1Department of Physiology, 2Department of Ophthalmology, and 3Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0723
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ABSTRACT |
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Kleiman, Robin J., Ning Tian, David Krizaj, Thomas N. Hwang, David R. Copenhagen, and Louis F. Reichardt. BDNF-Induced Potentiation of Spontaneous Twitching in Innervated Myocytes Requires Calcium Release From Intracellular Stores. J. Neurophysiol. 84: 472-483, 2000. Brain-derived neurotrophic factor (BDNF) can potentiate synaptic release at newly developed frog neuromuscular junctions. Although this potentiation depends on extracellular Ca2+ and reflects changes in acetylcholine release, little is known about the intracellular transduction or calcium signaling pathways. We have developed a video assay for neurotrophin-induced potentiation of myocyte twitching as a measure of potentiation of synaptic activity. We use this assay to show that BDNF-induced synaptic potentiation is not blocked by cadmium, indicating that Ca2+ influx through voltage-gated Ca2+ channels is not required. TrkB autophosphorylation is not blocked in Ca2+-free conditions, indicating that TrkB activity is not Ca2+ dependent. Additionally, an inhibitor of phospholipase C interferes with BDNF-induced potentiation. These results suggest that activation of the TrkB receptor activates phospholipase C to initiate intracellular Ca2+ release from stores which subsequently potentiates transmitter release.
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INTRODUCTION |
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Neurotrophins play pivotal roles in acute and
long-term changes in synaptic plasticity. Acute potentiation of
synaptic strength by neurotrophins is accomplished by increasing
neurotransmitter release (Kang and Schuman 1995;
Li et al. 1998
; Lohof et al. 1993
; Sala et al. 1998
) and by modulation of neurotransmitter
receptor sensitivity and ion channel conductance (Holm et al.
1997
; Levine et al. 1995
, 1998
).
Neurotrophins and their receptors can be up-regulated in response to
activity (Merlio et al. 1993
; Schmidt-Kastner et al. 1996
; Shieh et al. 1998
; Tao et al.
1998
), and neurotrophin release can be enhanced in response to
depolarization (Blöchl and Thoenen 1995
;
Xie et al. 1997
). Experimental increases in neurotrophins produce long-lasting changes in neuronal function (Cabelli et al. 1995
; Cohen-Cory and Fraser
1995
; McAllister et al. 1995
,
1997
). Reductions in neurotrophin levels produce
deficits in long-term potentiation (Patterson et al.
1996
) and other forms of activity-dependent synaptic plasticity
(Cabelli et al. 1997
).
Bath application of brain-derived neurotrophic factor (BDNF) acutely
potentiates neurotransmitter release in Xenopus motor neuron-myocyte co-cultures (Lohof et al. 1993).
Potentiation does not require protein synthesis and occurs without an
intact cell body but requires extracellular Ca2+
(Stoop and Poo 1995
, 1996
). It is not
known whether the need for extracellular Ca2+
stems from a requirement for a Ca2+ influx, a
Ca2+-sensitive activation of the TrkB receptor,
or some other Ca2+-sensitive process on the
extracellular surface of the cell. Although BDNF has been shown to
produce a rise in [Ca2+]i
(Stoop and Poo 1996
), the source for the rise in
[Ca2+]i triggered by BDNF
has not been elucidated.
Little is known about which TrkB-linked intracellular
signaling pathways are required for acute BDNF-induced synaptic
potentiation. Among the signal transduction pathways known to be
activated by Trk receptors are those leading to activation of MAP
kinase, PI3 kinase and phospholipase C (PLC
) (reviewed in
Kaplan and Miller 1997
). Activation of PLC
is one
attractive candidate to mediate synaptic potentiation because its
activation would result in intracellular Ca2+
release via the second messenger IP3 (Obermeier et al.
1993
). Changes in cytoplasmic Ca2+
concentrations can regulate a wide variety of cellular processes, including neurotransmitter release (reviewed in Matthews
1996
) and transcriptional activity (reviewed in Gallin
and Greenberg 1995
). To investigate the source of the
Ca2+ required for BDNF-induced potentiation and
whether there is an essential link between TrkB receptor activation and
the PLC
pathway, we developed a video assay for synaptic activity.
We demonstrate that, although extracellular
Ca2+ is required to produce BDNF-induced
potentiation, a Ca2+ influx through voltage-gated
Ca2+ channels is not required. We show that an
inhibitor of PLC prevents BDNF-induced synaptic potentiation,
suggesting an essential role for the TrkB-induced activation of PLC and
subsequent release of Ca2+ from intracellular stores.
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METHODS |
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Reagents
Recombinant human BDNF was generously provided by Amgen (Thousand Oaks, CA).
