Odor exposure paradigms

Novel odor stimulation. The IEG response of granule neurons was assessed in mice that had no previous experience with the test odors, “odor battery A.” Mice were administered BrdU for 4 d and allowed to mature for 1, 2, 3, 4, 5, 7, or 16 weeks. The mice were exposed to the odors as described below (see below, Odor stimulation) and perfused 1 h later (Fig. 1 A).

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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.

Extended odor familiarization. To examine whether the population response of adult-born neurons is modified by experience, we familiarized mice with a battery of odors and examined the IEG response to those same odors (Fig. 1 B). Mice were repetitively exposed to either odor battery A or odor battery B from 2 to 6 weeks after BrdU administration. At 7 weeks, the population response of adult-born and preexisting granule neurons to the same battery of familiarized odors was examined. Odor battery A was used in the novel odor familiarization and extended familiarization experiments, making the results directly comparable.

Distractor odor familiarization. To examine whether extended familiarization increases the response of adult-born neurons specifically or nonspecifically, we familiarized the mice with odor battery B before testing their response to odor battery A (Fig. 1C). Mice were repetitively exposed to odor battery B from 2 to 6 weeks after BrdU administration. At 7 weeks, the population response of adult-born and preexisting granule neurons to odor battery A was examined.

Early odor familiarization. To examine whether the effects of odor familiarization are long lasting, we examined the responses of adult-born neurons after an early, brief period of odor familiarization (Fig. 1 D). Mice were exposed to odor battery A on D15, D16, and D17, after BrdU administration. They were allowed to mature until 3, 4, 5, or 7 weeks, and then their response to odor battery A was assessed.

Odor stimulation

In the experiments assessing the effects of novel and familiarized odors on adult-born granule neuron activation, mice were housed in groups of 3-5 mice per cage, and they were familiarized to the odorants in the presence of other mice. Mice were housed under standard conditions, with routine exposure to a variety of animal colony odors between periods of stimulation (except for the experiments described in Fig. 5B). To better control the odor environment, the awake and alert mice underwent the final odor stimulation individually in the absence of food and water. Mice were housed individually in microisolator cages receiving 12 L/min of humidified, HEPA-filtered air for 8-24 h to reduce background activity caused by previous odor exposure. In experiments using c-fos as a measure of activation, mice received 8-12 h of clean air. In experiments using c-jun and EGR-1/zif268 as measures of activation, mice received 24 h of clean air. EGR-1/zif268 and, in particular, c-jun had higher levels of background expression than c-fos and required a significantly longer period of clean air exposure. Control and experimental mice were odor stimulated in parallel, and their brains were fixed, sectioned, and stained together to reduce variability.

Mice were exposed to eight sets of three odors. They were exposed to each set of odors for two intervals of 1 min separated by 1 min of exposure to clean air, to reduce potential effects of desensitization. 12 L/min of HEPA-filtered humidified air was blown over filter paper moistened with the odorants. The test odors (odor battery A) consisted of McCormick (Hunt Valley, MD) food flavorings, food-based odorants, and aliphatic aldehydes, including the following: (1) McCormick Imitation Banana Flavor, Campbell's (Camden, NJ) Beef Broth, and Campbell's Chicken Broth; (2) 2 g/ml yeast extract (Sigma, St. Louis, MO), Imitation Butter, and Imitation Coconut; (3) vanilla extract, anise extract, and almond extract; (4) Imitation Rum, Imitation Brandy, and Imitation Orange; (5) lemon extract, peppermint extract, and Imitation Strawberry flavor; (6) oyster-flavored sauce (China Bowl, Westport, CT), sesame oil (China Bowl), and Root Beer Flavor; (7) Pentanal (Sigma), hexanal (Sigma), and heptanal (Sigma); (8) octanal (Sigma), nonanal (Sigma), and decanal (Sigma). Aliphatic aldehydes were dissolved 1:10 in mineral oil. Three hundred microliters of the food-based odorants and 15 μl of the aliphatic aldehydes were used to stimulate the mice.

The second battery of odors (odor battery B), used in both the alternate familiarization and the distractor familiarization experiments, consisted of qualitatively distinct floral odors of an intensity similar to the test odors. They predominantly included perfume odors from The Body Shop (Wake Forest, NC), including the following: (1) Boyajian (Canton, MA) chili oil, basil oil, and garlic oil; (2) The Body Shop Juba, Mango, and Jasmin; (3) Japanese Musk, White Musk, and Mari Mari; (4) Satsuma, balsamic vinegar, and soy sauce; (5) Cedarwood, Woody Sandal-wood, and Cucumber; (6) Oceanus, Fuzzy peach, and Tea Rose; (7) Dewberry, Lilac, and Spirit of Moonflower; (8) Patchouli, Ananya, and olive oil.

