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First published online 18 May 2005
doi: 10.1242/dev.01867

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Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway

Department of Neurology and Program for Neuroscience, University of Michigan Medical Center, Ann Arbor, MI 48109-0585, USA

View larger version (109K): [in a new window]   Fig. 1. Retinoid receptors and RA effects in P15 mouse SVZ neurosphere (NS) cultures. (A) RT-PCR reveals that secondary NS expanded for 6 days express mRNA for RAR, ß, and RXR, but not RXR retinoid receptor subtypes. (B,C) Differentiation of NS for 7 DIV with RA increases ß-tubulin-immunoreactive neurons (C) compared with vehicle (B). (D-G) BrdU (red)/ß-tubulin (ß-tub; green) double labeling (arrows) demonstrates that RA treatment of differentiating NS for 4 DIV increases neuroblast proliferation (E,G) versus vehicle (D,F). (H,I) Activated caspase-3 (casp 3; green) immunolabeling (arrows) shows no influence of RA treatment (I) on cell death versus control (H). Bis-benzimide counterstain in blue. Scale bars: in I, 100 µm for A,B and 50 µm for D,E,H,I; in G, 20 µm for F,G.

View larger version (74K): [in a new window]   Fig. 2. Retinoid signaling increases neurogenesis in P15 mouse SVZ NS cultures. (A) RA treatment of differentiating NS for 7 DIV increases the percentage of neurons and decreases that of astrocytes in a concentration-dependent manner; **P<0.0001 versus vehicle or 0.2 µM RA groups; *P<0.001 versus vehicle or 0.2 µM RA groups. Oligodendrocyte differentiation is unchanged by RA exposure. (B) RA (1 µM) or retinol (Ret; 1 µM) treatment increases differentiating neurons after 7 DIV versus vehicle (Veh); the addition of disulfiram (DS) inhibits the retinol effect. *P< 0.0001 versus Veh or Ret/DS; +, P<0.001 versus Ret; #P<0.0005 versus Veh or Ret/DS. (C,D) At 4 DIV, 1 µM RA increases the proportion of proliferating neuroblasts (BrdU/ß-tubulin double-labeled) compared with total or BrdU-positive cells, and decreases the proportion of BrdU/GFAP double-labeled astrocytes. The proportion (%) of cells incorporating BrdU also significantly increases after RA exposure (C). For C and D, *denotes P<0.05 and **denotes P<0.005. (E) RA does not influence numbers of activated caspase-3-immunoreactive (dying) cells. (F,G) Secondary NS cultures were treated with vehicle (A) or 1 µM RA (B) for 6 days in expansion conditions. RA-treated NS are smaller and appear more differentiated with irregular morphology and process outgrowth (arrows). (H,I) When RA- or vehicle-treated secondary NS were passaged and expanded without RA, the tertiary NS not previously exposed to RA were larger and more numerous (H) compared with those treated with RA before passaging (I). Scale bar: 50 µm.

View larger version (93K): [in a new window]   Fig. 3. Slice culture model of SVZ-olfactory bulb neurogenesis. (A) DiI labeling of cells migrating from SVZ to olfactory bulb (OB) in a P10 explant 3 days after DiI crystal placement in SVZ. The inset shows the bipolar appearance of migrating cells (arrows) at higher magnification. (B) BrdU labeling of cells in the rostral migratory stream (RMS) of a P10 slice culture. Arrows in B show scattered, putative glia that incorporated BrdU. (B') Higher magnification of the boxed area in B. BrdU was given in vivo 6 hours before slice preparation and culture for 3 days. (C) Doublecortin-expressing neuroblasts in the SVZ and RMS of a P10 explant after 3 DIV. (C') Higher magnification of the boxed area in C. (D) Schematic of the electroporation technique. Plasmid DNA was placed in the open lateral ventricle of a hemisphere, electroporated, and parasagittal slices prepared. (E) GFP labeling of cells in the SVZ (arrows) and RMS (arrowheads) of a P2 slice 3 days after electroporation of a GFP reporter. Propidium iodide counterstain (PI). (F) Four days after electroporation of a GFP reporter, labeled cells appear in the SVZ-olfactory bulb pathway. The inset is a higher magnification view of the boxed area showing a cell with migratory morphology. LV, lateral ventricle; Cx, frontal cortex. (G,H) To examine dual plasmid electroporation efficiency, P2 slices were co-electroporated with GFP and dsRed reporters, cultured for 4 DIV, fixed and resectioned at 50 µm. Nearly 100% co-electroporation efficiency was found in the SVZ. All explant images in this and subsequent figures display parasagittal sections with anterior to the right and dorsal at top. Scale bars: in A, 150 µm for A,B',C, 250 µm for B,F and 500 µm for E; in inset in A, 75 µm for C' and insets in A and F; and 100 µm for G,H.

