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The Journal of Neuroscience, July 1, 1998, 18(13):4985-4992
Localized Synaptic Actions of Neurotrophin-4
Xin-hao
Wang,
Benedikt
Berninger, and
Mu-ming
Poo
Department of Biology, University of California at San Diego, La
Jolla, California 92093-0357
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ABSTRACT |
Neurotrophins secreted by the postsynaptic target cell may
participate in activity-dependent synaptic modification during development and in the mature brain. A fundamental question of how
neurotrophins can sculpt synaptic connections is whether
neurotrophin-induced synaptic changes are spatially restricted to the
site of neurotrophin secretion or whether they can spread to
neighboring synapses. Using a model system of nerve-muscle coculture
in which neurotrophin-4 (NT-4) is overexpressed in a subpopulation of
postsynaptic myocytes, we demonstrated that presynaptic potentiation is
restricted to synapses on myocytes overexpressing NT-4 without
affecting nearby synapses formed by the same neuron on control
myocytes. Likewise, postsynaptic modulation of acetylcholine channels
by secreted NT-4 is spatially restricted to <60 µm from the site of
NT-4 secretion. Therefore, activity-dependent secretion of
neurotrophins can result in highly localized modification of neuronal
connections.
Key words:
Xenopus; synaptic transmission; neuromuscular
junction; embryo injection; acetylcholine receptor; TrkB receptor
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INTRODUCTION |
Target-derived neurotrophic factors
are essential for the survival and differentiation of developing
neurons (Purves and Lichtman, 1985 ; Barde, 1989; Oppenheim, 1991).
Recent evidence indicates that neurotrophins, a family of proteins
related to nerve growth factor (NGF), may also participate in
activity-dependent modification of synaptic connections (Lo, 1995;
Thoenen, 1995; Berninger and Poo, 1996; Bonhoeffer, 1996; Cellerino and
Maffei, 1996; Katz and Shatz, 1996; Lewin and Barde, 1996). The
expression of NGF and brain-derived neurotrophic factor (BDNF) in
central neurons can be regulated by electrical or synaptic activity
(Gall and Isackson, 1989; Lu et al., 1991; Zafra et al., 1991).
Furthermore, secretion of NGF, BDNF, and NT-4 can be enhanced by
neuronal or synaptic activity (Blöchl and Thoenen, 1995; Goodman
et al., 1996; Wang and Poo, 1997). On the other hand, neurotrophins
have been shown to modulate the efficacy of synaptic transmission. Exogenous application of BDNF or neurotrophin-3 (NT-3) leads to a rapid
potentiation of synaptic transmission in several cell culture
preparations (Lohof et al., 1993; Kim et al., 1994; Lessmann et al.,
1994; Levine et al., 1995) and brain slices (Kang and Schuman, 1995).
In hippocampal slices from BDNF knock-out mice, long-term potentiation
(LTP) in the CA1 region is severely impaired and can be restored by
reintroduction of BDNF into the CA1 region (Korte et al., 1995, 1996;
Patterson et al., 1996). Similarly, scavenging of BDNF by TrkB-IgG was
shown to interfere with either the induction or maintenance of LTP in
hippocampal slice, depending on the precise induction protocol (Figurov
et al., 1996; Kang et al., 1997). These results have led to the
hypothesis that neurotrophins released from postsynaptic cells can
modify synaptic function, and such modification can be regulated by
synaptic activity, thereby contributing to the activity-dependent
refinement of synaptic connections.
In a previous study (Wang and Poo, 1997), we found that neuromuscular
synapses made on NT-4-overexpressing myocytes in nerve-muscle cultures
exhibit a higher level of spontaneous transmitter release, as well as
an enhanced postsynaptic response to the transmitter. Furthermore,
repetitive stimulation of the presynaptic neuron resulted in a
transient potentiation of evoked synaptic transmission and enhanced
postsynaptic responses at synapses formed on NT-4-overexpressing myocytes. These results strongly supported the notion that
neurotrophins secreted from the postsynaptic cell can indeed serve for
retrograde as well as autocrine modulation of synaptic function in an
activity-dependent manner.
