A method for the quantification of in vivo BrdU labeling from adult murine brain tissue using flow cytometry was characterized. The assay was sensitive enough to detect BrdU incorporated into cells of the hippocampus and SVZ, as well as the amygdala and cerebellum, regions with less proliferative activity. Moreover, flow cytometry was able to quantify cells labeled with BrdU up to three weeks prior, allowing measurement of cell proliferation and survival. Several manipulations known to down or up-regulate neurogenesis were performed in an effort to validate the utility of the assay. A decrease in proliferation was produced by the induction of experimental type-I diabetes or following the depletion of hippocampal norepinephrine content. Increases in cell proliferation were measured following repeated administration of ECS. The magnitude of all these effects measured using flow cytometry were similar to what has been reported in the literature using immunohistochemistry. Moreover, chronic administration of fluoxetine for 14 and 21 days, but not 7 days, robustly elevated cell proliferation in the hippocampus. The results obtained by flow cytometry after fluoxetine administration for 21 days were correlated with those obtained from manual counting.
The first step in the characterization of this method of measuring proliferating cells was to determine the dose of BrdU that would produce maximal labeling, without producing toxic side effects that have been associated with high doses of BrdU (Taupin, 2007). The 100 and 200 mg/kg doses produced comparable labeling of hippocampal progenitors when given once daily for four days. Therefore, the lower dose was used for all validation experiments. The 200 mg/kg dose produced maximal labeling in a single-injection protocol, which is more appropriate for some experiments, and was therefore used in the fluoxetine time-course experiment.
The generation of new neurons into adulthood occurs most prominently in the dentate gyrus of the hippocampus and in the SVZ, which lines the lateral ventricles. In addition to these two neurogenic zones, other brain regions retain proliferative capacities into adulthood, albeit to a lesser extent than the hippocampus and SVZ. These include the hypothalamus, striatum, neocortex, and amygdala, although whether functioning neurons are created in these regions is still uncertain (Gould, 2007). Flow cytometry was utilized to make regional comparisons of BrdU incorporation. The rate of proliferation in the SVZ was seven-fold higher than in the hippocampus, which agrees with immunohistochemical comparisons (Kim et al., 2006
). Moreover, this method was sensitive enough to detect BrdU incorporation in the amygdala and cerebellum, areas of much lower adult cytogenesis (Gould, 2007). In these brain regions, the progenitor cells are diffuse and not isolated to discrete neurogenic zones, a characteristic that makes manual counting difficult. The cellular resolution provided by flow cytometry makes this technology ideal for measuring proliferation in tissues with sparsely distributed cells.
Numerous physiologic factors regulate adult hippocampal neurogenesis (Balu and Lucki, 2008
; Zhao et al., 2008
). One such process is glucose homeostasis. Diabetes, which is characterized by elevated levels of circulating glucose, produces reductions in hippocampal neurogenesis that are associated with deficits in hippocampal-dependent tasks (Beauquis et al., 2006; Beauquis et al., 2008). Administration of STZ and induction of type-1 diabetes, reduced hippocampal cell proliferation as measured with flow cytometry.
Neurotransmitter systems are also involved in regulating the various stages of neurogenesis (Balu and Lucki, 2008
; Zhao et al., 2008
). There are extensive noradrenergic projections to the hippocampus originating from the locus ceruleus. In the rat, destruction of these noradrenergic cell bodies with DSP-4 reduced cell proliferation, but not survival, of hippocampal progenitors (Kulkarni et al., 2002
). Quantification of BrdU by flow cytometry revealed a significant reduction in hippocampal cell proliferation following the administration of DSP-4. This is the first demonstration of noradrenergic tone regulating cell proliferation in the mouse. The decrease in proliferation was accompanied by the selective depletion of norepinephrine in the hippocampus. This suggests that hippocampal norepinephrine may in part, be involved in maintaining a basal rate of neurogenesis.
Hippocampal neurogenesis has been implicated in depression (Dranovsky and Hen, 2006
), and is thought of as a potential target for therapeutic intervention. Although consistent with evidence for morphological deficits associated with depression, the generation of new neurons in the adult hippocampus might be more important for the therapeutic response produced by antidepressant treatments (Sahay and Hen, 2007). Flow cytometry successfully quantified elevations in BrdU incorporation following repeated administration of ECS, a somatic treatment for depression. Chronic treatment with the pharmacologic antidepressant fluoxetine produced robust elevations in cell proliferation. Moreover, the levels of cell proliferation after fluoxetine treatment obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. This demonstration is similar to that provided by Bilsland et al. (2006)
in their original characterization studies.
Hippocampal neurogenesis is a candidate biochemical readout for novel drug discovery in a number of disease areas, including depression, schizophrenia, epilepsy, diabetes and Alzheimer’s disease (Balu and Lucki, 2008
; Zhao et al., 2008
). However, conventional methods of BrdU quantification require the immunohistochemical detection and manual counting of BrdU positive cells from fixed tissue. This process is time consuming, labor intensive, intrinsically difficult when cell clustering is present, and therefore does not make neurogenesis an attractive target for the screening of potentially active compounds. Unlike immunohistochemistry, which depending on the sample size, can take weeks to months to analyze data, flow cytometry allows for the analysis of data in one to several days. The speedy analysis afforded by this technique makes hippocampal cell proliferation and survival a practical target in the drug discovery arena.
Inherent in the drug discovery process, is the requirement to know the treatment time required for compounds to produce their effects, as well as the time when maximum efficacy is reached. This type of experimental design requires large numbers of animals, which is made manageable with the use of flow cytometry. This study demonstrated that 14 days of fluoxetine administration was required to increase cell proliferation, as 7 days of treatment had no effect. This time-course for the onset of action of fluoxetine is similar to what has been reported in the mouse (Santarelli et al., 2003). Interestingly, 21 days of treatment did not produce a maximal effect, suggesting that longer durations of treatment might produce even larger augmentations of proliferative activity.
One important technical development of the method will be the ability to determine the phenotype of BrdU-positive cells. There are many immunohistochemically-validated antibodies used in conjunction with BrdU to identify cells from various lineages and in different stages of development (von Bohlen und Halback, 2007). These same cellular markers can be applied to flow cytometry. In addition to identifying cell phenotypes, flow cytometry also has the potential to measure other proliferative markers, such as the cell cycle associated protein Ki-67 and the transcription factor Sox-2.
In conclusion, this study has demonstrated the ability of flow cytometry to expeditiously and reliably measure cell proliferation and survival from adult murine brain tissue. Its inherent speed makes it an attractive method by which neurogenesis can be utilized as a platform for novel drug discovery. Moreover, it has the potential to greatly advance the neuroscience community’s understanding of the biological underpinnings of adult neurogenesis.