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Department of Physiology and Pharmacology and the Robert F. Furchgott Center for Neural and Behavioral Sciences, State University of New York Downstate Medical Center, Brooklyn, New York
Submitted 17 April 2007; accepted in final form 7 January 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Chuang et al. 2001, 2005; Lee et al. 2002; Sayin and Rutecki 2003; Young et al. 2004; Zhao et al. 2004) and long-term potentiation (LTP; Anwyl 1999; Bortolotto et al. 1999). The mechanisms of the mGluR contribution to these phenomena are understood to some extent, and may involve suppression of the post action potential afterhyperpolarization (AHP). Group I mGluR-induced epileptiform burst generation is facilitated by suppression of the AHP and by induction of a voltage-dependent inward current (Chuang et al. 2001; Martin et al. 2001; Young et al. 2004). LTP can be facilitated, or primed, by the excitability increase that follows from mGluR block of the AHP (Cohen et al. 1999; Sourdet et al. 2003). Moreover, the importance of AHP suppression by various transmitters to increased excitability and learning in a number of brain areas is widely reported (Moyer Jr. et al. 1996; Oh et al. 2003; Saar et al. 1998, 2001; Thompson et al. 1996). In particular, prolonged AHP suppression has been associated with hippocampus-dependent learning (Ohno et al. 2006; Zelcer et al. 2006; Zhang and Linden 2003). Thus it is of considerable interest that group I mGluR-induced AHP suppression may persist for extended periods after an agonist has been washed out (Cohen et al. 1999; Ireland and Abraham 2002). At present, the signaling pathways that lead to long-term group I mGluR-mediated effects are not well understood. This paper attempts to further that understanding by experimentally distinguishing between transient and persistent AHP suppression by group I mGluRs and by showing that persistent block of the AHP involves additional steps that need not be activated when the AHP is suppressed in the presence of agonist.
Both medium AHPs (mAHPs), which, in CA3, are mediated predominantly by the voltage-sensitive, intracellular Ca2+-insensitive M-current, and slow AHPs (sAHPs), which are mediated by an unidentified Ca2+-sensitive channel, are present in CA3 pyramidal cells (Storm 1990; Vogalis et al. 2003). They contribute to spike frequency adaptation and the suppression of both AHPs has been implicated in epileptogenesis (Cooper et al. 2000; Fernandez de Sevilla et al. 2006; Martin et al. 2001; Peña and Alavez-Pérez 2006; Tallent et al. 2001). The mAHP is readily apparent in CA3 pyramidal cells following single action potentials, whereas sAHPs become prominent only after repeated spikes. We examined the suppression of both mAHPs and sAHPs by the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) and addressed the conditions that led to their persistent suppression.
We found that persistent AHP suppression was induced by extended agonist exposures (30 min, but not 5 min) and that persistence was selectively inhibited at reduced temperature. Pretreatment of slices with protein synthesis inhibitors did not affect the suppression of AHPs during DHPG exposure but did prevent persistence of the AHP suppression. The p38 MAP kinase inhibitor SB 203580 acted similarly and persistent AHP suppression appeared to be selectively sensitive to the mGluR5-specific blocker MPEP. We conclude that there are two components of group I mGluR-mediated AHP suppression with different time courses and having different mechanisms. The first component appeared on DHPG application. It was reversible and was insensitive to temperature variations between 25–26 and 30–31°C. The second component appeared only after sufficiently prolonged activation of mGluR5 and was highly sensitive to reduced temperature. This second component was protein synthesis and p38 mitogen-activated protein (MAP) kinase dependent and locked in the group I mGluR-induced AHP suppression.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Animal use procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the State University of New York Downstate Medical Center. Transverse hippocampal slices (
300 µm thick) were prepared as previously described (Bianchi and Wong 1995). Briefly, guinea pigs (250 to 350 g) were anesthetized with halothane by inhalation and decapitated in conformity with the IACUC guidelines (protocol number 0619404). The heads were removed. Hippocampi were dissected and sliced in ice-cold artificial cerebrospinal fluid (aCSF; see following text) using a vibratome (Vibratome, St. Louis, MO). Slices were stored in aCSF at 30°C for 0.5 h and then at room temperature for 
0.5 h before use. One slice at a time was placed, submerged, in a recording chamber that was perfused at 3 to 5 ml/min with aCSF and maintained at 30–31°C (or at 25–26°C for low-temperature recordings). Artificial CSF consisted of (in mM): 124.0 NaCl, 26.0 NaHCO3, 5.0 KCl, 1.6 MgCl2, 2.0 CaCl2, and 10.0 D-glucose, and was maintained at pH 7.4 by bubbling with 95% O2-5% CO2. Slices were held against the coverslip bottom of the chamber by nylon threads that were stretched across a platinum ring. Slices were thus prevented from moving while allowing solution exchange on the underside of the slice. The recording chamber was held in a steel plate attached to the mechanical stage of a Nikon Diaphot microscope. The various micromanipulators were attached to the same plate.
