The Journal of Neurophysiology Vol. 81 No. 1 January 1999, pp. 72-84
Copyright ©1999 by the American Physiological Society

Muscarine Modulates Ca²⁺ Channel Currents in Rat Sensorimotor Pyramidal Cells Via Two Distinct Pathways

Ansalan E. Stewart, Zhen Yan, D. James Surmeier, and Robert C. Foehring

Department of Anatomy and Neurobiology, University of Tennessee at Memphis, Memphis, Tennessee 38163

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Stewart, Ansalan E., Zhen Yan, D. James Surmeier, and Robert C. Foehring. Muscarine modulates Ca²⁺ channel currents in rat sensorimotor pyramidal cells via two distinct pathways. J. Neurophysiol. 81: 72-84, 1999. We used the whole cell patch-clamp technique and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to study the muscarinic receptor-mediated modulation of calcium channel currents in both acutely isolated and cultured pyramidal neurons from rat sensorimotor cortex. Single-cell RT-PCR profiling for muscarinic receptor mRNAs revealed the expression of m1, m2, m3, and m4 subtypes in these cells. Muscarine reversibly reduced Ca²⁺ currents in a dose-dependent manner. The modulation was blocked by the muscarinic antagonist atropine. When the internal recording solution included 10 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), the modulation was rapid (_onset ~ 1.2 s). Under conditions where intracellular calcium levels were less controlled (0.0-0.1 mM BAPTA), a slowly developing component of the modulation also was observed (_onset ~17 s). Both fast and slow components also were observed in recordings with 10 mM EGTA or 20 mM BAPTA when Ca²⁺ was added to elevate internal [Ca²⁺] (~150 nM). The fast component was due to a reduction in both N- and P-type calcium currents, whereas the slow component involved L-type current. N-ethylmaleimide blocked the fast component but not the slow component of the modulation. Preincubation of cultured neurons with pertussis toxin (PTX) also greatly reduced the fast portion of the modulation. These results suggest a role for both PTX-sensitive G proteins as well as PTX-insensitive G proteins in the muscarinic modulation. The fast component of the modulation was reversed by strong depolarization, whereas the slow component was not. Reblock of the calcium channels by G proteins (at 90 mV) occurred with a median  of 68 ms. We conclude that activation of muscarinic receptors results in modulation of N- and P-type channels by a rapid, voltage-dependent pathway and of L-type current by a slow, voltage-independent pathway.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Cholinergic afferents from the nucleus basalis and the reticular nucleus provide one of the major extrathalamic inputs to the neocortex (Douglas and Martin 1990; Foote and Morrison 1987). On reaching the cortex, these fibers ramify most densely in the overlapping regions of the primary motor and primary somatosensory areas of the cortex, where they are distributed within all six layers. Muscarinic receptors also are distributed densely throughout these areas of the cortex (Paxinos 1995).

Based on the fiber projections and receptor distribution, acetylcholine has been proposed to have an important role in regulating cortical activity and cognition (Foote and Morrison 1987; Gallagher and Columbo 1995; Jacobs and Juliano 1995). Additionally, acetylcholine has been shown to enhance the effectiveness of other inputs to the cortex and unmask receptive fields in response to peripheral stimuli (Metherate et al. 1988). Acetylcholine also is required for plasticity in response to peripheral injury (Juliano et al. 1991). In vitro studies have demonstrated that acetylcholine, acting through muscarinic receptors, enhances the excitability of neocortical pyramidal cells by modulating voltage- and Ca²⁺-dependent K⁺ channels (Lorenzon and Foehring 1992; McCormick and Prince 1986; Schwindt et al. 1988) as well as by activating a nonselective cation current (Haj-Dahmane and Andrade 1996). Acetylcholine also has been shown to cause regular spiking in burst-firing pyramidal cells by activating muscarinic receptors and depolarizing the cells (Wang and McCormick 1993). These changes in excitability may be involved in learning and memory (Howard and Simons 1994) and state-dependent behaviors (Bal and McCormick 1996; Marrosu et al. 1995). To better understand the Ca²⁺-dependent mechanisms underlying cholinergic modulation of excitability in cortical neurons, it is important to determine cholinergic effects on Ca²⁺ currents.

Muscarinic receptors can be divided into M1 and M2 classes based on their differential sensitivity to the antagonist pirenzipine (Hammer et al. 1980). Molecular cloning has revealed the existence of five receptor subtypes (m1-m5), which are grouped according to sequence homology; m1, m3, and m5 into the M1 class and m2 and m4 into the M2 class (Bonner et al. 1987). The M1- and M2-class receptors also couple to different second messengers and G proteins (Brauner-Osborne and Brann 1996; Felder 1995). M1-class muscarinic receptors preferentially couple to Gq-class G proteins (Blin et al. 1995; Hille 1994), which when activated can result in the hydrolysis of phosphotidylinositol (Blin et al. 1995). M2-class muscarinic receptors typically couple to Gi/Go-class G proteins, which when activated can reduce adenylate cyclase activity or directly inhibit voltage-gated Ca²⁺ channels (Hille 1994). All five muscarinic receptor subtypes (m1-m5) are present in the cortex (Wei et al. 1994).

Five pharmacologically distinct high-voltage-activated (HVA) calcium channels have been described in central neurons (Birnbaumer et al. 1994), including neocortical pyramidal cells (Brown et al. 1994; Lorenzon and Foehring 1995; Regan et al. 1991; Sayer et al. 1993; Ye and Akaike 1993; unpublished observations). These have been classified as L, N, P, Q, and R type (Birnbaumer et al. 1994). Muscarinic modulations of HVA calcium currents have been described in various neuron types (Allen and Brown 1993; Hille 1994; Howe and Surmeier 1995; Toselli et al. 1989; Wanke et al. 1987; Yan and Surmeier 1996).

In rat sympathetic neurons, muscarinic activation was found to reduce calcium current via three distinct mechanisms that were coupled to two separate classes of G proteins (Beech et al. 1992; Shapiro et al. 1994). One pathway targeted N- and L-type calcium channels via a pertussis toxin (PTX)-insensitive G protein coupled to an unknown second-messenger pathway. N-type channels were targeted by the remaining two membrane-delimited pathways, one of which interacted with a PTX-sensitive G protein and the other with a PTX-insensitive G protein (Beech et al. 1992; Mathie et al. 1992). Similar results were reported in central neurons (Howe and Surmeier 1995). The pathway targeting L-type channels was found to require the release of intracellular Ca²⁺ in medium spiny neostriatal neurons (unpublished observation) but not in rat sympathetic neurons (Beech et al. 1991).

