Differential Long-lasting Potentiation of the NMDA and Non

0 European Neuroscience Association
European Journal of Neuroscience, Vol. 8, pp. 1182-1189, 1996
Differential Long-lasting Potentiation of the NMDA and
Non-NMDA Synaptic Currents Induced by Metabotropic
and NMDA Receptor Coactivation in Cerebellar Granule
Paola Rossi, Egidio D’Angelo and Vanni Taglietti
lstituto di Fisiologia Generale, Via Forlanini 6, 1-27100, Pavia, Italy
Keywords: cerebellum, LTP, metabotropic receptors, NMDA receptors, patch-clamp, rat
Whole-cell patch-clamp recordings in rat cerebellar slices were used to investigate the effect of metabotropic
glutamate receptor activation on mossy fibre-granule cell synaptic transmission. Transient application of 20 pM
1S,3R-1-aminocyclopentane-1,Sdicarboxylic acid simultaneously with low-frequency NMDA receptor activation
induced long-lasting non-decrementalpotentiation of both NMDA and non-NMDA receptor-mediated synaptic
transmission. Potentiation could be prevented by application of the metabotropic glutamate receptor antagonist
(+)-Omethyl-4-carboxyphenyl-glycineat 500 pM. Characteristically, NMDA potentiation was two to three times
as large as non-NMDA current potentiation, occurred only in a slow subcomponent, and was voltageindependent. This result demonstrates a pivotal role of NMDA receptors in the metabotropic potentiation of
transmission, which may be important in regulating cerebellar information processing.
Materials and methods
Many central neurons express both ionotropic and metabotropic
Slice preparation
glutamate receptors. Metabotropic glutamate receptors (mGluRs) can
Patch-clamp whole-cell recordings were carried out in granule cells
be differentiated into several subtypes (mGluRI-mGluR8) based on
of the internal granular layer of acutely isolated cerebellar slices.
their structure, pharmacological sensitivity and signal transduction
Cerebellar slices were obtained from 20- to 26-day-old rats (Wistar
mechanism (Watkins and Collingridge, 1994; Pin and Duvoisin,
strain, day of birth = postnatal day 1) as reported previously
1995). While N-methyl-o-aspartate (NMDA) and non-NMDA (or
(D’Angelo et al., 1993, 1994, 1995). Rats were anaesthetized with
AMPAkainate) ionotropic receptors mediate phasic transmission at
halothane (Aldrich) before being killed by decapitation. Krebs solution
excitatory synapses, activation of mGluRs regulates synaptic efficacy,
for slice cutting and recovery contained (mh4): NaCl 120, KCI 2,
contributing to the generation of long-term potentiation (LTP) or
MgS04 1.2, NaHC03 26, KH2PO4 1.2, CaClz 2, glucose 11. This
long-term depression of the excitatory postsynaptic currents (EPSCs)
solution was equilibrated with 95% O2 and 5% C 0 2 (pH 7.4). Slices
(Bliss and Collingridge, 1993; Linden and Connor, 1993). Of particular
were maintained at room temperature before being transferred to the
interest are the interaction of mGluRs and NMDA receptors in
recording chamber (1.5 ml) mounted on the microscope stage. The
generating plasticity and the possibility that NMDA receptors thempreparations were superfused at a rate of 2 4 mumin with Krebs
selves are a target of potentiation (O’Connor er al., 1994). We
solution to which 10 pM glycine and 10 pM bicuculline (Sigma) had
investigated the actions of the mGluR agonist 1S,3R-1-aminocyclopenbeen added, and maintained at 30°C with a feed-back Peltier device.
tane-l,3-dicarboxylic acid (t-ACPD) and of the antagonist (+)-0methyl-4-carboxyphenyl-glycine(MCPG) (Watkins and Collingridge,
Data recording and analysis
1994) on NMDA and non-NMDA phasic synaptic transmission in
Recordings were performed in the whole-cell configuration of the
cerebellar granule cells (Garthwaite and Brodbelt, 1989; Silver er al.,
patch clamp (Edwards er al., 1989). Patch pipettes were pulled from
1992; D’Angelo et al., 1993, 1994). Coactivation of mGluRs and
borosilicate glass capillaries (Hingelberg, Malsfeld, Germany) and
NMDA, but not non-NMDA receptors, induced a long-lasting potentihad 7-11 MIR resistance before a seal was formed with a filling
ation in an NMDA current subcomponent and, to a smaller extent,
solution having the following composition (a
):81, NaCl
in the non-NMDA current. In view of the key role played by Nh4DA
4, MgS04 2, CaC120.02, BAPTA 0.1, glucose 10, ATP-Mg 3, HEPES
receptors in regulating granule cell excitability (D’Angelo et al.,
15 (pH was adjusted to 7.2 with CsOH). This solution buffered
1995), this form of potentiation may be important in regulating
intracellular Ca2+at 100 nM, similar to the resting Ca2+concentration
cerebellar information processing (Eccles et al., 1967; Linden and
Connor, 1993).
measured in granule cells (Irving et al., 1992). BAPTA (tetrapotassium
Correspondence to: Egidio D’Angelo, as above
Received 22 September 1995. revised 22 December 1995, accepted I0 January 1996
Potentiation of an NMDA current subcomponent
-70 mV
+60 mV
20 ms
FIG.1. EPSCs before and after t-ACPD potentiation. EPSCs (averages of four
tracings) are shown at the holding membrane potentials of -70 and +60 mV.
Vertical lines indicate the position at which amplitudes of the non-NMDA
(A,) and NMDA currents (AN) were measured.
