This work was supported by grants from the Pharmaceutical Research and Manufacturers Association of America (PhRMA) Foundation (to B. Gulbransen), National Institutes of Health (Building Interdisciplinary Research Careers in Women&#8217;s Health) grant K12 HD065879 (B. Gulbransen) and start-up funds from Michigan State University (B. Gulbransen).

NOTA: Le procedure seguenti che coinvolgono il tessuto da animali da laboratorio sono coerenti con le linee guida AVMA per l&#39;eutanasia degli animali 2013 e sono state approvate in anticipo dal Michigan State University IACUC. 1. Preparazione del tessuto <ol><li> Anestetizzare animali ricerca in una camera contenente 2,5% isoflurano in ossigeno o mettendo 3-5 ml di isoflurano liquido su un materiale assorbente sul pavimento della camera, in modo che una barriera fisica impedisce animali dal contatto diretto con l&#39;isoflurano. Test per la profondità dell&#39;anestesia pizzicando la zampa. NOTA: La profondità di anestesia è ritenuto appropriato quando non c&#39;è prelievo riflesso dell&#39;arto posteriore. Una volta adeguatamente anestetizzato, eutanasia il mouse dislocazione cervicale </li><li> Posto l&#39;animale in posizione supina e pulire la pelle addominale con etanolo al 70%. Utilizzare pinze per pizzicare la pelle addominale in linea mediana e usare le forbici chirurgiche per fare un supporto 6 centimetril incisione lungo la linea alba di esporre gli organi digestivi interni. </li><li> Utilizzare pinze smussato per individuare ed esporre l&#39;ileo all&#39;interno del peritoneo. Tagliare il mesentere ileale / colon con le forbici e iniziare a svelare l&#39;intestino. </li><li> Una volta che la lunghezza dell&#39;intestino è adeguatamente dipanato, intercettato distale dell&#39;intestino allo stomaco e prossimale al cieco per una preparazione ileo. Per una grande preparazione intestinale, tagliare il distale colon al cieco e prossimale al retto. </li><li> Rimuovere rapidamente il segmento intestinale e metterlo in un bicchiere con i media DMEM / F12 addizionato con 3 micron nicardipina cloridrato e 1 micron scopolamina cloridrato (di seguito denominato &quot;media&quot;) su ghiaccio. L&#39;aggiunta di questi inibitori facilita microdissezione e immagini successive paralizzando la muscolatura liscia dell&#39;intestino. </li><li> Segmento Taglio di interesse (ad esempio, digiuno, ileo, colon distale o prossimale) sulla base di marcatori anatomici stabiliti. Tipicamente utilize tessuto dal ileo distale o del colon distale. Tuttavia, utilizzare la stessa procedura di base per isolare, di carico e di immagine neuroni e glia mienterici in tutte le regioni intestinali. </li><li> Rimuovere un piccolo segmento (4-6 cm) di un segmento dell&#39;intestino desiderato e posto in una capsula di Petri Sylgard rivestite pieno di mezzi refrigerati. </li><li> Fissare l&#39;estremità prossimale e distale del segmento intestinale con perni di insetti e aprire il tubo intestinale facendo una dritta, longitudinale tagliare lungo il confine mesenterica. </li><li> Tessuto piatto sotto leggera tensione con il lato mucosa e attenzione sezionare via strato mucoso utilizzando una pinza sottile (5 # e # 5/45 di lavoro migliore) e forbici primavera molto fini Pin. NOTA: La rimozione della mucosa può essere molto traumatico per il ENS se non fatto correttamente. Per i preparati di qualità, fare attenzione a limitare brusca eliminazione della mucosa dalla secchezza o raschiamento. La pratica migliore è quello di sollevare la mucosa e tagliare con le forbici sotto pregiati. </li><li> Tagliare il tessuto in preparazione più piccolos (circa 0,5 cm2) e pin in piatti di imaging (4 angoli con strato muscolare circolare verso l&#39;alto) immessi sul ghiaccio con mezzi freschi. </li><li> Sezionare con attenzione via muscolo circolare da prendere in giro a parte con le pinzette sottili per esporre il plesso mioenterico. Evitare eccessivo allungamento. </li><li> Porre piatto di imaging indietro soluzione ghiaccio e il cambiamento con mezzi freschi. </li><li> Preparare 2 ml di miscela enzimatica per piatto [dispasi 1 U / ml (4.48 mg / 8 ml), Collagenase Tipo II 150 U / ml (5,45 mg / 8 ml) nei media]. </li><li> Rimuovere piatti da ghiaccio e aggiungere il mix enzima dal punto 1.13. </li><li> Incubare piatti a temperatura ambiente per 15 minuti con 5% di CO 2/95% di aria. </li><li> Preparazioni di tessuto Lavare con i media 3 volte e angoli re-pin. </li></ol> 2. Loading Fluo-4 Dye NOTA: Evitare photobleaching lavorando con la luce limitata durante la manipolazione di coloranti fluorescenti e tessuto carichi di indicatore coloranti. <ol><li> Preparare 4 mM Fluo-4 soluzione di carico. <ol><li> Aggiungere 1,5 ml media e 1,2 ml di una probenecid magazzino 250 mm ad un&#39;aliquota di 1,5 ml di 4 mm Fluo-4 stock. 