1Department of Neuroscience, University of Turin, I-10125 Turin; and 2Department of Internal Medicine, Section of Human Physiology, University of Perugia, I-06126 Perugia, Italy
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ABSTRACT |
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Anchisi, Davide,
Bibiana Scelfo, and
Filippo Tempia.
Postsynaptic Currents in Deep Cerebellar Nuclei.
J. Neurophysiol. 85: 323-331, 2001.
Postsynaptic
currents were studied by whole cell recordings in visually identified
large neurons of the deep cerebellar nuclei (DCN) in slices of 4- to
11-day-old mice. Spontaneous postsynaptic currents were abolished by
the GABAA receptor antagonist bicuculline and had
a single-exponential decay with a mean time constant of 13.6 ± 3.2 (SD) ms. Excitatory postsynaptic currents (EPSCs) were evoked in 48/56 neurons recorded. The addition of AMPA and
N-methyl-D-aspartate (NMDA) receptor antagonists
together completely abolished all synaptic responses. In 1 mM
[Mg2+]o and at a holding
potential of 60 mV, the peak amplitude of the NMDA component of the
EPSC (NMDA-EPSC) was 83.2 ± 21.2% of the AMPA component
(AMPA-EPSC). This indicates that in DCN neurons, at a physiological
[Mg2+]o and at the
resting membrane potential, NMDA receptors contribute to the synaptic
signal. AMPA-EPSCs had a linear current-voltage relationship with a
reversal potential of +2.3 ± 0.4 mV and a single-exponential
decay with a voltage-dependent time constant that at
60 mV was
7.1 ± 3.3 ms. In 10 µM glycine and 1 mM
[Mg2+]o, the
I-V relationship of NMDA-EPSCs had a reversal potential of
0.5 ± 3.3 mV and a maximal inward current at
33.4 ± 5.8 mV. The apparent dissociation constant
(KD) of Mg2+ for
the NMDA receptor-channel at
60 mV, measured by varying [Mg2+]o, was 135.5 ± 55.3 µM, and when measured by fitting the I-V curves
with a theoretical function, it was 169.9 ± 119.5 µM. Thus in
the DCN, NMDA receptors have a sensitivity to
Mg2+ that corresponds to subunits that are weakly
blocked by this ion (
3 and
4) of which the DCN express
4.
NMDA-EPSCs had a double-exponential decay with voltage-dependent time
constants that at
60 mV were 20.2 ± 8.9 and 136.4 ± 62.8 ms. At positive voltages, the time constants were slower and their
contributions were about equal, while in the negative slope conductance
region of the I-V curve, the faster time constant became
predominant, conferring faster kinetics to the EPSC. The weak
sensitivity to Mg2+ of NMDA receptors, together
with a relatively fast kinetics, provide DCN neurons with strong
excitatory inputs in which fast dynamic signals are relatively well preserved.
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INTRODUCTION |
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Deep cerebellar nuclei (DCN)
neurons provide the main output of the cerebellum and receive
collaterals from the two main cerebellar afferent systems, climbing
fibers and mossy fibers. In addition, they receive GABAergic synapses
from Purkinje cell axons, which are the only efferents of the
cerebellar cortex. Therefore neurons of the DCN perform the final
processing of signals leaving thereafter the cerebellum (Ito
1984). DCN contains three main types of neurons: large
projection neurons that send their axons outside the cerebellum and are
the final cerebellar output element, and smaller inhibitory neurons
including GABAergic cells that project to the inferior olive and local
interneurons. It is possible that all these types of neurons receive
all three main afferents, but the final signal integration takes place
in the large projection neurons. The nature of DCN signal processing is
still matter of debate. Most of the attention has been devoted recently
to the Purkinje cell-DCN neuron synapse, which was the first GABAergic
synapse shown to undergo both long-term depression (Aizenman et
al. 1998
; Morishita and Sastry 1993
) and
long-term potentiation (Aizenman et al. 1998
).
Mossy fibers and climbing fibers, which on cortical neurons act through
glutamate receptors, send collateral branches to the DCN. Since DCN
neuron axons are the cerebellar efferent fibers, the pathway of mossy
and climbing fiber collateral branches-DCN-cerebellar efferent fibers
constitutes a cerebellar loop that could in principle work
independently of the cortex. It is likely that such a subcortical cerebellar loop adds to all motor commands a basal cerebellar contribution that can be shaped by cortical activity. The contribution of the trans-cortical loop is not fixed but is endowed with
several modifiable synapses, including the synapse between Purkinje
cell axons and DCN neurons (Aizenman et al. 1998;
Morishita and Sastry 1993
). It has been shown that DCN
neurons have functional glutamate receptors of both AMPA and
N-methyl-D-aspartate (NMDA) type (Audinat et al. 1990
) and glutamatergic fibers directed to DCN neurons evoke excitatory postsynaptic potentials (EPSPs) with components mediated by both receptors (Audinat et al. 1992
). In
view of the pivotal role of glutamatergic synapses on DCN neurons, it
is very important to throw more light on the relative contributions of the different receptors and on their functional properties.
