Department of Molecular Biomedical Sciences, Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University
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Jacquet, B. V., Ruckart, P., Ghashghaei, H. T. An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain. J. Vis. Exp. (46), e2486, doi:10.3791/2486 (2010).
Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.
The following techniques should be performed under sterile conditions, in a laminar flow hood, using sterilized tools.
Preparation of glass bottom dishes for organotypic slices
Extraction of early postnatal brains
The best results are obtained when slices are prepared from young postnatal mice (P1-P10).
Sectioning of the host brain
Donor brain sectioning and RMS transplantation
Time-lapse imaging of neuronal migration
Preparation of glass bottom dishes for organotypic slices
Brain extraction and embedding
Brain sectioning and RMS transplantation
Time-lapse imaging of organotypic slices
Buffer solution for tissue dissection and slice preparation (tissue preparation buffer)
|Stock solution||Volume||Final Concetration|
|10X HBSS||50 mL||1X|
|1M Hepes (pH 7.4)||1.25 mL||2.5mM|
|1M D-Glucose||15 mL||30mM|
|1M CaCl2||0.5 mL||1mM|
|1M MgSO4||0.5 mL||1mM|
|1M NaHCO3||2 mL||4mM|
Filter sterilize with a 0.2 μm filter and store at 4°C.
Culture medium for organotypic slices, tissue transplantation and imaging (slice medium)
|Stock solution||Volume||Final Concentration|
|Basal Medium Eagle||35 mL|
|Tissue preparation buffer||12.9 mL|
|1M D-Glucose||1.35 mL||20mM|
|200mM L-glutamine||0.25 mL||1mM|
|Penicillin-streptomycin||0.5 mL||100units/ mL penicillin and 0.1mg/ mL streptomycin|
Filter sterilize with a 0.2 μm filter and store at 4°C.
Preparation of Low-melting point agarose gel
Low-melting-point agarose is diluted in tissue preparation buffer at 0.3g/ mL in a 50 mL conical tube (see recipes). The tube is microwaved in increments of 5-10 seconds at high-power. Number of increments is dependent on total volume; for 10 mL, three increments (10-8-5 seconds, each) should suffice. The tube cap is carefully unscrewed between heating increments to release air pressure and avoid explosion of the tube. Caution must be taken as the tube content will be very hot. Once the agarose is completely dissolved, the tube is maintained in a 37°C water bath for at least 5 minutes to allow the temperature to stabilize prior to use. Prolonged exposure to room temperature will harden the gel. Although this must be avoided as much as possible, hardened gel can be reheated and re-melted for immediate use within 24 hours of initial preparation.
Immunohistochemistry on organotypic slices
Following imaging on the confocal microscope, slices may be fixed overnight at 4°C with 4% formaldehyde in PBS. Sections are then blocked overnight at 4°C, in 10% goat serum with 1% Triton X (Sigma, Cat. # S26-36-23) in PBS followed by overnight incubation with primary antibodies at 4°C. Fluorescently-tagged goat secondary antibodies are used for visualization (all diluted 1:1000, 1 hour incubation at room temperature). Labeled slices are thoroughly washed 5-6 times with ice cold PBS prior to mounting on glass slides and coverslipping.
IV. Representative Results
Our organotypic slice culture protocol has been thoroughly tested and optimized over that last few years for consistency in migration pattern and orientation. Analysis of cells emigrating from explants obtained from mice in which the expression of the red fluorescent protein, Td-tomato, is induced under the Nestin promoter (Nestin-Td tomato), reveals highly oriented and rapid migration of tdTomato+ neuroblasts into the host RMS (Figure 4A). High-magnification time-lapse analysis illustrates excellent resolution of the entire length of a migrating neuroblast during a 20-minute imaging session (Figure 4B).
Slices with donor Td-tomato+ cells were fixed and immunostained for different cellular components within the RMS. GFAP+ astrocytes and CD31+ blood vessels were revealed using fluorescent immunohistochemistry (Figure 5A). High-magnification analysis of slices stained for the cytoskeletal proteins actin and tubulin reveal non-uniform expression of these components by a cell in the midst of migration (Figure 5B).
Antibodies used in these examples: rabbit anti-RFP (Abcam, 1:250), rabbit anti-GFAP (Dako, 1:1000), rat anti-CD31(BD Pharmigen, 1:100), mouse anti-actin (Santa Cruz, 1:500), rabbit anti-tubulin (Sigma, 1:1000), goat anti-mouse Cy3 (Chemicon, 1:1000), goat anti-rabbit AlexaFluor 647 (Invitrogen, 1:1000), goat anti-rat AlexaFluor 488 (Invitrogen, 1:1000), goat anti-rabbit AlexaFluor 488 (Invitrogen, 1:1000).
Figure 1. Preparation of glass-bottom dishes for organotypic slices. Multiple spots of glue are placed around the circular glass-bottom component of the dish (red), leaving one side open for exchange of medium from underneath the filter. A 150μL drop of slice medium is placed in the center of the glass coverslip. A nucleopore membrane (blue) is then applied, shiny side down, while ensuring that no air bubbles are trapped between the glass coverslip and the membrane. One milliliter of slice medium (grey) is spread on top of membrane, and dishes are incubated at 37° C prior to use.
Figure 2. Brain extraction and preparation for sectioning. (A) The skull is exposed by incising the scalp from the neck to the snout (dotted line along the midline). The skull is then cut longitudinally and anteriorly starting at the cisterna magna, by making 1 medial and 2 lateral cuts (one on each side; 2A). (B) The lateral-most aspects of the cortex and the caudal aspect of the CNS are resected to improve stability of the tissue during vibratome sectioning. (C-D) The two hemispheres are then separated and placed medial face down in an embedding mold prior to application of 3% agarose gel dissolved in tissue preparation buffer.