Tissue culture
Oocyte-positive female Xenopus (NASCo) were injected
with 1,000 units of human chorionic gonadotropin (Sigma, St. Louis, MO; cat CG-10) the night before fertilization to induce egg
laying. Eggs were collected in 1 times modified frog Ringer (MMR; 0.1 M
NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM
CaCl2, 5 mM HEPES, and 0.1 mM EDTA). Eggs were
drained of all solution and incubated with crushed testes, freshly
harvested from euthanized male Xenopus. After 5 min, 0.1 times MMR was added to the embryos, which were allowed to develop to
the two-cell stage. Embryos were dejellyed by gently swirling the
embryos in a solution of 2% cysteine (Sigma C-7755), pH 8.0 for a few
minutes and were then incubated at 24°C until they reached Neiuwkoop
and Faber stage 21-22 (roughly 20 h) (Nieuwkoop and
Faber 1967).
Cultured Xenopus spinal motor neurons and myocytes were
prepared essentially as described previously (Tabti and Poo
1991). After the vitelline membrane was removed with fine
forceps, the embryos were washed in five changes of sterile 0.1 times
Ringer solution [1 times Ringer (in mM): 115 NaCl, 2.6 KCl, 2 CaCl2, and 10 HEPES, pH 7.6]. The embryos were
then transferred to Ca2+ and
Mg2+-free Ringer [CMF Ringer (in mM): 115 NaCl,
2.6 KCl, 10 HEPES, and 0.4 EDTA], where the neural tube and associated
myotomal tissue was dissected from the dorsal surface of the embryo.
The epithelial layer was removed and the tissue partially dissociated
after 20-30 min in CMF Ringer. The dissected tissue was drawn up into
a finely drawn Pasteur pipette and plated onto autoclaved 22 mm × 22 mm glass coverslips (Gold Seal No. 1 3306), which were submerged in
frog medium [1 part Ringer solution to 1 part L-15 (GIBCO) with 1%
fetal bovine serum]. Cultures were plated at a density of three
embryos per 35-mm dish. Cultures were left undisturbed for at least 30 min before moving them into a Tupperware container lined with wet paper
towel. Cultures were grown at least 20-24 h at room temperature before
use in experiments.
Excitatory postsynaptic potential (EPSP) recordings
The culture dish was mounted on the stage of an upright
microscope (Zeiss Axioskop, Carl Zeiss, Oberkochen, Germany). A ×40 water immersion objective lens was used for visualizing the cells and
recording pipettes using, Nomarski DIC optics. We recorded from
visually identified twitching myocytes. Patch pipettes were made of
borosilicate glass (type 7502, Garner Glass Company, Claremont, CA) on
a Brown-Flaming horizontal puller (model P-80/PC, Sutter instruments,
CA). The pipette-to-bath resistance ranged from 3 to 5 M. The
electrodes were tip-filled with a small volume (300-500 µm in
length) of gramicidin-free pipette solution (in mM: 1 NaCl, 150 KCl, 2 CaCl2, 1 MgCl2, and 10 mM
Na-HEPES, pH 7.3) and were then back-filled with pipette solution
containing gramicidin to avoid interference of gramicidin with seal
formation. The stock solution of gramicidin [gramicidin D (Dubos),
Sigma, St. Louis, MO] was prepared by dissolving 25 mg gramicidin into
500 µl dimethylsulfoxide (DMSO). This solution was aliquoted and
stored frozen. Each day before an experiment, 2 µl of stock solution
was added to 1 ml of pipette solution. The final concentration of
gramicidin in the pipette was 100 µg/ml (0.2% DMSO). During
experiments, cells were held at
70 mV once the G
seal was formed.
Access resistance and cell conductance was monitored every 5-10 min.
Recordings began after the access resistance reached a stable plateau
(
50 M
), that generally took 20-30 min. Electrode capacitance was compensated by 80-90% after the G
seal. Cell capacitance was not
compensated. Membrane potentials were recorded with an Axopatch 1D
amplifier (Axon Instruments, Burlingame, CA) in current-clamp mode
without injection of current, and data were collected using a
Macintosh-based interface (ITC-16 Mac computer interface, Instrutech, Great Neck, NY) run by HEKA software (Pulse + PulseFit, HEKA Elektronik GmbH, Germany). The signals were filtered at 1 kHz. The movement of
recorded myocytes was imaged simultaneously.
