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April 24, 2002
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The Journal of Neuroscience, 2002, 22:RC220:1-6
RAPID COMMUNICATION
Transforming Growth Factor  1 Alters Synapsin Distribution and
Modulates Synaptic Depression in Aplysia
J.
Chin1,
A.
Angers1,
L. J.
Cleary1,
A.
Eskin2, and
J. H.
Byrne1
1 Department of Neurobiology and Anatomy, W. M. Keck Center for the Neurobiology of Learning and Memory, University of
Texas-Houston Medical School, Houston, Texas 77030, and
2 Department of Biology and Biochemistry, University of
Houston, Houston, Texas 77204
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ABSTRACT |
Transforming growth factor  1 (TGF-1) induces long-term
synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has
acute effects as well. Treatment of pleural-pedal ganglia with
TGF-1 for 5 min activated mitogen-activated protein kinase (MAPK)
and stimulated the phosphorylation of synapsin in a MAPK-dependent
manner. This phosphorylation appeared to modulate synapsin distribution
in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-1-treated sensory neurons showed a significant reduction in the number of these puncta,
an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that
TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.
Key words:
synapsin; Aplysia; TGF-1; synaptic
depression; short-term plasticity; mobilization; phosphorylation; synaptic vesicles; MAPK
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INTRODUCTION |
Neurotrophins
and cytokines are signaling molecules that play an important role in
development. Recently, it has become clear that neurotrophins have
modulatory effects on synaptic transmission and neuronal plasticity in
mature organisms (for review, see Lu and Gottschalk, 2000 ).
Interestingly, some of these modulatory effects are acute. For example,
brain-derived neurotrophic factor (BDNF), a member of the neurotrophin
family, reduces synaptic depression in a mitogen-activated protein
kinase (MAPK)-dependent manner in rat hippocampal slices (Gottschalk et
al., 1999). Because synaptic depression has been attributed to the
depletion of the releasable pool of synaptic vesicles, the reduction in
synaptic depression is likely to be caused by a presynaptic mechanism. Indeed, phosphorylation of synapsin I through a MAPK pathway appears to
mediate the enhancement of neurotransmitter release induced by BDNF in
rat and mouse synaptosomes (Jovanovic et al., 2000). Although acute
modulatory effects of the neurotrophin family of growth factors have
been examined extensively, little is known about acute modulatory
effects of the transforming growth factor (TGF-) family of
growth factors that play important roles in early development of the
nervous system (Böttner et al., 2000). Because TGF-1 mediates
some forms of long-term neuronal plasticity in Aplysia
(Zhang et al., 1997; Chin et al., 1999; Farr et al., 1999), we examined
whether it had acute affects as well. In addition, we examined whether
TGF-1, like neurotrophins, might exert its acute effects via
phosphorylation of synapsin.
We found that TGF-1 activated MAPK and stimulated the
phosphorylation of synapsin in Aplysia sensory neurons. In
addition, TGF-1 altered the distribution of synapsin in sensory
neurons in a MAPK-dependent manner. Furthermore, TGF-1 decreased
low-frequency synaptic depression.
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MATERIALS AND METHODS |
MAPK phosphorylation assay. Aplysia
californica (100-200 gm; Alacrity, Redondo Beach, CA, and
Marinus, Long Beach, CA) were housed in cages within aquaria maintained
at 15°C on a 12 hr light/dark cycle. Animals were anesthetized with
isotonic MgCl2, and paired pleural-pedal ganglia
were removed and divided into control and experimental groups. Ganglia
were treated in groups of three to provide enough protein for analysis.
The pleural ganglia were desheathed to expose the ventral-caudal
cluster of sensory neurons in a 1:1 solution of isotonic
MgCl2 and artificial seawater (ASW) containing
(in mM): 460 NaCl, 10 KCl, 11.4 CaCl2, 27 MgCl2, and 10 HEPES. The ganglia "rested" in ASW for 2 hr at 15° C before recombinant human TGF-1 (1 ng/ml; R & D Systems) or BSA
control (Pentex) was applied for 5 min. The MEK inhibitor U0126 (20 µM; Promega) was applied for 1 hr before
treatment with TGF-1/BSA. Immediately after treatment, the pleural
sensory clusters were excised and homogenized in ice-cold buffer
containing (in mM): 10 EDTA, 20 Tris, pH 7.5, 1 Na orthovanadate, 1 DTT, 2 NaF, 2 NaPPi, 0.5 okadaic acid, 1 PMSF, 1%
SDS, 1% protease inhibitor cocktail (Sigma, St. Louis, MO). After
determination of protein content by Bradford assay, equal protein from
each group was resolved by SDS-PAGE and transferred to a nitrocellulose
membrane. The membrane was probed with an antibody specific to the
dually phosphorylated form of the ERK1/2 isoforms of MAPK (1:2500;
Promega V8031) and then exposed to
I125-conjugated Protein A (1:1000).
