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.     

Fine structure of the pinealopetal innervation of the mammalian pineal gland.

The mammalian pineal gland is innervated by peripheral sympathetic and parasympathetic nerve fibers as well as by nerve fibers originating in the central nervous system (central innervation). The perikarya of the sympathetic fibers are located in the superior cervical ganglia, while the fibers terminate in boutons containing small granular vesicles and a few large granular vesicles. Both noradrenaline and neuropeptide Y are contained in these neurons. The parasympathetic fibers originate from perikarya in the pterygopalatine ganglia. The neuropeptides, vasoactive intestinal peptide and peptide histidine isoleucine, are present in these fibers, the boutons of which contain small clear transmitter vesicles and larger granular vesicles. The fibers of the central innervation originate predominantly from perikarya located in hypothalamic and limbic forebrain structures as well as from perikarya in the optic system. These fibers terminate in boutons containing small clear and, in certain fibers, an abundant number of large granular vesicles. In rodents, the majority of the central fibers terminate in the deep pineal gland and the pineal stalk. From these areas impulses might be transmitted further caudally to the superficial pineal gland via neuronal structures or processes from pinealocytes. Several hypothalamic neuropeptides and monoamines might be contained in the central fibers. The intrapineal nerve fibers are located both in the perivascular spaces and intraparenchymally. The majority of the intraparenchymally located fibers terminate freely between the pinealocytes. However, some nerve terminals make synaptic contacts with the pinealocytes and in some species with intrapineal neurons. In fetal mammals, sympathetic, parasympathetic, and central fibers are also present. In addition, an unpaired nerve, connecting the caudal part of the pineal gland with the extreme rostral part of the mesencephalon, is present. This nerve is a homologue to the pineal nerve (nervus pinealis) observed in lower vertebrates.     

The ultrastructure of neuronal-pinealocytic interconnections in the monkey pineal.

Recent ultrastructural studies of neuronal-pinealocytic interconnections in the monkey pineal are reviewed. The pinealocytes in the adult monkey show almost all of the cytological specializations known in subprimate mammals. Adjacent pinealocytes are functionally coupled through ribbon synapses on cell bodies and gap junctions on cell bodies and cell processes. The pinealocytes receive direct synpatic contacts of nerve fibers with cholinergic terminal morphology. Nerve cells restricted to the central portion of the pineal receive synaptic contacts with more than three different morphologically defined types of nerve terminals. In addition to nerve terminals containing small clear vesicles or vesicles of pleomorphic morphology, a pinealocyte's terminal process containing the synaptic ribbon forms a true synaptic contact on the nerve cell body. The diversity of synapses on these nerve cells strongly suggests multiple origins of these neurons rather than a single peripheral parasympathetic origin. The possible involvement of pineal neurons in an intrinsic circuit that regulates the function of pinealocytes and integrates the neural input from the central as well as the peripheral nervous systems is discussed.     

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.     

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.     

Ultrastructure of Paneth cells in the intestine of various mammals

Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron-dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in peri- or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100–200 nm diameter) were more-or-less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense-cored vesicles (150–200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.     

Technique to study three-dimensional spatial arrangement of synaptic vesicles using data from single sections

Synaptic vesicles are membrane-bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three-dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model-derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus.     

3D analysis of synaptic vesicle density and distribution after acute foot‐shock stress by using serial section transmission electron microscopy

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter‐containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot‐shock stress and (2) rats with sham stress as control group. Two‐dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in‐section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three‐dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one‐dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot‐shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.     

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.     

Editorial

Soft X-ray (2.05–4.4nm) contact microscopy, using synchrotron radiation, of ultrasections of lead-polluted chloragogenous tissue of the earthworm Dendrobaena rubida has been achieved. The positive resist, after etching to leave areas corresponding to high absorbance in the tissue, is viewed by SEM and the imaging compared with the same section viewed, after irradiation, by TEM. No radiation damage as evident and the lead deposits associated with the chloragosomes and within the debris vesicles imaged clearly. The characteristics of the broad beam have thus produced a lead ‘map’. Resolution is in the order of 70 nm. The results are compared with the markedly different imaging of control tissue containing insignificant lead levels.     

