,

*Department of Neuroscience, Universita Politecnica delle Marche, Ancona, Italy

�Department of Medical Pharmacology and CNR Institute of Neuroscience, University of Milano, Italy

�Department of Experimental Medicine, Universita di Genova/Istituto Nazionale di Neuroscienze and Department of Neuroscience

and Brain Technologies, The Italian Institute of Technology, Genova, Italy

§Fondazione di Medicina Molecolare, Universita Politecnica delle Marche, Ancona, Italy

Glutamate and GABA mediate most of the excitatory andinhibitory synaptic transmission in the mammalian brain.Before their release into the cleft, glutamate and GABA aretaken up and accumulated in synaptic vesicles by specificvesicular transporters (Liu and Edwards 1997; Eiden 2000).Glutamate is accumulated in synaptic vesicles by the actionof the vesicular transporters VGLUT1-3; VGLUT1 isexpressed mainly in telencephalon, VGLUT2 in diencepha-lon and lower brainstem, and VGLUT3 in striatum, hippo-campus, neocortex and brainstem (Gras et al. 2002; Fremeauet al. 2004; Takamori 2006). GABA is transferred intosynaptic vesicles by the vesicular transporter VGAT which isexpressed throughout the brain (McIntire et al. 1997; Sagneet al. 1997; Takamori et al. 2000b). Early studies showedthat VGAT is localized to axon terminals (AxT) formingsymmetric synapses, VGLUT1 and VGLUT2 to AxTforming asymmetric synapses, whereas VGLUT3 is localized

to both asymmetric and symmetric synapses (Chaudhry et al.1998; Dumoulin et al. 1999; Fremeau et al. 2002; Gras et al.2002; Kaneko et al. 2002).

It is generally assumed that glutamate and GABAvesicularuptake and release mechanisms are totally segregated, butrecent studies suggest that exceptions do exist. AxTco-expressing VGLUT3 and VGAT have been documented

Received March 20, 2009; revised manuscript received June 15, 2009;accepted June 16, 2009.Address correspondence and reprint requests to Fiorenzo Conti,

Department of Neuroscience, Universita Politecnica delle Marche,Ancona, Italy and Fondazione di Medicina Molecolare, UniversitaPolitecnica delle Marche, Ancona, Italy. E-mail: f.conti@univpm.itAbbreviations used: AxT, axon terminals; EM, electron microscopy;

GAD, glutamic acid decarboxylase; GP, guinea pig; ir, immunoreactivity;PBS, phosphate-buffered saline; PFA, paraformaldehyde; TBST, Tris-buffered saline with Tergitol.

Abstract

Glutamate and GABA mediate most of the excitatory and

inhibitory synaptic transmission; they are taken up and accu-

mulated in synaptic vesicles by specific vesicular transporters

named VGLUT1-3 and VGAT, respectively. Recent studies

show that VGLUT2 and VGLUT3 are co-expressed with

VGAT. Because of the relevance this information has for our

understanding of synaptic physiology and plasticity, we

investigated whether VGLUT1 and VGAT are co-expressed in

rat cortical neurons. In cortical cultures and layer V cortical

terminals we observed a population of terminals expressing

VGLUT1 and VGAT. Post-embedding immunogold studies

showed that VGLUT1+/VGAT+ terminals formed both sym-

metric and asymmetric synapses. Triple-labeling studies

revealed GABAergic synapses expressing VGLUT1 and

glutamatergic synapses expressing VGAT. Immunoisolation

studies showed that anti-VGAT immunoisolated vesicles

contained VGLUT1 and anti-VGLUT1 immunoisolated vesi-

cles contained VGAT. Finally, vesicles containing VGAT res-

ident in glutamatergic terminals undergo active recycling. In

conclusion, we demonstrate that in neocortex VGLUT1 and

VGAT are co-expressed in a subset of axon terminals forming

both symmetric and asymmetric synapses, that VGLUT1 and

VGAT are sorted to the same vesicles and that vesicles at

synapses expressing the vesicular heterotransporter partici-

pate in the exo-endocytotic cycle.

Keywords: axon terminals, co-expression, synaptic vesicles,

VGAT, VGLUT1.

