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J Neurophysiol (January 1, 2003). 10.1152/jn.00342.2002
Submitted on Submitted 6 May 2002; accepted in final form 5 September 2002.
1Department of Pharmacology and Toxicology, College of Pharmacy and 2Anticonvulsant Screening Project, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Chapman, David E., Kristen A. Keefe, and Karen S. Wilcox. Evidence for Functionally Distinct Synaptic NMDA Receptors in Ventromedial Versus Dorsolateral Striatum. J. Neurophysiol. 89: 69-80, 2003. N-methyl-D-aspartate receptors (NMDARs) are comprised of different subunits. NR2 subunits confer different pharmacological and biophysical properties to NMDARs. Although NR2B subunit expression is uniform throughout striatum, NR2A subunit expression is greater laterally. Pharmacologically isolated NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) were elicited using minimal local stimulation and recorded in the whole cell configuration to test the hypothesis that biophysical and pharmacological properties of NMDAR-EPSCs of striatal neurons would vary as a function of their location in adult rat striatum. We observed that the decay-time kinetics of NMDAR-EPSCs are significantly slower in neurons of ventromedial versus dorsolateral striatum. Whereas ifenprodil did not differentially affect NMDAR-EPSCs in these regions, application of either glycine or D-serine increased the peak current of NMDAR-EPSCs selectively in dorsolateral striatum. These data provide evidence for functionally distinct NMDARs in the same neuron type in the same brain region of the adult rodent brain. These data thus suggest that the nature of synaptic processing of excitatory input is different in the ventromedial and dorsolateral striatum of the adult rodent brain, regions differentially involved in limbic versus sensorimotor processes, respectively.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The striatum is the main
input nucleus of the basal ganglia, a group of subcortical structures
critically involved in motor and cognitive function. Spiny efferent
neurons comprise approximately 95% of all neurons in striatum. These
neurons receive excitatory afferents from cerebral cortex and thalamus
(Kemp and Powell 1971
; Sadikot et al.
1992), which activate postsynaptic
N-methyl-D-aspartate receptors (NMDARs) and
non-NMDARs (Herrling et al. 1983).
NMDARs consist of an NR1 subunit and any of four NR2 subunits (NR2A-D)
(Dingledine et al. 1999). Whereas the NR1 subunit is expressed ubiquitously throughout the brain, expression of NR2 subunits
is under spatial and temporal regulation (Monyer et al. 1994). Although all spiny efferent neurons express NR2A and
NR2B subunits (Standaert et al. 1999), the relative
expression of NR2A subunit mRNA is greater laterally (Ganguly
and Keefe 2001; Watanabe et al. 1993). Despite
the unknown stoichiometry of native NMDARs, the differential expression
of the NR2A subunit mRNA in medial and lateral striatum suggests that
the subunit composition of NMDARs will vary between these regions.
Incorporation of different ratios of NR2 subunits into postsynaptic
receptors across striatum is likely to be important for information
processing, as many functional properties of NMDARs are governed by NR2
subunit composition. For example, NMDARs containing the NR2A subunit
demonstrate the highest affinity for competitive antagonists
(Buller et al. 1994). Receptors containing the NR2B subunit, meanwhile, have greater affinity for agonists, including glycine (Buller et al. 1994; Kutsuwada et al.
1992; Stern et al. 1992). Consistent with these
properties, receptor binding studies conducted on striatal tissue show
greater binding of competitive antagonists in lateral striatum, a
region rich in NR2A mRNA expression. In comparison, agonist binding is
uniform throughout striatum, paralleling the NR2B receptor mRNA
distribution (Buller et al. 1994; Monaghan et al.
1988). Thus there appear to be distinct NMDAR populations in striatum.
While the data reviewed above suggest the presence of distinct NMDARs
in different regions of striatum, it is not currently known whether the
properties of the postsynaptic receptors vary. Although deactivation
kinetics of NMDARs were previously characterized in striatum
(Götz et al. 1997), this study did not report the striatal subregion from which the recordings were conducted.
Furthermore, the study by Götz and colleagues used the nucleated
patch and rapid agonist exchange techniques, and therefore surveyed
primarily extrasynaptic receptors at a single, early developmental time point. Developmental studies and experiments in expression systems show
that the decay-time kinetics of NMDAR-mediated excitatory postsynaptic
currents (NMDAR-EPSCs) and deactivation kinetics, respectively,
become faster as the ratio of NR2A:NR2B increases (Flint et al.
