Muscarinic acetylcholine receptor-dependent induction of persistent synaptic enhancement in rat hippocampus in vivo

Neuroscience 144 (2007) 754 –761

MUSCARINIC ACETYLCHOLINE RECEPTOR-DEPENDENT INDUCTION OF PERSISTENT SYNAPTIC ENHANCEMENT IN RAT HIPPOCAMPUS IN VIVO S. LI,a W. K. CULLEN,a R. ANWYLb AND M. J. ROWANa*

Key words: excitatory postsynaptic potential, long-term potentiation, cholinergic autoreceptor, muscarinic receptor.

a

Department of Pharmacology and Therapeutics, Biotechnology Building, Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland

Human and animal studies indicate that cholinergic deficits produce an array of profound cognitive impairments (Sarter and Bruno, 1997; Parent and Baxter, 2004). The actions of acetylcholine (ACh) are mediated by two main classes of receptors, the G-protein-coupled muscarinic family and the ligand-gated nicotinic family, with different subtypes potentially playing different roles in the modulation of memory mechanisms. Although antagonism of these receptors generally disrupts the acquisition and recall of information, M2/M4 selective muscarinic ACh receptor antagonists can improve cognitive performance (Packard et al., 1990; Baratti et al., 1993; Ohno et al., 1994; Pike and Hamm, 1995; Quirion et al., 1995; Aura et al., 1997; Vannucchi et al., 1997; Kopf et al., 1998; Galli et al., 2000; Carey et al., 2001; Lazaris et al., 2003; Rowe et al., 2003), but also see (Messer and Miller, 1988; Ferreira et al., 2003; Tzavaral et al., 2003; Seeger et al., 2004). The cognitive enhancing effect of M2/M4 receptor antagonists has been attributed to disinhibition of endogenous ACh release by blocking activation of inhibitory autoreceptors, thereby facilitating mnemonic processes through as yet unknown mechanisms (Stillman et al., 1993, 1996; Ohno et al., 1994; Quirion et al., 1995; Rouse et al., 1997, 2000; Vannucchi et al., 1997; Kitaichi et al., 1999; Carey et al., 2001; Zhang et al., 2002). Since long-lasting changes in excitatory synaptic transmission in brain areas such as the hippocampus are thought to underlie certain types of learning and memory (Morris et al., 2003), and memory is known to be modulated by ACh, it is of interest to elucidate the synaptic effects of M2/M4 receptor antagonists and the factors influencing their persistence. In the hippocampus ACh has been shown to exert multiple, often opposing, cellular actions that can affect synaptic plasticity (Markram and Segal, 1992; Caulfield, 1993; Marino et al., 1998; Zheng et al., 1998; Hamilton and Nathanson, 2001; Kim et al., 2002; Fernandez de Sevilla and Buno, 2003). Although exogenously applied muscarinic ACh receptor agonists can facilitate the induction of long-term potentiation (LTP) at CA1 synapses (Burgard and Sarvey, 1990; Huerta and Lisman, 1993; Auerbach and Segal, 1996; Shinoe et al., 2005), muscarinic receptor antagonists lack consistent effects on LTP induction in vitro (Tanaka et al., 1989; Sokolov and Kleschevnikov, 1995; Yun et al., 2000; Lin et al., 2004) and have statedependent effects in vivo (Kikusui et al., 2000; Leung et al.,

b

Department of Physiology, Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland

Abstract—Presynaptic terminal autoinhibitory muscarinic acetylcholine (ACh) receptors are predominantly of the M2/M4 subtypes and antagonists at these receptors may facilitate cognitive processes by increasing ACh release. The present study examined the ability of the M2/M4 muscarinic ACh receptor antagonist N,N=-bis [6-[[(2-methoxyphenyl) methyl]amino]hexyl]-1,8-octane diamine tetrahydrochloride (methoctramine) to induce and modulate synaptic plasticity in the CA1 area of the hippocampus in urethane-anesthetized rats. Both methoctramine and another M2/M4 antagonist, {11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} (AF-DX 116), caused a rapid onset and persistent increase in baseline synaptic transmission after i.c.v. injection. Consistent with a requirement for activation of non-M2 receptors by endogenously released ACh, the M1/M3 receptor selective antagonists 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and 4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]10H-thieno[3,4-b][1,5]benzodiazepin-10-one dihydrochloride (telenzepine) prevented the induction of the persistent synaptic enhancement by methoctramine. The requirement for cholinergic activation was transient and independent of nicotinic ACh receptor stimulation. The synaptic enhancement was inhibited by the prior induction of long-term potentiation (LTP) by high frequency stimulation but induction of the synaptic enhancement by methoctramine before high frequency stimulation did not inhibit LTP. Unlike high frequency stimulation-evoked LTP, the synaptic enhancement induced by methoctramine appeared to be NMDA receptor-independent. The present studies provide evidence for the rapid induction of a persistent potentiation at hippocampal glutamatergic synapses by endogenous ACh in vivo following disinhibition of inhibitory M2 muscarinic autoreceptors. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Fax: ⫹35-3-1-6081466. E-mail address: [email protected] (M. J. Rowan). Abbreviations: ACh, acetylcholine; AF-DX 116, {11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one}; CPP, 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1phosphonic acid; fEPSP, field excitatory postsynaptic potential; HFS, high frequency stimulation; LTP, long-term potentiation; mecamylamine, (N,2,3,3-tetramethylbicyclo(2.2.1)heptan-2-amine) hydrochloride; methoctramine, N,N=-bis [6-[[(2-methoxyphenyl)methyl]amino] hexyl]-1,8-octane diamine tetrahydrochloride; telenzepine, 4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H-thieno[3, 4-b][1,5]benzodiazepin-10-one dihydrochloride; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.10.001

