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The Journal of Neuroscience, December 15, 1999, 19(24):10603-10610
Distinct Synaptic and Extrasynaptic NMDA Receptors in Developing
Cerebellar Granule Neurons
Gavin
Rumbaugh1 and
Stefano
Vicini2
Departments of 1 Pharmacology and
2 Physiology and Biophysics, Georgetown University School
of Medicine, Washington, DC 20007
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ABSTRACT |
In rat cerebellar granule neurons, mRNA and protein levels of the
NR2A and NR2C subunits of the NMDA receptor increase during the second
postnatal week. At this time, mRNA and protein levels of the NR2B
subunit begin to fall. To investigate targeting of NMDA receptor
subunits, we performed whole-cell recordings from rat cerebellar
granule neurons at different times during development and investigated
the pharmacological and biophysical properties of mossy fiber-evoked
NMDA EPSCs. Isolated NMDA EPSCs from newly formed synapses in the first
postnatal week exhibited partial block by the NR2B subunit-specific
antagonist
(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606). By the end of the second postnatal week, NMDA
EPSCs were virtually unaffected by the NR2B antagonist. In parallel, NMDA EPSC decay times decreased over a similar developmental time course. We compared properties of synaptic NMDA receptors with extrasynaptic receptors that are present on the cell body with rapid
application of glutamate to excised nucleated patches. Deactivation of
patch responses accelerated with development and closely resembled evoked NMDA EPSCs in rats of the same age. However, patch responses were highly sensitive to CP 101,606 through the second postnatal week,
and sensitivity was seen in some neurons up to the fourth postnatal
week. Spermine potentiated peak NMDA patch responses from postnatal
days 10-14 rats but had little effect on evoked NMDA EPSCs. Our
data suggest selective targeting of a distinct NMDA receptor subtype to
synaptic receptor populations in cerebellar granule neurons. Later in
development, similar changes occur in the extrasynaptic receptor population.
Key words:
glutamate receptor; development; cerebellum; EPSC; spermine; patch clamp
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INTRODUCTION |
NMDA receptors are involved in a
variety of processes, such as long-term potentiation, neuronal
migration, and synaptogenesis (McBain and Mayer, 1994 ; Sheetz and
Constantine-Paton, 1994). In the cerebellum, NMDA receptors are
required for normal motor coordination because lack of these receptors
in rats yields a phenotype that is noticeably uncoordinated and taxed
during locomotion (Kadotani et al., 1996).
NMDA receptors are composed of the obligatory NR1 subunit and at least
one copy of NR2A, NR2B, NR2C, or NR2D (McBain and Mayer, 1994;
Dingledine et al., 1999). The presence of specific NR2 subunits determines properties of the receptor, including agonist affinity, magnesium sensitivity, deactivation kinetics, modulation by polyamines, and channel conductance (McBain and Mayer, 1994; Dingledine et al.,
1999). For example, recombinant NMDA receptors comprising NR1/NR2A
heteromers exhibit fast deactivation, little sensitivity to ifenprodil
derivatives, and are not modulated by the polyamine spermine. In
contrast, NR1/NR2B receptors decay more slowly, are blocked by
ifenprodil derivatives, and are potentiated by micromolar levels of
spermine (Monyer et al., 1994; Williams et al., 1994; Mott et al.,
1998; Vicini et al., 1998). Moreover, NR1/NR2C or NR1/NR2D heteromers
have a smaller unitary single-channel conductance and are less
sensitive to block by magnesium (Feldmeyer and Cull-Candy, 1996).
In cerebellar granule neurons, the NR1 subunit is ubiquitously present
throughout development. In contrast, the NR2 subunits go through
variable expression patterns (Watanabe et al., 1994; Wang et al.,
1995). NR2B subunit protein expression begins in late embryonic stages
but gradually disappears during the second postnatal week (Wang et al.,
1995; Takahashi et al., 1996). In parallel to the drop in NR2B subunit
expression during the second postnatal week, the NR2A subunit, which is
completely absent at birth, begins to appear (Watanabe et al., 1994;
Wang et al., 1995). As development continues, the NR2C subunit also
begins to appear (Akazawa et al., 1994) and incorporates itself into
both synaptic and extrasynaptic NMDA receptors as observed in a NR2A
subunit gene knock-out study (Takahashi et al., 1996). In cerebellar
granule neurons, synaptic receptors are restricted to the glomerulus
and are therefore separated from extrasynaptic receptors in the cell body (Palay and Chan-Palay, 1974). Recently, several reports have suggested that cortical and hippocampal synaptic receptors are comprised of unique subunit combinations (Stocca and Vicini, 1998; Tovar and Westbrook, 1999). In particular, the NR2A subunit is inserted
into synaptic receptor populations soon after activity begins in
hippocampal neurons in culture (Tovar and Westbrook, 1999). By using
patch-clamp recordings from cerebellar slices combined with a selective
NR1/NR2B receptor antagonist, we present evidence for NR2A subunit
insertion at cerebellar mossy fiber-granule neuron synapses soon after
activity begins. We also show that the extrasynaptic receptor
population undergoes a similar, albeit much delayed, developmental change.
