Elsevier Editorial System(tm) for Neuroscience Manuscript Draft Manuscript Number: NSC-12-578R2

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Elsevier Editorial System(tm) for Neuroscience
Manuscript Draft
Manuscript Number: NSC-12-578R2
Title: Pre- and postsynaptic localization of NMDA receptor subunits at hippocampal mossy fibre
synapses
Article Type: Research Paper
Section/Category: Cellular and Molecular Neuroscience
Keywords: electron microscopy, immunocytochemistry, rat, nerve terminal, postsynaptic density
Corresponding Author: Dr. Vidar Gundersen, PhD
Corresponding Author's Institution: University of Oslo
First Author: Lars Krogh-Berg
Order of Authors: Lars Krogh-Berg; Max Larsson; Cecilie Morland; Vidar Gundersen, PhD
Abstract: The NMDA type of glutamate receptors is involved in synaptic plasticity in hippocampal
mossy fibre-CA3 pyramidal neuron synapses. The ultrastructural localization of NMDA receptor
subunits at this synapse type is not known. By postembedding electron microscopic immunogold
cytochemistry we show that the NMDA receptor subunits GluN1, GluN2A, GluN2B, GluN2C and GluN2D
are located in postsynaptic membranes of mossy fibre as well as CA3 recurrent associational
commissural synapses. In the mossy fibres the GluN1, GluN2B and GluN2D labelling patterns suggested
that these subunits were located also presynaptically in nerve terminal membranes and in mossy fibre
axons. GluN3B was predominantly present in mossy fibre synapses as compared to recurrent
associational commissural synapses, showing a presynaptic labelling pattern. In conclusion, while the
postsynaptic localization of GluN1, GluN2A, GluN2B, and GluN2D is in good agreement with the recent
finding of NMDA receptor dependent long term potentiation at CA3 mossy fibre synapses, we propose
that presynaptic GluN1, GluN2B, GluN2D and GluN3B subunits could be involved in plastic phenomena
such as certain types of long term potentiation and recurrent mossy fibre growth.
Response to Reviewers: RESPONSE TO REVIEWER
Reviewer #1
I have read through the paper. While I don't have any additional concerns, I still see problems related
to the points that I made in my first review and the authors' responses.
I will discuss these points here:
Concerning antibody specificity, it is not clear what the authors mean by "shadows" on blots .I
consulted with an expert on Western blots and that person also did not know about "shadows." Unless
the authors can provide evidence in the literature for this, I would assume that these light bands are
some other protein or a breakdown product or variant of the specific protein. Also, GluN2A and
GluN2B antibodies often show some cross reaction, depending on the part of the protein from which
the antibodies are made. Testing this is a fairly simple procedure: the subunits are transfected into
heterologous cells and these are run on a Western blot. In any case, the authors do have that notable
difference in labeling of the mossy fiber axons. But they need to acknowledge the problems with the
antibodies in the methods - both the possibilities of some other protein and of cross-reaction - or
provide specific publications where these things were tested.
All GluN antibodies produced strong bands at the appropriate molecular weights. However, some of
the GluN antibodies produced some very weak additional bands. We agree that we cannot rule out the
possibility that some of the very weak bands could represent labelling of cross-reactive proteins or
breakdown products of specific proteins, but the labeling could also represent light unspecific
attachment of the antibodies to sites with high protein concentration on the blot.
Based on the referee’s comment we have included a discussion of these points in the manuscript (p. 11,
last para). We replaced “.. produced single bands.., with “… produced strong staining of bands at..”, and
added “For some of the GluN subunit antibodies there were some very weak additional bands on the
Western blots, which may represent light background staining or breakdown products of the specific
proteins”.
Searching the literature we could not find any description of cross-reactivities of GluN2A and GluN2B
antibodies. Hence, we have not commented on this in the manuscript.
As Western blotting has much higher sensitivity than postembedding immunogold labeling, we would
like to point out that the very weak additional bands as detected on the Western blots should not bring
about any significant unspecific labeling when the antibodies were used in the immunogold method.
Concerning my concerns about overanalyzing the data, the authors state on p. 13 that in rA/C
synapses, a GluN2D labeling was most evident. That implies that GluN1 should be there also, based on
what is known about NMDARs. Also, the authors show modest evidence for presynaptic labeling for
GluN1 in rA/C in figure 4. The bottom line is that the distinctions in pre and post synaptic distribution
shown in figure 4 do not look that definitive, and the authors need to acknowledge this.
Based on the referee’s comment we have modified the description of the distribution of pre-and postsynaptically located receptors (p. 13, bottom). As the distribution of none GluN subunits across the
rA/C synapse were significantly different from the distribution of GluN2C, we deleted the sentence
“Across the rA/C synapses a presynaptic gold particle distribution was most evident for GluN2D (Fig.
4)”, and instead added “Although across the rA/C synapse gold particles for GluN1 and GluN2D showed
a tendency to be located at the presynaptic side, at the rA/C synapse there were no significant
differences between the GluN2C distribution and the other subunit distributions (Fig. 4)”.
Concerning the immunogold examples, the new figure 2 is a definite improvement over the old one.
We thank the referee for appreciating the new fig. 2.
Concerning the lateral resolution of 30 nm, the authors changed their citation from Bergersen et al.,
2008 to 2012 in the methods but did not change it in the results on p. 12. They also have not included
the 2012 reference in their reference list.
We have now corrected this.
Concerning my suggestion to rewrite the sentence beginning with "Despite," I made a mistake when I
said that it was in the last paragraph of the Discussion - it is in the last paragraph of the Results. I
mainly meant that it is grammatically incorrect. It should be rewritten.
The first part of sentence was redundant, and thus deleted.
Cover Letter
Dear Prof Hirsch / Prof Witter,
We resubmit our ms with ms no NSC-12-578R1.
We have made a point-to-point answer to the comments raised by the referee and
changed the text accordingly.
We now hope that the ms is suitable for publication in Neuroscience.
Looking forward to hearing from you soon.
