Masters of communication: The brain of the banded cleaner shrimp Stenopus hispidus (Olivier, 1811) with an emphasis on sensory processing areas.

The pan-tropic cleaner shrimp Stenopus hispidus (Crustacea, Stenopodidea) is famous for its specific cleaning behavior in association with client fish and an exclusively monogamous life-style. Cleaner shrimps feature a broad communicative repertoire, which is considered to depend on superb motor skills and the underlying mechanosensory circuits in combination with sensory organs. Their most prominent head appendages are the two pairs of very long biramous antennules and antennae, which are used both for attracting client fish and for intraspecific communication. Here, we studied the brain anatomy of several specimens of S. hispidus using histological sections, immunohistochemical labeling as well as X-ray microtomography in combination with 3D reconstructions. Furthermore, we investigated the morphology of antennules and antennae using fluorescence and scanning electron microscopy. Our analyses show that in addition to the complex organization of the multimodal processing centers, especially chemomechanosensory neuropils associated with the antennule and antenna are markedly pronounced when compared to the other neuropils of the central brain. We suggest that in their brains, three topographic maps are present corresponding to the sensory appendages. The brain areas which provide the neuronal substrate for these maps share distinct structural similarities to a unique extent in decapods, such as size and characteristic striated and perpendicular layering. We discuss our findings with respect to the sensory landscape within animal's habitat. In an evolutionary perspective, the cleaner shrimp's brain is an excellent example of how sensory potential and functional demands shape the architecture of primary chemomechanosensory processing areas.

Birth, survival and differentiation of neurons in an adult crustacean brain.

Life-long neurogenesis is a characteristic feature of many vertebrate and invertebrate species. In decapod crustaceans, new neurons are added throughout life to two cell clusters containing local (cluster 9) and projection (cluster 10) interneurons in the olfactory pathway. Adult-born neurons in clusters 9 and 10 in crayfish have the anatomical properties and chemistry of mature neurons by 6 months after birth. Here we use 5-bromo-2'-deoxyuridine (BrdU) incorporation to pulse label mitotically active cells in these cell clusters, followed by a survival time of up to 8 months, during which crayfish (Cherax destructor) were sacrificed at intervals and the numbers of BrdU-labeled cells quantified. We find a decrease in the numbers of BrdU-labeled cells in cell cluster 10 between the first and second weeks following BrdU exposure, suggesting a period of cell death shortly after proliferation. Additional delayed cell divisions in both cell clusters are indicated by increases in labeled cells long after the BrdU clearing time. The differentiation time of these cells into neurons was defined by detection of the first immunoreactivity for the transmitter SIFamide in cluster 10 BrdU-labeled cells, which begins at 4 weeks after BrdU labeling; the numbers of SIFamide-labeled cells continues to increase over the following month. Experiments testing whether proliferation and survival of Cluster 10 cells are influenced by locomotor activity provided no evidence of a correlation between activity levels and cell proliferation, but suggest a strong influence of locomotor activity on cell survival.

Adult neurogenesis in the crayfish brain: the hematopoietic anterior proliferation center has direct access to the brain and stem cell niche.

Neuronal stem cells residing in a niche on the surface of the adult crayfish (Procambarus clarkii) brain are not self-renewing. However, the neuronal precursors in the niche are not depleted despite continued neurogenesis and the exit of precursor cells from the niche throughout the organism's life. The neurogenic niche is therefore not a closed system, and we have previously proposed that the stem cell pool is replenished from the hematopoietic system. Noonin et al. (2012) demonstrated that the hematopoietic system in the crayfish Pacifastacus leniusculus includes an anterior proliferation center (APC) lying near the brain; they suggest that multipotent stem cells are concentrated in this region, and that the APC may provide neuronal stem cells for adult neurogenesis. The present study extends this work by describing the location and cellular organization of hematopoietic tissues in P. clarkii. We find that the APC lies within the cor frontale, or auxiliary heart, which pumps hemolymph to the brain and eyes through the cerebral and ophthalmic arteries, respectively. Vascular extensions of the cerebral artery converge on the neurogenic niche. APC cells lie in layered sheets within the cor frontale and form rosette-like structures reminiscent of stem cells in other developing tissues. We confirm here that APC cells in P. clarkii have characteristics of multipotent stem cells, and that their location within the cor frontale allows direct access to regions in the central nervous system in which adult neurogenesis occurs.

