The development of thalamocortical connections studied by using carbocyanine dyes in the early ontogeny of rats]

We studied the differentiation of neurons and development of their connections in the occipital cortex and thalamic areas of the brain in early ontogenesis of rats: from day 11 of embryogenesis until day 19 of postnatal development. We used the method of staining of brain tissues by carbocyanine dyes after its preliminary fixation in aldehydes. Three carbocyanine dyes were used: DiI, DiO and DiA. We showed the dynamics of structural differentiation of the cortical neurons and lateral geniculate body of the thalamus and the specificity of formation of the axonal pathways between the neocortex and thalamic areas. The results obtained confirmed the hypothesis on ordered spatial-temporal growth of the cortical and thalamic fibers in early embryogenesis and revealed synchronous development of both classes of neurons of the lateral geniculate body. Retrograde and anterograde staining of the nerve cells processes by DiI and DiO showed fine morphological details of their structure. DiI provided for a good staining of the cells until day 19 of postnatal ontogenesis and DiO, until the end of embryogenesis, while DiA was not capable of diffusion in the fixed tissue.     

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes

Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.     

Blastomere ablation and the developmental origin of identified monoamine-containing neurons in the leech

Ablation of different identifiable blastomeres of the early embryo of the leech Helobdella triserialis was found to lead to the absence of different sets of segmentally iterated monoamine-containing neurons in subsequent development. Thus the ablation of one of the paired N ectoteloblasts leads to the absence of one member of each of the three bilateral pairs of serotonin-containing neurons (one of which is the Retzius cell) from each segmental ganglion. The ablation of one of the paired OP blastomeres (precursors of the paired O and P ectoteloblasts) leads to the absence of one member of each of the two bilateral pairs of lateral dopamine-containing neurons that lie in the body wall of each segment. And the ablation of one of the paired Q ectoteloblast leads to the absence of one member of the bilateral pair of medial dopamine-containing neurons that lie in the body wall of each segment. These results suggest that each of these sets of monoamine-containing neurons is derived from a particular blastomere. Upon ablation of that blastomere the set does not develop from any other source.     

Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes

Eight pairs of chemosensory neurons in Caenorhabditis elegans take up fluorescein dyes entering through the chemosensory organs. These are amphid neurons ADF, ASH, ASI, ASJ, ASK, and ADL and phasmid neurons PHA and PHB. When filled with dye, the processes and cell bodies of these neurons can be examined in live animals by fluorescence microscopy. Using this technique, we have identified five genes, unc-33, unc-44, unc-51, unc-76, and unc-106, that affect the growth of the amphid and phasmid axons. These genes were found to affect the axons of the mechanosensory PDE neurons as well. The unc-33 mutation specifically affects neuronal microtubules. Sensory dendrites in this mutant have a superabundance of microtubules. Moreover, many of these microtubules are abnormal in diameter, and some form hooks or multiple tubules.     

Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem

Serotonin neurons in 14-d embryonic rat brain stem were identified by peroxidase-antiperoxidase immunocytochemistry with an affinity-purified antiserotonin antibody. Brain-stem tissue was dissected from 14- or 15- d embryonic rats, dissociated and grown in cell culture for up to 5 wk, and serotonin neurons were identified by immunocytochemistry. Within 24 h of plating, serotonin immunoreactivity was present in 3.3% of neurons. Immunoreactivity in neuronal cell bodies decreased with time, whereas staining of processes increased. The number of serotonin- immunoreactive neurons remained constant at 3-5% over the first 14 d in culture. From 14 to 28 d, the total number of neurons decreased with little change in the number of serotonin neurons, such that, by day 28 in culture, up to 36% of surviving neurons exhibited serotonin immunoreactivity. Similar percentages of cultured brain stem neurons accumulating 3H-serotonin were identified by autoradiography. Uptake was abolished by the serotonin-uptake inhibitor, clomipramine, but was unaffected by excess norepinephrine, or by the norepinephrine-uptake inhibitor, maprotiline. Synthesis of 3H-serotonin was detected after incubation of cultures with 3H-tryptophan, and newly synthesized serotonin was released by potassium depolarization in a calcium- dependent manner. More than 95% of serotonin neurons were destroyed after incubation of cultures with 5,6-dihydroxytryptamine. Brain-stem cultures contained virtually no neurons with the ability to accumulate 3H-norepinephrine or 3H-dopamine. Approximately 40% of brain-stem neurons were labeled with gamma-aminobutyric acid (3H-GABA). However, there was almost no overlap in the surface area of neurons accumulating 3H-serotonin or 3H-GABA.     

