Synapse Formation by Neuroblastoma Hybrid Cells

M. NIRENBERG, S.P. WILSON, H. HIGASHIDA, A. ROTTER, K. KREUGER, N. Buss, R. RAY,
            J. KENIMER, M. ADLER, AND H. FUKUI
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health,
                   Bethesda, Maryland 20205

The most widely held hypothesis concerning the
mechanism of synapse formation during the last 20
years has been the chemoaffmity hypothesis of Sperry
(1963), i.e., that neurons distinguish appropriate from
inappropriate synaptic partner cells by interactions be-
tween molecules that code for cell recognition. Al-
though Sperry avoided detailed models, many in-
vestigators have assumed that neurons or other cells
that possess different cell-recognition molecules are
generated and that the cells then sort out in appropriate
sequences by differential adhesiveness before synaptic
connections form. Another quite different hypothesis is
that neurons may form relatively stable intercellular
attachments during differentiation and that environmen-
tal factors and/or transynaptic signals may regulate the
expression of genes and thereby determine the final
pathway for differentiation and the type of neuron and
synapse to be expressed. These hypotheses are not
mutually exclusive; both may be involved in the
assembly of synaptic circuits.

We have established clonal lines of neuroblastoma
and somatic hybrid cells that express neural properties
and form synapses with cultured striated muscle cells,
and we have used the cells as model systems for studies
on the effects of extracellular molecules on the expres-
sion of neural properties and on synaptogenesis. We
find that the expression of stimulus-secretion coupling
and synaptogenesis can be regulated in neuroblastoma
hybrid cells by receptor-mediated events that are cou-
pled to the activation of adenylate cyclase. We also find
that many ?S-labeled glycoproteins are affected when
cells are shifted to a synapse competent state.

RESULTS

Synapse Competent and Defective Cell Lines

Twenty-six neuroblastoma or hybrid cell lines were
tested for their ability to synthesize and release acetyl-
choline (ACh) and form synapses with cultured rat
striated muscle cells (Higashida et al. 1978; Wilson et
al. 1978). Synapses were detected by intracellular
recording of miniature end-plate potentials and evoked
muscle responses. Photomicrographs of cells from four
of the five cell lines found that form abundant synapses
are shown in Figure 1. NBrlO-A and NBr20-A cells
originated by fusion of mouse neuroblastoma N 18TG-2
(Minna et al. 1972) with clonal BRL-30E rat liver cells;
NCB-20, by fusion of N18TG- 2 cells with fetal
Chinese hamster brain cells; and NGl08-15 (Nelson et
al. 1976), by fusion of Nl8TG-2 with C6BU-I rat gli-

oma cells (Amano et al. 1974). Muscle responses to
ACh secreted by the neural cells were inhibited rever-
sibly by 5 pM d-tubocurarine and irreversibly by 10 nM
cr-bungarotoxin. Omission of Ca+ + ions in the me-
dium reduced the frequency of miniature end-plate po-
tentials (Nelson et al. 1978).

The expression of some neural properties by the cells
is dependent on prolonged elevation of cellular CAMP.
For example, untreated cells have few or no neurites
and relatively small soma. However, treatment of cells
for 7 days with 1 mM dibutyryl CAMP promotes
neurite extension, and decreasing the concentration of
fetal bovine serum reduces neurite retraction (Seeds et
al. 1970). Neurites are produced in profusion under
these conditions as. shown in Figure 1.

Other cell lines were found that synthesize ACh and
adhere well to myotubes, but do not form synapses.
The phenotypes of the cell lines are summarized in
Table 1. Cells from five lines synthesize ACh, take up
Ca+ + ions from the medium and secrete ACh when
depolarized by 80 mM K+ ions, possess small clear
vesicles 60 nm in diameter and large dense-core
vesicles 180 nm in diameter, synthesize and secrete a
protein that stimulates the aggregation of nicotinic ACh
receptors (AChR) on myotube plasma membranes, and
form many synapses with cultured myotubes. Cells
from three lines take up Ca+ + ions slowly and to only
a small extent, secrete relatively little ACh when
depolarized, and form few synapses with muscle cells.
Two cell lines lack functional voltage-sensitive Ca' +
channels and therefore do not take up 45Ca+ + or
secrete more ACh when depolarized and do not form
synapses. Cells from five lines take up Ca+ + when
depolarized, but do not respond to the increase in
cytoplasmic Ca+ + concentration by secreting ACh,
and form few or no synapses. These cells release ACh
into the medium in the basal unstimulated state but lack
a Ca + +-dependent ACh secretion reaction(s). Three
cell lines possess small clear vesicles, but lack both
large dense-core vesicles and functional AChR ag-
gregation protein, and form few or no synapses. Nine
additional cell lines have little or no choline
acetyltransferase activity and thus synthesize little or no
ACh and do not form synapses with striated muscle
cells.

     Regulation of Synaptogenesis
Neuroblastoma and hybrid cells have PGEr receptors
that mediate the activation of adenylate cyclase (Sharma

707


708                       NIRENBERG ET AL.

Figure 1. Neuroblastoma hybrid cells from lines that form many synapses with cultured myotubes were treatea for 7 days with
I mM dibutyryl CAMP, and the concentration of fetal bovine serum was reduced from 5% to I % between the fifth and seventh
days (H. Higashida et al., in prep.). Bar, 50 Gm.

