Exp Brain Res (2001) 136:523–534
DOI 10.1007/s002210000600
R E S E A R C H A RT I C L E
Linda Bergersen · Ola Wærhaug · Johannes Helm
Marion Thomas · Petter Laake · Andrew J. Davies
Mariangela C. Wilson · Andrew P. Halestrap
Ole P. Ottersen
A novel postsynaptic density protein: the monocarboxylate transporter
MCT2 is co-localized with δ-glutamate receptors
in postsynaptic densities of parallel fiber–Purkinje cell synapses
Received: 4 July 2000 / Accepted: 5 October 2000 / Published online: 15 December 2000
© Springer-Verlag 2000
Abstract Confocal immunofluorescence microscopy
showed strong monocarboxylate transporter 2 (MCT2)
labeling of Purkinje cell bodies and punctate labeling in
the molecular layer. By immunogold cytochemistry, it
could be demonstrated that the MCT2 immunosignal
was concentrated at postsynaptic densities of parallel
fiber–Purkinje cell synapses. The distribution of MCT2
transporters within the individual postsynaptic densities
mimicked that of the δ2 glutamate receptor, as shown by
use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of
other monocarboxylate transporters (MCT1 and MCT4).
The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the
synaptic cleft of the parallel fiber-spine synapses. The
postsynaptic density (PSD) molecules identified so far
can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we
provide evidence that this list of molecules must now be
extended to comprise an organic molecule transporter:
the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions reL. Bergersen · O. Wærhaug · J. Helm · M. Thomas
O.P. Ottersen (✉)
Department of Anatomy, Institute of Basic Medical Sciences,
University of Oslo, POB 1105 Blindern, 0317 Oslo, Norway
e-mail: o.p.ottersen@basalmed.uio.no
Tel.: +47-22851270, Fax: +47-22851299
A.J. Davies · M.C. Wilson · A.P. Halestrap
Department of Biochemistry, School of Medical Sciences,
University of Bristol, Bristol BS8 1TD, UK
P. Laake
Section of Medical Statistics, University of Oslo,
POB 1122 Blindern, 0317 Oslo, Norway
L. Bergersen · O. Wærhaug
The Norwegian University of Sport and Physical Education,
POB 4014 Ullevål Hageby, 0806 Oslo, Norway
lated to the synaptic cleft and that this transporter may
allow an influx of lactate derived from perisynaptic glial
processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.
Keywords Monocarboxylate transporters · Cerebellum ·
Parallel fiber-spine synapses · Immunocytochemistry ·
Glutamate receptors
Introduction
The monocarboxylate transporters (MCTs) are a family
of proton-linked transporters with a number of substrates, including branched-chain oxo acids derived from
leucine, valine, and isoleucine; the keton bodies acetoacetate, β-hydroxybutyrate, and acetate; as well as pyruvate and lactate (Halestrap and Price 1999). Lactate (cotransported with protons) is the most important substrate.
Several isoforms of MCT, all with 12 predicted transmembrane regions (Halestrap and Price 1999), have been
characterized with respect to function and tissue distribution. The picture that has emerged is that the kinetic
properties and substrate specificity of a given isoform
are related to the unique metabolic requirements of the
tissue in which it is localized (Halestrap and Price 1993,
1999) MCT1, -2, and -4 are the monocarboxylate transporters that have been studied most extensively. The first
of these was cloned from hamster ovary cells by Garcia
et al. (1994) and found to be widely expressed, with particularly high concentrations in the heart and red muscle.
The expression level in muscle was upregulated in response to work, suggesting a special role coupled to lactic-acid oxidation. The second isoform (MCT2) was isolated from a Syrian hamster liver library and was shown
to have a ~60% amino acid sequence identity with
MCT1 (Garcia et al. 1995). Later Jackson et al. (1997)
cloned a rat MCT2 homologue. MCT2 has a ten-fold
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higher affinity for substrates than MCT1 and MCT4 and
is found in cells where rapid uptake at low substrate concentrations is required, including proximal kidney-tubule
cells and sperm tails. By contrast, MCT4 is most evident
in white muscle and other cells with a high glycolytic
rate, such as tumor cells and white blood cells, suggesting that it is expressed at sites with a predominant lacticacid efflux (Halestrap and Price 1999).
