Coactivation of M(1) muscarinic and alpha1 adrenergic receptors stimulates extracellular signal-regulated protein kinase and induces long-term depression at CA3-CA1 synapses in rat hippocampus

Abstract

Intact cholinergic innervation from the medial septum and noradrenergic innervation from the locus ceruleus are required for hippocampal-dependent learning and memory. However, much remains unclear about the precise roles of acetylcholine (ACh) and norepinephrine (NE) in hippocampal function, particularly in terms of how interactions between these two transmitter systems might play an important role in synaptic plasticity. Previously, we reported that activation of either muscarinic M(1) or adrenergic alpha1 receptors induces activity- and NMDA receptor-dependent long-term depression (LTD) at CA3-CA1 synapses in acute hippocampal slices, referred to as muscarinic LTD (mLTD) and norepinephrine LTD (NE LTD), respectively. In this study, we tested the hypothesis that mLTD and NE LTD are independent forms of LTD, yet require activation of a common Galphaq-coupled signaling pathway for their induction, and investigated the net effect of coactivation of M(1) and alpha1 receptors on the magnitude of LTD induced. We find that neither mLTD nor NE LTD requires phospholipase C activation, but both plasticities are prevented by inhibiting the Src kinase family and extracellular signal-regulated protein kinase (ERK) activation. Interestingly, LTD can be induced when M(1) and alpha1 agonists are coapplied at concentrations too low to induce LTD when applied separately, via a summed increase in ERK activation. Thus, because ACh and NE levels in vivo covary, especially during periods of memory encoding and consolidation, cooperative signaling through M(1) and alpha1 receptors could function to induce long-term changes in synaptic function important for cognition.

Figure 1.

mLTD does not require activation of adrenergic receptors, and NE LTD does not require activation of muscarinic receptors. A, Application of 50 μm CCh for 10 min results in LTD at hippocampal CA3–CA1 synapses measured in extracellular dendritic field potential recordings in acute slices prepared from 3- to 4-week-old rats (n = 7). B, Application of 40 μm Methox, a selective α1 agonist, for 10 min also induces LTD at these synapses (n = 4). C, mLTD induced with CCh (50 μm) is not altered by 10 μm prazosin, an α1 adrenergic receptor antagonist (n = 5). D, NE LTD induced with the Methox (40 μm) is not altered by 1 μm atropine, a broad spectrum muscarinic receptor antagonist (n = 5). Waveform traces are averages of 20 sweeps from 5 min before (thin line), during (dotted line), and 40 min after (thick line) agonist application. Error bars indicate SEM. Calibrations: 0.5 mV, 10 ms.

Figure 2.

ERK activation is required for induction, but not expression, of mLTD and NE LTD. A, mLTD is prevented in the presence of the MEK inhibitor U0126 (20 μm; n = 4; p < 0.05). B, ERK activation is required during induction (red squares; n = 5; p < 0.01), but not expression of mLTD (n = 5; p > 0.05; black circles). C, CCh-induced increase in ERK2 phosphorylation (pERK2) is blocked by preincubation with the M₁ antagonist, MTx-7 (100 nm; n = 3); *p < 0.02 vs control). D, NE LTD is prevented in the presence of U0126 (20 μm; n = 7; p < 0.05). E, ERK activation is required during NE LTD induction (n = 4; p < 0.03; red squares), but not during expression (n = 4; p > 0.05; black circles). F, Methox-induced increase in ERK2 phosphorylation is blocked by preincubation with the α1 antagonist prazosin (10 μm; n = 6; *p < 0.01 vs control). Phosphorylated ERK2 was normalized to total ERK2; treatments caused no change in total ERK2 levels. Error bars indicate SEM.

Figure 3.

Activation of the Src kinase family, but not the PLC pathway, is required for induction of mLTD and NE LTD and increase in pERK. A, mLTD induction is unaffected by blockade of PLC activation using the PLC inhibitor U73122 (10 μm; n = 5 in U73122; n = 4 interleaved control slices; p > 0.05). B, NE LTD induction is unaffected by the PLC inhibitor U73122 (10 μm; n = 5 in U73122; n = 3 interleaved control slices; p > 0.05). C, mLTD is prevented by the Src kinase inhibitor PP2 (10 μm; n = 5; p < 0.01). D, NE LTD is also prevented by the Src kinase inhibitor PP2 (10 μm; n = 5; p < 0.02). All statistical comparisons are made to the amount of LTD induced in interleaved DMSO-treated control slices. E, CCh-induced increase in ERK2 phosphorylation (pERK2) is significantly decreased by the Src kinase inhibitor, PP2 (n = 4; *p < 0.001 vs control; CCh + PP2, p < 0.01 vs control; CCh vs CCh + PP2, p < 0.03). F, Methox-induced increase in ERK2 phosphorylation is significantly decreased by the Src kinase inhibitor PP2 (n = 4; *p < 0.05 vs control; Methox + PP2, p < 0.01 vs control; Methox vs Methox + PP2, p < 0.01). Error bars indicate SEM.

Figure 4.

Strong coactivation of M₁ and α1 receptors does not induce LTD of greater magnitude. A, Waveform traces are averages of 20 sweeps from 5 min before (thin line) and 40 min after (thick line) agonist application. B, CCh (50 μm) and Methox (40 μm) do not induce a greater magnitude of LTD when coapplied (n = 6) than when applied separately (CCh, n = 4; Methox, n = 3). Error bars indicate SEM. Calibration: 0.5 mV, 10 ms.

Figure 5.

Coapplication of low concentrations of selective M₁ and α1 receptor agonists induces LTD. A, Application of 0.3 μm McN-A-343 (McN), a selective M₁ receptor agonist, does not induce mLTD (n = 4). B, Application of 3 μm Methox does not induce NE LTD (n = 5). C, Coapplication of 0.3 μm McN and 3 μm Methox induces LTD (n = 5). Waveform traces are averages of 20 sweeps from 5 min before (solid line) and 30 min after (dashed line) agonist application (A–C, insets). Calibrations: 0.5 mV, 10 ms. D, Application of either 0.3 μm McN or 3 μm Methox alone does not increase ERK2 phosphorylation (pERK2), but coapplication of the two agonists significantly increases ERK2 phosphorylation (n = 4; *p < 0.02 vs control). Phosphorylated ERK2 was normalized to total ERK2; treatments caused no change in total ERK2 levels.

Figure 6.

Proposed model of mLTD and NE LTD interaction. A, B, Our data demonstrates that a weak M₁ receptor (A) or a weak α1 receptor (B) activation is insufficient to activate Src kinase, phosphorylate ERK, and induce LTD. C–E, However, when combined, weak stimulation at both receptors (C) sufficiently activates Src, which phosphorylates ERK and induces LTD at the same magnitude as that induced with strong M₁ or α1 receptor stimulation alone (D, E). F, However, a combined strong activation of M₁ and α1 receptor activation does not induce LTD of a greater magnitude than that observed with either agonist when applied alone, demonstrating that maximal activation of one receptor saturates the mechanisms required for LTD such that LTD induced by activation of the other receptor is occluded.