Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098
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ABSTRACT |
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Tovar, Kenneth R.,
Kathleen Sprouffske, and
Gary L. Westbrook.
Fast NMDA Receptor-Mediated Synaptic Currents in Neurons From
Mice Lacking the 2 (NR2B) Subunit.
J. Neurophysiol. 83: 616-620, 2000.
The
N-methyl-D-aspartate (NMDA) receptor has
been implicated in the formation of synaptic connections. To
investigate the role of the
2 (NR2B) NMDA receptor subunit, which is
prominently expressed during early development, we used neurons from
mice lacking this subunit. Although
2
/
mice die soon
after birth, we examined whether NMDA receptor targeting to the
postsynaptic membrane was dependent on the
2 subunit by rescuing
hippocampal neurons from these mice and studying them in autaptic
cultures. In voltage-clamp recordings, excitatory postsynaptic currents
(EPSCs) from
2
/
neurons expressed an NMDA
receptor-mediated EPSC that was apparent as soon as synaptic activity
developed. However, compared with wild-type neurons, NMDA
receptor-mediated EPSC deactivation kinetics were much faster and were
less sensitive to glycine, but were blocked by Mg2+ or AP5.
Whole cell currents from
2
/
neurons were also more
sensitive to block by low concentrations of Zn2+ and much
less sensitive to the
2-specific antagonist ifenprodil than
wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation
kinetics and the pharmacological profile from
2
/
neurons are consistent with the expression of
1/
1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the
2
subunit. Thus
1 can substitute for the
2 subunit at synapses and
2 is not required for targeting of NMDA receptors to the postsynaptic membrane.
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INTRODUCTION |
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N-methyl-D-aspartate (NMDA)
receptors are composed of two subunit types, and
(or NR1 and
NR2 in the rat). In the hippocampus,
1 and
2 are the
predominantly expressed
subunits in excitatory neurons
(Monyer et al. 1994
; but see Das et al.
1998
). Mice lacking the
2 subunit (
2
/
) die soon after birth but can survive for a
few days with hand feeding. Hippocampal slices obtained from these mice
at postnatal day 3 lack a synaptic NMDA receptor-mediated component
(Kutsuwada et al. 1996
), indicating that early in
development
2-containing receptors are required either for formation
of functional NMDA receptors or synaptic localization of receptors.
Mice lacking the intracellular C-terminal domain of
2 show a similar
phenotype (Sprengel et al. 1998
), providing further
evidence for a role of the
2 subunit in targeting to the
postsynaptic density (PSD). In contrast, mice lacking the
1 subunit
are viable but have attenuated long-term potentiation (LTP)
(Sakimura et al. 1995
). The
1 subunit is detected at
very low levels in early development (Li et al. 1998
;
Monyer et al. 1994
), but NMDA receptors containing the
1 subunit are incorporated into the synaptic receptor complement soon after synapses have begun to form in vitro (Stocca and
Vicini 1998
; Tovar and Westbrook 1999
). We now
report that neurons cultured from
2
/
mice
express NMDA receptor-mediated EPSCs with unusually rapid deactivation
kinetics and distinctive pharmacological properties, consistent with
expression of
1/
1 diheteromeric receptors. Thus the
2 subunit
is not required for the formation of dual-component EPSCs. Likewise,
incorporation of NMDA receptors at synapses can occur independently of
the
2 subunit.
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METHODS |
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Breeding and cell culture
Because mice lacking both 2 alleles die soon after birth,
heterozygous (
2+/
) mice in a C57BL/6
background were used for generating
2
/
mice. The heterozygous mouse colony was derived from
2+/
mice (Kutsuwada et al.
1996
). Hippocampal neurons from neonatal
2
/
mice were rescued and grown in
microisland cultures to examine the postsynaptic phenotype of
2
/
neurons. Litters from heterozygous
matings delivered 19-19.5 days after mating plugs were detected and
ranged from 5-11 pups. Because mice lacking the
2 subunit are
unable to nurse (Kutsuwada et al. 1996
) and
2+/
mothers rarely nursed their pups,
neurons from each newborn pup were cultured individually with tissue
preserved for genotyping (see Genotyping). All cultures were
done using newborn animals. As previously described (Bekkers and
Stevens 1991
) hippocampi from these animals were removed,
enzymatically (papain; Collaborative Research) and mechanically
dissociated and plated in microisland culture. The microisland
substrate was prepared by spraying agarose-coated glass coverslips with
polylysine and collagen. Neurons were plated at low density on this
substrate to promote the formation of "autaptic" synapses.
Excitatory postsynaptic currents (EPSCs) were apparent 6-7 days after plating.