Electrophysiological recordings
Conventional electrophysiological recording techniques were used and have been described previously (Bianchi et al. 1999). Glass micropipettes pulled from thin-walled capillaries (TW100F; World Precision Instruments, Sarasota, FL) and filled with 2 M potassium acetate (resistances typically 30–50 M
) were used for current-clamp recordings of CA3 pyramidal cells. Voltage signals were amplified and intracellular current was injected using an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA). Recordings were displayed on an oscilloscope (DSO 400, Gould Instruments, Valley View, OH) and chart recorder (Gould TA240), stored on FM tape (Store 4DS, Racal, Southampton, UK), low-pass filtered (eight-pole Bessel, –3 dB at 1 kHz), and sampled at 5 kHz for storage and later analysis by computer (pCLAMP6, TL-1; Molecular Devices). Each cell was maintained at a given preset hyperpolarized membrane potential (–67.1 ± 0.7 mV, mean ± SE; n = 31) with current injection so that the increased activity following introduction of DHPG would not raise spontaneous spike frequency to the point that AHPs were difficult to isolate. For each independent experiment, the membrane potential was preset to a value that varied within 2 mV (membrane potentials are labeled next to the traces shown in all figures). In cases where the membrane potential chosen prior to addition of DHPG did not prevent high spontaneous spike rates during and after DHPG, or where doublet or triplet spiking following DHPG could not be avoided, the data were not used. Hyperpolarizing the cells also reduced the appearance of a voltage-dependent, group I mGluR-induced afterdepolarization (Young et al. 2004). Single action potentials, used to elicit mAHPs, were triggered by 3- to 4-ms square-current pulses of 0.2 to 2.5 nA. Action potential bursts, used to stimulate sAHPs, were triggered by 100-ms square-wave current injections in the same amplitude range. Slow AHPs were monitored less frequently than mAHPs, but because sAHPs were less affected by spontaneous activity (see Fig. 3A), they could often be monitored for a longer time (Figs. 3 and 4). Hyperpolarizing current pulses (–0.2 to –0.5 nA; 150 ms) were applied periodically to monitor cell input resistance and to adjust the bridge balance. Input resistance (in M) was calculated as: amplitude of the voltage deflection at the end of a 150-ms hyperpolarizing current injection (mV) divided by the amplitude of the current (nA).