4 mM Fluo-4 stock viene preparato aggiungendo 11,4 ml di Pluronic F-127 (20% in DMSO; integrato con 0,25% cremaphor-EL) a 50 mg di Fluo-4, AM. </li></ol></li><li> Incubare preparazioni in Fluo-4 soluzione di carico per 45 minuti in un incubatore buio a 37 ° C. </li><li> Togliere dal incubatore e lavare preparazioni di media 3 volte. </li><li> Mezzi di scambio per supporti contenente 200 mM probenecid e incubare 15 min a 37 ° C prima di imaging. NOTA: Probenecid è un farmaco che inibisce trasportatori di resistenza ai farmaci in neuroni. L&#39;aggiunta di questo farmaco inibisce la capacità dei neuroni di estrudere coloranti e migliora etichettatura neuronale. Bulk-caricamento di Ca 2+ indicatori coloranti in assenza di probenecid produce principalmente gliali loading cellule. L&#39;aggiunta di probenecid permette la visualizzazione delle risposte neuronali e gliali. </li><li> Preparare Modified KreBuffer bs. <ol><li> Fare un tampone Krebs modificato tale che le concentrazioni finali (in mM) dei componenti sono come segue: 121 NaCl, KCl 5,9, CaCl 2 2,5, 1,2 MgCl 2, 1.2 NaH 2 PO 4, 10 HEPES, 21.2 NaHCO 3, 1 piruvico acidi e 8 glucosio (pH aggiustato a 7,4 con NaOH). Aggiungere 3 micron nicardipina e 1 micron scopolamina per inibire le contrazioni muscolari durante Ca 2+ imaging e tutto il montaggio dissezioni. </li></ol></li></ol> 3. Imaging e analisi NOTA: utilizzare almeno un impianto di imaging di base con una sorgente di luce fluorescente, microscopio, una camera CCD di qualità e adeguato software di acquisizione. Variare l&#39;aggiunta di altri componenti a seconda della sorgente di luce e l&#39;applicazione specifica. Una ruota del filtro e otturatore devono essere usati con una sorgente di luce allo xeno ad arco tradizionale. Tuttavia, fonti di luce LED e sistemi di illuminazione non richiedono tali componenti. <ol><li> Posizionare il recording camera sotto il microscopio e utilizzando un sistema di perfusione a gravità con più serbatoi siringa riscaldati stabilire un tasso perfusione continua di 2-3 ml / min a 37 ° C tampone Krebs. Assicurati di evitare la formazione di bolle d&#39;aria sia nella linea di ingresso e di aspirazione collegato ad una trappola del vuoto. </li><li> Portare il plesso desiderati a fuoco in condizioni di illuminazione in campo chiaro. Evitare sovraesposizione del tessuto, che può portare a foto sbiancamento. </li><li> Esaminare il Fluo-4 di carico all&#39;interno dei gangli e selezionare gangli sano per l&#39;imaging. Malsano gangli / danneggiati esporranno autofluorescenza o puntata morfologia e non devono essere utilizzati per l&#39;imaging. </li><li> Una volta selezionato ganglio, deviare il percorso della luce per fotocamera e di ottenere immagini dal vivo con il software di acquisizione delle immagini. Assicurarsi che ganglio è a fuoco e impostare l&#39;immagine di tasso di acquisizione e di esposizione volte. NOTA: i tassi di acquisizione delle immagini e tempi variano a seconda degli eventi investigatori vogliono registrare. Per la maggior parte degli esperimenti, le immaginisono tradizionalmente acquisita a 0,5-1 Hz per le cellule gliali e fino a 2-10 Hz per i neuroni, perché gliali Ca 2+ risposte non sono così rapidi come Ca 2+ transitori nei neuroni. </li><li> Inizia esperimento e stabilire attività di base per 30 sec. </li><li> Applicare farmaci preriscaldate di interesse come agonisti e antagonisti del recettore utilizzando il sistema di perfusione a gravità ad una velocità di 2-3 ml / min. Seguire applicazione di agonisti / antagonisti ritornando ad una perfusione di tampone normale e consentire un periodo di wash / recupero di almeno 10 min. NOTA: I tempi Drug Application varieranno a seconda del composto individuale e disegno sperimentale. In generale, un&#39;applicazione 20-30 sec di agonista è sufficiente per attivare G-proteine ​​recettori accoppiati in neuroni e cellule gliali. Tuttavia, i canali ionici ligando-dipendenti (come i recettori nicotinici) richiedono tempi di applicazione di non più di 5-10 sec. Ulteriori esposizioni durata causerà canali ionici ligando gated a Dese rapidamentensitize. Antagonisti dovrebbero essere applicate per circa 3-15 minuti per garantire la completa blocco delle vie del recettore. Tuttavia, questa è una generalizzazione lordo e investigatori deve sempre ottimizzare qualsiasi farmaco sperimentale in loro particolare paradigma. </li><li> Interrompere la registrazione e visualizzazione di film time-lapse dell&#39;esperimento. Selezionare con attenzione regioni di interesse (ROI) utilizzando il software di analisi di immagine appropriata. </li><li> Utilizzare software per normalizzare e confrontare l&#39;intensità fluorescenza ROI contro il suo valore di base fluorescente iniziale. Le variazioni nella fluorescenza normalizzata sono direttamente proporzionali alle variazioni [Ca 2+]. <ol><li> Utilizzare una modifica di un metodo descritto in precedenza 26 con Af / F = ((F 1 - F 0) / F 0) ROI - ((F 1 - F 0) / F 0) sfondo, dove F1 è la fluorescenza in un dato point e F0 è la fluorescenza basale, per migliorare la precisione di valutazione. Questo aiuti modificanella riduzione del rumore da variazioni di fluorescenza in preparati tissutali che mostrano il movimento dello strato muscolare sottostante plesso mioenterico. </li></ol></li></ol>