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2003). Somewhat paradoxically, given the predominantly cognitive enhancing properties of M2 receptor antagonists, mice deficient in M2 receptors are cognitively impaired and have reduced in vitro theta-burst induced LTP due to increased GABAergic inhibitory transmission (Tzavaral et al., 2003; Seeger et al., 2004). Furthermore, in hippocampal slices agonists at postsynaptic muscarinic ACh receptors can induce a slowly developing persistent synaptic potentiation (termed LTPm) that is absent in these M2 receptor deficient mice (Segal and Auerbach, 1997; Seeger et al., 2004). The main aim of the present study was to elucidate how presynaptic muscarinic receptor antagonists that increase endogenous ACh release affect transmission at apical– dendritic excitatory synapses in the CA1 area in vivo. First, the actions of the relatively selective M2/M4 receptor antagonists N,N=-bis [6-[[(2-methoxyphenyl) methyl]amino]hexyl]-1,8-octane diamine tetrahydrochloride (methoctramine) and {11-[[2-[(diethylamino)methyl]-1piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} (AF-DX 116) (Doods et al., 1987; Michel and Whiting, 1988; Caulfield, 1993) were examined after i.c.v. injection in urethane-anesthetized rats. We then determined the ability of M1/M3 subtype selective muscarinic receptor antagonists (4,9-dihydro-3-methyl-4-[(4-methyl-1piperazinyl)acetyl]-10H-thieno[3,4-b][1,5]benzodiazepin10-one dihydrochloride (telenzepine) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP)) (Galvan et al., 1989; Michel et al., 1989; Caulfield, 1993) to prevent the induction of synaptic potentiation by methoctramine. Next, we examined the dependence of the methoctramineinduced synaptic potentiation on nicotinic and NMDA receptor activation, and synaptic stimulation. Finally, we assessed the interaction between the methoctramine-induced synaptic potentiation and high frequency stimulation (HFS)-induced LTP.

EXPERIMENTAL PROCEDURES Electrophysiological recording Experiments were performed on adult male Wistar rats (250 – 350 g) under urethane anesthesia (1.5 g/kg, i.p.) in a manner similar to that described previously (Li et al., 2000). The experiments were licensed by the Department of Health and Children, Ireland, in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC). Experiments were designed to minimize the number of animals used and their suffering. Two stainless steel screws (1.5 mm diameter) were placed in the skull without piercing the dura. One served as the ground electrode (7 mm posterior to bregma and 5 mm left of midline) and the other served as the reference electrode (8 mm anterior to bregma and 5 mm left of midline). Pairs of twisted tungsten wires (0.05 mm diameter), used as the active recording electrode (3.4 mm posterior to bregma and 2.5 mm right of midline) and the stimulating electrodes (4.2 mm posterior to bregma and 3.8 mm right of midline), were inserted through drill holes located above the CA1 area of the dorsal hippocampus. The optimal depth of these wire electrodes in the stratum radiatum was determined using electrophysiological and postmortem morphological criteria. After optimizing the wire electrode positions the relationship between stimulus intensity and the field excitatory postsynaptic potential (fEPSP) amplitude was assessed. The test pulse intensity was

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set to evoke a half maximum fEPSP amplitude. Double-pulse stimulation with an inter-pulse interval of 40 ms using square wave pulses (0.1 ms duration) was applied at a rate of 0.033 Hz. The baseline was recorded for at least 1 h in order to ensure that it was stable. During HFS the pulse intensity was increased to evoke a 75% maximum fEPSP amplitude and was applied as a set of 10 trains of 20 stimuli, inter-stimulus interval 5 ms (200 Hz) and inter-train interval 2 s. The EEG was simultaneously monitored (from the hippocampal recording electrode) during the experiments in order to ensure that no abnormal activity was evoked by the conditioning HFS.

I.c.v. injection A stainless steel guide cannula (0.45 mm inner diameter) containing a stylet was implanted into the right lateral cerebral ventricle (coordinates: 0.5 mm lateral to midline at bregma; 4.00 mm below the surface of the skull). A stainless steel internal cannula (0.4 mm outer diameter) connected to a 25 ␮l Hamilton microsyringe (Hamilton Co., Reno, NV, USA) via polythene tubing replaced the stylet for microinjections. A volume of 2.5 ␮l of vehicle or drug was delivered over 30 s and the internal cannula was left in place for 1 min prior to being replaced with the stylet. Verification of the placement of the cannula was carried out postmortem by checking the spread of ink dye after i.c.v. injection.