The goal of this study was to examine the effects of muscarine on Ca²⁺ channel currents in pyramidal cells of the sensorimotor cortex. In particular, we studied the dose dependence of the effect and identified the receptor types present and the calcium channels targeted. Furthermore we tested which G proteins were involved, and we characterized the kinetics and voltage dependence of the modulation.

	METHODS

Abstract Introduction Methods Results Discussion References

Acute isolation

Two- to 6-week-old Sprague-Dawley rats were anesthetized with methoxyflurane. Under anesthesia, the rats were decapitated. The brains were extracted and then sliced into 400 µM sections using a vibrating tissue slicer (Campden Instruments) in an oxygenated high sucrose solution (4°C). The high sucrose solution contained (in mM) 250 sucrose, 2.5 KCl, 1 NaH₂PO₄, 11 glucose, 4 MgSO₄, 0.1 CaCl₂, and 15 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.3 adjusted with 1 N NaOH; 300 mOsm/l). The primary motor and primary somatosensory cortices (hereafter referred to as sensorimotor cortex) were dissected from these slices with the aid of a stereomicroscope after the slices were held for a minimum of 1 h (at 32°C) in a carboxygen (95% O₂-5% CO₂)-bubbled artificial cerebral spinal fluid (ACSF), which contained (in mM) 125 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 20 glucose, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitro-arginine, and 0.05 glutathione (pH 7.4 adjusted with 1 N NaOH; 310 mOsm/l). The dissected tissue then was incubated for 20-30 min in an oxygenated ACSF containing Pronase E (Sigma protease type XIV, 1.0 mg/ml at 32°C) (Lorenzon and Foehring 1995). After the incubation period, the tissue first was rinsed in a Na isethionate solution (4°C) that contained (in mM) 140 Na isethionate, 2 KCl, 1 MgCl₂, 23 glucose, 15 HEPES, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitro-arginine, and 0.05 glutathione (pH 7.3 adjusted with 1 N NaOH; 310 mOsm/l). The tissue then was triturated in the same solution using fire-polished Pasteur pipettes. The supernatant was collected and then poured into a plastic petri dish (Lux) positioned on the stage of an inverted microscope (Nikon Diaphot 300). The cells were allowed several minutes to adhere to the petri dish, and then the background flow of HEPES-buffered saline solution was initiated (~1 ml/min). This solution contained (in mM) 10 HEPES, 138 NaCl, 3 KCl, 1 MgCl₂ and 2 CaCl₂; pH 7.3 with 1 N NaOH, 300 mOsm/l.

Cultured cell preparation

Pyramidal cells from E19 rat embryos were cultured for 2 wk according to the procedure outlined in Bargas et al. (1991). The cells were maintained in 5% CO₂ at 37°C. PTX-treated cultures were incubated with 50 ng/ml of the toxin for 24 h before recording. Simultaneously prepared untreated cultures were used as controls.

Recording solutions and pharmacological agents

The external recording solution used to isolate the Ca²⁺ channel currents consisted of (in mM) 125 NaCl, 20 CsCl, 1 MgCl₂, 10 HEPES, 5 BaCl₂, 0.001 tetrodotoxin, and 10 glucose (pH 7.3 with tetraethylammonium-OH; 300-305 mOsm/l). The internal recording solution included the following (in mM): 180 N-methyl-D-glucamine, 4 MgCl₂, 40 HEPES, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 0.1 leupeptin, 0.4 guanosine 5'-triphosphate (GTP), and 2 ATP, and 0.01 phosphocreatine; pH 7.2 (adjusted with 0.1 N H₂SO₄; 265-275 mOsm/l). EGTA was replaced with 0.1 mM BAPTA (or no chelator) for experiments where minimal chelation was desired. In some experiments, basal [Ca²⁺]_i was buffered to ~150 nM (estimated with software written by Dr. E. McCleskey) by combining 3 mM Ca²⁺ with 10 mM EGTA or 5.3 mM Ca²⁺ with 20 mM BAPTA.

The stock solutions of muscarine chloride, atropine sulfate, and the calcium channel antagonists (with the exception of nifedipine) were dissolved in water. Stock solutions of the calcium channel antagonists -conotoxin-GVIA (CgTX: 500 µM; Bachem, Torrance, CA), -conotoxin-MVIIC (MVIIC: 500 µM; Bachem) and -agatoxin-IVA (AgTX: 100 µM; a gift from Dr. Niccolas Saccomano, Pfizer, Groton, CT) were aliquoted and frozen. Each of the stocks were diluted to the appropriate concentrations in the external recording solution immediately before the experiment. Nifedipine first was dissolved in 95% ethanol before being added to the external solution resulting in a final ethanol concentration of <0.05%. This concentration of ethanol had no effect on these cells (Bargas et al. 1994; Lorenzon and Foehring 1995). Nifedipine was protected from ambient light. Cytochrome C (0.01%) was added to solutions containing AgTX to prevent nonspecific binding of AgTX to glass and plastic (Bargas et al. 1994; Lorenzon and Foehring 1995).

Single-cell reverse transcription-polymerase chain reaction (RT-PCR)

The methods used for the single-cell RT-PCR were similar to those described previously (Surmeier et al. 1996; Yan and Surmeier 1996). Electrodes contained ~5 µl of diethyl pyrocarbonate (DEPC)-treated water. The capillary glass used for making electrodes had been heated to 200°C for 4 h. Sterile gloves were worn during the procedure to minimize RNase contamination.

After aspiration, the electrode was broken and contents ejected into a presiliconized, 0.5 ml Eppendorf tube containing 5 µl DEPC-treated water, 0.5 µl RNAsin (28,000 U/ml), and 0.5 µl dithiothreitol (DTT) (0.1 M). One microliter of either oligo dT (0.5 µg/µl) or random hexanucleotides (50 ng/µl) was added and mixed before the mixture was heated at 70°C for 10 min and incubated on ice for >1 min. Single-strand cDNA was synthesized from the cellular mRNA by adding SuperScript II RT (1 µl, 200 U/µl), 10× PCR buffer [2 µl, 200 mM tris(hydroxymethyl)aminomethaneCl, pH  8.4], KCl (500 mM), MgCl₂ (2 µl, 25 mM), RNAsin (0.5 µl, 28,000 U/ml), DTT (1.5 µl, 0.1 M), and mixed dNTPs (1 µl, 10 mM). The reaction mixture (20 µl) was incubated at 42°C for 50 min. The reaction was terminated by heating the mixture to 70°C for 15 min and then icing. The RNA strand in the RNA-DNA hybrid then was removed by adding 1 µl RNase H (2 U/µl) and incubating for 20 min at 37°C. All reagents except RNAsin (Promega, Madison, WI) were obtained from GIBCO BRL (Grand Island, NY). The cDNA from the RT of RNA in single cortical neurons was subjected to PCR to detect the expression of mRNAs coding for muscarinic receptors.