J. Neurochem. (2009) 110, 1538–1546.

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1538 Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546� 2009 The Authors

in many brain regions, including the cerebral cortex (Seal andEdwards 2006). In addition, VGLUT2-VGAT co-localizationhas also been described in nearly all anteroventral peri-ventricular nucleus neurons (Ottem et al. 2004) and in a subsetof pre-synaptic terminals forming both asymmetric and,unexpectedly, symmetric synapses in the dentate gyrus(Boulland et al. 2009).

Here we report that in the cerebral cortex of adult ratsVGLUT1 and VGAT are co-expressed in a subset of AxTforming both symmetric and asymmetric synapses. We alsoshow that VGLUT1 and VGATare sorted to the same synapticvesicles, and that vesicles at synapses expressing the vesicularheterotransporter participate in the exo-endocytotic cycle.

Materials and methods

Animals and tissue preparationAdult male Sprague–Dawley albino rats (190–220 g; Charles River,

Milan, Italy) were used for this study. Their care and handling were

approved by the local ethical committee for animal research.

Experiments were carried out in accordance with European

Community Council Directive 86/609 and approved by the local

veterinary service. Animals were kept under a dark–light cycle of

12 h and permitted food and water ad libitum.Adult rats were transcardially perfused with a flush of saline

followed by freshly depolymerized 4% paraformaldehyde (PFA) and

0.5% glutaraldehyde in phosphate-buffered (0.1 M) saline (PBS)

[for electron microscopy (EM) studies; n = 2] or by 4% PFA only

(for confocal studies; n = 8). Brains were removed, post-fixed in the

same fixative for 3 days and cut with a vibratome into 50-lm-thick

sections.

AntibodiesSource, concentrations, and data on the characterization of primary

antibodies used in this study are listed in Table 1.

Cortical cell culturesPrimary neuronal cultures from cerebral cortex were obtained from

E18 Sprague–Dawley rats as described by Banker and Cowan

(1977) and Bartlett and Banker (1984). Pregnant animals were killed

Table 1 Primary and secondary antibodies used in the present study

Primary antibodies Host Dilution Source Characterization

VGAT R 1 : 1000 (IF)

1 : 500 (EM)

RH Edwards (UCSF) Chaudhry et al. (1998)

VGAT R 1 : 2500 (II and WB)

1 : 250 (IF)

Synaptic System/131002 Takamori et al. (2000b)

VGAT M 1 : 100 (IF and WB) Synaptic System/131011 Baer et al. (2007)

VGATecto R 1 : 30 (VR) R Jahn (Gottingen) Martens et al. (2008)

VGLUT1 R 1 : 1000 (IF) RH Edwards (UCSF) Bellocchio et al. (1998)

VGLUT1 GP 1 : 1000 (IF)

1 : 500 (EM)

Chemicon/AB5905 Melone et al. (2005)

VGLUT1 R 1 : 2500 (II & WB) Synaptic System/135303 Takamori et al. (2001)

VGLUT1 M 1 : 1000 (WB) Synaptic System/135311 Tafoya et al. (2006)

VGLUT2 R 1 : 1000 (WB) Synaptic System/135403 Herzog et al. (2006)

VGLUT2 M 1 : 1000 (WB) Chemicon/MAb5504 Wassle et al. (2006)

RAB3A R 1 : 1000 (WB) Synaptic System/107003 Schluter at al. (2002)

GABAAa1 R 1 : 25 (IF) Phosphosolutions/812-GA1N http://www.phosphosolutions.com

GAD65–67 H 1 : 100 (IF) Human serum/M Solimena (Dresden) Solimena et al. (1990)

PSD-95 M 1 : 100 (IF) NeuroMab (UC Davies, NIH)/K28-48 Kim et al. (1995)

