Ultrastructural description of glutamate-, aspartate-, taurine-, and glycine-like immunoreactive terminals from five rat brain regions

The ultrastructural localization of putative excitatory (glutamate, aspartate) and inhibitory (taurine, glycine) amino acid neurotransmitters is described in several selected rat brain regions. In general, axon terminal profiles immunoreactive for excitatory amino acids formed asymmetric synapses with non-immunoreactive small diameter dendritic profiles or dendritic spines. In the cerebellum, both mossy fiber terminals and parallel fiber terminals were immunoreactive for glutamate and aspartate. In the hippocampus, mossy fiber terminals within the stratum lucidum of the CA3 region were immunoreactive for glutamate. Localization of glutamate and aspartate to cerebellar parallel and mossy fibers, as well as the identification of glutamate in hippocampal mossy fibers, is consistent with the excitatory nature of these fibers as described in previous physiological studies. Glutamate-like immunoreactive terminals were also identified in subnucleus caudalis of the spinal trigeminal nucleus and in the dorsal horn of the spinal cord. Immunoreactive axon terminals for two putative inhibitory neurotransmitters, glycine and taurine, displayed a greater number of morphological variations in synaptic structure. In the cerebellum, taurine-like immunoreactivity was present in both basket cell axon terminals which formed symmetric synapses with Purkinje cell neurons, and in a few mossy fiber terminals which formed asymmetric synapses with dendritic spines. In the area dentata of the hippocampus, taurine-like immunoreactive profiles formed asymmetric synapses with dendritic elements. Glycine-like immunoreactive terminals formed symmetric synapses with cell perikarya in both the ventral horn of the spinal cord and in the cochlear nuclei, and on axon terminals in the spinal trigeminal and cochlear nuclei. In contrast, some glycine-like immunoreactive terminals formed asymmetric synapses with distal dendritic profiles in the spinal cord and spinal trigeminal nucleus. The localization of taurine to cerebellar basket cell axons and glycine to axon terminals that synapse on ventral horn motor neuron perikarya is consistent with the hypothesis that these amino acids are functioning as inhibitory neurotransmitters at these synapses. Taurine localization to cerebellar mossy fibers and to fibers in the molecular layer of the dentate gyrus may be more consistent with a proposed neuromodulator role of taurine.     

A review of recent observations concerning the synaptic organization of the basilar pontine nuclei

Ultrastructural studies are described that have identified in the basilar pontine nuclei (BPN), the synaptic boutons formed by the corticopontine, cerebellopontine, tectopontine, and dorsal column nucleipontine afferent projection systems. In addition, immunocytochemical studies visualized neuronal somata, dendrites, and synaptic boutons that contain immunoreactivity for GABA or the synthesizing enzyme glutamic acid decarboxylase (GAD). Based upon differences in the mode of degeneration and postsynaptic locus of degenerative synaptic boutons in the BPN, it is suggested that two types of cortical neurons and three classes of deep cerebellar nuclear cells project to the BPN. For similar reasons, it appears that two types of neurons in the dorsal column nuclei project to the BPN while only one type of afferent synaptic bouton takes origin from the superior colliculus. Furthermore it appears that the population of BPN neurons projecting to the paramedian lobule receives convergent inputs from the cutaneous periphery and the corresponding region of sensorimotor cortex. Studies employing GAD immunohistochemistry indicate that GABA-ergic neurons and axon terminals are present in the BPN and thus support the suggestion that a local inhibitory interneuron is present within the BPN. Taken together these observations suggest that basilar pontine neurons might play a more active role in the integration of various types of information destined for the cerebellar cortex than has previously been recognized.     

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.     

Chemical synapses in the mammalian central nervous system: An introduction

This paper provides a brief review of the historical development of our understanding of synaptic structure in the central nervous system. The basic structure of the synapse is reviewed as well as the development of ultrastructural techniques that have allowed the details of synaptic organization to be elucidated.     

Lateral magnocellular nucleus of the anterior neostriatum (LMAN) in the zebra finch: neuronal connectivity and the emergence of sex differences in cell morphology.

