In vivo and in vitro Approaches for the Study of Adult Neurogenesis in Light, Confocal ... ·...

In vivo and in vitro Approaches for the Study of Adult Neurogenesis in Light, Confocal, and Electron Microscopy N. Canalia, M. Armentano, G. Ponti and L. Bonfanti* Department of Veterinary Morphophysiology and Rita Levi Montalcini Centre for Brain Repair, University of Turin, 10095 Grugliasco, Italy Since their beginning, morphological and functional studies of the mammalian central nervous system must deal with the complexity of the nervous tissue. Recent studies, by revealing the existence of neurogenesis throughout life, introduced a new aspect of complexity. Adult neurogenic sites are heterogeneous systems involving stem cell niches which contain actively dividing cells, migration routes, and processes of cell differentiation/integration within the target regions. Since the location of neurogenic sites is restricted to specific brain regions, their study in vivo requires detailed morphological analyses. Although this complexity can be reduced in many in vitro approaches, stem cells and their progeny strongly depend on the tissue environment. Thus, ex vivo approaches such as tissue explants and organotypic cultures can be employed as an alternative way to investigate stem cell niches.

Keywords nervous system; neuronal precursors; astrocyte; neural stem cells; structural plasticity

1. Introduction

The mammalian central nervous system (CNS) is a highly complex structure made up of a huge number of neurons (1011), an even higher number of glial cells (1012) and extremely heterogeneous anatomical and functional relationships among its constitutive elements [1]. In addition to (and in relation to) the high specificity of its structure and hardwiring, the nervous tissue is known to be highly static and non-renewable, what accounts for its uncapability to undergo regeneration and repair (reviewed in [2]). Nevertheless, during the last decades various examples of adult structural plasticity have progressively been shown to occur, ultimately exiting in the demonstration of endogenous proliferative activity and production of new neurons at least within restricted brain sites [3]; reviewed in [4,5]. The elaborate architecture of mature CNS is the result of subsequent cell divisions and precise cell-cell and cell-substrate interactions starting from a small amount of undifferentiated cells in the embryonic neural tube, then growing and assemblying throughout development, including a relatively short postnatal period [6]. In this process, peri-ventricular germinative layers harbouring neural stem cells are the source of most neuronal and glial cell precursors. After CNS assembly, the protracted neurogenic processes are granted by neural stem cell compartments which persist in restricted domains [4]. Two main active germinative layers are present in the mammalian brain: the subventricular zone (SVZ), associated with the anterior part of the forebrain ventricular cavities, and the subgranular zone (SGZ), along the inner layer of the dentate gyrus, within the hippocampal formation (Fig. 1). Among them, only the SVZ maintains direct contact with the lateral ventricles. In addition, SVZ neuronal precursors undergo long-distance migration to reach their final site of destination in the olfactory bulb whereas those generated within the dentate gyrus differentiate locally. Notwithstanding differences in the cytoarchitecture, a common pattern can be found in the cell composition and functional profile of both neurogenic sites (Fig. 1).

