Learning and survival of newly generated neurons: when time matters

Abstract

New interneurons are continually added to the olfactory bulb (OB), the first central relay for processing olfactory information, throughout life. It remains unknown how these adult-generated interneurons integrate into preexisting networks or die. We used immunohistochemical approaches to quantify adult neurogenesis in mice subjected to olfactory training. We identified a critical period in the life of an adult-generated OB interneuron, during which learning triggers distinct consequences. Using a discrimination learning task performed at various times after the birth of new interneurons, we found that olfactory training could increase, decrease, or have no effect on the number of surviving newly generated neurons. Cell survival and elimination depend on both the age of the cell and its location within the granule cell layer. This study provides new insight into the contribution of the newly generated interneurons to OB function. It demonstrates that neuronal elimination is an active process, rather than a simple consequence of nonuse.

Figure 1.

Two weeks of olfactory discrimination learning increases neuronal survival in the GCL but not in the EPL and GL. A, New cells were labeled with BrdU, and their survival was evaluated 24 d later. Trained mice underwent discrimination learning when BrdU⁺ cells were between 8 and 22 d old (n = 6). B, Go/no-go procedure. C, Mean percentage of correct responses in each block of the first 2 d (T1 and T2) of training (n = 6). S+ was amyl acetate, and S− was mineral oil. The dashed line represents chance level (50%), and the dotted line represents performance criterion (85%). D, A coronal section of the left OB stained with the nuclear dye TOTO3 to visualize the different layers. E, BrdU⁺ cell density in the GL, EPL, and GCL after training. *p = 0.005 (n = 6). F, Representative images showing BrdU⁺ cells in the GCL of control and trained animals 24 d after BrdU administration. G, Double-labeled cells in the GCL 24 d after BrdU administration, showing the colocalization of BrdU (red) with NeuN (green). Scale bars: D, 100 μm; F, G, 15 μm. Error bars indicate the SEM.

Figure 2.

Opposite effects of olfactory learning on neuronal survival. A, Changes in BrdU⁺ cell density over time in naive animals after BrdU administration (day 0) (n = 6–7). B, New cells were labeled with BrdU, and their survival was evaluated 11, 18, 23, 30, 38, and 65 d later. Nine days before perfusion, trained mice were subjected to 1 week of olfactory training with a single odorant (n = 6–7). C, Mean percentage of correct responses in each block of the 7 d (T1–T7) of training (n = 6–7). S+ was linalool (decreasing concentrations), and S− was mineral oil. The dashed line represents chance level (50%), and the dotted line represents performance criterion (85%). D, BrdU⁺ cell density in the GCL after training. Values were normalized with respect to controls. C, Control mice, T, trained mice. **p ≤ 0.001 (n = 6–7). Error bars indicate the SEM.

Figure 3.

Olfactory learning has no effect on the first steps of neurogenesis. A, A sagittal section of the forebrain showing the different neurogenic zones labeled with BrdU. Inset, Higher magnification of BrdU staining. B, BrdU⁺ cell density in the SVZ, RMSv, and RMSh after training (n = 5). C, A sagittal section of the forebrain showing the SVZ–OB pathway labeled with DCX (red) and DAPI (blue) (left) and a higher magnification of the DCX/DAPI staining (right). D, Area of the SVZ, RMSv, and RMSh after training (n = 5). Scale bars: A, 250 μm; A, inset, 20 μm; C (left), 1 mm; C (right), 10 μm. Error bars indicate the SEM. LV, Lateral ventricle, Ctx, cortex.

Figure 4.

Newly generated deep GCs are more sensitive to olfactory learning than superficial GCs. A, A schematic diagram of the wiring of the OB, showing two populations of GCs as a function of location within the GCL. B, New cells were labeled with BrdU, and their survival was evaluated 30 and 38 d later. Nine days before perfusion, trained mice underwent olfactory training (n = 5). S+ was linalool, β-ionone, or amyl acetate, and S− was mineral oil. C, Quantification of BrdU⁺ cell distribution along the entire internal–external axis of the GCL (D, dorsal; L, lateral; V, ventral; M, medial). D, Distribution of BrdU⁺ cell density along the internal–external axis of the GCL, after training, for animals perfused 30 d after BrdU administration. Inset, Comparison of BrdU⁺ cell density for deep (rings 1–5) and superficial (rings 6–10) GCs. **p = 0.001 (n = 5). E, Distribution of BrdU⁺ cell density along the internal–external axis of the GCL, after training, for animals perfused 38 d after BrdU administration. Inset, Comparison of BrdU cell density for deep and superficial GCs. **p < 0.001 (n = 5). Scale bars, 100 μm. Error bars indicate the SEM. MCL, Mitral cell layer.