Presynaptic modulation of sensory neurons in the segmental ganglia of arthropods

The afferent terminals of arthropod sensory neurones receive abundant input synapses, usually closely intermingled with the sites of synaptic output. The majority of the input synapses use the neurotransmitter GABA, but in some afferents there is a significant glutamatergic or histaminergic component. GABA and histamine shunt afferent action potentials by increasing chloride conductance. Though glutamate can also have this effect in the arthropod central nervous system, its action on afferent terminals appears to be mediated by increases in potassium conductance or by the action of metabotropic receptors. The action of the presynaptic synapses on the afferents are many and varied. Even on the same afferent, they may have several distinct roles that can involve both tonic and phasic patterns of primary afferent depolarisation. Despite the ubiquity and importance of their effects however, the populations of neurones from which the presynaptic synapses are made, remain largely unidentified.     

GABAergic synapses in the brain identified with antisera to GABA and its synthesizing enzyme,glutamate decarboxylase

GABA is a known inhibitory neurotransmitter in the mammalian brain. The site of GABAergic synapses can be determined with immunocytochemical methods that localize either GABA or its synthesizing enzyme, glutamate decarboxylase (GAD). In general, GABAergic axon terminals contain pleomorphic synaptic vesicles and form symmetric synapses. However, a small number of GABAergic axon terminals in selected brain regions (spinal cord, cerebellum, superior colliculus, striatum, globus pallidus, inferior olive, and substantia nigra) form asymmetric synapses. GAD- and GABA-immunoreactive processes that contain synaptic vesicles participate in every known morphological type of chemical synapse. These include axosomatic, axodendritic, axospinous, initial segment, axoaxonic, dendrodendritic, serial, reciprocal, and ribbon synapses. Although GABAergic synapses form a heterogeneous group, they most commonly form axosomatic, axodendritic, and initial segment synapses in the brain and spinal cord. These findings provide helpful guidelines for the identification of GABAergic synapses in future studies.     

Erythrophagosomal haemolytic degradative pathway in rat brown adipocytes induced by hyperinsulinaemia: an ultrastructural study

The phagocytosis and degradation of erythrocytes were studied in brown adipose tissue of experimentally hyperinsulinaemic rats. We found that insulin induces intensive erythrophagocytosis by brown adipocytes and their degradation by haemolytic pathway. Ultrastuctural study revealed that haemolytic degradation of erythrophagosomes was characterized by progressive and uniform decrease of erythrocyte matrix density. At the beginning of the degradative process small, clear vesicles resembling primary lysosomes were visible inside the erythrophagosome. With time, the erythrocyte structure totally disappeared and transformed into a fine, granular material within the erythrophagosomal vacuole. Finally, the erythrocyte membrane detached from the phagosomal and clumped into the vacuolar space forming one or several small myelin‐like figures. In conclusion, brown adipocytes are capable of performing intensive erythrophagocytic activity when brown adipose tissue is stimulated and blood flow is enhanced. The molecular basis for favouring a haemolytic instead of more common granular erythrophagosomal degradative pathway remains unknown.     

Electron microscopic study of the morphology and morphogenesis of virus of hemorrhagic fever with renal syndrome

The morphology and morphogenesis of the virus of hemorrhagic fever with renal syndrome (HFRS) and the associated ultrastructural changes in neurons of the infected mouse brain were examined by electron microscopy. The primary location of the infection in large neurons was in the Golgi apparatus, which had highly proliferated laminar and vesicular profiles. A small number of matured virus particles were found later individually or in small groups within the distended Golgi cisternae and vesicles. Most of the virus particles were round, oval, or elongated and measured about 70–110 nm in diameter. A lipid bilayered viral envelope with an external fringe of surface projections could be resolved at high magnification. The maturation (budding) of the virus occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Viral inclusion bodies containing fine filamentous material were seen frequently in close proximity to sites of virus maturation. The known morphological and morphogenetic characteristics of the virus particles observed in infected mouse brain gave further evidence for taxonomic identification of HFRS virus as a member of the family of Bunyaviridae.     