et al. 1975b, 1977). Cells were cultured in the presence  dibutyryl CAMP or theophylline, on the percentage of
of PGEi to increase the endogenous rate of synthesis  myotubes tested that were innervated and the frequency
of CAMP and elevate intracellular levels of CAMP  of miniature end-plate potentials of myotubes. The
(Matsuzawa and Nirenberg 1975; Sharma et al. 1975a).  cells were cocultured and treated with the compounds
Treatment of cells with PGEi has no immediate effect  shown in Table 2 for 5-7 days before myotubes were
on cell membrane potential or rate of ACh secretion  assayed for synapses by intracellular microelectrode
(McGee et al. 1978).                recording. Treatment of cells with 1 mM dibutyryl
In Table 2 are shown the effects of treating  CAMP. 1 inM theophylline, or 10 PM PGE, increased
NG108-15 cells and rat myotubes with PGEi, or a  the percentage of muscle cells tested that were inner-
cyclic nucleotide phosphodiesterase inhibitor such as  vated from 15% to approximately 60% and also

                  Table 1. Cell Line Phenotypes
                  K+ -dependent        Vesicles       AChR
                   ACh                      large    aggrega-
            Cell    forma-   45ca+ +   13H]ACh    small    dense     don
            lines     tion    uptake     release    Cl.%%     core    protein    Synapse
    :       +      +++    +++     +      +       +       -I-++
                    +       +       +       +      +       +        +
            2       +                      +      +       +
            5       +      ++              +      +       +      - or +
            3       +      ++      f       +                    - or +
           9 -

Data from Higashida et al. (1978). Rotter et al. (1978). Wilson et al. (1978). and N.A. Busis, M.P.
Daniels. H.C. Bauer, P.A. Pudimat, P. Sonderegger. A.E. Schaffner. and M. Nirenherg.


SYNAPTOGENESIS BY NEUROBLASTOMA CELLS              709

Table 2. Effect of Culture Conditions
on Synaptogenesis and ACh Secretion
    by NG108-15 Cells

Culture
conditions

  %     Synaptic
myotubes   responses/
with      mini
synapses   myotube

Control
Dibutyryl CAMP (1 mM)
Theophylline (1 mM)
PGE, (10 PM)
PGEl (10 FM) +
theophylline (1 mM)

I5       0.7
55      14
64      10
63      11

98      32

Each value is the mean of values obtained from more
rhan 75 myotubes (Higashida er al. 1978).

resulted in 14- to 20-fold increases in the mean
miniature end-plate-potential frequencies (Higashida et
al. 1978). Presumably, each miniature end-plate poten-
tial reflects the response of a myotube to the spon-
taneous release of ACh from a single NG108-15 vesi-
cle. The effects of PGE, and theophylline were ad-
ditive or synergistic since 98% of the myotubes tested
were innervated and a 45fold increase in the miniature
end-plate-potential frequency was observed.

As shown in Figure 2, the effects of PGE,,
theophylline, or dibutyryl CAMP on synaptogenesis are
expressed slowly. Half-maximal increases in myotube
innervation required l-2 days of treatment. In other ex-
periments not shown here, cells were incubated with
PGEl, theophylline, dibutyryl CAMP, or PGEl and
theophylline for 5-7 days; the cells were then incubated
for an additional 4-14 days in the absence of the com-

ti                   I ' ' I '
a_  @ REGULATION OF SYNAPSE FORMATION @

2
& IOO-  PGEI tTpEOPHYLLlNE

F
zj ao-
Y
= 60-
0

DIWTYRYL CAMP 1
J) -I

Figure 2. Effects of PGEl, theophylline, or dibutyryl CAMP
on the percentage of myotubes innervated by NGl08-15 cells
are shown as a function of time of coculture. At zero time,
2 x lo5 mechanically dissociated NG108-15 cells were added
to each 35-mm petri dish, which contained well-differentiated
myotubes that had formed from myoblasts dissociated from
newborn rat hind-limbs during 9 days of culture. The cells
were cocultured in Dulbecco's modified Eagle's medium, 5 %
horse serum, 100 pM hypoxanthine, 16 pM thymidine. and the
following when indicated: Control, no addition; 1 mM theo-
phylline; 10 tiM PGEI; 10 PM PGE, and 1 mM theophylline;
or I mM dibutyryl CAMP. Synapses were detected by in-
tracellular microelectrode recording of miniature end-plate
potentials. Each point is the mean of values obtained from
IO-20 myotubes. (Data from Higashida et al. 1978.)

pounds to determine whether the effects on synapses
were reversible. On cessation of treatment, synapses
and ACh secretion rates slowly returned to control
values over a period of 7-10 days. Thus, the effects of
the compounds on synaptogenesis were expressed slow-
ly, and on withdrawal of the compounds, the effects
were reversed slowly. Treatment of NG108-15 cells
with PGEl and R020-1724, a CAMP phosphodiesterase
inhibitor, also increased the number of synapses. How-
ever, no reversal was detected due to withdrawal of
these compounds.

Effects of Culture Conditions on ACh Storage
         and Secretion

As shown in Figure 3A, intracellular ACh levels of
NG108-15 cells increased eightfold and threefold when
cells were treated for 3 or more days with 10 pM PGE,
and 1 mM theophylline or with 1 mM dibutyryl CAMP,
respectively. The cells were incubated with 30 pM
[3H]choline for 17 hours to label intracellular ACh.
Cells were then washed, and intracellular [3H]ACh was
extracted and quantitated. More than 90% of the in-
tracellular [3H]ACh was found to be particulate. Treat-
ment of cells for 5 or more days with dibutyryl CAMP
(Daniels and Hamprecht 1974) or with PGEI and theo-
phylline (Wilson et al. 1978) resulted in marked in-
creases in the abundance of small clear vesicles and
large dense-core vesicles. These results suggest that the
increase in intracellular ACh is due, at least in part, to

