bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphometric evaluation of populations of neuronal profiles (cell bodies, dendrites, and nerve terminals) in the central nervous system
Authors: Zoli Michele , Guidolin Diego , Agnati Luigi F.
Published: February 2005

Morphometric techniques have been developed to quantitatively characterize groups of transmitter‐identified neuronal profiles, such as cell groups, dendrite and nerve terminal fields. These morphometric techniques will be illustrated by introducing some general tools for image analysis which can be considered as a background for the present specific applications. The following methods have been included: (1) methods to identify and quantitatively characterize, from both numerical and geometrical standpoints, groups of profiles in a two‐ and three‐dimen‐sional frame; (2) methods to evaluate the evenness of a certain distribution of profiles in the plane; (3) methods to identify subgroups of profiles based on their different spatial or optical density; and (4) methods to compare the distributions of two or more groups of profiles. The applications of these general tools to some neuroanatomical problems, such as cell group definition and description, have been illustrated. Practical examples performed on immunocytochemical preparations of neuronal profile populations are also given. Finally, the potentiality of numerical classification to classify and compare morphometric data has been shown. As an example, numerical classification methods have been applied to the morphometric and microdensitometric analysis of adrenaline/neuropeptide Y costoring neuronal systems of the brainstem in adult and aged rats. © 1992 Wiley‐Liss, Inc.
A new procedure for handling impervious biological specimens
Authors: Lindley V. A.
Published: February 2005

A new application of techniques for preparing impervious biological specimens for light microscopy (LM) and transmission electron microscopy (TEM) has been developed. Microwave irradiation was used to facilitate fixation. A priming technique was used to increase the bonding of the outer surface of the specimens with the resin. Priming the waxy or cuticular surface with Z‐6040 (gamma‐glycidoxypropyl trimethoxysilane) solved the problem of specimen “pull out” from the resin. Insect specimens with various types of cuticles (waxy or chitinous) and seeds were successfully studied ultrastructurally using this technique. © 1992 Wiley‐Liss, Inc.
Fine structure of olfactory epithelia of gastropod molluscs
Authors: Emery Dennis G.
Published: February 2005

Among gastropod molluscs the chemical senses are most important for location of distant objects. They are used in food finding, locating mates, avoiding predators, trail following, and homing. Chemoreceptors are commonly associated with the oral area, the tentacles, and the osphradium, which lies in the mantle cavity.

Most chemosensory neurons are primary sensory neurons, although secondary sensory cells have been reported in the osphradium of some prosobranch gastropods. Most chemosensory organs contain sensory cells with ciliated sensory endings that are in contact with the external environment. Some sensory endings have only microvilli or have no surface elaborations. Cilia on sensory endings are commonly of the conventional type, but some species have modified cilia; some lack rootlets, some have an abnormal microtubular content, and some have paddle‐shaped endings. The perikarya of sensory neurons may be within the sensory epithelium, below it, or in ganglia near the sensory surface. In some groups of gastropods there are peripheral ganglia in the olfactory pathway; in others chemosensory axons appear to pass directly to the CNS.

Olfactory epithelia of terrestrial pulmonates have modified brush borders with long branching plasmatic processes and a spongy layer of cytoplasmic tubules which extend from the epithelial cells. Sensory endings of the olfactory receptors are entirely within this spongy layer. Aquatic pulmonates may have a similar spongy layer in their olfactory epithelia, but the cilia of sensory endings, as well as motile cilia of epithelial cells, extend well beyond the spongy layer. © 1992 Wiley‐Liss, Inc.
Ultrastructural histopathology of human olfactory dysfunction
Authors: Moran David T. , Jafek Bruce W. , Eller Pamela M. , Rowley J. Carter
Published: February 2005

This paper presents electron‐microscopic observations on biopsies of the olfactory mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post‐traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post‐viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics' olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The postviral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post‐viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent—or greatly reduced in area—in these individuals. © 1992 Wiley‐Liss, Inc.
Immunocytochemical localization of cell adhesion molecules in the developing and mature olfactory system
Authors: Miragall Fernando , Dermietzel Rolf
Published: February 2005

The localization of Ca++‐independent cell adhesion molecules (CAMS) in the developing and mature olfactory epithelium and bulb is reviewed. The CAMs included in this article are the neural cell adhesion molecule (N‐CAM), the 180 kD component of N‐CAM (N‐CAM 180), the embryonic form of N‐CAM (E‐N‐CAM), L1 glycoproteins, J1 glycoproteins, and the adhesion molecule on glia (AMOG). In addition, the expression of the L2‐HNK‐1 carbohydrate epitope, shared by N‐CAM, L1, J1 and myelin‐associated glycoprotein (MAG) in the adult olfactory epithelium and bulb has also been documented. For the localization of these molecules at the light and electron microscopic levels, immunocytochemical techniques were used and are described in detail.