Several pieces of evidence have shown that changes in the efficacy of
synaptic transmission induced by synaptic activity, including LTP or
long-term depression (LTD), are not restricted to the activated synapse
but can spread over considerable distance (Bonhoeffer et al., 1989;
Schuman and Madison, 1994; Cash et al., 1996; Engert and Bonhoeffer,
1997; Fitzsimonds et al., 1997; Schuman, 1997). For instance, in
Xenopus nerve-muscle cultures, LTD induced at one
neuromuscular synapse can spread to distant synapses made by the same
neuron onto other myocytes (Cash et al., 1996). This spread of LTD was
apparently mediated by signaling in the presynaptic cytoplasm. In the
present study, we have examined whether presynaptic modulation induced
by postsynaptic release of NT-4 at Xenopus neuromuscular
synapses can similarly spread to neighboring synapses or whether it is
spatially restricted to the site of secretion. Likewise, we have
determined the spatial range within which autocrine modulation of
postsynaptic responses by secreted NT-4 can spread in a muscle
cell.
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MATERIALS AND METHODS |
In vitro transcription and embryo injection. In
vitro transcription and embryo injection were performed as
described previously (Wang and Poo, 1997). Synthetic mRNAs for NT-4 and
green fluorescent protein (GFP) were mixed 1:1, and 16 ng of mRNA was
injected into one cell of two-cell stage embryos using a
stimulator-gated pressure ejection system (Picospritzer; General Valve,
Fairfield, NJ). The expression of NT-4 in nerve and muscle cells in
1-d-old cultures made from the injected embryos was confirmed by
immunocytochemical staining using NT-4 antibodies (Wang and Poo, 1997).
That the expression of GFP provides a reliable indicator of NT-4
expression in living cells was also confirmed by the colocalization of
NT-4 immunocytochemical staining and green fluorescence (Wang and Poo, 1997).
Culture preparation. Preparation of Xenopus
nerve-muscle cultures followed the method described previously
(Spitzer and Lamborghini, 1976; Tabti and Poo, 1991). The cells were
plated on clean glass coverslips and were used for experiments after 24 hr incubation at room temperature (20-22°C). The culture medium
consisted of (v/v) 50% Leibovitz's medium (L-15; Life Technologies,
Gaithersburg, MD), 1% fetal bovine serum (Life Technologies), and 49%
Ringer's solution (in mM: 115 NaCl, 2 CaCl2, 2.5 KCl, and 10 HEPES, pH 7.3).
Electrophysiology. Synaptic currents were recorded from
innervated muscle cells by the whole-cell recording method (Hamill et
al., 1981; Evers et al., 1989) using a patch-clamp amplifier (Axopatch
1A, Axon Instruments). The solution inside the recording pipette
contained (in mM): 150 KCl, 1 NaCl, 1 MgCl2, and 10 HEPES, pH 7.2. Recordings were made at
room temperature in culture medium. Extracellular stimulation of the
presynaptic neuron was made by a patch electrode at the cell body under
loose-seal conditions. To study postsynaptic action of NT-4 on the
kinetics of ACh-induced currents, perforated patch recording was made
from the myocyte to prevent washout of the cell content during
prolonged whole-cell recording. The pipette solution for perforated
patch recording contained: 130 mM potassium gluconate, 20 mM KCl, 1 mM NaCl, 1 mM
MgCl2, 10 mM HEPES, pH 7.2, and 200 µg/ml Amphotericin B (Sigma, St. Louis, MO). For iontophoretic
application of ACh, sharp microelectrodes (resistance, 100-200 M )
filled with 3 M ACh chloride (Sigma) were used (Poo, 1982).
Braking currents of 4-5 nA were used to prevent ACh leakage from the
pipette, and constant iontophoretic current pulses of 10-20 nA
amplitude and 1 msec duration were delivered through a microelectrode
amplifier (WPI) to the ACh pipette. The tip of the ACh pipette was bent
slightly to facilitate the visualization of the tip and positioned
tangentially into contact with the surface of the myocyte (see Fig. 4).