1997; Kirson and Yaari 1996; Vicini et
al. 1998). Furthermore, it has been proposed that
NMDAR-EPSCs which have faster decay-time kinetics shift the
probability and degree of induction of long-term potentiation (LTP)
(Philpot et al. 2001; Tang et al. 1999).
However, whether such differences are evident in the same neuronal type
of the adult brain expressing different ratios of NR2A:NR2B is unknown.
The purpose of this study was therefore to test whether the biophysical and pharmacological properties of NMDAR-EPSCs would differ based on location of the recorded neuron within the striatum and the developmental time point at which the slice was prepared. We report that the decay-time kinetics of the NMDAR-EPSCs were significantly faster in dorsolateral striatum in slices obtained from adult animals. In addition, application of either glycine or D-serine selectively enhanced the peak amplitude of NMDAR-EPSCs in dorsolateral striatum. These data indicate that there are functionally distinct synaptic NMDARs in ventromedial versus dorsolateral striatum in the adult rodent brain. Furthermore, these results suggest that processing of excitatory input through NMDARs is different in the ventromedial and dorsolateral striatum, two regions that differ in the processing of limbic and sensorimotor information.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male Sprague-Dawley rats (approximately 150 g) were used in adult experiments. P4-6 rats were used in all neonatal experiments. Rats were housed in groups in a room controlled for temperature and lighting. Rats had free access to food and water. All animal care and experimental manipulations were approved by the Institutional Animal Care and Use Committee of the University of Utah and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Striatal slice preparation
Acute brain slices were obtained as previously described, with
slight modifications (Wilcox et al. 1996). Briefly, rats
were anesthetized with pentobarbital (50 mg/kg) and decapitated. The brains were rapidly removed and placed into an ice cold, oxygenated (95% O2-5% CO2) sucrose
Ringer solution, pH = 7.4, containing (in mM) 200 sucrose, 3 KCl,
1.4 NaH2PO4, 2 MgSO4, 26 NaHCO3, 10 glucose, and 2 CaCl2. The brain was divided along
the midline. The brain hemisphere was then glued caudal-side down to a
Vibraslicer chuck (Campden Instruments). Coronal sections (300-350
µm) containing the striatum were collected and placed in a holding
chamber at room temperature containing oxygenated Ringer solution with
126 mM NaCl in place of the sucrose, pH = 7.37-7.41. The osmolality of
the Ringer solution was 295-305 mOsm. Sections remained in the Ringer
solution for 
1 h before recording.
Patch-clamp recordings
Slices were transferred into the recording chamber, which was
perfused with fresh, oxygenated, Mg2+-free Ringer
solution at room temperature (21-23°C) via a gravity-feed system at
approximately 4 ml/min. The whole cell patch-clamp technique was used
to record from single striatal neurons. Neurons were patch-clamped
using the blind technique (Blanton et al. 1989). Microelectrodes with a resistance of 3-6 M
were pulled using a P-87
micropipette puller (Sutter Instruments). For the majority of the
experiments, the internal solution in the borosilicate glass
microelectrodes consisted of (in mM) 130 K gluconate, 10 KCl, 10 HEPES,
1 EGTA, and 0.1 CaCl2. The pH was 7.28-7.31 and the osmolality was 285-295 mOsm. In a subset of experiments, CsCl (140 mM) was substituted for K gluconate and KCl, and QX-314 (10 mM) was
added to either internal solution.
EPSCs were elicited using local, minimal stimulation to mitigate
voltage- and space-clamp errors (Stevens and Wang 1994;
Wilcox et al. 1996). Briefly, a bipolar stimulating
electrode was placed in the vicinity of the recording electrode (<300
µm) either in the dorsolateral or the ventromedial striatum. The
stimulating electrode was used to deliver current pulses (100 µs) of
sufficient amplitude to produce the smallest EPSC (25-40 pA),
which could be reliably evoked at low frequency (0.1 Hz). To isolate
NMDAR-EPSCs, the Ringer solution also contained 10 µM
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block the non-NMDA
component of glutamate neurotransmission, 50 µM picrotoxin to block
activation of GABAA receptors, and 10 µM
glycine to maximally activate NMDARs. For experiments under nominal
glycine conditions, no glycine was included in the Ringer solution and
10 µM 6-nitro-7-sulfamoylbenzo(f)-quinoxaline-2,3-dione (NBQX) was
substituted for CNQX.
