Sensory deprivation disrupts homeostatic regeneration of newly generated olfactory sensory neurons after injury in adult mice

Abstract

Although it is well known that injury induces the generation of a substantial number of new olfactory sensory neurons (OSNs) in the adult olfactory epithelium (OE), it is not well understood whether olfactory sensory input influences the survival and maturation of these injury-induced OSNs in adults. Here, we investigated whether olfactory sensory deprivation affected the dynamic incorporation of newly generated OSNs 3, 7, 14, and 28 d after injury in adult mice. Mice were unilaterally deprived of olfactory sensory input by inserting a silicone tube into their nostrils. Methimazole, an olfactotoxic drug, was also injected intraperitoneally to bilaterally ablate OSNs. The OE was restored to its preinjury condition with new OSNs by day 28. No significant differences in the numbers of olfactory marker protein-positive mature OSNs or apoptotic OSNs were observed between the deprived and nondeprived sides 0-7 d after injury. However, between days 7 and 28, the sensory-deprived side showed markedly fewer OSNs and mature OSNs, but more apoptotic OSNs, than the nondeprived side. Intrinsic functional imaging of the dorsal surface of the olfactory bulb at day 28 revealed that responses to odor stimulation were weaker in the deprived side compared with those in the nondeprived side. Furthermore, prevention of cell death in new neurons 7-14 d after injury promoted the recovery of the OE. These results indicate that, in the adult OE, sensory deprivation disrupts compensatory OSN regeneration after injury and that newly generated OSNs have a critical time window for sensory-input-dependent survival 7-14 d after injury.

Keywords: apoptosis; homeostatic regeneration; olfactory epithelium; olfactory sensory neuron; sensory deprivation.

Figure 1.

Nostril occlusion for 28 d induces no histological changes in uninjured olfactory epithelium. A, Method for unilateral nostril occlusion. Left, Schematic diagram of a 10 mm silicone tube. A hair protrudes from the tube to confirm silicone tube insertion and the inner cavity of the silicon tube is packed with glue. Right, Schematic diagram of the unilateral nostril occlusion method. B, Nasal airflow on days 3 (d 3) and 28 (d 28) after silicone tube insertion for both open (top) and occluded sides (bottom). The nasal airflow on days 3 and 28 after silicone tube insertion is completely abolished on the occluded side but remains unaffected on the open side. Scale bar, 3 s. C, Images of the dorsal surface of the olfactory bulb 3 and 28 d after silicone tube insertion on both occluded (left) and open sides (right). Top image shows odorant-induced intrinsic imaging with TMT and 2-MBA 3 d after silicone tube insertion on both the occluded (left) and open sides (right); bottom image shows both occluded (left) and open sides (right) on day 28 after silicone tube insertion. The odor-induced signals are detected in the open side, but clear signals are not observed in the occluded side on days 3 and 28 after silicone tube insertion. Scale bar, 500 μm. D, Time course and experimental design. Unilateral nostril occlusion (unilateral n.o.) was performed on day 0 and perfusion with a fixative (fix.) was conducted on days 3 (n = 3 mice), 7 (n = 3), 14 (n = 4), or 28 (n = 3) after unilateral nostril occlusion. E, Analyzed area in the OE. Left, 3D reconstructed mouse nose. Red area shows OE and blue shows OB. Bold arrow indicates the caudal edge of the silicone tube; three small arrows indicate the positions of the selected coronal sections. Right, Coronal section from the three selected coronal sections of the OE. Rectangle shows nasal septum, which was the area analyzed. Scale bar, 500 μm. F, Photomicrographs of representative coronal sections through the nasal septum on days 3, 7, 14, and 28. Top, Lowest magnification; bottom, higher magnification of that portion of the OE in the top images captured at the squares. Left OE corresponds to open side and right OE occluded side. Scale bars, 100 μm at lower magnifications, 20 μm for the magnified views. G–I, Thickness of the OE (G), number of OSNs (H), and number of supporting cells (I) 3, 7, 14, and 28 d after unilateral nostril occlusion. Significant changes are not observed histologically during the sensory deprivation over 28 d (n.s., not significant, Mann–Whitney test). J, Photomicrographs of representative coronal sections through the nasal septum stained with anti-OMP antibody on days 3, 7, 14, and 28 after unilateral nostril occlusion. Brown cells stained by DAB correspond to OMP-positive cells. Scale bar, 20 μm. K, Number of OMP-positive cells 3, 7, 14, and 28 d after unilateral nostril occlusion.

