वयस्क माउस अग्रमस्तिष्क के तीव्र स्लाइस में समय चूक Neuroblast प्रवासन की इमेजिंग
Summary
हम माउस अग्रमस्तिष्क में neuronal प्रवास के videoimaging वास्तविक समय के लिए एक प्रोटोकॉल का वर्णन. virally लेबल या grafted neuronal व्यापारियों के प्रवास तीव्र जीना एक अपेक्षाकृत तेजी से अधिग्रहण के अंतराल के साथ व्यापक क्षेत्र फ्लोरोसेंट इमेजिंग का उपयोग करने के लिए स्थिर और प्रवास चरणों durations और की गति सहित, सेल प्रवास के विभिन्न चरणों का अध्ययन स्लाइस में दर्ज की गई थी प्रवास.
Abstract
There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.
Protocol
Discussion
neuronal व्यापारियों की सही लक्ष्यीकरण उपयुक्त मस्तिष्क क्षेत्रों के लिए एक मौलिक उचित और तंत्रिका circuitry के विधानसभा समारोह अंतर्निहित प्रक्रिया है. कोशिकाओं के विशाल बहुमत भ्रूण विकास के दौरान विस्थापित…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम के स्वास्थ्य अनुसंधान अनुदान (CIHR) की एक कनाडाई ASJK आंशिक रूप से एक Université Laval फैलोशिप द्वारा समर्थित संस्थानों द्वारा समर्थित किया गया. के रूप प्रसवोत्तर neurogenesis में एक कनाडा अनुसंधान चेयर के प्राप्तकर्ता है.
Materials
| Name of the reagent | Company | Catalogue number |
| Sucrose | Sigma | S9378 |
| Glucose (ACSF) | EMD | DX0145-3 |
| NaCI | Sigma | S9625 |
| KCI | Sigma | P9541 |
| MgCI2x6H2O | Sigma-Aldrich | M2670 |
| NaHCO3 | Sigma | S5761 |
| NaH2PO4xH2O | EMD | SX0710-1 |
| CaCI2x2H2O | Sigma-Aldrich | C3881 |
| Dextran TexasRed | Invitrogen | D1864 |
| Dextran CascadeBlue | Invitrogen | D1976 |
| Glucose (40X solution) | Sigma | G8769 |
| Sodium pyruvat | Gibco | 11360-070 |
| HEPES | Sigma | H3375 |
| HBSS | Gibco | 14170-112 |
| DNase I | Sigma | D-5025 |
| Trypsin-EDTA | Gibco | 25300-054 |
| Neurobasal medium | Gibco | 21103-049 |
| BSA | EMD | 2930 |
| Pen/Strep | Life Technologies | 15140-122 |
| Ketamine/Xylazine | CDMV | 5230 |
| Pasteur pipette | VWR | 14672-380 |
| 15 ml conical tube | Sarstedt | 62.553.205 |
| 50 ml conical tube | Sarstedt | 62.547.205 |
| Glass capillaries (stereotaxic injection) | WPI | 4878 |
| Paraffin oil | EMD | PX0045-3 |
| Proviodine | Rougier | 65655-1370 |
| Suture | Stoelting | 50487 |
| Anafen | CDMV | 11508 |
| 20 cc Syringe | VWR | SS-20L2 |
| Petri dish | VWR | 25384-094 |
| Agar | Laboratoire Mat | AP-0108 |
| Glue | Permabond | 910 |
| 95% O2/5% CO2 | Linde | 24068835 |
| Blade | WPI | 501901 |
| Nylon mesh | Warner Instruments | 64-0198 |
| Centrifuge | Eppendorf | 5702 000.019 |
| Pipette puller | Sutter Instrument | P-97 |
| Nanoliter injector | WPI | B203MC4 |
| Stereotaxic injection apparatus | WPI | 502900 |
| Micro drill system | WPI | 501819 |
| Vibratome | Thermo Scientific | 920110 |
| Wide-field fluorescent microscope | Olympus | BX61WIF |
| CCD camera | Photometrics | CS-HQ2-D |
| Ultra-quiet imaging chamber | Harvard Apparatus | 64-1487 |
| PH-1 Series 20 heater platform | Harvard Apparatus | 64-0284 |
| Heating system | Warner Instruments | TC-344B |
| 40X water immersion objective | Olympus | 1-UM587 |
| 10X water immersion objective | Olympus | 1-UM583 |
| Lambda DG-4 | Sutter Instruments | DG-4/OF |
| MetaMorph software | Molecular Devices | 40000 |
| Imaris software | Bitplane | BPI-IM70-F1 |
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