Dopamine receptors and groups I and II mGluRs cooperate for long-term depression induction in rat prefrontal cortex through converging postsynaptic activation of MAP kinases

Abstract

Tetanic stimuli to layer I-II afferents in rat prefrontal cortex induced long-term depression (LTD) of layer I-II to layer V pyramidal neuron glutamatergic synapses when tetani were coupled to bath application of dopamine. This LTD was blocked by the following metabotropic glutamate receptor (mGluR) antagonists coapplied with dopamine: (S)-alpha-methyl-4-carboxyphenylglycine (MCPG; group I and II antagonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; group I antagonist), or (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE; group II antagonist). This suggests that the dopamine-facilitated LTD requires synaptic activation of groups I and II mGluRs during tetanus. LTD could also be induced by coupling tetani to bath application of groups I and II mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). In the next series of experiments, coapplication of dopamine and 1S,3R-ACPD, but not application of either drug alone, consistently induced LTD without tetani or even single test stimuli during drug application, suggesting that coactivation of dopamine receptors and the mGluRs is sufficient for LTD induction. Immunoblot analyses with anti-active mitogen-activated protein kinases (MAP-Ks) revealed that D1 receptors, D2 receptors, group I mGluRs, and group II mGluRs all contribute to MAP-K activation in prefrontal cortex, and that combined activation of dopamine receptors and mGluRs synergistically or additively activate MAP-Ks. Consistently, LTD by dopamine + 1S, 3R-ACPD coapplication, as well as the two other forms of LTD (LTD by dopamine + tetani and LTD by 1S,3R-ACPD + tetani), was blocked by bath application of MAP-K kinase inhibitor PD98059. LTD by dopamine + 1S,3R-ACPD coapplication was also blocked by postsynaptic injection of synthetic MAP-K substrate peptide. Our results suggest that dopamine receptors and groups I and II mGluRs cooperate to induce LTD through converging postsynaptic activation of MAP-Ks.

Fig. 1.

Dopaminergic facilitation of LTD induction in layer I-II to layer V pyramidal neuron glutamatergic synapses in rat prefrontal cortex. A, Photo image of a representative neuron stained with biocytin. B, Schematic representation of experimental protocol used for electrical measurement. C, Application of 50 Hz tetanic stimuli to layer I-II fibers did not induce lasting synaptic changes (−0.7 ± 1.0% over baseline measured at 35–40 min after tetani,n = 11). D, Bath application of dopamine (100 μm in 20 μm ascorbic acid, 10–15 min) acutely depressed the synaptic responses, but the depression fully recovered within 30 min after dopamine washout (4.7 ± 7.0% over baseline measured at 35–40 min after washout,n = 5). E, Application of tetanic stimuli in the presence of dopamine induced LTD [−22 ± 7.6% over baseline measured at 35–40 min after tetani–washout,n = 14, p < 0.002 vs control (C)]. Averaged synaptic responses taken from the indicated time points in this condition are superimposed and shown in the inset in E.

Fig. 2.

Dopaminergic facilitation of LTD is inhibited by the presence of MCPG, the antagonist of group I and group II mGluRs.A, MCPG (300–500 μm) was coapplied in the bath with dopamine. The rest of the protocols are identical to those in Figure 1E. MCPG blocked induction of LTD [−5.2 ± 6.5% 35–40 min after tetani–drug washout,n = 5, p > 0.3 vs control (Fig. 1C)]. Top traces are averaged responses taken from the indicated time points. B, The same tetanic stimuli in the presence of MCPG alone did not induce lasting synaptic changes (−0.3 ± 3.8% 35–40 min after tetani–MCPG, n = 5, p > 0.9 vs control), suggesting that the block of dopamine-facilitated LTD by MCPG (A) was not a masking of LTD. Top traces are averaged synaptic responses taken from the indicated time points.

Fig. 3.