NMDA receptors of DCN neurons possess an interesting subunit repertoire
since they express the subunits NR1, NR2A, NR2B, and NR2D, which in the
mouse are called 1,
1,
2, and
4 (Akazawa et al.
1994
; Watanabe et al. 1994
). For the formation
of a functional NMDA receptor channel, it is necessary to have the
co-assembly of subunits of both NR1/
and NR2/
types, but the
functional properties of the channel, like the deactivation rate or the
block by Mg2+, seem to depend mainly on the
latter type of subunits (Hollmann and Heinemann 1994
).
In DCN neurons, the presence of NR2D/
4 is especially significant
since this subunit forms, together with NR1/
1, channels that
deactivate very slowly and are relatively resistant to the block by
Mg2+ while channels containing only NR1/
1 plus
the subunits NR2A/
1 and/or NR2B/
2 deactivate more rapidly and are
more potently blocked by Mg2+ (Kuner and
Schoepfer 1996
; Monyer et al. 1994
;
Vicini et al. 1998
). The strength of
Mg2+ block of NMDA receptor channels with
different NR2/
subunits can be distinguished by the degree of
rectification of their current-voltage relationship in the presence of
a physiological concentration of external Mg2+.
Thus in the presence of an external concentration of 1 mM
Mg2+, NR2A- or NR2B-containing channels are
almost completely blocked at
100 mV and pass maximal current at about
25 mV, while NR2C or NR2D containing channels are incompletely
blocked at
100 mV and pass maximal current at about
35 mV
(Kuner and Schoepfer 1996
). In addition, at membrane
voltages close to the resting potential there is a four- to fivefold
difference in the apparent dissociation constant of the channels for
Mg2+ with values of about 20 µM for NR2A- or
NR2B-containing channels and of 80 or 100 µM, respectively, for NR2C-
or NR2D-containing channels (Kuner and Schoepfer 1996
).
This is due to a different voltage dependence of the block, which is
expressed by the parameter
of the function (see
METHODS: Eq. 1) describing the current-voltage relationship of NMDA receptor currents (Kuner and Schoepfer
1996
; Woodhull 1973
).
A stronger block by Mg2+ confers to the NMDA
channel the capability to function as a coincidence detector because at
the resting membrane potential almost no inward current is allowed to
flow even when the agonist is bound to the receptor/channel
(Ishii et al. 1993; Kutsuwada et al.
1992
; Monyer et al. 1992
). Only the coincidence
of agonist binding plus membrane depolarization permits a flow of ions,
including Ca2+, through the channel. In contrast,
a weak Mg2+ block of NMDA receptor channels
formed by NR1/
1 and NR2D/
4 determines a relevant
Ca2+ influx even at resting or at slightly
depolarized potentials. Since DCN neurons express NR1/
1, NR2A/
1,
NR2B/
2, and NR2D/
4 (Akazawa et al. 1994
;
Watanabe et al. 1994
), in principle they can form both
types of NMDA receptors (NMDA-R), respectively, with faster kinetics
and a stronger Mg2+ block (with NR2A/
1,
NR2B/
2) or with slower kinetics and a weaker Mg2+ block (with NR2D/
4).
The results of our present investigation show that, in DCN neurons, the
NMDA-R component of EPSCs has fast kinetics and a weak
Mg2+ block, suggesting that NR2A/1 and
NR2B/
2 combinations are dominant for the former property while
NR2D/
4 determines the effect of Mg2+. In
addition, we show that, in the DCN, EPSCs display a significant NMDA
component at the resting membrane potential, indicating that in these
neurons NMDA-Rs have a physiological role in normal synaptic signaling
instead of working merely as coincidence detectors.
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METHODS |
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Experiments were performed on 4- to 11-day-old CD-1 mice of
either sex. The preparation of cerebellar slices was performed according to previously described techniques (Edwards et al.
