Light Microscopic Preparations Research Articles

Genetic variation in the African rodent subfamily Otomyinae (Muridae). II. Chromosomal changes in some populations of Otomys irroratus.

Chromosome-banding studies have been carried out on 31 specimens of Otomys irroratus from six localities. Light-microscope preparations of chromosomes were obtained from cultures of fibroblasts, spleen lymphocytes, peripheral blood lymphocytes, and directly from bone marrow. Karyotypic variability, both numerical and morphological, was detected in three populations. Diploid numbers ranged from 2n = 23 to 2n = 32. Intrapopulation differences were chiefly caused by variation in the number of copies in two pairs of small, biarmed, partly heterochromatic autosomes suggestive of B chromosomes. A major morphological variation in the karyotypes involved the presence of seven pairs of biarmed autosomes with totally heterochromatic short arms in the populations distributed to the west of 26 degrees 57' E. To the east of this longitude, populations of this species exhibited mostly acrocentric autosomes. G-banding patterns of these karyotypes and those of a karyotype from a previous study (Robinson and Elder, 1987) were compared. A chromosome originating from a tandem fusion, possibly leading to partial reproductive isolation, was found in one population. Possible implications of these results for mechanisms of speciation are discussed.

Nucleolar organizer regions in the normal, hyperplastic and carcinomatous epithelium of endometrium

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose Dmax and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AGNOR staining technique is of diagnostic importance in distinguishing between these two groups.

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10–20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10–20% sucrose was observed t to ave no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis “bays,” visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as “spot welds” between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.

Alterations of the Intestinal Innervation in Mice Infected with Schistosoma mansoni

Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation.

Distribution of glutamine-like immunoreactivity in the cerebellum of rat and baboon (Papio anubis) with reference to the issue of metabolic compartmentation

The cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehyde-fixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabeled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.

Plasticity of spinal systems after unilateral lumbosacral dorsal rhizotomy in the adult rat

Plasticity of spinal systems in response to lumbosacral deafferentation has previously been described for the cat, by using immunocytochemistry to demonstrate plasticity of tachykinin systems and degeneration methods to demonstrate plasticity of descending systems. In this study, we describe the response to lumbosacral deafferentation in the adult rat. Application of immunocytochemical methods to visualize tachykinins (predominantly substance P magnitude of SP), serotonin (5-HT), and dopamine B-hydroxylase (DBH), the synthesizing enzyme for norepinephrine, permits us to compare the response of SP systems in rat and cat spinal cord and to examine the response of two descending systems, serotoninergic and noradrenergic, to deafferentation. We used image analysis of light microscopic preparations to quantify the immunoreaction product in the spinal cord in order to estimate the magnitude, time course and localization of changes induced by the lesion. The distribution of SP, serotoninergic (5-HT), and noradrenergic staining in the spinal cord of rat is very similar to that of the cat. Unilateral lumbosacral rhizotomy elicits a partial depletion, followed by a partial replacement of tachykinin immunoreactivity in laminae I and II. This response was similar to that described for the cat, although characterized by a longer time course, and, as in the cat, is likely due to plasticity of tachykinin containing interneurons. The same lesion elicits no depletion but a marked and permanent increase in 5-HT immunoreactivity in laminae I and II, which develops more rapidly than the response by the SP system. These results indicate sprouting or increased production of SP and 5-HT in response to deafferentation. No change was seen in DBH immunoreactivity, indicating that the noradrenergic system does not show plasticity in response to deafferentation. Our results demonstrate that dorsal rhizotomy evokes different effects in different systems in the adult spinal cord of the rat and thus suggests that the response of undamaged pathways to partial denervation of their target is regulated rather than random.

Substance P-Containing Neurons Innervating LHRH-Containing Neurons in the Septo-Preoptic Area of Rats

Neuroanatomical attempts have been made to determine the synapses between luteinizing hormone-releasing hormone (LHRH)-containing neurons and substance P (SP)-containing neurons in the hypothalamus of female rats. Wheat germ agglutinin was injected into the septo-preoptic area (SPA) and found to be incorporated into certain SP-containing neurons within the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus. Hence, we used a preembedding double immuno-staining technique in demonstrating LHRH and SP neurons in the SPA. In light-microscopic preparations LHRH was labeled with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen while SP was labeled with silver-gold particles; brown LHRH cells appeared to be surrounded by black silver-gold dots. In electron-microscopic preparations, the labelings for LHRH and SP were made reversely; SP was localized with DAB chromogen, and SP-containing axonal terminals appeared to make synaptic contacts on silver-gold-labeled LHRH cell bodies and dendritic processes. The terminals contained numerous small clear vesicles and some large dense-cored vesicles, and the synaptic membrane specialization appeared to be symmetric and asymmetric. These findings indicate that certain SP neurons existing in the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus may project fibers to make synaptic contact with LHRH neurons in the SPA in the rat.

