Flow cytometric analysis of BrdU incorporation as a high-throughput method for measuring adult neurogenesis in the mouse

Abstract

Introduction-The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze.Methods-Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day.Results-The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Discussion-These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology. NIH Public Access

Indomethacin reverses decreased hippocampal cell proliferation in streptozotocin-induced diabetic mice

“…Background signal was accounted for using stained tissue from untreated animals that did not receive BrdU injections. We have previously shown that experimental results obtained with this method are essentially similar to those obtained using immunohistochemistry (Balu et al 2009). …”

“…; Roche Applied Sciences, Indianapolis, IN), dissolved in 0.9% saline, 24 hours prior to sacrifice. Labeling of BrdU was measured in dissociated hippocampal cells displaying the nuclear marker 7-aminoactinomycin D (7-AAD) by flow cytometry as previously described and validated by immunohistochemistry (Balu et al 2009). In brief, mice were decapitated and both hippocampal lobes were dissected on ice and then underwent mechanical trituration to make a single-cell suspension.…”

Neurotransmitter-mediated control of neurogenesis in the adult vertebrate brain

“…Further studies have revealed that a single intrahippocampal injection of a β 3 -adrenergic receptor (β 3 -AR) agonist into the brain or systemic administration over 7 days of isoproterenol, a nonselective β-AR agonist, increases proliferation in the DG and leads to higher number of nestin + GFAP + cells (Jhaveri et al, 2010). Ablation of noradrenergic neurons leads to reduced proliferation of progenitor cells in the SGZ, although proliferation in the SVZ remains unchanged after such injury (Kulkarni et al, 2002;Balu et al, 2009). These studies indicate different responses of progenitor cells to noradrenaline signaling, depending on their location in the brain.…”

Sex-Specific Effects of Chronic Fluoxetine Treatment on Neuroplasticity and Pharmacokinetics in Mice

“…It is postulated that the changes in the mobilization of neurotrophins in turn lead to increased neurogenesis thought to repair damage and restore hippocampal function. A minimum of 2 weeks of continuous treatment with antidepressants is necessary to increase neurogenesis in the hippocampus because a single acute treatment or continuous treatment up through 7 days does not alter cell proliferation (Balu et al, 2009b;Wu and Castrén, 2009). Long-term treatment with fluoxetine or other antidepressants is also necessary to alter brain-derived neurotrophic factor (BDNF) levels as acute administration does not alter protein or mRNA levels of BDNF in the hippocampus (Nibuya et al, 1995;Balu et al, 2008).…”