* Corresponding author: e-mail: [email protected], Phone: +39 011 6709115

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Fig. 1 Neuroanatomical correlates of adult neurogenesis in the subgranular zone (SGZ) and subventricular zone (SVZ) of rodents. Neural stem cell niches are within rectangles; asterisks: putative stem cells. Genesis of new neurons occurs in two brain areas (middle): the olfactory bulb (OB) and the hippocampus (H). Their germinative layers are respectively the SVZ (bottom and right) and the SGZ (left). Neuronal precursors generated within the SVZ migrate tangentially in the rostral migratory stream (RMS) and radially in the olfactory bulb. The SVZ is composed of two main compartments: astrocytic glial tubes, and tangential chains of neuronal precursors, the latter sliding within the glial ensheathment [7-9]. The SVZ chains dissolve in the olfactory bulb, whereby single neuroblasts move radially through the main (MOB) and accessory (AOB) olfactory bulb layers (Gr, granular layer; GL, glomerular layer). RE, rostral extension. The SVZ stem cell niche involves astrocytes (type B cells, b), neuroblasts (type A cells, a), transit amplifying cells (type C cells, c), and ependymal cells (e). The hippocampal SGZ is located in the inner layer of the dentate gyrus (DG, left top) and gives rise to granule cells (GC) with dendrite arborisation in the molecular layer (ml) and an axon projecting to the Ammon's horn (Ah). Even in the absence of glial tubes and chains, cell types similar to those described in the SVZ form a stem cell niche in the SGZ (left, bottom). Modified from Bonfanti and Ponti, Vet. J., 2007 [5]. In vivo studies, carried out by combining different techniques, led to hypothesize that astrocytes of the SVZ [10] and SGZ [11] are the neural stem cells. The identification of brain stem cells in the glial lineage [12] came in parallel with evidence that embryonic radial glia can act as stem cells, being capable of asymmetric divisions leading to the genesis of both astrocytes and neurons [13,14]. Radial glia, an early generated, elongated cell type spanning the thickness of the assemblying CNS and serving as scaffolding for radial migration of neuronal precursors [15], is a transient cell population which disappears by progressive transformation into astrocytes in the adult [16,17]. The indirect demonstration that radial glial cells somehow persist in the SVZ [18-20], confirmed by a more direct proof obtained by employing a lox-Cre-based technique to mark and follow radial glia through time [21], provided a link between these embryonic glial cells and adult neural stem cells (reviewed in [22,23]).

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The persistence of neurogenesis throughout life introduces a new level of complexity in the CNS structure and function. Adult neurogenic sites are heterogeneous systems involving stem cell niches which contain different cell types interacting each other and with the extracellular matrix, some of them actively dividing at different rates. In addition, the stem cell progeny undertake migration leading to cell differentiation/integration within the target regions. The complex interplay among multiple aspects of adult neurogenesis within restricted regions immersed in the mature brain parenchyma makes this type of study quite problematic in vivo. In addition, it has been recently shown that adult neurogenesis can vary among different vertebrates [24] and different mammals [5] and that embryonic/perinatal germinative layers undergo profound molecular and structural changes in the postnatal period, progressively shaping the adult neurogenic sites [18-20,22]. In this mini-review we will analyse different in vivo and in vitro morphological approaches to study postnatal/adult neurogenesis and neural stem cell niches at the light, confocal, and electron microscopic level.

2. In vivo morphological approach

The morphological study of adult neurogenic sites is made more difficult by some intrinsic histological features of their embryonic-like tissue. Cells in these sites are very small and tightly-packed. Astrocytes of the SVZ show a complex pattern of intermingled cell processes, derived from the simpler arrangement of peri-natal radial glia by increase of ramifications. Finally, since they contain hydrated tissue enriched in embryonic forms of adhesion (e.g. the anti-adhesive polysialylated neural cell adhesion molecule – PSA-NCAM) [2] and extracellular matrix (e.g. tenascin-C) [7,20] molecules, in neurogenic sites is difficult to obtain optimal fixation, particularly for electron microscopy [25]. This feature results in the occurrence of wide clefts among neuroblasts, even in very well fixed brains showing no clefts in the mature parenchyma (Fig. 4,A’’’ [7-9,25]). In the postnatal SVZ, the loose contact within the huge mass of tangentially-migrating neuroblasts can lead to partial tissue disgregation during cutting. In spite of these technical problems, the morphology of neurogenic sites can be studied both in light/confocal and electron microscopy. Due to a topographically well organized arrangement of neuronal and glial cell compartments, involving tangentially-oriented chains of neuroblasts surrounded by astrocytic glial tubes in the SVZ [9] (see Fig. 1), and rows of radial and horizontal astrocytes regularly intermingled among newly genereted granule cells in the SGZ [26], the correct orientation of tissue samples at cutting is very important for interpretation of microscopic images and results.

