1Department of Molecular Physiology and Biophysics and 2Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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ABSTRACT |
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Tang, Ka-Choi and David M. Lovinger. Role of Pertussis Toxin-Sensitive G-Proteins in Synaptic Transmission and Plasticity at Corticostriatal Synapses. J. Neurophysiol. 83: 60-69, 2000. The role of pertussis toxin (PTX)-sensitive G-proteins in corticostriatal synaptic transmission and long-term synaptic depression (LTD) was examined using extracellular field potential and whole cell voltage-clamp recordings in striatal slices. High-frequency stimulation (HFS) produced LTD, defined as long-lasting decreases both in synaptically driven population spikes (PSs) measured with field potential recording and in excitatory postsynaptic currents (EPSCs) measured with whole cell recording. Striatal LTD could not be induced in slices obtained from rats that had received a unilateral intrastriatal injection of PTX. However, LTD could be induced in slices obtained from paired control slices. Furthermore, striatal LTD was prevented by pretreatment with N-ethylmaleimide (NEM), another compound that disrupts the function of PTX-sensitive G-proteins. NEM, itself, also potentiated PS and EPSC amplitudes. In addition, NEM increased the frequency and amplitude of both spontaneous and miniature EPSCs and decreased the paired-pulse facilitation ratio, suggesting that it may act on both pre- and postsynaptic sites. The findings suggest that PTX-sensitive G-proteins have multiple roles at corticostriatal synapses, including regulation of synaptic transmission at both pre- and postsynaptic sites, and a key role in striatal LTD.
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INTRODUCTION |
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The striatum is an important brain region for
regulation of body movement as well as certain cognitive functions.
Striatal neurons are strongly activated by afferents from the cortex
that use glutamate as their neurotransmitter (Fonnum et al.
1981; Girault et al. 1986
; Reubi and
Cuenod 1979
). High-frequency activation of these glutamatergic
afferents induces a long-term depression (LTD) of synaptic transmission
at corticostriatal synapses, both in vivo (Garcia-Munoz et al.
1992
,1996
; Wickens and Reynolds 1997
) and in
vitro (Calabresi et al. 1992
, 1994
; Choi and
Lovinger 1997a
,b
; Lovinger et al. 1993
;
Walsh 1993
). Striatal LTD has been proposed to be a
cellular mechanism underlying motor learning and memory. In a recent
study (Choi and Lovinger 1997b
), evidence for a role of
LTD in the development of corticostriatal synapses was presented.
The induction of LTD is blocked by L-type calcium channel blockers and
intracellular dialysis of calcium chelators (Calabresi et al.
1992, 1994
; Choi and Lovinger 1997b
). These
findings suggest that LTD induction after high-frequency synaptic
activation involves postsynaptic membrane depolarization and increases
in postsynaptic calcium concentration brought about by L-type calcium
channel activation. The maintenance of LTD involves a decrease in the probability of neurotransmitter release at corticostriatal synapses (Choi and Lovinger 1997a
,b
).
At least two types of G-protein-coupled receptors have been implicated
in striatal LTD, dopamine receptors and metabotropic glutamate
receptors (Calabresi et al. 1992). In addition, a number of G-protein-coupled receptors have been shown to modulate
transmission at corticostriatal synapses. Many of these receptors may
be linked to pertussis toxin (PTX)-sensitive G-proteins. Thus, we
sought to better understand the role of PTX-sensitive
G-protein-coupled receptors in synaptic transmission and plasticity at
corticostriatal synapses. To this end we examined the effects on
corticostriatal synaptic transmission and LTD of PTX and
N-ethylmaleimide (NEM), two agents that inhibit the function
of these G-proteins. Some of these data have appeared in a recent
abstract (Tang and Lovinger 1998
).
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METHODS |
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Acute brain slices were prepared from 14-28-day-old rats. Rats were killed by decapitation, and the brains were quickly removed and placed in ice-cold, modified artificial cerebrospinal fluid (aCSF) containing (in mM) 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. Modified aCSF was adjusted to pH 7.4 by bubbling with 95% O2-5% CO2. Coronal sections (400 µm in thickness) were cut in ice-cold modified aCSF using a manual vibroslice (World Precision Instruments, New Haven, CT). Slices were then transferred to a nylon net submerged in normal aCSF containing (in mM) 124 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. Normal aCSF was oxygenated and maintained at pH 7.4 by bubbling with 95% O2-5% CO2 at room temperature (22-24°C). After incubation for 1 hour, a hemislice containing the cortex and striatum at the level of the head of the caudate was completely submerged in a Plexiglas recording chamber and continuously superfused with normal aCSF at a flow rate of 2-3 ml/min. Normal aCSF or drugs in aCSF were delivered to the recording chamber by superfusion driven by gravity flow. The temperature of the bath solution was kept at 32-35°C and stable within ±1oC in a given experiment.