In the experiments presented in Figure 5B, investigating extended familiarization and survival, we housed mice in a reduced-odor environment. Mice were housed individually in positive-pressure microisolator cages receiving ~5 L/min of HEPA-filtered air from days 7 to 49 after BrdU administration, limiting their odor exposure. Standard rodent colony conditions contain a variety of odors from other animals and strongly aromatic cleaning and antiseptic solutions, which could potentially confound the survival experiments.

Retroviral labeling of adult-born neurons

A retrovirus encoding wheat germ agglutinin (WGA), a trans-synaptic tracer, and green fluorescent protein (GFP) was used to elucidate the morphology of adult-born neurons and attempt to examine whether they formed synapses with their putative synaptic partners. WGA was cloned into the chicken matrix metalloproteinase vector (CMMP)-internal cytomegalovirus (ICMV)-internal ribosomal entry site (IRES)-enhanced GFP (eGFP) vector [gift from R. Mulligan, Harvard Medical School, Boston, MA (Klein et al., 2000)] at the 5′SacII and 3′BglII restriction sites to yield CMMP-ICMV-lectin-IRES-eGFP. The internal CMV promoter within the 5′ viral long-terminal repeat and the internal ribosome entry sequence yields bicistronic expression of both WGA and eGFP after integration into the target cell genome. For viral production, 150 mm plates of 293T cells underwent standard calcium phosphate transfection with 35 μg of pMD-gag-pol, 35 μg of pMD-G, which encodes vesicular stomatitus virus protein G (Niederman et al., 2002), and 40 μg of CMMP-ICMV-lectin-IRES-eGFP. Viral supernatant was passed through a 0.45 μm filter (Nalge, Hereford, UK) 28 h after transfection, concentrated by centrifugation (SW28 rotor at 25,000 × g, 4°C, for 1.5 h), and resuspended in 250 μl of a solution containing Tris-HCl, NaCl, and ethylenediaminetetraacetic acid. The titer of concentrated viral stock on NIH 3T3 cells was ~3 × 10⁸ infectious particles per milliliter as determined by fluorescence-activated cell sorter analysis.

Concentrated retroviral particles (2.5 μl) were injected bilaterally into each lateral ventricle 2-6 weeks before analysis. A Drummond Scientific (Broomall, PA) Nanoject Variable oocyte injector with a glass micropipette was used to inject the retrovirus 1 mm lateral to midline, 0 mm anterior/posterior to bregma, and 1.8 mm below the surface of cortex.

Immunocytochemistry

Immunocytochemistry was performed using primary antibodies against GFP (A11122; anti-rabbit; 1:1000; Invitrogen, Eugene, OR); synaptophysin (MAB368; anti-mouse; 1:1000; Chemicon, Temecula, CA); c-fos (ab-5; anti-rabbit; 1:2500; Oncogene Research, San Diego, CA); c-jun (sc-1694; rabbit, 1:750; Santa Cruz Biotechnology, Santa Cruz, CA); EGR-1/zif-268 (sc-110; anti-rabbit; 1:1500; Santa Cruz Biotechnology); BrdU (OBT0030; anti-rat, 1:400; Accurate Chemical, Westbury, NY); and neuronal-specific nuclear protein (NeuN) (MAB 377; anti-mouse; 1:500; Chemicon). Highly cross-adsorbed secondary antibodies were used to minimize cross-reactivity. We used Invitrogen secondary antibodies, including Alexa 488 anti-rabbit (A11034; 1:250); Alexa 546 antimouse (A11030; 1:750); Alexa 488 anti-rat (A11006; 1:250); Alexa 546 anti-rabbit (A11035; 1:750); Alexa 633 anti-mouse (A21052; 1:333). Vibratome-cut sections (30-μm-thick) were incubated in primary antibodies overnight and secondary antibodies for 2 h at 4°C. Antibodies were diluted in blocking solution containing 8% goat serum, 0.3% bovine serum albumin, and 0.3% Triton-X 100 dissolved in PBS. The BrdU antigen was exposed by incubating sections in 2 m HCl for 2 h. We used Hoechst 33258 (Sigma) as a nuclear stain. We stained control and experimental sections in parallel 24 h after perfusion. Sections were mounted on gel-subbed slides and coverslipped in PBS, which preserved the three-dimensional structure of the sections and avoided compression artifacts.

