Coactivation of beta-adrenergic and cholinergic receptors enhances the induction of long-term potentiation and synergistically activates mitogen-activated protein kinase in the hippocampal CA1 region

Abstract

Interactions between noradrenergic and cholinergic receptor signaling may be important in some forms of learning. To investigate whether noradrenergic and cholinergic receptor interactions regulate forms of synaptic plasticity thought to be involved in memory formation, we examined the effects of concurrent beta-adrenergic and cholinergic receptor activation on the induction of long-term potentiation (LTP) in the hippocampal CA1 region. Low concentrations of the beta-adrenergic receptor agonist isoproterenol (ISO) and the cholinergic receptor agonist carbachol had no effect on the induction of LTP by a brief train of 5 Hz stimulation when applied individually but dramatically facilitated LTP induction when coapplied. Although carbachol did not enhance ISO-induced increases in cAMP, coapplication of ISO and carbachol synergistically activated p42 mitogen-activated protein kinase (p42 MAPK). This suggests that concurrent beta-adrenergic and cholinergic receptor activation enhances LTP induction by activating MAPK and not by additive or synergistic effects on adenylyl cyclase. Consistent with this, blocking MAPK activation with MEK inhibitors suppressed the facilitation of LTP induction produced by concurrent beta-adrenergic and cholinergic receptor activation. Although MEK inhibitors also suppressed the induction of LTP by a stronger 5 Hz stimulation protocol that induced LTP in the absence of ISO and carbachol, they had no effect on LTP induced by high-frequency synaptic stimulation or low-frequency synaptic stimulation paired with postsynaptic depolarization. Our results indicate that MAPK activation has an important, modulatory role in the induction of LTP and suggest that coactivation of noradrenergic and cholinergic receptors regulates LTP induction via convergent effects on MAPK.

Fig. 1.

Coactivation of β-adrenergic and cholinergic receptors enhances the induction of LTP by a short train of 5 Hz stimulation. A, A 5 sec train of 5 Hz stimulation (delivered at time 0) by itself induced only a small potentiation of synaptic transmission (open symbols; fEPSPs potentiated to 120.5 ± 7.12% of baseline;n = 6; p < 0.05, pairedt test comparison to pre-5 Hz baseline) but induced robust LTP (filled symbols; fEPSPs potentiated to 175.6 ± 11.2% of baseline; n = 6) when delivered at the end of a 10 min bath application of ISO plus carbachol (CCh) (200 nm each; presence in bath indicated by the bar). Traces show fEPSPs recorded during baseline and 45 min after 5 Hz stimulation in a control experiment (5 Hz stimulation alone, lefttraces) and in an experiment in which 5 Hz stimulation was delivered in the presence of ISO plus carbachol (right traces). Calibration: 2 mV, 5 msec. B, Summary of the effects of 5 Hz stimulation on synaptic strength when 5 Hz stimulation was delivered alone (open bars; data from the experiment shown in A) or at the end of a 10 min application of ISO (n = 6), carbachol (n = 6), or ISO plus carbachol (data from the experiment shown in A). Although coapplication of ISO plus carbachol significantly enhances the amount of LTP induced by 5 Hz stimulation (*p < 0.05), neither ISO alone nor carbachol alone significantly enhances LTP induction (fEPSPs were potentiated to 125.4 ± 7.9% and 112.9 ± 4.1% of baseline, respectively).

Fig. 2.

Carbachol does not modulate ISO-induced increases in cAMP. The histogram shows average ± SEM cAMP levels (in picomoles per milligram of protein) from five separate experiments in which hippocampal slices (from the same animal) either were untreated (open bar) or were exposed to a 10 min bath application of 200 nm ISO alone, 200 nm carbachol (CCh) alone, or ISO plus carbachol (filled bars). Compared with basal levels of cAMP seen in untreated control slices, only ISO alone and ISO plus carbachol induced significant increases in cAMP levels (*p < 0.05). In four of these experiments a pair of slices also was exposed to a 10 min bath application of 50 μm forskolin. In these slices cAMP levels were increased to 92.5 ± 13.1 pmol/mg of protein.

Fig. 3.

