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The Journal of Neuroscience, August 15, 1998, 18(16):6558-6567
NMDA EPSCs at Glutamatergic Synapses in the Spinal Cord Dorsal
Horn of the Postnatal Rat
Rita
Bardoni1,
Pier
Cosimo
Magherini1, and
Amy B.
MacDermott2
1 Department of Biomedical Sciences, University of
Modena, 41100 Modena, Italy, and 2 Department of Physiology
and Cellular Biophysics and the Center for Neurobiology and Behavior,
Columbia University, New York, New York 10032
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ABSTRACT |
In rat dorsal horn, little is known about the properties of
synaptic NMDA receptors during the first two postnatal weeks, a period
of intense synaptogenesis. Using transverse spinal cord slices from
postnatal day 0-15 rats, we show that 20% of glutamatergic synapses
tested at low-stimulation intensity in spinal cord laminae I and II
were mediated exclusively by NMDA receptors. Essentially all of the
remaining glutamatergic EPSCs studied were attributable to the
activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at
pure and mixed synapses showed similar sensitivity to membrane
potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs
from pure and mixed synapses were measured at +50 mV. The 10-90% rise
times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay
 values were faster (1, 24 vs 42 msec; 2, 267 vs 357 msec)
than NMDA EPSCs at mixed synapses. Our results indicate that NMDA
receptors are expressed at glutamatergic synapses at a high frequency,
either alone or together with AMPA receptors, consistent with the
prominent role of NMDA receptors in central sensitization (McMahon et
al., 1993 ).
Key words:
NMDA receptor; spinal cord; dorsal horn; development; synaptic transmission; pure NMDA synapse
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INTRODUCTION |
Glutamate is the principal
fast excitatory neurotransmitter in the rat superficial dorsal horn.
Synaptically released glutamate activates postsynaptic AMPA and NMDA
receptors (Yoshimura and Jessell, 1990; Yoshimura and Nishi, 1993).
However, the frequency with which NMDA receptors are present at
glutamate synapses in the superficial dorsal horn and their basic
kinetic and voltage-dependent properties have not been investigated.
Despite this lack of specific information, it is known that NMDA
receptors in the dorsal horn are important in the generation and
maintenance of several forms of central sensitization associated with
allodynia and hyperalgesia (Dickenson et al., 1997). Furthermore, in
the adult rat after peripheral nerve injury, NMDA receptors have been
shown to be required for reestablishing narrowed receptive fields after
peripheral regeneration of afferents (Lewin et al., 1994). Thus, these
receptors appear to have multiple roles in the regulation of sensory
transmission in the spinal cord dorsal horn, yet little information is
available concerning the synaptic currents mediated by NMDA receptors
there.
NMDA receptors have a role in spinal cord development. Synaptic NMDA
receptor activation may help regulate dendritic outgrowth and establish
final synaptic connections in developing rat dorsal horn, as has been
suggested for ventral horn neurons (Kalb and Hockfield, 1990; Kalb,
1994). NMDA receptors may influence or mediate the dramatic changes in
receptive field size that evolve over the first few postnatal weeks
(Fitzgerald, 1985). One form of glutamatergic EPSC that has been
proposed to be developmentally important is the pure NMDA EPSC at which
only NMDA receptors are present, often called silent synapses (Isaac et
al., 1995; Liao et al., 1995; Durand et al., 1996). Some of the
earliest evidence for silent or ineffective synapses was obtained by
recordings made in the spinal cord (Wall, 1977; Malenka and Nicoll,
1997). Evidence for pure NMDA synapses was first found by Dale and
Roberts (1985) in a study on the spinal cord ventral horn of
Xenopus embryos. The presence of pure NMDA EPSCs in the
developing rat dorsal horn has only recently been reported (Bardoni et
al., 1997b; Zhuo and Li, 1997).
We have investigated the basic properties of NMDA receptor-mediated
synaptic currents in the rat superficial dorsal horn during the first
two postnatal weeks. NMDA receptors were found to be expressed both
alone and together with AMPA receptors, and the kinetics and voltage
dependence of NMDA EPSCs were measured. All synaptic NMDA receptors
were strongly voltage-dependent. The kinetics of the NMDA
receptor-mediated synaptic currents at synapses with NMDA and AMPA
receptors compared with those with NMDA receptors alone were
significantly different. No change in synaptic current kinetics was
detectable over 2 weeks of postnatal development.
The results of this work have been presented in preliminary form
(Bardoni et al., 1996, 1997a,b).