Drugs The following drugs were used: methoctramine, (N,2,3,3-tetramethylbicyclo(2.2.1)heptan-2-amine) hydrochloride (mecamylamine), scopolamine hydrobromide, 4-DAMP, telenzepine and 3-((R,S)-2carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were obtained from Sigma (St. Louis, MO, USA). AF-DX 116 was obtained from Tocris (Bristol, UK). With the exception of AF-DX 116, which was initially dissolved in dimethylsulphoxide (DMSO), all of the above drugs were dissolved and diluted in saline.

Data analysis The initial slope of the fEPSP was averaged across 10 sweeps (5 min epochs) and the magnitude of potentiation was expressed as the percentage pre-HFS or pre-drug baseline. Similar results were obtained when the fEPSP amplitude was measured. Statistical comparisons were assessed using two-tailed Student’s t-test and P values ⬍0.05, were considered significant.

RESULTS The M2/M4 muscarinic receptor antagonist methoctramine caused a dose-dependent rapid (usually ⬍5 min onset time) and persistent (⬎3 h) enhancement of excitatory synaptic transmission after i.c.v. injection (Fig. 1). Thus the fEPSP slope increased to 145.7⫾12.3% baseline in the first 10 min after the administration of 17 nmol (12.5 ␮g in 2.5 ␮l per rat, n⫽8) methoctramine (P⬍0.05 compared with pre-methoctramine baseline, 101⫾2.8%, or to saline vehicle-injected controls, 97.2⫾1.9%, n⫽5). Thereafter the enhancement was relatively stable, measuring 161.6⫾14.6% at 1 h (P⬍0.05 compared with pre-methoctramine baseline and to vehicle-injected controls, 96.3⫾6%) and remained elevated for the duration of recording (168.2⫾26.7% at 3 h, data not shown). Lowering the dose in 50% decrements (8.5, 4.2 and 2.1 nmol, n⫽5 per group) revealed that the enhancement was dose-dependent, with a threshold dose between 2.1 (P⬎0.05) and 4.2 (P⬍0.05) nmol (Fig. 1B). Consistent with the facilitatory effect of methoctramine being due to inhibition of M2/M4 ACh receptors, another

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excitatory synaptic transmission by methoctramine. In order to determine the role of nicotinic and non-M2/M4 muscarinic cholinoceptors animals were pretreated with a general nicotinic receptor antagonist or M1/M3 muscarinic receptor antagonists. Systemic injection with a dose (2 mg/kg, i.p.) of mecamylamine that is known to prevent activation of hippocampal nicotinic receptors (Tani et al., 1998), given 30 min prior to the injection of methoctramine (17 nmol, i.c.v.), did not affect baseline transmission and failed to prevent the methoctramine-induced rise in fEPSP slope (153.9⫾8.7% at 1 h after methoctramine administration, P⬎0.05, n⫽6, Fig. 2A). The requirement for muscarinic ACh receptor activation in the induction of synaptic potentiation by methoctramine was evaluated using telenzepine and 4-DAMP, antagonists with relatively higher affinity for non-M2 (mainly M1 and M3 receptors) over M2 muscarinic receptors (Galvan et al., 1989; Michel et al., 1989; Caulfield, 1993) (Fig.

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Dose (nmol) Fig. 1. Muscarinic M2/M4 receptor antagonists induce a rapid onset and persistent enhancement of excitatory synaptic transmission in the CA1 area of the anesthetized rat hippocampus. (A) Time course of the enhancement induced by i.c.v. injection (arrow) of either methoctramine (17 nmol, n⫽8) or AF-DX 116 (20 nmol, n⫽5), P⬍0.05 compared with saline (n⫽5) at 1 h. Insets show representative traces of field potentials before (1) and after (2) the injections. Horizontal scale bar⫽10 ms; vertical scale bar⫽1 mV. (B) Dose-dependence of the enhancement induced by methoctramine measured at different times after the injection. Values are the mean⫾S.E.M. fEPSP slope.

M2/M4 receptor antagonist AF-DX 116 also rapidly and persistently increased the fEPSP slope (Fig. 1A). After i.c.v. injection of AF-DX 116 (20 nmol), the enhancement measured 139.2⫾7.1% during the first 10 min and 147.7⫾8.9 at 1 h (n⫽5, P⬍0.05 compared with pre-injection baseline of 96.2⫾0.9% or to vehicle-injected animals). We hypothesized that since the M2/M4 muscarinic receptors are considered to be the predominant cholinergic inhibitory autoreceptors in the hippocampus (Vannucchi et al., 1997; Kitaichi et al., 1999; Rouse et al., 2000; Zhang et al., 2002; Tzavaral et al., 2003) and since muscarinic receptor antagonists, including methoctramine, increase hippocampal ACh release in vivo (Stillman et al., 1993, 1996) that activation of non-M2/M4 muscarinic receptors by endogenously released ACh might be necessary for the induction of the persistent enhancement of

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Time (min) Fig. 2. Inhibition of the methoctramine-induced persistent synaptic enhancement by muscarinic but not nicotinic receptor blockade. (A) The induction of the synaptic enhancement by methoctramine (17 nmol, i.c.v.) was not affected by pre-treatment with the nicotinic receptor antagonist mecamylamine (2 mg/kg, i.p., n⫽6, P⬎0.05). (B) The non-M2 subtype selective muscarinic receptor antagonists 4-DAMP (14 nmol, i.c.v., n⫽7, P⬍0.05) and telenzepine (28 nmol, i.c.v., n⫽9, P⬍0.05) prevented the effect of methoctramine. Insets show representative traces of field potentials before (1) and after (2) the injections. Horizontal scale bar⫽10 ms; vertical scale bar⫽1 mV. Values are the mean⫾S.E.M. fEPSP slope.

