The FASEB Journal article fj.12-206300. Published online December 6, 2012.
The FASEB Journal • Research Communication
Vesicular uptake and exocytosis of L-aspartate is
independent of sialin
Cecilie Morland,*,† Kaja Nordengen,*,† Max Larsson,*,†,‡ Laura M. Prolo,§,储
Zoya Farzampour,§,储 Richard J. Reimer,§,储 and Vidar Gundersen*,†,¶,1
*Department of Anatomy and †Center for Molecular Biology and Neuroscience, University of Oslo,
Oslo, Norway; ‡Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; §Department
of Neurology and Neurological Sciences and 储Graduate Program in Neuroscience, Stanford
University School of Medicine, Stanford, California, USA; and ¶Department of Neurology, Oslo
University Hospital, Rikshospitalet Oslo, Oslo, Norway
The mechanism of release and the role
of L-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for L-aspartate
has been difficult to prove, as no vesicular L-aspartate
transporter was identified until it was found that sialin
could transport L-aspartate and L-glutamate when reconstituted into liposomes. We sought to clarify the release
mechanism of L-aspartate and the role of sialin in this
process by combining L-aspartate uptake studies in
isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled L-aspartate was taken up into synaptic vesicles. The vesicular L-aspartate uptake, relative to the
L-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by
L-glutamate. We further show that sialin is not essential
for exocytosis of L-aspartate, as there was no difference
in ATP-dependent L-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in
significant vesicle uptake of L-aspartate, and depolarization-induced depletion of L-aspartate from hippocampal nerve terminals was similar in hippocampal
slices from sialin-knockout and wild-type mice. Further,
there was no evidence for nonvesicular release of
L-aspartate via volume-regulated anion channels or
plasma membrane excitatory amino acid transporters.
This suggests that L-aspartate is exocytotically released
from nerve terminals after vesicular accumulation by a
transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z.,
Reimer, R. J., Gundersen, V. Vesicular uptake and
exocytosis of L-aspartate is independent of sialin.
FASEB J. 27, 000 – 000 (2013). www.fasebj.org
ABSTRACT
Abbreviations: EAAT, excitatory amino acid transporter;
mCherry, monomeric Cherry; NCX(rev), reversed Na⫹-Ca2⫹ exchanger; NMDA, N-methyl d-aspartate; NPPB, 5-nitro-2-(3phenylpropylamino) benzoic acid; RFP, red fluorescent protein;
TFB-TBOA, (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid; TRPC, transient receptor potential
cation; VGLUT1, vesicular glutamate transporter 1; VRAC, volumeregulated anion channel
0892-6638/13/0027-0001 © FASEB
Key Words: synaptic transmission 䡠 VRAC 䡠 excitatory amino
acid transporters
Besides serving a role as a precursor of Krebs’ cycle
intermediates and as a building block of proteins,
l-aspartate has been proposed to act as a transmitter in
the brain. This was first suggested in the hippocampus
(1), and later in the cerebellum (2). In favor of
aspartergic neurotransmission is the evidence for exocytotic release of l-aspartate from nerve terminals
(3–5) and that l-aspartate stimulates the N-methyl
d-aspartate (NMDA) type of l-glutamate receptors (6),
producing NMDA-receptor-dependent postsynaptic responses (7, 8). A major argument against aspartergic
neurotransmission is the conflicting evidence on uptake of l-aspartate into synaptic vesicles. While there
seems to be consensus that l-aspartate does not inhibit
the vesicular uptake of l-glutamate (9 –11), only a few
studies have directly measured the uptake of l-aspartate into synaptic vesicles. Using isolated cortical synaptic vesicles, Naito and Ueda (12) found a significant
l-aspartate uptake. Other studies show negligible vesicular accumulation of l-aspartate (11, 13).
Recently sialin, which is known to transport sialic
acid out of lysosomes (14), was reported to also carry
l-aspartate (and l-glutamate) into reconstituted proteoliposomes (15). However, whether sialin has a role
in the release of l-aspartate from nerve terminals in
intact brain tissue has not been tested. To clarify these
ambiguities in aspartergic neurotransmission, we here
used brain tissue from the hippocampus, the brain
region showing the most robust evidence for synaptic
release of l-aspartate (3–5). We first measured the
ATP-dependent uptake of l-aspartate in synaptic vesicles isolated from the hippocampus and compared it to
the uptake in vesicles isolated from the whole brain and
3 other forebrain regions. We then used sialin-knockout mice to analyze the sialin-dependent l-aspartate
vesicle uptake. As a second control for sialin depen1
Correspondence: Department of Anatomy and the
CMBN, University of Oslo, POB 1105 Blindern, 0317 Oslo,
Norway. E-mail: vidar.gundersen@medisin.uio.no
doi: 10.1096/fj.12-206300
1
dency, we measured l-aspartate uptake in vesicles isolated from native PC12 cells and PC12 cells overexpressing sialin. By immunogold cytochemistry of hippocampal
slices from sialin-knockout and wild-type mice, we then
determined whether sialin was necessary for release of
l-aspartate in intact brain tissue. Our results show that
l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a sialin-independent mechanism.
MATERIALS AND METHODS
Animals
All animals were treated in strict accordance with the guidelines of the Norwegian Committees on Animal Experimentation (Norwegian Animal Welfare Act and European Communities Council Directive of 24 November 1986-86/609/EEC)
and the Stanford Institutional Animal Care and Use Committee. Male Wistar rats were from Scanbur (Sollentuna, Sweden). Male heterozygous sialin-knockout mice (B6; 129S5–
Slc17a5tm1Lex) were obtained from the Mutant Mouse
Regional Resource Centre (http://www.mmrrc.org) and
bred to homozygosity (16). Mouse genotypes were determined by PCR (16).
Preparation of synaptic vesicles
Synaptic vesicles from rat whole brain, hippocampus, striatum, entorhinal cortex, and frontal cortex, as well as from the
whole brain of sialin-knockout and wild-type mice, were
isolated as described by Erickson et al. (17). For isolation of
synaptic vesicles from the forebrain subregions, the dissected
tissue was kept on ice until all brain regions from 10 Wistar
rats (⬃200 g body weight) had been dissected out (⬍30 min).
