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The Journal of Physiology logoLink to The Journal of Physiology
. 2006 Oct 5;577(Pt 3):829–840. doi: 10.1113/jphysiol.2006.117119

Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex

Nicola Origlia 1,2, Nicola Kuczewski 2, Eugenio Aztiria 2, Dinesh Gautam 3, Jürgen Wess 3, Luciano Domenici 1,2,4
PMCID: PMC1890385  PMID: 17023506

Abstract

In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long-term synaptic plasticity. Specifically, we investigated long-term potentiation (LTP) and long-term depression (LTD) expression elicited by theta-burst stimulation (TBS) and low-frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M1/M3 double KO mice, while LTP was impaired in the M2/M4 double KO animals. On the other hand, LFS induced LTD in M2/M4 double KO mice, but failed to do so in M1/M3 KO mice. Interestingly, LFS produced LTP instead of LTD in M1/M3 KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M2 and M4 receptors. A LFS-dependent shift from LTD to LTP was also observed in slices from M1 KO mice, while LTD was simply abolished in slices from M3 KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of Gi/o proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M1 single KO and M1/M3 double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long-term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms.

A great number of studies conducted at physiological, morphological and behavioural levels have clearly demonstrated the involvement of the cholinergic system in the regulation of several brain functions. Cortical ACh release affects synaptic transmission (Krnjevic & Ropert, 1981; Gil et al. 1997; Kuczewski et al. 2005a), development of neuronal circuitry (Hohmann et al. 1991; Siciliano et al. 1997; Robertson et al. 1998; for a review see Gu, 2002) and high cognitive functions such as attention, learning and memory (Damasio et al. 1985; Casamenti et al. 1998; Steriade et al. 1991; Leanza et al. 1995; Everitt & Robbins, 1997; Sarter & Bruno, 2000; Dalley et al. 2001; Conner et al. 2003). The activity of the cholinergic system is also involved in the plastic reorganization of neuronal connectivity in the cerebral cortex (Aztiria et al. 2004). Interestingly, long-term potentiation (LTP) and long-term depression (LTD), two forms of synaptic plasticity believed to participate in the shaping of neuronal connections as well as in learning and memory, are regulated by cholinergic transmission (Brocher et al. 1992; Kirkwood et al. 1999; Pesavento et al. 2000). The fact that the reduction of cortical cholinergic innervation produces a shift from LTP to LTD that can be prevented by exogenous application of ACh (Kuczewski et al. 2005b) suggests a regulatory action of ACh on the direction of synaptic plasticity (LTP versus LTD). However, the role played by the different types of cholinergic receptors in these forms of plastic modifications remains to be determined (for a review see Bear, 2003). In the present report, we focused our attention on the role played by the muscarinic ACh receptors (mAChRs), and in particular, whether different mAChRs subtypes have different roles in the induction of LTP and LTD. Using mutant mice together with pharmacological tools, we investigated the role of different mAChRs in LTP and LTD. In particular, LTP induction and maintenance, as well as LTD, were studied in occipital slices from mice lacking specific mAChR subtypes. In addition, the roles of Gi/o proteins and phospholipase C (PLC) activation on long-term synaptic plasticity were evaluated by using pharmacological tools.

Methods

All experiments were carried out with mice that were at least 6 weeks old, and conducted following the European Community Council Directive for animal treatment. The generation of homozygous M1–M5 receptor single knockout (KO) mice (genetic background: 129/SvEv × CF1 (M1, M3, M4 and M5) or 129J1 × CF1 (M2)) has been previously described (Gomeza et al. 1999a, b; Yamada et al. 2001a, b; Fisahn et al. 2002). The generation and genetic background of the M2/M4 and M1/M3 double KO mice has been previously described (Duttaroy et al. 2002; Gautam et al. 2004). For each KO strain, wild-type (WT) mice of the same mixed genetic background were used in parallel as controls. In a subset of experiments, we also used mAChR KO mice that had been backcrossed for 10 generations onto the C57BL/6NTac background (Taconic Farms, Germantown, NY, USA).