Data were acquired with an Axopatch 1D amplifier and the CLAMPEX8 software package interfaced to a Digidata 1200 acquisition board (Axon Instruments). Signals were filtered at 5 kHz and sampled at 10 kHz.
Inclusion criteria
Only recordings that did not exhibit substantial changes in
either holding current or resistance at the electrode tip were used for
analysis. All cells required <100 pA to be clamped to 
70 mV. Cells
with resting membrane potentials above 55 mV were omitted from data
analysis. For analysis in current-clamp mode, the membrane potential
was monitored in response to current injection. In all cases, this
measurement was taken near the middle of the current injection. In
depolarizing current injections, during which an action potential was
generated, the measurement was taken immediately prior to the action
potential. A total of 153 cells meeting these inclusion criteria were
used in the present study.
Data analysis
The following parameters were determined for averaged EPSCs
in all experiments: risetimes, peak amplitudes, decay time constants, and weighted 
(w). All data are presented
as mean ± SE. Data were tested for significance using a
Student's t-test. Statistical significance was set at
P 
0.05. To evaluate differences in peak amplitude,
statistics were done on peak amplitudes expressed as percent of control
using a two-way ANOVA. For ifenprodil studies, control values were
defined as the peak amplitude of the NMDAR-EPSC prior to
administration of ifenprodil. For experiments where the concentration
of glycine or D-serine was manipulated, control values were
defined as the peak amplitude of the NMDAR-EPSC in the presence of
no added glycine. The decay time constants were fit with a double
exponential equation: I(t) = If × exp(t/f) + Is × exp(t/s), where
If is the amplitude of the fast
component, Is is the amplitude of the
slow component, and f and
s are the fast and slow time constants,
respectively. Weighted time constants were calculated using the
equation: w = [If/(If + Is)] × f + [(Is/(If + Is)] × s
(Stocca and Vicini 1998).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Basic properties of striatal neurons and isolation of synaptic NMDAR-EPSCs
In recordings from cells in both the ventromedial and dorsolateral
striatum, basic cell membrane properties were very similar between
regions and consistent with previous reports for spiny efferent neurons
(Tepper et al. 1998). Prolonged injection of current
produced the signature depolarization ramp (Fig.
1A). Additionally, the change
in membrane potential as a function of current injection was nearly
indistinguishable between the two regions of striatum (Fig.
1B) and showed decreased input resistance in response to
hyperpolarizing current injection (Tepper et al. 1998).
Isolated EPSCs were confirmed to be mediated by NMDARs, as shown by
the reversible block of the EPSCs in all cells tested by bath
application of the NMDAR antagonist
D,L-2-amino-5-phosphonovaleric acid
(D,L-APV; 50 µM; peak amplitude was 3.1 ± 2.8% of control; n = 6; Fig. 1C).
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Kinetics of NMDAR-EPSCs in ventromedial and dorsolateral adult striatum
Previous studies examining different timepoints in
postnatal development have shown that NMDARs comprised of different
subunits give rise to NMDAR-EPSCs with different decay-time
kinetics (Flint et al. 1997; Kirson and Yaari
1996). Additionally, heterologous expression systems expressing
NMDARs comprised of different subunits exhibit different
deactivation kinetics following rapid application of agonist
(Chen et al. 1999; Vicini et al. 1998).
However, differences in NMDAR decay-time kinetics have not previously
been demonstrated in a single neuronal type in a given structure in the
adult CNS in which different levels of NR2 subunits are expressed.
