The neuro-glandular brain of the Pyramicocephalus phocarum plerocercoid (Cestoda, Diphyllobothriidea): Immunocytochemical and ultrastructural study.

А novel type of a complex neuro-glandular brain structure including both nervous and glandular elements and associated with sensory ones is detected in Pyramicocephalus phocarum plerocercoid (Cestoda: Diphyllobothriidea), parasite of Gadus morua from the White Sea. The brain has two lateral lobes connected by a long cellular median commissure. The brain is tightly surrounded by glandular cells, which receive numerous synapses from the brain neurons. A complex of sensory organs associated with ducts and terminal pores of the frontal glands lies in the scolex tegument. Serotonin, FMRFamide- and GABA-like immunoreactive (IR) neurons are found in the brain, the main nerve cords, and the plexus of the plerocercoid. The innervation of the frontal gland ducts by FMRFamide-IR neurites is detected for the first time proving that they function under control of the nervous system and thus evidencing the eccrine nature of the secretion mechanism. Ultrastructural data show that light, dark and neurosecretory neurons are present in the brain lobes. The median commissure consists of loosely arranged thin parallel axons and several giant and small neurons. The commissure is stratified and penetrated by frontal glandular cells and their processes. Such neuro-glandular morpho-functional brain complex is suggested as a model for Diphyllobothriidae family. Five structural types of sensory organs are described in the scolex of P. phocarum; their colocalization with eccrine gland terminals is supposedly specific for Diphyllobothriidae family. Within the order Diphyllobothriidea, there are significant differences in the architecture of the plerocercoid brain at the family level. We suppose homology of giant commissural neurons among Diphyllobothriidea. Differences between diphyllobothriidean nervous system and that of other cestodes are discussed.

In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

Specialised rootlets of Sacculina pilosella (Rhizocephala: Sacculinidae) used for interactions with its host's nervous system.

Parasitic rhizocephalan barnacles induce morphological, physiological, and behavioural changes in their hosts. The mechanisms of these intimate host-parasite interactions remain unknown. We have shown previously that rootlets of the internae of Peltogasterella gracilis and Peltogaster paguri penetrate the ganglion's envelope of their hermit crab hosts and form specialised structures in the ganglion periphery, the so-called goblet-shaped organs. Here, we examine the gross morphology and ultrastructure of these goblet-shaped organs in the interna of Sacculina pilosella. They consist of three layers of cells; in the intermediate layer of the organs, unusual lamellar bodies and muscle cells were found. Extensive degeneration of the host nervous tissue was observed in the funnel of the goblet-shaped organs. We conclude that the ability to penetrate into the host's nervous tissue could be a common trait in rhizocephalans. The goblet-shaped organs may play a key role in the host-parasite relationships by enabling the parasite to influence the host via hormones and neurotransmitters.

Amperometry in Single Cells and Tissue.

The release from cells of signaling molecules through the controlled process of exocytosis involves multiple coordinated steps and is essential for the proper control of a multitude of biological pathways across the endocrine and nervous systems. However, these events are minute both temporally and in terms of the minute amounts of neurotransmitters, hormones, growth factors, and peptides released from single vesicles during exocytosis. It is therefore difficult to measure the kinetics of single exocytosis events in real time. One noninvasive method of measuring the release of molecules from cells is carbon-fiber amperometry. In this chapter, we will describe how we undertake such measurements from both single cells and in live tissue, how the subsequent data are analyzed, and how we interpret these results in terms of their relevant physiology.

Novel data on the innervation of the lophophore in adult phoronids (Lophophorata, Phoronida).

The structure of the lophophore nervous system may help clarify the status of the clade Lophophorata, whose monophyly is debated. In the current study, antibody labeling and confocal laser scanning microscopy revealed previously undescribed main nerve elements in the lophophore in adult phoronids: Phoronis australis and Phoronopsis harmeri. In both species, the nervous system includes a dorsal ganglion, a tentacle nerve ring, an inner nerve ring, intertentacular groups of perikarya, and tentacle nerves. The dorsal ganglion and tentacle nerve ring contain many serotonin-like immunoreactive perikarya of different sizes. The inner nerve ring is described for the first time in adult phoronids with complex lophophore. It contains a thin bundle of serotonin-like immunoreactive neurites. The tentacles possess abfrontal, frontal, and laterofrontal nerves. The abfrontal nerves originate from the tentacle nerve ring; the frontal tentacle nerves extend from the inner nerve ring in P. harmeri and from the intertentacular frontal nerves in P. australis. The intertentacular groups of perikarya are found in phoronids for the first time. These small nerve centers connect with neither the tentacle nerve ring nor the inner nerve ring, giving rise to the laterofrontal tentacle nerves. The discovery of the inner nerve ring in adult phoronids makes the architecture of the lophophore nervous system similar in all lophophorates and thereby supports the monophyly of this group. The presence of intertentacular nerves, perikarya, and groups of perikarya is a typical feature of the nervous system in lophophorate presumably coordinating movements of the tentacles and thereby increasing the efficiency of lophophore functioning.