Slice preparation and electrophysiology

Animals were anaesthetized by intraperitoneal injection of urethane (1.2 g kg−1; Sigma, St Louis, MO, USA) and decapitated immediately after disappearance of tail pinch reflex. Cortical coronal sections (400 μm thick) of the occipital pole were sliced with a vibratome. All steps were performed in ice-cold standard artificial cerebrospinal fluid (ACSF) solution (mm: NaCl, 126; KCl, 2.5; CaCl2, 2.5; MgCl2 1.25; NaH2PO4, 1.2; NaHCO3, 19; and glucose, 11) bubbled with 95% O2–5% CO2 unless otherwise stated. Prior to recording, slices were stored for at least 1 h in a recovery chamber containing oxygenated ACSF, at 33 ± 1°C. During electrophysiological recordings, slices were perfused at 3–4 ml min−1 with oxygenated ACSF, at 33 ± 1°C.

In a separate group of slices, the composition of ACSF used to perfuse slices was modified to block the AMPA glutamate receptor and to isolate NMDA receptor (NMDAr)-mediated field potential (FP). Modified ACSF contained high calcium (3 mm CaCl2) and low magnesium (0.1 mm MgCl2) in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Sigma; 20 μm; see drugs) and glycine (1 μm).

Extracellular FPs were evoked via a tungsten concentric bipolar stimulating electrode placed in layer IV. The recording electrode (pulled glass capillaries, o.d. 1.0 mm, i.d. 0.78 mm) was filled with ACSF solution and placed in layer II/III. The amplitude of the FPs in layer II/III was used as a measure of the evoked population excitatory current (see Kuczewski et al. 2005a, b). Baseline responses were obtained with a stimulation intensity that yielded 50–60% of maximal amplitude. FP amplitudes were monitored every 20 s and averaged every three responses. Theta-burst stimulation (TBS; 10 bursts of 5 pulses at 100 Hz; 250 ms between bursts) was used for LTP induction; low-frequency stimulation (LFS; 900 pulses at 1 Hz) was used for LTD induction. At least 10 min of stable basal FPs were recorded before TBS or LFS. LTP and LTD amplitudes were measured as relative values obtained by averaging the FP amplitudes during the last 10 min of recording after TBS and LFS, respectively, and normalized with respect to the average of FP amplitudes during 10 min of basal stimulation. Values are expressed as mean amplitude percentage change (±s.e.m.).

Drugs

The following drugs were used: atropine (Sigma) to block mAChRs; CNQX to block AMPA glutamate receptors (AMPAr); 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; Alexis Biochemicals) to block PLC; pertussis toxin (PTx; Sigma), a Gi/o-protein inhibitor; forskolin (Sigma) to stimulate adenylate cyclase; Ro 20-1724 (Sigma), an inhibitor of cAMP phosphodiesterase. Atropine (1 μm), U73122 (4 μm), forskolin (40 μm) and Ro 20-1724 (50 μm) were bath applied through the perfusion medium for 10 or 20 min, starting 5 min before TBS or LFS, respectively. CNQX (20 μm) was dissolved in modified ACSF and bath applied. Briefly, at the beginning slices were bath perfused with standard ACSF for at least 10 min and FPs were recorded. After 10 min, standard ACSF was substituted with modified ACSF plus CNQX. This step was followed by wash out and recovery of slices in standard ACSF.

In experiments involving PTx treatment, slices were incubated in ACSF containing PTx (5 μg ml−1) for at least 6 h before recordings.

