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Muscarinic control of intracortical connections to layer II in rat entorhinal cortex slice
Neuroscience Letters 273 (1999) 200±202 www.elsevier.com/locate/neulet
Muscarinic control of intracortical connections to layer II in rat entorhinal cortex slice Michael Richter, Tom Schilling, Wolfgang MuÈller* AG Molekulare Zellphysiologie, Johannes-MuÈller-Institut fuÈr Physiologie der ChariteÂ, Humboldt UniversitaÈt zu Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany Received 9 August 1999; accepted 11 August 1999
Abstract The cholinergic system is critically involved in oscillatory network activity and synaptic plasticity in the entorhinal cortex (EC) hippocampal formation. Here we demonstrate robust inhibition of ®eld potentials in layer II of the medial EC evoked by stimulation in the deep EC or in the lateral layer II by carbachol (CCh, 0.1±100 mM, KD ~1 mM). This effect appears not to be mediated by suppression of presynaptic Ca 21-signals since paired pulse facilitation was increased by CCh. Blockade of the effect by the muscarinic antagonists atropine and pirenzepine demonstrates mediation by muscarinic receptors, most likely of the M1 subtype. The effect is characterized by absence of desensitization and should be important for laminar shaping of oscillatory activity and synaptic plasticity during acetylcholine-dependent theta-rhythmic activity. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Muscarinic; Carbachol; Pirenzepine; Entorhinal cortex; Field potential; Paired pulse; Rat; Brain slice
Acetylcholine is known to exert profound control over the excitability and synaptic plasticity in many neurons of the entorhinal cortex-hippocampal formation. Sensory information is transferred via the layer II of the entorhinal cortex (EC) to the dentate gyrus and activity is fed back via the hippocampal cell layers and the deep layers of the EC to layers II and III of the EC [1,5,17]. The EC and the hippocampus are known to receive profuse cholinergic input from the basal forebrain and the septum, respectively. Muscarinic activation increases excitability and augments postsynaptic Ca 21-responses in principal neurons, thereby facilitating induction of synaptic plasticity [5,8,11]. On the network level, muscarinic activation induces synchronous population activity in the theta and less strong, in the gamma frequency range that depends on glutamatergic transmission [1,5]. In the present study we address cholinergic modulation of synaptically evoked ®eld potentials in layer II of the EC that is supposed to strongly in¯uence synchronous population activity within the EC. Horizontal entorhinal cortex-hippocampus slices (400 mm thick) were prepared from adult ether anesthetized Wistar rats (180±200 g) using a Campden manual vibratome * Corresponding author. Tel.: 149-30-2802-6121; fax: 149-302802-6669. E-mail address: [email protected] (W. MuÈller)
slicer (Loughborough, England) and standard techniques [6,7,12]. The slices were continuously perfused with oxygenated (95% O2/5% CO2) arti®cial cerebrospinal ¯uid (ACSF) containing (in mM): NaCl 129, KCl 3, CaCl2 1.6, MgCl2 1.8, NaHCO3 21, NaH2PO4 1.25, glucose 10 (358C, pH 7.4). Extracellular recordings were obtained in the interface con®guration. Sharp microelectrodes were pulled on a Brown±Flaming puller (Sutter Inst. Novato, CA, USA) from 1.5 mm borosilicate glass and ®lled with 4 M NaCl. Recordings were made with a Neuro Data IR-283 current-clamp ampli®er. Presynaptic ®bers were stimulated over a width of about 100 mm by bipolar glass-insulated platinum electrodes (0.1±0.2 ms, 3±15 V). Electrophysiological data were recorded with an Apple Macintosh Computer and a MacADIOS II AD interface and SuperScope II software (GW Instruments, Sommerville, MA, USA). Carbachol (CCh, 1±100 mM), atropine (1±10 mM) and pirenzepine (1±10 mM, all from Sigma, Deisenhofen, Germany) were bath applied by continuous perfusion [14]. Field potentials in layer II of the medial entorhinal cortex were evoked by bipolar stimulation either in layer V of the medial entorhinal cortex or in layer II of the lateral entorhinal cortex. These ®eld potentials had amplitudes of 1±3 mV and were completely blocked by a combined bath application of the ionotropic glutamate receptor antagonists NBQX (10 mM) and APV (30 mM), demonstrating mediation of
