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 approximately 70% of juxtaglomerular neurons (JGNs), a population that undergoes adult neurogenesis. Using in vivo two-photon microscopy, time-lapse analysis of identified JGN cell bodies revealed a neuronal turnover rate of approximately 3% 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.

Fig. 1.

GFP expression in the OB of Thy-1–GFP–K12 mice. (A) Fluorescent micrograph (montage of three images) of a coronal slice from an adult Thy-1–GFP–K12 mouse OB. GFP expression is diffuse in the GC layer (GCL) but confined to cell bodies in the mitral cell layer (MCL) and glomerular layer (GL). (Inset) High magnification of the GL and MCL showing that GFP is expressed in cell bodies and primary dendrites but not in higher order dendrites. The Inset and the dotted red box of the lower magnification image are the same region. (Scale bar: 100 μm.) (B) Confocal micrographs of the GL from the bulb of a Thy-1–GFP–K12 mouse processed with an antibody to the neuronal marker NeuN (red). GFP (green) is expressed by the transgene. Some neurons express both GFP and NeuN (solid circles), and some neurons express NeuN but not GFP (dotted circle). The vast majority of cells expressing GFP also expressed NeuN, indicating that the GFP is expressed in a subset of neurons. (Scale bar: 10 μm.)

Fig. 2.

Quantification of GFP expression in different JGN phenotypes. (A–D) Images in Left are GFP fluorescence (green), Middle images denote the neurochemical phenotype (red), and Right images are a merge of the green and red images. Cells were assigned to one of three categories: (i) GFP+/Phenotype− (white arrowheads; green in pie chart), (ii) GFP−/Phenotype+ (red arrowheads; red in pie chart), and (iii) GFP+/Phenotype+ (yellow arrowheads; yellow in pie chart). Four different phenotypes were tested: calbindin (A), calretinin (B), tyrosine hydroxylase (TH) (C), and glutamic acid decarboxylase (GAD6) (D). The pie charts show the quantitative analysis of the different cellular categories of the experiment, and the numbers correspond to the total number of neurons that were counted from several such micrographs. (Scale bar: 10 μm.)

Fig. 3.

In vivo imaging of the JGN population. (A) Single optical slice from the GL of a Thy-1–GFP–K12 mouse. The image is a raw data image and shows JGN cell bodies at a depth of ≈80 μm beneath the pial surface. The complete image stack (showing other image planes above and below this section) is shown in Movie 1. (Scale bar: 50 μm.) (B) Time-lapse in vivo imaging. (Upper) Blood vessel pattern of the dorsal surface of the OB. Note that the large blood vessels remained similar and are used for coarse alignment. (Lower) High-magnification optical sections (raw data of single optical planes) of the same glomerular region populated with JGNs. All neurons in this region can be identified at the two time points (yellow arrowheads) and are used for fine alignment. (Scale bar: 10 μm.)

Fig. 4.

In vivo analysis of JGN turnover. (A–F) Representative examples of in vivo time-lapse images of the JGN population. (A–C) Examples from two different mice imaged at 30-day intervals (A and B) and from a 100-day-interval experiment (C). All cell bodies can be re-identified in this field of view. All images are unprocessed raw data showing single optical sections. (D–F) Examples of JGN dynamics from a 30-day interval (D), added cell body highlighted with a yellow arrowhead), and 100-day experiments (E). In D and F, a new cell is gained (yellow arrowhead), whereas in E, a previously present cell was lost (yellow arrowhead). Stable cells are denoted with red circles. (G) Histogram of the average value of stable JGN cell bodies in both 30- and 100-day-interval experiments. (H) Statistical breakdown of the gain and loss of JGN cell bodies in the 30- and 100-day experiments. Both the level of stability and dynamics are significantly different between the 30- and 100-day experiments (Student’s t test). ∗, P < 0.05; ∗∗, P < 0.01. (Scale bars in A–F: 10 μm.)

Fig. 5.

Control experiments. (A and B) Time-lapse imaging of M/T cell bodies (≈300 μm beneath the pial surface). Two representative examples of the same population of M/T cell bodies imaged 100 days apart. Compared with JGNs, both the number and position of M/T cells are stable (red dots are shown for alignment between images). (C) Confocal micrograph of a BrdUrd-labeled cell (red) and a GFP-positive neuron (green). The overlay of three different views (XY, XZ, and YZ) show that the BrdUrd label is encompassed within this GFP-labeled cell body. (Scale bars: A and B, 20 μm; C, 10 μm.)