Postnatal development of the light and electron microscopic features of basket cells in the hippocampal dentate gyrus of the rat

Light and electron microscopic preparations were used to analyze the postnatal development of the basket cells of the rat dentate gyrus. The basket cells, located at the hilar border, were recognized in 2-day-old rats in Golgi preparations, where they displayed immature dendrites and a small axon arbor in the granule cell layer. At 5 days, the basket cells were found to have a large perikaryal cytoplasm, a round nucleus, an axon that forms symmetric synapses with granule cells, and dendrites and somata that are contacted by other axon terminals. The 10-day basket cells display more mature features, such as Nissl bodies and well-developed Golgi complexes. The basket cells from 16-day-old rats are mature in terms of their ultrastructural features, in that the nuclei are highly indented and display intranuclear rods or sheets, the perikaryal cytoplasm is packed with organelles, and the axon has developed an extensive arborization with the somata and dendrites of granule cells at the border with the molecular layer. This arborization will continue to expand as more granule cells are generated and added to the hilar border. These data correlate well with the immunocytochemical and biochemical development of GABAergic neurons in the dentate gyrus. Furthermore, the maturation of the structure of basket cells appears to precede the appearance of adult-like electrical activity in the hippocampus.

Cholinergic synapses in the central nervous system: Studies of the immunocytochemical localization of choline acetyltransferase

Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT-labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.

Local circuit neurons in both the dentate gyrus and Ammon's horn establish synaptic connections with principal neurons in five day old rats: a morphological basis for inhibition in early development.

Glutamate decarboxylase (GAD)-positive and Golgi impregnated local circuit neurons of the hippocampal formation of five day old rats were examined in light and electron microscopic preparations. The ultrastructural features of these neurons were similar in both the dentate gyrus and CA1 area of Ammon's horn. Somata displayed a perikaryal cytoplasm rich in organelles but lacked organized Nissl bodies. Most nuclei showed intranuclear infoldings of varying degrees but no intranuclear sheets or rods were found. Somata and dendrites were contacted by relatively immature axon terminals that formed mainly symmetric synapses. The axons of local circuit neurons in both the dentate gyrus and Ammon's horn formed symmetric synapses with somata and dendrites of the principal neurons in these regions. Thus, both GAD-positive and Golgi-impregnated terminals of local circuit neurons were observed to form synapses with pyramidal and granule cells. These terminals were usually small and contained relatively few pleomorphic synaptic vesicles. The results show that a circuitry for inhibition is established in the 5 day old dentate gyrus and Ammon's horn, even though the local circuit neurons lack some of the typical adult ultrastructural features at this age.

Electron microscopical and histochemical observations on melanized sperm and spermatophores of pond-cultured shrimp, Penaeus vannamei

Black spermatophores were collected by manual ejaculation of pond-cultured Penaeus vannamei males and were prepared for light and electron microscopical study. Light microscopy revealed varying numbers of individual and aggregated yellow-brown to black polymorphic pigment droplets measuring 2 to 400 μm in diameter in spermatophore capsules. Some black spermatophores contained mostly degenerate sperm, while others contained mixtures of normal-appearing unistellate sperm, abnormal spikeless sperm, and degenerating sperm. Yellow-brown to black oval pigment droplets measuring 1 to 2.5 μm in diameter were encountered in the nuclei both of normal-appearing unistellate sperm and of abnormal spikeless sperm. Degenerating sperm exhibited no spikes and no pigment droplets in the remains of the sperm cell bodies. Oval pigment droplets occurred also among sperm in the extracellular matrix of spermatophores. Capsular, matrical, and intranuclear pigment droplets all gave histochemical reactions characteristic of melanin; i.e., pigment droplets were bleached by acidified permanganate, gave positive Schmorl and methenamine silver reactions, and adsorbed ferrous ions. Acid phosphatase activity was not detected in the sperm or spermatophores. Electron microscopy revealed oval, electron-dense droplets within sperm nuclei of both unistellate and spikeless sperm. These droplets corresponded in size to the pigment droplets seen in sperm by light microscopy. Intranuclear droplets were bounded by a single membrane, exhibited fairly homogeneous internal contents, and were frequently enmeshed in strands of intranuclear chromatin threads. Spikeless sperm, in addition to containing oval electron-dense intranuclear droplets, exhibited arrays of small, electron-dense particles packed in the region of the sperm where one would expect to have encountered the spike, suggesting that these particles play a role in sperm spike depolymerization. It is suggested that the melanized condition reduces fecundity in this species by promoting the depolymerization of sperm spikes and the degeneration of sperm. The mechanism by which sperm and spermatophores become melanized is unknown and remains to be determined. No bacteria and no hemocytes were encountered in either light or electron microscope preparations of melanized spermatophores.

