Newborn neurons in the olfactory bulb selected for long-term survival through olfactory learning are prematurely suppressed when the olfactory memory is erased

Abstract

A role for newborn neurons in olfactory memory has been proposed based on learning-dependent modulation of olfactory bulb neurogenesis in adults. We hypothesized that if newborn neurons support memory, then they should be suppressed by memory erasure. Using an ecological approach in mice, we showed that behaviorally breaking a previously learned odor-reward association prematurely suppressed newborn neurons selected to survive during initial learning. Furthermore, intrabulbar infusions of the caspase pan-inhibitor ZVAD (benzyloxycarbonyl-Val-Ala-Asp) during the behavioral odor-reward extinction prevented newborn neurons death and erasure of the odor-reward association. Newborn neurons thus contribute to the bulbar network plasticity underlying long-term memory.

Figure 1.

a, Timing of the experiment. BrdU was administered 13 d before behavioral training. Mice were trained using an olfactory cue from D1 to D5, and then using a visual cue from D6 to D10. Retention of the olfactory task was tested on D14, and the animals were killed 1 h after the test. b, Behavioral suppression of an olfactory associative memory trace. Mice were first conditioned using an olfactory cue and then using a visual cue in the presence of the randomly presented odorant. Latency declined with sessions for olfactory (F_(4,45) = 7.86, p < 0.0001) and visual learning (F_(4,45) = 51.25, p < 0.0001), indicating that both were effective. Upon retention of the olfactory task, latency was higher than at D5 (D5 vs D14, p < 0.0001). A control group was similarly conditioned with the odorant (decrease in latency, F_(4,45) = 15.36, p < 0.0001) but underwent pseudo-conditioning with the visual cue in the absence of any odor (no decrease in latency, F_(4,45) = 0.21, p = 0.92). Latency during the retention test was not different from the last day of olfactory conditioning (D5 vs D14, p > 0.05), indicating that the olfactory task was remembered. c, Olfactory pseudo-conditioned mice did not learn the associative task (no decrease in latency, F_(4,45) = 0.54, p = 0.7), while the second visual conditioning task in the presence of the random odorant was acquired (F_(4,45) = 86.84, p < 0.0001). Latency during the retention test was similar to the end of pseudo-conditioning (D5 vs D14, p > 0.05). d, Effect of visual conditioning in the absence of the random odor on retention of the olfactory task. Olfactory and visual conditioning were acquired (F_(4,40) = 1.62, p < 0.0001; and F_(4,40) = 45.15, p < 0.0001, respectively). Latency during the retention test was similar to that on the last day of olfactory conditioning (D5 vs D14, p > 0.05), indicating that the olfactory task was remembered.

Figure 2.

a, Experimental groups (same as in Fig. 1). b, Representative BrdU-positive cell (bi) and newborn (BrdU-positive) cell counts (bii) in the granule cell layer of the OB. Between-group differences were found (F_(3,14) = 12.22, p < 0.0001). BrdU-positive cell density was higher in the groups remembering the associative olfactory task (groups 2 and 4) compared with the groups that forgot the task or had not learned it (groups 1 and 3). *p < 0.05; **p < 0.01; ***p < 0.001. c, Neuronal differentiation of newborn cells was assessed by BrdU/NeuN double labeling (ci) and was similar in all four experimental groups (cii) (F_(3.8) = 0.74 p = 0.56). d, Example of BrdU/Zif268 double labeling (di) and double-labeled cell counts (dii) showed differential involvement of newborn neurons during the retention test (F_(3,10) = 12.2, p < 0.001). *p < 0.05;**p < 0.01; ***p < 0.001. e, Example of BrdU/c-fos double labeling (ei) and double-labeled cell counts (eii) showed differential involvement of newborn neurons during the retention test (F_(3,10) = 21.5, p < 0.0001). *p < 0.05; ***p < 0.001. Scale bars: bi, ci, di, ei, 5 μm.

Figure 3.

a, Behavioral suppression of the odor–reward association using +carvone as the olfactory cue. Mice were first submitted to the +carvone–reward conditioning then to extinction (visual cue in the presence of the random odorant) (Extinction). Latency declined with sessions for olfactory (F_(4,36) = 8.83, p < 0.0001) and visual (F_(4,36) = 15.99, p < 0.0001) learning, indicating that both types of learning occurred. Upon retention test of the olfactory task, latency was higher than at D5 (D14 vs D5, p < 0.05). A control group was similarly conditioned but in the absence of any odor during visual conditioning (Control). Both olfactory (decrease in latency, F_(4,32) = 7.43, p < 0.0001) and visual conditioning (F_(4,32) = 6.14, p < 0.001) were effective. Latency during the retention test was not different from the last day of olfactory conditioning (D14 vs D5, p > 0.05), indicating that the olfactory task was remembered. b, BrdU-positive cell density was higher in the Control group, which remembered the associative olfactory task, compared with the Extinction group, which forgot the task. c, BrdU-positive cell density was higher in the ZVAD-treated group, which remembered the associative task, compared with the saline group, which forgot the task. *p < 0.05.

Figure 4.

Behavioral suppression of the olfactory associative memory trace was prevented by ZVAD infusions in the OB. a, Animals were implanted beforehand with bilateral cannulae in the OB, and ZVAD was infused after each session of extinction. b, Behavioral data. In the two groups, olfactory (decrease in latency: Saline, F_(4,36) = 8.53, p < 0.0001; ZVAD, F_(4,36) = 23.28, p < 0.0001) and visual (decrease in latency: Saline, F_(4,36) = 5;17, p < 0.005; ZVAD, F_(4,36) = 3.20, p < 0.05) conditioning were effective. Latency in the retention test increased compared with the last day of olfactory training in the Saline group (D14 vs D5, p < 0.05) but not in the ZVAD group (D5 vs D14, p > 0.05).