View larger version (48K): [in a new window]   Fig. 4. RA increases SVZ cell proliferation in explants. (A,B) Bis-benzimide staining of P2 slices cultured for 2 DIV with vehicle (Veh, A) or 10 µM RA (B) and re-sectioned at 40 µm. The SVZ and RMS stain as a dense band that expands after RA treatment. (C-F) BrdU immunostaining of the sections in A and B; slices were exposed to BrdU for 2 hours prior to fixation. More SVZ cells incorporated BrdU with RA treatment (D,F) compared with control (C,E). (E,F) Show regions of SVZ approximated by the boxed area in C. (G) Quantification of BrdU-positive cell numbers per fixed SVZ area (boxed region in C). **Denotes P<0.01. (H,I) TUNEL staining of control (H) and RA-treated (I) explants. No difference in TUNEL-stained cells (arrows) was seen (see text for quantitation). CTX: cortex. OB: olfactory bulb. Asterisk marks lateral ventricle in A. Scale bar: in B, 100 µm for A-D and 25 µm for E,F; in I, 50 µm for H,I.

View larger version (142K): [in a new window]   Fig. 5. Exposure of explants to RA increases neurogenesis in the SVZ-olfactory bulb pathway. (A,B) Explants were treated with vehicle (Veh; A) or RA (10 µM; B) for 4 DIV and then immunostained for calretinin. Note increased calretinin immunoreactivity and thicker RMS (arrows) in the RA-treated explant (B) compared with Veh (A). (C,D) Confocal images of re-sectioned explants (50 µm thick slices) double immuntstained for calretinin (green) and BrdU (red) after a 4-day exposure to Veh (C) or 10 µM RA (D). Explants were incubated with BrdU from 24-48 hours in vitro. Most BrdU-labeled cells in the RMS (arrows) express calretinin; the RMS is expanded in the RA treated slice. Insets in panels C and D show double labeled cells (arrows; BrdU at top; calretinin middle; merged at bottom). OB: olfactory bulb. *Denotes the lateral ventricle. Scale bar: 200 µm for A,B; 100 µm for C,D; 40 µm for insets.

View larger version (81K): [in a new window]   Fig. 6. Retinol stimulates SVZ neurogenesis in explants. (A-D) BrdU immunolabeling of SVZ cells (arrows) in slices incubated with vehicle (Veh; A), retinol (Ro; B), disulfiram (Ds, C), or Ds and Ro (D). Explants were treated for 2 days, exposed to BrdU for 2 hours before fixation, and then resectioned at 40 µm. Note the increased BrdU labeling in the Ro-treated slice (B) compared with vehicle (A) or Ds (C), and the lack of increased BrdU-positive cells in the explant cultured with both Ro and Ds (D). E-H: Confocal images of explants treated as in A-D and immunostained with an anti-calretinin antibody. Ro treatment (F) expanded the RMS (arrows) compared with the other groups, and the effect was blocked by co-treatment with Ds (H). I: Quantification of BrdU-positive cell numbers per fixed SVZ area (see Fig. 4C). **Denotes P<0.01. CTX: cortex. RMS: rostral migratory stream. OB: olfactory bulb. Scale bar: 100 µm.