Therefore to test the hypothesis that the differential expression of
the NR2A subunit in medial versus lateral striatum would lead to
differences in the decay-time kinetics of NMDAR-EPSCs in these two
regions, we used local, minimal stimulation to assess the decay-time
kinetics of NMDAR-EPSCs in neurons in ventromedial and dorsolateral
striatum (Figs. 2 and
3). As shown in Fig. 2A,
neurons recorded in ventromedial striatum had evoked NMDAR-EPSCs
that had significantly longer decay-time constants when compared with
neurons recorded in the dorsolateral striatum. A double exponential was
required to fit the decay of the NMDAR-EPSCs in both regions of the
striatum. The average values of the fast and slow components of the
decay-time constant (f and
s, respectively) of the NMDAR-EPSCs were
significantly faster in recordings from dorsolateral striatum (Table
1). Furthermore, the relative fractional
contribution of the fast component of the decay (%Fast) was
significantly greater in EPSCs from the dorsolateral striatum than
from the ventromedial striatum (Table 1). Taken together, these values
produced an average w of the decay-time
constant that was significantly faster for NMDAR-EPSCs in
dorsolateral cells (Fig. 3; Table 1). Additionally, the risetime kinetics of NMDAR-EPSCs of neurons in the dorsolateral striatum were significantly faster than those in the ventromedial striatum (Fig.
2C; Table 1). When QX-314 (10 mM) was included in the
internal pipette solution to block voltage-activated
Na+-channels and postsynaptic
GABAB receptor-mediated effects, the differences
in w of the decay-time constant of
NMDAR-EPSCs in ventromedial versus dorsolateral striatum were still
observed (w =373 ± 41 ms vs. 173 ± 26 ms; P = 0.002; n = 4 and 6, respectively) (Nathan et al. 1990). Furthermore, using a
CsCl internal solution with QX-314, we voltage clamped the membrane
potential of the neurons in both regions to +40 mV and observed that
the profound difference in decay time kinetics of NMDAR-EPSCs in
each region persisted (w =315 ± 3 ms vs.
237 ± 26 ms in ventromedial vs. dorsolateral striatum,
respectively; P = 0.04; n = 3, Fig.
2B).
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Kinetics of NMDAR-EPSCs in ventromedial and dorsolateral neurons in neonatal striatum
Expression of NR2 subunits is developmentally regulated
(Monyer et al. 1994). Whereas the NR2B subunit is
expressed in the striatum at birth, levels of NR2A subunit protein are
not detectable until P7 (Sheng et al. 1994; Wang
et al. 1995). To confirm the role of different NR2 subunit
combinations in the biophysical properties of NMDAR-EPSCs in
ventromedial versus dorsolateral striatum, we conducted whole cell
patch-clamp experiments under the same conditions (low frequency,
local, minimal stimulation) in slices from neonatal rats. Unlike adult
striatum (Fig. 2), recordings from neurons in ventromedial and
dorsolateral striatum from P4-6 rat pups revealed no significant
differences in f, s,
%Fast, and w decay-time kinetics, as well as
rise time kinetics (Fig. 4; Table 1).
However, the rise times (Table 1) and decay-time kinetics are
significantly slower in both regions of neonatal striatum than they are
in the corresponding regions of adult striatum, suggesting
incorporation of NR2A subunit into NMDARs throughout adult striatum
(w = 688 ± 53 vs. 488 ± 22 ms in
ventromedial; P < 0.001; 583 ± 41 vs. 231 ± 15 ms in dorsolateral; P < 0.0001). The slower
decay-time and risetime kinetics of EPSCs evoked in cells of both
regions of striatum in slices obtained from pups support the hypothesis
that the differences between NMDAR-EPSCs in ventromedial versus
dorsolateral striatum in adults arise as a consequence of differences
in NR2 subunit incorporation into the NMDARs.
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Effects of ifenprodil on NMDAR-EPSCs in ventromedial and dorsolateral neurons in adult striatum
Ifenprodil is an NMDAR antagonist that has a 400-fold greater
affinity for NR1/NR2B receptors than for NR1/NR2A (Williams 1993). We therefore used this agent to investigate the
pharmacological properties of synaptic NMDAR-EPSCs in neurons in
ventromedial and dorsolateral striatum. The low levels of NR2A mRNA
expression in ventromedial striatum led to the hypothesis that
ifenprodil would have a greater effect on NMDAR-EPSCs in the
NR2B-predominate ventromedial striatum. Figure
5A shows the averages
of 35 NMDAR-EPSCs from cells of the ventromedial and dorsolateral
adult striatum in the presence or absence of ifenprodil. In all cases,
drug administration was followed by a wash period, where NMDAR-EPSC
peak amplitudes returned to >95% of control amplitudes. At a number
of different concentrations (1, 3, and 10 µM), ifenprodil did not
differentially inhibit NMDAR-EPSCs in ventromedial versus
dorsolateral striatum of adult rats (Fig. 5A). Particularly interesting
was the fact that 3 µM ifenprodil had little effect on NMDAR-EPSC
peak amplitudes (Fig. 5A), because this concentration of ifenprodil
produces a profound decrease in NMDAR-EPSC amplitude at early
timepoints in development (no NR2A), and loses efficacy across
development as NR2A expression increases (Kew et al.