Membrane currents were filtered at 10 kHz and stored by a videotape
recorder for later playback onto a storage oscilloscope (2201, Tektronix) or an oscillographic recorder (RS3200, Gould) and for
analysis by a computer. The frequency, amplitude, and time course of
synaptic currents were analyzed by using the SCAN program, kindly
provided by Dr. J. Dempster (University of Strathclyde, Glasgow,
Scotland).
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RESULTS |
The expression of NT-4 was elevated in a subpopulation of
Xenopus myotomal myocytes by injection of NT-4 mRNA into one
of the blastomeres of two-cell stage Xenopus embryos. The
synaptic actions of NT-4 were examined by whole-cell patch-clamp
recording in nerve-muscle cultures prepared from embryos 1 d
after injection. By co-injecting mRNA for GFP together with NT-4
message, NT-4-overexpressing cells were identified by the appearance of
green fluorescence. As described previously, in these
Xenopus nerve-muscle cultures synapses made on
NT-4-overexpressing myocytes (M+ synapses) exhibited a higher level of
spontaneous ACh secretion, more stable evoked ACh secretion, and an
enhanced postsynaptic response to ACh compared with those made on
control myocytes not expressing exogenous NT-4 mRNA (M synapses)
(Wang and Poo, 1997). These effects were attributable to NT-4 secretion
from postsynaptic myocytes, because they were completely abolished by
adding TrkB-IgG, a recombinant scavenger protein that binds to secreted
NT-4, to the culture medium (Wang and Poo, 1997).
Localized presynaptic action of NT-4
In the present study, we first addressed the question of the
spatial range of presynaptic action of NT-4 by using cultures that
contained "triplet" circuits, which were single neurons innervating a pair of spherical myocytes of which only one was overexpressing NT-4
(Fig. 1A). We examined
whether presynaptic modulation at M+ synapses can spread to M
synapses within the triplet. Using whole-cell recording of synaptic
currents from such paired synapses (n = 17), we found
that the frequency of miniature EPSCs (MEPSCs) was in general higher at
M+ synapses than at M synapses (see Figs. 1B,
3A). However, the mean MEPSC amplitude at M+ synapses (558 ± 79 pA) was not statistically different
(p > 0.3, t test) from that at M
synapses (475 ± 53 pA). The distributions of MEPSC amplitudes
were also not significantly different between M+ and M synapses (Fig.
2). In nine triplets from which
successful paired recordings of evoked EPSCs from both synapses were
obtained, the mean ± SEM amplitude at M+ synapses (1.14 ± 0.23 nA) was higher than that at M synapses (0.66 ± 0.20 nA),
although the difference was not statistically significant
(p = 0.15, t test). However, the
coefficient of variation (CV) of the EPSC amplitude was significantly reduced at M+ synapses compared with that at M synapses (Fig. 3B). CV is defined as the
ratio of the SD to the mean value of the peak EPSC amplitude, and a
reduction in CV indicates a reduced fluctuation of evoked responses.
Overall, the effects on spontaneous and evoked synaptic currents at M+
synapses are consistent with a presynaptic enhancement of transmitter
secretion resulting from postsynaptic secretion of NT-4 (Wang and Poo,
1997). Furthermore, we found that the mean values of MEPSC frequency
and the CV of EPSCs at M synapses in these triplets were not
significantly different from those found at singly innervated myocytes
not expressing any exogenous message or expressing GFP message only
(Fig. 3). Thus, M+ synapses had no apparent influence on M synapses
formed by the same neuron. The consistent differences in the synaptic properties observed between M+ and M synapses in the same culture, regardless of whether they were made by the same neuron, clearly indicated that the concentration of secreted NT-4 in the culture medium
was ineffective in modifying the synapse.
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Figure 1.
Localized presynaptic action of NT-4.
