5-HT receptors mediate lineage-dependent effects of serotonin on adult neurogenesis in Procambarus clarkii

<p>Abstract</p> <p>Background</p> <p>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 (<it>Procambarus clarkii</it>) brain to determine which generation(s) is influenced by serotonin, and to identify and localize serotonin receptor subtypes underlying these effects.</p> <p>Results</p> <p>RT-PCR shows that mRNAs of serotonin receptors homologous to mammalian subtypes 1A and 2B are expressed in <it>P. clarkii </it>brain (referred to here as 5-HT_1αand 5-HT_2β). <it>In situ </it>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 <it>P. clarkii </it>5-HT_1α-specific agonist quipazine maleate salt (QMS) increases the number of bromodeoxyuridine (BrdU)-labeled cells in cluster 10; the <it>P. clarkii </it>5-HT_2β-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.</p> <p>Conclusion</p> <p>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-HT_1αand 5-HT_2βmRNAs 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-HT_1αand 5-HT_2βreceptors underlie these effects.</p

Neurogenesis in the central olfactory pathway of adult decapod crustaceans: development of the neurogenic niche in the brains of procambarid crayfish

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Adult Neurogenesis: Ultrastructure of a Neurogenic Niche and Neurovascular Relationships

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

From Blood to Brain: Adult-Born Neurons in the Crayfish Brain Are the Progeny of Cells Generated by the Immune System

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

Primary Neuronal Precursors in Adult Crayfish Brain: Replenishment from a Non-neuronal Source

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.</p

Electronmicrographs of Type I cells.

<p>(A) Region of the niche near the vascular cavity (vc) showing Type I cells lining the edge of the cavity. Note the microvilli (mv) projecting into the cavity. <i>Zonulae adherens</i> join adjacent cells (double arrow head). (B) Type I niche cells near the emergence of the streams have elongated nuclei containing heterochromatin and euchromatin. A nucleolus (n) is apparent. Connective tissue (ct) is adjacent to the dorsal surface of the niche facing the accessory lobe (AL); a cell with features of a crustacean hemocyte (asterisk; hyaline cell, e.g., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone.0039267-HoseJ1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone.0039267-ChavesdaSilva1" target="_blank">[57]</a>) is located within this tissue, although a blood vessel is not apparent. (C) Type I cells contain an abundance of mitochondrial profiles (white arrow), rough endoplasmic reticulum (black arrow), polysomes (arrowhead) and vesicles. (D) Cytoplasm of type I cells in high magnification: Observe the numerous mitochondria (arrow) and microtubules, seen in detail in the insert, lower left. (E) Another view of the apical surface of a Type I cell, showing a <i>zonula adherens</i> between adjacent cells, as well as microvilli projecting into the lumen of the vascular cavity (vc). The insert (upper right) shows a higher magnification of a <i>zonula adherens</i>. (F) Type I cells united by a septate junction. Scale bars: (A) 0.5 µm; (B) 5 µm; (C) 1 µm; (D, E) 0.5 µm; Inserts in (D) 0.1 µm and (E) 0.5 µm; (F) 0.2 µm.</p

Ultrastructure of the neurogenic niche cells.

<p>(A) Type I cells have nuclei that are frequently elongated; these contain both heterochromatin and euchromatin, and a nucleolus (n) is often seen. Mitochondrial profiles (white arrow) and rough endoplasmic reticulum (double arrowhead) are abundant. In the upper part of the image note the processes (arrows) projecting into the vascular cavity (vc).The vascular cavity is colorized yellow to highlight the processes of the niche cells projecting into this area. (B) Type II cells have a nucleus containing abundant euchromatin and sparse heterochromatin. Note mitochondrial profiles (arrow) and many vesicles in the cytoplasm. (C) Type III cells display a nucleus composed of clumps of heterochromatin. Their cytoplasm contains vesicles, some of them with membranes in their interior (black arrow), as well as a Golgi apparatus (outlined in C and magnified in the insert). (D) Type IV cells are found in pairs, suggestive of cell division, however, no shared plasma membrane between the two nuclei is visible. In the cytoplasm observe mitochondrial profiles (white arrow), dilated vesicles and cisternae of the endoplasmic reticulum (double arrowhead). Scale bars: (A–D) 2 µm. Insert in (C), 0.5 µm.</p

Stacked confocal scans of a 100 µm sagittal section in a <i>P. clarkii</i> brain show the neurogenic niche (A–D), stained for GS (blue), protruding from the ventral surface (arrow, red outline) of the accessory lobe (AL).

<p>Serotonergic labeling (5-HT) in the olfactory lobe (OL) and AL (A–D, green) reveals fibers from the Dorsal Giant Neuron (DGN) innervating these lobes (A and Aii; white arrows). 5-HT labeling of individual glomeruli (dotted outlines in Aii) and DGN fibers (Aii, arrows) are clearly shown in this section of the AL. Magnifications of this section, including images deeper into the tissue (Ai, B, C) display the three separate laser channels representing the nuclear stain, propidium iodide (PI, red), serotonin (5-HT, green) and glutamine synthetase (GS, blue); PI and GS labeling together reveal the multi-layered aspect of the niche when the brain is sectioned sagittally (B, C, D). In C and D, white arrows indicate the GS-labeled cytoplasm (blue) of the niche cells, demonstrating that the deepest, most dorsal niche layer (outline in D) does not label for GS (dotted line in C, lower panels). The PI labeling cells also delineates the trail of cells, composing a blood vessel that extends from within the AL into the niche itself (Ai, white square; B,white square; Bi, arrow). Higher magnifications show a serotonin immunoreative “crown” of terminals within the outer, ventral layer of the niche (C, asterisk); a weakly immunoreactive serotonergic fiber(s) (arrowheads) is observed connecting to this serrated-like region. Scale bar: (A) 200 µm; (Ai, Aii, B) 20 µm; (D) 10 µm; (Bi, C) 5 µm.</p