MEK inhibitors suppress low-frequency, but not high-frequency, stimulation-induced LTP. A, The amount of LTP induced by 30 sec of 5 Hz stimulation (delivered at time0) in slices continuously bathed in 20 μmU0126 (filledsymbols; fEPSPs were 122.2 ± 5.1% of baseline; n = 6) was reduced significantly (p < 0.01) as compared with that seen in interleaved vehicle control experiments (open symbols; 0.2% DMSO; fEPSPs were potentiated to 167.7 ± 9.9% of baseline; n = 5). B, Summary of the effects of three different MEK inhibitors on 5 Hz stimulation-induced LTP. Values show the amount of LTP present 40–45 min after 5 Hz stimulation. Similar amounts of LTP were induced by 5 Hz stimulation in interleaved vehicle control experiments (0.1–0.33% DMSO; n = 17), and the combined results are shown by the open bar. Results for U0126 are from the same experiments shown in A. fEPSPs in PD98059 (PD)-treated slices were 109.9 ± 9% of baseline (n = 5) after 5 Hz stimulation (**p < 0.01 compared with 0.33% DMSO control experiments), whereas fEPSPs were 118.4 ± 13.4% of baseline (n = 7) after 5 Hz stimulation in SL327 (SL)-treated slices (*p < 0.05 compared with 0.1% DMSO control experiments). In interleaved control experiments the fEPSPs were 155.1 ± 5.9% of baseline (0.33% DMSO; n = 5) and 165.1 ± 17.4% of baseline (0.1% DMSO; n = 7), respectively.C, High-frequency stimulation-induced LTP is not inhibited by U0126. Two 1-sec-long trains of 100 Hz stimulation (intertrain interval, 10 sec; delivered at time 0) induced similar amounts of LTP in vehicle control experiments (0.2% DMSO; open symbols; n = 6) and in slices continuously bathed in 20 μm U0126 (filled symbols; n = 5). In control experiments 55–60 min after 100 Hz stimulation the fEPSPs were potentiated to 185.9 ± 20.5% of baseline, whereas in U0126-treated slices the fEPSPs were 188.7 ± 43.2% of baseline.D, Summary of the effects of MEK inhibitors on 5 Hz stimulation-induced complex spike bursting. The plot shows the percentage of EPSPs during the 5 Hz stimulation train that evoked complex spike bursts (CSB, defined as two or more negative-going spikes after EPSP onset; see Thomas et al., 1998) in vehicle control experiments (left) and in the presence of MEK inhibitors (right). Measurements were taken from the experiments shown in B. Although complex spike bursting tended to be reduced in slices pretreated with MEK inhibitors, none of the inhibitors produced a statistically significant suppression of complex spike bursting when compared with interleaved vehicle control experiments (PD98059 vs 0.33% DMSO controls:t₍₈₎ = 1.78, p = 0.11; U0126 vs 0.2% DMSO controls:t₍₉₎ = 1.16, p = 0.28; SL327 vs 0.1% DMSO controls:t₍₁₁₎ = 1.12, p = 0.29).

Fig. 4.

SL327 does not inhibit the induction of LTP by low-frequency synaptic stimulation paired with postsynaptic depolarization. A, One hundred EPSPs (evoked at 2 Hz) were paired with tonic postsynaptic depolarization to between 0 and +20 mV at time 0 in vehicle control experiments (open circles; 0.2% DMSO in electrode-filling solution;n = 12) and in experiments in which SL327 was present either in the bath (filled circles; 10 μm, 0.1% DMSO; n = 7) or in the recording electrode-filling solution (filled triangles; 100 μm, 0.2% DMSO;n = 12). The 25–30 min postpairing EPSPs were potentiated to 241.5 ± 26.9% of baseline in control experiments, 255.5 ± 28.7% of baseline in experiments in which SL327 was bath-applied, and 234.1 ± 27.5% of baseline in experiments in which SL327 was injected into CA1 pyramidal cells via the recording electrode. The inset shows EPSPs (average of three responses) recorded during baseline and 30 min postpairing (larger response) in a control experiment (lefttraces) and in an experiment in which 100 μm SL327 was present in the recording electrode (righttraces). Calibration: 5 mV, 15 msec. B, Cumulative probability distribution showing results from all of the experiments depicted in A. Results from experiments in which SL327 was bath-applied (filled circles) and applied via the recording electrode (filled triangles) have been combined into one distribution.

Fig. 5.

MAPK activation may underlie the effects of ISO plus carbachol on 5 Hz stimulation-induced LTP. A, U0126 inhibits the induction of LTP by 5 sec of 5 Hz stimulation delivered during the coapplication of ISO and carbachol. A 5 sec train of 5 Hz stimulation delivered at the end of a 10 min bath application of ISO plus carbachol (200 nm each; the presence of agonists in the bath indicated by the bar) induced robust LTP in vehicle (0.2% DMSO) control experiments (open symbols; fEPSPs were potentiated to 163.9 ± 6.8% of baseline;n = 5) but had little effect on synaptic strength in slices continuously bathed in 20 μm U0126 (filled symbols; fEPSPs were 112.8 ± 7.3% of baseline; n = 5). The traces show superimposed fEPSPs recorded during baseline and 45 min post-5 Hz stimulation in a control experiment (left) and in a slice bathed in U0126 (right). Calibration: 1 mV, 5 msec. B, Synergistic activation of MAPK by coactivation of β-adrenergic and cholinergic receptor agonists. B1, Representative Western immunoblots showing protein bands visualized with antibodies to dually phosphorylated p42/44 MAPK (PP) and total p42/44 MAPK (Total) in control, untreated slices (Con), and slices bathed for 10 min in aCSF containing 200 nm ISO, 200 nm carbachol (CCh), or ISO plus carbachol (ISO + CCh). B2, Average results ± SEM from seven separate experiments like that shown inB1. Only coapplication of ISO plus carbachol induced a statistically significant (*p < 0.05) increase in phospho-p42 MAPK levels.