The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.
I. Preparation of Glass Micropipettes Filled with Fluorescent Microbeads for Ependymal Flow Assay (these steps may be skipped if preparing wholemounts for staining purposes only).
II. Wholemount Dissection and Fixation
III. Ependymal Flow Analysis using Fluorescent Microbeads
IV. Immunostaining Wholemounts
V. Mounting Immunostained Wholemounts onto Slides for Confocal Microscopy
Representative Results
Wholemount approaches have provided several key insights into the germinal activity of the adult SVZ. The network of chains of migrating young neurons in the SVZ was first observed after wholemounts of the lateral wall of the lateral ventricle were immunostained with antibodies to polysialylated neural cell adhesion molecule (PSA-NCAM) 1. These chains of migrating neuroblasts can also be seen after immunostaining wholemounts with doublecortin antibodies (Figure 1). Remarkably, the network of chains has a stereotyped pattern, with two general streams of cells, one running dorsally over and one running ventrally around the adhesion point. Wholemounts of the SVZ also provide a comprehensive view of the proliferative activity of progenitors in this region, as seen with Ki67 staining in Figure 2. Interestingly, two recent studies suggest a close interaction between dividing SVZ cells and the local vasculature 2,3 (Figure 2).
When examined under high power confocal microscopy, the en-face view provided by wholemounts allows a unique perspective of the apical surface of cells lining the ventricular system. This en-face perspective has recently revealed that SVZ type B1 cells, the adult neural stem cells, are part of a mixed neuroepithelium with non-dividing differentiated ependymal cells 4. The apical surface of type B1 cells contacts the lateral ventricle and is surrounded by large apical surfaces of ependymal cells in a pinwheel configuration (Figure 3, arrows indicate B1 apical surfaces). Furthermore, close examination of the apical surface of ependymal cells has revealed that the translational position and rotational orientation of their basal bodies are indicators of their planar polarity 5. Ependymal cell basal bodies are clustered in a patch on the apical surface. This patch is displaced from the center of the apical surface in the downstream” direction with respect to CSF flow (translational polarity); within this patch, each basal body is rotated about its long axis such that the basal foot, an accessory of the basal body, points in the direction of flow (rotational polarity). Neighboring ependymal cells have their basal bodies oriented in the same direction. Importantly, videomicrographs of the ependymal flow assay can be used to directly compare the flow in a specific region of the lateral wall to the orientation of ependymal cell basal bodies in that region (Figure 4).
In addition to providing a panoramic perspective of the largest germinal region in the adult brain, with higher power imaging, wholemounts allow a more complete and detailed analysis of individual cellular morphologies in the SVZ. High power confocal imaging of GFAP immunostaining on wholemounts has revealed that type B1 cells, in addition to their short ventricle-contacting apical process, have a long basal process in contact with blood vessels (Figure 5) 4. This cytoarchitecture had not been appreciated previously in coronal sections because the basal process runs mostly parallel to the ventricular wall. Serial sectioning therefore cuts individual cells into small fragments, making it nearly impossible to reconstruct a cell s complete morphology, or to understand its relationship to other cell types in the SVZ. The wholemount approach has several advantages over classical sectioning techniques, both in providing panoramic views with low power microscopy and a complete perspective of individual cells with high power microscopy. This technique will continue to be an important complement to future studies of this adult brain germinal zone.
Figure 1. Network of migratory neuronal chains in the SVZ. Tiled confocal images reconstruct a lateral wall wholemount that was stained with antibodies to doublecortin, which labels migrating neuroblasts throughout the SVZ. There are two general streams of migration, one running dorsally over and one running ventrally around the adhesion point, indicated by the asterisk (*). Arrows indicate anterior (a) and dorsal (d) directions. Scale bar = 1 mm.
Figure 2. Relationship between the vasculature and dividing cells in the SVZ. This lateral wall wholemount was immunostained with antibodies for Ki67, to label dividing cells in green, and antibodies against mouse immunoglobulins, to label the vasculature in red. Because this wholemount was not perfused with saline prior to staining, the endogenous mouse IgG molecules remain within blood vessels and are stained by secondary anti-mouse antibodies. Recent work suggests that dividing SVZ precursors (green) are located in close proximity to blood vessels (red) {Shen, 2008 #6523}{Tavazoie, 2008 #6522}. Arrows indicate anterior (a) and dorsal (d) directions. Scale bar = 1 mm.
Figure 3. The apical surface of ventricle-contacting cells on the lateral wall. High power confocal image of a wholemount immunostained for β-catenin, to label cell membranes in green, and γ-tubulin, to label basal bodies in red, reveals the planar organization of these epithelial cells. Type B1 cells, the adult neural stem cells, have a small apical surface with a single basal body, indicated by arrows. The apical surface of these cells is surrounded by the large apical surface of ependymal cells in a pinwheel configuration. Ependymal cells have planar polarity indicated by the position of their multiple basal bodies on the apical surface. Neighboring ependymal cells have their basal body clusters located on the same side of the apical surface (downward and leftward in this region), corresponding to the direction of CSF flow {Mirzadeh, 2010 #6573}. Scale bar = 10 μm.
Figure 4. The ependymal flow assay. Composite image created by merging 100 sequential frames from a movie taken during the ependymal flow assay. Fluorescent microbeads deposited dorsal and posterior to the adhesion area were propelled by ependymal cilia in two oriented streams, one over and one under the adhesion, towards the foramen of Monro. This oriented flow reveals the functional planar polarity of ependymal cells. Each flow line depicts the position of a single bead at consecutive points in time. Scale bar = 0.5 mm.