<ol>
	<li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+of+neurons+in+the+enteric+nervous+system.">Types of neurons in the enteric nervous system.</a> Journal of the Autonomic Nervous System. 81 (1-3), 87-96 (2000).</li><li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+autonomic+nervous+system:+peripheral+connections.">The organization of the autonomic nervous system: peripheral connections.</a> Neuroscience. 130 (1-2), 1-5 (2006).</li><li>Pham, T. D., Gershon, M. D., Rothman, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+of+origin+of+neurons+in+the+murine+enteric+nervous+system:+sequence+in+relation+to+phenotype.">Time of origin of neurons in the murine enteric nervous system: sequence in relation to phenotype.</a> Journal of Comparative Neurology. 314 (4), 789-798 (1991).</li><li>Nasser, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+enteric+glia+in+intestinal+physiology:+effects+of+the+gliotoxin+fluorocitrate+on+motor+and+secretory+function.">Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function.</a> Am J Physiol Gastrointest Liver Physiol. 291, G912-927 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+in+the+gut.">Glial cells in the gut.</a> Neurogastroenterology &amp; Motility. 17 (6), 777-790 (2005).</li><li>Broadhead, M. J., Bayguinov, P. O., Okamoto, T., Heredia, D. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++transients+in+myenteric+glial+cells+during+the+colonic+migrating+motor+complex+in+the+isolated+murine+large+intestine.">Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.</a> J. Physiol. 590, 335-350 (2012).</li><li>Gulbransen, B. D., Bains, J. S., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+targets+of+the+sympathetic+innervation+of+the+myenteric+plexus+in+the+guinea+pig+distal+colon.">Enteric glia are targets of the sympathetic innervation of the myenteric plexus in the guinea pig distal colon.</a> J. Neurosci. 30, 6801-6809 (2010).</li><li>McClain, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++Responses+in+Enteric+Glia+Are+Mediated+by+Connexin-43+Hemichannels+and+Modulate+Colonic+Transit+in+Mice.">Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice.</a> Gastroenterology. 146 (2), 497-507 (2014).</li><li>Chandrasekharan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+motor+dysfunction+in+human+diabetes+is+associated+with+enteric+neuronal+loss+and+increased+oxidative+stress.">Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress.</a> Neurogastroenterology &amp; Motility. 23 (2), e131-e126 (2011).</li><li>Abdo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cells+protect+neurons+from+oxidative+stress+in+part+via+reduced+glutathione.">Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione.</a> FASEB J. 24, 1082-1092 (2010).</li><li>Aube, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+enteric+neurone+phenotype+and+intestinal+functions+in+a+transgenic+mouse+model+of+enteric+glia+disruption.">Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption.</a> Gut. 55, 630-637 (2006).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nature Reviews Molecular cell biology. 1 (1), 11-21 (2000).</li><li>Gulbransen, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+neuronal+P2X7+receptor-pannexin-1+mediates+death+of+enteric+neurons+during+colitis.">Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis.</a> Nat Med. 18, 600-604 (2012).</li><li>Bayguinov, P. O., Hennig, G. W., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+activity+in+different+classes+of+myenteric+neurons+underlying+the+migrating+motor+complex+in+the+murine+colon.">Calcium activity in different classes of myenteric neurons underlying the migrating motor complex in the murine colon.</a> J Physiol. 588, 399-421 (2010).</li><li>Okamoto, T., Bayguinov, P. O., Broadhead, M. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+transients+in+submucous+neurons+during+the+colonic+migrating+motor+complex+in+the+isolated+murine+large+intestine.">Ca(2+) transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine.