Adult neurogenesis is necessary to refine and maintain circuit specificity

Abstract

The circuitry of the olfactory bulb contains a precise anatomical map that links isofunctional regions within each olfactory bulb. This intrabulbar map forms perinatally and undergoes activity-dependent refinement during the first postnatal weeks. Although this map retains its plasticity throughout adulthood, its organization is remarkably stable despite the addition of millions of new neurons to this circuit. Here we show that the continuous supply of new neuroblasts from the subventricular zone is necessary for both the restoration and maintenance of this precise central circuit. Using pharmacogenetic methods to conditionally ablate adult neurogenesis in transgenic mice, we find that the influx of neuroblasts is required for recovery of intrabulbar map precision after disruption due to sensory block. We further demonstrate that eliminating adult-born interneurons in naive animals leads to an expansion of tufted cell axons that is identical to the changes caused by sensory block, thus revealing an essential role for new neurons in circuit maintenance under baseline conditions. These findings show, for the first time, that inhibiting adult neurogenesis alters the circuitry of projection neurons in brain regions that receive new interneurons and points to a critical role for adult-born neurons in stabilizing a brain circuit that exhibits high levels of plasticity.

Keywords: interneuron; intrabulbar map; neurogenesis; olfactory deprivation; rostral migratory stream; subventricular zone.

Figure 1.

Intrabulbar axons contact preexisting and incoming GABAergic interneurons. A, Schematic illustrating the migration of neuroblasts from the SVZ, through the RMS, and into the OB to become GABAergic interneurons (green cells) that join or replace periglomerular cells or granule cells in the granule cell layer (GCL) and mitral cell layer (MCL). Intrabulbar axons (red) synapse onto granule cells within the IPL, connecting isofunctional olfactory columns (dashed rectangle) within the same OB. B, TMR tract tracing that labels intrabulbar axons in combination with GAD65-GFP expression indicates that intrabulbar projections target GAD65-positive GABAergic interneurons in the region of the IPL and MCL. C, Higher magnification of boxed region in B shows TMR-filled intrabulbar axon in close apposition to GAD65-GFP⁺ cell body with varicosities that are consistent with synaptic contacts (arrow). D–G, Intrabulbar axon labeling with TMR tract tracing combined with dual immunostaining for a postsynaptic marker (VGLUT2) and either a mature (CR) or an immature (DCX) granule cell marker. E, G, Orthogonal views of the boxed regions in D and F indicate that the juxtapositions between TMR-containing axons and either CR-positive (E) or DCX-positive (G) neurons are localized to VGLUT2-immunopositive puncta (white arrows), suggesting that intrabulbar axons target preexisting (D, E) and new (F, G) interneurons within the IPL. Scale bars: B, C, 20 μm; D, F, 10 μm; E, G, 5 μm.

Figure 2.

The influx of new neurons is completely eliminated in GfapTK mice and largely inhibited in NestinTK mice. After 6 weeks of vGCV treatment, proliferating BrdU⁺ profiles (red) were visible among GAD65-GFP⁺ neuroblasts (green) in GfapTK⁻ mice (A), but few BrdU⁺ or GAD65-GFP⁺ cells remained in GfapTK⁺ mice (B). This lack of proliferation within the RMS corresponded to an absence of DCX-immunoreactive immature neurons along this pathway in treated GfapTK⁺ mice (D) in contrast to large numbers of DCX-expressing neural progenitors in GfapTK⁻ controls (C). E, F, Immunostaining for GFAP (red) revealed that scaffold-forming astrocytes within the RMS persisted in GFAP-TK⁺ mice given vGCV (F) despite the absence of migrating GABAergic stem cells (green). Many BrdU⁺/GAD65-GFP⁺ cells were also present within the RMS of untreated (no vGCV) NestinTK control mice (G); however, all GAD65-GFP⁺ cells and most BrdU⁺ cells in treated NestinTK mice were eliminated (H). DCX immunostaining results supported this substantial, but incomplete, ablation because many incoming DCX⁺ immature neurons were visible in the RMS of untreated NestinTK⁺ controls (I), whereas few DCX⁺ cells remained in NestinTK⁺ mice treated with vGCV (J). A–J, Boxed areas along the RMS are shown in higher-magnification insets. Scale bars: A–J, 100 μm; insets, 20 μm.

Figure 3.

