Implantation af et kranievindue til gentagen In Vivo-billeddannelse i vågne mus
Summary
Præsenteret her er en protokol til implantation af et kronisk kranievindue til langsgående billeddannelse af hjerneceller i vågne, hovedbeherskede mus.
Abstract
For fuldt ud at forstå den cellulære fysiologi af neuroner og glia i at opføre dyr, er det nødvendigt at visualisere deres morfologi og registrere deres aktivitet in vivo i at opføre mus. Dette papir beskriver en metode til implantation af et kronisk kranievindue for at muliggøre langsgående billeddannelse af hjerneceller i vågne, hovedbeherskede mus. I kombination med genetiske strategier og virale injektioner er det muligt at mærke specifikke celler og regioner af interesse med strukturelle eller fysiologiske markører. Denne protokol viser, hvordan man kombinerer virale injektioner for at mærke neuroner i nærheden af GCaMP6-ekspressiv astrocytter i cortex til samtidig billeddannelse af begge celler gennem et kranievindue. Multifotonbilleddannelse af de samme celler kan udføres i dage, uger eller måneder i vågen, opførende dyr. Denne tilgang giver forskere en metode til at se cellulær dynamik i realtid og kan anvendes til at besvare en række spørgsmål inden for neurovidenskab.
Introduction
Evnen til at udføre in vivo multifotonfluorescensmikroskopi i musebarken er altafgørende for undersøgelsen af cellulær signalering og struktur 1,2,3,4,5,6,7,8,9, sygdomspatologi 10,11 og cellulær udvikling12,13 . Med implantation af kroniske kranievinduer er langsgående billeddannelse mulig, hvilket muliggør gentagen billeddannelse af kortikale områder i dage, uger eller måneder13,14 hos levende dyr. Multifotonmikroskopi er ideel til in vivo, gentagen billeddannelse på grund af forbedret dybdesondering og reduceret fotoskade forbundet med den anvendte infrarøde laser. Dette gør det muligt at studere molekylær og cellulær dynamik af specifikke celler i forskellige kortikale regioner.
Multifotonmikroskopi er blevet anvendt til in vivo-billeddannelse af neuronale og glialceller i mus 15,16,17,18,19,20. Forskellige strategier kan implementeres for at mærke bestemte celletyper og interesseområder. En almindelig tilgang er at drive ekspressionen af genetisk kodede fluorescerende proteiner på en cellespecifik måde ved hjælp af Cre-Lox-rekombinationssystemet. Dette kan udføres med genetisk modificerede mus, f.eks. krydsning af tdTomato “floxed” mus (Ai14) med en mus, der udtrykker Cre-recombinase under en promotor af interesse21. Alternativt kan celle- og stedspecifik mærkning opnås med virale injektioner. Her injiceres en virus, der koder for Cre-rekombinase under en cellespecifik promotor, og en virus, der koder for et floxet gen af interesse, i en defineret region. Passende celletyper, der modtager begge virale vektorer, vil derefter udtrykke det eller de ønskede gener. Disse gener kan være strukturelle markører, såsom tdTomato, for at se ændringer i cellulær morfologi22 eller genetisk kodede calciumindikatorer (GECIS’er), såsom GCaMP og / eller RCaMP, for at undersøge calciumdynamik23. Metoder til genetisk rekombination kan anvendes individuelt eller i kombination til mærkning af en eller flere celletyper. En tredje tilgang, der ikke kræver transgene mus eller virale konstruktioner (som har begrænset emballagekapacitet), er in utero elektroporation af DNA-konstruktioner24. Afhængigt af tidspunktet for elektroporationen kan forskellige celletyper målrettes 25,26,27.
Når du udfører multifotonbilleddannelse, kan mus afbildes, mens de er vågne eller anæstetiserede. Billeddannelse af vågne mus kan udføres ved at fastgøre musen via en vedhæftet hovedplade28. Denne fremgangsmåde gøres mindre stressende ved at tillade relativt fri bevægelighed for dyret ved hjælp af metoder, såsom fritsvævende, luftunderstøttede styrofoamkugler29, fritsvævende løbebånd1 eller et luftløftet hjemmebursystem, hvor mus fastgøres med en fastgjort hovedplade og får lov til at bevæge sig i et åbent kammer30. For hver af disse billeddannelsesbetingelser vil det først være nødvendigt at vænne musene til billeddannelsesopsætningen. Dette papir beskriver tilvænnings- og billeddannelsesproceduren ved hjælp af et luftløftet hjemmebursystem.
