Induction of Long-Term Depression in Cerebellar Purkinje Cells Requires a Rapidly Turned Over Protein

Laddawan Karachot,1 Yoshinori Shirai,1,2 Réjan Vigot,2 Tetsuo Yamamori,1,2 and Masao Ito1

 1Laboratory for Memory and Learning, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198; and  2Laboratory for Speciation Mechanisms I, National Institute for Basic Biology, Okazaki 444-8585, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Karachot, Laddawan, Yoshinori Shirai, Réjan Vigot, Tetsuo Yamamori, and Masao Ito. Induction of Long-Term Depression in Cerebellar Purkinje Cells Requires a Rapidly Turned Over Protein. J. Neurophysiol. 86: 280-289, 2001. Evidence is presented indicating that the induction of long-term depression (LTD) in Purkinje cells (PCs) requires a rapidly turned over protein(s) during a critical time period within 15 min after the onset of LTD-inducing stimulation and that synthesis of this protein is maintained by mRNAs supplied via transcription. LTD was induced in granule cell axon (GA)-to-PC synapses by stimulation of these synapses at 1 Hz for 5 min in conjunction with the climbing fibers (CFs) forming synapses on the same PCs and represented by a persistent reduction in the GA-induced excitatory postsynaptic potentials (EPSPs). Not only a prolonged but also a brief (5 min) pulse application of translational inhibitors (anisomycin, puromycin, or cycloheximide) effectively blocked the LTD induction. Pulses applied during the period from 30 min before to 10 min after the onset of conjunctive stimulation blocked the LTD induction, but those applied 15 min after were ineffective. The three translational inhibitors blocked the LTD induction similarly, suggesting that the effect is due to their common action of inhibiting protein synthesis. Infusion of a mRNA cap analogue (7-methyl GTP) into PCs also blocked LTD induction, ensuring that the postsynaptic protein synthesis within PCs is required for LTD induction. Transcriptional inhibitors, actinomycin D and 5,6-dichloro-l-beta -D-ribofuranosyl-benzimidazole, also blocked the LTD induction, but this effect was apparent when 5-min pulses of the transcriptional inhibitors preceded the conjunctive stimulation by 30 min or more. This time lag of 30 min is presumed to be required for depletion of the protein(s) required for LTD induction. The presently observed effects of translational and transcriptional inhibitors on the LTD induction are of temporal characteristics corresponding to their depressant effects on the type-1 metabotropic glutamate-receptor (mGluR1)-mediated slow EPSPs in PCs as we have reported recently. An antagonist of mGluR1s [(RS)-1-aminoindan-1,5-dicarboxylic acid], however, did not block LTD induction when it was applied during the 10-min period following conjunctive stimulation, where translational inhibitors effectively blocked LTD induction. This discrepancy in time course suggests that the rapidly turned over protein(s) required for LTD induction is involved in a process occurring downstream of the activation of mGluR1s.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Roles of protein synthesis in synaptic plasticity have been proposed based on the findings that translational and transcriptional inhibitors inhibit long-term potentiation (LTP) in hippocampal neurons (Frey and Morris 1997; Frey et al. 1988; Nguyen et al. 1994) and sensitization in Aplysia sensory neurons (Montarolo et al. 1986). Translational inhibitors also inhibit the neurotrophin-induced LTP in hippocampal neurons (Kang and Schuman 1996) and metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in hippocampal neurons (Huber et al. 2000). Protein synthesis and processing occur in ribosomes, the endoplasmic reticulum, and the Golgi complex, which are largely contained in neuronal somata. Recently, however, neuronal dendrites have drawn attention as local sites of protein synthesis and processing specific to synapses since dendrites contain various mRNAs (Gao 1998; Martone et al. 1998; Steward 1995; Tian et al. 1999) as well as ribosomes, polyribosomes, the endoplasmic reticulum, and part of the Golgi complex (Gardiol et al. 1999; Steward and Reeves 1988; Steward et al. 1996). Dendrites exhibit an increased expression level of certain proteins or their mRNAs following the induction of LTP in hippocampal and neocortical neurons (Klintsova and Greenough 1999; Osten et al. 1996; Roberts et al. 1998; Schuman 1999).

In cerebellar Purkinje cells (PCs), LTD is induced following conjunctive activation of two excitatory inputs to PCs, namely, granule cell axons (GAs) and climbing fibers (CFs) (Ekerot and Kano 1985; Ito and Kano 1982; Karachot et al. 1994; Sakurai 1987). The so-induced LTD is represented by persistent depression in the GA-evoked excitatory postsynaptic potentials (EPSPs) or currents (EPSCs). Inducing a persistent reduction in glutamate sensitivity of cultured PCs by conjunctive application of glutamate and membrane depolarization, Linden (1996) reported a depressant action of translational and transcriptional inhibitors on this reduced form of LTD. In the present study, we confirmed that translational inhibitors effectively blocked LTD in cerebellar slices, and the results were interesting in two respects. First, to block protein synthesis, neural tissues are usually incubated with translational inhibitors for 1-3 h (Frey et al. 1988; Linden 1996; Montarolo et al. 1986); however, a brief 5-min pulse application of translational inhibitors was found to be sufficient to block the induction of LTD. Second, translational inhibitors abolished the entire LTD including its early phase, contrary to the general presumption that protein synthesis is required for the late phase of synaptic plasticity but not for the early phase (Frey and Morris 1997; Frey et al. 1988; Linden 1996; Montarolo et al. 1986; Nguyen et al. 1994).