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MATERIALS AND METHODS |
Slice preparation. Postnatal day 0-15 (P0-P15) rats
were anesthetized and decapitated, and the cervical spinal cord was
removed and placed in oxygenated ice-cold Krebs' solution. Dorsal and ventral laminectomies were performed, and the spinal cord was isolated
and embedded in agarose (2.5% in Krebs' solution; 36-38°C). Transverse slices 300-400 µm thick were cut and then incubated in
oxygenated Krebs' solution at 35°C for 1 hr and used for
recording.
Solutions. Slices were perfused at room temperature with
95% O2-5% CO2 saturated Krebs' solution (in
mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1 NaH2PO4, 25 glucose, 1 MgCl2, and 2 CaCl2, pH 7.4, 320 mOsm). Bath solution (0 Mg2+) was obtained by
omitting MgCl2 from the Krebs' solution. Ten micromolar
bicuculline and 5 µM strychnine were added to the
perfusion bath in all experiments to block the strong GABAergic and
glycinergic input. The intracellular solution contained (in
mM): 130 Cs+-gluconate, 10 CsCl, 11 EGTA, 1 CaCl2, 10 HEPES, and 2 Mg2ATP, pH adjusted to 7.3 with NaOH, osmolarity adjusted
to 305-310 mOsm with sucrose. The drugs used included (in
µM): 10 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 6-nitro-7-sulfamoyl benzo[F]quinoxaline-2,3-dione (NBQX), 50 D( )-2-amino-5-phosphonopentanoic acid
(D-APV) (Tocris Cookson, Bristol, England), 10 bicuculline
methiodide, and 5 µM strychnine (Sigma, St. Louis, MO).
These compounds were superfused in the extracellular solution.
Recording and stimulation. Patch-clamp recordings in
whole-cell configuration were obtained from neurons in laminae I and II. The neurons were visually identified using a Zeiss (Oberkochen, Germany) Axioskop microscope fit with a 10× lens, a 63×
water-immersion lens, and a CCD video camera (Sony, Tokyo, Japan).
Lamina II was recognized as a translucent band in the external part of
the dorsal horn. Lamina I neurons were identified as the cells
localized in the thin region between lamina II and white matter.
The focal stimulation of the tissue around the postsynaptic neuron
under study was obtained by moving a glass pipette (5 µm tip
diameter) around the cell in a perimeter of ~100 µm from the cell
body until a synaptic response was evoked. Stimuli had intensities ranging from 2 to 20 V (stimulus isolation unit output) and a duration
of 0.3 msec. Stimulation frequency in most experiments was 0.5 Hz.
The recording electrodes were made from thick-walled borosilicate glass
and had a resistance of ~5 M . Whole-cell recordings were obtained
in voltage-clamp configuration; usually a 70% series resistance
compensation was introduced. Data were recorded and acquired using an
Axopatch-1D amplifier and pClamp 5.5 software (both from Axon
Instruments, Foster City, CA). Signals were filtered at 1 kHz and
sampled at 2-10 kHz. Peak amplitudes and rise time values were
measured from averaged EPSCs recorded at +50 mV and evaluated using
pClamp 5.5 software (Clampfit). EPSC decay fits and linear regressions
of the data were performed using Sigmaplot software (SPSS, Erkrath,
Germany).
Statistical analysis. The data in Figure 2 were expressed as
a percentage of the total number of cells (see Fig.
2A) or synapses (see Fig. 2B)
tested averaged by postnatal day. To test the data for a significant
trend over time, we used linear regression. However, the data were
required to first be transformed to the corresponding square root
values to normalize their distribution (Fleiss, 1986). In Figures 4, 6,
and 7, the data are represented with values averaged within 4-day bins.
Error bars represent SD in these figures. To evaluate differences
between NMDA receptor-mediated synaptic currents recorded from the two
synaptic types (mixed and pure), as well their age dependency, we used
the ANOVA test. One- and two-way ANOVA was obtained using SPSS
software.
Pure NMDA synapse identification procedure. Synapses
mediated by NMDA receptors alone, or pure NMDA EPSCs, were identified by recording EPSCs in voltage clamp in the presence of 10 µM bicuculline and 5 µM strychnine.
Initially, each cell was held at 70 mV, and focal stimulation was
applied at low frequency (0.2 Hz). In these conditions, high-intensity
focal stimulation (5-20 V) evoked EPSCs mediated mainly by AMPA
receptors. To establish the presence of pure NMDA EPSCs, the stimulus
intensity was progressively decreased (2-10 V) until AMPA EPSCs
disappeared. The membrane was then depolarized to +50 mV, and focal
stimuli were applied at the same frequency. The presence of a pure NMDA
EPSC was revealed by the appearance of a slow outward EPSC of 10-30 pA
amplitude. This minimal stimulation protocol to identify pure NMDA
synapses was first used by Liao et al. (1995) and Isaac et al. (1995).
