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The Journal of Neuroscience, September 1, 1998, 18(17):6803-6813
Calcium-Evoked Dendritic Exocytosis in Cultured Hippocampal
Neurons. Part I: Trans-Golgi Network-Derived Organelles Undergo
Regulated Exocytosis
Mirjana
Maletic-Savatic and
Roberto
Malinow
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
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ABSTRACT |
Exocytosis is a widely observed cellular mechanism for delivering
transmembrane proteins to the cell surface and releasing signaling
molecules into the extracellular space. Calcium-evoked exocytosis,
traditionally thought to be restricted to presynaptic specializations
in neurons, has been described recently in many cells. Here,
calcium-evoked dendritic exocytosis (CEDE) is visualized in living
cultured hippocampal neurons. Organelles that undergo CEDE are in
somata, dendrites, and perisynaptic regions, identified by using
immunocytochemistry and correlative light and electron microscopy. CEDE
is regulated developmentally: neurons <9 d in vitro do
not show CEDE. In addition, CEDE is blocked by tetanus toxin, an
inhibitor of regulated exocytosis, and nocodazole, an inhibitor of
microtubule polymerization. Organelles that undergo CEDE often are
found on the base of spines, putative sites of synaptic plasticity.
CEDE therefore could be involved in structural and functional
modification of spines and could play a role in synaptic plasticity,
where it might involve changes in receptor/channel density, release of
active compounds having effect on pre- and postsynaptic function,
and/or growth of synaptic structures.
Key words:
exocytosis; trans-Golgi network; dendrite; pyramidal
neurons; hippocampal culture; time-lapse imaging; FM1-43; immunocytochemistry; tetanus toxin; microtubules
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INTRODUCTION |
Constitutive exocytosis is
ubiquitous in eukaryotic cells. It is responsible for the recycling and
renewal of plasma membrane components and for the secretion of
molecules into the extracellular space (Alberts et al., 1989 ). In
neurons the exocytosis of neurotransmitter molecules from presynaptic
terminals represents the fundamental mechanism for the transfer of
information. Although presynaptic vesicle exocytosis has been examined
extensively, exocytosis has not yet been demonstrated directly in the
dendritic regions of neurons, where it might provide a regulated means
for effecting synaptic plasticity and/or homeostasis. Evidence exists
that nonfunctional pools of postsynaptic membrane proteins are present
(Hampson et al., 1992; Baude et al., 1995; Rubio and Wenthold, 1997)
and that postsynaptic exocytosis is required for some forms of synaptic plasticity (Lledo et al., 1998). Calcium-evoked exocytosis has been
demonstrated in neuronal somata (Huang and Neher, 1996) as well as in
non-neuronal cells (Morimoto et al., 1995). In addition, lysosomes in
fibroblasts, epithelial cells, and myoblasts undergo calcium-regulated
exocytosis, which is temperature- and ATP-dependent (Rodriguez et al.,
1997). In this study we demonstrate calcium-evoked exocytosis of
dendritic compartments, consistent with the view that some forms of
synaptic plasticity might occur via the regulated exocytosis of
dendritic organelles.
In the past several years the mechanisms of presynaptic vesicle
exocytosis have been examined in detail, including studies using
fluorescent membrane probes. The styryl dye FM1-43 has been used
effectively as a probe to monitor at the light microscope level the
dynamics of presynaptic vesicles in living cells (Betz et al., 1992). A
brief exposure to FM1-43, combined with the depolarization of the cell,
induces selective internalization of the dye into the presynaptic
terminals (Ryan et al., 1993). With triggered exocytosis the dye is
released into the extracellular space, and the loss of fluorescence is
used as a measure of exocytosis (Ryan et al., 1993).
FM1-43 nonspecifically attaches to the whole surface of a cell, and on
endocytosis the dye becomes internalized. In fully polarized
hippocampal neurons, endocytosis occurs over the entire somatodendritic
surface (Parton and Dotti, 1993). Using these properties, we developed
a method to label dendritic membranous compartments with FM1-43. This
method allowed us to visualize the behavior of labeled compartments in
living cultured hippocampal neurons and to determine the cellular
mechanisms controlling their exocytosis. Furthermore, because there is
evidence for mixing between endocytic and biosynthetic pathways
(Trowbridge et al., 1993), studies monitoring the fate of endocytosed
FM1-43 may shed light on the mechanisms by which newly synthesized
membrane-bound proteins, like receptors, are transported and inserted
into synapses.
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MATERIALS AND METHODS |
Cell culture
Cortical astrocytes were derived from 1-d-old rat pups and
plated onto poly-L-lysine-coated coverslips. Hippocampal
neurons were generated from 19-d-old rat embryos (Banker and Goslin,
1990) and plated onto a confluent monolayer of astrocytes. Astrocytes and neurons were plated at 50,000 and 30,000 cells per 18-mm-round glass coverslip (Fisher Scientific, Pittsburgh, PA), respectively. For
relocation experiments the astrocytes and neurons were plated at
100,000 and 85,000 cells per 25-mm-square gridded glass coverslip (Bellco Glass, Vineland, NJ), respectively. Cultures were maintained in
a serum-free medium (Banker and Goslin, 1990).
Labeling neurons with FM1-43
Acute FM1-43 loading. Cultures were exposed to 15 µM FM1-43 combined with 90 mM KCl for 1 min
while on the microscope stage and washed for 10 min with constantly
perfusing bath solution [containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3,
and 11 glucose].
Overnight FM1-43 loading. Cultures were exposed 16-36 hr to
1.5 µM FM1-43 at 35.5°C before transfer to the
microscope stage. Cultures were rinsed with 0.1% DMSO for 1 min and
maintained in constantly perfusing bath solution for 1 hr. The bath
solution (see above) contained 1 µM tetrodotoxin to
abolish action potentials.
Time-dependent loading. Mature cultures (>9 d in
vitro, DIV) were loaded with 15 µM FM1-43 for 1-16
hr. After loading, they were transferred to a microscope stage and
washed with the bath solution for 30 min. They were challenged with
vehicle (0.1% DMSO) or 1 µM A23187 for 1 min.
Calcium-evoked dendritic exocytosis (CEDE) was monitored for at least
15 min.
Calcium-evoked dendritic exocytosis
After overnight loading with 1.5 µM FM1-43, the
cultures were transferred to the microscope stage and maintained in
constantly perfusing bath solution for 1 hr. Calcium ionophore A23187
(1 µM) was applied directly onto the coverslip for 1 min.
