Serial Ultrathin Sections Research Articles

Morphology and function of reproductive organs in Neodasys chaetonotoideus (Gastrotricha: Neodasys) with a phylogenetic assessment of the reproductive system in Gastrotricha

The reproductive system of the important basal gastrotrich Neodasys chaetonotoideus is described and reconstructed on the basis of light microscopy, serial ultrathin sections (ultrastructure) and scanning electron microscopy. Starting frontally, the hermaphroditic reproductive system consists of paired and tube shaped lateral testes that do not possess elongated seminal ducts but most likely open directly via paired ventral pores. The unpaired, medio‐dorsal ovary region contains early oogenic stages that mature caudally towards the uterus region, where the most mature egg is positioned laterally to the midgut. The ovary region is not covered with an epithelial lining whereas the uterus region possesses a distinct epithelial wall. Between ovary and uterus region, we have detected a conspicuous section of the female gonad, the vitellogenic oviduct that consists of a thick epithelial wall which forms cellular protuberances into the developing oocytes passing the oviduct. We interpret this as a special, hitherto undescribed mode of vitellogenesis in Gastrotricha. Further caudally, the uterus continues with the fronto‐caudal organ, a complex of two substructures that are apparently homologous to the frontal organ and the caudal organ of many species of the Gastrotricha Macrodasyida. Neodasys chaetonotoideus obviously engages in spermatophore formation and transfer. In this study we develop a morpho‐functional scenario for the gonads and accessory organs in terms of spermatophore production, exchange and oviposition.We compare our newly obtained data with already published results on the reproductive organs of several species of Gastrotricha by means of a species‐character matrix and provide a computer aided evaluation by a parsimonious character optimization. A reconstruction of the reproductive system of the stem species of Gastrotricha on the basis of three recent phylogenetic analyses is presented. These reconstructions give support for a Neodasys‐like reproductive system in the ground pattern of Gastrotricha with slight morphological differences and direct transfer of spermatozoa rather than spermatophore transfer. The evolution of selected characters is traced thus revealing some incidents of convergent evolution as well as the evolutionary replacement of the ancestral frontal organ by the derived frontal sac in at least two separated lineages.

Larval apical sensory organ in a neritimorph gastropod, an ancient gastropod lineage with feeding larvae

The Neritimorpha is an ancient clade of gastropods that may have acquired larval planktotrophy independently of the evolution of this developmental mode in other gastropods (caenogastropods and heterobranchs). Neritimorphs are therefore centrally important to questions about larval evolution within the Gastropoda, but there is very little information about developmental morphology through metamorphosis for this group. We used immunolabeling (antibodies binding to acetylated α-tubulin and serotonin) and serial ultrathin sections for transmission electron microscopy to characterize the apical sensory organ in planktotrophic larvae of a marine neritimorph. The apical sensory organ of gastropod larvae is a highly conserved multicellular sensory structure that includes an apical ganglion and often an associated ciliary structure. Surprisingly, the apical ganglion of Nerita melanotragus (Smith, 1884) does not have typical ampullary neurons, a type of sensory neuron consisting of a cilia filled inpocketing that has been described in all other major gastropod groups. N. melanotragus has cilia-filled pockets embedded within the apical ganglion, but these so-called “sensory cups” are cassettes of multiple cells: one supporting cell and up to three multiciliated sensory cells. We suggest that an internalized pocket that is filled with cilia and open to the exterior via a narrow pore may be essential architectural features for whatever sensory cues are detected by ampullary neurons and sensory cups; however, morphogenesis of these features at the cellular level has undergone evolutionary change. We also note a correlation between the number of sensory elements consisting of cilia-filled pockets within the larval apical sensory organ of gastropods and morphological complexity of the velum or length of the trochal ciliary bands.

Synapse‐to‐synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function

Synaptic vesicle (SV) size is one parameter that controls the amount of neurotransmitter released from individual SVs and, therefore, is fundamental to our understanding of synaptic function. The recently discovered variability of mean SV size among excitatory hippocampal synapses -- if actively regulated -- is a potential mechanism for the regulation of transmitter release. Here, we investigated which parameters influence mean SV size. First, we revealed that synapse-to-synapse variability of SV size is a general phenomenon in several species and brain regions. In addition, we determined the relationship between mean SV size and synaptic morphology. In three-dimensional reconstructions from serial ultrathin sections, we found that SV size did not correlate with the area of the postsynaptic density (a measure for synaptic size and synaptic cleft volume) nor with the total number of SVs within a bouton or bouton volume. We tested the long-held hypothesis that a change in osmotic pressure (potentially caused by a change in neurotransmitter concentration) affects SV size. When we reduced the osmotic pressure, SVs became significantly smaller; however, an increase in osmotic pressure had no effect on SV size. Furthermore, we found that SV size does not adapt to chronic changes in activity and that the SV cycle is capable of providing constant SV size during long-lasting, high-frequency stimulation.