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 64 3- 6
M. Richter et al. / Neuroscience Letters 273 (1999) 200±202
201
synaptic ®eld potentials by ionotropic glutamate receptors. To address a possible involvement of modulation of presynaptic Ca 21-signaling [15], we tested for paired pulse facilitation of ®eld potentials at pulse intervals of 5±100 ms. Paired pulse stimulation revealed a signi®cant paired pulse facilitation of the population spike (PS) of up to 1110% at an interval of 30 ms (Fig. 1Al,B1). Fig. 1 demonstrates that bath application of CCh (1±100 mM) dose-dependently suppressed both these population responses by up to 65% (population spike amplitude). The least square ®t of the dose-response relation to the data for cholinergic inhibition gives a KD of 1:3 ^ 0:01 mM for the ®rst ®eld potential, but 4:1 ^ 0:02 mM for the second, facilitated ®eld potential. This reduced sensitivity of the second ®eld potential may
Fig. 2. Inhibition of cholinergic inhibition of ®eld potentials by muscarinic antagonists atropine and pirenzepine. (A) Field potentials are suppressed during superfusion with CCh (10 mM) and recover during combined perfusion of CCh and atropine (10 mM). (B) Field potentials are suppressed during superfusion with CCh (1 mM) and recover during combined perfusion of CCh and pirenzepine (1 mM). (C) Graph of time course of population spike (PS) amplitudes demonstrates continued suppression of the two PS for 60 min without adaptation for the ®rst and only minor adaptation for the second PS. Perfusion of pirenzepine (10 mM) in addition to CCh recovers about 90% of the PS in agreement with binding data for the muscarinic M1-receptor while atropine recovers the small residual component.
Fig. 1. Suppression of evoked ®eld potentials by carbachol. (A) Paired stimulation (*) in the deep EC at an interval of 30 ms evokes ®eld potentials with signi®cant paired pulse facilitation. Both ®eld potentials are reversibly inhibited by CCh (10 mM). (B) Mean values ^ SD of population spikes (PS) in control, during application of CCh (10 mM) and wash demonstrate a statistically signi®cant effect for the ®rst and second PS (P , 0:001, Student's t-test, n 9). (C) The paired pulse facilitation index (PPI) shows a slight but not signi®cant increase of facilitation by CCh (n 9). (D) Dose response relation for inhibition of the ®rst and second population spike by CCh with least square ®ts (lines, n 35).
be due to a cholinergic augmentation of the residual presynaptic calcium [2,4,11,15] and facilitation of transmitter release or a Ca 21-dependence of the inhibitory muscarinic effect. Cholinergic inhibition of ®eld potentials (FPs) was indistinguishable for stimulation in layer V of the medial EC or in layer II of the lateral EC. The ®rst and the facilitated second ®eld potential recovered completely with washout of CCh within 20 min (Fig. 1A3,B3). To address involvement of muscarinic receptors in the cholinergic suppression of glutamatergic ®eld responses, we used the speci®c muscarinic receptor antagonist atropine. Blockade of muscarinic receptors with atropine (1± 10 mM) completely reversed inhibition of ®eld potentials by CCh (Fig. 2A). To discriminate between muscarinic receptor subtypes for modulation of responses evoked from the deep EC, we applied the M1-receptor preferring antagonist pirenzepine. Due to a preference of ACh and CCh for M2- over M1-receptors, appropriate concentration
202
M. Richter et al. / Neuroscience Letters 273 (1999) 200±202
