HaydonMcCobb&Kater1984 - In order to determine the...

Unformatted text preview: In order to determine the stability of the tetraploid state in culture, we estab- lished single cell clones of tetraploid cells. Flow cytometry of serially subcul- tivated tetraploid clones demonstrated that the cells remained tetraploid without reversion to the diploid state [Fig. '1, E and F]. A diploid set of chromosomes is the usual composition of the eukaryotic genome. The diploid state is maintained by the reproduction of DNA and separa- tion of chromosomes during the mitotic cycle. The emergence of an increasing percentage of nuclei with 4C DNA con- tent in association with normal aging and with hypertension may be due either to arrest at the G2 stage of the mitotic cycle or to the development of true tetraploi- dy. The presence of reproductively via- ble tetraploid cells in the normal rat aorta could represent a stem cell population that proliferates preferentially during normal aging and that can be significant- ly expanded by hypertension. Alterna- tively, the increased frequency of these cells may be due to continuous conver- sion of diploid cells with an abnormal mitotic mechanism to the state of tetra- ploidy (5). The role of tetraploid smooth muscle cells in normal growth, aging, and disease is still unknown. Further characterization of the tetraploid cell population including its growth kinetics and interaction with diploid cells may increase our understanding of cellular polyploidy and of vascular physiology. ITZHAK D. GOLDBERG ELIOT M. ROSEN Joint Center for Radiation Therapy, Harvard Medical School, Boston, Massachusetts 02115 HOWARD M. SHAPIRO Center for Blood Research, ' Harvard Medical School LAWRENCE C. ZOLLER Department of Anatomy, Boston University School of Medicine, Boston 02118 KYL MYRICK STEPHEN E. LEVENSON Joint Centerfor Radiation Therapy, Harvard Medical School LISA CHRISTENSON Center for Blood Research, Harvard Medical School References and Notes 1. R. Ross and J. Glomset, N. Engl. J. Med. 295, 369 (1976). 2. T. B. Barrett, P. Sampson, G. K. Owens, S. M. Schwartz, E. P. Benditt, Proc. Natl. Acad. Sci. U.S.A. 80, 882 (1983). 3. I. D. Goldberg, H. Shapiro, M. B. Stemerman, J. Wei, D. Hardin, L. Christensen, Ann. N. Y. Acad. Sci., in press. 4. G. K. Owens and S. M. Schwartz, Circ. Res. 51, 280 (1982). 5. W. Y. Brodsky and I. V. Uryvaeva, Int. Rev. Cytol. 50, 275 (1977). 2 NOVEMBER 1984 6. Sorting was performed with a 5—W coherent argon ion laser (Innova Series) with ultraviolet optics set to a 350- to 354-nm broadband output reflector line, confocally focused to a 15-min wide spot. The photomultiplier tube was set to 330 at a gain of 10, with efiective volts 660 for deflection. A 418-nm long—pass fluorescent filter was used. 7. The cells were subcultured every 6 to 8 days in standard fashion. The medium was removed, and the dish was trypsinized [1:250 trypsin EDTA in buffered'saline (Gibco)]. The trypsini- zation was stopped by addition of fresh medium, and the cell suspension was counted. Cells were plated in fresh medium at an inoculation density Of 1 X 104 cells per square centimeter. Cultures were incubated at 37°C with 5 percent C02 and 95 percent humidified air. ‘ 8. H. M. Shapiro, D. M. Feinstein, A. S. Kirsch, L. Christensen, Cytometry 4, 11 (1983). Cells were centrifuged at 200g for 6 minutes and then suspended in nuclear isolation medium (0.6 per- cent NP-40, 1 mM CaClz, 21 mM MgC12, 0.2 percent bovine serum albumin in tris-bufiered isotonic saline, and 8.0 uM Hoechst dye 33342). The nuclear suspension was vortexed gently and cooled on ice for 10 minutes. Samples were filtered through a 50-pin pore size Nitex cloth, and the nuclei were syringed through a 25—gauge needle before flow cytometry. Flow cytometry was performed with a multiple—illumination wavelength, multiparameter flow c'ytometer sys- tem. 9. S. Gelfant, Symp. Int. Soc. Cell Biol. 2, 229 (1963); S. B. Fand, in Introduction to Quantita- tive Cytochemistry, G. F. Weid and