First-generation neuronal precursors in the crayfish brain are not self-renewing.

Adult-born neurons in crayfish (Procambarus clarkii) are the progeny of 1st-generation precursor cells (functionally analogous to neuronal stem cells in vertebrates) that are located in a neurogenic niche on the ventral surface of the brain. The daughters of these precursor cells migrate along the processes of bipolar niche cells to proliferation zones in the cell clusters where the somata of the olfactory interneurons reside. Here they divide again, producing offspring that differentiate into olfactory local and projection neurons. The features of this neuronal assembly line, and the fact that it continues to function when the brain is isolated and perfused or maintained in organotypic culture, 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. Further, we have determined that the 1st-generation precursor cells are not a self-renewing population, and that the niche is, nevertheless, not depleted as the animals grow and age. We conclude, therefore, that the niche is not a closed system and that there must be an extrinsic source of neuronal stem cells. Based on in vitro studies demonstrating that cells extracted from the hemolymph are attracted to the niche, as well as the intimate relationship between the niche and vasculature, we hypothesize that the hematopoietic system is a likely source of these cells.

Differential uptake of MRI contrast agents indicates charge-selective blood-brain interface in the crayfish.

This study provides a new perspective on the long-standing problem of the nature of the decapod crustacean blood-brain interface. Previous studies of crustacean blood-brain interface permeability have relied on invasive histological, immunohistochemical and electrophysiological techniques, indicating a leaky non-selective blood-brain barrier. The present investigation involves the use of magnetic resonance imaging (MRI), a method for non-invasive longitudinal tracking of tracers in real-time. Differential uptake rates of two molecularly distinct MRI contrast agents, namely manganese (Mn(II)) and Magnevist® (Gd-DTPA), were observed and quantified in the crayfish, Cherax destructor. Contrast agents were injected into the pericardium and uptake was observed with longitudinal MRI for approximately 14.5 h. Mn(II) was taken up quickly into neural tissue (within 6.5 min), whereas Gd-DTPA was not taken up into neural tissue and was instead restricted to the intracerebral vasculature or excreted into nearby sinuses. Our results provide evidence for a charge-selective intracerebral blood-brain interface in the crustacean nervous system, a structural characteristic once considered too complex for a lower-order arthropod.

Adult neurogenesis in the decapod crustacean brain: a hematopoietic connection?

New neurons are produced and integrated into circuits in the adult brains of many organisms, including crustaceans. In some crustacean species, the first-generation neuronal precursors reside in a niche exhibiting characteristics analogous to mammalian neurogenic niches. However, unlike mammalian niches where several generations of neuronal precursors co-exist, the lineage of precursor cells in crayfish is spatially separated allowing the influence of environmental and endogenous regulators on specific generations in the neuronal precursor lineage to be defined. Experiments also demonstrate that the first-generation neuronal precursors in the crayfish Procambarus clarkii are not self-renewing. A source external to the neurogenic niche must therefore provide cells that replenish the first-generation precursor pool, because although these cells divide and produce a continuous efflux of second-generation cells from the niche, the population of first-generation niche precursors is not diminished with growth and aging. In vitro studies show that 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. We propose that, in crayfish, the hematopoietic system may be a source of cells that replenish the niche cell pool. These and other studies reviewed here establish decapod crustaceans as model systems in which the processes underlying adult neurogenesis, such as stem cell origins and transformation, can be readily explored. Studies in diverse species where adult neurogenesis occurs will result in a broader understanding of fundamental mechanisms and how evolutionary processes may have shaped the vertebrate/mammalian condition.

Primary neuronal precursors in adult crayfish brain: replenishment from a non-neuronal source.

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.

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

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.

Brain architecture of the largest living land arthropod, the Giant Robber Crab Birgus latro (Crustacea, Anomura, Coenobitidae): evidence for a prominent central olfactory pathway?

