Ultrastructural study of neuronal cells and localization of ghrelin-like peptide and its receptor in the ganglia of the golden apple snail (Pomacea canaliculata).

Pomacea canaliculata is an invasive snail species causing major problems in agriculture. The snail biology was then investigated. The main objective of the present study was to investigate the nervous system of the snail. The nervous system comprises pairs of cerebral, buccal, pedal, pleural, parietal ganglia and an unpaired visceral ganglion. Most neurons were concentrated at the periphery of the ganglia. The neurons were classified into four types: NR1, NR2, NR3, and NR4. The percentages of the NR3 and NR4 in the pleural and pedal ganglia were significantly higher than those of other ganglia. Ultrastructural study revealed that nuclei of all neuronal types exhibited mostly euchromatins. Many organelles including ribosomes and endoplasmic reticulum were found in their cytoplasm. However, various mitochondria were found in the NR2 and NR3. The immunohistochemistry revealed immunoreactivity of ghrelin-like peptide in the neurons of the cerebral, pleural and pedal ganglia. However, immunoreactivity of GHS-R1a-like peptide existed only in the neurons of the pleural and pedal ganglia. The present study is the first to demonstrate the existence of ghrelin-like peptide and its receptor in P. canaliculata nervous system.

Localization of neurons expressing choline acetyltransferase, serotonin and/or FMRFamide in the central nervous system of the decapod shore crab Hemigrapsus sanguineus.

Although it is now established that neurons in crustacea contain multiple transmitter substances, little is know about patterns of expression and co-expression or about the functional effects of such co-transmission. The present study was designed to characterize the distributions and potential colocalization of choline acetyltransferase (ChAT), serotonin (5-HT) and neuropeptide H-Phe-Met-Arg-Phe-NH2 (FMRFamide) in the central nervous system (CNS) of the Asian shore crab, Hemigrapsus sanguineus using immunohistochemical analyses in combination with laser scanning confocal microscopy. ChAT was found to be expressed by small, medium-sized, and large neurons in all regions of the brain and ventral nerve cord (VNC). For the most part, ChAT, FMRFamide, and 5-HT are expressed in different neurons, although some colocalization of ChAT- with FMRFamide- or 5-HT-LIR is observed in small and medium-sized cells, mostly neurons that immunostain only weakly. In the brain, such double immunolabeling is observed primarily in neurons of the protocerebrum and, to a particularly great extent, in local olfactory interneurons of the deutocerebrum. The clusters of neurons in the VNC that stain most intensely for ChAT, FMRFamide, and 5-HT, with colocalization in some cases, are located in the subesophageal ganglia. This colocalization appears to be related to function, since it is present in regions of the CNS characterized by multiple afferent projections and outputs to a variety of functionally related centers involved in various physiological and behavioral processes. Further elucidation of the functional significance of these neurons and of the widespread process of co-transmission in the crustaceans should provide fascinating new insights.

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.

Patterns and distribution of presynaptic and postsynaptic elements within serial electron microscopic reconstructions of neuronal arbors from the medicinal leech Hirudo verbana.

Microscale connectomics involves the large-scale acquisition of high-resolution serial electron micrographs from which neuronal arbors can be reconstructed and synapses can be detected. In addition to connectivity information, these data sets are also rich with structural information, including vesicle types, number of postsynaptic partners at a given presynaptic site, and spatial distribution of synaptic inputs and outputs. This study uses serial block-face scanning electron microscopy (EM) to collect two volumes of serial EM data from ganglia of the medicinal leech. For the first volume, we sampled a small fraction of the neuropil belonging to an adult ganglion. From this data set we measured the proportion of arbors that contained vesicles and the types of vesicles contained and developed criteria to identify synapses and to measure the number of apparent postsynaptic partners in apposition to presynaptic boutons. For the second data set, we sampled an entire juvenile ganglion, which included the somata and arbors of all the neurons. We used this data set to placd our findings from mature tissue in the context of fully reconstructed arbors and to explore the spatial distribution of synaptic inputs and outputs on these arbors. We observed that some neurons segregated their arbors into input only and mixed input/output zones, that other neurons contained exclusively mixed input/output zones, and that still others contained only input zones. These results provide the groundwork for future behavioral studies. J. Comp. Neurol. 524:3677-3695, 2016. © 2016 Wiley Periodicals, Inc.