Neurite regeneration of long-term cultured adult insect neurosecretory cells identified as DUM neurons

A distinctive group of neurons having cell bodies located along the midline of the dorsal surface of the sixth abdominal (A6) ganglion of the adult cockroach Periplaneta americana has been characterized by direct anterogradc cobalt chloride staining. These neurons identified as dorsal unpaired median (DUM) neurons, present a T-shaped morphology. The soma gives rise to a single primary neurite running anteriorly in the ganglion before dividing into two lateral neurites which run into the left and the right side of the ganglion. A characteristic dendritic arborization arises from the lateral neurites within the ganglion. This major branching pattern is mainly located at the periphery of the A6 ganglion and forms a symmetrical complicated network. A new culture procedure of these same adult DUM neurons has been developed from the dissociation of the median parts of the A6 ganglia. In our experimental conditions, we show that cultured adult DUM neurons can survive for several weeks, and regenerate a single primary neuritc dividing into two symmetrical lateral neurites with a number of fine processes radiating from the endings. This corresponds to the typical DUM neuron morphology revealed in situ on the same preparation using the cobalt chloride staining technique. This culture system developed for the first time on A6 ganglia adult DUM neurons will allow a better understanding of the physiological intracellular mechanisms involved in the neurosecretory functions of DUM neurons, which are currently unknown.     

Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes

When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.     

The importance of identified neurons in gastropod molluscs to neuroscience

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Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.     

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.     

RNA in single identified neurons of aplysia

—An investigation of the types of RNA present in the whole abdominal ganglia of Aplysia and in single identified neurons was made. Because of the long in vitro functioning of the ganglion and large size of the neurons, adequate labelled precursor was incorporated to allow gel electrophoretic or sedimentation analysis of RNA isolated from a single nucleus or from cytoplasm. When labelled RNA is analysed a number of distinct peaks are found. Most of these are related to rRNA. In an effort to discern possible precursor-product relations and localization of these RNA species, RNA from the nucleus and the cytoplasm of neuron R2 was analysed after varying lengths of labelling time with tritiated precursors, in some cases followed by incubation with actinomycin. The results support the notion of a large transcribed precursor which is subsequently processed to form finished rRNA. Differences in rate of rRNA maturation among neurons are indicated and a previously unreported 14 S molecule is described.     

Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily

The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P < 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 ± 18.6%; SLC-Inc, 53.3 ± 17.1%; SLC-Large, 44.9 ± 18.3%) and were found to be equivalent in quality (motility: 88.0 ± 8.8%, 84.0 ± 3.5%, 90.0 ± 5.4%; normal morphology: 69.4 ± 17.0%, 69.4 ± 12.7%, 63.9 ± 15.6%; viability: 78 ± 16.7%, 83.8 ± 12.5%, 80.05 ± 14.6%; DNA fragmentation index: 14.7 ± 10.9%, 12.8 ± 8.1%, 11.6 ± 7.6%, respectively). The processing of an “average” stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use.     

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.     

Fluorescent tags of protein function in living cells

A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000.     

Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein

The oligopeptide permease (Opp) of Escherichia coli is an ATP-binding cassette transporter that uses the substrate-binding protein (SBP) OppA to bind peptides and deliver them to the membrane components (OppBCDF) for transport. OppA binds conventional peptides 2-5 residues in length regardless of their sequence, but does not facilitate transport of the cell wall component murein tripeptide (Mtp, L-Ala-γ-D-Glu-meso-Dap), which contains a D-amino acid and a γ-peptide linkage. Instead, MppA, a homologous substrate-binding protein, forms a functional transporter with OppBCDF for uptake of this unusual tripeptide. Here we have purified MppA and demonstrated biochemically that it binds Mtp with high affinity (K(D) ～ 250 nM). The crystal structure of MppA in complex with Mtp has revealed that Mtp is bound in a relatively extended conformation with its three carboxylates projecting from one side of the molecule and its two amino groups projecting from the opposite face. Specificity for Mtp is conferred by charge-charge and dipole-charge interactions with ionic and polar residues of MppA. Comparison of the structure of MppA-Mtp with structures of conventional tripeptides bound to OppA, reveals that the peptide ligands superimpose remarkably closely given the profound differences in their structures. Strikingly, the effect of the D-stereochemistry, which projects the side chain of the D-Glu residue at position 2 in the direction of the main chain in a conventional tripeptide, is compensated by the formation of a γ-linkage to the amino group of diaminopimelic acid, mimicking the peptide bond between residues 2 and 3 of a conventional tripeptide.     

Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium

Summary We describe a method for labeling cultured endothelial cells (ECs) and smooth muscle cells (SMCs) by letting the cells grow for three days in culture medium containing a low concentration of the fluorescent carbocyanine dyes DiI and DiO. We show that good labeling can be obtained with considerably lower concentrations (2.5 g/ml) than has previously been described. With optimal concentration the labeling is very strong and seems to label all membranous structures in the cells. It was possible to clearly distinguish differentially pre-labeled cells both in coculture and seeded on denaturated vascular grafts. The cells remain fluorescent for more than seven days and may be passaged with retained proliferative capability. We suggest that DiI/DiO-labeling using dye-containing medium may be used for several cell types and is applicable in tissue culture and in the detection of implanted cells in vivo.     

Rapid in vivo labeling of identified zebrafish neurons

We report a simple and rapid method to label individual neurons in live zebrafish embryos and to examine their gene expression profiles. Injection of plasmid DNA encoding an alpha-tubulin promotor driving GFP expression results in mosaic embryos containing a limited number of GFP-positive neurons. Labeled neurons express GFP in their soma and axon, providing the opportunity to analyze pathfinding behaviors of identified neurons in vivo. Moreover, the presence of only a small subset of GFP tagged neurons permits the rapid anatomical identification of these neurons based on soma position and axonal trajectory. Analysis of injected embryos reveals that most, if not all, spinal cord cell types and many other neuronal cell types elsewhere in the nervous system can be GFP tagged. Finally, by combining GFP labeling of individual neurons with fluorescent in situ hybridization, we demonstrate the potential of this method to elucidate gene expression patterns at single cell resolution.     

Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium.

We describe a method for labeling cultured endothelial cells (ECs) and smooth muscle cells (SMCs) by letting the cells grow for three days in culture medium containing a low concentration of the fluorescent carbocyanine dyes DiI and DiO. We show that good labeling can be obtained with considerably lower concentrations (2.5 micrograms/ml) than has previously been described. With optimal concentration the labeling is very strong and seems to label all membranous structures in the cells. It was possible to clearly distinguish differentially pre-labeled cells both in coculture and seeded on denaturated vascular grafts. The cells remain fluorescent for more than seven days and may be passaged with retained proliferative capability. We suggest that DiI/DiO-labeling using dye-containing medium may be used for several cell types and is applicable in tissue culture and in the detection of implanted cells in vivo.     

Fluorescent labelling of intracellular bacteria in living host cells

The fluorescent reagent, CellTracker, labels metabolically-active cells and was used here to label Chlamydia in vivo during their exponential phase of growth in infected cells. HeLa cells infected with C. psittaci were labelled with the CellTracker reagents between 15 and 48 h post-infection. The fluorescent label accumulated in the host-cell membrane compartment (inclusion) within which Chlamydia reside and replicate, and was also incorporated by the bacteria. Labelling with the CellTracker affected neither the growth nor the differentiation of the chlamydiae, and labelled chlamydiae isolated from infected cells were infectious. Our results demonstrate that the CellTracker could become a valuable tool for in vivo labelling of obligate intracellular parasites for which no genetic tools exist.