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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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