Twitching recordings
Neuron-myocyte cultures were imaged on either an inverted Nikon microscope or an Axiophot microscope with a ×40 objective. A charge-coupled device (CCD; Hamamatsu C2400) camera was used to transmit the images to a VCR, which recorded cell movement in real time. Videotapes were played back into a PC-compatible computer (Dell Optiplex GXMT 5166) and were analyzed using a program called "TWITCH," which utilizes a frame-grabber to measure changes in pixel intensities to detect cell movement. The digitized data were downloaded into the software program Igor Pro (Wavemetrics, Portland, OR). TWITCH was used to monitor up to nine separate regions of the screen simultaneously. A macro was written for Igor Pro that counted the twitching events and plotted frequency histograms of the data. The threshold for detecting a twitch was set at twice the magnitude of the mean to average peak amplitude of the noise.
To determine the fold change in frequency of muscle cell movement in
response to BDNF, we plotted frequency histograms of each experiment
using 5-min binwidths. The average frequency per 5 min was determined
during the control recording period by dividing the total number of
twitching events during the control period by the number of 5-min
intervals recorded. The peak frequency following BDNF application was
determined from frequency histograms of each experiment using a 5-min
binwidth. The fold change in the frequency was determined by comparing
the average frequency per 5 min before BDNF application to the peak
frequency after BDNF application. The fold change in frequency was
calculated by (PF ACF)/ACF, where PF is the peak frequency
after treatment and ACF is the average control frequency.
BDNF and other drugs were introduced into the cultures as 2 times solutions in frog medium. Over several minutes, half the volume of the dish (2 ml) was slowly drawn out of the dish using a sterile 6-ml syringe with a 18-gauge needle and attached tubing and was replaced slowly with the BDNF and/or drug containing medium. Medium change alone sometimes produced up to a 3.5-fold increase in the frequency of twitching. Therefore increases in the frequency of <3.5-fold were not considered to represent potentiation. Some recordings were done in Ca2+-free medium (in mM: 115 NaCl, 2 MgCl2, 2.6 KCl, and 3 EGTA, 0.1% BSA and 10 mM Na-HEPES, pH 7.3).
Immunoprecipitations
PC12 cell lines that stably express the rat TrkB receptor were
grown on collagen (Vitrogen 100, diluted 1:100 in PBS; Collagen, Fremont, CA) coated 100-mm tissue culture plates (Fisher Scientific, Houston, CA, No. 08772E) (Ip et al. 1993). Cells were
washed three times with Dulbecco's phosphate-buffered saline (PBS) or
with Ca2+-free, Mg2+-free
Dulbecco's PBS plus 2 mM MgCl2 and 3 mM EGTA.
Cells were incubated in PBS (with or without
Ca2+) for 10 min before adding 100 ng/ml
recombinant human BDNF. After incubation of the cells in BDNF for 10 min, cells were rinsed with PBS containing 0.018 mg/ml sodium
orthovanadate, scraped, and homogenized in 500 µl of rapid
immunoprecipitation assay (RIPA) buffer [50 mM NaCl, 50 mM
NaF, 5 mM EDTA, 10 mM Tris, pH 7.5, 1% Triton plus 2 µg/ml
aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, and
100 µg/ml phenylmethylsulfonyl fluoride (PMSF)] containing 0.18 mg/ml sodium orthovanadate per plate. Supernatants of 10,000 × g extracts were used for protein assays and
immunoprecipitations. Protein assays were carried out in duplicate
using the Bio-Rad DC protein assay kit (Bio-Rad, Richmond, CA).
Immunoprecipitations were carried out using 100 µg of extract with 3 µl of affinity purified anti-TrkB antibody (Huang et al.
1999
) and protein A coupled sepharose beads
(Amersham-Pharmacia, Piscataway, NJ). Proteins were eluted from the
beads in loading buffer and separated by SDS PAGE on 7.5% acrylamide
gels. Blots were probed with anti-phosphotyrosine (monoclonal 4G10
cat 05-310, Upstate Biotechnology, Lake Placid, NY) or
anti-rat TrkB. Alkaline phosphatase-conjugated secondary antibodies
(anti-mouse and anti-rabbit, respectively) were used with ECF
substrate (Amersham-Pharmacia, Piscataway, NJ) to visualize and enable
quantitation of signals on a Fuji Multiimager FLA 2000.
Fura imaging
Cells were loaded with (7-10 µM) Fura-2 AM (Molecular Probes,
Eugene, OR) in frog culture medium for 10 min and rinsed for an
additional 20 min before imaging in a laminar flow perfusion stage
(Warner Instruments, Hamden, CT) on an inverted microscope equipped
with a cooled CCD camera (PXL, Photometrics, Tuscon, AZ) using a ×40
objective. [Ca2+]i was
estimated by determining the 340:380 ratio using standard techniques
(Grynkiewicz et al. 1985). Background subtracted ratios were acquired every 5 s. Metafluor software (Universal Imaging, West Chester, PA) was used to control the image acquisition.
