Distinct Muscarinic Receptor Subtypes Suppress Excitatory and Inhibitory Synaptic Responses in Cortical Neurons

Fumitaka Kimura and Robert W. Baughman

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Kimura, Fumitaka and Robert W. Baughman. Distinct muscarinic receptor subtypes suppress excitatory and inhibitory synaptic responses in cortical neurons. J. Neurophysiol. 77: 709-716, 1997. Simultaneous whole cell recordings from monosynaptically connected cortical cells were performed with the use of two patch pipettes to determine the effect of acetylcholine (ACh) on both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in cultured neurons from rat visual cortex. For 96% of EPSPs and 73% of IPSPs, ACh potently suppressed postsynaptic potentials in a dose-dependent manner. The estimated effective concentrations to produce half maximal response (EC50s) were 30 and 210 nM for EPSPs and IPSPs, respectively. To identify what subtypes of ACh receptors are involved in the suppression of postsynaptic potentials, three different, partially selective muscarinic receptor antagonists were used. According to the comparison of estimated Schild coefficients for each of the three antagonists against the suppression by ACh, EPSPs are most likely mediated by m4 receptors, and IPSPs by m1 receptors. When cells were treated with pertussis toxin, which inactivates m2 and m4 receptors while leaving m1, m3, and m5 receptors intact, 7 of 8 EPSPs were resistant to ACh whereas 8 of 12 IPSPs were still suppressed by ACh. This result supports the interpretation that the suppression of EPSPs was mediated by m4 receptors and that of IPSPs by m1 receptors. To obtain an indication as to whether ACh works presynaptically or postsynaptically, 1/CV2 analysis was carried out. The resultant diagonal alignment of the ratio of 1/CV2 plotted against the ratio of the amplitude of postsynaptic potentials suggests a presynaptic mechanism for the suppression of both EPSPs and IPSPs. In addition, in many cases a large synaptic suppression was observed without an obvious change in the input resistance. Furthermore, in one case where a single inhibitory driver cell was recorded with three different follower cells sequentially, none of the three IPSPs was suppressed by ACh, providing additional support for the presynaptic localization of ACh action. These results suggest that in cerebral cortex ACh has, in addition to its direct facilitatory effect via m3 pharmacology, a suppressive effect on EPSPs and IPSPs via m1 and m4 muscarinic receptors, respectively, probably with a presynaptic site of action. Separation of the actions of ACh into different receptor-second messenger pathways with potential for independent interactions with other neuromodulatory systems may be an important aspect of the mechanism of cholinergic regulation of functional state in cortex. Separation of cholinergic effects at different receptors might also offer a means for selective pharmacological intervention in disorders of sleep or memory.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

In mammalian cortex, acetylcholine (ACh) influences the functional and behavioral state, but how it acts at the synaptic level is unclear. Cholinergic innervation in cortex arises from magnocellular neurons in the basal forebrain (Eckenstein et al. 1988; Houser et al. 1985; Rye et al. 1984). The innervation is diffuse and only roughly topographic, and it seems likely to play a neuromodulatory rather than a direct synaptic role (Umbriaco et al. 1994). Application of exogenous ACh in cortical areas increases the excitability of many neurons, although some are inhibited (reviewed in Caulfield 1993; McCormick 1992). The increase in excitability appears to be associated with reduction in K+ conductances (Caulfield 1993; McCormick 1992). Somewhat surprisingly, given the increase in excitability, ACh also has been shown to suppress excitatory synaptic responses in forebrain neurons (Bröcher et al. 1992; Dutar and Nicoll 1988; Hasselmo and Bower 1992; Kahle and Cotman 1989; Murakoshi 1995; Williams and Constanti 1988; Yamamoto and Kawai 1967), perhaps by presynaptic mechanisms (Hounsgaard 1978; Nickell and Shipley 1993; Segal 1989; Segal et al. 1989; Yamamoto and Kawai 1967). Evidence also exists for muscarinic suppression of inhibitory synaptic responses (Sugita et al. 1991). Muscarinic inhibition of Ca2+ conductances (Bernheim et al. 1992; Caulfield 1993; Segal et al. 1989) and increase of K+ conductances (Caulfield 1993) have been observed, which could account for synaptic suppression.