Figure 3. Brain sectioning and cross-transplantation. (A) The host tissue is sectioned at 150μm thickness, and the RMS-containing sections are carefully positioned flat on the nucleopore membrane of cold glass-bottom dishes. (B) Donor brains (brains expressing fluorescent reporters in the RMS) are sectioned at 250μm thickness, and the slices are collected in ice-cold tissue preparation buffer. The donor RMS is microdissected and cut into small explants. Using a pipettor equipped with a 20 μL tip, individual RMS explants are transferred to an incised site in the host RMS. (C) After 1-2 hours of incubation, dishes are transferred to an incubated stage on a confocal microscope and migration is captured using time-lapse imaging. The photomicrograph is a representative low-magnification image of a typical slice (grey) set up 1 hour after transplantation (red explant from tdTomato+ RMS of a donor mouse; red dotted lines outline the RMS in the host slice).
Figure 4. Migration of neuroblasts from explants into the host RMS. (A) Nestin-tdTomato+ neuroblasts (red) migrate from the explants into the host RMS (dotted green line) 1 hour after transplantation. tdTomato+ cells invading the RMS of the host organotypic slices move in a highly oriented and rapid manner away from the SEZ and toward the OB. (B) The migratory cycle can be observed in high-power time-lapse images of a neuroblast over approximately a 20 minute period. Scale bar = 10 μm.
Figure 5. Immunohistochemical assessment of organotypic slices. Explanted neuroblasts (red) were fixed in the midst of migration 12 hours post transplantation. (A) Fluorescent immunohistochemical staining of the slice reveals a dense pool of GFAP+ astrocytes (blue) and scattered CD31+ blood vessels (green) within the RMS of the host slice. (B) The cytoskeleton of an isolated tdTomato+ migrating cell (red) in a host RMS is revealed by co-immunostaining using antibodies against actin (blue) and tubulin (green).
Neuronal migration in the RMS is an essential component of postnatal neurogenesis in the olfactory bulbs 1. Migration through the RMS occurs in a plane tangential to the surface of the brain. Tangentially migrating neuroblasts are distinct from radially migrating cells based on the location of their progenitor source, as well as the divergent fate of their final neuronal products 1, 2, 3. The relatively pure population of tangentially migrating cells in the postnatal RMS makes this anatomically definable region an optimal platform for studying the mechanisms of tangential migration. Deciphering the cellular and molecular mechanisms of neuronal migration is critical for understanding many neurodevelopmental diseases (e.g., Ref. 4). This knowledge may further facilitate the identification of mechanisms that underlie the formation and spread of brain tumors (e.g., Ref. 5), and is essential for directing reprogrammed neuroblasts to sites of injury or neurodegeneration in future neuronal replacement strategies.
Despite enormous progress in the last few years, the current understanding of genetic and molecular mechanisms underlying neuronal migration, and their link to specific subcellular events remains fragmented. Much of the difficulty in making headways in this pursuit is due to the reliance of most migration studies on tissue fixation and immunohistochemistry, which have been used to draw indirect inferences regarding mechanisms of migration in the RMS and other migration streams in the embryonic brain. The 'fixed' approach is suboptimal since it only provides a snapshot of a cell, whereas temporal resolution is a key requirement for understanding a highly dynamic process such as cellular migration. Some insightful studies have addressed mechanisms of cellular migration using an in vitro method whereby explants, similar to those used in our protocol, are plated on plastic or glass substrates coated with components of the extracellular matrix (e.g.,Refs. 6, 7). Although studies utilizing this assay have identified a number of mechanisms with clear roles in migration, the relevance of their findings to in vivo conditions is unclear. More recently, direct in vivo imaging of neuroblasts through the use of fibered confocal microscopy and MRI have been conducted 8-10. However, such techniques yield low resolution images and thus are insufficient in subcellular assessment of neuronal migration. Another approach, the whole mount preparation devised by the Alvarez-Buylla group 11, has been utilized to generate important physiological insights in the regulation of the subependymal stem cell niche 12-14. Yet, this elegant approach cannot be utilized to study neuroblast migration in the RMS. Previous reports utilizing organotypic slices for analysis of migration in the embryonic brain 15 and the postnatal RMS have been published 16-18. We have perfected this approach and provide here the protocol which allows for high-throughput recording of orientation, speed, and intra- and intercellular dynamics of migration in the RMS at high resolution.
The presented protocol poses some challenges that require practice, patience, and dedicated time to both the preparation and the analysis of results. The following suggestions will assist with carrying out this protocol:
To our knowledge, the presented organotypic slice assay offers the best current approach for deciphering genetic and molecular mechanisms of neuroblast migration from the SEZ to the OB. Given the emerging information regarding the expression of signaling networks in the RMS 19, future experiments to link the identified signaling cascades to neuronal migration will massively benefit from our assay. Our protocol will facilitate the identification of candidate signaling molecules which may regulate neuroblast migration autonomously, while also allowing investigators to assess the role of chemokines and secreted factors in the regulation of migration in a non-autonomous manner, as we have recently demonstrated 18, 20. Our presented technique can easily be modified for study of stem cell transplantation, dendritic/axonal outgrowth, and a whole host of other topics in development neurobiology and adult neurogenesis.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by North Carolina State University and its College of Veterinary Medicine. No conflicts of interest declared.
We thank Dan McWhorter for narrating the protocol in the video. This work is supported by NIH Grant 5R01NS062182, a grant from American Federation for Aging Research, and institutional funds awarded to HTG.