C57BL/6J mice were obtained from the Animal Resources Centre (Canning Vale, WA, Australia; n=33 for females, n=42 for males, 11~12 weeks old). Upon arrival, mice were kept in standard polysulfone cages (3~5 mice/cage) under a 12:12 h light:dark cycle with food and water available ad libitum. Each standard cage (dimensions at 36 cm×15 cm×13 cm) featured a polycarbonate igloo, nesting material, a metal ring attached to the cage grill, and wooden blocks (Fig. 1A). Mice were allowed to acclimatize to the animal facility for 7 days before the experiments started. Animal experimental procedures were in accordance with the Australian Code for the Care and Use of Animals in Research and were approved by the Animal Care and Ethics Committee of The University of New South Wales (Approved ACEC No. 13/115A).

Environmental enrichment and odor exposure have been reported as effective methods to increase neurogenesis in the SGZ/hippocampus and the SVZ/rostral migration stream/olfactory bulb neurogenic systems respectively [17,18,19]. To test our hypothesis, an odor exposure experiment was undertaken with one cohort of mice to evaluate its effect on hippocampal neurogenesis. The effect of enriched environment on hippocampal neurogenesis was tested in the other cohort of mice, as a control experiment performed in the same animal facility. Specific odors were first tested to determine whether they were pleasant to mice (Fig. 1B). Mice were introduced to an opened bottle containing a piece of filter paper impregnated with 10 µl of an odorous extract (Sydney essential oil companies). The exposure to one type of novel odor was 30 mins per day for three continuous weeks. Odors were provided in a roster for the treatment group (female, n=22), and a bottle containing a non-treated filter paper was provided to the control mice (female, n=11). An enriched environment was set up in cages with larger dimensions (60 cm×38 cm×19 cm) fitted with a balcony, a loft, running wheels, ball, seesaw, tunnels (for making simple and complex mazes), and different types of dome and bedding (Fig. 1C). The environment was rearranged/renewed every second day for three weeks. All treated mice (male, n=21) were exposed to the enriched environment. In contrast, control mice (male, n=21) were housed in standard cages. The experimental scheme is shown in Fig. 2.

Mice were euthanized by injection of a lethal dose of pentobarbitone sodium (0.24 mg/g body weight), and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS. For isotropic fractionator method, brains were post-fixed in the same fixative at 4℃ for 4 days before olfactory bulbs and hippocampi were dissected and weighted. For immunofluorescence, brains were post-fixed in the same fixative at 4℃ overnight then cryoprotected in 30% sucrose for sectioning.

Numbers of neuronal, non-neuronal, and proliferating cells were counted using the isotropic fractionator method [20], the accuracy of which has been found comparable with unbiased stereology [21]. Tissue was manually homogenized in a dissociation solution (1% Triton X-100 in 40 mM trisodium citrate) to ensure the release of intact cell nuclei and the total cell number was determined by labeling the cell nuclei with 4′,6-diamidino-2-phenylindole dilactate (DAPI) (Invitrogen, Mulgrave, VIC, Australia) [14,22,23]. The neuronal cell number was determined using neuronal nuclear protein (NeuN)-specific labeling (1:1000; MAB377, Merck Millipore, Bayswater, Vic, Australia) and Alexa594-conjugated secondary antibody (1:300; A11032, Invitrogen). The following parameters were determined in each olfactory bulb and hippocampus: mass, total cell number, neuronal cell number, non-neuronal cell number (i.e., the total number of cell nuclei less the number of NeuN positive nuclei; mainly glia), and the ratio of non-neuronal/neuronal cell counts (referred to as ‘cellular constitution’). The number of proliferating cells was determined using Ki-67-specific labeling (1:1000; ab15580, Abcam, Melbourne, Vic, Australia) and Alexa594 conjugated secondary antibody (A11037, Invitrogen). The number of Ki-67 positive nuclei was determined in 10 µL homogenized sample (using 2~4 loadings from each case) and extrapolated to the total volume.

Brain regions at the planes of Bregma +2.96 mm (containing the rostral migration stream with proliferating cells migrating to the olfactory bulb) and Bregma −1.46 mm (containing the dentate gyrus with proliferating cells in situ) were sectioned in the coronal plane at 40 µm thickness. Sections were incubated with antibodies detecting doublecortin (DCX; 1:200; sc-8066, Santa Cruz Biotechnology, Dallas, TX, USA) and Ki-67 [14,23]. Immunofluorescence-labeled sections were examined and digitised with a confocal microscope (Nikon Eclipse 90i; Nikon, Tokyo, Japan) equipped with a Nikon D-ECLIPSE C1 high-resolution camera. Brain structures were identified using The Mouse Brain in Stereotaxic Coordinates [24].

All data are presented as mean±S.E.M. Pairwise comparisons between treated and control groups were performed. Statistical significance was established using Student's t-test. In all instances, the level of significance was set at p<0.05. Statistical analysis was performed using the IBM SPSS Statistics program (v22; IBM Corporation, Armonk, NY, USA), and graphs were made using Prism 6 (v6.0b; GraphPad Software, La Jolla, CA, USA).

We first analyzed the type of odor to be used in our study. Odors were categorized as “pleasant” if the mice displayed an interest by one or more of the following behaviors: drag the bottle containing a filter paper impregnated with an odor into the dome, attempt to pull out the filter paper, or repeatedly sniff at the opening of the bottle. Pleasant odors included food flavors of green apple, vanilla, orange, and strawberry as well as bergamot, lemongrass, lemon cold and lavender aromatic oils . The mice displayed strong interest towards these pleasant odors. As a negative control, mice were also exposed to a bottle containing a filter paper without any supplemental odor. The mice showed no interest to the control bottle.

With the daily exposure of novel odor for three weeks, the number of mature neurons significantly increased in the hippocampus (Fig. 3A). This change was not accompanied by a significant increase in the hippocampal mass, glial cell number, or total cell number. As a result, the glial versus neuronal cell number ratio was decreased. Because neither the number of proliferating (i.e., Ki-67-positive) cells changed significantly, nor the density of differentiating (i.e., DCX-positive) cells showed an obvious difference when the same anatomical plane was examined (Fig. 3B), the increased number of neuronal cells was most likely associated with an increased turnover of newly generated neurons or partly from the decrease of the apoptosis of the orginal resident neurons. To precisely describe the effect of novel odor exposure on hippocampal neurogenesis, we compared the results of odor stimulation to those of the ‘traditional’ enrichment method by exposing the other cohort of mice to the enriched housing environment for the same length of time. Results from the enriched housing were found to be similar to those of the odor exposure; i.e., the neuronal number was increased whereas the glia to neuron ratio decreased (Fig. 4).

After a three-week exposure to novel odors, the mature neuronal cell number was markedly increased in the olfactory bulb (Fig. 5A). This change was accompanied by an increase in the total cell number. In contrast, the number of glial cells did not change significantly indicating that the increased total cell number was associated with the increased neuronal cell number. The increased number of neurons and the unchanged number of non-neuronal cells caused a significant alteration in the non-neuronal/neuronal cell number ratio. Further cell counting results revealed an increase in the number of proliferating cells, suggesting that novel, pleasant odors directly facilitate cell proliferation in addition to increasing the turnover of neurons. An increased number of proliferating cells was also visible in the rostral migration stream in the sections (Fig. 5B).