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Abstract Background In the decapod crustacean brain, neurogenesis persists throughout the animal's life. After embryogenesis, the central olfactory pathway integrates newborn olfactory local and projection interneurons that replace old neurons or expand the existing population. In crayfish, these neurons are the descendants of precursor cells residing in a neurogenic niche. In this paper, the development of the niche was documented by monitoring proliferating cells with S-phase-specific markers combined with immunohistochemical, dye-injection and pulse-chase experiments. Results Between the end of embryogenesis and throughout the first post-embryonic stage (POI), a defined transverse band of mitotically active cells (which we will term 'the deutocerebral proliferative system' (DPS) appears. Just prior to hatching and in parallel with the formation of the DPS, the anlagen of the niche appears, closely associated with the vasculature. When the hatchling molts to the second post-embryonic stage (POII), the DPS differentiates into the lateral (LPZ) and medial (MPZ) proliferative zones. The LPZ and MPZ are characterized by a high number of mitotically active cells from the beginning of post-embryonic life; in contrast, the developing niche contains only very few dividing cells, a characteristic that persists in the adult organism. Conclusions Our data suggest that the LPZ and MPZ are largely responsible for the production of new neurons in the early post-embryonic stages, and that the neurogenic niche in the beginning plays a subordinate role. However, as the neuroblasts in the proliferation zones disappear during early post-embryonic life, the neuronal precursors in the niche gradually become the dominant and only mechanism for the generation of new neurons in the adult brain.

Abstract Background Adult neurogenesis, the production and integration of new neurons into circuits in the brains of adult animals, is a common feature of a variety of organisms, ranging from insects and crustaceans to birds and mammals. In the mammalian brain the 1st-generation neuronal precursors, the astrocytic stem cells, reside in neurogenic niches and are reported to undergo self-renewing divisions, thereby providing a source of new neurons throughout an animal's life. In contrast, our work shows that the 1st-generation neuronal precursors in the crayfish (Procambarus clarkii) brain, which also have glial properties and lie in a neurogenic niche resembling that of vertebrates, undergo geometrically symmetrical divisions and both daughters appear to migrate away from the niche. However, in spite of this continuous efflux of cells, the number of neuronal precursors in the crayfish niche continues to expand as the animals grow and age. Based on these observations we have hypothesized that (1) the neuronal stem cells in the crayfish brain are not self-renewing, and (2) a source external to the neurogenic niche must provide cells that replenish the stem cell pool. Results In the present study, we tested the first hypothesis using sequential double nucleoside labeling to track the fate of 1st- and 2nd-generation neuronal precursors, as well as testing the size of the labeled stem cell pool following increasing incubation times in 5-bromo-2'-deoxyuridine (BrdU). Our results indicate that the 1st-generation precursor cells in the crayfish brain, which are functionally analogous to neural stem cells in vertebrates, are not a self-renewing population. In addition, these studies establish the cycle time of these cells. In vitro studies examining the second hypothesis show that Cell Tracker™ Green-labeled cells extracted from the hemolymph, but not other tissues, are attracted to and incorporated into the neurogenic niche, a phenomenon that appears to involve serotonergic mechanisms. Conclusions These results challenge our current understanding of self-renewal capacity as a defining characteristic of all adult neuronal stem cells. In addition, we suggest that in crayfish, the hematopoietic system may be a source of cells that replenish the niche stem cell pool.