G. F. Bahr, Eds. (Academic Press, New York, ed. 2, 1970), pp. 209—211; J. R. Shea, J. Histochem. Cyto- chem. 18, 143 (1970). Deoxyribonucleic acid content was measured with a Vickers M85 scan- ning and integrating microdensitometer in indi— vidual smooth muscle cells stained by the Feul- gen technique. Measurements ’of staining inten- sity were made at 565 nm, with a spot size of 2, delineating mask of A-2 (enclosing one cell per measurement), bandwidth of 10, and objective of 40. For each cell population, 200 cells were measured. The data shown are presented as integrated extinction, which represents absolute absorbance divided by a constant neutral densi- ty reading. 10. Subconfluent cultures were incubated for 30 minutes in the presence of Colcemid (Gibco) at a final concentration of 0.1 rig/ml. Cells were dislodged from the flasks by treating with 0.25 percent trypsin—EDTA and then centrifuged at 150g for 7 minutes. The supernatant was dis- carded, and the cell pellet was suspended in 75 mM KC1 solution and allowed to stand at room temperature for 10 minutes. The 'cells were centrifuged and then suspended in a 3:1 (by volume) methanol—acetic acid fixative. After 1 hour, two changes of fixative were made. Air- dried slides were prepared and were stained for 7 minutes in a solution (50 pug/ml) of quinacrine mustard (Sigma). Slides were mounted in tris- maleate buffer (pH 5.6) and were observed un- der a Leitz orthoplan fluorescence microscope equipped with an orthomat camera. Well banded (Q bands) metaphases were photographed. 1]. We thank R. Tantravahi of the Cytoge’netics Laboratory, Dana—Farber Cancer Institute, for the karylogic analysis, and S. DeMarco for manuscript preparation. This work was support- ed by NCI grant 5-P01-CA—12662, and NIH grant A600599. 4 May 1984; accepted 19 July 1984 Serotonin Selectively Inhibits Growth Cone Motility and Synaptogenesis of Specific Identified Neurons Abstract. The motile activity of growth cones of specific identified neurons is inhibited by the neurotransmitter serotonin, although other identified neurons are unaffected. As a consequence, affected neurons are unable to form electrical synapses, whereas other neurons whose growth is unafiected can still interconnect. This result demonstrates that neurotransmitters can play a prominent role in regulating neuronal architecture and connectivity in addition to their classical role in neurotransmission. The characteristic morphology and re- sultant connectivity of adult neurons is due to the cOmbined action of precisely timed intrinsic and extrinsic signals on individual neurons (1). Extrinsic signals arising from neighboring neurons can regulate neuronal architecture (2), al- though proximate regulatory agents are not yet defined. One suggestion is that “trophic” substances released from some nerve terminals can control the growth of adjacent neurons (3). In light of the demonstration that neurotrans- mitter can be released from growth cones of growing neurons in culture (4), a candidate for such a regulatory agent is the classical chemical transmitter itself (5). We now report that the neurotransr mitter serotonin can inhibit neurite out- growth. We demonstrate a growth inhibi- tion specific to individual growth cones by a time-lapse study of the large identi- fied neurons of the snail Helisoma. We also demonstrate that this inhibitory ac- tion of serotonin prevents the formation of electrical synapses between specific identified neurons with overlapping out- growth, while connections between neu- rons whose growth cones are unrespon— sive to serotonin continue to form (6). These experiments were performed on buccal ganglion neurons 5 and 19 and on pedal ganglion neuron P5, all of which have been studied in terms of growth and connectivity (6, 7). Individual neurons were removed from ganglia of adult snails and plated in cell culture (8, 9), where neurons undergo a characteristic sequence of outgrowth. Growth cones arise from the cell body and elaborate an extensive network of