The song system of birds provides a model system to study basic mechanisms of neuronal plasticity and development underlying learned behavior. Song learning and production involve discrete sets of interconnected nuclei in the avian brain. One of these nuclei, the lateral magnocellular nucleus of the anterior neostriatum (LMAN), is the output of the so-called anterior forebrain pathway known to be essential for learning and maintenance of song, both processes depending on auditory feedback. In zebra finches, only males sing and this sexually dimorphic behavior is mirrored by sexual dimorphism in neuronal structure that develops during ontogeny. Female zebra finches are not able to sing and nuclei of the song system are strongly reduced in size or even lacking, when compared to male brains. Only LMAN can be delineated as easily in females as in males. Since female zebra finches, despite being unable to sing, recognize song just as males do and form a memory for song (model acquisition) early in life, LMAN is a putative candidate for song acquisition in both sexes. Therefore, development of LMAN was studied at the cellular and ultrastructural level in both male and female zebra finches. Regressive development of dendritic spines, enlargement of neuronal cell body and nuclei size, as well as changes at the nucleolar level are events all occurring exclusively in males, when song learning progresses. The decline in synapse number and the augmentation in synaptic contact length at synapses in LMAN in males are indicative for synaptic plasticity, whereas in females synapse number and synaptic contact length remain unchanged.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.     

Synapses on the mauthner cell of the goldfish: Thin section,freeze-fracture,and immunocytochemical studies

Large myelinated club ending and small-vesicle bouton synapses on the distal part of the lateral dendrite of the goldfish Mauthner cell were investigated with thin section, freeze-fracture, and immunocytochemical electron microscopic methods. Large myelinated club endings form mixed synapses, having both gap junctions and chemical synaptic junctions. The correlation of the number of gap junction particles (connexons) and the data from electrophysiological studies of single large myelinated club ending synapses suggest that only a small fraction of gap junction channels are open at any given time during electrical synaptic transmission. The chemical synaptic junctions at the large myelinated club ending synapse have large, round synaptic vesicles, indicating that they are excitatory. This result is in agreement with electrophysiological data demonstrating the excitatory nature of this chemical synapse. Freeze-fracture of these excitatory chemical synaptic junctions reveals the presence of the intramembrane particle aggregates in the postsynaptic E face. Small-vesicle boutons form chemical synaptic junctions with small, flat or oval synaptic vesicles. These structural data, in combination with previous electrophysiological studies, suggest that the small-vesicle bouton synapses are inhibitory. In support of this theory, the cytoplasmic side of the postsynaptic membrane of some of these synapses show positive immunocytochemical reaction to monoclonal antibodies against the rat glycine receptor. Freeze-fracture data reveal intramembrane particle aggregates in the postsynaptic P face of some small-vesicle bouton synapses which could possibly represent glycine receptor aggregates.     

Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis

Growth of new micrometre sized projections called dendritic spines in neurones has been linked to the encoding of long‐term memories in vertebrates. Numerous studies have been carried out at both the light and electron microscopy level to quantify dendritic spine densities in brain tissue in laboratory animals. Currently, such efforts using light microscopy have relied on manual counting of spines in confocal or two‐photon optical slice images of tissue containing fluorescently labelled spines. This manual approach can be slow and tedious, especially for samples with high spine densities. We introduce an alternative way of performing spine counting that uses an applied image intensity threshold followed by spatial image correlation spectroscopy (ICS) analysis. We investigated the effect of particle sizes above the diffraction limit on the autocorrelation analysis as well as the influence of background fluorescence. Our results show that, for well labelled cerebellar tissue samples imaged with a signal‐to‐noise ratio of 5 or greater, ICS‐based spine counts can be conducted with the same 15–20% precision as manual counting, but much more rapidly.     

Application of the Golgi/electron microscopy technique for cell identification in immunocytochemical, retrograde labeling, and developmental studies of hippocampal neurons.

