DA &#x795E;&#x7ECF;&#x5143;&#xFF0C;&#x548C;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E; &alpha;6 * nAChRs &#x53EF;&#x80FD;&#x5BF9;&#x70DF;&#x78B1;&#x578B;&#x80C6;&#x78B1;&#x80FD;&#x5BA3;&#x4F20;&#x6B64;&#x9014;&#x5F84;&#x7684;&#x91CD;&#x8981;&#x3002;

&Alpha;6 * &#x70DF;&#x78B1;&#x578B;&#x4E59;&#x9170;&#x80C6;&#x78B1;&#x53D7;&#x4F53;&#x7684;&#x8868;&#x8FBE;&#x548C;&#x529F;&#x80FD;&#x7684;&#x4E00;&#x79CD;&#x89C6;&#x89C9;&#x7A81;&#x51FA;&#x7535;&#x8DEF;&#x3002;

Nicotinic acetylcholine receptors (nAChRs) containing Î±6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of Î±6-containing nAChRs, we created and studied transgenic mice expressing a variant Î±6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In Î±6-GFP transgenic mice, Î±6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong Î±6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found Î±6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive Î±6* nAChRs, we confirmed functional, postsynaptic Î±6* nAChR expression. Further, sSC GABAergic neurons expressing Î±6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive Î±6* nAChRs by ACh. Î±6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of Î±6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that Î±6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that Î±6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.

Î±6* nicotinic acetylcholine receptor expression and function in a visual salience circuit.

R&eacute;cepteurs de l&#39;ac&eacute;tylcholine nicotinique (nAChR) contenant les sous-unit&eacute;s &alpha;6 sont exprim&eacute;s dans seulement quelques r&eacute;gions du cerveau, y compris des neurones de dopamine (DA) m&eacute;senc&eacute;phale, les neurones noradr&eacute;nergiques du locus coeruleus et cellules ganglionnaires r&eacute;tiniennes. Pour mieux comprendre le mod&egrave;le d&#39;expression r&eacute;gionale et subcellulaire de nAChR contenant &alpha;6, nous avons cr&eacute;&eacute; et &eacute;tudi&eacute; des souris transg&eacute;niques exprimant un variante &alpha;6 sous-unit&eacute; avec la prot&eacute;ine fluorescente verte (GFP) fondu dans le cadre dans la boucle intracellulaire M3-M4. Chez les souris transg&eacute;niques &alpha;6-GFP, la lib&eacute;ration de DA &alpha;6-d&eacute;pendante et exp&eacute;riences de liaison des radioligands a confirm&eacute; l&#39;expression correcte et fonction in vivo. En plus de la CNCDH expression forte &alpha;6 * dans les axones r&eacute;tinienne glutamatergiques, qui se terminent par superficiel colliculus sup&eacute;rieur (sSC), nous avons &eacute;galement constat&eacute; des expression sous-unit&eacute; &alpha;6 dans un sous-ensemble de corps cellulaires GABAergique dans cette r&eacute;gion du cerveau. Dans les enregistrements de patch-clamp de sSC de neurones dans des tranches de cerveau de souris exprimant une hypersensibilit&eacute; &alpha;6 * nAChR, nous avons confirm&eacute; la CNCDH expression fonctionnelle, postsynaptique &alpha;6 *. De plus, les neurones GABAergiques sSC exprimant &alpha;6 * nAChR pi&egrave;ce une conductance tonique m&eacute;di&eacute;e par l&#39;activation permanente de nAChR hypersensible &alpha;6 * ach. NAChR &Alpha;6 * appara&icirc;tre &eacute;galement dans une sous-population de neurones SC dans les couches de sortie. Enfin, l&#39;activation s&eacute;lective de &alpha;6 * nAChR in vivo induite sSC activation neuronale mesur&eacute;e avec l&#39;expression de c-Fos. Ensemble, ces r&eacute;sultats d&eacute;montrent que nAChR &alpha;6 * est particuli&egrave;rement bien plac&eacute;es pour m&eacute;diation cholinergique modulation du glutamate et de la lib&eacute;ration de GABA dans SC. Le SC est apparue comme une zone potentielle de cl&eacute;s du cerveau responsable de la saillance de courte latence de transmission des signaux au thalamus et m&eacute;senc&eacute;phale neurones dopaminergiques, et ces r&eacute;sultats sugg&egrave;rent que nAChR &alpha;6 * peut &ecirc;tre important pour la sensibilisation cholinergique nicotinique de cette voie.