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MATERIALS AND METHODS |
Electrophysiological recordings and slices
preparation. Sagittal slices of cerebellum (180-250 µm) were
prepared from postnatal days 6-30 (P6-P30) Sprague Dawley rats, as
described previously (Puia et al., 1994).
The recording chamber was continuously perfused at 5 ml/min with an
oxygenated extracellular medium composed of: 120 mM
NaCl, 3.1 mM KCl, 1 mM MgCl, 1.25 mM K2HPO4, 26 mM NaHCO3, 2.0 mM
CaCl2, 1.25 mM glucose, and 10 µM D-serine. The solution was maintained at pH 7.4 by
bubbling with 5% CO2-95%
O2. NMDA receptor-mediated synaptic
responses were pharmacologically isolated by picrotoxin (15 µM; Sigma, St. Louis MO), strychnine (10 µM; Sigma), and
2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) (5 µM; Tocris Cookson, Ballwin MO).
(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606) (Chenard et al., 1995) was a gift from Dr. Richard Woodward (Acea Pharmaceutical, Irvine, CA). CP 101,606 and NBQX were
dissolved in dimethylsulfoxide (<0.1% final concentration). Spermine tetrahydrochloride (Sigma), (+)-MK-801 maleate (Dizocilpine; Tocris Cookson), and all other drugs were dissolved in water. All drugs
were perfused through parallel inputs to the perfusion chamber.
Electrodes were pulled from borosilicate glass capillaries (Wiretrol
II; Drummond, Broomall, PA). Nucleated patches and whole-cell recordings from cerebellar granule neurons were obtained under visual
control with an Axioskop FS microscope (Zeiss, Oberkochen, Germany) equipped with Nomarski optics and an electrically insulated water immersion 40× objective with a long working distance. Typical pipette resistance was 6-8 M . Intracellular (patch pipette)
solutions contained (in mM): K-gluconate 145, EGTA 5, MgCl2 5, ATP-Na 5.0 GTP-Na 0.2, and HEPES 10, adjusted to pH 7.2 with KOH. Whole-cell recordings were performed with
a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City,
CA). We discarded cells in which a change in series resistance greater
than 15% was observed.
Evoked NMDA EPSCs were acquired by stimulation of the mossy fiber
inputs located in the vicinity of the recorded granule cell. To evoke
EPSCs, a tungsten bipolar electrode was placed on the white matter
located near the recorded cell. Stimulus durations ranged from 50 to
100 µsec, and stimulus intensity ranged from 200 to 300 µA. To
isolate NMDA EPSCs, NBQX (5 µM), strychnine (20 µM), and picrotoxin (15 µM) were perfused
during the entire recording.
For fast application of L-glutamate, we used a
computer-driven piezoelectric translator (PZ 150M; Burleigh
Instruments, Fishers, NY) to which double-barrel theta tubing was
attached. Nucleated outside-out membrane patches were excised from the
neurons immediately after establishing the whole-cell configuration and
positioned in front of the double-barrel applicator (Puia et al.,
1994). In each barrel, we used extracellular medium containing 5 µM NBQX with and without 1 mM
L-glutamate (flow rate of 0.25 ml/min). After each
recording, on and off rates, as well as pulse duration, were measured
by "blowing off" the patch and recording currents generated by the
liquid junction potential caused by a 50:1 dilution of the
L -glutamate-containing solution (Lester and Jahr, 1992). For fast application of L-glutamate in combination with
other drugs, we rapidly exchanged the solutions in both barrels by
means of solenoid valves connected to a vacuum. Drugs were added in both control and L-glutamate-containing solutions.
Data collection and analysis. Currents were filtered at 1 kHz with an eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA), digitized at 5-10 kHz using an IBM-compatible microcomputer equipped with Digidata 1200 data acquisition board (Axon
Instruments) and pClamp 8 software (Axon Instruments). Off-line data
analysis, curve fitting, and figure preparation were performed with
Clampfit 8 (Axon Instruments) and Origin 4.1 (Microcal, Northampton, MA) software. Fitting of the decay phase of currents recorded from
excised patches and NMDA EPSCs was performed using a simplex algorithm
for least-squares exponential fitting routines. Decay times of averaged
currents derived from fitting to double exponential equations of the
form I(t) = If *
exp( t/ f) + Is *
exp(t/s), where
If and
Is are the amplitudes of the
fast and slow decay components, and f and
s are their respective decay time constants used to fit the data. To compare decay times, we used a weighted mean
decay time constant: w = [If/(If + Is)] *
tf + [Is/(If + Is)] *
ts. Data values are expressed as
mean ± SEM, and p values represent the results of
paired or independent two-tailed t tests (as indicated).
