Five-colour in vivo imaging of neurons in Caenorhabditis elegans

In the last few years variants of the ‘green fluorescent protein’ (GFP) with different spectral properties have been generated. This has greatly increased the number of possible applications for these fluorochromes in cell biology. The significant overlap of the excitation and emission spectra of the different GFP variants imposes constraints on the number of variants that can be used simultaneously in a single sample. In particular, the two brightest variants, GFP and YFP, are difficult to separate spectrally. This study shows that GFP and YFP can be readily separated with little spectral overlap (cross‐talk) with the use of a confocal microscope equipped with an acusto‐optical beam splitter and freely adjustable emission windows. Under optimal recording conditions cross‐talk is less than 10%. Together with two other fluorescent proteins and the lipophilic dye DiD a total of five different colours can now be used simultaneously to label in vivo distinct anatomical structures such as neurons and their processes. Spatial resolution of the confocal microscope is sufficient to resolve the relative position of labelled axons within a single axon bundle. The use of five distinct marker dyes allows the in vivo analysis of the Caenorhabditis elegans nervous system at unprecedented resolution and richness in detail at the light microscopic 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.     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

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.     

Synaptic circuitry identified by intracellular labeling with horseradish peroxidase

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase as the dye which is injected since it diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.     

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.     

Analysis of nerve fibers and their distribution in histologic sections of the human brain

The field of quantitative analysis and subsequent mapping of the cerebral cortex has developed rapidly. New powerful tools have been applied to investigate large regions of complex folded gyrencephalic cortices in order to detect structural transition regions that might partition different cortical fields of disjunct neuronal functions. We have developed a new mapping approach based on axoarchitectonics, a method of cortical visualization that previously has been used only indirectly with regard to myeloarchitectonics. Myeloarchitectonic visualization has the disadvantage of producing strong agglomerative effects of closely neighbored nerve fibers. Therefore, single and neurofunctional-relevant parameters such as axonal branchings, axon areas, and axon numbers have not been determinable with satisfying precision. As a result, different staining techniques had to be explored in order to achieve a suitable histologic staining for axon visualization. The best results were obtained after modifying the Naoumenko-Feigin staining for axons. From these contrast-rich stained histologic sections, videomicroscopic digital image tiles were generated and analyzed using a new fiber analysis framework. Finally, the analysis of histologic images provided topologic ordered parameters of axons that were transferred into parameter maps. The axon parameter maps were analyzed further via a recently developed traverse generating algorithm that calculated test lines oriented perpendicular to the cortical surface and white matter border. The gray value coded parameters of the parameter maps were then transferred into profile arrays. These profile arrays were statistically analyzed by a reliable excess mass approach we recently developed. We found that specific axonal parameters are preferentially distributed throughout granular and agranular types of cortex. Furthermore, our new procedure detected transition regions originally defined by changes of cytoarchitectonic layering. Statistically significant inhomogeneities of the distribution of certain axon quantities were shown to indicate a subparcellation of areas 4 and 6. The quantification techniques established here for the analysis of spatial axon distributions within larger regions of the cerebral cortex are suitable to detect inhomogeneities of laminar axon patterns. Hence, these techniques can be recommended for systematic and observer-supported cortical area mapping and parcellation studies.     

Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean

Despite several reports on the morphology and functions associated with the morphometry of the vertebrate axoplasm cytoskeleton, the subject has not been thoroughly explored in invertebrates. In vertebrates, among many other functions, microtubules (MTs) serve as scaffolding for axon assembly, and neurofilaments (NFs) as the elements that determine the axon caliber. Intermediate filaments have never been described by electron microscopy in arthropods, although NF proteins have been revealed in the MT side-arms of the axoplasm of certain species, such as the crab Ucides cordatus. Thus, it is not known which elements of the cytoskeleton of invertebrates are responsible for determination of the axon caliber. We studied, by electron microscopy and morphometric analyses, the MT and axon area variability in differently sized axons of the protocerebral tract of the crab Ucides cordatus. Our results revealed differences in the distance between MTs, in MT density and number, and in the areas of differently sized axons. The number of MTs increases with the axon area, but this relationship is not directly proportional. Therefore, MT density is greater in smaller axons than in medium axons, similar to the morphometry of the vertebrate axon MT. The distance between MTs is, however, directly related to the axonal area. On the basis of the results shown here, and on previous reports by us and others, we suggest that MTs may be involved in the determination of the axon caliber, possibly due to the presence of NF proteins found in the side-arms.     