Brain tissues were homogenized in 0.32 M sucrose (5%, w/v)
and centrifuged for 10 min at 1000 g and 4°C. The supernatants were collected and centrifuged for 30 min at 21,000 g
and 4°C. The resulting synaptosomal pellets (P2 fractions),
were osmotically shocked by resuspension in ice-cold purified
water (7.5%, w/v) and centrifuged for 30 min at 21.000 g to
remove the crude synaptosomal membranes. Osmolarity was
restored with 0.1 M potassium tartrate and 25 mM Hepes (pH
7.4), before the mixtures were centrifuged for 1 h at 100,000
g and 4°C. The pellets (LP2 fractions), containing crude
synaptic vesicles, were gently resuspended in 0.32 M sucrose,
frozen in liquid N2, and stored at ⫺80°C or used for uptake
assay immediately.
Vesicular uptake of L-aspartate and L-glutamate
Vesicular uptake of l-glutamate and l-aspartate was determined as described by Fykse et al. (18) with slight modifications. Synaptic vesicles from ⬃35 mg brain tissue of the whole
brain, the hippocampus, the striatum, the frontal cortex, and
the entorhinal cortex were preincubated at 30°C for 15 min
in an incubation mixture containing 10 mM K-HEPES (pH
7.4), 4 mM KCl, 4 mM MgSO4, 0.25 M sucrose, and 1 mM
reduced glutathione. For experiments with Rose Bengal,
preincubation time was 25 min in the presence or absence of
Rose Bengal, 5 ␮M. The uptake was started when a substrate
solution containing (final concentrations) 2 mM ATP and 1
mM l-[3,4-3H]glutamic acid or 1 mM l-[2,3-3H]aspartic acid
(1 ␮Ci; both from PerkinElmer Life and Analytical Sciences,
Boston, MA, USA) was added. To determine whether the
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vesicular uptake of l-aspartate could be blocked by l-glutamate, in some experiments the vesicles were exposed to
l-glutamate (1 mM) both during preincubation and incubation. In these experiments, ATP was included also in the
preincubation step, to allow complete filling of the vesicles
with l-glutamate prior to incubation with [3H]l-aspartate.
The uptake of [3H]l-glutamate and [3H]l-aspartate was
stopped after 5 min by addition of 7 ml of ice-cold 0.15 M KCl
to the 300 ␮l samples. Blanks were treated as described above,
except that ATP was omitted from the incubation buffer. As a
negative control, some samples were incubated in ice-cold
buffer without ATP. The samples were immediately filtered
(Skatron filterMAT 11731; Skatron Instruments, Newmarket,
UK). The filters were washed with 3 ⫻ 7 ml of 0.15 M KCl,
dissolved in Filter-Count (PerkinElmer) and counted for
retained radioactivity in a Packard Tri-Carb 300 (Packard
Instrument Co., Meriden, CT, USA). The radioactivity in the
blanks (incubated at 30°C without ATP) was 20% higher than
the values in samples incubated on ice without ATP. ATPdependent uptake was defined as the radioactivity in the
samples (incubated at 30°C with ATP) minus the radioactivity
in the blanks and presented as means ⫾ se, n ⫽ 3–5
individual experiments for each observation. In each experiment, ATP-dependent uptake was measured in triplicates.
Due to small volumes of the subregional vesicular fractions,
the protein concentrations of these were not measured, and
thus most of the data are given as percentage of the l-glutamate uptake in the same region. However, in one experiment, the protein content in the samples was analyzed (BC
assay; Interchem, Montfuçon, France) and the absolute uptake values (pmol/min/mg protein) were calculated. Whether the
uptake values in the hippocampus were significantly different
from the values in the whole brain and the other subregions
was tested by 1-way ANOVA post hoc test for multiple comparisons (SPSS, Chicago, IL, USA). Uptake in sialin-knockout
and wild-type vesicles was statistically evaluated by Student’s t
test, 2 tails (SPSS).
PC12 cell vesicle preparation
The rat vesicular glutamate transporter 1 (VGLUT1) expression plasmid was derived as described previously (19). The rat
sialin cDNA (20) was subcloned into the plasmid expression
vector pcDNA3-Amp (Invitrogen, Carlsbad, CA, USA). Subsequently, a cDNA encoding the red fluorescent protein (RFP)
monomeric Cherry (mCherry) was subcloned at the 5= end of
the sialin sequence to generate a chimeric protein. The
plasmids were introduced into PC12 cells using Lipofectamine 2000 (Invitrogen). The cells were then selected in
800 mg/ml G418 (effective concentration; Life Technologies,
Gaithersburg, MD, USA), and the resulting clones were
screened by direct fluorescence microscopy for sialin-expressing cells and immunofluorescence microscopy for VGLUT1expressing cells to identify those with the highest level of
expression. Cells were maintained in DMEM (Life Technologies) supplemented with 10% equine serum (HyClone,
South Logan, UT, USA), 5% calf serum (HyClone), and
penicillin-streptomycin (Life Technologies). Vesicle membranes were prepared from PC12 as follows: cells were
harvested by trituration washed once in PBS, and cells from
each 15-cm plate were resuspended in 1 ml of 0.3 M sucrose
and 10 mM HEPES-KOH, pH 7.4 (SH buffer), containing 0.2
mM diisopropylfluorophosphate (DFP), 1 mg/ml pepstatin, 2
mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml E64, and 1.25
mM MgEGTA. The cells were then disrupted by homogenization at 4°C through a ball-bearing device with a clearance of
10 ␮m (Isobiotec, Heidelberg, Germany). The nuclear debris
was sedimented at 1000 g for 5 min, and heavier membranes
were eliminated by centrifugation at 20,000 g for 20 min (P2).
The FASEB Journal 䡠 www.fasebj.org
MORLAND ET AL.