Twenty-five-day-old rats were used in the PTX injection experiment.
After rats were anesthetized with ketamine (80 mg/kg, ip), they were
fixed in a stereotaxic instrument (Kopf Instruments, Tujunga, CA). An
incision was made in the skin on the head using a scalpel to expose the
skull. A small hole was drilled in the skull at coordinates AP1.5 mm
anterior to bregma, L2.5 mm from the midline. The position of the nose
clamp was 2.0 mm. PTX (1 µg) was dissolved in 2 ml of aCSF.
Intrastriatal injections were performed stereotaxically by lowering a
Hamilton syringe filled with PTX to a depth of 3.5 mm below the skull.
PTX was injected at a rate of 1 µl/min. After completion of the
injection, the syringe was left in place for 2 min and then slowly
removed. Sham-lesioned rats were injected with 2 µl of vehicle (aCSF)
alone at the same coordinates. One or 2 days later, rats were killed
for electrophysiological studies. The injection tract was clearly
visible during slice preparation. Only those striatal slices within 400 µm (rostral to caudal) of the injection tract were used for
recording. We were able to obtain good quality field potential
recordings from slices containing the injection site in both PTX- and
vehicle-treated brains, indicating that injection itself did not damage
the neurons or synaptic connections.
Extracellular field potential recordings were performed to record the
PSs evoked by afferent stimulation. Eighteen- to 28-day-old rats were
used in this experiment. Recordings were performed in the lateral half
of the dorsal striatum. Recordings were obtained using pipettes pulled
on a Flaming-Brown micropipette puller (Sutter Instrument, Novato, CA).
Pipettes with resistances <1 M when filled with 0.9% sodium
chloride were used. To evoke the PS, stimuli were delivered by an S88
stimulator (Grass Instruments, Quincy, MA) and a PSIU6 optical
isolation unit (Grass) every 20 s through bipolar twisted tungsten
electrodes placed in the white matter dorsal to the striatum. The
position of the recording electrode (1-2 mm ventral to the stimulating
electrode) was optimized by recording responses to single stimuli, and
the electrode was set at the depth where the maximal PS amplitude was
observed. Stimulus intensity was then reduced to evoke a PS with an
amplitude half the maximum that could be evoked. Once a PS of
half-maximal amplitude triggered by 0.05-Hz stimuli had been stably
maintained for 10 to 15 min, LTD was induced by high-frequency
stimulation (HFS) consisting of four 100 Hz trains of 1 s duration
delivered at a frequency of one train every 10 seconds. During HFS, the
stimulus intensity was increased to the level producing the maximal
amplitude PS. Signals were amplified 1,000 times using a differential
AC amplifier (A-M Systems, Olympia, WA), high-pass filtered at 10 Hz
and low-pass filtered at 1-5 kHz. Amplified signals were digitized at
20 kHz using a TL-1-125 interface (Axon Instruments, Foster City, CA)
and stored on a Dell Dimension (Dell, Austin, TX) Pentium microcomputer
using commercially available software (pClamp5.5, Axon).
Whole cell voltage-clamp recordings were performed to record the
stimulus-evoked excitatory postsynaptic currents (EPSCs) in the lateral
portion of the dorsal striatum. Fourteen- to 21-day-old rats were used
in this experiment. Tight-seal whole cell recordings were made using an
Axopatch 1-D amplifier (Axon). Recordings were obtained using pipettes
pulled on a Flaming-Brown micropipette puller (Sutter). Pipettes had
resistances ranging from 3 to 5 M when filled with an internal
solution containing (in mM) 120 CsMeSO3, 5 NaCl,
10 tetraethylammonium chloride, 10 HEPES, 3-5 QX-314
(Br2+ salt), 1.1 EGTA, 4 ATP
(Mg2+ salt), and 0.3 GTP
(Na+ salt), pH adjusted to 7.2 with CsOH,
osmolarity adjusted to 297-300 mOsm with sucrose. Recordings from
medium-sized neurons within three or four layers below the surface of
slices were made under differential interference contrast
(DIC)-enhanced visual guidance. Neurons were voltage-clamped at
60 or
70 mV during recording periods before and after application of HFS.