A, Phase-contrast (left) and fluorescence
(right) images of a spinal neuron innervating two
myocytes, one derived from a blastomere injected with NT-4 and GFP mRNA
(M+) and the other (M) from a
uninjected blastomere of the same Xenopus embryo. Scale
bar, 10 µm. B, Continuous traces depict simultaneous
whole-cell recordings from a myocyte overexpressing NT-4
(M+) and a control myocyte (M)
innervated by the same neuron. Downward deflections are MEPSCs or
EPSCs, samples of which are shown below at a higher time
resolution. EPSCs were elicited at a low frequency (at times indicated
by tick marks). Calibration: slow traces, 1 nA, 1 min;
fast traces, 1 nA, 8 msec.
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Figure 2.
Comparison of MEPSC amplitude distribution
recorded from pairs of M+ (filled circles) and
M (open circles) synapses innervated by the same
neuron. The cumulative probability refers to the fraction of total
events with amplitudes smaller than a given amplitude. Data points
represent mean ± SEM (n = 17 triplets). There
was no significant difference between the two distributions
(p > 0.05, Kolmogorov-Smirnov test).
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Figure 3.
Summary of paired recordings of synaptic currents
at M+ (closed circles) and M (open
circles) synapses made by the same neuron. A,
left, MEPSC frequencies at M+ and M synapses observed
at 17 triplets; data from the same triplet are connected by a
line. Middle, All data in the
left graph for M synapses are plotted against the
center-to-center distance between the two myocytes of each triplet
(open circles) and against the presynaptic cytoplasmic
distance between the M+ and M synapses (open
triangles). Dashed and solid
lines represent the best linear fit of the data for
center-to-center and cytoplasmic distances, respectively.
Right, NT-4 + GFP, The
mean values of MEPSC frequency at M+ and M synapses of 17 triplets
obtained from cultures made from embryos injected with both NT-4 and
GFP messages; GFP, data from cultures made from embryos
injected with GFP mRNA only (n = 16 for M
synapses; n = 13 for M+ synapses).
Bars represent mean ± SEM. Asterisk
marks data that were significantly different from corresponding control
values (M+ vs M values; p < 0.05, paired
t test). B, CV of EPSCs recorded from M+
and M myocytes in nine triplets. CV of EPSCs was calculated as the
ratio of the SD to the mean value of the peak EPSC amplitude.
C, Half-decay times of MEPSCs recorded from paired M+
and M myocytes in 17 triplets. Graphs in B and
C are plotted in the same manner as that described in
A.
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To define the spatial range quantitatively, we studied the relationship
of the distance between M+ and M synapses and several synaptic
parameters at M synapses. In these experiments, the direct
extracellular "center-to-center" distance between
NT-4-overexpressing myocytes and control myocytes innervated by the
same neuron ranged from 50 to 230 µm, whereas the presynaptic
cytoplasmic distance between the two synapses ranged from 60 to 360 µm. We reasoned that if the neurotrophin action may spread over a
distance >50 µm, then we should observe a neurotrophin effect on M
synapses in a distance-dependent manner. When values for various
synaptic parameters at M synapses were plotted according to their
extracellular or presynaptic cytoplasmic distances to the paired M+
synapses, no apparent distance dependence was observed (Fig. 3). This
suggests that NT-4 secreted from the NT-4-overexpressing myocyte was
ineffective in modulating synapses on a nearby myocyte as close as 50 µm away and that cytoplasmic changes leading to potentiated
transmitter secretion in the presynaptic neuron were also spatially
restricted within a distance of ~60 µm. However, these results only
provide an upper limit for the spatial range of retrograde neurotrophin action, because it is possible that secreted NT-4 may indeed affect synapses at distances smaller than those studied here. It is known that
NT-4 secreted from an NT-4-overexpressing myocyte also causes an
increase in the decay time of MEPSCs and EPSCs (Wang and Poo, 1997). An
increase in the decay time of synaptic currents can be attributed to an
increase in the mean bursting duration of ACh channels caused by
autocrine regulation of ACh channels by NT-4 (Wang and Poo, 1997). In
these triplet studies, we found that the half-decay time of MEPSCs was
significantly increased at M+ synapses but not at M synapses formed
by the same neuron (Fig. 3C). This result further supports
the conclusion that the effective range of NT-4 secreted from a myocyte
is restricted to 50 µm.