During development and organogenesis, the olfactory system exhibits a pattern of CAM expression similar to the general pattern described for the developing nervous system. In the adult olfactory system, however, a significant retention of CAMs characteristic for developmental and morphogenetic processes, such as E‐N‐CAM, AMOG, as well as the high molecular weight components of J1 glycoproteins, can be observed. The retention of these embryonic features are most likely associated with the cell turnover and high plasticity of this system. Moreover, the predominance of N‐CAM 180 with respect to other components of N‐CAM, as well as the absence of the L2/HNK‐1 carbohydrate epitope, are also particular traits of the primary olfactory system which could be associated with its exceptional properties. © 1992 Wiley‐Liss, Inc.
Comparison of bromine and permanganate as ultrastructural stains for lignin in plants infected by the fungus Colletotrichum lagenarium
Authors: Stein Barry D. , Klomparens Karen L. , Hammerschmidt Raymond
Published: February 2005

Transmission electron microscopy (TEM) and energy dispersive X‐ray microanalysis (EDS) were used to localize manganese from KMnO4, and bromine, as ultrastructural stains for lignin in an herbaceous plant. The Spookie cultivar of pumpkin is susceptible to infection by the fungus Colletotrichum lagenarium and served as a model system to compare the Br and KMnO4 techniques. Bromine was used in a fixation/staining procedure, and in separate experiments, KMnO4 was used as either a fixative or as a postsection stain. The technique for using bromine was modified from the woody plant procedure by adding a paraformaldehyde prefixation step. With the bromine procedure, cell walls were well‐preserved, but the cytoplasm was heavily extracted. The KMnO4 procedures produced well‐fixed cytoplasm, but with some staining artifacts. With all procedures, EDS dot mapping demonstrated lignin deposition in the cell walls specifically associated with sites of fungal infection. Lignin was also localized in secondary walls of tracheary elements, sites known to be highly lignified. The bromine procedure provided the most specific localization of lignin with a minimum of artifact. The specific applications of these stains provided data on the ultrastructural localization of lignin which contributed to the elucidation of its role in the interactions between pathogenic fungi in both their resistant and susceptible plant hosts. © 1992 Wiley‐Liss, Inc.
A technique for exposure of the glycoproteic matrix (zona pellucida and mucus) for scanning electron microscopy
Authors: Familiari G. , Nottola S. A. , Macchiarelli G. , Familiari A. , Motta P. M.
Published: February 2005

The fine structure of the zona pellucida (ZP) covering the oocyte and of the mucus covering the surface of the intestinal villi was investigated by using a new method employing ruthenium red (RR), saponin, and osmium‐thiocarbohydrazide impregnation.

The glycoproteic matrices both appeared constituted by thin filaments (ranging from 22 to 50 nm in thickness) anastomosed to form a very fine network.

RR prevented the dissolution and/or alteration of glycoproteins and polyanionic carbohydrates induced by acqueous fixatives. Saponin was a detergent of the soluble proteins. Osmium‐thiocarbohydrazide preserved the glycoproteic matrix filaments from the mechanical stress induced by dehydration and critical point drying and reduced filaments packing and shrinkage. The technical improvement was demonstrated by the following results: 1) a regular arrangement of the filaments network; 2) a thickness of mucus filaments smaller than that obtained with other methods of preparation; 3) a homogeneous thickness of ZP filaments.

This method allowed a very detailed study of the fine structural organization of the ZP and intestinal mucus. Therefore, this technique can be useful for a better evaluation of the morphodynamic of these and other glycoproteic matrices. © 1992 Wiley‐Liss, Inc.
Electron microscopy of in vivo recorded and intracellularly injected inferior olivary neurons and their GABAergic innervation in the cat
Authors: Ruigrok Tom J. H. , De Zeeuw Chris I.
Published: February 2005

This paper reports on the detailed morphology of inferior olivary neurons in the cat following electrophysiological examination, intracellular injection with horseradish peroxidase, and gamma aminobutyric acid (GABA) immunocytochemistry.

The activity of olivary cells was recorded intracellularly in vivo and their response to mesodiencephalic stimulation was tested. In a number of cases their response to stimulation of the contralateral superior cerebellar peduncle was also tested. Mesodiencephalic stimulation resulted in monosynaptic, and superior peduncle stimulation in disynaptic activation of cells in the medial accessory and principal olivary subdivisions. Rebound olivary activity was usually only found after mesodiencephalic stimulation.

Light microscopic investigation of osmicated and Araldite embedded Vibratome sections was facilitated considerably when performing the osmication in a glucose solution. Peroxidase labeled olivary cells, like that earlier described for Golgi‐impregnated material, possess a complex globular dendritic geometry. Especially, and unlike Golgi material, the abundance of exceptionally long and complex spiny appendages could be appreciated. The axons usually stemmed from first order dendrites and did not give rise to recurrent axon collaterals.

The ultrastructural analysis of this material, mainly from serial sections, was combined with postembedding GABA immunohistochemistry. In this way, GABAergic as well as non‐GABAergic profiles were studied in conjunction with HRP labeled cellular elements.