S. Li et al. / Neuroscience 144 (2007) 754 –761

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2B). Thirty minute pretreatment with a dose of telenzepine (28 nmol, i.c.v.) that did not affect baseline transmission, significantly reduced the increase in the fEPSP slope evoked by methoctramine (110.9⫾6.6% at 1 h, P⬍0.05 compared with methoctramine alone; P⬎0.05 compared with the pre-methoctramine baseline, 99.7⫾2.5%, n⫽9). The methoctramine-induced facilitation of excitatory synaptic transmission was also significantly reduced by 4-DAMP at a dose (14 nmol, i.c.v.) that did not affect baseline transmission, the fEPSP slope measuring 117.5⫾5.8% 1 h after methoctramine administration (P⬍0.05 compared with methoctramine alone, P⬎ 0.05 compared with the pre-methoctramine baseline, 108.3⫾3.3%, n⫽7). The above data indicate that the enhancement of excitatory transmission by methoctramine requires the activation of non-M2/non-M4 muscarinic receptors by endogenously released ACh. To determine if endogenous ACh is necessary for the maintenance, as well as the induction, of the persistent increase in the fEPSPs, the general muscarinic receptor antagonist scopolamine was administered after the application of methoctramine (Fig. 3A). Scopolamine (13 nmol, i.c.v.) failed to affect the potentiation when applied 1 h after methoctramine (175⫾30.1% at 2 h postmethoctramine, n⫽5, P⬍0.05 compared with pre-methoctramine baseline, 105⫾5.7%; P⬎0.05 compared with 2 h post-methoctramine alone, 163.5⫾13.5%, n⫽8). This dose of scopolamine on its own had no significant effect on baseline synaptic transmission and inhibited the methoctramine-induced persistent enhancement when given 30 min prior to methoctramine (data not shown, n⫽7). Next, the necessity for evoked synaptic activation of glutamate receptors for induction of the potentiation was assessed by ceasing electrical stimulation at the time of administration of methoctramine (Fig. 3B). In addition, to help rule out the possibility that any methoctramine that may still be present 1.5 h after its injection might trigger a late facilitation at the time of recommencing stimulation, scopolamine was administered at this time using the dose (13 nmol) found to inhibit the induction of the facilitation. Under these conditions methoctramine triggered a persistent enhancement that was similar to that observed when stimulation was carried out throughout the experiment (159.8⫾18.8% at 2 h post-injection of methoctramine, n⫽8, P⬍0.05 compared with pre-methoctramine baseline; P⬎0.05 compared with 2 h post-methoctramine alone with continuous stimulation, 163.5⫾13.5%, n⫽8). Although evoked excitatory synaptic activation was not a requirement for the induction of the methoctramine potentiation the possible involvement of background NMDA receptor activation was assessed using the NMDA receptor antagonist CPP (Fig. 3C). Injection of a dose of CPP (10 mg/kg, i.p.) that blocked HFS-induced LTP (113.4⫾13.2% at 1 h, n⫽8, data not shown) failed to affect the persistent potentiation induced by methoctramine. Thus the administration of methoctramine 45 min after the injection of CPP caused a large increase in the fEPSP slope (141.8⫾11.3% at 1 h, P⬎0.05 compared with

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methoctramine alone; P⬍0.05 compared with pre-methoctramine baseline, 100.4⫾1.7%, n⫽4). The relationship between HFS-induced LTP and methoctramine-induced long-lasting facilitation was examined first by pre-injection of methoctramine (Fig. 4A).

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methoctramine, there was evidence of inhibition (Fig. 4B). Thus tetanic stimulation induced a stable and robust LTP measuring 141.3⫾9.6% at 2 h after the tetanus (n⫽6). Subsequent injection of methoctramine (17 nmol i.c.v.) caused only a small further increase, the magnitude of potentiation reaching 155.8⫾10.9% 1 h later. This level of potentiation was not significantly different from that elicited by methoctramine alone. The LTP conditioning stimulation protocol was at or near the maximum to saturate LTP (Fig. 4C). Thus tetanus-induced LTP inhibited or occluded the facilitatory effect of methoctramine whereas prior treatment with methoctramine did not reduce the induction of LTP.

