Multiple Birthdating Analyses in Adult Neurogenesis: A Line-Up of the Usual Suspects

And more explanations of the different subgroups of neurons.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3107564/

Analyzing the variation in different subpopulations of newborn neurons is central to the study of adult hippocampal neurogenesis. The acclaimed working hypothesis that different subpopulations of newborn, differentiating neurons could be playing different roles arouses great interest. Therefore, the physiological and quantitative analysis of neuronal subpopulations at different ages is critical to studies of neurogenesis. Such approaches allow cells of different ages to be identified by labeling them according to their probable date of birth. Until very recently, only neurons born at one specific time point could be identified in each experimental animal. However the introduction of different immunohistochemically compatible markers now enables multiple subpopulations of newborn neurons to be analyzed in the same animal as in a line-up, revealing the relationships between these subpopulations in response to specific influences or conditions. This review summarizes the current research carried out using these techniques and outlines some of the key applications.

Keywords: neurogenesis, dentate gyrus, dual birthdating, CldU, IdU, BrdU

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Introduction

The accurate labeling of newborn cells in the adult brain poses a fundamental challenge in the study of adult neurogenesis. Adult brain neurogenesis is closely linked with learning and memory, and it has also been implicated in anxiety and depression (for recent reviews, see Aimone et al., 2010; Deng et al., 2010). Furthermore, many parameters associated with adult neurogenesis are altered in models of Alzheimer's and other neurodegenerative diseases, and in models of ischemia or schizophrenia. Adult neurogenesis has attracted considerable attention due to the cellular properties observed during this period, which may be adapted to rescue neuronal loss due to aging or neurodegenerative processes. In the adult hippocampus, newborn neurons are granule neurons of the dentate gyrus, whose precursors reside locally in the inner side of the granular layer, known as the subgranular zone (SGZ). The cells born in the adult dentate gyrus are derived from neural stem cells (NSC; type-1 cells); these cells are either quiescent (a small proportion) or divide slowly, to generate another group of rapidly dividing cells known as intermediate progenitor cells (type-2a cells). The progeny of these cells are neuroblasts that either continue to proliferate or exit the cell cycle to mature and differentiate into granule neurons, apparently indistinguishable from the rest of the dentate gyrus granule neurons. It will be important to note here that these cell populations can be traced using relatively specific markers (Figure ​(Figure1).1). These markers can be easily used in conjunction with “birth-marking” labels to determine that the different subpopulations were actually born in the adult brain.

Figure 1 Summary of the cell types in adult hippocampal neurogenesis and the expression of the main markers.

In recent years, the labeling of newly born cells in the adult brain has been almost overwhelmingly ruled by the use of 5-bromo-3′-deoxy-uridine (BrdU). “The underlying principle is straightforward: a permanent marker is brought into a cell of interest at the time point of division and the later fate of this cell is studied. Because the marker is persistent, it is possible to retrospectively conclude with confidence that a marked cell must have undergone cell division at the time when the marker was injected (sic)” (Kempermann, 2006). The protocol involves the administration of the thymidine analog (normally by intraperitoneal injection, and to a lesser extent via drinking water) which incorporates into the dual helix of any cell actively synthesizing DNA during the period the product remains systemically available and active in the body (usually around 2h or less). After the animal is sacrificed, staining is detected by immunohistochemistry using antibodies specifically directed against the analog.

BrdU has been the marker of choice in recent years for several reasons, in part because this method requires no radioactivity unlike the use of tritiated thymidine, which for decades was used to label dividing cell populations during brain development. Furthermore, BrdU staining is readily detected by immunohistochemistry. The only significant drawback of this technique is the requirement to unmask the epitopes recognized by the primary antibody against BrdU using hydrochloric acid. Naturally there some other minor issues are involved in its use, though these have largely been solved over the years.

One of the most interesting parameters that can be assessed by the labeling of dividing cells is survival time, defined as the time between the incorporation of the BrdU and that of the animal's sacrifice. The age of labeled cells is equivalent to the survival time (i.e., cells are 1 month old if the animal injected with BrdU was sacrificed 1 month after BrdU injection).

A key limitation of this method is its ability to recognize only a single pool of BrdU incorporated into the body, regardless of when and how it was administered. This has significant implications: all incorporated BrdU is detected as a single signal, and hence, the cell populations that have incorporated BrdU are indistinguishable. Therefore, BrdU administration must be sufficiently discrete in time (depending on the experimental design) so that cell populations to be marked by BrdU are also consistent in terms of age. This issue is particularly important in short experiments (i.e., days). If the animal is injected with BrdU over n days, and sacrificed 1 week after the last injection, the cohort of labeled cells is considered to be between 7 and 7−n days old. Therefore, the injection rate in conjunction with the survival time can produce large differences, and in the worst cases, cell populations that are markedly heterogeneous in terms of age may be labeled equally. Within the framework of the experimental design, the investigator must judge what heterogeneity in terms of age can be accepted in the target cell population. In contrast, in a long-term survival experiment, several injections administered over consecutive days can label a cohort of cells that are subsequently identified as a single group, not taking into account the age difference between the cells marked by the first and the last injection.