The GABAergic terminals usually contained pleomorphic vesicles and made symmetrical synapses whereas non‐GABAergic terminals nearly always formed asymmetrical synapses and contained round or oval vesicles. Most, if not all, HRP labeled spiny appendages were incorporated in glomeruli. A particular spiny appendage may contribute more than one spine head to a glomerular core, which, on average, consisted of spiny elements of six different neurons. A glomerular core is surrounded by approximately the same amounts of GABAergic and non‐GABAergic boutons. Also, all spiny appendages, and most of their individual spine heads, are contacted by GABAergic as well as non‐GABAergic boutons. Spiny appendages on the axon hillock may be incorporated in dendritic glomeruli, however, most synapses with the hillock were made by GABAergic boutons.

The combined physiological and morphological observations imply that (1) the cerebellar nucleican excert an excitatory influence on inferior olivary neurons through a mesodiencephalic relay, (2) the GABAergic nucleo‐olivary input seems to be capable of diminishing the oscillatory tendencies of olivary neurons, and (3) the mesodiencephalic (non‐GABAergic) and cerebellar (GABAergic) input may subserve a timing function since these inputs systematically impinge upon the same olivary spines. © 1993 Wiley‐Liss, Inc.
Glial cells in the nerve fiber layer of the main olfactory bulb of embryonic and adult mammals
Authors: Doucette R.
Published: February 2005

This article provides a detailed description of the glial cell types in the nerve fiber layer of the main olfactory bulb during embryonic development, in adult mammals, and at the nerve entry zone of the first cranial nerve. In adult mammals, the glial cell types of the olfactory nerve fiber layer include intrafascicular ensheathing cells, which have the exclusive role of ensheathing the olfactory axons in both the PNS and CNS, and interfascicular astrocytes, which occupy the spaces between adjacent olfactory fascicles. The ensheathing cells are particularly interesting because they possess a mixture of Schwann cell and astrocytic phenotypic features, are more likely to be of placodal than of CNS origin, and have the exclusive role of forming the glia limitans at the PNS‐CNS transitional zone. It is proposed that one important function of ensheathing cells is to modulate the growth of olfactory axons within the CNS; this modulation is probably mediated by selective cell adhesion molecules, extracellular matrix molecules, and chemotropic agents. © 1993 Wiley‐Liss, Inc.
Functional organization of the main olfactory bulb
Authors: Scott John W. , Wellis David P. , Riggott Marcia J. , Buonviso Nathalie
Published: February 2005

Complete understanding of the role of the mammalian main olfactory bulb in sensory processing has remained elusive despite many detailed studies on its anatomy and physiology. Several lines of recent evidence viewed in the context of earlier knowledge have provided new insights into the bulbar mechanisms of olfactory coding. The output cells of the olfactory bulb receive a localized olfactory nerve input and interneuronal input via dendrodendritic synapses on distinct sets of dendrites. The spatial arrangement of granule cell contacts on output cell basal dendrites suggests that lateral inhibitory interactions may occur between neighboring output cells. The input from olfactory receptor cell axons to the bulb also has spatial order, but does not represent a precise map of the receptor surface. Recent studies with antibodies and lectins suggest that different groups of axons from chemically similar receptor cells collect into certain glomeruli, even if the axons originate from cells that are not contiguous in the mucosa. Electrophysiological studies have begun to explore the participation of spatially organized circuits in olfactory processing. The degree to which neighboring output cells respond similarly to odor stimulation, for example, depends on the distance between the cells, with those further apart showing complementary responses. Also, a single output cell can show 2 or more different temporal response patterns when different odors are presented. Intracellular recordings indicate that these responses are shaped by IPSPs. Electrical stimulation during such recordings shows that some mitral cells are excited by nerve inputs close to their glomerular tufts, while they are inhibited by nerve inputs to other parts of the bulb. Finally, recordings from granule and periglomerular cells indicate their potential in mediating components of output cell odor responses. These considerations suggest that the olfactory bulb performs a spatially based analysis on the information coming from the receptor cells. While the spatial organization of the olfactory bulb is probably not faithfully represented in the projections to the olfactory cortex, bulbocortical projections are not random. The fact that spatial factors exist at each of these levels in the olfactory system must be considered in developing models of central olfactory processing. © 1993 Wiley‐Liss, Inc.
Are there structural and functional modules in the vertebrate olfactory bulb?
Authors: Kauer John S. , Cinelli Angel R.
Published: February 2005