Usually five different locations around the postsynaptic cell were
tested for the presence of pure NMDA synapses. In a few instances, the
increase of the stimulus intensity did not recruit mixed synapses, and
only pure NMDA EPSCs were seen.
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RESULTS |
Synapses with only NMDA receptors present
When recording from lamina II neurons that were held under voltage
clamp at 70 mV, focal stimulation of the surrounding tissue or
primary afferent stimulation evoked fast excitatory synaptic transmission. The evoked EPSCs were mediated predominantly by AMPA
receptors with a variable contribution from NMDA receptors (Yoshimura
and Jessell, 1990; Bardoni et al., 1996). Because of the voltage
dependence of NMDA receptor current flow, however, more systematic
tests for synaptic NMDA receptor activation in the presence of
Mg2+ were made at positive membrane potentials. To
detect pure NMDA EPSCs, stimulation intensity at the focal stimulating
electrode was decreased until no fast AMPA receptor-mediated EPSCs were apparent at 70 mV (Fig.
1A). When the membrane
potential was then changed to +50 mV, a slow outward EPSC often became
evident. This slow synaptic current at +50 mV was completely and
reversibly blocked by D-APV (Fig. 1A).
Thus, the slow synaptic currents were identified as being mediated by
NMDA receptors as defined by the blocking effect of D-APV,
effective on all eight neurons tested, and by the strong voltage
dependence of the EPSC amplitude. These are referred to here as pure
NMDA synapses or pure NMDA EPSCs.
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Figure 1.
Some glutamatergic synapses evoked in lamina II
neurons are mediated by NMDA receptors only. A, EPSCs
were recorded from a P10 lamina II neuron in 10 µM
bicuculline and 5 µM strychnine at different holding
potentials to test for the presence of pure NMDA EPSCs. Stimulation of
the neuron held at 70 mV produced no detectable AMPA EPSC. EPSCs
recorded at +50 mV were completely blocked by 50 µM
D-APV. After washout of D-APV for 5 min, the
NMDA EPSCs tested at +50 mV recovered. All currents are averages of
three traces. B, EPSCs were recorded from a P3 lamina II
neuron in 10 µM bicuculline and 5 µM
strychnine. Application of 10 µM NBQX did not affect the
EPSC recorded at +50 mV. When the rising phase of the EPSCs recorded in
control (solid line) and in NBQX (dashed
line) are superimposed, no effect of NBQX is detectable. Adding
D-APV to the NBQX containing extracellular solution blocks
the EPSC. All currents are averages of five traces.
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As an additional control to ensure that no AMPA receptors contributed
to the pure NMDA synaptic responses, we tested the effect of the AMPA
receptor antagonist NBQX on currents considered to be mediated by NMDA
receptors alone. Synaptic events were recorded at 70 and +50 mV in
the absence and presence of 10 µM NBQX (Fig. 1B). There was no change in the synaptic current
after addition of NBQX (n = 2). To be certain that NBQX
was not blocking a small fast AMPA current at +50 mV, the averaged
currents with and without NBQX were overlaid, revealing no change in
current rise time or peak associated with NBQX application, indicating
that no AMPA receptor-mediated component was present. However, as
expected, the synaptic current at +50 mV was completely blocked by 50 µM D-APV.
The frequency of occurrence of pure NMDA synapses was shown to decrease
with maturation in several regions of the nervous system (Durand et
al., 1996; Wu et al., 1996; Isaac et al., 1997). Therefore, we tested
whether the presence of pure NMDA EPSCs was developmentally regulated
in lamina II of the spinal cord. As shown in Figure
2A, a high percentage
(44%) of lamina II neurons exhibited at least one pure NMDA synapse of
the three to five synaptic inputs tested. Figure 2B
shows the percentage of pure NMDA synapses over the total number of
glutamatergic EPSCs (i.e., pure NMDA receptor-mediated and NMDA plus
AMPA receptor-mediated synapses) as a function of age. Averaged over
the 2 week test period, 20% of the total number of synapses tested
were mediated by NMDA receptors alone. No significant change in the
frequency of neurons expressing pure NMDA synapses or in the relative
proportion of pure NMDA synapses was observed in lamina II over the
first 2 postnatal weeks.