&Alpha;6 * l&#39;expression des r&eacute;cepteurs nicotiniques de l&#39;ac&eacute;tylcholine et la fonction dans un circuit de pr&eacute;gnance visuelle.

Nikotinische Acetylcholin-Rezeptoren (nAChRs), enth&auml;lt &alpha;6 Untereinheiten sind in nur wenigen Hirnareale, einschlie&szlig;lich Mittelhirn Dopamin (DA) Neuronen, noradrenergic Neuronen des Locus Ceruleus und retinalen Ganglienzellen ausgedr&uuml;ckt. Zum besseren Verst&auml;ndnis der regionalen und subzellulare Ausdrucksmuster der &alpha;6-haltigen nAChRs wir erstellt und studierte transgene M&auml;use einen variant &alpha;6 Untereinheit mit gr&uuml;n fluoreszierendes Protein (GFP) verschmolzen in Frame in der M3-M4 intrazellul&auml;ren Schleife zum Ausdruck zu bringen. &Alpha;6-GFP transgene M&auml;use best&auml;tigt &alpha;6-abh&auml;ngige synaptosomal DA Release und Radioligand Bindung Experimente korrekten Ausdruck und in-vivo-Funktion. Neben starken &alpha;6 * nAChR Ausdruck in Glutamat retinale Axone, wodurch in oberfl&auml;chliche superior Colliculus (sSC) beendet, fanden wir auch &alpha;6 Untereinheit Ausdruck in einer Teilmenge des GABAergic Zelle K&ouml;rper in diesem Bereich des Gehirns. In Patch-Clamp-Aufnahmen vom sSC Neuronen im Gehirn Scheiben von M&auml;usen, die &uuml;berempfindlich &alpha;6 * nAChRs zum Ausdruck zu bringen haben wir funktionelle, postsynaptisches &alpha;6 * nAChR Ausdruck bekr&auml;ftigt. Dar&uuml;ber hinaus zeigen sSC GABAergic Neuronen ausdr&uuml;ckt &alpha;6 * nAChRs ein Tonikum Leitwert von st&auml;ndigen Aktivierung von &uuml;berempfindlichen &alpha;6 * nAChRs von ACh vermittelt. &Alpha;6 * nAChRs erscheinen auch in einer Subpopulation von SC-Neuronen im Ausgang Schichten. Selektive Aktivierung &alpha;6 * nAChRs in-vivo induziert schlie&szlig;lich sSC neuronalen Aktivierung mit c-Fos Expression gemessen. Zusammen zeigen diese Ergebnisse, dass &alpha;6 * nAChRs einzigartig gelegen sind, vermitteln cholinerge Modulation von Glutamat und GABA-Freisetzung in SC. Der SC ist als ein m&ouml;gliche wichtige Gehirn-Bereich verantwortlich f&uuml;r die &Uuml;bertragung von kurzen Latenz hervortreten zum Thalamus und Mittelhirn DA Neuronen Signale, und diese Ergebnisse legen nahe, dass &alpha;6 * nAChRs m&ouml;glicherweise wichtig f&uuml;r nikotinische cholinerge Sensibilisierung von diesem Weg hervorgegangen.

&Alpha;6 * nikotinische Acetylcholin-Rezeptor-Ausdruck und Funktion in einem Stromkreis visuelle hervortreten.