Conventional PCR was carried out with a thermal cycler (MJ Research, Watertown, MA). Thin-walled plastic tubes (Perkin Elmer, Norwalk, CT) were used. PCR primers were developed from GenBank sequences with commercially available software OLIGO (National Biosciences, Plymouth, MN) and have been described previously (Yan and Surmeier 1996). To detect individual mRNAs, 2.5 µl of the single-cell cDNA was used as a template for conventional PCR amplification. Reaction mixtures contained 2-2.5 mM MgCl₂, 0.5 mM of each of the dNTPs, 1 µM primers, 2.5 U Taq DNA polymerase and buffer (Promega). The thermal cycling program was 94°C for 1 min, 58°C for 1 min, and 72°C for 1.5 min for 45 cycles.

PCR products were separated by electrophoresis in 1.5-2% agarose gel and visualized by staining with ethidium bromide. In representative cases, amplicons were purified from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilder, Germany) and sequenced with a dye termination procedure by the University of Tennessee Molecular Resource Center or St. Jude Children's Research Hospital Molecular Resource Center. These sequences were found to match published sequences.

PCR reactions were carried out following procedures designed to minimize the chances of cross-contamination (Cimino et al. 1990). Negative controls for contamination from extraneous and genomic DNA were run for every batch of neurons. To ensure that genomic DNA did not contribute to the PCR products, neurons were aspirated and processed in the normal manner except that the reverse transcriptase was omitted. Contamination from extraneous sources was checked by replacing the cellular template with water. Both controls were consistently negative in these experiments.

Whole cell recordings

Whole cell recordings were acquired at room temperature (21-24°C) using a DAGAN 8900 or an Axopatch 200A electrometer. The recordings were monitored and controlled by pClamp6 (Axon Instruments, Foster City, CA) installed on a 486 computer. The electrodes were pulled from 7052 glass (Garner) and fire polished. Typically, series resistance compensation of 70-80% was employed. Cells were not included in the comparisons of biophysical properties if the estimated series resistance error was >5 mV. Voltage control also was assessed by observing tail currents after brief voltage steps (see Lorenzon and Foehring 1995). A gravity-fed parallel array of glass tubes was used to apply the drugs to the cell being studied.

SYSTAT (SYSTAT, Evanston, IL) was used to carry out all statistical calculations. The population data are represented as median, means ± SE and box plots (Tukey 1977). In the box plots, the internal vertical line represents the median, whereas the outer edges of the box represent the inner quartiles of the data set. The horizontal bars extending from the box depict the two outer quartiles of the data set. Data points more than two times the difference between the interquartiles were considered outliers and are indicated by an asterisk in the plots. Statistical differences were determined with the Mann-Whitney U test ( = 0.05), unless otherwise noted.

	RESULTS

Abstract Introduction Methods Results Discussion References

Muscarinic effect

Our initial experiments were carried out using an internal recording solution which included 10 mM EGTA (EGTA internal). A 30-ms voltage step from 90 to +10 mV (repeated every 5 s) was used as the test command for all experiments unless otherwise stated. Muscarine (0.005-100 µM) reversibly reduced HVA Ca²⁺ channel currents in 122 of 150 sensorimotor pyramidal cells tested. On average, 1 µM muscarine blocked 13 ± 1% of the total current (median: 12%; n = 50; Fig. 1A) and 5 µM blocked 26 ± 4% (median 26%, n = 7). The kinetics of the modulation (5 µM muscarine) were quantified by measuring the peak current obtained in 5-ms voltage steps from 90 to 0 mV, repeated every 0.8-1.0 s. With this protocol and drug-delivery system, we can resolve changes in the 0.25-0.5 s range (Foehring 1996). The modulation was rapid in onset under these conditions, reaching its maximum value within 6 s. The _onset averaged 1.3 ± 0.2 s (n = 9; Fig. 2A). Similar data were obtained with 10 mM BAPTA in the recording electrode (6 of 6 cells showed the fast modulation only).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Muscarine reduces Ba2+ currents in a reversible manner. A: recorded with the 10 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) internal: 1 µM muscarine rapidly reduced the current by ~12% (current generated by a 30-ms voltage step from 90 to +10 mV). Left: peak current vs. time plot. Inset: box plot for the effect of 1 µM muscarine (median reduction ~12%). Right: representative traces depicting the current reduced by muscarine. B: in recordings with the low (0.1 mM) bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) internal an additional slow phase to the modulation was noted. Left: peak current vs. time plot. , initial rapid decrease in the current (in the first 5 s) is designated the fast component. Subsequent slowly developing decrease in the current is defined as the slow component. Inset: box plot for the effect of 1 µM muscarine with minimal chelation (median reduction ~ 20%). Right: representative traces depicting the current reduction by muscarine. A and B were taken at the times indicated in the plot at left.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Modulation could be separated into 2 kinetically different components. A: recorded with the 10 mM EGTA internal. Current was generated by a 10-ms voltage step from 90 to 0 mV repeated every second. Fast component had a onset of 1.6 s in this cell (median = 1.3 s). Inset: box plot for onset. B: recorded with the 0.1 mM BAPTA internal. Currents were obtained by a 30-ms step from a holding potential of 80 to +10 mV, repeated every 5 s. Slow component had a onset of 10 s in this cell (median = 17 s). Inset: box plots for onset.

In 15 of the 150 cells recorded from using the EGTA, a slowly developing component of the modulation also was noted (>15 s for maximum current reduction). This slow component was not seen in the six cells recorded from with 10 mM BAPTA. The initial block of the current, occurring within 5 s, was designated the fast component, whereas the subsequent slowly developing reduction in current amplitude from that point until the maximum block was defined as the slow component (see Fig. 1B).

Modulations with slow kinetics that are also sensitive to the level of chelation have been reported previously (Beech et al. 1991; Bernheim et al. 1991; Cardenas et al. 1997; Howe and Surmeier 1995), therefore we tested whether minimal chelation altered the proportion of the modulation displaying a fast versus a slow reduction in current (10 mM EGTA replaced with 0.1 mM BAPTA). We found that in 32 of 38 cells tested, a slow component was observed in cells recorded using the low BAPTA internal. Under these conditions, 1 µM muscarine blocked 22 ± 3% of the current (median = 20%) in cells where both the fast and the slow component were present (n = 14: 37% of the cells; Fig. 1B) and 13 ± 1% (median = 12%) in cells displaying only the slow phase (n = 18: 47% of the cells). Six cells displayed only the fast component (16% of the cells). Six additional cells were tested using an internal without any chelator present. Both the slow and fast components were seen in five of these cells (1 cell only showed a slow component).

