Impaired migration in the rostral migratory stream but spared olfactory function after the elimination of programmed cell death in Bax knock-out mice

Abstract

Rats and mice exhibit neurogenesis of olfactory bulb (OB) interneurons throughout adulthood. To homeostatically maintain stable neuron numbers, it is necessary to continuously remove a subset of OB neurons by programmed cell death (PCD). Here we demonstrate that Bax is critical for the elimination of OB neurons by showing that Bax-KO mice exhibit greatly reduced PCD in the OB. Despite the reduction of PCD, however, proliferation of progenitors and the size of the OB were virtually unaffected in Bax-knock-out (KO) mice. However, reducing PCD by Bax deletion affected the migration of a subset of adult-produced neurons by the disruption of glial tube formation as well as by premature detachment of neuroblasts from the migratory chain. Rescued cells aberrantly remained in the subventricular zone (SVZ)-rostral migratory stream (RMS), in which they differentiated into calretinin+ or GABA-expressing interneurons. Because of the migratory deficit, OB cell homeostasis involving new cell entry and PCD (neuronal turnover) was virtually absent in adult Bax-KO mice. Despite this, Bax-KO mice exhibited normal olfactory behaviors such as odor discrimination and olfactory memory which are thought to be influenced by adult neurogenesis. These results demonstrate that PCD is involved in the regulation of RMS migration and differentiation after OB neurogenesis, but that animals maintain normal olfactory function in the absence of PCD.

Figure 1.

PCD is reduced in the olfactory system of adult Bax-KO mice. Dying cells visualized by TUNEL in the SVZ (A, D), RMS (B, E), and OB (C, F) of WT (A–C) and Bax-KO (D–F) mice. Arrows indicate TUNEL⁺ cells, and blue dotted lines in B and E indicate the margins of the RMS. LV, Lateral ventricle. G, H, Double-immunofluorescence labeling of OB with activated caspase-3 (red) and NeuN (green) with Hoechst nuclear counterstaining (blue). Note that most caspase-3⁺ cells in WT mice are also NeuN⁺ (G), whereas caspase3⁺ cells lack NeuN-IR in Bax-KO (H). I, Quantification of TUNEL⁺ cells in SVZ, RMS, and olfactory granule cell layers (OGL) of WT and Bax-KO mice. Mean ± SEM, n = 4. *p < 0.05, Student's t test.

Figure 2.

The distribution of proliferating (PCNA⁺) neuroblasts in the adult olfactory system. A–F, Proliferating neuroblasts were visualized by PCNA labeling in the SVZ (A, D), RMS (B, E), and OB (C, F) of 2-month-old WT (A–C) and Bax-KO (D–F) mice. G, Quantification of PCNA⁺ cells. Mean ± SEM, n = 4. H–K, Nissl-stained coronal sections of WT (H) and Bax-KO (I) OB exhibit similar histology, except for a significant accumulation of cells in the subependyma (SE; insets show higher magnification of boxed area). The overall size of the OB was also comparable in WT (J) and Bax-KO (K) mice. L, Measurement of OGL area in serially cut sections. Note that the red shaded region exhibits an apparent increase, which is likely attributable to the expansion of the SE. LV, Lateral ventricle.

Figure 3.