The remaining light membrane vesicles (crude synaptic-like
microvesicles) were sedimented at 100,000 g for 1 h, and the
resulting pellet (P3) was resuspended in SH buffer containing
the same protease inhibitors, at a final protein concentration
of 10 ␮g/␮l.
Transport assay for PC12 vesicles
To initiate the reaction, a 10-␮l aliquot of membranes was
added to 190 ␮l SH buffer containing 4 mM MgCl2, 4 mM
KCl, 4 mM ATP, 100 ␮M unlabeled substrate, and 2 ␮Ci
3
H-substrate (American Radiolabeled Chemicals, St. Louis,
MO, USA). Incubation was performed at 29°C for 5 min, and
the reaction was terminated by rapid filtration (DAWP, 0.65
␮m; Millipore, Billerica, MA, USA), followed by immediate
washing with 6 ml cold SH buffer. Background uptake was
determined by parallel assays done in the presence of nigericin (5 ␮M) and valinomycin (20 ␮M) to dissipate the proton
electrochemical gradient. The trapped radioactivity was measured by scintillation counting in 2.5 ml Cytoscint (ICN
Biomedicals, Inc., Aurora, OH, USA). Transport measurements were repeated ⱖ3 times using ⱖ2 different membrane
preparations.
For Western blotting, P2 and P3 fractions from homogenized PC12 cells (30 ␮g of protein/lane) were separated by
SDS-PAGE and subsequently electrotransferred to polyvinylidene fluoride membranes. Blots were blocked with Blotto
(PBS with 5% nonfat dry milk and 0.1% Tween-20) for 1 h
and then incubated with an antibody directed against sialin
(Alpha Diagnostics, Owings Mills, MD, USA) at a 1:500
dilution or against RFP at 1:5000 overnight at 4°C. Blots were
washed, incubated with anti-rabbit horseradish peroxidasecoupled secondary antibody (1:10,000; Pierce, Rockford, IL,
USA) in Blotto for 1 h. Proteins were visualized using an ECL
Western blotting detection system (Amersham Biosciences,
Piscataway, NJ, USA) and exposure of the blot to autoradiography film (MidSci, St. Louis, MO, USA).
Preparation and incubation of hippocampal slices
Hippocampal slice experiments were performed as described
previously (21–23). Wistar rats were anesthetized with halotane or isoflurane and decapitated. The brains were removed and the hippocampi dissected out on ice and chopped
into 300 ␮m slices. The slices were preincubated in oxygenated Krebs’ solution (126 mM NaCl; 3 mM KCl; 10 mM
sodium phosphate buffer, pH 7.4; 1.2 mM CaCl2; 1.2 mM
MgSO4; and 10 mM glucose) for 45 min at 30°C. Then the
slices were incubated for 45 min at 30°C in Krebs’ solution
containing physiological (3 mM) or depolarizing (55 mM,
Na⫹ reduced to 80 mM) concentrations of K⫹. To ensure
that l-aspartate was not released through excitatory amino
acid transporters (EAATs), all incubations were done in
the presence of the EAAT inhibitor (3S)-3-[[3-[[4(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic
acid (TFB-TBOA; 5 ␮M; Tocris Bioscience, Ellisville, MO,
USA), except in one experiment where we incubated the
slices without TFB-TBOA at basal conditions (3 mM K⫹) and
with and without TFB-TBOA at depolarizing conditions (55
mM K ⫹ ). Some slices were exposed to 5-nitro-2-(3phenylpropylamino) benzoic acid (NPPB, 100 ␮M; Sigma, St
Louis, MO, USA), which inhibits volume-regulated anion
channels, or KB-R7943 (50 ␮M; Tocris), which inhibits Ca2⫹
influx via the reversed Na⫹-Ca2⫹ exchanger (NCX(rev)),
NMDA receptors, and transient receptor potential cation
(TRPC)-Ca2⫹ channels. All inhibitors were present for the
last 10 min of preincubation and for the whole incubation
period. Some slices were incubated at 55 mM K⫹ with 0.1 mM
Ca2 ⫹ (Mg2⫹ increased to 10 mM).
L-ASPARTATE
TRANSMISSION IS INDEPENDENT OF SIALIN
Slice experiments using sialin-knockout and wild-type mice
(3.5 wk of age) were performed essentially as described above,
but with the following modifications: 300-␮m-thick frontal
slices were vibrosliced into oxygenated carbonate-buffered
Krebs’ solution (126 mM NaCl, 26 mM NaHCO3, 3 mM KCl,
1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, and 10 mM
glucose). Then the slices were incubated for 1 h at 32°C in the
same buffer at 3 or 55 mM K⫹, without TFB-TBOA.
After incubation, the slices were fixed in 2.5% glutaraldehyde and 1% formaldehyde for 1 h at room temperature
(⬃20°C). The slices were stored in fixative diluted 1:10 at 4°C
until embedding and sectioning.
Electron microscopic immunocytochemistry
The slices were embedded in Lowicryl HM20 (mouse slices;
EMS, Hatfield, PA, USA) or Durcupan (rat slices; EMS) as
described previously (24). After the embedding, ultrathin
sections (80 –100 nm) were cut and labeled with the 435
l-aspartate (diluted 1:300), 607 l-glutamate (diluted 1:40,000
for single labeling and 1:5,000 for double labeling), and 990
GABA (diluted 1:300) antisera. The 435 l-aspartate, 607
l-glutamate, and 990 GABA antisera have been characterized
(22, 23, 25–27). To avoid possible cross-reactivities, the l-glutamate, l-aspartate, and GABA antisera were used with the
addition of 0.2 mM complexes of glutaraldehyde/formaldehyde-treated l-aspartate, asparagine plus glutamine and asparagine, l-glutamate plus GABA, and l-glutamate plus ␤-alanine, respectively. As a specificity test, ultrathin test sections
containing conjugates of amino acids fixed to brain proteins
by formaldehyde and glutaraldehyde (28) were run along
with the immunogold experiments. This test system showed
that the primary antibodies labeled only the conjugate containing the amino acid against which the antibodies were
raised (data not shown).