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Figure 2.
Analysis of the proportion of pure NMDA synapses
during the first 2 weeks of postnatal development. A,
Bars at each postnatal day represent the percentage of
lamina II neurons in which pure NMDA EPSCs were observed over the total
number of tested cells. The number over each bar is the
number of cells tested at that age. Inset, Linear
regression fit to the square root of the percentage of cells at each
age (r = 0.33; p > 0.05).
B, Bars represent the percentage of pure
NMDA synapses over the total number of glutamatergic (i.e., NMDA plus
AMPA) EPSCs for each age evoked by focally stimulating lamina II
neurons. The number over each bar is the number of
synapses tested at that age. Inset, Regression fit to
the square root of the percentage of synapses at each age
(r = 0.39; p > 0.05).
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It is important to note that the percentages of synapses reported in
these experiments (Fig. 2) are estimates. Under our stimulation conditions we could only detect pure NMDA EPSCs that had thresholds for
stimulation lower than those of mixed receptor EPSCs. In addition, the
synaptic responses defined as mixed may actually be attributable to
activation of both mixed and pure synapses by the same stimulus intensity (or, less likely, by the activation of pure NMDA and pure
AMPA synapses only). From our data, however, we can conclude that a
substantial proportion of glutamatergic synapses in lamina II are
mediated by NMDA receptors alone.
The amplitude, kinetics, and voltage dependence of pure
NMDA EPSCs
To understand the role of NMDA EPSCs in the transmission of
sensory signals in the dorsal horn, it is important to establish the
basic parameters of their kinetics and voltage dependence. These
variables will strongly influence the activity dependence of synaptic
NMDA receptor activation. Examples of pure NMDA EPSCs recorded from a
P1 and a P10 neuron held at +50 mV are shown in Figure
3. On average, peak amplitudes of pure
NMDA EPSCs were small, 26.9 ± 14.4 pA (n = 17),
with a slow rise time and current decay time course that was fit by one
(data not shown) or two exponentials (Fig. 3). The average rise time of
EPSCs at pure NMDA synapses was 15.9 ± 3.2 msec
(n = 14). Rise times did not change significantly over
the postnatal period under study (see Fig. 7). EPSCs from two neurons
were best fit with a single-exponential function giving decay values of 40 and 65 msec. EPSCs from 14 cells were best fit with two
exponentials with average decay: 1, 24.4 ± 6.9 msec and 2,
267.2 ± 95.3 msec. The amplitude of the first component was
15.9 ± 9.7 pA, and the amplitude of the second component was
11.6 ± 5.7 pA.
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Figure 3.
Kinetic analysis of pure NMDA EPSCs.
A, EPSCs were recorded from a P1 lamina II neuron held
at +50 mV, averaged, and fit with a double-exponential function. The
current is the average of five traces. The double-exponential curve is
superimposed on the EPSC decay phase (solid line),
whereas the two components of the double-exponential function are
represented by dashed lines (1, 21.4; 2, 209 msec). B, EPSCs were recorded from a P10 lamina II
neuron held at +50 mV, averaged, and fit with a double-exponential
function (1, 25.3; 2, 292 msec). The current is the average of
five traces.
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Pure NMDA EPSCs were strongly suppressed at negative membrane
potentials, suggesting potent block by Mg2+.
I-V relationships obtained from three lamina II neurons at
different ages (P1, P4, and P13) are represented in Figure
4A. In all three examples, maximum peak inward current for the pure NMDA EPSC is at
approximately 30 mV. Figure 4B shows the ratio of
pure NMDA EPSC amplitude recorded at 70 and at +50 mV as a function
of age (n = 22). One-way ANOVA of these data indicates
no age dependence of the ratio value. Thus, no change in voltage
dependence of the synaptic current at pure NMDA synapses occurred over
the first 2 weeks of postnatal development, indicating that the
Mg2+ sensitivity of NMDA EPSCs at pure NMDA synapses
remains strong throughout postnatal development.
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Figure 4.
Pure NMDA EPSCs are strongly voltage-dependent.
A, I-V relationships obtained by
plotting the peak amplitude of averaged pure NMDA EPSCs recorded in
bicuculline and strychnine from lamina II neurons as a function of
holding potential at different ages (P1, P4, and P13). Each point was
determined by averaging five EPSC traces. B,
Voltage dependence of NMDA receptors at pure NMDA synapses was assessed
by calculating the ratio of peak synaptic current amplitudes recorded
at 70 mV and +50 mV in bicuculline and strychnine from lamina II
neurons. Average ratio values and SDs are plotted in 4 d bins and
compared as a function of age. One-way ANOVA revealed no relationship
between voltage dependence and age (F = 0.91;
p = 0.457).