Presynaptic inhibition of olfactory receptor neurons in crustaceans

Presynaptic inhibition of transmitter release from primary sensory afferents is a common strategy for regulating sensory input to the arthropod central nervous system. In the olfactory system, presynaptic inhibition of olfactory receptor neurons has been long suspected, but until recently could not be demonstrated directly because of the difficulty in recording from the afferent nerve terminals. A preparation using the isolated but intact brain of the spiny lobster in combination with voltage-sensitive dye staining has allowed stimulus-evoked responses of olfactory receptor axons to be recorded selectively with optical imaging methods. This approach has provided the first direct physiological evidence for presynaptic inhibition of olfactory receptor neurons. As in other arthropod sensory systems, the cellular mechanism underlying presynaptic afferent inhibition appears to be a reduction of action potential amplitude in the axon terminal. In the spiny lobster, two inhibitory transmitters, GABA and histamine, can independently mediate presynaptic inhibition. GABA- and histaminergic interneurons in the lobster olfactory lobe (the target of olfactory receptor neurons) constitute dual, functionally distinct inhibitory pathways that are likely to play different roles in regulating primary olfactory input to the CNS. Presynaptic inhibition in the vertebrate olfactory system is also mediated by dual inhibitory pathways, but via a different cellular mechanism. Thus, it is possible that presynaptic inhibition of primary olfactory afferents evolved independently in vertebrates and invertebrates to fill a common, fundamental role in processing olfactory information.     

Receptor autoradiography and 2-deoxyglucose techniques in neuroscience

The use of the receptor autoradiography and 2-deoxyglucose (2-DG) techniques in neuroscience are reviewed. Receptors and other binding sites can be visualized autoradiographically in microtome tissue sections after labelling with radioligand in vivo or in vitro. Autoradiograms are generated by apposition of the labelled tissue to photographic emulsions. Combined with computerized image analysis, this technique can be used to analyse and quantify the microscopic distribution of receptors and receptor alterations associated with lesions or disease in human and animal tissues. The 2-DG technique permits microscopic analysis of modifications in brain glucose utilization induced by physiological and pharmacological manipulations. Limitations of these techniques and attempts to optimize their resolution are also discussed.     

Immunocytochemistry and calcium cytochemistry of the mammalian pineal organ: a comparison with retina and submammalian pineal organs.

Morphologically the mammalian pineal organ is a part of the diencephalon. It represents a neural tissue histologically ("pineal nervous tissue") and is dissimilar to endocrine glands. Submammalian pinealocytes resemble the photoreceptor cells of the retina, and some of their cytologic characteristics are preserved in the mammalian pinealocytes together with compounds demonstrable by cyto- and immunocytochemistry and participating in photochemical transduction. In our opinion, the main trend of today's literature on pineal functions--only considering the organ as a common endocrine gland--deviates from this structural and histochemical basis. In mammals, similar to the lower vertebrates, the pinealocytes have a sensory cilium developed to a different extent. The axonic processes of pinealocytes form ribbon-containing synapses on secondary pineal neurons, and/or neurohormonal terminals on the basal lamina of the surface of the pineal nervous tissue facing the perivascular spaces. Ribbon-containing axo-dendritic synapses were found in the rat, cat, guinea pig, ferret, and hedgehog. In the cat, we found GABA-immunoreactive interneurons, while the secondary nerve cells, whose axons enter the habenular commissure, were GABA-immunonegative. GABA-immunogold-labeled axons run between pinealocytes and form axo-dendritic synapses on intrapineal neurons. There is a similarity between the light and electron microscopic localization of Ca ions in the mammalian and submammalian pineal organs and retina of various vertebrates. Calcium pyroantimonate deposits--showing the presence of Ca ions--were found in the outer segments of the pineal and retinal photoreceptors of the frog. In the rat and human pineal organ, calcium accumulated on the plasmalemma of pinealocytes and intercellularly among pinealocytes. The formation of pineal concrements in mammals may be connected to the high need for Ca exchange of the pinealocytes for their supposed receptor and effector functions.     