Fine structure of the nervous system of Cercaria parvicaudata Stunkard & Shaw, 1931 (Digenea, Renicolidae).

The biology of free-living and parasitic Platyhelminthes is diverse. Taking into account the widespread prevalence of parasitic flatworms, Digenea is the least studied group regarding the fine structure of nervous system especially of the cercarial life stage. Here, we present a description of the fine structure of central nervous system (CNS) and two types of uniciliate sensory papillae of xiphidiocercaria Cercaria parvicaudata (Microphalloidea, Renicolidae). The present study documents that C. parvicaudata has a complex nervous system that includes a well-developed ganglion with a cortex of perikarya and glia-like sheaths, myelin-like structures within one of the dorsal nerve cords and four types of polarized synapses between neurites. Different types of neurons in the CNS could not be distinguished on ultrastructural level due to high similarity in their fine structure. Shared polarized synapses with high electron density of presynaptic components are numerous in the neuropile and nerve cords of this larva. Within the larval body, we detected specialized "support" processes that relate to different tissues. Some "support" processes are also closely related to the nervous system of C. parvicaudata, where they are considered as glia-like structures. In this case, the fine structure of glia-like "support" cells of C. parvicaudata differs from those described as glia-like cells in adult flatworms. We suggest a wide prevalence of glia-like cells among cercariae, as well as the fact that glia-like structures in digenean nervous systems can develop from various nonneuronal tissues.

Complex insight on microanatomy of larval "human broad tapeworm" Dibothriocephalus latus (Cestoda: Diphyllobothriidea).

BACKGROUND: In Europe, the tapeworm Dibothriocephalus latus (syn. Diphyllobothrium latum) is a well-known etiological agent of human diphyllobothriosis, which spreads by the consumption of raw fish flesh infected by plerocercoids (tapeworm's larval stage). However, the process of parasite establishment in both intermediate and definitive hosts is poorly understood. This study was targeted mainly on the scolex (anterior part) of the plerocercoid of this species, which facilitates penetration of the parasite in intermediate paratenic fish hosts, and subsequently its attachment to the intestine of the definitive host. METHODS: Plerocercoids were isolated from the musculature of European perch (Perca fluviatilis) caught in Italian alpine lakes. Parasites were examined using confocal microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunofluorescence tagging was held on whole mount larvae. RESULTS: The organisation of the central and peripheral nervous system was captured in D. latus plerocercoids, including the ultrastructure of the nerve cells possessing large dense neurosecretory granules. Two types of nerve fibres run from the body surface toward the nerve plexus located in the parenchyma on each side of bothria. One type of these fibres was found to be serotoninergic and possessed large subtegumental nerve cell bodies. A well-developed gland apparatus, found throughout the plerocercoid parenchyma, produced heterogeneous granules with lucent core packed in a dense layer. Three different types of microtriches occurred on the scolex and body surface of plerocercoids of D. latus: (i) uncinate spinitriches; (ii) coniform spinitriches; and (iii) capilliform filitriches. Non-ciliated sensory receptors were observed between the distal cytoplasm of the tegument and the underlying musculature. CONCLUSIONS: Confocal laser scanning microscopy and electron microscopy (SEM and TEM) showed the detailed microanatomy of the nervous system in the scolex of plerocercoids, and also several differences in the larval stages compared with adult D. latus. These features, i.e. well-developed glandular system and massive hook-shaped uncinate spinitriches, are thus probably required for plerocercoids inhabiting fish hosts and also for their post-infection attachment in the human intestine.

First evidence of serotoninergic components in the nervous system of the monogenean Chimaericola leptogaster (Chimaericolidae, Polyopisthocotylea), a gill parasite of the relict holocephalan fish.