E l
salt) was obtained from Molecular Probes (Eugene, OR). Recordings
were performed with an Axopatch 200A amplifier (Axon Instruments,
Foster City, CA), and data were converted from analogue to digital
at 20 or 2 kHz to resolve the different time courses of the nonNMDA and NMDA current components respectively.
Electrical stimulation was performed using a bipolar tungsten
electrode (Clark Instruments, Pangbourne, UK) placed over the mossy
fibre bundle. A local flow of solution was delivered over the recording
site through a four-barrel micropipette. The control solution was
perfused before sealing; thereafter different compounds were applied
by switching between the perfusion pathways, as specified for each
experimental protocol. A typical experimental protocol is illustrated
in Figures 1 and 2.
Data were analysed with p-CLAMP software. Data are reported as
mean ? SEM, and statistical comparison of means was made with
paired Student’s r-tests [differences were considered not significant
at P > 0.051.
Time (min)
FIG. 2. Metabotropic EPSC potentiation in a cerebellar granule cell.
Representative EPSC tracings recorded at -70 mV or +60 mV at different
times during the experiment are shown at the top. Relative A, values at -70
mV (open symbols) and A N values at +60 mV (filled symbols) are plotted at
the bottom. Values of AN are reversed and scaled up to demonstrate the similar
time course of potentiation in both EPSC components. In this and the following
figures, (i) time is set to 0 at the beginning of t-ACPD perfusion; (ii) a
horizontal bar represents perfusion, and application of 20 pM r-ACPD is
indicated in black; and (iii) the inset shows superimposed passive current
transients recorded at -70 mV (scale bars 50 PA, 0.5 ms). Here, four transients
are shown in the control 10, 20 and 30 min after r-ACPD perfusion.
Monitoring of recording conditions
A major problem for the kinetic analysis of membrane currents over
extended periods of time is the stability of the whole-cell configuration.
In order to monitor the whole-cell condition, a passive current
transient was elicited by a 10 mV hyperpolarizing voltage step just
before each EPSC and sampled at 20 lcHz (D’Angelo et aZ., 1994).
The constancy of the transient was assumed as a confirmation of
stability, as shown in the insets in Figures 1-4. Assuming that the
granule cell can be treated as a lumped somatodendritic compartment,
these transients were also used to measure the passive properties of
the electrode/cell system (Silver et al., 1992; D’Angelo et al., 1993,
1995). In the 42 cells covered in this study, at -70 mV membrane
resistance was R, = 3.7 ? 0.3 GSl and membrane capacitance was
C , = 3.4 ? 0.2 pF, reproducing typical values of cerebellar granule
cells, and series resistance was R, = 15.5 % 0.9 MQ. From similar
data, in each individual granule cell the cut-off frequency of the
voltage-clamp system was estimated as fVc = (2x.RSC,)-’. The
reliability of EPSC measurement was then assessed by comparison
offvc with the maximum frequency content of the EPSC, which was
estimated as fEPsc= 0.35/RT1&90(RTlCg0is the rise time between
10 and 90% of peak amplitude at -70 mV). The mean of values
obtained in individual neurons were as follows: control conditions,
fvc = 3.65 TL 0.52 ~ HandfEpsC
= 0.5 5 0.07 ~ H(n
Z = 35); 10
min after the beginning of t-ACPD perfusion, fVc = 3.56 ? 0.45
kHz and fEPsc = 0.6 ? 0.08 kHz (n = 29). indicating that the
voltage-clamp was six times faster than the fastest (non-NMDA)
EPSC component. EPSC recordings were considered in the present
analysis for as long as fVcand fEPscremained stable.
Another problem when modulatory processes are being investigated
is the integrity of intracellular signal transduction pathways. In three
experiments the perforated-patch technique was used (unpublished
results), preventing wash-out of Ca2+ and organic cytoplasmic constituents (Horn and Marty, 1988). In the perforated-patch experiments,
t-ACPD induced long-term EPSC changes similar to those obtained
using the conventional whole-cell recording technique, indicating that
modifications in cytoplasmic composition caused by the whole-cell
pipette solution were not critical. However, the system response was
slower in the perforated-patch than in the conventional whole-cell
recording configuration (fvc = 1.2 -C 0.4 kHz, n = 3). The wholecell recording configuration was subsequently used for detailed
analysis of EPSC changes.
It should also be noted that no run-down of synaptic transmission
has been reported in cerebellar granule cells in control recording
conditions (Fig. 4B in D’Angelo et aZ., 1995).
D-2-amino-5-phosphonovalericacid (APV),6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 7-chlorokynurenic acid (7-Cl-kyn), t-ACPD
and MCPG were obtained from Tocris Cookson (Bristol, UK).
1184 Potentiation of an NMDA current subcomponent
t60 mV
-70 mV
+(AW+ 7kyn)
Tim (min)
Tim (min)
FIG.3. General properties of t-ACPD potentiation. The time courses of EPSC
amplitude changes (A, at -70 mV) are shown at the left (mean t SEM).
Examples of EPSCs (averages of four traces) with the corresponding passive
current transients (insets) are shown at the right. (A) Application of t-ACPD
for 120 s (n = 7). (B) Protracted 1-ACPD application ( n = 5 ) . (C) Coapplication
of f-ACPD and 500 p M MCPG for 120 s in slices which had been preincubated
for at least 1 h with 500 p M MCPG ( n = 4). (D) Application of I-ACPD for
120 s simultaneously with interruption of mossy fibre stimulation (n = 5 ) . as
indicated by a reference line (stim).