neurites for 3 to 4 days until a morphological steady state is attained (6). , The behavior of growth cones of indi- vidual identified neurons is readily ana- lyzed by time-lapse low-light video mi- croscopy (10). The activity of growth cones from Helisoma neurons character- istically consists of a probing of the environment by filopodia and a ruffling action of lamellipodia. Concurrently, the neurite extends continuously at a nearly 561 constant rate (Fig. 1A). At the end of the growth period a quiescent phase nerve terminal results that is characteristically bright and shows essentially no motile activity. Serotonin (Sigma) has a neuron-specif- ic inhibitory effect on neurite outgrowth. Initially we examined this phenomenon by adding a 40-511 dose of serotonin (final concentration of 10‘8 to 5 X 10—5M) to the culture medium. In the neurons 19 studied by this method, nine of ten cells treated with a range of concentrations from 10‘6 to 5 X 10—5M serotonin showed an abrupt cessation of filopodial probing, a decrease in ruffiing action, a decrease in the surface area of the growth cone, and, most strikingly, an inhibition of neurite elongation (Fig. 1A). Serotonin significantly reduced (t = 5.55, P < 0.0005) neuron 19’s rate of outgrowth from 11.32 i 4.67 rim/hour E .5 x: E 2 o .1: h 3 o 2 100 0 (mean : standard deviation, n = 11 growth cones) to —0.12 i 4.99 um/hour (n = 11 growth cones) (ll). Exposure to carrier medium (50 percent L-15) (8) or to medium adjusted to the pH of the serotonin solution did not cause any of these growth inhibitory efiects. Neuron 19in cell culture may have several dozen growth cones on its different neurites. Serotonin causes a systemic inhibition of a11 growth cones when applied to culture medium at concentrations at or above 10—7M. In contrast, serotonin has no elfect on neuron 5 (n = 10 growth cones from seven neurons) even at concentra- tions of 5 X 10‘5M. The growth cones of neuron 5 retained their normal structural features and continued to advance over the substrate; the rate of elongation (15.75 i 2.99 rim/hour, n = 10 growth cones) being unafiected by serotonin (15.55 i 3.64 rim/hour, n = 10 growth Neuron 5‘ [—3 40 minute Fig. 1. Photomicrographs of the behavior of intact (A) and isolated (B) growth cones from identified neurons in cell culture. (A) Intact growth cones that are connected to the neuron (not shown) displayed at frame intervals of 40 minutes. Intact growth cones produce neurite outgrowth at a constant rate before serotonin treatment. Application of serotonin (arrows) inhibits the neurite outgrowth of neuron 19 (top) but has no effect on neuron 5 (bottom). (B) A growth cone of neuron 19 isolated by severing the interconnecting neurite with a micropipette (scratch, Bl). Isolated growth cones are inhibited by pipette application of serotonin (82), but after withdrawal of the pipette they resume their characteristic activity (B3). Serotonin’s effects on isolated and intact growth cones are virtually indistinguishable, indicating that the growth cones of neuron 19 are directly responsive to 562 serotonin. Calibration bar, 10 um. cones) (Fig. 1A). Thus, serotonin specifi- cally inhibits the motile activity of neu- ron 19’s growth cones without afiecting the growth cones of neuron 5. To determine the site responsible for mediating the growth inhibition seroto- nin was focally applied to specific areas of membrane of neuron 19 while the motile activity of its growth cones was monitored. In these experiments it was important to apply serotonin for short periods of time (less than 50 minutes) to minimize its dispersal throughout the culture medium and thus effectively re- tain the focal nature of application. Be- cause of this time constraint it was not possible to analyze the rate of neurite elongation quantitatively; instead the in- hibitory actions of serotonin were as- sessed by observing the accompanying structural changes of neuron 195 growth cones. A growth-inhibitory response to serotonin (applied from a micropipette