</a> Neurogastroenterol Motil. 24 (8), 769-778 (2012).</li><li>Kunze, W. A., Clerc, N., Furness, J. B., Gola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soma+and+neurites+of+primary+afferent+neurons+in+the+guinea-pig+intestine+respond+differentially+to+deformation.">The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation.</a> J Physiol. 526, 375-385 (2000).</li><li>Schemann, M., Michel, K., Peters, S., Bischoff, S. C., Neunlist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+the+gastrointestinal+tract:+mapping+the+human+enteric+nervous+system.">Imaging and the gastrointestinal tract: mapping the human enteric nervous system.</a> Am J Physiol. 282, G919-G925 (2002).</li><li>Hennig, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spread+of+pacemaker+activity+in+through+ICC+in+guinea-pig+antrum.">Visualization of spread of pacemaker activity in through ICC in guinea-pig antrum.</a> Neurogastro Motil. 14, 575 (2001).</li><li>Stevens, R. J., Publicover, N. G., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+organization+of+Ca2++waves+by+enteric+neural+reflexes.">Induction and organization of Ca2+ waves by enteric neural reflexes.</a> Nature. 399, 62-66 (1999).</li><li>Stevens, R. J., Publicover, N. G., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+and+neural+regulation+of+calcium+waves+in+longitudinal+and+circular+muscle+layers+of+guinea-pig+small+intestine.">Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea-pig small intestine.</a> Gastroenterology. 118, 982-984 (2000).</li><li>Jessen, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-like+glia+in+the+peripheral+nervous+system:+an+immunohistochemical+study+of+enteric+glia.">Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia.</a> Journal of Neuroscience. 3 (11), 2206-2218 (1983).</li><li>Gulbransen, B. D., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+functional+roles+for+enteric+glia+in+the+gastrointestinal+tract.">Novel functional roles for enteric glia in the gastrointestinal tract.</a> Nat Rev Gastroenterol Hepatol. 9, 625-632 (2012).</li><li>Gomes, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-dependent+paracrine+communication+between+enteric+neurons+and+glia+in+a+primary+cell+culture+derived+from+embryonic+mice.">ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.</a> Neurogastroenterology &amp; Motility. 21 (8), e870-e862 (2009).</li><li>Gulbransen, B. D., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+neuron-to-glia+signaling+in+the+enteric+nervous+system.">Purinergic neuron-to-glia signaling in the enteric nervous system.</a> Gastroenterology. 136, 1349-1358 (2009).</li><li>Ren, J., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptors+and+synaptic+transmission+in+enteric+neurons.">Purinergic receptors and synaptic transmission in enteric neurons.</a> Purinergic Signal. 4, 255-266 (2008).</li><li>Takahashi, A., Camacho, P., Lechleiter, J. D., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+calcium.">Measurement of intracellular calcium.</a> Physiol Rev. 79, 1089-1125 (1999).</li><li>Dongcheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+regionalisation+for+enteric+nervous+system+formation:+implications+for+Hirschsprung's+disease+and+stem+cell+therapy.">Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy.</a> Developmental Biology. 339 (2), 280-294 (2010).</li><li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behind+an+enteric+neuron+there+may+lie+a+glial+cell.">Behind an enteric neuron there may lie a glial cell.</a> J Clin Invest. 121, 3386-3389 (2011).</li><li>Boesmans, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuron-glia+interactions+in+the+enteric+nervous+system.">Imaging neuron-glia interactions in the enteric nervous system.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li></ol>

