摘要:

AbstractThe purpose of this study was to determine the structural basis for the hypermotility and impaired growth cone elongation of the homozygous weaver (wv/wv) mouse cerebellar granule cell neurons in culture. Two‐day cultures of dissociated week‐old normal (+/+) andwv/wvcerebellum were processed for electron microscopy of intact cells and cytoskeleton. Serial sections parallel to and starting from the substrate were examined. Fine‐caliber neurites of normal granule cells are packed with parallel arrays of microtubules at all levels. Microfilament‐packed microspikes are present at substrate level emanating from a cortical microfilament lattice at the terminus of neurites of varying length. Homozygous weaver granule cells at substrate level have lateral cytoplasmic extensions along the neurite. Microtubules that curve throughout the neurite are separated by cytoplasm. The lateral extensions and growth cone cytoplasmic projections contain microfilaments and occasionally microtubules. Microfilament‐packed microspikes are not observed. Immunofluorescent detection of actin confirms the ultrastructural picture. A hallmark of thewv/wvcytopathology is the presence of large numbers of coated vesicles throughout the neurite shaft at the cell‐substratum interface. These are rare at similar locations in +/+ neurites. We hypothesize that reduced tension in the growth cone and neurite owing to the presence of lateral extensions and absence of stable microspikes are responsible for the impaired elongation and hypermotility of mut

ISSN:0360-4012    

DOI:10.1002/jnr.490130112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractThe object of this study was to document and analyze local regulation by nerve growth factor (NGF) of neuronal growth cone properties and to explore the possible diversity of this effect in various NGF‐responsive preparations. In particular, scanning electron microscopy was used to characterize the morphology of neuronal growth cones in cultures of dissociated chick embryo dorsal root ganglia (DRG) under conditions of continuous NGF exposure, withdrawal of NGF for 5–6 hr, and restoration of NGF for various times. Comparison was made with similarly manipulated cultures of dissociated newborn rat sympathetic ganglia and neurite‐bearing PC12 pheochromocytoma cells. The growth cones of most of the continuously NGF‐treated DRG neurons (cultured on poly‐L‐lysine or collagencoated glass coverslips) had relatively compact central flattened areas and numerous prominent filopodia. Withdrawal of NGF resulted in a marked spreading of the central growth cone area so that the average maximum width of this structure increased by about threefold as compared to nondeprived cultures. The mean number and lengths of filopodia were unaffected. Restoration of NGF brought about, over a time course of tens of minutes, a return of the original type of growth cone morphology. Rather different responses were observed for the sympathetic neuron and PC12 cultures. Here, surface ruffles, only rarely seen in the chick cultures, were a major feature of the growth cones, whereas filopodia, though present, were less prominent. Removal of NGF led to loss of ruffles and to rounding up of the growth cones; NGF readdition elicited a rapid (<30 sec) reinitiation of ruffling and a more gradual (over tens of minutes) respreading of growth cones. These findings illustrate not only that NGF can regulate growth cone properties, but also that there is a diversity as to how this is manifested. Possible mechanisms and biological roles for this regulation ar

ISSN:0360-4012    

DOI:10.1002/jnr.490130113

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractThe response of growth cones from embryonic chick dorsal root ganglia to a patterned substrate of adsorbed nerve growth factor (NGF) was studied. The patterned substrate presented growth cones with an adsorbed NGF pattern and NGF‐free substrate. NGF‐responsive growth cones from 7 and 9 day ganglia could not proceed onto NGF‐free substrate, reproducing the adsorbed NGF pattern. NGF‐unresponsive growth cones from 17 day ganglia did not display any preference for adsorbed NGF or NGF‐free substrata, which resulted in neurites not reproducing the adsorbed NGF pattern. Neurite outgrowth from NGF responsive 7‐day ganglia onto a patterned NGF substrate, in NGF‐containing medium, was radially symmetrical, exhibiting no growth cone response to the patterned NGF substrate. The lack of NGF‐responsive growth cone extension onto NGF‐free substrate indicates that NGF is a requirement for neurite elongation. If NGF is withdrawn from growth cones by microperfusion, neurite elongation ceases. Thus, an adsorbed pattern of NGF may be duplicated because growth cones are not able to extend onto NGF‐free substrate, since NGF is a requirement for neurite elongation. These results indicate that substrate adsorbed NGF can support neurite formation and elongation as well as guide the direction of

ISSN:0360-4012    

DOI:10.1002/jnr.490130114

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractNeurite outgrowth is guided by narrow pathways of bioactive laminin. These pathways are created by ultraviolet light irradiation of laminin‐coated coverslips masked with electron microscope grids. Patterned outgrowth of neurites is independent of gross mechanical guidance and guidance caused by substrate limitation. Cells on unirradiated laminin are less readily displaced by shear forces than cells on irradiated laminin. This study suggests that ultraviolet light alters the adhesive properties of laminin and that differential cell‐substratum adhesion may guide extending neurites on the purified naturally occurring substance, lami