Stimuli were delivered every 20 s through bipolar twisted tungsten
electrodes placed in the white matter dorsal to the striatum, as
described. The series resistance, which was not compensated and was
typically between 5 and 10 M
, was monitored continuously. Once a
stable recording with an EPSC of 100-300 pA amplitude triggered by
0.05 Hz stimuli had been maintained for 10 to 15 min, LTD was induced by pairing HFS, consisting of four 100-Hz trains of 1 s duration delivered at a frequency of one train every 10 seconds, with
simultaneous 1 s depolarization of the postsynaptic neuron to
10
or 0 mV. Signals were filtered at 5 kHz, digitized at
20 kHz using a
DigiData 1200 interface (Axon) and stored on a Dell Dimension (Dell)
Pentium microcomputer using commercially available software (pClamp
6.0, Axon).
The PS and EPSC amplitudes were analyzed using peak-detection software provided in pClamp. These data were simply obtained by subtracting the peak values from the preresponse baseline values. For this measurement, a minimum was measured within a time window bounded by the increasing and decreasing phases of the synaptic responses. The other two cursors were placed at time points just before the occurrence of synaptic responses to obtain the baseline values. The amplitudes of synaptic responses were calculated by subtracting the peak values from the baseline values. Changes in PS and EPSC amplitudes during a time window 20-30 min after HFS or drug treatment are expressed as the percentage of the baseline response just before treatment. The representative PS and EPSC waveforms shown in the text are the average of 15-30 individual responses in a given recording. LTD was defined as a decrease in response amplitude (more than 20% below the baseline response amplitude) lasting at least 20 min after HFS. The magnitude of LTD was measured during a 10-min time window 20-30 min after the cessation of HFS. Paired-pulse facilitation (PPF) was elicited by paired stimuli with an interstimulus interval of 50 ms. Responses used for the PPF ratio were calculated as the ratio of the amplitude of the second EPSC to that of the first EPSC. All values are averaged from data obtained over 5-10 min and presented as mean ± SE. The statistical significance of changes in synaptic responses was measured using a two-tailed Student's t-test. The statistical criterion for significance was P < 0.05.
PTX and NEM were purchased from Sigma. TTX was purchased from Alomone Labs and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)was purchased from RBI.
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RESULTS |
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Effect of inhibition of PTX-sensitive G-proteins on LTD and synaptic transmission
Figure 1A illustrates that LTD could not be induced in eight of eight PTX-treated hemislices 24-48 h after PTX injection (PS = 123.9 ± 10.6% of baseline, P > 0.05, paired t-test, n = 8). In four of the eight hemislices, PS amplitudes were not significantly changed (<20% change relative to the baseline amplitude, P > 0.1, paired t-test, n = 4) after HFS, whereas in the remaining four hemislices, PS amplitudes were significantly potentiated (PS = 156.3 ± 11.6% of baseline, P < 0.01, paired t-test, n = 4). In these experiments, we used the opposite hemislices from the same animal as one control to minimize variability among animals or because of the anterior-posterior position of the slice. In opposite hemisphere control slices, LTD was induced in six of eight preparations (PS = 48.6 ± 9.8% of baseline, P < 0.01, paired t-test, n = 6). In one of the remaining two control hemislices, LTD was not induced after HFS. The PS amplitude was too small (maximal amplitude <0.2 mV) in the other control hemislice, and it was discarded.
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Parallel experiments were performed in vehicle-injected animals of the same ages to ensure that the blockade of LTD induction was due to the action of PTX. Vehicle (2 µl) was unilaterally injected into striatum. In these vehicle-injected animals, LTD could be induced in either vehicle-injected (PS = 44.3 ± 8.9% of baseline, P < 0.01, paired t-test, n = 5) or noninjected (PS = 43.6 ± 8.4% of baseline, P < 0.01, paired t-test, n = 5) hemislices (Fig. 1B). The maximal PS amplitude recorded from PTX-treated hemislices was significantly smaller than that obtained in control hemislices (PS amplitude from PTX-treated hemislices: 0.55 ± 0.1 mV, n = 6; control hemislices: 0.89 ± 0.2 mV, P < 0.01, paired t-test, n = 6). The magnitude of afferent stimulation needed to elicit a maximal amplitude PS did not differ between the PTX-exposed and control slices. No difference in PS amplitude between control and injected hemislices was observed in tissue from vehicle-injected animals. This suggests that prolonged PTX exposure decreases synaptic responses. In a subset of hemislices from PTX-treated animals we observed PS amplitudes that were within the range of those observed in control slices. These PTX-treated slices did not exhibit LTD after HFS. Thus, we do not believe that the absence of LTD induction in PTX-treated slices was related to the smaller response amplitude after PTX exposure.
We attempted to examine PTX effects using whole cell recording. Three animals were unilaterally injected with 1 µg of PTX in striatum, and whole cell recordings were performed 24 h later. However, the amplitude of evoked EPSCs was generally quite small (maximal amplitude <50 pA) in these PTX-injected animals, and thus we were unable to obtain accurate measures of synaptic plasticity in these experiments. The current levels at the holding potential did not differ in these neurons relative to neurons that were not exposed to PTX, indicating that the PTX-treated neurons were healthy and had input resistances similar to the control cells.