Of the five known muscarinic receptor genes, four (m1-m4) are expressed in cortex at significant levels (Buckley et al. 1988; Vilaro et al. 1990) in neocortex. We use the classification of muscarinic subtypes based on genotype (Dorje et al. 1991). These receptors are expressed differentially in different cortical layers and possibly in different cell types (Levey et al. 1991; Lidow et al. 1989; Mrzljak et al. 1993; Rossner et al. 1993), but the precise role of the different receptor subtypes is unclear. We have found that m1 and m4 receptors play very specific roles in regulating the strength of synaptic connections.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Culture preparation

Blocks of posterior dorsal cortices including the oc1/oc2 region (Paxinos and Watson 1982) of 1- to 5-day-old rat pups were enzymatically dissociated with papain (Baughman et al. 1991). The cell suspension was plated on cortical glia grown on collagen islands (300-400 µm ID) prepared by evaporating droplets of rat tail collagen (Collaborative Research) placed on a thin sheet of aragose on a 22-mm glass cover slip. A film of Sylgard (Dow Corning) was used to attach the cover slip over a 1.2-cm hole drilled in the bottom of the dish. After 24-48 h the cultures were treated with cytosine arabinoside (10-5 M) to inhibit cell division. Recordings were made from neurons maintained in culture from 7-141 days. Before 7 days in culture, synaptic contacts were fewer in number.

Electrophysiology

Electrophysiological recordings were made from neurons in culture with whole cell patch pipettes. The pipette solution contained (in mM) 150.0 KCH3SO3, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5.0 KCl, 0.5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5.0 MgATP, and 1.0 Na2GTP (reagents from Sigma), pH 7.3. Membrane potentials were recorded in current-clamp mode with an Axon Instruments Axoclamp 2A amplifier and digitized at 5-8 kHz (Indec Systems). Stimulation and recording were carried out simultaneously on two channels with CTWO software (K. Jones, Indec Systems). The normal bathing solution consisted of minimal essential medium (Gibco) modified by adding 10 µM glycine and reducing the NaHCO3 concentration to 1.3 mM. When equilibrated with atmospheric CO2, this medium had a pH of 7.4. The NaHCO3 subtracted from the minimal essential medium was replaced with NaCl.

Drug application

ACh and other drugs were applied through micropipes (200 µm ID) whose tips were positioned to bathe the recorded cell completely (Fig. 1). In some experiments with excitatory postsynaptic potentials (EPSPs), tetrodotoxin (Calbiochem) was added at a concentration of 5-30 nM to the bathing and micropipe solution to reduce polysynaptic activity. In experiments with inhibitory postsynaptic potentials (IPSPs), 1.0 mM kynurenic acid (Sigma) was added to all solutions. Data were analyzed off-line with CTWO, KaleidaGraph, StatView, and custom software written by F. Soo. Sources of drugs were as follows: ACh and methoctramine from Sigma, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) and pirenzepine from Research Biochemicals International.


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FIG. 1. Schematic diagram of preparation for recording from synaptic pairs. Whole cell patch recordings are made from 2 synaptically connected neurons in culture. An action potential in the 1st neuron produces a postsynaptic potential (PSP) in the 2nd neuron. Drugs are applied via micropipes positioned to bathe both neurons completely. ACh, acetylcholine; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Recordings of postsynaptic potentials (PSPs) were made from neurons from rat visual cortex maintained in cell culture. Seventy excitatory synaptic pairs and 56 inhibitory synaptic pairs were recorded with the preparation shown in Fig. 1. In each case the monosynaptic nature of the connection was confirmed. Criteria included short (1-4 ms) as well as constant synaptic latencies and ability to follow high-frequency (30-50 Hz) action potentials. For excitatory pairs, polysynaptic excitation was commonly observed, and in these cases tetrodotoxin was added at a concentration (5-30 nM) sufficient to reduce the response to a monosynaptic EPSP. As reported earlier (Huettner and Baughman 1988), stimulation of inhibitory PSPs sometimes led to polysynaptic EPSPs, presumably as a result of disinhibition, so for inhibitory cells, 1 mM kynurenic acid was added to the medium, which left only a monosynaptic IPSP. The membrane potential of the postsynaptic cell was adjusted by current injection to approximately -65 to -70 mV for EPSPs and -50 to -40 mV for IPSPs, which gave sufficient driving force to produce readily measurable PSPs in each case.