Immunoreactive bands were visualized by autoradiography.
Synapsin phosphorylation assay. Pleural-pedal ganglia were
isolated and desheathed as above. After overnight incubation in ASW
containing 10 µCi/ml 32P (specific
activity of 4500 Ci/mmol) (Homayouni et al., 1995), ganglia were
treated for 5 min with 1 ng/ml TGF-1 or BSA. Ganglia were treated in
groups of three to provide enough protein for analysis. Immediately
after treatment, pleural ganglia were homogenized as above. Equal
amounts of protein were subjected to immunoprecipitation in 5 vol of
immunoprecipitation buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, and protease
inhibitors), with 1:200 vol of antisera against Aplysia
synapsin (anti-apSyn) and 1:10 vol of protein-A Sepharose.
Immunoprecipitated material was resolved by SDS-PAGE and transferred to
nitrocellulose. 32P signal was quantified
with a phosphorimager. The membranes were then immunoblotted with
anti-apSyn antibody to quantify the amount of synapsin. The amount of
phosphorylation was calculated as the ratio of the treated and control
samples for each experiment, after normalization to protein content.
Immunocytochemistry. Culturing procedures followed those
described by Rayport and Schacher (1986). Neurons were removed
individually by microelectrodes with fine tips and plated on
poly-L-lysine-coated glass coverslips in Petri
dishes containing 50% hemolymph and 50% isotonic L15. After 5 d
in culture, neurons were treated with TGF-1 or BSA for 5 min and
immediately fixed in 4% paraformaldehyde in PBS containing 30%
sucrose. Cells were rinsed with PBS and incubated in 2% normal goat
serum in PBS containing 0.1% Triton X-100 before incubation with
anti-apSyn (1:200) overnight at 4°C. Cultures were rinsed with PBS
and incubated with tetramethylrhodamine-conjugated goat anti-rabbit
IgG. Immunofluorescence was viewed with confocal microscopy. A z-series
of optical sections through the neurites (0.15 µm increments through
~10 µm) were taken with a Bio-Rad 1024 MP confocal microscope.
The individual performing the quantification of immunoreactivity did
not know whether the sensory neuron had been treated with TGF-1 or
BSA. Stacked images of the arborizations of each cell were used for
quantification. The extent of the arborization visible in the
micrograph was traced using MetaMorph software to obtain a measure of
the total neurite length. Aggregates, or puncta, of intense apSyn
immunoreactivity were manually marked and automatically counted by the software.
Electrophysiology. Experiments were performed using a single
sensory neuron cocultured with a single motor neuron. The motor neuron
was impaled with an electrode filled with 3 M KAc
(10 M resistance), and resting potential and input resistance were
measured. A single EPSP was evoked in the motor neuron by extracellular stimulation of the sensory neuron. The cocultures were then divided into groups such that the two groups consisted of cultures having similar distributions of EPSP amplitudes (BSA, 29.5 ± 4.4 mV; mean ± SEM; TGF-1, 33.9 ± 7.5 mV). At least 2 hr later,
1 ng/ml TGF-1 or BSA was applied for 5 min before the motor neuron
was re-impaled for measurements. All treatments were performed in a
blinded manner. The sensory neuron was stimulated with a 1 Hz stimulus for 20 sec. EPSPs were recorded in the motor neuron with resting membrane potential current clamped at -80 mV.
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RESULTS |
TGF-1 induces synapsin phosphorylation
Because MAPK mediates neurotrophic factor-induced phosphorylation
of synapsin (Jovanovic et al., 2000), we examined the ability of
TGF-1 to induce phosphorylation of Aplysia synapsin via
MAPK. The Aplysia homolog of synapsin was recently cloned
(Angers et al., 1999). Aplysia synapsin shows the same
domain arrangement as other vertebrate and invertebrate synapsin I
molecules and exhibits MAPK consensus phosphorylation sites. Although
little is known about TGF-1 signaling in neurons, recent results
indicated that the MAPK cascade is engaged by TGF-1. For example,
TGF-1 results in the transient activation of the MAPK pathway in
chick ciliary ganglion neurons, which is necessary for the acute and sustained effects of TGF-1 on the expression of calcium-activated potassium channels (Lhuillier and Dryer, 2000).