Secondary antibodies conjugated to React Dilution Source

TRITC R 1 : 100 Jackson ImmunoResearch, West Grove, PA/111-025-003

TRITC M 1 : 100 Jackson ImmunoResearch, West Grove, PA/115-025-003

TRITC R 1 : 100 Molecular Probes, Eugene, OR/T2769

FITC GP 1 : 100 Vector Laboratories, Burlingame, CA/FI-7000

Alexa Fluor 488 H 1 : 200 Molecular Probes, A-11013

Cy5 R 1 : 100 Jackson ImmunoResearch, West Grove, PA/111-175-003

Cy5 M 1 : 100 Jackson ImmunoResearch, West Grove, PA/115-175-003

12 nm gold particles R 1 : 20 Jackson ImmunoResearch, West Grove, PA/111-205-144

18 nm gold particles GP 1 : 20 Jackson ImmunoResearch, West Grove, PA/706-215-144

GP, guinea pig; M, mouse; R, rabbit; H, human; IF, immunofluorescence; WB, western blotting; II, immunoisolation; EM, electron microscopy; VR,

vesicle recycling.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546

VGLUT1-VGAT co-localization | 1539

by cervical dislocation under CO2 anesthesia, and the fetuses

removed and put into ice-cold Hanks balanced salt solution. After

dissection of the cortices, cells were dissociated and plated on

poly-L-lysine-treated coverslips in Neurobasal (Invitrogen, Carlsbad,

CA, USA; Gibco, Rockville, MD, USA) with 2% B27 containing

100 U/mL penicillin, 100 lg/mL streptomycin, 200 mM glutamine,

10 nM glutamate. At 3 days in vitro, half of the culture medium was

replaced with fresh medium without glutamate.

At 21 days in vitro, neurons were fixed in ice-cold methanol for

2.5 min and processed for immunocytochemistry. Fixed cells were

detergent-permeabilized and labeled with a mixture of anti-

VGLUT1 [guinea pig (GP)], anti-VGAT (rabbit, R; or mouse, M),

anti-glutamic acid decarboxylase (GAD; 65 and 67; human serum),

anti-PSD-95 and anti-GABAAa1. Appropriate secondary antibodies

are reported in Table 1. After fixation and staining, images were

acquired using a Bio-Rad (Hercules, CA, USA) MRC-1024

confocal microscope equipped with Bio-Rad LaserSharp 3.2

software (pixel size 0.15 lm).

Co-localization studies in brain sectionsSections were incubated for 1 h in newborn calf serum (10% in

phosphate-buffered with 0.2% Triton X-100) and then for 2 h at

22�C plus overnight at 4�C in a solution containing a mixture of

VGAT (R) and VGLUT1 (GP) primary antibodies. The next day,

sections were incubated in a mixture of the appropriate secondary

fluorescent antibodies (Table 1). Sections were then mounted, air-

dried and coverslipped using Vectashield mounting medium (H-

1000; Vector, Burlingame, CA, USA). Images were acquired using a

Bio-Rad Microradiance confocal microscope equipped with Laser-

Sharp 3.2 software (pixel size 0.15 lm). Control experiments with

single-labeled sections and sections incubated with either two

primary and one secondary antibody or one primary and two

secondary antibodies revealed no appreciable FITC/tetramethylrho-

damine isothiocyanate bleed-through or antibody cross-reactivity.

Images from the fronto-parietal cortex were acquired from

randomly selected subfields in layer V (4–6; 2–6 sections/animal),

and from a plane in which the resolution of the two stains was

optimal and never > 1.8 lm from the surface (Melone et al. 2005).To improve signal/noise ratio, 15 frames/image were averaged by

Kalman filtering. In addition, to minimize the fusion of puncta, the

contrast of each image was adjusted manually within the maximum

range of levels for each color channel. Then, without reducing the

image resolution, each channel was examined separately to identify

and count manually immunopositive puncta; the two channels were

then merged and the number of co-localizing puncta was counted

manually (for further details, see Bragina et al. 2007).

Electron microscopy studiesSections were processed for osmium-free embedding method

according to Phend et al. (1995). Chips including layer V were

cut from the wafers and glued onto blank resin blocks with

cyanoacrylate. Thin (60–80 nm) sections were cut and collected on

nickel mesh grids and processed for immunogold labeling. After

treatment with 4% para-phenyenediamine in Tris-buffered saline

[0.1 M Tris, pH 7.6, with 0.005% Tergitol N P-10 (TBST)], grids

were incubated overnight in a solution of TBST (pH 7.6) containing

a mixture of anti-VGAT (R) and anti-VGLUT1 (GP) primary

antibodies. Grids were then incubated in TBST (pH 8.2) containing

a mixture of secondary antibodies conjugated to 12 and 18 nm gold

particles (Table 1), and counter-stained with uranyl acetate and

Sato’s lead. When primary antiserum was omitted, virtually no gold

particles could be detected on sections; when normal serum was

substituted for immune serum, sparse gold particles were scattered

across sections, but they did not show any evident relationship to

AxT (Fig. S1).