ratios of CCh and pirenzepine will hardly activate M1receptors (,10%) while a large fraction of M2-receptors will be activated (.80%; [12±14]). Bath application of pirenzepine (1 or 10 mM) in addition to bath application of CCh (1 or 10 mM, respectively) effectively reversed the effect of perfusion of CCh alone (1 or 10 mM, respectively) by some 90%, indicating that the muscarinic suppression of the layer II ®eld potential is mediated by the M1-receptor subtype (Fig. 2B,C, n 7). In good agreement with binding constants, atropine recovers a small residual fraction of the ®eld potential [13] (Fig. 2C). Many M1-receptor mediated effects are characterized by a strong desensitization [10,14]. Fig. 2C demonstrates that with continued application of CCh for 60 min inhibition of the ®eld potentials remained on a stable level without any sign of desensitization or adaptation for the ®rst and only a small adapting component for the second, facilitated ®eld potential. Our results demonstrate a strong muscarinic control in layer II of the EC of ®eld responses to stimulation in the lateral layer II as well as in the deep EC. This effect is most likely mediated by pirenzepine-sensitive M1-receptors that are coupled via G-proteins and phospholipase C to protein kinase C and IP3-mediated release of intracellular Ca 21. Absence of a signi®cant inhibitory effect on paired pulse facilitation suggests that muscarinic inhibition of these evoked ®eld responses is not mediated by a modulation of presynaptic Ca 21-signaling. Muscarinic inhibition of presynaptic Ca 21-signals appears to require application of a much higher concentration of CCh [3]. Presynaptic muscarinic inhibition of transmitter release has been demonstrated for hippocampal synaptosomes and the lateral amygdala, nucleus accumbens and striatum [9,16]. Rather excitatory postsynaptic effects of CCh onto EC layer II principal cells [5] suggest inhibition of ®eld potentials to be presynaptic. This will be tested in the near future with intracellular recording. With respect to the receptor subtype our data support inhibition of EC layer II ®eld potentials by activation of Ml-receptors. In contrast to other M1-receptor mediated effects, we did not observe desensitization, probably due to a direct effect of G-protein subunits onto the release machinery. Muscarinic inhibition of excitatory synaptic transmission adds an important component to muscarinic induction of synchronous population activity in the EC. Inhibition of inputs to layer II would tend to dampen oscillatory activity in this area, thereby reducing the likelihood of activitydependent synaptic modi®cation in this layer and, in pathophysiological conditions, neurodegeneration. We thank K. Stenkamp for contributions to initial experiments, K. Stenkamp, U. Heinemann and T. Gloveli for helpful discussions and A. DuÈerkop for excellent technical assistance. This study was supported by DFG through grant Mu 809/6±2, a Heisenberg-stipend to W.M. and a Charite grant.
[1] Dickson, C.T. and Alonso, A., Muscarinic induction of synchronous population activity in the entorhinal cortex. J. Neurosci., 17 (1997) 6729±6744. [2] Egorov, A.V., Gloveli, T. and MuÈller, W., Muscarinic control of Ca 21-signalling in CA1 pyramidal neurons in rat hippocamel slice. J. Neurophysiol., 82 (1999) in press. [3] Egorov, A.V., Heinemann, U. and MuÈller, W., Muscarinic activation reduces changes in [Ca 21]o evoked by stimulation of Schaffer collaterals during blocked synaptic transmission in rat hippocampal slices. Neurosci. Lett., 214 (1996) 187±190. [4] Egorov, A.V. and MuÈller, W., Subcellular muscarinic enhancement of excitability and Ca 21-signals in CA1dendrites in rat hippocampal slice. Neurosci. Lett., 261 (1999) 77±80. [5] Gloveli, T., Egorov, A.V., Schmitz, D., Heinemann, U. and MuÈller, W., Muscarinic control of excitability and [Ca 21]isignaling in layer II and III projection neurons of the medial entorhinal cortex. Eur. J. Neurosci., 11 (1999) 1±12. [6] Jones, R.S., Complex synaptic responses of entorhinal cortical cells in the rat to subicular stimulation in