Adult-born stem cell ablation does not alter OSN proliferation in the olfactory epithelium or glomerular targeting. After 6 weeks of vGCV treatment (A), proliferation within the OSN stem cell population appeared unaffected in GfapTK and NestinTK models, as evidenced by BrdU staining (red) within the olfactory epithelia of treated GfapTK⁺ and NestinTK⁺ mice (C and E, respectively), which did not differ from treated GfapTK⁻ and untreated NestinTK⁺ controls (B and D, respectively). Immunohistochemistry for β-gal in OB sections from vGCV-treated GfapTK/M71-IRES-taulacZ and GfapTK/P2-IRES-taulacZ mice was conducted to assess potential effects of adult stem cell ablation on the glomerular map (F–I). As illustrated by confocal images of β-gal-ir in representative dorsal M71 (F, G) and ventromedial P2 (H, I) glomeruli, OSN axonal convergence and glomerular targeting did not differ in control GfapTK⁻/M71-IRES-taulacZ (F) and GfapTK⁻/P2-IRES-taulacZ (H) control mice compared with GfapTK⁺/M71-IRES-taulacZ (G) and GfapTK⁻/P2-IRES-taulacZ (I) that experienced stem cell ablation for 6 weeks. PI, Propidium iodide. Scale bar, 20 μm.

Figure 4.

Ablation of adult-born neuroblasts does not cause morphological anomalies in OB tissue. Incoming GAD65-GFP-containing neuroblasts were visible in the SEL of treated GfapTK⁻ (A) and untreated NestinTK⁻ (C) controls. These populations of GAD65-GFP⁺ cells corresponded to dense clusters of Nissl-stained cells that were immunoreactive for DCX (A, C). After 6 weeks of treatment with vGCV in GfapTK⁺ and NestinTK⁺ mice, GAD65-GFP⁺ cells were absent from the SEL (B, D). Nissl-stained profiles, however, were present in this region, though less dense; and the basic organization of the OB did not contain any structural abnormalities. DCX-immunopositive cells were either completely absent or very sparse in the SEL of treated GfapTK⁺ or NestinTK⁺ mice, respectively (B, D). Quantification of DCX-containing processes extending radially into the OB further confirmed this observation in the GfapTK (t = 55.56, *p < 0.001) and NestinTK (t = 29.88, p < 0.001) models (E–I). However, the lack of SVZ stem cells did not change the densities of the general population of GAD65-GFP-containing cells in GfapTK and NestinTK mice (J–N). Scale bars: A–D, 200 μm; insets, 50 μm.

Figure 5.

Adult neurogenesis is necessary for the restoration of intrabulbar specificity after activity deprivation. A, Schematic illustrates a discrete iontophoretic tracer injection of TMR that labels tufted cells within an olfactory column and allows for visualization of associated axonal projection tufts within the IPL of the isofunctional column on the opposite side of the OB. B, Timelines of experimental design show that transgenic (+), wild-type (−) littermate, or mixed genotype mice experienced naris block followed by a recovery period after removal of the block when some mice were given vGCV to eliminate adult neurogenesis. Intrabulbar projections were visualized with tracer injections conducted 18–20 h before death. At 10 weeks of age, untreated GfapTK control mice exhibited control levels of intrabulbar projection specificity (C). However, naris closure from 7 to 10 weeks resulted in a significant broadening of these projections (D). When GfapTK⁻ mice experienced naris block followed by normal neurogenesis during recovery, intrabulbar axon tufts appeared refined as illustrated in representative images of injection and projection sites (E). However, inhibition of neurogenesis during recovery from sensory deprivation resulted in broad intrabulbar axonal projections (F). G, Graph of average projection to injection ratios for GfapTK groups (mean ± SEM). *p < 0.001 (one-way ANOVA). Scale bar, 50 μm.

Figure 6.

Incomplete ablation of SVZ stem cells also prevents the restoration of intrabulbar projection refinement after sensory block. A, Timelines represent groups of NestinTK mice that served as controls or experienced naris closure with or without an incomplete inhibition of neuroblasts from the RMS. As in GfapTK and wild-type mice, intrabulbar projection specificity appeared refined in NestinTK controls (B) and broadened in NestinTK mice that underwent naris block from 7 to 10 weeks of age (C). When normal levels of neurogenesis occurred after removal of the block, intrabulbar axons appeared refined once again (D). However, when adult-born stem cells were largely, but not completely, eliminated in NestinTK⁺ mice treated with vGCV after naris block, intrabulbar projections failed to refine to control levels of projection specificity (E). F, Graph represents average projection to injection ratios in NestinTK groups. *p < 0.001. Scale bar, 50 μm.

Figure 7.

Neurogenesis is required for the maintenance of intrabulbar projection specificity. A, Timelines illustrating experimental paradigm: neurogenesis was blocked in naive adult GfapTK and NestinTK mice via vGCV treatment for 6 weeks. Intrabulbar projection specificity was then assessed with discrete tracer injections. B, C, Images of representative TMR injection and projection sites (red) show that control mice lacking GfapTK expression had discrete intrabulbar projections, whereas GfapTK⁺ mice exhibited broad projection tufts. D, E, Intrabulbar projections were also refined in NestinTK mice that were not treated with vGCV to inhibit adult neurogenesis, whereas projections of treated NestinTK⁺ mice were expanded, as indicated by images of typical injection and projection sites. F, Graph represents average projection to injection ratios for GfapTK and NestinTK groups (mean ± SEM). *p < 0.005 (one-way ANOVA). Scale bar, 50 μm.