  NG,08-,5 CELLS B CELLULAR CAM C PROTEIN/FLASK
~~~~~~--:?

Figure 3. Effects of dibutyryl CAMP or PGEl and theophyl-
line on intracellular [3H]ACh levels of NG108-15 cells (A)
and intracellular CAMP (8); (C) cell proteins per flask are
shown as a function of time (Wilson et al. 1978). (A)
NG 1 OS- 15 cells were incubated in culture medium containing
approximately 30 FM (merItyl-3H]choline chloride for 17 hr to
enable cells to synthesize [`H]ACh. Cells were washed 3
times with isotonic buffer, and then intracellular r3H]ACh
was extracted (Toru and Aprison 1966) and separated from
other radioactive compounds by high-voltage paper electro-
phoresis (Potter and Murphy 1967). Where indicated, the
medium was supplemented with the following: Control, no
addition; 1 mM dibutyryl CAMP; or 10 CM PGEl and 1 mM
theophylline. (B) CAMP was extracted from cells and purified
as described by Matsuzawa and Nirenberg (1975); the amount
of CAMP in each sample was determined by inhibition of the
binding of [3H]cAMP to CAMP-dependent protein kinase.
(C) Protein was determined by a modification of the method
of Lowry et al. (1951).


710                       NIRENBERG ET AL.

an increase in the number of ACh storage vesicles in
ceils.

In the presence of 10 PM PGEI and 1 mM
theophylline, CAMP levels of NG108-15 cells increased
markedly and then decreased somewhat during the re-
mainder of the incubation period due to partial desen-
sitization of PGEl receptors (Fig. 3B). However,
CAMP levels of treated cells were higher than those of
control cells throughout the `I-day period examined.
The rate of cell division also decreased after the first
day of treatment with PGE, and theophylline; thus, the
rate of accumulation of protein per flask decreased in
the presence of PGE, and theophylline (Fig. 3C).

The effect of treating NG108-15 cells for 0, 1, 3, or
5 days with 1 mM dibutyryl CAMP on the ability of
cells to secrete ACh in response to a depolarizing
stimulus is shown in Figure 4A. The ceils were in-
cubated with 27 pM [3H]choline for 1 hour, washed by
perfusion, and then depolarized with 80 mM K+
(replacing 80 mM Na+) during the period shown.
[`H]ACh secreted into the medium was separated from
other 3H-labeled compounds and quantitated as
described by McGee et al. (1980). Logarithmically
`dividing, untreated control cells did not respond to
depolarization by secreting .ACh. However, cells
treated 1, 3, or 5 days with 1 mM dibutyryl CAMP
became increasingly responsive to depolarization with
respect to the amount of ACh secreted (McGee et al.
1978). Cells treated with PGE, and theophylline for 7
days secreted twice as much ACh when depolarized as
did cells that had been treated with dibutyryl CAMP (Fig.
4B).

REC+ILATm OF K'-CEFENCENT ~]ACH RELEASE FFCt.4 CELLS

LLS TREATED WITH MBUTYRIL- 8' '
CAMP FCft 0-5@AYS         CELLS TREATED VJTH Ffx,

MINUTES

Figure 4. [3H]ACh secretion by untreated control NG108-15
cells or by cells treated for 1, 3, or 5 days with I mM
dibutyryl CAMP (A) or by cells cultured for 7 days with or
without IO pM PGEl and I mM theophylline (B) is shown in
response to depolarization of cells with 80 mM K+ (replacing
80 rnr+i Na+ in the medium). The cells were incubated with
[merhyl-3H]choline chloride to enable them to synthesize
[3H]ACh. and then washed and depolarized. (Data from
McGee et al. [1978]; the figure was redrawn.) t3H]ACh was
separated from other radioactive compounds and quantitated
as described by McGee et al. (1980). The basal rate of
[3H]ACh secretion by unstimulated cells (5.4 mM K+) was
subtracted from each value shown.

Conditional Expression of Stimulus-Secretion
    Coupling in NBrlO-A Ceils

Depolarization of neuronal terminals is known to ac-
tivate voltage-sensitive Ca+ + channels; Ca + + ions
then flow into the neuronal terminals and increase the
rate of secretion of neurotransmitter. Thus, the effects
of treating cells for 7 days with PGEl and theophylline
or dibutyryl CAMP on Ca+ + uptake via voltage-
sensitive Ca+ + channels were determined. 45Ca+ + ions
rapidly bound to NBrlO-A cells initially and/or entered
cells incubated in medium containing 5.4 mM K+ ions,
but uptake soon plateaued. Depolarization of cells with
80 mM K+ resulted in a marked increase in 45Ca+ + up-
take. Half-maximal inhibition of depolarization-
dependent uptake of 45Ca+ + was obtained with
9x lo-' M D600; La3+, Co++, or Ni++ ions also in-
hibited K + dependent 45Ca+ + by cells. Electrophysio-
logical studies revealed Ca+ + spikes; action potentials
also were obtained when strontium or barium ions were
substituted for Ca+ + ions (not shown).

As shown in Figure 5, logarithmically dividing; un-
treated control NBrlO-A cells lacked functional
voltage-sensitive Ca + + channels and did not take up
4SCa+ + ions when cells were depolarized. However,
cells treated for 7 days with 10 pM PGEl and 1 mM
theophylline or with 1 mM dibutyryl CAMP took up
45Ca+ + via voltage-sensitive Ca+ + channels when cells
were depolarized with 80 mM K+ (Rotter et al. 1979).

<+-DEPENDENT SPECIFIC 45C02+
JPTAKE BY CELLS EXPOSED
TO DIFFERENT CONDITIONS

     PGEI +THEOPHYLLINE

