The FASEB Journal • Research Communication

Vesicular uptake and exocytosis of L-aspartate isindependent of sialin

Cecilie Morland,*,† Kaja Nordengen,*,† Max Larsson,*,†,‡ Laura M. Prolo,§,�

Zoya Farzampour,§,� Richard J. Reimer,§,� 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; §Departmentof Neurology and Neurological Sciences and �Graduate Program in Neuroscience, StanfordUniversity School of Medicine, Stanford, California, USA; and ¶Department of Neurology, OsloUniversity Hospital, Rikshospitalet Oslo, Oslo, Norway

ABSTRACT The mechanism of release and the roleof L-aspartate as a central neurotransmitter are contro-versial. A vesicular release mechanism for L-aspartatehas been difficult to prove, as no vesicular L-aspartatetransporter was identified until it was found that sialincould transport L-aspartate and L-glutamate when recon-stituted into liposomes. We sought to clarify the releasemechanism of L-aspartate and the role of sialin in thisprocess by combining L-aspartate uptake studies inisolated synaptic vesicles with immunocyotchemical in-vestigations of hippocampal slices. We found that ra-diolabeled L-aspartate was taken up into synaptic vesi-cles. The vesicular L-aspartate uptake, relative to theL-glutamate uptake, was twice as high in the hippocam-pus as in the whole brain, the striatum, and the ento-rhinal and frontal cortices and was not inhibited byL-glutamate. We further show that sialin is not essentialfor exocytosis of L-aspartate, as there was no differencein ATP-dependent L-aspartate uptake in synaptic vesi-cles from sialin-knockout and wild-type mice. In addi-tion, expression of sialin in PC12 cells did not result insignificant vesicle uptake of L-aspartate, and depolar-ization-induced depletion of L-aspartate from hip-pocampal nerve terminals was similar in hippocampalslices from sialin-knockout and wild-type mice. Further,there was no evidence for nonvesicular release ofL-aspartate via volume-regulated anion channels orplasma membrane excitatory amino acid transporters.This suggests that L-aspartate is exocytotically releasedfrom nerve terminals after vesicular accumulation by atransporter other than sialin.—Morland, C., Norden-gen, K., Larsson, M., Prolo, L. M., Farzampour, Z.,Reimer, R. J., Gundersen, V. Vesicular uptake andexocytosis of L-aspartate is independent of sialin.FASEB J. 27, 000–000 (2013). www.fasebj.org

Key Words: synaptic transmission � VRAC � excitatory aminoacid transporters

Besides serving a role as a precursor of Krebs’ cycleintermediates and as a building block of proteins,l-aspartate has been proposed to act as a transmitter inthe brain. This was first suggested in the hippocampus(1), and later in the cerebellum (2). In favor ofaspartergic neurotransmission is the evidence for exo-cytotic release of l-aspartate from nerve terminals(3–5) and that l-aspartate stimulates the N-methyld-aspartate (NMDA) type of l-glutamate receptors (6),producing NMDA-receptor-dependent postsynaptic re-sponses (7, 8). A major argument against aspartergicneurotransmission is the conflicting evidence on up-take of l-aspartate into synaptic vesicles. While thereseems to be consensus that l-aspartate does not inhibitthe vesicular uptake of l-glutamate (9–11), only a fewstudies have directly measured the uptake of l-aspar-tate into synaptic vesicles. Using isolated cortical synap-tic vesicles, Naito and Ueda (12) found a significantl-aspartate uptake. Other studies show negligible vesic-ular accumulation of l-aspartate (11, 13).

Recently sialin, which is known to transport sialicacid out of lysosomes (14), was reported to also carryl-aspartate (and l-glutamate) into reconstituted pro-teoliposomes (15). However, whether sialin has a rolein the release of l-aspartate from nerve terminals inintact brain tissue has not been tested. To clarify theseambiguities in aspartergic neurotransmission, we hereused brain tissue from the hippocampus, the brainregion showing the most robust evidence for synapticrelease of l-aspartate (3–5). We first measured theATP-dependent uptake of l-aspartate in synaptic vesi-cles isolated from the hippocampus and compared it tothe uptake in vesicles isolated from the whole brain and3 other forebrain regions. We then used sialin-knock-out mice to analyze the sialin-dependent l-aspartatevesicle uptake. As a second control for sialin depen-

1 Correspondence: Department of Anatomy and theCMBN, University of Oslo, POB 1105 Blindern, 0317 Oslo,Norway. E-mail: vidar.gundersen@medisin.uio.no

doi: 10.1096/fj.12-206300

Abbreviations: EAAT, excitatory amino acid transporter;mCherry, monomeric Cherry; NCX(rev), reversed Na�-Ca2� ex-changer; NMDA, N-methyl d-aspartate; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; RFP, red fluorescent protein;TFB-TBOA, (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phe-nyl]methoxy]-l-aspartic acid; TRPC, transient receptor potentialcation; VGLUT1, vesicular glutamate transporter 1; VRAC, volume-regulated anion channel

10892-6638/13/0027-0001 © FASEB

The FASEB Journal article fj.12-206300. Published online December 6, 2012.

dency, we measured l-aspartate uptake in vesicles iso-lated from native PC12 cells and PC12 cells overexpress-ing sialin. By immunogold cytochemistry of hippocampalslices from sialin-knockout and wild-type mice, we thendetermined whether sialin was necessary for release ofl-aspartate in intact brain tissue. Our results show thatl-aspartate is exocytotically released from nerve termi-nals after vesicular accumulation by a sialin-indepen-dent mechanism.