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Time (min) Fig. 4. Relationship between methoctramine induced synaptic facilitation and HFS-induced LTP. (A) Prior induction of synaptic potentiation by methoctramine injection did not inhibit HFS-induced LTP (n⫽7, P⬍0.05). (B) Prior induction of LTP partly occluded the synaptic potentiation induced by methoctramine injection (n⫽6, P⬎0.05). (C) HFS-induced LTP is partly occluded by prior induction of LTP with the protocol used in (B) (n⫽6, P⬎0.05). Insets show representative traces of field potentials at the times indicated on the graphs. Horizontal scale bar⫽10 ms; vertical scale bar⫽1 mV. Values are the mean⫾S.E.M. fEPSP slope.

The methoctramine-induced facilitation was followed by a robust HFS-induced LTP (341.9⫾39.9% 1 h postHFS, P⬍0.05 compared with 178.6⫾10.9% just prior to HFS; equivalent to 203.2⫾25.4% of the post-methoctramine enhanced responses, n⫽7). In contrast, when the methoctramine and tetanus were applied in the opposite sequence, inducing LTP prior to the administration of

The present studies elucidate a novel means of inducing synaptic plasticity in the hippocampus in vivo. Here, we report how cholinergic dependent processes can trigger a persistent enhancement of glutamatergic transmission independent of NMDA receptor activation. An agent that increases endogenous ACh release by reducing presynaptic inhibition, the M2/M4 muscarinic ACh receptor antagonist methoctramine, rapidly triggered a persistent enhancement of transmission at CA3 to CA1 synapses in the anesthetized rat. The induction of the synaptic strengthening required transient activation of M1/M3 muscarinic receptors but not nicotinic receptors by endogenous ACh and was NMDA receptor-independent. The mechanisms of the persistent synaptic enhancement induced by methoctramine were examined initially using a variety of different muscarinic receptor subtype selective antagonists. Although both methoctramine and AFDX-116 are selective for M2/M4 receptors, given their higher affinity for M2 receptors (Doods et al., 1987; Michel and Whiting, 1988; Caulfield, 1993) and the evidence of the greater importance of M2 over M4 receptors in regulating ACh release in the hippocampus (Zhang et al., 2002), it is likely that antagonism at M2 receptors plays a more significant role in the induction of hippocampal synaptic potentiation by these agents. A requirement for a transient activation of muscarinic receptors by endogenously released ACh was shown by the inability of the general muscarinic receptor antagonist scopolamine to reverse the synaptic enhancement when applied after methoctramine. Given that both telenzepine, an M1 muscarinic receptor preferring antagonist, and 4-DAMP, an M3/M1 receptor selective antagonist, prevented the methoctramine-induced synaptic enhancement, it is likely that the transient requirement for increased muscarinic receptor activation is mediated through M1 and possibly M3 receptor subtypes. An M1/M3 receptor-triggered phospholipid C-mediated IP3 production and subsequent Ca2⫹ release from intracellular stores provides an attractive putative signaling mechanism for the induction of the synaptic enhancement which warrants further investigation. Overall the present findings strongly indicate that antagonism of inhibitory M2 autoreceptors leads to activation of M1 or M3 muscarinic receptors by endogenous ACh which then trig-

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gers a persistent potentiation of glutamatergic transmission in vivo. It is unclear how the receptor mechanisms for muscarinic receptor-dependent induction of persistent synaptic potentiation in vivo observed in the present studies relate to in vitro findings. Bath application of M2 receptor antagonists to rat hippocampal slices has not been reported to affect baseline transmission at CA3 to CA1 synapses in rats (Auerbach and Segal, 1996; Segal and Auerbach, 1997; Tombaugh et al., 2002) or guinea pigs (Shimoshige et al., 1997). However, in mice bath application of an M2 receptor antagonist caused a significant increase in glutamate-mediated transmission that was abrogated in M2 receptor knockout animals (Seeger et al., 2004). As these authors did not report if the enhancement persisted after drug washout its relevance to the present in vivo findings is difficult to assess. Remarkably, in both rats and mice a muscarinic receptor-dependent slow onset synaptic potentiation induced by bath application of a low concentration of the non-subtype selective ACh receptor agonist carbachol to hippocampal slices was prevented by M2 but not M1/M3 receptor antagonism (Segal and Auerbach, 1997; Seeger et al., 2004). In in vitro studies cholinergic inputs from the septum are usually severed in the preparation of hippocampal slices which will greatly diminish the background activation of M2 receptors by endogenous ACh. Septohippocampal cholinergic neurons are known to be still active in the anesthetized rat (Apartis et al., 1998; Brazhnik and Fox, 1999) and should therefore provide a background source of ACh. Thus difference in basal tone to presynaptic M2 autoreceptors may help explain the difference between the previous in vitro and the current in vivo findings. Another major difference is the spread of methoctramine to extrahippocampal M2 receptors after i.c.v. administration which could indirectly affect hippocampal function, for example, by changing the firing patterns of the septo-hippocampal system. Future studies should examine the effect of local administration of methoctramine to the hippocampus in vivo. The activity dependence of the methoctramine-induced synaptic enhancement was relatively weak. Unlike HFS-induced LTP, the persistent enhancement did not require strong activation of glutamatergic transmission or significant NMDA receptor activation. Thus ceasing electrical stimulation of the afferent pathways or pretreatment with an NMDA receptor antagonist did not prevent the induction of synaptic plasticity by methoctramine. This clearly does not exclude an involvement of background activation of other glutamate receptor subtypes due to spontaneous activity that is widespread in vivo. We also cannot rule out a requirement for M2 receptor antagonistinduced increased glutamate release due to a direct block of inhibitory heterosynaptic M2 receptors on glutamatergic terminals (Nikbakht and Stone, 1999) or postsynaptic enhancement of glutamate-mediated depolarization via inhibition of endogenous cholinergic activation of M2 receptors positively coupled to inwardly rectifying K⫹ channels (Seeger and Alzheimer, 2001). In preliminary experiments we found that the methoctramine-induced enhancement is not

