Adult-born and preexisting olfactory granule neurons undergo distinct experience-dependent modifications of their olfactory responses in vivo

Abstract

Neurogenesis continues throughout adulthood in the mammalian olfactory bulb and hippocampal dentate gyrus, suggesting the hypothesis that recently generated, adult-born neurons contribute to neural plasticity and learning. To explore this hypothesis, we examined whether olfactory experience modifies the responses of adult-born neurons to odorants, using immediate early genes (IEGs) to assay the response of olfactory granule neurons. We find that, shortly after they differentiate and synaptically integrate, the population of adult-born olfactory granule neurons has a greater population IEG response to novel odors than mature, preexisting neurons. Familiarizing mice with test odors increases the response of the recently incorporated adult-born neuron population to the test odors, and this increased responsiveness is long lasting, demonstrating that the response of the adult-born neuron population is altered by experience. In contrast, familiarizing mice with test odors decreases the IEG response of developmentally generated neurons, suggesting that recently generated adult-born neurons play a distinct role in olfactory processing. The increased IEG response is stimulus specific; familiarizing mice with a set of different, "distractor" odors does not increase the adult-born neuron population response to the test odors. Odor familiarization does not influence the survival of adult-born neurons, indicating that the changes in the population response of adult-born neurons are not attributable to increased survival of odor-stimulated neurons. These results demonstrate that recently generated adult-born olfactory granule neurons and older, preexisting granule neurons undergo contrasting experience-dependent modifications in vivo and support the hypothesis that adult-born neurons are involved in olfactory learning.

Figure 1.

Olfactory stimulation and familiarization. A, Novel odor stimulation. Mice were administered BrdU and allowed to mature, and their response to the test odors was assessed from 1 to 16 weeks later. B, Extended odor familiarization. Mice were repetitively exposed to the test odors from 2 to 6 weeks. At 7 weeks, their response to those same test odors was assessed. The black nose indicates test odors. C, Distractor odor familiarization. Mice were exposed to a distractor battery of odors, odor battery B (red nose), from 2 to 6 weeks before their response to odor battery A was assessed at 7 weeks. D, Early odor familiarization. Mice were exposed to the test odors on D15, D16, and D17 and allowed to mature for 3, 4, 5, or 7 weeks, and then their response to the test odors was assessed. E, Odor stimulation. Individually housed, alert mice received 12 L/min of clean, HEPA-filtered air (see Materials and Methods) for 8-24 h. They were exposed to eight sets of three odors. They were exposed to each set of odors for two intervals of 1 min separated by a 1 min exposure to clean air, to reduce potential effects of desensitization. They were perfused 1 h after odorant exposure to allow expression of IEGs. Odor battery A consisted of 24 primarily food-based odors, and odor battery B, the distractor odors, consisted of 24 primarily floral odors. Control mice received no olfactory stimulation.

Figure 2.

Adult-born granule neurons differentiate into neurons and appear to form synapses both in the external plexiform layer and the granule cell layer itself. A, Hoechst (blue) stains nuclei (arrowheads) and reveals the granule cell layer (GCL), the mitral cell layer (MCL), and glomeruli (Glom). Intraventricular injection of a GFP-encoding (green) retrovirus labels adult-born granule neurons (arrow), which extend processes into the external plexiform layer (EPL) and the GCL and form boutons (B, arrowheads) that are closely apposed to synaptophysin (red)-rich presynaptic densities (C, dashed circles), suggesting the existence of synapses.

Figure 3.

Olfactory stimulation specifically increases the number of granule neurons expressing c-fos. A, Mice receiving only clean, HEPA-filtered air have very few c-fos-positive (white) granule neurons. The specks in the ventral glomerular layer are autofluorescent fixation artifacts and are easily distinguished from genuine cellular labeling at higher magnification. B, Novel odor exposure dramatically increases the number of c-fos-positive granule, mitral, and periglomerular neurons. C-E, Exposure to a single odorant, such as methyl-isovalerate, reproducibly activates specific bilaterally symmetric subregions of the olfactory bulb. D, An activated subregion at high magnification. E, An unactivated subregion immediately adjacent to D. F-I, Confocal imaging reveals that BrdU⁺ (green) adult-born cells in the granule layer (GCL) differentiate into mature NeuN-expressing neurons (blue) and express c-fos (red) in response to olfactory stimulation. The arrows indicate activated adult-born neurons. The arrowhead indicates an inactive adult-born neuron. Scale bar, 10 μm.

Figure 4.

Odor familiarization specifically increases the response of adult-born neurons but depresses the response of the overall population of granule neurons. A, Adult-born neurons are activated by odor stimulation, assessed by c-fos expression. B, Adult-born neurons are most responsive soon after they synaptically integrate; as they mature, they become less responsive. Preexisting granule neurons maintain a stable response. C, Extended odor familiarization (Ext. familiar.) increases the percentage of adult-born neurons that respond to the same odors, indicating that in vivo experience modifies the response of the adult-born neuron population. In contrast, the population of preexisting neurons becomes less responsive after odor familiarization, indicating that adult-born and preexisting neurons undergo distinct modifications in response to olfactory experience. D, Assessing activity with alternate IEG markers of activation confirms that extended odor familiarization increases the response of adult-born neurons. Independent experiments, using c-jun or EGR-1/zif268, demonstrate that extended odor familiarization increases the percentage of adult-born neurons activated by the familiarized odors. E, These experience-dependent changes are not unique to one set of odors; an alternate battery of qualitatively different odors also increases the response of the population of adult-born neurons. F, Alternate odor familiarization does not increase the response of the adult-born neuron population to the test odors, demonstrating that the change in response is specific to the familiarized odors. G, Brief, early odor familiarization yields a long-lasting increase in the response of the adult-born granule neuron population. Adult-born neurons exposed to novel odors on days 15, 16, and 17, during their period of synaptic integration, have an increased response to those same odors at least 5 weeks later. H, Early familiarization immediately depresses the response of the overall granule neuron population. IEG expression examined in n = 3 mice per data point (unless otherwise indicated) in ∼1100 BrdU⁺ cells and ∼950 NeuN⁺ cells per mouse in a blinded, systematic manner (see Materials and Methods). Two sets of data are displayed as components of multiple panels for clarity of presentation; these data are illustrated in either red or green. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001. Error bars indicate SD.

Figure 5.

Odor exposure does not influence the long-term survival of adult-born olfactory granule neurons in normal, wild-type mice, indicating that the enhanced population response observed after odor familiarization is not attributable to increased survival of odor-stimulated neurons. A, The number of adult-born granule neurons decreases during the first 2 months but is relatively stable thereafter. B, Extended familiarization to either odor battery A or odor battery B does not enhance the survival of adult-born olfactory granule neurons (data shown at 7 weeks). Control and experimental mice were housed individually under clean air conditions to limit their exposure to external odors. C, Repeated odor familiarization does not influence the proliferation of endogenous progenitors in the SVZ, RMS, or olfactory bulb. Error bars indicate SD. n = 3 mice examined, unless otherwise indicated.