Group I mGluR antagonist AIDA inhibits dopaminergic facilitation of LTD. A, AIDA (200 μm) was bath-applied with dopamine for 10–15 min, and tetanic stimuli were delivered (identical to Fig.1E except the presence of AIDA). No LTD was induced [−1.8 ± 6.0% 35–40 min after tetani–drug washout,n = 5, p > 0.1 vs control (Fig. 1C), but p < 0.025 versus dopamine + 50 Hz group (Fig. 1E)].B, In a separate group of neurons, AIDA alone was bath-applied, and tetani were delivered. Short-term changes occurred, but no lasting synaptic changes were induced (−1.3 ± 7.0% 35–40 min after tetani–AIDA, n = 6,p > 0.9 vs control), suggesting that the block of LTD by AIDA (A) was not a masking of LTD.Top traces are averaged synaptic responses taken from the indicated time points.

Fig. 4.

Group II mGluR antagonist MSOPPE inhibits dopaminergic facilitation of LTD. A, MSOPPE (200 μm) was bath-applied with dopamine for 10–15 min, and tetanic stimuli were delivered (identical to Fig.1E except the presence of MSOPPE). Tetani failed to induce LTD [−5.3 ± 3.1% 35–40 min after tetani–drug washout, n = 7, p > 0.1 vs control (Fig. 1C), but p < 0.04 versus dopamine + 50 Hz group (Fig. 1E)].B, Tetanic stimuli in the presence of MSOPPE alone induced some degree of post-tetanic changes in five of nine cases but did not induce LTP on average. Mean percentage increase 35–40 min after tetani–drug washout calculated from eight experiments was 0.6 ± 3.4% over baseline (recording of one cell that showed STP discontinued 25 min after tetani; p > 0.7 vs control depicted in Fig. 1C). Top tracesare averaged synaptic responses taken from the indicated time points.

Fig. 5.

MSOPPE, the group II mGluR antagonist, augments synaptic responses during 50 Hz tetanic stimuli, compared with control condition (see Fig. 4B for plots of EPSP slope before and after tetani in this MSOPPE condition). Traces shown in this figure are the synaptic responses evoked by the first of four episodes of tetanic stimuli in two different cells. On average, MSOPPE (200 μm, n = 8) increased the number of spikes (p < 0.05 in all four train episodes) and the number of the EPSPs whose amplitudes were larger than 50% of the first EPSP evoked in the given train episode (p < 0.05 in the first train episode,p < 0.1 in the second and fourth episodes). Ninety percent decay time from peak membrane depolarization was, on average, not different from control, but if five cells that showed clear post-tetanic changes (see Results) are taken into account, MSOPPE increased 90% decay over control in the first tetanus episode (2217 ± 606 msec vs 726 ± 228 msec, p< 0.02). The MSOPPE-treated cell shown in this figure is a cell that showed clear increases in all three of these parameters. MSOPPE did not reduce spike-train adaptation and afterhyperpolarization tested with postsynaptic current injection (data not shown). It is unlikely, however, that this augmentation of synaptic responses during tetanus by MSOPPE per se is the mechanism by which MSOPPE blocked LTD in the presence of dopamine (Fig. 4A), because dopamine itself augmented synaptic responses during tetanus (Otani et al., 1998b) and occluded the effect of MSOPPE. Insets show the same responses by different scales of amplitude and time.

Fig. 6.

Simultaneous activation of group I and group II mGluRs by 1S,3R-ACPD facilitates LTD induction. A, Tetanic stimuli in the presence of 1S,3R-ACPD (100 μm, 10–15 min) induced LTD (n = 4). Mean LTD occurring 35–40 min after tetani–drug washout was −37 ± 5.7% [(n = 4, p < 0.001 vs control (Fig. 1, top right inset)]. Top tracesare averaged responses taken from time points 1 and2. A superimposed representation of the responses (1+2) is also shown. B, Bath application of 1S,3R-ACPD (100 μm) alone only transiently depressed synaptic responses, which fully recovered within 30 min (n = 4; mean percentage change of the EPSP slope 35–40 min after drug washout, 3.4 ± 4.2%).

Fig. 7.