MATERIALS AND METHODS

Animals

All animals were treated in strict accordance with the guide-lines of the Norwegian Committees on Animal Experimenta-tion (Norwegian Animal Welfare Act and European Commu-nities Council Directive of 24 November 1986-86/609/EEC)and the Stanford Institutional Animal Care and Use Commit-tee. Male Wistar rats were from Scanbur (Sollentuna, Swe-den). Male heterozygous sialin-knockout mice (B6; 129S5–Slc17a5tm1Lex) were obtained from the Mutant MouseRegional Resource Centre (http://www.mmrrc.org) andbred to homozygosity (16). Mouse genotypes were deter-mined by PCR (16).

Preparation of synaptic vesicles

Synaptic vesicles from rat whole brain, hippocampus, stria-tum, entorhinal cortex, and frontal cortex, as well as from thewhole brain of sialin-knockout and wild-type mice, wereisolated as described by Erickson et al. (17). For isolation ofsynaptic vesicles from the forebrain subregions, the dissectedtissue was kept on ice until all brain regions from 10 Wistarrats (�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 superna-tants were collected and centrifuged for 30 min at 21,000 gand 4°C. The resulting synaptosomal pellets (P2 fractions),were osmotically shocked by resuspension in ice-cold purifiedwater (7.5%, w/v) and centrifuged for 30 min at 21.000 g toremove the crude synaptosomal membranes. Osmolarity wasrestored with 0.1 M potassium tartrate and 25 mM Hepes (pH7.4), before the mixtures were centrifuged for 1 h at 100,000g and 4°C. The pellets (LP2 fractions), containing crudesynaptic vesicles, were gently resuspended in 0.32 M sucrose,frozen in liquid N2, and stored at �80°C or used for uptakeassay immediately.

Vesicular uptake of L-aspartate and L-glutamate

Vesicular uptake of l-glutamate and l-aspartate was deter-mined as described by Fykse et al. (18) with slight modifica-tions. Synaptic vesicles from �35 mg brain tissue of the wholebrain, the hippocampus, the striatum, the frontal cortex, andthe entorhinal cortex were preincubated at 30°C for 15 minin an incubation mixture containing 10 mM K-HEPES (pH7.4), 4 mM KCl, 4 mM MgSO4, 0.25 M sucrose, and 1 mMreduced glutathione. For experiments with Rose Bengal,preincubation time was 25 min in the presence or absence ofRose Bengal, 5 �M. The uptake was started when a substratesolution containing (final concentrations) 2 mM ATP and 1mM 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

vesicular uptake of l-aspartate could be blocked by l-gluta-mate, in some experiments the vesicles were exposed tol-glutamate (1 mM) both during preincubation and incuba-tion. In these experiments, ATP was included also in thepreincubation step, to allow complete filling of the vesicleswith l-glutamate prior to incubation with [3H]l-aspartate.The uptake of [3H]l-glutamate and [3H]l-aspartate wasstopped after 5 min by addition of 7 ml of ice-cold 0.15 M KClto the 300 �l samples. Blanks were treated as described above,except that ATP was omitted from the incubation buffer. As anegative control, some samples were incubated in ice-coldbuffer 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 forretained radioactivity in a Packard Tri-Carb 300 (PackardInstrument Co., Meriden, CT, USA). The radioactivity in theblanks (incubated at 30°C without ATP) was 20% higher thanthe values in samples incubated on ice without ATP. ATP-dependent uptake was defined as the radioactivity in thesamples (incubated at 30°C with ATP) minus the radioactivityin the blanks and presented as means � se, n � 3–5individual experiments for each observation. In each experi-ment, ATP-dependent uptake was measured in triplicates.Due to small volumes of the subregional vesicular fractions,the protein concentrations of these were not measured, andthus most of the data are given as percentage of the l-gluta-mate uptake in the same region. However, in one experi-ment, the protein content in the samples was analyzed (BCassay; Interchem, Montfuçon, France) and the absolute up-take values (pmol/min/mg protein) were calculated. Whether theuptake values in the hippocampus were significantly differentfrom the values in the whole brain and the other subregionswas tested by 1-way ANOVA post hoc test for multiple compar-isons (SPSS, Chicago, IL, USA). Uptake in sialin-knockoutand wild-type vesicles was statistically evaluated by Student’s ttest, 2 tails (SPSS).

PC12 cell vesicle preparation

The rat vesicular glutamate transporter 1 (VGLUT1) expres-sion plasmid was derived as described previously (19). The ratsialin cDNA (20) was subcloned into the plasmid expressionvector pcDNA3-Amp (Invitrogen, Carlsbad, CA, USA). Subse-quently, a cDNA encoding the red fluorescent protein (RFP)monomeric Cherry (mCherry) was subcloned at the 5= end ofthe sialin sequence to generate a chimeric protein. Theplasmids were introduced into PC12 cells using Lipo-fectamine 2000 (Invitrogen). The cells were then selected in800 mg/ml G418 (effective concentration; Life Technologies,Gaithersburg, MD, USA), and the resulting clones werescreened by direct fluorescence microscopy for sialin-express-ing cells and immunofluorescence microscopy for VGLUT1-expressing cells to identify those with the highest level ofexpression. Cells were maintained in DMEM (Life Technol-ogies) supplemented with 10% equine serum (HyClone,South Logan, UT, USA), 5% calf serum (HyClone), andpenicillin-streptomycin (Life Technologies). Vesicle mem-branes were prepared from PC12 as follows: cells wereharvested by trituration washed once in PBS, and cells fromeach 15-cm plate were resuspended in 1 ml of 0.3 M sucroseand 10 mM HEPES-KOH, pH 7.4 (SH buffer), containing 0.2mM diisopropylfluorophosphate (DFP), 1 mg/ml pepstatin, 2mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml E64, and 1.25mM MgEGTA. The cells were then disrupted by homogeni-zation at 4°C through a ball-bearing device with a clearance of10 �m (Isobiotec, Heidelberg, Germany). The nuclear debriswas sedimented at 1000 g for 5 min, and heavier membraneswere eliminated by centrifugation at 20,000 g for 20 min (P2).