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.     

A brief update on lung stereology

Lung stereology has a long and successful tradition. From mice to men, the application of new stereological methods at several levels (alveoli, parenchymal cells, organelles, proteins) has led to new insights into normal lung architecture, parenchymal remodelling in emphysema‐like pathology, alveolar type II cell hyperplasia and hypertrophy and intracellular surfactant alterations as well as distribution of surfactant proteins. The Euler number of the network of alveolar openings, estimated using physical disectors at the light microscopic level, is an unbiased and direct estimate of alveolar number. Surfactant‐producing alveolar type II cells can be counted and sampled for local size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storage organelles, lamellar bodies, can be estimated using physical disectors at the EM level. By immunoelectron microscopy, surfactant protein distribution can be analysed with the relative labelling index. Together with the well‐established classical stereological methods, these design‐based methods now allow for a complete quantitative phenotype analysis in lung development and disease, including the structural characterization of gene‐manipulated mice, at the light and electron microscopic level.     

Use of lipophilic dyes in studies of axonal pathfinding in vivo

Fluorescent lipophilic dyes are an ideal tool to study axonal pathfinding. Because these dyes do not require active axonal transport for their spreading, they can be used in fixed tissue. Here, we describe the method we have used to study the molecular mechanisms of commissural axon pathfinding in the embryonic chicken spinal cord in vivo. Based on in vitro studies, different families of molecules had been suggested to play a role in the guidance of developing axons. In order to test their function in vivo, we used the commissural neurons that are located at the dorsolateral border of the chicken spinal cord as a model system [Stoeckli and Landmesser (1995) Neuron 14:1165-1179]. Axonin-1, NgCAM, and NrCAM, three members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs), were shown to be important for the correct growth pattern of commissural axons. We studied the effect of perturbations of specific CAM/CAM interactions by injection of function-blocking antibodies into the central canal of the spinal cord in ovo. After 2 days, the embryos were sacrificed and fluorescent tracers, such as Fast-DiI, were used to visualize commissural axons, and thus, to analyze their response to these perturbations in two different types of fixed preparations: transverse vibratome sections and whole-mount preparations of the spinal cord. Both pathfinding errors and defasciculation of axons were observed as a result of the perturbation of CAM/CAM interactions.     

Presence and distribution of FMRFamide-like immunoreactivity in the cyprid of the barnacle Balanus amphitrite (Cirripedia, Crustacea)

The presence and distribution of FMRFamide-like peptides (FLPs) in the cyprid larvae of the barnacle Balanus amphitrite were investigated using immunohistochemical methods. Barnacles are considered to be one of the most important constituents of animal fouling communities, and the cyprid stage is specialized for settlement and metamorphosis in to the sessile adult condition. FLPs immunoreactive (IR) neuronal cell bodies were detected in both the central and the peripheral nervous system. One bilateral group of neurons somata was immunodetected in the brain, and IR nerve fibers were observed in the neuropil area and optic lobes. Intense immunostaining was also observed in the frontal filament complex: frontal filament tracts leaving the optic lobes and projecting towards the compound eyes, swollen nerve endings in the frontal filament vesicles, and thin nerve endings in the external frontal filament. Thin IR nerve fibers were also present in the cement glands. Two pairs of neuronal cell bodies were immunodetected in the posterior ganglion; some of their axons appear to project to the cirri. FLPs IR neuronal cell bodies were also localized in the wall of the dilated midgut and in the narrow hindgut; their processes surround the gut wall and allow gut neurons to synapse with one another. Our data demonstrated the presence of FLPs IR substances in the barnacle cyprid. We hypothesize that these peptides act as integrators in the central nervous system, perform neuromuscular functions for thoracic limbs, trigger intestinal movements and, at the level of the frontal filament, play a neurosecretory role.     

