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Volume 16, Number 24,
Issue of December 15, 1996
pp. 7859-7867
Copyright ©1996 Society for Neuroscience
Motor Discoordination Results from Combined Gene Disruption of
the NMDA Receptor NR2A and NR2C Subunits, But Not from Single
Disruption of the NR2A or NR2C Subunit
Hiroshi Kadotani1,
Tomoo Hirano2,
Miwako Masugi1,
Kenji Nakamura3,
Kazuki Nakao3,
Motoya Katsuki3, and
Shigetada Nakanishi1
Departments of 1 Biological Sciences and
2 Physiology, Kyoto University Faculty of Medicine, Kyoto
606, Japan, and 3 Institute of Medical Science, University
of Tokyo, Tokyo 108, Japan
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
NMDA receptors consist of two distinct classes of subunits. The NR1
subunit possesses all properties of the NMDA receptor-channel complex,
whereas four NR2 subunits (NR2A-2D) potentiate and differentiate NMDA
receptor responses by heteromeric assemblies with NR1. The mRNAs for
the five NMDA receptor subunits are expressed in the cerebellum in a
distinct temporospatial manner. To study functions of the NMDA
receptors in the cerebellum, we generated knockout mice deficient in
either NR2A or NR2C or both of these subunits. All three mutant mice
developed normally and showed normal overall morphology of the
cerebellum. The NMDA receptor-mediated components of EPSCs in granule
cells, as assessed by whole-cell recordings of cerebellar slices, were
reduced in NR2A- and NR2C-deficient mice and nearly abolished in mice
lacking both NR2A and NR2C. The NR2A- and NR2C-deficient granule cells
were different in the current-voltage relationship and time course of
NMDA receptor responses. The NR2A and NR2C subunits thus contribute to
distinct NMDA receptor-mediated excitatory transmission in mossy
fiber-granule cell synapses in the mature cerebellum. Both NR2A- and
NR2C-deficient mice showed no impaired movements in the motor
coordination tasks tested. The mutant mice deficient in both NR2A and
NR2C could also manage simple coordinated tasks, such as staying on a
stationary or a slowly rotating rod, but failed more challenging tasks
such as staying on a quickly rotating rod. These data demonstrate that the NMDA receptors play an active role in motor coordination.
Key words:
NMDA receptor;
glutamatergic transmission;
gene
targeting;
whole-cell patch recording;
EPSC;
cerebellar granule cell;
heteromeric receptor;
motor coordination
INTRODUCTION
Glutamate receptors mediate most excitatory
synaptic transmission in the CNS and play important roles in synaptic
plasticity, neuronal development, and neurodegeneration (Mayer and
Westbrook, 1987 ; Nakanishi, 1992; Hollmann and Heinemann, 1994;
Nakanishi and Masu, 1994). There are two general groups of glutamate
receptors. Ionotropic glutamate receptors contain ligand-gated ion
channels and are subdivided into NMDA receptors and non-NMDA receptors, whereas metabotropic glutamate receptors are G-protein-linked receptors
that modulate the production of intracellular second messengers
(Nakanishi, 1992; Hollmann and Heinemann, 1994; Nakanishi and Masu,
1994; Pin and Duvoisin, 1995).
NMDA receptors are thought to be essential in many central actions of
glutamate, including learning, memory, cognitive processes, and
neurodegenerative disorders. They have unique properties, including
high permeability of Ca2+, modulation by glycine,
voltage-dependent Mg2+ block, and inhibition by
Zn2+ and specific channel blockers (Mayer and Westbrook,
1987; Nakanishi, 1992; Hollmann and Heinemann, 1994; Nakanishi and
Masu, 1994). NMDA receptors comprise heteromeric assemblies of
different subunits, which are classified into two distinct groups
(Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992;
Ishii et al., 1993). The NR1 subunit is a fundamental subunit that
shows all the properties of the NMDA receptor-channel complex
(Moriyoshi et al., 1991). In contrast, none of the four NR2 subunits
(NR2A-2D) show any receptor-channel activity in a homomeric
configuration; however, the heteromeric assemblies of NR1 with one or
more NR2 subunits seem to be required for functional NMDA receptor
activities in vivo. These heteromeric assemblies markedly
potentiate NMDA receptor activity and vary certain NMDA receptor
properties, such as affinity for agonists, gating kinetics, unitary
conductance, and sensitivity to antagonists and Mg2+ block
(Kutsuwada et al., 1992; Monyer et al., 1992; Ishii et al., 1993).