I recettori nicotinici acetilcolina (nAChRs) contenenti le subunit&agrave; &alpha;6 sono espressi in solo alcune aree del cervello, tra cui i neuroni della dopamina (DA) mesencefalo, noradrenergico neuroni del locus ceruleus e cellule gangliari della retina. Per meglio comprendere il modello di espressione regionale e sottocellulare di nAChRs contenenti &alpha;6, abbiamo creato e studiato topi transgenici che esprimono una variante &alpha;6 subunit&agrave; con proteina fluorescente verde (GFP) fuse in-frame in loop intracellulare M3-M4. Nei topi transgenici &alpha;6-GFP, rilascio DA synaptosomal &alpha;6-dipendente ed esperimenti di binding radioattivo confermarono espressione corretta e la funzione in vivo. Oltre alla forte &alpha;6 * nAChR espressione in assoni retinici glutamatergico, che terminano nel tubercolo superiore superficiale (sSC), abbiamo anche trovato un espressione subunit&agrave; &alpha;6 in un sottoinsieme dei corpi cellulari GABAergico in questa area del cervello. Nelle registrazioni di patch-clamp da sSC neuroni in fettine di cervello di topi che esprimono ipersensibili &alpha;6 * nAChRs, abbiamo confermato nAChR espressione funzionale, postsinaptici &alpha;6 *. Ulteriormente, sSC GABAergico neuroni che esprimono &alpha;6 * nAChRs esibiscono una conduttanza tonico mediata dall&#39;attivazione permanente di nAChRs ipersensibili &alpha;6 * di ACh. &Alpha;6 * nAChRs appaiono anche in una sottopopolazione di neuroni SC in livelli di output. Infine, attivazione selettiva di &alpha;6 * nAChRs in vivo indotta da attivazione neuronale sSC come misurata con espressione di c-Fos. Insieme, questi risultati dimostrano che &alpha;6 * nAChRs sono situati in modo univoco per mediare modulazione colinergica di glutammato e GABA release in SC. La SC &egrave; emersa come una potenziale zona chiave del cervello responsabile della trasmissione breve latenza salienza segnali al talamo e mesencefalo DA neuroni, e questi risultati suggeriscono che &alpha;6 * nAChRs pu&ograve; essere importante per la sensibilizzazione colinergica nicotina di questo percorso.

&Alpha;6 * espressione dei recettori nicotinici acetilcolina e funzione in un circuito di rilevanza visiva.