A number of different recording methods have shown that odorants elicit patterns of neuronal activity widely distributed across cells of the olfactory receptor epithelium, olfactory bulb, and piriform cortex in the vertebrate olfactory system. These findings suggest that the physicochemical properties of odorant molecules are processed by distributed coding mechanisms activated in parallel in olfactory circuits in order to characterize a single, “monomolecular” odorant. These findings also suggest that the response patterns seen at higher levels are set up by differential responses in peripheral receptor cells of the olfactory epithelium. One requirement for understanding the details of this proposed encoding scheme is correlation of odor‐generated patterns with the components of these circuits. In this paper, results from 2‐deoxyglucose and voltage‐sensitive dye studies suggest that certain components of these responses may relate to patterns established in reproducibly identifiable aggregates of bulbar cells. These findings are consistent with previous observations suggesting that columnar groups of periglomerular, mitral/tufted and granule cells, oriented perpendicular to the laminae of the bulb, are functionally related to one another. Such cell groups or modules, when activated in parallel, could serve as building block components of the complete ensemble response. According to this hypothesis, different sets of such modules would be activated with different odorant stimuli and modules could be shared to the degree to which the physicochemical properties of the different stimuli overlap. © 1993 Wiley‐Liss, Inc.
Contrast enhancement and polymer identification in the electron microscope by the formation and staining of unsaturated double bonds
Authors: Parker M. A. , Vesely D.
Published: February 2005

A new technique for the identification of phases contained within a polymer blend is described in this paper. The technique utilises the beam damage which occurs when polymers are irradiated in an electron microscope. It has been found that during the irradiation process isolated double bonds are formed which can be revealed by staining with osmium tetroxide. The density of staining and its relationship to electron exposure is shown to be a characteristic feature of a particular chemical structure. It allows for polymer phase identification with a high spatial resolution and also for contrast enhancement and preservation. This technique offers a unique way of studying a fine dispersion of phases in polymer blends, even where only low atomic number elements such as C, H, and O are present. © 1993 Wiley‐Liss, Inc.
Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure
Authors: Bridgman Paul C. , Lewis Annette K. , Victor John C.
Published: February 2005

In order to test the ability of freeze substitution to accurately preserve the ultra‐structure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical‐point‐drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical‐point‐dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right‐handed) two start helix was not affected by the different methods of preparation. However, the left‐handed “genetic” helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical‐point‐drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical‐point‐dried, filaments appeared to intersect at greater angles and seemed more “taut.” These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing steps. © 1993 Wiley‐Liss, Inc.
Intermediate voltage electron microscopy of transverse tubules at myotendinous junctions
Authors: Sonoda Masaki , Moriya Hideshige , Shimada Yutaka
Published: February 2005

The 3‐dimensional distribution of transverse (T) tubules at myotendinous junctions (MTJs) was studied by intermediate voltage (400 kV) electron microscopy of thick sections of rat vastus intermedius and chicken pectoralis muscles stained with lanthanum nitrate. Transversely oriented T tubules were seen to run at the level of A‐I junctions (the rat vastus intermedius) and Z bands (the chicken pectoralis), but were absent from such levels adjacent to the end of MTJ processes. These tubules opened to the lateral wall of sarcolemmal infoldings of MTJs and to the lateral cell surface. Longitudinally running T tubules were seen to connect with the transverse T tubules and to open at the bottom of junctional folds. The lack of T tubules at the final sarcomeric regions seems to indicate that the terminal sarcomeric half in close proximity to MTJs may be activated from the sarcoplasmic reticulum which forms couplings with the MTJ sarcolemma and/or longitudinal tubules in the MTJ processes. © 1993 Wiley‐Liss, Inc.
Application of cryotechniques in cartilage tissue preservation an immunoelectron microscopy: Potentials and problems
Authors: Hunziker Ernst B.
Published: February 2005

Cryotechnical processing of cartilage has the potential to solve many of the tissuespecific problems associated with various routine chemical fixation protocols. This is particularly the case with respect to extracellular matrix architecture, the distortion or destruction of which (caused by extraction and/or precipitation of proteoglycan molecules) may be prevented. Adoption of such techniques also permits high‐sensitivity immunoelectron‐microscopy of the extracellular matrix space (carbohydrate epitopes). However, a number of difficulties still remain to be resolved, particularly that of matrix‐cell interface separation occurring during freeze substitution and low temperature embedding. These problems are briefly addressed and possible solutions outlined. © 1993 Wiley‐Liss, Inc.
Improved preservation of ultrastructure in difficult‐to‐fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos
Authors: McDonald Kent , Morphew Mary K.
Published: February 2005

In this study, we have applied the techniques of high pressure freezing and freeze substitution to embryonic cell types which are usually difficult to fix properly for electron microscopy. In both Drosophila and Strongylocentrotus purpuratus, we see improved preservation of both membrane systems and cytoskeleton when compared to published results on the same cells using conventional electron microscope (EM) fixation methods. Finally, we have seen that postembedding labelling of sections is possible even after light osmium fixation during freeze substitution. © 1993 Wiley‐Liss, Inc.
Rapid freezing replica studies of membrane specializations in the proximal tubules of mouse kidney
Authors: Shibata Yosaburo , Hatae Tatsuhiko , Kuraoka Akio , Iida Hiroshi , Izumi Toshihiro
Published: February 2005