NMDA receptors are believed to be critical in the formation of neural
networks and the development and maturation of the cerebellum (Rabacchi
et al., 1992; Komuro and Rakic, 1993). In the cerebellum, expressions
of the mRNAs for the five NMDA receptor subunits are regulated in a
distinct temporospatial manner (Akazawa et al., 1994; Watanabe et al.,
1994). The NR1 mRNA is expressed in most neurons in the developing and
mature cerebellum. The NR2A mRNA is present in granule cells from the
early postnatal period to adulthood, whereas the NR2B mRNA is expressed
transiently in these cells during the early postnatal period. The
expression of the NR2D mRNA also occurs transiently in Purkinje cells
during the postnatal period. The NR2C mRNA appears in granule cells in
the third postnatal week and is predominantly expressed in mature granule cells. The biological significance for cerebellar function of
the diversity of NMDA receptor subunits, however, remains to be
understood.
As a first step for understanding the function of the different NMDA
receptor subunits, we generated knockout mice deficient in either NR2A
or NR2C or both of these subunits and studied NMDA receptor function in
the cerebellum by examining NMDA receptor activity in cerebellar
granule cells and the motor behaviors of the three mutant mice. The
results demonstrate that a deficiency of either the NR2A or NR2C
subunit causes no remarkable behavioral abnormality, but the disruption
of both NR2A and NR2C results in impairment of motor coordination.
MATERIALS AND METHODS
Generation of NR2A- and/or NR2C-deficient mice. The
NR2A and NR2C genes were isolated from a mouse genomic library prepared from 129/SvJ mice DNA (Stratagene, La Jolla, CA) by hybridization with
the rat NR2A cDNA probe (residues 1483-2072) and the rat NR2C cDNA
probe (residues 1833-2637), respectively (Ishii et al., 1993). For the
construction of the targeting vector of the NR2A gene, we incorporated
13.9 kb of genomic sequence and replaced an internal 2.1 kb
BamHI-BgII fragment encoding a part of the NR2A
transmembrane segments with the neomycin resistance gene. For the NR2C
gene, we incorporated 13.1 kb of genomic sequence and replaced an
internal 1.5 kb SmaI fragment encoding a part of the NR2C
transmembrane segments with the neomycin resistance gene. A 2.1 kb
herpes simplex virus thymidine kinase gene fragment was attached to the
3 end of each targeting vector for negative selection. Electroporation
of the targeting vector into embryonic stem (ES) cells, selection of ES
cells containing the properly targeted gene, generation of chimeric
mice, and germline transmission for the generation of heterozygous
mutant mice were carried out according to the procedures described by
Masu et al. (1995). The genotypes of 257 offspring obtained by NR2A
heterozygote matings were 24.5% (63/257) homozygous mutant, 26.8%
(69/257) wild type, and 48.7% (125/257) heterozygous mutant. The
genotypes of 362 offspring obtained by NR2C heterozygote matings were
22.7% (82/362) homozygous mutant, 25.1% (91/362) wild type, and
52.2% (189/362) heterozygous mutant. To generate homozygous mutant
mice lacking both NR2A and NR2C, homozygous NR2A- and NR2C-deficient
mice were first mated with each other, and the resultant mutant mice,
heterozygous for both NR2A and NR2C, were inbred with each other. The
genotypes of 248 offspring derived from NR2A and NR2C heterozygote
matings were 5.2% (13/248) homozygous mutant lacking both NR2A and
NR2C, 6.0% wild type (15/248), and 88.8% (220/248) heterozygous
mutant containing at least one intact NR2 allelic gene.
Southern blot analysis of the NR2A and NR2C genes was carried out using
tail DNA and hybridization with the 5 and 3 probe, respectively, as
illustrated in Figure 1. Northern blot analysis of NR2A and NR2C mRNAs
was performed by hybridization of total cerebellar RNA (20 µg) with
the rat NR2A cDNA probe (residues 1770-1982) and the rat NR2C cDNA
probe (residues 1895-2263), respectively (Ishii et al., 1993). Western
blot analysis was carried out with cerebellar membrane fractions.
Antibodies used were a monoclonal antibody (23F6) directed against
residues 439-453 of the NR2A subunit (Streit et al., 1995; a gift of
Molnár and Streit), a polyclonal antibody against residues
1227-1237 of the NR2C subunit (Chazot et al., 1994), and a polyclonal
antibody that reacts with the NR2C subunit as well as the NR2A and NR2B
subunits (Calbiochem-Novabiochem, La Jolla, CA). The NR2A antibody
preferably reacts with the deglycosylated NR2A subunit. Cerebellar
membrane fractions were thus treated with peptide
N-glycohydrolase F (Genzyme, Cambridge, MA) (6 U/ml) for 4 hr before Western blot analysis.
Fig. 1.
Targeted disruption of the NR2A and NR2C genes.