BACKGROUND: Several lineages within the Crustacea conquered land independently during evolution, thereby requiring physiological adaptations for a semi-terrestrial or even a fully terrestrial lifestyle. Birgus latro Linnaeus, 1767, the giant robber crab or coconut crab (Anomura, Coenobitidae), is the largest land-living arthropod and inhabits Indo-Pacific islands such as Christmas Island. B. latro has served as a model in numerous studies of physiological aspects related to the conquest of land by crustaceans. From an olfactory point of view, a transition from sea to land means that molecules need to be detected in gas phase instead of in water solution. Previous studies have provided physiological evidence that terrestrial hermit crabs (Coenobitidae) such as B. latro have a sensitive and well differentiated sense of smell. Here we analyze the brain, in particular the olfactory processing areas of B. latro, by morphological analysis followed by 3 D reconstruction and immunocytochemical studies of synaptic proteins and a neuropeptide. RESULTS: The primary and secondary olfactory centers dominate the brain of B. latro and together account for ca. 40% of the neuropil volume in its brain. The paired olfactory neuropils are tripartite and composed of more than 1,000 columnar olfactory glomeruli, which are radially arranged around the periphery of the olfactory neuropils. The glomeruli are innervated ca. 90,000 local interneurons and ca. 160,000 projection neurons per side. The secondary olfactory centers, the paired hemiellipsoid neuropils, are targeted by the axons of these olfactory projection neurons. The projection neuron axonal branches make contact to ca. 250.000 interneurons (per side) associated with the hemiellipsoid neuropils. The hemiellipsoid body neuropil is organized into parallel neuropil lamellae, a design that is quite unusual for decapod crustaceans. The architecture of the optic neuropils and areas associated with antenna two suggest that B. latro has visual and mechanosensory skills that are comparable to those of marine Crustacea. CONCLUSIONS: In parallel to previous behavioral findings that B. latro has aerial olfaction, our results indicate that their central olfactory pathway is indeed most prominent. Similar findings from the closely related terrestrial hermit crab Coenobita clypeatus suggest that in Coenobitidae, olfaction is a major sensory modality processed by the brain, and that for these animals, exploring the olfactory landscape is vital for survival in their terrestrial habitat. Future studies on terrestrial members of other crustacean taxa such as Isopoda, Amphipoda, Astacida, and Brachyura will shed light on how frequently the establishment of an aerial sense of olfaction evolved in Crustacea during the transition from sea to land. Amounting to ca. 1,000,000, the numbers of interneurons that analyse the olfactory input in B. latro brains surpasses that in other terrestrial arthropods, as e.g. the honeybee Apis mellifera or the moth Manduca sexta, by two orders of magnitude suggesting that B. latro in fact is a land-living arthropod that has devoted a substantial amount of nervous tissue to the sense of smell.

Brain photoreceptor pathways contributing to circadian rhythmicity in crayfish.

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.

Adult neurogenesis in the crayfish brain: proliferation, migration, and possible origin of precursor cells.

The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life-long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein-binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life-long growth and maintenance of the crustacean neurogenic niche.

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

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.

Omega-3 fatty acids upregulate adult neurogenesis.

Omega-3 fatty acids play crucial roles in the development and function of the central nervous system. These components, which must be obtained from dietary sources, have been implicated in a variety of neurodevelopmental and psychiatric disorders. Furthermore, the presence of omega-6 fatty acids may interfere with omega-3 fatty acid metabolism. The present study investigated whether changes in dietary ratios of omega-3:omega-6 fatty acids influence neurogenesis in the lobster (Homarus americanus) brain where, as in many vertebrate species, neurogenesis persists throughout life. The factors that regulate adult neurogenesis are highly conserved among species, and the crustacean brain has been successfully utilized as a model for investigating this process. In this study, lobsters were fed one of three diets that differed in fatty acid content. These animals were subsequently incubated in 5-bromo-2'-deoxyuridine (BrdU) to detect cells in S-phase of the cell cycle. A quantitative analysis of the resulting BrdU-labeled cells in the projection neuron cluster in the brain shows that short-term augmentation of dietary omega-3 relative to omega-6 fatty acids results in significant increases in the numbers of S phase cells, and that the circadian pattern of neurogenesis is also altered. It is proposed that the ratio of omega-3:omega-6 fatty acids may alter neurogenesis via modulatory influences on membrane proteins, cytokines and/or neurotrophins.

Adult neurogenesis: a common strategy across diverse species.