Brain tissue is heterogeneous in terms of energy metabolism. Several lines of evidence indicate that there is
an exchange of energy substrates and metabolites between neurons and glia and that this cellular interaction
plays a critical role in the brain’s energy homeostasis
(Poitry-Yamate et al. 1995; Pellerin et al. 1998a;
Magistretti et al. 1999). However, the nature of this interaction has not been worked out in any detail. The aim
of the present study was to assess the distribution of
three major MCTs (MCT1, 2, and 4) in brain neuropil,
using the cerebellum as a model. Extrapolating from the
situation in peripheral tissues, we assumed that cellular
and subcellular heterogeneities in energy metabolism
would be reflected by differences in the complement and
expression levels of monocarboxylate transporters. We
show that MCT4 is mainly expressed by astrocytes,
while MCT2 is selectively expressed in Purkinje cell
spines, with an enrichment corresponding to the postsynaptic density (PSD). MCT1 was located in endothelial
cells, as described previously (Gerhart et al. 1997). The
accumulation of MCT2 at the PSD suggests that the
postsynaptic membrane is not only involved in mediating synaptic transmission, but that it also contributes to
transport of energy substrates and to the regulation of
lactate and proton concentrations in the synaptic cleft.
Materials and methods
Animals and preparation of tissues
Adult male Wistar rats (250–300 g; n=5, from Møllegaard, Ejby,
Denmark) were used. They had been allowed free access to food
and drinking water. All experiments were performed in accordance with the guidelines of the Norwegian Committee on Animal
Experimentation. The animals were killed with an intraperitoneal
injection of Equithesin (0.4 ml per 100 g b.w.) and subjected to
transcardiac perfusion with 2% dextran (MW 70,000) in 0.1 M sodium-phosphate buffer (PB; pH 7.4, 4°C, 15 s), followed by a
mixture of glutaraldehyde (0.1%) and formaldehyde (4%; freshly
depolymerized from paraformaldehyde) in the same buffer (room
temperature, 50 ml/min for 20 min). The brain was left in situ
overnight (4°C). Specimens from the cerebellum (lobule VI) were
isolated, cryoprotected in graded concentrations of phosphatebuffered glycerol, and rapidly frozen in liquid propane (–170°C)
in a cryofixation unit (Reichert KF80, Vienna, Austria). The specimens were transferred to 0.5% uranyl acetate dissolved in anhydrous methanol (–90°C) in a cryosubstitution unit (AFS;
Reichert). The temperature was raised stepwise to –45°C. The
samples were infiltrated with Lowicryl HM20 resin (Lowi, Waldkraiburg, Germany), and polymerization was induced by UV light
for 48 h. A detailed description of the procedure has been published (van Lookeren Campagne et al. 1991; Hjelle et al. 1994).
Expression of MCT2 in COS-cells
Expression of MCT2 in COS-cells was performed as previously
described (Kirk et al. 2000). In brief, the coding region of MCT2
was subcloned into a mammalian expression vector (Promega),
and the response plasmid used in the Tet-Off gene-expression
system (Clontech). The resulting constructs were expressed in
COS cells by a liposome-mediated transfection procedure. After
transfection, the cells were fixed and stained with the MCT2 antibodies for confocal microscopy. Western blots of membrane preparations were derived from transfected and nontransfected cells.
Immunoblots of total brain extracts
Proteins were isolated as previously described (Han et al. 1995;
McCullagh et al. 1996). Protein samples of cerebellum and forebrain were separated on 8% SDS-polyacrylamide gels (130 mA,
9 h). Proteins were then transferred to Immobilon polyvinylidene
difluoride membranes (100 V, 60 min). The membranes were incubated with the MCT2 antibody (0.1 µg/ml overnight), followed by
donkey anti-rabbit IgG conjugated to horseradish peroxidase
(Amersham, 0.1 µg/ml, 1 h). MCT2 was detected using the enhanced chemiluminescence method (Hyperfilm-ECL, Amersham).
Molecular-weight markers were included.
Immunofluorescence
Cerebellum was dissected out after fixation, as described above.