The membrane specializations of the fresh unfixed kidney cortex of adult and neonatal ICR mice were examined by using rapid freezing replica methods. In proximal tubular cells, numerous apical intracellular tubules exhibited helical patterns on the E face with a pitch of about 12 nm. This regular pattern was often continuous with similar striped indentations on the edge of the vacuoles connecting with the tubules. On the luminal surface (ES) of these vacuoles, membrane surface particles were arranged regularly in striped patterns with a center‐to‐center spacing of about 12 nm. We could not identify differentiations on the PF or PS of the same membrane systems. Another membrane specialization was a plaque or patch of clear pits in tilted lattice alignments on the P face of the large vacuoles with a center‐to‐center spacing of about 20 nm. This type of specialization was often observed in the neonatal mice proximal tubular cells. These membrane specializations may indicate the active membrane functions in the proximal tubules and suggest the functional continuity and structural relationship of these apical endocytic membrane systems. © 1993 Wiley‐Liss, Inc.
Ultrastructural evidence for the axonal localization of caudodorsal cell hormone mRNA in the central nervous system of the mollusc Lymnaea stagnalis
Authors: Dirks Roeland W. , van Dorp Annette G. M. , van Minnen Jan , Fransen Jack A. M. , van der Ploeg Mels , Raap Anton K.
Published: February 2005

The technique of in situ hybridization has been used to evaluate the expression of an ovulation hormone mRNA (caudodorsal cell hormone; CDCH) in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. Hybridization with radioactive as well as with nonradioactive labeled oligonucleotide and plasmid probes revealed a specific labeling on cell bodies of caudodorsal cells (CDCs), which are known to produce CDCH, on the light microscopical level. In addition, specific labeling was observed outside the cell bodies, as far as the cerebral commissure, where CDCH is released in the haemolymph. To investigate whether these signals represent an axonal localization of the CDCH mRNA, we performed in situ hybridization at the electron microscopical (EM) level. The results showed an intraaxonal localization of CDCH mRNA with digoxigenin labeled oligonucleotide and plasmid probes. Gold labeling was observed in secretion granules, and double labeling experiments showed that these granules also contain CDCH. This specific intragranular localization suggest that CDCH mRNA is transported through the axon and released by exocytosis in the haemolymph. © 1993 Wiley‐Liss, Inc.
An assessment of the efficacy of in situ hybridization as a quantitative method by variance components estimation
Authors: McCabe Joseph T. , Kao Tzu‐Cheg , Volkov Marina L.
Published: February 2005

One serviceable feature of in situ hybridization is its potential for assessing relative levels of mRNA in specific regions of tissues and organs. To determine its efficacy as a quantitative technique, we applied a nested factorial design to a multifactorial experiment. Estimates of the magnitude of variance components then allowed an assessment of variation over samples of sections from the same tissue source, variation in label over 2 anatomical sites within the same section of tissue, as well as experiment‐to‐experiment variation. We found ∼ 51% of the total variance arose from experiment‐to‐experiment variation, while ∼ 21% of the total variance was due to variation in autoradiography grain density over neurons in the same brain region. Rat‐to‐rat variation accounted for approximately 11%. About 10% of the variance was due to variation between sections of tissue that were derived from the same tissue source and were hybridized in the same hybridization experiment. Variation between 2 homologous, bilaterally located brain regions located on the same tissue section (the right and left supraoptic nucleus), accounted for ∼ 5% of the total variance. The remaining unaccounted error variance was ∼ 2% of the total variance. Since an expected change in cellular content of a particular mRNA was observed as a function of experimental treatment, results suggest in situ hybridization is a useful quantitative method. Findings also indicate, however, the importance of experimental design: the use of multiple samples of tissue from the same tissue source, a sufficient number of tissue sources (animals, batches of cultured cells) to account for variations in sample sources, and the need to assess experiment‐to‐experiment and rat‐to‐rat variations. Results suggest the utility of analyzing the data of in situ hybridization experiments from the perspective of experimental design. © 1993 Wiley‐Liss, Inc.
Receptivity is a polarity dependent special function of hormonally regulated uterine epithelial cells
Authors: Glasser Stanley R. , Mulholland Joy
Published: February 2005

Useful knowledge of the mechanisms which regulate ovoreceptivity and implantation remains elusive in spite of increasing efforts to apply the technology of biochemistry and to a lesser extent, cellular and molecular biology to the analysis of the problem. Existing models used to analyze interactions of the blastocyst and endometrial cells of the uterus have been unable to account for nongenotypic embryonic losses, particularly those following in vitro fertilization and embryo transfer.