Figure 2.

Existing OSNs through the nasal septum are disrupted by methimazole administration but recover to preinjury states within 28 d. A, Time course of the experimental design after methimazole (a) and saline (b) administration. Methimazole and saline were administered by intraperitoneal (i.p.) injections on day 0. Fixation (fix.) was performed on days 3 (d3), 7 (d7), 14 (d14), and 28 (d28) after methimazole administration and on day 28 (d28) after saline administration. B, Photomicrographs of representative coronal sections through the nasal septum on days 3, 7, 14, and 28 after methimazole administration. Top, Lower magnification; bottom, higher magnification views of the portion of the OE in top photographs outlined by the square. Left side of the OE in the image corresponds to the left nostril and the right side of the OE image corresponds to the right nostril. Scale bar, 100 μm at lower magnification, 20 μm at higher magnification. C–E, Thickness of the OE (C), number of OSNs (D), and number of supporting cells (E) on days 3, 7, 14, and 28 after methimazole administration and on day 28 after saline administration. On day 28 after methimazole administration, the thickness of the OE and the number of OSNs are restored to those levels observed after saline administration (n.s., not significant; Mann–Whitney test). F, Photomicrographs of representative coronal sections stained with anti-OMP antibody through the nasal septum 3, 7, 14, and 28 d after methimazole administration, and 28 d after saline administration. Left images, left nostril; right images, right nostril. Scale bar, 20 μm. G, Numbers of OMP-positive cells on both left and right sides on days 3, 7, 14, and 28 after methimazole administration and on day 28 after saline administration. On day 14 after methimazole-induced injury, the number of OMP-positive cells is significantly reduced compared with that on day 28 after saline administration (***p < 0.001, Mann–Whitney test), whereas, on day 28 after methimazole administration, the number of OMP-positive cells is restored to that observed on day 28 after saline administration (n.s., not significant; Mann–Whitney test).

Figure 3.

Sensory deprivation inhibits complete replacement of newly generated OSNs after methimazole-induced injury. A, Time course of the experimental design. Methimazole administration (intraperitoneal, i.p.) and unilateral nostril occlusion (unilateral n.o.) were performed at day 0 (d 0) and fixation (fix.) was conducted on days 3 (d3), 7 (d7), 14 (d14), and 28 (d28). (B) Photomicrographs of representative coronal sections through the nasal septum on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.). Top, Lower magnification images; bottom, higher magnification of the portion of the OE depicted in top photographs indicated by the square. Left side of the image corresponds to the open side and the right side to the occluded side. Scale bars, 100 μm at low magnification, 20 μm at higher magnification. C–E, Thickness of the OE (C), number of olfactory sensory neurons OSNs (D), and number of supporting cells (E) on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.) and on days 14 and 28 after methimazole-induced injury only (injury only). On days 14 and 28 after injury + n.o, the OE thickness and number of OSNs in the occluded side are reduced significantly compared with those in the open side (*p < 0.05; ***p < 0.001; n.s., not significant; Mann–Whitney test). On days 14 and 28 after injury + n.o, the thickness of the OE and the number of OSNs in the OE are restored to those levels on the same days after methimazole-induced injury only (n.s., not significant; Mann–Whitney test). F, Photomicrographs of representative coronal sections stained with anti-OMP antibody through the nasal septum in the open (left) and the occluded side (right) on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.). Scale bar, 20 μm. G, Number of OMP-positive cells on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.) and on days 14 and 28 after methimazole-induced injury (injury only). On days 14 and 28 after injury + n.o., the number of OMP-positive cells on the occluded side is significantly reduced compared with that on the open side (***p < 0.001; Mann–Whitney test). On days 14 and 28 after injury + n.o, the number of OMP-positive cells is restored to those levels observed after methimazole-induced injury only at the corresponding periods (n.s., not significant; Mann–Whitney test). H, Immunohistological staining of the anti-neutrophil antibody in the OE (right) and bone marrow (left) on day 28 after methimazole-induced injury and unilateral nostril occlusion for both open and occluded sides. The OE is not stained with anti-neutrophil antibody, whereas the bone marrow as a positive control tissue is stained. Scale bars, 20 μm. I, Photomicrographs of representative coronal sections of the OE on day 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.). Left, Lower magnification; right, higher magnification images of the areas indicated by the squares in the left image. Left and right sides of the lower magnification image correspond to the open and occluded sides, respectively. Scale bars, 200 μm at low magnification, 20 μm at higher magnification. J–L, Thickness of the OE (J), number of OSNs (K), and number of OMP-positive cells (L) obtained in each area (l1, l2, m1, and m2) on day 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.). The OE is thinner and the number of OSNs and OMP-positive cells are significantly fewer on the occluded side in all subareas compared with those on the open side (*p < 0.05; **p < 0.01; ***p < 0.001; Mann–Whitney test).