Fluorescence was monitored for 30 min before and 30 min after A23187
application, at 15 min intervals. An additional image was taken ~5
min after ionophore application. All images in a given experiment were
taken with the same exposure time (100-500 msec, chosen so that the fluorescent signal did not saturate the camera detection limit). A
number of cells (>20) were tested for viability with 0.4% trypan blue. More than 90% of the neurons excluded the dye after A23187 challenge (data not shown), indicating that ionophore application did
not have toxic effects. Viability also was confirmed in a number of
experiments (>5) in which the subsequent application of A23187
combined with FM1-43 produced loading (data not shown).
EGTA treatment. Mature cultures (>9 DIV) were loaded with
1.5 µM FM1-43 overnight and washed as above. Cells were
incubated in 5 mM EGTA, pH 7.0, in
Ca2+-free artificial CSF for 10 min before A23187
challenge and for 15 min thereafter.
Tetanus toxin treatment. Mature cultures (>9 DIV) were
loaded with 1.5 µM FM1-43 for 14 hr and then exposed for
5 hr to 10 nM tetanus toxin (generously provided by Dr.
Giampetro Schiavo, Rockefeller University, New York, NY) and 1.5 µM FM1-43 at 35.5°C. Experiments were performed as
above.
Nocodazole treatment. Mature cultures (>9 DIV), loaded
overnight with 1.5 µM FM1-43, were exposed for 45 min to
2.5 µg/ml nocodazole and 1.5 µM FM1-43 at 35.5°C.
Experiments were performed as above.
Field electrical stimulation. Mature cultures (>9 DIV),
loaded overnight with 1.5 µM FM1-43, were stimulated with
repetitive field stimuli. The current was delivered between the two
silver wires placed 3 mm apart onto the coverslip with cultured
neurons. Different frequencies (1, 10, 50, and 100 Hz) and numbers of
stimuli were applied.
Measurement of released FM1-43. Neurons loaded overnight
with 1.5 µM FM1-43 were washed in the same manner as for
imaging experiments. They were incubated for 15 min in HEPES buffer, pH 7.2. An aliquot of this bathing solution
(Apre) was recovered. Then the neurons
were exposed to 1 µM A23187 for 1 min, followed by fresh
HEPES buffer for 15 min. An aliquot from this bathing solution
(Apost) was recovered.
Apre and Apost solutions
were pooled from 10 cultures. FM1-43 was extracted from these aqueous solutions with an equal volume of water-saturated butanol. The butanol
fraction was recovered and evaporated.
(3-[(3Cholamidopropyl)dimethylammonio]-1-propane-sulfonate) (CHAPS) (2%) was added (FM1-43 binds to CHAPS and
increases fluorescence; Henkel et al., 1996). This solution was put in
a quartz rectangular capillary, and FM1-43 fluorescence was measured
(FITC filters; Zeiss Axioskop, Oberkochen, Germany). We measured the
extracted fluorescence of HEPES buffer, Apre and
Apost. The amount of released FM1-43 was
calculated as [Apost  buffer]/[
Apre buffer]. Calibration curves were
generated from 1.5 nM to 1.5 µM FM1-43 before
each experiment (n = 7).
Microscopy
Images were acquired with a computer-controlled cooled CCD
camera (Photometrics, Tucson, AZ), using FITC filters (peak at 450 nm)
on a Zeiss Axioskop epifluorescence microscope (50 W mercury lamp), and
processed with Photometrics-supplied software (PMIS). Time-lapse images
of mature neurons loaded with FM1-43 were acquired every 2 min over a 1 hr period, using FITC filters on a Zeiss Axiovert inverted microscope
(50 W mercury lamp; 100× lens with additional 2.5×
magnification).
Immunocytochemistry
Mature neurons (>12 DIV) were loaded with 1.5 µM
FM1-43 overnight and then washed for 1 hr with the bathing solution.
Cultures were fixed in 4% paraformaldehyde in 0.1 M PBS
containing 0.12 M sucrose for 20 min at +4°C and
immediately permeabilized with 0.3% Triton X-100 in PBS for 5 min at
room temperature. FM1-43-labeled compartments were imaged after
fixation (100× lens, FITC filter, Zeiss Axioskop, 75 W xenon lamp) to
avoid confounding results if the labeled organelles moved before
fixation. Imaging with Texas Red filters (Omega Optical, Brattleboro,
VT) did not reveal any FM1-43 fluorescence. Cultures were incubated in
blocking solution (10% horse serum, 1% goat-serum, and 0.1% Triton
X-100 in PBS) for 1 hr at room temperature and then immunostained
overnight with the primary antibody [polyclonal anti-synapsin I
antibody: 1:10, polyclonal rabbit sera number 6246, generously provided by Dr. Pietro De Camilli (Yale University, New Haven, CT); polyclonal anti-transferrin receptor antibody raised in sheep: 215 µg/ml, Sigma
(St. Louis, MO); polyclonal anti-mannose-6-phosphate receptor antibody:
1:50, generously provided by Dr. W. J. Brown (Cornell University,
Ithaca, NY); polyclonal anti-cathepsin D antibody: 1:100, generously
provided by Dr. W. J. Brown (Cornell University); polyclonal
anti-inositol triphosphate (IP3) receptor antibody: 1:50, generously provided by Dr. T. Jayaraman (Mount Sinai School of
Medicine, New York, NY); monoclonal anti-BiP antibody: 16 µg/ml, Stress Gen (Sidney, Canada)]. Primary antibody was washed out three times for 10 min with the blocking solution. Then the cells were
incubated with the appropriate secondary biotinylated antibody (2.5 µg/ml; Cappel, West Chester, PA) for 1 hr at room temperature. After
the washout of the biotinylated antibody, the cultures were incubated
with Texas Red-conjugated avidin (25 µg/ml; Cappel) for 1 hr at room
temperature. Immunoreactivity was analyzed immediately after the Texas
Red-avidin was washed out with PBS (100× lens, Texas Red filter).
Neurons were relocalized by using gridded coverslips (Bellco).
NBD C6-ceramide labeling. Mature neurons
(>12 DIV) were loaded with 1.5 µM FM1-43 for 4 hr (see
Fig. 6B) or overnight (see Fig. 3) and washed for 1 hr before fixation as described above. FM1-43-labeled compartments were
imaged, and the area was exposed to UV light to bleach the FM1-43
fluorescence. When no FM1-43 fluorescence was detected, the neurons
were incubated with 40 µM NBD C6-ceramide
(Molecular Probes, Eugene, OR) for 1 hr at 37°C and washed with 2.5%
fat-free bovine serum albumin three times for 20 min at room
temperature. Fluorescence was analyzed with FITC filters.