Treatment of pleural-pedal ganglia with 1 ng/ml TGF-1 for 5 min
increased synapsin phosphorylation by 91 ± 23% (mean ± SEM; n = 4; t3 = 3.9;
p < 0.05) compared with BSA control (Fig.
1A1,A2). The amount of phosphorylation was calculated as the ratio of the treated and control samples for each experiment, after normalization to
protein content. After autoradiography, Western blot analysis was
performed using an antibody specific to Aplysia synapsin to quantify the amounts of total protein. Overall, no significant difference was found in the protein levels of synapsin between BSA- and
TGF-1-treated samples (data not shown). The TGF-1-induced increase in phosphorylation was blocked by preincubation with 20 µM U0126, a specific (Davies et al., 2000)
inhibitor of MEK (9 ± 19% change in phosphorylation compared
with U0126 + BSA control; n = 5;
t4 = 0.48; p = 0.65)
(Fig. 1A1,A2). We verified that U0126 inhibited MEK in Aplysia. U0126 decreased basal MAPK
phosphorylation in sensory neurons (n = 3;
t2 = 4.41; p < 0.05)
(Fig. 1B1,B2). These results indicate that
TGF-1 induces synapsin phosphorylation through a MAPK-dependent
pathway.
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Figure 1.
TGF-1 activated MAPK and induced synapsin
phosphorylation. A1, Autoradiographs (top
panels) show levels of phosphorylated synapsin
(phospho apSyn) in ganglia treated with BSA
(control), TGF-1, U0126 + BSA, or U0126 + TGF-1. Western
blots (bottom panels) of the same membranes show total
protein levels. For quantification of effects of TGF-1 on synapsin
phosphorylation, phosphorylation signal was normalized to total
synapsin determined by Western blot. A2, Summary data
showing that TGF-1 increased synapsin phosphorylation by 91%.
Treatment with U0126 blocked the increase in synapsin phosphorylation
by TGF-1. B, C, Western blots show
levels of dually phosphorylated MAPK (phospho MAPK) in
sensory neuron clusters after specified treatments. Equal amounts of
protein were loaded from control and experimental extracts and probed
with the antibody to dually phosphorylated, active MAPK.
B1, B2, U0126 alone (20 µM,
1 hr) decreased basal levels of dually phosphorylated MAPK by 56%.
C1, C2, TGF-1 increased levels of
dually phosphorylated, active MAPK by 37%. U0126 blocked the ability
of TGF-1 to increase levels of dually phosphorylated MAPK over
levels found in U0126 + BSA controls. *p < 0.05.
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TGF-1 activates MAPK in sensory neurons
Because MAPK activity appeared to be necessary for
TGF-1-induced synapsin phosphorylation, we investigated whether
TGF-1 activated MAPK in sensory neurons. Although many species
contain ERK1 and ERK2 isoforms of MAPK, only one immunoreactive band
(43 kDa) was identified by Western blot analysis in the
Aplysia CNS (Michael et al., 1998). In confirmation of these
results, the antibody to dually phosphorylated, activated ERK1/2
recognized a single band of 43 kDa in sensory neuron extracts (Fig.
1B1,C1). Furthermore, TGF-1 (1 ng/ml
for 5 min) increased the levels of dually phosphorylated MAPK compared
with BSA-treated controls (n = 7;
t6 = 2.66; p < 0.05)
(Fig. 1C1,1C2). The increase in active MAPK by
TGF-1 was completely blocked in the presence of 20 µM U0126 (n = 4;
t3 = 1.05; p = 0.37)
(Fig. 1C1,1C2). These results demonstrate that
TGF-1 activates MAPK and supports the hypothesis that TGF-1 leads
to synapsin phosphorylation via MAPK.
TGF-1 alters the distribution of synapsin in
sensory neurons
Phosphorylation of synapsin alters its binding properties to both
synaptic vesicles and cytoskeletal elements. After high-frequency stimulation of the frog neuromuscular junction, 30% of synapsin dissociated from synaptic vesicles (Torri Tarelli et al., 1992). Recently, this result was confirmed in cultured hippocampal neurons by
the finding that synaptic activity induced the dispersion of synapsin
in a phosphorylation state-dependent manner (Chi et al., 2001).
Moreover, in Aplysia sensory neurons, the neuromodulatory transmitter serotonin (5-HT) induced the dispersion of synapsin (Angers
et al., 2000). Thus, the distribution of synapsin was examined after
treatment with BSA or TGF-1 for 5 min. In control cultures, synapsin
immunoreactivity was present in a punctate pattern distributed along
neurites (n = 6) (Fig.
2A, left
panels). However, in TGF-1-treated neurons, ~54% fewer
puncta could be detected (n = 6) (Fig.