View larger version (90K): [in a new window]   Fig. 7. Inhibition of RA signaling alters SVZ cell morphology and impairs migration to the olfactory bulb. (A-D) Images of GFP (green) and calretinin (red) immunofluorescence double labeling after electroporating SVZ cells with EGFP (A), or EGFP plus dominant-negative (dn) RAR (B), dnRXR (C), or both dominant-negative constructs (D) as depicted in Fig. 3D. Explants were cultured for 4 DIV after electroporation and resectioned at 50 µm. After electroporation of EGFP (Con; A) or EGFP plus dnRXR (C), GFP-labeled cells appear in the SVZ and RMS and overlap with calretinin-expressing cells. Expression of EGFP with dnRAR (B) or dnRAR plus dnRXR (D) decreases the migration of GFP-positive cells; many appear in the septum and are calretinin-negative. Arrows in A-D outline the RMS. (E,F) Higher magnification images of GFP+ SVZ cells near the regions marked with arrowheads in A and B, respectively. GFP-labeled cells in the Con explant show a radial cell-like morphology (E), while those co-expressing dnRAR appear disorganized (F). (G) Quantification of migration distance from the SVZ to the most rostral GFP-positive cell in the distal RMS/olfactory bulb. Maximum migration distance was calculated by measuring the linear distance from the anterior border of the lateral ventricle to the most rostral GFP-positive cell (see inset and methods). GFP+ cells that express dnRAR or dnRAR/dnRXR migrate significantly shorter distances. (H) Quantification of GFP-positive cell number in the distal RMS and olfactory bulb (rostral to the line shown in the inset) reveals significantly fewer GFP+ cells migrating to the bulb in explants expressing dominant-negative retinoid receptors. For G and H, single asterisk denotes P<0.05, and double asterisk P<0.01. (I,J) P2 slices were electroporated with a dnRAR-IRES-GFP plasmid (J) or IRES-GFP control (I) and cultured for 4 DIV, fixed and resectioned at 50 µm. After electroporation of IRES-GFP vector alone (Vector), GFP-labeled cells migrated in the RMS toward the olfactory bulb (OB). SVZ cell expression of dnRAR-IRES-GFP (dnRAR) inhibits the rostral migration of GFP-positive cells. Arrows in I and J point to the RMS. Scale bar: in D, 200 µm for A-D; in E, 25 µm for E,F; in J, 200 µm for I,J.

View larger version (83K): [in a new window]   Fig. 8. Inhibition of RA signaling alters SVZ cell morphology and differentiation. Confocal images of 50 µm-thick resectioned explants immunolstained for GFP (green) and nestin (red in C, F) or PSA-NCAM (red in I, L). In the EGFP-electroporated control (A-C), some GFP-labeled SVZ cells express nestin (arrows) and have typical radial cell morphology. Others express GFP only (arrowheads). After GFP reporter co-electroporation with dnRAR (D-F), GFP-labeled cells lack radial morphology, have shorter processes, and more co-express nestin (arrows). Arrowheads show GFP single-labeled cells. G-L: In a typical control explant (G-I), most GFP+RMS cells express PSA-NCAM (arrows) and have a migratory morphology (fusiform shape and leading/lagging processes); in contrast, RMS cells co-expressing GFP and dnRAR (J-L) have more irregular cell bodies (arrows); many appear outside of the RMS and lack PSA-NCAM expression (arrowheads). Scale bar: 25 µm.

View larger version (75K): [in a new window]   Fig. 9. Blockade of endogenous RA synthesis inhibits forebrain SVZ cell proliferation. (A,B) Coronal sections through the anterior SVZ show confocal images of BrdU immunostaining after treatment of mice with vehicle (Veh, A) or disulfiram (DS; B) daily from P7-10. A single BrdU injection was administered 6 hours after the first DS/Veh injection. DS treatment decreases SVZ BrdU labeling compared with Veh. Insets in A and B are high magnification images of PSA-NCAM (left panels) or merged PSA-NCAM/BrdU double immunolabeling (right panels) showing that most BrdU-positive cells (red) in the SVZ are PSA-NCAM-positive (green) neuroblasts. (C) Quantification of BrdU immunoreactivity in a fixed dorsolateral SVZ area shows decreased BrdU labeling in the SVZ of DS-treated mice compared with controls. **Denotes P<0.01. Scale bars: 50 µm for A,B; 20 µm for insets.