Electron microscopy
The 14 DIV hippocampal neurons, loaded with 1.5 µM
FM1-43 overnight, were washed with PBS for 1 hr before fixation with
3% paraformaldehyde in 0.2 M cacodylate buffer, pH 7.30, for 45 min at room temperature. After being washed with 0.2 M cacodylate buffer, secondary fixation was performed,
using 1% OsO4 with 1.5% K4Fe(CN)6
in cacodylate (90 min at room temperature), followed by dHOH
washes and en bloc uranyl acetate staining. Cells were dehydrated through a graded series of ethanol and embedded in Epon-Araldite resin. Sections were collected on Formvar-coated cupri
slot grids, counterstained with uranyl acetate and Reynold's lead
citrate, and examined in the Hitachi H-7000 at 75 kV.
Fluorescent and electron micrographs were superimposed in Adobe
Photoshop 4.0. To align fluorescent and electron micrographs, we used
fiduciary points chosen on the basis of regions clearly identified on
both photographs (e.g., the intersection of dendrites and axons, and at
the dendritic branching points). Electron micrographs were scanned
(ScanJet Plus, Packard, Meriden, CT), and the image sizes of both
micrographs were matched. Images were aligned with respect to fiduciary
points. After alignment, the fluorescent image was pseudocolored for
better visualization of the FM-labeled structures.
Data analysis
For quantitative measurements of CEDE, several regions of
interest (2-5) were placed on dendrites (locations were chosen before post-treatment images were analyzed), and FM1-43 fluorescence was
integrated. Background fluorescence, measured in regions next to the
neurons, was subtracted. FM1-43 fluorescence values were normalized to
the intensity observed immediately before A23187 application.
Statistical analysis was done by one-way ANOVA.
The degree of colocalization between FM1-43 fluorescent sites and other
fluorescent markers was quantified with PMIS software (Photometrics).
Regions of interest from images of FM1-43 fluorescent sites were
identified, and pixel intensity along lines traversing ~50 µm was
measured [IF(x)]. Pixel intensities along the
same lines placed in the same regions from the image detecting another fluorescent marker were measured also [IM(x)].
We plotted IF(x) versus
IM(x) and calculated a correlation coefficient,
R (using Origin 4.0 software, Microcal, Amherst, MA). Each
neuron that was examined had two to eight lines analyzed. Locations
were chosen from FM1-43-loaded neurons before the corresponding
marker-labeled image was analyzed.
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RESULTS |
FM1-43 labels dendritic compartments
Brief exposure of cultured neurons to FM1-43, combined with
depolarizing stimuli, can produce a selective loading of presynaptic vesicles (Fig. 1A,C)
(Ryan et al., 1993). In contrast, we found that cultured hippocampal
neurons incubated with FM1-43 for 16-36 hr in the absence of
stimulation loaded intracellular compartments in all processes and the
soma (Figs. 1D, 2, 4). We focused our analysis on the
characterization of FM1-43-labeled compartments in processes,
anticipating that these may be more likely to be associated with
synaptic plasticity.
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Figure 1.
FM1-43 fluorescent probe labels presynaptic or
dendritic compartments in cultured hippocampal neurons, depending on
the loading protocol. A, Location of FM1-43 dye after
exposing 14 DIV neurons to FM1-43, combined with 90 mM KCl,
for 1 min. Scale bar, 10 µm. B, Expression of synapsin
I in characteristic punctate labeling pattern indicates presynaptic
axon terminals that are closely apposed to the neuronal soma and major
dendrite. The majority of FM1-43-labeled sites in A
corresponds to synapsin I immunoreactivity (arrow).
C, Time-lapse images of a region after acute FM1-43
loading of 20 DIV cultured neuron. Little movement of spots is
apparent. Spontaneous destaining (~20%) over a 1 hr observation
period was typical (display scale maximum was changed from 110 to 95 arbitrary units to facilitate the comparison of spot locations). The
time is indicated in minutes. Scale bar, 2 µm. D,
Overnight exposure of 14 DIV neurons to FM1-43 extensively labels
compartments in soma and dendrites. Scale bar, 10 µm.
E, Expression of synapsin I, again labeling presynaptic
sites closely apposed to the major dendrite. FM1-43-labeled
compartments and synapsin I do not overlap in the majority of sites,
indicating dendritic (arrowheads) and presynaptic
(arrows) localization of labeled sites, respectively.
F, Time-lapse images of a region after overnight FM1-43
loading of 20 DIV cultured neuron. Note the location of FM1-43 spots at
the base of small filopodial structures and coordinate movement of the
filopodia and spots. The time is indicated in minutes. Scale bar, 2 µm.
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To determine the distribution of FM1-43 fluorescence in pre- and
postsynaptic compartments after the two labeling protocols, we compared
its labeling pattern with that of a presynaptic marker protein,
synapsin I (Fig. 1B,E; n = 3). After
standard acute exposure and depolarizing stimuli, FM1-43 and synapsin I
showed significant colocalization, confirming the presynaptic
localization of FM1-43 with this loading protocol (Fig.
1A,B). However, with long exposure and no
stimulation, FM1-43 and synapsin I showed little colocalization (Fig.
1D,E). This indicates that the longer exposure
selectively labels intracellular compartments that are not presynaptic
terminals. Quantitated immunocytochemical analysis (see Materials and
Methods) confirmed that, with overnight exposure to the dye, FM1-43
fluorescence in synapsin I-labeled compartments accounts for only 10%
of the dye (Fig. 3A; R = 0.13 ± 0.06;
n = 3).
Dendritic compartments labeled with FM1-43 often were seen at the base
of small filopodial structures, proposed to be synaptic spine
precursors (Fig. 1F) (Ziv and Smith, 1996).
Time-lapse imaging over 1 hr revealed that FM1-43-loaded compartments
maintained this association although the filopodial structures moved
over time (~2 µm/min; Fig. 1F). In contrast,
presynaptic vesicles labeled with acute loading of FM1-43 did not move
within 1 hr (n = 3; Fig. 1C), again
supporting the conclusion that overnight FM1-43 exposure loads
compartments that are not presynaptic terminals.
The dendritic localization of FM1-43-labeled compartments was confirmed
further by correlative electron and fluorescent microscopy (Fig.
2). Neurons loaded with FM1-43 overnight
were imaged under high magnification (100× lens; Fig.