2 Vol. 27 March 2013 MORLAND ET AL.The FASEB Journal � www.fasebj.org

The remaining light membrane vesicles (crude synaptic-likemicrovesicles) were sedimented at 100,000 g for 1 h, and theresulting pellet (P3) was resuspended in SH buffer containingthe same protease inhibitors, at a final protein concentrationof 10 �g/�l.

Transport assay for PC12 vesicles

To initiate the reaction, a 10-�l aliquot of membranes wasadded to 190 �l SH buffer containing 4 mM MgCl2, 4 mMKCl, 4 mM ATP, 100 �M unlabeled substrate, and 2 �Ci3H-substrate (American Radiolabeled Chemicals, St. Louis,MO, USA). Incubation was performed at 29°C for 5 min, andthe reaction was terminated by rapid filtration (DAWP, 0.65�m; Millipore, Billerica, MA, USA), followed by immediatewashing with 6 ml cold SH buffer. Background uptake wasdetermined by parallel assays done in the presence of nigeri-cin (5 �M) and valinomycin (20 �M) to dissipate the protonelectrochemical gradient. The trapped radioactivity was mea-sured by scintillation counting in 2.5 ml Cytoscint (ICNBiomedicals, Inc., Aurora, OH, USA). Transport measure-ments were repeated �3 times using �2 different membranepreparations.

For Western blotting, P2 and P3 fractions from homoge-nized PC12 cells (30 �g of protein/lane) were separated bySDS-PAGE and subsequently electrotransferred to polyvi-nylidene fluoride membranes. Blots were blocked with Blotto(PBS with 5% nonfat dry milk and 0.1% Tween-20) for 1 hand then incubated with an antibody directed against sialin(Alpha Diagnostics, Owings Mills, MD, USA) at a 1:500dilution or against RFP at 1:5000 overnight at 4°C. Blots werewashed, incubated with anti-rabbit horseradish peroxidase-coupled secondary antibody (1:10,000; Pierce, Rockford, IL,USA) in Blotto for 1 h. Proteins were visualized using an ECLWestern blotting detection system (Amersham Biosciences,Piscataway, NJ, USA) and exposure of the blot to autoradiog-raphy film (MidSci, St. Louis, MO, USA).

Preparation and incubation of hippocampal slices

Hippocampal slice experiments were performed as describedpreviously (21–23). Wistar rats were anesthetized with ha-lotane or isoflurane and decapitated. The brains were re-moved and the hippocampi dissected out on ice and choppedinto 300 �m slices. The slices were preincubated in oxygen-ated Krebs’ solution (126 mM NaCl; 3 mM KCl; 10 mMsodium phosphate buffer, pH 7.4; 1.2 mM CaCl2; 1.2 mMMgSO4; and 10 mM glucose) for 45 min at 30°C. Then theslices were incubated for 45 min at 30°C in Krebs’ solutioncontaining physiological (3 mM) or depolarizing (55 mM,Na� reduced to 80 mM) concentrations of K�. To ensurethat l-aspartate was not released through excitatory aminoacid transporters (EAATs), all incubations were done inthe presence of the EAAT inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-asparticacid (TFB-TBOA; 5 �M; Tocris Bioscience, Ellisville, MO,USA), except in one experiment where we incubated theslices without TFB-TBOA at basal conditions (3 mM K�) andwith and without TFB-TBOA at depolarizing conditions (55mM K�). Some slices were exposed to 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 100 �M; Sigma, StLouis, MO, USA), which inhibits volume-regulated anionchannels, 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 thelast 10 min of preincubation and for the whole incubationperiod. Some slices were incubated at 55 mM K� with 0.1 mMCa2 � (Mg2� increased to 10 mM).

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 frontalslices were vibrosliced into oxygenated carbonate-bufferedKrebs’ solution (126 mM NaCl, 26 mM NaHCO3, 3 mM KCl,1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, and 10 mMglucose). Then the slices were incubated for 1 h at 32°C in thesame buffer at 3 or 55 mM K�, without TFB-TBOA.

After incubation, the slices were fixed in 2.5% glutaralde-hyde and 1% formaldehyde for 1 h at room temperature(�20°C). The slices were stored in fixative diluted 1:10 at 4°Cuntil embedding and sectioning.

Electron microscopic immunocytochemistry

The slices were embedded in Lowicryl HM20 (mouse slices;EMS, Hatfield, PA, USA) or Durcupan (rat slices; EMS) asdescribed previously (24). After the embedding, ultrathinsections (80–100 nm) were cut and labeled with the 435l-aspartate (diluted 1:300), 607 l-glutamate (diluted 1:40,000for single labeling and 1:5,000 for double labeling), and 990GABA (diluted 1:300) antisera. The 435 l-aspartate, 607l-glutamate, and 990 GABA antisera have been characterized(22, 23, 25–27). To avoid possible cross-reactivities, the l-glu-tamate, l-aspartate, and GABA antisera were used with theaddition of 0.2 mM complexes of glutaraldehyde/formalde-hyde-treated l-aspartate, asparagine plus glutamine and as-paragine, l-glutamate plus GABA, and l-glutamate plus -al-anine, respectively. As a specificity test, ultrathin test sectionscontaining conjugates of amino acids fixed to brain proteinsby formaldehyde and glutaraldehyde (28) were run alongwith the immunogold experiments. This test system showedthat the primary antibodies labeled only the conjugate con-taining the amino acid against which the antibodies wereraised (data not shown).