To visualize aspartate/glutamate and aspartate/GABA in
the same nerve terminals, we performed double-labeling
experiments, in which the ultrathin sections were first treated
with the l-aspartate antibodies and then with the l-glutamate
or GABA antibodies. Between the first and the second step,
the sections were exposed to formaldehyde vapor (80°C, 1 h)
to prevent interference between the sequential incubations
(29). Secondary antibodies coupled to colloidal 10- and
15-nm gold particles (British Biocell International, Cardiff,
UK) were used in the first and second step, respectively.
The sections were studied in a Tecnai 12 electron microscope (FEI, Hillsboro, OR, USA). Electron micrographs were
randomly taken from the CA1 stratum radiatum, granule, or
pyramidal cell layer. Excitatory terminals were defined as
those making asymmetric synapses with dendritic spines.
Inhibitory terminals were defined as those making symmetric
synapses with stem dendrites or granule cell bodies and
containing GABA immunogold particles. The densities (gold
particles/␮m2) of l-aspartate and l-glutamate gold particles
in excitatory terminals and of l-aspartate gold particles in
inhibitory nerve terminals, as well as the spatial relation
between gold particles signaling l-aspartate and l-glutamate
and synaptic vesicles in excitatory terminals in sialin-knockout
and wild-type slices incubated at 3 mM K⫹, were calculated
(30). A plug-in for ImageJ (U.S. National Institutes of Health,
Bethesda, MD, USA; http://rsb.info.nih.gov/ij/) was used to
outline the plasma membrane of nerve terminals, and to
record the distance between the center of a gold particle or
points randomly placed over the excitatory terminals (the
number of random points were approximately the same as
the number of gold particles in the terminals) and the center
of the closest synaptic vesicle. Recorded coordinates were
submitted to a program written in Python (http://www.
python.org) for computation of particle density (gold parti3
cles/␮m2; ref. 31). The source code of the ImageJ plug-in and
the Python program are available online (http://www.neuro.ki.
se/broman/maxl/software.html). The gold particle-synaptic
vesicle distances were sorted into bins of 5 nm, and the
frequencies of the intercenter distances for each bin were
calculated (Excel; Microsoft, Redmond, WA, USA). The
quantitative results were statistically evaluated by ␹2 test, 1-way
ANOVA post hoc test for multiple comparisons (SPSS), and
Student’s t test, 2 tails (SPSS).
RESULTS
L-Aspartate
is taken up into synaptic vesicles
We compared the ATP-dependent uptake of l-aspartate in isolated synaptic vesicles from the hippocampus
with the uptake in vesicles from the whole brain and
different other brain regions. The ATP-dependent vesicular l-aspartate uptake was assessed relative to the ATPdependent vesicular l-glutamate uptake in the same
vesicle fraction. In hippocampal vesicles, the ATP-dependent l-aspartate uptake was 18.1% of the l-glutamate
uptake (Fig. 1A). In comparison, vesicles isolated from the
whole brain showed an ATP-dependent l-aspartate uptake of 9.1% of the l-glutamate uptake. In the entorhinal
cortex, the frontal cortex, and the striatum, ATPdependent l-aspartate uptake was 9.6, 10.1, and 11.9%
(relative to the ATP-dependent l-glutamate uptake),
Figure 1. l-Aspartate is taken up into synaptic vesicles.
A) ATP-dependent uptake of l-aspartate relative to the
l-glutamate uptake (average⫾sem, number of experiments
indicated at bottom of each bar) in isolated rat synaptic
vesicles from the whole brain (brain), hippocampus (HC),
entorhinal cortex (EC), frontal cortex (FC), and striatum
(Stri). In each experiment, the ATP-dependent uptake was
analyzed in triplicate. Brackets indicate that the value in the
hippocampus is significantly different from the values in the
whole brain, entorhinal cortex. and frontal cortex (P⬍0.05,
1-way ANOVA post hoc; SPSS). B) Vesicular uptake of l-aspartate
without (Asp) and with l-glutamate (Asp⫹Glu) (average⫾
sem, number of experiments indicated at bottom of each
bar). The individual aspartate uptake values of each experiment without and with l-glutamate were normalized to the
average aspartate uptake without l-glutamate (0.0113 pmol/
min/mg tissue, n⫽3 experiments). sem was calculated based
on the normalized values for the individual experiments.
There was no significant difference between uptake values in
Asp and Asp⫹Glu (P⬎0.05, Student’s 2-tailed t test; SPSS).
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March 2013
respectively (Fig. 1A). The vesicular uptake of l-aspartate was not inhibited by l-glutamate, as addition of 1
mM l-glutamate during the preincubation and incubation steps did not alter the uptake of radiolabeled
l-aspartate in hippocampal vesicles (Fig. 1B). The
l-aspartate and l-glutamate uptake values reported
were obtained from frozen vesicles; however, to validate
the vitality of these vesicles, a control experiment (n ⫽
6 –9 replicates from 2–3 experiments) was performed
where we compared l-aspartate and l-glutamate uptake into frozen and fresh hippocampal vesicles. The
uptake into frozen vesicles (59.2⫾1.8 pmol/min/mg
protein for l-glutamate and 7.8⫾1.3 pmol/min/mg protein for l-aspartate) were only slightly lower that the
uptake in freshly prepared vesicles (67.3⫾7.0 pmol/
min/mg protein for l-glutamate and 9.8⫾1.9 pmol/
min/mg protein for l-aspartate, means⫾sd). l-Glutamate uptake, but not l-aspartate uptake, could be
almost completely blocked by the vesicular glutamate
uptake blocker Rose Bengal (5 ␮M, data not shown;
for characterization of Rose Bengal; see ref. 32),
supporting the notion that l-aspartate is accumulated into synaptic vesicles by another transporter
than the VGLUTs (33).