Genotyping
All genotyping was done using polymerase chain reaction (PCR)
amplification of genomic DNA prepared from mouse tissue. Tissue samples
were incubated in proteinase K (0.5 mg/ml; GibcoBRL) at 55°C for 12
h. Samples were centrifuged and the supernatant was added to an equal
volume of isopropanol to precipitate genomic DNA. The samples were spun
again to pellet the DNA. The pellet was washed in ice cold 70% ethanol
and allowed to dry. Genomic DNA was resuspended in TE buffer (10 mM
Tris, 1 mM EDTA, pH 8.0) and added to the PCR mixture of each of two
reactions. All samples were subjected to two separate reactions (see
Fig. 1 for details). PCR analysis was
done using Taq polymerase and Promega (Madison, WI)
reagents. Reaction products were run on 2% agarose gels and visualized
using ethidium bromide. Gel purification and sequencing of the reaction
products yielded the expected DNA sequences (data not shown). Primer
sequences were thus: Primer 1; 5'-ATgAAgCCCA gCgCAgAgTg-3';
Primer 2; 5'-ATggAAgTCAT CTTTCTCgTg-3'; Primer 3;
5'-ggCTACCTgC CCATTCgACC ACCAAgCgAA AC-3'; and Primer 4;
5'-AggACTCATC CTTATCTgCC ATTATCATAg-3'. The cycling conditions
(Perkin-Elmer GeneAmp 2400, Foster City, CA) were 30 cycles of 94°C
(melting) for 30 s, 67°C (annealing) for 40 s, and 72°C
(extension) for 50 s. The reaction solution contained
Mg2+ (2 mM), dNTPs (0.2 mM each), oligonucleotide
primers (0.01 mg/ml each), Taq polymerase (2.5 units),
reaction buffer (5 µL), and 2 µl of solubilized genomic DNA (50 µl final volume) in HPLC water. Each animal was genotyped using two
reactions, as shown in Figure 1B.
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Electrophysiology
The extracellular recording solution contained (in mM) 168 NaCl,
2.4 KCl, 10 HEPES, 10 D-glucose, 0.2-2.6
CaCl2, 0.01-0.02 glycine, 0.01 bicuculline
methiodide (BMI), and 0.005 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2, 3-dione (NBQX) (325 mmol/kg final osmolality; pH 7.4) in HPLC-grade water. The intracellular recording solution contained (in mM) 150 K-gluconate, 1.418 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 2 Na2ATP, and 0.2 GTP (320 mmol/kg final
osmolality; pH 7.4). Recording electrodes (TW150F-6; World Precision
Instruments, Sarasota FL) had resistances of 1-5 M. Autaptic EPSCs
were evoked using a depolarization (to +10 mV for 0.5 ms). Fast
solution exchanges were made with quartz flow pipes mounted to a
piezo-electric bimorph driven by a stimulus isolation unit (Winston
Electronics, Palo Alto, CA). Flow pipes were placed 50-100 µm from
the cell body. Recordings were done using an Axopatch 2C amplifier and
pCLAMP acquisition software (Axon Instruments, Foster City, CA). Data were low-pass filtered at 2.5 kHz, collected at 5 kHz and, analyzed using Axograph (Axon Instruments). Data from dose-response experiments were fitted with I = Imax/[1+(EC50/A)n],
where I is the current response,
Imax is the maximum response, A is the glycine concentration, and n is the Hill
coefficient. Each concentration tested represents data from
4
neurons. For glycine and antagonist experiments, neurons were
preequilibrated with the drug at the concentration being tested.
Control (NMDA, 1 mM) and experimental responses (NMDA plus drug) were
interleaved to control for possible run-down of the whole cell current.
Whole cell current amplitudes were measured by averaging three points on either side of the absolute peak current. All data are reported as
mean ± SE and were compared using an unpaired Student's
t-test. All data are from neurons
10 days in vitro (DIV).
Because the occurrence of wild-type and
2
/
animals in the same litter was rare, data from wild-type neurons was
obtained primarily from congenic animals rather than littermates of
2
/
animals. All salts and drugs were from
Sigma (St. Louis, MO) except for BMI, NBQX, NMDA, and ifenprodil (RBI,
Natick, MA). Statistical comparisons were made using Student's
t-test, with significance set at P < 0.05.
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RESULTS |
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Genotyping 2-targeted mice
Figure 1A shows the organization of 2 wild-type and
targeted alleles and the alignment of the PCR primers. The primers were designed to amplify the designated genomic DNA regions of wild-type and
targeted alleles across insert/wild-type junctions. Two separate PCRs
were used to determine the genotype of each animal. Each PCR was
designed to amplify a different pair of reaction products (bracketed
lanes in Fig. 1B). This resulted in a unique pair of reactions for
2+/+ and
2
/
animals, whereas
2+/
animals had four reaction products.