Denne protokol beskriver implantationen af et kronisk kranievindue til langsgående in vivo-billeddannelse i cortex. Her vil vi bruge mus, der betinget udtrykker GCaMP6f i astrocytter til at overvåge calciumsignaldynamikken. Endvidere beskriver dette papir proceduren for virale injektioner ved hjælp af tdTomato som en etiket for neuroner. Dette gør det muligt at bestemme ændringer i neuronal synaptisk struktur og / eller tilgængeligheden som en strukturel markør, der muliggør gentagen billeddannelse af den samme astrocyt. I hele protokollen vil afgørende trin blive fremhævet for at sikre den bedst mulige kvalitet af billeder opnået ved multifotonmikroskopi.
Protocol
Representative Results
Discussion
Her har vi præsenteret en protokol for implantation af kroniske kranievinduer til in vivo-billeddannelse af kortikale astrocytter og neuroner i vågne, hovedbeherskede mus på et luftløftet hjemmebur. Der er givet specifikke eksempler på kranievinduesapplikationen til billeddannelse af astrocytter, der udtrykker GECIS’er og neuronale synaptiske strukturer. Ved brug af multifotonmikroskopi kan astrocytisk calciumsignaldynamik og strukturel synaptisk dynamik registreres gentagne gange over dage.
<p class="j…Materials
| 15o Pointed Blade | Surgistar | 6500 | Surgery Tools |
| 19 G Needles | BD | 305186 | Surgery Supply |
| AAV1-CAG-FLEX-tdTomato | Addgene | 28306-AAV1 | Viral Vector |
| AAV1-CaMKII-0-4-Cre | Addgene | 105558-AAV1 | Viral Vector |
| Acteone | Fisher Scientific | A16P4 | Reagent |
| Alcohol Prep Pads | Fisher Scientific | Covidien 5750 | Surgery Supply |
| Beveler | Narishige | Equipment | |
| Borosilicate Glass | World Precision Instruments | TW100F-4 | Surgery Supply |
| Carbide Burs | SS White Dental | 14717 | Surgery Tools |
| Carprofen (Rimadyl), 50 mg/mL | Zoetis Mylan Institutional, LLC. | Drug | |
| Compressed Air | Fisher Scientific | 23-022-523 | Surgery Supply |
| Cotton Tip Applicators | Puritan | 836-WC NO BINDER | Surgery Supply |
| Cover Glass, No. 1 thickness, 3 mm/5 mm | Warner Instruments | 64-0720, 64-0700 | Surgery Supply |
| Dental Drill | Aseptico | Equipment | |
| Dexamethasone, 4 mg/mL | Mylan Institutional, LLC. | Drug | |
| Dissecting Microscope | Nikon | Equipment | |
| Duralay Liquid (dental cement liquid) | Patterson Dental | 602-8518 | Reagent |
| Duralay Powder (dental cement powder) | Patterson Dental | 602-7932 | Reagent |
| Enrofloxacin, 2.27% | Bayer | Drug | |
| Eye Ointment | Dechra | 17033-211-38 | Surgery Supply |
| Fiber Lite High Intensity Illuminator | Dolan-Jenner Industries | Equipment | |
| Forceps (Large) | World Precision Instruments | 14099 | Surgery Tools |
| Forceps (Small) | World Precision Instruments | 501764 | Surgery Tools |
| GCaMP6f B6; 129S-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/J | The Jackson Laboratory | Stock No: 024105 | Mouse line |
| Germinator | Fisher Scientific | Equipment | |
| GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J | The Jackson Laboratory | Stock No: 012586 | Mouse Line |
| Headplate | Neurotar | Model 1, Model 3 | Surgery Supply |
| Hemostatic forceps | World Precision Instruments | 501705 | Surgery Tools |
| Holder for 15o Pointed Blade | World Precision Instruments | 501247 | Surgery Tools |
| Holder for Scalpel Blades | World Precision Instruments | 500236 | Surgery Tools |