Impairment of RMS migration in Bax-KO mice. A–D, Parasagittal view of DCX-IR in 2-month-old WT (A, B) and Bax-KO (C, D) mice. B and D (and insets; scale bar, 20 μm) show higher-magnification views of boxed areas in A and C, respectively. Scale bar, 50 μm. Note that a subset of DCX⁺ cells project radial fibers (inset in D, white arrowheads). Boundaries of sections are indicated by dashed lines in A and C. E, Quantification of the area of the DCX⁺ region in SVZ and RMS of 2- and 12-month-old animals. The area containing DCX⁺ cells in anatomically matched sagittal sections (40 μm) was measured under a 10× objective lens. We excluded the area containing DCX⁺ radial fibers in Bax-KO RMS in these measurements. Mean ± SEM, n = 4. *p < 0.05, Student's t test comparisons between WT versus Bax-KO mice; **p < 0.05, Student's t test comparisons between 2- versus 12-month-old age group. F–Q, Distribution of BrdU⁺ cells in SVZ (F, G, L, M), RMS (H, I, N, O), and OGL (J, K, P, Q) at 1 month (F–I) after a single injection of BrdU on P4 in WT (F, H, J, L, N, P) and Bax-KO (G, I, K, M, O, Q) mice; BrdU is labeled red, and the nucleus is labeled blue (Hoechst33342). Dotted lines indicate the boundary of the RMS and subependyma (SE). Scale bar, 50 μm. R–T, Quantification of BrdU⁺ cells in SVZ (R), RMS (S), and OGL (T). For the SVZ and RMS, the total number of BrdU⁺ cells in anatomically matched sections was counted; for OGL, BrdU⁺ cells per unit area are shown. Mean ± SEM, n = 4. *p < 0.05, Student's t test.

Figure 4.

Differentiation of migration-impaired neuroblasts in Bax-KO RMS. A, B, Immunofluorescence staining for the mature granule cell marker NeuN (green) and BrdU (red) in WT (A) and Bax-KO (B) RMS. Nuclei were counterstained with Hoechst33342 (blue). Dotted lines indicate the boundary of the RMS. Inset in B shows complete overlap (yellow) of NeuN-IR (green) and BrdU-IR (red) in Z-sectioned confocal images. C, D, Immunostaining of the mature granule cell marker calretinin (green) in WT (C) and Bax-KO (D) RMS. E, F, Immunostaining of the interneuron marker GABA (green) in WT (E) and Bax-KO (F) RMS; insets show GABA-IR in the OB. G, H, TH immunostaining in WT (G) and Bax-KO (H) OB; insets in G and H show a higher-magnification view of OGL. I–L, Phosphorylated CREB immunostaining (green) in SVZ (I, J) and RMS (K, L) of WT (I, K) and Bax-KO (J, L) mice; insets in I and J show pan-CREB immunostaining (red) in adjacent sections. Scale bar, 50 μm. LV, Lateral ventricle.

Figure 5.

A–E, In vitro migration of explants from P4 (A, B, E) and 12-month-old (C, D) SVZ in WT (A, C) and Bax-KO (B, D) mice. SVZ explants were isolated from P3–P4 mice and embedded in Matrigel. Forty-eight hours later, the outward migration of neuroblasts was observed, and these formed typical cellular chains (insets in A and B). SVZ explants from 12-month-old mice were cultured on laminin-coated dishes, and the outward migrated neuroblasts were visualized by TuJ1 (red) 5 d after culture (C, D). Note that neuronal migration was preferentially observed on the extended GFAP⁺ glial beds (green). Nuclei were counterstained with Hoechst33342 (blue). E, Quantification of the migration distance from P4 explants. n = 7–8 explants from three to four mice for each genotype. F–J, WT (F, G) and Bax-KO (H, I) SVZ microexplants from P5 animals were grafted in the posterior RMS of 4-month-old WT mice. The position of the transplants (blue arrowhead) and observations (red arrows) are schematically shown in J. Before transplantation, SVZ neuroblasts were infected by GFP retrovirus, and the grafted cells were identified by the GFP signal. Three weeks after WT or Bax-KO SVZ grafts, GFP⁺ cells from WT and Bax-KO grafts were only infrequently observed in the RMS (F, H) and OB (G, I). Dotted lines in F and H indicate the borders of RMS. Scale bar, 50 μm. K, WT SVZ microexplants were grafted in the SVZ of 12-month-old Bax-KO mice. GFP⁺ cells remained at the transplant site (yellow dotted line), and no GFP⁺ cells were observed in the RMS or OB 6 weeks after transplantation.

Figure 6.

