Single-cell Labeling Research Articles

Robo and Frazzled/DCC mediate dendritic guidance at the CNS midline.

Neuronal connectivity is established by the axo-dendritic polarity, correct guidance and targeting of neurons. Unlike for axons, the mechanisms responsible for directed outgrowth of dendrites are not well understood. Using single-cell labeling, we describe specific guidance defects in dendrites of identified neurons in frazzled, robo, netrin and commissureless mutant embryos of Drosophila melanogaster. We found that the cell-surface molecules Frazzled and Robo work as guidance molecules not only for axons but also for dendrites as they navigate within the CNS. Furthermore, we report that each neuron showed a cell-autonomous and independent use of guidance molecules.

Axonal collateralization in primate basal ganglia and related thalamic nuclei

This paper provides an overview of the major organizational features of the basal ganglia and related thalamic centers, as delineated by the application of single-axon or single-cell labeling procedures in primates. These studies have revealed that the striatum, the external pallidum and the subthalamic nucleus harbor several types of projection neurons endowed with a highly collateralized axon that allows these neurons to interact with most components of the basal ganglia. In contrast, the internal pallidum, which is a major output structure of the basal ganglia, contains only two types of projection neurons. First, there is a minority of “limbic” pallidal neurons with a poorly branched axon that arborized profusely within the lateral habenula, which stands out as the most densely innervated pallidal target. Second, there is a majority of pallidal “motor” neurons with a long (total axonal length up to 27 cm) and highly branched axon that provides collaterals to the ventral tiers thalamic nuclei, the brainstem pedunculopontine nucleus and the centre médian/parafascicular thalamic complex. This type of axon allows internal pallidal neurons to send efferent copies of the same information to the thalamus and brainstem and hence influence various neuronal systems scattered throughout the neuraxis. Pallidal information is conveyed to the cerebral cortex and the striatum via the thalamus, while it is projected back to different components of the basal ganglia via the numerous reentrant pathways that arise from the pedunculopontine nucleus. Virtually all neurons in the centre médian thalamic nucleus innervate massively the striatum and less prominently the primary motor cortex, which in turn projects to the striatum directly or via a collateral from long-range corticofugal pyramidal axons. The results call for a reappraisal of our current concept of the anatomical and functional organization of basal ganglia, which play a crucial role in sensorimotor integration. Our data indicate that basal ganglia and related thalamic nuclei form a widely distributed neuronal network, whose elements are endowed with a highly patterned set of axon collaterals. This morphological feature allows a complex and exquisitely precise interaction between the various basal ganglia and related thalamic nuclei. The elucidation of this finely tuned network is needed to understand the complex spatiotemporal sequence of neural events that ensures the flow of cortical information through the basal ganglia and thalamus.

Axonal collateralization in primate basal ganglia and related thalamic nuclei

This paper provides an overview of the major organizational features of the basal ganglia and related thalamic centers, as delineated by the application of single-axon or single-cell labeling procedures in primates. These studies have revealed that the striatum, the external pallidum and the subthalamic nucleus harbor several types of projection neurons endowed with a highly collateralized axon that allows these neurons to interact with most components of the basal ganglia. In contrast, the internal pallidum, which is a major output structure of the basal ganglia, contains only two types of projection neurons. First, there is a minority of “limbic” pallidal neurons with a poorly branched axon that arborized profusely within the lateral habenula, which stands out as the most densely innervated pallidal target. Second, there is a majority of pallidal “motor” neurons with a long (total axonal length up to 27 cm) and highly branched axon that provides collaterals to the ventral tiers thalamic nuclei, the brainstem pedunculopontine nucleus and the centre médian/parafascicular thalamic complex. This type of axon allows internal pallidal neurons to send efferent copies of the same information to the thalamus and brainstem and hence influence various neuronal systems scattered throughout the neuraxis. Pallidal information is conveyed to the cerebral cortex and the striatum via the thalamus, while it is projected back to different components of the basal ganglia via the numerous reentrant pathways that arise from the pedunculopontine nucleus. Virtually all neurons in the centre médian thalamic nucleus innervate massively the striatum and less prominently the primary motor cortex, which in turn projects to the striatum directly or via a collateral from long-range corticofugal pyramidal axons. The results call for a reappraisal of our current concept of the anatomical and functional organization of basal ganglia, which play a crucial role in sensorimotor integration. Our data indicate that basal ganglia and related thalamic nuclei form a widely distributed neuronal network, whose elements are endowed with a highly patterned set of axon collaterals. This morphological feature allows a complex and exquisitely precise interaction between the various basal ganglia and related thalamic nuclei. The elucidation of this finely tuned network is needed to understand the complex spatiotemporal sequence of neural events that ensures the flow of cortical information through the basal ganglia and thalamus.