Lm        CONTROLCELLS
11-1,1 1 I
2 4M1N:TES8 lo

Figure 5. Effect of culture conditions on the expression of func-
tional voltage-sensitive Ca+ + channels of NBrlO-A cells.
4sCa+ + uptake was due to activation of voltage-sensitive Ca+ +
channels of untreated, IogarithmicaJly dividing control NBrlO-A
cells and cells cultured for 6 days with 1 mM dibutyryl CAMP
or 10 FM PGEl and I mM theophylline. The cells were
depolarized with 80 mM K+ (in place of 80 mM Na+). Values
for 45Ca++ binding to cells and/or uptake at 5.4 mM K+,
which were not inhibited by 100 CM D600 and were not
mediated by voltage-sensitive Ca+ + channels were subtracted.
Ca+ + uptake dependent on depolarization was completely
inhibited by 100 pM D600. (Data from Rotter et al. 1979)


SYNAPTOGENESIS BY NEUROBLASTOMA CELLS

711

Figure 6. Acquisition of functional voltage-sensitive Ca' +
channels by NBrlO-A cells cultured with or without 1 mM
dibutyryl CAMP is shown as a function of time of treatment.
Ca+ + action potentials were detected by intracellular rnicro-
electrode recording; each point is the m&n of values obtained
from 20 NBrlO-A cells. Logarithmically dividine NBrlO-A cells
reached confluency on app;oximately ihe fourth day of culture.
The medium contained 5 pM tetrodotoxin to inhibit voltage-
sensitive Na+ channels. (Data from Rotter et al. 1979.)

Four methods were used to measure Ca+ + uptake de-
pendent on cell depolarization: 45Ca+ + flux was
measured; net uptake of Ca+ + ions from the medium
by cells was determined using a Ca+ +-specific elec-
trode; Ca+ + ion concentrations were determined by a
spectrophotometric assay with murexide (P. Darvenezia
and M. Nirenberg, in prep.); and Ca++ action potentials
were demonstrated by electrophysiological methods
with intracellular microelectrode recording. Results
obtained by each method showed that the expression of
voltage-sensitive Ca+ + channels is increased by treat-
ment of cells with compounds that elevate cellular
CAMP levels.

The acquisition of voltage-sensitive Ca+ + channel
activity by NBrlO-A cells measured with intracellular
microelectrodes is shown in Figure 6 as a function of
days of culture of cells with or without 1 mM dibutyryl
CAMP. Cells were stimulated electrically with
depolarizing pulses of current in the presence of 5 pM
tetrodotoxin to inhibit voltage-sensitive Na+ channels.
Voltage-sensitive Ca+ + channel activity was deter-
mined at the resting membrane potential and at a mem-
brane potential of - 90 mV adjusted with steady cur-
rent. Only 10% of logarithmically dividing, untreated
cells at low density possessed functional voltage-
sensitive Ca+ + channels, and the Ca+ + spikes that
were detected were relatively weak. The proportion of
cells with Ca+ + spikes increased from 10% to 50% as
untreated cells multiplied and formed confluent
monolayers, whereas 100% of the cells tested that were
treated for 3 days with dibutyryl CAMP generated Ca+ +
spikes when stimulated electrically. The mean maximum
rate of rise of Ca+ + spikes, a measure of Ca+ + chan-
nel activity, increased approximately 20-fold when cells
were treated with dibutyryl CAMP (M. Adler and M.
Nirenberg, iri prep.) (Fig. 6B). A smaller increase was
observed when logarithmically dividing, untreated con-

trol cells formed confluent monolayers, revealing a
small cell concentration or contact-dependent regula-
tion of Ca+ + channel expression. These and the
previous results show that when CAMP levels are
elevated, cells acquire at similar rates voltage-sensitive
Ca+ + channels, depolarization-dependent ACh secre-
tion, and synapses.

Nitrendipine and other dihydropyridines have been
shown to reduce voltage-sensitive Ca+ + channel activ-
ity in smooth muscle (for review, see Triggle and
Swamy 1983), and specific binding sites for [`H]ni-
trendipine have been found in smooth muscle (Bolger et
al. 1982). cardiac muscle (Ehlert et al. 1982), and brain
(Murphy and Snyder 1982). The receptors for nitren-
dipine are thought to be either part of the voltage-sen-
sitive Ca+ + channel complex or regulators of channel
activity. As shown in Table 3, few or no specific bind-
ing sites for [3H]nitrendipine were detected in washed
membranes prepared from logarithmically dividing
control NBrlO-A ceils, whereas specific binding sites
were found in membranes prepared from NBrlO-A
cells that had been treated for 8 days with 10 pM PGE,
and 1 mM theophylline (M. Nirenberg, L. Anderson,
and A. Rotter, in prep.). Scatchard analysis revealed a
single class of specific binding sites for [3H]nitrendi-
pine with a dissociation constant of 2 x lo-lo M, which
agrees well with values reported for other tissues
(Bolger et al. 1982; Murphy and Snyder 1982). The
maximum number of specific nitrendipine-binding sites
in membranes from NBrlO-A cells treated with PGE,
and theophylline was found to be 61 fmoles/mg of
membrane protein, which corresponds to approximate-
ly 16,000 sites for nitrendipine per cell. SB37-B
neuroblastoma x L cell hybrid cells synthesize ACh but
have little or no voltage-sensitive Ca+ + channel activ-
ity and thus do not secrete ACh when cells are depolar-