1998; Kirson and Yaari 1996; Williams
1993). Even at a higher concentration of ifenprodil (10 µM),
there was still no significant difference between the amount of
inhibition of the NMDAR-EPSCs in ventromedial and dorsolateral striatal neurons (Fig. 5A; 38% inhibition in ventromedial
striatum vs. 46% in dorsolateral striatum, P = 0.3).
At each of these concentrations of ifenprodil, the kinetics of
NMDAR-EPSCs were not altered. This minimal decrease was not due to
an ifenprodil-mediated decrease in glutamate release, because 3 µM
ifenprodil had no significant effect on non-NMDAR-EPSCs (peak
amplitude of non-NMDA component of EPSC was 99 ± 2.3% of
control, n = 3; data not shown).
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Previous studies conducted on hippocampal cells in culture have shown
that decreasing the concentration of the required coagonist glycine
results in increased sensitivity of NMDA-evoked currents to blockade by
ifenprodil (Kendrick et al. 1998). However, elimination of exogenously added glycine failed to alter the inhibition of NMDAR-EPSCs by 10 µM ifenprodil in either location of striatum (41.0 ± 3.8% inhibition in ventromedial striatum vs. 38.7 ± 6.3% in dorsolateral striatum, P = 0.5, n = 3 for both ventromedial and dorsolateral striatum;
data not shown).
Effects of ifenprodil on NMDAR-EPSCs in ventromedial and dorsolateral neurons in neonatal striatum
Ifenprodil has been shown to effectively block NMDAR-EPSCs in
the brain slices prepared from neonatal animals (Kirson and Yaari 1996). Again, this is a time point at which no NR2A
subunit is expressed in brain. In the present studies, administration of 3 µM ifenprodil to P4-6 neonatal rats resulted in dramatic attenuation of NMDAR-EPSC peak amplitudes in both ventromedial and
dorsolateral striatum (Fig. 5B). The marked sensitivity of NMDAR-EPSCs to ifenprodil in recordings from neonatal striata, regardless of cell location, provides further evidence that the presence of the NR2A subunit in adult striatal NMDARs alters the function of these channels.
Effects of glycine and D-serine on NMDAR-EPSCs in ventromedial and dorsolateral striatum
Glycine is a required coagonist at NMDARs. NMDARs containing the
NR2B subunit have an approximately 10-fold greater affinity for this
ligand (Kutsuwada et al. 1992; Stern et al.
1992). Additionally, it has been shown previously in expression
systems and cell culture, as well as acute hippocampal brain slices,
that exogenous application of glycine increases the amplitude of
agonist-evoked currents and NMDAR-EPSCs, respectively
(Bergeron et al. 1998; Kutsuwada et al.
1992; Stern et al. 1992; Wilcox et al.
1996). Therefore we were interested in testing the effects of
different glycine concentrations on NMDAR-EPSCs from neurons in
ventromedial versus dorsolateral striatum. These experiments were
conducted in nominal (no added) glycine, and 10 µM NBQX was used in
place of CNQX to block all non-NMDAR-EPSCs. As seen previously in
high glycine (10 µM) conditions, low frequency stimulation (0.1 Hz)
of the ventromedial striatum in nominal glycine evoked EPSCs with a
longer decay-time constant relative to EPSCs elicited by the same
stimulation paradigm in dorsolateral striatum (Table
2). As seen in recordings from the in
vitro hippocampal brain slice preparation (Wilcox et al.
1996), the decay-time kinetics of NMDAR-EPSCs did not
change following the switch from nominal to 10 µM glycine (Fig.
