The key findings of this study are: (i) activation of M1 mAChR increased the neuronal AP firing via blockade of M-current, (ii) activation of M2 mAChR increased the paired-pulse ratio of field potentials and decreased both components of GABAergic inhibition, and (iii) activation of M4 mAChR depressed glutamatergic transmission presumably via activation of presynaptic receptors.
The previous studies investigating the mAChRs in slice preparations used either sharp microelectrode recordings [20, 30] or whole cell patch clamp recordings [22, 23, 31, 32]. The latter disturb the intracellular milieu and a massive rundown of M-current (modulated by mAChRs) was observed , precluding a reliable pharmacology. Intracellular recordings in cortical slices with sharp microelectrodes hence present a valuable method to quantify the effects at native mAChRs in slices.
However, reports in the neocortex focussed on the role of mAChRs or M-current either on the neuronal firing [20, 30, 32], on the GABAergic transmission [23, 31] or on the glutamatergic transmission [22, 34]. Only few studies in cortex e.g.  or in hippocampus e.g.  investigated the role of mAChRs on both neuronal firing and synaptic transmission. The comparison of data obtained in different preparations using different experimental conditions is problematic, though. Therefore, in the present study we investigated the 3 aspects, namely change of firing, depression of EPSPs and of IPSPs, in the same preparation under identical experimental conditions.
Although some authors used different concentrations of mAChRs agonists/antagonists, only one or few stimulus intensities e.g.  or current injections e.g.  were used. Therefore, the results were mostly qualitative from a physiological point of view, precluding quantitative comparisons of different agonists/antagonists.
Effect of mAChR activation on neuronal excitability
The consistent increase in the slope of neuronal firing by CCh in the somatosensory cortex is similar to the hippocampus  and the nucleus gracilis . However, our determination of slope and intercept provides a more quantitative analysis of changes of firing compared to previous observations [35, 36]. In particular, these authors compared the neuronal firing in response to a single current intensity before and during substance application. The interpretation of such data would be complicated by concomitant alterations of membrane potential and/or neuronal input resistance.
In any case, alterations of firing are mediated by mAChRs, as evidenced by the antagonism by atropine. Moreover, the antagonism by pirenzepine (M1/M4 mAChRs antagonist) but not by AFDX (M2/M4 mAChRs antagonist) further suggests an effect via M1 mAChR, similar to the M1 mAChR-mediated repetitive spontaneous neuronal firing observed in rat prefrontal cortex . Our conclusion is in line with previous data indicating that ACh application increases the neuronal firing of neocortical pyramidal neurones in mice lacking M3 or M5 mAChRs but not in mice lacking M1 mAChR, confirming further an involvement of the M1 mAChR in this effect .
The cellular mechanisms underlying the CCh-induced increase of AP firing can be inferred from the effect of linopirdine which is an established blocker of KV7 channels . Linopirdine increased the neuronal firing as in hippocampus  and entorhinal cortex . Together, these data indicate a M1 mAChR mediated depression of M-current  as the underlying mechanism of CCh-induced firing increase.
Effect of mAChR activation on GABAergic transmission
We studied the mAChR mediated effect on neurotransmitter release by using the paired-pulse protocol with field potentials. CCh increased the PPR for interpulse intervals ranging from 10 to 400 ms. This suggests that the CCh effect involves both GABAA and GABAB receptor-mediated effects. The CCh-induced increase of the PPR is most likely due to mAChR-mediated depression of GABA release , and evoked IPSCs , during the first stimulus. Normally, GABA would attenuate the response to a second stimulus by decreasing release via presynaptic GABAB receptors [39, 40]. Hence a mAChR-mediated depression of GABA release might contribute to the altered paired pulse properties of synaptic responses.
The involvement of M2 mAChR in the CCh-induced depression of GABA release underlying increase of PPR can be inferred from the antagonism by AFDX (M2/M4 mAChRs antagonist) and lack of antagonism by pirenzepine (M1/M4 mAChRs antagonist). Similar to the pharmacology at M1 mAChR, the effects of atropine and AFDX on this M2 mAChR-mediated increase of PPR were identical whether the antagonist was applied in the presence of, or before, CCh.
Further evidence was obtained by evaluating the alterations in inhibitory conductance mediated by GABAA and GABAB receptors. Firstly, the similarity in the depression of both gIPSP-A and gIPSP-B, indicates a common denominator for both effects i.e. depression of GABA release. The effects of the selected compounds revealed which mAChR is involved in this depression of GABA release. The CCh effects were considerably antagonized by the M2/M4 mAChRs antagonist AFDX but slightly by the M1/M4 mAChRs antagonist pirenzepine. Moreover, the M1/M4 mAChRs agonist xanomeline had no effects on gIPSP-A or gIPSP-B, yet subsequent addition of CCh reduced gIPSP-A and gIPSP. This observation strongly suggests that neither M1 nor M4 mAChRs are involved in the modulation of GABA release. Together these data support our view that M2 mAChR are involved in the modulation of GABA release.
This view is also supported by histological data. M2 mAChRs are predominantly localized at presynaptic axons of GABAergic neurones and are associated with asymmetric as well as symmetric cortical synapses [16–18, 23], suggesting that M2 mAChR can modulate GABA release in the neocortex. In addition, M1 mAChRs are mainly expressed on cortical pyramidal neurones rather than by GABAergic neurones .