Suppressive effect of ACh

When ACh was perfused locally through a micropipe, the amplitude of both EPSPs and IPSPs was suppressed, although the action of ACh on EPSPs was more potent. An example of suppression of an EPSP is shown in Fig. 2A; the effect was dose dependent (Fig. 2B). The EC50 for ACh-mediated suppression of EPSPs was ~30 nM (Fig. 2D). An example of suppression of an IPSP is shown in Fig. 3A; it is also dose dependent (Fig. 3B), with an EC50 of ~210 nM (Fig. 3D). For EPSPs in ~96% of the cases (67 of 70), and for IPSPs in ~73% of the cases (41 of 56), the responses were sensitive to suppression by ACh. Examples of cells with insensitive PSPs are indicated by the triangles in Figs. 2D and 3D. The data for responsive cells were fitted to the logistic equation, and the resulting parameters are listed in Table 1. EPSPs were ~7 times more sensitive to suppression by ACh than were IPSPs, and the maximum suppression achieved with EPSPs was slightly greater than for IPSPs.


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FIG. 2. Effect of ACh on an excitatory synaptic pair. A: depolarization of the cell in the top traces produces an action potential, which elicits an excitatory PSP (EPSP) in a nearby cell shown in the bottom traces. ACh (10-5 M) greatly suppresses the EPSP; the EPSP recovers with washout. Seventeen days in culture. Resting membrane potential: -77 mV (top) and -70 mV (bottom). B: increasing concentrations of ACh produce increased suppression in a dose-dependent manner. Eighty-one days in culture. Resting membrane potential: -65 mV. C: reversal of ACh (10-6 M) suppression of an EPSP by the muscarinic antagonist 4-DAMP (10-5 M). D: filled circles: dose-response curve for ACh suppression of EPSP pairs (mean ± SE, n = 7). Curve fit to logistic equation with nonlinear regression. Filled triangles: dose-response curve for an EPSP pair that was unaffected by ACh. EC50, effective concentration to produce half maximal response.


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FIG. 3. Effect of ACh on an inhibitory synaptic pair. A: action potential in the cell shown in the top trace elicits an inhibitory PSP (IPSP) in a nearby cell shown in the bottom traces. ACh (10-6 M) greatly suppresses the IPSP; the IPSP recovers with washout. One hundred three days in culture. Resting membrane potential: -54 mV (top) and -50 mV (bottom). B: increasing concentrations of ACh produce increased suppression in a dose-dependent manner. Forty-eight days in culture. Resting membrane potential: -61 mV. C: reversal of ACh (10-6 M) suppression of an IPSP by the muscarinic antagonist pirenzepine (10-5 M). D: filled circles: dose-response curve for ACh suppression of IPSP pairs (mean ± SE, n = 10). Curve fit to logistic equation with nonlinear regression. Filled triangles: dose-response curve for an IPSP pair that was unaffected by ACh.

 
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TABLE 1. Logistic parameters of ACh suppression of EPSPs and IPSPs

Distinct muscarinic subtypes involved in the suppression of EPSPs and IPSPs

The suppression of PSPs by ACh was reversed by muscarinic antagonists (Figs. 2C and 3C), which indicates that muscarinic cholinergic receptors are involved. Because m1-m4 muscarinic receptors are expressed in cortex (Buckley et al. 1988; Levey et al. 1991; Vilaro et al. 1990), we selected a series of antagonists to attempt to determine whether specific muscarinic receptor subtypes are involved. None of the available antagonists is completely selective, and we therefore chose a series of three antagonists, pirenzepine, 4-DAMP, and methoctramine, that together provide relatively specific discrimination of m1-m4. Each of these antagonists was able to reverse the suppression of EPSPs and IPSPs produced by ACh, but the potency was different in each case. Dose-response curves for each antagonist are shown for EPSPs in Fig. 4A and for IPSPs in Fig. 4B. Estimates of the affinities for the different antagonists were calculated from the Schild equation (Lazareno and Birdsall 1993a,b), and the results are summarized in Table 2. The approach described by Lazareno and Birdsall (1993a,b) permits estimation of the Schild coefficients with a single agonist concentration if the logistic parameters are known. The pKb (Kb defined as the antagonist dissociation constant) values calculated for each antagonist, for both EPSPs and IPSPs, were compared with the values observed for cloned human m1-m4 muscarinic receptors (Dorje et al. 1991). As indicated in Table 2, for EPSPs the antagonist pKb values observed were not significantly different from those reported for the m4 receptor, and for IPSPs the antagonist pKb values were not significantly different from those of the m1 receptor. In other words, the observed pKb values most closely resemble those for the m4 receptor for EPSPs and the m1 receptor for IPSPs.