Figure 5. GFAP+ type B1 cells have a long basal fiber with end-feet on blood vessels. Maximum projection of a high power confocal stack taken from a lateral wall wholemount immunostained with GFAP antibodies to label SVZ astrocytes. This staining labels adult neural stem cells, or type B1 cells, which have an apical ending on the ventricular surface, and as shown here, a long GFAP+ basal fiber that ends on blood vessels (arrows). Blood vessels are stained here because the secondary antibody used to visualize mouse anti-GFAP antibodies recognize endogenous mouse IgG within the vasculature. Scale bar = 50 μm.
Most studies of neurogenesis in ventricular and subventricular zones have relied on classical sectioning techniques to examine the microanatomy and cellular relationships in these regions. Here we describe an alternative technique, first used to analyze the network of migratory chains of neuroblasts generated in the SVZ 1, then used to study regeneration of the SVZ progenitor population following anti-mitotic treatment 6, and most recently used to study the precise apical and basal cell-cell interactions of adult SVZ neural stem cells 2,3,4. Interestingly, this technique has revealed that the neural stem cells, or type B1 cells, of the adult SVZ are part of a mixed neuroepithelium with differentiated non-dividing ependymal cells. En-face imaging using wholemounts has shown that this mixed neuroepithelium has pinwheel architecture consisting of the apical endings of type B1 cells surrounded by large apical surfaces of ependymal cells 4. This en-face analysis has clarified our understanding of the lineage of neural stem cells in embryonic and adult brains as consisting of cells with apical endings at the ventricle surface and basal processes contacting a vascular niche. These findings would have been nearly impossible using classical sectioning techniques. Wholemounts also facilitate the identification of neural stem cells via their ventricle-contacting apical process. As more specific markers for these stem cells are found, wholemounts will be an integral part of identifying and analyzing neural stem cell behavior.
Wholemounts of the lateral ventricle walls also provide the ideal perspective for studying the planar polarity of ependymal cells. Ependymal cells are multiciliated cells lining the ventricles that function to propel CSF in a coordinated manner. With the wholemount technique, the entire ependymal epithelium is exposed en-face and can be stained and studied comprehensively from its anterior to posterior and dorsal to ventral boundaries. Furthermore, ependymal flow assays performed on acutely dissected, live wholemounts robustly demonstrate the planar polarized flow generated by ependymal cilia. Recent work using wholemount approaches has uncovered cellular determinants of this ependymal planar polarity 5. Interestingly, wholemount studies have also suggested that ependymal-generated CSF flow establishes gradients of chemorepellents that guide the migration of young neurons in the SVZ 7. Wholemount approaches that initially identified the network of migratory neuronal chains are therefore continuing to provide insights into mechanisms regulating chain migration.
Analysis of the VZ and SVZ by wholemount imaging adds a new approach for both future studies and a way to clarify our understanding of existing studies. For example, a recent study suggested that neural stem cells in the adult SVZ were CD133+/CD24- cells in contact with the ventricle 8. Based on their immunostaining in sections, these authors claimed that these cells were a subpopulation of multiciliated ependymal cells. However, in our study using the wholemount approach, which gives a more comprehensive view of the entire ependymal epithelium, we found that all ependymal cells express CD24 and the only ventricle-contacting cells that were CD133+/CD24- were a subset of the type B1 cells 4. Furthermore, the wholemount technique promises to be useful in future studies examining the recently described mosaic organization of neural stem cells in the adult brain 9. Several studies have shown that neural stem cells in the adult brain are not a homogeneous population, but are regionally specified and normally produce only specific subtypes of olfactory bulb interneurons. These studies have proposed that different subpopulations of neural stem cells may be distinguished either by the expression of specific transcription factors 10,11,12,13,14 and/or by their regional localization along the dorsal-ventral and anterior-posterior extents of the lateral wall 9,15,16. As more molecular markers of the regionally specified subpopulations of adult neural stem cells are identified, wholemount imaging should provide a comprehensive view of the parcelation of these different progenitor domains along the ventricular wall.
The wholemount dissection and imaging techniques presented here may also be used to analyze the ventricular walls in the embryo. The dissection of the embryonic lateral wall is performed, step-by-step, in the same manner. There are only slight differences in the level of difficulty; the embryonic ventricles are relatively larger making the dissection easier, but the tissue is softer making manipulation more difficult. In particular, a similar exposure of the lateral ventricle can be used in embryos to dissect the cortical wall of the ventricle to study cortical neurogenesis. Recent evidence suggests that asymmetric centrosome inheritance maintains radial glia at the ventricular surface during cortical neurogenesis 17. En-face imaging of radial glial apical surfaces may provide insights into how centrosomes within these dividing cells are asymmetrically inherited.
As with most techniques, especially those involving precise dexterity, mastery requires practice. There are, however, a few elements in the dissection that are key to better results: 1) lighting adjusting the illumination of the sample to create shadows provides invaluable contrast during the dissection of tissue that is otherwise relatively homogeneous, 2) using the forceps like two insect pins the forceps in this technique are never used to pinch together or pick up tissue, but are used as maneuverable pins that can be continually readjusted to stabilize the tissue while cutting, 3) a balance of gentle retraction and cutting the knife should not only be used to cut but also to provide gentle retraction to separate the medial and lateral walls, remembering that the majority of this dissection is actually performed through gentle retraction with only intermittent cutting.
Work supported by NIH grant HD-32116, the Sandler Family Supporting Foundation, The John Bowes Stem Cell Fund, MEXT, MHLW, and HFSP. Z.M. supported by the Carlos Baldoceda Foundation and UCSF Krevans Fellowship. A.A.-B. holds the Heather and Melanie Muss Endowed Chair.