Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system

The peripheral nervous system of the ascidian tadpole larva comprises a distributed population of isolated receptor neurons, most of unproved function, organized along the trunk or tail epithelium. Previous reports using immunocytochemical methods failed to resolve the detailed morphology of the neurons and their axon pathways. Precleavage embryos of Ciona intestinalis transfected with the promoter of the neuron-specific synaptotagmin gene fused to a green fluorescent protein (GFP) gene yielded clearly labelled GFP profiles. These we examined in confocal image stacks of 31 larvae. Anchor cells, at least eight in each adhesive apical papilla, contribute axons to the papillar nerves that terminate in the sensory vesicle of the central nervous system. Two nerve bundles projected from each papilla, suggesting that at least two subpopulations of papillar neurons exist. Each bundle fasciculated with axons of the rostral trunk epidermal neurons (RTEN) in a stereotyped pattern. The RTEN had a hitherto unreported elaborate arbor of sensory dendrites within the tunic, suggesting that each has an extended sensorial field. Two subpopulations of apical trunk epidermal neurons (ATEN), anterior and posterior, were distinguished. As with the RTEN, these neurons extended dendritic arbors into the tunic. Two additional types of tail neuron, the caudal epidermal neurons (dorsal and ventral) as well as a novel bipolar interneuron, were identified. These identified neuron types are the substrate for the ascidian larva's entire peripheral sensory input, important during larval swimming and settlement.     

Cell counts and maps in the larval central nervous system of the ascidian Ciona intestinalis (L.)

Although the ascidian tadpole larva harbors a prospectively valuable prototype of the chordate nervous system, with extensively characterized neural plate cell lineages, the simple cellular composition of the resultant central nervous system (CNS) is not documented in detail. The average total number of cells in the larval CNS of Ciona intestinalis is 335 (range +/- 4, n = 3), 65 or 66 of which reside in the nerve cord of the tail. The estimates were made by tracing and counting the number of nuclei in serial semithin (1 micron) sections cut longitudinally through three larvae, fixed no later than 2 hours after hatching. Within a single fourth larva, L4, 266 cells constituted the CNS in the trunk region of the larva, 45 of which occurred within the visceral ganglion, 215 in the sensory vesicle, and 6 in the neck between the two. Each cell was assigned to one of thirteen categories. Most (182, roughly 68%) are classified as ependymal, a specialized non-neural cell peculiar to embryonic and larval chordates, from their position lining the cavities of the neural tube's elaborations or from clear similarities in the cytological appearance to those that do. Five cells are accessory cells of the sensory structures: three lens cells and a pigment-cup cell in the ocellus, and a single pigment cell in the otolith. Of the remaining 79 cells, 36 are sensory, 17 receptors in the ocellus and 19 presumed hydrostatic pressure receptors; these lie on the right and left sides of the sensory vesicle, respectively. Eighteen of the visceral ganglion cells have been tentatively classified as neurons, as have the remaining 25 cells which form two clusters in the posterior region of the sensory vesicle.     

Ablation of central nervous system progenitor cells in transgenic rats using bacterial nitroreductase system

Specific ablation of central nervous system (CNS) progenitor cells in the brain of live animals is a powerful method to determine the functions of these cells and to reveal novel avenues for the treatment of several CNS-related disorders. To achieve this goal, we generated a line of transgenic rats expressing a bacterial enzyme, Escherichia coli nitroreductase gene (NTR), under control of the nestin promoter. In this system, NTR(+) cells are selectively eliminated upon application of prodrug CB1954, through activation of programmed cell death machineries. At 5 days of age, which is a time when cerebellar development is occurring, transgenic rats bearing the nestin-NTR/green fluorescent protein (GFP) gene are overtly normal and express NTR/GFP in neuronal stem cells, without any toxicity in these cells. The functional consequence of progenitor cell ablation was demonstrated by administering prodrug CB1954 into the cerebellum at this 5-day time point. Stem cell ablation in these neonates resulted in sensorimotor abnormalities, cerebellar degeneration, overall reduction in cerebellar seize, and manifestation of ataxia. In adult rats, GFP expression was not seen in the hippocampal progenitor cells and seen only at very low levels in the lateral ventricles, indicating a different NTR/GFP expression pattern between neonates and adults. In addition, application of CB1954 by intraventricular delivery reduced the number of 5-bromo-2'-deoxyuridine-labeled proliferating cells in the lateral ventricle but not hippocampus of NTR/GFP rats. These findings shows that targeted expression of NTR under a specific promoter might be of significant value in addressing the function of distinct cell population in vivo.     