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sensitive to inhibition of voltage-dependent Ca2⫹ channels (Li et al., unpublished observations) which differentiates it from another NMDA receptor-independent form of plasticity at CA3–CA1 synapses (Grover and Teyler, 1990). A somewhat complex picture emerges regarding the relationship between the methoctramine-induced synaptic enhancement and HFS-induced LTP. Although HFS-induced LTP and the methoctramine-induced synaptic enhancement differed in their dependence on NMDA receptor activation, we found that prior LTP induction inhibited the persistent enhancement by the M2 antagonist. This is consistent with possible shared post-receptor downstream signaling mechanisms. However, prior induction of the synaptic enhancement by methoctramine did not occlude HFS-induced LTP. One explanation is that the methoctramine did not saturate the signaling mechanisms. Alternatively, HFS-induced LTP may require additional mechanisms to those recruited by methoctramine. In previous in vitro studies bath application of an M2 receptor antagonist has been reported to facilitate LTP induction by relatively weak conditioning stimulation in the CA1 area in hippocampal slices from aged, but not young adult, rats (Tombaugh et al., 2002). In contrast, ‘theta burst’-induced LTP in vitro can be inhibited by M2 receptor antagonism in young adult mice, possibly by occlusion, but more likely a consequence of enhanced GABA-ergic inhibition (Seeger et al., 2004). Furthermore, cholinergic agonists can facilitate HFS-induced LTP via M2 receptor activation (Segal and Auerbach, 1997; Shimoshige et al., 1997). In the present studies the apparent partial occlusion of the methoctramine-induced synaptic enhancement by the prior induction of LTP by HFS points to a partial saturation of overlapping mechanisms of potentiation. Alternatively, a metaplastic effect caused by the conditioning stimulation might mediate the apparent occlusion. Regardless of mechanisms, the sequence of induction of the two forms of synaptic plasticity had a profound effect on the nature of the interaction between them. Thus intense glutamatergic activation inhibits the subsequent induction of synaptic strengthening by cholinergic activation whereas cholinergic stimulation does not inhibit the subsequent induction of synaptic potentiation by intense glutamatergic activation. Such a sequence-dependence may have important implications for how cholinergic and glutamatergic systems interact in vivo. Elucidation of the processes underlying this new form of synaptic plasticity induced by indirectly activating cholinergic transmission at muscarinic receptors may help explain how cholinergic modulation of glutamatergic transmission and plasticity of that transmission contribute to mechanisms underlying learning and memory. The present finding of M2 muscarinic receptor antagonist-induced potentiation in vivo may be relevant to the cognitive enhancing effects of these compounds. The data are consistent with the proposal that block of M2 muscarinic autoreceptors facilitates learning via increased endogenous ACh activation of non-M2 muscarinic receptors.

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Acknowledgments—This research was supported by grants from Science Foundation Ireland, the Health Research Board of Ireland, Enterprise Ireland (Embark), the Irish Higher Education Authority (PRTLI) and the Wellcome Trust.