Effect of NEM on LTD and synaptic transmission
We also used NEM to block PTX-sensitive G-protein action in acute
slice experiments. NEM is a sulfhydryl alkylating agent that can
selectively inhibit PTX-sensitive G-protein-mediated effects in
central (Morishita et al. 1997) and peripheral
(Shapiro et al. 1994
) mammalian neurons, invertebrate
neurons (Fryer 1992
), and HEK 293 cells (McCool
et al. 1996
). The advantage of using NEM was that it allowed us
to examine synaptic transmission before and after inhibition of
PTX-sensitive G-proteins within a given recording. The main concern
with the use of NEM was drug specificity, because NEM has been reported
to inhibit a number of processes, such as membrane fusion reactions, in
the mM concentration range (Lledo et al. 1998
;
Macaulay and Forbes 1996
; Meffert et al.
1996
). In the present study, a 200 µM concentration of NEM
was used, which is in the range of concentrations previously shown to
block PTX-sensitive G-protein effects in slice preparations
(Morishita et al. 1997
). We examined the ability of NEM
to block inhibition of synaptic transmission produced by bath
application of 100 micromolar adenosine as a positive control for NEM
blockade of a response mediated by a PTX-sensitive G-protein
(Munshi et al. 1991
; Scholz and Miller
1992
). In two slices we observed that adenosine inhibited synaptic transmission in the absence of NEM (PSs decreased to 30.4% of
control in the presence of adenosine), but not in the presence of NEM
(PS = 98.2% of control; data not shown).
Induction of LTD was prevented in slices treated with NEM (200 µM) before and during HFS, and in fact the PS amplitude was larger after HFS in these animals (PS = 144.8 ± 12.2% of baseline, P < 0.05, paired t-test, n = 5; Fig. 2B). In contrast, LTD (PS = 55.0 ± 3.3% of baseline, n = 5; Fig. 2A) was induced in paired control slices that did not receive NEM treatment. The data shown in Fig. 2 were obtained using extracellular field potential recording. Similar results were obtained in experiments using whole cell voltage-clamp recording (Fig. 3). The induction of LTD was blocked in slices treated with NEM before and during HFS, and a significant potentiation in EPSC amplitude was observed after HFS in these slices (EPSC = 150.3 ± 13.2% of baseline, P < 0.01, paired t-test, n = 5; Fig. 3B). On the other hand, LTD (EPSC = 68.0 ± 3.2% of baseline, n = 5) was inducible in control slices (Fig. 3A).
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Application of NEM 30 min after LTD induction increased synaptic response amplitude to the point that responses were potentiated relative to pre-HFS baseline values. This effect of NEM was observed in both field potential (PS = 153.7 ± 14.8% of baseline, P < 0.01, paired t-test, n = 5; Fig. 2C) and whole cell (EPSC = 118.4 ± 9.9% of baseline, P > 0.05, paired t-test, n = 4; Fig. 3C) recordings. Interestingly, we also found that treatment with NEM alone potentiated the amplitudes of the PS (PS = 146.8 ± 13.4% of baseline, P < 0.01, paired t-test, n = 5; Fig. 2 D) in field potential recordings and the EPSC (EPSC = 131.2 ± 17.0% of baseline, P < 0.05, paired t-test, n = 5; Fig. 3D) in whole cell recordings. Application of NEM did not alter the baseline current amplitude at the holding potential in the whole cell voltage-clamp experiments. Thus, it is unlikely that NEM effects on synaptic transmission are due to alteration of the passive membrane properties of the postsynaptic neuron.
In past studies we observed that blockade of adenosine A1
receptors potentiates synaptic transmission in striatal slices
(Lovinger and Choi 1995). NEM may potentiate
transmission solely by preventing synaptic depression produced by
endogenous adenosine acting on A1 receptors. We examined this
possibility by first applying the A1 receptor antagonist DPCPX
(1 µM) to slices during field potential recording and then
determining whether NEM produces potentiation of the PS response after
stable potentiation by DPCPX. In two slices treated in this manner, we
still observed robust potentiation by 200 µM NEM after DPCPX
treatment (PS amplitude increased by 63% after NEM exposure), even
though DPCPX produced an increase in PS amplitude of 15%. This finding
indicates that the potentiation by NEM is not due solely to prevention
of the actions of endogenous adenosine acting on A1 receptors.