Localized postsynaptic action of NT-4
To further examine the postsynaptic action of NT-4, we performed a
series of experiments in which the timing and the site of NT-4
secretion were more precisely defined. Localized NT-4 secretion was
triggered by repetitive iontophoretic application of ACh pulses at a
high frequency to the surface of isolated NT-4-overexpressing myocytes
while the myocyte was whole-cell voltage-clamped at its resting
potential. The secretion of NT-4 was monitored indirectly as an
increase in the decay time of ACh-induced currents elicited by
low-frequency test ACh pulses at the site of tetanic ACh stimulation. As shown in Figure 4, within minutes
after a train of tetanic ACh pulses (4 Hz, 1 min), the half-decay time
of ACh currents induced by test ACh pulses (0.05 Hz) was significantly
prolonged in NT-4-overexpressing myocytes (M+) but not in control
myocytes (M). No significant change was found for the rise time and
amplitude of ACh currents after tetanic ACh stimulation (Fig. 4). The
time course and magnitude of ACh-induced membrane currents were
qualitatively similar to synaptic currents observed at developing
synapses, and the effect of tetanic ACh stimulation on the decay time
of ACh-induced currents was similar to that found for synaptic currents after repetitive presynaptic stimulation (Wang and Poo, 1997). The
effect of tetanic ACh stimulation on the decay time of ACh-induced currents can be attributed to the stimulation-induced secretion of NT-4
from the myocyte, because in the presence of TrkB-IgG (1 µg/ml), no
change in the decay time was observed (Fig.
5). The blocking effect of TrkB-IgG on
the NT-4 action on the decay time was specific because TrkA-IgG was not
effective (Wang and Poo, 1997). Furthermore, bath application of K252a
(200 nM), an inhibitor of the tyrosine kinase of Trk
receptors, abolished the effect (Fig. 5). This indicates that TrkB
signaling is required for the NT-4 action on the ACh channel. Finally,
the effect was not observed in myocytes expressing GFP message only
(Fig. 5). Thus, iontophoretic ACh application is an effective means in
triggering NT-4 secretion and in providing a physiological assay of the
action of secreted NT-4.
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Figure 4.
Modulation of ACh-induced currents in myocytes by
activity-induced secretion of NT-4. Half-decay time, peak amplitude,
and rise time of ACh-induced currents at various times before and after
tetanic ACh stimulation. Tetanic ACh pulses (4 Hz, 1 min) were applied
iontophoretically at time 0 to the myocyte surface
(arrow). Sample traces (average of 12 events) for
ACh-induced currents before (1) and after
(2) tetanic ACh stimulation are shown above.
Calibration: 0.25 nA, 5 msec. All data are normalized to the mean value
during the control period before the tetanic stimulation (mean ± SEM; n = 9 for M+; n = 7 for
M). Asterisks mark data that were significantly
different from corresponding control values (M+ vs M values;
*p < 0.05; **p < 0.01, t test).
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Figure 5.
Half-decay time of ACh-induced currents during
5-20 min after tetanic ACh application at NT-4-overexpressing
(M+) and control (M) myocytes. Data
(mean ± SEM) are normalized to the mean value during the control
period for each myocyte. The number associated with each
bar refers to the total number of myocytes examined.
NT-4 + GFP, Cultures prepared from
embryos injected with NT-4 and GFP mRNAs; NT-4 + GFP(TrkB-IgG), the same as the group of NT-4 + GFP,
except that TrkB-IgG (1 µg/ml) was applied to the culture medium
during recording; NT-4 + GFP(K252a), the
same as the group of NT-4 + GFP, except that K252a (200 nM)
was applied to the culture medium during recording; GFP,
cultures prepared from embryos injected with GFP mRNA alone. **Data
significantly different from their corresponding control values (M+ vs
M values; p < 0.01, t
test).