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The amplitude, kineticism, and Mg2+
sensitivity of NMDA EPSCs pharmacologically isolated from mixed
synapses
Although substantial proportions of glutamatergic synapses are
composed solely of NMDA receptors, other glutamatergic synapses in
laminae I and II are mediated by AMPA and NMDA receptors. Focal stimulation was applied at high intensity, evoking synaptic currents that included both AMPA and NMDA components. Under these conditions, the recorded NMDA EPSCs could be attributable to the activation of
receptors expressed both at pure NMDA synapses and at mixed synapses
(AMPA plus NMDA or AMPA alone). Thus, the synapses contributing to the
kinetics of NMDA EPSCs from mixed synapses are more heterogeneous than
those of pure NMDA EPSCs. To establish the properties of synaptic NMDA
receptors activated under these conditions and to compare them to those
expressed at pure NMDA synapses, we determined the voltage dependence
and kinetics of the associated synaptic currents. As shown in Figure
5, stimulation of a lamina II P4 neuron
held at 70 mV evoked a fast EPSC mediated by AMPA receptors, as
indicated by strong block with 10 µM CNQX. A small NMDA
receptor-mediated component of the EPSC was apparent at 70 mV in the
presence of CNQX (Fig. 5A) but was much more clearly seen by
depolarizing the membrane to +50 mV (Fig. 5B). Fifty
micromolar D-APV completely and reversibly blocked the
remaining NMDA EPSC. Nearly all glutamatergic synapses tested in this
way revealed evidence of an NMDA component to the EPSC.
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Figure 5.
NMDA EPSCs isolated from mixed glutamatergic
synapses in laminae I and II over the first 2 postnatal weeks.
A, EPSCs were recorded from a P4 lamina II neuron held
at 70 mV and assessed for sensitivity to different antagonists.
Glutamatergic EPSCs evoked in bicuculline and strychnine are mediated
by AMPA receptors, blocked by 10 µM CNQX and NMDA
receptors, blocked by 50 µM D-APV. The
antagonist effects were reversible after a 5 min wash.
B, EPSCs were recorded from the same neuron under the
same conditions as in A, except the membrane potential
was held at +50 mV. All currents are the average of five traces.
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Similar to pure NMDA EPSCs, NMDA EPSCs from a mixture of glutamatergic
EPSCs were strongly sensitive to membrane potential from birth. Figure
6A,B
shows NMDA EPSCs recorded from a lamina II P3 neuron. In the presence
of 1 mM extracellular Mg2+ (Fig.
6A), almost no current was evoked when membrane
potential was held at 70 mV, although a large outward EPSC was
apparent at +50 mV, indicating strong voltage dependence of these
EPSCs. When the bath was switched to one with nominally 0 Mg2+, a large EPSC was present at both 70 and +50
mV (Fig. 6,B1). By removal of bath
Mg2+, the voltage-dependent block of NMDA EPSCs was
diminished, as shown by the loss of negative slope conductance (Fig.
6A1,B2). NMDA receptor
Mg2+ sensitivity at mixed glutamatergic synapses was
assessed as a function of age by calculating the ratio of the peak
amplitudes of synaptic NMDA currents recorded from lamina I and II
neurons at 70 and at +50 mV, as illustrated in Figure 6C
(n = 33). Similar to pure NMDA EPSCs, the voltage
dependence of NMDA receptors at mixed glutamatergic synapses was
unchanged throughout the first two postnatal weeks. Furthermore, when
the voltage-dependent block of NMDA EPSCs for lamina I neurons was
considered separately from lamina II neurons by regression analysis
performed on raw data, there was no significant change with age in
either group (data not shown). When the ratio of NMDA EPSC amplitudes
measured at 70 and +50 mV was compared between mixed and pure NMDA
synapses, they were found to be similar (one-way ANOVA,
p = 0.772), indicating that pure and mixed NMDA
synapses show comparable Mg2+ sensitivity.
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Figure 6.
NMDA receptors at mixed glutamatergic synapses on
lamina I and II neurons are strongly blocked by Mg2+
during the first 2 postnatal weeks. A, Voltage
dependence of the NMDA EPSC was determined for a P3 lamina II neuron in
the presence of 1 mM Mg2+.
A1, NMDA EPSCs recorded at 70 and +50 mV.