After fixation, the cerebellum was rinsed in PB and incubated sequentially in 10% (3 h), 20% (3 h), and 30% (overnight) sucrose
in PB for cryoprotection. The cerebellum was sectioned at
12–15 µm thickness on a cryostat. Sections were collected on gelatine-coated slides and stored at –20°C before use in indirect immunofluorescence experiments, which were carried out as described (Veruki and Wässle 1996). The antibodies were diluted
1:5000 (0.4 µg/ml; MCT1), 1:2000 (0.1 µg/ml; MCT2), 1:5000
(0.4 µg/ml; MCT4), and 1:2000 (1 µg/ml; δ2 glutamate receptor)
in 0.1 M PB with 3% normal goat serum, 1% bovine serum albumin, 0.5% Triton-X-100, and 0.05% sodium azide, pH 7.4. The
primary antibodies were revealed by a carboxymethylindocyanine
(Cy3)-coupled secondary antibody (1:1000, Jackson Immuno Research Laboratories, West Grove, Penn., USA). Secondary antibodies were diluted in the same solution as the primary antibodies.
Cerebellar sections were viewed with a confocal microscope
(Leica TCS SP, Tektronix Phaser 440, dye sublimation).
Postembedding immunogold cytochemistry
Ultrathin sections were mounted on nickel grids and processed for
immunogold cytochemistry as described by Matsubara et al.
(1996). Briefly, the sections were treated with saturated solutions
of NaOH in absolute ethanol (2–3 s), rinsed, and incubated sequentially in; (1) 0.1% sodium borohydride and 50 mM glycine in
Tris buffer containing 0.05 M NaCl and 0.1% Triton X-100
(TBNT); (2) 2% human serum albumin (HSA) in TBNT; (3) antibodies to MCT1 (dilution: 2 µg/ml), MCT2 (dilution: 1 µg/ml),
MCT4 (dilution: 2 µg/ml), δ2 glutamate receptor (dilution:
1 µg/ml), GluR2/3 (dilution: 2 µg/ml), in TBNT and 2% HSA; (4)
2% HSA in TBNT; and (5) goat anti-rabbit immunoglobulins coupled to 10-nm gold particles (Amersham, Arlington Heights, Ill.,
USA) and diluted 1:20 in TBNT with 2% HSA and 5 mg/ml polyethyleneglycol. In double-labeling experiments (Ottersen et al.
1992), the sections were first treated with antibodies against
MCT2 (dilution: 1 µg/ml) and, then, with antibodies against δ2
glutamate receptors (dilution: 1 µg/ml). Formaldehyde vapor
(80°C, 1 h) was used between the sequential incubations to prevent interference (Wang and Larsson 1985). The MCT2 and δ2glutamate receptors were distinguished by means of different gold
particle sizes (10 nm for MCT2 and 15 nm for the δ2 receptor).
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at least 200 nm in pre- and postsynaptic directions from the
middle of the postsynaptic membrane were recorded. The length
of the synaptic profiles, the thickness of the postsynaptic densities,
and the width of the synaptic clefts were measured
Statistics
Fig. 1 Upper part: Sequence alignment of the C-termini of different monocarboxylate transporter (MCT) isoforms and proteins
known to be associated with postsynaptic specializations of parallel fiber–Purkinje cell synapses. [Kir2.1 is also included; this K+
channel has been demonstrated in Purkinje cells, but its synaptic
localization remains to be determined (Miyashita and Kubo
1997)]. Lower part: MCT4 and MCT1 (bottom part of figure)
have extrasynaptic localizations in the molecular layer of the cerebellum. The terminal TXV/SXV sequence of the latter MCT isoforms is known to interact with PSD 95 in other systems. PSD 95
is not expressed by Purkinje cells
The sections were examined in a Philips CM10 transmission electron microscope.
Antibodies
Antibodies against rat MCT1, MCT2, and MCT4 were raised in
New-Zealand white rabbits against C-terminal peptides, conjugated to keyhole limpet haemocyanin (KLH), and subjected to affinity purification as described previously (Poole et al. 1996; Jackson
et al. 1997; Wilson et al. 1998). The peptide sequence used for immunization was CPQQNSSGDPAEEESPV for MCT1, NTHNPPSDRDKESSI for MCT2, and CEPEKNGEVVHPPETSV for
MCT4. A search in the Swiss-Prot sequence database revealed no
proteins with significant homologies to the peptide sequence used
for immunization (Fig. 1). The δ2-glutamate-receptor antibody
was raised against a synthetic peptide QPTPTLGLNLGNDPDRGTSI, corresponding to the C-terminus of the rat δ2-receptor
subunit (Mayat et al. 1995). This antibody also recognizes the δ1receptor subunit, but does not label other glutamate receptors, including α-amino-3-hydoxy-5-methyl-4-isoxazole propionic acid
(AMPA) and N-methyl-D-aspartate (NMDA) receptors (Mayat
et al. 1995). The AMPA receptor antibody (Ab25; Wenthold et al.