To visualize aspartate/glutamate and aspartate/GABA inthe same nerve terminals, we performed double-labelingexperiments, in which the ultrathin sections were first treatedwith the l-aspartate antibodies and then with the l-glutamateor 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- and15-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 micro-scope (FEI, Hillsboro, OR, USA). Electron micrographs wererandomly taken from the CA1 stratum radiatum, granule, orpyramidal cell layer. Excitatory terminals were defined asthose making asymmetric synapses with dendritic spines.Inhibitory terminals were defined as those making symmetricsynapses with stem dendrites or granule cell bodies andcontaining GABA immunogold particles. The densities (goldparticles/�m2) of l-aspartate and l-glutamate gold particlesin excitatory terminals and of l-aspartate gold particles ininhibitory nerve terminals, as well as the spatial relationbetween gold particles signaling l-aspartate and l-glutamateand synaptic vesicles in excitatory terminals in sialin-knockoutand 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 tooutline the plasma membrane of nerve terminals, and torecord the distance between the center of a gold particle orpoints randomly placed over the excitatory terminals (thenumber of random points were approximately the same asthe number of gold particles in the terminals) and the centerof the closest synaptic vesicle. Recorded coordinates weresubmitted to a program written in Python (http://www.python.org) for computation of particle density (gold parti-

3L-ASPARTATE TRANSMISSION IS INDEPENDENT OF SIALIN

cles/�m2; ref. 31). The source code of the ImageJ plug-in andthe Python program are available online (http://www.neuro.ki.se/broman/maxl/software.html). The gold particle-synapticvesicle distances were sorted into bins of 5 nm, and thefrequencies of the intercenter distances for each bin werecalculated (Excel; Microsoft, Redmond, WA, USA). Thequantitative results were statistically evaluated by 2 test, 1-wayANOVA post hoc test for multiple comparisons (SPSS), andStudent’s t test, 2 tails (SPSS).

RESULTS

L-Aspartate is taken up into synaptic vesicles

We compared the ATP-dependent uptake of l-aspar-tate in isolated synaptic vesicles from the hippocampuswith the uptake in vesicles from the whole brain anddifferent other brain regions. The ATP-dependent vesic-ular l-aspartate uptake was assessed relative to the ATP-dependent vesicular l-glutamate uptake in the samevesicle fraction. In hippocampal vesicles, the ATP-depen-dent l-aspartate uptake was 18.1% of the l-glutamateuptake (Fig. 1A). In comparison, vesicles isolated from thewhole brain showed an ATP-dependent l-aspartate up-take of 9.1% of the l-glutamate uptake. In the entorhinalcortex, the frontal cortex, and the striatum, ATP-dependent l-aspartate uptake was 9.6, 10.1, and 11.9%(relative to the ATP-dependent l-glutamate uptake),

respectively (Fig. 1A). The vesicular uptake of l-aspar-tate was not inhibited by l-glutamate, as addition of 1mM l-glutamate during the preincubation and incuba-tion steps did not alter the uptake of radiolabeledl-aspartate in hippocampal vesicles (Fig. 1B). Thel-aspartate and l-glutamate uptake values reportedwere obtained from frozen vesicles; however, to validatethe vitality of these vesicles, a control experiment (n �6–9 replicates from 2–3 experiments) was performedwhere we compared l-aspartate and l-glutamate up-take into frozen and fresh hippocampal vesicles. Theuptake into frozen vesicles (59.2�1.8 pmol/min/mgprotein for l-glutamate and 7.8�1.3 pmol/min/mg pro-tein for l-aspartate) were only slightly lower that theuptake 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-Gluta-mate uptake, but not l-aspartate uptake, could bealmost completely blocked by the vesicular glutamateuptake blocker Rose Bengal (5 �M, data not shown;for characterization of Rose Bengal; see ref. 32),supporting the notion that l-aspartate is accumu-lated into synaptic vesicles by another transporterthan the VGLUTs (33).

Vesicular accumulation of L-aspartate issialin-independent

Previous studies have demonstrated that sialin, whenreconstituted into proteoliposomes, can catalyze trans-membrane l-aspartate transport. This activity was com-petitively inhibited by l-glutamate. To determinewhether sialin is an important vesicular transporter forl-aspartate, we measured ATP-dependent accumula-tion of l-aspartate in synaptic vesicles from sialin-knockout and wild-type whole brain. We found that thelack of sialin did not reduce the ability of the vesicles toaccumulate l-aspartate (Fig. 2A) or l-glutamate (datanot shown). This was further confirmed in another testsystem, in which we overexpressed sialin (tagged withthe RFP mCherry) in PC12 cells and isolated synapticlike microvesicles (P3 fraction) from wild-type cells andcells overexpressing sialin. Western blotting with anti-bodies against sialin showed that wild-type cells con-tained endoegenous sialin in the crude synaptosomefraction (containing lysosomes), while sialin was notdetected in synaptic-like microvesicles (P3 fraction; Fig.2B). In PC12 cells overexpressing sialin, antibodiesagainst sialin, as well as against RFP, recognized recom-binant sialin in both the P2 and the P3 fraction (Fig.2B). There was essentially no l-aspartate uptake in P3vesicles from wild-type or sialin-overexpressing PC12cells (Fig. 2C), while vesicles from cells expressing thevesicular l-glutamate transporter VGLUT1 displayedrobust uptake of l-glutamate (Fig. 2C). Together thesefindings strongly suggest that sialin is not capable ofmediating l-aspartate transport in native vesicles.