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Control solution contained 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) and 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 µM) in aCSF to block ionotropic glutamate receptors. Group I mGluRs were activated using the specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 10–50 µM), which was applied by addition to the superfusing solution. DHPG applications were for either 1–5 min (brief, <5 min), beginning when DHPG entered the recording chamber (evidenced by depolarization and cell firing), or 30 min. A few intermediate exposure times yielded intermediate durations of AHP suppression but were not investigated further. In some experiments (Fig. 5), protein synthesis was blocked using 4-[2-(3,5-dimethyl-2-oxo-cyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione (cycloheximide) or (2R,3S,4S)-2-[(4-methoxyphenyl)methyl]-3,4-pyrrolidinediol 3-acetate (anisomycin) at final concentrations of 100 and 15 µM, respectively. Anisomycin was stored as 15 mM aliquots at –20°C in DMSO. Slices were incubated in either cycloheximide or anisomycin for 1 h prior to recording and remained in the drug solution for the duration of the recording. We used 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 98059) to block the p42/44 MAP kinase pathway (Fig. 6). PD 98059 was made up as a stock solution in DMSO (25 mM), stored at –20°C, and used at 50 µM in aCSF. The p38 MAP kinase pathway was blocked using the selective inhibitor 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imadazol-4-yl]pyridine hydrochloride (SB 203580, 5–20 µM; Cuenda et al. 1995; Wang et al. 2007). Slices were pretreated with kinase blockers for 1 h prior to recording. Block of mGluR1 or mGluR5 was accomplished using (S)-(+)-
-amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385, 100 µM) or 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP, 50 µM), respectively, in aCSF. DHPG, CNQX, CPP, cycloheximide, anisomycin, PD 98059, SB 203580, LY367385, and MPEP were purchased from Tocris Bioscience (Ellisville, MO). All other chemicals were from Sigma–Aldrich (St. Louis, MO).
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All measurements were made in the presence of CNQX and CPP. Medium AHPs were measured by subtracting the baseline membrane potential prior to the depolarization from the membrane potential 150 ms postspike (see arrows in Figs. 1, 2 and 5–7
). Slow AHPs were elicited by action potential bursts triggered by 100-ms depolarizations. The sAHP was measured by subtracting baseline membrane potential from the minimum membrane potential between 200 and 700 ms postpulse (see Figs. 3–7). These definitions of mAHPs and sAHPs are not mutually exclusive. A small sAHP occurs after single action potentials (–1 mV at 700 ms postspike, returning to baseline in 2.8 s; Young et al. 2004) and mAHPs also occur following bursts. However, most of the mAHP had decayed by 200 ms post spike (mAHP half-width = 185 ± 53 ms; Young et al. 2004) and the sAHP did not peak until 299 ± 21 ms (see RESULTS), allowing for temporal separation of the potentials. More important, however, is that the multiple action potentials during a burst selectively augmented the sAHP over the mAHP. AHP amplitudes, reported as percentage of control, were normalized by control AHP amplitudes in their respective cells. Control AHP amplitude was the mean of measurements made prior to DHPG application. Afterdepolarizations that had not fully returned to baseline by the point of measurement were recorded as negative percentages. Data pooled across cells are reported as means ± SE.
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). These time courses were analyzed using a general linear mixed model (SPSS v. 13; SPSS, Chicago, IL). AHP amplitude as a function of drug washout time and its interaction with temperature, or duration of DHPG exposure, were tested using a repeated-measures ANOVA (reported as P values). Comparisons of AHP amplitudes between groups at particular time points were made using post hoc tests within the ANOVA (reported as P values). Additionally, within the ANOVA, a repeated-measures regression was performed and an estimation made of regression parameters (slopes and y-intercepts). The slopes of regressions of AHP recovery over time confirmed the interaction of washout time with temperature or length of DHPG treatment. In all cases, the y-intercepts supported the comparison by t-test of the degree of suppression by DHPG. Statistical comparisons of data shown in the bar graphs of Figs. 6, 7, and 8 were made using a two-way ANOVA followed by Newman–Keuls post hoc analysis. P values of <0.05 were regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; n = 28). Stimulus duration dependence
We first exposed guinea pig hippocampal slices to varying concentrations of DHPG from 10 to 50 µM and to exposure times from 1 to 30 min. DHPG at any concentration between 10 and 50 µM caused suppression of the AHP (both medium and slow) and there was no marked relationship between DHPG concentration and persistence of AHP suppression after drug washout. In contrast, varying the duration of DHPG treatment had a dramatic effect on duration of the AHP block. After a brief (<5 min) exposure to DHPG, suppressed AHPs often recovered within 20 to 30 min after agonist washout. When DHPG treatments were extended to 30 min, AHP suppression persisted after agonist washout for the duration of the recording (
100 min; see e.g. Fig. 1B). Figure 1A illustrates the block and subsequent recovery of mAHPs following single action potentials in a cell exposed to brief DHPG treatment, as contrasted to the AHP block and absence of recovery in a cell treated with DHPG for 30 min. Figure 1B shows the time course of mAHP amplitude (as percentage of control) during control, DHPG exposure, and DHPG washout, for the same two cells. Figure 1C summarizes the data from 21 cells: 11 cells treated with DHPG for <5 min and 10 cells treated for 30 min. AHPs were triggered by single action potentials produced by 3- to 4-ms depolarizing pulses. Medium AHP amplitude was measured at 150 ms after the spike (METHODS) and was expressed as percentage of control mAHP amplitude for each cell.
