Response of olfactory axons to loss of synaptic targets in the adult mouse

Abstract

Glomerular convergence has been proposed to rely on interactions between like olfactory axons, however topographic targeting is influenced by guidance molecules encountered in the olfactory bulb. Disruption of these cues during development misdirects sensory axons, however little is known about the role of bulb-derived signals in later life, as new axons arise during turnover of the olfactory sensory neuron (OSN) population. To evaluate the contribution of bulb neurons in maintaining topographic projections in adults, we ablated them with N-methyl-d-aspartate (NMDA) in P2-IRES-tauLacZ mice and examined how sensory axons responded to loss of their postsynaptic partners. NMDA lesion eliminated bulb neurons without damage to sensory axons or olfactory ensheathing glia. P2 axons contained within glomeruli at the time of lesion maintained convergence at these locations; there was no evidence of compensatory growth into the remnant tissue. Delayed apoptosis of OSNs in the target-deprived epithelium led to declines in P2 neuron number as well as the gradual atrophy, and in some cases complete loss, of P2 glomeruli in lesioned bulbs by 3 weeks. Increased cell proliferation in the epithelium partially restored the OSN population, and by 8 weeks, new P2 axons distributed within diverse locations in the bulb remnant and within the anterior olfactory nucleus. Prior studies have suggested that initial development of olfactory topography does not rely on synapse formation with target neurons, however the present data demonstrate that continued maintenance of the sensory map requires the presence of sufficient numbers and/or types of available bulbar synaptic targets.

Figure 1

Bulb sections stained with neutral red (top panels) or Fluoro-Jade (FJ, lower panels), showing neuronal degeneration after NMDA injection. Normal sections are shown in A and G. Treated bulbs are shown at 24 hrs (B), 4 days (C and H), 1 week (D and I), 2 weeks (E), and 3 weeks (F). Most mitral cells are lost within 24 hrs. By 4 days, glomerular layer (gl) organization breaks down, with some dying neurons still evident here (arrow) and in the granule cell layer (gcl). Degeneration is largely complete by 1 week, with the exception of some granule cells (arrow in I). Laminar organization is disrupted, though irregular, glomerular-like structures (asterisks) can still be observed at 1–3 weeks. Increasing cell density in the bulb remnant reflects reactive gliosis. onl, olfactory nerve layer; epl, external plexiform layer; mcl, mitral cell layer. Bar= 100 μm.

Figure 2

A–C. Bulb sections showing hybridization of ³⁵S-labeled NPY cRNA at 1 day (A), 1 week (B), and 2 weeks (C) after NMDA treatment. Normal bulbs are on the right and lesioned bulbs to the left. Most neurons expressing NPY are eliminated by NMDA within 24 hrs, but olfactory ensheathing glia in the nerve layer persist. The asterisk in B indicates the lesion cavity. D–F. NeuN-immunoreactivity (IR) in the normal bulb (D) and in lesioned bulbs at 1 week (E) and 3 weeks (F). Most neurons are eliminated by NMDA within the first week. Boxed areas in E–F indicate regions where stained nuclei remain; insets show these at higher magnification (arrows). G–I. GFAP-IR in the normal bulb (G), and in lesioned bulbs at 4 days (H) and 3 weeks (I). The arrowhead in G indicates a normal astrocyte in the external plexiform layer (epl). Reactive astrocytes are seen throughout the bulb 4 days after NMDA injection (H, open arrow). At 3 weeks, shrinkage of the lesioned bulb has reduced the distance between the olfactory nerve layer (onl) and the lesion cavity (asterisk in J), which is surrounded by GFAP+ processes. gcl, granule cell layer; gl, glomerular layer. Bar =1.0 mm in A–C and 100 μm in D–I.

Figure 3

X-gal staining showing the positions of medial P2 glomeruli in normal (left side) and NMDA-injected bulbs (right). Staining is shown at 1 week (A–B), 2 weeks (C–D), and 3 weeks (E–F). Note that though stained glomeruli become smaller, their relative positions within lesioned bulbs remain constant out to 3 weeks (arrows in F). Open arrows indicate glomerular structures, not stained by X-gal. The asterisk in F indicates the lesion cavity. aob, accessory olfactory bulb; gl, glomerular layer; gcl, granule cell layer; m, mitral cell layer; onl, olfactory nerve layer. Bar= 500 μm.

Figure 4

X-gal staining showing P2 foci (arrows) in NMDA-lesioned bulbs. A. A horizontal section showing the position of a medial glomerulus in the lesioned bulb (right, arrow) relative to the same glomerulus in the normal bulb (left) at 3 weeks. B–D. Sections from mice that survived 8 weeks. P2 foci are seen in some atypical locations in the bulb remnant, including one located dorsomedially in B. Four small foci are shown in a single section from another mouse in C. The section in D was taken from a mouse in which part of ventral bulb was spared. P2 axons converge in an abnormal, ventral location. E–H. P2 axon convergence in the normal bulb (E), and in lesioned bulbs (F–H). Convergence is maintained 2 weeks after glomerular layer (gl) neuronal targets are lost (F). Numbers of stained axons decline and foci become smaller by 3 weeks (G). The open arrow indicates a second area of convergence in a mouse that contained two medial P2 glomeruli in the normal bulb. X-gal stained axons do not appear to stray from the core group after lesion. H. Small P2 foci are seen in neuron-depleted bulbs at 8 weeks (arrow). Other, unstained foci can be seen nearby (asterisks). epl, external plexiform layer; gcl, granule cell layer; onl, olfactory nerve layer. Bar = 400 μm in A–D, and 100 μm in E–J.