2-1,6) and then processed for electron
microscopy (EM). The same regions were identified on the EM
preparations (Fig. 2-3,8) and aligned with the
light microscope images by using fiduciary points (Fig.
2-4,9). Under EM, FM1-43-fluorescent sites were
dendritic and often close to synapses. The sites included different
organelles, such as coated pits, endosomes, multivesicular bodies,
endoplasmic reticulum, Golgi apparatus, and mitochondria (Fig.
2-5,10). No dense core granules were
observed.
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Figure 2.
Identification of FM1-43-labeled compartments by
electron microscopy (EM). Shown are two example sets
(1-5, 6-10) of correlative fluorescent
and electron microscopy of mature neurons exposed overnight to FM1-43.
Fluorescent micrographs (1, 6)
show FM1-43-labeled compartments. Scale bars, 10 µm. Outlined
regions are enlarged (2,
7) and are represented on the electron
micrographs (3, 8). Scale bars: 1 µm in
3; 1.8 µm in 8.
Asterisks indicate fiduciary points used to superimpose
fluorescent and electron micrographs. FM1-43-labeled compartments in
dendrites (white arrowheads), opposite the presynaptic
sites (arrows), and in areas containing SER/TGN
organelles (black arrowheads) are evident on the
superimposed pseudocolored fluorescent micrographs (4,
9). Note the lack of FM1-43 in regions corresponding to
presynaptic terminals. EM sections at other levels in the same region
also failed to show presynaptic terminals in fluorescent regions.
Outlined regions on the electron micrographs are
enlarged in 5 and 10 for better
identification of postsynaptic SER/TGN-like structures. Scale bars: 0.3 µm in 5; 0.5 µm in 10. Figure
continues.
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To identify which dendritic organelles are labeled by overnight
exposure to FM1-43, we performed high-resolution immunocytochemical studies on FM1-43-loaded neurons (Fig.
3A). After FM1-43 imaging, the
cultures were fixed and labeled with different organelle-specific markers: transferrin receptor (Moos, 1996), cathepsin D (Nakanishi et
al., 1994), and mannose-6-phosphate receptor (Couce et al., 1992),
specific for endosome/lysosome organelles; IP3 receptor (Moschella et al., 1995) and BiP (Huovila et al., 1992), specific for
endoplasmic reticulum; and NBD C6-ceramide (Pagano et al., 1989), a trans-Golgi fluorescent marker. Quantitative
immunocytochemical analysis (see Materials and Methods) indicated
little colocalization between FM1-43-labeled compartments and
transferrin receptor (R = 0.2 ± 0.14;
n = 3), cathepsin D (R = 0.4 ± 0.16; n = 3), mannose-6-phosphate receptor
(R = 0.21 ± 0.1; n = 3),
IP3 receptor (R = 0.14 ± 0.07; n = 3), and BiP (R = 0.3 ± 0.09;
n = 3). However, we observed a consistently high
correlation of FM1-43-labeled dendritic sites only with NBD
C6-ceramide (R = 0.77 ± 0.11;
n = 5; Fig. 3). This indicates that, after overnight
exposure to FM1-43, dendritic organelles loaded with the FM1-43 are
primarily dendritic Golgi-like or Golgi-derived structures.
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Figure 3.
Organelles labeled by overnight exposure to FM1-43
colocalize with NBD C6-ceramide in dendrites, a label of
SER/TGN-like structures. A, Quantitative analysis of the
correlation between FM1-43-labeled compartments and specific organelles
identified by immunocytochemistry. Bars show mean ± SEM of the correlation coefficient analyzed in two to eight regions per neuron. Significant correlation was
observed only with a trans-Golgi network marker NBD
C6-ceramide. B, Colocalization of
FM1-43-labeled compartments and trans-Golgi network, identified by the
fluorescent marker NBD C6-ceramide. Most FM1-43 label
colocalizes with the trans-Golgi marker. Quantitative analysis of the
correlation between FM1-43-labeled compartments and NBD
C6-ceramide was performed along the indicated
lines. Scale bar, 10 µm. C,
Quantitative analysis of the correlation between FM1-43-labeled
compartments and NBD C6-ceramide for the neuron in
B. For each pixel on the indicated lines,
intensity in the channel measuring FM1-43 (ordinate) was
plotted against intensity in the channel measuring NBD
C6-ceramide (abscissa). The correlation
coefficient, R, was calculated.
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Dendritic organelles undergo regulated exocytosis
To determine whether FM1-43-labeled compartments exhibit
calcium-evoked exocytosis, we challenged neurons loaded with
FM1-43 for 16-36 hr with the calcium ionophore A23187 (calcimycin) for 1 min. This produced a marked loss of FM1-43 fluorescence (Fig. 4A) that was
attributable to the exocytosis of internalized dye (see below). We
measured this CEDE as the percentage of loss of background-subtracted
FM1-43 fluorescence as compared with pre-ionophore background-subtracted fluorescence (Fig. 4B).
Exposure to the carrier solution alone (0.1% DMSO) produced no loss of
fluorescence (Fig. 5; n = 12). Similarly, calcimycin did not have any effect when it was applied
in the presence of 5 mM EGTA (Fig. 5; n = 6). A23187 induced CEDE only in the presence of 2.5 mM
calcium. It could be recorded within 5 min after ionophore application and persisted for 15 min.
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Figure 4.
Calcium-evoked dendritic exocytosis (CEDE).
A, Fluorescent images of a representative culture
showing CEDE in response to ionophore application. Cultured hippocampal
neurons (12 DIV) loaded overnight with 1.5 µM FM1-43 were
challenged with 1 µM calcium ionophore A23187
(arrow) for 1 min after a prechallenge wash of 1 hr. The
three images shown were taken at the times indicated, after the removal
of FM1-43. Scale bar, 10 µm. B, Plot of the mean ± SEM of CEDE analyzed in two regions of interest
outlined in A, with respect to
fluorescence observed immediately before A23187 application.
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Figure 5.
CEDE is age-dependent and blocked by tetanus toxin
(TT) and nocodazole (noc). CEDE
was analyzed in developing cultured neurons (5-21 DIV;
EGTA, TT, and noc were
tested at >9 DIV). Bar graph shows mean ± SEM of CEDE 15 min
after A23187 application. Vehicle, 0.1% DMSO; *p < 0.05; **p < 0.01. Sample size in
parentheses refers to the number of cultures
examined.
