Olfactory discrimination learning increases the survival of adult-born neurons in the olfactory bulb

Abstract

In the olfactory bulb (OB), new neurons are added throughout life, forming an integral part of the functioning circuit. Yet only some of them survive more than a month. To determine whether this turnover depends on olfactory learning, we examined the survival of adult newborn cells labeled with the cell division marker BrdU, administered before learning in an olfactory discrimination task. We report that discrimination learning increases the number of newborn neurons in the adult OB by prolonging their survival. Simple exposure to the pair of olfactory cues did not alter neurogenesis, indicating that the mere activation of sensory inputs during the learning task was insufficient to alter neurogenesis. The increase in cell survival after learning was not uniformly distributed throughout angular sectors of coronal sections of the OB. Monitoring odor activation maps using patterns of Zif268 immediate early gene expression revealed that survival was greater in regions more activated by the non-reinforced odorant. We conclude that sensory activation in a learning context not only controls the total number of newborn neurons in the adult OB, but also refines their precise location. Shaping the distribution of newborn neurons by influencing their survival could optimize the olfactory information processing required for odor discrimination.

Figure 1.

Decline in newborn OB cells between 15 and 30 d after BrdU administration. A, A confocal microscope image of a coronal section through the OB of a 2-month-old MOR23-IRES (internal ribosome entry site)-tau-GFP mouse. Axon terminals of the GFP⁺ (MOR23) sensory neurons converge in two medial glomeruli (asterisks). Arrows indicate axons reaching the OB. Neurons were identified with anti-NeuN staining (red). B, New cells were labeled with BrdU and their survival evaluated either 15 or 30 d after intraperitoneal injections. C, BrdU⁺ cell density significantly decreased between 15 and 30 d, in both GL and GCL. ***p < 0.05, Student's t test (n = 8–9). D, Representative images showing BrdU⁺ cells in the GL and GCL at 15 and 30 d. Asterisk indicates the MOR23-GFP medial glomerulus; arrows point to BrdU⁺ cells. E, Percentage of double-labeled BrdU/NeuN⁺ cells (with respect to total BrdU⁺ cells) in the GL and GCL (n = 3). F, Double-labeled cells (arrows) in the GL and GCL 30 d after BrdU injections showing colocalization of BrdU (red) with NeuN (green). Some newborn cells are NeuN⁻ (arrowhead). In the GL, GFP⁺ axon terminals of MOR23-sensory neurons converged in one medial glomerulus (asterisk, pink). Scale bars, 50 μm. Error bars indicate SE.

Figure 2.

Olfactory discrimination learning between lyral and acetophenone in MOR23 mice. A, Experimental design for this study. B, Olfactory discrimination learning. Values are expressed as percentage of first correct responses during a daily session. **p < 0.01; ***p < 0.001 between paired/unpaired groups with Bonferroni's test after r-mANOVA (n = 15–16). C, Spontaneous exploration of odorants used in the learning task was not significantly different. Data are expressed as the percentage of lyral (pure) and acetophenone (10⁻³) sniffing time normalized to the total sniffing time (n = 7–8). D, Total exposure to the olfactory cues was similar in both groups. Data are expressed as the total number of visits during the entire training period (n = 15–16). ns, Not significant. Error bars indicate SE.

Figure 3.

Local increase of newborn cell survival after olfactory discrimination learning. A, B, Quantification of BrdU⁺ and Zif268⁺ cell distributions, through angular sectors of coronal sections, in the GL and GCL. A, A representative image showing the distinct layers and the medial position of the MOR23 glomerulus. GFP⁺ glomeruli were stained by immunohistochemistry with an anti-GFP antibody and VIP substrate as a chromogen (asterisk). B, The position of the MOR23 glomerulus was used to define the MOR23 axis (red dotted line) and all subsequent angular sectors. The defined sectors were grouped into four areas related to their location in the OB: lateral (sectors 12, 1, and 2), dorsal (sectors 3, 4, and 5), medial (sectors 6, 7, and 8), and ventral (sectors 9, 10, and 11). C, D, BrdU⁺ cell density in the GL (A) and GCL (B) after training. Values were normalized with respect to controls perfused 30 d after BrdU injections. Discrimination learning significantly increased the total density of BrdU⁺ cells when compared with unpaired and control animals in the GCL and with controls in the GL. *p < 0.05 and ^##p < 0.01 with Fisher's PLSD test after ANOVA (n = 9–15). E, Distribution of normalized BrdU⁺ cell density throughout all angular sectors after discrimination training in GCL. *p < 0.05; ***p < 0.001 with Fisher's PLSD test after r-mANOVA. F, Distribution of normalized sensory inputs driven by odorant cues. Spatial distribution of normalized Zif268⁺ cells throughout the radial axis of coronal OB sections after lyral (pure) or acetophenone (10⁻³) exposure. *p < 0.05 between two odorants with Fisher's PLSD test after r-mANOVA. G, H, Survival of newborn neurons was highest where odor activation maps differed most. G, Color-coded representations showing learning-induced survival maps throughout MOR23-containing sections (supplemental methods and Fig. 4, available at www.jneurosci.org as supplemental material). H, A representation similar to that in G showing the acetophenone-specific activation map. EPL, External plexiform layer; L, lateral; D, dorsal; M, medial; V, ventral. Scale bar, 200 μm. Error bars indicate SE.