2.1 Light and confocal microscopy

A technical survey of light/confocal microscopic techniques is not the aim of this review article, rather, a critical discussion about how these tools can be adapted to the study of adult neurogenesis could be useful in order to chose the appropriate approach. Due to complexity of intermingled cell bodies and processes in neurogenic sites, electron microscopy is preferred to study cell shape and cell-to-cell contact, whereas light microscopic techniques are mainly employed for localization and co-localization of specific antigens. Postnatal and adult neurogenic sites being an extension in time of developmental processes occurring within the mature brain parenchyma, the identification of specific stage markers and markers of cell differentiation/lineage is fundamental. In parallel, the detection of extracellular matrix and adhesion molecules is important in defining the extra-cellular milieu, with particular reference to the neural stem cell niches. Thus, double/triple immunofluorescence stainings as well as the use of confocal microscopy for their detection are required to ascertain the effective colocalization of markers in the same cell and/or subcellular compartment [5]. In addition to antigen localization, the existence of cell genesis and cell migration involves dynamic events such as cell proliferation. In the past, the only way to identify actively dividing cells in vivo consisted of morphological demonstration of mitotic figures in tissue sections. With the advent of autoradiographic, immunocytochemical, and viral/genetic manipulation techniques a number of markers have become available for selective detection of proliferating cells in vivo as well as for making possible


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to follow the fate of their differentiated progeny through time. Cell proliferation markers are both endogenous molecules specifically expressed during the cell cycle and exogenously-administered markers that are incorporated by the dividing cells. Endogenous markers are molecules expressed in specific steps of the cell cycle but absent or very low in post-mitotic cells, whose presence can be detected in vivo immunocytochemically. They include cyclins, DNA-associated enzymes and proteins. The most widely used are the Ki67 antigen [27] and the proliferating cell nuclear antigen (PCNA) [28]. The use of these markers has the advantage of not introducing variables in the experiment, but their very brief expression within cells hampers the studies on the temporal/spatial fate of newly born elements. Exogenous markers are nucleotide analogs that are incorporated within the nucleus of dividing cells during the DNA synthetic phase of the cell cycle. Previously used tagged thymidine analogs which must be revealed using autoradiographic techniques (e.g. tritiated thymidine; reviewed in [5]) have mostly been replaced by others that can be detected immunocytochemically by using specific antibodies (e.g. bromodeoxyuridine, BrdU, iododeoxyuridin, IdU, and clorodeoxyuridine, CldU [29]). Viral vectors such as retrovirus and lentivirus tagged with a reporter gene, allow to follow the progeny of newly generated cells without undergoing dilution [30,31]. The gene reporter product being a cytoplasmic protein it is possible to study cell lineage, cell migration and morphology of long-term marked cells [30,32]. To study neurogenesis in vivo, exogenous markers can be either systemically administered or injected within restricted sites by using stereotaxic co-ordinates. A single pulse of BrdU followed by two hours survival can visualize the location of a proliferating cell population. The spatial distribution and fate of the cell progeny can be followed for extended time (months) by exploiting the property of exogenous markers to persist in cells for subsequent generations. Nevertheless, the percentage of cells visualized with these techniques can be affected by diverse factors influencing the availability of marker within tissues, including both experimental (doses employed and ways of delivery) and individual variables (age, metabolism, blood brain barrier efficiency, etc.; [33]). Since BrdU and IdU can be visualized prior tissue treatment with chloridric acid in order to expose the antigen, this procedure could reduce the antigenicity for subsequent immunocytochemical staining to be performed on the same section, although successful double staining for most of the neurogenesis-related antigens have been obtained [34-36]. While studying adult neurogenesis in vivo, a critical analysis of the results obtained is required in the perspective to avoid false positive conclusions. Since nucleotide analogs are indicators of DNA synthesis rather than markers of cell division (37), some of them can also be expressed during certain phases preceding cell death (38), and their incorporation in the nucleus could be linked to DNA repair [37,39]. However, the incidence of the above mentioned drawbacks is minimal when techniques are employed appropriately [32], and further evidence that the elements visualized with cell proliferation markers do correspond to vital cells can be provided by their detection in association with specific molecules typically expressed by neural cell precursors [2,40,41]. With these concepts in mind, the best way to attest the existence of cell proliferation consists of employing different independent techniques and/or a combination of them [37], for instance by adding information obtained through ultrastructural visualization of mitoses and the use of both endogenous and exogenous markers [36].