1992) used for control reacts with GluR2 as well as GluR3.
Quantitative analysis
Ultrathin sections were either singly labeled with 10-nm gold particles to reveal the localization of MCT2, or double labeled with
10-nm gold particles and 15-nm gold particles for simultaneous
localization of MCT2 and δ2 receptors, respectively. Synapses
throughout the different layers of the cerebellum were identified in
the electron microscope according to morphological criteria (Palay
and Chan-Palay 1974). Transversely cut synaptic profiles with
clearly visible postsynaptic membrane and postsynaptic density
were selected for photography, and quantitative analysis was performed on photomicrographs magnified ×63 000, ×86 250, or
×97 500. At synapses between parallel fibers and Purkinje cell
spines, the positions of gold particles were determined along two
axes; an axis perpendicular to the postsynaptic membrane and an
axis tangential to this. All particles lying within a field limited by
The data were entered in Statview v.4.5 (Abacus Concept) and
Systat v.9.0 (SPSS) for statistical treatment. The analysis was performed only on synaptic profiles longer than 150 nm (Landsend et
al. 1997) To ensure that all immunoreactivity possibly related to
the synapse was detected, particles occurring within 50 nm of the
middle of the postsynaptic membrane and within 50 nm of the peripheral edge of the PSD were included (Matsubara et al. 1996). In
addition to routine descriptive statistics, two-dimensional q-q plots
and two-sample Kolmogorov-Smirnov nonparametric tests were
used to compare the distribution of gold particles and to examine
the significance of difference between the distributions. The level
of significance was determined to be 0.05.
A q-q plot is a graphical display where the quantiles of the distributions of the two variables are plotted in two dimensions. If the
two distributions are equal, the two quantiles will lie on a straight
line. Discrepancies from a straight line will point to specific differences in the distributions. The two-sample KolmogorovSmirnov test gives an overall comparison of the two distributions.
The comparison is based on the maximum of the vertical distances
between the two distributions and gives no specific indication of
where the differences between the distributions are.
Control experiments
In addition to the transfection experiments (see above), control experiments included replacement of the antibodies with nonimmune IgG or pre-adsorption with the peptide used for immunization. Such controls were performed for Western blot, immunofluorescence, and electron-microscopic experiments.
Results
Cells that had been transfected with MCT2 showed
strong immunolabeling with the MCT2 antibody, whereas control cells were unlabelled (Fig. 2A). Immunoblots
of membrane preparations from the transfected cells
showed a single band (Fig. 2B).
The blots that had been prepared from membrane
preparations of rat brain, liver, and kidney (Fig. 2C) also
showed single bands, except for a weak additional band
in the kidney fraction. The latter band had a molecular
weight of about 80 kDa, suggesting that it was due to dimerization. The results obtained with our own antibody
were compared with those obtained with the antibody of
Gerhart et al. (1998). The latter antibody produced some
extra bands, notably in the brain and liver (Fig. 2C).
Immunoblots of total brain extracts (Fig. 3) revealed a
single, strong MCT2-immunoreactive band with an approximate MW of about 40 kDa in both the cerebellum
and forebrain. This corresponds to the estimated size of
the cloned MCT2 transporter (Jackson et al. 1997). A
weak band was observed at a higher molecular weight
(Fig. 3). The peptide block of the MCT2 antibody resulted in no significant labeling (data not shown)
Confocal immunofluorescence microscopy showed
MCT2 labeling of Purkinje cell bodies (Fig. 4). There
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Fig. 2A–C Control experiments.
A Confocal microscopy of
COS-7 cells transiently transfected with rat monocarboxylate
transporter 2 (MCT2), as described in Kirk et al. (2000).