Neurons and Glia in the Midline of the Higher Crustacean Orchestia cavimana Are Generated via an Invariant Cell Lineage That Comprises a Median Neuroblast and Glial Progenitors

Midline cells are a common feature of both insects and crustaceans. Midline cells in the insects Schistocerca americana and Drosophila melanogaster have been shown to give rise to pairs of either neurons or glial cells (midline precursor) as well as to repeatedly generate neurons (median neuroblast) or both neurons and glia (median neuroglioblast). This study addresses midline cell lineages in a higher crustacean, the amphipod Orchestia cavimana. In vivo labeling of single midline cells shows that the resulting cell lineage is invariant and that these cells act as progenitors for sets of three glial precursors and one median neuroblast. The progeny are restricted to parasegmental units. The glial precursors give rise to three pairs of glial cells; two of them enwrap the commissures. The median neuroblast gives rise to about 10 cells that differentiate into 3 classes of neurons. The presence of median neuroblasts is also shown for another higher crustacean, the isopod Porcellio scaber using BrdU labeling. This is the first study to analyze the cell lineage of crustacean neurons generated by early ectodermal precursors. A comparison with those of insects demonstrates both conservation and change during the evolution of arthropods.

Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.

The organization of the striatal output system: a single-cell juxtacellular labeling study in the rat.

The aim of the present study was to provide a detailed account of the axonal branching pattern of striatal projection neurons in the rat. Seventy-seven striatofugal neurons were singly labeled following juxtacellular injection of biotin dextran amine. Their axons were entirely reconstructed along the sagittal plane with the help of a light microscope equipped with a camera lucida. The major findings of this study can be summarized as follows, (1) the striatofugal system originates from medium-sized spiny neurons that project only to globus pallidus (GP, type I, 36.4%), to both GP and substantia nigra pars reticulata (SNr, type II, 26%), or to globus pallidus, entopeduncular nucleus (EP) and SNr (type III, 37.6%); (2) the striatofugal system displays a high degree of axonal collateralization; about two-thirds of its axons arborize into two or three striatal target structures; (3) virtually all striatofugal axons send collaterals to the GP and none project exclusively to the EP and or SNr; (4) the three types of striatal projection neurons share similar somatodendritic morphology and have no preferential distribution in the dorsal striatum. These data, together with those of previous investigations, indicate that the striatofugal system can no longer be considered to be a simple dual (direct indirect) projection system. Instead, it stands out as a complex and widely distributed neuronal network whose elements are endowed with a highly patterned set of axon collaterals, which allows them to control in an exquisitely precise manner the flow of information along the main axis of the basal ganglia.

Formation of antennal lobe and mushroom body neuropils during metamorphosis in the honeybee,Apis mellifera

The projections to the mushroom bodies (mbs) have been clearly described in the brain of adult honeybees (Apis mellifera). Olfactory projection neurons arborize in the lip of the calyceal neuropil, whereas visual projection neurons project to the collar. To study the maturation of this pattern of innervation, as well as the development of uniglomerular projection neurons within the antennal lobes (als), we conducted the following three studies focused on the first four stages of pupal development: mass staining of olfactory projection neurons, single cell labeling of olfactory projection neurons, and simultaneous labeling of olfactory projection neurons and visual projection neurons. Examination of whole-mount preparations with the confocal laser scanning microscope revealed that the olfactory projection neurons achieved their adult arborization pattern within their main output region, the lip of the mb calyces, earlier during development (pupal stage 1) than their dendritic processes within their main input region, the al (pupal stage 2). Simultaneous labeling experiments showed further that the fiber terminals of olfactory projection neurons and visual projection neurons did not overlap but instead occupied their respective projection areas within the mb calyces as early as pupal stage 1. We conclude that selective innervation of different subregions of the calycal neuropil precedes the segregation of glomerular units within the antennal lobe neuropil, and that the Kenyon cells themselves provide a template for the innervation of olfactory and visual projection neurons.

Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.

AbstractClassical fate mapping approaches in any embryo typically involve labeling of single cells with nondiffusible dyes by pressure or iontophoretic microinjection followed by tracing the fate(s) of those cells and their descendants throughout development. The limitation of any approach that involves microinjection as a means of cell labeling is that, as development progresses, cells typically decrease in size and become less accessible to mechanical manipulation. The approach presented here utilizes local activation of a photoactivatable (caged) dye that becomes fluorescent when illuminated with specific wavelengths of ultraviolet light (1).KeywordsZebrafish EmbryoAcid Ethyl EsterEpifluorescent IlluminationShield StagePhotographic ExposureThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Successive patterns of clonal cell dispersion in relation to neuromeric subdivision in the mouse neuroepithelium.

We made use of the laacz procedure of single-cell labelling to visualize clones labelled before neuromere formation, in 12.5-day mouse embryos. This allowed us to deduce two successive phases of cell dispersion in the formation of the rhombencephalon: an initial anterior-posterior (AP) cell dispersion, followed by an asymmetrical dorsoventral (DV) cell distribution during which AP cell dispersion occurs in territories smaller than one rhombomere. We conclude that the general arrest of AP cell dispersion precedes the onset of morphological segmentation and is not imposed by the interface between adjacent rhombomeres. This demonstrates a major change in the mode of epithelial growth that precedes or accompanies the formation of neuromeres. We also deduced that the period of DV cell dispersion in the neuroepithelium is followed by a coherent growth phase. These results suggest a cell organization on a Cartesian grid, the coordinates of which correspond to the AP and DV axis of the neural tube. A similar sequence of AP cell dispersion followed by an arrest of AP cell dispersion, a preferential DV cell dispersion and then by a coherent neuroepithelial growth, is also observed in the spinal cord and mesencephalon. This demonstrates that a similar cascade of cell events occurs in these different domains of the CNS. In the prosencephalon, differences in spatial constraints may explain the variability in the orientation of cell clusters. Genetic and clonal patterning in the AP and DV dimensions follow the same spatial sequence. An interesting possibility is that these successive patterns of cell growth facilitate the acquisition of positional information.

Intracortical Axonal Projections of Lamina VI Cells of the Primary Somatosensory Cortex in the Rat: A Single-Cell Labeling Study

A sample of 84 neurons in lamina VIa of rat somatosensory cortex (S1) was juxtacellularly labeled with biocytin, and the axons of the neurons were traced. Three classes of cells were identified as corticothalamic, corticocortical, and local circuit neurons. Corticothalamic cells (46%) are small, short pyramids projecting either to the ventral posteromedial nucleus alone or to the posterior group as well. The former are in upper lamina VI, have apical dendrites terminating in layer IV, and have intracortical collaterals ascending to layer IV as a narrow column about the size of a barrel. The latter are in the lower half of lamina VI, have apical dendrites terminating in layer V, and have a more extensive network of collaterals terminating in the upper part of lamina V. Corticothalamic cells do not project to distant cortical targets through branching axons. Corticocortical cells (44%) are small, short pyramids, inverted or modified pyramids, or bipolar spiny neurons. They send collaterals principally to infragranular layers of S1 and branches to the second somatosensory cortex, the motor cortex, or the corpus callosum. Local circuit neurons (10%) are basket cells, concentrated in upper lamina VI, having smooth, beaded dendrites and a rich collateral network densely covered with varicosities in layers V and VI. We conclude that (1) dendritic morphology and axonal arborizations of corticothalamic cells relate to the projection target; (2) many apparently diverse layer VI cells project to other cortical fields; and (3) lamina VI is a network for corticothalamic and corticocortical communication.