We have recently reported that 5-min pulse applications of translational or transcriptional inhibitors effectively suppress slow EPSPs mediated by type 1 metabotropic glutamate receptors (mGluR1-EPSPs) in PCs (Karachot et al. 2000). Since activation of mGluR1s is known to be required for the induction of LTD (Aiba et al. 1994; Conquet et al. 1994; Hartell 1994; Shigemoto et al. 1994), it is possible that the protein required for the generation of mGluR1-EPSPs is a key factor responsible for the unique protein synthesis dependence of LTD. To test this possibility, we measured the time course of the depressant effect of translational and transcriptional inhibitors on LTD by applying 5-min inhibitor pulses and found that it is consistent with the previously reported time course of their effects on mGluR1-EPSPs (Karachot et al. 2000). However, we also found that the effect of translational inhibitors of depressing LTD induction outlasted the inactivation of mGluR1s by an antagonist. This suggests that the rapidly turned over protein synthesis is not directly involved in activation of mGluR1s but probably in a process occurring downstream of the activation of mGluR1s.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Sagittal slices (0.4-mm thick) were obtained from Wistar rat cerebella dissected under ether anesthesia. Adult (postnatal week 6) rats were used for intracellular recording from PCs and young rats (postnatal week 3-4) were preferred for whole cell patch-clamp recording. The standard perfusate (pH = 7.3) containing (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 25.0 NaHCO3, 1.18 KH2PO4, 1.19 MgSO4, and 11.0 glucose plus 20 µM picrotoxin was equilibrated with 95% O2-5% CO2 gas and was warmed to maintain the temperature of the perfusion chamber at 31-32°C. GAs were bipolarly stimulated near the pial surface at an intensity sufficient to induce EPSPs of 8-10 mV (Fig. 1A). CFs were bipolarly stimulated in the white matter at an intensity slightly higher than the threshold for evoking CF responses from each impaled PC (Fig. 1C). In most experiments, PCs were impaled at their dendritic shafts using glass microelectrodes (60-80 MOmega ) filled with 3 M KCl solution. In the experiments illustrated in Fig. 6, EPSPs were recorded with a whole cell configuration current-clamp using a whole cell clamp pipette (5-8 MOmega ) containing (in mM) 134 K-gluconate, 6 KCl, 4 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP, 0.3 Tris-GTP, and 14 phosphocreatine; the pH was adjusted to 7.25 with KOH. In part of these experiments, the pipette also contained 250 µ M 7-methyl GTP (m7GpppG), a mRNA cap analogue (Chu and Rhoades 1980; Huber et al. 2000).



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Fig. 1. Long-term depression (LTD) in Purkinje cells (PCs) of control and anisomycin-perfused cerebellar slices. Inset: arrangement of recording (M) and stimulating electrodes (S1 and S2) on rat cerebellar slices. MF, mossy fiber; GR, granule cell; GA, granule cell axon; CF, climbing fiber; IO, inferior olive. A: GA-evoked excitatory postsynaptic potentials (EPSPs) in a control PC recorded at the times indicated before and after the onset of 5-min conjunctive stimulation. *, spike components superposed on the EPSPs. B: superposition of the traces in A in a faster sweep. C: a and b are CF responses recorded at the times indicated. D: superposition of a and b. E-H: illustrated similarly to A-D except for another PC continuously perfused with anisomycin. Voltage and time scales are indicated. Note that the time scale is shown above in A, C, E, and G and below in the others.

To observe LTD, fast EPSPs mediated by alpha -amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors were evoked by stimulating GAs every 5 s, and five successive measurements were averaged. In Figs. 2, 3, 6-8, and 10, the initial slope of the GA-evoked EPSPs is plotted at 1-min intervals. Their 100% values were determined by averaging the five measurements during the 5-min period prior to conjunctive stimulation. No plot was made during conjunctive stimulation and the succeeding 1-min period to avoid the effect of the transitional change of the GA-stimulating conditions from the 0.2-Hz-test stimulation to 1-Hz conjunctive stimulation and vice versa. LTD was induced using the protocol of Karachot et al. (1994) of repetitive simultaneous stimulation of GAs and CFs at 1 Hz for 5 min (300 pulses). ANOVA was adopted for statistical analysis, and Duncan's new multiple range test was used for comparison between data obtained with different timings of conjunction and drug applications.