Smart specimen preparation for freeze substitution and serial ultrathin sectioning of yeast cells

A smart and efficient method for freeze substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 mum above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.

Amyloid Deposits Show Complexity and Intimate Spatial Relationship with Dendrosomatic Plasma Membranes: An Electron Microscopic 3D Reconstruction Analysis in 3xTg-AD Mice and Aged Canines

Little is known about how amyloid-beta (Abeta) is deposited in relation to the complex ultrastructure of the brain. Here we combined serial section immunoelectron microscopy with 3D reconstruction to elucidate the spatial relationship between Abeta deposits and ultrastructurally identified cellular compartments. The analysis was performed in a transgenic mouse model with mutant presenilin-1, and mutant amyloid-beta protein precursor (AbetaPP) and tau transgenes (3xTg-AD mice) and in aged dogs that develop Abeta plaques spontaneously. Reconstructions based on serial ultrathin sections of hippocampus (mice) or neocortex (dogs) that had been immunolabeled with Abeta (Abeta(1-42)) antibodies showed that the organization of extracellular Abeta deposits is more complex than anticipated from light microscopic analyses. In both species, deposits were tightly associated with plasma membranes of pyramidal cell bodies and major dendrites. The deposits typically consisted of thin sheets as well as slender tendrils that climbed along the large caliber dendritic stems of pyramidal neurons. No preferential association was observed between Abeta deposits and thin dendritic branches or spines, nor was there any evidence of preferential accumulation of Abeta around synaptic contacts or glial processes. Our data suggest that plaque formation is a precisely orchestrated process that involves specialized domains of dendrosomatic plasma membranes.

Complexity of contacts between synaptic boutons and dendritic spines in adult rat hippocampus: Three‐dimensional reconstructions from serial ultrathin sections in vivo

The morphological relationship between a synapse and dendritic spine is an important determinant of synaptic function, and has been studied extensively with the electron microscope. This has revealed that the universal idea of one synapse per spine is no longer tenable; dendritic spines may on occasions form synapses with two or more axonal boutons. To elucidate the detailed structure and relationship of spines and their synaptic contacts in the molecular layers of the dentate gyrus of the adult rat, a study using 3-dimensional (3D) reconstructions was made from serial ultrathin sections (up to 150 sections per series), of both pre- and postsynaptic segments of axons and dendrites. We found that synapses from all unmyelinated axons examined were en passant and although most contacts between synapse and spine are one-to-one, all reconstructions examined contained both multisynaptic axonal segments, and multisynaptic spines. In the dentate gyrus, 3.48% mushroom spines were multisynaptic, synapsing with more than two axons, and 0.46% of the total population of synapses were multisynaptic. Although these values appear small, they actually comprise millions of contacts per cubic millimeter of tissue. Axonal varicosities can contact more than three dendritic spines originating from different dendritic branches while spines may branch with up to three heads, and spine heads may receive two or more synaptic contacts. Our data confirm the complexity of relationships between spines and synapses and show that a one-one relationship between synapse and spine cannot be assumed automatically.

Important factors for the three-dimensional reconstruction of neuronal structures from serial ultrathin sections

Quantitative analysis of anatomical synaptic connectivity in microcircuits depends upon accurate three-dimensional (3D) reconstructions of synaptic ultrastructure using electron microscopy of serial ultrathin sections. Here we address two pitfalls in current methodology that lead to inaccurate reconstructions and compromise conclusions drawn from the data. The first pitfall is inaccurate determination of ultrathin section thickness, which negatively affects the 3D shape of reconstructions and therefore impairs quantitative measurement of synaptic structures. Secondly, current methodology significantly underestimates the number of synaptic junctions, with only two-thirds or less of genuine synaptic contacts being identified in dendrites that radiate within the plane of section. Here we propose a new methodology utilizing precise optical measurements of section thickness and successive observations of synaptic elements across serial ultrathin sections that corrects for these limitations to allow accurate 3D reconstruction of synaptic ultrastructure. We use this methodology to reveal that parvalbumin-expressing cortical interneurons have a much higher synaptic density than previously shown. This result suggests that this technique will be useful for re-examining synaptic connectivity of other cell types.