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Ionophore-induced exocytosis from neuronal cultures loaded overnight
with FM1-43 was confirmed by recovering dye in the extracellular medium
(Henkel et al., 1996). Extracellular medium aliquots from 10 pooled
cultures were obtained before (Apre) and
15 min after (Apost) A23187 application.
Dye from pooled samples was extracted with butanol and dissolved in 2%
CHAPS. FM1-43 fluorescence intensity was measured in microcapillaries.
The relative FM1-43 fluorescence intensity of solution after A23187
treatment was 3.32 ± 0.7 (n = 7;
p < 0.05). This shows directly that a significant
amount of FM1-43 was released by calcium ionophore treatment and could be recovered in the extracellular medium. Calibration curves generated from 1.5 nM to 1.5 µM FM1-43 indicate that
the observed exocytosis corresponds to ~35 nM FM1-43.
This is ~100 times more than the estimated FM1-43 release measured
after similar experiments conducted on presynaptic exocytosis (0.3 nM; Henkel et al., 1996).
If the organelles undergoing CEDE are derived from the trans-Golgi
network (TGN), then one may expect that endocytosed FM1-43 may require
several hours before reaching such compartments. To examine this
possibility, we measured the amount of CEDE as a function of the FM1-43
loading time (Fig. 6A).
Mature cultures were exposed to FM1-43 for different periods of time
(1-16 hr) and challenged with A23187. Only when neurons were exposed
to FM1-43 for >8 hr was CEDE observed (Fig. 6A).
Notably, FM1-43 showed little colocalization with NBD
C6-ceramide fluorescence after 4 hr of loading
(n = 2) and significant colocalization after 16 hr of
FM1-43 loading (Fig. 6B). These results indicate that the passive loading of FM1-43 requires 8 hr before the dye reaches CEDE-competent organelles, at which time the dye colocalizes with NBD
C6-ceramide. This result supports the view that organelles undergoing CEDE are TGN-like or TGN-derived structures.
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Figure 6.
FM1-43 reaches CEDE-competent compartments only
after 8 hr of loading. A, Plot of mean ± SEM of
CEDE observed 15 min after vehicle (0.1% DMSO; dotted
line) or A23187 (solid line) application in
mature neurons (>9 DIV) loaded for 1-16 hr with FM1-43 (FM1-43 at 1 hr, n = 13; all other time points,
n = 5). B, Quantitative analysis of
the correlation between FM1-43-labeled compartments and NBD
C6-ceramide in neurons loaded with the FM1-43 for 4 or 16 hr. High correlation is observed only in neurons loaded for 16 hr with
FM1-43 (n = 5), but not in neurons loaded for 4 hr
(n = 2).
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We tested if CEDE uses exocytotic machinery with tetanus toxin, a
specific inhibitor of exocytosis (Montecucco and Schiavo, 1994). CEDE
was blocked completely by pretreatment with 10 nM tetanus
toxin (see Fig. 5; n = 7), indicating regulated
exocytosis rather than some nonspecific loss of the dye from
intracellular compartments.
We found that CEDE is regulated developmentally. Although neurons of
all ages that were examined (5-21 DIV) showed comparable loading after
overnight exposure to FM1-43, only neurons that were 9 DIV or older
were capable of producing CEDE (see Fig. 5; n = 11;
p < 0.05). At this stage the cultured neurons have
already established synaptic contacts and are fully differentiated. At earlier ages A23187 occasionally induced regrouping of labeled sites or
intensified movement of fluorescently labeled organelles but caused no
detectable CEDE.
Dendritic exocytosis of FM1-43 also could be elicited with repetitive
field electrical stimulation (Fig. 7). We
tested different numbers and frequencies of stimuli. Low-frequency
stimuli (1 Hz) did not produce any detectable loss of fluorescence
(n = 3). Medium-frequency stimuli (10 Hz) could produce
loss of fluorescence, but only if sufficient stimuli were delivered.
High-frequency stimuli (50 Hz) generally produced exocytosis
(n = 10). Figure 7 shows a pattern of CEDE observed in
a neuron stimulated with different parameters. This pattern generally
was observed in the tested neurons. In addition, the application of 1 mM glutamate for 1 min could evoke the exocytosis of some
dendritic structures (n = 2; data not shown). We
characterized CEDE by using calcium ionophore as the stimulating agent,
because it gave the most robust effect.
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|
Figure 7.
Field electrical stimulation elicits dendritic
exocytosis. A, Fluorescent images of a neuron loaded
overnight with FM1-43 before (left) and after
(right) field electrical stimulation (50 Hz, 500 stimuli). Note the marked loss of fluorescence indicating dendritic and
somatic exocytosis. B, Plot of dendritic exocytosis in
the neuron (A) as a function of stimulation
frequency. Note the requirement for higher frequencies and repeated
stimuli, reminiscent of the requirements for LTP.
|
|
| |
DISCUSSION |
FM1-43 labels dendritic compartments
In this study we report on a novel biological process of regulated
dendritic exocytosis. Cultured hippocampal neurons were exposed to
FM1-43 for >16 hr with no stimulation. Because FM1-43 immerses into
the outer leaflet of the plasma membrane, it is internalized inside the
cells whenever there is endocytosis. On washout of the uninternalized
dye from the outer surface, the remaining FM1-43 fluorescence marks the
inner membrane of intracellular membranous compartments. Via the fusion
of endosomes and endosome-derived compartments, over
time, the dye has access to numerous intracellular compartments.
Because the neurons are exposed continuously to FM1-43 for >16 hr,
some FM1-43 can be found in intracellular compartments that are derived
from endocytic organelles like early endosomes (expressing transferrin
receptor), late endosomes and lysosomes (expressing cathepsin D and
mannose-6-phosphate receptor), and endoplasmic reticulum in soma
(expressing IP3 receptor and BiP). However, quantitative
analysis of the colocalization between FM1-43-labeled structures and
intracellular markers of various organelles shows the greatest degree
of colocalization with NBD C6-ceramide, indicating the
labeling of TGN-like or TGN-derived structures (see Fig. 3).
The immunocytochemical analysis of labeled compartments is supported by
correlative electron and light microscopy. Electron microscopy of
FM1-43-labeled regions indicates that the majority of labeled sites
clearly is in dendrites and not presynaptic terminals, has no
dense core vesicles, has smooth endoplasmic reticulum (SER)/TGN organelles, and often is close to synapses. SER/TGN-like structures have been identified in spine apparatus and parent dendrites (Spacek and Harris, 1997). In addition, numerous organelles potentially involved in endocytosis and exocytosis have been visualized in spines
under electron microscopic analysis (Harris and Kater, 1994; Spacek and
Harris, 1997), although their function has only been hypothesized.