INTERLABORATORY EVALUATION OF THE QUANTIFICATION OF RAT SPLENIC LYMPHOCYTE SUBTYPES USING IMMUNOFLUORESCENT STAINING AND FLOW CYTOMETRY

The quantification of rat splenic lymphocyte populations using immunofluorescent staining and flow cytometry was evaluated in an interlaboratory study involving 6 independent facilities employing a common protocol. The study had three specific aims: (1) to establish baseline values for rat splenic lymphocyte populations, (2) to examine variability in flow cytometry data both within and am ong laboratories, and (3) to evaluate single versus dual cell labeling of T-cell subpopulations, as well as quadrant and histogram analysis procedures. Splenic lymphocytes were enumerated following the lysis of red blood cells (RBC) with ammonium chloride. B-cells (anti-rat kappa light chain; clone OX12) were exam ined using a single im munofluorescent label, whereas T-cells (anti-CD5; OX19), T (T) (anti-CD4; W3/ 25), and T (T) helper h cytotoxic/ su ppressor cyt/ su p (anti-CD8; OX8) cells were examined using both single and dual labeling. Cell recoveries and viabilities reported by each laboratory following the lysis o...

A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin

We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: ≈ 1 μm), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and ‘tickled’ by the juxtamembranous iontophoretic pulses. Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate (> 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.

The fate of the CNS midline progenitors in Drosophila as revealed by a new method for single cell labelling

We present a new method for marking single cells and tracing their development through embryogenesis. Cells are labelled with a lipophilic fluorescent tracer (DiI) in their normal positions without impaling their membranes. The dye does not diffuse between cells but is transferred to the progeny, disclosing their morphology in all detail. Behaviour of labelled cells can be observed in vivo (cell divisions, morphogenetic movements and differentiation). Following photoconversion of the dye, fully differentiated clones can be analyzed in permanent preparations. We apply this method for cell lineage analysis of the embryonic Drosophila CNS. Here we describe the fate of the CNS midline cells. We present the complete lineages of these cells in the fully differentiated embryo and show that variability exists in segmental numbers of the midline progenitors as well as in the composition of their lineages.

Single cell analysis of commitment in early embryogenesis

ABSTRACT Fate maps of amphibian embryos tell us the destination of certain areas at later stages of development. After studying Vogt’s fate maps, Spemann wrote (in 1938) that “the question which at once calls for an answer is whether this pattern of presumptive primordia in the beginning gastrula is the expression of a real difference of these parts, whether they are already more or less predestined or ‘determined’ for their ultimate fate, or whether they are still indifferent and will not receive their determination until a later time.” Until recently answers to this question have relied upon explant experiments, which indicate that by the late blastula stage ectoderm, mesoderm and endoderm regions are distinctly determined. By using a method involving single cell labelling and transplantation, it is now possible to pinpoint more accurately the time during early embryogenesis at which individual blastomeres become committed. In the vegetal pole, determination towards endoderm is a gradual process beginning during the middle blastula stage (stage 8) and completed by the beginning of gastulation (stage 10). This method offers the possibilities of comparing the committed and the uncommitted state and studying at a molecular level the mechanisms of cell determination.

Alterations in population dynamics of arterial smooth muscle cells during atherogenesis: IV. Evidence for a polyclonal origin of hypercholesterolemic diet-induced atherosclerotic lesions in young swine

The purpose of this study was to determine whether atherosclerotic lesions developing in young swine fed hypercholesterolemic (HC) diets were monoclonal or polyclonal in origin. The approach involved labeling arterial cells with tritiated thymidine prior to feeding the HC diet. Baseline swine were sacrificed and radioautography studies carried out to determine baseline labeling indices and grain number distributions in media and in intimal cellular masses (cushions). Remaining swine were then fed an HC diet for 30–60 days before sacrificing and making the same type determinations as with the baseline swine on putative active lesions and media. If a lesion arose from a single cell (or multiple unlabeled cells) labeling indices would be expected to be greatly reduced without a change in grain number patterns. This was not observed in any of the 14 putative lesions that were studied. If the lesion arose from divisions by multiple cells including labeled cells we would expect the proportion of cells with a high number of grains to be decreased and those with low numbers to be increased since with division half the isotope goes to each daughter cell and this is reflected in the grain count. This occurred in nine of the putative lesions, indicating multicellular origin. In five there was neither change in grain number distributions nor reduction in labeling indices. Hence, these showed no division activity during the period of study; they were classified as inactive lesions or pre-existing cushions and provide no pertinent information for this study. The final conclusion was that all active lesions observed were polyclonal in origin. Further information was obtained by mathematical analysis on number of divisions made by the originally labeled cells; and considerable heterogeneity was observed, with some not dividing at all and others' with progeny going through as many as four divisions.