In this study the Golgi/electron microscopy (EM) technique has been used for an analysis of the fine structure, specific synaptic connections, and differentiation of neurons in the hippocampus and fascia dentata of rodents. In a first series of experiments the specific synaptic contacts formed between cholinergic terminals and identified hippocampal neurons were studied. By means of a variant of the section Golgi impregnation procedure, Vibratome sections immunostained for choline acetyltransferase, the acetylcholine-synthesizing enzyme, were Golgi-impregnated in order to identify the target neurons of cholinergic terminals in the hippocampus. It could be shown with this combined approach that cholinergic septohippocampal fibers form a variety of synapses with different target structures of the Golgi-impregnated and gold-toned hippocampal neurons. In this report cholinergic synapses on the heads of small spines, the necks of large complex spines, dendritic shafts, and cell bodies of identified dentate granule cells are described. The variety of cholinergic synapses suggests that cholinergic transmission in the fascia dentata is a complex event. Next, the Golgi/EM technique was applied to Vibratome sections that contained retrogradely labeled neurons in the hilar region of the fascia dentata following horseradish peroxidase (HRP) injection into the contralateral hippocampus. With this combined approach some of the hilar cells projecting to the contralateral side were identified as mossy cells by the presence of retrogradely transported HRP in thin sections through these Golgi-impregnated and gold-toned neurons. Our findings suggest that the mossy cells are part of the commissural/associational system terminating in the inner molecular layer of the fascia dentata. They are mainly driven by hilar collaterals of granule cell axons that form giant synapses on their dendrites. Finally, the Golgi/EM procedure was used to study the differentiation and developmental plasticity of hippocampal and dentate neurons in transplants and slice cultures of hippocampus. Under both experimental conditions, the differentiating neurons are deprived of their normal laminated afferent innervation but develop their major cell-specific characteristics including a large number of postsynaptic structures (spines). As revealed in thin sections of gold-toned identified cells, all these spines formed synapses with presynaptic boutons suggesting sprouting of the transplanted and cultured neurons, respectively. Altogether, the present report demonstrates the usefulness of the Golgi/EM technique, particularly of the section impregnation procedure, for a variety of studies requiring the identification of individual neurons at the ultrastructural level.     

Quantitative analysis of synapse structure with a semiautomatic interactive computer system: A study on identified pyramidal tract neurons in the sensory-motor cortex of cat

Pyramidal tract (Pt) neurons in the sensory-motor cortex of cat were labeled by injection of HRP into the spinal cord. Ultrastructural and quantitative analysis of the synaptic covering of their soma and apical dendrite (up to 100 μm from soma) was undertaken. We intercalated a visual-manual treatment between composite electron micrographs and a fully automated computer system and developed specific programs for evaluation of the morphometric data. Programs are included. A total of 412 synaptic boutons were examined that were found in contact with large Pt neurons. The mean linear percentage of the surface area covered by boutons was 26.2 ± 8.4% and the mean contacting length and cross-sectional area of the bouton profiles were 1.28 ± 0.58 μm and 0.89 ± 0.59 μm², respectively. All types of boutons with active zones accounted for 41.2% of the total. The distribution of two types of bouton (S- and F-type boutons, showing asymmetric and symmetric contacts, respectively) was examined quantitatively. The mean proportion of F-type boutons was 89.1% with a soma and S-type boutons contacting apical dendrites was 10.9%. In addition, GABAergic boutons were identified with the soma by immunocytochemistry with antibodies against glutamic acid decarboxylase. They formed symmetric synaptic contacts with the Pt cells that were identical to those formed by F-type boutons. The quantitative analysis revealed that synaptic clefts are narrower and synaptic vesicles are smaller in symmetric F-type boutons than in S-type boutons forming asymmetric contacts. These data establish that at least three parameters (postsynaptic density, synaptic cleft, and size of vesicles) can be utilized singly or in combination to identify GABAergic inhibitory synapses in neocortex.     