Non-transfected cells (C) and
transfected cells (T) are shown in
the same field. B Western blot of
membrane preparation derived
from transfected (T) and nontransfected (C) cells. C Western
blot of membrane preparations
derived from rat brain (B), liver
(L), and kidney (K) probed with
our own antibody (Halestrap Ab)
and with the antibody of Gerhart
et al. (1998) (Chemicon Ab)
was also distinct labeling in the molecular layer, which
was punctate or confluent depending on the intensity of
the immunoreaction (Fig. 4A, C). No labeling was found
in the interstices between the Purkinje cells, corresponding to the location of the Golgi epithelial cells. The labeling in the granule-cell layer was not completely suppressed by absorption with the immunogenic peptides
and may partly represent a background signal. Antibodies to the δ2-glutamate receptor produced an almost
identical labeling pattern to MCT2 (Fig. 4E). The
Purkinje-cell labeling for MCT2 and δ2-glutamate receptor was abolished by absorption with the respective immunogenic peptides (Fig. 4), while cross-adsorption
(anti-MCT2 adsorbed with δ2 receptor peptide and vice
versa) had no effect on the pattern or intensity of the immunostaining (not shown).
By immunogold cytochemistry, it could be shown that
the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber–Purkinje cell synapses
(Fig. 5A–C). Gold particles were distributed along the
entire mediolateral extent of the PSDs (Fig. 6), and very
few particles were associated with the spine plasma
membrane lateral to these. The distance between gold
particles and the postsynaptic membrane varied, but an
analysis of the perpendicular distribution of gold particles revealed a distinct peak corresponding to the PSD
(Fig. 7). Climbing-fiber synapses were not labeled
(Fig. 5E), nor were mossy fiber synapses in the granule
cell layer (Fig. 5D). Gold particles were frequently observed within Purkinje-cell dendrites (Fig. 5E).
The immunogold labeling pattern of MCT2 was compared with that of the δ2-glutamate receptor, which has
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Fig. 3 Immunoblots of total
brain extracts from the cerebellum (cb, right lane) and
forebrain (fbr, left lane;
50 µg/lane). The antibody to
monocarboxylate transporter
2 (MCT2) produced a single
band at the appropriate molecular weight, about 40 kDa, and a
weak band at higher molecular
weight
been shown to be selectively localized to parallel fiber–spine synapses (Landsend et al. 1997). The two proteins were revealed in the same ultrathin sections by use
of two different gold-particle sizes. Confirming previous
data, immunoparticles signaling the δ2-glutamate receptor were virtually restricted to the PSDs (Fig. 8C, D),
and the gold-particle density analysis revealed a peak coinciding with that of MCT2 (Fig. 7). The tangential distribution was also similar between the two particle sizes
(Fig. 6). Antibodies to the AMPA receptors, GluR2/3,
produced labeling of parallel fiber–spine synapses with a
gold-particle distribution mimicking that of MCT2 and
δ2 glutamate receptor (Fig. 8A). In agreement with previous results, GluR2/3 also occurred in climbing-fiber
and mossy-fiber synapses (not illustrated), i.e., at sites
that were not labeled with the MCT2- or δ2-glutamatereceptor antibody. No MCT2 immunogold signal was
found after adsorption with the immunizing peptide
(Fig. 8B).
Since the MCT2- and δ2-glutamate-receptor immunogold patterns appeared to be closely similar, the two
gold-particle distributions were compared more directly
by use of q-q plots (Fig. 9). If the two distributions are
equal, the two quantiles must lie on a straight line. Only
small and insignificant deviations from a straight line
were observed, irrespectively of whether the analysis
Fig. 4 Micrographs of monocarboxylate-transporter 2 (MCT2)
(A, C) and δ2-receptor (E) immunofluorescence in coronal sections through the rat cerebellum. The MCT2 and δ2-receptor antibodies labeled the Purkinje cell bodies (p) and neuropil, particularly in the molecular layer (m). The MCT2 immunolabeling was
abolished by pre-adsorption with the immunizing peptide (B, D).
Experimental and control sections are shown from two different
experiments (A and B, C–E, respectively) with different staining
intensities. g Granule cell layer, w white matter. Scale bar 100 µm
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Fig. 5 A–C Distribution of
monocarboxylate-transporter
2 (MCT2) immunoreactivity
at synapses between parallel
fibers (pf) and Purkinje cell
spines (s). Asterisks indicate
glial lamellae (A and C). Gold
particles occurred along the entire postsynaptic density (delimited by arrowheads; A, B.
m mitochondrion). D Synapses
of mossy fiber terminal (mf) are
devoid of labeling. d Granule
cell dendritic digits. Arrowheads indicate extent of postsynaptic density. E Synapses
between climbing fibers (cf)
and Purkinje cell dendritic
spines (s) are unlabelled (inset)
or associated with a maximum
of one gold particle. Note attachment plaques (open arrows)
between climbing-fiber terminal and Purkinje-cell dendritic
stem. The latter profile and the
interior of spines contain scattered gold particles (arrowheads). Scale bars 200 nm
was performed along a vertical axis (perpendicular to the
synaptic specialization; cf. Fig. 7) or along a tangential
axis (cf. Fig. 6).