1989; Llinás and Sugimori 1980
). Briefly,
the animals were anesthetized with halothane (Fluothane, Zeneca,
London, UK) and decapitated. The cerebellar vermis was rapidly
removed and placed in an ice-cold extracellular saline solution, that
contained (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose and was bubbled with 95%
O2-5% CO2 so that the pH
was 7.4. Some experiments were performed with varying concentrations of
extracellular Mg2+
([Mg2+]o) ranging from
0.01 to 3 mM. In these cases, the composition of the extracellular
solution was modified only with regard to [Mg2+]o without
readjusting the concentration of the other ions. Parasagittal cerebellar slices (300 µm thick) were prepared using a vibrotome (Vibroslice 752, Campden Instruments, Sileby, UK) and kept for the
first hour at 35°C and then at 25°C for the rest of the day. After
the first hour of incubation, a single slice at a time was transferred
to a recording chamber and continuously perfused at room temperature
(22-26°C) with the saline solution bubbled with the 95%
O2-5% CO2 mixture. A DCN
neuron soma was visualized using a ×63 water-immersion objective of an
upright microscope (Axioscop, Zeiss, Jena, Germany), and its upper
surface was cleaned by gently blowing and sucking saline solution from
a cleaning pipette (Edwards et al. 1989
). The pipettes
used for cleaning and for extracellular stimulation were pulled from
sodalime glass to a tip diameter of 10-15 µm. For both cleaning and
stimulation, the pipette was filled with extracellular saline solution.
For stimulation, the pipette was gently pressed into the tissue
surrounding the DCN neuron, and negative current pulses ranging from 3 to 90 µA with durations of 100 µs were delivered. For patch-clamp
recording, pipettes of borosilicate glass with tip diameters of 2-3
µm were used. When filled with an intracellular solution the
resistance was 1.4-2.5 M
. In most experiments, the internal pipette
solution had the following composition (in mM): 143 CsCl, 2 MgCl2, 10 HEPES, 4 Na2ATP,
0.4 Na3GTP, and 10 EGTA; in the experiments in
which different [Mg2+]o
were used, the composition was (in mM) 137 K-gluconate, 2 MgCl2, 10 HEPES, 4 Na2ATP,
0.4 Na3GTP, and 10 EGTA. The pH was adjusted to
7.3 with CsOH, for the CsCl solution, or with KOH, for the K-gluconate
solution. In eight cells, the fluorescent dye Lucifer yellow (1 mM) was
added to the internal solution and after recording the slice was fixed
with 4% paraformaldehyde, dehydrated, and mounted on glass. Then
micrographs were taken with an Axiophot (Zeiss) microscope at different
planes of focus, digitized, and stored in the hard disk of a Pentium II
PC. Then the best focused regions were assembled (Fig.
1). The somatic diameter was measured in
16 neurons: 12 of them were measured in fresh tissue by a graded scale
mounted in an ocular of the microscope; another 4 neurons were measured
after labeling with Lucifer yellow.
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After a tight seal (>5 G) was formed, the membrane was broken by
suction to achieve the whole cell configuration. All recordings were
performed in voltage-clamp using an EPC-7 patch-clamp amplifier (HEKA
Elektronik, Lambrecht/Pfalz, Germany). The holding potential was set at
60 mV and varied during I-V experiments, and the series resistance was compensated. Data were filtered at 3 kHz, digitized at
10 kHz with an A/D converter (ITC 16, Instrutech, Port Washington, NY),
and stored on a Macintosh computer (Quadra 650, Apple Computer, Cupertino, CA) using the Pulse Control software kindly provided by Dr.
J. D. Herrington and Dr. R. J. Bookman of the University of
Miami. Data were analyzed off-line by the commercial program IgorPro
(Wavemetrics, Lake Oswego, OR). Drugs were applied by changing the
perfusion line (exchange time in the chamber: 10-20 s), at the
following concentrations: 40 µM bicuculline, 10 µM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), 50 µM D (
)-2-amino-5-phosphonopentanoic acid
(D-AP5), 1 µM strychnine, and 10 µM glycine. NBQX and
D-AP5 were purchased from Tocris Cookson (Langford, UK);
bicuculline, strychnine, and glycine were bought from Sigma Chemical
(St. Louis, MO) and Lucifer yellow from Molecular Probes Inc. (Eugene, OR).
The current-voltage relationship of NMDA-EPSCs was fitted by the
following equation
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(1) |
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(2) |
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(3) |
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RESULTS |
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DCN contain different classes of neurons. Neurons projecting to
premotor structures have a larger somatic diameter [22.3 ± 4.8 (SD) µm] (De Zeeuw and Berrebi 1995), while GABAergic
neurons including those projecting to the inferior olive and local
interneurons have smaller soma (11 ± 2.6 µm) (De Zeeuw
and Berrebi 1995
). The neurons recorded in this study had a
somatic size across the major axis ranging from 14.5 to 32.0 µm
(mean = 19.8 ± 4.8 SD, n = 16) and across
the minor axis between 8.0 and 31.0 µm (mean = 14.7 ± 5.8 SD, n = 16). Therefore the majority of the neurons
recorded were projection neurons that send the cerebellar efferent
signals to premotor nuclei. It is possible that a few of them were
GABAergic neurons projecting to the inferior olive. Since dendrites are not clearly visible in living tissue, in eight cells, the dendritic tree was labeled by Lucifer yellow filling via the recording pipette (Fig. 1). The dendritic morphology of the recorded neurons was characterized by several thin dendrites arising from the soma and with
sparse dendritic spines as has been shown previously (Chan-Palay
1977
).