Kind regards
Vidar Gundersen
(on behalf of all the authors)
Neurologist, Associate Professor, MD, PhD
Department of Anatomy and the CMBN
University of Oslo
POB 1105 Blindern
0317 Oslo
Norway
tel: 47 22 85 14 96
fax: 47 22 85 12 78
e-mail: [email protected]
and
Department of Neurology
Oslo University Hospital, Rikshospitalet
POB 4950 Nydalen,
0424 OSLO
Norway
*Response to Reviews
RESPONSE TO REVIEWER
Reviewer #1
I have read through the paper. While I don't have any additional concerns, I still see problems related to the
points that I made in my first review and the authors' responses.
I will discuss these points here:
Concerning antibody specificity, it is not clear what the authors mean by "shadows" on blots .I consulted with
an expert on Western blots and that person also did not know about "shadows." Unless the authors can
provide evidence in the literature for this, I would assume that these light bands are some other protein or a
breakdown product or variant of the specific protein. Also, GluN2A and GluN2B antibodies often show some
cross reaction, depending on the part of the protein from which the antibodies are made. Testing this is a fairly
simple procedure: the subunits are transfected into heterologous cells and these are run on a Western blot. In
any case, the authors do have that notable difference in labeling of the mossy fiber axons. But they need to
acknowledge the problems with the antibodies in the methods - both the possibilities of some other protein
and of cross-reaction - or provide specific publications where these things were tested.
All GluN antibodies produced strong bands at the appropriate molecular weights. However,
some of the GluN antibodies produced some very weak additional bands. We agree that we
cannot rule out the possibility that some of the very weak bands could represent labelling of
cross-reactive proteins or breakdown products of specific proteins, but the labeling could also
represent light unspecific attachment of the antibodies to sites with high protein concentration
on the blot.
Based on the referee’s comment we have included a discussion of these points in the
manuscript (p. 11, last para). We replaced “.. produced single bands.., with “… produced
strong staining of bands at..”, and added “For some of the GluN subunit antibodies there were
some very weak additional bands on the Western blots, which may represent light background
staining or breakdown products of the specific proteins”.
Searching the literature we could not find any description of cross-reactivities of GluN2A and
GluN2B antibodies. Hence, we have not commented on this in the manuscript.
As Western blotting has much higher sensitivity than postembedding immunogold labeling,
we would like to point out that the very weak additional bands as detected on the Western
blots should not bring about any significant unspecific labeling when the antibodies were used
in the immunogold method.
Concerning my concerns about overanalyzing the data, the authors state on p. 13 that in rA/C synapses, a
GluN2D labeling was most evident. That implies that GluN1 should be there also, based on what is known
about NMDARs. Also, the authors show modest evidence for presynaptic labeling for GluN1 in rA/C in figure 4.
The bottom line is that the distinctions in pre and post synaptic distribution shown in figure 4 do not look that
definitive, and the authors need to acknowledge this.
Based on the referee’s comment we have modified the description of the distribution of preand post-synaptically located receptors (p. 13, bottom). As the distribution of none GluN
subunits across the rA/C synapse were significantly different from the distribution of GluN2C,
we deleted the sentence “Across the rA/C synapses a presynaptic gold particle distribution
was most evident for GluN2D (Fig. 4)”, and instead added “Although across the rA/C synapse
gold particles for GluN1 and GluN2D showed a tendency to be located at the presynaptic side,
at the rA/C synapse there were no significant differences between the GluN2C distribution
and the other subunit distributions (Fig. 4)”.
Concerning the immunogold examples, the new figure 2 is a definite improvement over the old one.
We thank the referee for appreciating the new fig. 2.
Concerning the lateral resolution of 30 nm, the authors changed their citation from Bergersen et al., 2008 to
2012 in the methods but did not change it in the results on p. 12. They also have not included the 2012
reference in their reference list.
We have now corrected this.
Concerning my suggestion to rewrite the sentence beginning with "Despite," I made a mistake when I said that
it was in the last paragraph of the Discussion - it is in the last paragraph of the Results. I mainly meant that it is
grammatically incorrect. It should be rewritten.
The first part of sentence was redundant, and thus deleted.
Graphical Abstract (for review)
Click here to download high resolution image
*Highlights (for review)
HIGHLIGHTS
NMDA glutamate receptor subunits are located in mossy fibres
Most of the NMDA receptor subunits are present in the postsynaptic membrane
GluN1, GluN2B, GluN2D and GluN3B have a presynaptic localisation
*Manuscript
Click here to view linked References
Pre- and postsynaptic localization of NMDA receptor subunits at hippocampal mossy
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fibre synapses
Lars Krogh Berg1¶, Max Larsson1,3¶, Cecilie Morland1, Vidar Gundersen1,2*
1.Department of Anatomy, Institute of Basic Medical Sciences, and Centre for Molecular
Biology and Neuroscience (CMBN), University of Oslo, Norway.
2.Department of Neurology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
3.Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
¶
These authors contributed equally to the work.
*Correspondence and requests for materials should be addressed to V.G.: Department of
Anatomy and the CMBN, University of Oslo, POB 1105 Blindern, 0317 Oslo, Norway.
Telephone: 47 22851496. Fax: 47 22851278. Email: [email protected]
ABSTRACT
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The NMDA type of glutamate receptors is involved in synaptic plasticity in hippocampal
mossy fibre-CA3 pyramidal neuron synapses. The ultrastructural localization of NMDA
receptor subunits at this synapse type is not known. By postembedding electron microscopic
immunogold cytochemistry we show that the NMDA receptor subunits GluN1, GluN2A,
GluN2B, GluN2C and GluN2D are located in postsynaptic membranes of mossy fibre as well
as CA3 recurrent associational commissural synapses. In the mossy fibres the GluN1,
GluN2B and GluN2D labelling patterns suggested that these subunits were located also
presynaptically in nerve terminal membranes and in mossy fibre axons. GluN3B was
predominantly present in mossy fibre synapses as compared to recurrent associational
commissural synapses, showing a presynaptic labelling pattern. In conclusion, while the
postsynaptic localization of GluN1, GluN2A, GluN2B, and GluN2D is in good agreement
with the recent finding of NMDA receptor dependent long term potentiation at CA3 mossy
fibre synapses, we propose that presynaptic GluN1, GluN2B, GluN2D and GluN3B subunits
could be involved in plastic phenomena such as certain types of long term potentiation and
recurrent mossy fibre growth.