To induce mGluR1-EPSPs, brief tetanic stimulations (50 Hz, 8 pulses at 30-s intervals) were applied to GAs bipolarly near the pial surface in the presence of 10 µM 2,3-dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), an antagonist for AMPA-selective glutamate receptors. An antagonist of N-methyl-D-aspartate (NMDA)-selective receptors, namely, 30 µM D(-)-2-amino-5-phosphonopentanoic acid (D-AP-5), was also added routinely. Even though adult PCs themselves do not form NMDA receptors, D-AP-5 eliminated a possible NMDA receptor-mediated activation of granule cells due to spread of stimuli to mossy fibers. The mGluR1-EPSPs thus obtained were confirmed to be blocked by 500 µM (RS)-a-methyl-4-carboxyphenyl glycine (MCPG), a nonspecific antagonist of mGluRs. A mGluR1-specific antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (Bachelor et al. 1997; Pellicciari et al. 1995) was used for the purpose of testing the involvement of mGluR1-EPSPs in LTD induction (Fig. 10). 7-Hydroxyiminocyclopropan[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (Pagano et al. 2000) was also tested, but DMSO used to dissolve it showed by itself a depressant effect, gradually developing in 10-20 min, on mGluR1-EPSPs even at a concentration as low as 0.01-0.05%; CPCCOEt was not used further for testing LTD.

NBQX, D-AP-5, and AIDA were purchased from Tocris, and other chemicals were from Sigma. The concentrations of translational and transcriptional inhibitors in the perfusates were selected according to the results of our previous study (Karachot et al. 2000): anisomycin, 25 µM; puromycin, 50 µM; cycloheximide, 30 µM, actinomycin D, 25 µM; 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB), 30 µM. These were dissolved in the perfusates, except for DRB whose dissolution required sonication. AIDA was dissolved in concentrations of 200-650 µM, and pH was adjusted to 7.3 with NaOH.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In recording from PCs, LTD was characterized by a reduction in the rising slope of the GA-evoked EPSP, which followed conjunctive stimulation, as shown in Fig. 1, A and B. That no significant change occurred in CF responses was taken as evidence indicating that the reduction in GA-evoked EPSPs was not due to deterioration of the examined PCs (Fig. 1, C and D). The plot of the magnitude of the rising slope of GA-evoked EPSPs in Fig. 2 (open circles) shows a gradual development of LTD over 60 min. To determine the magnitude of LTD, we measured the average reduction in the EPSP slopes at two phases: 7-16 min (early phase) and 35-44 min (late phase) after the onset of conjunctive stimulation (Fig. 2, i and ii). In 15 control PCs examined in this study, the magnitude of LTD was 20.1 ± 1.0% at 7-16 min and 37.8 ± 1.2% at 35-44 min (means and SE). The latter values are comparable with those reported previously (Karachot et al. 1994; Miyata et al. 1999).



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Fig. 2. Time courses of LTD in control PCs in contrast with anisomycin-perfused PCs. Plot of the magnitude of rising slopes of GA-induced EPSPs at 1-min intervals against time. Open circles, average from 15 control PCs. The 100% level is obtained from the average of the 5 plots preceding the onset of conjunction. Oblique shade (CONJ), 5-min period of conjunctive stimulation. Vertical bars shown on 1 side of the circles, SE. Dotted line passing through open circles represents the regression line for control: y = 96.206-1.5982X + 0.024705X2 - 0.00016539X3 (R = 0.9846). Closed circles, average from 5 PCs continuously perfused with anisomycin. A thick horizontal band indicates continuous application of anisomycin (ANI). Thin horizontal bars indicate the 10-min time periods during which the EPSP slope values are averaged for representing the magnitudes of LTD at its early (i) and late (ii) phases. The numberical figures attached on the left to the shaded column (5 and 15) indicate the number of PCs included in the measurements. The numerical figure 4 attached to a closed circle indicates that the number of PCs decreased at the time of this plot from 5 to 4 due to the failure of recording in 1 PC.

Effects of translational inhibitors

We used three translational inhibitors, namely, anisomycin (Grollman 1968), puromycin (Stanton and Sarvey 1984), and cycloheximide (Deadwyler et al. 1987; Stanton and Sarvey 1984). Emetine (Bennett et al. 1964) was also tested, but since it induced sustained depression of GA-evoked EPSPs by itself, its effect on LTD was difficult to evaluate; the data obtained with emetine were therefore excluded. The translational inhibitors presently used did not affect the membrane resting potential, input resistance, GA-evoked EPSPs, or CF responses of PCs, but they exerted a clear inhibitory effect on LTD. Figure 1, E-H, similarly shows GA-evoked EPSPs and CF responses under continuous perfusion with 25 µM anisomycin that started no less than 30 min before conjunctive stimulation, and GA-evoked EPSPs no longer exhibited LTD. As illustrated in Fig. 2 (closed circle), LTD was abolished at both the early (3.6 ± 1.3%) and late phases (1.4 ± 1.4%, 5 PCs) after conjunctive stimulation.