Sole activation of group I mGluRs with the agonist DHPG is insufficient to facilitate LTD. A, In the first group (n = 4), DHPG (100 μm, 10–15 min) was applied alone in the bath without tetani. DHPG induced a transient synaptic depression that was accompanied by a mild (a few millivolts) postsynaptic membrane depolarization and a reduction in spike-train adaptation and afterhyperpolarization (shown in top traces; membrane potential set at resting level). Mean change of the EPSP slope 35–40 min after drug washout was 2.0 ± 4.5% (n = 4). B, Tetanic stimuli in the presence of DHPG failed to facilitate LTD induction. Mean change of the EPSP slope 35–40 min after tetani–DHPG was 4.8 ± 7.1% [n = 5, p > 0.1 vs control (Fig. 1C)].

Fig. 8.

Sole activation of group II mGluRs with potent agonist DCG IV induces postsynaptic Ca²⁺-dependent LTD without tetanic stimuli. A, Bath application of DCG IV (50–100 nm) acutely depressed synaptic response that is followed by a lasting depression. Mean change of the EPSP slope 35–40 min after drug washout was −21 ± 3.8% (n = 6). Top traces show that DCG IV did not change spike-train adaptation and afterhyperpolarization. Superimposed averaged synaptic responses taken from time points 1 and2 are also shown. B, DCG IV-induced LTD was absent in cells injected with BAPTA. BAPTA did not block acute depressant action of DCG IV on the synaptic responses. Mean change of the EPSP slope 35–40 min after DCG IV washout was 0.1 ± 2.2% (n = 4, p < 0.005 vs above DCG IV group). In a separate group of neurons (n = 4), a late application of MSOPPE did not reverse the expression of DCG IV-induced LTD (see Results for details), further suggesting that DCG IV-induced LTD is not a result of insufficient washout of the drug.

Fig. 9.

Simultaneous activation of dopamine receptors and mGluRs is sufficient for LTD induction. A, Bath application (10–15 min) of dopamine (100 μm in 20 μm ascorbic acid) and 1S,3R-ACPD (100 μm) induced synaptic depression that remained after washout of the drugs [−27 ± 2.6% 35–40 min after drug washout,n = 5, p < 0.005 vs dopamine-alone group (Fig. 1D) andp < 0.001 vs 1S,3R-ACPD-alone group (Fig.6B)]. B, Induction of LTD by dopamine + ACPD coapplication did not require 0.033 Hz single test synaptic stimuli (−29 ± 4.2% 35–40 min after drug washout,n = 4, p < 0.01 vs dopamine-alone group and p < 0.005 vs 1S,3R-ACPD-alone group). Test stimuli were halted just before application of the drugs until 30 min after the beginning of drug washout. Halting of test stimuli itself did not change synaptic responses (see Results). C, LTD was also induced by coapplication of dopamine and group I agonist DHPG (100 μm) in four of five cells. The plots were made from all five experiments (−29 ± 8.9% 35–40 min after drug washout,n = 5, p < 0.025 vs both DHPG and dopamine groups). Thus, although LTD induced by 50 Hz tetani in the presence of dopamine requires synaptic activation of group Iand group II mGluRs (Figs. 2A, 3A, 4A), and although 50 Hz tetani in the presence of groups I and II agonist 1S,3R-ACPD, but not DHPG, induce LTD (Figs. 6A, 7B), pharmacological activation of group I mGluRs alone with DHPG, if combined with dopamine application, can induce LTD. However, this LTD induction is somewhat less consistent than that after groups Iand II mGluRs are coactivated with 1S,3R-ACPD (nine LTD of nine cases) (Fig.9A,B).

Fig. 10.