Figure 4.

Sensory deprivation results in the projection of axons from fewer newly generated OSNs. A, Analysis area of the OB. Left, coronal section of the OB. Square shows seven analyzed glomeruli in the OB. Scale bar, 500 μm. Middle, representative images of the left and right OB captured at day 28 after saline administration. Right, Intensity histograms of the OMP-immunostained area within glomeruli on both left and right sides. OMP-immunostained areas are shown in red for the left side and in blue for the right side of the OB. B, Representative coronal sections stained with anti-OMP antibody in the open (left) and the occluded (right) sides at days 3 (d3), 7 (d7), 14 (d14), and 28 (d28) after methimazole-induced injury and unilateral nostril occlusion. Each circled area corresponds to a glomerulus. Scale bar, 50 μm. C, Summary for the ratio of areas immunostained and unstained with OMP. The percentage of OMP-stained areas on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.) and on days 14 and 28 after methimazole-induced injury (injury only). OMP-stained area on the occluded side is significantly reduced compared with that on the open side on days 14 and 28 after methimazole-induced injury with unilateral nostril occlusion (*p < 0.05, ***p < 0.001; Mann–Whitney test). However, the OMP-immunostained area does not differ for these two conditions on days 14 and 28 (Mann–Whitney test). D, Schematic showing the quadrants of a coronal section through the OB (dorsolateral, d-l; dorsomedial, d-m; ventrolateral, v-l; and ventromedial, v-m). Scale bar, 500 μm. E, Representative coronal sections of each OB quadrant stained with anti-OMP antibody on the open (left) and the occluded (right) sides on day 28 (d28) after methimazole-induced injury and unilateral nostril occlusion (injury + n.o.). Each circled area corresponds to a glomerulus. Top, Dorsolateral quadrant (d-l); middle, dorsomedial quadrant (d-m); bottom, ventrolateral quadrant (v–l). Scale bar, 50 μm. F, Summary for the ratio of areas immunostained and unstained with OMP in each quadrant of the OB. The percent of the OMP-stained areas in each quadrant of the occluded side is significantly bottom than that of the open side (***p < 0.001; Mann–Whitney test).

Figure 5.

Sensory deprivation disrupts functional incorporation of newly generated OSNs after injury. A, Schematic diagram of in vivo intrinsic imaging. Intrinsic neural activity after stimulation with odors was recorded through a CCD camera. B, Representative functional images showing the dorsal view of the OB in three mice (#1, #2, and #3). Administration of TMT and 2-MBA was used for activating the dorsal zone of the OB. A silicone tube was inserted and remained in the left nostril for 28 d; the tube was removed 12 h before imaging. Left, Reopened side; right, open side. The intensity of the imaging signals are shown as pseudocolors. C, Percentage of the area responsive to stimulation with the odorants TMT and 2-MBA in the reopened and open sides shows that the responsive area of the reopened side is significantly reduced (*p < 0.05; Wilcoxon t test). D, Mean intensity of imaging signals after stimulation with TMT and 2-MBA in the reopened and the open sides. The mean intensities of the signals induced by both TMT and 2-MBA are significantly greater than those induced by air (**p < 0.01; Steel–Dwass test), whereas TMT- and 2-MBA-induced responses on the reopened side are significantly reduced compared with those on the open side (*p < 0.05; **p < 0.01; Steel–Dwass test). E, Odor-induced c-fos expression in the OB. Representative OB coronal sections stained with anti-c-fos antibody on day 28 after methimazole-induced injury and unilateral nostril occlusion (left, reopened side; right, open side). Right, Higher magnification images captured from the areas indicated by the squares on the left panel (dorsolateral, d-l; ventrolateral, v-l; ventromedial, v-m). Lactones, esters, and aldehydes were selected as stimulus odorants to induce c-fos immunoreactivity in the OB neurons. Scale bars, 500 μm at low magnification, 100 μm at higher magnification. F, Labeling of c-fos-positive cells within the glomerular layer in the caudal, middle, and rostral regions of the OB. The c-fos positive cells were plotted with a computer-assisted mapping system. Reactive cells are scattered throughout the dorsal and ventral regions of the OB. Caudal region of the OB corresponds to the image shown in E. Top half of the OB, dorsal region; bottom half of the OB, ventral region. Scale bar, 500 μm. G, Comparison of the number of c-fos-positive cells between the reopened and open sides in the dorsal and ventral regions of the OB. The number of c-fos-positive cells (per mm²) in the dorsal and ventral OB of the open side was significantly greater than that on the reopened side (*p < 0.05, ***p < 0.001; Mann–Whitney test). H, Nasal airflow in the reopened (black) and the open (gray) sides. Top traces, Frequency of respirations during 30 s. Middle traces, Nasal airflow. Bottom traces, Averaged airflows during 30 s. Black line indicates mean airflow; gray range, mean ± SEM. I, Frequency of the nasal airflow (n = 10 mice) in the reopened and open sides. Significant differences are not detected (n.s., not significant; Wilcoxon t test). J, Amplitude of the nasal airflow (n = 10 mice) in the reopened and open sides. Significant differences are not detected (n.s., not significant; Wilcoxon t test).