We compared the magnitude of the increases in PS and EPSC amplitude after NEM alone and under conditions in which NEM was applied before and during HFS. There was no difference in potentiation by NEM under these two conditions (unpaired t-test, P > 0.1 for both PS and EPSC measures).
Because NEM alone potentiated synaptic transmission, it was difficult to determine whether the blockade of LTD induction resulted from the slow development of the NEM facilitatory effect overriding LTD expression or from NEM inhibition of specific mechanisms of LTD induction. To address this issue, we examined whether LTD could be induced by HFS applied in a state of stable potentiation after NEM exposure. We consistently observed that NEM alone potentiated PS amplitudes. A stable potentiated PS was observed ~45 min after NEM application (average potentiated PS = 172.8 ± 8.8% of baseline, P < 0.001, paired t-test, n = 4) as shown in Fig. 4, A and B. In two of these slices, responses were recorded for 1 h after NEM exposure before delivery of HFS. Application of HFS during this stable potentiation by NEM could not induce LTD (Fig. 4, A and B). We also performed an experiment in which the stimulus amplitude was reduced after potentiation by NEM to elicit a PS with an amplitude similar to that recorded before NEM exposure. HFS was then delivered at the original stimulus intensity and the responses after HFS were recorded at the lower stimulus intensity. No consistent evidence of LTD was observed after NEM exposure using this paradigm (percent of pre-HFS PS amplitude = 98%, n = 2 slices).
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Mechanism(s) of NEM effects on transmission: presynaptic versus postsynaptic actions
Next, we assessed the mechanism by which NEM potentiates synaptic transmission using analysis of spontaneous EPSCs, miniature mEPSCs (mEPSCs) measured in the presence of the sodium channel blocker TTX, and measurement of paired-pulse facilitation (PPF). Figure 5A shows that NEM alone (200 µM) increased the frequency of spontaneous EPSCs (before NEM: 1.8 ± 0.6 Hz; after NEM: 42.4 ± 7.5 Hz, P < 0.01, paired t-test, n = 5). This effect could be due to NEM actions on either axons or axonal terminals of the presynaptic neuron. To investigate, we examined NEM's effects on mEPSCs by applying TTX (200 nM), which blocked evoked action potentials before NEM treatment and determined whether the change in frequency persisted when mEPSCs were isolated in this manner. Figure 5B shows that the NEM-induced increase in spontaneous EPSC frequency persisted even in the presence of TTX (200 nM; before TTX: 1.3 ± 0.6 Hz; after TTX alone: 1.0 ± 0.4 Hz, after TTX + NEM: 34.2 ± 9.4 Hz, n = 4). The spontaneous EPSC frequency was significantly increased in TTX + NEM relative to the either the control (before TTX) or TTX alone (P < 0.01, paired t-test, n = 4). We also examined the effect of TTX exposure after NEM application and observed that TTX did not significantly reduce the frequency of spontaneous EPSCs after NEM exposure (data not shown). These findings suggest that all the spontaneous EPSCs we recorded in this preparation either in the absence or presence of NEM are action potential-independent mEPSCs and that NEM increases mEPSC frequency.
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Another piece of evidence suggesting that NEM could act on a presynaptic site was provided by the observation that NEM decreased PPF ratio (ratio before NEM: 1.33 ± 0.04; ratio after NEM: 0.9 ± 0.07, P < 0.01, paired t-test, n = 5). The current traces in Fig. 5C show that the amplitude of the EPSC was significantly increased and PPF ratio was decreased after NEM application.
The amplitude of spontaneous EPSCs was also increased by 200 µM NEM (before NEM: 11.4 ± 2.1 pA; after NEM: 17.5 ± 1.6 pA, P < 0.01, paired t-test, n = 5) in conjunction with the increase in frequency as shown in Fig. 5A. This observation suggests that NEM could enhance synaptic transmission partly via postsynaptic actions. Figures 5B and 6A show that application of NEM also increased the amplitude of TTX-insensitive mEPSCs (control before TTX: 9.2 ± 1.3 pA; TTX alone: 8.8 ± 0.9 pA; TTX + NEM: 15.8 ± 1.9 pA). The increase in mEPSC amplitude was significant in the TTX + NEM condition relative to the control (before TTX) or TTX alone (paired t-test, P < 0.05, n = 4) conditions. When TTX was applied after NEM exposure, the amplitude of spontaneous EPSCs was not significantly decreased (paired t-test, P > 0.05, n = 5; inset in Fig. 6A). NEM increased the amplitude of mEPSCs as shown in a cumulative plot (Fig. 6A) in which the amplitude distribution of mEPSCs shifted to the right in the presence of TTX + NEM. Examining the data with a histogram distribution (Fig. 6B) also revealed that the mEPSC amplitude was increased in the presence of NEM. Our findings indicate that the spontaneous EPSCs we observed under all conditions were mEPSCs and that the amplitudes of the mEPSCs were enhanced by NEM.