Almost no synaptic current was apparent in 10 µM CNQX
when membrane potential was held at 70 mV (average of 5 traces),
whereas under the same conditions, an EPSC was evoked at +50 mV.
A2, I-V curve representing the
peak amplitudes of the average of five consecutive traces at different
membrane potentials. B, Voltage dependence of the NMDA
EPSCs for the same lamina II neuron was determined in a bath with no
added Mg2+. B1, In low
Mg2+, EPSCs evoked at 70 mV in CNQX were readily
apparent. EPSCs are averages of five traces. B2,
I-V curve was obtained by averaging five consecutive
traces recorded at different potentials in 0 Mg2+.
C, Mg2+ sensitivity of NMDA receptors
at mixed synapses in lamina I and II neurons was assessed by
calculating the ratio of NMDA current peak amplitudes recorded at 70
and +50. Ratio values are binned over 4 d and plotted as function
of age. Error bars represent SD. One-way ANOVA revealed no dependence
of Mg2+ sensitivity on age (F = 0.8; p = 0.457).
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Kinetic properties of NMDA EPSCs at mixed synapses were determined as a
function of postnatal age and compared with those of pure NMDA EPSCs.
In Figure 7A, rise times of
NMDA EPSCs isolated from mixed and pure synapses are plotted as a
function of age. Mean rise time measured at mixed synapses for both
lamina I and II neurons was 10.5 ± 2.7 msec (n = 23). As was the case for the pure NMDA EPSCs, there was no change in
rise time detected over the first two postnatal weeks. However, rise
times of mixed NMDA EPSCs were significantly faster than those of pure
NMDA EPSCs (two-way ANOVA, p  0.01). The significant
difference in rise times between the two types of NMDA EPSCs indicates
that some aspect of the synapses, such as molecular composition of the
NMDA receptors or source of the transmitter, must be different (see Discussion).
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Figure 7.
Kinetic analysis of NMDA EPSCs at mixed synapses
and comparison with pure NMDA EPSCs. A, Ten to 90% rise
time values determined from NMDA EPSCs at mixed and pure NMDA synapses
are binned and plotted as a function of age. The first two bins are
4 d, and the third bin includes data from 5 d. Two-way ANOVA
indicates no dependence of rise time on age for either type of synapse
(F = 1.43; p = 0.254) and a
highly significant difference in rise time of pure and mixed NMDA
synapses (F = 27.57; p
0.01). B, The decay phase of NMDA EPSCs at mixed
and pure synapses was fit with a double-exponential function. 1
values are plotted in bins as a function of age. The decay 1 values
of the two types of NMDA EPSCs are significantly different (two-way
ANOVA, F = 25.44; p 0.01),
although neither changes significantly as a function of age
(F = 1.032; p = 0.37).
C, 2 values determined from NMDA EPSCs at mixed and
pure synapses are binned and plotted against age. 2 values for mixed
and pure synapses are not significantly different (two-way ANOVA,
F = 3.4; p = 0.074) nor are
they dependent from age (F = 0.661;
p = 0.524). All kinetic parameters were determined
from averaged EPSCs (5-15 traces) recorded at +50 mV. Error bars
indicate SD.
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The decay phase of NMDA EPSCs at mixed synapses was best fit by a
double-exponential function in 23 of 24 neurons from laminae I and II
recorded at +50 mV. The decay for the one cell with a
single-exponential fit was 58 msec. Figure
7B,C shows the values of 1 and
2 as a function of age (1, 41.9 ± 11.25 msec; 2, 357.14 ± 132.96 msec; n = 23) for the mixed
synapse NMDA EPSCs. The corresponding amplitudes of the two components
are 32.0 ± 23.6 and 18.4 ± 13.4 pA. The values obtained
for NMDA EPSCs at mixed synapses were compared with those determined
for pure NMDA EPSCs that also required fitting with a
double-exponential function. 1 and 2 values of mixed and pure
NMDA EPSCs are plotted in Figure
7B,C. Using a two-way ANOVA, decay
remained constant over time for both pure or mixed synapses.
However, there was a significant difference between mixed and pure NMDA
EPSC groups for 1 (p 0.01), whereas no
significant difference was detected for 2. The differences in rise
times and decay 1 both suggest a fundamental difference in the NMDA
EPSCs at the two different types of synapses.
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DISCUSSION |
We have investigated the properties of synaptic NMDA receptors of
the rat spinal cord dorsal horn during postnatal development and found
that postsynaptic NMDA receptors contribute to synaptic transmission at
many glutamatergic synapses in laminae I and II by birth. At some
synapses, the NMDA receptors are the only glutamate receptors present
(Bardoni et al., 1997a,b; Zhuo and Li, 1997), whereas other synapses
are likely to include combinations of AMPA and NMDA receptors.
