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Volume 16, Number 10,
Issue of May 15, 1996
pp. 3256-3264
Copyright ©1996 Society for Neuroscience
Synaptic Modulation by Neurotrophic Factors: Differential and
Synergistic Effects of Brain-Derived Neurotrophic Factor and Ciliary
Neurotrophic Factor
Ron Stoop and
Mu-ming Poo
Department of Biological Sciences, Columbia University, New York,
New York 10027
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Extracellular application of brain-derived neurotrophic factor
(BDNF) and ciliary neurotrophic factor (CNTF) to developing
neuromuscular junctions in Xenopus nerve-muscle cultures
resulted in an increase in the frequency of spontaneous synaptic
currents (SSCs) and in the amplitude of nerve-evoked synaptic currents.
Analyses of the amplitude and time course of the SSCs suggest that
these effects are attributable to elevation of presynaptic transmitter
release. The actions of these two factors on the transmitter secretion
process, however, are distinctly different. Fura-2
Ca2+ imaging showed that an increase in
presynaptic cytosolic Ca2+
([Ca2+]i) accompanied the
synaptic potentiation by BDNF, whereas no change in
[Ca2+]i was observed
during synaptic potentiation by CNTF. Removing external
Ca2+ also abolished the potentiating effect of
BDNF but did not influence the CNTF effect. Moreover, the two factors
exerted different effects on the short-term synaptic plasticity.
Paired-pulse facilitation normally found at these synapses was reduced
by BDNF but unaffected by CNTF; CNTF, but not BDNF, reduced the extent
of synaptic depression during high-frequency tetanic stimulation.
Finally, the potentiation effect of BDNF and CNTF on spontaneous
transmitter release was additive when both factors were applied
together to the synapse at saturating concentrations (100 ng/ml) and
was highly synergistic when low doses (1 and 10 ng/ml) of both factors
were used. These results suggest that because of their differential
effects on the secretory machinery, BDNF and CNTF may act cooperatively
in modulating the development and functioning of synapses.
Key words:
BDNF;
CNTF;
neurotransmitter release;
Fura-2 imaging;
paired-pulse facilitation;
synaptic plasticity;
synergism
INTRODUCTION
Neurotrophic factors are known to play important
roles in the survival and differentiation of many types of neurons
during development. Brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), and NT-4/5, members of the neurotrophin family,
promote motor neuron survival in vitro and rescue motor
neurons from naturally occurring or axotomy-induced cell death
(Sendtner et al., 1992 ; Yan et al., 1992; Henderson et al., 1993;
Koliatsos et al., 1993). Mice carrying a null mutation in the BDNF gene
show deficits of sensory neurons, although motor neuron survival was
not affected (Conover et al., 1995; Liu et al., 1995). Ciliary
neurotrophic factor (CNTF), a member of the cytokine family of
neurotrophic factors, also promotes survival of embryonic motor neurons
in vitro (Arakawa et al., 1990) and in vivo
(Oppenheim et al., 1991; Forger et al., 1993). A null mutation in the
CNTF gene results in progressive motor neuron atrophy and postnatal
neuron loss (Masu et al., 1993).
Most observations on the survival and differentiation effects of
neurotrophic factors relate to long-term trophic functions of these
factors. On the other hand, the activities of downstream kinases and
effector proteins are known to be induced soon after binding of the
factors to the neuron (for reviews, see Heumann, 1994; Greene and
Kaplan, 1995). These cytosolic activities may lead to immediate
alterations of neuronal functions. For example, marked morphological
changes of nerve growth cones were induced within minutes after
exposure of PC12 cells to nerve growth factor (Connolly et al., 1985,
1987). In Xenopus nerve-muscle cultures, Lohof et al.
(1993) showed that within 10-20 min after addition of the
neurotrophins BDNF and NT-3, both spontaneous and evoked transmitter
release at developing neuromuscular synapses were potentiated. Similar
potentiation effects were also found after application of CNTF to these
developing synapses (Stoop and Poo, 1995). Because these two factors
are known to exert their effects via different receptors and
intracellular signal transduction cascades, it is of interest to
examine further whether synaptic potentiation by BDNF and CNTF was
attributable to modulation of the same cellular loci of the secretory
pathway within the nerve terminal. In the present study, the synaptic
effects of BDNF and CNTF under various conditions were compared further
to reveal differences in their presynaptic actions. The potential
additive or synergistic actions of these two factors at the synapse
were also examined.