Figure 6.

Ki67-positive cells on the occluded side decrease 14 and 28 d after injury. A, The number of Ki67-positive cells through the nasal septum in the open and occluded sides on days 3 (d3), 7 (d7), 14 (d14), and 28 (d28) after unilateral nostril occlusion. On days 14 and 28 after unilateral nostril occlusion, the number of Ki-67-positive cells on the occluded side is significantly decreased compared with that on the open side (**p < 0.01; Mann–Whitney test). B, Number of Ki67-positive cells through the nasal septum at 3, 7, 14, and 28 d of sensory deprivation. The number of Ki67-positive cells peaks at day 3 after injury and then gradually decreases. Methimazole administration does not induce significant changes in the number of Ki67-positive cells between the left and right sides at any time (n.s., not significant; Mann–Whitney test). C, Coronal sections stained with anti-Ki67 antibody in the open and occluded sides on days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion. Scale bar, 20 μm. D, Number of Ki67-positive cells through the nasal septum in the open and occluded sides at days 3, 7, 14, and 28 after methimazole-induced injury and unilateral nostril occlusion. On days 14 and 28 after methimazole-induced injury and unilateral nostril occlusion, the total number of Ki67-positive cells is significantly decreased on the occluded side compared with that on the open side (***p < 0.001; Mann–Whitney test).

Figure 7.

Caspase-3-activated immature cells increase 14 and 28 d after methimazole-induced injury combined with nostril occlusion. A, Number of caspase-3-positive cells through the nasal septum on the open and occluded sides at days 7 (d7), 14 (d14), and 28 (d28) after unilateral nostril occlusion. Nostril occlusion does not induce a significant change in number of caspase-3-activated cells between the open and occluded sides at any time point (n.s., not significant; Mann–Whitney test). B, Number of caspase-3-positive cells through the nasal septum on days 7, 14, and 28 after injury. Methimazole administration does not induce a significant change in the number of caspase-3-activated cells between the left and right sides at any time (n.s., not significant; Mann–Whitney test). C, Coronal sections through the nasal septum stained with anti-caspase-3 antibody in the open and occluded sides at days 7, 14, and 28 after methimazole-induced injury with unilateral nostril occlusion. Scale bar, 20 μm. D, Number of caspase-3-positive cells through the nasal septum in the open and occluded sides on days 7, 14, and 28 after methimazole-induced injury with unilateral nostril occlusion. On days 14 and 28, the total number of caspase-3-positive cells on the occluded side is significantly higher than that on the open side (***p < 0.001; Mann–Whitney test). E, Coronal sections through the nasal septum stained with anti-caspase-3 (red) antibody and anti-OMP (green) antibody from the occluded side 14 d after methimazole-induced injury combined with unilateral nostril occlusion. Two representative images of the OE are shown in the top and bottom. The middle row of images shows a higher magnification of the OE captured from the boxed regions shown in the top row of images. White arrowheads indicate caspase-3-positive apoptotic cells that are not costained with an anti-OMP antibody, whereas the yellow arrowhead indicates a caspase-3-positive apoptotic cell costained with the anti-OMP antibody. A majority of caspase-3-positive cells were not costained with the anti-OMP antibody. Scale bar, 20 μm.