The presence of a considerable dendritic compartment required an
assessment of the passive electrical membrane properties to establish
the voltage-clamp conditions for distal dendritic regions. For the
measurement of passive membrane properties, DCN neurons were recorded
in voltage-clamp mode, and small voltage steps of 5 mV were delivered
from a holding potential of 70 mV. With recording conditions
including a series resistance
(Rs) < 12.5 M
with 30-40%
compensation, the capacitive transients produced by voltage steps had a
decay that was always well fitted by a single-exponential function with
a decay time constant of 2.0 ± 0.5 (SD) ms (n = 10). The input resistance calculated from the same voltage steps was
159.9 ± 68.3 (SD) M
(n = 10) with the intracellular solution based on CsCl (see METHODS). The
relatively fast rate of decay and the single-exponential shape indicate
that DCN neurons under these recording conditions have passive
electrical properties that can be described by a single-compartment
electrical circuit, excluding the presence of a less efficient
voltage clamp in distal dendritic compartments.
Spontaneous postsynaptic currents
After entering the whole cell configuration, spontaneous inward
currents were present as previously reported (Momiyama and Takahashi 1994). Such spontaneous currents were completely and reversibly blocked by the GABAA receptor
antagonist bicuculline (40 µM; n = 56; Fig.
2A). The inward direction of
the spontaneous currents is in agreement with the chloride equilibrium
potential close to 0 mV due to the composition of the intracellular
solution (see METHODS). The decay of such spontaneous
inhibitory postsynaptic currents (sIPSCs) was well fitted by a
single-exponential function (Fig. 2B). The decay time
constant was measured in each DCN neuron for 5-19 sIPSCs and its
average value was 13.6 ± 3.2 ms (n = 7), and
within each cell, its distribution was unimodal. The decay time
constant had no correlation with the amplitude of the current, indicating that also the largest currents, possibly giving rise to
larger voltage errors, were under adequate voltage-clamp control to
assess the decay kinetics.
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Evoked excitatory postsynaptic currents
Monopolar electrical stimuli were delivered to the tissue
surrounding the neuron during recording under block of
GABAA and glycine receptors (bicuculline, 40 µM; strychnine, 1 µM). In these conditions, no spontaneous activity
was present. Stimulation evoked excitatory synaptic currents
(EPSCs) in 48/56 neurons (86%). The addition of blockers of
AMPA and NMDA receptors together completely abolished all synaptic
response (Fig. 3, A and
B; NBQX, 10 µM, D-AP5, 50 µM;
n = 3). In some cells, to dissect the single components of the evoked EPSCs, initially the NMDA-mediated current was blocked with D-AP5. The NMDA antagonist was then washed out until
the EPSC recovered to the control amplitude, and next the AMPA receptor antagonist NBQX was applied (Fig. 3, C and D).
The peak amplitude of the two components was compared in each cell in
the presence of 1 mM
[Mg2+]o and at a holding
voltage (VH) of 60 mV, which is
close to the resting membrane potential of these neurons
(Aizenmann et al. 1998
; Jahnsen 1986
;
Llinás and Muhlethaler 1988
). The NMDA component
of the EPSC (NMDA-EPSC) was 83.2 ± 21.2% (mean ± SD, n = 5) of the AMPA component (AMPA-EPSC). Since
NMDA-EPSCs decay slower than AMPA-EPSCs, their contribution to the
charge transfer across the membrane is even larger. The net amount of
charge that accumulates in the cell during an EPSC can be calculated by
constructing the time integral of the membrane current. From the data
shown in Fig. 3C, NMDA receptors allowed the entry of 11.2 pC of net positive charge in the first 100 ms from the stimulation,
while at the same time point AMPA receptors contributed only for 3.5 pC. Thus in DCN the NMDA component of the EPSC is responsible for a net
charge transfer that is more than threefold relative to the AMPA
component. Taken together, these data indicate that in DCN neurons, at
a physiological [Mg2+]o
and at the resting membrane potential, there is a significant contribution of NMDA receptors to synaptic signals.