MATERIALS AND METHODS
Culture preparations and chemicals.
Xenopus cultures were prepared as described previously
(Spitzer and Lamborghini, 1976; Tabti and Poo, 1991). Briefly, neural
tubes and the associated myotomal tissue of 1-d-old Xenopus
embryos (stage 20-22 according to Nieuwkoop and Faber, 1967) were
dissociated in
Ca2+/Mg2+-free saline
supplemented with EDTA (115 mM NaCl, 2.6 mM KCl, 0.5 mM EDTA, 10 mM HEPES, pH 7.60) for 15-20 min. The cells were
plated on glass coverslips and were used for experiments after 24 hr
incubation at room temperature. The culture medium consisted of
(vol/vol) 50% Leibovitz L-15 medium (Sigma, St. Louis, MO), 1% fetal
calf serum (Gibco, Gaithersburg, MD), and 49% Ringer's solution (115 mM NaCl, 2 mM
CaCl2, 2.5 mM KCl, 10 mM HEPES, pH 7.6). For experiments performed
under zero external Ca2+ conditions, the culture
medium was replaced with a solution containing 115 mM NaCl, 2 mM
MgCl2, 2.5 mM KCl, 10 mM HEPES, 3 mM EGTA, and
0.1% bovine serum albumin (BSA), pH 7.3. Escherichia
coli-derived rat recombinant CNTF and BDNF were obtained from
Regeneron (Tarrytown, NY) and aliquoted and stored at a concentration
of 0.78 mg/ml in PBS at  70°C before use. The
tetra(acetoxymethyl)-ester form of
bis-(o-aminophenoxy)-ethane-N,N,N ,N-tetra-acetic acid
(BAPTA-AM) was obtained from Calbiochem (San Diego, CA).
Electrophysiology. Whole-cell patch recordings (Hamill and
Sackmann, 1981) were made in culture medium after 1 d of incubation at
room temperature (20-22°C). The solution inside the whole-cell
recording pipette contained 150 mM KCl, 1 mM NaCl, 1 mM
MgCl2, and 10 mM HEPES, pH
7.2. The membrane current in all recordings was monitored by a
patch-clamp amplifier (EPC-7, List, Great Neck, NY). To monitor
long-term (>1 hr) changes in synaptic currents at the same synapse,
the whole-cell recording pipette was removed, and repeated recording
from the postsynaptic myocyte was carried out later with a different
pipette to avoid excessive ``washout'' of cytosolic components. Data
were stored on a videotape recorder for later playback onto a storage
oscilloscope (5113, Tektronix, Beaverton, OR) and a chart recorder
(EasyGraf Recorder TA240, Gould, East Rutherford, NJ) and for analysis
by the SCAN program (kindly provided by Dr. J. Dempster, Strathclyde
University, Glasgow, UK). Unless indicated otherwise, BDNF and CNTF
were added to the culture medium, and the recording was made in the
presence of the factor throughout the entire course of the experiment.
Both spherical and flat myocytes were used for these experiments,
because there is no apparent difference in the physiological properties
of synapses on these two types of cells (Evers et al., 1989).
Fura-2 [Ca2+]i imaging. Fura-2 was
loaded into the presynaptic neuron in 1-d-old Xenopus
nerve-muscle cultures using the whole-cell patch-recording pipette.
Fura-2 (Molecular Probes, Eugene, OR) was dissolved in intracellular
pipette solution containing 150 mM potassium
gluconate, 1 mM sodium gluconate, 1 mM MgCl2, and 10 mM HEPES, pH 7.2, and whole-cell patch recording
was achieved at the soma of the neuron for a brief period (2 sec)
before the pipette was detached. This allowed enough Fura-2 to diffuse
into the neuron and attained a fluorescence level (at 380 nm)
comparable to that observed after 30 min incubation with 3-6
µM Fura-2AM (Zheng et al., 1994). The cultures
were then mounted on the stage of an inverted microscope (DIAPHOT,
Nikon) equipped with a cooled charge-coupled device (CCD)-based imaging
system (CH220 CCD camera, Photometrics, Tucson, AZ). A Nikon 40×/1.3
numerical aperture Fluor DL objective lens was used throughout the
experiments. Fura-2 was excited at 340 and 380 nm wavelengths
alternately through a computer-controlled shutter and filter wheel. The
exposure time at each wavelength was 100 msec. Paired digital images at
340 and 380 nm excitation were collected with background subtracted.
