Adult neurogenesis in the central olfactory pathway in the absence of receptor neuron turnover in Libinia emarginata

Abstract

Life-long neurogenesis is a characteristic feature of the olfactory pathways of a phylogenetically diverse array of animals. In both vertebrates and invertebrates, the life-long addition of olfactory interneurons in the brain occurs in parallel with the continuous proliferation of olfactory receptor neurons in the olfactory organ. It has been proposed that these two processes are related functionally, with new olfactory interneurons being added to accommodate the new olfactory receptor neurons added in the periphery. While this has not been tested directly because the two processes are not readily separable, this question can be addressed in the olfactory pathway of the crab, Libinia emarginata. Unlike most decapod crustaceans, which moult and grow throughout life, L. emarginata has a terminal, maturational moult after which animals become anecdysic (stop moulting). Because the addition of new receptor neurons in crustaceans is associated with moulting, a comparison of neurogenesis in immature and mature L. emarginata provides an opportunity to examine the interdependence of central and peripheral neurogenesis in the olfactory pathway. This study demonstrates that the continuous addition of olfactory receptor neurons in L. emarginata ceases at the terminal moult but that proliferation and differentiation of olfactory interneurons in the brain continues in mature animals. Contrary to the general assumption, therefore, continuous neurogenesis in the central olfactory pathway of this species does not occur as part of a process involving the coregulation of central and peripheral neurogenesis. These findings suggest that peripheral neurogenesis is not a requirement for continuous neurogenesis in the central olfactory pathway.

Fig. 1

Schematic diagram outlining the olfactory pathway of the crab Libinia emarginata. The axons of the olfactory receptor neurons project ipsilaterally to the brain where they terminate within the olfactory lobe. The olfactory lobe is also innervated by populations of local interneurons and projection neurons whose somata reside within soma clusters 9 and 10, respectively. The main output pathway from the olfactory lobe is provided by the projection neurons, whose axons form the olfactory globular tract. The olfactory globular tract bifurcates at the midline of the brain before projecting bilaterally to the lateral protocerebral neuropil in the eyestalk. The lateral protocerebral neuropil is innervated by a large population of local interneurons whose somata reside in a cluster, known as soma cluster A.

Fig. 2

BrdU labelling in the olfactory pathway of immature animals. ORN, olfactory receptor neuron. (A and B) Clusters of BrdU-labelled cells (arrows) at (A) the distal and (B) proximal margins of the olfactory organ. (C–F) Clusters of BrdU-labelled cells (arrows) within (C and D) soma cluster 10, (E) soma cluster 9 and (F) soma cluster A. The olfactory lobe glomeruli (C) and lateral protocerebral neuropil (F) are labelled with an antibody against Drosophila synapsin. (G) BrdU (blue) and crustacean-SIFamide (green) labelling in cluster 10 1 day after the injection of BrdU. Note the absence of crustacean-SIFamide-immunoreactive cells in and around the proliferation zone of cluster 10 (*), indicating that crustacean-SIFamide labelling is specific to mature neurons. The inset shows a higher magnification view of BrdU-labelled cells within the proliferation zone, showing the absence of crustacean-SIFamide labelling in these cells. (H) BrdU (blue) and crustacean-SIFamide (green) labelling in cluster 10, 6 months after the injection of BrdU. The arrows identify neurons double-labelled for BrdU and crustacean-SIFamide. (I) BrdU (blue) and dextran (green) labelling in cluster 10, 6 months after the injection of BrdU. The dextran dye was applied to the olfactory lobe to label the somata of interneurons innervating this neuropil. The arrows identify double-labelled neurons. Scale bars, 50 μm (A, B, D and G), 100 μm (C, E and F), 10 μm (H and I, and inset in G).

Fig. 3

BrdU labelling in the olfactory pathway of mature animals. ORN, olfactory receptor neuron. (A and B) Clusters of BrdU-labelled cells were not observed either (A) distally or (B) proximally within the olfactory organs of mature animals, though numerous labelled glial cells (*) could be observed. The inset in A shows the elongated nucleus of a labelled glial cell (*) surrounded by the smaller, round nuclei of unlabelled olfactory receptor neurons. (C) BrdU-labelled cells observed in the olfactory organ of a mature crab (n = 1 of 8) (D) Cluster of BrdU-labelled cells in the olfactory organ of a mature crab following aesthetasc ablation, a procedure designed to examine regenerative responses. This cluster was located midway along the olfactory organ. (E) BrdU labelling in soma cluster 10 of a mature animal injected with BrdU both 4 months and 24 h before being killed. Cells labelled by the first injection (arrowheads) are present amongst the other cells in the cluster while those labelled by the second injection (arrow) occur within the proliferation zone, adjacent to the olfactory lobe. (F and G) Clusters of BrdU-labelled cells (arrows) in (F) soma cluster 9 and (G) soma cluster A of mature crabs. (H) BrdU (blue) and crustacean-SIFamide (green) labelling in cluster 10 of a mature crab injected 6 months earlier with BrdU. The arrow identifies the soma of a double-labelled neuron. (I) BrdU (blue) and dextran (green) labelling in cluster 10 of a mature animal 6 months after the injection of BrdU. The dextran dye was applied to the olfactory lobe. The soma of a double-labelled neuron is identified by the arrow and its primary neurite by the arrowheads. Scale bars: 100 μm (A–C and G), 50 μm (D–F), 10 μm (H and I), 20 μm (inset in A).