Adult neurogenesis, the generation of new neurons from adult precursor cells, occurs in the brains of a phylogenetically diverse array of animals. In the higher (amniotic) vertebrates, these precursor cells are glial cells that reside within specialized regions, known as neurogenic niches, the elements of which both support and regulate neurogenesis. The in vivo identity and location of the precursor cells responsible for adult neurogenesis in nonvertebrate taxa, however, remain largely unknown. Among the invertebrates, adult neurogenesis has been particularly well characterized in freshwater crayfish (Arthropoda, Crustacea), although the identity of the precursor cells sustaining continuous neuronal proliferation in these animals has yet to be established. Here we provide evidence suggesting that, as in the higher vertebrates, the precursor cells maintaining adult neurogenesis in the crayfish Procambarus clarkii are glial cells. These precursor cells reside within a specialized region, or niche, on the ventral surface of the brain, and their progeny migrate from this niche along glial fibers and then proliferate to form new neurons in the central olfactory pathway. The niche in which these precursor cells reside has many features in common with the neurogenic niches of higher vertebrates. These commonalities include: glial cells functioning as both precursor and support cells, directed migration, close association with the brain vasculature, and specialized basal laminae. The cellular machinery maintaining adult neurogenesis appears, therefore, to be shared by widely disparate taxa. These extensive structural and functional parallels suggest a common strategy for the generation of new neurons in adult brains.

Magnetic resonance imaging at 9.4 T as a tool for studying neural anatomy in non-vertebrates.

This report describes magnetic resonance imaging (MRI) methods we have developed at 9.4 T for observing internal organs and the nervous system of an invertebrate organism, the crayfish, Cherax destructor. We have compared results acquired using two different pulse sequences, and have tested manganese (Mn(2+)) as an agent to enhance contrast of neural tissues in this organism. These techniques serve as a foundation for further development of functional MRI and neural tract-tracing methods in non-vertebrate systems.

Ecological, evolutionary, and functional correlates of sensilla number and glomerular density in the olfactory system of decapod crustaceans.

One of the features common among olfactory systems for vertebrate and invertebrate species is the division of the primary processing area into distinct clumps of synaptic neuropil, called glomeruli. The olfactory glomeruli appear to serve as functional units of olfaction and are the location of the primary processing between chemosensory afferents and second-order neurons. Although glomeruli are found across all phyla, their numbers and size appear to be characteristic for each species, giving rise to the speculation that there is a relationship between glomerular number and function. It has been hypothesized, for example, that animals with more glomeruli may be able to resolve a wider range of odors. Crustacean species are distributed among freshwater, marine, and terrestrial habitats in arctic, temperate, and tropical climates. They also exhibit a variety of lifestyles and behaviors in which olfaction may play a dominant role. Feeding, for example, ranges from carnivorous, through subaquatic and terrestrial omnivorous scavenging, to filter feeding. Mating and territorial behaviors also are known to involve chemical signals. The current study examines glomerular numbers in the olfactory lobes of 17 crustacean species from six of the seven taxa now included in the reptantian decapods. Estimates of the glomerular numbers were obtained from the analysis of sectioned material treated immunocytochemically with an antibody against synapsin that labels proteins contained in neuronal terminals. The numbers of glomeruli found in the different species were then compared with the volume of the glomerular neuropil, numbers of olfactory sensilla, life styles, habitat, and phylogenetic affinities. The picture that emerges from these correlations is that the decapod crustaceans have exploited various strategies in the construction of their olfactory systems in which the problems of size, sensitivity, and selectivity have all interacted. We find a continuum across the groups ranging from those that favor a high convergence of receptor neurons onto a few glomeruli to those that share a small number of receptor neurons among many glomeruli. The potential functional consequences of these differences are discussed.

Regulation of life-long neurogenesis in the decapod crustacean brain.

This article provides an overview of our understanding of life-long neurogenesis in the decapod crustacean brain, where the proliferation of sensory and interneurons is controlled by many of the same factors as is neurogenesis in the mammalian brain. The relative simplicity, spatial organization and accessibility of the crustacean brain provide opportunities to examine specific neuronal pathways that regulate neurogenesis and the sequence of gene expression that leads to neuronal differentiation.