The cytosolic-free Ca2+ concentration was
determined from the ratio (F340/F380) calculated from the pairs using
the ratio method (Grynkiewicz et al., 1985, Tsien and Poenie, 1986).
Calibration of Rmin (the limiting value
that the ratio can have at zero [Ca2+]) and
Rmax (the limiting value that the ratio can
have at saturating [Ca2+]) was carried out
using standard Ca2+ buffers with 0 and 39.8 µM free [Ca2+]
(Molecular Probes).
RESULTS
Synaptic potentiation by BDNF and CNTF
The efficacy of synaptic transmission at developing neuromuscular
junctions in Xenopus nerve-muscle cultures was measured by
whole-cell recording of spontaneous synaptic currents (SSCs) and
impulse-evoked synaptic currents (ESCs) from innervated muscle cells.
In agreement with previous reports (Lohof et al., 1993; Stoop and Poo,
1995), we observed an increase in the frequency of the SSCs within
5-10 min after application of either BDNF or CNTF (final
concentration, 100 ng/ml) to the culture medium. The increase in SSC
frequency was accompanied by an increase in the amplitude of the ESCs
for both BDNF and CNTF (Fig. 1). The rate of recovery
from the potentiated levels after removal of BDNF and CNTF was examined
further. As shown in Figure 2, 1 hr after removal of the
factor the potentiation by BDNF of both the SSC frequency and the ESC
amplitude had essentially disappeared, whereas the potentiation induced
by CNTF remained unchanged. Six hours after removal of the factor,
potentiation by both BDNF and CNTF reversed completely to the initial
control levels before application of the factors.
Fig. 1.
Potentiation of SSCs and ESCs by brain-derived
neurotrophic factor (BDNF) and ciliary neurotrophic factor
(CNTF) at developing Xenopus neuromuscular
synapses. The continuous traces depict membrane currents recorded from
innervated myocytes under voltage-clamp (Vh = 70 mV) in a 1-d-old culture. Downward events are inward currents.
SSCs occurred at random and ESCs were elicited at a frequency of 0.05 Hz at the times marked by the brackets. The arrow
marks the time of addition of either BDNF or CNTF to the culture medium
(final concentration 100 ng/ml). Insets above depict
computer-averaged ESCs and samples of SSCs at higher time resolution
for the recording periods pointed to by the lines.
Calibration: slow traces, 2 nA, 5 min; fast traces, 1 nA, 20 msec.
[View Larger Version of this Image (26K GIF file)]
Fig. 2.
Time course of reversal of BDNF and CNTF effects.
Mean SSC frequency and ESC amplitude were determined immediately
(0), 1 hr (1 hr), and 6 hr (6 hr)
after a 30 min treatment with CNTF or BDNF (both at 100 ng/ml). The
cells were washed thoroughly and incubated with fresh culture medium at
the end of the 30 min treatment. The SSC frequency and mean ESC
amplitude were normalized for each synapse to the mean values before
treatment with the factor. All data are represented as mean ± SEM;
values marked with * are significantly different from the control
values at the untreated synapse (p < 0.05, t
test).
[View Larger Version of this Image (56K GIF file)]
Synaptic currents may be enhanced by an increased presynaptic release
of transmitter or by an increased postsynaptic sensitivity to the
transmitter. An increased postsynaptic acetylcholine (ACh) sensitivity
could explain the increase in ESC amplitude as well as an increase in
the SSC frequency, because previously undetectable small ACh quanta may
emerge after exposure to the factor. As shown in Figure
3, we found no detectable change in the mean, the range,
and the distribution of the SSC amplitude, suggesting that it is
unlikely that ACh sensitivity had increased by the factor. The absence
of any change in the rise time and the decay time of the SSCs after
significant elevation of SSC frequency had occurred suggests that these
factors did not affect the properties of postsynaptic ACh channels
(Fig. 3). Thus the primary action of BDNF and CNTF at these synapses
seems to be a presynaptic modulation of transmitter secretion
mechanisms.
Fig. 3.
Absence of any change in the properties of SSCs.
The amplitude, rise time, and decay time distributions of SSCs before
and after BDNF and CNTF treatment were compared. Values observed for
each synapse were normalized to the maximal value that included 95% of
all events. The cumulative probability refers to the fraction of total
events with values smaller than a given value before (closed
circles) and after (open circles) addition of the
factor. Synapses in the left column were treated with BDNF
(n = 9), and synapses in the right column were
treated with CNTF (n = 8). Data points represent mean ± SEM.