Ca2+ levels were calibrated in motor neurons
using the equation
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RESULTS |
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BDNF increases the frequency of spontaneous twitching in innervated myocytes
BDNF increases the frequency of spontaneous synaptic events
recorded under voltage clamp from innervated myocytes (Lohof et al. 1993; Stoop and Poo 1995
,
1996
; unpublished observations). Potentiation of
transmitter release begins within minutes and peaks approximately 15 min after BDNF application, as evidenced by an increase in the
frequency of spontaneous synaptic events (Lohof et al.
1993
). Because there is no change in the amplitude of these
spontaneous events, this is strong evidence that the effects of BDNF
are largely on the presynaptic motor neuron. However, there is evidence
that TrkB receptors on the muscle cell may also mediate additional
effects postsynaptically on acetylcholine receptor function
(Wang and Poo 1997
).
Xenopus myocytes contacted in culture by motor neurons
often contract spontaneously (Fig. 1).
Figure 1, A and B, shows images of five myocytes
as marked in Fig. 1C. All five myocytes twitched spontaneously in this preparation. A and B show
the morphology of myocyte 1 captured at rest and during
twitching, respectively. Figure 1C outlines the edges of the
cells and reveals changes in the boundaries of myocyte 1. Unlike cardiac myocytes, cultured myocytes do not contract in the
absence of contact with neurons (Peng et al. 1991;
unpublished observations). Spontaneous twitching is readily observed in
the presence of the sodium channel blocker TTX, suggesting that action
potentials are not required to produce this activity (Fig. 3,
C and D). Moreover, consistent with this conclusion, we rarely observed synchronous twitching from multiple muscle cells contacted by a single motor neuron (Fig. 1).
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To validate that spontaneous twitching activity resulted from presynaptically driven EPSPs, we recorded spontaneous synaptic events under current clamp and videotaped the cells simultaneously. Figure 2 illustrates that every recorded-twitching movement detected by image analysis (see METHODS) corresponded to a recorded EPSP. These dual recordings revealed that not every EPSP resulted in a twitching event. In particular, small EPSPs often failed to elicit a twitching event, while larger ones consistently produced movement. Figure 2B is an enlarged version of the data outlined in the box drawn in Fig. 2A, and the arrows denote the positions of small amplitude EPSPs that did not result in detectable twitching events. These data suggest that the frequency of spontaneous twitching events represents the frequency of a subset of EPSPs that are large enough to elicit a twitching response. Thus our image analysis computer program justifiably provides a simple assay for a synaptic activity in cultured motor neuron-myocyte pairs.
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Figure 3A shows an example of a BDNF-induced increase in the frequency of spontaneous twitching of an innervated myocyte. The upward deflections show the timing of twitching events. Following 30 min in control medium, 100 ng/ml BDNF was added to the medium. An increase in the frequency of twitching was evident approximately 20 min after BDNF application and peaked after approximately 30 min. Although the timing of twitches can be determined with an accuracy of <100 ms, the amplitude of these deflections is not a good indicator of the strength of contraction because the amplitude of the events varied with the placement of the detection window. Since this approach is useful for examining changes in relative frequency, but not changes in amplitude, twitching frequency histograms were used as our measure of synaptic activity. Figure 3B shows an example of the frequency histograms, which are plotted using a 5-min binwidth. The average frequency per 5 min during the control recording period was 18.6 twitches. The peak frequency per 5 min, after BDNF application was 127 twitches. This represented a 5.8-fold increase in the frequency of twitching.
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We considered the twitching frequency to be potentiated by BDNF when the peak twitching frequency following the application of BDNF was more than 3.5-fold that of the control frequency. The rationale for this criterion was based on the finding that occasionally just switching media elicited an increase. The average increase for exchanging the media alone was 0.65-fold, and the largest increase we saw from exchanging the media alone was 3.5-fold. Twenty-one of 31 myocytes (68%) observed to twitching in control solution responded to BDNF with a more than 3.5-fold increase in frequency (Fig. 3C). Including all cells, the average increase in the frequency of twitching was approximately 15-fold (Fig. 3D) and peaked approximately 25 min after the BDNF application, although individual cells varied considerably with respect to the magnitude and timing of maximal potentiation. The delay between BDNF application and potentiation seemed to vary within a 5- or 10-min range from cell to cell and may vary somewhat from one batch of BDNF to another, although we did not examine this systematically.
Addition of TTX (1 µM) significantly decreased the number of cells that exhibited a potentiated twitching response in response to BDNF (11 of 28 cells; Fig. 3C). The cells that exhibited BDNF-induced potentiation in the presence of TTX did so with a similar time course to BDNF alone (not shown). Including all cells, the average increase in frequency was lower (3.3-fold in TTX plus BDNF vs. 14.9-fold in BDNF alone).