&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30B3;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53; &alpha;6 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3092;&#x542B;&#x3080; (nAChRs) &#x306E;&#x307F;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x8133;&#x9818;&#x57DF;&#x306E;&#x4E2D;&#x8133;&#x30C9;&#x30FC;&#x30D1;&#x30DF;&#x30F3; (DA) &#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3001;&#x795E;&#x7D4C;, &#x3068;&#x7DB2;&#x819C;&#x795E;&#x7D4C;&#x7BC0;&#x7D30;&#x80DE;&#x306E;&#x30CE;&#x30EB;&#x30A2;&#x30C9;&#x30EC;&#x30CA;&#x30EA;&#x30F3;&#x4F5C;&#x52D5;&#x6027;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3092;&#x542B;&#x3080;&#x3067;&#x8868;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x826F;&#x3044; &alpha;6 &#x3092;&#x542B;&#x3080; nAChRs &#x306E;&#x5730;&#x57DF;&#x304A;&#x3088;&#x3073;&#x7D30;&#x80DE;&#x30EC;&#x30D9;&#x30EB;&#x4E0B;&#x306E;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x4F5C;&#x6210;&#x3001;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8; &alpha;6 &#x7DD1;&#x8272;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; (GFP) &#x3092;&#x878D;&#x5408;&#x3057;&#x305F;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3067;-&#x30D5;&#x30EC;&#x30FC;&#x30E0;&#x306B; M3 M4 &#x7D30;&#x80DE;&#x5185;&#x30EB;&#x30FC;&#x30D7;&#x3059;&#x308B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x3092;&#x52C9;&#x5F37;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&Alpha;6 GFP &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x3067;&#x306F;&#x3001;&#x6B63;&#x3057;&#x3044;&#x5F0F;&#x3068;&#x95A2;&#x6570;&#x306E; in vivo &alpha;6 &#x4F9D;&#x5B58;&#x30B7;&#x30CA;&#x30D7;&#x30C8;&#x30BD;&#x30FC;&#x30E0; DA &#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3068;&#x653E;&#x5C04;&#x6027;&#x30EA;&#x30AC;&#x30F3;&#x30C9;&#x306E;&#x7D50;&#x5408;&#x5B9F;&#x9A13;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x5F37;&#x3044; &alpha;6 &#x203B; nAChR &#x5F0F;&#x6D45;&#x4E18; (sSC) &#x3092;&#x7D42;&#x4E86;&#x3001;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x4F5C;&#x52D5;&#x6027;&#x7DB2;&#x819C;&#x8EF8;&#x7D22;&#x3067;&#x306B;&#x52A0;&#x3048;&#x3066;&#x6211;&#x3005; &#x3082; &alpha;6 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x5F0F; gaba &#x4F5C;&#x52D5;&#x6027;&#x7D30;&#x80DE;&#x4F53;&#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x306B;&#x3053;&#x306E;&#x8133;&#x9818;&#x57DF;&#x3067;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002;SSC &#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x904E;&#x654F; &alpha;6 &#x25CB; nAChRs &#x767A;&#x73FE;&#x30DE;&#x30A6;&#x30B9;&#x304B;&#x3089;&#x8133;&#x30B9;&#x30E9;&#x30A4;&#x30B9;&#x304B;&#x3089;&#x30D1;&#x30C3;&#x30C1;&#x30AF; &#x30E9;&#x30F3;&#x30D7;&#x8A18;&#x9332;&#x3067;&#x3001;&#x6A5F;&#x80FD;&#x6027;&#x3001;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5F8C; &alpha;6 &#x203B; nAChR &#x5F0F;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&alpha;6 &#x25CB; nAChRs &#x3092;&#x8868;&#x73FE;&#x3059;&#x308B; sSC Gaba &#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x904E;&#x654F; &alpha;6 &#x25CB; ACh &#x306B;&#x3088;&#x308B; nAChRs &#x306E;&#x7ACB;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x30C8;&#x30CB;&#x30C3;&#x30AF; &#x30B3;&#x30F3;&#x30C0;&#x30AF;&#x30BF;&#x30F3;&#x30B9;&#x3092;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&Alpha;6 &#x203B; nAChRs &#x306F;&#x3001;SC &#x51FA;&#x529B;&#x5C64;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x4E9C;&#x96C6;&#x56E3;&#x306B;&#x3082;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&alpha;6 &#x25CB; nAChRs in vivo &#x3067;&#x306E;&#x9078;&#x629E;&#x7684;&#x6D3B;&#x6027;&#x5316;&#x8A98;&#x8D77; sSC &#x795E;&#x7D4C;&#x6D3B;&#x6027;&#x3068;&#x3057;&#x3066; C-fos &#x767A;&#x73FE;&#x3092;&#x6E2C;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x304B;&#x3089; &alpha;6 &#x25CB; nAChRs &#x304C;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x304A;&#x3088;&#x3073; GABA &#x653E;&#x51FA; sc &#x30B3;&#x30EA;&#x30F3;&#x4F5C;&#x52D5;&#x6027;&#x5909;&#x8ABF;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x4E00;&#x610F;&#x306B;&#x4F4D;&#x7F6E;&#x3057;&#x3066; &#x3053;&#x3068;&#x3092;&#x793A;&#x3059;SC &#x306F;&#x3001;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30AD;&#x30FC;&#x3092;&#x8133;&#x306E;&#x30A8;&#x30EA;&#x30A2;&#x3092;&#x77ED;&#x6F5C;&#x6642;&#x9855;&#x73FE;&#x9001;&#x4FE1;&#x3059;&#x308B;&#x8CAC;&#x4EFB;&#x8996;&#x5E8A;&#x3068;&#x4E2D;&#x8133;&#x306B;&#x4FE1;&#x53F7;&#x3092; DA &#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3068; &alpha;6 &#x25CB; nAChRs &#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30B3;&#x30EA;&#x30F3;&#x4F5C;&#x52D5;&#x6027;&#x611F;&#x4F5C;&#x3053;&#x306E;&#x7D4C;&#x8DEF;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3068;&#x3057;&#x3066;&#x6D6E;&#x4E0A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&Alpha;6 &#x203B; &#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30B3;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x767A;&#x73FE;&#x3068;&#x8996;&#x899A;&#x7684;&#x306A;&#x9855;&#x73FE;&#x56DE;&#x8DEF;&#x5185;&#x306E;&#x95A2;&#x6570;&#x3002;