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We attempted to examine this apparent postsynaptic effect of NEM further by perfusing the postsynaptic neuron with NEM (200 µM) in the patch pipette. In these recordings, we observed an increase in spontaneous EPSC frequency compared with cells without NEM perfusion (basal EPSC frequency: 1.5 ± 1.0 Hz, n = 11) in 4 of 11 cells perfused with NEM (EPSC frequency: 6.3 ± 0.4 Hz, P < 0.0001, unpaired t-test). In these four cells we also observed a slight increase in the spontaneous EPSC amplitude to 11.2 ± 2.4 pA, but this increase was not significant (compared with the predrug control (10.3 ± 1.7 pA, P > 0.1, unpaired t-test, n = 9). A loss of PPF (PPF ratio = 0.89 ± 0.1, P < 0.01, unpaired t-test) in the presence of NEM was also observed in these four cells. In addition, we tried to induce LTD in two of the NEM-perfused cells showing a loss of PPF. LTD could not be induced in these two cells. In the remaining seven cells, no increase in spontaneous EPSC frequency (2.0 ± 0.5 Hz, P > 0.4, unpaired t-test), or amplitude (9.4 ± 0.8 pA, P > 0.2, unpaired t-test), or loss of PPF (PPF ratio = 1.25 ± 0.04, P > 0.2, unpaired t-test) was observed. Moreover, we also tried to induce LTD in two NEM-perfused cells that showed no change in PPF ratio. LTD could be induced in these two cells. Bath application of NEM (200 mM) after a 30-min intracellular perfusion of NEM (200 mM) still increased the spontaneous EPSC frequency (before NEM bath application: 3.5 Hz; after NEM bath application: 48.3 Hz) and amplitude (before NEM bath application: 10.4 pA; after NEM bath application: 16.5 pA) in two NEM-perfused cells. We also perfused four cells with 5 mM NEM to examine whether a stronger effect of NEM could be observed. NEM at the 5 mM concentration produced a larger increase in the spontaneous EPSC frequency (34.2 ± 8.1 Hz, P < 0.001, unpaired t-test) and amplitude (14.5 ± 1.5 pA, P < 0.01, unpaired t-test). After synaptic perfusion 5 mM NEM also changed PPF to paired-pulse depression (0.45 ± 0.04, P < 0.001, unpaired t-test).
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DISCUSSION |
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We observed evidence that PTX-sensitive G-proteins have multiple roles in corticostriatal synaptic transmission and plasticity. G-protein inhibition on an acute time scale (i.e., during recording from a single-slice preparation) produces potentiation of synaptic transmission that appears to involve both pre- and postsynaptic mechanisms. Prolonged inhibition produced by PTX injection appears to decrease synaptic transmission, at least on average. Striatal LTD could not be induced after either PTX injection or acute NEM application. This finding indicates that PTX-sensitive G-proteins play a role in LTD.
Although LTD was not observed in PTX-treated slices, it was observed
under several control conditions, suggesting that a PTX-sensitive G-protein signaling pathway is necessary for LTD induction or expression. Because PTX was injected locally into the striatum, it may
inactivate G-proteins on both presynaptic terminals and postsynaptic
cell bodies. Thus, it is difficult to determine the synaptic location
of the receptors and G-proteins involved in LTD at this time. LTD
induction is known to involve both dopamine receptors and metabotropic
glutamate receptors (Calabresi et al. 1992). There is
ample evidence for both pre- and postsynaptic metabotropic glutamate
receptors (Calabresi et al. 1993
; Lovinger and
McCool 1995
; Testa et al. 1998
) and dopamine D2
receptors in the striatum (Freund et al. 1984
;
Smith and Bolam 1990
). The location of the G-proteins
and G-protein-coupled receptors involved in LTD will have to be
determined in future experiments.
In agreement with the findings obtained in PTX-treated slices, LTD
could not be induced by HFS in the presence of NEM. This observation
supports our conclusion that activation of PTX-sensitive G-proteins is
necessary for LTD induction. However, the mechanism of blockade of LTD
induction by NEM is not entirely certain from this study. The blockade
of PTX-sensitive G-protein activity by PTX or NEM may interfere with
signaling cascades activated by receptors such as the dopamine D2
receptors that are required for LTD induction (Calabresi et al.
1992). Our positive control experiment with adenosine indicates
that NEM can block adenosine A1 receptor-mediated inhibition of
transmission, a process that likely involves a PTX-sensitive G-protein
(Munshi et al. 1991
; Scholz and Miller
1992
). Other mechanisms, such as indirect interactions of NEM
on voltage-dependent calcium channels (Shapiro et al.