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RESULTS |
Pharmacological and kinetic properties of synaptic
NMDA receptors
Cerebellar granule neurons were identified by their anatomical
location in the granule layer and by their small cell bodies. After the
formation of a gigaseal, cells were voltage clamped at 60 mV in the
whole-cell recording configuration. After a short latency period during
which the cell was allowed to stabilize, a stimulating electrode was
placed on the mossy fibers, and EPSCs were evoked. NBQX, picrotoxin,
and strychnine were included in Mg2+-free
perfusion solution to pharmacologically isolate the NMDA component of
the EPSC. To ensure that the resulting component of the EPSC was caused
solely by the activation of NMDA receptors, the competitive NMDA
antagonist (±)-3-(2-carboxy piperazin-4-yl)-propyl-1-phosphonic acid
(CPP) was bath applied. In all cells tested, 10 µM
CPP completely and reversibly blocked the resulting evoked current
(n = 4; data not shown). The decay time of NMDA
EPSCs at all developmental stages was best fitted with a dual
exponential function. To allow a more direct comparison among EPSC
decay kinetics, we described the dual exponential decay using a
weighted time constant (w).
To verify the change in decay kinetics during development, we analyzed
NMDA EPSCs from rats ranging from 6 to 30 d old (Fig. 1A, Table
1). As reported previously, decay times
from more mature animals exhibited much faster decay times (Takahashi
et al., 1996). During the second postnatal week (P10-P14), NMDA
current from granule neurons showed intermediate decay kinetics (Table
1), a time when mRNAs for NR2A, NR2B, and NR2C subunits are all present (Watanabe et al., 1994; Wang et al., 1995).
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Figure 1.
Comparison of kinetic and pharmacological
properties of evoked NMDA EPSCs in developing granule neurons.
A, Top, Evoked NMDA EPSCs from granule
neurons in rats at P7 and P21. The averages of 5-10 consecutive sweeps
are shown with a superimposed double exponential curve. The individual
exponential curves are also shown. Bottom, The two
responses are shown superimposed after scaling with an indication of
the weighted time constant (w) to emphasize
differences in deactivation. B, Top,
Average of five to seven traces taken from a P7 rat before
(control) and in the presence of 5 µM CP 101,606 (CP). Bottom,
Average of six to seven traces taken from a P14 rat before
(control) and in the presence of 5 µM CP 101,606 (CP). C, Relationship
between percentage control response in the presence of CP
101,606 (5 µM) as a function of the weighted time
constant of NMDA EPSCs in P6-P8 and P10-P14 rats.
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During the first postnatal week, when mossy fiber-granule cell
synapses form (Arsenio Nunes and Sotelo, 1985), the primary NR2 subunit
detected by Western blot analysis is NR2B (Takahashi et al., 1996). We
were able to evoke NMDA EPSCs in the cerebellum as early as P6.
Currents at this age yielded large weighted time constants and
exceedingly long, slow tails (Fig. 1). NMDA EPSCs during this period
(P6-P8) were partially blocked by CP 101,606 (5 µM)
(Fig. 1B,C), an antagonist
selective for NR1/NR2B heteromers (Mott et al., 1998) that has been
shown to be devoid of presynaptic action in cortical neurons (Stocca
and Vicini, 1998). However, the extent of blockade was never as strong
as that observed with recombinant receptors (Mott et al., 1998). Evoked
NMDA EPSCs from granule neurons in rats ranging from P10 to P14 showed
greater variability in CP 101,606 blockade (5 µM) (Fig.
1B,C). This greater variability of
blockade together with a decrease in decay times (Fig. 1C,
Table 1) parallels the reported increase of NR2A subunit expression in
this age group (Watanabe et al., 1994). A nonsynchronized developmental
expression of the NR2A receptor subunit between different cells as
reported for NR2C subunit (Ozaki et al., 1997) could underlie the large
variability of CP101,606 effect in Figure 1B.
NMDA responses in excised nucleated patches
To make a direct comparison of synaptic to extrasynaptic
receptors, we chose to study nucleated outside-out patches (Puia et
al., 1994). These patches have a greater surface area than conventional
outside-out patches and therefore a greater number of NMDA receptors.
This allowed for a reliable measure of deactivation kinetics from a
smaller number of repetitive L-glutamate applications. Deactivation of nucleated patch responses was best described by double
exponential curves with similar time constants to those measured for
NMDA EPSCs (Table 1). However, patch responses had on average larger
peak amplitudes than NMDA EPSCs (Table 1).
Occasionally, responses from nucleated patches were quite small. This
allowed us to observe the behavior of a few NMDA channels during the
tail of patch responses (Fig.