The localisation and distribution of the serotoninergic nerve elements was studied for the first time in the flatworm Chimaericola leptogaster (Leuckart, 1830) using immunocytochemical methodology and confocal laser scanning microscopy. The musculature was investigated by histochemical staining of actin filaments; scanning electron microscopy was used to identify the sensory structures on the worm's surface. Uniciliated, bi-ciliated and multiciliated sensory endings have been described on the worm's surface. The morphological data demonstrate the presence of circular, longitudinal and diagonal muscles that comprise the musculature of C. leptogaster in the anterior, median and posterior body regions. Well-developed radial and circular muscle fibres were also observed surrounding the genital pore, two vaginae and in clumps of the haptor. The study revealed the presence of biogenic amine, serotonin, in the central and peripheral nervous systems of C. leptogaster: in the neurons and fibres of the cephalic ganglia and ventral nerve cord, in the innervation of reproductive system compartments. The localised sites of the serotoninergic elements point to important roles of serotonin in monogenean reproductive processes and, possibly, in the regulation of muscle function.

New data on the nervous system of Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae): revisiting old hypotheses.

Data on the interposition of the immunoreactive nerve cords in Cercaria parvicaudata Stunkard & Shaw, 1931 (Trematoda: Renicolidae) and its chaetotaxy were obtained. The nervous system of C. parvicaudata was described using immunostaining of 5-hydroxytryptamine and FMRFamide immunoreactive nerve elements. The morphology and distribution of sensory receptors were analysed using scanning electron microscopy and the silver nitrate impregnation technique. Our integrated approach to the study of the nervous system revealed a clear colocalization of surface papillae with nerve cords and commissures in C. parvicaudata. The structure of the nervous system in C. parvicaudata differs partly from the classical model that defines the entire nomenclature of chaetotaxy.

Homozygous TBC1 domain-containing kinase (TBCK) mutation causes a novel lysosomal storage disease - a new type of neuronal ceroid lipofuscinosis (CLN15)?

Homozygous mutation of TBC1 domain-containing kinase (TBCK) is the cause of a very recently defined severe childhood disorder, which is characterized by severe hypotonia, global developmental delay, intellectual disability, epilepsy, characteristic facies and premature death. The link between TBCK loss of function and symptoms in patients with TBCK deficiency disorder (TBCK-DD) remains elusive. Here we demonstrate for the first time the histopathological characteristics of TBCK deficiency consisting of 1) a widespread and massive accumulation of lipofuscin storage material in neurons of the central nervous system without notable neuronal degeneration, 2) storage deposits in few astrocytes, 3) carbohydrate-rich deposits in brain, spleen and liver and 4) vacuolated lymphocytes. Biochemical examinations ruled out more than 20 known lysosomal storage diseases. These investigations strikingly uncover TBCK-DD as a novel type of lysosomal storage disease which is characterized by different storage products rather than one specific type of accumulated material. Due to the clear predominance of intraneuronal lipofuscin storage material and the characteristic clinical presentation we propose to classify this disease as a new subtype of neuronal ceroid lipofuscinosis (CLN15). Our results and previous reports suggest an autophagosomal-lysosomal dysfunction caused by enhanced mTORC1-mediated autophagosome formation and reduced Rab-mediated autophagosome-lysosome fusion, thus disclosing potential novel targets for therapeutic approaches in TBCK-DD.

The neuroanatomy of the siboglinid Riftia pachyptila highlights sedentarian annelid nervous system evolution.

Tracing the evolution of the siboglinid group, peculiar group of marine gutless annelids, requires the detailed study of the fragmentarily explored central nervous system of vestimentiferans and other siboglinids. 3D reconstructions of the neuroanatomy of Riftia revealed that the "brain" of adult vestimentiferans is a fusion product of the supraesophageal and subesophageal ganglia. The supraesophageal ganglion-like area contains the following neural structures that are homologous to the annelid elements: the peripheral perikarya of the brain lobes, two main transverse commissures, mushroom-like structures, commissural cell cluster, and the circumesophageal connectives with two roots which give rise to the palp neurites. Three pairs of giant perikarya are located in the supraesophageal ganglion, giving rise to the paired giant axons. The circumesophageal connectives run to the VNC. The subesophageal ganglion-like area contains a tripartite ventral aggregation of perikarya (= the postoral ganglion of the VNC) interconnected by the subenteral commissure. The paired VNC is intraepidermal, not ganglionated over most of its length, associated with the ciliary field, and comprises the giant axons. The pairs of VNC and the giant axons fuse posteriorly. Within siboglinids, the vestimentiferans are distinguished by a large and considerably differentiated brain. This reflects the derived development of the tentacle crown. The tentacles of vestimentiferans are homologous to the annelid palps based on their innervation from the dorsal and ventral roots of the circumesophageal connectives. Neuroanatomy of the vestimentiferan brains is close to the brains of Cirratuliiformia and Spionida/Sabellida, which have several transverse commissures, specific position of the giant somata (if any), and palp nerve roots (if any). The palps and palp neurite roots originally developed in all main annelid clades (basally branching, errantian and sedentarian annelids), show the greatest diversity in their number in sedentarian species. Over the course of evolution of Sedentaria, the number of palps and their nerve roots either dramatically increased (as in vestimentiferan siboglinids) or were lost.