Optimal concentration of primary antibodies was sought by

testing several dilutions; the concentration yielding the lowest level

of background labeling and immunopositive elements was used to

perform the final studies (for details, see Melone et al. 2009). Grids(three grids/rat) were examined on a Zeiss EM 900 electron

microscope (Carl Zeiss, Oberkochen, Germany). AxT forming

symmetric or asymmetric synaptic contacts and containing at least

(b)

(a)

Fig. 1 In vitro and in vivo evidence for VGLUT1 and VGAT co-local-

ization in cortical synapses. (a) VGLUT1 (green) and VGAT (blue)

immunoreactivity (ir) in cortical cultures. Arrow points to a double-

labeled punctum enlarged and shown on separate channels in the

insets. (b) VGLUT1 (green)-VGAT (red) co-localization in perisomatic

axon terminals of cortical sections. A layer V neuron outlined by

VGAT+ axon terminals. Arrow indicates VGAT/VGLUT1 co-localiza-

tion. Only puncta showing a very large overlap and morphological

similarity were considered double-labeled, whereas puncta exhibiting

only partial overlap were discarded. Scale bars: 5 lm; insets, 2 lm.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546� 2009 The Authors

1540 | G. Fattorini et al.

one 12 nm or one 18 nm gold particle were selected and

photographed at 50 000 and 85 000X. All positive profiles

examined displayed a particle density for both VGLUT1 and VGAT

significantly higher (p < 0.0001) as compared to the nuclear non-

specific labeling (0.30 ± 0.04 for 12 nm and 0.30 ± 0.05 for 18 nm;

n = 12). Statistical analysis was performed using a two tailed t-testwith GraphPad Prism software (v.4.0, GraphPad Software Inc., San

Diego, CA, USA).

Studies of immunoisolated vesiclesEupergit C1Z methacrylate microbeads (1 lm in diameter; Rohm

Pharmaceuticals, Darmstadt, Germany) were either blocked with

glycine or conjugated with affinity-purified goat anti-rabbit anti-

bodies (IgG; Sigma, Milan, Italy) as previously described (Burger

et al. 1991). Affinity-purified anti-VGAT (R) and anti-VGLUT1 (R)

antibodies (Table 1) were conjugated with the covalently bound

secondary antibodies, generating IgG-coated anti-VGAT or anti-

VGLUT1 beads. Beads coated with glycine or secondary antibodies

were used as negative controls (mock beads). The LS1 fraction

(150–300 lg protein) obtained from osmotic lysis of rat cerebro-

cortical synaptosomes prepared from either adult (3–4 months old)

or newborn (P0) rats (Huttner et al. 1983) was incubated for 2–4 h

at 4�C in PBS with the various bead preparations (50–70 lL settled

beads in 500 lL final volume) under constant rotation. Beads were

sedimented from the supernatant fraction by centrifugation at

1000 g for 1 min followed by repeated washes in PBS. Corre-

sponding amounts of bead pellets and supernatant fractions were

solubilized in sample buffer and subjected to sodium dodecyl

sulfate–polyacrylamide gel electrophoresis on 10% polyacrylamide

gels. Gels were electrophoretically transferred to nitrocellulose

membranes and immunoblotted with polyclonal or monoclonal anti-

VGAT, anti-VGLUT1, and anti-VGLUT2 antibodies (Table 1).

Specific immunoreactivity (ir) was revealed using peroxidase-

conjugated secondary antibodies and the chemiluminescence detec-

tion system as described (Bragina et al. 2007). Quantification of

recovered ir was performed by densitometric analysis of the

fluorograms and by data interpolation into a standard curve of rat

brain LS1 fraction (starting material) run in parallel with the

unknown samples. Experiments were repeated at least five times on

different days using different starting material and/or combinations

of primary antibodies.