Depletion of L-aspartate from hippocampal terminalsis independent of sialin

To investigate whether sialin is required for exocytoticrelease of l-aspartate in intact brain tissue, we exposed

Figure 1. l-Aspartate is taken up into synaptic vesicles.A) ATP-dependent uptake of l-aspartate relative to thel-glutamate uptake (average�sem, number of experimentsindicated at bottom of each bar) in isolated rat synapticvesicles from the whole brain (brain), hippocampus (HC),entorhinal cortex (EC), frontal cortex (FC), and striatum(Stri). In each experiment, the ATP-dependent uptake wasanalyzed in triplicate. Brackets indicate that the value in thehippocampus is significantly different from the values in thewhole brain, entorhinal cortex. and frontal cortex (P�0.05,1-way ANOVA post hoc; SPSS). B) Vesicular uptake of l-aspartatewithout (Asp) and with l-glutamate (Asp�Glu) (average�sem, number of experiments indicated at bottom of eachbar). The individual aspartate uptake values of each experi-ment without and with l-glutamate were normalized to theaverage aspartate uptake without l-glutamate (0.0113 pmol/min/mg tissue, n�3 experiments). sem was calculated basedon the normalized values for the individual experiments.There was no significant difference between uptake values inAsp and Asp�Glu (P�0.05, Student’s 2-tailed t test; SPSS).

4 Vol. 27 March 2013 MORLAND ET AL.The FASEB Journal � www.fasebj.org

hippocampal slices from sialin-knockout and wild-typemice 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 l-glutamate immunogold particles in excitatory terminalsin the CA1 stratum radiatum (Fig. 3A, C) and withGABA immunogold particles in inhibitory terminals inthe granule and pyramidal cell layers (Fig. 3E, G). Ondepolarization with 55 mM K�, gold particles signalingl-aspartate were depleted from both types of terminals(Fig. 3A vs. B; E vs. F). If sialin carries l-aspartate intosynaptic vesicles before release, we would expect thatthe absence of sialin would greatly reduce vesicularrelease of l-aspartate from nerve terminals. However,there was no difference in l-aspartate labeling ofexcitatory terminals between sialin knockouts (Fig. 3C,D) and wild types (Fig. 3A, B), either in the basalcondition (Fig. 3A, C) or after depolarization (Fig. 3B,D). Quantitative analysis showed that excitatory nerveterminals in wild-type and knockout slices displayeda similar reduction in the density of gold particlesrepresenting l-aspartate (73 and 83%, respectively) inresponse to depolarization (Fig. 3I). Correspondingly,sialin was not necessary for the depolarization-induceddepletion of l-glutamate from excitatory nerve termi-nals (Fig. 3A–D, J). Likewise, in inhibitory terminals,there was no difference in l-aspartate labeling betweensialin-knockout (Fig. 3G, H) and wild-type slices (Fig.2E, F), either in the basal condition (Fig. 3E, G) or afterdepolarization (Fig. 3F, H). Quantifications showedthat the depolarization-induced decrease in l-aspartatelabeling was similar in wild-type and knockout slices (73and 80%, respectively; Fig. 2K).

To confirm that l-aspartate is contained withinsynaptic vesicles in sialin-knockout hippocampus, wemeasured the distance between the center of goldparticles signaling l-aspartate and the center of theclosest synaptic vesicle in excitatory terminals inwild-type and sialin-knockout slices. The l-aspartateintercenter distances were compared to those pro-duced by the l-glutamate antibodies. The quantifica-tions showed that most l-aspartate and l-glutamate

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 anepitope; ref. 24) from the vesicle center and that thisrelation was similar in hippocampus from wild-type andsialin-knockout mice (Fig. 4). In wild-type hippocam-pus, 85 and 60% of l-aspartate and l-glutamate goldparticles were closer to the nearest synaptic vesicle than30 nm, while in sialin-knockout slices, these values were80 and 77% (Fig. 4). Thus, both in wild-type andsialin-knockout hippocampus, the large majority ofl-aspartate and l-glutamate gold particles representsynaptic vesicle epitopes.

L-Aspartate release from nerve terminals is Ca2�

dependent

As we could not find any evidence of sialin-dependentrelease of l-aspartate from nerve terminals, we wantedto clarify the Ca2� dependency of l-aspartate depletionfrom hippocampal nerve terminals (21, 22). Duringischemic conditions, the release of excitatory aminoacids can occur through reversal of EAATs (36–41). Asthis release can be blocked by inhibiting the EAATs(36–38, 40, 42–44), we incubated the slices in thepresence of the nontransportable EAAT blocker TFB-TBOA (45) to determine whether EAATs contribute tothe release of l-aspartate from nerve terminals duringdepolarization. As shown previously (23), addition ofTBOA per se at high K� did not significantly reduce thedepletion of gold particles signaling l-aspartate fromexcitatory nerve terminals (Fig. 5A–D), suggesting thatl-aspartate is not released via EAATs during membranedepolarization.