The MCT2 distribution was also compared with the
distributions of other monocarboxylate carriers (Fig. 10).
The MCT1 immunofluorescence signal was localized in
the endothelial cells (Fig. 10A), while MCT4 immunolabeling was found mainly in slender processes (Fig. 10D)
that were identified as glial profiles in the electron microscope (Fig. 10B). The parallel fiber–spine synapses
were devoid of gold particles signaling MCT4 (Fig. 10B)
or MCT1 (not shown). The monocarboxylate carrier
MCT3 (Philp et al. 1998) is thought to be restricted to
the retina and was not examined here.
Discussion
Excitatory synapses in the CNS are characterized by a
zone of increased electron density just underneath the
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Fig. 6A–C Distribution of gold particles along the postsynaptic
densities (PSDs) of parallel fiber–spine synapses. Each bin
corresponds to 20% of the PSD radius, as seen in a single profile.
Zero corresponds to the midpoint of the profile, 100% to its lateral
margin. Gold particles lateral to the PSD (bin value >100%) may
represent epitopes within the PSD, since epitope and gold par-ticle may be separated by up to 20 nm (Matsubara et al. 1996).
Only profiles longer than about 150 nm were included to ensure
that they represented central as well as peripheral parts of the synapse. A Single labeling of monocarboxylate transporter MCT2.
B, C Labeling of MCT2 (B) and δ2 receptor (C) in double-labeled
sections. Number of particles (n) are indicated. The data in A are
obtained from three animals and 72 synapses, in B and C from
two animals and 40 synapses
Fig. 7 Distribution of gold particles at the parallel fiber–spine synapses. Particles were recorded along an axis perpendicular to the
postsynaptic density (PSD). Values along the abscissa represent distance (in nanometers) between the center of gold particles and the
midpoint of the postsynaptic membrane (negative values denote
postsynaptic direction) A Single labeling of monocarboxylate transporter 2 (MCT2). B, C Labeling of MCT2 and δ2 receptor in double-labeled sections. The average widths of postsynaptic densities
(psd) and synaptic clefts (sc) are indicated. n indicates number of
gold particles. The data in A were obtained from three animals and
81 synapses, those in B and C from two animals and 44 synapses
postsynaptic membrane. This zone – designated the postsynaptic density (PSD) in early electron-microscopic
studies – was purified in the early 1980s. The first protein to be identified as a component of the PSD was
CaMKII (Goldenring et al. 1983; Kennedy et al. 1983),
but subsequent studies have revealed that the PSD of excitatory synapses contains several types of ionotropic
glutamate receptor as well as molecules responsible for
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Fig. 8A–D Electron micrographs showing immunogold
labeling at synapses between
parallel fiber terminals (pf) and
Purkinje cell spines (s). The
sections were labeled with antibodies to GluR2/3 (A), antibodies to monocarboxylatetransporter 2 (MCT2) and δ2
receptors (C, D), and antibody
to MCT2 following absorption
with the immunizing peptide
(B). In the double-labeled preparations, the size of gold particles were 15 nm for δ2 receptor
and 10 nm for MCT2. m Mitochondrion. Arrowheads indicate gold particles. Scale bars
200 nm
the anchoring of these (reviews: Kennedy 1997, 1998;
Hsueh and Sheng 1998).
The picture that has emerged over the last few years
is that these anchoring molecules serve as scaffolds, not
only for the receptors themselves, but also for molecules
that interact with the cytoskeleton and for molecules that
mediate downstream effects of glutamate receptors
(Craven and Bredt 1998). The multifunctionality of the
anchoring molecules is dependent on their contents of
several binding motifs. Notably, anchoring molecules
with an affinity to ionotropic glutamate receptors contain
a number of PDZ domains, each of which has preference
for a distinct molecule species (Sheng and Pak 1999).