Functional studiesRecycling of GABAergic vesicles was monitored using rabbit

polyclonal antibodies directed against the intravesicular domain

of VGAT (VGATecto; Table 1). Cultures were incubated with

VGATecto antibodies for 4 min at 22�C in HEPES-buffered Krebs–

Ringer solution (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,

2 mM CaCl2,10 mM glucose and 25 mM HEPES/NaOH, pH 7.4)

in the presence of 55 mM KCl. Cells were then fixed with 4% PFA

and 4% sucrose in 0.12 M phosphate buffer for 25 min at 37�C.Fixed cells were detergent permeabilized and labeled with

tetramethylrhodamine isothiocyanate-conjugated anti-rabbit anti-

bodies (Table 1). Cultures were counter-stained with monoclonal

antibodies directed against PSD-95 and GP anti-VGLUT1 poly-

clonal antibodies, followed by a mixture of the appropriate

(c)

(b)

(d)

(a)

Fig. 2 Electron microscopic evidence for

VGLUT1 and VGAT co-localization in cor-

tical synapses. Post-embedding immuno-

gold labeling for VGLUT1 (large dots) and

VGAT (small dots) in layer V. (a and b)

Axon terminals displaying both VGLUT1

and VGAT coding particles make symmetric

synaptic contacts (arrowheads). (c and d)

Axon terminals exhibiting VGAT and

VGLUT1 immunogold labeling form asym-

metric synaptic contacts (arrowheads).

Scale bars: (a and b), 0.12 lm; (c and d),

0.15 lm.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546

VGLUT1-VGAT co-localization | 1541

secondary fluorescent antibodies (Table 1). Coverslips were

mounted in Vectashield. After fixation and staining, images were

acquired with a Bio-Rad MRC-1024 confocal microscope equipped

with Lasersharp 3.2 software and analyzed using the Image J

software (v1.39c; NIH).

Results

In cortical cultures stained with antibodies directed againstVGLUT1 and VGAT, we found that 10.4 ± 1.2% ofVGLUT1+ puncta (n = 70598) and 9.1 ± 0.9% of VGAT+puncta (n = 69827) were double-labeled (Fig. 1a). We alsostudied VGLUT1/GAD65–67 co-localization, and found that4.4 ± 0.4% of VGLUT1+ puncta (n = 91670) wereGAD65–67+ (Fig. S2), in line with the observation that notall GABAergic terminals express GADs (e.g. Loscher et al.1998; Oliva et al. 2000; Chattopadhyaya et al. 2004; Lopez-Bendito et al. 2004; Ma et al. 2006; Bragina et al. 2008).

To verify whether VGLUT1/VGAT co-localization wasdetectable also in vivo, we studied cortical AxT in layer V,where both VGLUT1 and VGAT are highly expressed(Minelli et al. 2003a,b). Analysis of 24 sections (six animals)revealed that 14.0 ± 1.5% of VGLUT1+ puncta (n = 9137)and 9.6 ± 1.2% of VGAT+ puncta (n = 13195) co-expressedVGLUT1 and VGAT (Fig. 1b). Post-embedding EM studiesconfirmed that a large fraction of positive profiles (n = 279)exhibited exclusively gold particles coding for eitherVGLUT1 (58.7%) or VGAT (31.5%), with VGLUT1 andVGAT mainly localized to pre-synaptic terminals makingasymmetric and symmetric synaptic contacts, respectively(Chaudhry et al. 1998; Kaneko et al. 2002). However, insome AxT (9.7%) both VGLUT1 and VGAT gold particleswere detectable; AxT containing both VGLUT1 and VGATmade both symmetric and asymmetric synapses (Fig. 2a–d).

Next, we studied the expression of VGLUT1 in GABA-ergic synapses and of VGAT in glutamatergic synapsesby studying triple-labeled cortical cultures stained withVGLUT1, VGAT, and GABAAa1 or with VGLUT1, VGAT,and PSD-95, the anchoring protein for NMDA receptors.Analysis of 6156 VGAT+ puncta facing GABAAR positiveelements (GABAergic synapses) and of 19 749 VGLUT1+puncta facing PSD-95+ elements (glutamatergic synapses)revealed that 10.4 ± 2.0% of GABAergic synapses wereVGLUT1+ (Fig. 3a) and 6.3 ± 1.0% of glutamatergic syn-apses were VGAT+ (Fig. 3b).