Microscopy

Confocal images were obtained using a Zeiss (Oberkochen, Germany) LSM 510 laser-scanning confocal microscope. Fluorescence images were obtained using a Zeiss Axioplan and a SPOT cooled CCD digital camera.

Odor familiarization increases the population response of adult-born neurons

To examine whether the population response of adult-born neurons is modified by experience, we familiarized mice to odor battery A, consisting of food-based odors, and examined the IEG response of granule neurons to those same odors. Control mice were not familiarized with the odors before testing their response to the odors; thus, the odors were novel for these mice. The same battery of odors was used in the novel odor-exposure experiments described above and in these familiarization experiments, making the results directly comparable.

Extended odor familiarization significantly increases the percentage of adult-born neurons that express c-fos in response to the test odors (Fig. 4C) (two-tailed t test, t = 9.619, p = 0.0001). By 7 weeks, 14.5 ± 2.5% of adult-born neurons respond to the battery of novel odors; however, after familiarization, 25.8 ± 2.1% of adult-born neurons respond (n = 8 mice; 1218 ± 224 cells/mouse). The response of both control and experimental mice was assessed at 7 weeks, controlling for variation in IEG expression resulting from differences in developmental state. A qualitative inspection of the samples suggested that adult-born granule neurons appear to have brighter, more intense c-fos immunostaining in response to stimulation after familiarization than they do to novel odor exposure.

In contrast to adult-born neurons, the overall granule neuron population becomes less responsive after extended odor familiarization (Woo et al., 1996; Allingham et al., 1999) (two-tailed t test, t = 5.217, p = 0.0001) (Fig. 4C). The overall population of granule neurons consists predominantly of perinatally generated and already mature adult-born neurons; recently generated adult-born neurons form a small fraction of all granule neurons (Petreanu and Alvarez-Buylla, 2002; Winner et al., 2002). This overall reduction in olfactory response after familiarization, which has been characterized previously using both IEG-based (Woo et al., 1996; Allingham et al., 1999) and electrophysiological techniques (Buonviso and Chaput, 2000), is thought to represent tuning of olfactory receptive ranges, resulting in a reduction of nonspecific activity. Consistent with this interpretation, we observed that, after familiarization, the pattern of activation in the granule and periglomerular layers is more distinct and less diffuse (S. S. P. Magavi, unpublished results). Our results indicate that adult-born olfactory granule neurons and preexisting neurons have contrasting responses to familiarization.

To assess the effects of familiarization on adult-born neurons using different measures of activity, we examined the effects of extended odor familiarization using both c-jun and EGR-1/zif268 expression, IEGs that are also upregulated by electrophysiological activation (Cole et al., 1989; Baba et al., 1997). Experiments using these independent markers of activation confirmed that a significantly greater percentage of adult-born neurons are activated in odor-familiarized mice than in control mice (Fig. 4D); thus, odor familiarization increases the adult-born granule neuron population response (two-tailed t test, c-jun, t = 5.972, p = 0.0094; EGR-1/zif268, t = 4.465, p = 0.0209). These experiments were performed independently of the c-fos experiments, in separate groups of mice. The different IEGs have different thresholds for detection and/or protein expression, contributing to the different numbers of cells activated in each case (Fig. 4D). These data demonstrate that the adult-born olfactory granule neuron population undergoes experience-dependent modification of its IEG response to physiological odorant stimuli in awake mice.

To examine whether familiarization with different types of odors can also increase the IEG response of adult-born neurons, we familiarized mice with an alternate battery of odors and examined their IEG response to that alternate battery of odors. The alternate set of odors, odor battery B, consists primarily of perfume-based floral odors (see Materials and Methods) that smell qualitatively different from the food- and spice-based odors in odor battery A. Theoretically, the odors used in odor battery A could have had unique properties that contribute to the changes in the adult-born neuron population response induced by familiarization. We observed the same result with battery B as with battery A: familiarization with odor battery B increased the response of the adult-born neurons to odor battery B (Fig. 4E) (two-tailed t test, t = 7.307, p < 0.0001; n = 5 mice per group). The dramatic qualitative differences between the two batteries of odors suggest that the increase in population IEG response observed in recently generated adult-born neurons can be induced with a variety of odors, not only those used in our experiments. Attributable simply to differences between the two odor batteries, odor battery B activated a greater percentage of the adult-born neuron population than did odor battery A in the control, novel odor exposure paradigm (unpaired t test, t = 6.372, df = 10. p < 0.001). These experiments indicate that experience-dependent modification of the adult-born neuron population response generalizes across a broad range of odors; familiarization with a variety of odors increases the IEG response of adult-born neurons to those same odors.