| Iodine Prep Pads | Avantor | 15648-926 | Surgery Supply |
| Isoflurane | Piramal | Surgery Supply | |
| Isoflurane table top system with Induction Box | Harvard Apparatus | Equipment | |
| Isoflurane Vaporizer | SurgiVet | Equipment | |
| Krazy Glue | Office Depot | KG517 | Reagent |
| Loctite 401 | Henkel | 40140 | fast-curing instant adhesive |
| Loctite 454 | Fisher Scientific | NC9194415 | cyanoacrylate adhesive gel |
| Micropipette Puller | Sutter Instruments | Equipment | |
| Multiphoton Microscope | Equipment | ||
| Nitrogen | Matheson | NI M200 | Gas |
| Oxygen | Matheson | OX M250 | Gas |
| Picospritzer | Parker | intracellular microinjection dispense system | |
| Pipette Tips | Rainin | 17014340 | Surgery Supply |
| Rodent Hair Trimmer | Wahl | Equipment | |
| Saline (0.9% Sodium Chloride) | Med Vet International | RX0.9NACL-30BAC | Surgery Supply |
| Scalpel Blades, Size 11 | Integra | 4-111 | Surgery Tools |
| Scissors | World Precision Instruments | 503667 | Surgery Tools |
| Stereotaxic Instrument | Stoelting | Equipment | |
| Sugi Sponge Strips (sponge strips) | Kettenbach Dental | 31002 | Surgery Supply |
| SURGIFOAM (gel foam) | Ethicon | 1972 | Surgery Supply |
| Syringe with 26 G Needle | BD | 309625 | Surgery Supply |
| Tamoxifen | Sigma Aldrich | T5648-1G | Reagent |
| Ti:Sapphire Laser | Coherent | Equipment | |
| Transfer Pipettes | Fisher Scientific | 13-711-9AM | Surgery Supply |
| Water Blanket | Fisher Scientific | Equipment | |
| Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL | Fresenius Kabi USA | Drug |
References
- Cichon, J., Gan, W. B. Branch-specific dendritic Ca2+ spikes cause persistent synaptic plasticity. Nature. 520 (7546), 180-185 (2015).
- Goncalves, J. T., et al. Circuit level defects in the developing neocortex of Fragile X mice. Nature Neuroscience. 16 (7), 903-909 (2013).
- Padmashri, R., et al. Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome. Journal of Neuroscience. 33 (50), 19715-19723 (2013).
- Peters, A. J., Chen, S. X., Komiyama, T. Emergence of reproducible spatiotemporal activity during motor learning. Nature. 510 (7504), 263-267 (2014).
- Poskanzer, K. E., Yuste, R. Astrocytes regulate cortical state switching in vivo. Proceedings of the National Academy of Sciences of the United States of America. 113 (19), 2675-2684 (2016).
- Srinivasan, R., et al. Ca2+ signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo. Nature Neuroscience. 18 (5), 708-717 (2015).
- Takata, N., et al. Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo. Journal of Neuroscience. 31 (49), 18155-18165 (2011).
- Yang, G., Pan, F., Gan, W. B. Stably maintained dendritic spines are associated with lifelong memories. Nature. 462 (7275), 920-924 (2009).
- Zuo, Y., et al. Development of long-term dendritic spine stability in diverse regions of cerebral cortex. Neuron. 46 (2), 181-189 (2005).
- Grutzendler, J., Gan, W. B. Two-photon imaging of synaptic plasticity and pathology in the living mouse brain. NeuroRx. 3 (4), 489-496 (2006).
- Isshiki, M., et al. Enhanced synapse remodelling as a common phenotype in mouse models of autism. Nature Communications. 5, 4742 (2014).
- Cruz-Martin, A., Crespo, M., Portera-Cailliau, C. Delayed stabilization of dendritic spines in fragile X mice. Journal of Neuroscience. 30 (23), 7793-7803 (2010).