The mAHP was suppressed by DHPG and the degree of mAHP suppression was not significantly different between short and long DHPG exposures. AHPs were suppressed to –27 ± 18.5% of control (n = 11) for the 5-min treatment and to –8.5 ± 16.9% of control (n = 10) for the 30-min treatment. Baseline membrane potentials were –65.6 ± 0.8 mV (n = 11) for 5-min DHPG and –66.8 ± 0.8 mV (n = 10) for 30-min DHPG treatments. Control values of mAHP amplitude also did not differ significantly in the two cases [–2.5 ± 0.3 mV (n = 11) for 5-min DHPG treatment; –2.5 ± 0.4 mV (n = 10) for 30-min DHPG treatment]. However, the recovery of the mAHP after brief DHPG treatment was significantly faster than that after 30-min DHPG treatment (interaction of drug washout time and DHPG treatment duration, P < 0.001; METHODS). By 25 min after DHPG washout, and from that point on, mAHP amplitude was significantly larger (P 0.007) after a brief DHPG treatment than it was after 30 min of DHPG. Medium AHP amplitudes during DHPG washout were also fitted with linear regressions (METHODS). The recovery rate after 5 min, but not that after 30 min of DHPG, was significantly greater than zero (P < 0.001 vs. P = 0.07). Thus after a 5-min DHPG treatment, the suppressed mAHP had fully recovered by about 30 min of washout. After a 30-min treatment with DHPG, the mAHP was similarly suppressed and did not significantly recover.
Temperature dependence
The results illustrated in Fig. 1 were obtained from experiments performed at 30–31°C. To test whether reduced temperature might point to differences in the signaling mechanisms of transient and persistent AHP suppression, we compared these data with results from another block of experiments done at 25–26°C. In these experiments, DHPG exposure time was fixed at 30 min. Figure 2A shows examples of AHPs from one cell following single action potentials before, during, and after a 30-min DHPG treatment at 25–26°C. Figure 2B plots the time course of the changes in mAHP amplitude for that cell. Figure 2C shows summary data from DHPG treatments at 25–26 and 30–31°C. At 25–26°C, mAHP amplitude recovered from suppression by DHPG significantly faster than in cells maintained at 30–31°C (interaction of drug washout time and temperature, P = 0.01). Medium AHP amplitudes recorded at the two temperatures were significantly different from 15 min of washout onward (P 0.01). Control values of mAHP amplitude were not significantly different between 25–26 and 30–31°C. At 25–26°C, the control mAHP amplitude was –2.9 ± 0.4 mV (n = 10). At 30–31°C, the control mAHP was –2.5 ± 0.4 mV (n = 10). Baseline membrane potentials were –67.7 ± 1.1 mV (n = 10) at 25–26°C and –66.8 ± 0.8 mV (n = 10) at 30–31°C. The degree of suppression by DHPG was also not significantly different at the two temperatures. During DHPG treatment, the mAHP was 8.7 ± 11.2% of control (n = 10) at 25–26°C and –17.4 ± 15.9% (n = 10) at 30–31°C. Thus lowering the temperature by 5°C did not affect suppression of the mAHP during DHPG exposure, but markedly decreased the persistence of mAHP suppression following agonist washout.