The authors have nothing to disclose.

The methodologies described in this manuscript provide a consistent approach to effectively study neurons and enteric glia in the ENS. Although imaging neurons and enteric glia in culture has yielded a wealth of insight into the function of individual cells, studying these cells in their native, multi-cellular environment is crucial for our understanding their physiology and pathophysiology. Fluorescence microscopy is a crucial technique for assessing multidirectional interactions of cells in the ENS. Loading cells of the ENS with selective fluorescent markers and image acquisition with high-resolution microscopy permits quantitative observations of cellular activity in the ENS. Imaging live tissue samples of the ENS is performed relatively quickly and generates large amounts of in-depth functional and spatial data. Mouse myenteric and submucosal plexus preparations used in these experiments allow for molecular and genetic manipulation approaches. Ca2+ imaging in whole-mount preparations provides a useful tool for the assessment of neuron-glia interactions.
In advanced experimental paradigms, several probes can be combined to obtain information about different events within the cells. Fluorescence microscopy can record images with enhanced contrast of specific molecules, if an appropriate fluorescent label is used. Fluo-4 was chosen because it possesses a large dynamic range. Sufficient incubation time is vital when using the AM dyes in ENS. Dye concentration and loading method may need to be adjusted to achieve best results. Ideal preparations should be loaded with sufficient dye to visualize changes in fluorescence but not so much so that the dye chelates the target ions and interferes with intracellular signaling. Exposure to fluorescent light should be limited to prevent phototoxicity in cells and photobleaching of dyes. 
Investigators must be careful with several steps of this experiment, especially solution and tissue preparation. Particular care has to be taken during processing and dissection of ENS tissue in order to maintain cellular functions. The GI tract contains several layers and tissue varieties, which pose challenges for dissection and imaging quality in these whole-mount preparations 27. Furthermore, the interconnecting fiber tracts of the MP are wider and ganglia are larger than those of the SMP 2. The neuronal density of the myenteric plexus is higher compared to that of the submucosal plexus 28. Slow and imprecise dissections will have detrimental effects on the quality of the plexus preparations and thus the overall success of the experiments. Therefore, clean/undamaged tools, practice and manual dexterity are critical to this procedure. 
In whole-mount tissue preparations, careful consideration should be taken when drawing the regions of interest (ROI) to correctly assess the kinetics and degree of observed change in fluorescence intensity of the desired cell type. As the ganglia are located on a contractile muscle layer, motion artifacts caused by gut motility are a primary concern during in situ imaging. Thus, suppressing these motion disturbances through re-pinning tissue preparations after incubation with enzymes and the addition of pharmacological inhibition (nicardipine/scopolamine) to buffers permits clear and reliable image acquisition. Aside from pharmacology and mechanical approaches to prevent tissue movement, recent studies illustrate the application of advanced software methodologies and cell type response characteristics to correct for residual tissue movement in the recordings and improve the accuracy of analysis 29. Barring these technical hurdles, this method provides physiologically relevant conditions to assess morphologic and quantitative characteristics of neurons and enteric glia in the ENS.