A, Exons of the NR2A (top) and NR2C
(bottom) genes around the disrupted sites are shown by
closed boxes. Homologous recombination resulted in replacement of the 2.1 kb (NR2A) or 1.5 kb (NR2C) fragment encoding the
putative transmembrane region (M2 and M3
for NR2A; M1 and M2 for NR2C) with the
neomycin resistance gene (NEO). The herpes simplex virus
thymidine kinase gene (TK) was attached to the 3 end of each targeting vector for negative selection. Restriction sites
indicated are as follows: D, DraII;
E, EcoRI; B,
BamHI; Bg, BglI;
Sa, SalI; and Sm,
SmaI. The locations of the probes used for Southern blot
analysis are indicated. B, Southern blot analysis of
genomic DNAs from the Wild-type, NR2A-disrupted
(NR2A /), NR2C-disrupted
(NR2C/), and both NR2A- and
NR2C-disrupted
(NR2A//NR2C/)
mice. DraII-digested DNAs were hybridized with
probe A for the analysis of NR2A gene disruption
(left), and BamHI-digested DNAs were
hybridized with probe B for the analysis of NR2C gene
disruption (right). C, Northern blot
analysis of expression of the NR2A mRNA (left) or NR2C
mRNA (right) in the cerebellum of the wildtype and
homozygous NR2A/, NR2C/, and
NR2A//NR2C/ mutant mice. The
NR2A mRNA is absent in the NR2A/ and
NR2A//NR2C/ mice, whereas the NR2C mRNA
is absent in the NR2C/ and
NR2A//NR2C/ mice. D,
Western blot analysis using an antibody that preferably reacts with the
deglycosylated NR2A subunit (~170 kDa) (left) and an
antibody that reacts with the NR2C subunit (~140 kDa) as well as the
NR2A and NR2B subunits (~180 kDa) (right). NR2A
immunoreactivity is absent in cerebellar membrane fractions from the
NR2A/ and
NR2A//NR2C/ mice, whereas NR2C
immunoreactivity is absent in cerebellar membrane fractions from the
NR2C/ and
NR2A//NR2C/ mice.
[View Larger Version of this Image (31K GIF file)]
Histological analysis. Animals (5-6 weeks old) were deeply
anesthetized with sodium pentobarbital, and the brains were dissected rapidly. The tissue was immersed in a vial containing 45 ml of 99%
ethanol. The vial was stored at room temperature for a week, and 99%
ethanol was changed every other day. The tissue was bisected on the
midline, dehydrated, and embedded in paraffin. Sagittal sections were
cut on a microtome at 4.5 µm. Nissl staining was carried out by
immersing deparaffinized slides in 0.1% toluidine blue at 58°C for 1 hr. The cell density and sizes of granule cells were calculated from
Nissl-stained sections.
Electrophysiology. Transverse slices 150 µm thick were
prepared from mice at postnatal days 18-20 using a microslicer and kept at room temperature for at least 1 hr in the normal external solution containing (in mM): 124 NaCl, 26 NaHCO3, 1.8 KCl, 1.24 KH2PO4, 2.5 CaCl2, 1.3 MgCl2, and 10 glucose, saturated
with 95% O2/5% CO2. All experiments were
performed with whole-cell configuration of the patch-clamp technique
(Edwards et al., 1989) with borosilicate pipettes (resistance of 5-15
M when filled with an intracellular solution: 120 mM
CsF, 27 mM CsCl2, 5 mM EGTA, 10 mM Hepes; pH adjusted to 7.3 with CsOH). The recording
chamber was perfused continuously with a solution of the following
constituents (in mM): 109 NaCl, 26 NaHCO3, 16.8 KCl, 1.24 KH2PO4, 2.5 CaCl2, 0.1 MgCl2, 10 glucose; and 20 µM (+)-bicuculline
(Sigma, St. Louis, MO) (saturated with 95% O2/5%
CO2). The external K+ concentration was
increased to enhance the frequency of spontaneous EPSCs, and 0.1 mM MgCl2 was added to manifest a different
sensitivity of Mg2+ block of the NR1/NR2A and NR1/NR2C
assemblies at hyperpolarized potentials (Monyer et al., 1992; Ishii et
al., 1993); (+)-bicuculline was added to suppress IPSCs. To record
evoked synaptic responses, a slice was perfused with the normal
external solution in which 5 µM glycine and 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were
supplemented and MgCl2 was reduced to 0.1 mM;
glycine and CNQX were added to enhance NMDA receptor-mediated currents
and suppress non-NMDA receptor-mediated currents, respectively. A glass
pipette filled with the above external solution was placed around the
recording granule cells, and 100 µsec voltage pulses were applied to
stimulate mossy fibers. Data were discarded when the drift of a
junction potential during recording was >5 mV. Recorded currents were
filtered at 5 kHz, stored in a DAT recorder, and analyzed with QP-110J
software (Nihon Kohden, Tokyo, Japan) on an NEC computer. All
experiments were carried out at 20-23°C.