vitro: demonstration of an NMDA receptor-mediated component. Neurosci. Lett., 81 (1987) 209±214. [7] Jones, R.S. and Heinemann, U., Synaptic and intrinsic responses of medial entorhinal cortical cells in normal and magnesium-free medium in vitro. J. Neurophysiol., 59 (1988) 1476±1496. [8] Klink, R. and Alonso, A., Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons. J. Neurophysiol., 77 (1997) 1829±1843. [9] Marchi, M. and Raiteri, M., Interaction acetylcholine-glutamate in rat hippocampus: involvement of two subtypes of M-2 muscarinic receptors. J. Pharmacol. Exp. Ther., 248 (1989) 1255±1260. [10] Misgeld, U., MuÈller, W. and Polder, H.R., Potentiation and suppression by eserine of muscarinic synaptic transmission in the guinea-pig hippocampal slice. J. Physiol. Lond., 409 (1989) 191±206. [11] MuÈller, W. and Connor, J.A., Cholinergic input uncouples Ca 21 changes from K 1 conductance activation and ampli®es intradendritic Ca 21 changes in hippocampal neurons. Neuron, 6 (1991) 901±905. [12] MuÈller, W. and Misgeld, U., Slow cholinergic excitation of guinea pig hippocampal neurons is mediated by two muscarinic receptor subtypes. Neurosci. Lett., 67 (1986) 107±112. [13] MuÈller, W. and Misgeld, U., Carbachol and pirenzepine discriminate effects mediated by two muscarinic receptor subtypes on hippocampal neurons in vitro. Experientia, 57 (Suppl.) (1989) 114±122. [14] MuÈller, W., Heinemann, U. and Misgeld, U., Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration. Exp. Brain Res., 72 (1988) 287±298. [15] Regehr, W.G. and Tank, D.W., The maintenance of LTP at hippocampal mossy ®ber synapses is independent of sustained presynaptic calcium. Neuron, 7 (1991) 451±459. [16] Sugita, S., Uchimura, N., Jiang, Z.G. and North, R.A., Distinct muscarinic receptors inhibit release of gammaaminobutyric acid and excitatory amino acids in mammalian brain. Proc. Natl. Acad. Sci. USA, 88 (1991) 2608±2611. [17] Witter, M.P., Groenewegen, H.J., Lopes da Silva, F.H. and Lohman, A.H., Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region. Prog. Neurobiol., 33 (1989) 161±253.
Muscarinic control of intracortical connections to layer II in rat entorhinal cortex slice Michael Richter, Tom Schilling, Wolfgang MuÈller* AG Molekulare Zellphysiologie, Johannes-MuÈller-Institut fuÈr Physiologie der ChariteÂ, Humboldt UniversitaÈt zu Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany Received 9 August 1999; accepted 11 August 1999
Abstract The cholinergic system is critically involved in oscillatory network activity and synaptic plasticity in the entorhinal cortex (EC) hippocampal formation. Here we demonstrate robust inhibition of ®eld potentials in layer II of the medial EC evoked by stimulation in the deep EC or in the lateral layer II by carbachol (CCh, 0.1±100 mM, KD ~1 mM). This effect appears not to be mediated by suppression of presynaptic Ca 21-signals since paired pulse facilitation was increased by CCh. Blockade of the effect by the muscarinic antagonists atropine and pirenzepine demonstrates mediation by muscarinic receptors, most likely of the M1 subtype. The effect is characterized by absence of desensitization and should be important for laminar shaping of oscillatory activity and synaptic plasticity during acetylcholine-dependent theta-rhythmic activity. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Muscarinic; Carbachol; Pirenzepine; Entorhinal cortex; Field potential; Paired pulse; Rat; Brain slice
