Adult neurogenesis and cell cycle regulation in the crustacean olfactory pathway: from glial precursors to differentiated neurons

Abstract

Adult neurogenesis is a characteristic feature of the olfactory pathways of decapod crustaceans. In crayfish and clawed lobsters, adult-born neurons are the progeny of precursor cells with glial characteristics located in a neurogenic niche on the ventral surface of the brain. The daughters of these precursor cells migrate during S and G(2 )stages of the cell cycle along glial fibers to lateral (cluster 10) and medial (cluster 9) proliferation zones. Here, they divide (M phase) producing offspring that differentiate into olfactory interneurons. The complete lineage of cells producing neurons in these animals, therefore, is arranged along the migratory stream according to cell cycle stage. We have exploited this model to examine the influence of environmental and endogenous factors on adult neurogenesis. We find that increased levels of serotonin upregulate neuronal production, as does maintaining animals in an enriched (versus deprived) environment or augmenting their diet with omega-3 fatty acids; increased levels of nitric oxide, on the other hand, decrease the rate of neurogenesis. The features of the neurogenic niche and migratory streams, and the fact that these continue to function in vitro, provide opportunities unavailable in other organisms to explore the sequence of cellular and molecular events leading to the production of new neurons in adult brains.

Fig. 1

(A) Diagram of the crayfish brain including the optic ganglia. The soma clusters 9 and 10 (circled) flank two prominent neuropil regions of the deutocerebrum, the olfactory (OL) and accessory (AL) lobes. Cluster 9 contains interneurons that are local to the OL and AL. Cluster 10 contains the projection neurons that branch in either the OL or AL and have axons extending through the brain to neuropils of the lateral protocerebrum, situated in the eyestalks. (B) BrdU-labelled nuclei in the migratory stream (MS), the lateral proliferation zone (LPZ) and the adjacent cluster 10 (CL10). (C) BrdU-labelled cells in the migratory stream, the medial proliferation zone (MPZ) and cluster 9 (CL9) which is separated from the MPZ by a stream of migrating cells. Scale bar: B, C, 20 μm

Fig. 2

(A) Counts of BrdU-labelled cells in clusters 9 and 10 on both sides of the crayfish brain (Cherax destructor) in animals exposed to impoverished (I) and enriched (E) conditions over a period of 5 weeks. A decline in the numbers of cells in animals in the impoverished conditions is recorded after 2 weeks. (B) Counts of surviving BrdU-labelled cells in clusters 9 and 10 on both sides of the brain 2 weeks after immersion in BrdU for 24 h, and 4 weeks of subjection to the different environmental conditions. Asterisks in (A) and (B) indicate significant differences. (C) Counts of BrdU-labelled cells in cluster 10 of the lobster brain (Homarus americanus) are correlated with the time of day, with proliferation levels being highest at dusk and lowest at dawn. D. Levels of serotonin (5-HT) influence neurogenesis in the LPZ of the lobster brain in vitro. Dissected brains from 6 to 7th stage juvenile lobsters were incubated in serotonin (10⁻¹⁴–10⁻⁴ M; n = 8 brains/condition) dissolved in an enriched lobster saline solution containing 0.2 mg/ml BrdU at 13°C for 4 h. In comparison to control brains (C; no serotonin added), serotonin at 10⁻¹⁰–10⁻⁶ M significantly increased BrdU incorporation in the LPZ (asterisks; ANOVA followed by a Tukey’s post-hoc)

Fig. 3

The proliferative system maintaining adult neurogenesis in the central olfactory pathway of the crayfish Procambarus clarkii. (A) Left side of the brain of P. clarkii labelled immunocytochemically for BrdU (green). Labelled cells are found in the lateral proliferation zone (LPZ) contiguous with cluster 10 and in the medial proliferation zone (MPZ) near cluster 9. The two zones are linked by a chain of labelled cells in a migratory stream that originates in the oval region labelled “niche”. Labelling for Drosophila synapsin (blue) and propidium iodide (red) is also shown. (B) Both the LPZ and MPZ are contacted by the processes of a specialized population of glial cells immunoreactive to glutamine synthetase (green). The somata of these cells form a cluster, the niche (red box), on the ventral surface of the brain. (C) Glial cells (green) in the niche labelled by intracellular injection of Lucifer yellow, have short processes projecting to the vascular cavity and longer fibers that fasciculate together to form the tracts projecting to the LPZ and MPZ. (blue: glutamine synthetase; red: propidium iodide). (D) The vascular cavity in the centre of the glial soma cluster in a brain in which the brain vascular system was filled by injecting a dextran dye solution into the cerebral artery. The cavity, outlined in green by its reactivity to an antibody to Elav, contains the dextran dye (red) which is also contained within a larger blood vessel that runs along beneath the niche. Propidium iodide (blue) labelling of the glial cell nuclei is also shown. (E)Differential interference contrast image of a living niche dissected from the ventral surface of the brain. The glial clusters, vascular cavity, migratory stream and blood vessel shown in the labelled preparation in (D) are all clearly distinguishable in the living system. (F) BrdU (cyan) and IodU (blue) labelling in the lateral migratory stream and LPZ in a double nucleoside analogue-labelling experiment. Animals were exposed to BrdU for 6 h and then removed and maintained in fresh pond water for 6 days after which they were immersed in IodU for 6 h, killed and processed with antibodies to BrdU and IodU. The nuclei in the migratory stream after 6 days label only with the IodU indicating that the initially present BrdU labelled nuclei had moved out to the LPZ where they predictably label for both BrdU and IodU. (G) The proliferation zones and the stream labelled with antibodies raised against BrdU (green) and the M-phase marker, phospho-histone H3 (ser10) (red). Histone labels cells exclusively in the lateral and medial proliferation zones. Scale bars: A, 100 μm; B, 75 μm; C, 20 μm; D, 20 μm; E, 25 μm; F, 40 μm; G, 50 μm