Abstract Background Serotonin (5-HT) is a potent regulator of adult neurogenesis in the crustacean brain, as in the vertebrate brain. However, there are relatively few data regarding the mechanisms of serotonin's action and which precursor cells are targeted. Therefore, we exploited the spatial separation of the neuronal precursor lineage that generates adult-born neurons in the crayfish (Procambarus clarkii) brain to determine which generation(s) is influenced by serotonin, and to identify and localize serotonin receptor subtypes underlying these effects. Results RT-PCR shows that mRNAs of serotonin receptors homologous to mammalian subtypes 1A and 2B are expressed in P. clarkii brain (referred to here as 5-HT1α and 5-HT2β). In situ hybridization with antisense riboprobes reveals strong expression of these mRNAs in several brain regions, including cell clusters 9 and 10 where adult-born neurons reside. Antibodies generated against the crustacean forms of these receptors do not bind to the primary neuronal precursors (stem cells) in the neurogenic niche or their daughters as they migrate, but do label these second-generation precursors as they approach the proliferation zones of cell clusters 9 and 10. Like serotonin, administration of the P. clarkii 5-HT1α-specific agonist quipazine maleate salt (QMS) increases the number of bromodeoxyuridine (BrdU)-labeled cells in cluster 10; the P. clarkii 5-HT2β-specific antagonist methiothepin mesylate salt (MMS) suppresses neurogenesis in this region. However, serotonin, QMS and MMS do not alter the rate of BrdU incorporation into niche precursors or their migratory daughters. Conclusion Our results demonstrate that the influences of serotonin on adult neurogenesis in the crayfish brain are confined to the late second-generation precursors and their descendants. Further, the distribution of 5-HT1α and 5-HT2β mRNAs and proteins indicate that these serotonergic effects are exerted directly on specific generations of neuronal precursors. Taken together, these results suggest that the influence of serotonin on adult neurogenesis in the crustacean brain is lineage dependent, and that 5-HT1α and 5-HT2β receptors underlie these effects.

Two decades after the discovery of adult‐born neurons in the brains of decapod crustaceans, the deutocerebral proliferative system (DPS) producing these neural lineages has become a model of adult neurogenesis in invertebrates. Studies on crayfish have provided substantial insights into the anatomy, cellular dynamics, and regulation of the DPS. Contrary to traditional thinking, recent evidence suggests that the neurogenic niche in the crayfish DPS lacks self‐renewing stem cells, its cell pool being instead sustained via integration of hemocytes generated by the innate immune system. Here, we investigated the origin, division and migration patterns of the adult‐born neural progenitor (NP) lineages in detail. We show that the niche cell pool is not only replenished by hemocyte integration but also by limited numbers of symmetric cell divisions with some characteristics reminiscent of interkinetic nuclear migration. Once specified in the niche, first generation NPs act as transit‐amplifying intermediate NPs that eventually exit and produce multicellular clones as they move along migratory streams toward target brain areas. Different clones may migrate simultaneously in the streams but occupy separate tracks and show spatio‐temporally flexible division patterns. Based on this, we propose an extended DPS model that emphasizes structural similarities to pseudostratified neuroepithelia in other arthropods and vertebrates. This model includes hemocyte integration and intrinsic cell proliferation to synergistically counteract niche cell pool depletion during the animal's lifespan. Further, we discuss parallels to recent findings on mammalian adult neurogenesis, as both systems seem to exhibit a similar decoupling of proliferative replenishment divisions and consuming neurogenic divisions. [ABSTRACT FROM AUTHOR]

New neurons continue to be born and integrated into the brains of adult decapod crustaceans. Evidence in crayfish indicates that the 1st-generation neural precursors that generate these adult-born neurons originate in the immune system and travel to the neurogenic niche via the circulatory system. These precursors are attracted to the niche, become integrated amongst niche cells, and undergo mitosis within a few days; both daughters of this division migrate away from the niche toward the brain clusters where they will divide again and differentiate into neurons. In the crustacean brain, the rate of neuronal production is highly sensitive to serotonin (5-hydroxytryptamine, 5-HT) levels. These effects are lineage-dependent, as serotonin's influence is limited to late 2nd-generation neural precursors and their progeny. Experiments indicate that serotonin regulates adult neurogenesis in the crustacean brain by multiple mechanisms: via direct effects of serotonin released from brain neurons into the hemolymph or by local release onto target cells, or by indirect influences via a serotonin-mediated release of agents from other regions, such as hormones from the sinus gland and cytokines from hematopoietic tissues. Evidence in crayfish also indicates that serotonin mediates the attraction of neural precursors generated by the immune system to the neurogenic niche. Thus, studies in the crustacean brain have revealed multiple roles for this monoamine in adult neurogenesis, and identified several pathways by which serotonin influences the generation of new neurons. [ABSTRACT FROM AUTHOR]