2A). Channel behavior was strikingly different when comparing responses from younger (P7-P8)
with older [greater than P18 (P>18)] animals. Patch responses from immature neurons resulted in channel bursting many hundreds of
milliseconds after glutamate application, whereas responses in older
animals had noticeably fewer openings during the same time period. As
seen for NMDA EPSCs, decay times of average responses in nucleated
patches also decreased with age (Fig. 2B, Table 1). In nucleated patches from animals further along in development (P>18),
glutamate activated currents decayed faster when compared with immature
neurons (P6-P8) (Fig. 2C). Weighted time constants of
deactivation from responses in nucleated patches were significantly faster with development but were not significantly different from NMDA
EPSCs in rats at P7-P8 (Table1). In mature animals (P>18), the
weighted time constants from patch responses were not significantly different from NMDA EPSCs despite the presence of two
uncharacteristically slow responses (Fig. 2C). The average
weighted time constants of patch responses were virtually identical to
NMDA EPSCs when these two patches were excluded from the older group.
Weighted time constants of patch responses from animals in the P10-P14 age group had intermediate values and were significantly different from
NMDA EPSCs in the same age group (Table 1).
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Figure 2.
Deactivation of patch responses accelerate with
development. A, Single response to
L-glutamate (1 mM, 4 msec) in nucleated patches
from P7 and P24 rats. B, Top, Average
L-glutamate (1 mM, 4 msec) activated currents
in two nucleated patches from P7 and P24 rats. The average of seven
consecutive L-glutamate applications are shown with a
superimposed double exponential curve. The individual exponential
curves are also shown. Bottom, The two responses are
shown overlapped after scaling with an indication of the weighted time
constant (w) to emphasize differences in
deactivation. C, Summary of all individual weighted time
constants from NMDA responses in nucleated patches at three
developmental age groups (P7-P8, P10-P14, and greater than P18).
Horizontal bar located among responses indicates
arithmetic mean.
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Pharmacological antagonism of nucleated patch responses
Our findings suggest that extrasynaptic NMDA receptors undergo
similar kinetic changes throughout development when compared with
synaptic receptors. Therefore, it was reasonable to assume that
sensitivity of glutamate responses in nucleated patches to NR2B-specific antagonists could be similar to NMDA EPSCs from the same
developmental period. We investigated the effect of 5 µM
CP 101,606 on L-glutamate responses in excised nucleated
patches from cerebellar granule neurons from rats in the three age
groups. Efficacy of CP 101,606 blockade was tested on 200 msec
applications of L-glutamate to excised nucleated patches.
Contrary to NMDA EPSCs, patch responses were highly sensitive to
application of the NR1/NR2B antagonist. Application of CP 101,606 to
nucleated patches in rats at P6-P8 resulted in an almost complete
abolishment of peak NMDA current in all cells tested (n = 6) (Fig.
3A,C).
Likewise, in patches from P10-P14 rats (n = 7), CP
101,606 also blocked peak NMDA current efficaciously (Fig. 3). CP
101,606 was less efficacious in P>18 rats (n = 6) (Fig. 3B,C). As reported previously
(Stocca and Vicini, 1998), only a partial recovery from NR2B-specific
antagonists was observed (Fig. 3A). Figure 3C
demonstrates the relationship between the weighted time constant of
patch responses and the magnitude of block by the NR1/NR2B antagonist.
In these experiments, patch currents were elicited by 4 msec glutamate
pulses to assess deactivation. CP 101,606 was less effective on patch
responses with exceedingly fast weighted time constants
(w < 100 msec), indicating a possible correlation with NR2A subunit expression (Fig. 3C).
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Figure 3.
Effect of NR1/NR2B antagonists on nucleated patch
responses. A, Top, Responses to 1 mM L-glutamate before
(control), during (CP), and after
(wash) application of 5 µM CP 101,606 in a
P7 rat. In each panel, at least four traces were averaged.
Bottom, As above, with responses elicited from nucleated
patches in a P14 rat. In both panels, horizontal bars
indicate duration of L-glutamate applications (200 msec).
B, A comparison of the effect of NR1/NR2B-specific
antagonists between patch responses and NMDA EPSCs of the same age.
*p < 0.05, significant differences from
100% (independent t test); #p < 0.01, statistical significance between nucleated patches and NMDA EPSCs
of the same age group (independent t test).
C, The relationship between the percentage control after
CP 101,606 application and the weighted time constant of nucleated
patch responses (4 msec pulses) from rats in the three age
groups.
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We also directly compared the average blockade of patch responses by 5 µM CP 101,606 with average blockade of NMDA EPSCs (Fig. 3B). The effect of CP 101,606 on NMDA EPSC amplitude was
lost in some neurons as early as P10, and it was not significant in the
P10-P14 age group (Fig. 3B). In contrast, the effect of the NR2B antagonist on patch responses in this age group was not different from that in the P6-P8 age group. Even for NMDA EPSCs in rats at
P6-P8, when decay kinetics are at their slowest (Table 1), the CP
101,606 blockade was significantly less than patch responses of the
same age (Fig. 3B). Although the antagonist had no
significant effect on the P>18 age group as a whole, CP 101,606 sensitivity was still observed in some patches as late as P30 (Fig.