Figure 8.

Susceptibility to apoptosis in newly generated OSNs is enhanced 7–14 after injury and unilateral nostril occlusion. A, Mice in two experimental groups received unilateral nostril occlusions (n.o.) at different times after methimazole-induced injury. Top, Sensory deprivation 0–7 d, n.o. (d0–7); bottom, sensory deprivation 7–14, n.o. (d7–14). Mice in the n.o. d 0–7 group received both methimazole administration and unilateral nostril occlusion on day 0; however, the nostril occlusion was removed on day 7 (reopen). Mice in the n.o. d 7–14 group received methimazole administration on day 0 and unilateral nostril occlusion on day 7. Mice in both groups were perfused with fixative (fixed) on day 14 after the methimazole-induced injury. B, Photomicrographs of representative coronal sections of the olfactory nasal septum in the two groups. Higher magnification views of the OE captured from the area depicted by the square are illustrated in the bottom photomicrographs. Scale bars, 100 μm (lower magnification) and 20 μm (higher magnification). C–E, Thickness of the OE (C), number of OSNs (D), and number of supporting cells (E) in both the n.o. d 0–7 and n.o. d 7–14 groups. F, Representative coronal sections of the olfactory nasal septum stained with anti-OMP antibody in both the n.o. d 0–7 and n.o. d 7–14 groups. Scale bar, 20 μm. G, Number of OMP-positive cells through the nasal septum in both the n.o. d 0–7 and n.o. d 7–14 groups. The number of OMP-positive cells in the n.o. d 0–7 group does not differ between the open and the occluded sides, whereas for the n.o. d 7–14 group, the number of OMP-positive cells on the occluded side is significantly bottom than that on the open side (***p < 0.001; n.s., not significant; Mann–Whitney test). H, Photomicrographs of representative coronal sections from the olfactory nasal septum stained with anti-caspase-3 antibody in both the n.o. d 0–7 and n.o. d 7–14 groups. Arrowheads show caspase-3-positive cells. Scale bar, 20 μm. I, Number of caspase-3-positive cells in both the n.o. d 0–7 and n.o. d 7–14 groups. The number of caspase-3-positive cells in the open and closed sides of the n.o. d 0–7 group does not change (n.s., not significant; Mann–Whitney test), whereas the number of caspase-3-positive cells on the occluded side is significantly higher than that on the open side in the n.o. d 7–14 group (***p < 0.001; Mann–Whitney test). J, Two experimental groups: top, caspase inhibitor administration; bottom, DMSO administration. Both groups of mice received unilateral nostril occlusion 7–14 d after methimazole-induced injury. Mice were administered a caspase inhibitor or DMSO (control) on days 7, 9, 11, and 13 after the injury. On day 14 after injury, mice in both groups were perfused with fixative (fixed) for analysis. K, Photomicrographs of representative coronal sections from the olfactory nasal septum are shown for both groups (caspase inhibitor administration and DMSO administration). Higher magnification views of the OE captured from the areas depicted by the squares are illustrated in bottom images. Scale bars, 100 μm at lower magnification, 20 μm at higher magnification. L–N, Thickness of OE (L), number of OSNs (M), and number of supporting cells (N) in groups administered a caspase inhibitor or DMSO. The thickness of the OE and number of OSNs on the occluded side are significantly decreased compared with those on the open side in the group administered DMSO (***p < 0.001; Mann–Whitney test), whereas no histological differences were observed between the two sides in the group administered the caspase inhibitor (n.s., not significant; Mann–Whitney test).

Figure 9.

Schematic diagram for sensory-dependent replacement of newly generated OSNs after injury. A, Time course for the repair process after injury. Newly generated OSNs are promptly produced after injury and mature during the 7–14 d after injury. Mature OSNs gradually increase and the tissue repair is completed during the 28 d after injury. B, Time course of the repair process after injury and sensory deprivation. During sensory deprivation, newly generated OSNs are highly susceptible to apoptosis while mature OSNs are emerging 7–14 d after injury. As a result of enhanced apoptosis in the immature neurons, OE repair by new neurons is incomplete 28 d after injury.