To clarify whether omission of extracellular Ca2�

could inhibit l-aspartate release when the EAATs wereblocked, we exposed the slices to low Ca2�/high Mg2�

during depolarization in the presence of TFB-TBOA. At3 mM K�, there was strong l-aspartate labeling inexcitatory nerve terminals, which was robustly reducedon depolarization with 55 mM K� (Fig. 6A, B). Whenexternal Ca2� was replaced by Mg2� at 55 mM K�, thedepletion of l-aspartate immunogold particles from the

Figure 2. Vesicular uptake of l-aspartate isindependent of sialin. A) Measurement of ATP-dependent vesicular accumulation of 3H-l-as-partate in whole-brain synaptic vesicles fromsialin-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 and13.8 � 5.2 pmol/min/mg protein in the KO(means�se, n�4 experiments, P�0.05, Stu-dent’s t test; SPSS). B) Western blots of vesiclesderived from WT (WT PC12) and PC12 cellsexpressing recombinant sialin (A3) with anantibody directed against sialin (left panel) andRFP (right panel) demonstrate marked overex-

pression of sialin in the stable cell line. The mCherry tag (RFP) on the recombinant protein causes the primary band in therecombinant 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-likemicrovesicles 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 cellscompared to WT. Brackets indicate that the glutamate uptake was significantly higher in B1 VGLUT1 than in WT (P�0.05,Student’s t test).

5L-ASPARTATE TRANSMISSION IS INDEPENDENT OF SIALIN

terminals was blocked (Fig. 6A). Further highlighting thecalcium dependency of l-aspartate release from nerveterminals, we found that KB-R7943, a blocker of calciumentrance through, e.g., NCX(rev) (46), NMDA receptors,

(47), and TRPC-Ca2� channels (48, 49), weakly inhibiteddepletion of both l-aspartate (Fig. 6B) and l-glutamate(Fig. 6C) from nerve terminals (by 21 and 41%, respec-tively). Taken together, these data show that l-aspartate is

Figure 3. Depletion of l-aspar-tate from nerve endings is inde-pendent of sialin. Electron mi-crographs from wild-type (A, B,E, F) and sialin-knockout (C, D,G, H) slices incubated at 3 mMK� (A, C, E, G) and 55 mM K�

(B, D, F, H). A–D) Labeling forl-aspartate (small gold parti-cles) and l-glutamate (largegold particles) is shown in excit-atory terminals (te) makingasymmetric synapses with den-dritic spines (s) in CA1 stratumradiatum. E–H) Labeling for l-aspartate (small gold parti-cles)/GABA (large gold parti-cles) is shown in inhibitoryterminals (ti) making symmet-ric synapses with pyramidal cellbodies (P). I–K) Bar chartsshow the density (average�sem; gold particles/�m2) of im-munogold particles for l-aspar-tate (I) and l-glutamate (J) inexcitatory terminals and for l-aspartate in inhibitory termi-nals (K) in wild-type and sialin-knockout slices incubated at 3mM K� (basal) and 55 K� (de-pol). Asterisk indicate that thevalues at 3 mM K� are signifi-cantly different from the valuesat 55 mM K� (P�0.05, Stu-dent’s 2-tailed t test; SPSS).Quantifications were done in 3slices from wild-type basal slices(total of 84 excitatory and 71inhibitory terminals), 3 wild-type depol slices (total of 88excitatory and 70 inhibitory terminals), 3 knockout basal slices (total of 77 excitatory and 67 inhibitory terminals), and 3knockout depol slices (total of 79 excitatory and 63 inhibitory terminals).

Figure 4. l-Aspartate and l-glutamate are pres-ent in synaptic vesicles lacking sialin. Histo-grams show the frequency distribution of thedistances separating l-aspartate and l-gluta-mate gold particles and synaptic vesicles inexcitatory terminals in wild-type (WT) and sia-lin-knockout (KO) slices. Distances are put intobins of 5 nm (bars, x axis), and the number ofgold particles in each bin is given along the yaxis. Because of the lateral resolution of theimmunogold method (�30 nm), gold particlescould be separated by a distance of up to �30nm and still signal epitopes in the vesicle.Distributions of l-aspartate and l-glutamateintercenter distances in WT slices were notsignificantly different from the distributions insialin-KO slices (P�0.05, 2 test). These quan-tifications were done in 60 nerve terminalspositive for aspartate/glutamate in both WTand sialin KO CA1 radiatum.

6 Vol. 27 March 2013 MORLAND ET AL.The FASEB Journal � www.fasebj.org

released in a strictly Ca2�-dependent manner even whenthe EAATs were blocked, supporting the idea of exocy-totic release of l-aspartate.

L-Aspartate release from nerve terminals is independent ofvolume-regulated anion channels (VRACs)

As VRACs are proposed to release excitatory aminoacids (50, 51), and the activity of these channels isthought to be Ca2� dependent (52), we investigatedwhether VRACs could account for the release of l-as-partate from hippocampal nerve terminals. Hippocam-pal slices were exposed to physiological and depolariz-ing concentrations of K� in the presence or absence ofthe VRAC inhibitor NPPB. All incubations were done inthe presence of TFB-TBOA. NPPB did not affect thedensity of l-aspartate immunogold particles in nerveterminals at either basal or depolarizing conditions(Fig. 7A–D, I). The same was true for l-glutamateimmunogold particles (Fig. 7E–H, J).

DISCUSSION

Here we show that l-aspartate is transported intosynaptic vesicles in a sialin-independent manner. The

highest vesicular uptake of l-aspartate (relative to theuptake of l-glutamate) was found in hippocampalvesicles, which is consistent with slice data from thepresent and previous studies (21–23) showing thatl-aspartate is released by exocytosis from hippocampalnerve terminals. There was no difference in the ATP-dependent uptake of l-aspartate in vesicles from sialin-knockout compared to wild-type vesicles, and synaptic-like microvesicles from PC12 cells transfected withsialin were not capable of l-aspartate uptake. Consis-tent with this, our results from sialin-knockout miceshow that the vesicular l-aspartate release in the hip-pocampus is independent of sialin, indicating thatl-aspartate is packed into vesicles via a hitherto un-known transporter.