The effects of odor familiarization are odor-battery specific

We next examined whether the effects of odor familiarization are specific: whether extended familiarization increases the response of adult-born neurons specifically, only to the odors with which the mice are familiarized, or nonspecifically to all odors. To test the specificity of the increased adult-born neuron response, we familiarized the mice to an alternate battery of 24 different “distractor” odors, the primarily floral-based odors of odor battery B, before assessing their IEG response to the food-based test odors we used previously, in odor battery A (Fig. 1C). Although the two odor batteries may have some chemical overlap in component odorants, to human observers, the odor batteries smell quite different.

Extended familiarization with distractor odors does not increase the response of adult-born neurons to the test odors (t test, t = 1.088, p = 0.3081) (Fig. 4F). We found that 14.1 ± 3.7% of adult-born olfactory granule neurons in control mice responded to the test odors (n = 5 mice; 983 ± 59 cells examined/mouse), as did 12.1 ± 1.2% of adult-born neurons in mice familiarized with the distractor odors (n = 5 mice, 1033 ± 68 cells examined/mouse), a statistically insignificant difference (two-tailed t test, t = 1.088, p = 0.3081). The increased response of recently adult-born neurons after odor familiarization is restricted to the battery of odors used to familiarize the mice, demonstrating that adult-born neurons undergo a stimulus-specific form of experience-dependent modification.

Although adult-born neurons undergo a stimulus-specific modification after odor familiarization, preexisting granule neurons undergo a nonspecific reduction in their response after familiarization. Extended familiarization with the distractor odors results in a statistically significant decrease in the number of preexisting neurons that respond to the alternate set of “test” odors (two-tailed t test, t = 2.939, p = 0.0424) (Fig. 4F). This result, which is consistent with previous reports (Woo et al., 1996; Allingham et al., 1999; Buonviso and Chaput, 2000), indicates that exposure to a broad variety of odors nonspecifically decreases the response of the preexisting olfactory granule neurons. Although these effects on preexisting neurons are nonspecific, the experience-dependent modification of responses of recently generated adult-born neurons that are induced by odor familiarization is quite specific. These experiments demonstrate that recently generated adult-born neurons respond quite distinctly from preexisting granule neurons to olfactory stimuli in vivo.

The effects of odor stimulation are long lasting

To examine whether the effects of odor familiarization are long lasting, we examined the responses of adult-born neurons after an early, brief period of odor familiarization. These experiments also serve to characterize how the response of adult-born neurons evolves over time. An early, brief period of odor familiarization is sufficient to produce long-lasting changes in the population response of adult-born neurons (Fig. 4G). To examine the effects of early, brief olfactory familiarization, we administered BrdU to mice, familiarized them with the test odors on days 15, 16, and 17, and then examined the responses of adult-born neurons at weeks 3-7 (Fig. 1D). Adult-born neurons are highly responsive to novel odors on days 15-17, soon after they synaptically integrate. At week 3, there is no significant difference in the response of adult-born neurons to novel versus familiar stimuli (two-tailed t test, t = 1.129, p = 0.3221). However, as the adult-born neurons mature, their response to the familiarized odors becomes significantly greater than their response to novel odors (two-tailed t test, t = 4.403, p = 0.006). Even 7 weeks after early familiarization, 19.6 ± 2.1% of recently adult-born neurons still respond to the familiarized odors (n = 8 mice, 1195 ± 126 cells/mouse), compared with only 14.5 ± 2.6% of adult-born neurons responding to novel odors. These results demonstrate that even a brief period of odor familiarization induces long-lasting changes in the recently generated adult-born neuron population.

Early, brief odor familiarization results in an immediate decrease in the response of the overall population of granule neurons that lasts for several weeks (Fig. 4H). Just a few days after familiarization, at week 3, the response of the overall population of neurons is significantly decreased (t test, t = 4.520, p = 0.01). The responses of the overall population of granule neurons to novel and familiarized odors are no longer significantly different by week 7. Together, these data further illustrate that early, brief familiarization has opposite effects on recently generated adult-born neurons versus preexisting neurons.