The kinetics of NMDA EPSCs in the superficial dorsal horn
All of the NMDA EPSCs recorded from neurons in laminae I and II
decayed with fast kinetics at +50 mV. The average decay values
measured at pure NMDA synapses were 24 and 267 msec, whereas the
average decay values at mixed synapses were 42 and 357 msec. Decay
kinetics of NMDA EPSCs have been shown to be determined by channel
kinetics of the NMDA receptor (Lester et al., 1990; Rosenmund et al.,
1995) that in turn are influenced by the subunits that contribute to
receptor composition. Initially, the NR2D subunit appeared to be the
major NR2 subunit expressed in spinal cord at embryonic day 17 (Monyer
et al., 1994) and in mature dorsal horn neurons (Tölle et al.,
1993). However, single-channel recordings from dorsal horn neurons have
shown that channels with different conductance levels are present with
proposed subunit composition, including NR2A/B (higher conductance) and
NR2D (lower conductance) (Momiyama et al., 1996). Most recently,
in situ hybridization and single-cell PCR studies in dorsal
horn have reported that the most common subunit composition of NMDA
receptor at the cervical level is NR1, NR2A, B and D, with NR2A
predominant over B and D (Karlsson et al., 1997; Sjödin et al.,
1997). The rapid kinetics we have reported for dorsal horn neuron NMDA
EPSCs suggest a strong influence of the NR2A subunit on receptor
configuration, because receptors expressed in heterologous expression
systems (Monyer et al., 1994) and in developing neurons (Flint et al.,
1997) that include NR2A have rapid kinetics.
Developmental changes in NMDA EPSC kinetics have been observed in
several regions of the CNS (Carmignoto and Vicini, 1992; Hestrin, 1992;
Ramoa and McCormick, 1994; Khazipov et al., 1995; Shi et al., 1997). In
these studies, the decay kinetics became faster as development
progressed. The most common pattern has been that the decay of the
NMDA EPSC is fit by a single-exponential function at younger ages,
whereas a double-exponential function is required at older ages.
Khazipov et al. (1995) has proposed that the contribution of the fast
component increases with age. In dorsal horn neurons, fits of the NMDA
EPSC decay required two exponentials at all postnatal ages, and decay
values are comparable to the values obtained from NMDA EPSCs in
mature systems (Carmignoto and Vicini, 1992; Khazipov et al., 1995).
These observations further support the idea that the kinetics of the
NMDA EPSCs in superficial dorsal horn, and possibly the associated
subunit composition of the synaptic NMDA receptors, are in a relatively
mature configuration by birth.
Possible explanations for differences in NMDA EPSC kinetics
recorded as pure and mixed synapses
Kinetic analysis of pure NMDA EPSCs evoked in dorsal horn neurons
has revealed that these currents have slower rise times and faster
decay 1s than NMDA EPSCs at mixed synapses do. There are several
possible explanations for the observed differences in EPSC
kinetics.
If we make the assumption that rise time and decay values of NMDA
EPSCs are principally determined by channel kinetics (Lester et al.,
1990; Rosenmund et al., 1995) and that channel kinetics are mainly
determined by receptor subunit composition, then it is NMDA receptor
subunit composition that favors faster kinetics at pure synapses. NR2A
subunit has been shown to display fast kinetics (Monyer et al., 1994),
and NR2A expression correlates highly with fast NMDA EPSC decay values in developing neocortical neurons (Flint et al., 1997). Vicini
et al. (1998) have recently shown that NMDA receptors expressed in HEK
293 cells composed of NR1 and NR2A subunits have deactivation kinetics
similar to the decay values we report for pure NMDA EPSCs.
Specifically, they found that after 1 msec application of 1 mM Glu, currents decayed with a fast of 33 msec and a
slow of 247 msec (compared with 24 and 267 msec for pure NMDA
EPSCs, respectively). When cells were cotransfected with NR1,
NR2A, and NR2B, the fast and slow decay values became
proportionally slower as the relative amount of NR2B was increased. For
example, when NR1, NR2A, and NR2B receptor subunit cDNAs were added in
1:3:1 proportions, average decay values were 43 and 387 msec. This
compares favorably with the 42 and 357 msec decay values measured
from NMDA EPSCs from mixed synapses. These data provide a good
molecular hypothesis for the NMDA EPSC decay kinetics reported in our
studies. The hypothesis is that the decay values of the pure NMDA
EPSCs are attributable to receptors composed only of NR1 and NR2A
subunits. NMDA receptors at other synapses would include additional NR2 subunits, such as NR2B.