Histochemistry

Mice were anaesthetized by intraperitoneal injection of urethane (1.2 g kg−1; Sigma) and perfused with 4% paraformaldehyde/PBS (pH 7.4) after disappearance of tail pinch reflex. Samples were cryoprotected in 30% sucrose/PBS at 4°C, and coronal sections (40 μm thick) were cut on a freezing microtome and collected in four series. One of the series was subjected to Nissl staining. Another series of sections from each mouse group was processed for acetylcholinesterase (AChE) histochemistry, as described elsewhere (Hedreen et al. 1985), in order to determine whether the lack of mAChRs affected normal terminal cholinergic innervation of the cortex. In brief, sections were incubated in a medium containing sodium citrate, copper sulphate, potassium ferricyanide, and acetylthiocholine iodide, at pH 6. Non-specific esterases were inhibited by the addition of ethopropazine (Sigma) at a final concentration of 10−4m to the incubation mixture. The reaction product was finally intensified by 1 min incubation with ammonium sulphide and silver nitrate. Sections from control and KO mice were processed simultaneously in order to avoid artefacts in the estimation of the AChE-positive fibre density. Mounted sections were analysed in a blinded fashion under the microscope and the density of cholinergic fibres was determined using the NIH Image software (Rasband & Bright, 1995). The optical density of the corpus callosum, which normally contains no AChE-positive fibres, was used as a blank and was therefore subtracted from that of the visual cortex.

Statistics

Statistical comparison between FP amplitudes measured during baseline and during the last 10 min of recording following TBS or LFS was performed by applying Student's t test. The t test was also used for comparisons among different groups. Differences were considered significant with P < 0.05.

Results

In occipital slices of WT mice LTP and LTD were induced by TBS and LFS, respectively (LTP: 124 ± 7% of baseline after TBS, n = 7; Fig. 1A; LTD: 73 ± 9% of baseline after LFS, n = 6; Fig. 1B). In order to investigate whether LTP and LTD require the activation of mAChR, we bath applied the mAChR antagonist atropine; the duration of atropine application was 10 min, centred around TBS, and 20 min during LFS (Fig 1C and D). Atropine, 1 μm, prevented both LTP (98 ± 4%, n = 6; Fig. 1C) and LTD (98 ± 9%, n = 6; Fig. 1D) without interfering with basal responses (Fig 1E, n = 10). Therefore the activation of mAChRs is necessary to induce LTP and LTD.

Figure 1. Synaptic plasticity in the visual cortex of wild-type (WT) mice requires muscarinic acetylcholine receptor (mAChR) activation.

Figure 1

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A, theta-burst stimulation (TBS) of layer IV produces a stable long-term potentiation (LTP) in the visual cortex slices. B, low-frequency stimulation (LFS) of layer IV produces a stable long-term depression (LTD) in the visual cortex slices. C, LTP is prevented by bath application of 1 μm atropine. D, LTD is prevented by bath application of 1 μm atropine. E, bath application of 1 μm atropine has no effect on baseline synaptic responses. Black horizontal bars in B and D represent the time window of LFS. Grey horizontal bars represent atropine application time windows. Top inserts in A, B, C, D and E show representative field potentials (FPs). Scale bars for FPs are shown in E.