The kinetics of the slow portion of the modulation were quantified in 15 cells where only the slow component was evident. This modulation displayed an average _onset of 17 ± 4 (Fig. 2B).

We then tested whether the chelator effects are primarily due to buffering of transients or alteration of resting [Ca²⁺ ]_i by buffering the internal solution to ~150 nM with 10 mM EGTA or 20 mM BAPTA and added Ca²⁺ (see METHODS; Fig. 3) (Beech et al. 1991; Howe and Surmeier 1995). Under these conditions, a slow component of the modulation was seen in response to 1 µM muscarine in most cells tested (5 of 7 cells in EGTA plus Ca²⁺; 5 of 5 cells in BAPTA plus Ca²⁺). The median modulation for these recordings was 20%. These data suggest that the primary effect of BAPTA or EGTA is to lower [Ca²⁺ ]_i (Beech et al. 1991).

View larger version (30K): [in this window] [in a new window]   FIG. 3. Slow component of the modulation was observed in the presence of chelators if resting [Ca2+]i was raised to ~150 nM. Currents were obtained using the protocol in the insets, repeated every 5 s. A: recording solution contained 20 mM BAPTA plus 5.3 mM Ca2+ (see METHODS). Left: peak current vs. time plot illustrating an initial fast and subsequent slow phase to the modulation in response to 1 µM muscarine (maximum modulation was attained in ~30 s). Slow modulation was observed in 5 of 5 cells tested. Insets: voltage protocol (left) and box plot summary of data for percent modulation (right). Right: representative traces in control and 1 µM muscarine. B: similar data obtained with 10 mM EGTA and 3 mM Ca2+ in the recording pipette. Left: peak current vs. time plot illustrating an initial fast and subsequent slow phase to the modulation in response to 1 µM muscarine (maximum modulation was attained in ~30 s). Slow modulation was observed in 5 of 7 cells tested. Insets: voltage protocol (left) and box plot summary of data for percent modulation (right). Right: representative traces in control and 1 µM muscarine.

The magnitude of the muscarinic modulation was dependent on the concentration of muscarine used. We directly tested the dose dependence of the modulation in 11 cells. In seven cells the EGTA internal was used to isolate the fast component (Fig. 4A). In these cells, 0.005, 0.05, 0.5, 5, and 50 µM muscarine blocked 6 ± 1%, 12 ± 2%, 18 ± 3%, 26 ± 4%, and 32 ± 4 of the current, respectively. The maximum block was ~32% (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Muscarinic modulation was dose dependent. Current was generated by a voltage step from 90 to +10 mV repeated every 5 s. A and B: recorded with the 10 mM EGTA internal: A1: representative current traces illustrating the concentration-dependence of the modulation (each trace is the average of 3 records at the same dose). A2: Hill plot for pooled data. B: dose-response relationship of pooled data. Line is best fit of a single Langmuir isotherm raised to the Hill coefficient (see text for details).

We also examined the dose dependence in four cells where the low BAPTA internal was used and both components of the modulation were present. We found that 0.005, 0.05, 0.5, 5, and 50 µM muscarine blocked 7 ± 1%, 19 ± 5%, 30 ± 6%, 39 ± 7%, and 45 ± 8% of the current, respectively. Thus ~45% of the current was blockable (data not shown).

The dose-response data were well fit with a single Langmuir isotherm of the form The best fit (as determined by R² values) was obtained when n was the Hill coefficient (0.41 for the data gathered using the EGTA internal: Fig. 4A2) and 0.5 for the data gathered using the low BAPTA (data not shown). The EC₅₀s for the pooled data for the EGTA internal (7 cells) and the low BAPTA internal (4 cells: both fast and slow component combined) were 130 nM (Fig. 4B) and 136 nM (not shown), respectively.

In acutely dissociated cells (n = 4), atropine (3-40 nM) antagonized the muscarinic modulation. However, atropine alone also blocked current, making this data difficult to interpret. We hypothesized that this affect of atropine might be an artifact of enzyme treatment. Therefore we tested the effect of muscarine and atropine in cultured cortical pyramidal cells (EGTA internal). Atropine had no effect on the control current in cultured cells. In the five cells tested, muscarine blocked 17 ± 3% of the current before the application of atropine (data not shown). Atropine completely blocked the muscarinic modulation in four of five cells tested and reduced the modulation in the remaining cell (data not shown).

Receptor types

Muscarine's effect on pyramidal neurons could be mediated by any of the five muscarinic receptor subtypes. Recent studies in other cell types have linked a rapid membrane-delimited modulation to M2-class muscarinic receptors (m2, m4), whereas a kinetically slower second-messenger-mediated modulation was attributed to the activation of M1-class receptors (m1, m3, m5) (Bernheim et al. 1992; Howe and Surmeier 1995; Yan and Surmeier 1996).

Because there are no highly selective M1- and M2-class agonists and antagonists (Brauner-Osborne and Brann 1996; Hulme et al. 1990), pharmacological strategies were not used to identify the receptor subtypes. Rather, RT-PCR was used to determine which muscarine receptor mRNAs were expressed in cortical pyramidal cells. We first used RT-PCR on tissue samples of sensorimotor cortex. Consistent with Wei et al. (1994), we detected mRNA for all five receptor subtypes in sensorimotor cortex (data not shown). We then used single-cell RT-PCR to determine the pattern of expression of mRNA for the muscarinic receptor subtypes within individual pyramidal cells (Fig. 5A). In the 16 cells tested, the percent of cells expressing detectable levels of each receptor mRNA was as follows: m1, 69%; m2, 19%; m3, 44%; and m4, 56%. The m5 mRNA was not detected in these pyramidal cells (Fig. 5). Detectable levels of the M1- and M2-class mRNAs were colocalized in 35% of the cells, whereas 45% of the cells had detectable levels of only m1 or m3 receptor subtypes and the remaining 20% had only m4 receptor subtypes (Fig. 5B).

View larger version (62K): [in this window] [in a new window]   FIG. 5. Pyramidal cells express the muscarinic receptor subtypes m1, m2, m3, and m4. A: ethidium bromide gel showing the m1, m3, and m4 mRNAs detected in a single pyramidal cell. B: bar graph showing the pattern of coordinate expression for the 5 muscarine receptor subtypes in 16 pyramidal cells. M2-class receptor subtypes (m2, m4) are represented by densely stippled horizontal bars, whereas sparsely stippled horizontal bars indicate M1-class receptor subtypes (m1, m3). Overlap of the horizontal bars designates the percent coexpression of subtypes within single pyramidal cells.