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FIG. 4. Dose-response curves for the subtype-selective muscarinic antagonists 4-DAMP (bullet ), pirenzepine (black-square), and methoctramine (black-triangle) in reversing the suppressive effect of ACh on synaptic responses. One hundred percent response represents the maximum reduction in PSP amplitude observed with 10-6 M ACh. Curve fitting to Schild equation with nonlinear regression. A: antagonism of ACh effect on EPSPs (mean ± SE, n = 4 for 4-DAMP, n = 4 for pirenzepine, n = 5 for methoctramine). B: antagonism of ACh effect on IPSPs (mean ± SE, n = 8 for 4-DAMP, n = 7 for pirenzepine, n = 4 for methoctramine).

 
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TABLE 2. Estimates of pKb for antagonists inhibiting ACh-induced suppression of EPSPs and IPSPs

Another way of comparing the antagonists, which follows from the previous analysis, is to look at the relative affinities under given conditions. This is illustrated in Table 3, where the affinities of the different antagonists for the m1-m4 cloned receptors and for blocking ACh suppression of EPSPs and IPSPs are normalized to the affinity for 4-DAMP. This approach also illustrates the similarity of the values for EPSPs with m4 clones receptors, and of the values for IPSPs with m1 cloned receptors.

 
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TABLE 3. Relative affinities of different antagonists at muscarinic receptor subtypes and at EPSPs and IPSPs

Effect of pertussis toxin on the ACh suppression of PSPs

Additional support for this assignment of receptor subtype was obtained from second-messenger pharmacology. Muscarinic m1 and m4 receptors generally act via different second messengers; m4 inhibits adenylyl cyclase, whereas m1 is a potent phospholipase activator. Pertussis toxin selectively inactivates the G protein subunits associated with the m2 and m4 receptor (Higashida et al. 1990). If the m4 receptor actually mediates the action of ACh on EPSPs, then pretreatment with pertussis toxin should make the EPSP responses resistant to application of ACh. Of eight EPSP pairs tested after pretreatment with pertussis toxin, seven were insensitive to ACh (Fig. 5A) and only one still showed some suppression. In contrast, in untreated cultures, as mentioned above, ~96% of the pairs tested responded to ACh. This supports the interpretation that an m4 (or m2) receptor is involved in the muscarinic suppression of EPSPs. On the other hand, of 12 IPSPs tested after treatment with pertussis toxin, 8 (67%) were still suppressed by ACh (Fig. 5B), and 4 were unaffected, close to the fraction of IPSPs suppressed (73%) in control conditions, which suggests that IPSPs are not suppressed by an m4 (or m2)-receptor-based mechanism.


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FIG. 5. Effect of pertussis toxin (PTX) on ACh suppressive effect. Cultures were treated for 3 days with 50 ng/ml pertussis toxin. A: example of an EPSP that was not suppressed by ACh after toxin treatment (ACh effect eliminated). Forty-three days in culture. Resting membrane potential: -63 mV (top) and -66 mV (bottom). B: example of an IPSP that was suppressed after toxin treatment (ACh effect still present). Forty-six days in culture. Resting membrane potential: -56 mV (top) and -45 mV (bottom). In A and B the top calibration bars refer to the top trace (presynaptic cell) and the bottom calibration bars refer to the bottom trace (postsynaptic cell).