Il sistema nervoso enterico (ENS) è organizzata in due plessi ganglionated incorporato all&#39;interno della parete del tubo digerente 1. Questi circuiti neurali intramuscolari, plesso mienterico (MP) e sottomucosa plesso (SMP), sono composti da neuroni e glia enterica (Figura 1) 2. Il deputato e SMP regolano gastrointestinali (GI) funzioni come la motilità intestinale e l&#39;assorbimento epiteliale e la secrezione, rispettivamente, 3. Glia enterica si trovano in prossimità di neuroni all&#39;interno gangli ma le popolazioni di glia enterica esistono anche all&#39;interno di interconnessione tratti di fibre e porzioni extra-gangliari della parete intestinale 3,4. Glia enterici sono stati originariamente credeva di fornire solo il supporto nutritivo per i neuroni. Tuttavia, studi recenti suggeriscono che le interazioni neuroni-glia sono essenziali per ENS funziona 5,6. Ad esempio, i dati mostrano che glia enterica &quot;ascoltare&quot; per l&#39;attività neuronale 7e modulare circuiti neuronali 6,8, proteggere i neuroni enterici dallo stress ossidativo 9 e sono in grado di generare nuovi neuroni enterici in risposta al danno 10,11. Il protocollo presentato in questa tecnica offre un metodo semplice e robusto per esaminare la complessa interazione tra neuroni e glia enterica utilizzando in situ intracellulare Ca 2+ imaging. Ca 2+ è una molecola di segnalazione ubiquitario nelle cellule eccitabili e svolge un ruolo essenziale in eventi di segnalazione sinaptica nel sistema nervoso 12. Eccitazione dei neuroni o glia enterica suscita un aumento della concentrazione citoplasmatica di Ca 2+ sia afflusso attraverso Ca 2+ canali -permeable o Ca 2+ rilascio dai depositi intracellulari di calcio. Imaging Ca 2 + transitori in neuroni e glia con coloranti fluorescenti è un fondato e tecnica ampiamente utilizzata per studiare l&#39;organizzazione funzionale e dinamica dell&#39;ENS 13-17. Ca 2+ di imaging ha dimostrato di essere uno strumento importante per studiare i segmenti di tessuto GI intatte per chiarire la diffusione di eccitabilità attraverso ICC reti pacemaker 18 e intestino muscolatura liscia 19,20. Esso consente ai ricercatori di sondare un ampio spettro di parametri fisiologici e fornisce informazioni sia la loro distribuzione spaziale e la dinamica temporale. Le celle possono essere efficacemente colorati in modo minimamente invasivo utilizzando indicatori fluorescenti membrana permeabile e protocolli di colorazione ottimizzati 21. Ciò offre la possibilità di controllare un gran numero di neuroni e glia enterica in preparazioni funzionalmente conservati 14-16,22, così come in vivo 23. -Montaggio interi preparati di tessuto sono di massa caricato con un alta affinità Ca 2+ colorante indicatore come Fluo-4 che ne aumenta la fluorescenza quando si lega a Ca 2+. Le variazioni di fluorescenza sono registrate da una telecamera CCD e anazati digitalmente 6. L&#39;avvento di Ca 2+ ha fornito la possibilità di controllare neuroni e cellule gliali interazioni, risposta a vari stimoli, e il coinvolgimento di questi tipi di cellule nei processi gastrointestinali in tempo reale. In situ Ca 2+ di imaging ha dato grande intuizione dei meccanismi di segnalazione di neuroni enterici e glia e possiede diversi vantaggi rispetto ai modelli di coltura cellulare 6,24. In primo luogo, in situ preparati mantenere l&#39;ambiente di matrice nativa di neuroni e glia e lasciano la maggior parte delle loro connessioni con il tessuto intatto bersaglio. In secondo luogo, la genetica e la morfologia di colta glia enterica sono significativamente modificati rispetto al vivo 6,24. In terzo luogo, molte interazioni eterotipiche si perdono in colture cellulari primarie e questo limite valutazione interazioni cellula-cellula. Anche se le cellule coltivate sono particolarmente adatte per le indagini di proprietà fondamentali, la loro usefulness per studiare le interazioni complesse tra glia e neuroni enterici è limitata. Indagare interazione neuroni-glia utilizzando un approccio in situ è fisiologicamente più rilevante come i percorsi sinaptici rimangono intatti 25. Rispetto agli approcci di coltura cellulare, un approccio in situ offre migliori condizioni per la comprensione sistematica delle interazioni complesse tra i neuroni e glia enterica. Inoltre, l&#39;organizzazione planare del plesso ganglionated nei preparati tutto il montaggio è ideale per l&#39;imaging fluorescente intracellulari di Ca 2+ transitori e questa tecnica offre un approccio diretto per valutare l&#39;attività dei neuroni-glia nella ENS.