Our l-aspartate uptake data are in agreement withthe results of Naito and Ueda (12), who reportedl-aspartate uptake to be 7.5% of the vesicular l-gluta-mate uptake, but at odds with the studies of Fykse et al.(11) and Maycox et al. (13), who did not find evidencefor uptake of l-aspartate in synaptic vesicles. Thereason for this discrepancy is not clear, but in order toobtain near-saturation of the vesicles with transmitter(12), we used 5 min incubation time, while the otherstudies 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-aspar-tate from nerve endings is Ca2�

dependent also when excita-tory amino acid transportersare blocked. Quantification ofgold particles representing l-as-partate (A, B) and l-glutamate(C) in excitatory nerve termi-nals in CA1 stratum radiatum inslices incubated at 3 mMK�

(basal) and at 55 mM K� (de-pol), and under depolarizingconditions where exocytosis wasinhibited by low Ca2�/highMg2� (depol low Ca2�) or byKB-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 wasperformed in 3 basal slices (total of 59 terminals), 3 depol slices (total of 65 terminals), and 4 depol low Ca2� slices (total of176 terminals). Quantification in B and C was performed in 3 basal slices (total of 56 and 91 terminals, respectively), 3 and 5depol slices (total of 62 and 149 terminals) and 4 depol KB-R7943 slices (total of 101 and 133 terminals). *P � 0.05, Student’s2-tailed t test (SPSS).

Figure 5. l-Aspartate is not de-pleted from nerve terminalsthrough excitatory amino acidtransporters. A–C) Electron mi-crographs show gold particlesrepresenting l-aspartate (smallgold particles) and l-glutamate(large gold particles) in excit-atory nerve terminals (t) mak-ing asymmetric synapses withdendritic spines (s) in the CA1stratum radiatum. Slices were in-

cubated at 3 mM K� (A), 55 mM K� (B), or 55 mM K� with TFB-TBOA (C). D) Quantifications of gold particles representingl-aspartate in excitatory nerve terminals in slices incubated at 3 mM K� (basal), at 55 mM K� (depol), and at 55 mM K� withTFB-TBOA (depol TBOA). Bar charts indicate particle density (average�sem; gold particles/�m2). Quantification wasperformed in 4 basal slices (total of 53 terminals), 4 depol slices (total of 123 terminals), and 6 depol TBOA slices (total of 74terminals). *P � 0.05; 1-way ANOVA, Dunnett post hoc test.

7L-ASPARTATE TRANSMISSION IS INDEPENDENT OF SIALIN

rectly, but used acidification of the vesicles as anindirect measure of uptake. Relative to l-glutamate, thehighest l-aspartate uptake was found in the hippocam-pus. Because vesicular l-glutamate uptake is similar inthe hippocampus and the whole brain (53), this meansthat the l-aspartate uptake is about twice as high inhippocampal as in whole-brain vesicles. The fact thatthe uptake ratio of l-aspartate to l-glutamate variedbetween forebrain regions suggests that l-aspartate andl-glutamate are taken up into different pools of vesi-cles, or into partially overlapping vesicle pools, in whichsome 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-aspartateand l-glutamate is our finding that l-glutamate doesnot inhibit vesicular l-aspartate uptake, and previousfindings showing that l-aspartate does not inhibit l-glu-tamate uptake (11, 54). Also suggesting that l-aspartateis released from a different vesicular pool than l-gluta-mate is the fact that the vesicular release of l-aspartatecan be regulated differently from that of l-glutamate(7, 8). For instance, NMDA enhances l-aspartate, butnot l-glutamate, release from hippocampal slices (55),and reduced levels of 5-lipoxygenase metabolites selec-tively reduced the release of l-aspartate (56). Along the

same line, we recently showed that valproate reducesthe nerve terminal content of aspartate, but not that ofglutamate (35).

The lysosomal H�-coupled sialic acid transporter,sialin, was recently suggested to transport l-aspartateand l-glutamate into hippocampal synaptic vesicles(15). Here we show that synaptic vesicles from sialin-knockout and wild-type mice do not differ in theirability to take up l-aspartate. In line with the vesicularuptake data are our immunogold results indicating thatl-aspartate is present in synaptic vesicles from bothwild-type and sialin-knockout brains (Fig. 5). Furthersupporting the idea that vesicular uptake of l-aspartateis independent of sialin is that when we overexpressessialin in the vesicles of PC12 cells, they were still unableto accumulate l-aspartate. Finally, l-aspartate releasefrom hippocampal nerve terminals occurs in the ab-sence of sialin. As sialin transports l-aspartate andl-glutamate with similar affinities (15), and the intra-cellular concentration of l-glutamate is about 5 timesthe l-aspartate concentration (57), sialin should con-tribute to at least a part of the total vesicular uptake ofl-glutamate. Thus, at least a part of the vesicularl-glutamate uptake should be inhibited by addingexternal l-aspartate to isolated vesicles, which, how-

Figure 7. l-Aspartate is not de-pleted from nerve terminalsthrough volume-regulated an-ion channels. A–H) Electronmicrographs show gold parti-cles representing l-aspartate(A–D) or l-glutamate (E–H) inexcitatory nerve terminals (t)making asymmetric synapses withdendritic spines (s) in the CA1stratum radiatum. A, E) Terminalsincubated at 3 mM K�. C, G)Terminals incubated at 3 mMK� with NPPB. B, F) Terminalsincubated at 55 mM K�. D, H)Terminals incubated at 55 mMK� with NPPB. I, J) Quantifi-cation of gold particles repre-senting l-aspartate (I) and l-glutamate (J) in excitatorynerve terminals in slices incu-bated at 3 mM K� (basal), at 3mM K� with NPPB (basalNPPB) at 55 mM K� (depol),and at 55 mM K� with NPPB(depol NPPB). Bar charts indi-cate particle density (average�sem; gold particles/�m2). Quanti-fications in I and J were per-formed in 3 and 4 basal slices(total of 68 and 138 terminals,respectively), 3 basal NPPBslices (total of 132 and 91 termi-nals), 3–4 depol slices (total of62 and 97 terminals), and 3–4depol NPPB slices (total of 110and 158 terminals). *P � 0.05;Student’s 2-tailed t test (SPSS).