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond.

Para una reconsideración del legado de Ramón y Cajal a las neurociencias / A Reconsideration of the legacy of Ramón y Cajal to the neurosciences

Entre 1887 y 1897 Santiago Ramón y Cajal realizó una serie de estudios micrográficos que jalonarían el debate acerca de la estructura y la fisiología del sistema nervioso al suministrar substrato empírico y guía heurística al marco teórico dentro del cual serían las mismas investigadas en lo sucesivo. El significado del trabajo realizado por Cajal en ese decenio no puede ponderarse sin un serio intento de establecer los pertinentes vínculos entre el mismo, sus antecedentes y su contexto contemporáneo. A tal fin, los apartados primero y segundo abordan el contexto científico en que Cajal elaborara en el periodo indicado la teoría neuronal y la ley de la polarización dinámica, prestando atención a planteamientos convergentes y divergentes de contemporáneos y antecesores. El tercero, por su parte, se dedica a la discusión del marco metodológico de la formulación de las señaladas aportaciones, soslayando recientes conatos de polémica historiográfica en torno a la paternidad de las mismas e incidiendo en que el núcleo del legado de Cajal no ha de buscarse en discusiones de esta naturaleza, sino en el amplio cuerpo de observaciones que recogiera y, muy particularmente, en la profusión de hipótesis mediante las cuales tratara de incardinarlo cabalmente en los marcos teóricos sancionados en ciencias biológicas

Between 1887 and 1897, Santiago Ramón y Cajal carried out a series of micrographic studies that would mark the debate about the structure and the physiology of the nervous system, establishing the theoretical framework for its study in the 20th century. The nature and meaning of the task undertaken by Cajal could not be fully weighed without a serious attempt to establish the pertinent links between such task, its antecedents and its contemporary context. To this end, the first and second sections address the scientific context in which, during the aforementioned period, Cajal elaborated the neuron doctrine and the dynamic polarization law, focusing on the convergent and divergent approaches of previous and contemporary researchers. The third section, on the other hand, is devoted to the discussion of the methodological framework of the formulation of the abovementioned contributions, avoiding recent attempts of historiographical controversy about its paternity and emphasizing that the core of Cajal’s legacy does not have to be sought in discussions of this nature, but in the wide body of observations he collected and, particularly, in the profusion of hypotheses he used in order to incardinate those contributions within the theoretical frameworks sanctioned in biological sciences

Ground plan of the larval nervous system in phoronids: Evidence from larvae of viviparous phoronid.

Nervous system organization differs greatly in larvae and adults of many species, but has nevertheless been traditionally used for phylogenetic studies. In phoronids, the organization of the larval nervous system depends on the type of development. With the goal of understanding the ground plan of the nervous system in phoronid larvae, the development and organization of the larval nervous system were studied in a viviparous phoronid species. The ground plan of the phoronid larval nervous system includes an apical organ, a continuous nerve tract under the preoral and postoral ciliated bands, and two lateral nerves extending between the apical organ and the nerve tract. A bilobed larva with such an organization of the nervous system is suggested to be the primary larva of the taxonomic group Brachiozoa, which includes the phyla Brachiopoda and Phoronida. The ground plan of the nervous system of phoronid larvae is similar to that of the early larvae of annelids and of some deuterostomians. The protostome- and deuterostome-like features, which are characteristic of many organ systems in phoronids, were probably inherited by phoronids from the last common bilaterian ancestor. The information provided here on the ground plan of the larval nervous system should be useful for future analyses of phoronid phylogeny and evolution.

Musculoskeletal and nervous systems of the attachment organ in three species of Diplectanum (Monogenea: Dactylogyroidea).