These results demonstrate that the potentiating actions of BDNF on
synaptic activity are reflected in the frequency of myocyte twitching.
BDNF treatment produced an increase in the frequency of twitching that
paralleled the time course and magnitude of the increase in the
frequency of recorded spontaneous synaptic activity as well as the
percent of BDNF-responsive cells (Lohof et al. 1993;
unpublished data). Therefore we used the twitching assay to investigate
the signaling pathways involved in BDNF-induced potentiation.
Removal of extracellular Ca2+ severely retards the ability of BDNF to produce potentiated twitching
Previous studies have demonstrated a requirement for
extracellular calcium to produce BDNF-induced potentiation of
spontaneous synaptic currents (Stoop and Poo 1996). We
were able to observe a similar requirement for extracellular calcium to
produce BDNF-induced potentiation of twitching using our video assay.
Removing Ca2+ from the medium decreased the
frequency of spontaneous twitching (Fig.
4, A and B), but
the cells twitched at a stable rate for at least 1 h in the
absence of Ca2+ (not shown). The rate of
twitching was observed for 10 min in the absence of
Ca2+ before the addition of BDNF. In most cases
no BDNF-induced potentiation was seen in the absence of extracellular
Ca2+(16 of 18 cells; Fig. 4C). The
average fold increase in the absence of Ca2+ was
only 1-fold as compared with a increase of 15-fold in the presence of
Ca2+ (Fig. 4D).
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These results confirm that extracellular Ca2+ plays an important role in producing BDNF-induced potentiation of twitching activity. There are several Ca2+-dependent processes that could be affected by the removal of extracellular Ca2+. One possibility is that influx of extracellular Ca2+ is required. Alternatively, extracellular Ca2+ is required for some step in TrkB activation, such as ligand binding or dimerization. An alternative possibility is that in the absence of extracellular calcium, internal stores of calcium are depleted by leakage, and calcium entry via store-operated calcium channels cannot replace what is lost. Thus if calcium release from internal stores is required for potentiation, partial depletion of these stores could prevent potentiated transmitter release. To evaluate these putative mechanisms we designed the following experiments.
Voltage-gated Ca2+ channel blocker cadmium does not interfere with BDNF-induced potentiation of twitching
Neurotransmitter release is known to be triggered by
Ca2+ influx through voltage-gated
Ca2+ channels. The divalent cation
Cd2+ blocks Ca2+ entry
through all types of voltage-gated Ca2+ channels
found in this preparation (Barish 1991; reviewed
in Scott et al. 1991
). To evaluate the potential role of
Ca2+ influx through voltage-gated
Ca2+ channels in BDNF-evoked potentiation, we
examined the effects of BDNF in the presence of Cd2
+. Figure 5, A and
B, shows a typical response to BDNF in the presence of
Cd2+ ions. Addition of 100 µM
Cd2+ to the cultures had no effect on the ability
of BDNF to produce potentiation or on the time course of the
potentiation observed. The example shown in Fig. 5, A and
B, demonstrates that an increase in the frequency of
twitching was evident between 5 and 10 min after BDNF application and
peaked between 30 and 35 min after BDNF application. The average
frequency in the presence of Cd2+ for this cell
was 6 twitches per 5 min, while the peak frequency after BDNF
application increased to 80 twitches per 5 min. This represented a
12.3-fold increase in the frequency of twitching. Most of the cells
examined (71%) responded to BDNF with a >3.5-fold increase in the
frequency (Fig. 5C). On average there was a 13.7-fold increase in the frequency of twitching in response to BDNF in the
presence of this Ca2+ channel blocker, which was
not significantly different from the 14.9-fold increase observed in the
absence of Cd2+ (Fig. 5D). These
results indicate that the BDNF-dependent enhancement of twitching
frequency does not require a Ca2+ influx through
voltage-gated Ca2+ channels.
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TrkB receptor is capable of tyrosine phosphorylation in the absence of extracellular Ca2+
Because extracellular Ca2+ is required to
observe BDNF-induced synaptic potentiation, but
Ca2+ channel blockers have no significant
inhibitory effect, it seemed possible that the TrkB receptor is
sensitive to Ca2+ and unable to signal in its
absence (Zhou et al. 1997). To evaluate this
possibility, tyrosine phosphorylation of the TrkB receptor was
quantified in response to BDNF in the presence and absence of
Ca2+, using a PC12 cell line expressing TrkB
receptors. Figure 6B shows a
western blot with an antibody to phosphotyrosine of protein immunoprecipitated with antibodies directed against TrkB, except for
lane 1, which did not include any antibody in the
precipitation step. In response to 100 ng/ml BDNF, tyrosine
phosphorylation of TrkB was observed in the presence and absence of
extracellular Ca2+ (lanes 3 and
5 of Fig. 6), demonstrating that extracellular
Ca2+ is not essential for ligand-dependent
activation of TrkB. A shows the same blot in B
after it was stripped and blotted with antibodies to TrkB. C
shows graphically that quantitative estimates of the amount of tyrosine
phosphorylation of TrkB in response to BDNF were the same in the
presence and absence of extracellular Ca2+.