Three-dimensional wall structure and the innervation of dental pulp blood vessels.

The involvement of neural components in plasma extravasation and blood flow in the dental pulp has been established by pharmacological and physiological studies. We review here the segmental constitution of pulp vessels and the possible involvement of neural components in both the contractility and permeability of the pulp vessels from a morphological viewpoint. Six vascular segments can be identified based on the morphology of peri-endothelial cells, such as smooth muscle cells and pericytes. These are: muscular arterioles, terminal arterioles, precapillary arterioles, capillaries, postcapillary venules, and collecting or muscular venules. The perivascular nerve forms a mesh with numerous terminal varicosities, some of which attach directly to arteriolar smooth muscle cells. This mesh can be seen by scanning electron microscopy, and indicates the important role of neural components in regulating the pulpal circulation. After administering norepinephrine (0.2 mg/kg/dog), the surface texture of the smooth muscle cells of pulp arterioles reveals marked irregularities, which are correlated with arteriolar contraction. The pericytes in larger postcapillary venules (diameter 20 microm or larger) also show irregularities, whereas no changes are seen in the pericytes of either smaller postcapillary venules or capillaries. The intercellular spaces of pericytes in the postcapillary venules are wide enough for leukocytes to pass through, and the occasional extravasation of leukocytes through venule walls can be seen under electron microscopy. The microvessels of healthy human dental pulp react weakly to selectins, indicating that apparently healthy dental pulp may be weakly inflamed. In rat dental pulp, CGRP-immunoreactive nerves and nerve terminals containing many granular vesicles supply the postcapillary venules more densely than the arterioles, which suggests the involvement of postcapillary venules in neurogenic inflammation in the dental pulp.     

Synaptic morphology of inner and outer hair cells of the human organ of Corti

Innervations of inner and outer hair cells of the organ of Corti of the human cochlea were studied by serial section electron microscopy. At the base of inner hair cells, presumed afferent fibers were of varying size and demonstrated synaptic specialization consisting of a presynaptic body, vesicles, and asymmetrical synaptic membrane specialization. Two types of neurons, vesiculated presumably efferent and nonvesiculated presumably afferent, synapsed at the base of outer hair cells. The synaptic specialization of afferent fibers included presynaptic body, vesicles, and asymmetrical membrane thickening, whereas efferent synapses demonstrated presynaptic vesicles and a subsynaptic cisterna. Some presumably afferent nerve terminals formed a reciprocal synapse with outer hair cells in both the human and the chimpanzee. Such a synaptic relationship demonstrated morphologic specialization consistent with both hair cell-to-neuron and neuron-to-hair cell transmission between the same outer hair cell and nerve terminal. The innervation density of inner and outer hair cells and the comparative anatomy of the afferent and efferent innervation are discussed.     

Characterization of chemically defined neurons and their cellular relationships by combined immunocytochemistry and radioautographic localization of transmitter uptake sites

Radioautography and immunocytochemistry may be combined at the light and electron microscopic levels for simultaneously localizing uptake sites for exogenous transmitter molecules [such as (³H)monoamines or (³H)amino acids] and endogenous transmitter-related antigens (classical transmitters and their synthesizing enzymes as well as neuropeptides) in the central nervous system. Silver grain accumulations indicative of transmitter uptake sites are readily distinguishable from immunocytochemical labels of the peroxidase-antiperoxidase (PAP), avitin-biotin, or colloidal gold methods. The combination of uptake radioautography and immunocytochemistry may be applied to the investigation of (1) the chemical identity of (³H) transmitter-accumulating elements, (2) the coexistence of different neurotransmitters within the same neurons, and (3) the cellular basis of interactions between certain neurotransmitters, in particular monoamines, GABA, and neuropeptides. This article describes and evaluates the method and reviews the available experimental data derived from its application.     

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.     

Immunoelectron microscopic localization of neurotransmitters in the cochlea

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to γ-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.