Possible presynaptic site of ACh action

To obtain evidence concerning the pre- versus postsynaptic localization of the site of action, the effect of muscarinic suppression at different concentrations of ACh on the variance of the PSPs was analyzed. In the standard quantal model for synaptic transmission, the calculated value of 1/CV2 (where CV or coefficient of variation = SD/mean) of the response is affected by presynaptic changes in either the number of release sites (n) or the probability of release (p), but not by changes in the postsynaptic response (q). This can be visualized by plotting the ratio of experimental to control 1/CV2 against the ratio of experimental to control response amplitude (response ratio), where 1/CV2 = np/(1 - p) (Bekkers and Stevens 1990; Kamiya et al. 1991; Malinow and Tsien 1990; Manabe et al. 1993). A change in the ratio of 1/CV2 with changes in the response ratio indictates a presynaptic action, although it does not distinguish changes in n versus changes in p. If the suppression is produced only by a decrease in postsynaptic responsiveness, a horizontal plot would result, reflecting the independence of 1/CV2 from q. We found that for both EPSPs and IPSPs, 1/CV2 decreased approximately proportionally as the response was suppressed with ACh (Fig. 6). Thus for both EPSPs and IPSPs the results are most consistent with a presynaptic localization of the site of action of ACh, provided the assumption of a standard quantal model is valid (see DISCUSSION).


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FIG. 6. Plot of ratio of experimental to control 1/CV2 (CV or coefficient of variation) as a function of the response ratio (PSP response size in ACh/PSP response size in control) at an ACh concentration of 10-6 M. For each cell 1/CV2 and the response ratio were determined for 16 consecutive PSP responses. Solid lines: results expected for a directly proportional response. A: EPSP responses (n = 12 cells). B: IPSP responses (n = 9 cells).

In one case three different follower cells were obtained for a single inhibitory driver cell, and in all three cells the response was unaffected by ACh (Fig. 7). Similarly, in another case with two different follower cells for an inhibitory driver, both responses were suppressed by ACh. As described in the DISCUSSION, this provides an additional argument that the action of ACh in these cases was likely to have been presynaptic.


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FIG. 7. Three IPSP pairs with the same presynaptic cell are not effected by ACh. In the entire population of 56 IPSP pairs studied, 27% were not effected by ACh. If the action of ACh is postsynaptic, the chance that with 1 presynaptic cell 3 different postsynaptic cells would be unaffected is ~0.019. Top calibration bars: top 2 postsynaptic traces. Bottom calibration bars: bottom postsynaptic trace.

In addition to affecting synaptic strength, ACh had direct actions on the cells, sometimes producing a depolarization, sometimes producing a hyperpolarization or occasionally producing both. These effects, however, at least as detected by recording at the soma, could not account for the suppression of the synaptic responses. In many cases a large synaptic suppression was observed without a detectable direct effect. In most cells the input resistance increased or was unchanged; in the presence of ACh (10-6 M) it was 97.5 ± 10.0% (mean ± SE) of control for EPSP followers (n = 10 and 107.5 ± 7.1% of control for IPSP followers (n = 11).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Our results demonstrate a powerful muscarinic suppression of both EPSPs and IPSPs in cortical neurons. This is consistent with a number of previous studies in hippocampus (Ben-Ari et al. 1981; Hounsgaard 1978; Scanziani et al. 1995; Segal 1982; Valentino and Dingledine 1981; Yamamoto and Kawai 1967), cortex (Hasselmo and Bower 1992; Williams and Constanti 1988), striatum (Hsu et al. 1995), and basal forebrain (Szerb et al. 1994). These extensive demonstrations of muscarinic synaptic suppression in intact tissues support the likelihood that our studies in cell culture represent a reasonable model for in vivo responses. The maximum amount of suppression that we observe is large, equalling ~70-90% of the control response. The median effective concentration for the effect on EPSPs is ~7 times lower than for IPSPs, which suggests that excitatory transmission is essentially blocked by the time inhibitory transmission is effected.