The actions of the mGluR agonist r-ACPD on mossy fibre-granule
cell synaptic currents were investigated in 42 granule cells of the
internal granular layer of cerebellar slices in the whole-cell patchclamp recording configuration. The experiments were performed 2026 days after birth, when synaptic properties had developed mature
characteristics (Garthwaite and Brodbelt, 1989; D'Angelo et al., 1993;
Farrant et al., 1994; Monyer et al., 1994). EPSCs were activated by
0.1 Hz mossy fibre electrical stimulation of constant intensity.
Inhibitory synaptic currents were blocked by the GABA-A receptor
antagonist bicuculline (10 pM), and 10 pM glycine was added to
saturate the glycine site on the NMDA receptor.
The EPSCs showed a fast and a slow component (Fig. I ) ,
corresponding to the non-NMDA (AMPAkainate) and NMDA currents identified in previous work (Silver et al., 1992; D'Angelo et al.,
1993). The EPSCs were depressed during application of 20 pM
t-ACPD for 120 s; however, both the NMDA and the non-NMDA
component showed marked enhancement during subsequent washing
(Figs 1 and 2). NMDA and non-NMDA current potentiation followed
a similar time course (Fig. 2).
Amplitude estimates of the two EPSC components were obtained
by measuring the non-NMDA current peak (A,) and the NMDA
current 25 ms after the beginning of the EPSC (AN), as shown in
Figure 1. Although changes in A, and A N were observed at both
positive and negative membrane potentials, for convenience A, was
usually measured at -70 mV and AN at +60 mV (Fig. 2). In all the
reported experiments, the quality and stability of recordings were
FIG.4. Effects of t-ACPD on the NMDA current. Following control EPSC
recordings ( I ) , the NMDA EPSC was isolated by perfusing with 10 p M
CNQX (2) and potentiated following application of 20 p M I-ACPD for 120 s
(3). Finally. the NMDA-EPSC was blocked by 100 p M APV + 50 p M 7-CIkyn (4).( A ) EPSCs (averages of four tracings) in different phases of the
experiment at -70 and +60 mV. The inset shows two passive current transients
superimposed (phases I and 3). (B) Time course of potentiation in the NMDA
current (n = 6, mean t SEM). The plot shows normalized AN values measured
at +60 mV in the isolated NMDA EPSCs (filled circles) and in composite
EPSCs (open symbols).
monitored by activating passive current transients (inset), and by
calculating the cut-off frequency of the voltage-clamp system cfvc)
and the maximum frequency content of the EPSCs (fEpsc; see
Materials and methods).
General properties of t-ACPD potentiation
As reported in Figure 2, following application of t-ACPD for 120 s,
EPSC potentiation developed in 13 of the 14 neurons tested, attaining
its maximum level within 10 min (Fig. 3A). The potentiation then
lasted without any tendency to decrease for at least 45 min, i.e. the
time during which a constant whole-cell current transient could be
maintained in three cells. It should be noted that protracting the
period of t-ACPD perfusion caused an enduring depression in both
the NMDA and the non-NMDA EPSC component (n = 5/5; Fig. 3B).
Application of t-ACPD with the mGluR antagonist MCPG (500
FM) (Watkins and Collingridge, 1994) for 120 s did not induce any
subsequent potentiation (n = 4/4; Fig. 3C), the effect resembling that
first reported at hippocampal synapses (Bashir et al., 1993; O'Connor
et al., 1994). During t-ACPD and MCPG coapplication an EPSC
depression was usually observed, as with t-ACPD alone. In these
experiments, the slices were preincubated for at least 1 h with MCPG
to improve MCPG blockade.
Interestingly, potentiation did not arise when, after having interrupted synaptic stimulation during t-ACPD perfusion, stimulation
was restarted (n = 5/5, Fig. 3D). On restimulation, however, the
EPSCs were already maximally depressed.
In summary, the induction of potentiation depended on specific
MCPG-sensitive metabotropic stimulation and on simultaneous
ionotropic receptor coactivation, while subsequent expression was
independent of the presence of t-ACPD. Depression showed opposite
Potentiation of an NMDA current subcomponent
+(Aw+ W)
FIG. 6. Selective t-ACPD potentiation in a slow NMDA current
subcomponent. The decay phase of NMDA EPSCs at +60 mV (averages
of four traces) have been fitted to the sum of two exponential functions,
which are shown superimposed. Control: q = 75.5 ms. T, = 210.7 ms,
Af/(Af + A,) = 0.86. After r-ACPD potentiation: T~ = 61 ms, 7, = 438.4
ms. Af/(Af + A,) = 0.56. Note the increase in the slow. but not in the
fast, component after I-ACPD potentiation.
Tim (min)
FIG.5. Effects of I-ACPD on the non-NMDA current. The non-NMDA-EPSC
isolated with 100 pM APV + 50 FM 7-CI-kyn ( I ) was depressed during
application of 20 pM t-ACPD for 120 s (2). and no switch to potentiation
was observed during subsequent washing (3). Addition of 10 FM CNQX
blocked the non-NMDA-EPSC (4). (A) EPSCs (averages of four traces) in
different phases of the experiment at -70 mV. The inset shows three passive
current transients superimposed (phases 1-3). ( B ) Time course of changes in
the non-NMDA EPSC components (n = 8, mean f SEM). The plot shows
normalized A, values measured at -70 mV.