To assess the involvement of the different mAChR subtypes in synaptic plasticity, we first investigated the effects of TBS and LFS in M1/M3 and M2/M4 double KO mice. TBS evoked a normal LTP in M1/M3 double KO mice (125 ± 5% of baseline, n = 6; Fig. 2A), while LTP was impaired in the M2/M4 double KO animals (102 ± 7% of baseline, n = 8, P < 0.05 with respect to WT; Fig. 2A). On the other hand, LFS induced a normal LTD in M2/M4 KO mice (70 ± 9% of baseline, n = 6; Fig. 2B) but failed to do so in M1/M3 KO animals; interestingly, LFS produced LTP instead of LTD in M1/M3 KO mice (151 ± 12%, n = 7; Fig. 2B). To examine whether synaptic transmission was affected in mAChR KO mice, we assessed the relationship between the amplitude of the response and the stimulus intensity under basal conditions (input–output curve). In Fig. 2C we plotted the amplitude of FP as a function of increasing stimulus intensity; the input–output curves in WT (n = 7), M1/M3 (n = 6) and M2/M4 (n = 6) double KO mice clearly overlapped, thus excluding the possibility that the lack of mAChRs affected basal synaptic transmission. Further investigations were conducted on FP amplitude during induction protocols, i.e. TBS and LFS. Concerning TBS, we analysed the responses during the first burst, and we calculated the amplitude of the responses evoked by the fifth stimulus expressed as mean percentage with respect to the amplitude responses evoked by the first stimulus; the relative amplitude of fifth response was attenuated as result of synaptic fatigue without significant differences in magnitude between WT, and M1/M3 and M2/M4 double KO mice (see Table 1). For LFS, the responses evoked by the 10th stimulus during the stimulation protocol (900 pulses) were expressed in relative percentage changes with respect to the amplitude response evoked by the first stimulus and then averaged: the mean values were 57 ± 6% in WT mice, 53 ± 10% in M2/M4 double KO mice, and 62 ± 10% in M1/M3 double KO mice (Table 1; no significant differences were found between the three experimental groups). In addition, we investigated whether the responses induced by paired-pulse stimulation were modified in mAChR double KO mice. In WT, and M1/M3 and M2/M4 double KO mice, the effects of paired-pulse stimulation were examined. In these animals, we analysed the amplitude of responses induced by pairs of stimuli with different interstimulus intervals (ISIs; 50–200 ms), measuring the ratio between the second and first response amplitudes. Table 2 summarizes paired-pulse ratios in WT, and M1/M3 and M2/M4 double KO mice, showing no significant differences in magnitude between the three different experimental groups. We also studied the NMDA component of the FP synaptic response. Previous studies (Quinlan et al. 1999; Philpot et al. 2001; Huemmeke et al. 2004) showed that the FP recorded in layer II–III of visual cortical slices maintained in ACSF is dominated by the activity of AMPA glutamate receptors (see also Myme et al. 2003); indeed application of 10–20 μm CNQX blocked most of the FP response. Under our experimental conditions, NMDAr mediated FPs were pharmacologically isolated in modified ACSF containing high calcium and low magnesium in the presence of CNQX (20 μm) to block AMPAr (see Methods, ACSF modified composition). Figure 2D provides examples of FPs recorded in standard ACSF, and in modified ACSF plus CNQX (top row, left and right side, respectively). In agreement with Huemmeke et al. (2004), AMPAr-mediated FP had a latency of 5–5.5 ms, while NMDAr-mediated FP had a longer latency (range of variability, 6.5–7 ms) and was reduced in amplitude with respect to AMPAr-mediated FP (see histogram in Fig. 2D). The histogram in Fig. 2D indicates that the amplitudes of NMDAr-mediated FPs expressed as relative values with respect to FPs in standard ACSF were not significantly different between WT (29 ± 5%, n = 5), and M1/M3 (33 ± 3%, n = 4) M2/M4 (31 ± 4%, n = 4) double KO mice. Thus, the absence of mAChRs did not influence synaptic transmission.

Figure 2. LTP and LTD depend on different mAChR subtypes in mouse visual cortex.

Figure 2

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A, TBS stimulation failed to induce LTP in the visual cortex slices of M2/M4 but not M1/M3 double knockout (KO) mice. B, LFS produced a normal LTD in the visual cortex slices of M2/M4 KO animals, but produced an LTP in the visual cortex slices of M1/M3 KO mice (black horizontal bar represents the time window of LFS). Top inserts in A and B show representative FPs; horizontal scale bar, 10 ms; vertical scale bar, 0.5 mV. C, input–output curves in WT, M1/M3 and M2/M4 KO slices; FP amplitudes are normalized with respect to values at 10 V stimulation. D, top insets show representative FP recorded in standard artificial cerebrospinal fluid (ACSF) (left) and NMDAr-mediated FP in modified ACSF (high calcium, low magnesium, see Methods) plus CNQX (right); the asterisk indicates the non-synaptic component of the FP. The histogram shows the mean values of FP amplitude (NMDAr-mediated FP) in modified ACSF expressed as percentage of FP amplitude in standard ACSF. Briefly, FPs in layer II–III (frequency of stimulus 0.05 Hz) were recorded in slices bath perfused with standard ACSF; after 10 min, standard ACSF was substituted with modified ACSF containing high calcium and low magnesium plus CNQX. No significant differences in NMDAr FP amplitude were found between WT, M1/M3 and M2/M4 KO mice.

Table 1.