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Inhibitor of PLC prevents BDNF-induced potentiation of twitching
There does not seem to be a requirement for extracellular calcium to influx via voltage-gated calcium channels or to enable the Trk receptor to signal. One of the most parsimonious explanations for the inability of BDNF to produce a potentiated twitching response in the absence of extracellular calcium is a secondary depletion of intracellular stores of calcium that are required either for the potentiation of transmitter release or for the expression of the potentiated twitching by the muscle cell. Next, we investigated the possibility that TrkB activation regulates synaptic transmission via actions on an intracellular Ca2+ signaling pathway.
Neurotrophin receptors are capable of activating PLC, which
hydrolyses PIP2 to generate DAG and IP3. IP3 in
turn, interacts with IP3 receptors present on endoplasmic reticulum
(ER) membrane to release Ca2+ into the
cytoplasm. An IP3-induced rise in the cytoplasmic concentration of
Ca2+ could play an important role in producing
the BDNF-induced potentiation of synaptic activity. To evaluate the
potential contribution of PLC activity to the production of
BDNF-induced potentiation, we examined the effects of a specific
inhibitor of PLC (Smith et al. 1990
). Treatment of cells
with 5 µM of the PLC inhibitor U73122 (Calbiochem, San Diego, CA)
prevented potentiation of the twitching response by BDNF in 10 of 10 cells examined (Fig. 7, A, B,
D, and E). The average increase in response to BDNF was
0.65-fold in the presence of U73122 as compared with 14.9-fold in BDNF
alone (Fig. 7E). To test whether the myocytes' ability to
twitch had been impaired by the U73122 treatment, 300 µM ATP was
added to U73122-treated myocytes. A significant increase in the
frequency of twitching was observable in response to ATP treatment in 7 of 10 cells examined (Fig. 7C). This was consistent with
previous reports that ATP potentiates the frequency of spontaneous
synaptic currents in innervated Xenopus myocytes (Fu
and Poo 1991
) and suggests that the postsynaptic cell is
capable of expressing a potentiated response in the presence of this
PLC inhibitor. The severe disruption of BDNF-induced potentiation of
the twitching response by U73122 suggests that activation of PLC is
essential for potentiation.
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BDNF induces a rise in intracellular Ca2+ concentrations that can be blocked by inhibitors of intracellular stores
To examine the potential role of PLC-dependent Ca2+ release from intracellular stores using a second approach, Fura-2 imaging was used to monitor BDNF-induced changes in intracellular Ca2+ concentrations in the presence and absence of drugs that deplete intracellular stores. First, the effect of BDNF on intracellular Ca2+ concentrations was determined. Every neuron examined (7 of 7) exhibited an increase in intracellular Ca2+ with a time course that approximates that of the increase in spontaneous twitching. Before application of BDNF, the mean resting concentration of intracellular Ca2+ was 71 ± 18 nM (mean ± SE, n = 7). This increased to 258 ± 40 nM (n = 7) between 20 and 30 min following the BDNF application. In some cases, BDNF application was followed by very large transient increases in intracellular Ca2+ concentration superimposed on the already elevated intracellular Ca2+ concentration (Fig. 8A). Myocytes did not exhibit any rise in [Ca2+]i in response to BDNF treatment.
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Our twitching data suggest that since a Ca2+ influx is not required and inhibitors of PLC prevent the potentiation, the increase in intracellular Ca2+ that we observe in response to BDNF application is likely to be due to release from intracellular stores. To test this possibility further, cultures were preincubated for 3 h with a mixture of thapsigargin (2 µM) and cyclopiazonic acid (CPA; 5 µM) to deplete the intracellular stores of Ca2+. Pretreatment of neurons with these drugs prevented any significant increase in the cytoplasmic calcium concentration in response to 100 ng/ml BDNF application (n = 4). The basal cytoplasmic concentration of Ca2+ in these cells was 53 ± 24 nM. Following BDNF application the basal Ca2+ concentration was 69 ± 17 nM (n = 3; Fig. 8C). These results argue strongly for an essential BDNF-induced rise in intracellular Ca2+ from intracellular stores. Our results with the PLC inhibitor suggest that this rise in [Ca2+]i is mediated by a PLC-mediated pathway.