2
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Key words: electron microscopy, immunocytochemistry, rat, nerve terminal, postsynaptic
density
3
The classic view of synaptic transmission at glutamatergic synapses is that glutamate is
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released from the presynaptic terminal to act on postsynaptic glutamate receptors. Activation
of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors will depolarise the
postsynaptic membrane and remove the magnesium block of the N-methyl-D-aspartate
(NMDA) receptors (GluNs). Hence, cations, including Ca2+, will flow into the postsynaptic
dendrite, contributing to excitatory transmission and initiating a series of possible intracellular
cascades, as for instance those leading to synaptic plasticity. In the hippocampus, NMDA
receptors are involved in synaptic events, such as induction of long term potentiation (LTP) at
the CA1 Schaffer collateral commissural synapse (Collingridge et al 1983; Bashir et al.,
1991), the CA3 recurrent associational/commissural synapse (rA/C synapse) and the perforant
path - granule cell synapse (Morris et al., 1986; Ishihara et al., 1990). However, at the stratum
lucidum mossy fibre synapse, which is formed by giant mossy fibre terminals and proximal
dendrites of CA3 pyramidal neurons, the role of NMDA receptors is unclear. For instance,
LTP was long thought to be an NMDA receptor independent process at these synapses (Harris
and Cotman, 1986; Nicoll and Schmitz, 2005). In line with this were reports of very weak
immunocytochemical labelling for NMDA receptors of the terminal field of the mossy fibres
(the stratum lucidum) (Watanabe et al., 1998). However, the existence of NMDA receptors at
the mossy fibre synapse has been indicated by electrophysiological experiments (Jonas et al.,
1993; Weisskopf and Nicoll, 1995) and by electron microscopic immunogold cytochemistry
(Takumi et al., 1999; Gylterud Owe et al., 2005). Recently, NMDA dependent LTP was
demonstrated at the mossy fibre synapse (Kwon and Castillo, 2007; Rebola et al., 2008).
Contrary to the classical mossy fibre LTP, which is a presynaptic phenomenon (Nicoll and
Schmitz, 2005) the latter studies suggested that the NMDA receptor-dependent type of mossy
fibre LTP requires postsynaptic NMDA receptors.
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Three families of NMDA receptor subunits have been identified: GluN1, a family of
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GluN2 subunits (GluN2A, GluN2B, GluN2C, and GluN2D), and a pair of GluN3 subunits
(GluN3A and GluN3B) (Paoletti and Neyton, 2007). Native NMDA receptors are composed
of heterodimers of two GluN1 subunits and two GluN2 or GluN3 subunits (Cavara and
Hollmann, 2008; Ulbrich and Isacoff, 2008). GluN1 is thought to be necessary for assembly
of a functional GluN channel. The subunit composition at different types of excitatory
synapse is poorly characterized. Although previous studies have concluded that GluN1 and
GluN2A, but not GluN2B are located in proximal dendrites of CA3 pyramidal cells (Fritschy
et al., 1998; Watanabe et al., 1998), the GluN subunit composition of mossy fibre synapses is
not fully known. Generally, it is thought that GluN2A is concentrated in the postsynaptic
membrane, whereas GluN2B has a more widespread distribution at extrasynaptic sites (Köhr,
2006). Adding to the classical view of how an excitatory synapse works, a role for presynaptic
NMDA receptors in shaping the postsynaptic excitatory response has been proposed (for
review, see Corlew et al., 2008). For example, in the dentate gyrus GluN2B containing
receptors located in membranes of excitatory presynaptic nerve terminals can strengthen the
postsynaptic response (Jourdain et al., 2007). Whether the mossy fibre system expresses
presynaptic NMDA receptors is unknown.
To address the question of which NMDA receptor subunits that are present at synaptic
and extrasynaptic sites in the hippocampal mossy fibre system we used quantitative electron
microscopic immunogold cytochemistry of all the different NMDA receptor subunits in CA3
region of adult rat hippocampus. We compared the localization of NMDA receptor subunits at
mossy fibre synapses (located on the proximal dendrites of CA3 pyramidal neurons) with the
subunit localization at rA/C synapses (located on the distal dendrites of CA3 pyramidal
neurons).
5
1 EXPERIMENTAL PROCEDURES
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1.1 Tissue preparation
For postembedding immunogold cytochemistry, three adult male Wistar rats (150-250g,
Scanbur, Sollentuna, Sweden) were deeply anesthetized with pentobarbital and fixed by
transcardiac perfusion, with a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde in
phosphate buffer (pH 7.4; 50 mL/min for 20 min), after a brief flush of 5% dextran (MW
70 000) in the same buffer. Brains were removed and the hippocampus dissected out.
Specimens were cryoprotected in graded concentrations of glycerol (10%, 20% and 30%),
frozen in liquid propane, freeze-substituted with methanol and embedded in Lowicryl HM 20
(Lowi, Waldkraiburg, Switzerland). Ultrathin sections (50-100 nm) were cut and mounted on
nickel grids (300 mesh square, Electron Microscopy Sciences, USA). Animals used in this
study were treated in accordance with the guidelines of the Norwegian Committees on Animal
Experimentation (Norwegian Animal Welfare Act and European Communities Council
Directive of 24 November 1986-86/609/EEC). Formal approval to conduct the described
animal experiments has been obtained from the animal subjects review board of Institute of
Basic Medical Sciences, University of Oslo (Vit 05/03). All efforts were made to minimize
the number of animals used and their suffering.
1.2 Antibodies
Primary antibodies were: rabbit polyclonal anti-GluN1 (Chemicon, USA, AB1516, raised
against a rat C-terminal peptide containing amino acids 834-864), rabbit polyclonal antiGluN2A (AbCam, UK, AB 14596, raised against a mouse C-terminal fusion protein
containing amino acids 1265-1464); rabbit polyclonal anti-GluN2B (Molecular Probes, USA,
A-6474, raised against a fusion protein containing the rat C-terminal amino acids 984-1104),
rabbit polyclonal anti-GluN2B (Advanced ImmunoChemicals, USA, anti-GluN2B, raised
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against a C-terminal fusion protein), goat polyclonal anti-GluN2C (Santa Cruz, USA, sc1
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1470, raised against a mouse C-terminal peptide containing amino acids 1188-1238), goat
polyclonal anti-GluN2D (Santa Cruz, USA, sc-1471, raised against a mouse C-terminal
peptide containing amino acids 1272-1322) and rabbit polyclonal anti-GluN3B (Santa Cruz,
USA, sc-50474, raised against a mouse N-terminal peptide containing amino acids 81-380).