One of the findings in this study is that even 5-min pulse applications of anisomycin effectively block LTD when applied simultaneous to conjunctive stimulation (Fig. 3A, GA-evoked EPSPs at the late phase: -0.2 ± 3.1%, 6 PCs). Blockade of LTD occurred also when 5-min applications of anisomycin preceded conjunction by 10 min (Fig. 3B, 10.1 ± 4.1%, 9 PCs) or even 30 min (Fig. 3C, 15.1 ± 3.1%, 7 PCs). Blockade of LTD also occurred even when 5-min anisomycin pulses were delayed by 5 min (Fig. 3D, 9.0 ± 4.7%, 8 PCs) or 10 min (Fig. 3E, 19.3 ± 5.4%, 8 PCs) after the onset of conjunction. However, when 5-min pulse applications of anisomycin were delayed by 15 min, LTD occurred normally (Fig. 3F, 41.0 ± 5.9%, 5 PCs; P > 0.05 as compared with that of the control).



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Fig. 3. LTD in PCs applied with 5-min anisomycin pulses. Illustration similar to that in Fig. 2 but with varied intervals between conjunction (indicated by shaded columns) and 5-min pulse applications of anisomycin (indicated by horizontal thick bars): 0 min (A), -10 min (B), -30 min (C), 5 min (D), 10 min (E), and 15 min (F). Dotted curves represent the regression line for the 15 control PCs shown in Fig. 2.

Figure 4A summarizes similar measurements obtained using the three translational inhibitors on the late phase of LTD at 35-44 min after the onset of conjunction. It is noted that these inhibitors suppressed the magnitude of LTD statistically significantly (P < 0.01) when 5-min pulses of the translational inhibitors were applied 20 min before to 10 min after the onset of conjunctive stimulation. However, cycloheximide applied 30 min before conjunction was ineffective (P > 0.05), whereas anisomycin similarly applied was still effective. Figure 4B summarizes the measurements on the early phase of LTD. Differing from the inhibition of the late phase, the inhibition of the early phase of LTD was insignificant (P > 0.05) when translational inhibitors were applied 20-30 min before (Fig. 3C) or 15 min after (Fig. 3F) the onset of conjunction. The reason why translational inhibitors applied 10 min after the onset of conjunction reduced the magnitude of the early phase of LTD to a lesser degree than that of the late phase (compare Fig. 4, A and B) is presumably because the early phase of LTD has already developed before it was aborted by translational inhibitors. The difference obtained between Fig. 4, A and B, when translational inhibitors were applied 20 or 30 min before conjunction, however, would suggest that the early phase of LTD is less sensitive to translational inhibitors as compared with the late phases (DISCUSSION).



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Fig. 4. Effect of translational inhibitors on LTD induction as a function of time intervals between 5-min inhibitor pulse applications and conjunctive stimulation. Ordinates, magnitudes of LTD measured at 35-44 min (A) or 7-16 min (B) after the onset of conjunctive stimulation. Abscissae, time interval from the onset of 5-min inhibitor pulses to that of conjunctive stimulation. Positive values of time indicate that 5-min inhibitor pulse applications succeeded conjunctive stimulation as in Fig. 3, D-F, while negative values indicate that inhibitor pulse applications preceded conjunctive stimulation as in Fig. 2, B and C. c, control samples and p, those under continuous perfusion with anisomycin as shown in Fig. 2. Three types of columns were used for anisomycin (ANI), puromycin (PURO), and cycloheximide (CYCLO) as indicated above A. Vertical bars, SE. ANOVA (Duncan's new multiple range test for comparing the 15 control samples in Fig. 2). **P < 0.01; *P < 0.05.

When applied 10 min after the onset of conjunction, the inhibitors acted after the very initial development of LTD and aborted it (Fig. 3E). Events at this critical timing are illustrated in Fig. 5 with cycloheximide. Cycloheximide pulses applied 10 min, but not 15 min, after the onset of conjunctive stimulation effectively aborted the LTD induction. These measurements in Figs. 3-5 using 5-min pulse applications of translational inhibitors indicate that protein synthesis is required critically during the period within 15 min after the onset of 5-min conjunctive stimulation.



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Fig. 5. Critical time window for blocking LTD with cycloheximide. Illustration similar to that in Fig. 3 but for 2 sets of PCs applied with 5-min pulses of cycloheximide at different timings (10 and 15 min after the onset of conjunction) as indicated (). Regression lines: open circle , Y = 98.268 - 2.2347X + 0.060569X2 - 0.00069863X3 (R = 0.95097); , Y = 1.0163 - 4.1115X - 0.31381X2 - 0.0081618X3 + 6.7997e - 05X4 (R = 0.67759).

While anisomycin is widely used as a protein synthesis inhibitor, it is known also to activate 45- and 55-kDa protein kinases (p45/p55) at a concentration as low as 0.037 µM (10 ng/ml) (Cano et al. 1994). However, 5-min pulses of 0.1 µM anisomycin applied simultaneously with conjunctive stimulation did not inhibit the induction of LTD (LTD at 35-44 min: 29.1 + 2.6%, 4 PCs, P > 0.05 compared with the 15 control samples shown in Fig. 2). Anisomycin is also known to activate the 70- and 85-kDa S6 protein kinases (p70/85S6k), which are homologous of the mitogen-activated kinase-activated protein kinase-2 (MAPKAPK-2) (Cano et al. 1994, 1996). However, 25 nM rapamycin that blocks the anisomycin activation of p70/85S6k (Cano et al. 1994) did not alter the blocking effect on the LTD induction of the coapplied 25 µM anisomycin simultaneously with conjunctive stimulation (LTD at 35-44 min: 5.2 + 1.6%, 3 PCs, P > 0.05 compared with 6 samples subjected to 5-min 25 µM anisomycin pulses alone).