TABLE1. Potentiation of the NMDA and non-NMDA current
Composite EPSC
-70 mV
+60 mV
1 1 85
A, (%)
A N (%)
+43.2 2 2.7
+38.1 f 7.7
+81.1 f 9.9
+110.6 % 27
P < 0.01
P < 0.01
+ I 5 8 3 5 39.5
+I66 2 12.6
Amplitude changes induced by I-ACPD in composite EPSCs (n = 6) and in
the pharmacologically isolated NMDA current ( n = 6). Reported values are
the mean 2 SEM for increments in A, and A N relative to control measured
10 min after I-ACPD application and subsequent washing, at -70 and +60
mV. The statistical significance of paired Student’s 1-tests is reported (n.s.,
not significant).
properties, implying the involvement of different receptors and
Selective involvement of the NMDA current during induction
In six experiments continuous perfusion of 10 pM CNQX was used
to block non-NMDA receptors, obtaining the NMDA EPSC in
isolation (Fig. 4). During t-ACPD application the NMDA EPSC was
depressed, and during subsequent washing potentiation was observed
both at -70 and at +60 mV (Fig. 4A). The NMDA current potentiation
(evaluated as AN at -1-60 mV) developed following a similar time
course both in NMDA EPSCs and in composite EPSCs (Fig. 4B).
In eight experiments continuous perfusion of 100 pM APV + 50
pM 7-CI-kyn was used to block the NMDA receptors and obtain the
non-NMDA EPSC in isolation (Fig. 5). Coapplication of the two
drugs has been shown to enhance NMDA receptor blockade in the
presence of 10 pM glycine in this preparation (D’Angelo ef al., 1995).
During r-ACPD application the non-NMDA EPSC was depressed, but
no potentiation was observed during subsequent washing in any of
the eight cells tested (Fig. 5A, B).
The absence of any potentiation after blocking of the NMDA
receptors resembles the effect of suspending stimulation during
t-ACPD application, in striking contrast to the nearly 100% success
obtained both in control conditions and after blocking the nonNMDA receptors. Thus, simultaneous stimulation of metabotropic
and NMDA, but not of non-NMDA receptors, was required to induce
Differential potentiation of the NMDA and non-NMDA current
The analysis of A, and AN values in six cells allowed an evaluation
of r-ACPD actions on the non-NMDA and NMDA components.
As shown in Table I , following r-ACPD potentiation the NMDA
component increased 2-3 times more than the non-NMDA component
at both -70 and +60 mV (the differences were statistically significant,
P < 0.01). Similar amplitude changes occurred in the NMDA current
isolated pharmacologically (Table 1).
On closer inspection, NMDA current decay kinetics proved biphasic
both in composite EPSCs and in the NMDA current in isolation
(Fig. 6; cf. D’Angelo et al., 1994). At +60 mV, a membrane potential
at which the signal-to-noise ratio was favourable and the influence of
the non-NMDA component on NMDA current decay was negligible,
reliable fittings were obtained with the sum of two exponential
functions (with mean time constants q = 41.5 ms and T> = 285.6
ms) in all six EPSC decays examined (Table 2). In potentiated EPSCs,
neither zf nor 2, showed any significant change. Although amplitude
in the faster subcomponent (Af) remained stable, amplitude in the
slower subcomponent (A,) nearly doubled, changing the relative
amplitude ratio Ad(Af + A,) from 0.46 to 0.23 (P < 0.01). Similar
results were obtained in the six cells in which the NMDA current
was isolated pharmacologically (Fig. 6; Table 2).
These results indicate that following potentiation the NMDA current
increased more than the non-NMDA current, and that the NMDA
current increase was mostly related to a slow subcomponent.
Voltage-dependence of NMDA EPSC potentiation
In order to determine whether the NMDA current potentiation involved
a voltage-dependent conductance change, we used AN measurements
at different holding potentials. It should be noted that AN was sensitive
to changes in the slow NMDA current subcomponent at both extremes
of the membrane potential range examined (Fig. 5A and Table 1). In
the control, A N showed a characteristic negative slope in currentvoltage plots at negative membrane potentials (Fig. 7A, open circles).
Following t-ACPD potentiation, A N increased over the whole membrane potential range (Fig. 7A, filled circles), without any noticeable
change in reversal potential. Measurements of A N and a current
reversal potential of +10 mV (Fig. 7A) were then used to calculate
conductance. Normalized conductance plots (Fig. 7B) were fitted to
a Boltzmann equation of the form:
1186 Potentiation of an NMDA current subcomponent
TABLE2. Bi-exponential decay kinetic\ of the NMDA current
Composite EPSC
At (PA)
A, (PA)
+ A,)
41.5 f 6.4
42.1 2 6.8
285.6 2 38.3
262.5 f 25
6.9 f 0.6
47.9 2 1.5
8 2 3.6
25.7 f 9.8
P < 0.02
0.46 t 0.09
0.23 -C 0.08
P < 0.01
44.7 2 9
51.3 f 5.8
274 ? 38.5
317.4 2 63.1
14.7 2 2.8
13.4 2 2.2
5.6 f 3.5
16.8 2 2.5
P < 0.02
0.72 2 0.08
0.44 f 0.05
P < 0.01
Changes in bi-exponential decay induced by r-ACPD in the NMDA current of composite EPSCs (n = 5) and in the NMDA current isolated pharmacologically
( n = 6). Values of ~ f T, ~ Af,
, A, and AJAp + A,) at +60 mV are reported. The statistical significance of paired Student's t-tests is reported (n.s.. not significant).