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DISCUSSION |
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BDNF-induced potentiation is mediated by a PLC-sensitive pathway
Our studies suggest that PLC-regulated Ca2+
stores play a critical role in the induction of BDNF-induced
potentiation. Figure 9 shows a schematic
diagram of our proposed BDNF-evoked signaling pathway. We confirm
previous observations that extracellular Ca2+ is
required for BDNF-induced potentiation (Stoop and Poo
1996). Our data show, however, that the voltage-gated
Ca2+ channel blocker Cd2+
had no effect on the BDNF-induced potentiation, suggesting very strongly that Ca2+ influx, at least through
voltage-gated Ca2+ channels, is not required to
produce potentiation. This is very similar to what has been described
for BDNF-induced potentiation in cultured hippocampal neurons
(Li et al. 1998
). The somewhat paradoxical requirement
for extracellular Ca2+, but not for
Ca2+ influx can be explained either by a
Ca2+ requirement for the Trk receptor to signal,
or by a requirement for a secondary calcium influx, perhaps through
store-operated channels to replenish intracellular calcium stores. We
examined the former possibility by looking at BDNF-induced tyrosine
phosphorylation of the TrkB receptor in the presence and absence of
extracellular Ca2+ in a stable TrkB expressing
PC12 cell line. These experiments demonstrate that, in PC12 cells, BDNF
does not require extracellular Ca2+ to induce
tyrosine phosphorylation of the TrkB receptor. This suggests that,
although the Trk receptor signal transduction cascade is initiated in
the absence of extracellular Ca2+, there must be
an extracellular Ca2+ requirement downstream of
receptor activation. This would be possible if removal of extracellular
Ca2+ results in a secondary depletion of
intracellular Ca2+ stores in the neuron and/or
myocyte. To replenish intracellular Ca2+ stores,
an influx of Ca2+ through store-operated
Ca2+ channels (capacitative
Ca2+ channels) may be required. These
plasma-membrane Ca2+ channels are activated in
response to decreases in Ca2+ concentrations in
ER, such as those that occur when IP3 receptor-activated Ca2+ stores are released into the cytoplasm
(reviewed in Barritt 1999
). Although we have not
addressed this issue directly, a similar influx has been demonstrated
in other neurons (Li et al. 1999
; Simpson et al.
1995
).
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TRPC (TRP-Ca) channels are vertebrate homologues of the
Drosophila transient receptor potential (TRP) channels,
which represent the best candidates for mediating a store-operated
conductance. These channels mediate store-operated and/or PLC-dependent
cation conductance in nonexcitable cells. The role of the TRPC-mediated conductance in neurons has not been fully explored; however, most family members are expressed in mammalian brain, and TRPC3, TRPC4, and
TRPC5 are highly expressed in brain. A recent study by Li and
colleagues has described a role for TRPC3, which co-localized and
co-immunoprecipitated with TrkB, in mediating a PLC-dependent cation
influx in response to BDNF in mammalian neurons (Li et al.
1999). Thus one possibility is that activation of a
PLC-dependent cation influx via the TRPC3 channel is needed to produce
the BDNF-induced potentiation. Alternatively, it is possible that the
extended period in Ca2+-free medium reduced the
ability of the store-operated channels to replenish intracellular
calcium stores. Under these conditions, activation of PLC
-dependent
pathway may not have been able to release sufficient
Ca2+ from the intracellular stores to produce
potentiation. While our experiments in the absence of extracellular
Ca2+ cannot distinguish between an effect on the
presynaptic cell and the postsynaptic cell, our Fura-2 data argue for
some presynaptic locus of action.
A requirement for Ca2+ release from neuronal
internal stores is supported by Fura-2 experiments demonstrating that
BDNF treatment of cultured motor neurons results in a rise in the
intracellular concentration of Ca2+ (Stoop
and Poo 1996; unpublished observations). The release of Ca2+ from internal stores is likely to be at
least partly mediated by TrkB activation of PLC because we are able to
demonstrate that an inhibitor of PLC function prevents the BDNF-induced
potentiation of twitching. Activated TrkB is autophosphorylated at
tyrosine residues 670, 674, 675, and 785 and can phosphorylate and
activate PLC
(Guiton et al. 1994
; Middlemas et
al. 1994
). The TrkB phosphotyrosine residue 785 can complex
with phosphorylated PLC
via interaction with its SH2 domain
(Middlemas et al. 1994
). Activated PLC
stimulates the
generation of IP3, which opens the IP3 receptor-gated channels in the
ER. The resultant rise in the cytoplasmic concentration of
Ca2+ is likely to be critical for the
potentiation of the release of neurotransmitter (Guo et al.