[View Larger Version of this Image (26K GIF file)]
Effects of BDNF and CNTF on [Ca2+]i
The mechanism by which BDNF and CNTF potentiate presynaptic
transmitter secretion was examined further. It has been shown that a
rapid increase in [Ca2+]i
occurs after administration of BDNF to cultured hippocampal neurons
(Berninger et al., 1993). Such elevation of
[Ca2+]i may explain a
higher frequency of spontaneous transmitter secretion observed after
BDNF and CNTF. We thus examined the level of
[Ca2+]i in the
presynaptic neurons of Xenopus neuromuscular synapses, using
the Ca2+-sensitive fluorescence dye Fura-2,
before and during a period of 30 min after BDNF or CNTF administration.
To measure the level of
[Ca2+]i in the
presynaptic nerve terminal, free from interference of fluorescence from
the postsynaptic cell, the presynaptic neuron was loaded with Fura-2
through a whole-cell recording pipette at the soma. Synaptic currents
were monitored by another whole-cell recording pipette at the
postsynaptic myocyte before and after Fura-2 loading to observe the
effect of dye-loading on the level of spontaneous transmitter
secretion. To exclude artifacts introduced by the loading procedure,
only those synapses that showed no change in the SSC frequency after
presynaptic Fura-2 loading were used for Ca2+
imaging. Figure 4A shows an example of data
from a Fura-2-loaded neuron before and at different times after BDNF
treatment, using the standard ratio imaging method (Tsien and Poenie,
1986). An elevated
[Ca2+]i in the
presynaptic terminal of the Fura-2-loaded neuron was observed within a
few minutes after BDNF addition, and the concentration remained at a
high level in the presence of the factor for at least 30 min. An
example of quantitative measurements of
[Ca2+]i at several
different presynaptic sites of the same neuron is shown in Figure
5A. Recording of SSCs from this particular
synapse as well as two other synapses before and 30 min after the BDNF
treatment confirmed the potentiation effect. In total, five synapses
were examined for the BDNF effect on the presynaptic
[Ca2+]i, and the results
are summarized in Figure 5B. Similar measurements of
presynaptic [Ca2+]i were
also performed for synapses treated with CNTF. As shown in Figures
4B and 5, no change in
[Ca2+]i was observed
after CNTF treatment, although recording of the same synapses confirmed
that the SSC frequency at these synapses was increased by CNTF to the
same level as typically observed in the absence of Fura-2 loading.
Finally, we examined the dependence of BDNF-induced changes in
[Ca2+]i on the presence
of external Ca2+ by replacing culture medium with
a Ca2+-free saline (see Materials and Methods).
As shown in Figure 5C, depletion of external
Ca2+ reduced the basal level of
[Ca2+]i, and subsequent
addition of BDNF produced no further change in
[Ca2+]i. Thus, the
increased [Ca2+]i seemed
to be the result of an elevated influx of
Ca2+.
Fig. 4.
Fura-2 ratio imaging of
[Ca2+]i in the
presynaptic neurons treated with BDNF and CNTF. The neuron was loaded
with Fura-2 through a whole-cell recording pipette at the soma.
A, A neuron innervating a muscle cell was treated with 100 ng/ml BDNF at time 0 (numbers in minutes), resulting in an increase in
[Ca2+]i. The first
black and white images represent fluorescence observed at
380 nm at low and high resolution, respectively, indicating the outline
of presynaptic neurite surrounding a spherical postsynaptic myocyte.
Squares mark the sites for which quantitative measurements
of fluorescence intensity have been made (see Fig. 5).
Color images depict
[Ca2+]i in the
presynaptic neurite at various times after the BDNF treatment, shown in
pseudo-colors with the corresponding
Ca2+ concentration indicated by the
color bar. Insets shown at 0 and 32 min are
recordings of SSCs before and 32 min after BDNF administration. Bar, 10 µm. B, Same as in A, except that CNTF was
applied instead of BDNF. In the first black and white micrograph, the
fluorescence image at 380 nm was superimposed by a low-level
bright-light image of the same field to reveal the geometry of the
presynaptic neurite on an extended postsynaptic myocyte. Note the
absence of any change in
[Ca2+]i, whereas marked
increase in SSCs was observed at 32 min after the CNTF treatment.