The 1st-generation neural precursors in the crustacean brain are functionally analogous to neural stem cells in mammals. Their slow cycling, migration of their progeny, and differentiation of their descendants into neurons over several weeks are features of the neural precursor lineage in crayfish that also characterize adult neurogenesis in mammals. However, the 1st-generation precursors in crayfish do not self-renew, contrasting with conventional wisdom that proposes the long-term self-renewal of adult neural stem cells. Nevertheless, the crayfish neurogenic niche, which contains a total of 200-300 cells, is never exhausted and neurons continue to be produced in the brain throughout the animal's life. The pool of neural precursors in the niche therefore cannot be a closed system, and must be replenished from an extrinsic source. Our in vitro and in vivo data show that cells originating in the innate immune system (but not other cell types) are attracted to and incorporated into the neurogenic niche, and that they express a niche-specific marker, glutamine synthetase. Further, labeled hemocytes that undergo adoptive transfer to recipient crayfish generate cells in neuronal clusters in the olfactory pathway of the adult brain. These hemocyte descendants express appropriate neurotransmitters and project to target areas typical of neurons in these regions. These studies indicate that under natural conditions, the immune system provides neural precursors supporting adult neurogenesis in the crayfish brain, challenging the canonical view that ectodermal tissues generating the embryonic nervous system are the sole source of neurons in the adult brain. However, these are not the first studies that directly implicate the immune system as a source of neural precursor cells. Several types of data in mammals, including adoptive transfers of bone marrow or stem cells as well as the presence of fetal microchimerism, suggest that there must be a population of cells that are able to access the brain and generate new neurons in these species. [ABSTRACT FROM AUTHOR]

The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues [ABSTRACT FROM AUTHOR]

Background: Adult neurogenesis, the production and integration of new neurons into circuits in the brains of adult animals, is a common feature of a variety of organisms, ranging from insects and crustaceans to birds and mammals. In the mammalian brain the 1st-generation neuronal precursors, the astrocytic stem cells, reside in neurogenic niches and are reported to undergo self-renewing divisions, thereby providing a source of new neurons throughout an animal's life. In contrast, our work shows that the 1st-generation neuronal precursors in the crayfish (Procambarus clarkii) brain, which also have glial properties and lie in a neurogenic niche resembling that of vertebrates, undergo geometrically symmetrical divisions and both daughters appear to migrate away from the niche. However, in spite of this continuous efflux of cells, the number of neuronal precursors in the crayfish niche continues to expand as the animals grow and age. Based on these observations we have hypothesized that (1) the neuronal stem cells in the crayfish brain are not self-renewing, and (2) a source external to the neurogenic niche must provide cells that replenish the stem cell pool. Results: In the present study, we tested the first hypothesis using sequential double nucleoside labeling to track the fate of 1st- and 2nd-generation neuronal precursors, as well as testing the size of the labeled stem cell pool following increasing incubation times in 5-bromo-2'-deoxyuridine (BrdU). Our results indicate that the 1stgeneration precursor cells in the crayfish brain, which are functionally analogous to neural stem cells in vertebrates, are not a self-renewing population. In addition, these studies establish the cycle time of these cells. In vitro studies examining the second hypothesis show that Cell Tracker™ Green-labeled cells extracted from the hemolymph, but not other tissues, are attracted to and incorporated into the neurogenic niche, a phenomenon that appears to involve serotonergic mechanisms. Conclusions: These results challenge our current understanding of self-renewal capacity as a defining characteristic of all adult neuronal stem cells. In addition, we suggest that in crayfish, the hematopoietic system may be a source of cells that replenish the niche stem cell pool. [ABSTRACT FROM AUTHOR]