FIG.7. Voltage-dependence of NMDA-EPSCs in control (open circles) and
after f-ACPD potentiation (filled circles). (A) NMDA EPSC amplitude,
measured as AN. at different holding potentials (n = 6, mean f SEM).
(B)NMDA conductance plots normalized to maximum control conductance
obtained from data shown in A. Boltzmann fits gave the following parameters:
= I , g,i, = 0.025, V1/2 = 0.48 mV, K = 15 mV-' (R >
in control g,
0.99). after t-ACPD potentiation g,
= 2.06, g,,
= 0.045, V1/2 = 0.23 mV,
K = 15 mV-I (R > 0.99). (C) NMDA EPSC half-width at different holding
potentials (n = 6, mean f SEM).
Flc. 8. Hypothetical scheme illustrating the possible actions of glutamate and
t-ACPD in the present experiments. Glutamate released during low-frequency
mossy fibre transmission activates postsynaptic NMDA and non-NMDA
receptors, while exogenous t-ACPD stimulates postsynaptic and probably also
presynaptic mGluRs. On the postsynaptic side, coincidence of mGluR and
NMDA stimulation causes long-term enhancement of NMDA and non-NMDA
receptor-mediated transmission, which may involve a convergent action on
intracellular Ca" release or protein kinase C (PKC) activity. The shaded
areas indicate intracellular processes which, as well as presynaptic mCluRs.
have been demonstrated in different studies but remain speculative here. The
scheme is based on data in this work and in ( I ) Kinney and Slater (1993).
Larson-Prior et a/. (1995); (2) Aronica et a/. (1993). Shigemoto ef a/. (1992).
O'Connor et a/.(1995); (3) Irving et a/. (1992); (4) Bliss and Collingridge,
(1993); ( 5 ) Glaum and Miller (1994). Ohishi et d.(1994).
the membrane was depolarized (Fig. 7C, open circles), reflecting the
intrinsic voltage-dependence of NMDA current kinetics (D' Angelo
et al., 1994). Following t-ACPD potentiation, the half-width approximately doubled at all membrane potentials (Fig. 7C, filled circles). It
should be noted that, since the decay rate of neither of the two
NMDA EPSC subcomponents changed appreciably after potentiation,
the half-width increase should reflect the higher slow-to-fast amplitude
ratio between the two NMDA EPSC subcomponents.
These results indicate that the mechanism of the t-ACPD NMDA
EPSC potentiation was not voltage-dependent.
where g,, and gminare the maximum and minimum conductances,
V112is the half-activation potential and K is a slope factor (O'Connor
et aL, 1994). Although both g,,, and gminrevealed the expected twofold increase in conductance, Vl12 and K did not change appreciably
(Fig. 7B).
To evaluate the changes in the time course of the NMDA EPSC
before and after potentiation, we used half-width measures at different
holding potentials. In the control, half-width became greater the more
In this paper we first demonstrate that mGluR stimulation can induce
a long-lasting potentiation of transmission at the mossy fibre-granule
cell synapse of the rat cerebellum. Metabotropic potentiation was
differentially expressed in the non-NMDA current and in a slow
NMDA current subcomponent of the EPSCs, and depended on NMDA
receptor coactivation. A hypothetical scheme illustrating the processes
involved in this form of potentiation is illustrated in Figure 8 and is
discussed below. Changes in the holding current [usually a reduction
Potentiation of an NMDA current subcomponent
at positive membrane potentials (Rossi and D’Angelo, unpublished
observation)] suggested that non-synaptic membrane currents were
also modulated by t-ACPD. The present results, however, do not
provide evidence for slow metabotropic synaptic responses like those
observed in hippocampal CA3 pyramidal neurons (Charpak and
Gahwiler, 1991; Gerber et al., 1993) and in Purkinje neurons
(Batchelor et al., 1995).
General properties of metabotropic LTP
The EPSC potentiation consisted of (i) an induction phase requiring
coactivation of metabotropic and NMDA receptors, and (ii) an
expression phase outlasting the presence of r-ACPD. EPSC potentiation (iii) could be prevented by the mGluR antagonist MCPG.
Moreover, EPSC potentiation was (iv) long-lasting (at least 45 min)
and (v) non-decremental over the recording time. Thus, the t-ACPD
potentiation at the mossy fibre-granule cell synapse may be regarded
as a ‘metabotropic’ form of LTP (Bliss and Collingridge, 1993). Two
properties make the present t-ACPD potentiation more similar to that
observed in dentate granule cells than to that in pyramidal neurons
of the hippocampus. First, the present t-ACPD potentiation had a
rapid onset ( < I 0 min), as in dentate granule cells (O’Connor et al.,
1994), while that in CAI neurons is much slower (>30 min; Bortolotto
and Collingridge, 1993). Secondly, mGluR stimulation was insufficient
by itself to induce potentiation, unlike what occurs in hippocampal
pyramidal cells (Bashir et al., 1993; Bortolotto and Collingridge,
1993), but specific NMDA receptor coactivation was also required,
as recently observed in dentate granule cells (O’Connor et al., 1995).
It is interesting to note that, in the turtle cerebellum, metabotropic
potentiation in the NMDA receptor-dependent response of granule
cells was observed in the continuous presence of t-ACPD (Kinney
and Slater, 1993), a condition which induces depression in our
experiments in the rat. Although there may be a genuine difference
in the mechanism of mGluR potentiation, it should be noted that the
enhancement of transmission in the turtle has been observed indirectly
through the response of Purkinje cells to mossy fibre stimulation, so
that t-ACPD could also change granule cell excitability and hence
the response to mossy fibre stimulation.