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AMPA-EPSC properties
The current-voltage relationship (I-V relationship) of
AMPA-EPSCs, obtained in the presence of bicuculline, strychnine and D-AP5, was nearly linear with a slight outward
rectification and a reversal potential of +2.3 ± 0.4 (SD) mV
(n = 3; Fig. 4,
A and B). The AMPA-EPSCs had a decay that was
fitted by a single exponential with a time constant at the
VH of 60 mV of 7.1 ± 3.3 ms
(n = 3; Fig. 4, A and C). The
time constant was voltage dependent, becoming slower at positive
voltages (Fig. 4C).
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NMDA-EPSC properties
NMDA-EPSCs were recorded in the presence of bicuculline and
strychnine in the perfusate to block GABAA and
glycine receptors, respectively, and NBQX to block AMPA receptors and
with 10 µM glycine to saturate the binding site of NMDA receptors for
this amino acid (Kew et al. 1998; Wilcox et al.
1996
). With 1 mM
[Mg2+]o the
I-V relationship of NMDA-EPSCs showed the typical outward rectification with a region of negative slope conductance
(n = 13; Fig. 5,
A and B) as described for NMDA receptors-mediated currents (Mayer et al. 1984
; Nowak et al.
1984
). When the perfusion was changed to one with nominally
zero [Mg2+]o, the
I-V relationship of the NMDA-EPSCs tended to become linear (not shown) as expected from the known voltage-dependent block by
[Mg2+]o. To characterize
the properties of the NMDA-EPSC in these neurons, we fitted the
I-V curves obtained in 1 mM
[Mg2+]o with Eq. 1 (see METHODS). For all cells, this equation fitted the experimental data points very reliably. In Fig. 5B, the
fit on the cumulative data from 13 cells is shown. From the individual fits, it was possible to precisely measure the reversal potential of
the NMDA-EPSC, which was
0.5 ± 3.3 mV (n = 13),
and the maximal inward current, measured as the negative peak of the
function fitting the I-V relationship, which was attained at
a membrane potential of
33.4 ± 5.8 mV (n = 13).
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The parameters a and of the best fit curve for Eq. 1 allowed us to calculate the apparent dissociation constant
(KD) of Mg2+ for
the NMDA receptor channel by Eq. 2 (see
METHODS). The parameter a, which represents the
affinity of the channel for Mg2+ in absence of
transmembrane potential, was 5.3 ± 1.3 (SE) mM (n = 14). The parameter
, which represents the voltage dependence of
the block, was 0.69 ± 0.04 (n = 14). The
KD value calculated for a membrane
potential of
60 mV was 169.9 ± 119.5 (SD) µM
(n = 14). Since such
KD value was unusually high compared
with other types of neurons, to provide an independent assessment of
this parameter, the amplitude of the NMDA-EPSC was measured at
different extracellular concentrations of Mg2+
keeping the VH constant at
60 mV
(Fig. 5C). The results were fitted by Eq. 3 and
the Mg2+ concentration that gave an inhibition of
50% of the NMDA-EPSC was taken as the
KD for Mg2+ (see
METHODS). With this procedure, the value of
KD was 135.5± 55.3 µM
(n = 4), which is not significantly different from the measurement derived from the current-voltage relationship (Student's t-test: P > 0.05), confirming that in DCN
neurons NMDA receptor channels are only weakly blocked by external
Mg2+. This result is also in agreement with
the presence of a relevant inward current detected in these neurons
through NMDA receptors at the resting membrane potential of
60 mV
(see Fig. 3).
The rise time of NMDA-EPSCs was measured as the interval between the
beginning of the stimulation and the time at which the current reached
its peak amplitude. NMDA-EPSCs rise time was on average 12.5 ± 3.0 ms (n = 9). The decay of NMDA-EPSCs was fitted by a
double-exponential curve (Figs. 5A and
6A) with, at the
VH of 60 mV, a faster time constant
of 20.2 ± 8.9 ms (n = 9) and a slower one of
136.4 ± 62.8 ms (n = 9). Both exponential
components had a time constant that was voltage dependent, becoming
slower toward positive values (Fig. 6, A and B).
To analyze the contribution of each component, the amplitudes
extrapolated to the time origin (beginning of the stimulus artifact)
were plotted as a function of the holding voltage (Fig. 6C).
While at positive voltages, the contributions of the slow and of the
fast component are about equal: at
40 and
60 mV, the amplitude of
the faster component becomes larger than the slower one, so that at
negative potentials the slow component contributes less to the decay
(Fig. 6, A and C). This relative prevalence of
the faster time constant at negative potentials accounts for a further
increase of the decay velocity at such potentials. In fact, this effect
adds to the faster decay of each component at negative potentials (Fig.