Animal behavioral analysis. For measurements of locomotor
activity and the number of rearing events, individual animals were placed in a novel environment of the circular open-field (68 cm in
diameter), in which the floor was divided into nine spaces by painted
lines. The animal was observed directly, and the locomotor activity was
counted as the number of line crossings in the open-field during a
period of 3 min. Under the same conditions, the number of rearing
events was determined by counting the number of times the animal stood
upright on its hindlimbs during a period of 3 min, and the frequency of
defecation and urination was measured during the same period. For
analysis of the righting reflex, animals were lifted manually from the
supporting surface and placed on their backs. Righting responses were
recorded on video, and the time required for righting reflex was
calculated. The grip strength was measured according to the procedure
described by van Riezen and Boersma (1969), using similar equipment.
Footprint analysis was carried out as described by Aiba et al. (1994).
The fixed bar consisted of a wooden bar (either 6 mm or 20 mm in width, 80 cm in length, and 40 cm above the ground) held horizontally on both
ends. An animal was placed on the fixed bar, and the animal behavior
was recorded on videotape. The time the animal remained on the bar was
measured. The rota-rod consisted of a gritted metal roller (3 cm in
diameter). A mouse was placed on the roller, and the time it remained
on the rotating roller was measured. In both the fixed bar and rota-rod
tests, a maximum of 60 sec was allowed per animal. Statistical analysis
was carried out by ANOVA, and post hoc comparisons were made
with Scheffé test. The animals used for all behavioral
experiments were 5-6 weeks old.
RESULTS
Generation of NR2A- and/or NR2C-deficient mice
We cloned and mapped the mouse NR2A and NR2C genes and disrupted
them in the ES cell line derived from the 129/SvJ mouse strain (Wurst
and Joyner, 1993). Each targeting vector included the neomycin resistance gene for positive selection and the herpes simplex virus
thymidine kinase gene for negative selection (Fig.
1A). For targeted disruption of the
NR2A and NR2C genes, the region covering the second transmembrane
segment of the NR2 subunits was replaced with the neomycin resistance
gene (Fig. 1A). The targeting vector was
electroporated into ES cells, which were then selected with G418 and
the pyrimidine analog gancyclovir. Southern blot analysis was carried
out to identify targeted clones from G418- and gancyclovir-resistant
clones. Three NR2A-targeted and four NR2C-targeted clones were isolated
from 33 and 60 clones, respectively. Chimeric mice were generated by
injecting the targeted ES clones into C57BL/6J blastocysts. The
targeted mutations of both NR2A and NR2C genes were transmitted into
the germline, and heterozygous and homozygous mutant mice containing
the properly targeted NR2A or NR2C gene were identified by Southern
blot analysis (Fig. 1B). Homozygous NR2A
(NR2A/) and NR2C mutant mice (NR2C/)
were mated to generate double-mutant mice lacking both NR2A and NR2C
(NR2A//NR2C/).
As reported previously (Sakimura et al., 1995), homozygous
NR2A/ mutant mice developed and mated normally.
Similarly, both homozygous NR2C/ mutant mice and
NR2A//NR2C/ mutant mice showed no
apparent survival disadvantage or any obvious abnormality in their
appearance as compared with their wild-type littermates. Northern blot
analysis confirmed the lack of either NR2A mRNA or NR2C mRNA or both in
cerebellar RNAs of the corresponding homozygous mutant mice (Fig.
1C). The disappearance of immunoreactivity for NR2A or NR2C
or both was confirmed by Western blot analysis of cerebellar membrane
fractions of the respective homozygous mutant mice (Fig.
1D).
Histological analysis
The three different mutant mice showed no gross anatomical changes
in the brain. In the adult cerebellum, the NR2A mRNA is distributed in
granule cells and cerebellar nuclear neurons, whereas the NR2C mRNA is
predominantly expressed in granule cells (Akazawa et al., 1994;
Watanabe et al., 1994). Nonetheless, as viewed by Nissl staining, the
cellular organization of the cerebellum was unaltered in all three
mutant mice (Fig. 2). The sizes and density of granule
cells, as calculated by more magnified visualization (Fig. 2), were
indistinguishable among the wild-type and three mutant mice.
Fig. 2.
Histological analysis of the cerebellum.
Parasagittal sections of the cerebellum were treated with Nissl
staining (top), and magnified views covering the
granule cell layer are indicated (bottom). The number of
granule cells was calculated to be 2.9 × 104 ± 4.2 × 103/mm2
(Wildtype), 3.0 × 104 ± 2.1 × 103/mm2
(NR2A/), 3.0 × 104 ± 8.6 × 102/mm2
(NR2C/), and 3.1 × 104 ± 2.7 × 103/mm2
(NR2A//NR2C/).
