Sensory input regulates spatial and subtype-specific patterns of neuronal turnover in the adult olfactory bulb

Abstract

Throughout life, new neurons are added and old ones eliminated in the adult mouse olfactory bulb. Previous studies suggested that olfactory experience controls the process by which new neurons are integrated into mature circuits. Here we report novel olfactory-experience-dependent mechanisms of neuronal turnover. Using two-photon laser-scanning microscopy and sensory manipulations in adult live mice, we found that the neuronal turnover was dynamically controlled by olfactory input in a neuronal subtype-specific manner. Olfactory input enhanced this turnover, which was characterized by the reiterated use of the same positions in the glomeruli by new neurons. Our results suggest that olfactory-experience-dependent modification of neuronal turnover confers structural plasticity and stability on the olfactory bulb.

Figure 1.

In vivo three-time-point 2PLSM imaging of PGC turnover in VGAT-Venus mice. A–C, Coronal sections of the olfactory glomerular layer in VGAT-Venus mice stained for PGC subtype-specific markers TH, CB, and CR (red). All the TH+ and CB+ PGCs were labeled with Venus (A and B, respectively, green), whereas a small population of CR+ PGCs was not labeled with Venus (C, white arrows). See also Table 1. D, E, Detection of the addition (D) and apoptosis (E) of Venus+ PGCs using immunohistochemistry. Mice were injected with BrdU to label neuronal progenitors. The analysis was performed 28 d later, when the labeled cells had matured. Representative images of Venus-expressing BrdU+ (D, red) and ssDNA+ apoptotic (E, red) PGCs (D, E, green) are shown. F, In vivo imaging of PGC turnover in VGAT-Venus mice under normal physiological conditions on days 0, 28, and 56. PGCs labeled with a pink arrow had disappeared (pink circles, lost population) at the next imaging session. The PGC labeled with a pink arrowhead observed on day 0 had been lost on day 56 (pink circle). At the positions indicated by a yellow arrow, new added PGCs were observed 28 d later (yellow circles, added population). The PGC labeled with a yellow arrowhead was identified on day 28 and still observed on day 56. PGCs stably observed throughout the experimental period are numbered. Scale bars: A–C, 50 μm; D–F, 10 μm.

Figure 2.

Olfactory-input-dependent reiterated use of the same positions in the glomeruli by new PGCs. A, Experimental design. B, C, Representative in vivo images showing the patterns of PGC addition after naris occlusion and reopening in VGAT-Venus mice. Sequential images on days 0, 28, and 56 clearly show the stable (numbered), lost (pink arrows and circles), and added (yellow arrows and circles) PGCs in these areas. Note that the “replacement” cell (black asterisk, see F) was observed only after naris reopening in these areas (C). The PGC labeled with a pink arrowhead in C had been lost on day 56. D, Percentage of total identified PGCs that was added or lost during the 4 week naris occlusion (days 0–28). Compared with the control group, in the 4 week naris occlusion group, the population of added PGCs was significantly decreased, whereas that of the lost PGCs was increased (*p < 0.05, ***p < 0.000001). E, Percentage of total identified PGCs that was added or lost after naris reopening (days 28–56). Naris reopening significantly increased the population of added PGCs compared with the control and occlusion groups (***p < 0.0001). F, Classification of new PGC additions and the positions that had lost PGCs. PGCs labeled with pink arrows were eliminated during the 4 week naris occlusion (B, C, F). Of the newly added PGCs observed on day 56, the replacement cells are labeled with a black asterisk. The “filled” positions on day 56 indicate positions where lost cells were replaced by new PGCs (black asterisk). The pink circles indicate the territory of lost PGCs (B, C, F). G, Quantification on day 56 of the replacement cell additions and the filled positions. The percentages of replacement cells and of filled positions were strikingly increased (*p < 0.05, ***p < 0.005) by naris reopening. Scale bars: 10 μm. The data are presented as the mean ± SEM.

Figure 3.