containing 10—6 to 1075M serotonin; wa- ter pressure head, 7 cm; tip diameter <10 um was detected only when the serotonin-containing pipette was placed adjacent to the growth cone. This growth-inhibitory response characteristi- cally consisted of filopodial and lamelli- podial retraction and a decreased surface area of the growth cone. Application of serotonin directly to growth cones al- ways (n = 10) inhibited motile activity (12), an effect which was reversed on withdrawal of the pipette. Focal applica- tion of serotonin to a neurite or to the soma (n = 7), on the other hand, never caused such inhibitory efl‘ects. It is possible to show the autonomous reaction of the growth cone to serotonin more dramatically by isolating these or- ganelles from the cell proper. By sever- ing the interconnecting neurite with the tip of a glass micropipette, one produces a viable isolated growth cone (13). Pi- pette application of serotonin to such isolated growth cones of neuron 19 al- ways resulted in the reversible retraction of filopodia and lamellipodia and de- creased extension activity (n = 8) (Fig. 1B). Thus, the growth cone itself can detect serotonin and transduce this re- sponse into a growth-inhibiting efl‘ect. Although'this does not exclude addition- al contributions from the rest of the cell, it seems reasonable to regard the growth cone, in this respect, as an autonomous organelle. The processes of growth and synapto- genesis are intimately intertwined. Since Helisoma neurons must be in an active growth state to form electrical synapses (6), we reasoned that serotonin may pre- vent the formation of these connections SCIENCE, VOL. 226 by virtue of its growth-inhibitory charac- teristics. As a simple test, neurons 5 and 19 were plated in cell culture under con- ditions known to result in the formation of electrical connections (6); additionally serotonin (10‘6M) was added to the me— dium (day 1) specifically to inhibit fur- ther outgrowth of neuron 19. Later, after the unafi‘ected, growing neurites of neu- ron 5 had overlapped the steady-state neurites of neuron 19 (Fig. 2A), the re- sultant connectivity was determined (days 3 and 4). In the presence of seroto- nin, neuron 19 never formed electrical connections with neuron 5 (mean cou- pling coefficient 0.00 i 0.00, n = 7) (Fig. 2B) (11, 14), whereas in control cultures electrical connections always formed (0.18 i 0.04, n = 9) (Fig. ZB) (15). In contrast, serotonin did not pre— vent the formation of electrical connec- tions between pairs of neuron 5, as would be predicted from this neuron’s resistance to serotonin’s growth efi'ects. In the presence of serotonin, pairs of neuron 5 always formed electrical con- nections (0.31 i 0.09, n = 5) (Fig. 2B). Given the previous demonstration that Helisoma neurons must be in an active growth state to form electrical connec- tions (6) these data indicate that by inhib- iting neurite outgrowth, serotonin is able to prevent neuron 19 from forming elec- trical connections with other neurons that are themselves competent to inter- connect. Serotonin’s effects are not restricted to neuron 19. We have examined the response of another neuron, P5. Rather than a total immobilization, as with neu- ron 19, or no effect, as with neuron 5, serotonin can transiently inhibit P5’s mo- tile activity (16). This range of elfects makes it plausible that, as is the case in chemical synaptic transmission, the na- ture of the transmitter’s effect on out- growth resides largely in the target neu- ron. Perhaps as more neurons are exam- ined some will be found whose growth is even enhanced by serotonin. Although serotonin’s locus of action seems restricted to the growth cone, the precise linkage with motility could take several forms; it may act by second messengers such as adenosine 3',5'-mo- nophosphate (cyclic AMP) or by altering transmembrane ion fluxes. Given the un- certainties of pharmacologically manipu- lating molluscan neurons, the best reso- lution to this Question may come from direct