G-protein involvement

The involvement of G proteins in the modulation was explored by substituting for GTP in the internal recording solution with its nonhydrolyzable form, GTPS (400 µM), which should render a G-protein-mediated event irreversible. With GTP in the internal solution, both the fast (n = 15) and slow (n = 8) components of the muscarinic modulation were readily reversible and could be repeatably induced (Fig. 6A). With GTPS in the internal recording solution, both the fast (EGTA internal; n = 5) and slow components (low BAPTA internal; n = 4) of the modulation were seen in response to 1 µM muscarine. In the first minute after attaining whole cell mode, these responses were reversible (median reversibility = 100%; data not shown). After allowing GTPS to dialyze into the cell for 8 min, the fast component of the muscarinic effect became irreversible (Fig. 6B; median % reversal = 0%; significant difference: P < 0.006). The slow component also became irreversible under these conditions (Fig. 6C; n = 4; 0% median reversal).

View larger version (24K): [in this window] [in a new window]   FIG. 6. Modulations became irreversible in the presence of the nonhydrolyzable GTP analogue, GTPS. A: with GTP in the recording solution, both the fast and slow modulations were reversible and repeatable. Left: peak current vs. time plot showing that the 2nd response to muscarine was similar in amplitude to the 1st in this cell. Inset: box plots for percent modulation during the 1st and 2nd application of muscarine (n = 8 cells). Right: representative traces from the same cell as that providing the data shown on the left. Inset: voltage protocol, repeated every 5 s. B: data obtained with GTP replaced by GTPS (10 mM EGTA in electrode). Left: plot of peak current vs. time. After ~10 min perfusion, the response to muscarine (fast modulation isolated) became irreversible. Right: representative traces from same cell as that providing data on left. C: data obtained with GTP replaced by GTPS (0.1 mM BAPTA in electrode). Left: plot of peak current vs. time. After ~8 min perfusion, the response to muscarine (fast and slow modulations) became irreversible. Right: representative traces from same cell as that providing data on left.

Many calcium channel modulations have been shown to be mediated by the Gi/Go subclass of G proteins (Ehrlich and Elmslie 1995; Foehring 1996; Howe and Surmeier 1995; Shapiro et al. 1994; Toselli et al. 1989; Yan and Surmeier 1996; Yan et al. 1997). This subclass can be distinguished by PTX sensitivity (Simon et al. 1991). Isolated neurons must be incubated with PTX ~24 h to allow time for PTX to uncouple the G protein and the receptor (Simon et al. 1991). This was not feasible for acutely dissociated pyramidal cells because they are only viable for 1-2 h after being isolated. As a consequence, we used cultured pyramidal cells for these experiments. In four pyramidal cells cultured without PTX, 1 µM muscarine reduced the calcium current (median = 14%). No modulation was evident in four of six cells preincubated in PTX. The modulation was only 1 and 6% in the other cells (median = 0%; P < 0.002; n = 6; Fig. 7A).

View larger version (27K): [in this window] [in a new window]   FIG. 7. Fast component of the modulation is mediated by a pertussis toxin (PTX)-sensitive G protein. A: summary scatter plots of recordings (10 mM EGTA internal) from cultured cortical neurons. Fast modulation was much smaller after preincubation in PTX (median modulation before PTX = 14%; median modulation after PTX = 0%). In this and subsequent scatter plots, each cell is represented by . Numbers within a circle denote the number of cells with that percent modulation. B: scatter plots of data from recordings from dissociated cortical neurons with EGTA internal. N-ethylmaleimide (NEM; 50 µM) greatly reduced the fast modulation (median modulation before NEM = 11%; median modulation after NEM = 1%). C and D: slow component of the modulation is mediated by an NEM-insensitive G protein. C: recorded with 0.1 mM BAPTA internal: peak current vs. time plot showing that NEM can be used to separate the 2 components of the modulation. Fast component was defined as the initial jump in current amplitude (left arrow) and the slow component as the subsequent reduction until a steady state is attained (curved line). After NEM, the slow modulation remains but the fast modulation is gone. D: recorded with the 0.1 mM BAPTA internal. Scatter plots showing that NEM eliminates the fast component of the modulation but spares the slow portion. (medians: fast modulation before NEM = 8%; slow before NEM = 7%; fast modulation after NEM = 0%; slow modulation after NEM = 9%).

Another test for the involvement of Gi/Go subclass G proteins is the sensitivity of the modulation to N-ethylmaleimide (NEM) (Foehring 1996; Shapiro et al. 1994; Wollmuth et al. 1995; Yan and Surmeier 1996; Yan et al. 1997). This experiment was performed on acutely dissociated neurons. The amplitude of the modulation was compared before and after a 2-min application of 50 µM NEM. NEM alone blocked current (10 ± 2%; n = 9). When the EGTA internal was used, NEM prevented the modulation in three of four cells examined and partially blocked the modulation in the fourth cell (Fig. 7B; median modulation after NEM = 1 vs. 13% before NEM). In all five cells where the low BAPTA internal was used, the fast component (the initial fast block) was blocked by NEM, whereas the slow component (the difference between the total block and the fast component) was unaffected (Fig. 7, C and D). Thus NEM could be used to separate the two components of the modulation.

Calcium channels targeted

FAST COMPONENT (EGTA INTERNAL). N-type channels have been found to be the target of many fast membrane-delimited modulations (Boland and Bean 1993; Cardenas et al. 1997; Ehrlich and Elmslie 1995; Foehring 1996; Herlitze et al. 1996; Howe and Surmeier 1995; Ikeda 1996; Mathie et al. 1992), therefore a saturating dose of the selective N-type channel antagonist -conotoxin GVIA (CgTX; 1 µM) (Lorenzon and Foehring 1995) was used on 12 cells to test whether N-type channels were involved in the fast component of the modulation. If the modulation involved N-type channels, then it would be reduced by CgTX. The percent modulation was determined by calculating the absolute amplitude of current blocked by muscarine then dividing that value by the amplitude of control current. Before CgTX, 1 µM muscarine reduced the current by 10 ± 1% (median = 10%; n = 12 cells). CgTX reduced the current 34 ± 4% (median = 35%) in these cells. The modulation after CgTX was 5 ± 1% (median = 5%; P < 0.02). The percent reduction of the modulation by CgTX (and other agents) was calculated first by taking the difference between the percent modulation before and after CgTX, then dividing that value by the percent modulation before CgTX. In 10 of the 12 cells tested, the muscarinic modulation was reduced by between 14-100% in the presence of CgTX (in 2 cells, the modulation was unaffected). The median reduction in the modulation by CgTX was 49% (n = 12; Fig. 8A). Therefore, muscarine's effect on the calcium channel current was partially due to modulation of N-type channels.