Cerebral ganglion ultrastructure and differential proteins revealed using proteomics in the aplysiid (Notarcus leachii cirrosus Stimpson) under cadmium and lead stress.

Cadmium (Cd) and lead (Pb) are both highly toxic metals in environments. However the toxicological mechanism is not clear. In this study, the aplysiid, Notarcus leachii cirrosus Stimpson (NLCS) was subjected to Cd (NLCS-Cd) or Pb (NLCS-Pb). The cerebral ganglion of NLCS was investigated with a transmission electron microscope. Next the differential proteins were separated and identified using proteomic approaches. Eighteen protein spots in NLCS-Cd and seventeen protein spots in NLCS-Pb were observed to be significantly changed. These protein spots were further excised in gels and identified. A hypothetical pathway was drawn to show the correlation between the partially identified proteins. The results indicated that damage to the cerebral ganglion was follows: cell apoptosis, lysosomes proliferation, cytoskeleton disruption, and oxidative stress. These phenomena and data indicated potential biomarkers for evaluating the contamination levels of Cd and Pb. This study provided positive insights into the mechanisms of Cd and Pb toxicity.

Deep-tissue confocal imaging of the central projections of ovipositor sensory afferents in the Egyptian cotton leafworm, Spodoptera littoralis.

The pre-ovipositon behavior of moths is largely dependent upon the cues that a gravid female perceives while assessing potential oviposition sites. Assessment of such sites is accomplished, at least in part, by mechanosensory and gustatory sensilla located on the ovipositor whose sensory neurons project into the terminal abdominal ganglion (TAG). Using anterograde backfill staining, confocal laser scanning microscopy, and three dimensional reconstruction, we traced and analyzed the central projections of the sensory neurons housed in the sensilla located on the ovipositor papillae and explored the neuropilar composition of the TAG in the Egyptian cotton leafworm, Spodoptera littoralis. The TAG consists of three fused neuromeres (6-8th Ner) associated with the 6-8th abdominal segments. Within the TAG, and specifically in the 8th neuromere, four unstructured neuropilar compartments are present; the dorso-ipsilateral motor neuropil (MN), the medio-ipsilateral mechanosensory neuropil (MchN), the medio-ipsilateral small gustatory neuropil (GN), and the medio-contralateral posterior ovipositor glomerulus (Og). The Og appears quite compact, with a hollow core free of terminal arborizations. The MchN is further subdivided into 4 unstructured glomeruli in the 8th neuromere, whose afferents are subsequently extended into 3 glomeruli in the 7th and 6th neuromeres. Few neurites of the Og are populated with large dense varicosities reminiscent of neurosecretory vesicles. Given that all ovipositor nerves converge into a common ganglionic center, the TAG, we assume that this ganglion may be a center for coordination of oviposition behaviors, including movements of the ovipositor during assessment of oviposition substrates and egg laying in S. littoralis.

Functional and Morphological Correlates in the Drosophila LRRK2 loss-of-function Model of Parkinson's Disease: Drug Effects of Withania somnifera (Dunal) Administration.