Effects of a mRNA cap analogue

The mRNA cap analogue (m7GpppG) inhibits translation by competing with the endogenous capped mRNA for the cap-binding brain elF-4E (Gingras et al. 1999). Figure 6 illustrates that, without loading of m7GpppG in the whole cell clamp pipette, LTD developed as observed in intracellular recording shown in Fig. 2. The magnitudes of LTD at 7-16 and 35-44 min after the onset of conjunctive stimulation are 16.1 ± 3.8% (7 PCs) and 37.6 ± 3.2% (5 PCs), respectively, which are not significantly (P > 0.05) different from those obtained in the intracellularly recorded 15 control PCs (20.1 ± 1.0% at 7-16 min and 37.8 ± 1.2% at 35-44 min). When the pipette was loaded with m7GpppG, LTD was significantly depressed (P < 0.01) in both early and late phases, the magnitudes of LTD at 7-16 min being -1.0 ± 2.2% (4 PCs) and that at 35-44 min being -11.2 ± 3.9% (3 PCs). Hence, m7GpppG infused through the whole cell clamp pipette blocked LTD similarly to translational inhibitors.



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Fig. 6. Effect of a mRNA cap analogue on LTD. Data obtained with whole cell configuration current-clamp recording from PCs. Illustration similar to that in Fig. 2. open circle , control cases of conjunctive stimulation. , with infusion of 7-methyl guanosine 5' triphosphate (m7GpppG). Conjunctive stimulation started 17-25 min after the onset of giga sealing.

Effects of transcriptional inhibitors

We used two types of transcriptional inhibitors, actinomycin D and DRB. Perfusion of 25 µM actinomycin D for 5 min immediately decreased the amplitudes of GA-evoked AMPA-EPSPs (see the downward shift of open circle  shown in Fig. 7). This effect is too sudden to be ascribed to transcriptional inhibition; it presumably arose from a side effect of actinomycin D, which may have a direct effect on GA-to-PC transmission. However, this transient effect of actinomycin D did not affect our observation of LTD (Fig. 7, ). The magnitude of LTD measured 35-44 min after the onset of conjunctive stimulation, immediately succeeding 5-min pulse applications of actinomycin D (Fig. 7), was 31.2 ± 2.4% (9 PCs), which was not significantly different from that of the control LTD (37.8%, P > 0.05). LTD was suppressed only when 5-min pulse applications of actinomycin D preceded the conjunctive stimulation by 30 min or more (Fig. 8, ). DRB (30 µM) also blocked the LTD induction after a delay of 30 min (Fig. 8, open circle ). It is notable that DRB did not induce an immediate depression of the GA-evoked EPSPs during its application such as that caused by actinomycin D. It can be observed from Fig. 8 that actinomycin D and DRB blocked the entire LTD including its early phase. Figure 9 summarizes these observations and shows that conjunctive stimulation at 30-60 min after 5-min pulse applications of either actinomycin D or DRB induced significantly (P < 0.01) smaller LTD magnitude as compared with control LTD.



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Fig. 7. Effect of actinomycin D. Illustration similar to those in Fig. 2 and 3, A-F, but for 2 sets of PCs applied with actinomycin D. One set (open circle ) was applied with 5-min actinomycin D pulses alone. The other () was applied with 5-min pulses of actinomycin D followed by conjunctive stimulation (). Regression line for the 15 control cases shown in Fig. 2 is indicated for comparison.



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Fig. 8. Blockade of LTD induction by actinomycin D (ACT-D) and 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB). Illustration similar to that in Fig. 7 but for 2 sets of PCs, 1 being applied with 5-min actinomycin D pulses () 35 min before conjunctive stimulation and the other with DRB (open circle ) 30 min before conjunctive stimulation.



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Fig. 9. Effect of transcriptional inhibitors on LTD as a function of time intervals between 5-min inhibitor pulse applications and conjunctive stimulation. Illustration similar to that in Fig. 4A for LTD magnitudes 35-44 min after conjunction.

Effect of mGluR1 antagonist

We reported previously that a rapidly turned over protein plays a key role in the metabotropic glutamatergic transmission that produces characteristic slow mGluR1-EPSPs in PCs (Karachot et al. 2000). Since an antagonist (Hartell 1994), an antibody (Shigemoto et al. 1994), and gene deficiency (Aiba et al. 1994; Conquet et al. 1994) against mGluR1s block LTD induction, it is possible that translational and transcriptional inhibitors block LTD induction by interfering with mGluR1s. We, therefore compared the effect of a mGluR1 antagonist and protein synthesis inhibitors on LTD induction.