LTD induction in prefrontal cortex requires activation of MAP-Ks. A, LTD by 50 Hz tetani in the presence of dopamine (Fig. 1E) was blocked by bath application of PD98059 (20 μm), the specific inhibitor of MAP-K kinases (MEK1 and MEK2, which phosphorylate ERK1 and ERK2). Mean change of the EPSP slope measured 35–40 min after drug washout–tetani was 0.3 ± 4.1% (n = 6,p < 0.02 vs dopamine + tetani group depicted in Fig. 1E). B, LTD by 50 Hz tetani in the presence of 1S,3R-ACPD (Fig.6A) was also blocked by bath application of PD98059. Mean change of the EPSP slope measured 35–40 min after drug washout–tetani was −0.6 ± 3.9% [n = 4,p < 0.002 vs 1S,3R-ACPD + tetani group (Fig.6A)]. C, LTD by coactivation of dopamine receptors and groups I and II mGluRs with 1S,3R-ACPD (Fig. 9A) is blocked by PD98059. Mean change of the EPSP slope measured 35–40 min after drug washout was −4.9 ± 5.8% (n = 6,p < 0.02 vs the group depicted in Fig.9A). D, LTD by dopamine + 1S,3R-ACPD coapplication is also blocked by postsynaptic injection of specific synthetic MAP-K substrate peptide Ala-Pro-Arg-Thr-Pro-Gly-Gly-Arg-Arg (1 mm in electrode). This peptide, when abundantly present, acts as a specific competitive inhibitor against endogenous MAP-K substrates. Mean change of the EPSP slope 35–40 min after drug washout was 8.4 ± 6.2% [n = 5, p < 0.001 vs dopamine + 1S,3R-ACPD group (Fig.9A)]. Thus, critical MAP-K activation for LTD induction after coactivation of dopamine receptors and mGluRs occurs in postsynaptic sites.

Fig. 11.

Dopamine and mGluR agonists additively or synergistically activate MAP-Ks as detected with anti-active MAP-K antibody. Slices were incubated at 28°C in buffer containing 1 μm bicuculline without other drugs (CNT) or with dopamine (DA, 100 μm) or an mGluR agonist (1S,3R-ACPD, 100 μm; DHPG, 100 μm; DCG IV, 100 nm) or both for 2 or 5 min. Bicuculline itself did not change MAP-K activity (data not shown). Equal amounts of lysates (50 μg) were resolved on a 10% acrylamide gel. Activated MAP-Ks were detected with an antibody anti-active MAP-Ks (anti-active ERKs) that recognizes dually phosphorylated ERKs. After stripping, the immunoblot was reprobed with polyclonal anti-ERK2 (see Materials and Methods). A,B, Results with 2 and 5 min application, respectively. Note that phosphorylated protein bands are always denser after dopamine plus an mGluR agonist than either drug alone (see Results).C, Quantification by scanning densitometry of the autoradiogram of anti-active ERKs using NIH Image 1.6. The data are representative of at least three independent experiments. For the ERK2 activity (2 and 5 min), every drug condition showed statistical significance of at least p < 0.05 over control. For the ERK1 activity (5 min), the increase seen in the presence of dopamine and a given mGluR agonist was always significantly larger than the increase seen in the presence of that mGluR agonist alone (p < 0.05). The graph is plotted as percentage changes relative to CNT levels for p42/ERK2, and raw arbitrary numbers for p44/ERK1 because of the lack of p44/ERK1 phosphorylation in CNT.

Fig. 12.

Dopamine D1 and D2 receptors synergistically activate MAP-Ks as detected with anti-active MAP-K antibody. Methods are similar to those depicted in Figure 11 except the use of D1 antagonist SCH23390 (1 μm) and D2 antagonist sulpiride (50 μm). These antagonists were preincubated for 10 min before dopamine (100 μm) was applied. In all results in the figure, the duration of dopamine application was 2 min, but similar results were obtained after 5 min application. A, Phosphorylation bands of ERK1 and ERK2 after various conditions. Control tissue showed a relatively dense band of ERK2 as in Figure 11and also a light ERK1 band in this case. Dopamine increased phosphorylation of both ERK1 and ERK2. Note that phosphorylation bands are lighter in the presence of SCH23390 or sulpiride, and the band in the presence of two antagonists is at control level. B, Synergism between D1 and D2 receptors revealed by quantification by scanning densitometry of the autoradiogram of anti-active ERKs using NIH Image 1.6. Each group contains four observations. Dopamine increased ERK1 and ERK2 phosphorylation more than twofold (ERK1,p < 0.001; ERK2, p < 0.005). This dopamine effect was highly significantly reduced by the presence of the D1 antagonist SCH23390 (ERK1, p < 0.002; ERK2, p < 0.001) or the D2 antagonist sulpiride (ERK1, p < 0.002; ERK2, p < 0.05). Co-presence of SCH23390 and sulpiride blocked dopamine phosphorylation of MAP-Ks (ERK1 and ERK2, p < 0.001). These results suggest that sole activation of either D1 or D2 receptors by dopamine is insufficient to bring about activation of MAP-Ks. Two subtypes of dopamine receptors must be stimulated for MAP-K activation. The graph is plotted as percentage changes relative to CNT levels for both p42/ERK2 and p44/ERK1. For this purpose, control was taken as 100 for ERK2 and 12.5 for ERK1, to reflect roughly the ERK2/ERK1 densitometry proportion obtained by scanning.