Variation of field potential during TBS and LFS induction protocols

TBS (% change) LFS (% change)
WT 45 ± 4 57 ± 6
M2/M4 KO 48 ± 3 53 ± 10
M1/M3 KO 56 ± 3 62 ± 10
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TBS, theta-burst stimulation; LFS, low-frequency stimulation; WT, wild type; KO, knockout. Values in the TBS column represent the mean ±s.e.m. percentage change of the fifth pulse response with respect to the first during the first burst of stimulation. In the LFS column, values represent the mean ±s.e.m. percentage change of the 10th pulse response with respect to the first during the stimulation protocol (900 pulses, 1 Hz, n = 8 slices for each group).

Table 2.

Paired-pulse facilitation of FP with different interstimulus intervals (ISIs)

ISI WT M1/M3 KO M2/M4 KO
50 ms 124 ± 4 132 ± 7 129 ± 4
100 ms 109 ± 1 105 ± 1 111 ± 4
150 ms 93 ± 5 95 ± 6 94 ± 3
200 ms 95 ± 2 102 ± 2 99 ± 1
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Values represent the mean ±s.e.m. of the ratio between the second and the first pulse response amplitudes obtained for different ISI intervals (n = 6 slices for each group).

To investigate whether KO mice displayed abnormalities of cortical cholinergic innervation, we analysed AChE expression in the primary visual cortex of WT and mAChR double KO mice (Fig. 3A and B). Cortical cholinergic innervation, which is involved in mechanisms underlying LTP (Kuczewski et al. 2005b), was normal in KO mice, as suggested by AChE staining (Fig. 3C, n = 3 for control and KO mice). Thus, the impairment in long-term synaptic plasticity observed with mAChR KO mice was not due to morphological alterations of cortical cholinergic innervation.

Figure 3. Cortical slices from M1/M3 and M2/M4 double KO mice exhibit normal cholinergic fibre density and distribution.

Figure 3

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A, acetylcholinesterase (AChE) positive fibre density was measured from the boxed field shown in layers II–III of the visual cortex. High-magnification views of the equivalent squared area shown in A are shown in B for each of the double KO mice and the respective controls (calibration bar is 950 μm for A and 190 μm for B). C, results of densitometric analyses carried out on AChE-stained sections (3 animals per group).

In order to identify the contribution of each individual mAChR subtype on long-term synaptic plasticity, we investigated M1–M4 receptor single KO mice. We examined LTP expression only in slices obtained from M2 or M4 single KO mice, since LTP was not compromised in M1/M3 double KO animals. We found that TBS was able to induce a normal LTP in both M2 (132 ± 11%, n = 5; Fig. 4A, □) and M4 (133 ± 6%, n = 8, Fig. 4A, •) receptor single KO mice, suggesting that LTP is affected only when both M2 and M4 mAChRs are absent.

Figure 4. M2 and M4 mAChR single KO mice show normal LTP, while LTD is impaired in M1 and M3 mAChR single KO mice.

Figure 4

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A, in visual cortex slices of M2 and M4 single KO mice, TBS induced a normal LTP. B, LFS failed to induce LTD in the visual cortex slices of M3 single KO mice, and produced an LTP in M1 single KO mice (black horizontal bar represents the time window of LFS). Top inserts in A and B show representative FPs; vertical scale bar, 0.5 mV, horizontal scale bar, 5 ms.

As described above, the simultaneous lack of M1 and M3 receptors induced a shift from LTD to LTP following LFS. In M1 single KO mice, LFS produced a shift from LTD to LTP (150 ± 8%, n = 5; Fig. 4B, □), similar to the results obtained with the M1/M3 double KO mice. On the other hand, in M3 KO slices, LFS failed to induce LTD without any significant change with respect to baseline (105 ± 9%, n = 5; Fig. 4B, ▵). Thus, both M1 and M3 receptors contribute to LTD expression in mouse visual cortex.