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To further delineate the range of postsynaptic action of secreted NT-4,
we examined the spatial distribution of NT-4-induced modulation of ACh
channels in the myocyte membrane. Specifically, we measured the effect
of tetanic ACh pulses (4 Hz, 1 min) on the decay time of ACh-induced
currents at different distances from the site of tetanic ACh
stimulation. Two types of myocytes were used. For spherical myocytes of
different diameters, ACh-induced currents were measured by applying
test ACh pulses at the site of tetanic ACh application (stimulation
site) and at the pole opposite to the stimulation site (control site)
(Fig. 6A). Considering all myocytes that we have examined (range of diameter, 31-43 µm), the effect of tetanic ACh stimulation on the decay time of ACh currents
appeared with a slower time course at the control site (Fig.
6B). Plotting the data according to the myocyte
diameter revealed a dependence of the effect on the size of the myocyte (Fig. 6C). During the 5-20 min after the tetanic
stimulation, the decay time was similarly prolonged at both the
stimulation and control sites in myocytes with a diameter between 31 and 34 µm. The effect on the control site was significantly reduced
for myocytes with a diameter between 34 and 37 µm and completely
disappeared for myocytes with a diameter of >37 µm. Thus, both the
secretion of NT-4 induced by tetanic ACh pulses and the action of NT-4
on myocyte ACh channels did not spread beyond a distance of ~40 µm in the cytoplasm or ~60 µm in the extracellular space,
respectively. In the second set of experiments using spindle-shaped
myocytes, ACh-induced currents were measured at four different
distances (0, 25, 50, and 75 µm) from the site of tetanic ACh
stimulation (Fig. 7A). The
effect of tetanic ACh pulses in prolonging the decay time was clearly
observed at 0, 25, and 50 µm but not at 75 µm (Fig. 7B).
Together with the result obtained from spherical myocytes, we conclude
that after tetanic ACh stimulation, the site of NT-4 secretion, the
diffusion of NT-4 in the extracellular space, and the cytoplasmic
spread of NT-4-activated downstream signaling events responsible for
ACh channel modulation are all restricted within a range of ~60 µm.
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Figure 6.
Localized modulation of ACh-induced currents
by secreted NT-4 at spherical myocytes. A,
Phase-contrast image of a spherical Xenopus myocyte in
1-d-old culture. The membrane current of the myocyte was monitored by a
whole-cell recording pipette (R). The same
iontophoresis pipette (ACh) was used for tetanic ACh
stimulation at stimulation sites (S), as
well as for delivering ACh pulses at a low frequency as test pulses at
both S and control sites
(C). B, Half-decay time of
ACh-induced currents at control and stimulation sites after tetanic ACh
stimulation of NT-4-overexpressing (M+) and control
(M) myocytes. Tetanic ACh pulses (4 Hz, 1 min) were
applied at time 0. All data are normalized to the mean value obtained
during the control period before tetanic stimulation (mean ± SEM;
n = 21 for M+; n = 9 for M).
Asterisks mark data significantly different from values
at control sites measured in the same period (*p < 0.05; **p < 0.01, t test).
C, Dependence of the decay time on the myocyte diameter.
Normalized half-decay times of ACh-induced currents at 5-20 min after
tetanic ACh stimulation at control and stimulation sites were averaged
for groups of myocytes with diameters between 31 and 43 µm in 3 µm
bins. Asterisks mark data significantly different from
values at control sites measured in the same period
(p < 0.05, t test).
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Figure 7.
Localized modulation of ACh-induced currents by
secreted NT-4 at spindle myocytes. A, Phase-contrast
image of a Xenopus spindle myocyte. Tetanic ACh pulses
(4 Hz, 1 min) were applied to one site by an ACh iontophoresis pipette
(ACh), and ACh-induced test currents were monitored at
0, 25, 50, and 75 µm from the site of tetanic ACh stimulation.
B, Dependence of the decay time on the distance from the
site of tetanic stimulation. Normalized decay times 5-20 min after
tetanic ACh stimulation were averaged for seven NT-4-overexpressing
(M+) and six control (M) spindle
myocytes. Asterisks mark data significantly different
from values obtained from M myocytes (p < 0.05, t test).