The induction phase
During induction, a low-frequency (0.1 Hz) NMDA receptor activation, insufficient by itself to induce any potentiation, played a
permissive role in the metabotropic potentiation of transmission.
Induction may involve the convergence of NMDA receptors and
mGluR actions on a common intracellular mechanism in the postsynaptic element (Otani et al., 1993). Influx of Ca2+ through the
associated channel (Mayer and Westbrook, 1987) is the natural way
of NMDA-receptor coupling to intracellular modulatory systems
(Bliss and Collingridge, 1993). In the experiments reported in Figure
4, the NMDA EPSC charge at -70 mV was 103 5 31 fC (n = 4;
see also D’Angelo et al., 1993). Supposing that three synapses are
active, that just one-tenth of the NMDA channel charge is carried by
Ca” (Mayer and Westbrook, 1987), and that Ca2+ diffuses in a
dendritic terminal volume of 0.2 pm3 (Jakab and Hamory, 1987),
Ca2+ would eventually attain a theoretical free concentration of
80 pM (conversion from charge to moles is calculated using Faraday’s
constant). Thus, although the Ca2’ build-up is usually curtailed by
intracellular Ca2+ buffering, low-frequency synaptic activation of the
NMDA receptors at -70 mV may be sufficient to activate secondary
Ca2+-dependent processes. It is possible, for instance, that Ca2+
influx through the NMDA channels augments the effectiveness of
mGluRs in releasing Ca2+ from intracellular stores, as demonstrated
in cerebellar granule cells in culture (Irving et al., 1992), eventually
causing the cytoplasmic Ca2+ increase necessary for inducing potentiation (Bliss and Collingridge, 1993; Fig. 8). It is also possible that
the inositol triphosphate elevation caused by mGluRl stimulation
(Aronica et al., 1993) converges with Ca2+ elevation in activating
protein kinase C and subsequent potentiation (Bliss and Collingridge,
1993; O’Connor et al., 1995; Fig. 8).
During induction, EPSC depression tended to obscure the acute
r-ACPD enhancement of the NMDA current observed using bath
application of t-ACPD and NMDA in cerebellar granule cells (see
fig. 5 by N. T. Slater in Glaum and Miller, 1994).
The expression phase
During the expression phase, potentiation occurred both in the NMDA
and in the non-NMDA current (Fig. 8). That the NMDA current is a
target for plasticity has been reported before (Berretta et al., 1991;
Asztely et al., 1992; O’Connor et al., 1994; Weisskopf and Nicoll,
1995). In hippocampal granule cells the NMDA current potentiation
is similar to the non-NMDA current potentiation (O’Connor et al.,
1995). Here we demonstrate that, in cerebellar granule cells, NMDA
current potentiation is two to three times as large as non-NMDA
current potentiation, and that the NMDA current increases selectively
in a slow subcomponent.
The greater increase in the NMDA than in the non-NMDA current
and the selective change in an NMDA current subcomponent, although
not excluding presynaptic enhancement in glutamate release, suggest
the involvement of postsynaptic mechanisms of channel modulation
(Bliss and Collingridge, 1993). The enhancement in the slow NMDA
current subcomponent is more likely to involve a change in NMDA
channel deactivation (Jonas and Spruston, 1994) than a reduction in
Mg2+ block (Ben-Ari et al., 1992; Chen and Huang, 1992), since
potentiation was voltage-independent. The relationship of this finding
with the expression of two NMDA receptor channel subunits, NR2A and NR-2C, in mature cerebellar granule cells (Monyer et al.,
1994; see also Farrant et al., 1994) remains to be established.
Relationship to glutamate receptor subtypes
The observation that 500 pM MCPG prevented any further t-ACPD
potentiation suggests that either mGluRl or mGluR2, but not mGluR4
(Watkins and Collingridge, 1994; Pin and Duvoisin, 1995), induces
the EPSC potentiation. In granule cells in culture mGluRl stimulation
determines activation of the intracellular phospholipase C/inositol
triphosphate cascade and Ca2+ elevation (Aronica et al., 1993).
Stimulation of postsynaptic MCPG-sensitive mGluRs (Bashir et al.,
1993) linked to the phospholipase Chnositol triphosphate cascade is
indeed considered an important step in the induction of potentiation
in different preparations (Pin and Duvoisin, 1995). Thus, although
evidence for mGluR, in siru does not seem definitive at present
(Shigemoto et al., 1992). mGluRl is a likely candidate for the
induction of potentiation observed here (Fig. 8). No evidence for
mGluR2/mGluR~has been reported in granule cells either in in situ
hybridization or immunolabelling studies (Ohishi et al., 1994).
Differently from potentiation, EPSC depression during t-ACPD
perfusion was not prevented by 500 pM MCPG and should act,
therefore, through receptors other than mGluRl or mGluR2. Candidates
for EPSC depression are receptors like m G l u h , which, as at other
central synapses (Glaum and Miller, 1994), may cause presynaptic
inhibition of mossy fibre glutamate release (Fig. 8). No direct evidence
is currently available for any mGluRs in the mossy fibre, although
neurons potentially projecting to the cerebellum contain the mGlu&
mRNA transcript (Glaum and Miller, 1994). The m G l u h mRNA
1188 Potentiation of an NMDA current subcomponent
transcript is also present in cerebellar granule cells (Tanabe et a/..
1993), in which mGluR4 are known to mediate presynaptic inhibition
of transmission at the parallel fibre-Purkinje cell synapse (LarsonPrior et al., 1995).