We also analyzed whether VGLUT1-VGAT were ex-pressed in different vesicle populations or on the samevesicles. Immunoisolation of GABAergic and glutamatergicvesicles resulted in substantial enrichment in VGAT andVGLUT1 ir, respectively (Fig. 4). However, 20.2 ± 2.8% ofVGAT ir associated with anti-VGAT-immunoisolatedGABAergic vesicles co-purified with anti-VGLUT1-immunoisolated glutamatergic vesicles (Fig. 4). Conversely,18.9 ± 5.3% of VGLUT1 ir associated with anti-VGLUT1-

immunoisolated glutamatergic vesicles co-purified with anti-VGAT-immunoisolated GABAergic vesicles (Fig. 4). Theassociation of heterotransporters with GABAergic andglutamatergic vesicles appeared to be specific, as (i)VGLUT1 or VGAT ir was not detected in association withmock (either glycine- or secondary antibodies-coated) beads;(ii) VGLUT2 ir was virtually absent from VGAT immunois-olated vesicles, while it was associated in detectable amountswith VGLUT1 immunoisolated vesicles; and (iii) comparableamounts of the synaptic vesicle associated small G proteinRab3A were associated with both populations of immunois-olated vesicles. The extent of co-localization of VGLUT1and VGAT in glutamatergic and GABAergic vesicles appears

(b)

(a)

Fig. 3 Triple-labeling studies of VGAT expression in glutamatergic

synapses and of VGLUT1 expression in GABAergic synapses. (a)

VGLUT1 (green), VGAT (blue) and GABAAa1 (red) ir in rat cortical

cultures. Arrow indicate a GABAergic synapse (i.e. VGAT ir facing

GABAAa1 ir) expressing VGLUT1. Insets show VGAT/GABAAa1 ir

(top), VGLUT1 (middle), and triple labeling (bottom). (b) VGLUT1

(green), VGAT (blue) and PSD-95 (red) ir in cortical cultures. Arrow

indicates a glutamatergic synapse (i.e. VGLUT1 ir facing PSD-95 ir)

expressing VGAT. Insets show VGLUT1/PSD-95 ir (top), VGAT

(middle), and triple labeling (bottom). Scale bars: 5 lm; insets,

2 lm.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546� 2009 The Authors

1542 | G. Fattorini et al.

to be developmentally regulated. In fact, for both types ofvesicles, the co-localization of the two transporters was morepronounced at early stages of post-natal development (post-natal day 0; Fig. S3) than that observed in the adult brain(Fig. 4).

Finally, to investigate whether vesicles expressing theheterotransporter participate in the exo-endocytotic cycle, westudied glutamatergic synapes (i.e. VGLUT1+ puncta facingPSD-95+ elements) in live cortical neurons incubated with anantibody directed against the endovesicular domain of VGATunder depolarizing conditions. The analysis showed that8.0 ± 1.4% of the labeled glutamatergic synapses (n = 4380)were VGAT+, indicating that they had incorporated theantibody (Fig. 5) and thereby that synaptic vesicles contain-ing VGAT resident in glutamatergic terminals undergo activerecycling.

Discussion

These studies show that in the cerebral cortex of adult ratsVGLUT1 and VGAT are co-expressed in a subset of AxTforming both symmetric and asymmetric synapses, thatVGLUT1 and VGAT are sorted to the same population ofvesicles, and that vesicles containing VGAT resident inglutamatergic terminals undergo active recycling.