Five-minute pulse applications of AIDA at 500 µM, but not at 200-300 µM, rapidly suppressed the GA-evoked mGluR1-EPSPs, down to 30% within 2 min, as shown in Fig. 10A. Applications of AIDA simultaneous to conjunctive stimulation blocked LTD and instead induced potentiation of GA-induced AMPA-EPSPs as described previously (Hartell 1994) (Fig. 10B). However, 5-min pulse applications of AIDA shortly following conjunctive stimulation did not interfere with LTD induction as shown in Fig. 10, C and D. This contrasts to the effect of translational inhibitors, which blocked LTD induction even when applied 10 min after the onset of conjunctive stimulation (Figs. 3E and 5). The discrepancy suggests that the effect of translational inhibitors is not due to direct inactivation of mGluR1 receptors (DISCUSSION). A transient downward shift of the plotted points is noted in Fig. 10, C and D, during 5-min pulse application of AIDA. This may be due to a reversible effect of AIDA on GA-mediated AMPA-EPSPs, which, however, did not influence the present observation of the much longer lasting LTD.



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Fig. 10. Effects of a type 1 metabotropic glutamate receptor (mGluR1) antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). A (ordinate): peak amplitude of mGluR-excitatory postsynaptic potentials (EPSPs). AIDA (500 µM) was added to perfusates during the 5-min period indicated by a horizontal bar. a-c: specimen records of mGluR-EPSPs observed at the times indicated. B-D: illustrations similar to those in Fig. 3 but for 5-min application of AIDA with delay of 0 min (B), 7 min (C), and 10 min (D) after the onset of conjunction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Specificity of protein synthesis inhibitors

All the three types of translational inhibitors blocked the induction of LTD rapidly and persistently (Fig. 4). We previously reported that these translational inhibitors rapidly and persistently suppressed amino acid incorporation into cerebellar slices (Karachot et al. 2000). This general agreement in these two sets of data supports the view that protein synthesis is required for the induction of LTD. However, a discrepancy was noted in that translational inhibitors applied 20 or 30 min before the onset of conjunction did no reduce the magnitude of the early phase of LTD at 7-16 min (Fig. 4B), even while methionine incorporation into cerebellar slices continues to be inhibited for 30 min after 5-min pulse applications of translational inhibitors (Karachot et al. 2000). The discrepancy is smaller for the effect of translational inhibitors on the late phase of LTD at 35-44 min; yet, cycloheximide pulses applied 30 min after the onset of conjunction did not reduce significantly the late of phase of LTD (Fig. 4A). These observations suggest that the concentration of translational inhibitors gradually declines within 30 min after their pulse applications even though this recovery is not reflected in the methionine incorporation into cerebellar slices measured by Karachot et al. (2000). The discrepancy may not be surprising since the present LTD measurements reflect events only in PCs, while the methionine incorporation into cerebellar slices involves protein synthesis in not only PCs but also in other types of cerebellar neurons.

Translational inhibitors activate certain protein kinases independent of protein synthesis inhibition (Cano et al. 1994). However, all the three types of kinases known to be activated by anisomycin [p45/p55, p70/85S6k, and c-Jun NH2-terminal kinases/stress-activated protein kinases (JNK/SAPKs)] can be excluded as a cause of the blockade of LTD for the following reasons. Anisomycin did not block the LTD induction at a concentration of 0.1 µM (see RESULTS), which is subthreshold for inhibiting protein synthesis yet effective in activating p45/p55 (Cano et al. 1994). The report that puromycin is ineffective in activating P45/p55 (Cano et al. 1994) justifies the exclusion of p45/p55 because the LTD induction is blocked by puromycin similarly to anisomycin (Fig. 4). p70/85S6k kinases are unlikely to be involved in the LTD induction because rapamycin, an inhibitor of anisomycin-induced activation of p75/80S7k, did not prevent the anisomycin-induced blockade of LTD induction (see RESULTS). And finally, responses of JNK/SAPKs to application of anisomycin become distinct after 15 min (Cano et al. 1995), being too slow to account for the rapid anisomycin-induced inhibition of LTD induction (Fig. 4). While phosphorylation of JNK/SAPKs proceeds within 5 min of 10-µM-anisomycin application, it takes 60 min with 10-µM-cycloheximide application, and only slight phosphorylation occurs within 60 min of 10-µM-puromycin application (Sidhu and Omiecinski 1998), unlike the uniformly rapid inhibitory action of the three translational inhibitors on LTD induction (Fig. 4).

Actinomycin D blocks transcription by intercalating DNAs (Sobell 1973), while DRB inhibits mRNA synthesis by blocking the initiation of RNA chain elongation (Tamm and Sehgal 1978). The close similarity of the effects on the LTD induction in spite of the distinct mechanisms of action of the two transcriptional inhibitors supports the view that their common action of inhibiting mRNA synthesis underlies the blockade of LTD induction. The 30-min delay required for the action of both actinomycin D and DRB can be accounted as the time required for the gradual degradation of preexisting dendritic mRNAs to a level lower than that required for LTD induction (see following text).

