M2 muscarinic acetylcholine receptors regulate long-term potentiation at hippocampal CA3 pyramidal cell synapses in an input-specific fashion

Abstract

Muscarinic receptors have long been known as crucial players in hippocampus-dependent learning and memory, but our understanding of the cellular underpinnings and the receptor subtypes involved lags well behind. This holds in particular for the hippocampal CA3 region, where the mechanisms of synaptic plasticity depend on the type of afferent input. Williams and Johnston (Williams S, Johnston D. Science 242: 84-87, 1988; Williams S, Johnston D. J Neurophysiol 64: 1089-1097, 1990) demonstrated muscarinic depression of mossy fiber (MF) long-term potentiation (LTP) through a presynaptic site of action and Maeda et al. (Maeda T, Kaneko S, Satoh M. Brain Res 619: 324-330, 1993) proposed a bidirectional modulation of MF LTP by muscarinic receptor subtypes. Since then, this issue, as well as muscarinic regulation of plasticity at associational/commissural (A/C) fiber-CA3 synapses has remained largely neglected, not least because of the lack of highly selective ligands for the different muscarinic receptor subtypes. In the present study, we performed field potential and whole cell recordings from the hippocampal CA3 region of M(2) receptor knockout mice to determine the role of M(2) receptors in short-term and long-term plasticity at A/C and MF inputs to CA3 pyramidal cells. At the A/C synapse, M(2) receptors promoted short-term facilitation and LTP. Unexpectedly, M(2) receptors mediated the opposite effect on LTP at the MF synapse, which was significantly reduced, most likely involving a depressant effect of M(2) receptors on adenylyl cyclase activity in MF terminals. Our data demonstrate that cholinergic projections recruit M(2) receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner.

Fig. 1.

M₂ receptors enhance long-term synaptic plasticity at associational/commissural (A/C) synapses of CA3 pyramidal cells. A: theta burst stimulation (TBS) produced weaker long-term potentiation (LTP) of A/C field excitatory postsynaptic potentials (fEPSPs) in M₂−/− hippocampi (red circles) than in M₂+/+ hippocampi (black circles). Insets illustrate averaged traces of fEPSPs measured in CA3 stratum radiatum at the like-numbered time points before TBS (point 1, average of 30 traces) and after LTP had been introduced (point 2, average of 15 traces). B: a comparable decrease of LTP in M₂-deficient hippocampi was observed when high-frequency stimulation (HFS) instead of TBS was used for induction and could be reproduced in M₂+/+ hippocampi by the M₂ receptor antagonist gallamine (green squares). Insets depict averaged traces at the like-numbered time points as indicated in A. *P < 0.05.

Fig. 2.

M₂ receptors affect short-term plasticity at A/C synapses, but not at mossy fiber (MF) synapses, of CA3 pyramidal cells. A: reduced short-term plasticity of A/C excitatory postsynaptic currents (EPSCs) in M₂−/− hippocampus. Traces at top are from a CA3 pyramidal cell of M₂+/+ hippocampus, illustrating the facilitation of A/C EPSCs during a train of 4 stimuli before (control, black trace) and during (green trace) application of M₂ receptor antagonist gallamine (20 μM). To compare quantitatively the degree of facilitation in M₂+/+ hippocampi with and without gallamine (black and green circles, respectively) and in M₂−/− hippocampi (red circles), the amplitudes of the 2nd to 4th EPSCs were normalized to that of the 1st EPSC. B: responses of MF EPSCs to 4 stimuli at 20 Hz in the absence (black trace) and presence of gallamine (green trace) in M₂+/+ hippocampus. Amplitudes of the 2nd to 4th EPSCs during 20-Hz stimulation were normalized to amplitude of the 1st EPSC. Recordings from M₂+/+ hippocampi in the absence (black squares) and presence of gallamine (20 μM; green squares) or from M₂−/− hippocampi (red squares) showed that short-term plasticity was not sensitive to pharmacological suppression or genetic disruption of M₂ receptors. C: in M₂+/+ hippocampi, gallamine enhanced amplitude of the 1st EPSC at A/C synapses (left, black bar) but had the opposite effect at MF synapses (right, black bar). The inhibitory effect of gallamine on the 1st EPSC at MF synapses was reversed by the GABA_B receptor antagonist CGP-55845 (blue bar). Effects of gallamine were absent in M₂−/− hippocampi (red bars). D: comparison of normalized frequency facilitation during 1-Hz stimulation in M₂+/+ and M₂−/− hippocampi did not reveal a significant role of M₂ receptors in this paradigm. Inset shows averaged traces from like-numbered time points. *P < 0.05.

Fig. 3.

M₂ receptors inhibit MF LTP. A and B: scatter plots of MF EPSCs before and after HFS to induce LTP in M₂+/+ and M₂−/− hippocampi. At the end of the recordings, EPSCs were suppressed by the group II metabotropic glutamate agonist DCG IV (2.5 μM), confirming that they were produced by MF activation. Each data point represents a single EPSC evoked at 0.1 Hz. Insets above plots depict averaged traces taken at the like-numbered time points. C: normalized time courses of MF LTP in M₂+/+ hippocampi (black circles) and M₂−/− hippocampi (red circles) indicate a suppressive effect of M₂ receptors. D: normalized time courses of MF LTP in the absence (black squares) and presence of gallamine (green squares) show that the M₂ receptor antagonist gallamine replicates the effect of M₂ receptor knockout on MF LTP when examined in M₂+/+ hippocampus. *P < 0.05.

Fig. 4.

M₂ receptors act on adenylyl cyclase to inhibit MF LTP A: scatter plot of potentiation of MF EPSCs by forskolin (50 μM) in M₂+/+ hippocampus. Each data point represents a single EPSC evoked at 0.1 Hz. Inset above plots depicts averaged traces taken at the like-numbered time points. B: stronger potentiation of MF EPSCs by forskolin (50 μM) in M₂ −/− hippocampi (red circles) than in M₂+/+ hippocampi (black circles). By contrast, forskolin did not exhibit an appreciable effect at A/C synapses in either genotype (black and red squares) C: pretreatment with forskolin occluded subsequent induction of MF LTP by HFS regardless of genotype. D: pharmacological inhibition of adenylyl cyclase with DDOA reduced and equalized LTP in control (black squares) and M₂-deficient hippocampi (red squares). *P < 0.05.

Fig. 5.

GABA_B receptors do not appear to be involved in muscarinic regulation of MF LTP. Application of the GABA_B receptor antagonist CGP-55845 did not alter the time course or strength of MF LTP compared with control recordings (blue squares vs. black circles) and did not change enhancement of MF LTP by gallamine (green circles vs. red squares). *P < 0.05.

Fig. 6.

Nicotinic receptor activation does not account for stronger MF LTP in M₂-deficient hippocampi. A: the α7 nicotinic acetylcholine receptor antagonist α-bungarotoxin (αBTX; red bars) reduced MF EPSCs evoked at low stimulus frequency (0.1 Hz) when M₂ receptors were pharmacologically suppressed by gallamine (20 μM; right) but not when M₂ receptors were functional (left). B: αBTX did not impair the gallamine-induced augmentation of MF LTP. *P < 0.05.