Using distinct in vitro and ex vivo microscopy techniques,including immunogold post-embedding EM, we showed thatin the cerebral cortex about 10% of VGLUT1+ and about10% VGAT+ AxT contain both vesicular transporters.VGLUT1/VGAT co-expression has never been described inthe brain of adult mammals, although it has previously beenreported that a subset of cone bipolar cells in cat retinacontain VGLUT1 and VGAT (Kao et al. 2004) and some

(a) (b)

Fig. 4 VGLUT1-VGAT are expressed in the same vesicles at cortical

synapses. (a) Glutamatergic and GABAergic synaptic vesicles were

immunoisolated from the LS1 fraction of adult rat cerebral cortex using

beads coupled with either rabbit VGLUT1 (raVGLUT1-IP) or rabbit

VGAT (raVGAT-IP) antibodies. Beads coupled with anti-rabbit IgGs

(Mock) served as negative control. After immunoisolation, corre-

sponding amounts of pellet and supernatant (Sup) fractions were

subjected to immunoblotting with rabbit and mouse anti-VGAT anti-

bodies (raVGAT and maVGAT, respectively; upper panel), rabbit

and mouse anti-VGLUT1 antibodies (raVGLUT1 and maVGLUT1,

respectively; middle panel) or rabbit and mouse anti-VGLUT2 anti-

bodies (raVGLUT2 and maVGLUT2, respectively; lower panel). (b)

Quantification of the recovered immunoreactivity was carried out by

densitometric scanning and interpolation of the data into a standard

curve of rat brain LS1. The amounts of VGAT (upper left panel),

VGLUT1 (upper right panel), VGLUT2 (lower left panel) immuno-

reactivities (IR) detected in synaptic vesicles immunoisolated with

anti-VGAT (VGAT-SV), anti-VGLUT-1 (VGLT1-SV) or with anti-rabbit

IgGs (Mock) beads are shown as mean (±SEM) of five independent

experiments. Rab3A immunoreactivity in the same preparations is

shown for comparison (lower right panel).

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546

VGLUT1-VGAT co-localization | 1543

mossy fiber terminals express both transporters during earlypost-natal life (Safiulina et al. 2006). Immunoisolation ofglutamatergic and GABAergic synaptic vesicles showed thatabout 20% of both populations express VGAT and VGLUT1,respectively. This phenomenon is proportionally higher inearly stages of development and does not apply to VGLUT2,which appears to be completely excluded from GABAergicvesicles, whereas it associates with a subpopulation ofVGLUT1 vesicles, in agreement with recent reports (Na-kamura et al. 2005; Boulland et al. 2009). The higher ratioof VGAT and VGLUT1 co-isolation in immunoisolatedvesicles, as compared to the extent of immunocytochemicalco-localization may be in principle attributed to either somedegree of clustering of GABAergic and glutamatergicvesicles occurring following lysis of synaptosomes, thestringent parameters used in confocal studies to avoid false-positives or the lower sensitivity of post-embedding immu-nogold EM (e.g. Takumi et al. 1998). The observation that apopulation of cortical vesicles express both VGLUT1 andVGAT indicates that, in VGLUT1/VGAT-positive terminals,at least some vesicles are capable of taking up both glutamateand GABA. In this context, it is worth noting that inimmunoisolated vesicles expressing VGLUT1, GABAuptake is higher than in controls (Takamori et al. 2000a).Moreover, in glutamatergic synapses of live primary neurons,glutamatergic synaptic vesicles can be labeled with anantibody directed against the endovesicular domain ofVGAT, unambiguously demonstrating that the subpopulationof VGLUT1/VGAT-positive vesicles experience activerecycling.

Triple-labeling studies in vitro suggested that VGLUT1/VGAT co-expression occurs both at glutamatergic synapses,identified by VGLUT1-PSD-95 co-localization, and atGABAergic synapses, identified by VGAT-GABAAR. Post-embedding immunogold EM studies showed that AxTcontaining both VGLUT1 and VGAT formed either sym-metric or asymmetric synaptic contacts. Thus, the presentevidence points to the existence of cortical synapses in whichthe morphological features do not correlate entirely with thevesicular transporter expressed. The advent of techniquesenabling identification of neurotransmitters and their synthe-sizing enzymes, receptors, plasma membrane and vesiculartransporters has revealed that asymmetric, excitatory syn-apses use glutamate or aspartate as neurotransmitter andoriginate from principal cells, whereas symmetric, inhibitorysynapses use GABA and derive from aspiny interneurons.An obvious corollary is that glutamate and GABA arereleased by different sets of terminals originating fromdifferent sets of neurons. The demonstration that the mainvesicular GABA transporter is expressed at terminalsexpressing also VGLUT1 (present results; Kao et al. 2004;Safiulina et al. 2006), VGLUT2 (Ottem et al. 2004; Boul-land et al. 2009), or VGLUT3 (Hioki et al. 2004; Somogyiet al. 2004) indicates that the heterogeneity of cortical AxT isgreater than previously thought and opens new concepts inthe physiology of amino acidergic neurotransmission (Peterset al. 1991; see also Shepherd 1994).