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DISCUSSION |
Previous work has shown that local application of exogenous
neurotrophins can induce localized changes in the morphology of nerve
growth cones and in the secretion of neurotransmitters from presynaptic
nerve terminals (Campenot, 1977; Seeley and Greene, 1983; Stoop and
Poo, 1995). However, it is unknown over which distance neurotrophins
secreted locally from a cell can exert their influence on the structure
and function of the synapse. Using Xenopus nerve-muscle
cultures, we have quantitatively assayed the spatial range of
presynaptic and postsynaptic actions of secreted NT-4. For the
presynaptic action on the transmitter release mechanisms, our data
provided an upper limit of 50-60 µm for the spread of NT-4 action
within the presynaptic neuron. For the postsynaptic NT-4 action on ACh
channel kinetics, which we were able to monitor with higher spatial
resolution, we found that the NT-4 effect can spread up to 50 µm but
not >60 µm from the site at which NT-4 secretion was induced in the
myocyte. This result suggests that both the diffusion of secreted NT-4
molecules in the extracellular space and the spread of downstream
effectors of NT-4 signaling in the cytoplasm are restricted to ~50
µm.
Using an embryo injection technique, NT-4 levels were transiently
elevated in approximately half of the cells in these Xenopus nerve-muscle cultures. The levels of NT-4 overexpression and of NT-4
basal secretion achieved in the present study were nonsaturating, indicating that they were likely within a physiological range. As shown
previously, NT-4 release evoked by high-frequency synaptic stimulation
in this preparation was effective in further potentiating the
presynaptic transmitter release, as well as in modulating postsynaptic
ACh receptor properties (Wang and Poo, 1997).
Electrical or synaptic activity has been shown to regulate the
synthesis of BDNF and NGF in hippocampal cultures (Zafra et al., 1990;
Lu et al., 1991), of NT-4 in rat skeletal muscle (Funakoshi et al.,
1995), and of NT-3 in cultured Xenopus myocytes (Xie et al.,
1997). Our present and previous (Wang and Poo, 1997) findings indicate
that synaptic activity can regulate the secretion of NT-4 from
Xenopus myocytes, consistent with previous results on depolarization-induced NGF and BDNF secretion (Blöchl and
Thoenen, 1995; Goodman et al., 1996). Whereas activity-dependent
neurotrophin synthesis regulates the amount of available neurotrophin
with relatively slow kinetics, neurotrophin secretion induced by
synaptic activity provides a means for controlling the availability of neurotrophins at a synapse on a much more rapid time scale, thereby temporally coupling presynaptic activity to postsynaptic neurotrophin secretion. An increase in the rate of neurotrophin synthesis may also
contribute to replenishing the pool of releasable neurotrophins upon
secretion. Because neurotrophins have been shown to potentiate synaptic
transmission, regulation of both synthesis and secretion by activity
results in a positive feedback at active synapses, and such mechanisms
may be used to strengthen or stabilize these synaptic connections.
Overexpressing myocytes also appeared to secrete NT-4 constitutively.
This is suggested by the findings that an increase in the mean burst
duration of ACh receptors was observed in single-channel recordings
from isolated NT-4-overexpressing myocytes compared with that found in
control myocytes, and that this effect was blocked by the presence of
TrkB-IgG (Wang and Poo, 1997). Constitutive secretion of NT-4 may also
account for the elevated frequency of spontaneous transmitter release
and the reduced fluctuation of evoked transmitter release, although it
remains unclear to what extent the synaptic activity attributable to
spontaneous secretion of ACh also plays a significant part in
triggering NT-4 secretion from the myocyte. Secretion triggered by
spontaneous synaptic activity may also be considered constitutive
because it is independent of the action potential in the presynaptic
neuron. During development of the nervous system, constitutive
neurotrophin secretion may serve to maintain basal synaptic function,
whereas action potential-triggered neurotrophin secretion may be
responsible for activity-dependent modulation of synaptic
connections.
It has been proposed that during the process of synapse refinement by
activity, nerve terminals compete for limiting amounts of neurotrophic
factors secreted by the target cell (Purves and Lichtman, 1985). There
is evidence that neurotrophins are required for the formation of ocular
dominance columns (Maffei et al., 1992; Cabelli et al., 1995, 1997).