[View Larger Version of this Image (121K GIF file)]
Fig. 5.
Changes in presynaptic
[Ca2+]i induced by BDNF
or CNTF treatment. A,
[Ca2+]i was measured with
2 min intervals at different locations of the presynaptic terminal
shown in Figure 4 before and after the onset of BDNF or CNTF
application (at time 0). The measurement boxes were 5 × 5 pixels, and
their locations are indicated in Figure 4. The measurements made from
the boxes in Figure 4 are represented from top to bottom in the same
sequence by squares, triangles, and
diamonds, respectively. Filled and open
symbols represent synapses treated with BDNF and CNTF,
respectively. B, Average
[Ca2+]i changes over time
for data collected from five synapses, each treated with BDNF and CNTF.
The data points represent mean ± SEM. C, The
[Ca2+]i level was reduced
by substitution of the external medium with
Ca2+-free solution (supplemented with EGTA and
BSA; see Materials and Methods). Subsequent treatment of the culture
did not induce any elevation of
[Ca2+]i. The same
protocol as that described above was used to measure presynaptic
[Ca2+]i. Data points
represent mean ± SEM (n = 5 synapses).
[View Larger Version of this Image (20K GIF file)]
Dependence of synaptic potentiation on Ca2+
In a series of experiments, we replaced the culture medium with
Ca2+-free saline (see Materials and Methods) and
measured the effect of BDNF and CNTF on spontaneous transmitter
secretion. Depletion of external Ca2+
([Ca2+]o) caused a slight
decrease in the basal SSC frequency. Further addition of BDNF in the
absence of external Ca2+ was ineffective in
elevating the SSC frequency. In contrast, administration of CNTF still
led to a significant increase in the SSC frequency (Fig.
6). Cultures were also preincubated with BAPTA-AM (20 µM) for 30 min, which resulted in a drop in
[Ca2+]i to a level of
~70% of its normal value, as shown by Fura-2 imaging (Girod et al.,
1995). No elevated spontaneous release was observed after addition of
either BDNF or CNTF (Fig. 6). Thus, both factors potentiated the
exocytosis of a population of vesicles, which requires a basal level of
cytosolic Ca2+ for the exocytic process.
Fig. 6.
Dependence of BDNF and CNTF effects on
Ca2+. A, The frequency of SSCs was
monitored before and after the culture medium was substituted with
Ca2+-free saline at the time marked by the
lower bar. BDNF (100 ng/ml) was added to the
Ca2+-free saline at time marked by the
upper bar. The SSC frequency values recorded from each
synapse were normalized against the average value of the same synapse
before medium change. Data points represent mean ± SEM (n = 5 synapses). B, The same as A except that CNTF
(100 ng/ml) was added instead of BDNF (n = 6). In separate
cultures, the cells were incubated with BAPTA-AM for 30 min before and
throughout the experiment. For clarity, only results before and 30 min
after application of either BDNF (A) or CNTF (B)
were shown (open circles). Note that no potentiation of SSC
frequency was observed after treatment with either factor.
[View Larger Version of this Image (19K GIF file)]
Different effects on short-term synaptic plasticity
The difference in synaptic potentiation by BDNF and CNTF was
studied further by examining the effects of these factors on short-term
plasticity of transmitter release upon repetitive activation of the
synapse. First, we measured paired-pulse facilitation (PPF), the
increase in the amplitude of the postsynaptic response when the synapse
is activated by two successive presynaptic action potentials. This
facilitation is known to reflect an enhanced transmitter release
resulting from the residual Ca2+ in the
presynaptic terminal (Zucker, 1989). At control neuromuscular synapses
in 1-d-old Xenopus cultures, a slight PPF can be seen when
the second pulse is applied 25 msec after the first one (Fig.
7). After exposure of the culture to BDNF (100 ng/ml)
for 30 min, the amplitude of the ESC was potentiated, but this
potentiation was accompanied by a disappearance of the PPF at 25 msec.
Potentiation of the ESC amplitude by CNTF (100 ng/ml), on the other
hand, had no effect on the PPF at these synapses. Thus, the
potentiation effect of BDNF occludes PPF, whereas that of CNTF does
not.
Fig. 7.