The GluN1 antibodies (e.g. Sassoè-Pognetto and Ottersen, 2000; Glass et al., 2004;
Coleman et al., 2010), as well as the GluN2A and GluN2B antibodies (Jensen et al., 2009)
have been used in previous immunogold studies and found to specifically label glutamatergic
synapses. The GluN2C, GluN2D and GluN3B antibodies have been specificity characterised
and used in immunohistochemical studies (Marvizon et al., 2002; Karadottir et al., 2005;
Salter and Fern, 2005; Wee et al., 2010). It has been shown that the GluN2D antibodies used
here cross-react with GluN2C (but not vice versa) (Marvizon et al., 2002).
Secondary antibodies, conjugated to 10 nm gold particles, were: goat anti-rabbit
(GAR) IgG (British BioCell International, BBI), rabbit anti-goat (RAG) IgG (GE Healthcare,
UK), rabbit anti-goat (RAG) IgG (BBI) and goat anti-mouse (GAM) IgG (BBI).
1.3 Western blots
To characterise the GluN antibodies Western blots were made. Hippocampi from adult male
Wistar rats (150-250 g) were removed and homogenized in a homogenization solution,
containing 1% sodium dodecyl sulfate in PB, 1% EDTA and 1% phenylmethylsulphonyl
fluoride in dimethyl sulfoxide. The protein concentration obtained was 10 µg/µl. SDS-gel
electrophoresis (25 µg per lane) was performed on polyacrylamide gels (Criterion Bio Rad
7.5%-gel Tris HCl). Blots were transferred to nitrocellulose membranes and incubated over
night with primary antibodies. All GluN receptor antibodies were diluted 1:1000 (GluN1, 0.1
µg/ml; GluN2A, 1 µg/ml; GluN2B, 0.2 µg/ml; GluN2C, 0.2 µg/ml;GluN2D, 0.5 µg/ml;
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GluN3B, 0.2 µg/ml). HRP conjugated secondary antibodies (diluted 1:3000) and a
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chemiluminescent detection system (SuperSignal West Pico Chemiluminescent Substrate
(Pierce, USA)) was used to visualize immunoreactive proteins.
1.4 Postembedding immunogold electron microscopic immunocytochemistry
The immunogold protocol used has been thoroughly described (Bergersen et al., 2008), and
adapted with minor modifications. Briefly, to disclose antigenic sites, sections were first
etched with 2% H2O2 in phosphate buffer saline (PBS). Then, unspecific aldehyde binding
sites were blocked by incubating the sections in 50 mM glycine in PBS containing 0.1%
Triton X-100 (TBST). Thereafter, the sections were incubated with 2% human serum albumin
(HSA) in TBST, followed by an overnight incubation with the primary antibodies in 2% HSA
solution at the following dilutions: anti-GluN1 at 1:50 (2 µg/ml), anti-GluN2A at 1:100 (10
µg/ml), a mixture of the two GluN2B antibodies at 1:100 each (2 µg/ml), anti-GluN2C at
1:1000 (0.2 µg/ml), anti-GluN2D at 1:50 (10 µg/ml) and anti- GluN3B at 1:300 (0.7 µg/ml).
For visualization of the primary antibody-antigen binding, sections were incubated for 2 h
with secondary antibodies coupled to 10 nm colloidal gold particles (GAR, RAG and GAM)
diluted 1:50 in TBST with 2% HSA. Finally they were rinsed in ultra purified water and dried
before being counterstained with 2% uranyl acetate and 0.3% lead citrate. In addition, tissue
sections were incubated with the secondary antibodies only (without the primary GluN
antibodies). In these sections there was no labelling, suggesting that the secondary antibodies
were specific.
Sections were observed in the electron microscope (Philips CM 10, FEI Tecnai 12 or
FEI Morgagni) and micrographs were taken randomly in the stratum radiatum and the stratum
lucidum of the hippocampal CA3 region at magnifications of 18,000×, 34,000× or 43,000×
(depending on microscope). These terminals were identified by their large size and the
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presence of densely packed small clear spherical vesicles and by multiple asymmetric
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synaptic specializations with postsynaptic dendritic thorns. Small terminals forming
asymmetric synapses (recurrent associational/commissural synapses (rA/C synapses)) were
sampled in the CA3 stratum radiatum. Mossy fibre axons in stratum lucidum were identified
by their small diameter (about 200 nm), lack of surrounding myelin, the existence of
microtubules and by their location in bundles (Blackstad and Kjaerheim, 1961). Micrographs
were taken where the mossy fibre axons had been cut in the transverse direction.
1.5 Quantitative immunogold analysis
The quantifications were performed in three animals. Linear density of gold particles (number
of gold particles/μm membrane length) at synapses between mossy fibre terminals and
dendritic thorn complexes (multiple synaptic contacts per dendritic thorn), were determined
by measuring length of the postsynaptic membrane and counting the number of particles
belonging to the postsynaptic membrane (i.e. gold particles situated within 30 nm from the
postsynaptic membrane, taking into account that the lateral resolution of the present
immunogold method is about 30 nm (Bergersen et al., 2012). The same procedure was done
for the CA3 rA/C synapses. In each animal, the postsynaptic membrane lengths were summed
and divided by the total number of gold particles, giving an estimate of the mean linear
density of gold particles for each synapse type (mossy fibre and rA/C synapses) per animal.
For the mossy fibre axons, the linear density of gold particles in the axonal membrane
was determined by measuring the length of axonal membrane stretches and counting the
number particles that were situated within 30 nm in the axonal direction from the plasma
membrane. Particles outside the axon were excluded due to a significant overlap with
neighbouring axons. This may have slightly underestimated the densities of gold particles in
the axonal membrane compared to the densities that were estimated in the postsynaptic
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membranes. Like for the postsynaptic densities, the mean plasma membrane axonal densities
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were calculated in each animal by dividing the total membrane length by the total number of
gold particles that could be ascribed to the axonal plasma membrane.