The size of the granule cells was determined to be 4.55 ± 0.31 µm (Wild-type), 4.51 ± 0.29 µm
(NR2A/), 4.48 ± 0.31 µm
(NR2C/), and 4.44 ± 0.33 µm
(NR2A//NR2C/).
n = 3 animals for each genotype.
[View Larger Version of this Image (94K GIF file)]
Mossy fiber-granule cell EPSCs
In the mossy fiber-granule cell synapses of the cerebellum, the
activation of non-NMDA receptors and NMDA receptors
has been shown to evoke the fast and slow components of EPSCs,
respectively (Silver et al., 1992; D'Angelo et al., 1993). To
investigate NMDA receptor responses in mutant mice, we first
characterized spontaneous EPSCs of granule cells by whole-cell
patch-clamp recordings in cerebellar slice preparations from the
wild-type and three NR2 mutant mice at postnatal days 18-20.
Spontaneous EPSC traces of the four genotypes recorded at different
membrane potentials are presented in Figure
3A. In the wild-type mice, spontaneous EPSCs consisted of a fast component and the following slow component. The
fast component had a rapid rising phase and decayed in a few milliseconds, whereas the slow component lasted for hundreds of milliseconds. This pattern of EPSCs in mouse slice preparations agreed
with the pattern of the fast and slow components that are characteristic of the non-NMDA and NMDA receptors, respectively, in rat
cerebellar slice preparations (Silver et al., 1992; D'Angelo et al.,
1993). We also confirmed that the slow component, but not the fast
component, of EPSCs was abolished by the addition of the NMDA receptor
antagonist D-2-amino-5-phosphonovalerate (D-AP5) (n = 5; data not shown).
Fig. 3.
Spontaneous mossy fiber-granule cell EPSCs.
A, Spontaneous EPSCs were recorded from single granule
cells of the four genotypes under voltage clamp at 40,  40, 80, and
100 mV, and 5-10 traces are averaged and indicated.
B, Magnitudes of the fast component (open
circles, measured as the peak amplitude) and the slow component (filled circles, measured at 10 msec after the
peak of the fast component) of EPSCs of the four genotypes are plotted
against the clamping voltages. C, Relative amplitudes of
the slow component against the fast component measured at 80 mV are
indicated. Columns and error bars represent mean ± SD; wild type,
n = 8; NR2A/, n = 5; NR2C/, n = 8; and
NR2A//NR2C/,
n = 5; **p < 0.01. D, Relative amplitudes of the slow component measured at
80 mV against that measured at 40 mV are indicated; n = 5 for each genotype; **p < 0.01.
[View Larger Version of this Image (32K GIF file)]
In recordings of the three mutant genotypes, the fast component was
elicited in granule cells, but the magnitude of the slow component
differed from those of the wild-type genotype. It has been shown by
coexpression of recombinant NR1 and NR2 subunits in Xenopus
oocytes or transfected cells that the NR1/NR2A assembly is more
sensitive to Mg2+ block than the NR1/NR2C assembly at
hyperpolarized potentials (Kutsuwada et al., 1992; Meguro et al., 1992;
Monyer et al., 1992; Ishii et al., 1993). The differences of NMDA
receptor responses among the four genotypes were characterized more
quantitatively by comparing the slow components of EPSCs in the four
genotypes as presented in Figure 3B-D. The current-voltage
plots of the magnitude of the fast component (peak amplitude of the
fast component) were linear in all four genotypes, consistent with the
properties of non-NMDA receptor responses. In contrast, the amplitude
of the slow component (10 msec after the peak of the fast component) was reduced at hyperpolarized potentials in both the wild-type and
NR2A/ mutant mice (Fig. 3B). This reduction
was more marked in the NR2C/ mutant mice, and the slow
component nearly disappeared in the NR2A//NR2C/ mutant mice (Fig.
3B). When the relative magnitude of the fast and slow
components was calculated at 80 mV, this ratio in both the
NR2A/ and NR2C/ mutant mice was reduced
to about half of that of the wild-type mice (p < 0.01), and the ratio in the
NR2A//NR2C/ mutant mice was almost
negligible (p < 0.01) (Fig. 3C).
Furthermore, when the relative magnitudes of the slow component
measured at 80 mV against that of 40 mV were compared, this ratio
slightly increased in the NR2A/ mutant mice but greatly
decreased in the NR2C/ mutant mice as compared with
that of the wild-type mice (p < 0.01) (Fig.
3D).
It has been shown that the decay time of NMDA receptor responses of the
recombinant NR1/NR2A assembly is significantly faster than that of the
recombinant NR1/NR2C assembly (Monyer et al., 1992). We examined
pharmacologically isolated NMDA receptor-mediated EPSCs that were
evoked by mossy fiber stimulation in the presence of the non-NMDA
receptor antagonist CNQX. EPSCs were recorded from granule cells of the
wild-type and the NR2A/ and NR2C/
mutant mice, and these evoked EPSCs were abolished by the addition of
D-AP5 (Fig. 4). No such responses were
detected in any of the granule cells examined from the
NR2A//NR2C/ mutant mice
(n = 9). The rise time of the NMDA receptor-mediated EPSCs was similar among the three genotypes (Table 1).