ized and do not form synapses with muscle cells. Spe-
cific binding sites for [3H]nitrendipine were not
detected in membranes prepared from SB37-B cells
grown with or without PGEl and theophylline. These
results show that the CAMP-dependent expression of

Table 3. PGEl and Theophylline Regulate
the Number of Nitrendipine-binding Sites
   Expressed by NBrlO-A Cells

Treatment of cells    fmoles 13H]nitrendipine
   (5-8 days)     specifically boundlmg protein
NBr IO-A
control                 <2
10 FM PGEl
+ I mM theophylline          I7
SB37-B
control                 <2
lo PM PGE,
+ 1 mM theophylline          <2

Data from M. Nirenberg. L. Anderson. R. Rotter. and R.
Ray (in prep.). The reaction-mixture components and con-
ditions were similar to those reported by Murphy and
Snyder (1982). Each reaction mixture contained 0.2 nM
f3H]nitrendipine and approximately 250 rg of protein (a
washed membrane fraction pelleted at 48,COOg).


712                      NIRENBERG ET AL.

voltage-sensitive Ca+ + channels in NBrlO-A cells is
accompanied by an increase in the number of specific
binding sites for [3H]nitrendipine and suggest that
elevation of cellular CAMP results in an increase in
the number of Ca+ + channels in cells.

CAMP-dependent Changes in Glycoproteins
       of NGlOS-15 Cells

Elevation of CAMP levels of neuroblastoma or hybrid
cells gradually results in the acquisition by cells of
functional action potential channels for Na+, K+
(Nelson et al. 1978). and Ca++ (Rotter et al. 1979),
which suggests that CAMP regulates the synthesis or
catabolism of channel molecules. We therefore deter-
mined the effect of PGE, on glycoproteins expressed

  2ocl

  140

  100

   60

   40

  200

m 140
k loo
2-
   60

   40

  200

   140

   100

   60

   40

CONTROL         PGEl

by NC 10% I5 cells. Logarithmically dividing control
NG108-15 cells and cells treated with 10 pM PGE, for
7 days were incubated for 18 hours with [`?S]methi-
onine. %-labeled glycoproteins soluble in 1 o/c Triton
X-100 were fractionated by chromatography on wheat
germ agglutinin-. Lens cufinaris agglutinin-, and Ric-
inus communis agglutininii-agarose columns. %-la-
beled glycoproteins were eluted with 0.5 M N-acetyl-
glucosaminq, 0.2 M cr-methylmannoside, and 0.2 M
lactose, respectively, desalted, and subjected to two-
dimensional polyacrylamide gel electrophoresis and
autoradiography (Fig. 7). Autoradiographs of duplicate
two-dimensional gels (triplicates in other experiments),
exposed for 7 hours and 1, 2, and 4 days, were com-
pared with the aid of a computer and programs de-
signed for analysis of two-dimensional gels (Vo et al.
1981). Computer-generated plots based on the 48 auto-
radiographs analyzed in this experiment were obtained.

    I
WHEAT GERM

III 1    I                      I    I IIIJ
5.0  5.5      6.0   6.5   5.0  5.5      6.0   6.5
          PH

Figure 7. Two-dimensional electrophoresis of
?S-labeled glycoprotein fractions. NGl08-15
cells treated for 6 days with IO pM PGEr and
logarithmically dividing, untreated control cells
were incubated for 18 hr in Dulbecco's mod-
ified Eagle's medium that contained 2% fetal
bovine serum and 5 PM L-[3sS]methionine (106
&i/ml of medium). The ceils were harvested
and washed in isotonic phosphate-buffered
saline. The pelleted cells were then lysed. and
the proteins were solubilized by the addition of
a solution containing 10 mM HEPES (pH 7.4);
I50 mM NaCI; I mM CaClz, 1 mM MgClz, 1
mM MnClz; I mM phenylmethylsulfonyl
fluoride, and 1% Triton X-100. Cell lysates
were centrifuged at 100.6OOg for 1 hr at 3oC.
and glycoproteins in the supernatant portions
were fractionated by wheat germ agglutinin-.
L. culinaris agglutinin-, and R. communis ag-
glutininn-agarose column chromatography. 35S-
labeled glycoproteins were eluted with 0.5 M N-
acetylglucosamine, 0.2 M a-methylmannoside.
or 0.2 M lactose, respectively, dialyzed against
0.01 M NH~HCOJ, and lyophilized. Two por-
tions from each sample were subjected to two-
dimensional polyacrylamide gel electrophoresis
on duplicate 7.5% acrylamide gels according to
the method of O'Farrell (1975) with I .O x lo6
cpm (IO-20 rg of protein) applied to each gel.
Autoradiographs were exposed for 7 hr and I,
2. and 4 days; those shown were exposed for 2
days. Arrows indicate some of the CAMP-de-
pendent differences in 35S-labeled glycopro-
teins. i.e., control cells vs. cells treated with
PGEr. (Data from K.E. Krueger, M.I. Miller.
and M. Nirenberg, in prep.)


SYNAPTOGENESIS BY NEUROBLASTOMA CELLS               713

In most cases, different species of 35S-labeled glyco-
proteins were eluted from the three lectin columns.
Most species of 35S-labeled glycoproteins were syn-
thesized by both control cells and PGEi-treated cells.
However, numerous CAMP-dependent changes in the
35S-labeled glycoproteins were found as summarized in
Table 4. Twelve %-labeled glycoproteins were ex-
pressed by PGEi-treated cells that were not detected in
extracts of logarithmically dividing control cells. In
addition, the radioactivities of 29 %-labeled glycopro-
teins expressed by cells treated with PGEi were 2.5 to