Vesicular accumulation of L-aspartate is
sialin-independent
Previous studies have demonstrated that sialin, when
reconstituted into proteoliposomes, can catalyze transmembrane l-aspartate transport. This activity was competitively inhibited by l-glutamate. To determine
whether sialin is an important vesicular transporter for
l-aspartate, we measured ATP-dependent accumulation of l-aspartate in synaptic vesicles from sialinknockout and wild-type whole brain. We found that the
lack of sialin did not reduce the ability of the vesicles to
accumulate l-aspartate (Fig. 2A) or l-glutamate (data
not shown). This was further confirmed in another test
system, in which we overexpressed sialin (tagged with
the RFP mCherry) in PC12 cells and isolated synaptic
like microvesicles (P3 fraction) from wild-type cells and
cells overexpressing sialin. Western blotting with antibodies against sialin showed that wild-type cells contained endoegenous sialin in the crude synaptosome
fraction (containing lysosomes), while sialin was not
detected in synaptic-like microvesicles (P3 fraction; Fig.
2B). In PC12 cells overexpressing sialin, antibodies
against sialin, as well as against RFP, recognized recombinant sialin in both the P2 and the P3 fraction (Fig.
2B). There was essentially no l-aspartate uptake in P3
vesicles from wild-type or sialin-overexpressing PC12
cells (Fig. 2C), while vesicles from cells expressing the
vesicular l-glutamate transporter VGLUT1 displayed
robust uptake of l-glutamate (Fig. 2C). Together these
findings strongly suggest that sialin is not capable of
mediating l-aspartate transport in native vesicles.
Depletion of L-aspartate from hippocampal terminals
is independent of sialin
To investigate whether sialin is required for exocytotic
release of l-aspartate in intact brain tissue, we exposed
The FASEB Journal 䡠 www.fasebj.org
MORLAND ET AL.
Figure 2. Vesicular uptake of l-aspartate is
independent of sialin. A) Measurement of ATPdependent vesicular accumulation of 3H-l-aspartate in whole-brain synaptic vesicles from
sialin-knockout (KO) and wild-type (WT) mice.
ATP-dependent uptake of l-aspartate was 9.5 ⫾
1.8 pmol/min/mg protein in the WT and
13.8 ⫾ 5.2 pmol/min/mg protein in the KO
(means⫾se, n⫽4 experiments, P⬎0.05, Student’s t test; SPSS). B) Western blots of vesicles
derived from WT (WT PC12) and PC12 cells
expressing recombinant sialin (A3) with an
antibody directed against sialin (left panel) and
RFP (right panel) demonstrate marked overexpression of sialin in the stable cell line. The mCherry tag (RFP) on the recombinant protein causes the primary band in the
recombinant cells to migrate more slowly than the band in the WT cells. C) Measurement of vesicular accumulation (means⫾sd;
nmol/mg/5 min, n⫽2 experiments) of 3H-l-aspartate (open bars) and 3H-l-glutamate (solid bars) in crude synaptic-like
microvesicles derived from wild-type PC12 cells (WT PC12) and those expressing recombinant sialin (A3 sialin). There is a
⬎3-fold increase in accumulation of l-glutamate in synaptic-like microvesicles from VGLUT1 (B1 VGLUT1)-expressing cells
compared to WT. Brackets indicate that the glutamate uptake was significantly higher in B1 VGLUT1 than in WT (P⬍0.05,
Student’s t test).
hippocampal slices from sialin-knockout and wild-type
mice to physiological (3 mM) or depolarizing (55 mM)
concentrations of K⫹. As observed previously in rats
(22, 34, 35), in mouse slices incubated at 3 mM K⫹,
l-aspartate immunogold particles colocalized with lglutamate immunogold particles in excitatory terminals
in the CA1 stratum radiatum (Fig. 3A, C) and with
GABA immunogold particles in inhibitory terminals in
the granule and pyramidal cell layers (Fig. 3E, G). On
depolarization with 55 mM K⫹, gold particles signaling
l-aspartate were depleted from both types of terminals
(Fig. 3A vs. B; E vs. F). If sialin carries l-aspartate into
synaptic vesicles before release, we would expect that
the absence of sialin would greatly reduce vesicular
release of l-aspartate from nerve terminals. However,
there was no difference in l-aspartate labeling of
excitatory terminals between sialin knockouts (Fig. 3C,
D) and wild types (Fig. 3A, B), either in the basal
condition (Fig. 3A, C) or after depolarization (Fig. 3B,
D). Quantitative analysis showed that excitatory nerve
terminals in wild-type and knockout slices displayed
a similar reduction in the density of gold particles
representing l-aspartate (73 and 83%, respectively) in
response to depolarization (Fig. 3I). Correspondingly,
sialin was not necessary for the depolarization-induced
depletion of l-glutamate from excitatory nerve terminals (Fig. 3A–D, J). Likewise, in inhibitory terminals,
there was no difference in l-aspartate labeling between
sialin-knockout (Fig. 3G, H) and wild-type slices (Fig.
2E, F), either in the basal condition (Fig. 3E, G) or after
depolarization (Fig. 3F, H). Quantifications showed
that the depolarization-induced decrease in l-aspartate
labeling was similar in wild-type and knockout slices (73
and 80%, respectively; Fig. 2K).
To confirm that l-aspartate is contained within
synaptic vesicles in sialin-knockout hippocampus, we
measured the distance between the center of gold
particles signaling l-aspartate and the center of the
closest synaptic vesicle in excitatory terminals in
wild-type and sialin-knockout slices. The l-aspartate
intercenter distances were compared to those produced by the l-glutamate antibodies. The quantifications showed that most l-aspartate and l-glutamate
L-ASPARTATE
TRANSMISSION IS INDEPENDENT OF SIALIN
gold particles were situated within a distance of 30 nm
(which is similar to the immunogold lateral resolution,
i.e., the distance that separates a gold particle and an
epitope; ref. 24) from the vesicle center and that this
relation was similar in hippocampus from wild-type and
sialin-knockout mice (Fig. 4). In wild-type hippocampus, 85 and 60% of l-aspartate and l-glutamate gold
particles were closer to the nearest synaptic vesicle than
30 nm, while in sialin-knockout slices, these values were
80 and 77% (Fig. 4). Thus, both in wild-type and
sialin-knockout hippocampus, the large majority of
l-aspartate and l-glutamate gold particles represent
synaptic vesicle epitopes.
