Neural network in planarian revealed by an antibody against planarian synaptotagmin homologue.

In order to investigate the neural connection of planarian, it is imperative to produce an antibody that specifically stains axons. To identify axon-specific genes, we constructed a cDNA library from a single eye by using a single cell PCR method, in which visual neurons are major components, and sequenced one thousand independent clones. We succeeded in the identification of a planarian homologue of synaptotagmin, Djsyt, whose specific expression in neurons was confirmed by in situ hybridization. The antibody against DjSYT specifically stained axons although its mRNA is distributed in the cell bodies. By using anti-DjSYT, we succeeded in the visualization of neural connections in planarians by whole mount staining. The anti-DjSYT antibody will become a powerful tool to analyze the molecular mechanisms underlying neural network formation in planarian.     

Spinal projections of brainstem respiratory related neurons in the cat as revealed by retrograde fluorescent markers

By means of retrograde axonal transport of fluorescent tracers, connections between brainstem respiratory related regions and the spinal cord has been studied in the cat. Neurons at the pneumotaxic center project bilaterally (90% ipsi-, 10% contra-) to cervical and lumbar spinal cord and ipsilaterally to thoracic levels. The ventrolateral nucleus of the tractus solitarius project mainly contralaterally (85%) to cervical levels and only contralaterally to thoracic levels; no efferent projections were found to lumbar levels. The ventral respiratory group showed a great number of neurons projecting to the spinal cord especially from the nucleus retroambiguus. Both nuclei, ambiguus and retroambiguus, project mainly contralaterally (70%) to the spinal cord. The B?tzinger complex showed rather scarce bilateral projections to cervical and only ipsilateral projections to lower cervical, thoracic and lumber levels.     

A new planarian extrachromosomal virus-like element revealed by subtraction hybridization

A combination of suppression subtraction hybridization (SSH) and a new technique of mirror orientation selection (MOS) was used to compare the total DNA for two, sexual (SR) and asexual (AR), races of freshwater planarian Giradia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in AR. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed though the planarian body and is possibly replicated only in certain parenchymal cells.     

Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing

The continued addition of new neurons to mature olfactory circuits represents a remarkable mode of cellular and structural brain plasticity. However, the anatomical configuration of newly established circuits, the types and numbers of neurons that form new synaptic connections, and the effect of sensory experience on synaptic connectivity in the olfactory bulb remain poorly understood. Using in vivo electroporation and monosynaptic tracing, we show that postnatal-born granule cells form synaptic connections with centrifugal inputs and mitral/tufted cells in the mouse olfactory bulb. In addition, newly born granule cells receive extensive input from local inhibitory short axon cells, a poorly understood cell population. The connectivity of short axon cells shows clustered organization, and their synaptic input onto newborn granule cells dramatically and selectively expands with odor stimulation. Our findings suggest that sensory experience promotes the synaptic integration of new neurons into cell type-specific olfactory circuits.     

Search for the evolutionary origin of a brain: planarian brain characterized by microarray

The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural brain emerged, we analyzed the central nervous system (CNS) of a lower invertebrate, planarian. We conducted a large-scale screening of the head part-specific genes in the planarian by constructing a cDNA microarray. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians revealed 205 genes with head part-specific spikes, including essential genes in the vertebrate nervous system. The expression patterns of the top 30 genes showing the strongest spikes implied that the planarian brain has undergone functional regionalization. We demonstrate the complex cytoarchitecture of the planarian brain, despite its simple superficiality of the morphology.     

Localization of newly synthesized precursors of basal lamina in the regenerating planarian as revealed by autoradiography

Autoradiography has been carried out to investigate the site of synthesis of the basal lamina in the regenerating planarian, Dugesia japonica. Since the basic collagenous structures of the basal lamina arose from RR-positive amorphous precursor, [³H]proline, [³H]glucose and [³⁵S]sodium sulphate were used as radioactive precursors of collagen, unsulphated and sulphated GAG respectively. Cytoplasm of the most regenerating epidermal cells was heavily labeled with [³H]proline during epithelization. A quantitative uptake analysis of [³H]proline indicates a progressive decline in the amount of labeled precursor in the epidermis with a corresponding increase in deposition of the labeled collagen at the presumptive basal lamina. Several myoblasts at the subepidermal region were highly labeled with both [³H]glucose and [³⁵S]sodium sulphate. Silver grains of these labeled precursors were also present in the presumptive portion of basal lamina. These observations suggest that the regenerating epidermal cell is the only site of synthesis of the basal lamina collagen while the myoblast exclusively secretes extracellular GAG. Some of the GAG may be closely associated with the amorphous zone.     

Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes

We previously showed that liposomes composed of dioleoylphosphatidyl-ethanolamine and palmitoyl-homocysteine (8:2) are highly fusion competent when exposed to an acidic environment of pH less than 6.5. (Connor, J., M. B. Yatvin, and L. Huang, 1984, Proc. Natl. Acad. Sci. USA. 81:1715-1718). Palmitoyl anti-H2Kk was incorporated into these pH-sensitive liposomes by a modified reserve-phase evaporation method. Mouse L929 cells (k haplotype) treated with immunoliposomes composed of dioleoylphosphatidylethanolamine/palmitoyl-homocysteine (8:2) with an entrapped fluorescent dye, calcein, showed diffused fluorescence throughout the cytoplasm. Measurements by use of a microscope-associated photometer gave an approximate value of 50 microM for the cytoplasmic calcein concentration. This concentration represents an efficient delivery of the aqueous content of the immunoliposome. Cells treated with immunoliposomes composed of dioleoylphosphatidylcholine (pH-insensitive liposomes) showed only punctate fluorescence. The cytoplasmic delivery of calcein by the pH-sensitive immunoliposomes could be inhibited by chloroquine or by incubation at 20 degrees C. These results suggest that the efficient cytoplasmic delivery involves the endocytic pathway, particularly the acidic organelles such as the endosomes and/or lysosomes. One possibility is that the immunoliposomes fuse with the endosome membranes from within the endosomes, thus releasing the contents into the cytoplasm. This nontoxic method should be widely applicable to the intracellular delivery of biomolecules into living cells.     

Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers

Planarians are considered to be among the most primitive animals which developed the central nervous system (CNS). To understand the origin and evolution of the CNS, we have isolated a neural marker gene from a planarian, Dugesia japonica, and analyzed the structure of the planarian CNS by in situ hybridization. The planarian CNS is located on the ventral side of the body, and composed of a mass of cephalic ganglions in the head region and a pair of ventral nerve cords (VNC). Cephalic ganglions cluster independently from VNC, are more dorsal than VNC, and form an inverted U-shaped brain-like structure with nine branches on each outer side. Two eyes are located on the dorsal side of the 3(rd) branch and visual axons form optic chiasma on the dorsal-inside region of the inverted U-shaped brain. The 6(th)-9(th) branches cluster more closely and form auricles on the surface which may function as the sensory organ of taste. We found that the gross structure of the planarian CNS along the anterior-posterior (A-P) axis is strikingly similar to the distribution pattern of the "primary" neurons of vertebrate embryos which differentiate at the neural plate stage to provide a fundamental nervous system, although the vertebrate CNS is located on the dorsal side. These data suggest that the basic plan for the CNS development along the A-P axis might have been acquired at an early stage of evolution before conversion of the location of the CNS from the ventral to the dorsal side.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Membrane potentials in respiring and respiration-deficient yeasts monitored by a fluorescent dye

Changes in fluorescence of 3,3′-dipropylthiodicarbocyanine iodide which had been equilibrated with suspensions of the wild-type yeast Saccharomyces cerevisiae and of respiration-deficient mutants were followed. The changes have been attributed to changes of yeast membrane potentials, since the fluorescence with wild-type yeast could be affected in a predictable manner by uncouplers and the pore-forming agent nystatin. As in other systems, a rise of steady-state fluorescence was ascribed to depolarization and a drop of the fluorescence to hyperpolarization. (1) A considerable rise in steady-state fluorescence was brought about by addition of antimycin A or some other mitochondrial inhibitors to respiring cells. A major part of the composite membrane potential monitored in intact yeast cells appeared to be represented by the membrane potential of mitochondria. (2) Addition of D-glucose and of other substrates of hexokinase, including non-metabolizable 2-deoxy-D-glucose, induced a two-phase response of fluorescence, indicating transient depolarization followed by repolarization. Such a response was not elicited by other sugars which had been reported to be transported into the cells by a glucose carrier or by D-galactose in galactose-adapted cells. The depolarization was explained by electrogenic ATP exit from mitochondria to replenish the ATP consumed in the hexokinase reaction and the repolarization by subsequent activation of respiration. (3) In non-respiring cells only a drop in fluorescence was induced by glucose and this was ascribed to an ATP-dependent polarization of the plasma membrane. (4) Steady-state fluorescence in suspensions of respiration-deficient mutants, lacking cytochrome a, cytochrome b, or both, was high and remained unaffected by uncouplers and nystatin. This indicates that membranes of the mutants may have been entirely depolarized. A partial polarization, apparently restricted to the plasma membrane, could be achieved by glucose addition.     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

