An acetylcholinesterase inhibitor, eserine, induces long-term depression at CA3-CA1 synapses in the hippocampus of adult rats

Abstract

Studies in humans and rodents support a role for muscarinic ACh receptor (mAChR) and nicotinic AChR in learning and memory, and both regulate hippocampal synaptic plasticity using complex and often times opposing mechanisms. Acetylcholinesterase (AChE) inhibitors are commonly prescribed to enhance cholinergic signaling in Alzheimer's disease in hopes of rescuing cognitive function, caused, in part, by degeneration of cholinergic innervation to the hippocampus and cortex. Unfortunately, therapeutic efficacy is moderate and inconsistent, perhaps due to unanticipated mechanisms. M1 mAChRs bidirectionally control synaptic strength at CA3-CA1 synapses; weak pharmacological activation using carbachol (CCh) facilitates potentiation, whereas strong agonism induces muscarinic long-term depression (mLTD) via an ERK-dependent mechanism. Here, we tested the prediction that accumulation of extracellular ACh via inhibition of AChE is sufficient to induce LTD at CA3-CA1 synapses in hippocampal slices from adult rats. Although AChE inhibition with eserine induces LTD, it unexpectedly does not share properties with mLTD induced by CCh, as reported previously. Eserine-LTD was prevented by the M3 mAChR-preferring antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), and pharmacological inhibition of MEK was completely ineffective. Additionally, pharmacological inhibition of p38 MAPK prevents mLTD but has no effect on eserine-LTD. Finally, long-term expression of eserine-LTD is partially dependent on a decrease in presynaptic release probability, likely caused by tonic activation of mAChRs by the sustained increase in extracellular ACh. Thus these findings extend current literature by showing that pharmacological AChE inhibition causes a prolonged decrease in presynaptic glutamate release at CA3-CA1 synapses, in addition to inducing a likely postsynaptic form of LTD.

Keywords: Alzheimer's disease; acetylcholinesterase inhibitor; hippocampus; long-term depression; muscarinic AChR.

Fig. 1.

Acute eserine treatment induces long-term depression (LTD; eserine-LTD) at CA3-CA1 synapses. A: 10 min bath application of 100 nM eserine. LTD: 83 ± 8% of baseline field excitatory postsynaptic potentials (fEPSP) slope, 85–90 min (n = 4). B: 10 min bath application of 10 μM eserine. LTD: 80 ± 3% of baseline fEPSP slope, 60–65 min (n = 6).

Fig. 2.

Eserine-LTD is blocked by inhibiting 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP)-sensitive receptors. A: eserine-LTD (100 nM) is blocked by combined treatment with pirenzepine (Pir) and 4-DAMP. LTD: 74 ± 5% of baseline fEPSP slope in control solution (n = 5) vs. 1.02 ± 5% in pirenzepine + 4-DAMP (n = 5). B: eserine-LTD (100 nM) is unaffected by pirenzepine alone. LTD: 77 ± 3% of baseline fEPSP slope in control solution (n = 3) vs. 72 ± 4% in pirenzepine (n = 7). C: eserine-LTD (10 μM), blocked by treatment with 4-DAMP alone. LTD: 80 ± 2% of baseline fEPSP slope in control solution (n = 5) vs. 1.05 ± 5% in 4-DAMP (n = 5); Student's t-test. **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3.

