Activation of M2 muscarinic receptors leads to sustained suppression of hippocampal transmission in the medial prefrontal cortex

Abstract

Cholinergic innervation of the prefrontal cortex is critically involved in arousal, learning and memory. Dysfunction of muscarinic acetylcholine receptors and their downstream signalling pathways has been identified in mental retardation. To assess the role played by the muscarinic receptors at the hippocampal-frontal cortex synapses, an important relay in information storage, we used a newly developed frontal slice preparation in which hippocampal afferent fibres are preserved. Transient activation of muscarinic receptors by carbachol results in a long-lasting depression of synaptic efficacy at the hippocampal but not cortical pathways or local circuitry. On the basis of a combination of electrophysiological, pharmacological and anatomical results, this input-specific muscarinic modulation can be partially attributed to the M2 subtype of muscarinic receptors, possibly through a combination of pre- and postsynaptic mechanisms.

Figure 1. Acute and long-term suppression of EPSPs in the mPFC

A, schematic diagram showing a coronal slice preparation on which hippocampal projections to mPFC were preserved. The bold curvy line indicates the projection trajectory. PrL: prelimbic; IL: infralimbic region. B, recording configuration highlighting layer 2/3 (L2/3) pyramidal neurons that receive both cortical inputs at layer 1 (L1) and hippocampal afferents. Selective stimulation of these pathways resulted in input-specific EPSPs (C and D). C, representative cortical EPSPs before, during and 30 min after application of 20 μm carbachol (CCh). D, representative hippocampal EPSPs before, during and 30 min after application of 20 μm CCh. E, comparison of the time course of CCh-induced EPSP suppression at the cortical and hippocampal pathways. F, EPSP suppression at different phases (**P < 0.01).

Figure 2. Changes in PPF ratio associated with the hippocampal pathway

A, experimental procedure showing three phases: pre-CCh, CCh application and CCh washout. B, averaged EPSC traces in response to two consecutive stimulations of the hippocampal–mPFC pathway, taken from phases 1 and 3, were superimposed. Note that the first EPSC remained suppressed after CCh was washed out. C, traces of EPSCs in response to stimulation of cortical pathways. Note that the first EPSC was completely recovered from CCh-induced acute depression. D, comparison of the PPF ratio of hippocampal synapses before CCh application and after CCh washout (**P < 0.01). The PPF ratio was calculated as EPSC2/EPSC1. E, comparison of the PPF ratio of cortical/local circuitry synapses before CCh application and after CCh washout (N. S.: non-significant).

Figure 3. Involvement of different subtypes of mAChR in various phases of CCh-induced synaptic suppression of the hippocampal pathway

A, time course of changes in EPSP slope of the hippocampal synapses under various conditions: CCh alone, CCh + M1 antagonist pirenzepine (2 μm), CCh + M2 antagonist AF-DX 116 (2 μm), and CCh + M1 + M2 antagonists. B, summarized EPSP reduction in both acute and sustained phases (*P < 0.05; **P < 0.01).

Figure 4. Involvement of synaptic activation in CCh-LTD

A, comparison of CCh effects between two conditions: with and without concurrent synaptic stimulation during 10 min CCh application. B, time course of EPSP suppression when APV (50 μm) was co-applied with CCh. C, time course of EPSP suppression when nifedipine (10 μm) was co-applied with CCh. D, time course of EPSP suppression by CCh when BAPTA (5 μm) was included in recording pipettes throughout the experiment. E, summarized data showing the effects of concurrent stimulation, APV, nifedipine, or intracellular BAPTA on CCh-induced LTD. Statistical comparisons were made between each experimental condition and CCh alone (*P < 0.05).