The decay time, however, was slightly slower in the
NR2A/ mutant mice than in the wild-type mice and was
faster in the NR2C/ mutant mice (Table 1). The time
course of the NMDA receptor-mediated EPSCs of wild-type granule cells
could be fitted to the sum of two exponential functions, as observed
previously in rat granule cells (D'Angelo et al., 1994). The
NR2A/ and NR2C/ mutant mice also
displayed a double-exponential decay. The time constants of both fast
and slow components of decay ( Nfast and Nslow) increased in the NR2A/
mutant mice and decreased in the NR2C/ mutant mice as
compared with the wild-type mice (Table 1). Thus, taking into account
the difference in Mg2+ sensitivity at the hyperpolarized
potentials and the time course of the NMDA receptor currents between
the recombinant NR1/NR2A and NR1/NR2C heteromeric receptors (Kutsuwada
et al., 1992; Meguro et al., 1992; Monyer et al., 1992; Ishii et al.,
1993), the results indicate that in cerebellar granule cells, ablation
of either NR2A or NR2C alone retains NMDA receptor activity derived
from the NR1/NR2C and NR1/NR2A heteromeric assemblies, respectively, and that deficit of both NR2A and NR2C results in an almost complete loss of NMDA receptor activity.
Fig. 4.
Granule cell EPSCs elicited by mossy fiber
stimulation. EPSCs elicited by mossy fiber stimulation were recorded in
the presence of 10 µM CNQX, 5 µM glycine,
and 0.1 mM Mg2+ from single granule cells of
the wild-type and NR2A/ and NR2C/
mutant mice under voltage clamp at 80 mV. Fourteen to sixteen traces
recorded in the absence (top) and presence
(bottom) of 100 µM D-AP5 are
averaged and indicated.
[View Larger Version of this Image (14K GIF file)]
Motor coordination
None of the three mutant mice showed any ataxic gait or any
sign of tremor. These mutants walked normally on the ground, and analysis of hind footprints of the four genotypes showed no difference in either the walking line or the length of steps. The general locomotor activities of the four genotypes were compared by the open-field test (Fig. 5). There was no difference in the
horizontal locomotor activities of the four genotypes as quantified by
counting the number of line crossings in the open-field (Fig.
5A); however, the amount of vertical activity as measured by
rearing was slightly decreased in both the NR2A/ and
NR2C/ mutant mice and significantly lowered in the
NR2A//NR2C/ mutant mice
(p < 0.01) (Fig. 5B). Because the
frequency of either defecation or urination was unaltered among the
four genotypes (Table 2), the difference of the rearing
behavior does not seem to result from changes in emotional or
exploratory behavior. The grip strength and righting reflex were also
unchanged among the four genotypes (Table 2). It thus seems unlikely
that weakness of muscle strength is responsible for the decrease in
rearing behavior in the mutant mice.
Fig. 5.
Open-field test. A, A mouse was
placed in a novel environment of the open-field, and its locomotor
activity was determined by counting the number of line crossings in the
open-field during the first 3 min. B, Under the above
condition, the number of rearing events was determined by counting the
number of times the animal stood upright on its hindlimbs for the first
3 min. In both A and B,
n = 10 for wild type, n = 7 for
NR2A/ and NR2C/, and
n = 13 for
NR2A//NR2C/. Columns and error
bars represent mean ± SEM; **p < 0.01.
[View Larger Version of this Image (24K GIF file)]
To further assess the possibility of a motor discoordination, we
performed a fixed bar test. We initially used a wide wooden bar (20 mm
in width) and found that all four genotypes could walk smoothly and
stay on the bar with no difference (Fig.
6B). We then used a narrower wooden
bar (6 mm in width). The wild-type and NR2A/ and
NR2C/ mutant mice could again stand up and walk
smoothly on the narrow wooden bar (Fig. 6A). In
contrast, the NR2A//NR2C/ mice could
not stand up, and they shinnied along the bar by grasping and pulling
with their forepaws and dragging their hindlimbs (Fig. 6A). Furthermore, the
NR2A//NR2C/ mice fell off the bar more
quickly than the others (p < 0.01) (Fig.
6B), and this deficit could not be improved by
training for 5 d (data not shown).
Fig. 6.