Adult-born neuron response varies as a function of maturity

The population of adult-born neurons is most responsive to novel odors soon after the neurons undergo synaptic integration. Three weeks after their generation, adult-born neurons are more likely to express IEGs in response to novel odorants than are preexisting neurons. As they mature, adult-born neurons become less responsive, but they reach a plateau by ~2 months and continue to respond to novel odors for at least 4 months. Changes in the IEG response of recently generated adult-born neurons may reflect modifications including changes in neurotransmitter receptors, electrophysiological characteristics of the neurons, or the threshold for IEG activation. These experiments form a baseline for our other experiments; familiarization-induced changes in IEG responses of adult-born neurons were assessed at the same time after neuronal birth, reducing variability attributable to developmental state. Our data indicate that adult-born granule neurons have a unique role in olfactory processing; there is a striking contrast between the stable IEG response of the overall population of granule neurons and the early peak responsiveness of adult-born neurons. Because adult-born olfactory neurons are continuously being generated, there is always a cohort of highly responsive, recently generated neurons, providing a source of plasticity in olfactory circuitry.

Our results indicate that recently generated neurons and mature neurons have differing properties in vivo and are compatible with in vitro experiments showing that these populations of neurons have differing electrophysiological properties. Recently generated periglomerular olfactory neurons have a greater Na⁺ current than more mature neurons (Belluzzi et al., 2003). Recently generated adult-born hippocampal granule neurons have a lower threshold for initiating action potentials and LTP than more mature neurons (Schmidt-Hieber et al., 2004). Surgically induced olfactory deprivation preferentially modifies the morphology and increases the excitability of newly generated olfactory neurons in vitro (Saghatelyan et al., 2005). Adult-born neurons also differ from neonatal neurons in their tendency to be located deeper rather than more superficially within the granule cell layer (Lemasson et al., 2005). Thus, both previous data and our results, gathered in response to physiological odor stimulation in vivo, indicate that adult-born neurons have distinctive properties.

The population response of adult-born neurons is modified by experience

Odor familiarization modifies the response properties of adult-born olfactory granule neurons in vivo. Familiarizing mice with test odors increases the response of the recently generated adult-born neuron population to the test odors. Brief exposure to the test odors is sufficient to induce long-lasting changes in the adult-born neurons' response. In these experiments, we made comparisons between adult-born neurons at the same stages of maturity, eliminating potential differences resulting from maturational influences on IEG expression. These results demonstrate, in vivo, that the response of adult-born neurons is modified by olfactory experience in alert mice.

The increased response of adult-born olfactory granule neurons after familiarization with test odors might be specific to the battery of test odors, or it might be nonspecific. In theory, environmental enrichment could lower the threshold for activation in response to all odors, or otherwise allow them to respond more strongly to any odors. However, familiarization with distractor odors does not increase the IEG response of adult-born neurons to the original test odors. These results demonstrate that the increase in adult-born neuron responsiveness is stimulus specific, strongly supporting the interpretation that adult-born neurons become modified to preferentially respond to a particular set of stimuli.

Theoretically, the increased response of adult-born granule neurons after familiarization might be solely attributable to an increased response in neurons “upstream” of the olfactory granule neurons. Increased upstream activity would lead to increased activity of all olfactory granule neurons. However, the increased response after familiarization is unique to recently generated granule neurons, and the response of preexisting granule neurons is decreased. Because recently generated granule neurons and preexisting granule neurons have contrasting responses to familiarization with the same stimuli, the changes we observed must be occurring at the level of granule neurons. The two most likely possibilities for the changes we observe in the response of adult-born neurons are that there is (1) a modification in the adult-born granule neurons themselves or (2) a synaptic modification that occurs only between mitral, tufted, or centrifugal cells and adult-born granule neurons but not between these cells and preexisting granule neurons.

Adult neurogenesis might simply contribute new neurons with the same properties as preexisting, mature neurons. These new neurons might be simply replacing older, damaged neurons or contributing greater numbers of functionally identical units to the olfactory bulb. Alternatively, recently generated adult-born neurons might make distinct contributions to olfactory circuitry. In contrast to recently generated adult-born neurons, the overall granule neuron population becomes less responsive after odor familiarization. The decreased overall response after familiarization suggests that the receptive fields of granule neurons are being tuned, reducing nonspecific activity. Thus, it is likely that olfactory bulb plasticity occurs both via integration of adult-born neurons and via modification of preexisting neurons. The contrasting effects of odor familiarization on adult-born and preexisting neurons indicate that recently generated neurons have unique properties that distinguish them from older neurons.