ISSN:0360-4012    

DOI:10.1002/jnr.490130115

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractThe motility of fish epidermal cells (keratocytes) was examined in the presence and absence of DC electric fields. In fields of 0.5–15 V/cm, single epidermal cells, cell clusters, and cell sheets migrate toward the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. During locomotion, keratocytes extend broad lamellipodia, which contain a pervasive actomyosin network. The lamellipodial extension and locomotion of keratocytes are reversiby inhibited by a variety of calcium channel antagonists, whereas thir motility is unaffected by hyperpolarizing and depolarizing (low and high K+) media. Microtubule disassembly has no effect on cell morphology, motility or the ability of the cells to be guided by a DC electric field. Using these results, the role that membrane‐regulated Ca2+influx may play in generating cytoskeletal and protrusive activity in keratocytes and other cells is discussed in some detail. Mechanisms by which an external electric field may bias transmembrane ion fluxes and thereby control cell locomotion are also exami

ISSN:0360-4012    

DOI:10.1002/jnr.490130116

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractMonopolar electric current pulses were focally applied through a micropipette to the growth cone ofXenopusembryonic neurons in culture. Application of the current directly in front of the growth cone modulated the rate of growth cone extension: Negative (sink) currents increased the growth rate, while positive (source) currents reduced the growth rate. When the currents were applied in a direction perpendicular to the direction of the neurite growth, both negative and positive currents produced inhibitory effects. Application of a negative focal current at a 45° angle with respect to the direction of neurite growth resulted in an oriented growth of the neurite toward the current sink. However, after the growth cone had been attracted to the vicinity of a current sink, further extension of the neurite was inhibited. These current effects occur rapidly after the onset of the current application, and are at least partially reversible within 1 hr after the termination of the current. The magnitude of current density required to induce a growth cone response was found to be in the order of a few pA per μm2. Such current density is close to that which may be generated at the muscle cell surface by the acetylcholine molecules released from the growth cone during the early phase of nerve‐muscle cont

ISSN:0360-4012    

DOI:10.1002/jnr.490130117

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractThe rate and direction of neurite growth have been shown in a number of studies to be determined by the distribution of adhesive sites on the growth cone. Recent evidence showing that the application of extrinsic electric fields can redistribute membrane molecules and alter both the rate and direction of neurite growth have raised the question whether endogenous electric fields might be produced by steady currents in growth cones. To investigate this question, we have devised a novel circularly vibrating microprobe capable of measuring current densities in the range of 5 nA/cm2(near the theoretical limit of sensitivity), with a spatial resolution of 2 μm. The design of this device and the development of a novel algorithm for computing current vectors on‐line is described. Using this probe we have found that cultured goldfish retinal ganglion cell growth cones generate steady inward currents goldfish retinal ganglion cell growth cones generate steady inward currents at their tips. The measured currents, in the range of 10–100 nA/cm2, appear to flow into the filopodia at their tips and back outward near the junctures of the filopodia and the growth cone. The currents appear to be produced only during active growth. Ion substitution experiments support the conclusion that the majority of this current is carried by Ca2+ions, which we postulate flow through a population of activated voltage‐sensitive Ca2+channels located on the filopodial tips. Calculation of the transmembrane current density (4 × 10−6nA/cm2) leads to an estimate of channel density (10 channels/μm2) in close agreement with the measured density of Ca2+channels in other systems. The assumption that calcium channel proteins are conveyed to nerve terminals by active transport, whereas sodium channel proteins are conveyed passively by a slower somatofugal diffusion process [Strichartz et al, 1984], would explain why developing neurons tend to display Ca2+‐sensitive electrogenesis at their growing tips, and Na+‐sensitive action potentials later in development.In order to gain some insight into the possible role of these steady growth currents, we estimated the membrane depolarization and axial voltage gradient they produce. It is likely that the currents produce sufficient membrane depolarization (≅ 4 mV) to cause autogenous activation of ion channel permeabilities. Similarly, the axial voltage gradient (≅4 mV/cm) would be expected to move intracytoplasmic vesicles by electrophoresis at a rate (20–40 pm/hr) very close to that at which the filopodia are observed to grow. These considerations lead us to propose that growth cone currents set up both a Ca2+gradient and an electric field that play significant roles in growth. These are likely to include both the transport and exocytotic fusion of vesicles into growing membrane, as well as the alignment of molecules involved in contraction and adhesion. A steady calcium current would also be associated with the spontaneous release of neurotransmitter from the growth cone, as recently observed [Young and Poo, 19831] and a mechanism is proposed whereby this release might serve to autofocus receptor molecules at sites of contact with target cells.In order to determine whether the extracellular electric fields produced by the measured growth cone currents might be of sufficient magnitude to affect the distribution of surface membrane molecules by external lateral electrophoresis, we applied focal electric fields to growth cones with a micropipette. The observed field strengths necessary to affect filopodial orientation (70‐350 mV/cm) were several orders of magnitude larger than those produced by growth cone currents. We conclude that the physiological effects of growth cone currents are confined to the growth cone, wher'e they may play a significant role in the fusion of new membrane, the release of transmitter, and the transport and alignment