REFERENCES Apartis E, Poindessous-Jazat FR, Lamour YA, Bassant MH (1998) Loss of rhythmically bursting neurons in rat medial septum following selective lesion of septohippocampal cholinergic system. J Neurophysiol 79:1633–1642. Auerbach JM, Segal M (1996) Muscarinic receptors mediating depression and long-term potentiation in rat hippocampus. J Physiol 492:479 – 493. Aura J, Sirvio J, Riekkinen P (1997) Methoctramine moderately improves memory but pirenzepine disrupts performance in delayed non-matching to position test. Eur J Pharmacol 333:129 –134. Baratti CM, Opezzo JW, Kopf SR (1993) Facilitation of memory storage by the acetylcholine-M(2) muscarinic receptor antagonist-AFDX-116. Behav Neural Biol 60:69 –74. Brazhnik ES, Fox SE (1999) Action potentials and relations to the theta rhythm of medial septal neurons in vivo. Exp Brain Res 127: 244 –258. Burgard EC, Sarvey JM (1990) Muscarinic receptor activation facilitates the induction of long-term potentiation (LTP) in the rat dentate gyrus. Neurosci Lett 116:34 –39. Carey GJ, Billard W, Binch H, Cohen-Williams M, Crosby G, Grzelak M, Guzik H, Kozlowski JA, Lowe DB, Pond AJ, Tedesco RP, Watkins RW, Coffin VL (2001) SCH 57790, a selective muscarinic M-2 receptor antagonist, releases acetylcholine and produces cognitive enhancement in laboratory animals. Eur J Pharmacol 431:189 –200. Caulfield MP (1993) Muscarinic receptors: characterization, coupling and function. Pharmacol Ther 58:319 –379. Doods HN, Mathy MJ, Davidesko D, van Charldorp KJ, de Jonge A, van Zwieten PA (1987) Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M1, M2 and M3 receptors. J Pharmacol Exp Ther 242:257–262. Fernandez de Sevilla D, Buno W (2003) Presynaptic inhibition of Schaffer collateral synapses by stimulation of hippocampal cholinergic afferent fibres. Eur J Neurosci 17:555–558. Ferreira AR, Furstenau L, Blanco C, Kornisiuk E, Sanchez G, Daroit D, Castro e Silva M, Cervenansky C, Jerusalinsky D, Quillfeldt JA (2003) Role of hippocampal M1 and M4 muscarinic receptor subtypes in memory consolidation in the rat. Pharmacol Biochem Behav 74:411– 415. Galli RL, Fine RE, Thorpe BC, Hale BS, Lieberman HR (2000) Antisense oligonucleotide sequences targeting the muscarinic type 2 acetylcholine receptor enhance performance in the Morris water maze. Int J Neurosci 103:53– 68. Galvan M, Boer R, Schudt C (1989) Interaction of telenzepine with muscarinic receptors in mammalian sympathetic-ganglia. Eur J Pharmacol 167:1–10. Grover L, Teyler T (1990) Two components of long-term potentiation induced by different patterns of afferent activation. Nature 347:477– 479. Hamilton SE, Nathanson NM (2001) The M-1 receptor is required for muscarinic activation of mitogen-activated protein (MAP) kinase in murine cerebral cortical neurons. J Biol Chem 276:15850 –15853. Huerta PT, Lisman JE (1993) Heightened synaptic plasticity of hippocampal CA1 neurons during a cholinergically induced rhythmic state. Nature 364:723–725. Kikusui T, Aoyagi A, Kaneko T (2000) Spatial working memory is independent of hippocampal CA1 long-term potentiation in rats. Behav Neurosci 114:700 –706. Kim J, Isokawa M, Ledent C, Alger B (2002) Activation of muscarinic acetylcholine receptors enhances the release of endogenous cannabinoids in the hippocampus. J Neurosci 22:10182–10191.

Kitaichi K, Hori T, Srivastava LK, Quirion R (1999) Antisense oligodeoxynucleotides against the muscarinic m(2), but not m(4), receptor supports its role as autoreceptors in the rat hippocampus. Mol Brain Res 67:98 –106. Kopf SR, Boccia MM, Baratti CM (1998) AF-DX 116, a presynaptic muscarinic receptor antagonist, potentiates the effects of glucose and reverses the effects of insulin on memory. Neurobiol Learn Mem 70:305–313. Lazaris A, Cassel S, Stemmelin J, Cassel JC, Kelche C (2003) Intrastriatal infusions of methoctramine improve memory in cognitively impaired aged rats. Neurobiol Aging 24:379 –383. Leung LS, Shen BX, Rajakumar N, Ma JY (2003) Cholinergic activity enhances hippocampal long-term potentiation in CA1 during walking in rats. J Neurosci 23:9297–9304. Li S, Anwyl R, Rowan MJ (2000) A persistent reduction in short-term facilitation accompanies long-term potentiation in the CA1 area in the intact hippocampus. Neuroscience 100:213–220. Lin YW, Yang HW, Min MY, Chiu TH (2004) Heat-shock pretreatment prevents suppression of long-term potentiation induced by scopolamine in rat hippocampal CA1 synapses. Brain Res 999:222–226. Marino MJ, Rouse ST, Levey AI, Potter LT, Conn PJ (1998) Activation of the genetically defined m1 muscarinic receptor potentiates Nmethyl-D-aspartate (NMDA) receptor currents in hippocampal pyramidal cells. Proc Natl Acad Sci U S A 95:11465–11470. Markram H, Segal M (1992) Activation of protein-kinase-C suppresses responses to NMDA in Rat CA1 hippocampal-neurons. J Physiol 457:491–501. Messer WS, Miller MD (1988) Intrahippocampal injections of gallamine impair learning of a memory task. Neurosci Lett 89:367–372. Michel AD, Stefanich E, Whiting RL (1989) Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP. Eur J Pharmacol 166:459 – 466. Michel AD, Whiting RL (1988) Methoctramine, a polymethylene tetraamine, differentiates 3 subtypes of muscarinic receptor in direct binding-studies. Eur J Pharmacol 145:61– 66. Morris RGM, Moser EI, Riedel G, Martin SJ, Sandin J, Day M, O’Carroll C (2003) Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory. Philos Trans R Soc Lond B Biol Sci 358:773–786. Nikbakht M, Stone T (1999) Occlusive responses to adenosine A1 receptor and muscarinic M2 receptor activation on hippocampal presynaptic terminals. Brain Res 829:193–196. Ohno M, Yamamoto T, Watanabe S (1994) Blockade of hippocampal M(1) muscarinic receptors impairs working-memory performance of rats. Brain Res 650:260 –266. Packard MG, Regenold W, Quirion R, White NM (1990) Post-training injection of the acetylcholine M2 receptor antagonist AF-DX 116 improves memory. Brain Res 524:72–76. Parent MB, Baxter MG (2004) Septohippocampal acetylcholine: Involved in but not necessary for learning and memory? Learn Mem 11:9 –20. Pike BR, Hamm RJ (1995) Postinjury administration of BIBN-99, a selective muscarinic M(2) receptor antagonist, improves cognitive performance following traumatic brain injury in rats. Brain Res 686:37– 43. Quirion R, Wilson A, Rowe W, Aubert I, Richard J, Doods H, Parent A, White N, Meaney MJ (1995) Facilitation of acetylcholine-release and cognitive performance by an M(2)-muscarinic receptor antagonist in aged memory-impaired rats. J Neurosci 15:1455–1462. Rouse ST, Edmunds SM, Yi H, Gilmor ML, Levey AI (2000) Localization of M-2 muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus. Neurosci Lett 284:182–186. Rouse ST, Thomas TM, Levey AI (1997) Muscarinic acetylcholine receptor subtype, m2: Diverse functional implications of differential synaptic localization. Life Sci 60:1031–1038. Rowe WB, O’Donnell JP, Pearson D, Rose GM, Meaney MJ, Quirion R (2003) Long-term effects of BIBN-99, a selective muscarinic M2