Figure 5

Horizontal (A–B, I–J) and coronal (C–D, K–L) bulb sections showing changes in sensory innervation with target neuron loss. Lateral is to the left. A–D. OMP-immunoreactivity (IR; red) in sections through normal bulb (A), and at 1 (B), 3 (C) and 8 weeks after NMDA treatment. Note the irregular appearance of glomeruli at 1 week. By 3 weeks, OMP+ glomeruli have disappeared from most of the lesioned bulb (C) but at 8 weeks, the remnant contains numerous stained foci (D). Areas indicated by arrows are shown at higher magnification below in panels E–H. I–L. GAP-43-IR (green) in sections through normal (I) and lesioned bulbs at 2 (J), 3 (K) and 8 weeks (L) after NMDA. GAP-43+ fibers have penetrated into the remnant by 3 weeks (open arrow in K), and stained foci are seen at 8 weeks (arrow in L). M–P. Double labeling for OMP (red) and GAP-43 (green) in normal (M), and lesioned bulbs at 1 (N), 3 (O) and 8 weeks (P). At 3 weeks (O), GAP43+ fibers extend into the bulb parenchyma (open arrow), with some coalescing into small foci with OMP+ fibers (arrowhead). At 8 weeks, many foci contain both OMP+ and GAP-43+ fibers (yellow-orange; arrowhead in P). Q. Ectopic distribution of OMP+ foci (open arrow) within the anterior olfactory nucleus (aon) is seen in some mice at 8 weeks. Caudal medial bulb located to the right of this area (not shown). (Q). Asterisks indicate the lesion cavity. gl, glomerular layer; gcl, granule cell layer, onl, olfactory nerve layer. Bar = 500 μm in A–D and I–L, and 104 μm in E–H and M-, and 220 μm in Q.

Figure 6

A–E. X-gal staining in the epithelium of P2 mice. A. Coronal section showing P2 neurons in the epithelium ipsilateral (left) and contralateral (right) to bulb NMDA lesion at 3 weeks. A portion of the nasal septum and the medial tips of endoturbinate III are shown. Stained neurons in the septum of a saline-treated mouse (B), and mice that survived 4 days (C), 3 weeks (D) and 8 weeks (E) after lesion. F–H. OMP-IR in the septum of a saline mouse (F) and in the deprived epithelium at 3 weeks (G) and 8 weeks after NMDA (H). I–K. Double labeling showing the increase in GAP43+ neurons (green) that accompanies the decline in numbers of OMP+ neurons (red). A normal section is shown in I. Staining ipsilateral to lesion is shown at 3 weeks (J), and 8 weeks (K). Bar= 82 μm in A, 49 μm in B–E, 25 μm in F–H, 27 μm in I–K.

Figure. 7

Autoradiograms showing changes in OMP- (A–B) or GAP-43- (C–D) mRNA expression in the epithelium after NMDA lesion. The target-deprived epithelium is on the left in all panels. A and C show patterns of labeling at 3 weeks; B and D show labeling at 8 weeks. E. Line graph illustrating changes in the density of hybridization of OMP and GAP-43 cRNAs in epithelium after lesion. Measures made ipsilateral to NMDA injection are expressed as a percentage of measures from the contralateral side. Values from saline-injected mice are shown at the zero time point. Group means +/− S.E.M. are plotted. **p< 0.01, *p<0.001 vs. measures from saline mice; Student’s Neuman-Keuls test. Bar = 1.0 mm.

Figure 8

Apoptosis and proliferation in the target-deprived epithelium. A–B. TUNEL-labeling in the normal (A) and deprived epithelium one week after NMDA bulb lesion (B). Open arrows indicate labeled nuclei. C–F. Merged confocal microscope images showing TUNEL labeling and OMP-IR (C–D) or GAP-43-IR (E–F). Optical section thickness = 2.1 μm. After NMDA lesion, most TUNEL-labeled cells (open arrows) do not label for either OMP- (C) or GAP-43-IR (E), even though they are located near adjacent sensory neurons. Small numbers of double-labeled cells are seen at one week post-lesion (D, F, arrows). G. Ki-67-IR indicates increased cell proliferation within the ipsilateral septal epithelium (left) by 3 weeks. Arrows indicate labeled nuclei. Individual stained cells are shown at higher magnification in the inset. Bar = 25 μm in A–B; 20 μm in C–F, 84 μm in G, and 7 μm in inset.