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in situ ca2+ imaging of the enteric nervous system

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

L&#39;uso corretto di questa tecnica permette ai ricercatori di misurare con precisione intracellulare Ca 2+ [Ca 2+] i transitori nei neuroni enterici e glia in tutto il montaggio preparazioni di tessuto. Un esempio rappresentativo di un agonista-evocato Ca 2+ risposte in glia all&#39;interno di un ganglio myenteric dal colon del mouse è mostrato in Video 1. I risultati seguenti hanno lo scopo di illustrare alcuni risultati rappresentativi che abbiamo ottenuto con questo metodo. In primo luogo, la figura 2 illustra i risultati di un esperimento di misurazione gliali enteriche [Ca 2+] i cambiamenti in risposta alla stimolazione con ATP all&#39;interno cavia del colon longitudinale plesso muscolare mienterico (LMMP) preparati. Specificamente, questa figura mostra il metodo per la corretta analisi del protocollo sperimentale sopra elencati compreso il contorno del ganglio myenteric analizzato e asterischi denotano la posizione dei neuroni enterici. Questi risultati anche illustrate la dose ottimale di un centinaio micromolar ATP sulla mobilitazione del [Ca 2+] i nella cavia glia myenteric. Questa risposta può essere utilizzata per calibrare la stimolazione gliale enterica e normalizzare risposte per testare stimoli. Avanti, Figura 3 chiarisce come selezionare correttamente regioni di interesse (ROI) che circonda le cellule gliali, mostrato con circostante cerchi gialli. Questi risultati mostrano anche le variazioni di fluorescenza desiderati in condizioni basali e in risposta a stimoli farmacologici. Infine, la Figura 4 mostra le considerazioni spaziali per la scelta glia enterica e neuroni per [Ca 2+] i risposte in preparati tutto il montaggio. <img alt="Figura 1" src="/files/ftp_upload/52506/52506fig1highres.jpg" /> Figura 1. Organizzazione della ENS. L&#39;ENS contiene due grandi plessi ganglionated. Il plesso mienterico si trova tra l&#39;lstrati muscolari ongitudinal e circolari. Il plesso sottomucosa si trova tra la mucosa e lo strato muscolare circolare. L&#39;ENS è esclusivamente composto da neuroni e glia enterica. Tratti di fibre del nervo collegano gangli. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52506/52506fig2highres.jpg" /> Figura 2. enterica glia nella cavia del colon plesso mioenterico rispondere alle atp in situ. (A) Fluo-4 fluorescenza in un ganglio mienterici (illustrato a tratteggio) in condizioni basali. Le frecce indicano spessi tratti di fibre interganglionic. (B) Dopo stimolazione con 100 mmol / L ATP, le cellule gliali, ma non i neuroni, aumentano rapidamente Fluo-4 fluorescenza indica un aumento di [Ca 2+] <sub&gt; i. Si noti che le cellule che rispondono sono piccole e circondano i neuroni molto più grandi (spazi bui contrassegnati da un asterisco). (C) glia enterica rispondere alle ATP in modo dose-dipendente con 1 mmol / L suscitare risposte massime 24. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig2large.jpg" target="_blank">Cliccate qui per visualizzare un grande versione di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52506/52506fig3highres.jpg" /> Figura 3. murino S-100-GFP + cellule del colon plesso rispondere mienterico ATP in situ. (A) cellule gliali (verde) in un mouse del colon ganglio mienterico S-100-GFP + (delineati dalla linea tratteggiata). Sei regioni di interesse (ROI) che circonda le cellule gliali all&#39;interno dei gangli sono mostrate come cerchi gialli. Le frecce indicano tratti di fibre spesse portano nel ganglio. Asterischi dposizione enote di 2 neuroni enterici. (B) Stesso ganglio mostrando Rhod-2 fluorescenza in condizioni basali. (C) A seguito di stimolazione con 100 mmol / L ATP, le cellule gliali rispondono con un aumento [Ca 2+] i, come indicato da un aumento Rhod- 2 di fluorescenza. (D) Tracce corrispondente a ciascun ROI mostrato in A-C. (E) la risposta media (media ± SEM) delle 6 ROI mostrati in D 24. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/52506/52506fig4highres.jpg" /> Figura 4. In situ imaging comunicazione enterica neurone-to-glia. (A) Immagini rappresentative (pseudocolored) da un esperimento di imaging Ca 2+ in cui un intero-mountpreparazione del plesso mioenterico è stata impugnata con l&#39;agonista del recettore neuronale P2X7 BzATP (100 micron, 30 sec). Si noti che l&#39;agonista neuronale provoca un aumento della fluorescenza Fluo4 nel neurone (A &#39;) prima che le cellule circostanti enteriche gliali (A &quot;). (B) Analisi della variazione di fluorescenza nel tempo glia (blu) e neuroni (rosso ) in seguito all&#39;applicazione del agonisti neuronale, BzATP. (C) neuronale e risposte gliali a BzATP in tampone normale (linee continue) e in tampone contenente bassa Ca 2+ e Mg 2+ (linee tratteggiate) per potenziare i recettori P2X7 neuronali 13. <a href="https://www.jove.com/files/ftp_upload/52506/52506fig4large.jpg" target="_blank">Si prega clicca qui per vedere una versione più grande di questa figura.</a> Video 1. Agonist-evocato Ca 2+ risposta glia enterica insitu. Questo video mostra un ganglio myenteric dal mouse colon distale caricato con il Ca 2+ colorante indicatore, Fluo-4. L&#39;agonista cellule gliali, ADP, viene aggiunto al bagno quando indicato. ADP suscita un aumento intracellulare Ca 2+ in glia enterica come ha rilevato l&#39;innalzamento transitorio in Fluo-4 fluorescenza. <a href="https://www.jove.com/files/ftp_upload/52506/JOVE.mp4" target="_blank">Clicca qui per vedere il video.</a>

Watch this Scientific Journal Video about In Situ Ca2+ Imaging of the Enteric Nervous System at JoVE.com

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging del sistema nervoso enterico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

in-situ-ca2-imaging-of-the-enteric-nervous-system

Research

JoVE Journal

Neuroscience

In Situ Ca2+ Imaging of the Enteric Nervous System

17.0K Views.  Michigan State University. The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging. 

Video: In Situ Ca2+ Imaging of the Enteric Nervous System

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

La somministrazione orale di Rotenone utilizzando un Gavage e Image Analysis di alfa-sinucleina inclusioni nel sistema nervoso enterico

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Ricerca

Neuroscienze

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Imaging multimodale impianto di cellule staminali nel sistema nervoso centrale di topi

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Etichettatura di singole cellule nel sistema nervoso centrale di<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Trapianto di progenitori interneuroni corticali chemiogeneticamente ingegnerizzati nei primi cervelli dei topi postnatali

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Consegna intratecala di Oligonucleotidi Antisense nel Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imaging Dell'assorbimento mitocondriale di Ca2+ in astrociti e neuroni utilizzando indicatori Ca2+ geneticamente codificati (GECI)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...