Conclusions and functional implications
Potentiation in the slow NMDA current subcomponent seems particularly suited to enhance the input-output function of the granule cell
since, at high frequency, the NMDA current sustains temporal
summation (D’Angelo et al., 1995). Potentiation in the non-NMDA
current, by enhancing synaptic depolarization, favours the voltagedependent NMDA channel response (Fig. 7; D’Angelo er a / . , 1995).
Moreover, since the NMDA receptor is an effector as well as a target
of potentiation, the potentiated synapse may become highly susceptible
to further plastic changes (Bortolotto et al., 1994). A similar mechanism may be called into play during high-frequency transmission, as
reported at hippocampal synapses (Bashir et al., 1993; Bortolotto
et al., 1994; O’Connor er al., 1994, 1995). At present, the relationship
between this mechanism and the long-term enhancement in synaptic
transmission caused by high-frequency mossy fibre stimulation
(D’Angelo et al., 1995) remains to be established. There is evidence
that potentiation at the mossy fibre-granule cell synapse is retransmitted to Purkinje cells through increased granule cell excitation
(Kinney and Slater, 1993; Larson-Prior et al., 1995), suggesting an
important role for the present form of potentiation in regulating
information processing in the cerebellar cortex (Eccles et a/., 1967;
Linden and Connor, 1993).
This work was supported by grants from the Minister0 della Ricerca Scientifica
e Tecnologica and Consorzio Interuniversitario Nazionale di Fisica della
Materia, Italy.
NMDA current amplitude
non-NMDA current amplitude
excitatory postsynaptic current
( + )-O-methyl-4-carboxyphenyl-glycine
1 S.3R- 1 aninocyclopentane- I ,3-dicarboxylic acid
Aronica, E., Condorelli, D. F., Nicoletti, F., Dell’Albani, P., Amico, C. and
Balazs, R. ( 1993) Metabotropic glutamate receptors in cultured cerebellar
granule cells: developmental profile. J. Neurochein., 60, 559-565.
Asztely. F., Wigstrom. H. and Gustafsson, B. (1992) The relative contribution
of NMDA receptor channels in the expression of long-term potentiation in
the hippocampal CAI region. Eur: J. Neurosci., 4, 6 8 1 4 9 0 .
Bashir, Z. I., Bortolotto, Z. A., Davies, C. H., Berretta, N., Irving. A. J., Seal,
A. J., Henley, J. M., Jane, D. E., Watkins. J. C. and Collingridge, G. L.
(1993) Induction of LTP in the hippocampus needs synaptic activation of
glutamate metabotropic receptors. Narure, 363, 347-350.
Batchelor, A. M., Madge, D. J. and Garthwaite, J. (1995) Synaptic activation
of metabotropic glutamate receptors in the parallel fibre-Purkinje cell
pathway in rat cerebellar slices. Neuroscience, 63, 91 1-915.
Ben-An, Y.,Aniksztejn, L. and Bregestovski, P. (1992) Protein kinase C
modulation of NMDA currents: an important link for LTP induction. Trends
Neurosci., 15, 333-339.
Berretta, N., Berton, E, Bianchi, R., Brunelli, M., Capogna, M. and
Francesconi, W. (1991) Long-term potentiation of NMDA receptor-mediated
EPSP in guinea-pig hippocampal slices. Eur: J. Neurosci., 3, 850-854.
Bliss, T. V. P. and Collingridge, G. L. (1993) A synaptic model of memory:
long-term potentiation in the hippocampus. Nature. 361, 3 1-39.
Bortolotto, Z. A. and Collingridge. G. L. (1993) Characterisation of LTP
induced by the activation of glutamate metahotropic receptors in area CAI
of the hippocampus. Neuropkar~naColog?: 32. 1-9.
Bortolotto, Z. A.. Bashir, Z. 1.. Davies, C. H. and Collingridge. G. L. ( 1994)
A molecular switch activated by metabotropic glutamate receptors regulates
induction of long-term potentiation. Nature. 368. 740-743.
Charpak. S. and Gahwiler. B. H. (1991) Glutamate mediates a slow synaptic
response in hippocampal slice cultures. Proc. R. SOC. Lond. B. 243, 22 1-226.
Chen. L. and Huang, L.-Y. M. (1992) Protein kinase C reduces MgZt block
of NMDA-receptor channels as a mechanism of modulation. Nutirre, 356,
52 1-5 23.
D’Angelo. E.. Rossi, P. and Taglietti. V. (1993) Different proportions of
N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor currents at the
mossy fibre-granule cell synapse of developing rat cerebellum.
Neuroscience. 53, 12 I -I 30.
D’Angelo. E.. Rossi, P. and Taglietti, V. (1994) Voltage-dependent kinetics of
N-methyl-D-aspartate synaptic currents in rat cerebellar granule cells. Eur:
J . Neurosci.. 6, 640-645.
D’Angelo, E.. De Filippi. G., Rossi, P. and Taglietti. V. (1995) Synaptic
excitation of individual rat cerebellar granule cells in siru: evidence for the
role of NMDA receptors. J. Physiol. (Lond.). 484. 3 9 7 4 1 3.
Eccles, J. C.. Ito, M. and Szentagothai, J. (1967) The Cerehellutn (1.7 (I
Neumnal Machine. Springer Verlag, Berlin.
Edwards, F. A.. Konnerth. A.. Sakmann. B. and Takahashi, T. A. (1989) A
thin slice preparation for patch-clamp recordings from neurones of the
mammalian central nervous system. Pfiigers Arch.. 414. 600-612.