6B).
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The presence of a stronger rectification of the slow component at
negative voltages suggests that this component could be more sensitive
to [Mg2+]o than the fast
one. To test this hypothesis, we compared the amplitude of the two
components at a VH of 60 mV,
in 0 [Mg2+]o relative to
1 mM [Mg2+]o. In the
former condition, of Mg2+-free extracellular
solution, the slow component accounted for 44.7% of the
amplitude while the remaining 55.3% was due to the fast component
(n = 5). The addition of 1 mM
[Mg2+]o significantly
decreased the contribution of the slow component to 29.8% while
the fast component increased to 70.2% (Student's paired
t-test, P < 0.05).
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DISCUSSION |
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In the present study, we show that in large neurons of the DCN fast synaptic transmission is mediated by GABAergic synaptic currents with properties very similar to other neuronal types and by glutamatergic currents that, in contrast, present some unusual features.
The sIPSCs were completely abolished by the addition of bicuculline,
indicating that they were exclusively mediated by
GABAA receptors and therefore that DCN neurons do
not receive a significant number of spontaneously active glycinergic
inhibitory synapses. This result is in agreement with previous studies
in which the addition of bicuculline abolished miniature IPSCs
(Momiyama and Takahashi 1994) or spontaneous and evoked
inhibitory postsynaptic potentials (Aizenmann et al.
1998
; Audinat et al. 1992
; Mouginot and
Gahwiler 1995
).
The single-exponential decay of sIPSCs with relatively fast kinetics is
comparable to the characteristics of IPSCs of principal neurons in
slices of other regions of the CNS, including Purkinje cells
(Vincent et al. 1992) pyramidal neurons of the
hippocampus (Collingridge et al. 1984
) and neocortex
(Salin and Prince 1996
). This contrasts with the
double-exponential decay described for granule cells of the dentate
gyrus (Edwards et al. 1990
) and for cerebellar granule
cells (Puia et al. 1994
). It is interesting to note
that, as in the principal neurons of other regions, the principal
neurons of DCN, investigated in this study, also have sIPSCs with a
single-exponential decay.
A main finding of our experiments is a significant NMDA component in
EPSCs even at a membrane voltage corresponding to the resting
potential. Therefore in these neurons, NMDA receptors must be involved
in normal synaptic signaling and are responsible for a significant
portion of the EPSC. This NMDA contribution results in EPSCs that last
longer than those mediated by AMPA receptors as can be appreciated by
comparing the AMPA component with the total glutamatergic EPSC in the
same cell (see Fig. 3). This finding is in agreement with a previous
study in organotypic cultures, where, in the DCN, EPSPs recorded in 0.5 mM [Mg2+]o had AMPA and
NMDA components of comparable amplitude (Audinat et al.
1992). An explanation for the presence of a relevant
NMDA-mediated current at the resting potential is a relatively weak
effect of Mg2+ at negative potentials. Our
results indicate that this is the case: in fact, in DCN neurons, we
observed a maximal inward current at about
35 mV and the
KD for Mg2+ at
60 mV was 135.5 µM measured by the dose-response curve for [Mg2+]o. Such a high
KD value is confirmed by the
KD, calculated by Eq. 2, of
169.9 µM at the same VH, derived
from the data obtained by fitting Eq. 1 to the
I-V relationship. Both the maximal inward current at about
35 mV and the KD for
Mg2+ at
60 mV larger than 100 µM correspond
to the values of NMDA receptor channels with a weak
Mg2+ block (Kuner and Schoepfer
1996
). The weaker effect of Mg2+ on the
channels containing either NR2C/
3 or NR2D/
4 is due to a lower
voltage dependence of the block (Kuner and Schoepfer
1996
), which is described by the parameter
of Eq. 1. The channels containing either NR2A/
1 or NR2B/
2 have a
strong voltage dependence of the block by Mg2+
with a value of
of about 1, while the channels containing either NR2C/
3 or NR2D/
4 have a
of about 0.7 (Kuner and
Schoepfer 1996
). In DCN, the value of
of 0.69 indicates
that the NMDA receptor channels of these neurons have the same
voltage-dependent block by Mg2+ as NR2C/
3- or
NR2D/
4-contaning channels.
Our observation of a weak sensitivity to
[Mg2+]o of NMDA receptors
in DCN neurons is unexpected because these neurons express the 1
subunit and from P0 to P7 also the
2 subunit, which are both
strongly blocked by Mg2+ (Ishii et al.