2A, right panels, B). This
effect was statistically significant
(t10 = 2.38; p < 0.05). To test the hypothesis that MAPK mediates the effect of TGF-1
on the dispersion of synapsin, sensory neurons were incubated with 20 µM U0126 for 1 hr before treatment with either
TGF-1 or BSA. The MEK inhibitor blocked the ability of TGF-1 to
decrease the number of puncta (n = 10 for U0126 + BSA;
n = 9 for U0126 + TGF-1;
t17 = 0.6; p = 0.56) (Fig. 2A, bottom panels,
C).
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Figure 2.
TGF-1 induced dispersion of synapsin in cultured
sensory neurons. A, Confocal images of cultured sensory
neurons immunostained for synapsin. Control neuron (top left
panel) shows punctate pattern of staining.
TGF-1-treated neuron (top right panel)
exhibits fewer detectable puncta. The area defined by dashed
lines is seen in higher magnification in the middle
panels. Arrows denote puncta. There is no
difference in staining when neurons are treated with U0126 before BSA
(bottom left panel) or TGF-1 (bottom
right panel). Scale bar: 80 µm (40 µm for
middle panels). B, C,
Summary graphs showing the reduction in the number of synapsin puncta
per 100 µm neurite length in TGF-1-treated neurons compared with
BSA controls (B) and blockade of the effect of
TGF-1 by U0126 (C). MetaMorph software was
used to measure total neurite length and count the number of puncta.
*p < 0.05; Student's t test.
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TGF-1 modulates synaptic depression
The interactions of synapsin with vesicle membranes, actin, and
other cytoskeleton proteins are believed to tether synaptic vesicles
into a "reserve pool" of vesicles. It is thought that the abolition
of these interactions by phosphorylation of synapsin (Jovanovic et al.,
1996; Matsubara et al., 1996; Hosaka et al., 1999) results in the
release of synapsin-bound vesicles from the reserve pool, allowing
vesicle mobilization to the readily releasable pool of vesicles (Turner
et al., 1999). Augmentation of the readily releasable pool of vesicles
may then lead to an increase in synaptic efficacy. The results
presented above suggest that TGF-1 treatment may alter the
distribution of synapsin within the terminal, thereby altering the
availability of synaptic vesicles in the reserve pool for mobilization
to the readily releasable pool. If so, TGF-1-treated synapses would
be less susceptible to depression. Thus, to examine whether the effects
of TGF-1 on the phosphorylation state and distribution of synapsin
have a physiological consequence, we studied synaptic depression evoked
by low-frequency stimuli. To challenge the release machinery,
EPSPs were elicited at an interstimulus interval of 1 sec, a rate that
leads to significant homosynaptic depression (Byrne, 1982) and
partially depletes the synaptic vesicle pools (Gingrich and Byrne,
1985; Armitage and Siegelbaum, 1998; Royer et al., 2000).
TGF-1 (1 ng/ml for 5 min) reduced the magnitude of synaptic
depression (Fig.
3B1,B2). The
amplitude of the EPSP at steady state was ~50% greater in
TGF-1-treated cocultures (n = 5) compared with
controls (n = 6;
F(1,17) = 6.77; p < 0.05; repeated measures ANOVA on EPSPs 3-20). A repeated measures
ANOVA was also used to compare the amplitudes of pretreatment EPSPs
with the first EPSP in the train in BSA- and TGF-1-treated groups.
There was no significant between-group difference in the amplitudes
of EPSPs in the BSA- or TGF-1-treated groups
(F(1,9) = 0.03; p = 0.88). Although there was significant reduction of the EPSPs in both the BSA-
and TGF-1-treated groups
(F(1,9) = 11.8; p < 0.01), which is presumably caused by rundown over the 2 hr pretest
phase, there was no significant interaction effect between treatment (BSA vs TGF-1) and time (pretest vs first EPSP of train;
F(1,9) = 4.58; p = 0.06). Consequently, it is unlikely that the effect of TGF-1 on the
reversal of synaptic depression during a train of stimuli (Fig.
3A) is caused by a shift in baseline synaptic transmission.
These results are consistent with previous findings that TGF-1
affects long-term, but not short-term, synaptic efficacy tested with a
single EPSP (Zhang et al., 1997). These results also indicate
that TGF-1 alters the steady-state level of transmitter release,
which could be dependent on the mobilization of vesicles from the
reserve pool to the readily releasable pool of transmitter (Turner et
al., 1999; Thomson, 2000), an effect that would be expressed when the
synapse is challenged with a train of action potentials.
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Figure 3.