Scanning electron microscopy of teased intrafusal muscle fibers from rat muscle spindles

Scanning electron microscopic (SEM) studies on intrafusal muscle fibers and their various sensory and motor endings are not available thus far although SEM greatly increases the resolution power for analyzing surface structures in comparison to light microscopic preparations. From a total of 68 isolated muscle spindles in lumbrical muscles of Wistar rats, only one primary sensory ending could be isolated. Secondary sensory endings were of an annulospiral, a flat, cuff-shaped, or a more complicated branched type; they were always completely surrounding intrafusal muscle fibers. Motor terminals were more focally arranged not surrounding the muscle fibers. Three types could be distinguished. Collagen fibrils were seen to insert on capsular elements as well as on the surface of intrafusal muscle fibers. Thus SEM helps understanding the most complicated electromechanical system for motor control.

Feeding of the chewing louse Damalinia ovis (Schrank) (Phthiraptera:Trichodectidae) on sheep

The possibility that Damalinia ovis ingests intact epidermis could explain economically important irritation behaviour by infested sheep. To test this, feeding lice were observed on sheep by hand lens and macrophotography and on light and scanning electron microscope preparations. Aspects of feeding behaviour were described, particularly an epidermal ingestion posture. Louse ingesta were examined in paraffin and frozen sections. Lipid-covered stratum corneum squames were identified in crop, midgut, rectum and faeces. Nucleated keratinocytes from inner epidermal strata were not seen, neither were wool fibres. Vertical frozen sheep skin sections, with feeding lice cryofixed by liquid nitrogen, showed mandibles engaged in the outer stratum corneum of the epidermis. The epidermal origin of some ingesta was confirmed by confining lice over sheep whose skin was stained with Oil Red O, whose colour was seen in crop, midgut, rectum and faeces of harvested lice. Lipase was found in louse midgut. It was concluded that sebaceous secretions may form an important component of the D. ovis diet. The relative contribution from loose scurf or from stratum corneum squames was not determined. Mechanical stimuli by feeding lice did not seem to be the source of host irritation. The role of sensitising agents in louse brei filtrates was investigated with inconclusive results. Intradermal injections of louse faeces did not elicit a demonstrable response.

Correlation between NOR sizes and numbers in non-Hodgkin's lymphomas.

A series of 36 cases of non-Hodgkin's lymphomas (NHL) has been studied by means of the argyrophil (AgNOR) method for nuclear organizer regions (NORs). Morphometric analysis of highly magnified photographic images of light microscope preparations of the AgNORs was performed by means of an interactive image analysis system. It was observed that in the low-grade specimens (where NORs are less frequent than in those of high-grade histology), the AgNOR sites were highly significantly (P less than 0.001) larger than in high-grade NHL. In the low-grade lymphomas, the AgNOR maximum diameter (Dmax) ranged from 0.7 to 1.7 micron 2 (mean 1.11 micron 2) and area ranged from 0.48 to 1.99 micron 2 (mean 1.11 micron 2). In contrast, in the high-grade specimens, Dmax was from 0.33 to 0.51 micron (mean 0.41 micron) and the area ranged from 0.082 to 0.19 micron 2 (mean 0.13 micron 2). Thus, a well-defined inverse relationship was observed between AgNOR numbers and their sizes. There was total separation between low- and high-grade values in this series. This light microscope technique offers some advantages over ultrastructural morphometry of interphase NORs (fibrillar centres).

Types and spatial distribution of vasoactive intestinal polypeptide (VIP)-containing synapses in the rat visual cortex.

In the rat visual cortex vasoactive intestinal polypeptide (VIP)-containing structures were studied by means of light and electron microscopy and image analysis. VIP-immunoreactive axon terminals were found to form symmetric synapses with small dendritic shafts, dendritic spines and somata of pyramidal cells and interneurons. VIP-terminals often occurred in pairs with VIP-negative, asymmetric synapses on the same postsynaptic structure. VIP-immunostained dendrites and perikarya were contacted by a purely asymmetric and a mixed population of VIP-negative terminals, respectively. Synaptic connections between two VIP-neurons are seldom as compared to the other types of VIP-synapses. Quantitative studies obtained by the image analysis of VIP-stained boutons and dendritic particles in light microscopic preparations suggest a distinct laminar distribution. Dendritic particles are most frequent in layers I-II, whereas axonal boutons have three laminar accumulations: at the border of layers I-II, in layer IV and layer VI. Together with previous results, the present findings argue for a non-random spatial distribution of VIP-boutons.