To exclude the possibility that the mixed genetic background of the mutant mice used affected the outcome of the studies described above, we repeated key experiments with M1 single KO, M3 single KO and M2/M4 double KO mice that had been backcrossed for 10 generations onto the C57BL/6NTac background. We found that LFS induced the shift from LTD to LTP in backcrossed M1 single KO mice (127 ± 5%, n = 7), but failed to induce LTD in backcrossed M3 single KO mice (99 ± 11%, n = 5; Fig. 5A and B); TBS failed to induce LTP in backcrossed M2/M4 double KO mice (109 ± 8, n = 5; Fig. 5C). Thus, the results obtained with the backcrossed KO mice exclude the possibility that the observed changes in LTP and LTD are dependent on a specific (mixed) mouse genetic background.

Figure 5. Control experiments using cortical slices from backcrossed M1 single KO, M3 single KO, and M2/M4 double KO mice.

Figure 5

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Cortical slices were prepared from KO mice that had been backcrossed for 10 generations onto the C57BL/6NTac background. A, LFS induced LTP in M1 single KO mice. B, LFS failed to induce LTD in M3 single KO mice. C, TBS failed to induce LTP in M2/M4 double KO mice.

Downstream mechanisms involved in muscarinic-receptor-dependent LTP and LTD were studied using pharmacological tools interfering with different intracellular pathways activated by mAChRs. M2 and M4 mAChRs are coupled to the PTx-sensitive Gi and Go proteins, inducing the inhibition of adenylyl cyclase, whereas the M1 and M3 mAChRs preferentially interact with the PTx-insensitive Gq/11 and G13 proteins, leading to the activation of phospholipases C and D (for a review see Wess, 1996). We blocked Gi and Go proteins in control mice by pretreating the slices with 5 μg ml−1 PTx (Chen et al. 2001; DeBock et al. 2003); the aim of this treatment was to mimic the absence of M2 and M4 mAChRs. PTx blocked the induction of LTP by TBS (104 ± 1%, n = 5; Fig. 6A), leaving LTD unaffected (80 ± 9%, n = 6; Fig. 7B), which was similar to the results obtained with M2/M4 double KO mice. PTx was also used in M2/M4 double KO mice to exclude non-specific effects. In the presence of PTx, TBS was unable to elicit LTP, similar to the findings obtained with untreated slices from M2/M4 double KO mice (PTx-treated slices, FP amplitude = 99 ± 5%, n = 4, data not shown; untreated slices, FP = 102 ± 7%, see Fig. 2A). Moreover, LTD after LFS was unaffected by PTx in slices from M2/M4 double KO mice (PTx-treated slices, FP = 75 ± 7%, data not shown; untreated slices, FP = 70 ± 9%, see Fig. 2B). To investigate whether cAMP was involved in downstream processes triggered by Gi/o, we used bath application of forskolin (40 μm), which activates adenylate cyclase, and Ro 20-1724 (50 μm), an inhibitor of cAMP phosphodiesterase (Lu & Gean, 1999). We found that LTP was not affected by forskolin (145 ± 7, n = 5, Fig. 6B) or Ro 20-1724 treatment (136 ± 12, n = 6, Fig. 6C), suggesting that cAMP modulation is not required for LTP expression.

Figure 6. Inhibition of Gi/o proteins but not of phospholipase C (PLC) blocks TBS-induced LTP in WT cortical slices.

Figure 6

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A, preincubation of slices with 5 μg ml−1 pertussis toxin (PTx) inhibits LTP after TBS stimulation. B, bath application of forskolin (40 μm) does not influence TBS-induced LTP. C, bath application of Ro 20-1724 (50 μm) does not affect TBS-induced LTP, in analogy to the results shown in B. D, bath application of the PLC inhibitor U73122 (4 μm) has no effect on LTP induction. Grey horizontal bars in A, B, C and D represent drug application time windows.

Figure 7. LFS-induced LTP in M1 single KO mice is muscarinic receptor dependent, and requires activation of PTx-sensitive G proteins.

Figure 7

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A, in WT slices, bath application of U73122 (4 μm) during LFS mimics the absence of M1 receptors, inducing a shift to LTP. B, WT slices preincubated with PTx (5 μg ml−1) show normal LTD after LFS. C, bath application of atropine (1 μm) inhibits the LFS induced shift to LTP in M1 KO slices. D, the LFS-induced shift to LTP in M1 KO slices is blocked by PTx pretreatment. Grey horizontal bars in A and C represent the time windows of drug application.