Reversible changes in complex hippocampal synapses of CA3 thorny excrescences during hibernation of Siberian ground squirrel: a serial ultrathin section investigation in vivo

Reversible changes in complex hippocampal synapses of CA3 thorny excrescences during hibernation of Siberian ground squirrel: a serial ultrathin section investigation in vivo

The ultrastructure of a halobacterial cell in the region of the flagellum outlet

An intracellular structure previously identified in archaeal cells (Halobacterium salinarum) was studied by electron microscopy of single and serial ultrathin sections. This structure was localized under the cytoplasmic membrane near the cellular poles on both sides of the disc-like lamellar structure (DLS), which we found earlier in the motility apparatus of Hb. salinarum. The organization of this structure differs from that of DLS. Morphological characteristics of the new structure coincide with those of bacterial “polar organelle” (PO) described previously. Similar to PO, the new structure exhibited cytochemical staining that reveals the ATPase activity. The bacterial PO is compared with the analogous structure in archaea.

Visualizing the complex substructure of euglenid pellicle strips with SEM

H.J. Esson and B.S. Leander. 2008. Visualizing the complex substructure of euglenid pellicle strips with SEM. Phycologia 47: 529–532. DOI: 10.2216/08-26.1Comparative analysis of cytoskeletal diversity within the Euglenophyceae has provided important context for understanding the phylogenetic relationships and major evolutionary transitions within the group (e.g. switches in modes of nutrition and motility). Some ultrastructural characters used in earlier cladistic analyses of euglenids involved different states for the lateral projections that extend from the frame of each pellicle strip in photosynthetic lineages. Previously, the overall structure of ‘strip projections’ in different lineages was (arduously) reconstructed from a series of ultra-thin sections viewed with transmission electron microscopy (TEM). In this study, we were able to determine the structure of strip projections with greater precision, and without the laborious protocols associated with TEM (e.g. ultramicrotomy), by examining disrupted pellicles from three photosynthetic euglenids (Lepocinclis fusiformis, Phacus longicauda var. tortus, and P. segretii) using scanning electron microscopy (SEM). The structure of the strip projections observed here demonstrated that either (1) previous TEM studies of the pellicle overlooked certain ultrastructural features in some taxa or (2) the (prearticular) strip projections in L. fusiformis, P. segretii, and P. longicauda var. tortus represent a novel character state that could be phylogenetically informative.

Confocal/TEM Overlay Microscopy: A Simple Method for Correlating Confocal and Electron Microscopy of Cells Expressing GFP/YFP Fusion Proteins

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.

Three-dimensional structure of the ciliate Didinium nasutum nucleoli

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.

Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy

The mechanisms underlying hepatitis C virus (HCV) morphogenesis remain elusive, but lipid droplets have recently been shown to be important organelles for virus production. We investigated the interaction between HCV-like particles and lipid droplets by three-dimensional reconstructions of serial ultrathin electron microscopy sections of cells producing the HCV core protein. The budding of HCV-like particles was mostly initiated at membranes close to the lipid droplets rather than at membranes directly apposed to the lipid droplets. This may have important implications for our understanding of the complex relationship between HCV and lipids and may make easier to dissect out the HCV life cycle.

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.

WITHDRAWN: Dendritic Spine and Synapse Morphological Alterations Induced by a Neural Cell Adhesion Molecule (NCAM) Mimetic.

The neural cell adhesion molecule (NCAM) is a glycoprotein expressed on the surface of neurons and glial cells. It plays a key role in morphogenesis of the nervous system, regeneration of damaged neural tissue and synaptic plasticity. The extracellular domain of NCAM engages in homophilic interactions (NCAM binding to NCAM) and in heterophilic interactions between NCAM and other proteins such as the fibroblast growth factor (FGF) receptor. It promotes synaptogenesis and activity-dependent remodelling of synapses but less is know of its influence on synaptic and dendritic morphology. Recently, quantitative electron microscopy and 3-dimensional reconstruction (3-D) of ultrathin serial sections has been used to examine the morphology of synapses and dendritic spines in the hippocampus of rats treated with a neural cell adhesion molecule-derived fibroblast growth factor receptor agonist, FGL-peptide (an NCAM mimetic). These data show clearly that the FGL peptide has marked influences on both spine and synaptic form.

Ultrastructure and function of the mastax in Dicranophorus forcipatus (Rotifera: Monogononta)

Rotifers are characterized by a complex set of cuticularized jaw elements in the pharynx. The fine structure of the jaw elements has been the subject of SEM studies for some time, but only very limited information exists on the ultrastructure of the jaw elements and their function beyond taxonomic considerations. Drawing on SEM and TEM techniques, the present study presents a detailed analysis of the mastax in Dicranophorus forcipatus, a carnivorous monogonont rotifer species from freshwater habitats characterized by an extrusible, grasping jaw apparatus. Based on ultrathin serial sections, the jaw elements are reconstructed and, in total, nine paired and two unpaired muscles identified. Possibly homologous muscles in other rotifer species are discussed and functional considerations of the forcipate mastax are suggested.