Genotypes from progeny of heterozygous mice pairings were
22(+/+):70(+/
):24(
/
),
not significantly different from a Mendelian distribution (using
2 criteria). Genotyping of outbred litters
(resulting from
2+/
and wild-type pairings)
produced a wild-type to heterozygote ratio of 43:31, indicating that
our genotyping method did not favor the production of reaction products
from the targeted allele.
NMDA receptor-mediated EPSCs from 2
/
neurons
EPSCs in wild-type hippocampal neurons have two kinetically
distinct components. The fast component (deactivation : ca. 1-5 ms)
results from the activation of AMPA receptors whereas the slow
component (deactivation
: ca. 40 and 300 ms) results from NMDA
receptor activation (McBain and Mayer 1994
). In
2
/
neurons, EPSCs were apparent in
microisland cultures after 6-7 days. However, the decay of the EPSC
did not show two distinct kinetic components, characteristic of AMPA
and NMDA receptor-mediated EPSCs in wild-type neurons. The NMDA
receptor antagonist AP5 markedly shortened the decay of the
dual-component EPSC in wild-type neurons (Fig.
2A) but had a much less
profound effect on the decay in
2
/
neurons
(Fig. 2B). The AP5-sensitive components from wild-type and
2
/
neurons are superimposed in Fig.
2C. The deactivation kinetics of NMDA receptor-mediated
EPSCs from wild-type neurons (Fig. 2, A and C)
were well-fitted with two exponentials (
f = 42.4 ± 3.6 ms;
s = 288.8 ± 24.1 ms, n = 15), similar to cultured rat neurons (e.g.,
Tovar and Westbrook 1999
). In contrast, the deactivation kinetics of EPSCs from
2
/
neurons (Fig.
2B) were dramatically faster (
f = 20.6 ± 1.7 ms;
s = 100.8 ± 13.6 ms, n = 7). For NMDA receptor-mediated EPSCs from
2
/
neurons, the sum of squared errors
(SSE) for two-exponential fits was 9.9 ± 3.4 times better than
for a single exponential fit. In
2
/
neurons, the deactivation kinetics were dominated by
f. The ratio
(
f/
s) was 6.04 ± 1.09 (n = 7) for
2
/
neurons and 1.16 ± 0.15 (n = 15) for wild-type
neurons. Although the deactivation kinetics from
2
/
neurons were usually well-fitted with
two exponentials, in two cases the deactivation could be fitted with a
single exponential (t
34 ms). The fast deactivation kinetics
of NMDA receptor-mediated EPSCs in
2
/
neurons are consistent with deactivation kinetics expected of recombinant
1/
1 diheteromeric receptors (Vicini et al.
1998
) and are much faster than EPSC deactivations seen in other
preparations (McBain and Mayer 1994
; see however,
Bardoni et al. 1998
). Consistent with an NMDA
receptor-mediated EPSC, currents from
2
/
neurons became larger by increasing extracellular glycine
(n = 4) and were completely blocked by
Mg2+ (n = 4; Fig. 2D).
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Pharmacology of 2
/
NMDA receptors is consistent
with
1/
1 receptors
Experiments using recombinant receptors have provided
pharmacological tools that can be diagnostic for specific diheteromeric subunit combinations. We used glycine (Kutsuwada et al.
1992), Zn2+ (Paoletti et al.
1997
), and ifenprodil (Williams 1993
) to
investigate the subunit composition of NMDA receptors from
2
/
neurons.
Recombinant 1/
1 receptors are more than tenfold less sensitive to
glycine than
1/
2 receptors (Kutsuwada et al.
1992
). We measured the deactivation of whole cell
glycine-evoked currents from
2
/
and
wild-type neurons (Fig. 3A)
using fast application methods. Glycine deactivations from
2
/
neurons decayed quickly and were
well-fitted by single exponentials (0.11 ± 0.1 s,
n = 6) whereas the deactivations from wild-type neurons
were slower and required two exponential components to be well-fitted
(
f = 0.266 ± 0.2 s,
s = 1.38 ± 0.06 s,
n = 6). This is consistent with an eight- to tenfold
faster glycine microscopic unbinding rate in receptors from
2
/
neurons. We confirmed the lower glycine
affinity of receptors from
2
/
neurons by
comparing the glycine dose-response relationship for
2
/
(EC50 = 3.61 µM) with wild-type (EC50 = 0.14 µM) neurons
(Fig. 3A). The EC50 value for
2
/
NMDA receptors is consistent with that
of
1/
1-containing NMDA receptors (Kutsuwada et al.
1992
).