Comparison with other forms of synaptic plasticity

Protein synthesis inhibitors affect the late phase, but not the early phase, of LTP (Frey et al. 1988; Nguyen et al. 1994) or LTD (Kauderer and Kandel 2000) in hippocampal neurons or sensitization in Aplysia (Montarolo et al. 1986). When a translational inhibitor was continuously applied to cultured PCs immediately after conjunctive stimulation, LTD started to decline 45 min after the onset of the glutamate/depolarization conjunctive stimulation (Linden 1996). The phase of LTD within 45 min after the onset of conjunctive stimulation in cultured PCs is thus independent of protein synthesis in contrast to the present results in PCs of cerebellar slices that both of the early and late phases of LTD are dependent on protein synthesis. However, the present results obtained in PCs of cerebellar slices are in accordance with the reports on the neurotrophin-induced LTP (Kang and Schuman 1996) and mGluR5-mediated LTD (Huber et al. 2000) in hippocampal neurons, which are inhibited by translational inhibitors in both its early and late phases.

To explain the different effects of translational inhibitors on PCs in cerebellar slices and cultures, a question may arise as to whether translational inhibitors act on GAs, presynaptic to PCs in cerebellar slices, which are absent in cultured PCs used by Linden (1996). However, as far as we assume that the rapid protein synthesis required for LTD induction is the same as that required for the generation of mGluR1-EPSPs (Karachot et al. 2000), it must occur within PCs because translational inhibitors do not affect GA-mediated AMPA-EPSPs and because the characteristic delay of action of transcriptional inhibitors on LTD induction is in accordance with the slow decline of mGluR1-EPSPs (see following text). The present result that a mRNA cap analogue infused into PCs also blocked LTD induction (Fig. 6) strongly supports the view that postsynaptic protein synthesis is required for LTD induction.

There are three factors to be considered in relation to the different protein synthesis dependencies of PCs in cerebellar slices and cultures. 1) The LTD-inducing stimulating conditions are considerably different between the two types of PC preparations. In cerebellar slices, LTD induction requires application of 300 pulses to GAs and CFs, while two to six conjunctive stimulations by a glutamate pulse and a 3-s membrane depolarization to 0 mV are sufficient in cultured PCs. 2) The time courses of LTD are also considerably different. LTD in cerebellar slices develops slowly over 1 h (Fig. 2), while the reduced form of LTD in cultured PCs develops rapidly to reach a plateau within a few minutes (Linden 1996). And 3) signal transduction processes underlying LTD also show certain differences. It is known that LTD induction in tissue cultures does not require either nitric oxide (Linden et al. 1995) or inositol trisphosphate (Narasimhan et al. 1998), which are required for LTD induction in cerebellar slices (Boxall and Garthwaite 1996; Inoue et al. 1998; Shibuki and Okada 1991). The corticotropin-releasing factor (CRF) contained in CFs, which is indispensable for LTD induction in cerebellar slices (Miyata et al. 1999), could be absent in tissue cultures lacking CFs. Thus the two forms of LTD, which are induced under different stimulation conditions and which developed with different time courses, could be underlain by signal transduction processes to which protein synthesis contributes differently.

The protein-synthesis-dependency, however, may not uniform between the early and late phases of LTD in PCs of cerebellar slices because when translational inhibitors were applied 20-30 min before the onset of conjunction, the late phase of LTD was in most cases significantly depressed (Fig. 4A), whereas the early phase of LTD was insignificantly affected (P > 0.05, Fig. 4B). It appears that the early phase of LTD is less sensitive to translational inhibition.

Roles of a rapidly turned over protein in LTD induction

Since the translational inhibitors suppressed the entire LTD including its early phase (Figs. 2 and 3), one may assume that the protein required for LTD induction is rapidly synthesized during conjunctive stimulation. Such synaptically driven rapid protein synthesis is possible. For example, Weiler et al. (1997) have recently reported that stimulation of type-1 metabotropic glutamate receptors in brain tissues triggered a rapid association of mRNAs of the Fragile-X mental retardation protein (FMRP) with the translational machinery within 1-2 min, resulting in an increased expression level of FMRP near synapses. Huber et al. (2000) suggest that the synthesis of proteins required for the induction of mGluR5-mediated LTD in hippocampal neurons occurs within minutes after cessation of LTD-inducing stimulation.

An alternative possibility is that the protein(s) required for the initial development of LTD is constitutively present and maintained critically at a level required for the induction of LTD by an unusually high turnover rate against fast degradation. When translational inhibitors block protein synthesis, the level of the protein(s) would decrease, resulting in blockade of LTD induction. In either case, the protein(s) is rapidly turned over, however, the difference between the two possibilities is whether production of the protein(s) depends on the LTD-inducing stimulation or not.