Olfactory-input-dependent regulation on the survival of DAergic subtype of PGCs. A, Experimental design. Bold letters indicate the time points of the analyses shown in B, C, and H–M. B, C, Apoptosis (B) and the addition (C) of PGCs of each subtype upon naris occlusion and reopening. Naris occlusion and reopening significantly increased the number of apoptotic (ssDNA+) and newly added (BrdU+) TH+ DAergic PGCs, respectively, but not CB+ or CR+ PGCs (***p < 0.000005). BrdU was injected on day 28 (C). D, Diagram of the Cre-mediated recombination in TH+ DAergic PGCs used in this study. E–G, Coronal sections of the olfactory glomerular layer in TH-Cre;Rosa26R-CFP mice stained for TH, CB, and CR (red). The majority of the CFP+ PGCs (green) expressed TH. Only a small number of TH-/CFP+ PGCs and CFP-/TH+ PGCs (white arrows and arrowheads, respectively) were observed (E). The CB+ and CR+ PGCs never expressed CFP (F and G, respectively). See also Table 1. H–M, Olfactory-input-dependent dynamic alterations in the number of DAergic PGCs. Coronal sections of the olfactory glomerular layer in TH-Cre;Rosa26R-CFP mice (H–K) stained for GFP (green) and TH (red). The merged image (bottom right) shows that almost all the CFP+ DAergic PGCs also expressed TH in the control OB (H). Although the TH expression in these cells was mostly abolished by naris occlusion (day 28, I and day 56, J, red), these cells could be clearly detected by their stable and continuous CFP expression (green). The number of CFP+ DAergic PGCs was significantly reduced by 4 week naris occlusion (L, day 28, **p < 0.01), and increased by naris reopening (L, day 56, ***p < 0.0005), to a level comparable to that of the control OB (H). The number of DAT+ DAergic PGCs was also decreased by the 4 week naris occlusion (M, day 28, *p < 0.05) and increased by naris reopening (M, day 56, **p < 0.01). Scale bars: 50 μm. The data are presented as the mean ± SEM.

Figure 4.

Olfactory-input-dependent recruitment of new DAergic PGCs into the positions of functional DAergic PGCs eliminated by laser ablation. A–C, GFP-expression pattern in the olfactory glomerular layer of TH-GFP mice. Coronal sections of the glomerular layer were stained for TH, CB, and CR (A–C, red). All of the TH+ PGCs (A), but none of the CB+ or CR+ PGCs (B and C, respectively) were labeled with GFP (green). A portion of the GFP+ PGCs did not express TH (A, white arrows). See also Table 1. D, In vivo repeated imaging of GFP+ PGCs in TH-GFP mice. Sequential images on days 0, 28, and 56 clearly show the stable (numbered), lost (pink arrows and circles), and added (yellow arrows and circles) PGCs in this area. E, Classification of the positions that had lost PGCs by laser ablation. PGCs labeled with a red asterisk were eliminated by laser ablation (E–J). Positions filled by new PGCs on day 28 are indicated by a black asterisk (E, G, J). F–H, Representative in vivo images showing the positions that had lost PGCs by laser ablation in TH-GFP mice. There was no SR101-negative “shadow” (white dashed circle) at the site of the ablated PGCs (F). Note that positions filled by new PGCs (black asterisks) were observed in the ablation group (G) but not in the ablation and occlusion group (H) in these areas (day 28). Single optical sections and their z-stack projection images of two PGCs in G are shown in I and J. I, J, Analysis of the dendrites of the eliminated DAergic PGCs and the newly added ones at the same positions. Series of single optical sections at 2 μm intervals (μm: z-axis distance from the center of the PGC) and z-stack projection images showed that the eliminated and new DAergic PGCs extended their primary dendrites in distinct directions (Z-stack Projection, black shadows). In contrast, PGCs observed to be stable (cell 4 and 6) did not change their dendritic direction throughout the imaging period. The yellow open arrowheads indicate the primary dendrites of targeted DAergic PGCs. K, Quantification of the positions filled by new PGCs on day 28. The percentage of filled positions in the ablation group (red) was significantly greater than in the control group (white) (*p < 0.05), and the increase was suppressed by a 4 week naris occlusion (black) (*p < 0.05). The pink circles indicate the territory of lost PGCs (D–H). Numbers in F–J indicate PGCs that were consistently observed throughout the experimental period. Scale bars: A–C, 50 μm, D, F–H, 10 μm; I, J, 5 μm. The data are presented as the mean ± SEM.