measurements of both of these candidates. At the other extreme from questions of cellular mechanisms is the role of neurotransmitters in regulating growth in adult nervous systems. Seroto- 2 NOVEMBER 1984 nin has been proposed to have a regula- tory role in neurogenesis (17). Could appropriately located release sites of sero- tonin in situ also regulate the quantity of neurite outgrowth and the actual form of a dendritic tree? Perhaps this is the basis for the kinds of effects seen on the plas- ticity of neuronal connections in the ver- 19M tebrate visual system by another mono- aminergic neurotransmitter, noradrena- lin (I8). Numerous investigations have demon- strated that macromolecules can play important roles in the elaboration of neu- ronal architecture and connectivity. Our demonstration that a neurotransmitter is Fig. 2. Serotonin prevents the formation of specific electrical connections. (A) Serotonin (IO—(’M) was added to the medium (day l) to inhibit the outgrowth from the previously growing neuron l9. Serotonin was added before neuron 19 began to overlap with the growing neurites of neuron 5 (top). By day 3 there was little additional outgrowth of neuron 19 compared with a major elaboration of the arbor of the neurons 5 (bottom). This continued outgrowth of neuron 5 caused an extensive overlap of neurites between both neurons 5 and between neurons 5 and 19. (B) Direct current applied intracellularly to neuron 5 does not pass into neuron 19 but does pass into the paired neuron 5. Thus, serotonin‘s inhibition of outgrowth of neuron 19 thereby prevents the formation of specific electrical connections. Calibration: Horizontal, 2 seconds. Vertical left: neuron 19, 5 mV; neuron 5, 20 mV; vertical right: top neuron 5, 10 mV; bottom neuron 5, 20 mV. 563 able to regulate growth and, consequent- ly, connectivity indicates that rather common simple molecules may also play prominent roles in regulating the pattern of neuronal connectivity. P. G. HAYDON D. P. MCCOBB S. B. KATER Department onoology, University of Iowa, Iowa City 52242 References and Notes 1. R. W. Gunderson and J. N. Barrett, J. Cell. Biol. 87, 546 (1980); E. R. Peterson and S. M. Crain, Dev. Brain Res. 2, 341 (1982), 2. G. Lynch, B. Stanfield, C. W. Cotman, Brain Res. 59, 155 (1973); D. H. Hubel, T. N. Wiesel, S. Le Vay, Phil. Trans. R. Soc. London Ser. B 278, 377 (1977); M. Shankland, D. Bentley, C. S. Goodman, Dev. Biol. 92, 507 (1982); S. Denis- Donini, J. Glowinski, A. Prochiantz, J. Neuro~ sci. 3, 2292 (1983). 3. C. E. Aguilar, M. A. Bisby, E. Cooper, J. Diamond, J. Physiol. (London) 234, 449 (1973). 4. R. I. Hume, L. W. Role, G. D. Fischbach, Nature (London) 305, 632 (1983); S. H. Young and M. M. Poo, ibid., p. 634. 5. Experiments with high concentrations of seroto- nin (10"T‘M) suggest that this neurotransmitter may affect the initiation of outgrowth [M. A. Kostenko, V. S. Musienko, T. I. Smolikhina, Brain Res.»276, 43 (1983)]. 6. R, D. Hadley, S. B.-Kater, C. S. Cohan, Science 221, 466 (1983). The formation of electrical connections critically relies on a spatial and temporal coincidence of neurite outgrowth from both partner neurons. 7. A. G. M. Bulloch and S. B. Kater, J. Neurophy« siol. 48, 569 (1982); R. D. Hadley and S. B. Kater, J. Neurosci. 3, 924 (1983); P. G. Haydon and S. B. Kater, Soc. Neurosci. Abstr. 9, 371 (1983). , 8. R. G. Wong, R. D. Hadley, S.. B. Kater, G. C. Hauser, J. Neurosci. 1, 1008 (1981). , 9. Central ganglia were treated with trypsin, the sheaths were cut with a tungsten microknife, and identified neurons were removed and trans- ferred to culture dishes with a glass micropi- pette, 10. S. B. Kater and ,R. D. Hadley, Trends Neurosci. 5, 80 (1982). The rate of neurite elongation was quantified by measuring the advance of the leading edge of the growth cone either directly from the television monitor of the video micros— copy system or from monochrome photographs taken at 20-minute frame intervals. The effect of ...
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