3C).
Patch excision has been found to alter properties of NMDA receptor
channels (Lester and Jahr, 1992; Clark et al., 1997). Clark et al.
(1997) have shown that excising patches from granule neurons alters the
conductance of extrasynaptic NMDA receptors. However, we did not
observed changes in channel current amplitude in nucleated patches. At
a holding potential of 60 mV, NMDA channel current amplitude was
4.1 ± 0.1 pA in nucleated patches (n = 7) and
4.0 ± 0.1 pA in the whole-cell configuration (n = 15). This does not exclude the possibility that nucleated patch
excision does not alter other channel properties.
CP 101,606 blocks extrasynaptic NMDA receptors after MK-801
blockade of NMDA EPSCs
One of the most valuable characteristics of the high
signal-to-noise ratio of recordings in cerebellar granule neurons is the ability to observe channel current activity in the whole-cell recording configuration (Silver et al., 1992). As soon as whole-cell recordings were established in the presence of bicuculline and NBQX,
spontaneous NMDA channel currents (Fig.
4A) were observed in
all cells recorded (n = 15), as investigated previously
in detail by Rossi and Slater (1993). The extent of spontaneous
activity was variable from cell to cell and was abolished by 10 µM CPP in all cells tested (n = 4). CP 101,606 (5 µM), which had little effect
on NMDA EPSCs at P14, had no obvious effect on spontaneous NMDA
channels in the same age group (Fig. 4A). The average
integral current in one second (Ii)
was 645 ± 73 pA*msec in the absence and 661 ± 82 pA*msec in
the presence of CP 101,606 (n = 5). However, in three
of these neurons, perfusion of 10 µM CPP
resulted in blockade of spontaneous NMDA channel currents
(Ii = 109 ± 45 pA*msec).
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Figure 4.
CP 101,606 blockade of extrasynaptic receptors in
the intact granule neuron. A, Top,
Spontaneous NMDA channel currents recorded after establishing a
whole-cell recording from a P14 rat. Middle, Effect of
bath perfusion of 5 µM CP 101,606. Bottom,
Trace represents effect of 10 µM CPP on spontaneous NMDA
single channel events. CPP was bath applied immediately after CP
101,606. B, Evoked NMDA EPSCs in the presence of 10 µM MK-801 recorded in the same cell after washout of CPP.
Stimulus frequency was 0.1 Hz. Responses at different stimulus number
(Stim) from the beginning of MK-801 perfusion are
illustrated. C, Top, Current sweep taken
immediately after Stim 30 in B. MK-801
was allowed to wash for ~3 min. Middle, Bath perfusion
of 100 µM NMDA after MK-801 wash. Bottom,
Trace acquired 2 min after application of a solution containing both
100 µM NMDA and 5 µM CP 101,606.
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The lack of effect of CP 101,606 on background NMDA channel activity
and on NMDA EPSCs may relate to the lack of access in the brain slice
rather than distinct receptor populations. Therefore, we attempted to
block extrasynaptic NMDA receptors in the slice while recording in the
whole-cell configuration from a P14 rat. To specifically block synaptic
NMDA receptors, the open channel blocker MK-801 (10 µM)
was perfused until NMDA EPSCs were abolished (Fig.
4B). In this condition, receptors activated by bath
perfusion of NMDA should be primarily extrasynaptic in origin. Indeed,
as illustrated in Figure 4C, when 100 µM NMDA was perfused over the entire slice, a
small population of receptors were activated
(Ii = 702 ± 140 pA*msec). This
population of receptors was blocked when 5 µM
CP 101,606 was added to the NMDA solution
(Ii = 119 ± 47 pA*msec;
n = 3).
Spermine potentiates patch responses but not NMDA EPSCs
Previous reports indicate that polyamines have the ability to
potentiate recombinant NMDA receptors comprising NR2B subunits while
producing no potentiation in recombinant NMDA receptors comprising NR2A
subunits (Williams et al., 1994). To further investigate the apparent
differences in subunit composition between synaptic and extrasynaptic
NMDA receptors in cerebellar granule neurons, we applied spermine to
both NMDA EPSCs and nucleated patch responses.
Spermine (500 µM) was ineffective in potentiating the
peak of NMDA EPSCs in P6-P8 granule neurons (Fig.
5A,C).