Separation of endometrial uterine epithelial (UE) and uterine stromal (US) cells was used to demonstrate that each cell type responds independently and interdependently to the same regulatory signals. Cultured by classical techniques UE cells proved unable to respond to steroid hormone signals. For this reason UE cell cultures could not be used to develop an experimental cell system that mimicked growth and development of UE cells in utero. The failure of classical UE cell cultures derived from their inability to maintain epithelial cell polarity. Polarity, the spatial asymmetry of plasma membrane domains, is intrinsic to the structure and function of an epithelial cell. Apical and basolateral surfaces have different lipid and protein compositions which are correlates of the special functions of that epithelial cell. As epithelial cells differentiate they must, in response to regulatory cues, direct the flux of membrane components moving into and out of each surface in order to establish the polarity characteristic of each stage specific expression. The acquisition of receptivity by the apical surface of the UE cell may be considered to be such a special function.

To prove this hypothesis polarized cultures of primary UE cells had to be developed that were hormonally responsive. Such an experimental cell system could serve as a model for in vitro implantation. This essay describes such a culture system in which blastocysts cocultured with UE cells in the presence of estrogen, will as predicted, fail to attach. This polarized UE cell system provides a functional in vitro model to study ovoreceptivity. It is now feasible to initiate studies of hormonal regulation of the composition and function of UE cell plasma membranes as they reflect the nonreceptive, receptive, and refractory nature of its apical surface. © 1993 Wiley‐Liss, Inc.
Endometrial adenocarcinoma
Authors: Noumoff Joel S. , Faruqi Shamim
Published: February 2005

Adenocarcinoma of the endometrium is the most common gynecologic malignancy in the United States, accounting for some 36,000 cases of invasive cancer each year. Hyperplastic lesions of the endometrium follow a continuum, with the risk of progression to carcinoma being related to the severity of the disorder. Risk factors associated with the development of adenocarcinoma include hyperplasia, obesity, menstrual abnormalities, diabetes, hypertension, prior pelvic irradiation, sequential oral contraceptive use, diet, and exogenous estrogen use. There is also some evidence of genetic predisposition, and some data indicating the possibility of specific genetic abnormalities and activation of oncogenes as factors determining the etiology of the disease. At this time there is no accepted screening test for endometrial carcinoma, though the role of immunochemistry techniques for screening and follow‐up has just begun to be realized. Dilatation and curettage along with hysteroscopy remain the major means of diagnosis. A variety of prognostic variables including tumor cell type, histologic grade, depth of myometrial invasion, status of peritoneal cytology, presence of disease in preformed vascular spaces, presence of adnexal metastases, and presence of cervical involvement have been defined. Although the treatment plan for each patient must be individualized, the mainstay of treatment remains total abdominal hysterectomy with bilateral salpingo‐oophorectomy. Metastatic and recurrent disease is usually treated with hormonal therapy and systemic chemotherapy. Radiation therapy like surgery in recurrent disease is only applicable for the treatment of local recurrences. © 1993 Wiley‐Liss, Inc.
Characterization of agarose as an encapsulation medium for particulate specimens for transmission electron microscopy
Authors: Wood Jacqueline I. , Klomparens Karen L.
Published: February 2005

Agarose, agar, and gelatin were initially compared as encapsulation media for 3 structurally diverse particulate specimens: bacteria, yeast, and mitochondria. Agarose proved superior to both gelatin and agar for ease of handling and overall image quality (minimum background). All sample types exhibited high quality fixation and structural detail with no heat damage from the agarose medium. Based on this finding, we further characterized agarose encapsulation as affected by post‐fixation, en bloc staining and resin type. Osmium tetroxide post‐fixation, followed by en bloc uranyl acetate staining, could be performed without an increase in the electron density of the encapsulation medium. Agarose proved successful as an encapsulation medium regardless of the resin type or preparation protocol, thus providing flexibility in experimental design and excellent results over a range of variables. © 1993 Wiley‐Liss, Inc.
Software for quantitative immunogold and in situ hybridization
Authors: Bolender Robert P.
Published: February 2005

A well‐deserved criticism of stereology is that it is often too difficult to understand and use. Nevertheless, it is rapidly becoming one of the most effective ways of collecting and interpreting structural data in experimental biology. Recent breakthroughs in theory have produced a remarkable set of tools that can be used to engineer new methods. The dilemma remains, however, in that some of these new methods continue to be too difficult to understand and use. One solution to this growing problem of technology transfer might be to simplify the methods with computer software. To test this idea, programs were written for quantitative immunogold and in situ hybridization. Simulators were used to develop and test new experimental designs, which, in turn, were translated into step‐by‐step laboratory toolkits. This paper shows how these toolkits can turn two‐dimensional section data into estimates for the number of labeled molecules in three‐dimensional organelles, cells, tissues, and organs. The results indicate that software can identify the key data of an experiment and reduce the computational requirements of a new stereological method to entering constants and variables into data entry forms. © 1993 Wiley‐Liss, Inc.
Application of ESEM to environmental colloids
Authors: Nuttall H. E. , Kale Rahul
Published: February 2005

Environmental colloids are toxic or radioactive particles suspended in ground or surface water. These hazardous particles can facilitate and accelerate the transport of toxicants and enhance the threat to humans by exposure to pathogenic substances. The chemical and physical properties of hazardous colloids have not been well characterized nor are there standard colloid remediation technologies to prevent their deleterious effects. Colloid characterization requires measurement of their size distribution, zeta potential, chemical composition, adsorption capacity, and morphology. The environmental scanning electron microscope (ESEM) by ElectroScan, Inc., analyzes particle sizes, composition, and morphology. It is also used in this study to identify the attachment of colloids onto packing or rock surfaces in our development of a colloid remediation process.