Acetylcholine is known to exert profound control over the excitability and synaptic plasticity in many neurons of the entorhinal cortex-hippocampal formation. Sensory information is transferred via the layer II of the entorhinal cortex (EC) to the dentate gyrus and activity is fed back via the hippocampal cell layers and the deep layers of the EC to layers II and III of the EC [1,5,17]. The EC and the hippocampus are known to receive profuse cholinergic input from the basal forebrain and the septum, respectively. Muscarinic activation increases excitability and augments postsynaptic Ca 21-responses in principal neurons, thereby facilitating induction of synaptic plasticity [5,8,11]. On the network level, muscarinic activation induces synchronous population activity in the theta and less strong, in the gamma frequency range that depends on glutamatergic transmission [1,5]. In the present study we address cholinergic modulation of synaptically evoked ®eld potentials in layer II of the EC that is supposed to strongly in¯uence synchronous population activity within the EC. Horizontal entorhinal cortex-hippocampus slices (400 mm thick) were prepared from adult ether anesthetized Wistar rats (180±200 g) using a Campden manual vibratome * Corresponding author. Tel.: 149-30-2802-6121; fax: 149-302802-6669. E-mail address: [email protected] (W. MuÈller)
slicer (Loughborough, England) and standard techniques [6,7,12]. The slices were continuously perfused with oxygenated (95% O2/5% CO2) arti®cial cerebrospinal ¯uid (ACSF) containing (in mM): NaCl 129, KCl 3, CaCl2 1.6, MgCl2 1.8, NaHCO3 21, NaH2PO4 1.25, glucose 10 (358C, pH 7.4). Extracellular recordings were obtained in the interface con®guration. Sharp microelectrodes were pulled on a Brown±Flaming puller (Sutter Inst. Novato, CA, USA) from 1.5 mm borosilicate glass and ®lled with 4 M NaCl. Recordings were made with a Neuro Data IR-283 current-clamp ampli®er. Presynaptic ®bers were stimulated over a width of about 100 mm by bipolar glass-insulated platinum electrodes (0.1±0.2 ms, 3±15 V). Electrophysiological data were recorded with an Apple Macintosh Computer and a MacADIOS II AD interface and SuperScope II software (GW Instruments, Sommerville, MA, USA). Carbachol (CCh, 1±100 mM), atropine (1±10 mM) and pirenzepine (1±10 mM, all from Sigma, Deisenhofen, Germany) were bath applied by continuous perfusion [14]. Field potentials in layer II of the medial entorhinal cortex were evoked by bipolar stimulation either in layer V of the medial entorhinal cortex or in layer II of the lateral entorhinal cortex. These ®eld potentials had amplitudes of 1±3 mV and were completely blocked by a combined bath application of the ionotropic glutamate receptor antagonists NBQX (10 mM) and APV (30 mM), demonstrating mediation of
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 64 3- 6
M. Richter et al. / Neuroscience Letters 273 (1999) 200±202
201
synaptic ®eld potentials by ionotropic glutamate receptors. To address a possible involvement of modulation of presynaptic Ca 21-signaling [15], we tested for paired pulse facilitation of ®eld potentials at pulse intervals of 5±100 ms. Paired pulse stimulation revealed a signi®cant paired pulse facilitation of the population spike (PS) of up to 1110% at an interval of 30 ms (Fig. 1Al,B1). Fig. 1 demonstrates that bath application of CCh (1±100 mM) dose-dependently suppressed both these population responses by up to 65% (population spike amplitude). The least square ®t of the dose-response relation to the data for cholinergic inhibition gives a KD of 1:3 ^ 0:01 mM for the ®rst ®eld potential, but 4:1 ^ 0:02 mM for the second, facilitated ®eld potential. This reduced sensitivity of the second ®eld potential may
Fig. 2. Inhibition of cholinergic inhibition of ®eld potentials by muscarinic antagonists atropine and pirenzepine. (A) Field potentials are suppressed during superfusion with CCh (10 mM) and recover during combined perfusion of CCh and atropine (10 mM). (B) Field potentials are suppressed during superfusion with CCh (1 mM) and recover during combined perfusion of CCh and pirenzepine (1 mM). (C) Graph of time course of population spike (PS) amplitudes demonstrates continued suppression of the two PS for 60 min without adaptation for the ®rst and only minor adaptation for the second PS. Perfusion of pirenzepine (10 mM) in addition to CCh recovers about 90% of the PS in agreement with binding data for the muscarinic M1-receptor while atropine recovers the small residual component.