In five of six cells tested, bath perfusion of spermine either had no
effect or slightly reduced the peak of NMDA EPSCs from animals in this age group. As for CP 101,606, we were concerned that spermine was not
able to access synaptic NMDA receptors. Therefore, we analyzed
single-channel currents from the tails of evoked NMDA responses before
and after bath perfusion of spermine (Fig. 5A). At a holding
potential of 60 mV, channel currents were 4.1 ± 0.1 pA in
control and 2.2 ± 0.3 pA when spermine was present
(n = 4), consistent with previous reports (Rock and
MacDonald, 1992). When spermine (500 µM) was
applied to nucleated patches from P10-P14 animals, we observed quite
different effects when compared with P7 NMDA EPSCs (Fig. 5), although
it also reduced NMDA single-channel current amplitude (data not
shown). In seven of eight cells tested, spermine potentiated
peak NMDA current from patch responses in animals through the second
postnatal week (Fig. 5B,C).
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Figure 5.
Spermine potentiates patch responses but not NMDA
EPSCs. A, Top, Evoked NMDA EPSCs from a
P7 granule neuron. Left trace represents average of 20 consecutive sweeps in control conditions. Right trace
represents the average of 15 sweeps in the presence of 500 µM spermine (sp 500).
Bottom, Left trace represents
single-channel currents from the tail of individual evoked NMDA EPSCs
in control conditions. Right trace represents NMDA
single-channel currents from the same cell in the presence of 500 µM spermine. In both traces, overlapped channel openings
were observed. B, Nucleated patch responses from a P14
granule neuron. Left trace is the average of five
consecutive 200 msec L-glutamate applications in control
conditions. Right trace is the average of five
consecutive 200 msec L-glutamate applications in the
presence of 500 µM spermine (sp 500).
Horizontal bars indicate duration of the
L-glutamate applications. C, Summary
illustrating peak NMDA EPSCs in rats at P7 (n = 6 cells) and peak patch currents in P14 rats (n = 8 cells) in the absence and the presence of 500 µM
spermine. *p < 0.05, statistical significance
between control and spermine responses (paired t
test).
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DISCUSSION |
Decay times of NMDA EPSC in rat cerebellar granule neurons
decrease during the first 3 weeks after birth, as reported previously (Feldemeyer and Cull-Candy, 1996; Takahashi et al., 1996). In parallel,
we report similar kinetic changes for deactivation of L-glutamate responses in nucleated patches excised from
these neurons. In contrast to NMDA EPSCs, nucleated patch responses remained sensitive to an NR1/NR2B-specific antagonist for a much longer
period during development. In addition, spermine, which potentiated
patch currents from rats through the second postnatal week, had no
effect on NMDA EPSCs, even at early stages of development.
NR2A subunit targeted to synaptic receptors in granule neurons
Recent studies suggest the incorporation of the NR2A subunit into
synaptic receptors in hippocampal and cortical neurons (Li et al.,
1998; Stocca and Vicini, 1998; Tovar and Westbrook, 1999). We extend
these findings to cerebellar granule neurons by suggesting that the
NR2A subunit is inserted into the synaptic receptor population at a
time when synaptogenesis and synaptic activity begins. Functional mossy
fiber-granule neuron synapses have been reported as early as P5
(Takahashi et al., 1996), as confirmed by anatomical data (Arsenio
Nunes and Sotelo, 1985). At this early age, mRNA and protein studies
indicate that the NR2B subunit is much more abundant than the NR2A
subunit (Akazawa et al., 1994; Wang et al., 1995; Takahashi et al.,
1996). However, we found that NMDA EPSCs in these young rats were
poorly sensitive to CP 101,606, indicating the contribution of NR2A
subunits to synaptic NMDA receptors. The report that synaptic receptors
are of uniform high conductance (59 pS) in rats at P12-P13 (Clark et
al., 1997) suggests the absence of the NR2C subunit early in
development. Furthermore, CP 101,606 sensitivity in newly formed
synapses was much different from patch responses in animals of the same
age. Additionally, through the second postnatal week, sensitivity to
the NR2B blocker was lost for synaptic receptors but changed only
slightly in patch responses. It is only later in development that
extrasynaptic receptors exhibit waning sensitivity to the antagonist.
NR2B subunit is slow in disappearing from cell bodies
during development
The extent of current blockade by CP 101,606 was indistinguishable
between recombinant NR1/NR2B receptors (Mott et al., 1998) and NMDA
receptors in the cell body of granule neurons in slices from rats
during the first 2 postnatal weeks. Furthermore, we observed NMDA
responses from nucleated patches that were sensitive to CP 101,606 in
older rats (P>18). At the same time, however, in this group the
majority of patches were insensitive to NR1/NR2B antagonists. Together,
these results indicate that, as the NR2B subunit disappears from
granule neurons, NMDA responses in both extrasynaptic and synaptic
populations are produced by receptors composed of subunits other than
NR2B. This is further supported by the presence of low conductance
channels in patches excised from granule cell bodies in wild-type and
NRA knock-out mice after the second postnatal week, indicating the
presence of the NR2C subunit (Takahashi et al., 1996).