6; Table 2). However, addition of 10 µM
glycine did result in differential effects on the amplitude
of NMDAR-EPSCs in ventromedial and dorsolateral striatum (Fig. 6).
In ventromedial striatum, there was no change in the peak amplitude of
the NMDAR-EPSCs (Fig. 6, A and C; 103.9 ± 3.3% of control; n = 9 in 9 different slices). In
dorsolateral striatum, however, bath application of 10 µM glycine
resulted in an increase in the amplitude of the NMDAR-EPSCs (Fig.
6, B and C; 145.0 ± 10.4% of control;
n = 6 in 6 different slices). A two-way ANOVA revealed
a significant interaction between the concentration of glycine and the
location of the cell (P = 0.003). On post hoc analysis,
the enhancement of the NMDAR-EPSC amplitude following bath
application of 10 µM glycine was significant in dorsolateral striatum
(P = 0.017), but not in ventromedial striatum (P = 0.79; Fig. 6C).
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Although 10 µM glycine produced a selective enhancement of
NMDAR-EPSCs in dorsolateral striatum in our experiments, there is
evidence that glycine uptake regulates synaptic levels of glycine and
therefore the ability to modulate NMDAR activation (Berger et
al. 1998). Although a glycine transporter is expressed in
striatum (Borowsky et al. 1993; Zafra et al.
1995), it is currently unknown if there are regional variations
in either expression or function of the transporter. To control for any
potential effect of regional function of gycine transporters, we
examined the effect of D-serine on NMDA-R EPSCs in
dorsolateral versus ventromedial striatum. D-serine is an
agonist at the glycine site of the NMDA receptor that, like glycine,
also displays a difference in affinity for NMDA receptors that are
comprised of different NR2 subunits. In addition, D-serine
does not serve as a substrate for the glycine transporter
(Schell et al. 1995; Supplisson and Bergman
1997). As seen with glycine, application of 3 µM
D-serine showed no enhancement of EPSCs in ventromedial
striatum (102.5 ± 3.4% of control; P = 0.48;
n = 3; Fig. 7,
A and C), but a significant enhancement in
dorsolateral striatum (124.9 ± 7.0% of control;
P = 0.016; n = 4; Fig. 7, B
and C). These data provide additional support for distinct
NMDARs in differential regions in adult striatum.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present experiments provide important new evidence for both temporal and spatial regulation of functionally distinct synaptic NMDARs in brain slices of the rodent striatum. Not only do the risetimes and decay times of NMDAR-EPSCs become faster as a consequence of development, but there is a regional disparity in the EPSC kinetics as well. NMDAR-EPSCs in the dorsolateral striatum have significantly faster risetimes as well as substantially faster decay times than those recorded in the ventromedial region of the striatum. Additionally, application of the NMDA receptor coagonists glycine and D-serine significantly increases the NMDAR-EPSC peak amplitude only in the dorsolateral region of the striatum. These findings suggest strongly that properties of NMDARs, and thus processing of excitatory input across ventromedial to dorsolateral adult striatum, are not uniform. Such variation likely will give rise to differences in signal processing in these specific regions of striatum, and might be relevant to the different functions of ventromedial (limbic) versus dorsolateral (sensorimotor) striatum.
The differences in the NMDAR-EPSC rise- and decay-time kinetics
observed regionally in adult striatum likely arise as a consequence of
differences in NR2A subunit incorporation into the synaptic NMDARs.
First, irrespective of location in striatum, basic membrane properties
of the cells were very similar. Second, the level of NR2A subunit mRNA
expression is substantially greater in lateral than medial striatum
(Ganguly and Keefe 2001; Watanabe et al. 1993), and this regional distribution of subunit expression
matches the regional distribution of the kinetic differences. Third,
the activation and deactivation kinetics of NR1/NR2A receptors studied in expression systems are faster than those of NR1/NR2B receptors (Chen et al. 1999; Monyer et al. 1994;
Vicini et al. 1998). Likewise, as NR2A subunit
expression increases across development, the rise times (Kirson
and Yaari 1996) and decay kinetics of the NMDAR-EPSC, both
here and in a number of other brain regions, decrease
(Carmignoto and Vicini 1992; Flint et al.