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Recombinant 1/
1 receptors are also selectively antagonized in a
voltage-independent manner by nanomolar Zn2+
(Paoletti et al. 1997
). Because the
IC50 for high-affinity Zn2+
antagonism is near the levels of contaminating
Zn2+ (Paoletti et al. 1997
), the
high-affinity Zn2+ chelator TPEN (1 µM) was
included in the control solution. Whole cell NMDA (1 mM) currents from
wild-type neurons (Fig. 3B) were reduced slightly in
Zn2+ (500 nM; 80.8 ± 3.0% of control,
n = 6) whereas currents from neurons lacking
2
were almost completely blocked (18.1 ± 1.8% of control,
n = 6). In contrast, the selective
1/
2 receptor antagonist ifenprodil (3 µM: Williams 1993
) reduced whole cell NMDA
(1 mM) currents from wild-type (33.3 ± 6.0% of control,
n = 8) to a much greater extent than currents from
2
/
neurons (85.2 ± 1.7%,
n = 5; Fig. 3C). Thus the pharmacological profile of
2
/
NMDA receptors is consistent with that
expected from
1/
1 heteromers.
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DISCUSSION |
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NMDA receptor-mediated EPSCs from cultured hippocampal neurons
lacking the 2 subunit have very fast deactivation kinetics compared
with those from wild-type neurons. Fast NMDA receptor-mediated EPSC
kinetics have been reported in wild-type animals but seem to be the
exception rather than the norm. EPSC deactivation kinetics similar to
those reported here occur during synapse formation in the rat spinal
cord (Bardoni et al. 1998
) and cerebellar granule cells
(D'Angelo et al. 1993
). Additionally, EPSC deactivation kinetics recorded from mature synapses on cerebellar granule neurons from mice lacking the
3 (NR2C) subunit are very similar to those that we report from
2
/
neurons
(Ebralidze et al. 1996
). NMDA receptor-mediated EPSC deactivation kinetics in other preparations are comparable to our
results from wild-type neurons. The slow kinetics of the NMDA receptor-mediated EPSC have been thought to account for rhythmic, pacemaker activities like breathing, locomotion, and suckling, arising
from central pattern generators in brainstem nuclei and the spinal
cord. However, the neonatal death in mice lacking the
2 subunit most
likely results from the absence of synaptic NMDA receptors in regions
otherwise expressing the
2 subunit, rather than changes in
deactivation kinetics.
Our data indicates that the 2 subunit is not required for synaptic
localization of NMDA receptors. Previous work with these animals
reported a lack of the NMDA receptor-mediated component of the field
EPSP in
2
/
hippocampi from animals at
postnatal day 2-3 (Kutsuwada et al. 1996
). This is
consistent with our observation that NMDA-evoked currents from
2
/
neurons often do not appear until 4-5
DIV and
1 is not expressed until after this time in vivo
(Monyer et al. 1994
; Sheng et al. 1994
).
The increase in expression of
1 is coincident with the acceleration
of the wild-type NMDA receptor-mediated deactivation kinetics
(Carmingnoto and Vicini 1992
; Hestrin
1992
).
On the basis of the characteristics of NMDA receptor-mediated EPSCs,
we propose that the NMDA receptors expressed in
2
/
neurons are
1/
1 diheteromeric
receptors. If this is true, this means that the signals required for
NMDA receptor targeting to the somato-dendritic membrane and/or
synaptic localization of NMDA receptors are found on
1 or
1. The
2 subunit is required early in development because of a need for
functional NMDA receptors rather than targeting (to the
somato-dendritic membrane) or localization (to the postsynaptic
membrane) of synaptic receptors. Targeting and localization events may
require different amino acid sequence motifs. The carboxy-terminal
intracellular regions of
1 and
2 have amino acid sequence
homologies in the region just after the final transmembrane segment and
at the PDZ recognition sites at the carboxy termini. Mice lacking the
PDZ recognition sequences in
2 show decreased levels of synaptic
NMDA receptors (Mori et al. 1998
). This suggests that
NMDA receptor targeting is retained but synaptic localization may be
reduced. Furthermore, proteins that bind to the PDZ domain such as
PSD-95 are not required for synaptic localization of these receptors.
The AMPA receptor-mediated component appears normal in animals without
the NMDA receptor-mediated component (Kutsuwada et al.
1996
), indicating that incorporation of AMPA receptors at
synapses is independent of NMDA receptor activation.
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ACKNOWLEDGMENTS |
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We thank Dr. Masayoshi Mishina for a gift of 2-targeted mice and
A. Miller for technical assistance with genotyping.
This work was supported by National Institute of Mental Health Grants MH-11204 to K. R. Tovar and MH-46613 to G. L. Westbrook. K. R. Tovar was also supported by the Scottish Rite Schizophrenia Research Program, Northern Masonic Jurisdiction, USA.
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FOOTNOTES |
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Address for reprint requests: G. L. Westbrook, Vollum Institute, L474, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201-3098.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 July 1999; accepted in final form 1 September 1999.
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REFERENCES |
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