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.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Determining the neuronal connectivity of Golgi-impregnated neurons: ultrastructural assessment of functional aspects.

The combined light and electron microscopic analysis of Golgi-impregnated neural tissue is a potent tool for determining the connectivity of neural networks within the brain. In the experimental paradigms commonly applied in these studies, the Golgi-impregnated neurons are typically examined as the postsynaptic neuronal components. The structural characteristics and the pattern of distribution of their synaptic connections with other groups of identified neurons are analyzed. Due to the high power of resolution of the Golgi-electron microscopic technique, the ultrastructural analysis of Golgi-impregnated neurons can be expanded to elucidate activity-dependent structural alterations in their cytoarchitecture. These structural alterations can then be correlated under different physiological conditions with changes in the functional efficacy of the subcellular neuronal components.     

Synaptic connections of neurones identified by Golgi impregnation: Characterization by immunocytochemical,enzyme histochemical,and degeneration methods

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi-impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi-impregnated neurones and their pre-and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre-and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP-filled neurones and Golgi-impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.     

New ways to look at axons in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to the compact arrangement of cell bodies and axons.     

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.     

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.     

Analysis of neuronal networks: A review of techniques for labeling axonal projections

In order to analyze connections between neurons in the vetebrate central nervous system, methods have been developed to label a given population of axons of known origin so that they can be differentiated from other, non-labeled structures. Three such methods are reviewed here: experimentally induced orthograde (Wallerian) degeneration, axon transport of radioactive proteins demonstrated by autoradiography, and axon transport of macromolecules that can be reacted histochemically to yield a visible reaction product. Each of the methods has particular strengths and weaknesses. Degeneration methods may differentiate between different functional classes of axons which have different fiber diameters. However, degeneration distorts the morphology of axon terminals, making them more difficult to interpret, and degenerating terminals may be removed rapidly by phagocytosis. Autoradiography of radioactive terminals preserves normal fine structure, but the necessary exposure times extend the method by weeks or months, and care must be exercised to distinguish labeled axons from other structures exhibiting background or transneuronal radioactivity. Histochemical methods, such as those used to demonstrate horseradish peroxidase conjugated to wheat germ lectin (WGA-HRP), are sensitive and rapid, but the injection site must be carefully characterized, and the presence of transneuronal label may make interpretation of the results difficult. Experimental methods of axonal labeling have been invaluable in studying neuronal networks. Each of the methods described here may be of particular value, given the nature of the system to be analyzed.     

Activity-related, dynamic neuron-glial interactions in the hypothalamo-neurohypophysial system.

Magnocellular neurons located in the supraoptic nucleus send their principal axons to terminate in the neurohypophysis, where they release vasopressin and oxytocin into the blood circulation. This magnocellular hypothalamo-neurohypophysial system is known to undergo dramatic activity-dependent structural plasticity during chronic physiological stimulation, such as dehydration and lactation. This structural plasticity is accompanied not only by synaptic remodeling, increased direct neuronal membrane apposition, and dendritic bundling in the supraoptic nucleus, but also organization of neurovascular contacts in the neurohypophysis. The adjacent glial cells actively participate in these plastic changes in addition to magnocellular neurons themselves. Many molecules that are possibly concerned with dynamic structural remodeling are highly expressed in the hypothalamo-neurohypophysial system, although they are generally at low expression levels in other regions of adult brains. Interestingly, some of them are highly expressed only in embryonic brains. On the basis of function, these molecules are classified mainly into two categories. Cytoskeletal proteins, such as tubulin, microtubule-associated proteins, and intermediate filament proteins, are responsible for changing both glial and neuronal morphology and location. Cell adhesion molecules, belonging to immunoglobulin superfamily proteins and extracellular matrix glycoproteins, also participate in neuronal-glial, neuronal-neuronal, and glial-glial recognition and guidance. Thus, the hypothalamo-neurohypophysial system is an interesting model for elucidating physiological significance and molecular mechanisms of activity-dependent structural plasticity in adult brains.     