The taxonomy of Diplectanum Diesing, 1858, a genus of monopisthocotylean monogeneans, remains unsettled and needs to be revised based on new morphological criteria. Recent studies in monopisthocotyleans have shown that the muscle arrangement in the posterior attachment organ (haptor) differs between congeneric species and can be used as an additional criterion in genus-level taxonomy. To explore the possibility of using the haptoral musculature and nervous system in the taxonomy of Diplectanum, we conducted a detailed confocal-microscopy study of three species of Diplectanum (D. aculeatum Parona et Perugia, 1889, D. sciaenae van Beneden et Hesse, 1863 and D. similis Bychowsky, 1957) with phalloidin staining for muscle and indirect immunostaining for 5HT and FMRFamide. A further goal was to clarify the functional mechanics of the haptor and the role of its essential components (squamodiscs and anchors) in attachment to the host. The system of connecting bars and gaffing anchors was found to have a complex musculature consisting of 23 muscles in D. aculeatum and D. sciaenae, and 21 muscles in D. similis. The squamodiscs were shown to be operated by several groups of muscles attached primarily to the area termed the squamodisc fulcrum. Most of the haptoral musculature is identical in D. aculeatum and D. sciaenae and these species differ only in the presence of a muscle sheath around the tissue strand between the squamodiscs in D. sciaenae and in the different patterns of superficial squamodisc muscles. Diplectanum similis shows more significant differences from the other two species: besides lacking two of the haptoral muscles, it also differs in the shapes and arrangement of several other muscles. The nervous system of all three species conforms to the general pattern typical for the Dactylogyroidea and shows little variation between species.

Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development.

An 'interactome' screen of all Drosophila cell-surface and secreted proteins containing immunoglobulin superfamily (IgSF) domains discovered a network formed by paralogs of Beaten Path (Beat) and Sidestep (Side), a ligand-receptor pair that is central to motor axon guidance. Here we describe a new method for interactome screening, the Bio-Plex Interactome Assay (BPIA), which allows identification of many interactions in a single sample. Using the BPIA, we 'deorphanized' four more members of the Beat-Side network. We confirmed interactions using surface plasmon resonance. The expression patterns of beat and side genes suggest that Beats are neuronal receptors for Sides expressed on peripheral tissues. side-VI is expressed in muscle fibers targeted by the ISNb nerve, as well as at growth cone choice points and synaptic targets for the ISN and TN nerves. beat-V genes, encoding Side-VI receptors, are expressed in ISNb and ISN motor neurons.

The first data on the innervation of the lophophore in the rhynchonelliform brachiopod Hemithiris psittacea: what is the ground pattern of the lophophore in lophophorates?

BACKGROUND: The nervous system in brachiopods has seldom been studied with modern methods. An understanding of lophophore innervation in adult brachiopods is useful for comparing the innervation of the same lophophore type among different brachiopods and can also help answer questions about the monophyly of the lophophorates. Although some brachiopods are studied with modern methods, rhynchonelliform brachiopods still require investigation. The current study used transmission electron microscopy, immunocytochemistry, and confocal laser scanning microscopy to investigate the nerve system of the lophophore and tentacles in the rhynchonelliform Hemithiris psittacea. RESULTS: Four longitudinal nerves pass along each brachium of the lophophore: the main, accessory, second accessory, and lower. The main brachial nerve extends at the base of the dorsal side of the brachial fold and gives rise to the cross nerves, passing through the extracellular matrix to the tentacles. Cross nerves skirt the accessory brachial nerve, branch, and penetrate into adjacent outer and inner tentacles, where they are referred to as the frontal tentacular nerves. The second accessory nerve passes along the base of the inner tentacles. This nerve consists of Ʊ-like parts, which repetitively skirt the frontal and lateral sides of the inner tentacle and the frontal sides of the outer tentacles. The second accessory nerve gives rise to the latero-frontal nerves of the inner and outer tentacles. The abfrontal nerves of the inner tentacles also originate from the second accessory nerve, whereas the abfrontal nerves of the outer tentacles originate from the lower brachial nerve. The lower brachial nerve extends along the outer side of the lophophore brachia and gives rise to the intertentacular nerves, which form a T-like branch and penetrate the adjacent outer tentacles where they are referred to as abfrontal nerves. The paired outer radial nerves start from the lower brachial nerve, extend into the second accessory nerve, and give rise to the lateroabfrontal tentacular nerves of the outer tentacles. CONCLUSIONS: The innervation of the lophophore in the rhynchonelliform Hemithiris psittacea differs from that in the inarticulate Lingula anatina in several ways. The accessory brachial nerve does not participate in the innervation of the tentacles in H. psittacea as it does in L. anatina. The second accessory nerve is present in H. psittacea but not in L. anatina. There are six tentacular nerves in the outer tentacles of H. psittacea but only four in all other brachiopods studied to date. The reduced contribution of the accessory brachial nerve to tentacle innervation may reflect the general pattern of reduction of the inner lophophoral nerve in both phoronids and brachiopods. Bryozoan lophophores, in contrast, have a weakened outer nerve and a strengthened inner nerve. Our results suggest that the ancestral lophophore of all lophophorates had a simple shape but many nerve elements.