High efficiency transfection of primary skeletal muscle cells with lipid-based reagents

Lipofection is a convenient method for gene transfer into muscle cells but reportedly is inefficient. We tested the efficacy of commercially available lipid-based and polyamine transfection reagents. Primary rat skeletal muscle cell cultures were transfected at three stages of development and assayed after fusion. Efficiency reached 30% during the proliferation stage and up to 23% when most myoblasts had fused into myotubes. Optimization of transfection conditions with three different vectors yielded efficiencies exceeding 50%. Thus, lipid-based transfection into primary skeletal muscle cells can be several times more efficient than previously reported.     

Long-lasting Potentiation of a Direct Central Connection between Identified Motor Neurons in the Locust

The plasticity of the direct central connection between the fast extensor and the posterior fast flexor tibiae motor neurons in the locust ( Schistocerca gregaria ) metathoracic ganglion was studied. An action potential in the fast extensor results in a monosynaptic excitatory postsynaptic potential (EPSP) in the flexor motor neuron. Antidromic stimulation of the fast extensor at 100 Hz for 3.5 s resulted in a long-lasting potentiation of the EPSP amplitude. The potentiation was not dependent on feedback caused by movement of the tibia, and was associated with an increase in the input resistance of the flexor motor neuron. The potentiation was heterosynaptic, and was not affected by bath application of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid. The potentiation was voltage dependent, as hyperpolarizing the flexor motor neuron during the stimulation blocked the development of the potentiation whereas depolarizing the flexor in the absence of presynaptic activity caused potentiation of subsequent fast extensor-evoked EPSPs. The depolarization-induced potentiation was calcium dependent. Antidromic stimulation of the fast extensor at 100 Hz for 3.5 s also caused modulation of the presynaptic action potential. The spike duration was increased and the amplitude of the afterhyperpolarization reduced. These effects were dependent on movement of the tibia. Bath application of the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin blocked the changes in the presynaptic spike. The modulation was probably due to the release of 5-HT from proprioceptive afferents that monitor movement of the tibia about the femur. The modulation of the presynaptic action potential increases transmitter release onto the flexor motor neurons, and this acts in synergy with the postsynaptic modulation to potentiate the connection.     

Pattern of substance P- and cholecystokinin-like immunoreactivity during regeneration of the neural complex in the ascidian Ciona intestinalis.

The neural ganglion of ascidians exhibits a novel and rapid pattern of regeneration whereby within approximately 28-35 days of total ablation an entirely new neural complex is formed. In normal adults, neuronal cell bodies expressing substance P- (SP-Li), neurokinin A-(NKA-Li), CCK/gastrin- (CCK-Li), and insulin-like immunoreactivity exhibit a clearly defined pattern of localization in the cortical rind of the ganglion with characteristic long processes arising from the perikarya running throughout the neuropile. CCK-Li cell bodies are particularly concentrated close to the points of exit of the main nerve trunks. We have used antisera raised against these peptides to monitor the process of regeneration up to postoperative (pa) day 35. Only SP and CCK antisera produced positive staining in the regenerating tissue. Immunoreactive cell bodies first appear following 14 days pa. At this time CCK-Li neurons are more abundant than SP-Li neurons and in contrast to the pattern found in the normal adult ganglion, immunoreactive cell bodies are located both peripherally and centrally in the core of the ganglion and processes were rarely seen. Later stages exhibited an increasing number of SP-Li neurons and at 35 days pa SP-Li cell bodies clearly predominate. CCK-Li neurons typically become clustered close to the points of emergence of the anterior nerve roots. The early expression of CCK-Li and SP-Li molecules during regeneration is considered in terms of their potential role in development and cell proliferation in the newly forming ganglion.     

Origin and distribution of bone marrow-derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.     