View larger version (34K): [in this window] [in a new window]   FIG. 8. Both N- and P-type channels were targets of the fast component of the modulation. A, left: representative traces demonstrating that muscarine reduces less current after N-type channels are blocked. Right: peak current vs. time plot illustrating the time course of the drug affects. Modulation is reduced from 16 to 7% of the current in the presence of 1 µM -conotoxin-GVIA (CgTX) in this cell (median reduction in modulation = 49%). Inset: box plot of percent reduction in modulation by CgTx for 12 cells tested. B: combination of 25 nM -agatoxin-IVA (AgTX) and 1 µM CgTX virtually eliminated the fast component. Left: representative traces showing that the combined effect of the toxins was to block the modulation (median block of modulation = 100%). In this cell, the modulation was reduced from 14 to 1% after N- and P-type channels were blocked by the toxins. Right: peak current vs. time plot illustrating the time course of the drug effect. Inset: box plot of percent reduction in modulation by AgTx + CgTx for 7 cells tested.

Because the block of N-type channels did not entirely prevent the muscarinic modulation, we tested for the involvement of a second channel type. The next likely candidate was P-type channels because they also have been found to be targets of membrane-delimited modulations (Foehring 1996; Howe and Surmeier 1995; Mintz and Bean 1993; Mogul et al. 1993). In 11 cells, we used 25 nM agatoxin IVA (AgTX); a relatively selective dose for P-type channels (Randall and Tsien 1995). Muscarine (1 µM) reduced the current by 12 ± 2% (median = 12%) before AgTX. AgTX blocked 22 ± 3% of the current. The modulation after AgTX was 4 ± 1% (median = 4%; significant difference from control: P < 0.003). In the presence of 25 nM AgTX, the modulation was reduced in 10 of 11 cells by 8-99% (no effect in 1 cell) with a median reduction of 55% (n = 11), suggesting that P-type channels also are modulated by muscarine (data not shown).

The calcium channel antagonists each blocked approximately half of the modulation. When both toxins were added simultaneously, they blocked 52 ± 7% of the total calcium channel current. In these eight cells, 1 µM muscarine reduced the current by 18 ± 4% (median = 17%) before the addition of AgTX + CgTX. After the toxins were applied, the modulation was reduced to 2 ± 1% (median = 0%; significant: P < 0.001; Fig. 8B). The combined toxins, therefore, blocked virtually all of the modulation (median reduction = 100%; n = 8).

The involvement of L-type channels was assessed using its antagonist nifedipine (5 µM) (Lorenzon and Foehring 1995). Nifedipine had no effect on muscarine's ability to reduce calcium current with the 10 mM EGTA internal (n = 4; data not shown).

SLOW COMPONENT (LOW BAPTA INTERNAL). Both the fast and the slow phases of the modulation could be examined in parallel by using the low BAPTA internal. The initial block of the current, occurring within 5 s, was designated the fast component while the subsequent slowly developing reduction in current amplitude from that point until the maximum block was defined as the slow component (see Fig. 1B). We previously showed that with the EGTA internal, AgTX + CgTX almost completely blocked the fast component of the modulation (n = 8, see preceding section). In three cells, we recorded with 0.1 mM BAPTA and applied AgTX + CgTX to examine the contribution of N- and P-type channels to the slow modulation component. The combination of 1 µM CgTX and 25 nM AgTX blocked the fast but not the slow portion of the modulation in these cells (1 µM muscarine; n = 3). Before applying the toxins, the fast modulation was 12 ± 3% (median = 15%) and the slow was 12 ± 3% (median = 12%; for a total modulation of 24 ± 4%; median = 24%). After the toxins, the fast modulation was eliminated (median = 0%), and the slow remained unchanged (median block = 12%; data not shown).

As described previously, the slow component also could be isolated by blocking the fast component with NEM (Fig. 5C). After 50 µM NEM, nifedipine blocked 100% of the modulation in every cell tested, suggesting that the slow phase of the modulation is due to the modulation of L-type channels (n = 5; data not shown).

For further confirmation of L-type channel involvement in the slow portion of the modulation, we used the dihydropyridine agonist BayK 8644 (BayK: 5 µM). BayK increases L-type current amplitude, shifts the voltage-dependence to more negative potentials, and enhances and slows the tail current (Jones and Jacobs 1990). Reduction of the BayK-enhanced slow tails would indicate that the modulator is acting on L-type currents. Muscarine (5 µM) reduced the BayK-enhanced tail by 24 ± 9% in the four neurons tested (median = 21%; n = 4; Fig. 9). The modulation of the BayK-enhanced tail was slow in onset (_onset was 13 ± 3 s, median = 14 s, n = 4) requiring ~25 ms (median) to reach steady state (Fig. 9A). When the EGTA internal was used, muscarine had no effect on the BayK-enhanced tail (n = 3; data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 9. Slow component targeted L-type channels. A: plot of peak current vs. time for tail current measured at ~4 ms (and 70 mV) after a 30-ms step to +10 mV. Addition of BayK 8644 caused an increase in tail current amplitude. Subsequent addition of muscarine led to a slow (in onset:  = 19 ms in this cell) reduction of the BayK-enhanced tail by 31% in this cell (median = 21%). Box plot summarizes data for onset from 4 cells. B: representative traces corresponding to the data in A. Box plot summarizes data for percent reduction in the tail current by 1 µM muscarine.

Voltage dependence

In many cell types, strong depolarization has been shown to enhance or facilitate HVA calcium currents (Dolphin 1996; Ikeda 1991; Kasai 1992; Song and Surmeier 1996) as well as to reverse neurotransmitter modulations (Ehrlich and Elmslie 1995; Foehring 1996; Howe and Surmeier 1995; Kasai 1992; Yan and Surmeier 1996). These are thought to be related phenomena resulting from the disruption of an interaction between the G protein and the channel (Bean 1989; Elmslie and Jones 1994; Golard and Siegelbaum 1993).

After blocking the slow portion of the modulation with the EGTA internal, the voltage dependence of the remaining fast component was examined by comparing the current elicited by a 15-ms test pulse to 30 mV to the current elicited by the same test pulse when it was preceded by a 30-ms positive prepulse to +100 mV (protocol in Fig. 8). The modulation was voltage dependent in the sense that the percent reduction in current in response to muscarine was greatly reduced by voltage steps to +100 mV (9 of 10 cells; Fig. 10A). On average, the rapid phase of the modulation was reduced from 15 ± 3% (median = 14%) to 2 ± 1% (median = 2%; significant: P < 0.001) by a prepulse to +100 mV (5 µM muscarine; Fig. 10B).