Compared with synaptic receptors, the insertion of subunits other than
NR2B in receptors located on cell body of granule neurons occurs with a
delay. This is apparent after seeing the differences in CP 101,606 sensitivity between extrasynaptic and synaptic receptors among all
groups tested. For instance, CP 101,606 sensitivity of individual
neurons from evoked NMDA EPSCs from P10-P14 rats is similar to
individual responses from nucleated patches in animals older than P18.
Some responses were reduced, whereas others exhibited no reduction or
slight potentiation. These results are further supported by the fact
that CP 101,606 blocks NMDA receptors in the intact granule neurons
after synaptic receptors were inhibited by MK-801 from P14 rats.
Spermine, which selectively potentiates peak NMDA current from
recombinant NR1a/NR2B receptors, did not increase current amplitude in
NR1a/NR2A receptors (Williams et al., 1994). Spermine potentiated patch
responses from P14 rats but was ineffective in potentiating the peak of
NMDA EPSCs at P7. These data further support the hypothesis that NR2B
is slow in disappearing from granule cell bodies during development.
Our results with spermine as a marker for the NR2B subunit have to be
taken with caution. It is possible that spermine may not efficaciously
increase the peak of synaptic NMDA responses because of inherent
differences between synaptic and extrasynaptic receptors other than
just latency of NR2A subunit expression and NR2B disappearance. One
possibility is that synaptic NMDA receptors contain NR1 splice variants
with exon 5. It has been shown that recombinant NR2B receptor
sensitivity to spermine is abolished when coexpressed with the NR1b
splice form (Durand et al., 1992). Also, we cannot rule out the effect
of spermine on presynaptic voltage-gated calcium channels or other
processes involved in synaptic transmission. Lastly, the effect of
spermine on channel conductance complicates the interpretation of the
effects on peak current.
Alternative interpretations of distinct effects of drugs in
synaptic and extrasynaptic receptors
The fact that mossy fiber-granule neuron synapses are enclosed in
the glomerulus and the inherent thickness of the slice may have
hindered the effective access of drugs to synaptic receptors. Experiments with CPP and spermine argue against this possibility. CPP
quickly and efficaciously blocked synaptic NMDA receptors in all cells
tested. Moreover, we were able to determine the presence of spermine in
the synapse by its effect on the channel current amplitude observed in
the tail of NMDA EPSCs. Lastly, experiments with MK-801 showed that CP
101,606 is indeed able to penetrate the tissue and inhibit
extrasynaptic NMDA receptors during whole-cell recordings in the slice.
A second concern is for artifactual alterations of CP 101,606 sensitivity. Patch excision as the reason for increased CP 101606 sensitivity of extrasynaptic receptors seems unlikely because it was
also observed during whole-cell recordings. Furthermore, some patch
responses were insensitive to this drug in mature rats.
Consequences for deactivation from changes in subunit composition
during development
There is clear evidence for distinct decay kinetics among
recombinant receptors containing NR1 and NR2 subunits, with NR1/NR2A heteromers being the fastest (Monyer et al., 1994; Vicini et al., 1998). Native receptors in the cerebellum also seem to have similar characteristics. Experiments with NR2C knock-out mice show that, in
P14-P18 animals, NMDA EPSCs are faster when compared with their wild-type counterparts (Ebralidze et al., 1996). Furthermore, NMDA EPSC
decay times from NR2C knock-out mice (Ebralidze et al., 1996) correlate
closely to deactivation kinetics measured from recombinant NR1/NR2A
receptors (Vicini et al., 1998). In this work, we confirm the decrease
in NMDA EPSC decay times during development. Early in development, when
synapses first form, NMDA EPSCs are at their slowest. This is
suggestive of a high proportion of receptors containing NR2B. However,
as development proceeds, decay times for EPSCs decrease. This is
suggestive of the disappearance of the NR2B subunit and the increased
expression of the NR2A subunit in synaptic populations. This phenomenon
parallels a gradual decrease in CP 101,606 sensitivity.
In extrasynaptic populations, CP 101,606 sensitivity of patch responses
does not significantly change during the first 2 weeks, but
deactivation is significantly faster during the second postnatal week.
This result indicates that deactivation of NMDA responses may not
exclusively be determined by the changes in expression of NR2 subunits.
Other factors may also influence deactivation kinetics. For example,
evidence from NR2A knock-out mice show that NMDA EPSC decay times still
increase with age (Takahashi et al., 1996), although recombinant NMDA
receptor data show little differences between NR1/NR2B and NR1/NR2C
current deactivation (Monyer et al., 1994; Vicini et al., 1998). Also,
although our data show that the reported decrease in channel
conductance in excised patches (Clark et al., 1997) does not occur in
nucleated patches, we cannot rule out that, during development, patch
excision may artificially accelerate deactivation kinetics.