ISSN:0360-4012    

DOI:10.1002/jnr.490130118

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractWhen plated in cell culture,Helisomaneurons extend new neurites with large growth cones. These growth cones progress from actively growing organelles to stable structures over a period of several days. The ability to distinguish morphologically between these growth states has provided the opportunity to test whether changes in growth status are correlated with changes in the ionic properties of the growth cone membrane. This investigation has begun to study these changes by examining single channel ionic currents using patch clamp recording techniques and has demonstrated the presence of at least one ion channel having a conductance of 70 pS in the membrane ofHelisomagrowth cones. Recordings from cell‐attached patches show that this channel is normally active in growing growth cones, whereas channel activity is absent in recordings from stable growth cones. The absence of channel activity in growth cones that have reached the stable state is not due to the loss of channels from these growth cones. Rather, the channel is present in an inactivated state. The activity of this channel is not dependent on the presence of Ca, as channel activity persists when Ca is removed from the solution that bathes the internal surface of the membrane. These data demonstrate a correlation between channel activity and the growth state of neuronal growth cone

ISSN:0360-4012    

DOI:10.1002/jnr.490130119

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractLarge growth cones were produced in vitro by nerve growth factor (NGF) treatment of multinucleate cells produced by chemical fusion of cells of the neuron‐like clone PC12. These endings were studied both at the light microscopic and ultrastructural levels. The activity of ionic channels at growth cones was recorded with intracellular microelectrodes, patch recording of single channels, and whole cone recording from mechanically isolated growth cones. Morphologically, these large growth cones were characterized by the presence of microspikes and filopodia, by the presence of actin demonstrated immunohistochemically, and by the presence of catecholamine fluorescence. At the ultrastructural level they contained a broad spectrum of organelles with a distribution characteristic of neuronal growth cones, including dense core vesicles, abundant smooth membrane cisternae, microtubules, and a filamentous network. The presence of channels capable of generating action potentials was revealed by intracellular microelectrode recording from the growth cone in the presence of locally applied tetraethylammonium (TEA). TEA appeared to block outward current channels that could effectively shunt inward current activated by depolarization. Action potentials elicited by depolarizing current in the presence of TEA could be blocked reversibly by Cd2+, a specific blocker of Ca channels. These action potentials were often followed by a long after‐hyperpolarization lasting hundreds of milliseconds. This after‐hyperpolarization was similar to that recorded in the cell body of PC12 cells where it appears to be mediated by Ca‐activated K current. Single channel recording from outside‐out excised patches of membrane from the growth cones perfused with KF revealed the presence of voltage sensitive Na channels, Ca‐activated K channels, and K channels resembling delayed rectifier K channels. Macroscopic currents recorded from mechanically isolated growth cones in the “whole cone” configuration showed rapid inward currents at potentials ≥ ‐40 mV, followed by delayed outward currents at more positive potentials, a finding providing additional evidence for the presence of Na and K chann

ISSN:0360-4012    

DOI:10.1002/jnr.490130120

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY

摘要:

AbstractIntracellular recordings were obtained from growing tips of regenerating giant axons in the lamprey spinal cord, the recording sites verified by Lucifer yellow injection. In the presence of extracellular Ba++(3–6 mM), tetraethylammonium (10–15 mM), and 4‐aminopyridine (4–6 mM), action potentials showed prolonged plateaus. The fast initial phase of the action potential, but not the plateau (Ba++‐spike), was blocked by tetrodotoxin (10−6gm/ml). The Ba++spike was associated with increased membrane conductance and could be terminated with hyperpolarizing current pulses. Normal axons did not generate similar Ba++spikes. However, TTX‐resistant, voltage‐dependant conductance changes could be elicited in normal axons if much higher concentrations of Ba++(18–30 mM) were used. Their rate of rise was slower than in regenerating axons (0.6 V/sec vs 3.2 V/sec; n = 5), and the response did not outlast the current pulse. The Ba++responses in normal and regenerating axons were blocked by ions known to block voltage‐gated Ca++conductances (Co++, Ni++, or Cd++). Therefore, these spikes probably represent Ba++entry through voltage‐dependent Ca++channels, suggesting the presence of a higher‐than‐average voltage‐dependent Ca++conductance in the growing axon. However, Ca++‐dependent spikes could not be obtained under any conditions in either normal or regenerating axons. Simultaneous intracellular recordings from growth cones and axons indicated that the Ba++spike was initiated, in most cases, at the growth cone. The Ba++spikes were recorded in regenerating axons for as long as 50 days following cord transection and were not correlatable with the “dying‐back” phenomenon in cut axons, which usually is over before day 6.The concept of a higher‐than‐average voltage‐dependent Ca++conductance in growing tips of regenerating axons is in agreement with the hypothesis that Ca++is important in regeneration and that regeneration may be related to the

ISSN:0360-4012    

DOI:10.1002/jnr.490130121

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1985

数据来源: WILEY