View larger version (31K): [in this window] [in a new window]   FIG. 10. Fast component was voltage dependent. A, left: in the absence of the prepulse, the modulation was 23% in this cell. Right: prepulse eliminated the modulation. Bottom, left: box plot showing the population data for voltage dependence of the fast component (isolated with the EGTA internal; n = 10 cells). Top box plot summarizes data with the prepulse, bottom box plot is for data without the prepulse. Bottom, right: box plots comparing the percent of the modulation that was eliminated by a prepulse to +100 mV (% voltage dependence). We compared cells recorded from using the 10 mM EGTA (n = 10) or the 0.1 mM BAPTA (n = 8) internals. In the presence of NEM, the slow component is isolated and is not voltage dependent. B: kinetics of reinhibition (at 90 mV) after a prepulse to +120 was characterized by fitting a single exponential to the decline in peak current seen at a test step to 30 mV after various intervals at 90 mV (see protocol in inset at right). Left: exponential fit to data measured from traces shown at right (Cd2+-subtracted). reinhibition in this cell was 81 ms. Inset: box plot illustrating reinhibition for 8 cells measured (median = 68 ms). Right: representative traces illustrating the time dependence of reinhibition. Inset: voltage protocol.

After the prepulse, the muscarinic inhibition of the calcium channel current was reestablished in a time-dependent fashion. To quantify this, we varied the duration of the interval spent at 90 mV between a prepulse to +120 mV and the test pulse to 30 mV. Reinhibition of the current occurred with a  of 69 ± 14 ms (median = 68 ms; n = 8; Fig. 10B). The kinetics and voltage dependence of the fast component of the modulation are consistent with the characteristics of a membrane-delimited response (Bean 1989).

To test the voltage dependence of the slow component in isolation, we blocked the fast component with 50 µM NEM. Before the prepulse, the median reduction by 5 µM muscarine was 15% (n = 15). After the prepulse, the modulation was unchanged (median = 15%). Therefore the slow component remaining after NEM was not reversed by the prepulse (i.e., was not voltage dependent; data not shown).

The voltage dependence of the two components also could be illustrated by comparing the percent of the modulation reversed by the prepulse. When the EGTA internal was used (fast component isolated) 83 ± 9% (median = 92%; n = 10) of the modulation was voltage dependent by this criteria, as compared with only 35 ± 17% (median = 20%; n = 8) when the low BAPTA internal was used (both components present). Additionally, when both the low BAPTA internal and NEM were used (slow component isolated), only 1 ± 1% of the modulation was affected by voltage (Fig. 10; median = 0%; n = 8).

Because the fast component of the modulation had rapid kinetics and was voltage dependent, both characteristics of membrane-delimited pathways, we tested for slowed activation kinetics, which is associated with many such modulations (Bean 1989; Elmslie and Jones 1994; Golard and Siegelbaum 1993; Jones 1991; but see Bargas et al. 1994; Brown 1993; Foehring 1996; Yan et al. 1997). Activation of the Ca²⁺ current elicited by a 30-ms voltage step from 90 to +10 mV was measured as an initial delay followed by a single exponential. Activations were well fit by a single exponential in both control and after muscarine (50 nM to 50 µM), and no significant differences were obtained for two dosage ranges (5 and 2 µM). The average _activation for control for the low dose group (n = 28 cells) was 1.3 ± 0.1 s (median = 1.2 s) as compared with that of muscarine which had an average _activation of 1.2 ± 0.1 s (median = 1 s; data not shown). In the high-dose group (n = 63 cells), the activation  was 1.1 ± 0.1 s (median = 0.9 s) in control versus 1.3 ± 0.2 (median = 1.1 s) in muscarine (data not shown). At a test step of 20 mV, similar data were obtained: the _activation was 1.9 ms in control solutions and 1.8 ms in 10 µM muscarine (n = 5 cells).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The excitability and firing behavior of cortical pyramidal cells can be altered by the activation of muscarinic receptors (Haj-Dahmane and Andrade 1996; Lorenzon and Foehring 1992; McCormick and Prince 1986; Schwindt et al. 1988; Wang and McCormick 1993). Part of this effect of muscarine is calcium dependent (Lorenzon and Foehring 1992; Schwindt et al. 1988). Therefore to gain insight into the mechanisms of cholinergic influence on pyramidal neurons, we studied the direct effect of muscarine on Ca²⁺ channel currents in acutely dissociated and cultured sensorimotor pyramidal cells.

Muscarine reversibly reduced the current in a dose-dependent manner, and the modulation was blocked by atropine in cultured neurons. Thus the effect studied was mediated by muscarinic receptors.

The modulation could be separated into two components based on sensitivity to internal [Ca²⁺]_i, kinetics of onset, calcium channels targeted, the G protein involved and voltage dependence. The fast portion of the modulation was characterized by rapid onset kinetics and voltage dependence and was not prevented by 10 mM EGTA or 10 mM BAPTA. The fast component was observed in 81% (122/150) of cells recorded from with 10 mM EGTA and 53% (20/38) of cells recorded from with 0.1 mM BAPTA, suggesting that the fast component may also be sensitive to [Ca²⁺]_i or chelators. The fast modulation also was characterized by NEM- and PTX-sensitive G proteins and block of N- and P-type channels. The slow phase was prevented by 10 mM EGTA (or 10 mM BAPTA), had slow onset kinetics, and was voltage independent. The slow phase also was observed in the presence of 10 mM EGTA or 10 mM BAPTA if [Ca²⁺]_i was buffered to ~150 nM, suggesting that the effect of chelators is mediated primarily through effects on resting [Ca²⁺]_i. The slow phase of the modulation used a NEM-insensitive G protein and targeted L-type channels.

We found that the dose-response data for the muscarinic modulation was best fit by a one site model. The Hill coefficients were <1, suggesting negative cooperativity (Taylor and Insel 1990).