Ascending efferent projections of the superior olivary complex

The superior olivary complex conveys information about binaural time and intensity to higher centers in the auditory pathway. This information is sent primarily to the subdivisions of the inferior colliculus and to the nuclei of the lateral lemniscus. Olivary projections are the predominant afferents to the central nucleus of the inferior colliculus. Electron microscopic observations of axonal endings in the central nucleus suggest that the ipsilateral medial superior olive and contralateral lateral superior olive make excitatory synapses. In contrast, the axons from the ipsilateral lateral superior olive to the central nucleus contain glycine and have a morphology consistent with inhibitory synapses. Little is known about the transmitter types used by olivary projections to the nuclei of the lateral lemniscus, but they are presumed to be similar to the collicular projections. Olivary ascending efferents are tonotopically organized and terminate in laminae in the inferior colliculus. They combine with other laminar afferents and postsynaptic neurons to create fibro-dendritic laminae in the colliculus. The key to the functional organization of the olivary efferents is the possible segregation of excitatory olivary efferents from each other in "synaptic domains" located on the laminae. This segregation may be the major determinant of response properties in the colliculus. Olivary efferents may converge with other non-olivary afferents on the same postsynaptic neurons in the colliculus. Inhibitory efferents from the lateral superior olive are essential in shaping the response properties of neurons in the colliculus. Olivary efferents to the nuclei of the lateral lemniscus are also key components of ascending pathways that inhibit neurons in the midbrain.     

Signal processing in a simple visual system: the locust ocellar system and its synapses

The neurons with the widest axons that carry information into a locust brain belong to L-neurons, the large, second-order neurons of the ocelli. L-neurons play roles in flight control and boosting visual sensitivity. Their morphology is simple, and their axons convey graded potentials from the ocellus with little decrement to the brain, which makes them good subjects in which to study transmission of graded potentials. L-neurons are very sensitive to changes in light, due to an abnormally high gain in the sign inverting synapses they receive from photoreceptors. Adaptation ensures that L-neurons signal contrast in a light signal when average light intensity changes, and that their responses depend on the speed of change in light. Neurons L1-3 make excitatory output synapses with third-order neurons and with L4-5. These synapses transmit tonically, but are unable to convey hyperpolarising signals about large increases in light. Graded rebound spikes enhance depolarising responses. L1-3 also make reciprocal inhibitory synapses with each other and transmission at these decrements so rapidly that it normally requires a presynaptic spike. The resolution with which graded potentials can be transferred has been studied at the inhibitory synapses, and is limited by intrinsic variability in the mechanism that determines neurotransmitter release. Electron microscopy has shown that each excitatory connection made from an L-neuron to a postsynaptic partner consists of thousands of discrete synaptic contacts, in which individual dense-staining bars in the presynaptic neuron are associated with clouds of vesicles. Acetylcholine is likely to be a neurotransmitter released by L-neurons.     

Supraspinal connections of the ovary: structural and functional aspects

This review summarizes our recent studies using the viral transneuronal tracing technique to identify sites in the central nervous system (CNS) that are connected with the ovary. A neurotropic virus (pseudorabies virus) was injected into the ovary and various times after the inoculation the spinal cord and brain were examined for virus-infected neurons identified by immunocytochemistry. Such neurons could be detected in well-defined cell groups of the spinal cord (intermediolateral cell column), brain stem (vagal nuclei, area postrema, parapyramidal nucleus, caudal raphe nuclei, A1, A5, A7 noradrenergic cell groups, locus coeruleus, Barrington's nucleus, periaqueductal gray), hypothalamus (paraventricular nucleus, anterior hypothalamus, arcuate nucleus, zona incerta), and, at longer survival time, in some telencephalic structures (amygdala, bed nucleus of the stria terminalis). These findings provided the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the brain and the ovary presumably involved in the neuronal control of the organ. The observations indicate that there is a significant overlap of CNS structures connected with the ovary, the testis, other organs and organ systems, suggesting similar neuronal circuitries of the autonomic nervous system innervating the different organs. The known descending neuronal connections between the CNS structures labeled from the ovary by the viral transneuronal tracing technique and the findings suggesting a pituitary independent interplay between certain cerebral structures such as the hypothalamus, the amygdala, and the ovary are also summarized in this review.     

Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta.

