XB-ART-731Dev Growth Differ 2006 Jan 01;481:15-24. doi: 10.1111/j.1440-169X.2006.00840.x.
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Functional regeneration of the olfactory bulb requires reconnection to the olfactory nerve in Xenopus larvae.
Larvae of the South African clawed frog (Xenopus laevis) can regenerate the telencephalon, which consists of the olfactory bulb and the cerebrum, after it has been partially removed. Some authors have argued that the telencephalon, once removed, must be reconnected to the olfactory nerve in order to regenerate. However, considerable regeneration has been observed before reconnection. Therefore, we have conducted several experiments to learn whether or not reconnection is a prerequisite for regeneration. We found that the olfactory bulb did not regenerate without reconnection, while the cerebrum regenerated by itself. On the other hand, when the brain was reconnected by the olfactory nerve, both the cerebrum and the olfactory bulb regenerated. Morphological and histological investigation showed that the regenerated telencephalon was identical to the intact one in morphology, types and distributions of cells, and connections between neurons. Froglets with a regenerated telencephalon also recovered olfaction, the primary function of the frog telencephalon. These results suggest that the Xenopus larva requires reconnection of the regenerating brain to the olfactory nerve in order to regenerate the olfactory bulb, and thus the regenerated brain functions, in order to process olfactory information.
PubMed ID: 16466389
Article link: Dev Growth Differ
Species referenced: Xenopus laevis
Genes referenced: rbfox3
Antibodies: Rbfox3 Ab1
Article Images: [+] show captions
|Fig. 1. Regeneration of the olfactory bulb required the reconnection of the olfactory nerves to it. The anterior half of the telencephalon was removed from stage 53 larvae, and the regenerating telencephalon was observed macroscopically at 20 days (A– C, dorsal view of the anterior head of an albino larva), 100 days (D– F, dorsal view of the brain) and 8 months (G–I, dorsal view of the brain) after removal. At 20 days, the two olfactory nerves had reconnected bilaterally to the regenerating hemispheres (A, red arrowheads) or unilaterally (B, red arrowheads) in many larvae. In some larvae, however, no connections were observed (C). Tadpoles were separately reared according to the presence or absence of reconnection of the olfactory nerves to the brain, and observation continued. After 100 days, a swelling was observed only in the hemisphere reconnected by the olfactory nerve (D,E, white arrowhead). After 8 months, the olfactory bulb was observed to regenerate only in the reconnected hemisphere (G,H, above the dotted line). Individuals without reconnection regenerated only the cerebrum but not the olfactory bulb (F,I). Red arrowheads indicate the reconnection between the olfactory nerves and the regenerating telencephalon. White arrowheads indicate the regenerating olfactory bulb. The area above the dotted line is the regenerated olfactory bulb. Bars, 1 mm.|
|Fig. 2. Layer structure in the regenerated olfactory bulb. The anterior half of the telencephalon (mainly the olfactory bulb) was removed from stage 53 larvae, and the regenerating telencephalon was then observed histologically on horizontal sections. The intact telencephalon is composed of an olfactory bulb and a cerebrum in both larvae (A) and froglets (C). Just after the operation, the lateral ventricles were open at the anterior end, and no olfactory bulb was left (B). At 8 months after the operation, with the larvae now frogs, the ventricles were closed and the olfactory bulb regenerated in the individuals in which the telencephalon was reconnected by the olfactory nerves (D, the section of the brain in Fig. 1G). As for the animals in which the telencephalon had not been reconnected by the olfactory nerves, the ventricles were closed but the olfactory bulb did not regenerate (E, the brain in Fig. 1I). The fourlayer structure (olfactory nerve layer, glomerular layer, mitral cell layer, and granule cell layer) was distinctly observed in the intact olfactory bulb (F). The same layer structure was also seen in the regenerated telencephalon that had been reconnected by the olfactory nerve (G). O, olfactory bulb; C, cerebrum; LV, lateral ventricle; ON, olfactory nerve; OL,olfactory nerve layer; GL, glomerular layer; MC, mitral cell layer; GR, granule cell layer. Bars, 200 μm (A,B,F,G); 500 μm (C–E).|
|Fig. 3. Effect of removal of the olfactory nerves on regeneration of the olfactory bulb. Olfactory nerves and the anterior half of the telencephalon were removed simultaneously from stage 53 larvae, then observed macroscopically for 20 days after the operation. In tadpoles with only partially removed telencephalons, the olfactory nerves were observed to reenter the skull, although it was hard to observe whether olfactory nerves reconnected to the regenerating telencephalon (A, red arrowheads). In larvae with unilateral removal of the left olfactory nerve and bilateral removal of the anterior half of the telencephalon, the remaining olfactory nerve reentered the skull (B, red arrowhead). In individuals with bilateral removal of the olfactory nerves and the anterior half of the telencephalon, although the olfactory nerves slightly regenerated from the olfactory organ, they did not reach the skull (C, white arrowheads). Red arrowheads indicate the points where the olfactory nerves entered the skull. White arrowheads indicate the regenerating olfactory nerves. Arrows indicate the anterior end of the regenerating telencephalon. Bar, 2 mm.|
|Fig. 4. Neurons and glial cells in the regenerated telencephalon. The distribution of neurons and glial (ependymal) cells was observed by immunohistochemistry in intact froglets (A,B,E,F,J) and froglets with regenerated telencephalon 3 months after the operation during the larval stage (C,D,G,H,K). Positive signals to anti-NeuN antibody indicate neurons (especially nuclei) (A–E,G); positive signals to anti-β-tubulin I + II antibody indicate neurons (especially axons) (F, H); positive signals to anti-GFAP antibody indicate radial projections of ependymal cells (J,K). Mitral/ tufted cells had large cell bodies and the NeuN-positive process in regenerated telencephalon (C), as was observed in intact froglets (A). In the granule cell layer, small dense cell bodies were crowded in both the regenerated (D) and intact froglets (B). Pyramidal cells had large nuclei and extended the β-tubulin-positive process peripherally in both regenerated (G,H) and intact froglets (E,F). Ependymal cells had radial projections in both the regenerated (K) and intact telencephalon (J). Ependymal layers in opposition to each other were observed touching each other because the lateral ventricle was lost by artifact (J,K). Schematic diagrams of the distribution were drawn for neurons (I) and ependymal cells (L). Bars, 20 μm.|
|Fig. 5. Mitral cells extended their axons through the lateral olfactory tract (LOT) in the regenerated telencephalon. The LOT was observed by DiI tracing in intact froglets (A,D,G) and froglets with regenerated telencephalon 3 months after the operation performed during the larval stage (B,E,H). The LOT in the cerebrum were anterogradely labeled red by the application of DiI to the mitral cell layer in the olfactory bulb, in both intact and regenerated froglets (A–C). Mitral cells (D–F, arrowheads) and the LOT in the anterior cerebrum (G–I) were retrogradely labeled by the application of DiI to the LOT in the cerebrum, in both regenerated and intact froglets. The neural nuclei were counterstained with Hoechst 33342 (blue). Bar, 20 μm.|
|Fig. 6. Froglets with regenerated telencephalon responded to the olfactory stimulus. Xenopus froglets showed sweeping action in response to the olfactory stimulus (food odor) (A). The sweeping action is a flicking of the forelimbs to the mouth several times in succession (B). All intact froglets made this action within 60 s (C, blue line), but froglets with their olfactory nerves cut did not respond to the stimulus at all (C, green line). While half of the froglets with regenerated telencephalon 3 months after removal showed the same action as the intact froglets, the other half took no action (C, red line).|