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.
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.
De enterisk nervesystem (ENS) er organisert i to ganglionated nervefletninger innleiret inne i veggen av fordøyelseskanalen 1. Disse intramuskulære nevrale kretser, den myenterisk plexus (MP) og submucosal plexus (SMP), er sammensatt av enteriske neuroner og glia (Figur 1) 2. MP og SMP regulere gastrointestinale (GI) funksjoner som intestinal motilitet og epitelial absorpsjon og sekresjon, henholdsvis 3. Ente gliaceller ligger i umiddelbar nærhet til nevroner innenfor ganglier, men bestander av ente gliaceller også eksisterer innenfor sammenhengende fiber traktater og ekstra ganglieblokkere deler av tarmveggen 3,4. Enteriske gliaceller ble opprinnelig antatt å bare gi næringsstøtte til nerveceller. Men nyere studier sterkt at nevron-gliaceller interaksjoner er avgjørende for ENS fungerer 5,6. For eksempel, data viser at mage gliaceller "lytte" til neuronal aktivitet 7og modulere neuronal kretser 6,8, beskytte enteriske neuroner fra oksidativt stress 9 og er i stand til å generere nye enteriske nerveceller i respons til skade 10,11. Protokollen som presenteres i denne teknisk gjennomgang gir en enkel og robust fremgangsmåte for å undersøke det komplekse samspill mellom neuroner og glia enterisk ved hjelp av in situ intracellulær Ca2 + avbildning.
Ca 2+ er en allestedsnærværende signalmolekyl i eksiterbare celler, og spiller en viktig rolle i synaptiske signal hendelser i nervesystemet 12. Eksitasjon av nevroner eller ente gliaceller utløser en høyde i cytoplasma Ca 2+ konsentrasjon enten ved tilstrømning gjennom Ca 2+ -permeable kanaler eller Ca 2+ løslatelse fra intracellulære kalsium butikker. Imaging Ca 2 + transienter i nevroner og gliaceller med fluorescerende fargestoffer er en etablert og mye brukt teknikk for å studere den funksjonelle organiseringen og dynamikkENS 13-17. Ca 2+ bildebehandling har vist seg å være et viktig verktøy i å studere intakte GI vev segmenter for å belyse spredningen av oppstemthet gjennom ICC pacemaker nettverk 18 og gut glatt muskulatur 19,20. Det gjør at forskerne å undersøke et bredt spekter av fysiologiske parametre og gir informasjon om både deres romlige fordeling og timelige dynamikk. Celler kan effektivt farget i en minimal invasiv måte ved hjelp av membran-gjennomtrengelig fluorescerende indikatorer og optimaliserte flekker protokoller 21. Dette gir mulighet for å overvåke et stort antall av neuroner og gliaceller i enterisk funksjonelt konserverte preparater 14-16,22, så vel som in vivo 23. Hele-mount vevspreparater er bulk lastet med en høy affinitet Ca 2+ indikatorfarge som Fluo-4 som øker sin fluorescens når bundet til Ca 2+. Endringer i fluorescens blir registrert av en CCD-kamera og analysert digitalt seks. Ankomsten av Ca 2+ gitt mulighet til å overvåke nevroner og gliaceller celle interaksjoner, reaksjonsevne til ulike stimuli, og involvering av disse celletypene i gastrointestinale prosesser i sanntid.
In situ Ca 2+ bildebehandling har gitt god innsikt i signalmekanismer enteriske nevroner og gliaceller og besitter flere klare fordeler i forhold til cellekultur modeller 6,24. Først i situ forberedelser opprettholde den opprinnelige matrise miljø av nevroner og gliaceller og la hoveddelen av sine forbindelser for å målrette vev intakt. For det andre er genetikk og morfologi av kultivert ente gliaceller vesentlig endret i forhold til in vivo 6,24. For det tredje er mange heterotypic interaksjoner tapt i primærcellekultur og dette begrenser vurdere celle-celle interaksjoner. Selv dyrkede celler er godt egnet for undersøkelse av grunnleggende egenskaper, deres usefulness for å studere komplekse interaksjoner mellom enteriske gliaceller og nevroner er begrenset. Gransker nevron-gliaceller samspill ved hjelp av en in situ tilnærming er mer fysiologisk relevant som synaptic trasé forbli intakt 25. I forhold til cellekultur tilnærminger, en in situ tilnærmingen gir bedre vilkår for systematisk forstå intrikate samspillet mellom nevroner og ente gliaceller. Videre er den plane organiseringen av ganglionated plexus i hele monterte preparater ideell for fluoriserende billeddannelse av intracellulære Ca 2 + transienter og denne teknikken tilveiebringer en grei metode for å vurdere neuron-glia aktivitet i ENS.
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.
The authors have nothing to disclose.
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’s Health) grant K12 HD065879 (B. Gulbransen) and start-up funds from Michigan State University (B. Gulbransen).
Name | Company | Catalog Number |
BubbleStop Syringe Heater | AutoMate Scientific | 10-4-35-G |
CaCl2 | Sigma | C3306 |
Collagenase, Type II, powder | Gibco | 17101-015 |
Dispase | Sigma-Aldrich | 42613-33-2 |
Dissection tools | Roboz | |
DMSO | Sigma-Aldrich | D5879 |
Fixed-stage microscope | Olympus | BX51WI |
Fluo-4 AM dye | Invitrogen | F-14201 |
Glucose | Sigma | G8270 |
Insect pins | Fine Science Tools | Minutien Pins |
iQ Live Cell Imaging Software | Andor | Andor iQ3 |
KCl | Sigma | P3911 |
MgCl2 | Sigma | M9272 |
NaCl | Sigma | S9888 |
NaH2PO4 | Sigma | S8282 |
NaHCO3 | Sigma | S6014 |
Neo sCMOS camera | Andor | Neo 5.5 sCMOS |
Nicardipine | Sigma | N7510 |
Perfusion chamber | Custom | |
Peristaltic pump | Harvard Apparatus | Model 720 |
Pluronic F-127 | Invitrogen | P3000MP |
Probenecid | Molecular Probes | P36400 |
Scopolamine | Sigma | S1013 |
Sutter Lambda DG-4 | Sutter | DG-4 |
Sylgard | Dow Corning | 184 |
Temperature Controller | Warner Instruments | TC-344C |