To give an estimate of the general tissue labelling, straight lines of 250 nm length
(approximately the mean length of mossy fiber- dendritic thorn post synaptic densities) were
projected randomly with respect to orientation and location onto random electron micrographs
of the neuropil in the CA3 stratum radiatum. Particles situated 30 nm or closer, to both sides
of the line, were included in the analysis (general neuropil labelling). If a line, with its 30 nm
margins, overlapped with axons, myelin, mitochondria or postsynaptic densities, it was
excluded and replaced with a new line. Mean densities for each NMDA receptor subunit over
the general neuropil were calculated in each animal by dividing the total length of the lines by
the total number of gold particles ascribed to the lines.
To determine the distribution of gold particles across the postsynaptic membrane for
each NMDA receptor subunit, the shortest distance from the centre of the gold particles
located within 120 nm in the postsynaptic and presynaptic direction from the postsynaptic
membrane was measured. The gold particle-postsynaptic membrane distances for each tissue
profile from all animals were pooled together and plotted in a frequency histogram.
Outlines of postsynaptic and axonal plasma membranes and the location of the center
of each gold particle were recorded using an ImageJ (http://rsb.info.nih.gov/ij/) plugin written
for the purpose (Larsson and Broman, 2005). A similar ImageJ plugin was used to project
random lines onto electron micrographs (see above). The different sets of coordinates were
submitted to a program written in Python (http://www.python.org) to calculate membrane
lengths and the perpendicular distances between gold particles and membranes/random lines.
The ImageJ plugin and Python software are available at
http://www.neuro.ki.se/broman/maxl/software.html. All gold particle linear densities are
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presented as the mean linear density (number of gold particles/µm membrane length) of three
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animals±SEM. We performed the quanticifations in one ultrathin section for each GluN
subunit in three animals. We labelled several sections with each GluN subunit antibody to
confirm that the labelling patterns were reproducible. The number of profiles used for
quantifying the density of GluN subunit labeling in mossy fibre synapses, mossy fibre axons
and rA/C synapses, as well as the average membrane lengths of these profiles are given in
each of three animals in Table1. The numbers of gold particles in mossy fibre synapses and
rA/C synapses, and the numbers of mossy fibre and rA/C synapses used for the
quantifications of each GluN subunit across the synaptic membrane in each of three animals
(animal no 1, 2, 3) are given un Table2.
Differences between tissue profile densities were evaluated by one-way ANOVA post
hoc test (Tukey’s, SPSS). Differences in frequency distribution of gold particles across the
postsynaptic membrane were evaluated by Chi squared (χ2) test.
2 RESULTS
2.1 Antibody description
To characterise the GluN antibodies Western blots of hippocampal homogenates were made.
The GluN1, GluN2A, GluN2B, GluN2C, GluN2D and GluN3B antibodies produced strong
staining of bands at appropriate molecular weights (Fig. 1). GluN1 and GluN3B gave bands
at approximately 120 kDa, while GluN2A, GluN2B, GluN2C and GluN2D were detected at
approximately 150-160 kDa. Our Western blots show similar GluN1-2D bands as those
presented by Marvizon et al. (2002). For some of the GluN subunit antibodies there were
some very weak additional bands on the Western blots, which may represent light background
staining or breakdown products of the specific proteins.
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2.2 Immunogold cytochemistry
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The purpose of this study was to examine the subunit localization of NMDA receptors in the
mossy fibre system of rat hippocampus and to compare it with that at the excitatory rA/C
synapses in the stratum radiatum of CA3. This task requires the use of a high resolution
morphological method. We have therefore used a quantitative electron microscopic
immunogold method, which can localise immunogold particles to individual plasma
membranes (Bergersen et al., 2008).
First, we investigated the localization of each NMDA receptor subunit at mossy fibre
and rA/C synaptic sites. Of the two GluN3 subunits we decided to concentrate on the
localisation of GluN3B, which is reported to be strongly expressed throughout the
hippocampus, as opposed to GluN3A, which is located at only low levels in the hippocampus
(Ciabarra et al., 1995; Ritter et al., 2002, Wong et al., 2002, Wee et al., 2008). Immunogold
labelling showed that GluN1, GluN2A, GluN2B, GluN2C, GluN2D were present both at the
mossy fibre and the rA/C synapses, while GluN3B labelling was located predominantly in
mossy fibre synapses with low levels in rA/C synapses (Fig. 2). We then quantified the
density of the NMDA receptor subunits in the postsynaptic membranes of these synapses. As
the lateral resolution of the immunogold method is about 30 nm (Bergersen et al., 2012), gold
particles situated within 30 nm from each side of the postsynaptic membrane were included in
the analysis. The quantifications showed that apart from GluN1 and GluN3B, the densities of
all NMDA receptor subunits tended to be higher in rA/C synaptic membranes than in the
synaptic membranes of mossy fibre synapses, although the differences only reached statistical
significance for GluN2B and GluN2C (Fig. 3). The density of GluN3B was instead
significantly higher at mossy fibre synapses than at rA/C synapses. At the latter type of
synapse the density of GluN3B labelling was not significantly higher than the background
labelling density.
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The quantitative analysis described above showed that NMDA receptors are present in
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synaptic mossy fibre membranes. As the width of the synaptic cleft is about 15 nm (this study;
Bergersen et al., 2003) the labelling described above could report epitopes situated both in the
pre- and postsynaptic membrane (cf. the lateral immunogold resolution). To clarify whether
the NMDA receptor subunits are situated in presynaptic or postsynaptic membranes of mossy
fibre and rA/C synapses, the position of the gold particles was determined along an axis
perpendicular to the synaptic membrane with the border between the postsynaptic density
(PSD) and the synaptic cleft as origin. From the frequency distribution histograms in Fig. 4 it
is evident that gold particles signalling GluN2A and GluN2C showed a skewed frequency
distribution towards the postsynaptic side, whereas gold particles for GluN1, GluN2B and
GluN2D were more equally distributed across the mossy fibre synaptic membranes. A
considerable fraction of gold particles representing GluN2D, but also those representing
GluN1 and GluN2B (and partly GluN2A, see Discussion), were situated at such a distance in
the presynaptic direction that they cannot signal receptors in the postsynaptic membrane (Fig.