This inherent limitation of BrdU labeling implies that each animal can only contain a population of cells marked, as homogeneous as possible in terms of age, precluding any distinction between cells of very different ages. BrdU injections at two times sufficiently separated would generate similarly labeled cells although they are very different. For this reason, the data from cell populations with qualitatively different ages has been achieved by using different experimental groups (different animals) that were subjected to different injection regimes of BrdU and/or different survival times (e.g., group A is sacrificed after 1 week to analyze 1 week old cells, while group B is sacrificed after 1 month to analyze 1 month old cells). This approach has generated a large amount of data. However, it is not a trivial matter that these comparisons between cell populations of different age, born in the adult brain, have been analyzed to date in different animals. It is clear that the study of possible direct relationships between subpopulations of qualitatively different age is strengthened considerably when performed in the same animal, avoiding potential confounding factors due to any intra-group variability between individuals. This problem can now be avoided by the use of a recent technique that permits two or three cell subpopulations of different ages to be labeled in the same animal.

This method involves the injection of the thymidine analogs 5-iodo-2′-deoxy-uridine (IdU) and 5-chloro-2′-deoxy-uridine (CldU), which can be unequivocally distinguished from one other using respective antibodies anti-CldU and anti-IdU, labeling two populations of different ages in the same animal.

It only takes a dual immunohistochemistry to detect the two cell populations of different age born in the adult brain, triple immunohistochemistry in the event that is also to characterize the phenotype of these two subpopulations.

The first attempts to cope with the problem were not so simple. Certainly, the first works reporting the separate detection of two different halogenated nucleotides that have been incorporated in DNA, used immunofluorescence dual-staining methods in vitro (Shibui et al., 1989; Bakker et al., 1991; Aten et al., 1992). However, these methods required complex histological procedures to distinguish between the two antibodies.

By using this technique, an early study by Manders et al. (1992) compared different replication patterns by using immunofluorescence dual staining of cell nuclei in vitro, after incorporating two independent markers of DNA replication in the same nucleus, and recording fluorescence signals with a dual-color confocal microscope. However, one of the first studies reporting the use of dual birthdate labeling in the brain used BrdU and IdU in human patients (Hoshino et al., 1992). The authors injected brain tumors patients with BrdU, followed 5h later by IdU. The percentage of BrdU⁺ cells was used to establish the S phase fraction, while the IdU/BrdU and BrdU/IdU ratios were used to establish the duration of the S phase and other parameters of cell cycle kinetics. Subsequently, Burns and Kuan (2005) were able to distinguish different cell populations depending on the embryonic day the cells were generated during cortical development, by using antibodies raised in different species (rat and mouse) to distinguish two deoxyuridines (again BrdU and IdU), and further incubation with species-specific secondary antibodies conjugated to different fluorophores. This method permits the separate evaluation of dual-labeled cells (BrdU and IdU signal) and single-labeled cells (BrdU signal). This way, the authors also demonstrated the length of the S phase of neural progenitor cells in the adult mouse dentate gyrus.

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Characterization and Set-Up

A number of papers have described different approaches to implement these protocols. In most of them, both thymidine analogs were injected in the same animal to a number of individuals. The first consideration must be the analog dose injected. For this purpose, three important factors have to be taken into account in the use of two thymidine analogs at a time: (i) the absence of cross-reactivity in the immunohistochemistry, (ii) the possibility to detect them along with markers of lineage/differentiation, making possible the identification of the maturational state of the newborn cell/neuron and nevertheless, (iii) the ability to quantify the number of these cells by means of stereology. Each of these requirements can be fulfilled by the use of equimolar administration of CldU and IdU to the animals, as demonstrated by Vega and Peterson (2005).

For this goal, we have used in mice doses of CldU and IdU that are equimolar to the BrdU dose. Specifically, we use to prepare it according to the 50mg/kg bw BrdU dose. This dose was selected because the cells with a nucleus completely filled by the labeling at this dose, or a nucleus containing easily identifiable patches of chromatin marked by the fluorophore at this dose, can clearly be identified as cells that were in S phase in time of administration of the thymidine analog, thus preventing to analyze cells with a weak incorporation possibly due to DNA repair or other unknown factors. The doses used were 42.75mg/kg bw for CldU and 56.75mg/kg bw for IdU. The use of saturating doses of BrdU, CldU, and IdU has been described previously by Leuner et al. (2009).

Nevertheless, the doses of thymidine analogs used in the literature vary considerably. One of the most common approaches is to administer a first analog over a 2–3 week period, and after a variable intermediate period without labeling (see Re-entering the cell cycle), then the second analog over a subsequent 2–3 week period. This protocol has usually been achieved by administering the nucleosides in drinking water (see for example Maslov et al., 2004; Gobeske et al., 2009) at 1mg/ml both nucleosides, while Bonaguidi et al. (2008) used 1.15mg/ml CldU and 0.85mg/ml IdU with 2.5% sucrose. Other approaches consist of intraperitoneal injection of the analogs. This way the doses used has been heterogeneous either, see for example Bauer and Patterson (2005) using 16.67mg/ml CldU in saline and 10mg/ml IdU in PBS/NaOH, pH 8.0, while Gobeske et al. (2009), Mira et al. (2010), and Stone et al. (2010) used 10mg/ml CldU in saline and IdU equimolar to CldU, what means to use doses adjusted volumetrically to the molar equivalent of 50mg/kg BrdU for each animal. The authors used 42.5mg/kg CldU and 57.5mg/kg IdU.