We then used the PLC inhibitor U73122, which inhibits mAChR-stimulated phosphoinositide hydrolysis in human neuroblastoma (Thompson et al. 1991) and rat pancreatic acinar cells (Yule & Williams, 1992), with an IC50 value of 3.7 μm (Thompson et al. 1991). Bath application of U73122 (4 μm) did not affect LTP induced by TBS in WT slices (138 ± 7%, n = 5; Fig. 6D), in agreement with previous studies with visual cortex slices (Edagawa et al. 2000). However, LFS induced an LTP instead of LTD in WT slices treated with U73122 (167 ± 12%, n = 5; Fig. 7A), similar to the results obtained with M1/M3 double KO and M1 single KO mice. The paradoxical LTP shift observed in M1/M3 double KO and M1 single KO following LFS was sensitive to atropine, indicative of the involvement of mAChRs; indeed, bath application of atropine (1 μm) blocked LTP after LFS in M1 single KO mice (95 ± 8%, n = 6; Fig. 7C). Finally, we tested whether LTP induced by LFS in the absence of M1 receptors requires PTx-sensitive G proteins. Remarkably, LFS failed to induce both LTD and LTP in M1 single KO slices treated with PTx (104 ± 7%, n = 6; Fig. 7D).

Thus, these results indicate that PTx-sensitive G proteins control LTP induced by TBS, while PTx-insensitive G proteins, via activation of PLC, are involved in LTD induced by LFS. The paradoxical shift from LTD to LTP induced by LFS in the absence of M1 receptors is a mixed effect due to failure of PLC activation coupled with the activation of PTx-sensitive G proteins.

Discussion

In the present paper, we showed that LTP and LTD rely on the activation of different mAChRs. The principal mAChR subtypes expressed in rodent cortex are M1, M2, M3 and M4 (Levey et al. 1991; Kuczewski et al. 2005a). These receptors can be grouped according to the type of G proteins that they activate. M2 and M4 mAChRs are coupled to the PTx-sensitive Gi and Go proteins, leading to the inhibition of adenylyl cyclase, whereas the M1 and M3 mAChRs preferentially interact with the PTx-insensitive Gq/11 and G13 proteins, leading to the activation of phospholipases C and D (for a review see Wess, 1996). We showed that different mAChRs control different forms of long-term synaptic plasticity. Indeed, an impairment of LTP but not of LTD was found in M2/M4 mAChR double KO mice, while M1/M3 KO mice lacked LTD but showed normal LTP. The analysis of mAChR single KO mice demonstrated that LTP was not affected in M2 or M4 single KO animals, while an impairment of LTD was observed in the absence of either M1 or M3 mAChRs. On the other hand, the expression of LTD requires M1- and M3-dependent stimulation of PTx-insensitive G proteins. In M1/M3 double KO and in M1 single KO mice, LFS induced a LTP instead of LTD.

The present data suggest that the expression of cortical synaptic plasticity depends on the activation of different G-protein-linked pathways activated by different mAChR. Our data suggest that M2- and M4-dependent activation of Gi and/or Go proteins is required for LTP but not for LTD. PTx, an inhibitor of Gi and Go proteins, blocked LTP induction by TBS in control mice, suggesting that M2 and M4 mAChRs are involved in the induction and/or maintenance of LTP, but not LTD, through activation of Gi/Go. We also examined whether modulation of intracellular cAMP levels is involved in LTP expression. To this aim we used two different compounds, forskolin and Ro 20-1724, both of which raise intracellular cAMP levels. LTP in WT slices was not affected by either compound, suggesting that inhibition of cAMP through Gi/Go is not required for LTP. Since Gi/Go activation leads to inhibition of adenylate cyclase through Gα subunits, as well as activation of other effector proteins such as PI3 kinase (PI3K) through Gβγ (Rosenblum et al. 2000), it is possible that Gi/o proteins coupled to M2/M4 receptors can activate Gβγ-dependent pathways converging on MAP kinases, such as extracellular signal regulated kinases (ERKs; Koch et al. 1994; Crespo et al. 1994). In agreement with this notion, LTP in visual cortex requires the activation of ERK (Di Cristo et al. 2001).

