In vivo imaging of juxtaglomerular neuron turnover in the mouse olfactory bulb

Abstract

As a consequence of adult neurogenesis, the olfactory bulb (OB) receives a continuous influx of newborn neurons well into adulthood. However, their rates of generation and turnover, the factors controlling their survival, and how newborn neurons intercalate into adult circuits are largely unknown. To visualize the dynamics of adult neurogenesis, we produced a line of transgenic mice expressing GFP in Ϸ70% of juxtaglomerular neurons (JGNs), a population that undergoes adult neurogenesis. Using in vivo twophoton microscopy, time-lapse analysis of identified JGN cell bodies revealed a neuronal turnover rate of Ϸ3% of this population per month. Although new neurons appeared and older ones disappeared, the overall number of JGNs remained constant. This approach provides a dynamic view of the actual appearance and disappearance of newborn neurons in the vertebrate central nervous system, and provides an experimental substrate for functional analysis of adult neurogenesis.neurogenesis ͉ olfaction N ewborn neurons in the adult mammalian brain continuously migrate into the hippocampus and the olfactory bulb (OB) (1, 2). In the OB, newborn neurons originate in the subventricular zone and differentiate into two distinct populations of interneurons: granule cells and juxtaglomerular neurons (JGNs). Adult neurogenesis in the mammalian brain was discovered and has been primarily analyzed by methods that label dividing cells by incorporation of labeled cyclic nucleotides, such as radiolabeled thymidine or the uridine analogue 5-bromo-2-deoxyuridine (BrdUrd). These methods have significant limitations. For example, neither radiolabeled thymidine nor BrdUrd are neuron-specific, and both can be analyzed only in fixed tissue after histological processing. These caveats and others (see discussions in refs. 3 and 4) limit direct analysis of the dynamics of neurogenesis, such as turnover rates and subtle changes in levels of neurogenesis in response to different experimental conditions and manipulations.To study adult neurogenesis using an alternative approach, we developed transgenic mice expressing GFP in a subset of JGNs and imaged adult neurogenesis over periods of up to 3 months, in vivo. We observed an Ϸ3%-per-month turnover rate of JGN in the OB. The number of newly appearing JGNs (representing newborn neurons) was balanced by the number of JGNs disappearing (presumably by cell death) thereby maintaining a stable population of JGN neurons. ResultsLabeling of JGNs in Transgenic Mice. Using a methodology based on transgenic GFP expression under control of the Thy-1 promoter, Feng et al. (5) created a bevy of mouse lines in which distinct neuronal subpopulations were strongly labeled by GFP and other fluorescent proteins. These lines have proven invaluable for numerous in vivo imaging experiments (6-10) enabling visualization of axonal and dendritic dynamics in developing and adult animals. However, none of the existing lines of Thy-1-XFP mice has labeling in the regenerating populations of the OB (granule and juxtaglomerular neu...

Distinct Modes of Neuron Addition in Adult Mouse Neurogenesis

“…For example, the low degree of disappearance of pre-existing neurons imaged in the GL (Mizrahi et al, 2006) is consistent with the net addition of newly arriving neurons there implying no or little cell death of previously generated neurons. Moreover, the transgenic GFP-labeling used in this study primarily targets calbindinϩ neurons that we find not to be generated in the adult at least as detected by the GLAST::CreERT2-mediated fate-mapping approach labeling 90% of all newly generated neurons.…”

“…Extrapolating these data from labeling a single or few cohorts would suggest a considerable contribution of adultgenerated neurons to the OB network, leading several studies to propose an increase in the size of the dentate gyrus (DG) and the OB attributable to adult neurogenesis (Kempermann et al, 2003;Merson et al, 2006). Although the majority of the BrdU analysis focused on the GCL, in vivo imaging could only access the GL where a turnover of only 3% of the neurons was observed (Mizrahi et al, 2006). Thus, a certain degree of inconsistency between different reports may be attributable to the relatively short time frame analyzed by imaging or the restriction to labeling few cohorts of adult-generated cells.…”

Migration, fate and in vivo imaging of adult stem cells in the CNS

“…Several techniques for in vivo cellular imaging are now available that allow the characterization of transplanted cells in the living organism, including magnetic resonance imaging (MRI), 23 bioluminescence, 24 positron emission tomography 25 and multiple photon microscopy. 26 All of these imaging methods, based on different principles, provide distinctive, usually complementary information. In this review, we will focus on cell labelling for MRI, as MRI is non-invasive, clinically translatable and displays good resolution, ranging from 50 mm in animals up to 300 mm in whole body clinical scanners.…”

Mechanisms of Neurogenesis in the Normal and Injured Adult Brain

“…92,93 Since this methodology enables the same region of olfactory glomeruli to be observed over time, it can be used to detect new additions and eliminations of neurons in addition to stably existing neurons. Using this method and a naris occlusion plug that can reversibly regulate olfactory input (Fig.…”