2.2 Electron microscopy

Most markers employed in light microscopy for detection of the newly generated cells focusing on single aspects such as cell proliferation, cell migration, antigen expression, frequently does not provide a visualization of the entire cell population. In other terms, immunofluorescent approaches usually visualize molecules rather than real cells.


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Fig. 2 Ultrastructure of rabbit cerebellar chains (coronal sections). Serial reconstruction of two subpial chains (Ch1 and Ch2; note that at the beginning they are fused in a single chain). (A) Semithin section at the beginning of the reconstruction. (B-D) Neuroblasts (indicated by numbers) with large nucleus and a thin halo of electrondense cytoplasm are regularly arranged in subpial position, separated from the basal lamina (bl) by glial endfeet (g). These cells are associated with large processes showing the same type of cytoplasm, thus forming aggregates reminiscent of SVZ tangential chains formed by elongated bipolar-shaped cells. (E) Some representative levels of total 43 drawings; each nucleated cell has a different gray shade. Numbers in italic on the left are the levels (three of them are showed in the micrographs B, C, D). Numbers within the cell nuclei identify the 5 cells reconstructed, both in drawings and in micrographs. bv, blood vessel; PC, pial cell; sa, subarachnoid space. Scale bars: 4µm. Reproduced from Ref. [36]. By contrast, conventional electron microscopy can really provide a visualization of cell populations that are cytologically recognizable. This is important as a complementary approach in systems such as neurogenic sites that retain developmental features, whereby antigens can be transiently expressed in the same cell type, and the identity itself of cell ‘types’ can vary at different stages of the cell life [12,22]. An example can be given by the identification of chains of neuroblasts, namely the tangential aggregates allowing fast cell migration in the adult SVZ, from the lateral ventricle to the olfactory bulb [7-9]. Serial reconstruction of these neuroblasts’ aggregates were used to show that they actually form chains [8], even when they leave the SVZ to enter the brain parenchyma, as in the rabbit parenchymal


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chains [34,35], or when they are very small on the rabbit cerebellar surface [36] (Fig. 2). Ultrastructurally, the leading and trailing processes of the spindle-shaped neuroblasts can be entirely reconstructed. In addition, their mutual contact as well as their relationships with the surrounding tissue can be studied in detail. For instance, ultrastructural reconstruction of SVZ chains and parenchymal chains allowed the evaluation of their contact with astrocytic glia and different substrates [34,35] (Fig. 3). As to the cytology of newly generated cells, it has been used to classify different morphological/functional cell types in the stem cell niche [42], confirming that they are recognizable as distinct neuronal and glial compartments [25]. Nevertheless, they do not fit exactly with the expression of specific markers, what makes more difficult the identification of stem cells in vivo. For instance, most SVZ astrocytes show very similar cytological features but display a cohort of glial antigens (as a population) that are not co-expressed in all single cells, and we know that only 1% of these astrocytes actually act as stem cells [43]. Thus, in order to gain more information concerning the dynamic and heterogeneous features of cells acting in the neural stem cell niche, it is possible to combine electron microscopy and antigen detection using immunoelectronmicroscopy [10,20,35,42]. In the case of molecules that retain their antigenic properties after processing for electron microscopy this can be done with post-embedding techniques [20]. Alternatively, using pre-embedding methods the tissue is included in resin after development of the immunocytochemical reaction (Fig. 4). These techniques allow the simultaneous visualization of the antigen and the cell cytology, although the preservation of the latter is of reduced quality in the pre-embedded tissue. In addition, in pre-embedded material the portions available for analysis are limited to the superficial layers of the block harbouring the immunoreaction product. The use of immunoelectronmicroscopy could be particularly suitable in defining the different stages of modification/maturation of the SVZ glial cell population [22]. Indeed, under the profile of cytology these modifications are limited to different degrees of electrondensity, with a progressive reduction in the shift from radial glia to astrocytes [20].