Based on our understanding of the role of vesicularneurotransmitter transporters, the most likely interpretationof the present results is that glutamate and GABA are co-localized and may be co-released from this subpopulation ofnerve terminals. Indeed, in the brain regions where VGLUT1and VGAT are co-expressed, glutamate and GABA are co-localized (Conti et al. 1987; Sandler and Smith 1991; Kaoet al. 2004). As far as glutamate/GABA co-release isconcerned, there is evidence that it occurs in hippocampalmossy fibers both in epileptic animals and under physiolog-ical conditions (Walker et al. 2001; Gutierrez 2005; Safiulinaet al. 2006). In addition, highly purified GABAergic synap-tosomes from cerebral cortex also release glutamate uponchemical depolarization (Docherty et al. 1987). The immu-noisolation studies indicate that VGLUT1 and VGAT areexpressed in the very same synaptic vesicles; since theactivities of VGAT and VGLUT1 are not inhibited byglutamate and GABA, respectively (Bellocchio et al. 2000;McIntire et al. 1997), it is possible that glutamate and GABAare packaged in the same vesicles and are co-released uponexocytosis. Since co-localization of GABAA receptors withGluR1, NR1 or PSD-95 has been described (Rao et al.2000), it is possible that co-released glutamate and GABAact post-synaptically. Alternatively, one of the transmittersreleased by synaptic vesicles coexpressing both transportersacts post-synaptically, whereas the other acts pre-synapticallyor diffuse extrasynaptically.

Fig. 5 Synaptic vesicles expressing the VGAT in glutamatergic syn-

apses participate in the exo-endocytotic cycle. Cultured cortical neu-

rons were incubated in the presence of anti VGAT-ecto Ab (red) under

depolarizing conditions and counter-stained with antibodies against

VGLUT1 (green) or PSD-95 (blu). Arrow points to a glutamatergic

synapse (i.e. VGLUT1 ir facing PSD-95 ir), expressing VGAT recycling

vesicles. The synapse is enlarged and shown on different channels in

the insets. Scale bar: 5 lm; insets, 2 lm.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1538–1546� 2009 The Authors

1544 | G. Fattorini et al.

Alternative possibilities to co-release do however exist,and they are not less attractive. Although the activity of onevesicular transporter does not rule out the possibility that thesecond is also active, whether VGAT or VGLUT1 is active atvesicles expressing both VGLUT1 and VGAT dependsultimately on the level of substrate, i.e. glutamate or GABA.This would imply that regulating the level of the substrate(s)might switch the activity of one of the two transporters, thusmodifying the content of synaptic vesicles and the nature ofthe transmitter released. Such a mechanism could provide anadditional way of modifying the activity of cortical synapsesand the excitation/inhibition balance which sets the activityof neural networks.

Acknowledgements

This work was supported by grants from Ministero Istruzione

Universita e Ricerca (PRIN) to FC and FB, from Compagnia di San

Paolo (2005.1964) to MM and FB, Cariplo 2006.0779/10.9251 and

Fondazione Monzino to MM. We thank R. Edwards (UCSF),

R. Jahn (Gottingen), and M. Solimena (Dresden) for antibodies, and

R. Fesce (Varese) for helpful discussions.

Supporting information

Additional Supporting Information may be found in the online

version of this article:

Figure S1. Substitution of immune serum with normal serum

yielded sparse gold particles scattered across sections; note that gold

particles did not show any evident relationship to axon terminals

(AxT).

Figure S2. VGLUT1 and GAD65–67 co-localization in cortical

cultures.

Figure S3. The co-localization of VGLUT1 and VGAT in

glutamatergic and GABAergic vesicles is higher at early stages of

post-natal development.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing files) should be

addressed to the authors.

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