1993
; Monyer et al. 1994
). A possible
explanation is an involvement of the other
subunit expressed by DCN
neurons,
4, which has a lower affinity for
Mg2+ (Ishii et al. 1993
;
Monyer et al. 1994
). Although in adult animals, NR2D/
4 expression is restricted to a few types of neurons, during development, especially in embryonic and neonatal age, this subunit is
strongly expressed throughout the CNS (Monyer et al.
1994
). In addition to a low sensitivity to
Mg2+, the NR2D/
4 subunit generates currents
that decay slowly with a time course of several seconds (Ishii
et al. 1993
; Kutsuwada et al. 1992
;
Monyer et al. 1992
). For these reasons, it has been suggested that such a long-lasting current is involved in the generation of Ca2+ signals important for the
development of the CNS (Momiyama et al. 1996
;
Monyer et al. 1994
). However, in DCN neurons we find that NMDA-EPSCs are weakly blocked by Mg2+, but
at the same time, they are relatively rapid, with kinetics that are
typical of the
1 and
2 subunits. However, the latter subunits are
potently blocked by Mg2+ so that normally they do
not contribute significantly to synaptic signaling unless the
postsynaptic cell is at the same time depolarized by another excitatory
synapse. Thus if we consider together the Mg2+
dependence and the decay rate of NMDA-EPSCs in DCN neurons, they do not
correspond to any single subunit known at present. It should be
emphasized that all our measurements of Mg2+
effects on NMDA-EPSCs have been obtained by taking the peak amplitude of the current, when possible slow channels, like those formed by
NR1/
1 with NR2D/
4, would not yet be significantly activated. In
fact, while NMDA receptor channels formed by NR1/
1 plus NR2A/
1 activate with a rise time constant of about 13 ms (Wyllie et al. 1998
), those formed by NR1/
1 plus NR2D/
4 have a slower
activation rate, with a time constant of about 45 ms (Wyllie et
al. 1998
). Since in DCN the peak of the NMDA-EPSC is reached
12.5 ms from the beginning of the stimulation of presynaptic fibers,
the activation kinetics better corresponds to NMDA receptor channels
containing the NR2A/
1 subunit. As a consequence, our finding of a
weak sensitivity to Mg2+ really refers to
currents with fast kinetic properties. Thus NMDA-EPSCs in DCN possess
novel physiological properties that cannot be explained simply by the
sum of the properties of the cloned subunits as studied by expression
in heterologous systems.
Recent evidence (Buller and Monaghan 1997; Dunah
et al. 1998
) indicates that NR2D/
4 can assemble with
NR2A/
1 or NR2B/
2 subunits but without affecting the conductance
levels of individual receptor channels, which are dominated by the
latter subunits (Cull-Candy et al. 1998
). However, the
kinetics and/or the degree of block by Mg2+ of
such heterotrimeric channels (
1,
1/2,
4) are not known. They
could have intermediate properties, or the features of one NR2/
subunit could dominate over the others. Our results of NMDA EPSCs,
which are at the same time rapid but weakly blocked by Mg2+, suggest that NMDA-Rs in DCN neurons are
formed by the co-assembly of both kinds of
subunits (1 or 2 together with 4). The possible formation of such a subunit composition
has been suggested by immunoprecipitation experiments (Dunah et
al. 1998
). If this is the case, our study points to the
possibility that in these neurons the
4 subunit is dominant for the
determination of the Mg2+ block with the result
of allowing more current flow at negative potentials, while the
1/2
subunits are dominant regarding the decay rate, keeping the kinetics
fast. More studies of functional expression are needed to test this
hypothesis. However, our results cannot exclude other explanations,
such as the involvement of a novel subunit, like for instance NR3A
(Das et al. 1998
; Sun et al. 1998
) or an
as yet undiscovered one, or posttranslational modifications altering
the functional properties of NMDA receptor channels.
In cerebellar Purkinje cells of young rats, NR2D is the only NR2
subunit expressed (Akazawa et al. 1994). In these cells, recordings from outside-out patches in young animals revealed a single
channel activity that was attributed to NR2D (Cull-Candy et al.
1998
; Momiyama et al. 1996
) and NMDA application
to Purkinje cells from newborn rats evoked an inward current clearly
attributable to NMDA-R (Krupa and Crepel 1990
;
Rosenmund et al. 1992
). However, whole cell recordings
in older animals failed to detect NMDA-R-mediated EPSCs (Llano
et al. 1991
; Perkel et al. 1990
). Taken
together, these data suggest the possibility that in Purkinje cells
NR2D/
4 is located extrasynaptically and that, as a consequence, it
does not contribute to the EPSC. In contrast, in DCN neurons that also express other NR2/
subunits in addition to NR2D/
4, our data suggest that this subunit contributes to the generation of EPSCs.