3. In vitro morphological approaches

Since their first isolation in 1992 [44] neural stem cells have been mainly studied in vitro by retrospective analysis with clonal essays which consist of a negative selection of self-renewing and expanding stem progenitor cells forming free-floating aggregates called neurospheres [45]. Although fundamental for studying the cell biology of neural stem cells, these methods provide an artificial environment that is quite different from the natural milieu of the neural stem cell niche, thus inducing the stem cell to act differently [46,47]. For instance, it is well known that the progeny of neural stem cells in vivo is prevalently committed to the neuronal lineage [3-5,25,42,45], whereas most cells obtained from in vitro expanded neurospheres, when induced to differentiate give rise to a large population of astrocytes [45]. On the other hand, primary cultures, although reflecting the cellular composition of the tissue of origin, do not retain their mutual relationships and do not allow to distinguish the potentially different contribution of progenitors and stem cells to the final differentiated progeny. In this context, we are trying to develop an ex vivo model using tissue explants and organotypic cultures of SVZ neurogenic site in order to study the postnatal modifications of the periventricular neural stem cell niche. Both these techniques use freshly-cut, thick brain slices maintained viable for some days in a culture medium and retaining a three-dimensional organization reminiscent of the tissue of origin [48,49]. With respect to the in vivo approach, the advantage of this method regards the possibility to perform experimental manipulations on a tissue that is organotypically organized. In the case of the neural stem cell niche, an important aspect could be the maintenance of the neuron-glial relationships. In order to obtain an optimal survival of glial cells, brain slices from P0-P5 animals can be maintaned in Neurobasal medium, whereas slices from P13-P21 mice are cultured for 1-2 DIV OPTIMEM/HBSS medium containing horse serum, and later transferred in Neurobasal medium.


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Fig. 3 Chain/substrate relationships in rodents and rabbit, evaluated with conventional electron microscopy. Chains of neuroblasts belonging to different species and different anatomical locations, when put in relationship with their substrates, including: different types of astrocytic glia organization (in black), blood vessels (black circles), reveal different types of morphological organization. The grey lines mark the direct contact between chains and brain parenchyma, without the interposition of astrocytic glia. (bottom), Histograms showing the percentages of direct contact between different types of chains and astrocytic glia.

Fig. 4 (A) A parenchymal chain (rectangle) within the white matter (WM) of the anterior forcep of the corpus callosum, in a vibratome slice processed for pre-embedding localization of doublecortin (DCX; higher magnification in A’). (A’’) Semi-thin section of the same tissue embedded in araldite. (A’’’) Immunoelectronmicroscopic visualization of doublecortin in the chain neuroblasts (a), but not in astrocytes (b) and parenchyma (P). Scale bars: A, 50 µm; A’,A’’, 20 µm; A’’’, 2 µm.