TGF-1, via MAPK, reduces synaptic depression
induced by low-frequency stimuli. A, Phase-contrast
image of cocultured sensory neuron (SN) and motor
neuron (MN) from which recordings were made. The
SN was stimulated (20 sec duration, 1 Hz) with an extracellular
electrode (right), and EPSPs were measured in the MN
with an intracellular electrode (left).
B1, Examples of EPSPs recorded from cocultures treated
with BSA or TGF-1. For clarity, only the 1st, 10th, and 20th EPSPs
of the train are shown. EPSPs of the TGF-1-treated neurons were
scaled by a factor of 89% to account for the initial difference in
amplitude in these examples. The dashed line was drawn
arbitrarily at half-maximal amplitude for comparison purposes.
B2, Group data. EPSP amplitudes are normalized to the
first EPSP in the train. TGF-1 ( ) reduced synaptic depression
compared with BSA controls ( ) (p < 0.05;
repeated measures two-way ANOVA, with an interaction between the number
of stimulations and treatment, p < 0.02).
C1, Examples of EPSPs recorded from cocultures treated
with U0126 + BSA or U0126 + TGF-1. EPSPs of the U0126 + TGF-1-treated
group were scaled by a factor of 109% to those of the U0126 + BSA-treated group to account for the initial difference in amplitude in
these examples. C2, U0126 () blocks the ability of
TGF-1 to modulate depression compared with control ().
Inset, Comparison of BSA and
U0+BSA groups demonstrates that basal MAPK activity
affects the amplitude of the second EPSP.
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We next examined whether MAPK played a role in the effect of TGF-1
on synaptic depression. Preincubation with U0126 for 1 hr blocked the
ability of TGF-1 to modulate the steady-state level of synaptic
depression (n = 5 for BSA; n = 4 for
TGF-1; F(1,17) = 0.82;
p = 0.39; repeated measures ANOVA on EPSPs 3-20) (Fig.
3C1,C2). In addition, treatment with U0126 alone,
which decreases the activity of endogenous MAPK (Fig. 1C),
affected the properties of synaptic depression. The amplitude of the
second EPSP of the train in U0126 + BSA-treated neurons was
significantly lower than in BSA-treated neurons, indicating that
inhibition of endogenous MAPK resulted in more pronounced synaptic
depression (t9 = 3.49;
p < 0.01) (Fig. 3C2, inset).
TGF-1 did not appear to have any obvious effects on the
biophysical properties of the motor neuron. There was no difference in
resting potential (BSA, before,  64 ± 1.1 mV, after, 62.3 ± 1.7 mV, t10 = 0.83,
p = 0.43; TGF-1, before, 62.8 ± 1.0 mV, after, 60.4 ± 1.7 mV, t8 = 1.21, p = 0.26) or input resistance (BSA, before,
11.0 ± 0.4 M, after, 10.8 ± 0.5 M,
t10 = 0.21; p = 0.21; TGF-1, before, 14.3 ± 1.8 M, after, 14.3 ± 1.6 M, t8 = 0.03, p = 0.97) of the postsynaptic cell after treatment.
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DISCUSSION |
Although growth factors are traditionally thought of as being
essential for the growth and differentiation of neuronal populations, several recent studies indicate that they play a critical role in the
acute modulation of synaptic transmission (for review, see Schinder and
Poo, 2000). At least one mechanism by which growth factors can alter
synaptic transmission is through the modulation of synapsin (Jovanovic
et al., 1996, 2000). The involvement of synapsin in the maintenance of
a reserve pool of synaptic vesicles in nerve terminals has been
suggested by several functional and morphological studies (Torri
Tarelli et al., 1992; Turner et al., 1999; Thomson, 2000; Chi et al.,
2001), providing further support for the hypothesis that synapsin
modulation may impact neurotransmitter release. The results presented
here are consistent with these findings and suggest a new role for
TGF-1 in synaptic plasticity.
Modulation of synapsin by phosphorylation
The observation that TGF-1 disrupts the normal punctate
distribution of synapsin supports the hypothesis that TGF-1-mediated phosphorylation of synapsin has a functional consequence in terms of
synapsin localization. The punctate staining pattern may represent high-concentration aggregates of synapsin such as might be expected when many synapsin molecules are bound to the reserve pool of synaptic
vesicles by interactions with synaptic vesicles or cytoskeletal elements. The reduction in detectable puncta after TGF-1 treatment may represent the phosphorylation of synapsin and subsequent dispersion of synapsin and synapsin-bound vesicles from high-concentration aggregates. Phosphorylation by MAPK controls interactions of synapsin with the cytoskeleton (Matsubara et al., 1996; Jovanovic et al., 2000).