This paper describes four investigations of the olfactory mucosa of the brown trout: 1) the ultrastructure of the olfactory mucosa as revealed by scanning (SEM), conventional transmission (TEM), and high voltage (HVEM) electron microscopy; 2) light and electron-microscopic investigations of retrograde transport of the tracer macromolecule horseradish peroxidase (HRP) when applied to the cut olfactory nerve; 3) SEM and TEM investigations of the effects of olfactory nerve transection on cell populations within the olfactory epithelium; and 4) ultrastructural investigations of reversible degeneration of olfactory receptors caused by elevated copper concentrations. The trout olfactory epithelium contains five cell types: ciliated epithelial cells, ciliated olfactory receptor cells, microvillar olfactory receptor cells, supporting cells, and basal cells. The ciliated and microvillar olfactory receptor cells and a small number of basal cells are backfilled by HRP when the tracer is applied to the cut olfactory nerve. When the olfactory nerve is cut, both ciliated and microvillar olfactory receptor cells degenerate within 2 days and are morphologically intact again within 8 days. When wild trout are taken from their native stream and placed in tanks with elevated copper concentrations, ciliated and microvillar cells degenerate. Replacement of these trout into their stream of origin is followed by morphologic restoration of both types of olfactory receptor cells. Ciliated and microvillar receptor cells are primary sensory bipolar neurons whose dendrites make contact with the environment; their axons travel directly to the brain. Consequently, substances can be transported directly from the environment into the brain via these "naked neurons." Since fish cannot escape from the water in which they swim, and since that water may occasionally contain brain-toxic substances, the ability to close off--and later reopen--this anatomic gateway to the brain would confer a tremendous selective advantage upon animals that evolved the "brain-sparing" capacity to do so. Consequently, the unique regenerative powers of vertebrate olfactory receptor neurons may have their evolutionary origin in fishes.     

Ultrastructural localization of fructose‐1,6‐bisphosphatase in mouse brain

Fructose‐1,6‐bisphosphatase has been studied in adult mouse brain of different ages using an antibody directed against the liver isoform. The presence of liver fructose‐1,6‐bisphosphatase in cerebellum, cerebral cortex, and hippocampus was assayed using Western blot and different immunocytochemical techniques. Immunocytochemistry with peroxidase reaction product was used to locate fructose‐1,6‐bisphosphatase in both neurons and astrocytes in the same areas, as well as in the rest of the brain, at light and electron microscope levels. Double immunofluorescence with neuronal or astrocytic markers confirmed the neuronal and astrocytic location of fructose‐1,6‐bisphosphatase in confocal microscope images. At the subcellular level, fructose‐1,6‐bisphosphatase was located in the nuclear and cytoplasmic compartments of both neurons and astrocytes, at all ages studied. Ultrastructurally, immunostaining appeared as small patches in the nucleus and the cytosol. In addition, immunostaining was present over the outer mitochondrial membrane, the plasma membrane, and the membranes of the rough endoplasmic reticulum and nuclear envelope, but not over Golgi membranes. In the neuropil fructose‐1,6‐bisphosphatase was located in dendritic spines, as well as in abundant astrocytic processes that, in some cases, surrounded immunopositive synapses. The possible role of fructose‐1,6‐bisphosphatase in neurons and astrocytes is discussed. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Synaptic structure, distribution, and circuitry in the central nervous system of the locust and related insects.

The Orthopteran central nervous system has proved a fertile substrate for combined morphological and physiological studies of identified neurons. Electron microscopy reveals two major types of synaptic contacts between nerve fibres: chemical synapses (which predominate) and electrotonic (gap) junctions. The chemical synapses are characterized by a structural asymmetry between the pre- and postsynaptic electron dense paramembranous structures. The postsynaptic paramembranous density defines the extent of a synaptic contact that varies according to synaptic type and location in single identified neurons. Synaptic bars are the most prominent presynaptic element at both monadic and dyadic (divergent) synapses. These are associated with small electron lucent synaptic vesicles in neurons that are cholinergic or glutamatergic (round vesicles) or GABAergic (pleomorphic vesicles). Dense core vesicles of different sizes are indicative of the presence of peptide or amine transmitters. Synapses are mostly found on small-diameter neuropilar branches and the number of synaptic contacts constituting a single physiological synapse ranges from a few tens to several thousand depending on the neurones involved. Some principles of synaptic circuitry can be deduced from the analysis of highly ordered brain neuropiles. With the light microscope, synaptic location can be inferred from the distribution of the presynaptic protein synapsin I. In the ventral nerve cord, identified neurons that are components of circuits subserving known behaviours, have been studied using electrophysiology in combination with light and electron microscopy and immunocytochemistry of neuroactive compounds. This has allowed the synaptic distribution of the major classes of neurone in the ventral nerve cord to be analysed within a functional context.     

