FORMATION OF SEROTONIN BY ISOLATED SEROTONIN-CONTAINING NEURONS AND BY ISOLATED NON-AMINE-CONTAINING NEURONS

Isolated giant serotonin-containing neurons of the cerebral ganglia of Helix pomatia were shown to produce serotonin when incubated with 5-hydroxytryptophan (5-HTP) whereas cells of the buccal ganglia, which are non-amine-containing cells did not. The rate of production was comparable to that for Ach in the isolated neurons of Aplysia. The significance of these results is discussed.     

FINE STRUCTURE OF NERVE FIBERS AND GROWTH CONES OF ISOLATED SYMPATHETIC NEURONS IN CULTURE

The leading tips of elongating nerve fibers are enlarged into "growth cones" which are seen in tissue culture to continually undergo changes in conformation and to foster numerous transitory slender extensions (filopodia) and/or a veillike ruffling sheet. After explantation of 1-day-old rat superior cervical ganglia (as pieces or as individual neurons), nerve fibers and tips were photographed during growth and through the initial stages of aldehyde fixation and then relocated after embedding in plastic. Electron microscopy of serially sectioned tips revealed the following. The moving parts of the cone, the peripheral flange and filopodia, contained a distinctive apparently filamentous feltwork from which all organelles except membranous structures were excluded; microtubules were notably absent from these areas. The cone interior contained varied forms of agranular endoplasmic reticulum, vacuoles, vesicles, coated vesicles, mitochondria, microtubules, and occasional neurofilaments and polysomes. Dense-cored vesicles and lysosomal structures were also present and appeared to be formed locally, at least in part from reticulum. The possible roles of the various forms of agranular membranous components are discussed and it is suggested that structures involved in both the assembly and degradation of membrane are present in the cone. The content of these growing tips resembles that in sensory neuron growth cones studied by others.     

PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Role of Non-coding RNAs in Axon Regeneration after Peripheral Nerve Injury

Peripheral nerve injury (PNI) may lead to disability and neuropathic pain, which constitutes a substantial economic burden to patients and society. It was found that the peripheral nervous system (PNS) has the ability to regenerate after injury due to a permissive microenvironment mainly provided by Schwann cells (SCs) and the intrinsic growth capacity of neurons; however, the results of injury repair are not always satisfactory. Effective, long-distance axon regeneration after PNI is achieved by precise regulation of gene expression. Numerous studies have shown that in the process of peripheral nerve damage and repair, differential expression of non-coding RNAs (ncRNAs) significantly affects axon regeneration, especially expression of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In the present article, we review the cellular and molecular mechanisms of axon regeneration after PNI, and analyze the roles of these ncRNAs in nerve repair. In addition, we discuss the characteristics and functions of these ncRNAs. Finally, we provide a thorough perspective on the functional mechanisms of ncRNAs in nervous injury repair, and explore the potential these ncRNAs offer as targets of nerve injury treatment.     

THE FINE STRUCTURE OF NEURONS

1. Thin sections of representative neurons from intramural, sympathetic and dorsal root ganglia, medulla oblongata, and cerebellar cortex were studied with the aid of the electron microscope. 2. The Nissl substance of these neurons consists of masses of endoplasmic reticulum showing various degrees of orientation; upon and between the cisternae, tubules, and vesicles of the reticulum lie clusters of punctate granules, 10 to 30 mµ in diameter. 3. A second system of membranes can be distinguished from the endoplasmic reticulum of the Nissl bodies by shallower and more tightly packed cisternae and by absence of granules. Intermediate forms between the two membranous systems have been found. 4. The cytoplasm between Nissl bodies contains numerous mitochondria, rounded lipid inclusions, and fine filaments.     

CHEMICAL CHARACTERIZATION OF THE ISOLATED CELL SURFACE OF AMOEBA

The cell surface has been isolated from uninucleate, freshwater, phagocytic amoebae by a new procedure. Several criteria were employed to demonstrate purity of the cell surface fraction. All morphological components of the tripartite surface were present in the isolated surface and the weight of the isolated surface was quantitatively accounted for by the components analyzed. Chemical analyses showed the presence of lipid, protein, and carbohydrate. Mannose was the predominant neutral sugar. Analyses for three different strains of Amoeba were similar. Phosphate was found to be the major anionic group in the cell surface material. Sulfate, uronic acid, sialic acid, muramic acid, and nonamidated glutamic acid and aspartic acid were absent. Evidence is presented suggesting that the phosphate is associated with an unidentified nonreducing polyol.     

Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.     

Volume Regulation of Nerve Terminals

Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.     

Axon Regrowth during Development and Regeneration Following Injury Share Molecular Mechanisms

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Highlights? Initial axon outgrowth and regrowth during remodeling are mechanistically distinct ? The nuclear receptor UNF is necessary for axon regrowth but not for initial growth ? TOR is also required for developmental regrowth but not for initial axon outgrowth ? UNF promotes axon regrowth via the TOR pathway and guidance via unknown mechanisms     

THE SURFACE PROPERTIES OF ISOLATED RAT BRAIN MYELIN: A MICROELECTROPHORETIC STUDY

Abstract— The surface properties of isolated rat brain myelin have been investigated by the method of particle microelectrophoresis. The mobility has been measured as a function of ionic strength and composition of the buffer, the results suggesting the presence of areas of non-ionogenic material. Antisera against ganglioside and myelin produce a fall in mobility whereas antiproteolipid apoprotein produces a slight increase but antibasic protein and anticerebroside sera had no effect on mobility. Of the lipases studied only phospholipase D produced a clear increase in mobility. From this preliminary study some indications of the asymmetric disposition of components in the myelin sheath may be derived. The ganglioside appears to be wholly located at intraperiod line interface, along with some fraction of the galactocerebroside and phosphatidylcholine. There appears to be few amino groups, but SH groups are present.     

THE FINE STRUCTURE OF SYMPATHETIC NEURONS IN X-IRRADIATED FROGS

The effects of whole body x-irradiation on the fine structure of sympathetic neurons were studied in 15 unanesthetized adult frogs (Rana pipiens), as seen at intervals ranging from 1 hour to 2 weeks after single exposures to 1000 r and 2000 r. Using standard procedures, the lumbar sympathetic ganglia of experimental and 20 control animals were prepared for electron microscope examination. Radiation produced conspicuous but irregular and variable deterioration, swelling, and clearing of neuronal lysosomes. These changes may have been due to an increased permeability of lysosomal membranes, causing the entry of fluid into lysosomes and their swelling and deterioration, but a pronounced escape of lysosomal enzymes into the cytoplasm was questionable. Less frequent were the dilatation and the parallel layering or complete fusion and tight packing of the rough-edged endoplasmic reticulum. The number of vacuoles, probably derived from Golgi cisternae, was somewhat increased. These vacuoles were conjectured to serve the "sequestration" of damaged cytoplasmic areas. Abnormal amounts of presumptive glycogen granules occupied some axons of myelinated and unmyelinated fibers, especially of presynaptic nerve fibers. This was assumed to be due to a decreased breakdown of glycogen and probably caused the interruption of the transmission of nerve impulses in presynaptic fibers. The maximal incidence of these alterations seemingly occurred 8 days after exposure to 1000 r, and 1 hour after x-irradiation with 2000 r. Signs of recovery appeared 2 weeks after exposure to 2000 r.     

Conserved Dopamine Neurotrophic Factor-Transduced Mesenchymal Stem Cells Promote Axon Regeneration and Functional Recovery of Injured Sciatic Nerve

Peripheral nerve injury (PNI) is a common disease that often results in axonal degeneration and the loss of neurons, ultimately leading to limited nerve regeneration and severe functional impairment. Currently, there are no effective treatments for PNI. In the present study, we transduced conserved dopamine neurotrophic factor (CDNF) into mesenchymal stem cells (MSCs) in collagen tubes to investigate their regenerative effects on rat peripheral nerves in an in vivo transection model. Scanning electron microscopy of the collagen tubes demonstrated their ability to be resorbed in vivo. We observed notable overexpression of the CDNF protein in the distal sciatic nerve after application of CDNF-MSCs. Quantitative analysis of neurofilament 200 (NF200) and S100 immunohistochemistry showed significant enhancement of axonal and Schwann cell regeneration in the group receiving CDNF-MSCs (CDNF-MSCs group) compared with the control groups. Myelination thickness, axon diameter and the axon-to fiber diameter ratio (G-ratio) were significantly higher in the CDNF-MSCs group at 8 and 12 weeks after nerve transection surgery. After surgery, the sciatic functional index, target muscle weight, wet weight ratio of gastrocnemius muscle and horseradish peroxidase (HRP) tracing demonstrated functional recovery. Light and electron microscopy confirmed successful regeneration of the sciatic nerve. The greater numbers of HRP-labeled neuron cell bodies and increased sciatic nerve index values (SFI) in the CDNF-MSCs group suggest that CDNF exerts neuroprotective effects in vivo. We also observed higher target muscle weights and a significant improvement in muscle atrophism in the CDNF-MSCs group. Collectively, these findings indicate that CDNF gene therapy delivered by MSCs is capable of promoting nerve regeneration and functional recovery, likely because of the significant neuroprotective and neurotrophic effects of CDNF and the superior environment offered by MSCs and collagen tubes.     