Fixed bar test. A, Photographs of
mouse behavior in the fixed bar test. A mouse was placed on the
midpoint of the 6-mm-wide fixed bar, and animal behavior was
videotaped. The wild-type, NR2A/, and
NR2C/ mutant mice walked normally on the narrow bar,
but the NR2A//NR2C/ mutant mouse
showed a characteristic movement of grasping and pulling with its
forepaws and dragging its hindlimbs. B, The time an
animal remained on the bar of either 20 mm (open column)
(n = 6-7) or 6 mm in width (column
with diagonal lines) (n = 17-27) was measured for a maximum of 60 sec. Columns and error bars
represent mean ± SEM.
NR2A//NR2C/ mice spent
significantly less time on the 6-mm-wide bar than did the other mice;
**p < 0.01.
[View Larger Version of this Image (51K GIF file)]
To further characterize the motor discoordination, we employed a
rota-rod test using a gritted roller (3 cm in diameter) (Fig. 7). All of the four genotypes could remain on the
stationary rota-rod for the period of time allowed (60 sec). When the
rota-rod was running at either 10 rpm or 20 rpm, all genotypes stayed
on the rota-rod with no significant difference (Fig. 7A);
however, when the rod was running more quickly (25 rpm), the
NR2A//NR2C/ mice failed to stay on the
rotating rod (P < 0.05) (Fig. 7A). Furthermore, a clear difference in improvement of performance at 20 rpm
was observed in the NR2A//NR2C/ mice
when the training proceeded from day 1 to day 5 (Fig. 7B). Thus, ablation of NR2A or NR2C alone causes no obvious motor
discoordination. Similarly, mice deficient in both NR2A and NR2C retain
the ability to manage simple coordinated tasks such as walking on the
ground or staying on the stationary or slowly running rota-rod. The
NR2A//NR2C/ mice cannot adapt, however,
to more challenging tasks such as walking on the narrow bar or staying
on the quickly running rota-rod.
Fig. 7.
Rota-rod test. A, The time an
animal remained on a rota-rod rotated at either 20 rpm (open
column) or 25 rpm (column with diagonal
lines) was measured on day 1 without training. Columns and
error bars represent mean ± SEM. The
NR2A//NR2C/ mice spent
significantly less time on the rota-rod rotated at 25 rpm than did the
others (*p < 0.05). At least 20 animals were tested for each genotype. B, The time an animal remained
on a rotating rota-rod (20 rpm) was measured during training by two trials per day. A maximum of 60 sec was allowed for each animal per
trial.  , Wild type (n = 33);  ,
NR2A/ (n = 30);  ,
NR2C/ (n = 35);  ,
NR2A//NR2C/
(n = 25). The
NR2A//NR2C/ mice spent
significantly less time than did the others
(p < 0.05), although their performance did
improve (F (4,96) = 9.98;
p < 0.01). Marks and error bars represent
mean ± SEM.
[View Larger Version of this Image (27K GIF file)]
DISCUSSION
In this investigation, we generated knockout mice deficient in
either NR2A or NR2C or both of the NMDA receptor subunits. This
permitted an examination of the function of the NR2A and/or NR2C
subunits in glutamatergic transmission between mossy fibers and
cerebellar granule cells as well as of the effects of the deficiency of
these subunits on motor coordination.
The mRNAs encoding the five NMDA receptor subunits show
differential expressions in developing and adult cerebellum (Akazawa et
al., 1994; Watanabe et al., 1994). In cerebellar granule cells, the NR1
mRNA is expressed at all developmental and mature stages. The NR2A and
NR2B mRNAs appear in the external granular layer from postnatal day 3, whereas the expression of NR2C mRNA begins at the third postnatal week.
In contrast, no NR2D mRNA is expressed at any developmental stage of
granule cells. The expression of the NR2A and NR2C mRNAs continues or
increases in mature granule cells, whereas the expression of the NR2B
mRNA disappears during maturation of granule cells. The present
examination by whole-cell patch-clamp recordings has indicated that
deficit of either NR2A or NR2C alone reduces the NMDA receptor-mediated
component of EPSC in granule cells of the mature cerebellum. Relative
amplitudes of the NMDA receptor activity measured at 80 mV and 40
mV are greatly reduced in NR2C/ granule cells but are
comparable between wild-type and NR2A/ granule cells.
Furthermore, the NMDA receptor activity of the NR2C/
granule cells decays faster than that of the wild-type and
NR2A/ granule cells. Thus, the properties of the NMDA
receptors in the NR2A/ and NR2C/
granule cells most closely resemble those of recombinantly expressed NR1/NR2C and NR1/NR2A assemblies, respectively. Ebralidze et al. (1995)
reported macroscopic and single-channel recordings of NMDA receptor
activities in cerebellar granule cells of the NR2C-deficient mice. They
showed that the NR2C deficiency results in the disappearance of NMDA
receptor channels of low unitary conductance that are normally
expressed in granule cells during developmental maturation. Takahashi
et al. (1995) reported single-channel recordings of NMDA
receptor-mediated currents in cerebellar granule cells of the
NR2A-deficient mice and indicated that NMDA receptor channels of low
unitary conductance predominate in the NR2A-deficient granule cells.