L-Aspartate
release from nerve terminals is Ca2ⴙ
dependent
As we could not find any evidence of sialin-dependent
release of l-aspartate from nerve terminals, we wanted
to clarify the Ca2⫹ dependency of l-aspartate depletion
from hippocampal nerve terminals (21, 22). During
ischemic conditions, the release of excitatory amino
acids can occur through reversal of EAATs (36 – 41). As
this release can be blocked by inhibiting the EAATs
(36 –38, 40, 42– 44), we incubated the slices in the
presence of the nontransportable EAAT blocker TFBTBOA (45) to determine whether EAATs contribute to
the release of l-aspartate from nerve terminals during
depolarization. As shown previously (23), addition of
TBOA per se at high K⫹ did not significantly reduce the
depletion of gold particles signaling l-aspartate from
excitatory nerve terminals (Fig. 5A–D), suggesting that
l-aspartate is not released via EAATs during membrane
depolarization.
To clarify whether omission of extracellular Ca2⫹
could inhibit l-aspartate release when the EAATs were
blocked, we exposed the slices to low Ca2⫹/high Mg2⫹
during depolarization in the presence of TFB-TBOA. At
3 mM K⫹, there was strong l-aspartate labeling in
excitatory nerve terminals, which was robustly reduced
on depolarization with 55 mM K⫹ (Fig. 6A, B). When
external Ca2⫹ was replaced by Mg2⫹ at 55 mM K⫹, the
depletion of l-aspartate immunogold particles from the
5
Figure 3. Depletion of l-aspartate from nerve endings is independent of sialin. Electron micrographs from wild-type (A, B,
E, F) and sialin-knockout (C, D,
G, H) slices incubated at 3 mM
K⫹ (A, C, E, G) and 55 mM K⫹
(B, D, F, H). A–D) Labeling for
l-aspartate (small gold particles) and l-glutamate (large
gold particles) is shown in excitatory terminals (te) making
asymmetric synapses with dendritic spines (s) in CA1 stratum
radiatum. E–H) Labeling for laspartate (small gold particles)/GABA (large gold particles) is shown in inhibitory
terminals (ti) making symmetric synapses with pyramidal cell
bodies (P). I–K) Bar charts
show the density (average⫾
sem; gold particles/␮m2) of immunogold particles for l-aspartate (I) and l-glutamate (J) in
excitatory terminals and for laspartate in inhibitory terminals (K) in wild-type and sialinknockout slices incubated at 3
mM K⫹ (basal) and 55 K⫹ (depol). Asterisk indicate that the
values at 3 mM K⫹ are significantly different from the values
at 55 mM K⫹ (P⬍0.05, Student’s 2-tailed t test; SPSS).
Quantifications were done in 3
slices from wild-type basal slices
(total of 84 excitatory and 71
inhibitory terminals), 3 wildtype depol slices (total of 88
excitatory and 70 inhibitory terminals), 3 knockout basal slices (total of 77 excitatory and 67 inhibitory terminals), and 3
knockout depol slices (total of 79 excitatory and 63 inhibitory terminals).
terminals was blocked (Fig. 6A). Further highlighting the
calcium dependency of l-aspartate release from nerve
terminals, we found that KB-R7943, a blocker of calcium
entrance through, e.g., NCX(rev) (46), NMDA receptors,
(47), and TRPC-Ca2⫹ channels (48, 49), weakly inhibited
depletion of both l-aspartate (Fig. 6B) and l-glutamate
(Fig. 6C) from nerve terminals (by 21 and 41%, respectively). Taken together, these data show that l-aspartate is
Figure 4. l-Aspartate and l-glutamate are present in synaptic vesicles lacking sialin. Histograms show the frequency distribution of the
distances separating l-aspartate and l-glutamate gold particles and synaptic vesicles in
excitatory terminals in wild-type (WT) and sialin-knockout (KO) slices. Distances are put into
bins of 5 nm (bars, x axis), and the number of
gold particles in each bin is given along the y
axis. Because of the lateral resolution of the
immunogold method (⬃30 nm), gold particles
could be separated by a distance of up to ⬃30
nm and still signal epitopes in the vesicle.
Distributions of l-aspartate and l-glutamate
intercenter distances in WT slices were not
significantly different from the distributions in
sialin-KO slices (P⬎0.05, ␹2 test). These quantifications were done in 60 nerve terminals
positive for aspartate/glutamate in both WT
and sialin KO CA1 radiatum.
6
Vol. 27
March 2013
The FASEB Journal 䡠 www.fasebj.org
MORLAND ET AL.
Figure 5. l-Aspartate is not depleted from nerve terminals
through excitatory amino acid
transporters. A–C) Electron micrographs show gold particles
representing l-aspartate (small
gold particles) and l-glutamate
(large gold particles) in excitatory nerve terminals (t) making asymmetric synapses with
dendritic spines (s) in the CA1
stratum radiatum. Slices were incubated at 3 mM K⫹ (A), 55 mM K⫹ (B), or 55 mM K⫹ with TFB-TBOA (C). D) Quantifications of gold particles representing
l-aspartate in excitatory nerve terminals in slices incubated at 3 mM K⫹ (basal), at 55 mM K⫹ (depol), and at 55 mM K⫹ with
TFB-TBOA (depol TBOA). Bar charts indicate particle density (average⫾sem; gold particles/␮m2). Quantification was
performed in 4 basal slices (total of 53 terminals), 4 depol slices (total of 123 terminals), and 6 depol TBOA slices (total of 74
terminals). *P ⬍ 0.05; 1-way ANOVA, Dunnett post hoc test.
released in a strictly Ca2⫹-dependent manner even when
the EAATs were blocked, supporting the idea of exocytotic release of l-aspartate.