Our assignment of an m4 receptor response for the EPSPs and an m1 receptor response for the IPSPs is based first on the binding affinities for the relatively selective antagonists pirenzepine, methoctramine, and 4-DAMP, as described by Dorje et al. (1991). This provides evidence that neither m2 or m3 receptors are likely to be involved, but the distinction between m1 and m4 is difficult on the basis of the antagonists alone (Fig. 4, Table 2). We therefore tested second-messenger involvement, because m4 responses are generally linked to pertussis-toxin-sensitive G proteins and m1 responses are not. The near elimination of EPSP sensitivity to ACh after pertussis toxin treatment supports the assignment of an m4 mechanism for EPSPs. On the other hand, the persistence of an ACh effect for IPSPs after pertussis toxin treatment is consistent with the assignment of an m1 mechanism for IPSPs. In an earlier study Segal (1982) oberved that pirenzepine was relatively ineffective at preventing ACh suppression of EPSPs in hippocampal neurons and suggested the action of a "non-M1" mechanism, which is consistent with our observations. On the other hand, Das et al. (1992) concluded that in slices of frontal cortex the lack of sensitivity of ACh suppression of EPSPs to himbacine, an m2-m4-selective antagonist, argued against an m4 and for an m1 receptor mechanism. On the basis of the available data, we cannot account for this difference. In amygdala, accumbens, and striatum, Sugita et al. (1991) felt that the action of selective antagonists on muscarinic suppression of excitatory responses was consistent with an M3 (m3) mechanism, although m4 or m5 could not be ruled out. Hsu et al. (1995) also suggested an M3 mechanism for muscarinic supression of EPSPs in striatum on the basis of antagonist rank order.

Support for m1 suppression of IPSPs is provided by the observation that gamma -aminobutyric acid release from cortical slices is suppressed by an m1-like mechanism (Hashimoto et al. 1994; Hasuo et al. 1988). Sugita et al. (1991) also concluded that in amygdala, accumbens, and striatum, muscarinic suppression of gamma -aminobutyric acid responses was likely to be M1 (m1) based.

What is known about the expression of muscarinic receptors in cortical neurons? Levels of mRNA message and antibody detection of expressed protein argue that m1-m4 muscarinic receptors are present in cortex in vivo (Buckley et al. 1988; Levey et al. 1991), and that m1, m3, and m4 continue to be expressed in culture (Eva et al. 1990). Immunocytochemical localization of m1 and m4 receptor protein (Levey et al. 1991) reveals a rather uniform distribution across all cortical layers that would fit with the present finding of a relatively ubiquitous presence in neurons in culture. An obvious question for future studies is whether m4 mRNA and protein is concentrated in excitatory neurons and m1 mRNA and protein is concentrated in inhibitory neurons, as our experiments would suggest.

Does ACh suppression of synaptic responses occur presynaptically or postsynaptically? On the basis of the correlation of changes in the variance of the response with the degree of suppression (Fig. 6), and assuming a model for quantal release in which changes in n or p occur only presynaptically, our results suggest a presynaptic site of action of ACh for both EPSPs and IPSPs. On the other hand, some experiments have suggested that changes in synaptic strength may occur as a result of the activation or inactivation of clusters of postsynaptic glutamate receptors, producing a change in CV without any change in presynaptic release (Isaac et al. 1995; Kullmann 1994; Selig et al. 1995; see also Faber and Korn 1991). In principle such postsynaptic changes might account for our results. An argument against effects on postsynaptic receptors, however, is that in the presence of ACh, the sensitivity of postsynaptic cells to exogenously applied glutamate or gamma -aminobutyric acid does not change (Segal 1982), which instead supports a presynaptic action for ACh. Another possible postsynaptic mechanism to suppress PSPs is a decrease in the input resistance of the postsynaptic cell, which would shunt and thus depress the PSPs. We observed, however, that in the majority of postsynaptic cells for both EPSPs and IPSPs, the input resistance increased or was unchanged during the application of ACh. Although the current pulses that we used to test the input resistance probably did not spread throughout the dendritic field, they most likely would have revealed any decreases in conductance large enough to account for the observed suppression.

An experiment that we carried out with a single inhibitory driver cell and three different followers provides an independent argument that at least for IPSPs, a presynaptic action is involved. In this case the response of all three postsynaptic cells was unaffected by ACh. As described above, the chance that any one inhibitory pair does not respond to ACh is ~27%. This means that if the ACh response is postsynaptic, the chance that three cells would not respond is only ~2%, which suggests that in this case the action of ACh was most likely presynaptic.