1997; Hurst et al. 2001; Stocca and
Vicini 1998). Finally, the present results show that there are
no differences in the rise times or decay kinetics of NMDAR-EPSCs in ventromedial versus dorsolateral striatum at a developmental timepoint (P4-6) at which there is no NR2A subunit expression in
striatum (Sheng et al. 1994; Wang et al.
1995). However, the immaturity of excitatory synapses on
striatal neurons at this timepoint (Butler et al. 1998)
may contribute to the slow NMDAR-EPSC decay-time kinetics in
both regions. Although other cellular mechanisms such as
phosphorylation and
[Ca2+]i (Shi et
al. 2000; Umemiya et al. 2001) can
influence NMDAR-EPSC decay-time kinetics, regional differences in
the striatum of either calcineurin or Ca2+
buffering have not been reported (for example, see Sola et al. 1999). In addition, we have demonstrated here that the kinetics for the EPSCs are still dramatically different at holding
potentials of +40 mV, a voltage at which Ca2+
influx through NMDA receptor-gated ion channels and voltage-sensitive Ca2+ channels is much reduced. This suggests that
the kinetics of the EPSCs are due to intrinsic differences of the
NMDA receptor rather than regional differences in calcium signaling.
Taken together, these findings strongly suggest that the differential
regional distribution of the NR2A subunit in adult striatum results in the presence of functionally distinct NMDARs in the same neuron type in
different regions of adult striatum.
Whereas the kinetic properties of NMDAR-EPSCs in ventromedial and
dorsolateral striatum differ, the present findings indicate that not
all of the pharmacological properties of the receptor vary in a similar
manner. In slices from adults, bath application of the NR2B-selective
antagonist ifenprodil did not differentially affect
NMDAR-EPSCs in ventromedial versus dorsolateral striatum. These
findings are consistent with our previous in vivo studies that failed
to find differential effects of pharmacologically distinct NMDAR
antagonists on immediate early gene expression in medial versus lateral
striatum (Adams et al. 2000; Keefe and Adams
1998; Keefe and Ganguly 1998). Not only did
ifenprodil not differentially affect NMDAREPSCs in dorsolateral
versus ventromedial striatum, it produced only limited blockade of the
NMDAR-EPSCs in the adult striatum. Consistent with these
observations, a number of reports using adult animals have found
ifenprodil to be diminished in its capacity to block synaptic
NMDAR-EPSCs (Cathala et al. 2000; Ramoa and
Prusky 1997; Tovar and Westbrook 1999). These studies suggest that incorporation of the NR2A subunit into the receptor complex in adult animals is responsible for this decreased sensitivity to ifenprodil (Tovar and Westbrook 1999;
Williams 2001). If this is the case, then it is likely
that all synaptic NMDA receptors in the adult striatum contain an NR2A
subunit. Further supporting this hypothesis are the findings herein
that the risetimes and decay-time kinetics of the NMDAR-EPSCs are
faster in adults than in neonates, irrespective of regional location within the striatum, and that NMDAR-EPSCs are potently blocked by
ifenprodil in striatal slices taken at a developmental time point
(P4-6) at which the NR2A subunit is not present (Sheng et al.
1994; Wang et al. 1995). Therefore based on the
observed developmental changes and regional differences in the kinetic
and pharmacological properties of the NMDAR-EPSCs, we hypothesize
that the majority of synaptic striatal NMDARs in adult likely contain
the NR2A subunit, but that the number of NR2A subunits per
receptor is greater in dorsolateral striatum than in ventromedial
striatum, giving rise to functionally distinct NMDARs in different
regions of the adult striatum.