Differential effects of BDNF and CNTF on PPF
facilitation. A, Samples of computer-averaged ESCs induced
by a pair of pulses at an interval of 25 msec or 100 msec are shown for
a control synapse and for synapses 30 min after treatment with CNTF or
BDNF (at 100 ng/ml). B, The ratio of the ESC amplitude
induced by the second stimulus to that of the first paired-pulse ratio
(PPR) for different interpulse intervals (25, 50, 75, and 100 msec) and
different treatments: control (filled circles), CNTF
(open circles), and BDNF (gray circles). For an
interpulse interval of 25 msec, the PPR for synapse not treated with
any factor (filled circle) and CNTF-treated synapses (100 ng/ml, 30 min incubation; open circle) was significantly
higher than that of BDNF-treated synapses (100 ng/ml, 30 min
incubation; gray circle).
[View Larger Version of this Image (16K GIF file)]
Tetanic stimulation of presynaptic neurons of many synapses results in
a gradual reduction in the average amplitude of evoked responses, a
form of synaptic depression that can be attributed to the depletion of
synaptic vesicles (Zucker, 1989). We examined the rate of
tetanus-induced synaptic depression before and after treatment with
either BDNF or CNTF. As shown in Figure 8, tetanic
stimulation at a frequency of 5 Hz before the treatment of these
factors resulted in a 50% reduction in the mean ESC amplitude within
30 sec, in agreement with that reported for tetanus-induced depression
at these developing Xenopus synapses (Alder et al., 1995).
After treatment with BDNF, the 30 sec tetanus induced a similar extent
of reduction in the ESC amplitude as compared with that observed during
the pretreatment period. After treatment with CNTF, however, there was
no obvious depression induced by the same 30 sec tetanus that had
produced a marked depression in untreated or BDNF-treated synapses.
Fig. 8.
The effects of BDNF and CNTF on synaptic
depression during tetanic stimulation. A, Examples of
membrane currents recorded from postsynaptic myocyte during a train of
tetanic stimulation at 5 Hz before (left) and 30 min after
(right) treatment with CNTF or BDNF (both at 100 ng/ml).
B, Changes in the mean ESC amplitude over time during
tetanic stimulation at 5 Hz at control synapses (before treatments with
the factors; filled circles; n = 6), BDNF-treated
synapses (100 ng/ml, 30 min incubation; gray circles;
n = 6), and CNTF-treated synapses (100 ng/ml, 30 min
incubation; open circles; n = 6). Mean ESC
amplitudes during 4 sec intervals were normalized to the mean ESC
amplitude during the first 4 sec after the onset of tetanus. The
normalized amplitudes at 18, 22, 26, and 30 sec (marked by *) are
significantly different from values observed at control and
BDNF-treated values (p < 0.05, t test).
[View Larger Version of this Image (27K GIF file)]
Synergistic actions of BDNF and CNTF
The above studies suggested that different intracellular targets
in the transmitter secretion machinery are affected by BDNF and CNTF.
We explored this possibility further by examining whether the effects
of these two factors are mutually exclusive by simultaneous application
of both factors at different dose levels. We first determined the
dose-response of each factor in potentiating the SSC frequency when
applied alone. Either BDNF or CNTF was added to the culture at
different concentrations, and the change in the SSC frequency was
measured after 30 min, when the effect had reached a plateau level
(Lohof et al., 1993; Stoop and Poo, 1995). As shown in Figure
9, we found that the extent of potentiation of the SSC
frequency was dose-dependent, and the effect apparently saturated at a
level of ~100 ng/ml for either BDNF or CNTF. When CNTF (100 ng/ml)
was added to synapses that had been treated previously with a
saturating dose of BDNF (100 ng/ml), a further increase in SSC
frequency was observed. The same additive effect was observed when the
order of treatment of the factors was reversed. Thus, a saturating
action of one factor did not occlude further action of the other. The
cooperative action of these factors in potentiating spontaneous
transmitter secretion was revealed further by experiments using lower
doses of BDNF and CNTF. As shown in Figure 9, treatment of the synapse
with a single factor at either 1 or 10 ng/ml produced insignificant or
small effects, respectively; however, combined treatments of both
factors at these concentrations markedly potentiated the spontaneous
transmitter secretion.
Fig. 9.
Synergistic effects by CNTF and BDNF in
potentiation of spontaneous ACh secretion. The mean SSC frequency
observed 30 min after application of different concentrations of CNTF
(open circles) or BDNF (gray circles) or both
(solid circles) to the culture. The frequency values were
normalized for each synapse by the mean value observed at the same
synapse before addition of the factor. The data points represent mean ± SEM (n = 8-12). The differences between values at 100 ng/ml CNTF and at 0, 2, or 10 ng/ml CNTF are statistically significant.