A cell adhesion molecule mimetic, FGL peptide, induces alterations in synapse and dendritic spine structure in the dentate gyrus of aged rats: a three‐dimensional ultrastructural study

The FGL peptide is a neural cell adhesion molecule (NCAM) mimetic comprising a 15-amino-acid-long sequence of the FG loop region of the second fibronectin type III module of NCAM. It corresponds to the binding site of NCAM for the fibroblast growth factor receptor 1. FGL improves cognitive function through enhancement of synaptic function. We examined the effect of FGL on synaptic and dendritic structure in the brains of aged (22-month-old) rats that were injected subcutaneously (8 mg/kg) at 2-day intervals until 19 days after the start of the experiment. Animals were perfused with fixative, brains removed and coronal sections cut at 50 microm. The hippocampal volume was measured, tissue embedded and ultrathin sections viewed in a JEOL 1010 electron microscope. Analyses were made of synaptic and dendritic parameters following three-dimensional reconstruction via images from a series of approximately 100 serial ultrathin sections. FGL affected neither hippocampal volume nor spine or synaptic density in the middle molecular layer of the dentate gyrus. However, it increased the ratio of mushroom to thin spines, number of multivesicular bodies and also increased the frequency of appearance of coated pits. Three-dimensional analysis showed a significant decrease in both post-synaptic density and apposition zone curvature of mushroom spines following FGL treatment, whereas for thin spines the convexity of the apposition zone increased. These data indicate that FGL induces large changes in the fine structure of synapses and dendritic spines in hippocampus of aged rats, complementing data showing its effect on cognitive processes.

Correlative imaging of ultrathin serial sections of zebra finch brain.

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Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections

In conventional transmission electron microscopy, uranyl acetate staining is used to enhance the cellular components. However, uranyl acetate is considered a radioactive material that is very toxic if ingested or inhaled and subject to restrictions in many countries. In an attempt to introduce a substitute for uranyl acetate, we evaluated oolong tea extract (OTE) for staining of ultrathin sections. Tissue sections from normal rat liver representing an ideal model organ were processed according to a routine electron microscopic fixation and embedding procedure. Serial ultrathin sections were cut and processed with either routine double electron staining or 0.2% OTE staining for 30-40 min at room temperature followed by lead citrate staining (OTE staining method). Transmission electron microscopy observations revealed that all sub-cellular structures in hepatocytes were clearly visible with OTE staining and the quality of staining was highly compatible with those of routine double staining methods. It is suggested that OTE could be used as a non-radioactive and hazard-free substitute for uranyl acetate in transmission electron microscopy staining.

Study of nuclear remodeling in reconstructed mouse embryos by optical and electron microscopy

To date, there is a large body of information on the structural composition of oocytes and embryonic cells of mammals, embryonal nucleogenesis, processes of gene expression, and specific protein synthesis during the preimplantation period. Different factors playing the key role in the transformation of the oocyte to the embryo after in the fertilization and activation of the embryonic genome [1–3] have been revealed. The number of such factors, revealed, as a rule, in the oocyte cytoplasm, constantly increases. In numerous studies on nucleus transplantation it was convincingly shown that oocytes contain specific factors causing genes highly specialized somatic cells to behave as the embryonic genome. It was established that these changes in gene expression are accompanied by considerable structural rearrangement of nuclei of these cells and their chromosome material [4–6]. As a result, the nuclei acquire the potential to direct and support embryonic development to term, though with an extremely low probability. The ability of mature oocytes to induce epigenetic reprogramming of nuclei and transform highly specialized cells ito stem cells is unique. Paradoxically, oocytes become unable to support these processes immediately after fertilization [6, 7]. Currently, the topical studies on derivation of embryonic stem cells from reconstructed embryos for therapeutic use challenge the problem of determination of the main strategy of cell reprogramming It is necessary to know in detail the spatiotemporal pattern of structural and functional rearrangements in donor nuclei and in reconstructed embryos to elucidate the mechanisms underlying these processes, and their influence on further embryo development. Among different methods of cell studies, the optical and electron microscopy are most adequate to provide a clear picture of localization of nuclei and chromatin structure and detect even very small changes in cells. Preparation of serial sections with subsequent three-dimensional reconstruction of objects on the basis of electron microscopy is a very useful approach to studying the rearrangements of nuclei in somatic cells being transferred into oocytes. No studies of this kind have been performed thus far. In this study, we obtained mouse embryos with the genome of cumulus cells and examined rearrangements in cumulus cell nuclei at early stages of their remodelling in the oocyte cytoplasm using serial ultrathin sections and three-dimensional reconstruction.