Although a number of authors used doses of CldU and IdU equimolar to that of BrdU, this BrdU dose itself varies in the literature from 50mg/kg bw BrdU (as used by ourselves), some others used equimolar doses to 100mg/kg bw (for example Breunig et al., 2007). A number of other authors have published the use of equimolar doses but not mentioning to what BrdU dose.

Despite the variability in the protocols used, and despite the completely different injection regimes used, all these studies report reliable and consistent labeling. In our studies, CldU can easily be prepared in 0.9% saline. IdU by using 0.1M PBS with 2 drops of 5N NaOH added per 10–15ml PBS. This solution was then heated 2–3 times in a microwave oven without boiling and vigorously stirred manually.

The next parameter to be taken into account is the survival time. Several groups have used different survival times in different groups of animals to characterize and set-up the technique. Specifically, we injected CldU and IdU at different time points in the same animals and they were then sacrificed either 24h or 2 weeks after the last injection.

Finally, mice were anesthetized with pentobarbital, transcardially perfused with saline followed by 4% paraformaldehyde in phosphate buffer (PB). The brains were removed, post-fixed overnight in the same fixative and sectioned with a vibratome (50μm thick sections). The slices were pre-incubated with 0.5% Triton X-100 and 0.1% bovine serum albumin, and then dual immunohistochemistry was performed. Briefly, we used rat anti-CldU antibody (Accuratechemicals 1:500) and a mouse anti-IdU antibody (BD Biosciences 1:500) overnight. Primary antibodies were detected by using secondary Alexa-conjugated antibodies from Molecular Probes (1:1,000): Alexa 488 conjugated donkey anti-rat for the anti-CldU antibody, Alexa 594 conjugated donkey anti-mouse for the anti-IdU antibody overnight, and the reverse fluorophore-conjugated antibodies for the control experiments switching the colors of the secondary antibodies. Sections were counterstained finally with DAPI (Calbiochem 1:1,000) for 10min.

We found and consistently replicate the result that cells were labeled in decreasing numbers with increasing survival time, as expected, and this was consistent for both analogs. In addition, the number of cells dual labeled for CldU and IdU (CldU⁺/IdU⁺ cells) in the same animal also decreases as the time between injections of each thymidine analog increased, as is expected (see Figure ​Figure22 taken from Llorens-Martin et al., 2010).

Figure 2 Representative pictures of CldU+ and IdU+ cells in set-up experiments. The thymidine analogs were injected in the same individuals separated by either 24h or 1 week; BrdU-equimolecular dosages of CldU and IdU were injected. Animals were then (more ...)

Other authors had previously demonstrated the feasibility and reliability of both analogs to produce a replicable and consistent labeling of dividing cells. In this way, Vega and Peterson (2005) showed that simultaneous equimolar delivery of both IdU and CldU co-labeled all cells with both markers. Next they demonstrated that the administration of equimolar concentrations of IdU and CldU 1 day apart is able to label three populations of proliferative cells (one of the markers or both together).

In a different experiment, we changed the order of the injections with respect to our design mentioned above; some animals were injected CldU first and later IdU, while other animals were injected in reverse order. The quality of aforementioned labeling unchanged whatever the order in which the analogs were injected. The number of cells obtained after 24h survival either with CldU or IdU, and the number of cells obtained after 2 weeks survival by injecting CldU or IdU, were consistently similar.

Bonaguidi et al. (2008) performed a number of interesting control experiments. By labeling with IdU and CldU separated in time by several weeks, the authors could see in the granule cell layer (GCL) of the hippocampal dentate gyrus that older cells were located farther within the GCL than younger cells, consistent with labeling two temporally distinct precursor populations. Besides, dual-labeled cells were found in very low numbers.

Our experiments have all been performed in mice, like many other authors. However, a number of studies have reported similar protocols, and importantly, similar results about the reliability of the technique by using rats. Problems of specificity, cross-reactivity, and feasibility of the technique, don't therefore seem to rely on the species the experiments have been carried out up to date.

We have never encountered cross-reactivity problems of the antibodies in tissue from mice. Cross-reactivity has not been a major problem frequently reported in the literature, except for the early works by Shibui et al. (1989) by using BrdU and IdU, or the work by Aten et al. (1992) by using CldU and IdU detected by means of anti-BrdU antibodies specific for BrdU-CldU or only BrdU. The former work required complicated histological procedures to ensure the specificity of staining, while the second one required the use of a high-salt buffer to differentially remove binding from each substrate. None of these problems are common today with the commercially available antibodies.