IO-fold higher than those of control cells. CAMP-
dependent decreases in the 35S-labeled glycoproteins
were also found. Forty-eight 35S-labeled glycoproteins
were expressed by control cells but not by cells treated
with PGEi. In addition, the radioactivities of 25 %-
labeled glycoproteins expressed by control cells were
2.5-6.5fold higher than those of cells treated with
PGEi. No changes in the 35S-labeled glycoproteins
were detected when cells were incubated with 10 pM
PGEi for 20 minutes (not shown here).

DISCUSSION

Neuroblastoma hybrid cells were obtained that syn-
thesize ACh and form synapses with cultured striated
muscle cells. Other cell lines were found that syn-
thesize ACh, adhere to myotubes, but form few or no
synapses. Different kinds of presynaptic defects were
identified, such as cell lines without voltage-sensitive
Ca+ + channel activity, large dense-core vesicles,
depolarization-dependent ACh secretion, or active pro-
tein that increases the aggregation of nicotinic AChR on
myotube plasma membranes.

No immediate effect of CAMP was detected on
voltage-sensitive Ca+ + channel activity, ACh secre-
tion, or the percentage of myotubes that were inner-
vated. In contrast, these properties and synaptogenesis
were regulated by prolonged elevation of CAMP levels
of hybrid cells. Activation of adenylate cyclase with 10
PM PGEl for 5 days, or treatment of cells with I mM
dibutyryl CAMP, resulted in lo-lOO-fold increases in
voltage-sensitive Ca+ + channel activity, 15-45-fold in-
creases in spontaneous secretion of ACh at synapses,
and 5-lo-fold increases in the number of muscle cells
innervated. Treated cells also contained considerably

more large dense-core vesicles and small clear vesicles
and had higher levels of intracellular ACh than did un-
treated cells.

Treatment of NBr'lO-A cells with 10 pM PGEi and
1 mM theophylline resulted in the appearance of speci-
fic binding sites for [jH]nitrendipine, a putative antag-
onist of voltage-sensitive Ca+ + channels. Specific ni-
trendipine-binding sites were not detected in SB37-B
cells, which lack voltage-sensitive Ca+ + channel activ-
ity. These results suggest that cells acquire more
voltage-sensitive Ca + + channels when cellular CAMP
is elevated for a number of days.

By regulating the expression of Ca+ + channels in
hybrid cells, CAMP would be expected to function as a
conditional switch that modulates the activities of many
Ca+ +-dependent reactions in the neural cells and
couples these reactions to the cell membrane potential
and to transynaptic or hormonal stimuli that affect
adenylate cyclase activity.

Glycoproteins of NG 1 OS- 15 hybrid cells grown in the
presence of [35S]methionine with or without 10 pM
PGEi were solubilized and fractionated by wheat germ
agglutinin, lentil lectin, or ricin column chromatog-
raphy and by two-dimensional gel electrophoresis. Ele-
vation of cellular CAMP levels resulted in the disap-
pearance of some glycoproteins, the appearance of new
glycoproteins of different relative molecular weights,
large increases or decreases in the radioactivities of
some "5S-labeled glycoproteins, and changes in the iso-
electric points of still other 35S-labeled glycoproteins.
These results agree with and extend previous reports of
differentiation-specific changes in neuroblastoma pro-
teins or glycoproteins (Truding et al. 1974, 1975;
Charalampous 1977; Garvican and Brown 1977;
Prashad et al. 1977; Akeson and Hsu 1978; Prashad
and Rosenberg 1978; Rosenberg et al. 1978; Zisapel
and Littauer 1979; Littauer et al. 1980; Atkinson and
Bramwell 1981; Sugiyama et al. 1980; Imada and Im-
ada 1982).

Treatment of neuroblastoma or hybrid cells with di-
butyryl CAMP has been shown to alter the levels of
some species of polysomal mRNA (Morrison et al.
1980; U.Z. Littauer, pers. comm.). When "undif-
ferentiated" neuroblastoma cells were shifted to a more
differentiated state by altering various growth condi-
tions, some species of polysomal poly(A)+ RNA were
no longer expressed (Felsani et al. 1978; Grouse et al.

Table 4. CAMP-dependent Changes in 35S-labeled Glycoproteins
            No. of "S-labeled glycoproteins

Effects of CAMP on         control         PGEI
`%-labeled glycoproteins      cells           cells
Increased expression        0           I2
Increased expression       29           29t
Decreased expression       48            0
Decreased expression       25           251
The experiment is described in Fig. 7 (K.E. Krueger, M. Miller, and M.
Nirenberg in prep.). The total number of 35S-labeled  roteins detected in
extracts of control cells was 317. Twelve additional b S-labeled proteins
were detected in extracts of cells that had been treated with PGEI,


714                       NIRENBERG ET AL.

1980) and many new species of poly(A)+ RNA ap-
peared (Grouse et al. 1980). Marked effects of CAMP
or dibutyryl CAMP on mRNA levels of other
eukaryotic cells have been reported (Derda et al. 1980;
Maurer 1981; Miles et al. 1981; Lamers et al. 1982;
Landfear et al. 1982; Wu and Johnson 1982;
Mangiarotti et al. 1983).

Twelve new 3SS-labeled glycoproteins were expressed