1994
), should be considered as having possible roles in the
synaptic actions of NEM in striatum.
Application of NEM enhances transmission before LTD induction, and it
could well be the case that this enhancement of transmission simply
occludes LTD induction and expression. In addition to the experiments
explicitly performed to examine this possibility, which are described
in the RESULTS section, three pieces of evidence argue
against this interpretation. First, a prolonged exposure to PTX leads
to a reduction in synaptic response amplitude and also blocks LTD (Fig.
1A). This finding indicates that LTD is blocked when
PTX-sensitive G-proteins are inhibited, regardless of whether baseline
transmission is enhanced or inhibited. The observation that some of the
responses observed in PTX-treated slices were as large in amplitude as
those observed in untreated slices, suggests that the inability to
induce LTD was not simply related to the small baseline response
amplitude in the PTX-treated slices. Second, NEM blockade of LTD
induction is observed regardless of the time between NEM exposure and
high-frequency stimulation (compare Fig. 2B and 4). This
indicates that LTD is not masked by the slowly developing NEM
potentiation. These findings also indicate that LTD induction is
blocked even before NEM potentiation is complete, indicating that
blockade of LTD is not due to the large response amplitude after the
full NEM effect. Third, other treatments that potentiate synaptic
transmission, including blockade of adenosine A1 receptors and
treatment with aniracetam (Lovinger and Choi 1995;
Lovinger et al. 1993
), do not prevent synaptic depression. That LTD can be observed even in the face of increased synaptic transmission supports the idea that the effects of NEM interact directly with mechanisms involved in LTD induction and/or maintenance.
We cannot, however, determine whether blockade of the function of
PTX-sensitive G-proteins prevents LTD by blocking a specific set of
molecular steps linking receptor activation to LTD induction or by
antagonizing the synaptic mechanisms involved in LTD expression. Indeed, the expression of LTD has been postulated to involve a decrease
in the probability of neurotransmitter release (Choi and
Lovinger 1997b), and NEM appears to have an opposite action. We
observed that NEM increased the probability of neurotransmitter release, as indicated by the increase in spontaneous EPSC frequency and
the loss of PPF observed after NEM treatment. This effect would
probably counteract the decrease in neurotransmitter release probability during LTD expression and contribute to the reversal of LTD
by NEM. It should be noted, however, that evidence of decreased probability of neurotransmitter release associated with LTD has not
been consistently observed in experiments on brain slices from adult
rat (Calabresi et al. 1999
). However, a recent report (Dos Santos Villar and Walsh 1999
) that an increase in
PPF ratio is observed during LTD expression is consistent with our
earlier findings. Thus, there may be two types of striatal LTD. One
type does not appear to involve a change in release probability,
whereas the other type involves decreased release probability and may be more highly expressed in developing striatum.
Potentiation by NEM in the absence of HFS suggests that tonic synaptic
inhibition involving PTX-sensitive G-proteins occurs at corticostriatal
synapses. This could be due to either agonist-independent receptor-mediated G-protein activation (Schütz and
Freissmuth 1992) or tonic activity of a G-protein-coupled
receptor by an endogenous agonist present within the slice. With
respect to the latter mechanism, it is well known that adenosine is
present in brain slices at concentrations that can activate inhibitory
adenosine A1 receptors. Indeed, inhibition of corticostriatal synaptic
transmission by endogenous adenosine has been observed in striatal
slices (Lovinger and Choi 1995
). Thus, it is tempting to
speculate that inhibition of endogenous adenosine receptor activity
contributes to the potentiation of transmission by NEM. However, our
observations in the experiments with combined DPCPX and NEM exposure
suggest that NEM effects on synaptic transmission are not due solely to
elimination of A1 receptor-mediated synaptic modulation. In addition,
NEM appears to have postsynaptic actions that contribute to
potentiation, whereas adenosine A1 receptor effects are believed to be
predominantly presynaptic (Malenka and Kocsis 1988
).
Furthermore, NEM reversal of adenosine A1-mediated inhibition cannot
account for the blockade of LTD induction because adenosine A1
receptors are not involved in this process (Lovinger and Choi
1995
). Thus, we believe that relief of tonic G-protein activity
is the most likely explanation for the effects of NEM on striatal
synaptic transmission and plasticity.