Smooth vesicles fusing with the plasma membrane were seen in some
spines, and exocytosis was suggested on the basis of these observations
(Spacek and Harris, 1997).
In contrast to acute loading protocols, overnight exposure of neurons
to FM1-43 appears to load little dye in presynaptic sites. This is
indicated by several observations. First, organelles labeled with the
overnight exposure to FM1-43 do not colocalize with synapsin I, a
marker for presynaptic vesicles (see Fig. 1D,E). In
addition, in associated electron micrographs the fluorescent regions
are in dendrites and only occasionally in presynaptic terminals (see
Fig. 2). Organelles loaded with overnight exposure to FM1-43 show
considerable movement (~2 µm/min) when observed during the 1 hr
period, whereas organelles loaded with brief exposure to high potassium
(which loads presynaptic terminals) do not move (see Fig.
1F). After the neurons are exposed to FM1-43 in the absence of depolarization, 8 hr are required before the dye reaches exocytosis-competent organelles (see Fig. 6A).
Presynaptic vesicles require only seconds after endocytosis to become
exocytosis-competent (Ryan, 1996). Finally, compartments labeled with
overnight exposure to FM1-43 colocalize with NBD
C6-ceramide, which is found only in soma and dendrites and
not in presynaptic terminals (see Figs. 3,
6B). The significantly lower labeling of presynaptic
sites during prolonged dye exposure may be attributable to their lower volume, mechanical perturbation (moving the coverslip to the recording chamber could induce presynaptic release), or extensive washing (generally >1 hr may preferentially induce presynaptic loss).
Calcium-evoked dendritic exocytosis
We find that a brief challenge of hippocampal neurons with calcium
ionophore A23187 produces a robust dendritic exocytosis. CEDE of FM1-43
was demonstrated directly by measuring dye fluorescence in the
extracellular medium. Calcium is required for CEDE, because ionophore
did not produce any effect in the presence of the calcium chelator EGTA
(see Fig. 5). CEDE was blocked by tetanus toxin, an agent known to
block exocytosis (Montecucco and Schiavo, 1994). This indicates that
the target of tetanus toxin, synaptobrevin, cellubrevin, or a homolog
(Yamasaki et al., 1994; Chilcote et al., 1995), is necessary for CEDE.
CEDE can be detected within 5 min after ionophore application and
reaches a plateau ~15 min after stimulation. Individual sites do not
disappear suddenly but destain continuously over this time. This is
comparable to what has been described with presynaptic destaining, in
which a single spot observed by light microscopy destains slowly rather
than abruptly (Betz et al., 1992). The slow time course is explained by
the presence of many small (50 nm) structures (labeled presynaptic
vesicles) that cannot be viewed individually by light microscopy (Betz
et al., 1992). The slow time course of CEDE may owe to a similar
underlying process. For instance, CEDE destaining is consistent with
the budding of small vesicles from dendritic SER/TGN-like structures
and their fusion with the local dendritic plasma membrane. Such a
process could occur over a time frame of minutes and would cause a
relative decrease in the fluorescence of the SER/TGN-like compartments, rather than their complete destaining. Although small vesicles may be
formed, they appear not to be transported for long distances along the
dendrite, because all regions along a dendrite lose fluorescence with a
similar time course. If vesicles were to move along a dendrite, then
one would expect bright regions to lose fluorescence and neighboring
dark regions (in the dendrite) to gain fluorescence. However, we see
homogeneous loss of FM1-43 fluorescence over time, which indicates a
delivery to the immediate surface. Therefore, organelles appear to be
released locally.
Intact microtubules are required for CEDE, because nocodazole, an agent
producing microtubule depolymerization (Goslin et al., 1989), prevents
CEDE (see Fig. 5). An association between CEDE-competent compartments
and microtubules is supported by the observation that these
compartments undergo rapid movements that appear synchronized to local
filopodial movements (see Fig. 1F). Thus,
microtubules may provide a delivery system for TGN-like organelles to
reach appropriate sites as well as a docking station for additional
components that may be necessary to execute the numerous processes
required for exocytosis (budding, docking, fusion, etc.).
Exocytosis of FM1-43-labeled sites also was observed in the soma, which
is consistent with the recent publication demonstrating calcium-dependent exocytosis of compounds from the cell body of dorsal
root ganglion cells (Huang and Neher, 1996).
Our experiments indicate that passive loading of FM1-43 requires 8 hr
before the dye reaches structures that are competent to undergo CEDE.
Studies of intracellular trafficking indicate that 8 hr is sufficient
for endocytosed material to reach biosynthetic pathways like SER/TGN in
cultured cells (Green and Kelly, 1992). Thus, these experiments further
support the view that the organelles undergoing CEDE are TGN-derived
structures and part of the biosynthetic pathway.
Under experimental conditions described in this study, in which calcium
is raised everywhere in the cell, CEDE is widespread. However, the
homogeneous loss of the dye in a nonhomogeneously labeled structure
indicates that exocytosis is local. These observations suggest the
existence of a general mechanism that is present throughout the cell.
It is well known that physiological stimuli can produce a localized
rise in postsynaptic Ca2+ concentration. Under such
conditions one may expect localized CEDE, thereby ensuring spatial
specificity to this process.
Possible Roles of CEDE
CEDE may underlie several previously described phenomena. Some of
them include the release of dopamine from dendrites in substantia nigra
neurons (Heeringa and Abercrombie, 1995) and somatic release of
substance P observed after strong stimulation (Huang and Neher, 1996).
Furthermore, there is evidence supporting a role for activity or
calcium in the release of growth factors and  -amyloid (Querfurth and
Selkoe, 1994; Blochl and Thoenen, 1995). It will be interesting to
determine whether CEDE releases such compounds.
In addition, CEDE may be involved in the activity-dependent expression
of transmembrane proteins. There are several reports on the
activity-dependent expression of membrane-bound proteins, like neural
cell adhesion molecules (NCAM) (Kiss et al., 1994). CEDE also might be
involved in the redistribution of substance P receptors in spinal
neurons after somatosensory stimulation (Mantyh et al., 1995) or the
homeostatic control of transmembrane glucose transporter proteins
(Cushman and Wardzala, 1980; Suzuki and Kono, 1980). Similarly, CEDE
might play a role in the distribution of ion channels at the surface
membrane, observed under different stimulus conditions (Harris et al.,
1991; Turrigiano et al., 1994).