Cellular distributions and functions of histamine, octopamine, and serotonin in the peripheral visual system, brain, and circumesophageal ring of the horseshoe crab Limulus polyphemus

The data reviewed here show that histamine, octopamine, and serotonin are abundant in the visual system of the horseshoe crab Limulus polyphemus. Anatomical and biochemical evidence, including new biochemical data presented here, indicates that histamine is a neurotransmitter in primary retinal afferents, and that it may be involved in visual information processing within the lateral eye. The presence of histamine in neurons of the central nervous system outside of the visual centers suggests that this amine also has functions unrelated to vision. However, the physiological actions of histamine in the Limulus nervous system are not yet known. Octopamine is present in and released from the axons of neurons that transmit circadian information from the brain to the eyes, and octopamine mimics the actions of circadian input on many retinal functions. In addition, octopamine probably has major functions in other parts of the nervous system as octopamine immunoreactive processes are widely distributed in the central nervous system and in peripheral motor nerves. Indeed, octopamine modulates functions of the heart and exoskeletal muscles as well as the eyes. A surprising finding is that although octopamine is a circulating neurohormone in Limulus, there is no structural evidence for its release into the hemolymph from central sites. The distribution of serotonin in Limulus brain suggests this amine modulates the central processing of visual information. Serotonin modulates cholinergic synapses in the central nervous system, but nothing further is known about its physiological actions.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

Quantification of axonally transported material using cytofluorimetric scanning

This paper describes in detail a cytofluorimetric scanning technique used for studying amounts of material axonally transported in antero- and retrograde direction in peripheral nerves. Operating procedures, preparation of tissues and instrumental set-up are described. The basis for quantification of material in a nerve section treated for immunofluorescence is discussed. The reliability of the method has been tested by comparing results with biochemical data. There are several advantages of the technique. (1) Many different substances can be studied in one single nerve segment, thus reducing biological variation and costs. (2) Both morphological data and quantitative figures can be obtained; following scanning the section can be photographed. (3) The method can also be used on studies in the central nervous system and on tissue cultures, since it is possible to scan on single axons or bundles of fibres.     

Basic techniques for long distance axon tracing in the spinal cord

The regeneration of axons after a spinal cord injury or disease is attracting a significant amount of interest among researchers. Being able to assess these axons in terms of morphology, length and origin is essential to our understanding of the regeneration process. Recently, two specific axon tracers have gained much recognition; biotinylated dextran amine (BDA) 10 kDa as an anterograde tracer and cholera toxin‐B as a retrograde tracer. However, there are still several complexities when using these tracers, including the volume that should be administered and the best administration site so that a significant amount of axons are labeled in the area of interest. In this article, we describe some simple procedures for injecting the tracers and detecting them. We also quantified the number of axons at different locations of the spinal cord. Our results show axons labeled from motor cortex injections traveled down to the lumbosacral spinal cord in 2 weeks, while BDA injections into the lateral vestibular nucleus and reticular formation took 3 weeks to label axons in the lumbosacral spinal cord. Moreover, this protocol outlines some basic procedures that could be used in any laboratory and gives insight into the number of axons labeled and how procedures could be tailored to meet specific researcher's needs. Microsc. Res. Tech. 76:1240–1249, 2013. © 2013 Wiley Periodicals, Inc.