Eserine-LTD does not depend on ERK1/2 activity. A: U0126 treatment for 20 min before carbachol (CCh) application completely blocks CCh-induced muscarinic LTD (mLTD) in 3- to 5-wk-old rats. LTD: 86 ± 3% of baseline fEPSP slope in control solution (n = 8) vs. 99 ± 5% of fEPSP slope in U0126 (n = 9). B: the blocking of mLTD in adult rats (age 3–4 mo) requires pretreatment with U0126, lasting at least 1 h. LTD: 76 ± 3% of baseline fEPSP slope in control solution (n = 6) vs. 76 ± 4% of fEPSP slope in U0126 < 1 h (n = 5). C: LTD: 76 ± 7% of baseline fEPSP slope in control solution vs. 1.05 ± 4% of baseline fEPSP slope in U0126 > 1 h (n = 6); P = 0.002, Student's t-test. D: pretreatment with U0126 ≥ 1 h does not block eserine-LTD (10 μM): 77 ± 6% of baseline fEPSP slope in control solution (n = 5) vs. 75 ± 2% of baseline fEPSP slope in U0126 > 1 h (n = 4). *P ≤ 0.05; **P ≤ 0.01.

Fig. 4.

The inhibition of p38 MAPK prevents CCh-LTD but not eserine-LTD. A: pretreatment with SB203580 (SB; 5 μM) for at least 1 h before agonist application completely blocks LTD induced by CCh in adult rats; LTD: 86 ± 2% of baseline fEPSP slope in control solution (n = 6) vs. 97 ± 3% of baseline fEPSP slope in SB203580 (n = 7); P = 0.003, Student's t-test. B: eserine-LTD (10 μM) remained intact, despite p38 MAPK blockade; LTD: 83 ± 3% of baseline fEPSP slope in control solution (n = 4) vs. 85 ± 10% of baseline fEPSP slope in SB203580 (n = 5). *P ≤ 0.05.

Fig. 5.

Eserine-LTD occurs independent of GABA channel conductance. A: picrotoxin, a GABA_AR channel blocker, does not prevent LTD induced by 10 μM eserine. LTD: 86 ± 7% of baseline fEPSP slope (n = 6). B: CGP 52432 (CGP), a GABA_BR-selective inhibitor, does not prevent eserine-LTD (10 μM). LTD: 86 ± 3% of baseline fEPSP slope in control solution (n = 4) vs. 89 ± 6% in CGP (n = 5); P > 0.05, Student's t-test.

Fig. 6.

An increase in paired-pulse ratio (PPR) strongly correlates with eserine-LTD expression. A: control eserine-LTD (10 μM) experiments, presented in Figs. 2C, 3, and 4, averaged and overlayed with normalized PPRs. Consistent with a presynaptic mechanism of expression, changes in fEPSP slope during eserine-LTD experiments are accompanied by temporally matched fluctuations in PPR (n = 16). B: linear regression plotting fEPSP slope as a function of PPR over the time course of eserine-LTD expression (25–65 min). A very strong, negative correlation exists between the normalized fEPSP slope and PPR. Linear regression: slope = −0.97, R² = 0.46, P = 0.0 × 10⁻³⁰ (n = 4,231 sweeps).

Fig. 7.

Atropine partially attenuates eserine-LTD but fully reverses an eserine-induced increase in PPR. A: atropine (1 μM), applied between 45 and 75 min of eserine-LTD (10 μM) expression. LTD: 78 ± 5% of baseline fEPSP slope preatropine (time point 1) vs. 86 ± 6% of baseline fEPSP slope postatropine (time point 2), n = 6; P = 0.02, Student's paired t-test. B: 30 min treatment with atropine alone. Postatropine slope: 1.01% of baseline (40–45 min, n = 7). C: paired-pulse facilitation ratios during atropine experiments shown in A, plotted with respect to fEPSP slope. Eserine-LTD and atropine-mediated attenuation of LTD are accompanied by an increase and normalization of PPR, respectively. PPR: 1.08 ± 3% of baseline PPR preatropine (time point 1) vs. 1.01 ± 2% of baseline PPR postatropine (time point 2), n = 6; P = 0.002, Student's paired t-test. D: LTD and PPR data from time points 1 and 2 (pre- and postatropine, respectively), presented in a bar chart. fEPSP slope remains significantly depressed, despite normalization of PPR by atropine. *P ≤ 0.05; **P ≤ 0.01.