The common fruit fly Drosophila melanogaster (Dm) is a simple animal species that contributed significantly to the development of neurobiology whose leucine-rich repeat kinase 2 mutants (LRRK2) loss-of-function in the WD40 domain represent a very interesting tool to look into physiopathology of Parkinson's disease (PD). Accordingly, LRRK2 Dm have also the potential to contribute to reveal innovative therapeutic approaches to its treatment. Withania somnifera Dunal, a plant that grows spontaneously also in Mediterranean regions, is known in folk medicine for its anti-inflammatory and protective properties against neurodegeneration. The aim of this study was to evaluate the neuroprotective effects of its standardized root methanolic extract (Wse) on the LRRK2 loss-of-function Dm model of PD. To this end mutant and wild type (WT) flies were administered Wse, through diet, at different concentrations as larvae and adults (L+/A+) or as adults (L-/A+) only. LRRK2 mutants have a significantly reduced lifespan and compromised motor function and mitochondrial morphology compared to WT flies 1% Wse-enriched diet, administered to Dm LRRK2 as L-/A+and improved a) locomotor activity b) muscle electrophysiological response to stimuli and also c) protected against mitochondria degeneration. In contrast, the administration of Wse to Dm LRRK2 as L+/A+, no matter at which concentration, worsened lifespan and determined the appearance of increased endosomal activity in the thoracic ganglia. These results, while confirming that the LRRK2 loss-of-function in the WD40 domain represents a valid model of PD, reveal that under appropriate concentrations Wse can be usefully employed to counteract some deficits associated with the disease. However, a careful assessment of the risks, likely related to the impaired endosomal activity, is required.

DLA based compressed sensing for high resolution MR microscopy of neuronal tissue.

In this work we present the implementation of compressed sensing (CS) on a high field preclinical scanner (17.2 T) using an undersampling trajectory based on the diffusion limited aggregation (DLA) random growth model. When applied to a library of images this approach performs better than the traditional undersampling based on the polynomial probability density function. In addition, we show that the method is applicable to imaging live neuronal tissues, allowing significantly shorter acquisition times while maintaining the image quality necessary for identifying the majority of neurons via an automatic cell segmentation algorithm.

[Heterochronies in the Formation of the Nervous and Digestive Systems in Early Postlarval Development of Opistobranch Mollusks: Organization of Basic Functional Systems of the Arctic Dorid Cadlina laevis].

For the first time using laser confocal microscopy and histochemical and immunocytochemical methods (detection of F-actine, catecholamines, acetylcholintransferase, substances of P and FM RFamide) in combination with classical histological methods and electron microscopy of total preparations, the general structure and regularities of formation of the main organs and the nervous, muscular, and digestive systems in early postlarval development (2 to 4 months) in the opistobranch mollusk Cadlina laevis were studied. Heterochronies manifested in positive allometry of the sensory organs, ganglia of the central nervous system, and the pharyngeal region of the digestive system in relation to general body sizes in juvenile individuals compared to adult animals were detected.

Monitoring Spiking Activity of Many Individual Neurons in Invertebrate Ganglia.

Optical recording with fast voltage sensitive dyes makes it possible, in suitable preparations, to simultaneously monitor the action potentials of large numbers of individual neurons. Here we describe methods for doing this, including considerations of different dyes and imaging systems, methods for correlating the optical signals with their source neurons, procedures for getting good signals, and the use of Independent Component Analysis for spike-sorting raw optical data into single neuron traces. These combined tools represent a powerful approach for large-scale recording of neural networks with high temporal and spatial resolution.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

Effects of andiroba (Carapa guianensis) oil in ticks: Ultrastructural analysis of the synganglion of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

The present study performed an analysis of the ultrastructural changes induced by andiroba seed oil in the synganglion of Rhipicephalus sanguineus female ticks, aiming to provide scientific grounds to help in the creation of more specific and efficient methods of control. The synganglion consists of a mass of fused nerves externally covered by the neural lamella, a uniform and acellular layer. Just below, the perineurium is found, formed by glial cells. Internally, the synganglion is subdivided into an outer cortical region (cortex), which contains the cellular bodies of the neural cells and an inner region, the neuropile, formed by a set of nerve fibers (extensions of the neural cells). The results showed that the synganglion of females exposed to andiroba oil showed structural changes, such as: irregular and apparently thinner neural lamella, perineurium glial cells presenting large cytoplasmic vacuoles, decrease in the extensions of glial cells, separation of cortex cells, which were formerly attached through their membranes, neural cells presenting irregular plasma membranes and cytoplasm with autophagic vacuoles and mitochondria with disorganized cristae and in process of degeneration. This study confirmed the neurotoxic action of the andiroba oil, which would probably be able to impair the neural functions. Thus, it is suggested that this product has the potential to be used as an alternative method to control ticks.