The highly localized action of neurotrophin may provide a mechanism for
retaining specificity of synaptic modification during the process of
synapse refinement. Thus, activity-dependent synthesis,
activity-triggered local secretion, and localized synaptic action of
neurotrophins may all contribute to the selective stabilization of
active synaptic connections. In sharp contrast to the localized
presynaptic action of NT-4, activity-induced long-term depression in
these Xenopus neuromuscular synapses can spread
presynaptically over a distance of 400 µm to neighboring synapses
formed by the same axon on other myocytes (Cash et al., 1996). The
coexistence of such opposite signaling mechanisms involved in synaptic
modulation may help ensure effective pruning of developing synapses
during the process of synapse elimination.
Previous studies have shown that presynaptic potentiation of
transmitter secretion at M+ synapses represents a long-lasting synaptic
modification that cannot be reversed by a 1 hr incubation of TrkB-IgG
(Wang and Poo, 1997). Thus, long-term synaptic actions of neurotrophins
can be localized. Such a localized effect indicates that the activated
intracellular effector molecules associated with the TrkB signaling
pathway must be spatially restricted within a range <60 µm from the
site of synaptic contact with the NT-4-overexpressing myocyte. On the
other hand, target-derived trophic factors are known to affect neuronal
survival and maintenance of global neuronal properties of the
presynaptic neuron (Barde, 1989; Oppenheim, 1991; Lewin and Barde,
1996), including the efficacy of synaptic inputs on its dendrites
(Purves and Nja, 1978). The present results thus underscore the
existence of two distinct modes of the action of neurotrophins as
retrograde factors: a local action involving spatially restricted
activation of Trk transduction pathways and their downstream
substrates, and a global somatic action that depends on axonal
transport of either internalized neurotrophin-receptor complexes or
downstream effector molecules in the Trk transduction pathway.
Using a cultured Aplysia sensory neuron with bifurcating
neurites synapsing on two separated motoneurons, Martin et al. (1997) have recently demonstrated a branch-specific long-term facilitation. When synapses formed by one branch of the sensory neuron axon were
locally perfused with serotonin, long-term facilitation was restricted
to the site of perfusion and did not spread to the synapses formed by
the unperfused branch, an effect that required local protein synthesis
and involved the growth of new synapses on the perfused branch.
Interestingly, long-term facilitation at Aplysia
sensory-motoneuron synapses seems to require endogenous TGF and is
occluded by treatment with exogenous TGF (Zhang et al., 1997). It is
possible that TGF and NT-4 exert their highly localized action on
the presynaptic neuron via similar cellular mechanisms.
LTP induced by activation of specific synaptic inputs to CA1 pyramidal
neurons in hippocampal slice cultures is accompanied by a spread of
synaptic potentiation to adjacent synapses made by other presynaptic
inputs to the same postsynaptic neuron (Engert and Bonhoeffer, 1997).
The distance of affected synapses was found to be <70 µm. Such
restricted spread of LTP could be attributed to the localized action of
secreted factors similar to that shown here. It is known that gene
expression and protein synthesis are required for the maintenance of
long-term synaptic modification (Nguyen et al., 1994; Kang and Schuman,
1996; Martin et al., 1997). Therefore, some local signal(s) must
persist to preserve synapse specificity. One intriguing possibility is
that a synaptic "tag" (Frey and Morris, 1997; Martin et al., 1997)
is established by the initial action of locally secreted neurotrophins
and that global gene activation results in long-term potentiation
specifically of those synapses carrying the tag.
| |
FOOTNOTES |
Received Jan. 9, 1998; revised March 27, 1998; accepted April 13, 1998.
This work was supported by National Institutes of Health Grant NS22764.
B.B. is supported by a fellowship from the Human Frontier Science
Program Organization. We thank Genentech Inc. for providing Trk-IgG
fusion proteins.
Correspondence should be addressed to Dr. Mu-ming Poo, Department of
Biology, University of California at San Diego, La Jolla, CA
92093-0357.
| |
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Top
Abstract
Introduction