The existence of specific anchoring proteins helps explain the precise compartmentation of glutamate receptors along the neuronal plasma membrane. Glutamate receptors are enriched up to several hundred fold in postsynaptic membranes facing glutamate-containing terminals (Landsend et al. 1997; Nusser et al. 1998). The
specificity extends further, in as much as some glutamate
receptors have been shown to be targeted to specific subpopulations of glutamate synapses on individual postsynaptic cells. This is the case for the δ2 glutamate recep-
tors, which cluster at the parallel fiber synapses onto
Purkinje cell spines, but are absent from climbing-fiber
synapses on the same cells (Landsend et al. 1997; Zhao
et al. 1997). PSD 93 was recently found to be responsible for the anchoring of the δ2 glutamate receptor
(Roche et al. 1999). In agreement, the δ2 glutamate receptor has a terminal TXI sequence that is known to favor binding to PSD 93 (Roche et al. 1999).
Here, we provide evidence that the monocarboxylate
carrier MCT2 is co-expressed with the δ2 glutamate receptor, at the synaptic as well as at the subsynaptic level.
MCT2 is, thus, the first example of an organic molecule
transporter that is enriched in the PSD. MCT2 has a terminal SXI sequence similar to the δ2 receptor, suggesting that it could be anchored to one of the PDZ domains
of PSD 93. In support of this view, a detailed analysis of
the tangential and perpendicular distribution of immunogold particles (signaling MCT2 and δ2 receptor, respectively) revealed almost identical distribution patterns.
Notably, the particle distribution peaked at ~10 nm postsynaptically in the case of MCT2 and ~8 nm postsynaptically in the case of δ2 receptor. This suggests that the
C-termini of the two proteins are attached at about the
531
Fig. 9 A series of q-q plots of gold-particle distributions along the
perpendicular (A) or tangential (B) axis of parallel fiber–spine
synapses (cf. Figs. 6 and 7). The distribution of monocarboxylatetransporter 2 (MCT2) immunogold particles in single-labeled
preparations (MCT2-single) was compared with that of MCT2 or
δ2 immunogold particles in double-labeled preparations (MCT2dual or δ2-dual). The latter two types of particle were compared
as well. If the two distributions are equal, the two quantiles will lie
on a straight line. Note that the difference between MCT2 and δ2
is no greater than that between MCT2-single and MCT2-dual
same depth within the postsynaptic specialization and,
possibly, to the same anchoring molecule.
The antibody used in the present study was raised
against the C-terminal 15 amino acids of MCT2, isolated
from rat testis (Jackson et al. 1997). The same pattern of
labeling, judged by light microscopy, was produced by
an antibody raised to a GST fusion protein containing
the last 53 amino acids of MCT2 (not shown). Using
RNA probes transcribed from a mouse cDNA sequence
that was about 90% identical to that of Jackson et al.
(1997), it was shown by in situ hybridization that MCT2
mRNA is strongly expressed in Purkinje cells (KoehlerStec et al. 1998). This finding is in agreement with the
present immunocytochemical data.
On this background, it needs to be explained why another antibody, raised against the same 15 C-terminal
amino acids, produced immunolabeling in glial cells, but
not in Purkinje cells of the rat cerebellar cortex (Gerhart
et al. 1998). We have confirmed this result in our own
laboratory. Why the two antibodies produce different labeling patterns is unknown, although it should be pointed out that the antibody of Gerhart et al. (1998) revealed
an extra, low-molecular weight band, and a relatively
weak band at the appropriate molecular weight for the
MCT2 monomer (Fig. 2). One must also consider the
possibility that the two antibodies recognize different
parts of the C-terminal amino-acid sequence. The phosphorylation status of this sequence (which includes several serine residues and one threonine residue; cf. Fig. 1)
may differ between glial and Purkinje cells and differentially affect antibody binding. The same mechanism may
underlie the discrepant findings in heart, skeletal muscle,
and retina, which were labeled with the antibody of
Gerhart et al. (1998), but not with that of Jackson et al.
(1997). In situ hybridization analyses have also produced
different patterns of labeling, depending on the probe
sequence (cf. Koehler-Stec et al. 1998 with Pellerin et al.
1998b).
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Fig. 10 Comparison between the immunofluorescence patterns
for monocarboxylate transporter 1 (MCT1) (A), MCT2 (C), and
MCT4 (D). MCT4 occurred in slender processes, which were
identified as astrocyte profiles (asterisks) by immunogold electron
microscopy (B). MCT1 was concentrated in the endothelial cells.