Farrant, M., Feldmeyer, D., Takahashi, T. and Cull-Candy. S. G. (1994)
NMDA-receptor channel diversity in the developing cerebellum. Nature,
368, 335-339.
Garthwaite, J. and Brodbelt, A. R. (1989) Synaptic activation of N-methyl-oaspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway
in adult and immature rat cerebellar slices. Neuroscience, 29, 401-412.
Gerber, U.. Luthi. A. and Gahwiler. B. H. (1993) Inhibition of a slow synaptic
response by a metabotropic glutamate receptor antagonist in hippocampal
CA3 pyramidal cells. Proc. R. Soc. Lund. B, 254. 169-172.
Glaum. S. R. and Miller, R. J. (1994) Acute regulation of synaptic transmission
by metabotropic glutamate receptors. In Conn, P. J. and Patel. J. (eds).
The Metabotropic Glutamate Receptors. Humana Press, Totowa. NJ. pp.
147- I 72.
Hom. R. and Marty, A. (1988) Muscarinic activation of ionic currents
measured by a new whole-cell recording method. J . Gem Ph\siol.. 92.
145-1 59.
Irving, A. J., Collingridge, G. L. and Schofield, J. G. (1992) L-Glutamate and
acetylcholine mobilise Ca” from the same intracellular pool in cerebellar
granule cells using transduction mechanisms with different Ca”
sensitivities. Cell Calcium, 13, 293-301.
Jakab, R. L. and Hamori. J. (1987) Quantitative morphology and synaptology
of cerebellar glomeruli in the rat. Anor. Embyol., 179, 81-88.
Jonas. P. and Spruston, N. (1994) Mechanisms shaping glutamate-mediated
excitatory postsynaptic currents in the CNS. Curr: Opin. Neurohiol.. 4.
Kinney. G. A. and Slater, N. T. (1993) Potentiation of NMDA receptormediated transmission in turtle cerebellar granule cells by activation of
metabotropic glutamate receptors. J. Neurophysiol.. 69. 585-594.
Larson-Prior, L. J., Morrison, P. D.. Bushey. R. M. and Slater, N. T. (1995)
Frequency dependent activation of a slow N-methyl-D-aspartate-dependent
excitatory postsynaptic potential in turtle cerebellum by mossy fibre
afferents. Neuroscience. 64.8 6 8 7 9 .
Linden, D. J. and Connor. J. A . (1993) Cellular mechanisms of long-term
depression in the cerebellum. Cum Opin. Neirrohiol., 3. 401406.
Mayer. M. L. and Westbrook, G. L. (1987) Permeation and block of N-methylo-aspartate acid receptor channels by divalent cations in mouse cultured
central neurons. J. Physiol. (Lond.). 394. 501-527.
Monyer, H.. Bumashev. N., Laurie, D. J.. Sakmann, B. and Seeburg. P. H.
(1994) Developmental and regional expression in the rat brain and functional
properties of four NMDA receptors. Neuron, 12. 529-540.
O’Connor. J. J.. Rowan, M. J. and Anwyl. R. ( 1994). Long-lasting enhancement
of NMDA receptoi-mediated synaptic transmission by metabotropic
glutamate receptor activation. Nature. 367, 557-559.
O’Connor, J. J., Rowan, M. J. and Anwyl, R. (1995). Tetanically induced LTP
involves a similar increase in the AMPA and NMDA receptor components
of the excitatory postsynaptic current: investigations of the involvement of
mGlu receptors. J. Neurosci.. 15. 2013-2020.
Potentiation of an NMDA current subcomponent
Ohishi, H., Ogawa-Meguro, R., Shigemoto, R., Kaneko, T., Nakanishi, S. and
Mizuno, N. ( 1994) Immunohistochemical localization of metabotropic
glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex. Neuron,
13, 55-66,
Otani, S.. Ben-Ari, Y. and Roisin-Lallemand, M.-P. ( 1993) Metabotropic
receptor stimulation coupled to weak tetanus leads to long-term potentiation
and a rapid elevation of cytosolic protein kinase C activity. Brain Res.,
613, 1-9.
Pin, J.-P. and Duvoisin, R. (1995) The metabotropic glutamate receptors:
structure and functions. Neuropharmacologv, 34, 1-26.
Shigemoto, R.. Nakanishi, S. and Mizuno, N. (1992) Distribution of the
mRNA for a metabotropic glutamate receptor (mGluRI) in the central
nervous system: an in situ hybridization study in adult and developing rat.
J. Comp. Neurol., 322, 121-135.
Silver, R. A,, Traynelis, S. F. and Cull-Candy, S. G. (1992) Rapid time-course
miniature and evoked excitatory currents at cerebellar synapses in situ.
Nature, 355, 163-166.
Tanabe, Y., Nomura, A,, Masu, M., Shigemoto, R.. Mizuno, N. and
Nakanishi, S. ( 1993) Signal transduction, pharmacological properties, and
expression patterns of two rat metabotropic glutamate receptors, mGluR3
and mGluR4. J. Neurosci., 13, 1372-1378.
Watkins, J. and Collingridge, G. ( 1994). Phenylglycine derivatives as
antagonists of metabotropic glutamate receptors. Trends Pharmacol. Sci.,
15, 333-342.
Weisskopf, M. G. and Nicoll, R. A. (1995). Presynaptic changes during mossy
fibre LTP revealed by NMDA receptor-mediated synaptic responses. Nature,
376, 25C259.
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