Fig. 1. Suppression of evoked ®eld potentials by carbachol. (A) Paired stimulation (*) in the deep EC at an interval of 30 ms evokes ®eld potentials with signi®cant paired pulse facilitation. Both ®eld potentials are reversibly inhibited by CCh (10 mM). (B) Mean values ^ SD of population spikes (PS) in control, during application of CCh (10 mM) and wash demonstrate a statistically signi®cant effect for the ®rst and second PS (P , 0:001, Student's t-test, n 9). (C) The paired pulse facilitation index (PPI) shows a slight but not signi®cant increase of facilitation by CCh (n 9). (D) Dose response relation for inhibition of the ®rst and second population spike by CCh with least square ®ts (lines, n 35).
be due to a cholinergic augmentation of the residual presynaptic calcium [2,4,11,15] and facilitation of transmitter release or a Ca 21-dependence of the inhibitory muscarinic effect. Cholinergic inhibition of ®eld potentials (FPs) was indistinguishable for stimulation in layer V of the medial EC or in layer II of the lateral EC. The ®rst and the facilitated second ®eld potential recovered completely with washout of CCh within 20 min (Fig. 1A3,B3). To address involvement of muscarinic receptors in the cholinergic suppression of glutamatergic ®eld responses, we used the speci®c muscarinic receptor antagonist atropine. Blockade of muscarinic receptors with atropine (1± 10 mM) completely reversed inhibition of ®eld potentials by CCh (Fig. 2A). To discriminate between muscarinic receptor subtypes for modulation of responses evoked from the deep EC, we applied the M1-receptor preferring antagonist pirenzepine. Due to a preference of ACh and CCh for M2- over M1-receptors, appropriate concentration
202
M. Richter et al. / Neuroscience Letters 273 (1999) 200±202
ratios of CCh and pirenzepine will hardly activate M1receptors (,10%) while a large fraction of M2-receptors will be activated (.80%; [12±14]). Bath application of pirenzepine (1 or 10 mM) in addition to bath application of CCh (1 or 10 mM, respectively) effectively reversed the effect of perfusion of CCh alone (1 or 10 mM, respectively) by some 90%, indicating that the muscarinic suppression of the layer II ®eld potential is mediated by the M1-receptor subtype (Fig. 2B,C, n 7). In good agreement with binding constants, atropine recovers a small residual fraction of the ®eld potential [13] (Fig. 2C). Many M1-receptor mediated effects are characterized by a strong desensitization [10,14]. Fig. 2C demonstrates that with continued application of CCh for 60 min inhibition of the ®eld potentials remained on a stable level without any sign of desensitization or adaptation for the ®rst and only a small adapting component for the second, facilitated ®eld potential. Our results demonstrate a strong muscarinic control in layer II of the EC of ®eld responses to stimulation in the lateral layer II as well as in the deep EC. This effect is most likely mediated by pirenzepine-sensitive M1-receptors that are coupled via G-proteins and phospholipase C to protein kinase C and IP3-mediated release of intracellular Ca 21. Absence of a signi®cant inhibitory effect on paired pulse facilitation suggests that muscarinic inhibition of these evoked ®eld responses is not mediated by a modulation of presynaptic Ca 21-signaling. Muscarinic inhibition of presynaptic Ca 21-signals appears to require application of a much higher concentration of CCh [3]. Presynaptic muscarinic inhibition of transmitter release has been demonstrated for hippocampal synaptosomes and the lateral amygdala, nucleus accumbens and striatum [9,16]. Rather excitatory postsynaptic effects of CCh onto EC layer II principal cells [5] suggest inhibition of ®eld potentials to be presynaptic. This will be tested in the near future with intracellular recording. With respect to the receptor subtype our data support inhibition of EC layer II ®eld potentials by activation of Ml-receptors. In contrast to other M1-receptor mediated effects, we did not observe desensitization, probably due to a direct effect of G-protein subunits onto the release machinery. Muscarinic inhibition of excitatory synaptic transmission adds an important component to muscarinic induction of synchronous population activity in the EC. Inhibition of inputs to layer II would tend to dampen oscillatory activity in this area, thereby reducing the likelihood of activitydependent synaptic modi®cation in this layer and, in pathophysiological conditions, neurodegeneration. We thank K. Stenkamp for contributions to initial experiments, K. Stenkamp, U. Heinemann and T. Gloveli for helpful discussions and A. DuÈerkop for excellent technical assistance. This study was supported by DFG through grant Mu 809/6±2, a Heisenberg-stipend to W.M. and a Charite grant.