by cells treated with PGEI, and 2.5lo-fold increases
were found in the radioactivities of 29 additional 3sS-
labeled glycoproteins. Concomitantly, neuroblastoma
or hybrid cells acquired voltage-sensitive Na+ (Cat-
terall et al. 1973). K+ (Nelson et al. 1969), and Ca++
channels; Ca+ +-dependent K + channels; extended long
neurites; and acetylcholinesterase specific activity in-
creased (Blume et al. 1970). These results suggest that
some of the 35S-labeled glycoproteins that are expres-
sed to a greater extent in PGEl-treated cells may play a
role in some of the newly expressed neuronal mem-
brane functions. Synapse-defective cell lines that lack
channel activity or the ability to extend neurites and
other variant cell lines may provide a means of identify-
ing functions of some membrane proteins.

In summary, the results show that CAMP regulates
synaptogenesis by regulating the expression of voltage-
sensitive Ca+ + channels and suggest that CAMP affects
posttranslational modifications of some species of
glycoproteins and/or regulates gene expression.

REFERENCES

AKESON. R. and W.C. Hsu. 1978. Identification of a high
molecular weight nervous system specific cell surface
glycoprotein on murine neuroblastoma cells. Exp. Cell
Res. 115: 367,

AWANO, T.. B. HAMPRECHT, and W. KEMPER. 1974. High
activity of choline acetyltransferase induced in neuroblas-
toma x glia hybrid cells. Erp. Cell Res. 85: 399.
ATKINSON, M.A.L. and M.E. BRAMWELL. 1981. Studies on
the properties of hybrid cells. III. A membrane glycopro-
tein found on the surface of a wide range of malignant
cells. J. Cell Sci. 48: 147.

BLUME: A., F. GILBERT, S. WILSON, J. FARBER, R. ROSEN-
BERG,  and M. NIRENBERG. 1970. Regulation of
acetylcholinesterase in neuroblastoma cells. Proc. Nor/.
Acad. Sci. 67: 786.

BOLGER, G.T., P.J. GENGO. E.M. LUCHOWSKI, H. SIEGEL,
D.J. TRIGGLE. and R.A. JANIS. 1982. High affinity bind-
ing of a calcium channel antagonist to smooth and cardiac
muscle. Biochem. Biophys. Res. Commun. 104: 1604.
CATTERALL, W.A. and M. NIRENBERG. 1973. Sodium uptake
associated with activation of action potential ionophores of
cultured neuroblastoma and muscle cells. Proc. Narl.
Acad Sci. 70: 3759.

CHARALAMPOUS. F.C. 1977. Differences in plasma mem-
brane organization of neuroblastoma cells grown in the
differentiated and undifferentiated states. Arch. Biochem.
Biophys. 181: 103.

DANIELS, M.P. and B. HAMPRECHT. 1974. The ultrastructure
of neuroblastoma glioma somatic cell hybrids. J. Cell
Biol. 63: 69 I.
DERDA. D.F., M.F. MILES, J.S. SCHWEPPE, and R.A.
JUNGMANN. 1980. Cyclic AMP regulation of lactate
dehydrogenase: Isoproterenol and N6, 0' `-dibutyryl cyclic

AMP increase the levels of lactate dehydrogenase-5
isozyme and its messenger RNA in rat C6 glioma cells. J.
Biol. Chem. 255: 11112.

EHLERT, F.J., E. IWGA, W.R. ROESKE, and H.I. YAMA-
MURA. 1982. The interaction of [3H]nitrendipine with
receptors for calcium antagonists in the cerebral cortex
and heart of rats. Biochem. Biophys. Res. Commun.
  104: 937.

FELSANI, A., F. BERTHELOT, F. GROS, and B. CROIZAT.
1978. Complexity of polysomal polyadenylated RNA of
undifferentiated and differentiated neuroblastoma cells.
Eur. J. Biochem. 92: 569.

GARVICAN, J.H. and G.L. BROWN. 1977. A comparative
analysis of the protein components of plasma membranes
isolated from differentiated and undifferentiated mouse
neuroblastoma cells in tissue culture. Eur. J. Biochem.
76: 251.

GROUSE, L.D.. B.K. SCHRIER, C.H. LETENDRE, M.Y.
ZUBAIRI,. and P.G. NELSON. 1980. Neuroblastoma dif-
ferentiation involves both the disappearance of old and the
appearance of new poly(A)+ messenger RNA sequences
in polyribosomes. J. Biol. Chem. 255: 387 I.
HIGASHIDA, H., S.P. WILSON, M. ADLER, and M.
NIRENBERG. 1978. Synapse formation by neuroblastoma
and hybrid cell lines. Sot. Neurosci. Absrr.4: 591.
IMADA, S. and M. IMADA. 1982. Increase of a surface
glycoprotein by cyclic AMP in Chinese hamster ovary
cells. Dependence on cell-cell interaction. J. Biol. Chem.
  257: 9108.

LAMERS, W.H., R.W. HANSON, and H.M. MEISNER. 1982.
CAMP stimulates transcription of the gene for cytosolic
phosphoenolpyruvate carboxykinase in rat liver nuclei.
Proc. Natl. Acad. Sci. 79: 5137.
LANDFEAR, S.M., P. LEFEBVRE, S. CHUNG, and H.F. LODISH.
1982. Transcriptional control of gene expression during
development of Dictyostelium discoideum. Mol. Cell. Biol.
2: 1417.

LITTAUER, U.Z., M.Y. GIOVANNI, and M.C. GLICK. 1980. A
glycoprotein from neurites of differentiated neuroblastoma
cells. J. Biol. Chem. 255: 5448.
LOWRY, O.H., N.J. ROSEBROUGH, A.L. FARR, and R.J. RAN-
DALL. 1951. Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193: 265.
MANGIAROTTI. G.. A. CECCARELLI, and H.F. LODISH. 1983.
Cyclic AMP stabilizes a class of developmentally
regulated Dictyostelium discoideum mRNAs. Nature
301: 616.

MATSUZAWA, H. and M. NIRENBERG. 1975. Receptor-
mediated shifts in cGMP and CAMP levels in
neuroblastoma cells. Proc. Nut/. Acud. Sci. 72: 3472.
MAURER, R.A. 1981. Transcriptional regulation of the pro-
lactin gene by ergocryptine and cyclic AMP. Nurure
  294: 94.