Although ifenprodil did not differentiate functionally distinct NMDARs
in ventromedial and dorsolateral striatum, manipulation of the glycine
or D-serine concentration did. NMDARs containing the NR2A
subunit have a lower affinity for glycine than do those containing the
NR2B subunit (Kutsuwada et al. 1992; Stern et al. 1992). Thus NMDARs in dorsolateral striatum, where NR2A
expression is greatest, should have the lowest affinity for glycine in
striatum and be most affected by exogenous glycine application. In
fact, addition of 10 µM glycine or 3 µM D-serine
significantly enhanced NMDAR-EPSCs in dorsolateral striatum. While
differences in endogenous glycine levels could exist, regional
differences in glycine transporter expression in striatum to date have
not been reported (Borowsky et al. 1993; Zafra et
al. 1995). The fact that D-serine had no effect on
NMDAR-EPSC peak amplitude in ventromedial striatum suggests that
differences in endogenous glycine levels due to regional differences in
transporter expression or function do not underlie the selective
enhancement of EPSCs in the dorsolateral striatum. These data
further support the idea that levels of ambient glycine in the slice
are not saturating for postsynaptic NMDA receptors expressed
in cells of the dorsolateral striatum, an area rich in NR2A subunit
expression. Indeed, receptors comprised of NR2A subunits have a lower
glycine affinity than those receptors comprised of NR2B subunits. Thus
the kinetic differences discussed above and the selective effect of
glycine and D-serine manipulations on NMDAR-EPSC peak
amplitude in neurons of dorsolateral striatum may reflect a greater
overall incorporation of NR2A-subunits into the NMDARs mediating these
EPSCs. In addition, these findings suggest that glycine or
D-serine availability is likely to significantly and
differentially impact NMDAR-mediated functions in different regions of striatum.
The functional relevance of subpopulations of NMDARs with distinct
biophysical and pharmacological properties in striatum is currently
unknown. However, Partridge et al. (2000) showed that
one can induce LTP at corticostriatal synapses in both medial and
lateral striatum early in development. As animals age, however, high-frequency stimulation of corticostriatal afferents results in
long-term depression in dorsolateral striatum, the area in which NR2A
mRNA expression increases notably during development. At this same
developmental timepoint, high-frequency stimulation of cortical
afferents terminating in the dorsomedial aspect of striatum, where NR2A
mRNA expression is not as abundant, continues to produce LTP. At a
variety of synapses, such as those in visual cortex and hippocampus,
changing the ratio of NR2A:NR2B is hypothesized to produce a shift in
the probability and degree of induction of LTP (Kirkwood et al.
1996; Philpot et al. 2001; Tang et al. 1999; but see also Barth and Malenka 2001;
Lu et al. 2001). Thus the relative level of NR2A
expression in different regions of adult striatum may underlie
different types of corticostriatal synaptic plasticity.
In conclusion, the present study provides electrophysiological and pharmacological evidence for different types of synaptic NMDARs in neurons in ventromedial versus dorsolateral striatum. These differences are most likely due to differential regional expression of NR2 subunits. NMDAR-EPSCs in neurons in dorsolateral striatum have substantially faster decay kinetics than those in ventromedial striatum, and the kinetics of NMDAR-EPSCs in both regions are significantly faster than the kinetics of NMDAR-EPSCs in brain slices of neonates. In addition, we have demonstrated that ambient concentrations of glycine are subsaturating in dorsolateral striatum, where a higher amount of NR2A subunit is expressed. These findings are consistent with both the temporal and spatial regulation of levels of expression of mRNA encoding the NR2A subunit in the striatum and may relate to known differences in the processing of cognitive and motor information in these two different regions of the striatum.
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ACKNOWLEDGMENTS |
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The authors thank the laboratory of the Anticonvulsant Screening Project at the University of Utah for the use of their facilities.
This work was supported by National Institutes of Health Grants NS-35579 and GM-07559 to K. A. Keefe, a College of Pharmacy Seed Grant to K. S. Wilcox, and predoctoral fellowships from the American Foundation for Pharmaceutical Education and National Institutes of Health DA-14859 to D. E. Chapman.
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FOOTNOTES |
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Address for reprint requests: K. S. Wilcox, 30 South 2000 East, Rm. 201, Salt Lake City, UT 84112 (E-mail: kwilcox{at}deans.pharm.utah.edu).
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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. Data for
dorsolateral cells are mean values ± SE (n = 9-15) and are represented by 
. Error bars that are not
visible are smaller than the symbol. C: reversible
blockade of NMDAR-EPSCs by the NMDAR antagonist
D,L-2-amino-5-phosphonovaleric acid
(D,L-APV; 50 µM; averages of 35 traces). Local
minimal stimulation of striatum in close proximity (<300 µm) to
recorded cells elicits a long-lasting EPSC. On bath application of
D,L-APV, there was a block of the EPSC, confirming
that it was mediated by the NMDAR. Recordings were conducted at a
membrane potential of 
, values from
individual ventromedial cells. 