S. Li et al. / Neuroscience 144 (2007) 754 –761 receptor antagonist, on improving spatial memory performance in aged cognitively impaired rats. Behav Brain Res 145:171–178. Sarter M, Bruno JP (1997) Cognitive functions of cortical acetylcholine: toward a unifying hypothesis. Brain Res Brain Res Rev 23:28 – 46. Seeger T, Alzheimer C (2001) Muscarinic activation of inwardly rectifying K⫹ conductance reduces EPSPs in rat hippocampal CA1 pyramidal cells. J Physiol 535:383–396. Seeger T, Fedorova I, Zheng F, Miyakawa T, Koustova E, Gomeza J, Basile AS, Alzheimer C, Wess J (2004) M-2 muscarinic acetylcholine receptor knock-out mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity. J Neurosci 24: 10117–10127. Segal M, Auerbach JM (1997) Muscarinic receptors involved in hippocampal plasticity. Life Sci 60:1085–1091. Shimoshige Y, Maeda T, Kaneko S, Akaike A, Satoh M (1997) Involvement of M2 receptor in an enhancement of long-term potentiation by carbachol in Schaffer collateral-CA1 synapses of hippocampal slices. Neurosci Res 27:175–180. Shinoe T, Matsui M, Taketo M, Manabe T (2005) Modulation of synaptic plasticity by physiological activation of M1 muscarinic acetylcholine receptors in the mouse hippocampus. J Neurosci 25: 11194 –11200. Sokolov MV, Kleschevnikov AM (1995) Atropine suppresses associative LTP in the CA1 region of rat hippocampal slices. Brain Res 672:281–284. Stillman MJ, ShukittHale B, Galli RL, Levy A, Lieberman HR (1996) Effects of M(2) antagonists on in vivo hippocampal acetylcholine levels. Brain Res Bull 41:221–226. Stillman MJ, Shukitthale B, Kong RM, Levy A, Lieberman HR (1993) Elevation of hippocampal extracellular acetylcholine levels by methoctramine. Brain Res Bull 32:385–389.

761

Tanaka Y, Sakurai M, Hayashi S (1989) Effect of scopolamine and HP-029, a cholinesterase inhibitor, on long-term potentiation in hippocampal slices of the guinea-pig. Neurosci Lett 98:179 –183. Tani Y, Saito K, Imoto M, Ohno T (1998) Pharmacological characterization of nicotinic receptor-mediated acetylcholine release in rat brain: an in vivo microdialysis study. Eur J Pharmacol 351: 181–188. Tombaugh G, Rowe W, Chow A, Michael T, Rose G (2002) Thetafrequency synaptic potentiation in CA1 in vitro distinguishes cognitively impaired from unimpaired aged Fischer 344 rats. J Neurosci 22:9932–9940. Tzavaral ET, Bymaster FP, Felder CC, Wade M, Gomeza J, Wess J, McKinzie DL, Nomikos GG (2003) Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice. Mol Psychiatry 8: 673– 679. Vannucchi MG, Scali C, Kopf SR, Pepeu G, Casamenti F (1997) Selective muscarinic antagonists differentially affect in vivo acetylcholine release and memory performances of young and aged rats. Neuroscience 79:837– 846. Yun SH, Cheong MY, Mook-Jung I, Huh K, Lee CJ, Jung MW (2000) Cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus of the rat. Neuroscience 97:671– 676. Zhang WL, Basile AS, Gomeza J, Volpicelli LA, Levey AI, Wess J (2002) Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice. J Neurosci 22:1709 –1717. Zheng F, Gingrich MB, Traynelis SF, Conn PJ (1998) Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition. Nat Neurosci 1:185–191.

(Accepted 2 October 2006) (Available online 13 November 2006)