Thus, phosphorylation of synapsin by MAPK may lead to alteration of
synapsin distribution by terminating the interaction between synapsin
and the cytoskeleton, allowing the dispersion of synapsin.
There is evidence that in cultured Aplysia neurons the
presence of an appropriate postsynaptic partner can differentially influence both synapse formation and modulation (Glanzman et al., 1989,
1990, 1991). Thus, it is possible that the results of these experiments
may reflect developmental effects of TGF-1 rather than an effect on
mature synapses. However, it is clear from images of isolated sensory
neurons that varicosities, morphological swellings that indicate
presynaptic specializations, exist in these cultures (Fig.
2A). In addition, varicosities of isolated sensory
neurons exhibit intense immunoreactivity for glutamate (Levenson et
al., 2000), indicating that the neurotransmitter is localized in
varicosities even in the absence of a postsynaptic neuron. Because
synapsin immunoreactivity is also localized to varicosities, it appears that vesicles and synapsin coexist in isolated sensory neurons. Thus,
it seems likely that synapsin may be regulated similarly in isolated
sensory neurons as well as in cocultured presynaptic and postsynaptic
neurons. Indeed, modulation of synapsin puncta, by the neuromodulator
5-HT, occurs similarly in isolated sensory neurons as well as in
sensory neurons cocultured with a postsynaptic neuron (Angers et al.,
2000). Thus, it does not seem likely that the effect of TGF-1 on
synapsin puncta in isolated sensory neurons is caused by a
developmental effect on neurons that would not be pertinent to mature synapses.
Synapsin modulation and synaptic depression
The finding that TGF-1 reduces synaptic depression via a
MAPK-dependent mechanism, in conjunction with the findings that TGF-1 leads to the phosphorylation of synapsin and alters its neuritic distribution, suggests that the reduction in synaptic depression is mediated by the phosphorylation of synapsin by
MAPK. These actions of TGF-1 are reminiscent of recently
described actions of BDNF in synaptic transmission. In addition to
phosphorylating synapsin and increasing neurotransmitter release
(Jovanovic et al., 2000), BDNF reduces synaptic fatigue in a
MAPK-dependent manner in rat hippocampal slices (Gottschalk et al.,
1999). Because synaptic fatigue has been attributed to the depletion of
the releasable pool of synaptic vesicles (Zucker, 1989; Larkman et al.,
1991; Dobrunz and Stevens, 1997), the reduction in synaptic fatigue induced by BDNF appears to be caused by a presynaptic mechanism, possibly by increased mobilization of vesicles via the modulation of synapsin.
MAPK modulation of synaptic depression
In these experiments, there was a difference in the kinetics of
synaptic depression between control cultures (cultures receiving only
BSA) and cultures treated with the MEK inhibitor U0126 (Fig. 3C2, inset). One possible explanation for the
transient decrease in the amplitude of the second EPSP of the train in
the presence of U0126 is that there are at least two mechanisms for
mobilization depending on the requirement for calcium (Rosenmund and
Stevens, 1996; Kuromi and Kidokoro, 2000).
Ca2+-dependent (activity-dependent)
mobilization is recruited after the second stimulus, whereas basal MAPK
activity may be important for
Ca2+-independent vesicle mobilization at
the beginning of the stimulus train. In the presence of U0126,
Ca2+-independent mobilization may be
impaired because of the lack of MAPK activity, and thus EPSP amplitude
decreases sharply, below the level of mobilization that is supported by
Ca2+-dependent mobilization. After the
second stimulus, Ca2+-dependent
mobilization may be recruited, and the subsequent rebound in EPSP
amplitude may reflect the mobilization of vesicles into the readily
releasable pool.
Because basal MAPK activity affects synaptic depression, it might be
expected then that inhibition of MAPK-induced phosphorylation of
synapsin may lead to increased numbers of synapsin puncta. However,
although the regulation of synapsin function by increased phosphorylation has been studied over the last several years, very
little information is known regarding how synapsin is regulated when
phosphorylation is decreased below basal levels. In our study, no
significant change in the number of synapsin puncta was observed in the
presence of the inhibitor of MAPK activity (Fig.
2A,C). In fact, there was a trend
for a slight decrease in synapsin puncta (Fig. 2, compare left
bars in B and C). Indeed, under some
circumstances, this trend becomes statistically significant, as
observed in another study (Fioravante et al., 2001). Therefore,
basal phosphorylation of synapsin may play an additional, thus far
uncharacterized, role in the regulation of synapsin. In addition, there
may be numerous MAPK substrate proteins affected by modulatory factors, and the net effect on puncta and transmitter release could be caused by
a complex interaction among them.