The differences between values at 100 ng/ml BDNF and at 0 or 2 ng/ml
BDNF are also statistically significant. All values observed for
combined BDNF and CNTF treatments are significantly different from
values for BDNF or CNTF treatment alone at the corresponding
concentration (p < 0.05, t test).
[View Larger Version of this Image (21K GIF file)]
DISCUSSION
Similar synaptic potentiation by BDNF and CNTF
In the present study, we have compared the effects of two
neurotrophic factors, BDNF and CNTF, on the physiological functions of
developing neuromuscular synapses under various conditions. Although
these two factors bind to distinctly different cell surface receptors
and activate different intracellular signaling pathways (Heumann,
1994), the physiological effects of the two factors are quite similar:
both caused increases in the frequency of SSCs and in the amplitude of
action-potential ESCs. The SSCs found at these developing synapses are
similar to miniature endplate currents at mature neuromuscular
junctions and are independent of action potentials in these neurons
(Xie and Poo, 1986). Elevation of SSC frequency was not attributable to
spontaneous action potentials induced by the factor, because ESCs are
usually of much large amplitudes than SSCs, and the amplitude
distribution of SSCs after the potentiation by the factor did not
exhibit any increase in the population of larger amplitudes. To
determine whether BDNF and CNTF produce any postsynaptic changes in the
response of the myocyte to the released transmitter, we have analyzed
the amplitude distribution and the rise and decay times of the SSCs.
The absence of any detectable changes in any of these parameters argues
that within the duration of these experiments, the potentiation effects
seem to be caused primarily by an elevated level of presynaptic ACh
release without any changes in the postsynaptic ACh sensitivity.
Acute synaptic modulation by neurotrophins has been observed recently
at a number of central synapses. Synaptic transmission is potentiated
by neurotrophins in rat hippocampal slices (Berzaghi et al., 1995; Kang
and Schuman, 1995a). At these hippocampal synapses, the effects of
neurotrophins did not interfere with tetanus-induced long-term
potentiation and seemed to be presynaptic in origin. In cultures of rat
hippocampal neurons, BDNF elevates excitatory synaptic activity by
potentiating glutamatergic synaptic transmission (Girod et al., 1994;
Levine et al., 1995). In cultures of rat cortical neurons, however,
NT-3 inhibited activities of GABAergic synapses with no apparent effect
on excitatory synaptic transmission (Kim et al., 1994).
The role of Ca2+ in BDNF and CNTF effects
It is well known that the frequency of SSCs is affected directly
by changes in intraterminal Ca2+ levels (Miledi,
1973). Using Fura-2 fluorescence imaging, Berninger et al. (1993)
observed a transient increase in
[Ca2+]i within 1 min
after application of BDNF to isolated hippocampal cells in culture.
After BDNF administration, we found a gradual increase in
[Ca2+]i from 100 nM to up to 350 nM during a
period of 10-15 min. In all three cases for which SSCs were monitored
before and after fluorescence measurements, an increase in SSC
frequency was observed. The elevations of
[Ca2+]i after
administration of BDNF (Fig. 6A) exhibited a time course
similar to that of the increase in SSC frequency reported earlier
(Lohof et al., 1993; Stoop and Poo, 1995). In contrast, we failed to
observe any change in
[Ca2+]i in all five cases
for which the SSC frequency was elevated by CNTF. Thus, an increase in
[Ca2+]i at the nerve
terminal induced by BDNF may account for the increase in SSC frequency,
whereas a change in
[Ca2+]i does not seem to
be involved in the CNTF effect.
The mechanism by which BDNF causes an elevation in
[Ca2+]i may involve the
action of phospholipase C (PLC). Both BDNF and NT-3 stimulate
phosphorylation of PLC and subsequent phosphatidyl-inositol-hydrolysis
in cultured rat cortical neurons (Widmer et al., 1993). BDNF also
increases the concentration of inositol triphosphate
(IP3) in rat hippocampal synaptosomes (Knipper et
al., 1993). Because the BDNF-induced rise in
[Ca2+]i was abolished by
removal of external Ca2+ (Fig. 5C),
the rise in [Ca2+]i may
be attributed directly to the BDNF-induced elevation of
Ca2+ influx. Alternatively, external
Ca2+ may be required for signal transduction by
BDNF, and the elevation of IP3 triggers
Ca2+ release from internal stores.