Scanning electron microscope analysis of structural changes and aberrations in human chromosomes associated with the inhibition and reversal of inhibition of ultraviolet light induced DNA repair.

Metaphase chromosomes appear decondensed in preparations from mitotic cells that have been irradiated with ultraviolet light (UV) and incubated with inhibitors of DNA synthesis; under these conditions DNA repair is inhibited and both single and double strand DNA breaks accumulate. After reversal of the inhibition chromosomes are condensed, but are often damaged. In this paper we show by scanning electron microscopy (SEM) that decondensed HeLa chromosomes are composed of fibre clusters similar to those previously described for the large chromosomes of the Indian muntjac. This suggests that the clusters may be a universal higher order packing unit in mammalian metaphase chromosomes. We also examine by SEM the nature of the aberrations that appear after the reversal of periods of inhibited repair; these include gaps, breaks, deletions and telomeric packing abnormalities. SEM analysis allows an extension and reconsideration of conclusions about chromatid continuity based on the study of conventional light microscope (LM) preparations.

A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster

Individuals expressing recessive mutations in the Deformed ( Dfd) locus of Drosophila melanogaster were examined for embryonic and adult defects. Mutant embryos were examined in both scanning electron microscope and light microscope preparations. The adult Dfd recessive mutant phenotype was assessed in somatic clones and in survivors homozygous for hypomorphic alleles of the gene. The time of Dfd + action was determined by studying a temperature conditional allele. Dfd + is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. Shift studies utilizing a temperature conditional allele have shown that the gene product is necessary during at least two periods of development, during embryonic segmentation and head involution and during the late larval and pupal stages. From these studies we conclude that Dfd is a homeotic gene necessary for proper specification of both the embryonic and the adult head.

A light and electron microscope study ofPicea glauca (White spruce) somatic embryos

Somatic embryos in embryogenic callus cultures derived from Immature zygotic embryos ofPicea glauca (White spruce) were examined by light and electron microscopy. Somatic embryos consist of an embryonic region of small densely cytoplasmic cells subtended by a suspensor consisting of long highly vacuolated cells. Mitotic figures are frequent in the embryonic cells but are not observed in the suspensor. Cell divisions in the embryonic region apparently produce rows of cells which elongate to form the suspensor. The presence of abundant polysomes, coated membranes and dictyosomes in the cytoplasm of embryonic and upper suspensor cells suggests rapid growth of the embryo. In contrast the basipetal suspensor cells appear to be senescing. While only a few scattered microfilaments are present in the meristematic cells, the upper suspensor cells contain numerous bundles of longitudinally oriented microfilaments. These bundles correspond to actin cables observed in light microscope preparations stained with rhodamine labelled phalloidin and are oriented parallel to the direction of active streaming in these cells.

Applications of a large-specimen resin-embedding technique in experimental cardiac pathology

This paper describes the large-specimen resin-embedding method which has been developed for use with cardiac specimens in our laboratory during the past seven years. The technical problems experienced are discussed, the excellent quality of the light and electron microscopic preparations obtained from the single large resin blocks is illustrated, and the major advantages of using this methodology in experimental heart research are briefly considered.

The cytology of human Pacinian corpuscles: evidence for sprouting of the central axon.

During the course of the studies on non-traumatized Pacinian corpuscles from normal human adults, we have frequently encountered corpuscles which have an 'apparently multiple' innervation in both light and electron microscopic preparations. On closer inspection of serial sections for both light and electron microscopy, these 'apparently multiple' axon terminals have been found in fact to be branches of the main central axon within the inner core of the corpuscle. Sprouting occurred at the trunk or at the extreme tip of the main axon, and such sprouts extended in various directions from the central axon throughout the inner core, producing tortuous and complex patterns of this 'multiple' innervation. These axonal sprouts do not have separate inner cores separated from one another, but rather are embedded in a common inner core. The presence of a common inner core thus differentiates normal axonal sprouts from the experimentally or pathologically produced multiple innervation that results form regeneration of axons in a previously denervated corpuscle. We conclude that the inner core of Pacinian corpuscles is a unique micro-environment promoting sprouting of sensory axon in the normal human adult as well as juvenile Pacinian corpuscles.