Topography of the thalamo-cortical projections on trunk representation demonstrated by fluorescent neuro-tracers

With the aim to study the detailed topography of the thalamo-cortical neurones projecting to the trunk representation zone of the first somatosensory area (SI), punctate injections of three different fluorescent tracers (Evans Blue, Nuclear Yellow and Fast Blue) were performed in the three physiologically defined subareas forming the trunk region of SI. These injections resulted in the labelling of three different cell aggregates, narrow in dorsoventral and mediolateral extent but elongated rostrocaudally, located in topographically distinct regions of the nucleus ventralis posterio-lateralis. The results suggest that the highly organized topography of the trunk representation of area SI is imposed by the thalamo-cortical input from VPL.     

Targeting and tracing antigens in live cells with fluorescent nanobodies

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.     

Innervation of the maxillary vibrissae in mice as revealed by anterograde and retrograde tract tracing

Vibrissae are a unique sensory system of mammals that is characterized by a rich and diverse innervation involved in numerous sensory tasks with the potential for species-specific differences. In the present study, indocarbocyanine dyes (DiI and PTIR271) and confocal microscopy were combined to study the innervation of the mystacial vibrissae and vibrissa-specific sensory neuron distribution in the maxillary portion of the trigeminal ganglion of the mouse. The deeper regions of the vibrissa cavernous sinus (CS) contained a dense plexus of free nerve endings, possibly of autonomic fibers. The superficial part of this sinus displayed a massive array of corpuscular endings. Innervation in the region of the ring sinus consisted of Merkel endings and different morphological variances of lanceolate endings. The region of the inner conical body had a circular plexus of free nerve endings. In addition to confirming previous observations obtained by a variety of other techniques and ultrastructural studies, our studies revealed denser terminal receptor endings in a different distribution pattern than previously demonstrated in studies using the rat. We also revealed the distribution of sensory neurons in the trigeminal ganglion using retrograde tracing with fluorescent tracers from two nearby vibrissae. We determined that the populations of sensory neurons innervating the two vibrissae were largely overlapping. This suggests that the somatotopic maps of vibrissal projections reported at the different levels in the neuraxis are not faithfully reproduced at the level of the ganglion.This work was supported by a grant from the NIDCD (RO1 DC 005590; BF), the Egyptian government (AM), and the NIH (ES00365-01 and RR-02-003; LH).     

Gap junctions between odontoblasts revealed by transjunctional flux of fluorescent tracers

Summary Cell communication between odontoblasts was investigated with the use of fluorescent-dye tracers; Lucifer Yellow CH (molecular weight = 457.3), and dextran-Lucifer Yellow CH (average molecular weight = 10000). Dyes were injected into cell bodies of individual odontoblasts via an intracellular microelectrode or into a group of cells through their processes, and passage to adjacent cells was examined with a fluorescence microscope. Lucifer Yellow CH appeared to diffuse very easily among odontoblasts, while dextran-Lucifer Yellow remained within the injected cell or cells. This efficient migration of Lucifer Yellow CH can be considered a functional manifestation of gap junctions between odontoblasts.     

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts

Transmembrane voltage-sensitive fluorescence measurements are limited by baseline drift that can obscure changes in resting membrane potential and by motion artifacts that can obscure repolarization. Voltage-dependent shift of emission wavelengths may allow reduction of drift and motion artifacts by emission ratiometry. We have tested this for action potentials and potassium-induced changes in resting membrane potential in rabbit hearts stained with di-4-ANEPPS [Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] using laser excitation (488 nm) and a two-photomultiplier tube system or spectrofluorometer (resolution of 500-1,000 Hz and <1 mm). Green and red emissions produced upright and inverted action potentials, respectively. Ratios of green emission to red emission followed action potential contours and exhibited larger fractional changes than either emission alone (P < 0.001). The largest changes and signal-to-noise ratio (signal/noise) were obtained with numerator wavelengths of 525-550 nm and denominator wavelengths of 650-700 nm. Ratiometry lessened drift 56-66% (P < 0.015) and indicated decreases in resting membrane potential. Ratiometry lessened motion artifacts and increased magnitudes of deflections representing phase-zero depolarizations relative to total deflections by 123-188% in intact hearts (P < 0.02). Durations of action potentials at different pacing rates, temperatures, and potassium concentrations were independent of whether they were measured ratiometrically or with microelectrodes (P > or = 0.65). The ratiometric calibration slope was 0.017/100 mV and decreased with time. Thus emission ratiometry lessens the effects of motion and drift and indicates resting membrane potential changes and repolarization.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.