FINE STRUCTURE OF CELLS ISOLATED FROM ADULT MOUSE LIVER

Suspensions of isolated cells in various media were prepared from mouse liver which had been perfused via the portal vein with a buffered medium containing 0.40 M sucrose, and the cells were fixed with osmium tetroxide. Their fine structure was compared with that of cells from perfused and unperfused intact liver. Perfusion brought about some separation of the cells with little or no damage to cell membranes. When cells were dispersed in 0.40 M sucrose medium the plasma membranes partially broke down, and this disintegration was increased by transfer of the cells to media of lower osmolarity. This is presumed to account for the loss of permeability barriers which occurs in isolated liver cells. The mitochondria in cells of perfused liver and in isolated cells remained elongated, but the layers of many mitochondrial cristae became separated by clear spaces. When cells were transferred to a medium containing 0.20 M sucrose, the mitochondria swelled and became spherical, often with displacement of the swollen cristae to the periphery. In a medium containing 0.06 M sucrose and 0.08 M potassium chloride the outer chamber of many mitochondria became swollen with displacement of the mitochondrial body to one side to give a crescent-shaped appearance. These changes in mitochondrial morphology are discussed in relation to the metabolic activity of isolated liver cells.     

DNA CONTENT OF PURIFIED PREPARATIONS OF MOUSE PURKINJE NEURONS ISOLATED BY A VELOCITY SEDIMENTATION TECHNIQUE

Abstract— The DNA content of mouse Purkinje neurons was investigated employing a biochemical approach. Material for the biochemical assay was provided by means of a sedimentation velocity separation technique which yields bulk quantities of well-preserved Purkinje perikarya in a high degree of purity. The same amount of DNA/cell was recorded for mixed cerebellar cell somata (7·6 ± 0±2 pg/cell), as for the Purkinje perikarya enriched fractions (7±2 & 0·2 pg/cell). No evidence could be found for the existence of a tetraploid DNA complement in mouse Purkinje neurons despite indications to the contrary from a parallel cytophotometric study.     

P物质对大鼠分离的DRG细胞GABA激活电流的抑制作用

本文就用全细胞膜片箝技术,在新鲜分离的大鼠ＤＲＧ细胞上证明,在部分细胞Ｐ物质（１０＾－７－１０＾－５ｍｏｌ／Ｌ）可引起浓度依赖性的内向流（４／２６）;在多数细胞虽未检测到ＳＰ引起的膜电流,但却能对ＧＡＢＡＡ受体激活介导的膜内向流产生抑制效应（１８／２２）,并有加速去敏感的作用。本文就有关ＳＰ以ＧＡＢＡ激活电流抑制效应的可能意义进行了讨论。     

Isolation of Nerve Terminals from Crustacean Muscle

A method for the isolation of gamma-aminobutyric acidergic (GABAergic) and glutamatergic terminals from crustacean muscle was developed, using differential centrifugation and sucrose density gradient centrifugation. Individual fractions were assessed using a variety of markers. One fraction was isolated which showed 40-fold purification of glutamate decarboxylase with a yield of 12%. This fraction was enriched in GABA, glutamate, glutamate dehydrogenase, and 5'-nucleotidase, but not in NADPH cytochrome c reductase. This fraction possessed an uptake system for GABA and glutamate with apparent kinetic constants of Km = 50 microM, Vmax = 250 pmol/min/mg of protein and Km = 183 microM, Vmax = 219 pmol/min/mg of protein, respectively. Electron microscopy showed nerve terminal profiles and a heterogeneous population of membrane vesicles. This fraction contained 3.4 nmol ATP/mg of protein which was stable for 30 min at 12 degrees C, and was also able to synthesise ATP from exogenous adenosine. The terminals released labelled GABA and glutamate in a Ca2+-dependent fashion on depolarisation. No release of ATP was detected. It is concluded that viable nerve terminals have been isolated which could be used as model systems for the study of GABAergic and glutamatergic neurochemistry.