Our data suggest that more complex mechanisms underlie LTD induction. As shown by the results obtained with single KO mice, the absence of M3 mAChRs results in the blockade of LTD, suggesting a role of M3 mAChRs in LTD. The lack of M1 mAChRs induced a paradoxical shift from LTD to LTP following LFS. Studies with atropine demonstrated that this shift involves signalling via mAChRs. Moreover, using an inhibitor of PLC (U73122; Thompson et al. 1991; Yule & Williams, 1992) in control slices we were able to reproduce the shift from LTD to LTP following LFS. Interestingly, LTP was enhanced in slices treated with U73122 with respect to single M1 and double M1/M3 KO mice, suggesting that the lack of M1 and M3 mAChRs is not sufficient to completely abolish PLC activation involved in long-term synaptic plasticity. Indeed, other neurotransmitter receptors such as the serotonin 5-HT2 receptor (Edagawa et al. 2000) and the metabotropic glutamate receptors (Otani et al. 2002) are involved in forms of cortical plasticity dependent on PLC activation. Using PTx in M1 single KO mice, we showed that the paradoxical LTP generation after LFS is blocked. Thus, the shift from LTP to LTD in the absence of M1 mAChRs is due to failure of PLC activation coupled to PTx-sensitive G protein action.

Regarding long-term synaptic plasticity, the present results suggest that the direction of synaptic plasticity depends on the combined activity of different mAChRs. An intriguing possibility is that ACh, by acting on different mAChR subtypes, regulates the Bienenstock-Cooper-Munro (BMC) threshold for synaptic modification. According to the BMC model, a given synaptic stimulus could produce either LTP or LTD depending on whether or not it is able to overcome a certain modification threshold (Θm; reviewed by Bear, 2003). According to this hypothesis, Θm would be affected by an imbalance between M1/M3 and M2/M4 receptor signalling in a way that the predominant activation of M2/M4 receptors in absence of M1 receptors, as occurring in M1 single KO mice, would lead to a decreased Θm, favouring the induction of LTP over LTD. Consistent with this concept, a recent report showed that Θm could vary according to the expression levels of different NMDA receptor subunits (Philpot et al. 2001). From previous studies, there is increasing evidence that mAChRs and NMDA receptors interact to modulate neuronal plasticity in the visual cortex (Boroojerdi et al. 2001) and in other brain areas (Liu et al. 2004; Massey et al. 2004; Young et al. 2004; Grishin et al. 2005). Thus, it remains to be investigated whether the interaction of mAChRs and NMDA receptors at the cellular level may lead to the modulation of Θm.

Another possibility is that in the absence of the M1 receptors, the M2 and M4 receptors that show higher affinity for ACh than the M3 receptor (Lazareno & Birdsall, 1995) would be preferentially activated leading to LTP instead of LTD.

In the present paper, we showed that LTP was normal in M2 and M4 single KO animals. Previous studies (for a review see Wess, 2004) have shown that the disruption of one specific mAChR gene has little effect on the expression levels of the remaining mAChRs. Our data therefore strongly suggest that LTP in the visual cortex is affected only when both M2 and M4 mAChRs are lacking. Our results differ from recent data obtained in the hippocampus where the absence of M2 receptors is sufficient to produce a pronounced impairment of LTP (Seeger et al. 2004), indicative of local differences in the dependence of LTP and LTD on single mAChRs. Thus, the local and selective activation of different mAChR subtypes by ACh may contribute to the multitude of actions that the cholinergic system exerts on brain functions, such as learning and memory, attention and sensory map plasticity, through modulation of the different forms of long-term synaptic plasticity.

Acknowledgments

We are grateful to Roberto Maggio and Tommaso Pizzorusso for helpful discussions. We acknowledge Carlo Orsini for online acquisition software and for technical assistance. This work was partially supported by a grant (05/140) from Fondazione Cassa di Risparmio, Pisa.

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