4). Interestingly, GluN3B immunogold particles showed a distribution indicative of a
predominantly presynaptic localisation (Fig.4) As almost all gold particles signalling GluN2C
could belong to the postsynaptic membrane, we compared the other subunit frequency
distributions with that of GluN2C. At the mossy fibre synapse the GluN1, GluN2D and
GluN3B gold particles frequency distributions were significantly different from the gold
particle distributions of GluN2C. Although across the rA/C synapse gold particles for GluN1
and GluN2D showed a tendency to be located at the presynaptic side, at the rA/C synapse
there were no significant differences between the GluN2C distribution and the other subunit
distributions (Fig. 4). In mossy fibre terminals there were evidence of GluN labelling over
cytoplasmic areas, especially for GluN1, GluN2B, GluN2D and GluN3B (Figs. 3 and 4; for
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an overview of GluN3B labelling within mossy fibre terminals, see Fig. 5, where vesicular
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structures were labelled for GluN3B).
In the extrasynaptic membrane of mossy fibre terminals the densities of gold particles
signalling GluN subunits were low and at the level of the densities in the general neuropil.
However, in mossy fibre axons the quantifications showed that the axonal plasma membrane
was associated with GluN1, GluN2B and GluN2D labelling (Fig. 2 and 3). In addition, these
subunits were located in the mossy fibre axoplasm (Fig. 3).We could not find any labelling for
GluN2A, GluN2C or GluN3B in mossy fibre axons.
3 DISCUSSION
Here we demonstrate by immunogold cytochemistry that NMDA receptors are present at
hippocampal mossy fibre and rA/C synapses. The NMDA receptor subunits GluN1, GluN2A,
GluN2B, GluN2C and GluN2D were observed in the postsynaptic membrane of these types of
synapse. Our data are compatible with findings showing that the NMDA receptor type of
mossy fibre LTP is dependent on postsynaptic NMDA receptors (Kwon and Castillo, 2007;
Rebola et al., 2008), and that NMDA receptors regulate the excitability of the CA3 pyramidal
cell recurrent network (Fukushima et al., 2009). In contrast, GluN3B was present in mossy
fibres, predominantly at presynaptic sites, suggesting that this subunit is especially involved
in regulation of mossy fibre terminal activity.
Morphological evidence of NMDA receptor subunits at mossy fibre synapses has
previously been given. With subunit specific antibodies against GluN1 (Petralia et al., 1994a;
Watanabe et al., 1998) and GluN2A (Watanabe et al., 1998; Janssen et al., 2005) labelling has
been localised at mossy fibre synapses. There are variable results concerning the presence of
GluN2B in stratum lucidum (comprising the proximal dendrites of CA3 pyramidal cells).
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Watanabe et al. (1998) conclude that the density of GluN2B in stratum lucidum is low, while
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the studies of Charton et al. (1999) and Janssen et al. (2005) showed quite distinct GluN2B
labelling in this layer. Moreover, we corroborate previous immunogold localizations of
NMDA receptors at mossy fibre synapses (Takumi et al., 1999; Gylterud Owe et al., 2005).
However, there are some differences between the previous immunogold studies and ours: (1)
The previous studies used a mixture of antibodies against GluN1, GluN2A and GluN2B (to
enhance the immunogold signal), while we used subtype specific antibodies, producing lower
labelling sensitivities. (2) The studies by Takumi et al. (1999) and Gylterud Owe et al. (2005)
showed that the density of NMDA receptors at mossy fibre synapses was lower than at CA1
Schaffer collateral synapses. We found that only GluN2B and GluN2C subunits were present
at significantly lower densities at the mossy fibre synapse compared to at the rA/C synapse,
which, like the Schaffer collateral synapses, have axons that stem from the CA3 pyramidal
neurons and make synapses known to possess functional NMDA receptors (Zalutsky and
Cotman, 1990). In light of the fact that GluN1/GluN2A are thought to have a synaptic
location, it was surprising that we did not find any difference in the densities of these subunits
between the two synapse types. This could mean that the level of GluN1/GluN2A is
approximately the same at mossy fibre and rA/C synapses, while GluN2B is more densely
packed at the rA/C synapse. If a similar situation is true at the mossy fibre synapse vs. the
Schaffer collateral synapse, this could still produce the result of Takumi et al. (1999) and
Gylterud Owe et al. (2005), because their NMDA receptor labelling did not distinguish
between GluN1, GluN2A and GluN2B subunits. (3) The previous studies did not examine the
distribution of NMDA receptors across the synaptic membrane and thus could not give any
information about the possibilities of presynaptically located receptors.
Thus, an important question is whether some of the receptor subunits are located at
presynaptic mossy fibre sites. In favour of a presynaptic localization for GluN1, GluN2B,
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GluN2D and GluN3B is that these subunits are present in the cytoplasm of mossy fibre
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terminals (see Fig. 2) and that the immunogold particles were located so far to the presynaptic
side that they are likely to be situated in the presynaptic active zone membrane (see Fig. 4).
The GluN2D antibodies have been shown to cross react with GluN2C (Marvizon et al., 2012).
As GluN2C showed a rather strict postsynaptic localization, the presynaptic GluN2D labelling
cannot be due to cross-reaction with GluN2C. Thus, even though GluN2D antibodies crossreact with Glun2C, this does not alter our conclusion that GluN2D is presynaptically located.
A special note should be made on GluN3B. These antibodies were directed against the
extracellular N-terminal part of the protein. This could contribute to the skewed presynaptic
immunogold distribution. However, the fact that 40% of the GluN3 gold particles were
located further away than 30 nm in the presynaptic direction from the postsynaptic membrane
cannot be explained by antibody reaction to an extracellular epitope and strongly suggest that
GluN3B have a presynaptic localisation. This notion is further supported by our finding of
GluN3B labelling in vesicular structures in the cytoplasm of mossy fibre terminals (see Fig.