Ebralidze et al. (1995) also determined that the NMDA receptors in
mature granule cells consist of the NR1/NR2A assembly with high
conductance, the NR1/NR2C assembly with low conductance, and the
NR1/NR2A/NR2C assembly in varying compositions with a wide range of
conductance. In the present investigation, we have shown that ablation
of both NR2A and NR2C results in an almost complete loss of both
spontaneous and evoked EPSCs in mature granule cells. Thus, these
results demonstrate that both NR2A and NR2C subunits contribute to
functional glutamatergic neurotransmission in mossy fiber-granule cell
synapses of the mature cerebellum.
In the rat cerebellum, granule cells migrate from the external granular
layer toward the internal granular layer during postnatal days 0-14
(Hager et al., 1995). The NMDA receptor has been reported to regulate
the migration of granule cells in slice preparations of the developing
mouse cerebellum; blockade of NMDA receptors by specific antagonists
(D-AP5 or MK-801) and the stimulation of NMDA receptors
decrease and increase the rate of cell movement, respectively (Komuro
and Rakic, 1993). None of the three groups of mutant mice
(NR2A/, NR2C/, and
NR2A//NR2C/) show an overall
morphological abnormality, including the cellular organization and the
size and cell density of granule cells in the mature cerebellum. This
finding in the NR2A//NR2C/ mutant mice
is particularly interesting because the NMDA receptor activity is
negligible in these cells at postnatal days 18-20. The NR2B mRNA is
transiently expressed in granule cells during postnatal days 3-14
(Akazawa et al., 1994). Thus, our observation of the
NR2A//NR2C/ mutant mice supports the
previous notion that the NR2A and NR2B subunits subserve different
physiological requirements during cerebellar development, and the
NR1/NR2B heteromeric receptor may have an active role in granule cell
migration (Komuro and Rakic, 1993; Rossi and Slater, 1993; Farrant et
al., 1994; Vallano et al., 1996).
Recently, three gene knockout mice that are relevant to
cerebellar function in motor coordination have been generated and characterized (Aiba et al., 1994; Conquet et al., 1994; Chen et al.,
1995; Kashiwabuchi et al., 1995). These are mice deficient in mGluR1,
the glutamate receptor  2 subunit (GluR2), and PKC , and these
mutant mice show an ataxic gait (Aiba et al., 1994; Chen et al., 1995;
Kashiwabuchi et al., 1995). The mGluR1 mutant mice also show intention
tremor (Aiba et al., 1994; Conquet et al., 1994). In contrast, the
NR2A//NR2C/ mutant mice never display
ataxia or any sign of tremor and walk normally on the ground.
Furthermore, in the rota-rod test, the mGluR1, GluR2, and PKC
mutant mice have been shown to fall off the slowly rolling rota-rod (8 or 10 rpm) (Aiba et al., 1994; Chen et al., 1995; Kashiwabuchi et al.,
1995). In contrast, the NR2A//NR2C/
mutant mice can manage to stay on the slowly running rota-rod (10 rpm)
and fail only the quickly running rota-rod (25 rpm). The motor
discoordination of the NR2A//NR2C/
mutant mice is thus clearly milder than that of the mGluR1, GluR2, or PKC knockout mice. It has been proposed that the NMDA receptors in cerebellar granule cells play an important role in determining the
efficacy of synaptic summation and the output spike frequency in the
mossy fiber-granule cell synapses during repetitive mossy fiber
activation (D'Angelo et al., 1995). The NMDA receptors in granule
cells could thus contribute to fine tuning the efficacy of information
transmission at the mossy fiber-granule cell synapses and may
influence its subsequent transfer to Purkinje cells (Larson-Prior et
al., 1995). It is thus tempting to speculate that a loss of NMDA
receptor activity in granule cells is directly involved in motor
discoordination by impairing mossy fiber information processing.
FOOTNOTES
Received May 13, 1996; revised Sept. 24, 1996; accepted Sept. 30, 1996.
This work was supported in part by research grants from the Ministry of
Education, Science and Culture of Japan, the Ministry of Health and
Welfare of Japan, the Uehara Memorial Foundation, and the Sankyo
Foundation. We thank Drs. E. Molnár and P. Streit for their gift
of the NR2A antibody, Drs. P. L. Chazot and F. A. Stephenson for their
gift of the NR2C antibody, Dr. G. Katsuura for advice and assistance,
Dr. S. R. Nash for reading this manuscript, and A. Uesugi, Y. Kikui,
and M. Fukao for technical assistance.
Correspondence should be addressed to Shigetada Nakanishi, Department
of Biological Sciences, Kyoto University Faculty of Medicine, Yoshida,
Sakyo-ku, Kyoto 606, Japan.
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