In the hippocampus, L-type Ca2+ channels may be
involved in the synaptic potentiation by BDNF, because administration
of nifedipine, a blocker of these channels, prevents potentiation by
BDNF (Kang and Schuman, 1995b). In addition to potential direct
modulation of Ca2+ channels in these
Xenopus neurons (O'Dowd et al., 1988; Ribera and Spitzer,
1990; Barish, 1991a,b), neurotrophins may affect
Ca2+ entry during the action potential by
altering voltage-dependent K+ conductances.
Although changes in the expression of voltage-dependent ion channels
can be induced by long-term application of neurotrophic factors (Lesser
and Lo, 1995; Nick et al., 1995), whether ion channel modulation is
involved in the acute actions of neurotrophic factors reported here
remains to be examined.
Effects of BDNF and CNTF on short-term synaptic plasticity
The differences in the mechanism by which BDNF and CNTF potentiate
the transmitter release were revealed further by the differential
effects of these factors on activity-dependent plasticity in
transmitter release. In PPF, the facilitated transmitter secretion
triggered by the second presynaptic stimulus is attributed to the
residual Ca2+ resulting from the first stimulus
(Kamiya and Zucker, 1994). We found that potentiation by BDNF reduced
the magnitude of the PPF that is normally seen at these synapses,
whereas CNTF had no effect. Elevated
[Ca2+]i resulting from
increased external Ca2+ concentration is known to
reduce PPF (Rahamimoff, 1968). The BDNF effect in reducing PPF is thus
consistent with the elevated
[Ca2+]i induced by this
factor.
Under repetitive stimulation, developing neuromuscular junctions
exhibit marked reduction of ESC amplitudes over time, presumably as a
result of depletion of synaptic vesicles. This synaptic depression
essentially was abolished by treatment with CNTF, suggesting that CNTF
may help to mobilize vesicle supply at these synapses. Because the
amplitude of the ESCs observed at a low stimulus frequency was
increased by CNTF, the immediate availability of vesicle and/or
probability of vesicle exocytosis was also increased by CNTF. Finally,
we noted that the effects of CNTF persisted for a longer duration than
those of BDNF after the factors were removed from the culture medium.
As shown in a previous study (Stoop and Poo, 1995), potentiation of
spontaneous ACh release by CNTF, in contrast to that by BDNF, requires
signaling with the cell body, because CNTF was ineffective in
potentiating synapses that were ``cut loose'' from the cell body.
Such a long-range somatic signaling induced by CNTF may involve an
increase in vesicle supply that results in a more persistent increase
of transmitter release and reduced synaptic depression during
high-frequency stimulation.
Synergistic actions of BDNF and CNTF
Dose-response studies of the potentiation effects of BDNF and
CNTF on spontaneous ACh secretion showed that near maximal potentiation
by one factor (obtained at a concentration of 100 ng/ml after 30 min
treatment) did not occlude subsequent potentiation by the other factor.
Thus, it seems that these two factors act on different sites of the
transmitter secretion machinery. At low concentrations of both BDNF and
CNTF, simultaneous exposure to both factors led to a marked
potentiation of the spontaneous release that cannot be explained purely
by the additive effects of the two factors. Long-term synergistic
actions of BDNF and CNTF have been reported for the induction of
choline acetyltransferase (Wong et al., 1993, 1995; Xu et al., 1995)
and for improving the performance of Wobbler mice (Klinkosz et al.,
1995). Our results show that acute synaptic effects of BDNF and CNTF
also can be synergistic. At the presynaptic nerve terminal, down-stream
effectors activated by BDNF and CNTF may act on two different but
sequential steps in the secretion machinery, leading to a synergistic
effect on transmitter secretion. Whether such synergistic action of
neurotrophic factors plays a role in regulating the development and
functioning of neuromuscular synapses in the nervous system remains to
be investigated.
FOOTNOTES
Received Nov. 13, 1995; revised Feb. 26, 1996; accepted Feb. 28, 1996.
This work was supported by a grant from the National Science Foundation
(IBN-92-22106). We thank Regeneron Inc. for kindly providing
recombinant BDNF and CNTF.
Correspondence should be addressed to Dr. Mu-ming Poo at his present
address: Department of Biology, University of California at San Diego,
La Jolla, CA 92093-0357.
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Substance via MeSH