CA1 pyramidal neurons in the first postnatal week express NR2A, NR2B,
and NR2D, like DCN neurons (Kirson et al. 1999): as can
be calculated from the data of Kirson et al. (1999)
, in
these neurons the KD for
Mg2+ of NMDA-evoked currents at
60 mV is 65.6 µM, which is intermediate between the value of the strongly blocked
subunits (20 µM) and that of NR2D (100 µM) (Kuner and
Schoepfer 1997
), but only about half of the
KD that we find for DCN. A difference
relative to our study is that NMDA application also activates
extrasynaptic receptors, which could have a different subunit
composition, like a larger proportion of NR2B subunits (Tovar
and Westbrook 1999
).
Another study on DCN (Cull-Candy et al. 1998), using
single-channel recordings, described a mixed population of
single-channel openings: "high conductance," which can be
attributed to NR2A or NR2B subunits, and "low conductance," which
can be attributed to NR2D subunits. But the latter behavior was always
found to be associated with high-conductance openings, so that it was
not possible to assess the
[Mg2+]o sensitivity of
low-conductance channels. However, in some recordings, high-conductance
channels could be observed alone: the percentage of block of the
single-channel charge transfer exerted by 0.1 mM
[Mg2+]o at
60 mV was
81%. This value corresponds to NR2A and NR2B (Kuner and
Schoepfer 1997
), as can be expected by the fact that high-conductance openings are due to these subunits.
The [Mg2+]o sensitivity
of the NR2D subunit in native neurons has been measured by
single-channel recording in P3 Purkinje cells, which only express this
subunit (Momiyama et al. 1996). In this study,
low-conductance channels are blocked only by about 34% by 0.1 mM
[Mg2+]o (Momiyama
et al. 1996
), which is in agreement with the value of 45%
obtained for the same subunit expressed in Xenopus oocytes (Kuner and Schoepfer 1997
) and with the value of 46%
found in the present study in DCN neurons.
Our study has been conducted on DCN neurons from 4- to 11-day-old mice,
an age at which the cerebellum is still developing. Actually, the
expression of the 2 subunit in DCN is strongly reduced between P7
and P14 (Watanabe et al. 1994
). In contrast, the
expression of
1 and
4 remains constant in the DCN throughout postnatal life (Watanabe et al. 1994
). Therefore our
conclusions on the physiological properties of NMDA-EPSCs are not
likely to change at later stages of development. Over the short range
of age in which our study has been conducted, we did not find any correlation with age for any of the parameters measured. However, it is
possible that at older ages the diminished expression of
2 could
cause some degree of reduction of the NMDA-EPSC relative to the AMPA component.
The physiological meaning of keeping the EPSCs faster could be related
to the nature of the dynamic signals that they represent in which a
duration of more than a few hundred milliseconds would reduce the
resolution of temporal information. Why do DCN neurons need an NMDA
component in their EPSC? A possible explanation could be the extremely
powerful GABAergic inhibition exerted by Purkinje cells on DCN neurons.
Adding an NMDA component to excitatory synaptic signals could be a
mechanism to obtain a stronger excitation in spite of the relatively
small number of excitatory synapses. In the medial vestibular nucleus,
the block of NMDA receptors decreases the spontaneous firing rate,
suggesting that in this structure NMDA receptors may contribute to the
resting activity of neurons (Smith et al. 1990). Our
results suggest that, in the DCN, NMDA receptors could have a similar
role and that they can contribute to synaptic signals.
A recent report (Aizenman and Linden 2000) has shown
that in these synapses the influx of Ca2+ through
NMDA-R channels triggers a novel form of plasticity resulting in a
long-term increase of intrinsic excitability not accompanied by changes
in synaptic efficacy. This form of plasticity can be induced also at
hyperpolarized potentials and therefore requires NMDA-Rs with a weak
sensitivity to Mg2+. Our results of a relatively
weak effect of Mg2+ on NMDA-R channels help to
explain how they can trigger such a form of plasticity also without a
concurrent membrane depolarization.
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ACKNOWLEDGMENTS |
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We thank Dr. Piergiorgio Strata for valuable help and advice and Dr. Nick Hartell for revising the manuscript.
The financial support of Telethon-Italy (Grant 1129) is gratefully acknowledged.
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FOOTNOTES |
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Address for reprint requests: F. Tempia, Dept. of Neuroscience, University of Turin, C.so Raffaello 30, I-10125 Turin, Italy (E-mail: tempia{at}medfarm.unito.it).
Received 1 March 2000; accepted in final form 21 September 2000.
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REFERENCES |
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