Abbreviations as in Fig. 4. Scale bar 50 µm in A, C, D and
200 nm in B
On the basis of the above data, one cannot exclude
the possibility that the glial cells express a pool of
MCT2 that is not visualized by the Jackson antibody
used here. However, our findings suggest that MCT4 is a
major monocarboxylate transporter in cerebellar astrocytes, while MCT2 is the predominant monocarboxylate
transporter in Purkinje cells. The possibility remains that
both cell types contain additional transporters, which
were not included among those that were investigated in
the present study.
MCT4 is known to be responsible for the export of
lactate from glycolytic muscle fibers (Juel and Halestrap
1999) and may have an analogous function in the brain.
There is ample evidence that there is a flux of lactate
from glial cells to neurons (Pellerin et al. 1998a). MCT4
and MCT2 could act in sequence to facilitate lactate
transport between the Bergmann glial processes and the
Purkinje cells that they ensheath. The Purkinje cell dendrites have an extraordinary high density of mitochondria (Palay and Chan-Palay 1974), pointing to an active
oxidative metabolism, and the high affinity of MCT2 for
lactate (Broer et al. 1999) make it ideally suited for uptake of this metabolic fuel. The surrounding glial pro-
cesses, in contrast, contain few mitochondria and may
depend largely on glycolytic pathways.
Glutamate uptake is coupled to a stoichiometric uptake of H+ (Danbolt 1994). This will rapidly create a deficit of H+ in the synaptic cleft, which has to be compensated for by outward movement of H+ from surrounding
cellular compartments. In the case of glia, the extracellular deficit of H+ could energize outward lactate/H+ transport, through MCT4, essentially leading to an exchange
of glutamate with lactate across the plasma membrane. It
has also been proposed that part of the glutamate could
be metabolically converted to lactate after uptake into
astrocytes (Hassel and Sonnewald 1995) and that glial
export of lactate would be favored by the increased glycolytic rate imposed by glutamate uptake (Pellerin and
Magistretti 1994, 1997). Lactate derived from glia could
be an important metabolic substrate for neurons (reviewed by Magistretti et al. 1999).
In the case of the Purkinje cells, the extracellular H+
deficit could drive the Na+/H+ exchange. Neurons are
known to express several subtypes of Na+/H+ antiporters
(Attaphitaya et al. 1999), and one of these (NHE3) is almost exclusively localized to Purkinje cells (Ma and
Haddad 1997). Exchange of H+ for Na+ could allow
MCT2 to operate in the inward direction (i.e., along the
supposed concentration gradient for lactate), allowing
the glial-derived lactate to be accumulated in the dendritic spines and stems.
If MCT2 serves as a point of entry of an energy substrate, why is it concentrated at the synapse? The spatial
533
coupling between glutamate exocytosis, glutamate uptake, and lactate transport provides a unique way of adjusting energy supply to excitatory drive. Uptake of glutamate by the glutamate transporter EAAT1, which is enriched at those astrocyte-membrane domains that face
the parallel fiber–spine synapse (Chaudhry et al. 1995),
will directly and indirectly promote lactate release
through MCT4, which is enriched in the same membrane
domains (cf. Fig. 10B). Released lactate will then be
transported into the Purkinje-cell spines by the synaptic
MCT2 molecules. Thus, the more glutamate that is released from the parallel fibers, the more lactate will be
released from the glial processes and be available for
postsynaptic uptake. Glutamate – itself an energy substrate – may also be taken up directly into the postsynaptic Purkinje cells, through EAAT4 (which is concentrated
in the spine membrane lateral to the PSD; Dehnes et al.
1998) and through EAAT3 (demonstrated in Purkinje
cells by enzyme-based immunocytochemistry; Rothstein
et al. 1995). We are thus beginning to realize that the
synapse not only serves as a site of interneuronal signal
transfer – it may also act as an important site of metabolic coupling.
Acknowledgements We thank Bjørg Riber, Karen M. Gujord,
Gunnar Lothe, Carina Knudsen, and Kari Ruud for technical assistance, and Dr. M. Krestchatisky for helpful discussions. This work
was supported by the Norwegian Research Council and Professor
Letten F. Saugstad’s Fund.
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