The degree of organotypic organization varies with the age of the donor animals and the type of tissue [50]. Greater disruption of three-dimensional arrangement is observed in tissues obtained from younger animals, whereby undifferentiated cells undergoing dynamic events are still prevalent. In the case of


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adult neurogenic sites, this feature is expected to persist at later postnatal stages. For example, in postnatal SVZ-derived tissue explants neuroblasts form chains that leave the explant core radially, in a glia-independent way [48] (Fig. 5). Apart from problems linked to tissue architecture, cultured brain slices have usually been obtained from embryonic or early post-natal donors since the survival of neurons is greatly reduced in tissue derived from older animals [50]. As concerns the nervous system, the technique of post-natal organotypic culture has been employed for studies on axonal growth both in co-culture experiment [51] and in single-slice culture systems [52], as well as for studies on toxic, metabolic effects [53,54], as a way to reproduce hyschemia in vitro [55] or to study cell migration [56]. Direct studies on neurogenesis have been addressed in the hippocampus, limitedly to 5-day-old mice [57]. Taking advantage of the properties of neurogenic sites, up to now we were able to culture viable mouse SVZ explants in which some chains of neuroblasts still migrate radially up to the first month of age, and organotypic cultures up to the end of the third postnatal week (Fig. 5). Both these approches can be exploited to study the crucial stages leading to the establishment of adult neurogenesis, namely the shift between radial glia and SVZ astrocytes (occurring during the first two postnatal weeks [22,23]) and the assembly of the adult SVZ stem cell niche (occurring within the first four postnatal weeks [18-20]). Organotypic cultures including the SVZ are forebrain slices 250-350 µm thick placed on a porous membrane and maintained at the interface between air and the culture medium [49]. Tissue explants are obtained by microdissection of the slice carried out under a steromicroscope in order to isolate just the SVZ tissue. Thus, SVZ explants contain the same stem cell niche but in the absence of the surrounding brain tissue (striatum grey matter and corpus callosum white matter). Tissue explants, other than from classical neurogenic sites can be obtained from non-neurogenic brain parenchymal tissue containing local progenitor cells [58]. Both explants and slices can be subsequently processed for immunofluorescence or embedded for either cryostat cutting or electron microscopy. The direct microscopic observation of the explant/slice surface can provide information about the behaviour of cells moving or changing their shape as a consequence of the culture conditions. Nevertheless, it has obvious limits: i) the disruption of tissue architecture mainly occurs at the slice surface, and ii) the portions of the slice available for immunocytochemical analyses are limited to those that can be reached by antibody penetration and by optics resolution. Thus, a further step could be the embedding of the entire culture to obtain thin sagittal sections to be processed for immunocytochemistry. This method allows the analysis of internal parts of the culture as well as the possibility to localize many antigens on the same specimen. In addition, tissue sections of standardized thickness are necessary when quantification studies are required.

4. Conclusions

The approach to the study of the CNS with the aim of understanding cell-cell interactions, which is commonly considered as a difficult task due to its complex architecture, has been further complicated by the discovery of structural plasticity and persistent neurogenesis. The occurrence of such processes within restricted brain regions implies the coexistence of various dynamic events usually linked to development and absent in the mature nervous tissue. The analysis of these processes, with particular reference to the relationships among cells acting herein, requires the combination of different morphological techniques to be applied in vivo as well as in three-dimensional, ex vivo models retaining the complex milieu and cell-cell contact typical of neural stem cell niches. Ultimately, the approaches described here can be used to unravel how stem cell niches and their progeny progressively change through the postnatal period, in order to adapt to the maturing brain tissue [20,22,23].


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Fig. 5 Organotypic cultures (A) and tissue explants (B) of the mouse SVZ. Tissues have been obtained from 10 days old (A) and 5 days old (B) animals. The SVZ area is indicated in pink in the schematic drowings; the area dissected to obtain SVZ explants is indicated by the red square. LV, lateral ventricle; WM, white matter; GM, grey matter; e, ependyma. Tissues cultured for two days in vitro using both techniques can be either directly immunostained for glial (vimentin), neuronal (β-tubulin) and cell proliferation (Ki67) antigens, or cryostat cut and subsequently immunostained (D).

Acknowledgements The support by Compagnia di San Paolo (Progetto NEUROTRANSPLANT), M.U.R.S.T., Regione Piemonte, and University of Turin, is gratefully acknowledged.

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