- Mostany, R., et al. Altered synaptic dynamics during normal brain aging. Journal of Neuroscience. 33 (9), 4094-4104 (2013).
- Trachtenberg, J. T., et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature. 420 (6917), 788-794 (2002).
- Agarwal, A., et al. Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron. 93 (3), 587-605 (2017).
- Bindocci, E., et al. Three-dimensional Ca2+ imaging advances understanding of astrocyte biology. Science. 356 (6339), (2017).
- Dana, H., et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nature Methods. 16 (7), 649-657 (2019).
- Han, S., Yang, W., Yuste, R. Two-color volumetric imaging of neuronal activity of cortical columns. Cell Reports. 27 (7), 2229-2240 (2019).
- Nimmerjahn, A., Kirchhoff, F., Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science. 308 (5726), 1314-1318 (2005).
- Stowell, R. D., et al. Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex. Nature Neuroscience. 22 (11), 1782-1792 (2019).
- Madisen, L., et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neuroscience. 13 (1), 133-140 (2010).
- Chen, S. X., et al. Subtype-specific plasticity of inhibitory circuits in motor cortex during motor learning. Nature Neuroscience. 18 (8), 1109-1115 (2015).
- Stobart, J. L., et al. Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons. Neuron. 98 (4), 726-735 (2018).
- Matsui, A., et al. Mouse in utero electroporation: controlled spatiotemporal gene transfection. Journal of Visualized Experiments: JoVE. (54), e3024 (2011).
- Roth, R. H., et al. Cortical synaptic AMPA receptor plasticity during motor learning. Neuron. 105 (5), 895-908 (2020).
- Stogsdill, J. A., et al. Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature. 551 (7679), 192-197 (2017).
- Suresh, A., Dunaevsky, A. Relationship between synaptic AMPAR and spine dynamics: impairments in the FXS mouse. Cerebral Cortex. 27 (8), 4244-4256 (2017).
- Yang, G., et al. Transcranial two-photon imaging of synaptic structures in the cortex of awake head-restrained mice. Methods in Molecular Biology. 1010, 35-43 (2013).
- Dombeck, D. A., et al. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron. 56 (1), 43-57 (2007).
- Kislin, M., et al. Flat-floored air-lifted platform: a new method for combining behavior with microscopy or electrophysiology on awake freely moving rodents. Journal of Visualized Experiments: JoVE. (88), e51869 (2014).
- Holtmaat, A., et al. high-resolution imaging in the mouse neocortex through a chronic cranial window. Nature Protocols. 4 (8), 1128-1144 (2009).
- Hauglund, N. L., et al. Meningeal lymphangiogenesis and enhanced glymphatic activity in mice with chronically implanted EEG electrodes. Journal of Neuroscience. 40 (11), 2371-2380 (2020).
- De Paola, V., et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron. 49 (6), 861-875 (2006).
- Cheng, A., et al. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nature Methods. 8 (2), 139-142 (2011).
- Yang, G., et al. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nature Protocols. 5 (2), 201-208 (2010).
- Shih, A. Y., et al. A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. Journal of Visualized Experiments: JoVE. (61), e3742 (2012).
- Helm, P. J., Ottersen, O. P., Nase, G. Analysis of optical properties of the mouse cranium–implications for in vivo multi photon laser scanning microscopy. Journal of Neuroscience Methods. 178 (2), 316-322 (2009).
- Stobart, J. L., et al. Long-term in vivo calcium imaging of astrocytes reveals distinct cellular compartment responses to sensory stimulation. Cerebral Cortex. 28 (1), 184-198 (2018).
- Pryazhnikov, E., et al. Longitudinal two-photon imaging in somatosensory cortex of behaving mice reveals dendritic spine formation enhancement by subchronic administration of low-dose ketamine. Scientific Reports. 8 (1), 6464 (2018).
- Thrane, A. S., et al. General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex. Proceedings of the National Academy of Sciences of the United States of America. 109 (46), 18974-18979 (2012).
- Paukert, M., et al. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron. 82 (6), 1263-1270 (2014).
- Delekate, A., et al. Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer’s disease mouse model. Nature Communications. 5, 5422 (2014).