5), probably reflecting trafficking of receptors to and from the plasma membrane. Moreover,
along with GluN1 and GluN2D, GluN3B showed a frequency distribution of gold particles
across the mossy fibre synapse that was significantly different from the postsynaptic
distribution of GluN2C. Although this was not the case for GluN2B, the presence of GluN2B
in presynaptic terminals and axons support the notion that this subunit has a presynaptic
localization in the mossy fibre system. GluN2B, together with GluN1 and GluN2D, were
present in the mossy fibre presynaptic axons. The immunogold method does not allow us to
definitely conclude that the receptors are actually inserted into the axolemma. It could be that
the axons serve as mere transport routes for receptor delivery to the terminals. If this is the
case, axolemma labelling could still be observed, because receptor subunits transported closer
to the plasma membrane than about 30 nm would be detected by us as belonging to the
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membrane. The observation that GluN1, GluN2B and GluN2D subunits are situated within
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axons and terminals suggests that these subunits move along the mossy fibre axon into the
terminals for insertion into the presynaptic active zone membrane. This would be analogous
to the situation for the CB1 cannabinoid receptor, which is present in presynaptic axons and
terminals (Nyíri et al., 2005; Katona et al., 2006). It should also be mentioned that the
GluN2A labelling showed a distribution pattern at mossy fibre synapses which could reflect
presynaptically located receptors (see Fig. 4). However, we did not find any evidence of
GluN2A subunits in the cytoplasm of mossy fibre terminals or axons, questioning the
presence of this receptor subunit at presynaptic mossy fibre sites (but see Aoki et al. (2003)
for a report of presynaptic GluN2A in cortical synapses).
To our knowledge this is the first study aiming at ultrastructural detection of most of
the known NMDA receptor subunits at glutamatergic synapses. GluN1, GluN2A, GluN2B,
GluN2C, GluN2D and GluN3B were found to be present at mossy fibre synapses. However,
whether all subunits are present at all release sites or whether there are unique sites expressing
receptors with different subunit compositions cannot be inferred from our study. Answering
this would require detailed double labelling experiments using serial sections, which is
beyond the scope of this study. However, we can conclude that presynaptic mossy fibre
membranes are likely to have a subunit composition consisting of GluN1 and/or GluN2B /
GluN2D / GluN3B. A presynaptic GluN 1 localization is in harmony with a previous electron
microscopic immunocytochemical study showing the presence of GluN1 in mossy fibre
presynaptic terminals and axons (Siegel et al., 1994). Presynaptic GluN1 subunits have also
been detected at the ultrastructural level in other types of central synapses (Aoki et al., 1994,
1997; Liu et al., 1994; DeBiasi et al., 1996; Paquet and Smith, 2000; Wang et al., 2000; Pickel
et al., 2006; Lu et al., 2003; Corlew et al., 2007). The presynaptic GluN2B finding supports
previous conclusions that this subunit is situated at extrasynaptic sites (Köhr, 2006), and in
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particular electron microscopic immunocytochemistry showing that GluN2B is present in
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presynaptic terminals in various brain areas (Charton et al., 1999; Fujisawa and Aoki, 2003),
including perforant afferents in the dentate molecular layer (Jourdain et al., 2007). Like
GluN2B, GluN2D is believed to have an extrasynaptic localization (Misra et al., 2000;
Brickley et al., 2003). In addition, our evidence of a presynaptic mossy fibre localization of
GluN2Ds is in line with a previous light microscopic study (Thompson et al., 2002).
Interestingly, the GluN2D subunit shows weak Mg2+ sensitivity (Monyer et al., 1994;
Momiyama et al., 1996), a property which is also proposed for GluN3B, at least when
expressed in heterodimers with GluN1 (Chatterton et al., 2002). Thus, if GluN2D or GluN3B
is a part of presynaptic NMDA receptors this means that the receptors would be less
dependent on a simultaneous membrane depolarisation (eg by AMPA receptor activation) for
their function than the postsynaptic NMDA receptors. This would be an ideal property for a
presynaptic receptor, ensuring that its function mainly depends on ligand binding. In fact, this
was the case for the GluN2B containing presynaptic NMDA receptors studied by Jourdain et
al. (2007), which were functional even in the presence of Mg2+. This raises the question of
whether extrasynaptic (including presynaptic) NMDA receptors may consist of a
heterotrimeric assembly of GluN1, GluN2B, GluN2D (Dunah et al., 1998; Brickley et al.,
2003) or GluN3B.
What could be the functional consequences of presynaptic mossy fibre NMDA
receptors? So far no role for presynaptic NMDA receptors has been disclosed in mossy fibre
LTP in the hippocampus. However, activation of presynaptic NMDA receptors, in particular
GluN2Bs, are shown to enhance the probability of glutamate release from excitatory terminals
of hippocampal perforant path synapses (Dalby and Mody, 2003; Jourdain et al., 2007), as
well as in the entorhinal cortex (Beretta and Jones, 1996; Woodhall et al., 2001, Yang et al.,
2006) and visual cortex (Sjöström et al., 2003; Corlew et al., 2007; Li et al., 2008; Brasier and
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Feldman, 2008). Interestingly, presynaptic NMDA receptors are thought to be involved in
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various forms of synaptic plasticity, such as LTP in the lateral amygdala when cortical and
thalamic afferent fibres are activated simultaneously (Humeau et al., 2003; Shaban et al.,
2006; Fourcaudot et al., 2008). Whether such a type of plasticity occurs at the mossy fibreCA3 pyramidal cell synapse, for example during concomitant stimulation of mossy fibre and
rA/C axons is not known. Previous studies have shown that GABA terminals contain NMDA
receptors (DeBiasi et al., 1996; Paquet and Smith, 2000) and that glutamate acting through
such receptors can modulate GABA release (Mathew and Hablitz, 2011). Thus, as not only
glutamate, but also GABA could be released from mossy fibre terminals (Walker et al., 2001;
Bergersen et al, 2003), it is possible that NMDA receptors are involved in the regulation of
GABA release from mossy fibre terminals. In addition, it has been suggested that increased
glutamate release through presynaptic NMDA receptors are involved in seizure maintenance
in epilepsy (Yang et al., 2006). As it has been shown that NMDA receptor antagonists could
regulate the growth of recurrent mossy fibres during epilepsy (Sutula et al., 1996; Wang et al.,
2004; Chen et al., 2007), it is tempting to speculate that presynaptic NMDA receptors could
also be involved in regulating mossy fibre sprouting.
CONCLUSION
By electron microscopic immunogold cytochemistry we found that NMDA glutamate
receptor subunits are located in mossy fibres, most of them are present in the postsynaptic
membrane, while GluN1, GluN2B, GluN2D and GluN3B have a presynaptic localisation.