L-Aspartate
release from nerve terminals is independent of
volume-regulated anion channels (VRACs)
As VRACs are proposed to release excitatory amino
acids (50, 51), and the activity of these channels is
thought to be Ca2⫹ dependent (52), we investigated
whether VRACs could account for the release of l-aspartate from hippocampal nerve terminals. Hippocampal slices were exposed to physiological and depolarizing concentrations of K⫹ in the presence or absence of
the VRAC inhibitor NPPB. All incubations were done in
the presence of TFB-TBOA. NPPB did not affect the
density of l-aspartate immunogold particles in nerve
terminals at either basal or depolarizing conditions
(Fig. 7A–D, I). The same was true for l-glutamate
immunogold particles (Fig. 7E–H, J).
DISCUSSION
Here we show that l-aspartate is transported into
synaptic vesicles in a sialin-independent manner. The
highest vesicular uptake of l-aspartate (relative to the
uptake of l-glutamate) was found in hippocampal
vesicles, which is consistent with slice data from the
present and previous studies (21–23) showing that
l-aspartate is released by exocytosis from hippocampal
nerve terminals. There was no difference in the ATPdependent uptake of l-aspartate in vesicles from sialinknockout compared to wild-type vesicles, and synapticlike microvesicles from PC12 cells transfected with
sialin were not capable of l-aspartate uptake. Consistent with this, our results from sialin-knockout mice
show that the vesicular l-aspartate release in the hippocampus is independent of sialin, indicating that
l-aspartate is packed into vesicles via a hitherto unknown transporter.
Our l-aspartate uptake data are in agreement with
the results of Naito and Ueda (12), who reported
l-aspartate uptake to be 7.5% of the vesicular l-glutamate uptake, but at odds with the studies of Fykse et al.
(11) and Maycox et al. (13), who did not find evidence
for uptake of l-aspartate in synaptic vesicles. The
reason for this discrepancy is not clear, but in order to
obtain near-saturation of the vesicles with transmitter
(12), we used 5 min incubation time, while the other
studies used 1.5 and 2 min. In addition, Maycox et al.
(13) did not measure vesicular l-aspartate uptake di-
Figure 6. Depletion of l-aspartate from nerve endings is Ca2⫹
dependent also when excitatory amino acid transporters
are blocked. Quantification of
gold particles representing l-aspartate (A, B) and l-glutamate
(C) in excitatory nerve terminals in CA1 stratum radiatum in
slices incubated at 3 mMK⫹
(basal) and at 55 mM K⫹ (depol), and under depolarizing
conditions where exocytosis was
inhibited by low Ca2⫹/high
Mg2⫹ (depol low Ca2⫹) or by
KB-R7943 (depol KB-R7943).
All slices were incubated in the
presence of TFB-TBOA. Bar charts indicate particle density (average⫾sem; gold particles/␮m2). Quantification in A was
performed in 3 basal slices (total of 59 terminals), 3 depol slices (total of 65 terminals), and 4 depol low Ca2⫹ slices (total of
176 terminals). Quantification in B and C was performed in 3 basal slices (total of 56 and 91 terminals, respectively), 3 and 5
depol slices (total of 62 and 149 terminals) and 4 depol KB-R7943 slices (total of 101 and 133 terminals). *P ⬍ 0.05, Student’s
2-tailed t test (SPSS).
L-ASPARTATE
TRANSMISSION IS INDEPENDENT OF SIALIN
7
Figure 7. l-Aspartate is not depleted from nerve terminals
through volume-regulated anion channels. A–H) Electron
micrographs show gold particles representing l-aspartate
(A–D) or l-glutamate (E–H) in
excitatory nerve terminals (t)
making asymmetric synapses with
dendritic spines (s) in the CA1
stratum radiatum. A, E) Terminals
incubated at 3 mM K⫹. C, G)
Terminals incubated at 3 mM
K⫹ with NPPB. B, F) Terminals
incubated at 55 mM K⫹. D, H)
Terminals incubated at 55 mM
K⫹ with NPPB. I, J) Quantification of gold particles representing l-aspartate (I) and lglutamate (J) in excitatory
nerve terminals in slices incubated at 3 mM K⫹ (basal), at 3
mM K⫹ with NPPB (basal
NPPB) at 55 mM K⫹ (depol),
and at 55 mM K⫹ with NPPB
(depol NPPB). Bar charts indicate particle density (average⫾
sem; gold particles/␮m2). Quantifications in I and J were performed in 3 and 4 basal slices
(total of 68 and 138 terminals,
respectively), 3 basal NPPB
slices (total of 132 and 91 terminals), 3– 4 depol slices (total of
62 and 97 terminals), and 3– 4
depol NPPB slices (total of 110
and 158 terminals). *P ⬍ 0.05;
Student’s 2-tailed t test (SPSS).
rectly, but used acidification of the vesicles as an
indirect measure of uptake. Relative to l-glutamate, the
highest l-aspartate uptake was found in the hippocampus. Because vesicular l-glutamate uptake is similar in
the hippocampus and the whole brain (53), this means
that the l-aspartate uptake is about twice as high in
hippocampal as in whole-brain vesicles. The fact that
the uptake ratio of l-aspartate to l-glutamate varied
between forebrain regions suggests that l-aspartate and
l-glutamate are taken up into different pools of vesicles, or into partially overlapping vesicle pools, in which
some vesicles take up both l-aspartate and l-glutamate,
while others take up only l-aspartate or l-glutamate.
Supporting incongruent vesicle pools for l-aspartate
and l-glutamate is our finding that l-glutamate does
not inhibit vesicular l-aspartate uptake, and previous
findings showing that l-aspartate does not inhibit l-glutamate uptake (11, 54). Also suggesting that l-aspartate
is released from a different vesicular pool than l-glutamate is the fact that the vesicular release of l-aspartate
can be regulated differently from that of l-glutamate
(7, 8). For instance, NMDA enhances l-aspartate, but
not l-glutamate, release from hippocampal slices (55),
and reduced levels of 5-lipoxygenase metabolites selectively reduced the release of l-aspartate (56). Along the
8
Vol. 27
March 2013
same line, we recently showed that valproate reduces
the nerve terminal content of aspartate, but not that of
glutamate (35).