TGF-1 has been shown previously to induce long-term synaptic
facilitation (Zhang et al., 1997). The present work provides the first
evidence that TGF-1 signaling produces acute effects on synaptic
transmission and that at least one target of TGF-1 is synapsin.
Because many neurons physiologically fire in bursts of action
potentials, modulation of synapsin and thus transmitter release by
TGF-1 may have profound effects on the efficacy of synaptic
communication. Furthermore, these results, together with the recent
findings that synapsin phosphorylation by MAPK is correlated with
BDNF-induced enhancement of neurotransmitter release in rat and mouse
synaptosomes (Jovanovic et al., 2000) and that BDNF reduces synaptic
fatigue via a MAPK-dependent mechanism (Gottschalk et al., 1999),
provide strong evidence for the importance of MAPK activity in the
acute modulation of neurotransmitter release.
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FOOTNOTES |
Received Jan. 16, 2002; revised Feb. 21, 2002; accepted Feb. 22, 2002.
This work was supported by National Institutes of Health (NIH)
Fellowship MH12107 (J.C.) and NIH Grants NS38100 (L.J.C.), NS28462
(A.E.), and NS19895 (J.H.B.). We thank D. Fioravante for comments on a
previous draft of this manuscript.
Correspondence should be addressed to John H. Byrne, Department of
Neurobiology and Anatomy, University of Texas-Houston Medical School,
6431 Fannin Street, Houston, TX 77030. E-mail:
john.h.byrne{at}uth.tmc.edu.
J. Chin's present address: Gladstone Institute of Neurological
Disease, University of California San Francisco, P.O. Box 419100, San
Francisco, CA 94141-9100.
A. Angers's present address: Department of Neurology and Neurosurgery,
Montreal Neurological Institute, McGill University, 3801 University
Street, Montreal, Quebec, H3A 2B4, Canada.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2002, 22:RC220 (1-6). The
publication date is the date of posting online at
www.jneurosci.org.
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REFERENCES |
-
Angers A,
Bean AJ,
Byrne JH
(1999)
Cloning and molecular characterization of Aplysia synaptic vesicle protein synapsin.
Soc Neurosci Abstr
25:1749.
-
Angers A,
Chin J,
Cleary LJ,
Byrne JH
(2000)
5-HT treatment induces redistribution of Aplysia synapsin.
Soc Neurosci Abstr
26:881.
-
Armitage BA,
Siegelbaum SA
(1998)
Presynaptic induction and expression of homosynaptic depression at Aplysia sensorimotor neuron synapses.
J Neurosci
18:8770-8779[Abstract/Full Text].
-
Böttner M,
Krieglstein K,
Unsicker K
(2000)
The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions.
J Neurochem
75:2227-2240[Abstract/Full Text].
-
Byrne JH
(1982)
Analysis of synaptic depression contributing to habituation of gill-withdrawal reflex in Aplysia californica.
J Neurophysiol
48:431-438[Medline].
-
Chi P,
Greengard P,
Ryan TA
(2001)
Synapsin dispersion and reclustering during synaptic activity.
Nat Neurosci
4:1187-1193[Medline].
-
Chin J,
Angers A,
Cleary LJ,
Eskin A,
Byrne JH
(1999)
TGF-1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TR-II-like immunoreactivity.
Learn Mem
6:317-330[Abstract/Full Text].
-
Davies SP,
Reddy H,
Caivano M,
Cohen P
(2000)
Specificity and mechanism of action of some commonly used protein kinase inhibitors.
Biochem J
351:95-105[Medline].
-
Dobrunz LE,
Stevens CF
(1997)
Heterogeneity of release probability, facilitation, and depletion at central synapses.
Neuron
18:995-1008[Medline].
-
Farr M,
Mathews J,
Zhu D-F,
Ambron R
(1999)
Inflammation causes a long-term hyperexcitability in the nociceptive neurons of Aplysia.
Learn Mem
6:300-317.
-
Fioravante D,
Chin J,
Angers A,
Cleary LJ,
Byrne JH
(2001)
Timecourse and kinase involvement in 5-HT-induced redistribution of synapsin in Aplysia.
Soc Neurosci Abstr
27:1016.
-
Gingrich KJ,
Byrne JH
(1985)
Simulation of synaptic depression, posttetanic potentiation, and presynaptic facilitation of synaptic potentials from sensory neurons mediating gill-withdrawal reflex in Aplysia.
J Neurophysiol
53:652-669[Medline].