We have examined in some detail the mechanism(s) by which NEM
facilitates synaptic transmission. NEM alone increased the frequency and amplitude of spontaneous EPSCs, and this effect was also seen for
mEPSCs recorded in the presence of TTX. According to classic interpretation of miniature synaptic response analysis (del
Castillo and Katz 1954), this finding would suggest that NEM
acts on presynaptic terminals at corticostriatal synapses to increase
the probability of glutamate release. This interpretation is also
consistent with the observation that NEM abolishes PPF. A change in the
number of postsynaptically "silent" synapses may also account for
the NEM-induced increase in spontaneous EPSC frequency (Isaac et
al. 1995
; Liao et al. 1995
). However, it is not
clear how this would lead to a loss of PPF.
We also obtained evidence that NEM increases the amplitude of
spontaneous and miniature EPSCs, a finding that would be interpreted classically as indicating a postsynaptic NEM effect. However, another
possible interpretation of these data is that NEM increases the amount
of neurotransmitter released per quantum (Liu et al. 1999), an effect that would also increase mEPSC amplitude. It is also possible that the increase in mEPSC amplitude could result from
simultaneous multiquantal release in the presence of NEM.
In the hippocampus, Morishita et al. (1997) observed that
NEM could only increase GABA receptor-mediated miniature inhibitory postsynaptic current (mIPSC) frequency but not mIPSC amplitude. They
observed an increase in mIPSC amplitude by NEM in medium containing 0 mM calcium and 8 mM magnesium. The difference between these findings
and our results could be due to the different brain regions and
neurotransmitters investigated. It has also been demonstrated that PTX
treatment can block the induction of long-term potentiation (LTP) at stratum radiatum CA1 synapses (Goh and
Pennefather 1989
). In addition, cerebellar LTD involves
activation of metabotropic glutamate receptors that most likely act
through PTX-sensitive G-proteins (Linden and Connor
1993
). Thus involvement of PTX-sensitive G-proteins is a common
feature of many forms of synaptic plasticity including striatal LTD.
Potentiation of transmission was also observed after NEM treatment combined with HFS in whole cell and field potential recording experiments. It is possible that this increase reflects the slow potentiation observed with NEM treatment alone and not any LTP produced by specific synergism between the effects of HFS and NEM. Indeed, we did not observe any difference in the magnitude of response potentiation by NEM alone compared with potentiation after NEM and HFS in data from either field potential or whole cell recording experiments. This finding is consistent with the idea that the increase in synaptic response amplitude after combined NEM and HFS is most likely due to the effects of NEM alone.
It is not clear why inhibition of synaptic responses was observed after
prolonged PTX exposure, whereas acute NEM exposure enhanced
transmission. It is possible that constant, prolonged inhibition of
Gi/o-type G-proteins leads initially to a tonic increase in
neurotransmitter release that eventually begins to deplete
neurotransmitter pools, because PTX-catalyzed ADP ribosylation and
inactivation of G-proteins are irreversible biochemical reactions (Katada et al. 1984; Ui 1984
). However,
we cannot rule out the possibility that the potentiation of
transmission produced by acute NEM exposure involves different
mechanisms than those produced by PTX exposure.
The effects of postsynaptic perfusion with NEM indicate that NEM must
have a local action at synapses, because the compound could not diffuse
too far and maintain an effective concentration, even if it escapes the
postsynaptic neuron. Several mechanisms could contribute to these
effects. First, NEM may act only at a postsynaptic site. Second, NEM
may initiate as yet unknown intracellular pathways in the postsynaptic
neuron to stimulate formation of a retrograde messenger. Third, NEM may
diffuse through the postsynaptic plasma membrane and across the
synaptic cleft to act on a presynaptic site. That NEM increased mEPSC
amplitude is consistent with a postsynaptic action. However, the
possibility of NEM's producing only postsynaptic actions appears
unlikely, given the increase in frequency of spontaneous EPSCs observed
after NEM perfusion. So far, there is no known pathway by which
inhibition of PTX-sensitive G-proteins by NEM can stimulate formation
of a retrograde messenger. However, activation of G-proteins has been
previously reported to be involved in the generation of a known
retrograde messenger at developing neuromuscular synapses
(Harish and Poo 1992). NEM is reasonably hydrophobic and
can pass through membranes when applied extracellularly. It is quite
possible that NEM could diffuse back to the presynaptic terminal after
postsynaptic perfusion. At present we cannot distinguish between these
different mechanisms, but the possibility that NEM possesses or
activates retrograde signals is intriguing and may indicate potential
use as an experimental tool for future studies.
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ACKNOWLEDGMENTS |
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We thank Dr. Richard W. Tsien for helpful comments on the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-30470 and NS-37615 and by the Tourette Syndrome Association.
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FOOTNOTES |
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Address for reprint requests: D. M. Lovinger, Room 724, MRB1, Dept. of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 July 1999; accepted in final form 10 September 1999.
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REFERENCES |
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