Alternatively, the slower rise time of the pure synapses could indicate
that they are located at electrotonically distant sites from the
recording electrode and that the slow rise time is simply attributable
to the low-pass filtering of the dendritic membrane (Rall and Segev,
1991). Because the decay values are faster at pure NMDA synapses,
dendritic filtering cannot account for the difference in decay
time. It has been suggested that the decay values of NMDA
EPSCs are voltage-dependent (Hestrin, 1992). Thus, it is possible to
account for faster decay values at these putatively remote pure
NMDA synapses because of the inability of the voltage clamp to bring
the membrane potential at the synapse to +50 mV. These EPSCs would
decay at a faster rate than synapses closer to the soma because of the
more negative membrane potential at the site of channel
opening. It is also possible that EPSC decay would be faster if
remote synapses were preferentially subject to shunting by activation
of other channels, although both GABAA and glycine
receptors were blocked in our experiments.
A third explanation for why pure NMDA EPSCs have a slower time to peak
than mixed EPSCs is that pure NMDA EPSCs are attributable to spillover
of synaptically released glutamate onto receptors on neighboring
neurons, either at synaptic sites (Kullmann et al., 1996) or even
extrasynaptic sites. If this is true, the diffusion of glutamate to
receptors mediating pure NMDA responses could become rate-limiting for
the EPSC rise time rather than channel kinetics, thus explaining the
slow rise time observed in our studies. A diffusion-limited rise time
would not, however, predict the faster decay we have observed at
pure NMDA synapses. In this case, the faster decay 1 would have to
be explained by some other mechanism such as one of those outlined
above.
Role of NMDA EPSCs in the spinal cord dorsal horn
Evidence for an important role for NMDA receptors in
nociceptive processing in dorsal horn is becoming increasingly strong. Glutamate is released from both primary afferents and at least some of
the local interneuronal connections (Willis and Coggeshall, 1991). The
synaptic activation of NMDA receptors appears to contribute to both the
induction and maintenance of many forms of central sensitization
associated with hyperalgesia and allodynia (Dickenson et al., 1997).
However, little is known about which synaptic connections express
postsynaptic NMDA receptors. Our data suggest that postsynaptic NMDA
receptors are ubiquitous throughout the superficial dorsal horn. They
were detected on every neuron tested in our study and activated under a
variety of stimulation conditions. Nearly all of the NMDA EPSCs decayed
with both fast and slow values, on the order of tens and several
hundreds of milliseconds, respectively. With pain-evoked action
potentials coming in to dorsal horn neurons at 5-20 Hz (Bessou and
Perl, 1969), these slow EPSPs driven by NMDA EPSCs are expected to show
strong summation. Furthermore, because of the strong voltage dependence
of the NMDA synaptic currents in the rat dorsal horn (Figs. 4, 6), this
summation is expected to be highly nonlinear and to powerfully
influence the output firing of the dorsal horn neurons. Finally,
tetanic orthodromic activation of some dorsal horn neurons initiates an
NMDA receptor-dependent sequence of events resulting in long-term
changes in synaptic transmission referred to as long-term potentiation
(LTP) and long-term depression (LTD) (Randic et al., 1993; Sandkuhler
et al., 1997). The voltage dependence of current flow through the NMDA
receptor is believed to provide the sensitivity to specific tetanic
activity in hippocampal NMDA receptor-dependent LTP and LTD. We have
shown that by birth, synaptic NMDA receptors throughout the dorsal horn express a strong voltage-dependent Mg2+ block,
endowing them with the ability to function as activity detectors in the
dorsal horn.
| |
FOOTNOTES |
Received March 27, 1998; revised May 29, 1998; accepted June 2, 1998.
This work was supported by the Whitehall Foundation, the National
Science Foundation (A.B.M.), and Ministero Dell'Università e
Della Ricerca Scientifica e Tecnologica (R.B. and P.C.M.). We thank Steve Siegelbaum, Gary Westbrook, and Jianguo Gu for helpful comments on an earlier version of this manuscript, and Bob Hawkins, Michael Meyers, and Emilia Bagiella for helpful discussions on data
analysis.
Correspondence should be addressed to Rita Bardoni, Department of
Biomedical Sciences, Section of Physiology, Via Campi, 287, I-41100,
Modena, Italy. E-mail: bardoni{at}unimo.it
| |
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Top
Abstract
Introduction