Nicotinic &#xC544; &#xC138; &#xD2F8; &#xCF5C;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4; (nAChRs) &alpha;6 &#xD558;&#xC704; &#xB2E8;&#xC704;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xB2E8; &#xBA87; &#xB1CC; &#xC601;&#xC5ED;, midbrain &#xB3C4;&#xD30C;&#xBBFC; (DA) &#xB274;&#xB7F0;, &#xB85C;&#xCEE4; &#xC2A4; ceruleus &#xB9DD;&#xB9C9; &#xC2E0;&#xACBD; &#xC808; &#xC138;&#xD3EC;&#xC758; noradrenergic &#xB274;&#xB7F0;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4;. &#xB354; &#xD3EC;&#xD568; &#xD558;&#xB294; &alpha;6 Nachrs&#xC758; &#xC9C0;&#xC5ED; &#xBC0F; subcellular &#xC2DD; &#xD328;&#xD134;&#xC744; &#xD30C;&#xC545;, &#xC6B0;&#xB9AC;&#xB294; &#xB9CC;&#xB4E4;&#xACE0; &#xBCC0;&#xD615; &alpha;6 &#xC735;&#xD569; &#xB179;&#xC0C9; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8; (GFP)&#xC640; &#xC18C; &#xB2E8;&#xC704;&#xC5D0;-&#xD504;&#xB808;&#xC784; M3 M4 &#xC138;&#xD3EC;&#xB0B4; &#xB8E8;&#xD504;&#xC5D0;&#xC11C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xB97C; &#xACF5;&#xBD80; &#xD588;&#xB2E4;. &Alpha;6 GFP &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xC5D0; &alpha;6 &#xC885;&#xC18D; synaptosomal DA &#xCD9C;&#xC2DC; &#xBC0F; radioligand &#xBC14;&#xC778;&#xB529; &#xC2E4;&#xD5D8; &#xC62C;&#xBC14;&#xB978; &#xD45C;&#xD604;&#xACFC; in vivo &#xAE30;&#xB2A5; &#xD655;&#xC778;. &#xD53C;&#xC0C1;&#xC801;&#xC778; &#xC6B0;&#xC218;&#xD55C; colliculus (sSC)&#xC5D0;&#xC11C; &#xC885;&#xB8CC; glutamatergic &#xB9DD;&#xB9C9; &#xCD95; &#xC0AD;&#xC5D0; &#xAC15;&#xD55C; &alpha;6 * nAChR &#xC2DD; &#xC774;&#xC678;&#xC5D0; &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xBC1C;&#xACAC; &alpha;6 &#xC18C; &#xB2E8;&#xC704; &#xC2DD; GABAergic &#xC140; &#xBC14;&#xB514;&#xC758; &#xBD80;&#xBD84; &#xC9D1;&#xD569;&#xC5D0;&#xC774; &#xB1CC; &#xC601;&#xC5ED;&#xC5D0;&#xC11C;. SSC &#xC2E0;&#xACBD; &#xACFC;&#xBBFC; &alpha;6 * nAChRs &#xD45C;&#xD604; &#xC950;&#xC5D0;&#xC11C; &#xB1CC; &#xC870;&#xAC01;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504; &#xB179;&#xC74C; &#xAE30;&#xB2A5;, postsynaptic &alpha;6 * nAChR &#xC2DD;&#xC744; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xB610;&#xD55C;, &alpha;6 * nAChRs &#xD45C;&#xD604; sSC GABAergic &#xC2E0;&#xACBD;&#xC758; &#xACFC;&#xBBFC; &alpha;6 * nAChRs Ach&#xC5D0; &#xC758;&#xD574; &#xC11C; &#xD65C;&#xC131;&#xD654; &#xD558; &#xC5EC; &#xC911;&#xC7AC; &#xD1A0; &#xB2C9; &#xC804;&#xB3C4;&#xB3C4; &#xC804;&#xC2DC;. &Alpha;6 * nAChRs SC &#xCD9C;&#xB825; &#xCE35; &#xB274;&#xB7F0; subpopulation&#xC5D0;&#xB3C4; &#xB098;&#xD0C0;&#xB0A9;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &alpha;6 * nAChRs vivo&#xC5D0;&#xC11C;&#xC758; &#xC120;&#xD0DD;&#xC801; &#xD65C;&#xC131;&#xD654; c-Fos &#xC2DD; &#xCE21;&#xC815;&#xC73C;&#xB85C; sSC &#xC2E0;&#xACBD; &#xD65C;&#xC131;&#xD654; &#xC720;&#xB3C4;. &#xD568;&#xAED8;, &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &alpha;6 * nAChRs &#xACE0;&#xC720; &#xC870;&#xBBF8;&#xB8CC;&#xC640; &#xCE90;&#xB864;&#xB77C;&#xC774;&#xB098;&#xC5D0;&#xC11C; GABA &#xB9B4;&#xB9AC;&#xC2A4; cholinergic &#xBCC0;&#xC870; &#xC911;&#xC7AC;&#xC5D0; &#xC704;&#xCE58; &#xD558; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC785;&#xC99D; &#xC0AC;&#xC6B0;&#xC2A4; &#xCE90;&#xB864;&#xB77C;&#xC774;&#xB098;&#xB294; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC8FC;&#xC694; &#xB450;&#xB1CC; &#xC601;&#xC5ED; &#xC804;&#xC1A1; &#xC9E7;&#xC740; &#xB300;&#xAE30; &#xC2DC;&#xAC04; &#xB3CC;&#xCD9C; &#xD558; &#xC2E0;&#xD638;&#xB97C; &#xC2DC;&#xC0C1; &#xBC0F; midbrain DA &#xB274;&#xB7F0;, &#xADF8;&#xB9AC;&#xACE0; &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &alpha;6 * Nachrs&#xC774; &#xACBD;&#xB85C;&#xC758; nicotinic cholinergic &#xBBFC;&#xAC10;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4; &#xB5A0;&#xC624;&#xB974;&#xACE0; &#xC788;&#xB2E4;.