Our work suggests that receptors primarily comprised of the NR2B
subunit can display relatively fast decay times yet still possess
characteristics of NR1/NR2B receptors, such as CP 101,606 and spermine
sensitivity. At the same time, slow NMDA EPSCs in immature rats display
a decreased sensitivity to NR2B antagonists (Stocca and Vicini,
1998; Tovar and Westbrook, 1999). Further studies will be necessary to
clarify whether the presumed formation of receptors containing more
than one type of NR2 subunit may underlie some of these findings
(Vicini et al., 1998).
Conclusions
Our results further support the hypothesis that NMDA receptors
containing the NR2A subunit can rapidly integrate into synapses around
the time of synaptogenesis (Tovar and Westbrook, 1999). However, we
also provide clear evidence for the targeting of the NR2A subunit to
extrasynaptic receptors later in development. What is the significance
of this phenomenon? One possibility is that the presence of the NR2A
subunit confers specific properties to extrasynaptic receptors, such as
an increase in calcium-dependent inactivation (Krupp et al., 1996) or a
decrease in glutamate and glycine sensitivity (Laurie and
Seeburg, 1994). In addition, NR1/NR2A heteromers with fast
deactivation have limited ability to undergo persistent channel
openings. This could result in an extrasynaptic receptor population
that is more suitable for physiological activation only during intense
neuronal activity.
| |
FOOTNOTES |
Received July 21, 1999; revised Aug. 27, 1999; accepted Sept. 28, 1999.
This work was supported by National Institute of Mental Health Grants
R01 MH58946 and KO2 MH01680. We are grateful to Dr. Barry Wolfe, Dr.
Deirdre O'Leary, and Stacie Grossman for critical reading of this manuscript.
Correspondence should be addressed to Dr. Stefano Vicini, Department of
Physiology and Biophysics, Georgetown University School of Medicine,
3900 Reservoir Road, NW, Washington, DC 20007. E-mail: svicin01{at}gusun.georgetown.edu.
| |
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A Hippocampal NR2B Deficit Can Mimic Age-Related Changes in Long-Term Potentiation and Spatial Learning in the Fischer 344 Rat
J. Neurosci.,
May 1, 2002;
22(9):
3628 - 3637.
[Abstract]
[Full Text]
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D. Billups, Y.-B. Liu, S. Birnstiel, and N. T. Slater
NMDA Receptor-Mediated Currents in Rat Cerebellar Granule and Unipolar Brush Cells
J Neurophysiol,
April 1, 2002;
87(4):
1948 - 1959.
[Abstract]
[Full Text]
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J. F. Otto, M. M. Kimball, and K. S. Wilcox
Effects of the Anticonvulsant Retigabine on Cultured Cortical Neurons: Changes in Electroresponsive Properties and Synaptic Transmission
Mol. Pharmacol.,
April 1, 2002;
61(4):
921 - 927.
[Abstract]
[Full Text]
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J. Nabekura, T. Ueno, S. Katsurabayashi, A. Furuta, N. Akaike, and M. Okada
Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy
J. Physiol.,
March 15, 2002;
539(3):
735 - 741.
[Abstract]
[Full Text]
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H. Guo and L-Y M Huang
Alteration in the voltage dependence of NMDA receptor channels in rat dorsal horn neurones following peripheral inflammation
J. Physiol.,
November 15, 2001;
537(1):
115 - 123.
[Abstract]
[Full Text]
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X. Luo, V. Heidinger, S. Picaud, G. Lambrou, H. Dreyfus, J. Sahel, and D. Hicks
Selective Excitotoxic Degeneration of Adult Pig Retinal Ganglion Cells In Vitro
Invest. Ophthalmol. Vis. Sci.,
April 1, 2001;
42(5):
1096 - 1106.
[Abstract]
[Full Text]
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J. D. Sinor, S. Du, S. Venneti, R. C. Blitzblau, D. N. Leszkiewicz, P. A. Rosenberg, and E. Aizenman
NMDA and Glutamate Evoke Excitotoxicity at Distinct Cellular Locations in Rat Cortical Neurons In Vitro
J. Neurosci.,
December 1, 2000;
20(23):
8831 - 8837.
[Abstract]
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L. Cathala, C. Misra, and S. Cull-Candy
Developmental Profile of the Changing Properties of NMDA Receptors at Cerebellar Mossy Fiber-Granule Cell Synapses
J. Neurosci.,
August 15, 2000;
20(16):
5899 - 5905.
[Abstract]
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H. Hirai and T. Launey
The Regulatory Connection between the Activity of Granule Cell NMDA Receptors and Dendritic Differentiation of Cerebellar Purkinje Cells
J. Neurosci.,
July 15, 2000;
20(14):
5217 - 5224.
[Abstract]
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S. Vicini and G. Rumbaugh
A slow NMDA channel: in search of a role
J. Physiol.,
June 1, 2000;
525(2):
283 - 283.
[Full Text]
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A. Momiyama
Distinct synaptic and extrasynaptic NMDA receptors identified in dorsal horn neurones of the adult rat spinal cord
J. Physiol.,
March 15, 2000;
523(3):
621 - 628.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