Modification of motor neuron size and position in the central nervous system of adult snapping shrimps

Cell bodies of claw closer motor neurons in snapping shrimp are dimorphic. Snapper claw motor neurons are larger than corresponding pincer claw motor neurons, but the relative sizes of these cells are reversed during claw transformation. An additional neuronal modification occurs early within this period, in that the pincer claw dorsal inhibitor cell body migrates within the nervous system, from a dorsal to a ventral position. These findings are evidence of rapid, reversible changes in the nervous system following the trigger for the transformation process.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

绿色荧光蛋白报道基因在鼠C6胶质瘤细胞中的应用研究

目的 :探讨绿色荧光蛋白 (GFP)报道基因转染C6鼠胶质瘤细胞的体内外表达及对细胞生物学性状的影响。方法 :荧光相差显微镜筛选稳定表达GFP的克隆 ,流式细胞术分析细胞周期。将已转染和未转染瘤细胞植入SD大鼠脑内 ,建立动物模型 ,定期随机处死大鼠 ,鼠脑标本行病理学、增殖与凋亡的检测 ,激光共聚焦显微镜检查肿瘤细胞内荧光强度及其分布。结果 :GFP在C6瘤细胞的体外和体内均获得长期稳定表达 ,检测肿瘤细胞的敏感性及特异性优于HE染色。结论 :GFP对肿瘤细胞生物学性状无影响是比较理想的报道基因     

Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA. J. Comp. Neurol. 382:247-259, 1997. © 1997 Wiley-Liss, Inc.     

Mitochondrial imaging in dorsal root ganglion neurons following the application of inducible adenoviral vector expressing two fluorescent proteins

Mitochondrial morphology and dynamics are known to vary considerably depending on the cell type and organism studied. The objective of this study was to assess the potential application of adenoviral-fluorescent protein constructs for long-term tracking of mitochondria in neurons. An adenoviral vector containing two fluorescent proteins, the enhanced green fluorescent protein (eGFP) targeted to the cytoplasm to highlight the neuronal processes, and the red fluorescent protein (RFP) directed to mitochondria under the control of an inducible promoter, facilitated an efficient and accurate method to study mitochondrial dynamics in long-term studies. Dorsal root ganglion neurons from rat embryos were cultured and infected. The infected neurons exhibited green fluorescence after 24h, while 16 h following induction with doxycycline, red fluorescence protein began to localize within mitochondria. The red fluorescent protein was transported into mitochondria at the cell body followed by distribution within processes. As the neurons aged, the expression of red fluorescent protein was confined to cytoplasmic vacuoles and not mitochondria. Further analysis suggested that the cytoplasmic vacuoles were likely of lysosomal origin. Taken together, the current study presents novel strategies to study the life history of cellular organelles such as mitochondria in long-term studies.     

Neuroanatomical organization of the cholinergic system in the central nervous system of a basal actinopterygian fish,the senegal bichir Polypterus senegalus

Polypterid bony fishes are believed to be basal to other living ray‐finned fishes, and their brain organization is therefore critical in providing information as to primitive neural characters that existed in the earliest ray‐finned fishes. The cholinergic system has been characterized in more advanced ray‐finned fishes, but not in polypterids. In order to establish which cholinergic neural centers characterized the earliest ray‐finned fishes, the distribution of choline acetyltransferase (ChAT) is described in Polypterus and compared with the distribution of this molecule in other ray‐finned fishes. Cell groups immunoreactive for ChAT were observed in the hypothalamus, the habenula, the optic tectum, the isthmus, the cranial motor nuclei, and the spinal motor column. Cholinergic fibers were observed in both the telencephalic pallium and the subpallium, in the thalamus and pretectum, in the optic tectum and torus semicircularis, in the mesencephalic tegmentum, in the cerebellar crest, in the solitary nucleus, and in the dorsal column nuclei. Comparison of the data within a segmental neuromeric context indicates that the cholinergic system in polypterid fishes is generally similar to that in other ray‐finned fishes, but cholinergic‐positive neurons in the pallium and subpallium, and in the thalamus and cerebellum, of teleosts appear to have evolved following the separation of polypterids and other ray‐finned fishes. J. Comp. Neurol. 521:24–49, 2013. © 2012 Wiley Periodicals, Inc.     

Cholinotoxicity of the ethylcholine aziridinium ion in primary cultures from rat central nervous system

The cytotoxic effects of ethylcholine aziridinium ion (AF64A) were studied in primary cultures prepared from either whole brain, septum, or midbrain of fetal rats. AF64A, at concentrations up to 22.5 microM, significantly reduced the number of acetylcholinesterase-stained cells without affecting the number of dopaminergic neurons or their ability to take up and release [3H]dopamine. Many of the survived acetylcholinesterase-stained cells appeared with intact somata but damaged processes, indicating a retrograde degeneration starting at the nerve terminal. Higher concentrations of AF64A (greater than 22.5 microM), caused general toxicity which was expressed by degeneration of various neuronal and glial cells. Choline (500 microM), significantly protected the cells from AF64A induced cytotoxicity. The results are consistent with a previously described kinetic model, that predicted a dual action of AF64A: selective cholinotoxicity at low concentrations and non-selective cytotoxicity at higher concentrations.     

