Adult neurogenesis: examples from the decapod crustaceans and comparisons with mammals

Abstract

Defining evolutionary origins is a means of understanding an organism's position within the integrated web of living beings, and not only to trace characteristics back in time, but also to project forward in an attempt to reveal relationships with more recently evolved forms. Both the vertebrates and arthropods possess condensed nervous systems, but this is dorsal in the vertebrates and ventral in the arthropods. Also, whereas the nervous system in the vertebrates develops from a neural tube in the embryo, that of the arthropods comes from an ectodermal plate. Despite these apparently fundamental differences, it is now generally accepted that life-long neurogenesis, the generation of functionally integrated neurons from progenitor cells, is a common feature of the adult brains of a variety of organisms, ranging from insects and crustaceans to birds and mammals. Among decapod crustaceans, there is evidence for adult neurogenesis in basal species of the Dendrobranchiata, as well as in more recent terrestrial, marine and fresh-water species. The widespread nature of this phenomenon in decapod species may relate to the importance of the adult-born neurons, although their functional contribution is not yet known. The many similarities between the systems generating neurons in the adult brains of decapod crustaceans and mammals, reviewed in this paper, suggest that adult neurogenesis is governed by common ancestral mechanisms that have been retained in a phylogenetically broad group of species.

Figure 1

An overview of the phylogenetic relationships among the decapod crustaceans, adapted from Sandeman et al., 1993 and Scholtz and Richter, 1995 (A) and Dixon et al., 2003 (B). The ellipses indicate those groups containing species in which studies have shown the presence of cell proliferation and the production of new neurons within sexually mature adults (see Schmidt and Harzsch, 1999). Evidence for the differentiation of newly born cells into neurons expressing the appropriate transmitters has been obtained only for the Astacida (Sullivan and Beltz, 2005a) and Achelata (Schmidt, 2001).

Figure 2

A. Diagram of the left side of the brain of Astacus leptodactylus, seen from the ventral side and showing the disposition of the deutocerebral organ first described by Bazin (1969a, b). This organ consists of a central cluster of cells that surround a homogeneously staining cavity. The central cluster of cells are connected by thin strands of fibers that run laterally over the surface of the accessory lobe to end amongst the projection neurons in Cluster 10 and medially around the nerve root of the nerve from the antennule to end in the local olfactory neurons in Cluster 9. B. Light micrograph of a section through the cavity in the center of the deutocerebral organ cell cluster of A. leptodactylus showing the amorphous contents and the surrounding cells with characteristically dark granules in their nuclei. C. Electron micrograph of a section through the same area as in B of A. leptodactylus which shows the membranes of the cells surrounding the cavity to be sculpted into many finger like processes that contact the structureless contents. D. Light micrograph of a semi-thin, toluidine blue stained section through the neurogenic niche of Procambarus clarkii. The components of the neurogenic system of P. clarkii, with a central niche consisting of a cluster of cells surrounding a central cavity and linked by fiber strands to the local and projection olfactory neurons, is homologous with the deutocerebral organ of Astacus. Abbreviations: amas cellulaire, A. C.; groupe olfactif antérieur, CEL.OLF.ANT; groupe olfactif postérieur, CEL.OLF.POST.; glomérule olfactif, GLOM.OLF; tractus antérieur, T. A.; tractus postérieur, T. P.; terminal bar, BT; Cluster 9, CL9; Cluster 10, CL10; nucleus, N; substance in the cavity, S; villi, V; vascular cavity, VC. Scale bars: B, 20 μm; C, 15 μm; D, 20 μm. (A, from Bazin, 1969a; B and C are previously unpublished images from Bazin, 1969b; D from Zhang et al., 2009)

Figure 3

Location of the components of the deutocerebral organ/neurogenic system on the ventral surface of the brains of the Astacida (A. leptodactylus), Anomala (G. squamifera, P. longicornus) and Brachyura (C. maenas). The central cell cluster/niche in Astacus leptodactylus lies on the ventral surface of the accessory lobe (AL) with a long medial migratory stream that curves around the nerve from the antennule and ends among the cells of the local olfactory interneurons in Cluster 9. The lateral migratory stream projects directly from the niche across the AL to end among the cells of the olfactory projection neurons in Cluster 10. B. Galathea squamifera is similar to A. leptodactylus, except that the niche lies more medial but is still separated from the local and projection neuron clusters by relatively long medial and lateral migratory streams. C. In Pisidia longicornis, the medial migatory stream is long, the lateral migratory stream very short and the niche is positioned close to the projection neuron cluster (CL10). D. The niche in Carcinus maenas is located within the olfactory projection neurons in Cluster 10. The accessory lobes in Galathea and Pisidia are relatively small in comparison with A. leptodactylus and reduced to a vestige in C. maenas. The migratory streams in these species therefore do not have to cross a large accessory lobe as in the astacids, and follow a path around the posterior and medial edges of the olfactory lobes (OL). Abbreviations: Cluster 9, CL9; Cluster 10, CL10; olfactory lobe, OL; accessory lobe, AL. Images are modified from Bazin 1969b. The diagrams are not to scale.