Double-immunofluorescence labeling for NCAM/GFAP (A, E), DCX/GFAP (B, F), TUJ-1/GFAP (C, G), MAP2/GFAP (D, H), and GFAP/Tenacin-C (I–N) in the RMS of 2-month-old WT (A–D, I–K) and Bax-KO (E–H, L–N) mice. Dotted lines indicate the boundary of the RMS. Insets in I–N show higher-magnification images of the boxed areas. O, Schematic summary of the distribution of immature and mature neuronal makers in the Bax-KO RMS. Within the RMS, there are two separate regions; one contains a glial tube (GFAP⁺) that is PSA-NCAM⁺, DCX⁺, TUJ-1⁺, but is NeuN⁻, MAP2⁻ (A-zone), whereas the N-zone is devoid of a glial tube and contains more mature neurons that were PSA-NCAM⁻, NeuN⁺ and MAP2⁺.

Figure 7.

The SVZ of both 2-month-old WT (A) and Bax-KO (B) mice exhibit a typical arrangement of niche components. A, Migrating neuroblast; B1, B2, astrocytes; C, precursor cell; e, ependymal cell; V, lateral ventricle. Scale bar, 10 μm. Arrow indicates cellular debris from normally occurring cell death. Cells were classified according to criteria established by Doetsch et al. (1997). C, The roof of the lateral ventricle immediately below the corpus callosum shows few neuroblasts migrating toward the RMS in the WT compared with the Bax-KO in which large numbers of cells are found (D, arrows); this accumulation also occurs at the base of the ventricle (data not shown). CPU, Caudate–putamen; LV, lateral ventricle.

Figure 8.

A, The RMS of 2-month-old WT mice is composed of migrating neuroblasts (A) interspersed with astrocyte-like glial cells (B). The arrow points to the remnants of a dying cell. A, B-type cell (box) is enlarged in A′, showing light filamentous cytoplasm and a nucleus heavily rimmed with chromatin typical of astrocytes. B, In the RMS of 2-month-old Bax-KO mice, some neuroblasts appear to have ceased migrating and begun differentiation (N). The boxed area in B is enlarged in B′ to show a typical dense A cell with very sparse cytoplasm surrounded by spaces characteristic of migrating cells, and an N cell, with more differentiated cytoplasm containing some mitochondria, ribosomes, and endoplasmic reticulum, and a nucleus that is lighter and less rimmed with chromatin than B cells and that appears to be an immature neuron. Note that, in the Bax-KO, the N cells are located to one side of the field across the RMS (N-zone), in which B cells are sparse, whereas A cells and one B cell are located at the other side (A-zone). Scale bars: A, B, 10 μm; A′, B′, 1 μm. The RMS of both 2-month-old WT and Bax-KO mice exhibit frequent puncta adherentia. In C, a group of WT A cells can be seen with several puncta (arrows); coated vesicle formation is often associated with these structures (*); D illustrates A-B contacts in WT. E, Bax-KO mice also exhibit A-N puncta in which these cells are in close proximity (arrow); the N cell also has a vesicle lined density that may be a forming synapse (double arrow). F, Puncta (arrows) between two N cells. Another striking feature of the Bax-KO is the absence of astrocytic end feet on blood vessels, seen in G and H, which are common in WT, as seen in G. *, Astrocytic process; As, astrocyte; Vs, blood vessel. Scale bars, 10 μm.

Figure 9.

Olfactory behaviors. A, Summary of the experimental design. Baseline activity was measured with two identical odorants (B), and 1 h later a novel odor was presented and the percentage of time approaching to the novel odor was measured (N). Animals were then reexposed to the odor after a 3-h interval (R). The next day, animals were exposed to the familiar odor and a novel odor, and odor preference was measured as an index of olfactory memory (M). B, C, Odor discrimination and the memory test, in 3-month-old (B, n = 6) or 12-month-old (C, n = 7) animals. D, E, The fine odor discrimination test in 3-month-old (D, n = 4) or 12-month-old (E, n = 5) WT and Bax-KO mice. Ratios of vanilla (VAN) and coconut (COC) mixtures in water ([+]) or denotonium benzoate (bitter, [−]) containing dishes are represented on the x-axis. Mean ± SEM; *p < 0.05, Student's t test.