MCGEE, R., P. SIMPSON, C. CHRISTIAN. M. MATA, P.
NELSON, and M. NIRENBERG. 1978. Regulation of
acetylcholine release from neuroblastoma- x glioma
hvbrid cells. Proc. Narl. Acud. Sci. 75: 1314.

MCKEE, R., C. SMITH, C. CHRISTIAN, M. MATA, P.
NELSON, and M. NIRENBERG. 1980. A new capillary tube
system for measuring the uptake and release of materials
from cultured cells. Anal. Biochem. 101: 320.
MILES, M.F., P. HUNG, and R.A. JUNGMANN. 1981. Cyclic
AMP regulation of lactate dehydrogenase: Quantitation of
lactate dehydrogenase M-subunit messenger RNA in iso-
proterenol and N6, 02'-dibutyryl cyclic AMP-stimulated
rat C6 glioma cells by hybridization analysis using a
cloned cDNA probe. J. Biol. Chem. 256: 12545.
MINNA. J., D. GLAZER, and M. NIRENBERG. 1972. Genetic
dissection of neural properties using somatic cell hybrids.
Nat. New Biol. 235: 225.

MORRISON, M.R., S. PARDUE. N. PRASHAD, D.E. CROALL.
and R. BRODEUR. 1980. Relative increase in polysomal
mRNA for RI CAMP-binding protein in neuroblastoma


SYNAF'TOGENESIS BY NEUROBLASTOMA CELLS               715

cells   treated   with 1,  ND-dibutyryl-adenosine
3',-5'-phosphate. Eur. J. Biochem. 106: 463.
MURPHY, K.M.M. and S.H. SNYDER. 1982. Calcium an-
tagonist receptor binding sites labeled with 13H]nitren-
dipine. Eur. J. Pharmacol. 77: 201.
NELSON, P.G., C. CHRISTIAN, and M. NIRENBERG. 1970.
Synapse formation between clonal neuroblastoma x
glioma hybrid cells and striated muscle cells. Proc. Nat/.
Acad. Sci. 73: 123.

NELSON, P.. W. RUFFNER, and M. NIRENBERG. 1969.
Neuronal tumor cells with excitable membranes grown in
vitro. Proc. Natl. Acad. Sci. 64: 1004.
NELSON, P.G.,  C.N. CHRISTIAN, M.P. DANIELS, M.
HENKART, P. BULLOCK, D. MULLINAX. and M.
NIRENBERG. 1978. Formation of synapses between cells of
a neuroblastoma x glioma hybrid clone and mouse
myotubes. Brain Res. 147: 245.
O'FARRELL, P.H. 1975. High resolution two-dimensional
electrophoresis of proteins. J. Eiol. Chem. 250: 4007.
POTTER, L.T. and W. MURPHY. 1967. Electrophoresis of
acetylcholine, choline, and related compounds. Biochem.
  Pharmacol. 16: 1386.

PRASHAD, N. and R.N. ROSENBERG. 1978. Induction of cyclic
AMP-binding proteins by dibutyryl cyclic AMP in mouse
neuroblastoma cells. Biochim. Biophys. Acra 539: 459.
PRASHAD, N., B. WISCHMEYER, C. EVETTS, F. BASKIN, and
R. ROSENBERG. 1977. Dibutyryl CAMP-induced protein
changes in differentiating mouse neuroblastoma cells. Cell
Differ. 6: 147.

ROSENBERG, R.N., C.K. VANCE, M. MORRISON, N.
PRASHAD, J. MEYNE, and F. BASKIN. 1978. Differentia-
tion of neuroblastoma, glioma, and hybrid cells in culture
as measured by the synthesis of specific protein species:
Evidence for neuroblast glioblast reciprocal genetic
regulation. J. Neurochem. 36: 1343.    .     -

ROTTER. A., R. RAY; and M. NIRENBERG. 1979. Regulation

of calcium uptake in neuroblastoma or hybrid &Is. A
possible mechansim for synapse plasticity. Fed. Proc.
38: 476.

SEEDS, N., A.G. GILMAN. T. AMANO, and M. NIRENBERG.
1970. Regulation of axon formation by clonal lines of a
neural tumor. Proc. Narl. Acad. Sci. 66: 160.
SHARMA, S.K., W.A. KLEE, and M. NIRENBERG. 1975a.
Dual regulation of adenylate cyclase accounts for narcotic

dependence and tolerance. Proc. Narl. Acad. Sci.
72: 3092.

-. 1977. Opiate-dependent modulation of adenylate

cyclase. Proc: Narl. A&d. Sci. 74: 3365.
SHARMA. S.K.. M. NIRENBERG. and W.A. KLEE. 1975b.

Morphine receptors as regulators of adenylate cyclase ac-
tivity. Proc. Natl. Acad. Sci. 72: 590.
SPERRY, R.W. 1963. Chemoaffinity in the orderly growth of
nerve fiber patterns and connections. Proc. Nafl. Acad.
  Sci. 50: 703.

SUGIYAMA, K., M. TOMIDA, Y. HONMA, and M. HOZUMI.
1980. Expression of a cell surface glycoprotein (~180)
related to cell-substratum adhesion during differentiation
of mouse myeloid leukemia cells. Cancer Res. 40: 3387.
TORU, M. and M.H. APRISON. 1966. Brain acetylcholine
studies: A new extraction procedure. J. Neurochem.
  13: 1533.

TRIGGLE, D.J. and V.C. SWAMY. 1983. Calcium antagonists.
Some chemical-pharmacologic aspects. Circ. Res. 52: 17
(Suppl. I.).

TRUDING, R., M.L. SHELANSKI, and P. MORELL. 1975.
Glycoproteins released into the culture medium of dif-
ferentiating murine neuroblastoma cells. J. Biol. Chem.
250: 9348.

TRUDING, R., M.L. SHELANSKI, M.P. DANIELS. and P. MOR-
RELL. 1974. Comparison of surface membranes isolated
from cultured murine neuroblastoma cells in the differen-
tiated and undifferentiated state. J. Eiol. Chem.
249: 3973.

Vo, K.P., M.J. MILLER, E.P. GEIDUSCHEK, C. NIELSON, A.
OLSON, and N.H. XUONG. 1981. Computer analysis of
two-dimensional gels. Anal. Biochem. li2: 258.  -

WILSON, S., H. HIGASHIDA. I. MINNA. and M. NIRENBERG.

1978. Defects in synapse format& and acetylcholine
release by neuroblastoma and hybrid cell lines. Fed. Proc.
37: 1784.

WV, J.R. and L.F. JOHNSON. 1982. Regulation of
dihydrofolate reductase gene transcription in methotrexate
resistant mouse ftbroblasts. J. Cell. Physiol. 110: 183.
ZISAPEL, N. and U.Z. LITTAUER. 1979. Expression of
external-surface membrane proteins in differentiated and
undifferentiated mouse neuroblastoma cells. Eur. J.
Biochem. 95: 51.