-
Glanzman DL,
Kandel ER,
Schacher S
(1989)
Identified target motor neuron regulates neurite outgrowth and synapse formation of Aplysia sensory neurons in vitro.
Neuron
3:441-450[Medline].
-
Glanzman DL,
Kandel ER,
Schacher S
(1990)
Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons.
Science
249:799-802[Medline].
-
Glanzman DL,
Kandel ER,
Schacher S
(1991)
Target-dependent morphological segregation of Aplysia sensory outgrowth in vitro.
Neuron
7:903-913[Medline].
-
Gottschalk WA,
Jiang H,
Tartaglia N,
Feng L,
Figurov A,
Lu B
(1999)
Signaling mechanisms mediating BDNF modulation of synaptic plasticity in the hippocampus.
Learn Mem
6:243-256[Abstract/Full Text].
-
Homayouni R,
Byrne JH,
Eskin A
(1995)
Dynamics of protein phosphorylation in sensory neurons of Aplysia.
J Neurosci
15:429-438[Abstract].
-
Hosaka M,
Hammer RE,
Südhof TC
(1999)
A phospho-switch controls the dynamic association of synapsins with synaptic vesicles.
Neuron
24:377-387[Medline].
-
Jovanovic JN,
Benfenati F,
Siow YL,
Sihra TS,
Sanghera JS,
Pelech SL,
Greengard P,
Czernik AJ
(1996)
Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.
Proc Natl Acad Sci USA
93:3679-3683[Abstract].
-
Jovanovic JN,
Czernik AJ,
Fienberg AA,
Greengard P,
Sihra TS
(2000)
Synapsins as mediators of BDNF-enhanced neurotransmitter release.
Nat Neurosci
3:323-329[Medline].
-
Kuromi H,
Kidokoro Y
(2000)
Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses.
Neuron
27:133-143[Medline].
-
Larkman A,
Stratford K,
Jack J
(1991)
Quantal analysis of excitatory synaptic action and depression in hippocampal slices.
Nature
350:344-347[Medline].
-
Levenson J, Sherry DM, Dryer L, Chin J, Byrne JH, Eskin
A (2000) Localization of glutamate and glutamate transporters
in the sensory neurons of Aplysia. 423:121-131.
-
Lhuillier L,
Dryer SE
(2000)
Developmental regulation of neuronal KCa channels by TGF-1: transcriptional and posttranscriptional effects mediated by Erk MAP kinase.
J Neurosci
20:5616-5622[Abstract/Full Text].
-
Lu B,
Gottschalk W
(2000)
Modulation of hippocampal synaptic transmission and plasticity by neurotrophins.
Prog Brain Res
128:231-241[Medline].
-
Matsubara M,
Kusubata M,
Ishiguro K,
Uchida T,
Titani K,
Taniguchi H
(1996)
Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions.
J Biol Chem
271:21108-21113[Abstract/Full Text].
-
Michael D,
Martin KC,
Seger R,
Ning MM,
Baston R,
Kandel ER
(1998)
Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia.
Proc Natl Acad Sci USA
95:1864-1869[Abstract/Full Text].
-
Rayport SG,
Schacher S
(1986)
Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization.
J Neurosci
6:759-763[Abstract].
-
Rosenmund C,
Stevens CF
(1996)
Definition of the readily releasable pool of vesicles at hippocampal synapses.
Neuron
16:1197-1207[Medline].
-
Royer S,
Coulson RL,
Klein M
(2000)
Switching off and on of synaptic sites at Aplysia sensorimotor synapses.
J Neurosci
20:626-638[Abstract/Full Text].
-
Schinder AF,
Poo M
(2000)
The neurotrophin hypothesis for synaptic plasticity.
Trends Neurosci
23:639-645[Medline].
-
Thomson AM
(2000)
Molecular frequency filters at central synapses.
Prog Neurobiol
62:159-196[Medline].
-
Torri Tarelli F,
Bossi M,
Fesce R,
Greengard P,
Valtorta F
(1992)
Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.
Neuron
9:1143-1153[Medline].
-
Turner KM,
Burgoyne RD,
Morgan A
(1999)
Protein phosphorylation and the regulation of synaptic membrane traffic.
Trends Neurosci
22:459-464[Medline].
-
Zhang F,
Endo S,
Cleary L,
Eskin A,
Byrne JH
(1997)
Role of transforming growth factor beta in long-term synaptic facilitation in Aplysia.
Science
275:1318-1320[Abstract/Full Text].
-
Zucker RS
(1989)
Short-term synaptic plasticity.
Annu Rev Neurosci
12:13-31[Medline].
Copyright © Society for Neuroscience 0270-6474//$05.00/0
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INTRODUCTION