3-acetylpyridine-induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells.

Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal ßIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.

Olfactory projection neuron pathways in two species of marine Isopoda (Peracarida, Malacostraca, Crustacea).

The neuroanatomy of the olfactory pathway has been intensely studied in many representatives of Malacostraca. Nevertheless, the knowledge about bilateral olfactory integration pathways is mainly based on Decapoda. Here, we investigated the olfactory projection neuron pathway of two marine isopod species, Saduria entomon and Idotea emarginata, by lipophilic dye injections into the olfactory neuropil. We show that both arms of the olfactory globular tract form a chiasm in the center of the brain, as known from several other crustaceans. Furthermore, the olfactory projection neurons innervate both the medulla terminalis and the hemiellipsoid body of the ipsi- and the contralateral hemisphere. Both protocerebral neuropils are innervated to a comparable extent. This is reminiscent of the situation in the basal decapod taxon Dendrobranchiata. Thus, we propose that an innervation by the olfactory globular tract of both the medulla terminalis and the hemiellipsoid body is characteristic of the decapod ground pattern, but also of the ground pattern of Caridoida.

Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada).

Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships of Tardigrada. Here, we present new insight into the morphology of the brain in a marine arthrotardigrade, Actinarctus doryphorus, based on transmission electron microscopy, supported by scanning electron microscopy, conventional light microscopy as well as confocal laser scanning microscopy. Arthrotardigrades contain a large number of plesiomorphic characters and likely represent ancestral tardigrades. They often have segmented body outlines and each trunk segment, with its paired set of legs, may have up to five sensory appendages. Noticeably, the head carries numerous cephalic appendages that are structurally equivalent to the sensory appendages of the trunk segments. Our data reveal that the brain of A. doryphorus is partitioned into three paired lobes, and that these lobes exhibit a more pronounced separation as compared to that of eutardigrades. The first brain lobe in A. doryphorus is located anteriodorsally, with the second lobe just below it in an anterioventral position. Both of these two paired lobes are located anterior to the buccal tube. The third pair of brain lobes are situated posterioventrally to the first two lobes, and flank the buccal tube. In addition, A. doryphorus possesses a subpharyngeal ganglion, which is connected with the first of the four ventral trunk ganglia. The first and second brain lobes in A. doryphorus innervate the clavae and cirri of the head. The innervations of these structures indicate a homology between, respectively, the clavae and cirri of A. doryphorus and the temporalia and papilla cephalica of eutardigrades. The third brain lobes innervate the buccal lamella and the stylets as described for eutardigrades. Collectively, these findings suggest that the head region of extant tardigrades is the result of cephalization of multiple segments. Our results on the brain anatomy of Actinarctus doryphorus support the monophyly of Panarthropoda.

Fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of Helicoverpa armigera (Insecta).

The fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of the cotton bollworm Helicoverpa armigera (Insecta, Lepidoptera) are investigated by scanning electron and transmission electron microscopy combined with confocal laser scanning microscopy. The pit organ located on the third segment of the labial palp is about 300 µm deep with a 60-µm-wide opening, each structure containing about 1200 sensilla. Two sensillum types have been found, namely hair-shaped and club-shaped sensilla, located on the upper and lower half of the pit, respectively. Most sensilla possess a single dendrite. The dendrite housed by the club-shaped sensilla is often split into several branches or becomes lamellated in the outer segment. As reported previously, the sensory axons of the sensilla in the labial pit organ form a bundle entering the ipsilateral side of the subesophageal ganglion via the labial palp nerve and project to three distinct areas: the labial pit organ glomerulus in each antennal lobe, the subesophageal ganglion and the ventral nerve cord. In the antennal lobe, the labial pit organ glomerulus is innervated by sensory axons from the labial pit organ only; no antennal afferents target this unit. One neuron has been found extending fine processes into the subesophageal ganglion and innervating the labial palp via one branch passing at the base of the labial palp nerve. The soma of this assumed motor neuron is located in the ipsilateral cell body layer of the subesophageal ganglion. Our results provide valuable knowledge concerning the neural circuit encoding information about carbon dioxide and should stimulate further investigations directed at controlling pest species such as H. armigera.

[On the nervous system of a parasitic cnidarian Polypodium hydriforme].

Nerve cells in a parasitic cnidarian Polypodium hydriforme at the parasitic and free-living stages of the life cycle have been localized immunocytochemically using antibodies to FMRF-amide, and their ultrastructure has been described. Ganglion cells form a net under epidermis consisting of bi- and tripolar neurons which cross the mesoglea and usually contact muscle cells and cnidocytes. Fusiform sensory and neurosecretory cells, especially characteristic to sensory tentacles, are interspersed among epidermal cells. All three types of nerve cells have dense cored vesicles about 80-120 nm in diameter. The sensory cells demonstrate a sensory flagellum-like immobile structure. Neurosecretory and sensory cells form septate junctions with epidermal cells. Ganglion cells show gap junctions between them. A centriole encircled by a fragment of nuclear envelope which is a marker of ectodermal lineage cells in Polypodium has been described in the cytoplasm of a sensory cell, thus proving the ectodermal nature of the nervous system.

The neuroanatomical and ultrastructural organization of statocyst hair cells in the pond snail, Lymnaea stagnalis.

The ultrastructure, neuroanatomy and central projection patterns, including the intercellular connections of the statocyst hair cells of the pond snail, Lymnaea stagnalis, were studied, applying different intra- and extracellular cellular staining techniques combined with correlative light- and electron microscopy. Based on the ultrastructure different hair cells could be distinguished according to their vesicle and granule content, meanwhile the general organization of the sensory neurons was rather uniform, showing clearly separated perinuclear and "vesicular" cytoplasmic regions. Following intra- and extracellular labeling with fluorescence dyes or HRP a typical, local arborization of the hair cells was demonstrated in the cerebral ganglion neuropil, indicating a limited input-output system connected to the process of gravireception. Correlative light- and electron microscopy of HRP-labeled hair cells revealed both axo-somatic and axo-axonic output contacts of hair cell varicosities, and input on sensory axons located far from the terminal arborizations. Our findings suggest (i) a versatile ultrastructural background of hair cells corresponding possibly to processing different gravireceptive information, and (ii) the synaptic (or non-synaptic) influence of gravireception at different anatomical (terminal, axonal and cell body) levels when processed centrally. The results may also serve as a functional morphological background for previously obtained physiological and behavioral observations.

[Distribution and ultrastructure of the tyrosine hydroxylase-positive neurons of the central nervous system of bivalve mollusc Megangulus venulosus under the influence of increased temperature and hypoxia].

Using immunocytochemistry combined with light and electron microscopy, the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive neurons in the CNS of bivalve mollusc, Megangulus venulosus, have been studied under the influence of increased temperature and hypoxia. It has been established, that the stress causes changes in the amount of TH and in the structure of TH-immunopositive neurons in all ganglia. The most essential changes in CNS of M. venulosus were revealed after 60 min exposure to increased temperature and hypoxia; degenerative changes in large neurons, reduction of the synapses and reduction of TH-immunoreactivity in neurons and neuropil.

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons.

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.