Slow AHP
Slow AHPs are small following single action potentials, but are robust and have a prolonged time course after action potential bursts. We triggered bursts with 100-ms depolarizations and measured peak sAHP amplitude between 200 and 700 ms postpulse. In contrast to the mAHP, where no marked differences were seen between amplitude or time course at 25–26 and 30–31°C, the time course of the sAHP was significantly slowed at the lower temperature. Time to peak was 299.0 ± 21.2 ms at 30–31°C and 600.0 ± 16.4 ms at 25–6°C (P < 0.001, n = 27). This slowing is consistent with previous reports (Lee et al. 2005; Li et al. 2002; Sah and McLachlan 1991). Lowering the temperature did not cause statistically significant changes in either control sAHP amplitude [at 30–31°C: –8.3 ± 0.8 mV (n = 12); at 25–26°C: –7.6 ± 0.5 mV (n = 10)] or input resistance [at 30–31°C: 45.3 ± 4.3 M (n = 13); at 25–26°C: 53.3 ± 4.5 M (n = 12)]. Figure 3 shows the suppression of sAHPs by 30-min treatment with DHPG. As with the mAHP, recovery of sAHP amplitude following DHPG washout was significantly faster at 25–26 than at 30–31°C (interaction of DHPG washout time with temperature, P = 0.04). Slow AHP amplitudes were significantly greater at 25–26 than at 30–31°C from 45 min of washout on (P 0.04). Also as with the mAHP, the degree of suppression during DHPG exposure was not significantly different between 25–26 and 30–31°C. Slow AHP amplitude during DHPG was 30.9 ± 4.4% of control (n = 9) at 25–26°C and 24.5 ± 13.4% (n = 9) at 30–31°C. Baseline membrane potentials were –67.3 ± 0.8 mV (n = 9) at 25–26°C and –66.9 ± 1.2 mV (n = 9) at 30–31°C.
Figure 4 compares recovery of the sAHP following suppression by 5- or 30-min DHPG treatments. Similar to the mAHP, sAHP suppression reversed following short DHPG treatments, but persisted following 30-min exposures (interaction of drug washout time and DHPG treatment duration, P = 0.008; Fig. 4B). The sAHPs in cells treated for 5 min with DHPG were significantly larger than those in cells treated for 30 min by 45 min of washout and remained larger thereafter (P 0.003). The degree of sAHP suppression produced by the two treatments did not differ significantly. Measurements at 15 min of washout were used to approximate the degree of suppression by DHPG. There was no significant difference in suppression between 5-min (19.0 ± 9.2% of control; n = 6) and 30-min (35.1 ± 15.4%; n = 9) treatment groups. Control sAHP amplitude was –8.9 ± 0.5 mV (n = 6) for the 5-min DHPG treatment and –9.0 ± 0.9 mV (n = 9) for 30-min treatments. Baseline membrane potentials were –66.9 ± 0.3 mV (n = 6) in cells treated for 5 min and –66.9 ± 1.2 mV (n = 9) in cells treated for 30 min.
Thus the mechanism(s) for persistent suppression appeared similar for the medium and slow AHPs, in that they shared a similar dependence on the duration of agonist exposure and a similar sensitivity to temperature.