1996
; Li et al. 1998
; Tse et al. 1997
). Our observation that both the rise in intracellular
calcium and the increased frequency of twitching often takes between 10 and 20 min to reach its peak suggests that the signal transduction cascade involved in producing these effects may have additional components. Similarly, Tanaka and colleagues (1997)
have
reported that BDNF treatment attenuates inhibitory postsynaptic
potentials (IPSPs) in cultured rat hippocampal neurons in a
U73122-dependent fashion, and this effect takes approximately 10-15
min to reach its peak. Other factors that would certainly influence the
time course of this response in our system include the rate of
association for human BDNF with the Xenopus TrkB receptor at
25°C. Trk receptors appear to associate with their ligands with
unusually slow kinetics (Mahadeo et al. 1994
).
BDNF-induced potentiation of twitching mimics BDNF-induced potentiation of spontaneous synaptic currents
The video assay used in this study has several attractive features. It is noninvasive and convenient and permits monitoring of cells over extended time. In addition, multiple cells can be monitored in a field of view simultaneously. The TWITCH software can monitor up to nine different locations within the field at the same time, allowing collection of data on more cells, more quickly. However, this video assay will not differentiate between presynaptic and postsynaptic effects since it only collects meaningful information on relative changes in the frequency of spontaneous activity.
The time course of the twitching response was similar but somewhat
slower than that reported for the changes in frequency of spontaneous
synaptic currents recorded (Lohof et al. 1993). Subtle
changes in the experimental preparation may account for these
differences, as our unpublished voltage-clamp data of myocytes is, on
average, somewhat slower than that of Lohof et al.
(1993)
.
BDNF-induced potentiation of twitching is enhanced by action potentials
We were able to detect BDNF-induced potentiation in the presence
of TTX, suggesting that sodium action potentials are not required for
this effect. However, we did not observe potentiation in as many cells
when TTX was present. This suggests that action potentials, while not
required, do enhance the likelihood that potentiation will develop.
Recent studies demonstrate that presynaptic depolarization paired with
concentrations of BDNF that are normally not sufficient to produce
BDNF-induced potentiation (10 ng/ml) will allow induction of
BDNF-induced potentiation (Boulanger and Poo 1999).
Together with our data, this suggests that presynaptic depolarization
greatly increases the likelihood that BDNF can produce potentiation. In
cultured rat motor neurons, depolarization and treatments that elevate
cAMP levels produce a marked increase in the amount of TrkB receptor
expressed on the cell surface within 30 min (Meyer-Franke et al.
1998
), suggesting that surface expression of the Trk B receptor
exhibits rapid and dynamic regulation. It is possible that under normal
circumstances, spontaneous action potentials may provide sufficient
presynaptic depolarization for BDNF to induce potentiation by promoting
the insertion of TrkB receptors into the plasma membrane. Blocking
action potentials for 30 min may decrease the spontaneous amount of
presynaptic activity enough to reduce the amount of TrkB receptor
expressed on the surface of the neurons, and decrease the likelihood of producing potentiation. We did not observe BDNF-induced potentiation from every cell even in the absence of TTX, which may reflect the
existence of a subpopulation of cells lacking sufficient surface TrkB
receptor (or any at all) to produce potentiation.
The role of BDNF in regulating synaptic function has emerged as a central theme in understanding activity-induced plasticity. TrkB receptors and BDNF are also required for trophic support of many neurons throughout the life of the organism. Understanding how TrkB receptor activation can orchestrate such complex cascades to result in each of its requisite functions are complicated questions, but some common themes are beginning to emerge. The pattern of Ca2+ transients induced by BDNF could play a significant role in mediating both acute and long-term changes in structure or function. The surface distribution of the TrkB receptors in combination with the subcellular localization of Ca2+ stores determines the range of patterns of Ca2+ transients that BDNF can produce in a particular neuron. Both surface expression of Trk receptors and neurotrophin release can be regulated in response to depolarization. Thus the activity-dependent regulation of both of these processes could produce associative properties of neurotrophin-induced plasticity. Key issues to address will be how the surface expression of TrkB receptor is regulated by activity and how this alters the dynamics of Ca2+ release from internal stores.
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ACKNOWLEDGMENTS |
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L. F. Reichardt is an investigator at the Howard Hughes Medical Institute. R. J. Kleiman was the recipient of an American Heart Association fellowship. We are grateful to P. Wang for help with data analysis.
This work was supported by National Institutes of Health grants to D. R. Copenhagen and L. F. Reichardt.
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FOOTNOTES |
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Address for reprint requests: L. F. Reichardt, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143-0723 (E-mail: lfr{at}cgl.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 1999; accepted in final form 5 April 2000.
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REFERENCES |
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