Fig. 13.

Schematic drawing of possible mechanisms for LTD induction in prefrontal layer I–II to layer V pyramidal neuron glutamatergic synapses. The coexistence of D1-like and D2-like dopamine receptors and that of D2-like and glutamate in the same synapse are still hypothetical. In classical protocol (Fig.1E), LTD is induced by 50 Hz electrical stimulation to the glutamatergic fibers in the presence of dopamine in the bath (shown in the figure as a dopaminergic synaptic terminal).(a) Dopamine alone acutely and reversibly depresses low-frequency glutamatergic transmission, including the NMDA receptor-mediated component (Law-Tho et al., 1994; Law-Tho, 1995; our unpublished observation), by presynaptic or postsynaptic action, or both, and accordingly, LTD induced by the classical protocol does not require NMDA receptor activation (Otani et al., 1998b).(b) During high-frequency (50 Hz) drive to glutamatergic synapses, dopamine augments the synaptic responses and increases postsynaptic depolarization (Otani et al., 1998). This effect of dopamine serves as a critical factor for the NMDA-independent, postsynaptic Ca²⁺-dependent induction of LTD (Otani et al., 1998b). (c) In the present study, it was shown that for this LTD induction, synaptic activation of both group I and group II mGluRs is necessary (Figs. 2A, 3A, 4A). Moreover, coapplication of dopamine and 1S,3R-ACPD consistently induced LTD without tetanus or single synaptic stimuli, suggesting that coactivation of dopamine receptors and the mGluRs is sufficient for LTD induction (Fig. 9A,B). We propose that a biochemical mechanism underlying this LTD induction is converging postsynaptic activation of MAP-Ks (ERK1 and ERK2) by groups I and II mGluRs and D1-like and D2-like dopamine receptors. Thus, dopamine D1 and D2 receptors synergistically activate MAP-Ks (Fig. 12), whereas group I mGluRs alone or group II mGluRs alone can activate MAP-Ks (Fig. 11). A combined activation of the dopamine receptors and mGluRs causes synergistic (ERK1) or additive (ERK2) increases of MAP-K activity (Fig. 11). Second messenger pathways involved in this converging MAP-K activation in prefrontal neurons (shown as pathwaysi, ii, iii, andiv) are yet to be demonstrated. However, the following lines of evidence support our hypothesis. (i andii) Activation of MAP-Ks by PKC and PKA has been demonstrated in various cell types including hippocampal cells (Nestler and Greengard 1994; Cobb and Goldsmith, 1995; Robinson and Cobb, 1997;Roberson et al., 1999). (iii) Lines of evidence suggest that D2 receptors are coupled positively to phospholipase A2 and production of arachidonic acid, which activates PKC (Piomelli et al., 1991; Vial and Piomelli, 1995; Nilsson et al., 1998). There may be a synergistic action between D1 and D2 receptors for arachidonic acid production (Piomelli et al., 1991). (iv) It has been reported that group II mGluR activation with DCG IV, as well as group I mGluR activation with DHPG, stimulates the phospholipase D pathway, which also leads to PKC activation (Klein et al., 1997).