On the longevity of resident endoneurial macrophages in the peripheral nervous system: a study of physiological macrophage turnover in bone marrow chimeric mice

Macrophages are intimately involved in the pathogenesis of peripheral nervous system (PNS) disorders. Recently, we characterized a resident endoneurial macrophage population, which contributes rapidly to the endoneurial macrophage response in PNS diseases. Unlike microglial cells, resident macrophages undergo a physiological turnover of 50% in the sciatic nerve and 80% in dorsal root ganglia (DRG) within 12 weeks. Further information about the dynamics of this turnover is not available. This study examined the macrophage turnover in the sciatic nerve and DRGs over a longer period and addresses the question whether the turnover of resident macrophages is complete or whether there is a truly resident endoneurial macrophage population. We used chimeric mice carrying GFP(+) bone marrow and immunohistochemistry to detect hematogenous (GFP(+)) endoneurial macrophages after turnover. Non-exchanged, resident macrophages were GFP(-). Quantification of GFP(+) and GFP(-) macrophages revealed a maximal turnover of 75%, reached in DRGs after 12 weeks and in sciatic nerves after 36 weeks. GFP(-) long-term resident macrophages were further characterized after sciatic nerve injury, where they participated in the early macrophage response of Wallerian degeneration. Our results point toward a small but truly resident PNS macrophage population. These macrophages are an interesting target for further characterization and might have a distinct role in peripheral nerve disease.     

Distribution of the iron-regulating protein hepcidin in the murine central nervous system

Iron serves as an essential trace element for all body tissues, including the central nervous system (CNS). Because iron deficiency as well as iron overload is known to cause damage to the mammalian brain, the maintenance of iron homeostasis is crucial. It has been discovered recently that hepcidin plays an essential role in iron metabolism outside the CNS. A defect in hepcidin expression is responsible for iron accumulation and mice over-expressing hepcidin die postnatally by a severe anemia. We have used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the cellular distribution of hepcidin mRNA and protein in brain, spinal cord, and dorsal root ganglia. Our results show a wide-spread distribution of hepcidin in different brain areas, including the olfactory bulb, cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon, cerebellum, pons, spinal cord, as well as in dorsal root ganglia of the peripheral nervous system. Hepcidin immunoreactivity is not restricted to neurons, but can be detected in both neurons and GFAP-positive glia cells. Because hepcidin action in organs outside the CNS is linked to iron homeostasis, we speculate that it is also involved in such processes in the CNS, putatively together with other iron regulating proteins. Cellular mechanisms and functions of hepcidin in the CNS remain to be elucidated.     

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.     

Ubiquitin, cell stress and diseases of the nervous system

Cells, including those of the nervous system, respond to damage by an increase in the synthesis of a family of proteins called 'stress proteins' which are amongst the most conserved gene products in evolution suggesting fundamental roles in cell metabolism. Stress-induced proteins have functions in normal cells, particularly for the importation of protein into membrane-limited organelles, and their up-regulation following stress is thought to be cytoprotective, by protecting proteins and organelles from damage. Ubiquitin is an important protein induced by cell stress. It is only found in nucleated cells and has several known functions; the most investigated being as a co-factor for the non-lysosomal intracellular degradation of abnormal or short lived proteins. Morphological studies using immunohistochemistry to localize ubiquitin protein conjugates have revealed that ubiquitin is a component of many of the filamentous inclusion bodies characteristic of neurodegenerative diseases, suggesting activation of a common neuronal response in this type of disease process. Immunohistochemical localization of ubiquitin conjugates has provided a new tool for the sensitive detection of such inclusions and has resulted in the identification of novel inclusion bodies in all cases of motor neuron disease. Preliminary work on enzymes involved in ubiquitin metabolism suggest that there are several possible mechanisms for the formation of inclusion bodies and may provide indirect evidence for the dynamics of inclusion body formation. Work in other areas of pathology indicate important roles for the stress proteins in immune surveillance and autoimmunity and it is likely that the general principles which are currently evolving will also have an impact in neuropathology.