The translational inhibitors still blocked LTD induction even when applied 10 min, but not 15 min or more, after the onset of conjunctive stimulation (Figs. 3E, 4, and 5). This indicates that protein synthesis is required for the initial development of LTD within 15 min after the onset of conjunctive stimulation but not for its further development or maintenance. Therefore contrary to the current presumption that protein synthesis is required for the development and maintenance of the late phase of LTP or LTD, the present results indicate that a rapidly turned over protein(s) is required for the initial development of LTD only within 15 min following the onset of conjunctive stimulation. During this critical period, there could be a regenerative process such as the autophosphorylation of CaM kinase type II that has been suggested to be involved in the initial phase of LTP (Giese et al. 1998). The self-regeneration must be transformed to a more stable process based on a change in receptors or related molecular structures in synapses (Lisman and Falton 1999). Thereafter, LTD would continually be enhanced even if the protein synthesis is inhibited due to the takeover by a more structurally based stable process, for example, the recently demonstrated clathrin-mediated endocytosis (Wang and Linden 2000).

Sites of translational and transcriptional inhibition

We have reported previously that translational inhibitors rapidly suppress mGluR1-EPSPs in PCs (Karachot et al. 2000); the peak amplitude of mGluR1-EPSPs decreased to 50-60% during 5-min applications of anisomycin (55%), puromycin (51%), and cycloheximide (62%). This rapid onset of the effect of translational inhibitors suggests that the translation required for the generation of mGluR1-EPSPs and presumably also for LTD induction occurs locally near the synapses under observation. Nevertheless the relative contribution of somatic and dendritic translations to mGluR1-EPSPs and LTD should be further investigated.

Our previous study (Karachot et al. 2000) also revealed that it took 30 min to obtain a similar extent of decrease to translational inhibitors after 5-min pulse applications of actinomycin D (57%) and DRB (52%). It appears that LTD induction is blocked when the peak amplitude of mGluR1-EPSPs decreases to 50-60% regardless of the time course of the decrease. This close correspondence between the observations on LTD and mGluR1-EPSPs is explicable if we assume that both mGluR1-EPSPs and LTD depend on the local concentrations of the same protein(s).

The time course of the depletion of the local protein(s) under transcriptional inhibition will depend on the rate of transportation of mRNAs from the transcription site in the nucleus to the translation site in somata and/or dendrites and that of proteins so produced from the latter site to the dendritic synapses under observation as well as on the rate of degradation of the mRNAs and protein(s). It is difficult to precisely relate the presently obtained time lag of 30 min to these factors because of lack of relevant data. It may, however, be pointed out that the rate 242 µm/h measured for dendritic transport of mRNAs (Muslimov et al. 1997) is too slow because it allows mRNAs to travel a distance of only 120 µm even if the entire 30-min lag time is spent for the transport. Since motor proteins, such as kinesin and dynein superfamily proteins, are transported along microtubules at rates of up to 4,320 µm/h (720 µm/10 min) (Hirokawa 1998), it is possible that the mRNAs and/or protein(s) required for LTD induction travel faster and reach remote sites of PC dendrites within a fraction of the 30-min time lag.

Relationship with metabotropic glutamate-mediated transmission

Because the translational inhibitors did not affect appreciably, the GA-evoked EPSPs or the CF-evoked responses consisting of EPSPs and Ca2+ spikes (Fig. 1, E-H), the maintenance of ionotropic glutamate receptors or voltage-dependent Ca2+ channels involved in the generation of these electric signals does not require rapidly turned over proteins. On the other hand, as reported previously (Karachot et al. 2000), generation of slow mGluR1-EPSPs in PCs requires a rapidly turned over protein(s). Because an antagonist (Hartell 1994), an antibody (Shigemoto et al. 1994) and gene deficiency (Aiba et al. 1994; Conquet et al. 1994) against mGluR1s block LTD induction, it may appear that translational and transcriptional inhibitors block LTD induction by inactivating mGluR1s. However, inactivation of mGluR1s with AIDA did not parallel the translational-inhibitor-caused blockade of LTD induction in terms of the time course of their sensitive phases following conjunctive stimulation (compare Figs. 3D and 5 and Fig. 10, C and D).

As suggested in the preceding text, rapid protein synthesis appears to occur commonly in the signal transduction pathways linking mGluR1s to both the generation of mGluR1-EPSPs and induction of LTD. The well-known pathway linking mGluR1s to phospholipase C (PLC) via Gq proteins in turn activates signal transduction cascades resulting in production of diacylglycerol and protein kinase C, as well as inositol trisphosphate, that eventually causes the release of Ca2+ ions from intracellular stores. These PLC-mediated pathways are involved in the induction of LTD (Inoue et al. 1998; Linden and Connor 1991), but not in the generation of mGluR1-EPSPs (Hirono et al. 1998; Tempia et al. 1998). Therefore it is unlikely that the PLC pathways involve the presently postulated rapid protein synthesis. The protein synthesis required for LTD induction should occur in another, yet unidentified pathway, at its stage of activation of G proteins or further downstream processes such as activation of effector molecules, second messengers, and/or their target molecules.


    FOOTNOTES

Address for reprint requests: M. Ito, Laboratory for Memory and Learning, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan (E-mail: Ito-BSI{at}brain.riken.go.jp).

Received 24 July 2000; accepted in final form 10 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society