What are possible presynaptic mechanisms by which muscarinic receptors could reduce the synaptic response? Block of presynaptic Ca2+ influx would certainly be a candidate (Bernheim et al. 1992; Caulfield 1993; Segal et al. 1989). Alternatively, an increase in K+ conductance (Caulfield 1993) could reduce presynaptic depolarization and thereby reduce release. Muscarinic block of Ca2+ conductances has been observed in neurons (Bernheim et al. 1992; Caulfield 1993; Segal et al. 1989), and inhibition of ACh release by presynaptic "autoreceptors" acting with "M2" receptor pharmacology to increase K+ conductance has been reported (Caulfield 1993). Recently an argument was made that the cholinergic autoreceptor in hippocampus is more likely to be m4 than m2 (McKinney et al. 1993).

What is the role of synaptic suppression by ACh? It is easy to see how increased excitability produced by ACh might be useful in activating cortical circuits, but what is the advantage of synaptic suppression? A theoretical argument, supported by evidence from in vitro tissue slice studies, suggests that suppression of intracortical signals may be important to permit learning of new information (Hasselmo 1995; Hasselmo and Bower 1992). This proposal also suggests that afferent inputs to cortex should not be suppressed by ACh, which would be interesting to test by coculturing cortical cells with thalamic afferent neurons.

What is the functional significance of different muscarinic receptors producing suppression of EPSPs versus IPSPs? Because m1 and m4 receptors activate different second-messenger pathways, an important advantage of the use of these receptors would be to permit independent regulation of the two types of response via other neuromodulators and second messengers. Such interactions between different neuromodulatory systems might be important in determining the functional state of the cortex. For example, during waking the cholinergic, adrenergic, and serotonergic systems are all active; during deep, slow-wave sleep all are inactive; and during rapid eye movement sleep only the cholinergic system is active (Marrosu et al. 1995). During changes in attention and during learning these systems are also differentially active (Inglis and Fibiger 1995; Muir et al. 1994). The cholinergic system must be active at the time of training for at least some types of learning to occur (Aigner et al. 1991). The massive and relatively selective degeneration of the basalocortical cholinergic system in Alzheimer's disease also supports a role for this system in memory. On the other hand, general enhancement of cholinergic function for example with anticholinesterase drugs provides rather limited improvement in Alzheimer's patients. The results reported here provide a rational basis for exploring a selective pharmacological approach targeting specific muscarinic receptor subtypes in controlling functional state in cortex.

In summary, we have found that by acting on different muscarinic receptor subtypes, ACh can suppress both excitatory and inhibitory synaptic responses in cortical neurons. Suppression of EPSPs is produced by an m4-like receptor, and suppression of IPSPs is produced by an m1-like receptor. Separation of the actions of ACh into different receptor-second messenger pathways with potential for independent interactions with other neuromodulatory systems may be an important aspect of the mechanism of cholinergic regulation of functional state in cortex. Separation of cholinergic effects at different receptors might also offer a means for selective pharmacological intervention in disorders of sleep ormemory.

    ACKNOWLEDGEMENTS

  We thank S. Lazareno and M. Eliasson for helpful discussions, C. Bader and I. Mintz for reading the manuscript, and L. Lewis for assistance in the laboratory. F. Soo provided software for some of the data analysis.

  This work was supported by grants from the National Institutes of Health to R. W. Baughman, from the Human Frontier Science Program to F. Kimura, and from Research Aid of the Inoue Foundation for Science to F. Kimura.

    FOOTNOTES

   Present address of R. W. Baughman: NINDS Div. of Fundamental Neuroscience and Developmental Disorders, FED 916, Bethesda, MD 20892-9170. E-mail: rb175{at}nih.gov

  Present address and address for reprint requests: F. Kimura, Dept. of Neurophysiology, Biomedical Research Ctr., Osaka University Medical School, 2-2 Yamadaoka, Suita, 565 Japan. E-mail: fkimura{at}nphys.med.osaka-u.ac.jp

  Received 1 July 1996; accepted in final form 2 October 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society