Could CEDE be involved in long-term potentiation (LTP)? Indeed, a
recent report indicates that postsynaptic exocytosis is required to
generate LTP (Lledo et al., 1998). Localized calcium entry during LTP
induction might trigger the insertion of glutamate receptors (stored in
intracellular compartments or perisynaptic regions) (Hampson et al.,
1992; Baude et al., 1995) into synapses by a CEDE-like process. This
would produce larger responses to a synaptically released transmitter.
A similar process triggered by insulin appears to deliver GABA
receptors to the cell surface (Wan et al., 1997). In addition to
delivering receptors to synapses, substances concentrated inside the
postsynaptic membranous structures could be released into the synaptic
cleft, potentially affecting presynaptic function.
Finally, CEDE could provide new membrane at postsynaptic sites,
allowing for the growth and formation of new synapses (Lisman and
Harris, 1993). Indeed, SER and smooth vesicles have been localized at
the base and inside dendritic spines (Spacek and Harris, 1997), but
their function has not yet been clarified.
Conclusion
In summary, in this manuscript we have described a novel process
of regulated exocytosis in cultured hippocampal neuronsCEDE. We have
shown that CEDE has a number of important, physiologically relevant,
properties: it is calcium-evoked and it requires mature neurons,
exocytotic machinery, and intact microtubules. Compartments that
undergo CEDE derive from SER/TGN-like organelles in dendrites and
postsynaptic sites. This phenomenon thus could play a role in synaptic
plasticity, where it might involve changes in glutamate sensitivity,
release of active compounds having effect on pre- and postsynaptic
function, and/or growth of the plasma membrane.
| |
FOOTNOTES |
Received March 23, 1998; revised June 18, 1998; accepted June 22, 1998.
This study was supported by the Mathers Foundation and National
Institutes of Health. We are grateful to Nancy Dawkins for preparing
the cultures, Tamara Howard for assistance with the electron
microscopy, Jason Kass for technical assistance with immunocytochemistry, and Irena Miloslavskaya for assistance in image
processing.
Correspondence should be addressed to Dr. Roberto Malinow, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY 11724.
| |
REFERENCES |
-
Alberts B,
Bray D,
Lewis J,
Raff M,
Roberts K,
Watson JD
(1989)
In: Molecular biology of the cell. Hamden, CT: Garland.
-
Banker GA,
Goslin K
(1990)
In: Culturing nerve cells. Cambridge, MA: MIT.
-
Baude A,
Nusser Z,
Molnar E,
McIlhinney RA,
Somogyi P
(1995)
High-resolution immunogold localization of AMPA-type glutamate receptor subunits at synaptic and nonsynaptic sites in rat hippocampus.
Neuroscience
69:1031-1055[Web of Science][Medline].
-
Betz WJ,
Mao F,
Bewick GS
(1992)
Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.
J Neurosci
12:363-375[Abstract].
-
Blochl A,
Thoenen H
(1995)
Characterization of nerve growth factor (NGF) release from hippocampal neurons: evidence for a constitutive and an unconventional sodium-dependent regulated pathway.
Eur J Neurosci
7:1220-1228[Web of Science][Medline].
-
Chilcote TJ,
Galli T,
Mundigl O,
Edelmann L,
McPherson PS,
Takei K,
De Camilli P
(1995)
Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.
J Cell Biol
129:219-231[Abstract/Free Full Text].
-
Couce ME,
Weatherington AJ,
McGinty JF
(1992)
Expression of insulin-like growth factor-II (IGF-II) and IGF-II/mannose-6-phosphate receptor in the rat hippocampus: an in situ hybridization and immunocytochemical study.
Endocrinology
131:1636-1642[Abstract/Free Full Text].
-
Cushman SW,
Wardzala LJ
(1980)
Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane.
J Biol Chem
255:4758-4762[Free Full Text].
-
Goslin K,
Birgbauer E,
Banker G,
Solomon F
(1989)
The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.
J Cell Biol
109:1621-1631[Abstract/Free Full Text].
-
Green SA,
Kelly RB
(1992)
Low-density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells.
J Cell Biol
117:47-55[Abstract/Free Full Text].
-
Hampson DR,
Huang XP,
Oberdorfer MD,
Goh JW,
Auyeung A,
Wenthold RJ
(1992)
Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody.
Neuroscience
50:11-22[Web of Science][Medline].
-
Harris Jr HW,
Strange K,
Zeidel ML
(1991)
Current understanding of the cellular biology and molecular structure of the antidiuretic hormone-stimulated water transport pathway.
J Clin Invest
88:1-8.
-
Harris KM,
Kater SB
(1994)
Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function.
Annu Rev Neurosci
17:341-371[Web of Science][Medline].
-
Heeringa MJ,
Abercrombie ED
(1995)
Biochemistry of somatodendritic dopamine release in substantia nigra: an in vivo comparison with striatal dopamine release.
J Neurochem
65:192-200[Web of Science][Medline].
-
Henkel W,
Lubke J,
Betz WJ
(1996)
FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.
Proc Natl Acad Sci USA
93:1918-1923[Abstract/Free Full Text].
-
Huang L-YM,
Neher E
(1996)
Ca2+-dependent exocytosis in the somata of dorsal root ganglion neurons.
Neuron
17:135-145[Web of Science][Medline].
-
Huovila A-P,
Eder AM,
Fuller SD
(1992)
Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.
J Cell Biol
118:1305-1320[Abstract/Free Full Text].
-
Kiss JZ,
Wang C,
Olive S,
Rougon G,
Lang J,
Baetens D,
Harry D,
Pralong WF
(1994)
Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.
EMBO J
13:5284-5292[Web of Science][Medline].
-
Lisman JE,
Harris KM
(1993)
Quantal analysis and synaptic anatomyintegrating two views of hippocampal plasticity.
Trends Neurosci
16:141-147[Web of Science][Medline].
-
Lledo PM,
Zhang X,
Sudhof TC,
Malenka RC,
Nicoll RA
(1998)
Postsynaptic membrane fusion and long-term potentiation.
Science
279:399-403[Abstract/Free Full Text].
-
Mantyh PW,
DeMaster E,
Malhotra A,
Ghilardi JR,
Rogers SD,
Mantyh CR,
Liu H,
Basbaum AI,
Vigna SR,
Maggio JE
(1995)
Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation.
Science
268:1629-1632[Abstract/Free Full Text].
-
Montecucco C,
Schiavo G
(1994)
Mechanism of action of tetanus and botulinum neurotoxins.
Mol Microbiol
13:1-8[Web of Science][Medline].