&Alpha;6 * nicotinic &#xC544; &#xC138; &#xD2F8; &#xCF5C;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4; &#xC2DD; &#xACE0; &#xC2DC;&#xAC01;&#xC801; &#xB3CC;&#xCD9C; &#xD68C;&#xB85C;&#xC5D0; &#xD568;&#xC218;.

Los receptores nicot&iacute;nicos de la acetilcolina (nAChRs) que contienen subunidades &alpha;6 se expresan en s&oacute;lo unas pocas &aacute;reas del cerebro, incluyendo las neuronas de dopamina (DA) del midbrain, las neuronas noradren&eacute;rgicas del locus ceruleus y las c&eacute;lulas ganglionares de la retina. Para comprender mejor el patr&oacute;n de expresi&oacute;n regional y subcelular de nAChRs que contienen &alpha;6, creamos y estudiaron ratones transg&eacute;nicos que expresaban un marco &alpha;6 variante subunidad con prote&iacute;na fluorescente verde (GFP) fusionado en el lazo intracelular M3-M4. En ratones transg&eacute;nicos &alpha;6-GFP, &alpha;6-dependiente synaptosomal DA liberaci&oacute;n y experimentos de uni&oacute;n de radioligando confirman correcta expresi&oacute;n y funci&oacute;n in vivo. Adem&aacute;s de fuerte &alpha;6 * nAChR expresi&oacute;n en axones retinianos glutamat&eacute;rgica, que terminan en el col&iacute;culo superior superficial (sSC), tambi&eacute;n encontramos &alpha;6 subunidad expresi&oacute;n en un subconjunto de los cuerpos celulares de GABA&eacute;rgico en esta zona del cerebro. En grabaciones de patch-clamp de neuronas de sSC en rebanadas del cerebro de los ratones que expresaban hipersensible &alpha;6 * nAChRs, confirmamos funcional, postsin&aacute;pticos &alpha;6 * nAChR expresi&oacute;n. Adem&aacute;s, las neuronas GABA&eacute;rgicas de la sSC expresando &alpha;6 * nAChRs exhiben una conductancia de t&oacute;nica mediada por la activaci&oacute;n permanente de hipersensible &alpha;6 * nAChRs por ACh. &Alpha;6 * nAChRs tambi&eacute;n aparecen en una subpoblaci&oacute;n de neuronas SC en capas de salida. Finalmente, la activaci&oacute;n selectiva de &alpha;6 * nAChRs in vivo inducida por activaci&oacute;n neuronal sSC medida con expresi&oacute;n de c-Fos. Juntos, estos resultados demuestran que &alpha;6 * nAChRs &uacute;nicamente est&aacute;n situados para mediar la modulaci&oacute;n colin&eacute;rgica de glutamato y la liberaci&oacute;n de GABA en SC. La SC ha surgido como un &aacute;rea clave del cerebro potencial responsable de transmitir la prominencia de latencia corto se&ntilde;ales al t&aacute;lamo y mesenc&eacute;falo DA las neuronas, y estos resultados sugieren que &alpha;6 * nAChRs puede ser importante para la sensibilizaci&oacute;n colin&eacute;rgica nicot&iacute;nica de esta v&iacute;a.

&Alpha;6 * expresi&oacute;n de receptores nicot&iacute;nicos de la acetilcolina y la funci&oacute;n en un circuito de prominencia visual.

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address . Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity.

Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures.

An emerging number of rare genetic disorders termed ciliopathies are associated with pediatric obesity. It is becoming clear that the mechanisms associated with cilia dysfunction and obesity in these syndromes are complex. In addition to ciliopathic syndromic forms of obesity, several cilia-associated signaling gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis including their roles in centrally mediated food intake as well as in peripheral tissues, many questions remain. Here, we briefly discuss the syndromic ciliopathies and monoallelic cilia signaling gene mutations associated with obesity. We also describe potential ways cilia may be involved in common obesity. We discuss how neuronal cilia impact food intake potentially through leptin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We highlight several recent studies that have implicated the potential for cilia in peripheral tissues such as adipose and the pancreas to contribute to metabolic dysfunction. Then we discuss the potential for cilia to impact energy homeostasis through their roles in both development and adult tissue homeostasis. The studies discussed in this review highlight how a comprehensive understanding of the requirement of cilia for the regulation of diverse biological functions will contribute to our understanding of common forms of obesity.

Cilia signaling and obesity.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl Syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay, and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased preweaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility, and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly, and pituitary abnormalities are only present when Bbs5 is disrupted prior to P7 indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity independent of the age of Bbs5 loss.

Staci E. Engle

Department of Medicinal Chemistry and Molecular Pharmacology

Purdue University

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Artificial Intelligence Approaches to Assessing Primary Cilia

Staci E. Engle

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicinal Chemistry and Molecular Pharmacology

Purdue University

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Artificial Intelligence Approaches to Assessing Primary Cilia

Department of Medicinal Chemistry and Molecular Pharmacology