Effects of protein synthesis inhibitors
Following pretreatment with, and in the presence of, cycloheximide or anisomycin, mAHPs were evoked by single action potentials and sAHPs by bursts of action potentials (Fig. 5). Mean AHP values recorded in the presence of cycloheximide (mAHP: –2.7 ± 0.7 mV, n = 5; sAHP: –9.4 ± 1.2 mV, n = 5; Fig. 5, A and B, Control) were not significantly different from those recorded in control solution in the absence of cycloheximide (mAHP: –3.0 ± 0.5 mV, n = 5; sAHP: –8.1 ± 1.0 mV, n = 6). Application of DHPG for 30 min at 30–31°C suppressed both AHPs (e.g., Fig. 5, A–D), comparably to experiments carried out in control solution. In the presence of DHPG, mAHPs in cycloheximide were suppressed by 106.2 ± 12.0% (n = 5; Fig. 5E, cycloheximide, black bar), a value not significantly different from that obtained in the absence of cycloheximide (94.4 ± 3.2%; n = 5; Fig. 5E, aCSF, black bar). Figure 5F shows that in cycloheximide DHPG suppressed sAHPs by 70.4 ± 7.6% (n = 5, cycloheximide, black bar) as opposed to 89.2 ± 16.5% in control solution (n = 6, aCSF, black bar). Likewise, in anisomycin, neither the mAHP nor the sAHP amplitudes before DHPG application differed from those recorded in control solution (mAHP: –3.2 ± 0.7 mV, n = 5; sAHP: –9.7 ± 0.7 mV, n = 5). The degree of AHP suppression by DHPG was also unaffected by the presence of anisomycin (mAHP: 108.2 ± 17.0%; n = 5; Fig. 5E, anisomycin, black bar; sAHP: 85.6 ± 5.5%; n = 5; Fig. 5F, anisomycin, black bar). However, persistence of AHP suppression by DHPG was blocked in cells exposed to cycloheximide or anisomycin (Fig. 5, E and F). The AHP suppression remaining at 30–60 min following DHPG washout in the presence of protein synthesis inhibitors at 30–31°C (Fig. 5E: mAHP in cycloheximide, gray bar: 16.7 ± 10.5%; n = 5; and mAHP in anisomycin, gray bar: 12.8 ± 9.5%; n = 5; Fig. 5F: sAHP in cycloheximide, gray bar: 7.1 ± 9.5%; n = 5; and sAHP in anisomycin, gray bar: 19.7 ± 8.6%; n = 5) was significantly smaller (P < 0.01) than that measured in the absence of inhibitors (Fig. 5E: mAHP in aCSF, gray bar: 86.5 ± 6.5%; n = 5; and sAHP in aCSF, gray bar: 89.2 ± 16.5%; n = 6). The remaining AHP suppression in protein synthesis inhibitors was not significantly different from that obtained in cells recorded at the reduced temperature of 25–26°C in control solution (mAHP suppression: 24.6 ± 6.9%; n = 5; and sAHP: 8.0 ± 6.5%; n = 6).
Effects of MAP kinase inhibitors
Having determined that the additional pathway elements leading from transient to persistent AHP suppression included synthesis of new protein, we next attempted to identify pathway intermediates that led to the required protein synthesis. Two sets of experiments were done in which MAP kinases were inhibited with either PD 98059 on the one hand, or SB 203580 on the other, which inhibit activities of p42/44 or p38 MAP kinases, respectively (Cuenda et al. 1995; Dudley et al. 1995; Wang et al. 2007). Pretreatment of slices for 1 h with PD 98059 had no significant effect on the persistence of AHP suppression caused by a 30-min exposure to DHPG (see following text). However, blocking p38 MAP kinase with SB 203580 significantly shortened the time course of AHP suppression (see following text).
Neither PD 98059 nor SB 203580 affected the amplitude of AHPs in control solution (for slices incubated in PD 98059, Fig. 6, A and B, Control: mAHP, –2.1 ± 0.4 mV, n = 5; sAHP, –7.1 ± 1.7 mV, n = 4; for slices incubated in SB 203580, Fig. 6, A and B, Control: mAHP, –3.8 ± 0.5 mV, n = 5; sAHP, –8.5 ± 0.7 mV, n = 6). Nor was the suppression of AHPs during DHPG treatment affected by either of the MAP kinase blockers (for slices incubated in PD 98059, Fig. 6, E and F, black bars: mAHP, 103.1 ± 9.4%, n = 5; sAHP, 91.8 ± 11.0%; n = 4; for slices incubated in SB 203580, Fig. 6, E and F, black bars: mAHP, 96.3 ± 13.8%, n = 5; sAHP, 84.3 ± 7.7%, n = 6). In addition, PD 98059 had no effect on AHP recovery during washout of DHPG. For slices incubated in PD 98059 (Fig. 6, E and F, gray bars), AHP suppression 30–60 min after washout of DHPG were: mAHP, 82.9 ± 6.4% (n = 5); sAHP, 68.6 ± 10.2% (n = 4). Inhibition of p38 MAP kinase, however, blocked persistence of AHP suppression (Fig. 6, E and F, SB 203580, gray bars). Suppression of both mAHPs and sAHPs was significantly reduced after DHPG washout in the presence of SB 203580: mAHP, 28.5 ± 2.5% (P < 0.01; n = 5); sAHP, 16.7 ± 6.5% (P < 0.01; n = 5).