ACh reduces the GABA release in neocortex by two separate mechanisms i) by decreasing Ca2+ currents via activation of a M2 mAChR/PI3K/Ca2+-independent PKC pathway and to a smaller extent by activation of a M1 mAChR/PLC/Ca2+-dependent PKC pathway . This combination might explain the relatively large contribution of the M2 mAChR and the absence of detectable effects of the M1 mAChR in the CCh-induced increase of PPR observed here.
Effect of mAChR activation on glutamatergic transmission
Our observation of CCh decreasing the amplitudes of evoked synaptic potentials is in line with a wealth of evidence from various areas of the rodent brain [22, 34–36]. Both field potentials and intracellularly recorded EPSPs were depressed by CCh to a similar degree. Considering the marginal changes in Em, this decrease cannot be accounted for by a membrane depolarization, confirming results obtained in entorhinal cortex . These authors reported that the depression of synaptic transmission persisted when neurones were returned to their initial resting potential. However, previous observations in hippocampus e.g.  or neocortex e.g. [21, 22] compared the synaptic response following a single stimulus intensity before and during substance application. Our determination of I50, EPSPmax and slope factor provide a more quantitative analysis of changes of synaptic transmission, corroborating and extending previous findings. In particular, the consistent effects over a wide range of stimulus intensities exclude the possibility of threshold phenomena.
Our data indicate that this CCh-induced depression of EPSP is 1) fully prevented when CCh is applied in the presence of atropine or AFDX and 2) only partially prevented when CCh was applied in the presence of pirenzepine. This corroborates and extends previous data from layer V of the somatosensory cortex . Unlike these authors, applying CCh in the presence of antagonists, we also applied the mAChRs antagonists in the presence of CCh, revealing marked differences with different sequence of application. The CCh-mediated depression of EPSP was fully reversed when pirenzepine or atropine was added in the presence of CCh, i.e. pirenzepine is more effective when applied in the presence of the agonist. AFDX, however, only partially reversed this depression, i.e. is less effective when applied in the presence of the agonist. This peculiarity may contribute to the discrepancies between previous reports. Usually, the authors have either applied the antagonist in the presence of agonist or vice versa, the agonist in the presence of antagonists. To the best of our knowledge, the two complementary protocols had not been used in the same study so far e.g. either addition or pre-application was used, see [22, 42].
We propose that pirenzepine, a M1/M4 prefering mAChRs antagonist , affects the EPSP amplitude via M4 mAChR for the following reasons:
1) The increase of neuronal firing by CCh was prevented and reversed by pirenzepine. However, the decrease of EPSPmax by CCh was reversed but not prevented by pirenzepine. This difference may suggest that the pirenzepine-sensitive effects on EPSPmax and on slope of neuronal firing are mediated by two distinct mAChRs, i.e. the pirenzepine-sensitive effect on EPSPmax is not mediated by M1 mAChR.
2) Only M2 and M4 mAChRs (but not M1, M3 and M5 mAChRs) are coupled to G-proteins Gi/Go family involved in the inhibition of neurotransmitter release [7, 44, 45]. In addition, recent evidence indicates that M4 mAChR is the major mAChR subtype responsible for direct cholinergic modulation of EPSP . Therefore, the pirenzepine effect on CCh-induced EPSP depression is probably due to an action of pirenzepine on M4 rather than M1 mAChRs . This view is supported by immunohistochemical data , showing that cortical M1 mAChRs are located exclusively at postsynaptic sites.
3) The effects of atropine and AFDX on the M2 mAChR-mediated increase of PPR were identical regardless of the sequence of application. However, the effects of pirenzepine and AFDX on the CCh-induced decrease of EPSP were different whether this antagonist is applied in the presence or before the CCh application. This difference may suggest that the CCh-induced decrease of EPSPmax and increase of PPR are mediated by two distinct mAChRs, i.e. the CCh-induced decrease of EPSPmax is not mediated by M2 mAChR. Moreover, the increase of PPR by CCh (mediated by M2 mAChR) was not prevented or reversed by pirenzepine. This suggests that pirenzepine is not active at M2 mAChR in our conditions.
4) AFDX applied before CCh or pirenzepine applied in the presence of CCh can both fully prevent and reverse, respectively, the CCh-induced depression of EPSPmax. The similar potency of pirenzepine and AFDX on CCh-induced EPSPmax depression therefore suggests an involvement of M4 mAChR as the most parsimonious explanation.
Cholinergic transmission and cognition
Ample evidence suggests that mAChR-mediated signalling in the cortex is critical for learning and memory, yet the mechanisms of ACh facilitating cognitive processes remained elusive. The effects reported here, namely the depression of EPSP and the increased neuronal firing mediated by different mAChRs would provide a very interesting system for “attention” at the cellular level. The M4 mAChR-mediated depression of EPSP reduces the noise of ongoing synaptic activity while the M1 mAChR-mediated increase of neuronal firing augments the neuronal response to a given synaptic input, i.e. these two effects would improve the signal to noise ratio. Moreover, the shallower input–output curve of synaptic responses induced by M4 mAChR observed here extends the subthreshold range for temporal summation, which is augmented by a CCh-induced reduction of Kir-type potassium current . In addition, the cholinergic depression of GABA release corresponds to a depression of inhibition by higher frequencies of stimulation  implicated in long-term potentiation . Together, these data corroborate and extend a theoretical framework proposed by Hasselmo and McGaughy .