Background: Serotonin (5-HT) is a potent regulator of adult neurogenesis in the crustacean brain, as in the vertebrate brain. However, there are relatively few data regarding the mechanisms of serotonin's action and which precursor cells are targeted. Therefore, we exploited the spatial separation of the neuronal precursor lineage that generates adult-born neurons in the crayfish (Procambarus clarkii) brain to determine which generation(s) is influenced by serotonin, and to identify and localize serotonin receptor subtypes underlying these effects. Results: RT-PCR shows that mRNAs of serotonin receptors homologous to mammalian subtypes 1A and 2B are expressed in P. clarkii brain (referred to here as 5-HT1α and 5-HT2β). In situ hybridization with antisense riboprobes reveals strong expression of these mRNAs in several brain regions, including cell clusters 9 and 10 where adult-born neurons reside. Antibodies generated against the crustacean forms of these receptors do not bind to the primary neuronal precursors (stem cells) in the neurogenic niche or their daughters as they migrate, but do label these second-generation precursors as they approach the proliferation zones of cell clusters 9 and 10. Like serotonin, administration of the P. clarkii 5-HT1α-specific agonist quipazine maleate salt (QMS) increases the number of bromodeoxyuridine (BrdU)-labeled cells in cluster 10; the P. clarkii 5-HT2β-specific antagonist methiothepin mesylate salt (MMS) suppresses neurogenesis in this region. However, serotonin, QMS and MMS do not alter the rate of BrdU incorporation into niche precursors or their migratory daughters. Conclusion: Our results demonstrate that the influences of serotonin on adult neurogenesis in the crayfish brain are confined to the late second-generation precursors and their descendants. Further, the distribution of 5-HT1α and 5-HT2β mRNAs and proteins indicate that these serotonergic effects are exerted directly on specific generations of neuronal precursors. Taken together, these results suggest that the influence of serotonin on adult neurogenesis in the crustacean brain is lineage dependent, and that 5-HT1α and 5-HT2β receptors underlie these effects. [ABSTRACT FROM AUTHOR]

Freshwater crayfish have three known photoreceptive systems: the compound eyes, extraretinal brain photoreceptors, and caudal photoreceptors. The primary goal of the work described here was to explore the contribution of the brain photoreceptors to circadian locomotory activity and define some of the underlying neural pathways. Immunocytochemical studies of the brain photoreceptors in the parastacid (southern hemisphere) crayfish Cherax destructor reveal their expression of the blue light-sensitive photopigment cryptochrome and the neurotransmitter histamine. The brain photoreceptors project to two small protocerebral neuropils, the brain photoreceptor neuropils (BPNs), where they terminate among fibers expressing the neuropeptide pigment-dispersing hormone (PDH), a signaling molecule in arthropod circadian systems. Comparable pathways are also described in the astacid (northern hemisphere) crayfish Procambarus clarkii. Despite exhibiting markedly different diurnal locomotor activity rhythms, removal of the compound eyes and caudal photoreceptors in both C. destructor and P. clarkii (leaving the brain photoreceptors intact) does not abolish the normal light/dark activity cycle in either species, nor prevent the entrainment of their activity cycles to phase shifts of the light/dark period. These results suggest, therefore, that crayfish brain photoreceptors are sufficient for the entrainment of locomotor activity rhythms to photic stimuli, and that they can act in the absence of the compound eyes and caudal photoreceptors. We also demonstrate that the intensity of PDH expression in the BPNs varies in phase with the locomotor activity rhythm of both crayfish species. Together, these findings suggest that the brain photoreceptor cells can function as extraretinal circadian photoreceptors and that the BPN represents part of an entrainment pathway synchronizing locomotor activity to environmental light/dark cycles, and implicating the neuropeptide PDH in these functions. [ABSTRACT FROM AUTHOR]

Mitotically active regions persist in the brains of decapod crustaceans throughout their lifetimes, as they do in many vertebrates. The most well-studied of these regions in decapods occurs within a soma cluster, known as cluster 10, located in the deutocerebrum. Cluster 10 in crayfish and lobsters is composed of the somata of two anatomically and functionally distinct classes of projection neurons: olfactory lobe (OL) projection neurons and accessory lobe (AL) projection neurons. While adult-generated cells in cluster 10 survive for at least a year, their final phenotypes remain unknown. To address this question, we combined BrdU labeling of proliferating cells with specific neuronal and glial markers and tracers to examine the differentiation of newborn cells in cluster 10 of the crayfish, Cherax destructor. Our results show that large numbers of adult-generated cells in cluster 10 differentiate into neurons expressing the neuropeptide crustacean-SIFamide. No evidence was obtained suggesting that cells differentiate into glia. The functional phenotypes of newborn neurons in cluster 10 were examined by combining BrdU immunocytochemistry with the application of dextran dyes to different brain neuropils. These studies showed that while the majority of cells born during the early postembryonic development of C. destructor differentiate in AL projection neurons, neurogenesis in adult crayfish is characterized by the addition of both OL and AL projection neurons. In addition to our examination of neurogenesis in the olfactory pathway, we provide the first evidence that adult neurogenesis is also a characteristic feature of the optic neuropils of decapod crustaceans. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [ABSTRACT FROM AUTHOR]