8 Vol. 27 March 2013 MORLAND ET AL.The FASEB Journal � www.fasebj.org

ever, was not found to be the case (9–11, 54). Takentogether, our findings strongly suggest that sialin isneither sufficient for vesicular storage nor necessary forrelease of l-aspartate in intact brain preparations. Thisindicates that either sialin is present in insufficientamounts in the vesicular membrane, or sialin does nottransport l-aspartate into synaptic vesicles under phys-iological conditions.

In Miyaji et al. (15), the researchers report to havedetected sialin and measured vesicular l-aspartate up-take in the hippocampal P2 fraction. According tostandard nomenclature, the P2 fraction refers to acrude synaptosomal fraction (see, e.g., refs. 18, 58, 59),which is likely to contain nonvesicular membranes, andpresumably also lysosomal membranes. Thus, the local-ization of sialin in synaptic vesicles could be ques-tioned. The notion that sialin is not an importantconstituent of synaptic vesicles, or present in lowamounts, is supported by a proteomic study that did notdetect sialin among the synaptic vesicle proteins (60).

In the course of this study we set out to localize sialinat the subcellular level using immunogold cytochemis-try. However, during antibody testing, the sialin anti-bodies (commercially available from Alpha Diagnosticsand from R.R.) produced several bands on Westernblots of brain tissue (unpublished results), hamperingthe use of these antibodies in immunolocalizationstudies. Two previous studies have reported sialin im-munolocalization in the brain (61, 62). However, noneof these give ultrastructural data, and thus whethersialin is present in synaptic vesicles could not be in-ferred from these studies.

VRACs could form an alternative release mechanismfor l-aspartate and l-glutamate, as these channels havebeen shown to be responsible for release of excitatoryamino acids from cultured astrocytes and from patho-logical brain tissue (44, 50, 51, 63, 64). Because theopening of these channels is reported to be Ca2�

sensitive (52) it was essential to determine whetherl-aspartate could escape from nerve terminals throughVRACs. Using the VRAC blocker NPPB, we found noevidence for reduced l-aspartate or l-glutamate releasefrom nerve terminals during membrane depolariza-tion. Thus, VRACs do not appear to contribute to thenerve terminal release of excitatory amino acids. This isa novel finding, as previous work on VRAC-mediatedrelease of excitatory amino acids from intact braintissue has not distinguished between astrocytic andneuronal release. Similarly, as EAATs are present onhippocampal nerve terminals (25, 65) and reported tomediate l-aspartate and l-glutamate release in patho-logical states (41, 43, 44, 66), we investigated whetherl-aspartate could be released from nerve terminals viathese transporters. In this respect, it should be notedthat blocking the EAATs effectively inhibits reversedtransport of excitatory amino acids (23, 34, 36–38, 40,41, 67) showing that TBOA blocks both the forwardand reverse aspartate/glutamate transport across termi-nal membranes. We found that the EAAT blockerTBOA did not inhibit the release of l-aspartate (orl-glutamate). This is in line with data showing that,under conditions of partial ischemia (51), as well asunder physiological conditions (23, 68, 69), the EAATs

are not responsible for any significant release of l-as-partate and l-glutamate. Hence, our results on inhibi-tion of EAATs and VRAC strongly suggest that nonve-sicular mechanisms do not play a role in the nerveterminal release of l-aspartate.

These results show that nerve terminals have anl-aspartate-containing pool of synaptic vesicles, whichundergo exocytosis during neuronal stimulation. Thesensitivity of l-aspartate release to Ca2� (present studyand refs. 8, 18, 21, 22, 55, 70–77) and tetanus toxin (22,69, 78, 79) reported in previous studies further supporta vesicular release mechanism. Low micromolar con-centrations of KB-R7943 have previously been shown toselectively inhibit aspartate release from synaptosomes(74) and organotypic cultures (7). In our study, KB-R7943, used at a concentration (50 �M) that probablyinhibits several receptors and channels that mediateCa2� influx (47–49, 80), reduced the nerve terminaldepletion of both l-aspartate and l-glutamate, high-lighting the calcium-dependency of l-aspartate/l-glu-tamate release. The higher concentration of KB-R7943might explain the discrepancy between our results andthose of Nadler and coworkers.

We conclude that l-aspartate is taken up into synap-tic vesicles by a yet unidentified vesicular transporterand is subjected to regulated exocytotic release.

The authors are grateful to Inger-Lise Bogen for advice onthe vesicular uptake assay and to Anna Torbjørg Bore fortechnical 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. andNS065664 to L.M.P. from the U.S. National Institute ofNeurological Disorders and Stroke. The content is solely theresponsibility of the authors and does not necessarily repre-sent the official views of the National Institute of NeurologicalDisorders and Stroke or the U.S. National Institutes ofHealth. L.M.P. is in the Medical Scientist Training Program atStanford University School of Medicine.

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Received for publication February 15, 2012.Accepted for publication November 26, 2012.

11L-ASPARTATE TRANSMISSION IS INDEPENDENT OF SIALIN