Autoradiographic and in situ hybridization studies have shown that all five receptor subtypes of muscarinic receptors (m1-m5) are present in the cortex (Buckley et al. 1988; Levey et al. 1994) with variations in the relative abundance of the subtypes depending on the area of cortex examined (Brann et al. 1993; Buckley et al. 1988; Levey et al. 1994). Our RT-PCR results provided evidence that mRNA for more than one receptor type was expressed in most pyramidal neurons. The single-cell RT-PCR detected the presence of mRNA for the muscarinic receptor subtypes m1 and detected the presence of mRNA for the muscarinic receptor types m1, m2, m3, and m4. Detectable levels of M1- and M2-class mRNAs colocalized in 35% of the cells. Although we did not directly link the receptor class to a particular phase of the modulation due to overlapping pharmacological profiles, previous studies in other cell types have linked a rapid modulation to the M2-class receptors and a slow component to the M1-class (Bernheim et al. 1992; Brann et al. 1993; Hille 1994; Yan and Surmeier 1996). Consistent with this possibility, the pattern of receptor subtype expression in neocortical pyramidal neurons was correlated with the electrophysiological data. Both the fast and the slow components of the modulation were observed in 37% of the cells recorded with the low BAPTA internal, and RT-PCR indicated that 35% of the neurons coexpressed M1- and M2-class receptor subtypes. Additionally, 16% of the cells displayed the fast component alone compared with 19% of the neurons expressing detectable levels of only M2-class receptor subtypes. Furthermore the slow modulation alone was observed in 47% of the cells, whereas 45% of the neurons expressed detectable levels of only M1-receptor subtypes.

For both the fast and slow components, a G protein served as the first link between the muscarinic receptor and the ion channel. This was demonstrated by rendering the modulation irreversible with GTPS. The rapid component of the modulation was mediated by a NEM- and PTX-sensitive G protein (Gi/Go). A similar rapid, membrane-delimited muscarinic modulation was found to be mediated by a PTX-sensitive G protein in rat sympathetic neurons, striatal medium spiny cells, and striatal cholinergic interneurons (Beech et al. 1992; Howe and Surmeier 1995; Shapiro et al. 1994; Yan and Surmeier 1996).

On the other hand, the G protein mediating the slow component was NEM insensitive and thus unlikely to be of the Gi/Go subclass. Studies of slow second-messenger-mediated muscarinic modulations due to M1 receptor activation in sympathetic neurons point to Gq as the most likely candidate (Hille 1994). Transfection studies in mammalian cell lines support this conclusion by showing that M1 class receptor subtypes preferentially associate with Gq class G-proteins (Brauner-Osborne and Brann 1996).

Previous studies of muscarinic effects in rat sympathetic and striatal neurons revealed that muscarine acts on N- and P-type channels in a membrane-delimited manner (Howe and Surmeier 1995; Mathie et al. 1992; Yan and Surmeier 1996; Yan et al. 1997). This is likely to be true in neocortical pyramidal cells as well. Membrane-delimited modulations are typically rapid, are voltage dependent, and display slowed activation kinetics, although modulations that do not fit all three criteria have been designated as membrane delimited (Bean 1989; Brown 1993).

The _onset of the fast phase of the modulation in neocortical pyramidal cells was rapid and within the range of membrane-delimited modulations (Boland and Bean 1993; Foehring 1996; Howe and Surmeier 1995; Jones 1991; Yan and Surmeier 1996; Yan et al. 1997). The elimination of the rapid phase of the modulation by strong depolarizing prepulses as well as a facilitation of the current in response to the same protocol are characteristic of voltage-dependent membrane-delimited modulations (Bean 1989; Elmslie and Jones 1994; Foehring 1996; Howe and Surmeier 1995; Yan and Surmeier 1996) as was the rate of reinhibition at 90 mV (Ehrlich and Elmslie 1995; Ikeda 1991). Slowed activation kinetics in the presence of muscarine were not obvious in our data at +10 or 20 mV. However, the membrane-delimited modulation of calcium currents in this same cell type by serotonin was only associated with slowed activation kinetics when high concentrations of the transmitter (i.e., 100 µM) was applied (Foehring 1996).

As seen in rat sympathetic and striatal neurons (Howe and Surmeier 1995; Mathie et al. 1992), we found that in neocortical pyramidal cells L-type channels were the target of the slow component of the modulation. This pathway was sensitive to the presence of millimolar concentrations of chelators in the internal recording solution and was voltage independent, which is characteristic of some cytoplasmic pathways (Hille 1994). We assume that the primary difference in our internal recording conditions was the difference in ability to chelate Ca²⁺, although we cannot completely rule out effects on intracellular Mg²⁺.

Thus we have shown divergence of action due to the activation of muscarinic receptors in neocortical pyramidal cells. The fast modulation of N- and P-type currents by muscarine converges with the effects of serotonin_1A receptors (Foehring 1996) and ₂-adrenergic receptors (Foehring and Lorenzon 1993) on these cells. Muscarinic receptor activation also initiates a slower, cytoplasmic modulation of L-type current.

These findings regarding muscarine's effect on calcium channels are likely to be significant for the function of neocortical pyramidal cells. Muscarine could regulate excitability in different directions by acting on N- and P-type or L-type calcium channels. The reduction of N- and P-type calcium currents by muscarine could increase the firing frequency of pyramidal cells secondary to a reduction in the Ca²⁺-dependent K⁺ currents that underlie the medium and slow afterhyperpolarization (Pineda et al. 1998). L-type channels do not activate Ca²⁺-dependent afterhyperpolarizations in these cells but contribute to the inward current underlying repetitive firing (Pineda et al. 1998). Thus a reduction in current through L-type channels could lead to reduced firing frequency (reduced excitability). L-type channels also may be involved in regulation of gene expression (Bito et al. 1997) or synaptic plasticity (Kullman et al. 1992). Modulation of HVA Ca²⁺ channels also may affect the integration of dendritic synaptic inputs. For example, HVA Ca²⁺ channels contribute to dendritic electrogenesis (Kim and Connors 1993), which is facilitated by muscarinic agonists in hippocampal pyramidal neurons (Tsubokawa and Ross 1997). These changes in excitability induced by muscarinic receptor activation may allow for the regulation of state-dependent behaviors. This mechanism by which muscarinic receptor activation can regulate cortical excitability would be compromised in disease states affecting cholinergic input to the cortex such as Alzheimer's disease.

	ACKNOWLEDGEMENTS

  We thank R. Scroggs for reading the manuscript and Birch Harms for excellent technical assistance.

  This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-33579 (to R. C. Foehring) and NS-34696 (to D. J. Surmeier) and an American Psychological Association Minority Fellowship in Neuroscience (to A. E. Stewart).

	FOOTNOTES

   Present address of D. J. Surmeier: Dept. of Physiology/NUIN, Northwestern University Medical School, Searle 5-447, 320 E. Superior St., Chicago, IL 60611.

  Address for reprint requests: R. C. Foehring, 855 Monroe Ave., Dept. of Anatomy and Neurobiology, University of Tennessee at Memphis, Memphis, TN 38163.

  Received 23 March 1998; accepted in final form 25 September 1998.

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