Strategies for studying microtubule transport in the neuron

Microtubules are prominent cytoskeletal elements within the neuron. They are essential for the differentiation, growth, and maintenance of axons and dendrites. The microtubules within each type of process have a distinct pattern of organization, and these distinct patterns result in many of the morphological and structural features that distinguish axons and dendrites from one another. There are a number of challenges that must be met in order for the neuron to establish the microtubule arrays of axons and dendrites. One attractive model invokes the active transport of microtubules from the cell body of the neuron into and down these processes. In support of this model, specific motor proteins have now been identified within neurons that have the necessary properties to transport microtubules into developing axons and dendrites with the appropriate orientation for each type of process. An important goal is to develop microscopic methods that permit the visualization of microtubule transport within different regions of the neuron. To date, achieving this goal has met with mixed success, probably as a result of the geometry of the neuron and the inherent complexity of the neuronal microtubule arrays. While some approaches have failed to reveal microtubule transport, other more recent approaches have proven successful. These approaches provide strong visual support for a model based on microtubule transport, and provide hope that future approaches can provide even clearer demonstrations of this transport.     

Review: Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.     

Distribution of NADPH-diaphorase in rat mesencephalon: A light and electron microscopical study

NADPH-diaphorase is a useful technique to reveal NO producing neurons at light microscopic level (LM). A modification of the technique using the tetrazolium salt BSPT as susbtrate, is useful to study the ultrastructure of NO neurons. The aim of this work was to perform a detailed analysis of NADPH diaphorase reactive neurons in rat mesencephalon both at light and electron microscopic levels.
NADPH-diaphorase reactive neurons were observed in superior colliculus, in central gray matter, in dorsal and medial raphe and in the pedunculopontine tegmental nucleus using two histochemical techniques at LM. Electron microscopy showed deposits on membranes of the endoplasmic reticulum, Golgi apparatus and nuclear envelope of dorsal raphe neurons. Presynaptic and postsynaptic terminals showed deposits on membranous elements but postsynaptic terminals also showed deposits on the inner surface of their membranes.
Further physiological studies are needed to clarify the meaning of the ultrastructural findings such as the putative interaction of NOS with postsynaptic proteins, receptors or membranous channels.     

Image analysis of Nissl-stained neuronal perikarya in the primary visual cortex of the rat: Automatic detection and segmentation of neuronal profiles with nuclei and nucleoli*

An image analysing procedure for the morphometric characterization of cortical neurons in Nissl-stained brain sections is described. It consists of the automatic detection of cellular profiles and their compartments: cytoplasm, nucleus and nucleolus. The algorithm was designed to cope with the large morphological spectrum of cortical perikarya (e.g. geometrical properties of perikarya, staining intensities of cell compartments and nucleo-plasmic area-ratio) including pyramidal (Golgi-category I) and non-pyramidal (Golgi-category II) neurons. Clusters of cells were separated and non-neuronal structures (e.g. glia, endothelial cells) as well as tangential, non-nucleolated sections through neuronal perikarya recognized and excluded from further analysis without requiring interactive procedures. The performance of the profile recognition procedure was evaluated using 426 nucleolated and non-nucleolated profiles of different types of neurons in the primary visual cortex of the rat. Nucleolated profiles were recognized as such with a 91% accuracy, non-nucleolated profiles were rejected correctly in 90% of cases. After automatic segmentation and selection of nucleolated neuronal profiles from the microscopic field, a large set of quantitative morphological features including geometrical, densitometrical and textural parameters can be measured using high power light microscopy. This permits quantitative morphometric characterization of different neuronal types. This procedure is the first part of a system for the automatic classification of Nissl-stained cortical neurons.     

Effect of age on the structure of Meissner corpuscles in murine digital pads

A light and electron microscopic study was performed to determine age changes in Meissner corpuscles. In forepaw digital pads of mice aged to their maximum life expectancy, corpuscles were found to increase in size and complexity until middle age, and then to become smaller, disorganized and lobulated with more advanced age. Nerve terminals at more advanced ages became attenuated with a loss of axonal processes, increased density of the axoplasm, and disordered arrangement of the organelles. Degeneration of axonal mitochondria accelerated with age. Lamellar cell processes investing the axons often become dense and attenuated with fewer plasmalemma-associated vesicles. Basal laminae remained where lamellar processes had disintegrated. Lipofuscin was seen in the lamellar cells only at extremely old age. Extracellular material composed of fine basal lamina substance and collagen fibrils increased remarkably with age. Increased growth and complexity of corpuscles until middle age perhaps compensated for age-associated loss of corpuscles and primary sensory neurons. Changes predominating at older ages are attributed to distal axonopathy and atrophy of the sensory neurons. The probable effect of these age changes on cutaneous sensitivity is considered in relation to current theory of mechanoelectric transduction. © 1996 Wiley-Liss, Inc.