The lysosomal H⫹-coupled sialic acid transporter,
sialin, was recently suggested to transport l-aspartate
and l-glutamate into hippocampal synaptic vesicles
(15). Here we show that synaptic vesicles from sialinknockout and wild-type mice do not differ in their
ability to take up l-aspartate. In line with the vesicular
uptake data are our immunogold results indicating that
l-aspartate is present in synaptic vesicles from both
wild-type and sialin-knockout brains (Fig. 5). Further
supporting the idea that vesicular uptake of l-aspartate
is independent of sialin is that when we overexpresses
sialin in the vesicles of PC12 cells, they were still unable
to accumulate l-aspartate. Finally, l-aspartate release
from hippocampal nerve terminals occurs in the absence of sialin. As sialin transports l-aspartate and
l-glutamate with similar affinities (15), and the intracellular concentration of l-glutamate is about 5 times
the l-aspartate concentration (57), sialin should contribute to at least a part of the total vesicular uptake of
l-glutamate. Thus, at least a part of the vesicular
l-glutamate uptake should be inhibited by adding
external l-aspartate to isolated vesicles, which, how-
The FASEB Journal 䡠 www.fasebj.org
MORLAND ET AL.
ever, was not found to be the case (9 –11, 54). Taken
together, our findings strongly suggest that sialin is
neither sufficient for vesicular storage nor necessary for
release of l-aspartate in intact brain preparations. This
indicates that either sialin is present in insufficient
amounts in the vesicular membrane, or sialin does not
transport l-aspartate into synaptic vesicles under physiological conditions.
In Miyaji et al. (15), the researchers report to have
detected sialin and measured vesicular l-aspartate uptake in the hippocampal P2 fraction. According to
standard nomenclature, the P2 fraction refers to a
crude synaptosomal fraction (see, e.g., refs. 18, 58, 59),
which is likely to contain nonvesicular membranes, and
presumably also lysosomal membranes. Thus, the localization of sialin in synaptic vesicles could be questioned. The notion that sialin is not an important
constituent of synaptic vesicles, or present in low
amounts, is supported by a proteomic study that did not
detect sialin among the synaptic vesicle proteins (60).
In the course of this study we set out to localize sialin
at the subcellular level using immunogold cytochemistry. However, during antibody testing, the sialin antibodies (commercially available from Alpha Diagnostics
and from R.R.) produced several bands on Western
blots of brain tissue (unpublished results), hampering
the use of these antibodies in immunolocalization
studies. Two previous studies have reported sialin immunolocalization in the brain (61, 62). However, none
of these give ultrastructural data, and thus whether
sialin is present in synaptic vesicles could not be inferred from these studies.
VRACs could form an alternative release mechanism
for l-aspartate and l-glutamate, as these channels have
been shown to be responsible for release of excitatory
amino acids from cultured astrocytes and from pathological brain tissue (44, 50, 51, 63, 64). Because the
opening of these channels is reported to be Ca2⫹
sensitive (52) it was essential to determine whether
l-aspartate could escape from nerve terminals through
VRACs. Using the VRAC blocker NPPB, we found no
evidence for reduced l-aspartate or l-glutamate release
from nerve terminals during membrane depolarization. Thus, VRACs do not appear to contribute to the
nerve terminal release of excitatory amino acids. This is
a novel finding, as previous work on VRAC-mediated
release of excitatory amino acids from intact brain
tissue has not distinguished between astrocytic and
neuronal release. Similarly, as EAATs are present on
hippocampal nerve terminals (25, 65) and reported to
mediate l-aspartate and l-glutamate release in pathological states (41, 43, 44, 66), we investigated whether
l-aspartate could be released from nerve terminals via
these transporters. In this respect, it should be noted
that blocking the EAATs effectively inhibits reversed
transport of excitatory amino acids (23, 34, 36 –38, 40,
41, 67) showing that TBOA blocks both the forward
and reverse aspartate/glutamate transport across terminal membranes. We found that the EAAT blocker
TBOA did not inhibit the release of l-aspartate (or
l-glutamate). This is in line with data showing that,
under conditions of partial ischemia (51), as well as
under physiological conditions (23, 68, 69), the EAATs
L-ASPARTATE
TRANSMISSION IS INDEPENDENT OF SIALIN
are not responsible for any significant release of l-aspartate and l-glutamate. Hence, our results on inhibition of EAATs and VRAC strongly suggest that nonvesicular mechanisms do not play a role in the nerve
terminal release of l-aspartate.
These results show that nerve terminals have an
l-aspartate-containing pool of synaptic vesicles, which
undergo exocytosis during neuronal stimulation. The
sensitivity of l-aspartate release to Ca2⫹ (present study
and refs. 8, 18, 21, 22, 55, 70 –77) and tetanus toxin (22,
69, 78, 79) reported in previous studies further support
a vesicular release mechanism. Low micromolar concentrations of KB-R7943 have previously been shown to
selectively inhibit aspartate release from synaptosomes
(74) and organotypic cultures (7). In our study, KBR7943, used at a concentration (50 ␮M) that probably
inhibits several receptors and channels that mediate
Ca2⫹ influx (47– 49, 80), reduced the nerve terminal
depletion of both l-aspartate and l-glutamate, highlighting the calcium-dependency of l-aspartate/l-glutamate release. The higher concentration of KB-R7943
might explain the discrepancy between our results and
those of Nadler and coworkers.
We conclude that l-aspartate is taken up into synaptic vesicles by a yet unidentified vesicular transporter
and is subjected to regulated exocytotic release.
The authors are grateful to Inger-Lise Bogen for advice on
the vesicular uptake assay and to Anna Torbjørg Bore for
technical support. The project was supported by grant 06/
5289 from the Faculty of Medicine, University of Oslo, to C.M.
and K.N., and awards NS050417 and NS045634 to R.J.R. and
NS065664 to L.M.P. from the U.S. National Institute of
Neurological Disorders and Stroke. The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological
Disorders and Stroke or the U.S. National Institutes of
Health. L.M.P. is in the Medical Scientist Training Program at
Stanford University School of Medicine.
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Received for publication February 15, 2012.
Accepted for publication November 26, 2012.
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