Involvement of mGluR1 and mGluR5 in persistent AHP suppression
The two group I mGluRs, mGluR1 and mGluR5, may activate different physiological responses (e.g., Chuang et al. 2002; Mannaioni et al. 2001). For example, mGluR5 is thought to be selectively responsible for mGluR-LTD in CA1 (Anwyl 2006; Fitzjohn et al. 1999). Thus a final set of experiments was performed to test whether mGluR1 and mGluR5 contribute equally to AHP suppression and its persistence following 30-min DHPG treatment. Either mGluR1 or mGluR5 was blocked using LY 367385 or MPEP, respectively (METHODS). Figure 7 shows that blocking mGluR5, but not mGluR1, significantly reduced persistence of AHP suppression.
Neither MPEP nor LY 367385 affected AHP amplitude in control solution or suppression by DHPG. AHP amplitudes in control solution (Fig. 7, A and B, Control) were: mAHP, –3.1 ± 0.8 mV, n = 5; sAHP, –7.9 ± 0.6 mV, n = 5, for slices in MPEP; mAHP, –2.9 ± 0.1 mV, n = 5; sAHP, –6.3 ± 1.6 mV, n = 5, for slices in LY 367385. During DHPG treatment, AHP suppression was not affected by either blocker (for slices in MPEP, Fig. 7, E and F, black bars: mAHP, 112.2 ± 13.7%, n = 5; sAHP, 93.6 ± 7.2%, n = 5; and for slices in LY 367385, Fig. 7, E and F, black bars: mAHP, 105.6 ± 13.0%, n = 5; sAHP, 88.6 ± 8.7%, n = 5). At 30–60 min of DHPG washout, AHP suppression was clearly reduced in slices treated with MPEP compared with slices either without blockers or treated with LY 367385 (for slices in MPEP, Fig. 7, E and F, gray bars: mAHP, 13.2 ± 9.3%, n = 5, P < 0.01; sAHP, 16.2 ± 4.1%, n = 5, P < 0.01; for slices in LY 367385, Fig. 7, E and F, gray bars: mAHP, 86.2 ± 15.1%, n = 5; sAHP, 69.8 ± 9.7%, n = 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Ireland and Abraham (2002) showed, in addition, that the AHP suppression remaining in the presence of tyrosine phosphatase inhibitors persisted after agonist washout. Here, we focus on stimulus conditions and signaling mechanisms underlying persistent AHP suppression. Unlike transient suppression, which was observed under all experimental conditions tested, persistent AHP suppression by DHPG occurred only when DHPG treatment duration was adequately long and the bath temperature was sufficiently high. The more stringent experimental conditions required for persistent AHP suppression were paralleled by additional requirements for p38 MAP kinase and protein synthesis signaling for its induction. Finally, we found that although either mGluR1 or mGluR5 could mediate transient AHP suppression, activation of mGluR5, but not mGluR1, was necessary and sufficient to elicit long-lasting AHP suppression.
Most previous studies have shown that agonist stimulation of group I mGluRs elicited transient suppression of AHPs, with AHPs recovering within minutes following the activating stimulus (Abdul-Ghani et al. 1996a,b; Desai and Conn 1991; Gereau 4th et al. 1995; Heuss et al. 1999; Hu and Storm 1991; Nouranifar et al. 1998). However, persistent group I mGluR-mediated AHP suppression has also been reported (Cohen et al. 1999; Ireland and Abraham 2002) and it has been suggested that persistent AHP suppression can be induced only with sufficiently long stimulus durations (Ireland and Abraham 2002). The present results support this suggestion. Moreover, our data suggest that the variations in temperature used by different investigators must also be taken into account to explain the variability regarding persistence of AHP suppression by DHPG. A majority of the studies were conducted at room temperature (19–22°C) or at <28°C (Abdul-Ghani et al. 1996a,b; Desai and Conn 1991; Gereau 4th et al. 1995; Heuss et al. 1999; Nouranifar et al. 1998), whereas others were conducted at 30 or 32°C (Cohen et al. 1999; Hu and Storm 1991; Ireland and Abraham 2002). Our data indicate that persistent AHP suppression could not be induced at the low temperature of 25–26°C. Indeed, data obtained by the groups listed earlier using temperatures <28°C reported transient AHP suppression following group I mGluR stimulation. Our results also suggest that experimental temperature is an important parameter in the study of group I mGluR-mediated plasticity processes.