Figure 4

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 labeled immunocytochemically for BrdU (green). Labeled 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 labeled cells in a migratory stream that originates in the oval region labeled “niche”. Labeling 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 neurons form a cluster, the niche (red box), on the ventral surface of the brain. C. Glial cells (green, asterisks) in the niche labeled by intracellular injection of Lucifer yellow, have short processes (single arrow heads) that project to, and end in small swellings around the vascular cavity (arrow) and longer fibers (double arrow heads) 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) labeling 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 labeled preparation are all clearly distinguishable in the living system. Scale bars: A, 100μm; B, 75μm; C and D, 20μm; E, 25μm. (Images from Sullivan et al., 2007b)

Figure 5

A. Distribution of BrdU-labelled cells within the glial tracts over time. B. The glial tracts are migratory pathways to the proliferation zones. BrdU (cyan) and IdU (blue) labelling in the LPZ and the adjacent region of the glial tract in a double-nucleoside labelling experiment in which crayfish were exposed initially to BrdU and then six days later to IdU. The double arrowheads show BrdU-labelled cells in the region of the glial tract immediately adjacent to the LPZ while the arrows indicate IdU-labelled occurring along regions of the tract closer to the glial soma cluster. Scale bars = 40 μm. (Images from Sullivan et al., 2007a)

Figure 6

Precursor cells in the niche express LIS1 protein. A. Western blot for LIS1 on protein preparations from the P. clarkii brain (lane 1, 35mm carapace length (CL); lane 2, 16mm CL; lane 3, 8mm CL and adult mouse brain (lane 4). B. Whole mount brains were double-labeled for BrdU (red) and LIS1 (green) and counterstained with propidium iodide (blue), and optically sectioned with the confocal microscope. The white box outlines the niche, which is magnified in C and D. C. LIS1 staining is found throughout the niche cells and their fibers that form the stream, but is particularly strong immediately around the vascular cavity (white arrow). D. The same magnification as in C, but projecting all three channels in a single image. Scare bars: B, 200μm; C, D, 50 μm. (Images from Zhang et al., 2009)

Figure 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 labeled red indicate that they passed through the cell cycle in the presence of BrdU. Cells labeled green indicate that they have terminals in the accessory lobe but did not pass through a cell cycle in the presence of BrdU. Double-labeled 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-labeled 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 labeling indicates that these cells were born in the adult animal and have differentiated into olfactory interneurons. Scale bars: A, 10μm; B, C, 20μm; C insert, 10μm.

Figure 8

Model summarizing our current view of events leading to the production of new olfactory interneurons in adult crayfish. Neuronal precursor (1^st generation) cells reside within a neurogenic niche where they divide symmetrically. Their daughters (2^nd generation precursors) migrate along tracts created by the fibers of the niche cells, towards either the LPZ or the MPZ. At least one more division will occur in the LPZ and MPZ before the progeny (3^rd and subsequent generations) differentiate into neurons.

Figure 9

Comparison of precursor cell total numbers and BrdU-labeled cells in the niches and the streams in different sizes of crayfish. The x-axis indicates the average carapace length (CL) in mm of the animals. Animals were exposed to BrdU for 8 hours prior to fixation, then immunolabeled for BrdU and glutamine synthetase and counterstained with propidium iodide. Total cell numbers and BrdU-labeled cells in the niches and streams were counted. Histograms A and B show the total numbers of cells and BrdU-positive cells in the niche respectively. Note a significant decrease in the numbers (B) of BrdU-labeled cells with increasing size. Significant difference between groups (Tukey multiple comparison) are marked with single (P<0.05), double (P< 0.01) or triple (P<0.001) asterisks. n = numbers of niches assayed in A-B. C. Triple-labeled M-phase cell close to the niche that has almost completed cytokinesis, immunolabeled with GS (cyan), phosphohistone-H3 (green) and BrdU (red). Images show a small area of cytoplasm that is still shared between the two emerging, geometrically symmetrical daughter cells. The cytoplasm of the dividing cells is GS-positive, a characteristic feature of the cells residing in the niche, thus confirming the ancestry of these cells. The asterisk marks the vascular cavity. Scale bar: C, 20μm; insets, 10μm.

Figure 10

Schematic representation of the cellular basis of adult neurogenesis in the olfactory midbrain of Panulirus argus. Large putative adult neuroblasts (NB) in the vicinity of proliferation zones act as primary neuronal stem cells. By rapid asymmetric cell divisions they self-renew and generate daughter cells that migrate into the proliferation zone and there divide once symmetrically on a much slower time scale. After this mitosis, both daughter cells are pushed out of the proliferation zone and either differentiate into neurons (the vast majority) or die by apoptosis (very few). In essence this process resembles closely neurogenesis in the nervous system of embryonic and larval crustaceans and insects, where large neuroblasts undergo a series of asymmetric divisions in which they self-renew and generate smaller ganglion mothers cells, which in turn divide once symmetrically generating two neurons. One significant difference is that the putative adult neuroblasts (NB) are associated with specialized cellular aggregates that likely are critical for their unusual life-long capacity to self-renew and proliferate. (Image and legend adapted from Schmidt, 2007; request for permission to reprint is pending with John Wiley and Son, Inc.).

Figure 11

Cell types and anatomy of the adult SVZ niche. Schema of frontal section of the adult mouse brain showing the SVZ (orange) adjacent to the lateral ventricle (LV). SVZ astrocytes in this region (B, blue) are stem cells which generate migrating neuroblasts (A, red) destined for the olfactory bulb via a rapidly dividing transit-amplifying cell (C, green). Region in box is expanded at right to show the relationship of cells in this region and some elements of the SVZ niche. Multi-ciliated ependymal cells (E, grey) line the walls of the lateral ventricle. Chains of neuroblasts travel through tunnels formed by processes of SVZ astrocytes. Transit-amplifying cells are found in small clusters adjacent to the chains. Signals released from axons (pink) regulate proliferation and survival in this region. A specialized basal lamina (BL, black) extends from perivascular cells and contacts all cell types. Endothelial cells, blood vessels (BV) and the basal lamina are all likely key components of the niche. (Image and legend from Riquelme et al., 2008; permission to reprint granted by The Royal Society).