High diversity in neuropeptide immunoreactivity patterns among three closely related species of Dinophilidae (Annelida).

Neuropeptides are conserved metazoan signaling molecules, and represent useful markers for comparative investigations on the morphology and function of the nervous system. However, little is known about the variation of neuropeptide expression patterns across closely related species in invertebrate groups other than insects. In this study, we compare the immunoreactivity patterns of 14 neuropeptides in three closely related microscopic dinophilid annelids (Dinophilus gyrociliatus, D. taeniatus and Trilobodrilus axi). The brains of all three species were found to consist of around 700 somata, surrounding a central neuropil with 3-5 ventral and 2-5 dorsal commissures. Neuropeptide immunoreactivity was detected in the brain, the ventral cords, stomatogastric nervous system, and additional nerves. Different neuropeptides are expressed in specific, non-overlapping cells in the brain in all three species. FMRFamide, MLD/pedal peptide, allatotropin, RNamide, excitatory peptide, and FVRIamide showed a broad localization within the brain, while calcitonin, SIFamide, vasotocin, RGWamide, DLamide, FLamide, FVamide, MIP, and serotonin were present in fewer cells in demarcated regions. The different markers did not reveal ganglionic subdivisions or physical compartmentalization in any of these microscopic brains. The non-overlapping expression of different neuropeptides may indicate that the regionalization in these uniform, small brains is realized by individual cells, rather than cell clusters, representing an alternative to the lobular organization observed in several macroscopic annelids. Furthermore, despite the similar gross brain morphology, we found an unexpectedly high variation in the expression patterns of neuropeptides across species. This suggests that neuropeptide expression evolves faster than morphology, representing a possible mechanism for the evolutionary divergence of behaviors.

Size and specimen-dependent strategy for x-ray micro-ct and tem correlative analysis of nervous system samples.

Correlative approaches are a powerful tool in the investigation of biological samples, but require specific preparation procedures to maintain the strength of the employed methods. Here we report the optimization of the embedding protocol of nervous system samples for a correlative synchrotron X-ray computed microtomography (micro-CT) and transmission electron microscopy (TEM) approach. We demonstrate that it is possible to locate, with the micrometric resolution of micro-CT, specific volumes of interest for a further ultrastructural characterization to be performed with TEM. This approach can be applied to samples of different size and morphology up to several cm. Our optimized method represents an invaluable tool for investigating those pathologies in which microscopic alterations are localized in few confined regions, rather than diffused in entire tissues, organs or systems. We present a proof of concept of our method in a mouse model of Globoid Cells Leukodistrophy.

A Molecular Reporter for Monitoring Autophagic Flux in Nervous System In Vivo.

The relevance of autophagy in neuronal health has been extensively reported in a plethora of conditions affecting the nervous system, such as neurodegenerative diseases, cancer, diabetes, and tissue injury, where altered autophagic activity may contribute to the pathological process. Autophagy is a dynamic pathway involving the formation of a membrane surrounding and enclosing cargoes that are delivered to lysosomal compartments for degradation. Cargoes can include large protein aggregates, organelles, or even pathogens. Traditionally, autophagy assessment relies on the measurement of LC3-II protein levels or the visualization of LC3-positive puncta. However, these approaches represent a static measurement of autophagy markers, making difficult the dissection of the actual changes in the autophagy process (activation, inhibition, or no effects), due to the dynamic regulation of LC3 viral levels. To circumvent this limitation, we previously developed an adeno-associated vector (AAV) to deliver a molecular autophagy sensor to the neuronal compartment in vivo. Here, we describe the detailed design and methods to use an engineered AAV harboring the monomeric tandem mCherry-GFP-LC3 to determine autophagic fluxes in the nervous system. Key methodological details to succeed in the use of this reporter are provided.