[1] Dickson, C.T. and Alonso, A., Muscarinic induction of synchronous population activity in the entorhinal cortex. J. Neurosci., 17 (1997) 6729±6744. [2] Egorov, A.V., Gloveli, T. and MuÈller, W., Muscarinic control of Ca 21-signalling in CA1 pyramidal neurons in rat hippocamel slice. J. Neurophysiol., 82 (1999) in press. [3] Egorov, A.V., Heinemann, U. and MuÈller, W., Muscarinic activation reduces changes in [Ca 21]o evoked by stimulation of Schaffer collaterals during blocked synaptic transmission in rat hippocampal slices. Neurosci. Lett., 214 (1996) 187±190. [4] Egorov, A.V. and MuÈller, W., Subcellular muscarinic enhancement of excitability and Ca 21-signals in CA1dendrites in rat hippocampal slice. Neurosci. Lett., 261 (1999) 77±80. [5] Gloveli, T., Egorov, A.V., Schmitz, D., Heinemann, U. and MuÈller, W., Muscarinic control of excitability and [Ca 21]isignaling in layer II and III projection neurons of the medial entorhinal cortex. Eur. J. Neurosci., 11 (1999) 1±12. [6] Jones, R.S., Complex synaptic responses of entorhinal cortical cells in the rat to subicular stimulation in vitro: demonstration of an NMDA receptor-mediated component. Neurosci. Lett., 81 (1987) 209±214. [7] Jones, R.S. and Heinemann, U., Synaptic and intrinsic responses of medial entorhinal cortical cells in normal and magnesium-free medium in vitro. J. Neurophysiol., 59 (1988) 1476±1496. [8] Klink, R. and Alonso, A., Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons. J. Neurophysiol., 77 (1997) 1829±1843. [9] Marchi, M. and Raiteri, M., Interaction acetylcholine-glutamate in rat hippocampus: involvement of two subtypes of M-2 muscarinic receptors. J. Pharmacol. Exp. Ther., 248 (1989) 1255±1260. [10] Misgeld, U., MuÈller, W. and Polder, H.R., Potentiation and suppression by eserine of muscarinic synaptic transmission in the guinea-pig hippocampal slice. J. Physiol. Lond., 409 (1989) 191±206. [11] MuÈller, W. and Connor, J.A., Cholinergic input uncouples Ca 21 changes from K 1 conductance activation and ampli®es intradendritic Ca 21 changes in hippocampal neurons. Neuron, 6 (1991) 901±905. [12] MuÈller, W. and Misgeld, U., Slow cholinergic excitation of guinea pig hippocampal neurons is mediated by two muscarinic receptor subtypes. Neurosci. Lett., 67 (1986) 107±112. [13] MuÈller, W. and Misgeld, U., Carbachol and pirenzepine discriminate effects mediated by two muscarinic receptor subtypes on hippocampal neurons in vitro. Experientia, 57 (Suppl.) (1989) 114±122. [14] MuÈller, W., Heinemann, U. and Misgeld, U., Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration. Exp. Brain Res., 72 (1988) 287±298. [15] Regehr, W.G. and Tank, D.W., The maintenance of LTP at hippocampal mossy ®ber synapses is independent of sustained presynaptic calcium. Neuron, 7 (1991) 451±459. [16] Sugita, S., Uchimura, N., Jiang, Z.G. and North, R.A., Distinct muscarinic receptors inhibit release of gammaaminobutyric acid and excitatory amino acids in mammalian brain. Proc. Natl. Acad. Sci. USA, 88 (1991) 2608±2611. [17] Witter, M.P., Groenewegen, H.J., Lopes da Silva, F.H. and Lohman, A.H., Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region. Prog. Neurobiol., 33 (1989) 161±253.