The ESEM has confirmed the composition of groundwater colloids in our studies to be generally the same material as the surrounding rock. The morphology studies have generally shown that colloids are simply small pieces of the rock surface that has exfoliated into the surrounding water. However, in general, the source and chemical composition of groundwater colloids is site dependent. We have found that an ESEM works best as a valuable analysis tool within a suite of colloid characterization instruments. © 1993 Wiley‐Liss, Inc.
Dynamics of soldering reactions: Microscopic observations
Authors: Singler Timothy J. , Clum James A. , Prack Edward R.
Published: February 2005

This paper provides a summary of some in situ, high‐resolution studies of solder spreading reactions on microelectronic circuit metallizations. Experiments are described that focus on the use of the environmental scanning electron microscope, or ESEM. Those experiments have been complemented by studies using optical hot‐stage microscopy and have been supplemented by additional analytical tools such as energy dispersive X‐ray microanalysis, Auger and ESCA to evaluate chemical processes. Two general results from dynamic scanning electron microscope observations are that (1) molten solder alloys undergo a segregation process during spreading in which a “precursor” film spreads in advance of the bulk solder and (2) the spreading front, which may be enriched in Sn from Pb‐Sn or Bi‐Sn solders, or In from Pb‐In solders, spreads along high‐reactivity features of the metallization surface as a reacting “precursor” film. A third observation from these tests is that, in unconfined geometries, the reactive metallization, if not sufficiently thick, can be dissolved by the solder before wetting is complete, leading to dewetting of the solder. Both the kinetics and extent of spreading of these films and the relationship of these phenomena to the commonly measured contact angle and wetting forces are currently being examined by a range of complementary techniques. Information gathered in these studies shows that process temperature as well as composition, reactivity, and relative amounts of the solder and metallization species should all be factors of interest to the those responsible for control of soldering processes. © 1993 Wiley‐Liss, Inc.
Aspects of vertebrate gustatory phylogeny: Morphology and turnover of chick taste bud cells
Authors: Ganchrow Judith R. , Ganchrow Donald , Royer Suzanne M. , Kinnamon John C.
Published: February 2005

The taste bud is a receptor form observed across vertebrates. The present report compares chick taste buds to those of other vertebrates using light and electron microscopy.

Unlike mammals, but common to many modern avians, the dorsal surface of chick anterior tongue lacks taste papillae and taste buds. Ultrastructurally, chick buds located in the anterior floor of the mouth (as in some reptiles and amphibians) and palate contain dark, intermediate, light, and basal cell types. Dark, intermediate, and light cells extend microvilli into intragemmal lumina and pores communicating with the oral cavity. As specialized features, dark cell apices lack dense granules and exhibit short microvilli relative to light and intermediate cells. Dark cell cytoplasmic fingers envelop intragemmal nerve fibers and cells as in other species, and sometimes contain abundant clear vesicles. Nerve profile expansions often are located adjacent to dark, intermediate, and light cell nuclei. Classical afferent synaptic contacts are rarely observed.

Taste cell turnover is suggested by mitotic and degenerating figures in chick buds. In addition, tritiated thymidine injected into hatchlings, whose anterior mandibular oral taste bud population approximates that in adults, reveals a turnover rate of about 4.5 days. This is about half that observed in altricial mammals, reflecting a species difference or developmental factor in precocial avians.

It is concluded that chick taste buds exhibit morphologic features common to other vertebrate buds with specializations reflecting the influences of niche, glandular relations, and/or age. © 1993 Wiley‐Liss, Inc.
A modified autowasher device for rapidly washing large numbers of EM‐grids
Authors: Chen Ming H.
Published: February 2005

This device consists of a siphon system and 5 to 10 grid disks, modified from the previous mode (Chen, 1973), for large numbers of grids with ultrathin sections. This method improves the ease of assembling grids onto the grid disk and also requires much less stain solution. This system only takes 5 min for one single stain washing, at a maximum of 100 grids, and also avoids stain contamination. The grid disk can also be used for immunocytochemistry work and for critical point drying of grids with biological specimens.
Cellular relations in mouse circumvallate taste buds
Authors: Murray Raymond G.
Published: February 2005