Three-dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Ultrastructural identification of neuropeptides in the central nervous system

A number of different neuropeptides have been described within presynaptic terminals at the ultrastructural level in the central nervous system. The majority of these neuropeptides share a common morphology with one another. Terminals containing neuropeptides have a population of small, clear vesicles associated with the active zone of the synapse and a lesser number of large, granular vesicles that are located at a distance from the active site of the synapse. It is believed that the large, granular vesicles act as a mechanism for the transport/storage of the neuropeptides, while the small, clear vesicles are thought to be acting as structures responsible for the release of the neurotransmitter/neuropeptide into the synaptic cleft. The neuropeptide containing terminals most often have asymmetrical junctions associated with their presynaptic membranes, although symmetrical junctions have been described with peptide containing terminals in a number of areas in the central nervous system. Neuropeptide containing terminals contact every part of the neuronal membrane; however, the majority of synaptic contacts involve portions of the dendritic shafts. Evidence is beginning to accumulate to indicate that for certain neuropeptides there is a specific spatial arrangement to their termination along the neuronal membrane.     

Monoamine synaptic structure and localization in the central nervous system

The monoamines dopamine, noradrenaline, adrenaline, and serotonin as well as the diamine histamine have a widespread distribution in the central nervous system within synaptic terminals and nonsynaptic varicosities. In certain regions of the central nervous system the monoamines are contained in varicosities that have no synaptic specialization associated with them, suggesting a possible neuromodulatory role for some of the monoamines. The majority of monoamine labelled structures are synaptic terminals which are characterized by the presence of small, clear vesicles (40–60 nm) and large, granular vesicles (70–120 nm) within the terminal. A third population of vesicles—small, granular vesicles—which are visible only after histochemical staining, are probably the equivalent of the small, clear vesicles present after either autoradiographic or immunohistochemical labelling. Most monoamine containing terminals contact dendrites and dendritic spines and, less frequently, neuronal somata and other axons. Both asymmetrical and symmetrical membrane specializations are associated with monoaminergic terminals; however, asymmetrical contacts are the most frequent type found. These ultrastructural results indicate that monoamine containing terminals and varicosities in general share many common morphological features, but still have diverse functions.     

Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

Ultrastructural features of the myotendinous junction of the sternomastoid muscle in Wistar rats: From newborn to aging

The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called “finger‐like processes” and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered “finger‐like processes,” thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and “finger‐like processes” due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 75:1292–1296, 2012. © 2012 Wiley Periodicals, Inc.     

Membrane and cytoplasmic structure at synaptic junctions in the mammalian central nervous system

Application of rapid freezing, freeze substitution fixation, and freeze fracture techniques to the study of synaptic junctions in the mammalian central nervous system has revealed new aspects of synaptic structure that are consistent with and partially explicate advances in synaptic biochemistry and physiology. In the axoplasm adjacent to the presynaptic active zone, synaptic vesicles are linked to large spectrin-like filamentous proteins by shorter proteins that resemble synapsin I in morphology. This mesh of presynaptic filamentous proteins serves to concentrate synaptic vesicles in the vicinity of the active zone. The affinity with which the vesicles are bound by the mesh is probably modulated by the extent of phosphorylation at specific sites on the constituent filamentous proteins, and changes in the binding affinity result in changes in transmitter release. The structural organization of the postsynaptic density in Purkinje cell dendritic spines consists of very fine strands with adherent, heterogeneous globular proteins. Some of these globular proteins probably correspond to protein kinases and their substrates. The postsynaptic density, positioned at the site of the maximal depolarization caused by synaptic currents, apparently serves as a supporting framework for a variety of proteins, which respond to and transduce postsynaptic depolarization. At least two classes of filamentous protein fill the cytoplasm of spines with a complex mesh, which presumably contributes to maintenance of the spine shape. Membrane bound cisterns are a ubiquitous feature of Purkinje cell dendritic spines. Studies of rapidly frozen tissue with electron probe microanalysis and elemental imaging reveal that these cisterns take up and sequester calcium, which is derived from the extracellular space, and which probably enters the spine as part of the synaptic current.     

Morphometric,quantitative, and three‐dimensional analysis of the heart muscle fibers of old rats: Transmission electron microscopy and high‐resolution scanning electron microscopy methods

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high‐resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A‐, I‐, and H‐bands and their M‐ and Z‐lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm², a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope

Studies of labeled neurons at the light-microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high-voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel-intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.