The temperature sensitivity of persistent AHP suppression may be due to the suppression of some biochemical processes necessary for its induction. The requirement of protein synthesis for persistent AHP suppression (Fig. 5) and the relatively complex biochemical process associated with group I mGluR-induced protein synthesis (Klann and Dever 2004) suggest that this is the temperature-sensitive step. Indeed, studies on hypothermia protection for hypoxic-ischemic brain injury have shown suppression of protein synthesis, at both the transcriptional and the translational levels. This attenuation of protein synthesis was considered to afford protection from hypoxic-ischemic injury (Fukui et al. 2006).
Protein synthesis plays a role in several forms of group I mGluR-induced plasticity in addition to suppression of the AHP. mRNA translation is required for the induction of group I mGluR-mediated epileptogenesis in hippocampus (Merlin et al. 1998). Similarly, group I mGluR-induced LTP in hippocampus (Raymond et al. 2000) and LTD in hippocampus (Huber et al. 2000, 2001) and cerebellum (Linden 1996; Zhang and Linden 2006) are translation dependent. Parallel studies showed that group I mGluR stimulation elicited activation of p42/44 MAP kinase (ERK 1/2) in hippocampus and cerebellar preparations and that inhibition of p42/44 MAP kinase prevented the plastic changes (Banko et al. 2006; Endo and Launey 2003; Gallagher et al. 2004). A general consensus is that group I mGluR induces protein translation by activating the p42/44 MAP kinase signaling pathway (Banko et al. 2006). Based on these studies, we examined whether protein synthesis underlying persistent AHP suppression was also activated via the p42/44 MAP kinase pathway. Unexpectedly, blocking p42/44 MAP kinase activation had no effect, but inhibition of p38 MAP kinase effectively prevented persistent AHP suppression (Fig. 6). Inhibition of p38 MAP kinase has also been shown to block DHPG and synaptic group I mGluR-induced LTD in neonatal area CA1 (Bolshakov et al. 2000; Huang et al. 2004) as well as LTD induced by DHPG in the hippocampal dentate gyrus (Rush et al. 2002). Since activation of p38 MAP kinase has been shown to stimulate translation or transcription underlying neuronal plasticity (Guan et al. 2003; Klann and Dever 2004), it is possible that activation of this kinase links group I mGluR stimulation to de novo protein synthesis for the induction of persistent AHP suppression (Fig. 8).
Finally, our data reveal that persistent AHP suppression was induced by mGluR5, but not mGluR1. In this regard, it is interesting to know that mGluR5, but not mGluR1, has been implicated in the induction of LTD in CA1 (Bolshakov et al. 2000; Huber et al. 2000, 2001; Palmer et al. 1997). Moreover, p38 MAP kinase has frequently (Bolshakov et al. 2000; Huang et al. 2004; but see Gallagher et al. 2004) been reported to play a necessary role in LTD in CA1. Our data do not provide any explanation for the specificity of mGluR5 in the induction of persistent AHP suppression. However, at least two speculations can be raised: a spatial proximity of mGluR5 (over mGluR1) to the signaling cascade for AHP suppression or a specific coupling of mGluR5 to p38 MAP kinase activation and associated downstream signaling over that of mGluR1.
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Address for reprint requests and other correspondence: S. R. Young, Department of Physiology and Pharmacology Box 29, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203 (E-mail: syoung{at}downstate.edu)
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