The fine structure of the taste buds of circumvallate papillae of two strains of mice was studied by electron microscopy. Mice anesthetized with ketamine were perfused through the heart with a double aldehyde mixture in cacodylate buffer and the tissues embedded in Epon. Semi‐serial sections were employed. The morphology and relationships of cell types are consistent with the majority of descriptions of mammalian taste buds served by the ninth cranial nerve. Cells of type II are particularly well documented, as the stages in their origin, maturation and degeneration could be followed. Significant differences, however, relate to cell type I. These cells contain large dense‐cored granules, contrasted with the more irregular and somewhat larger dark granules of the type I cells in the rabbit. These granules do not produce a dense homogenous product for the pore, as seen in the rabbit. Rather the pore substance consists of small, empty vesicles in a diffuse dark matrix. These granules are only moderately larger than the dense‐cored vesicles of the type III cells. All features of the type III cell were demonstrated, although no instance of a complete cell was seen in any section. No significant differences were noted between the two strains of mice. Intimate proximity of a nerve to a cell nucleolus, suggestive of a trophic pathway, is illustrated. © 1993 Wiley‐Liss, Inc.
Morphological types of neurons located at taste‐responsive sites in the solitary nucleus of the hamster
Authors: Whitehead Mark C. , McPheeters Martha , Savoy Larry D. , Frank Marion E.
Published: February 2005

HRP histochemistry and microelectrode mapping were combined to study the sizes, shapes, and orientations of neuronal cell bodies and dendrites located at sites of taste‐elicited single unit activity in the nucleus of the solitary tract (NST). Cells responding to sapid stimulation of the anterior tongue were extracellularly recorded using micropipettes containing HRP. Iontophoretic injection of the marker at the recording sites resulted in small (50–200 μm diameter) opaque zones bordered by a small number (2–15) of neurons with Golgi‐like filling of their cell bodies, dendrites, and to some extent, their axons. The cell bodies were near (50–250 μm) the injection sites into which they sent labelled dendrites. Two broad categories of neurons were typically filled. Elongate cells had oval‐ to spindle‐shaped cell bodies oriented mediolaterally. Two primary dendrites extended 100–300 μm from the cell body, one medially and one laterally, and branched within a cylindrical dendritic field oriented mediolaterally. A minority of the HRP‐filled elongate cells had unusually long rostrally or caudally directed dendritic branches. Stellate cells had oval, round, triangular, or polygonal cell bodies and 3–5 primary dendrites coursing 200–300 μm in all directions and branching as unoriented, spheroidal fields. A minority of stellate cells had relatively unbranched wavy dendrites, resembling tentacles, while others had unusually small cell bodies (10–15 μm diameter), small dendrites, and locally arborizing axons. Of 151 labelled cells, all but 12 were remarkably confined to the rostral NST. Nearly 90% were concentrated in the rostral central cytoarchitectonic subdivision, where stellate cells predominated, or in the rostral lateral subdivision, where elongate cells predominated. These morphological types of neurons, filled at neurophysiological recording sites, are compared with cell types identified in previous light and electron microscopic studies of the cytoarchitecture, connections, and synaptic organization of the gustatory NST. © 1993 Wiley‐Liss, Inc.
Interaction of lung surfactant protein a with alveolar macrophages
Authors: Ohmer‐Schröck Daniela , Schlatterer Christina , Plattner Helmut , Schlepper‐Schäfer Jutta
Published: February 2005

We analyzed the binding mechanism of human recombinant lung surfactant protein A (SP‐A) to rat alveolar macrophages using anti‐SP‐A antiserum and protein A coated onto gold particles. Results were compared with our recent data on binding and uptake of SP‐A‐coated colloidal gold particles. The rationale for the current approach was to avoid any possible steric effects on SP‐A binding to the cell surface. Binding of unlabeled SP‐A depends on the presence of calcium ions in the medium and involves a mannose‐specific mechanism. Binding is partly inhibited by the collagenase‐resistent fragment of SP‐A, representing mainly the globular part of SP‐A. Taken together, these facts indicate binding of SP‐A via the carbohydrate binding site on the globular region of SP‐A. On the other hand, a partial inhibition of SP‐A binding by fragments of C1q (representing the collagenous region of C1q) indicates a second binding site for SP‐A by the collagen‐like portion to the C1q receptor of macrophages. We conclude that two different mechanisms are probably involved in SP‐A binding to alveolar macrophages. Specificity of the binding was shown with fluorescein‐labeled SP‐A. Binding was inhibited by an excess of unlabeled SP‐A. Binding and uptake of SP‐A are seen only with alveolar macrophages and not with other macrophage populations isolated from rat, such as liver macrophages (Kupffer cells), resident peritoneal macrophages, and peritoneal macrophages activated by Corynebacterium parvum. Therefore, binding sites for SP‐A occur exclusively on alveolar macrophages. In addition, the intracellular Ca2+ concentration of the lung macrophages was determined by using the fluorescent dye fura‐2/AM. Intracellular [Ca2+] increased immediately after addition of SP‐A. This indicates immediate activation of macrophages by SP‐A. © 1993 Wiley‐Liss, Inc.