Fig. 4

(A) BrdU-labelled nuclei in the lateral migratory stream close to the LPZ. One large nucleus (arrow head) is typically separated from the LPZ by two or three smaller nuclei. (B) Measures of nuclear size in the migratory streams reveal their tendency to increase as they approach the proliferation zones. The abscissa shows the distance along the migratory stream from the MPZ to the LPZ divided into 10% bins, with 0% being at the MPZ and 100% at the LPZ. The graph shows that nuclei in the region of the niche tend to be smaller than those closer to the MPZ and LPZ. At the ends of the streams, close to the proliferation zones, the nuclei tend to decrease in size again as shown in A. Scale bar: A, 20 μm

Fig. 5

A model summarizing our current view of events leading to the production of new olfactory interneurons in adult crayfish, clawed lobsters and crabs. Relatively quiescent precursor cells exhibiting glial characteristics reside within a neurogenic niche where they divide asymmetrically, resting in G₁ phase of the cell cycle between divisions, to produce one self-renewed precursor cell and one daughter cell. Daughter cells begin migrating to the proliferation zones, progressing from S phase of the cell cycle to the G₂ phase as they migrate. Close to the proliferation zones these cells undergo mitosis to become third generation progenitors; one or more divisions of these cells will generate immature neurons in clusters 9 or 10. Many of these cells subsequently differentiate into olfactory interneurons and become incorporated into the nervous system as functional units

Fig. 6

BrdU-labelled cells in the proliferation zones and migratory streams of Cherax destructor (A) and Homarus americanus (B). Arrowheads in A and B mark the migratory stream labelled with β-tubulin (fibers) and BrdU (cells). The niche can be seen in A (arrow) but is hidden in the fold between the accessory lobe and the antenna 2 neuropil in B. The features of this system in C. destructor and H. americanus are virtually identical to those we have described in detail for P. clarkii. Scale bar: A, B, 100 μm

Fig. 7

(A) The left side of a brain of P. clarkii in which dextran was applied to the accessory lobe. The dextran (green) enters neurons that have their terminals in the accessory lobe and labels the corresponding cell bodies and axons. From this it is clear that both projection neurons in cluster 10, and local interneurons in cluster 9, have their terminals in the accessory lobes and the axons from the projection neurons lie in the olfactory globular tract. (B) Cluster 10 cell bodies from an animal that was exposed to BrdU for 12 days and then maintained in fresh pond water for 4 months. At this stage the animal was killed and dextran fluorescein 3000 MW was applied to the accessory lobe (Sullivan and Beltz 2005c). Cells labelled red indicate that they passed through the cell cycle in the presence of BrdU. Cells labelled green indicate that they have terminals in the accessory lobe but did not pass through a cell cycle in the presence of BrdU. Double-labelled cells (orange) are cells that passed through a cell cycle in the presence of BrdU and have differentiated into neurons with their terminals in the accessory lobe. (C) Cluster 10 cell bodies with BrdU (blue) and crustacean-SIFamide (green) label six months after being exposed to BrdU. Double-labelled cells, green and blue (arrowheads). Crustacean-SIFamide immunoreactivity is known to be expressed in olfactory interneurons in P. clarkii (Yasuda-Kamatani and Yasuda, 2006) and the presence of double labelling indicates that these cells were born in the adult animal and have differentiated into olfactory interneurons. Scale bars: A, 10 μm; B, C, 20 μm; C insert, 10 μm

Fig. 8

Live incubation of crayfish (P. clarkii) in 2 mM lithium chloride for seven days, followed by labelling for BrdU (orange) and glutamine synthetase (green), shows an unusual accumulation of BrdU-labelled cells along the migratory streams (arrows). Scale bar: 50 μm