-
Moos T
(1996)
Immunohistochemical localization of intraneuronal transferrin receptor immunoreactivity in the adult mouse central nervous system.
J Comp Neurol
375:675-692[Web of Science][Medline].
-
Morimoto T,
Popov S,
Buckley KM,
Poo MM
(1995)
Calcium-dependent transmitter secretion from fibroblasts: modulation by synaptotagmin I.
Neuron
15:689-696[Web of Science][Medline].
-
Moschella MC,
Watras J,
Jayaraman T,
Marks AR
(1995)
Inositol 1,4,5-trisphosphate receptor in skeletal muscle: differential expression in myofibres.
J Muscle Res Cell Motil
16:390-400[Web of Science][Medline].
-
Nakanishi H,
Tominaga K,
Amano T,
Hirotsu I,
Inoue T,
Yamamoto K
(1994)
Age-related changes in activities and localizations of cathepsins D, E, B, and L in the rat brain tissues.
Exp Neurol
126:119-128[Web of Science][Medline].
-
Pagano E,
Sepanski MA,
Martin OC
(1989)
Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.
J Cell Biol
109:2067-2079[Abstract/Free Full Text].
-
Parton RG,
Dotti CG
(1993)
Cell biology of neuronal endocytosis.
J Neurosci Res
36:1-9[Web of Science][Medline].
-
Querfurth HW,
Selkoe DJ
(1994)
Calcium ionophore increases amyloid beta peptide production by cultured cells.
Biochemistry
33:4550-4561[Medline].
-
Rodriguez A,
Webster P,
Ortego J,
Andrews NW
(1997)
Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.
J Cell Biol
137:93-104[Abstract/Free Full Text].
-
Rubio ME,
Wenthold RJ
(1997)
Glutamate receptors are selectively targeted to postsynaptic sites in neurons.
Neuron
18:939-950[Web of Science][Medline].
-
Ryan TA
(1996)
Endocytosis at nerve terminals: timing is everything.
Neuron
17:1035-1037[Web of Science][Medline].
-
Ryan TA,
Reuter H,
Wendland B,
Schweizer FE,
Tsien RW,
Smith SJ
(1993)
The kinetics of synaptic vesicle recycling measured at single presynaptic boutons.
Neuron
11:713-724[Web of Science][Medline].
-
Spacek J,
Harris KM
(1997)
Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat.
J Neurosci
17:190-203[Abstract/Free Full Text].
-
Suzuki K,
Kono T
(1980)
Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site.
Proc Natl Acad Sci USA
77:2542-2545[Abstract/Free Full Text].
-
Trowbridge IS,
Collawn JF,
Hopkins CR
(1993)
Signal-dependent membrane protein trafficking in the endocytic pathway.
Annu Rev Cell Biol
9:129-161[Web of Science].
-
Turrigiano G,
Abbott LF,
Marder E
(1994)
Activity-dependent changes in the intrinsic properties of cultured neurons.
Science
264:974-977[Abstract/Free Full Text].
-
Wan Q,
Xiong ZG,
Man HY,
Ackerley CA,
Braunton J,
Lu WY,
Becker LE,
MacDonald JF,
Wang YT
(1997)
Recruitment of functional GABAA receptors to postsynaptic domains by insulin.
Nature
388:686-690[Medline].
-
Yamasaki S,
Baumeister A,
Binz T,
Blasi J,
Link E,
Cornille F,
Roques B,
Fykse EM,
Sudhof TC,
Jahn R,
Niemann H
(1994)
Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin.
J Biol Chem
269:12764-12772[Abstract/Free Full Text].
-
Ziv NE,
Smith SJ
(1996)
Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.
Neuron
17:91-102[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18176803-11$05.00/0
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27 - 34.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y Zilberter
Dendritic release of glutamate suppresses synaptic inhibition of pyramidal neurons in rat neocortex
J. Physiol.,
November 1, 2000;
528(3):
489 - 496.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. U. Walkley, M. Zervas, and S. Wiseman
Gangliosides as Modulators of Dendritogenesis in Normal and Storage Disease-affected Pyramidal Neurons
Cereb Cortex,
October 1, 2000;
10(10):
1028 - 1037.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F.-M. Zhou and J. J. Hablitz
Activation of Serotonin Receptors Modulates Synaptic Transmission in Rat Cerebral Cortex
J Neurophysiol,
December 1, 1999;
82(6):
2989 - 2999.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Coco, G. Raposo, S. Martinez, J.-J. Fontaine, S. Takamori, A. Zahraoui, R. Jahn, M. Matteoli, D. Louvard, and T. Galli
Subcellular Localization of Tetanus Neurotoxin-Insensitive Vesicle-Associated Membrane Protein (VAMP)/VAMP7 in Neuronal Cells: Evidence for a Novel Membrane Compartment
J. Neurosci.,
November 15, 1999;
19(22):
9803 - 9812.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R A Lenz and B. Alger
Calcium dependence of depolarization-induced suppression of inhibition in rat hippocampal CA1 pyramidal neurons
J. Physiol.,
November 15, 1999;
521(1):
147 - 157.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Verderio, S. Coco, A. Bacci, O. Rossetto, P. De Camilli, C. Montecucco, and M. Matteoli
Tetanus Toxin Blocks the Exocytosis of Synaptic Vesicles Clustered at Synapses But Not of Synaptic Vesicles in Isolated Axons
J. Neurosci.,
August 15, 1999;
19(16):
6723 - 6732.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Aroniadou-Anderjaska, M. Ennis, and M. T. Shipley
Dendrodendritic Recurrent Excitation in Mitral Cells of the Rat Olfactory Bulb
J Neurophysiol,
July 1, 1999;
82(1):
489 - 494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Shi, Y. Hayashi, R. S. Petralia, S. H. Zaman, R. J. Wenthold, K. Svoboda, and R. Malinow
Rapid Spine Delivery and Redistribution of AMPA Receptors After Synaptic NMDA Receptor Activation
Science,
June 11, 1999;
284(5421):
1811 - 1816.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. Maletic-Savatic, T. Koothan, and R. Malinow
Calcium-Evoked Dendritic Exocytosis in Cultured Hippocampal Neurons. Part II: Mediation by Calcium/Calmodulin-Dependent Protein Kinase II
J. Neurosci.,
September 1, 1998;
18(17):
6814 - 6821.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Scott, M. S. Kruse, H. Forssberg, H. Brismar, P. Greengard, and A. Aperia
Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation
PNAS,
February 5, 2002;
99(3):
1661 - 1664.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