19
ACKNOWLEDGEMENT
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This work was funded by grants from the Research Council of Norway AND University of
Norway (no. 178821/v40; 170441/v40). The funding sources had no role in planning or
conducting the experiments.
None of the authors have any conflict of interest.
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Figure legends
Fig 1
Western blots of hippocampal homogenates probed with antibodies against GluN1, GluN2A,
GluN2B (from Advanced ImmunoChemicals (ac) and Molecular probes (mp)), GluN2C,
GluN2D and GluN3B.
Fig 2
26
Electron micrographs showing GluN1 (A-C), GluN2A (D-F), GluN2B (G-I), GluN2C (J-L),
1
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GluN2D (M-O) and GluN3B (P-R) labelling of CA3 hippocampus. All subunits are localised
both in mossy fibre (A, D, G, J, M, P) and rA/C (B, E, H, K, N, Q) synapses, while mossy
fibre presynaptic axons (C, F, I, L, O, R) only contain GluN1, GluN2B and GluN2D (C, I,
M). Symbols: mft, mossy fibre terminal; rA/C, recurrent associational-commissural terminal;
sp, dendritic spine; mfax, mossy fibre axon; m, mitochondrion. Scale bars, 200 nm.
Fig 3
Immunogold quantifications of GluN1, GluN2A, GluN2B, GluN2C, GluN2D and GluN3B
labelling of the postsynaptic membrane of mossy fibre (MF) and recurrent
associational/commissural (rA/C) synapses and of the mossy fibre axonal plasma membrane
(mfax) and in the general neuropil (GNL). The bar charts show the mean density of gold
particles (number of gold particles per µm membrane length ± SEM, n=3 animals) signalling
NMDA receptor subunits. Background labelling in mitochondrial outer membranes for the
GluN1-GluN3B antibodies was low and between 0.20-0.08 gold particles/µm. *, the values
MF is significantly higher from those in mfax and GNL; **, the values in rA/C are
significantly higher than those in MF, mfax and GNL; ***, the values in rA/C are
significantly higher than those in mfax and GNL; ****, the values in mfax are significantly
higher than those in GNL (p<0.05, one-way ANOVA post hoc test (Tukey’s).
Fig 4
Frequency distributions of GluN1, GluN2A, GluN2B, GluN2C, GluN2D and GluN3B across
the postsynaptic membrane of mossy fibre and rA/C synapses. The distance from the border
between the postsynaptic membrane (PSD) and the synaptic cleft (sc) to the centre of each
subunit gold particle was recorded along an axis perpendicular to the postsynaptic membrane
27
(and laterally confined by the extent of the latter). Positive values indicate postsynaptic
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direction. The average widths of the mossy fibre and rA/C synaptic cleft (sc) and postsynaptic
density (PSD) are indicated. The gold particle-membrane distances were sorted into bins of 30
nm. Note that GluN2A and GluN2C show a skewed frequency distribution in the postsynaptic
direction at mossy fibre synapses. Gold particles for GluN1, GluN2B and GluN2D are more
equally distributed across the mossy fibre postsynaptic membrane, while gold particles for
GluN3B are skewed towards the presynaptic direction. As the centre of a gold particle can at
most be separated by a distance of about 30 nm from the epitope a considerable fraction of
GluN1, GluN2B and GluN2D gold particles is located so far in the presynaptic direction that
they cannot signal receptors in the postsynaptic membrane. As the GluN2C gold particles
showed the strongest postsynaptic-like distribution across mossy fibre and rA/C synapses, we
compared the gold particle distribution of GluN2C with the distributions of the other receptor
subunits. At the mossy fibre synapse the GluN1, GluN2D and GluN3B distributions were
significantly different from the distribution of GluN2C (p<0.05, χ2 test), while across the rA/C
synapses there were no statistical difference between the subunit distributions. The
distributions of GluN1, GluN2D and GluN3B gold particles across the mossy fibre synapses
were statistically different from the distributions across rA/C synapses (p<0.05, χ2 test).
Fig. 5
Electron micrograph showing GluN3B labelling in a mossy fibre terminal (mft) making
multiple synapses with dendritic spines (sp). Gold particles within the terminal are associated
with vesicular structures (arrowheads). Insets: higher magnification showing the areas
indicated by broken lines. Scale: 100 nm, 50 nm in insets.
28
Table
GluN1
GluN2A
GluN2B
GluN2C
GluN2D
GluN3B
membrane length
Animal no
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Mf syn
Mf ax
rA/C syn
1
nm
2
nm
3
nm
40
45
41
40
40
41
41
40
41
40
44
40
41
40
42
42
40
42
40
87
42
40
87
43
42
88
42
40
40
40
40
40
40
42
100
46
40
41
41
40
43
40
40
43
42
53
65
65
38
45
35
41
133
57
210.4
250.8
222.3
220.5
300.1
240.6
213.6
287.9
236.7
Table1. The figures are the number of profiles for each GluN subunit in each of three animals
(animal no 1, 2, 3) used for the quantifications presented in Fig. 3. Mf syn, mossy fibre
synapses; Mf ax, mossy fibre axons; rA/C syn, rA/C synapses. Average membrane lengths are
given for each profile in each animal.
Table2
GluN1
GluN2A
GluN2B
GluN2C
GluN2D
GluN3B
Animal no
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Mf syn gold part
Mf syn number
rA/C syn gold part
rA/C syn number
34
9
19
5
36
10
25
6
34
8
24
7
23
8
14
6
22
6
18
5
23
7
16
5
31
8
29
7
33
7
30
7
35
8
27
6
41
9
51
12
48
11
53
11
47
10
49
13
22
7
10
4
23
7
15
6
25
7
11
5
82
20
15
9
86
18
12
8
84
17
16
11
Table2. The figures are the numbers of gold particles (gold part) in mossy
fibre synapses (Mf syn) and rA/C synapses (rA/C syn), and the numbers
of mossy fibre (Mf syn number) and rA/C (rA/C syn number) synapses
used for the quantifications presented in Fig. 4 for each GluN subunit in
each of three animals (animal no 1, 2, 3).
Figure 1
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Figure2
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Figure2
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Figure 3
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Figure 4
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Figure 4
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Figure 5
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