Muscarine Modulates Ca 2/ 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
INTRODUCTION
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).
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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 voltageand Ca 2/ -dependent K / channels (Lorenzon and Foehring
1992; McCormick and Prince 1986; Schwindt et al. 1988)
as well as by activating a nonselective cation current (HajDahmane 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
Ca 2/ -dependent mechanisms underlying cholinergic modulation of excitability in cortical neurons, it is important to
determine cholinergic effects on Ca 2/ 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
Ca 2/ 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
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Stewart, Ansalan E., Zhen Yan, D. James Surmeier, and Robert C. Foehring. Muscarine modulates Ca 2/ 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 Ca 2/ 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( b-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 ( tonset Ç 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 ( tonset Ç17 s). Both fast
and slow components also were observed in recordings with 10
mM EGTA or 20 mM BAPTA when Ca 2/ was added to elevate
internal [Ca 2/ ] ( Ç150 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 PTXinsensitive 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 090 mV) occurred with a median t 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.
MUSCARINE MODULATES CALCIUM CURRENTS
METHODS
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 mM sections using
a vibrating tissue slicer (Campden Instruments) in an oxygenated
high sucrose solution (47C). 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 327C) 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 327C) (Lorenzon and
Foehring 1995). After the incubation period, the tissue first was
rinsed in a Na isethionate solution (47C) 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 ( Ç1 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.
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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 377C. 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 Ca 2/ 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-methylD-glucamine, 4 MgCl2 , 40 HEPES, 10 ethylene glycol-bis( b-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 [Ca 2/ ] i was buffered to Ç150 nM
(estimated with software written by Dr. E. McCleskey) by combining 3 mM Ca 2/ with 10 mM EGTA or 5.3 mM Ca 2/ 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
v-conotoxin-GVIA (CgTX: 500 mM; Bachem, Torrance, CA), vconotoxin-MVIIC (MVIIC: 500 mM; Bachem) and v-agatoxin-IVA
(AgTX: 100 mM; 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 õ0.05%. 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 Ç5 ml of diethyl pyrocarbonate
(DEPC)-treated water. The capillary glass used for making electrodes had been heated to 2007C 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 ml DEPCtreated water, 0.5 ml RNAsin (28,000 U/ml), and 0.5 ml dithiothreitol (DTT) (0.1 M). One microliter of either oligo dT (0.5 mg/ ml)
or random hexanucleotides (50 ng/ ml) was added and mixed before
the mixture was heated at 707C for 10 min and incubated on ice
for ú1 min. Single-strand cDNA was synthesized from the cellular
mRNA by adding SuperScript II RT (1 ml, 200 U/ ml), 101 PCR
buffer [2 ml, 200 mM tris(hydroxymethyl)aminomethaneCl, pH
8.4], KCl (500 mM), MgCl2 (2 ml, 25 mM), RNAsin (0.5 ml,
28,000 U/ml), DTT (1.5 ml, 0.1 M), and mixed dNTPs (1 ml, 10
mM). The reaction mixture (20 ml) was incubated at 427C for 50
min. The reaction was terminated by heating the mixture to 707C
for 15 min and then icing. The RNA strand in the RNA-DNA
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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 secondmessenger 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
Ca 2/ 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 Ca 2/ 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.
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A. E. STEWART, Z. YAN, D. J. SURMEIER, AND R. C. FOEHRING
Whole cell recordings
Whole cell recordings were acquired at room temperature (21–
247C) 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 ú5 mV.
Voltage control also was assessed by observing tail currents after
brief voltage steps (see Lorenzon and Foehring 1995). A gravityfed 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 ( a Å 0.05), unless
otherwise noted.
RESULTS
Muscarinic effect
Our initial experiments were carried out using an internal
recording solution which included 10 mM EGTA (EGTA
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internal). A 30-ms voltage step from 090 to /10 mV (repeated every 5 s) was used as the test command for all
experiments unless otherwise stated. Muscarine (0.005–100
mM) reversibly reduced HVA Ca 2/ channel currents in 122
of 150 sensorimotor pyramidal cells tested. On average, 1
mM muscarine blocked 13 { 1% of the total current (median:
12%; n Å 50; Fig. 1A) and 5 mM blocked 26 { 4% (median
26%, n Å 7). The kinetics of the modulation (5 mM muscarine) were quantified by measuring the peak current obtained
in 5-ms voltage steps from 090 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 tonset
averaged 1.3 { 0.2 s (n Å 9; Fig. 2A). Similar data were
obtained with 10 mM BAPTA in the recording electrode (6
of 6 cells showed the fast modulation only).
In 15 of the 150 cells recorded from using the EGTA, a
slowly developing component of the modulation also was
noted ( ú15 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 mM muscarine blocked 22 { 3% of the current
(median Å 20%) in cells where both the fast and the slow
component were present (n Å 14: 37% of the cells; Fig.
1B) and 13 { 1% (median Å 12%) in cells displaying only
the slow phase (n Å 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 tonset of 17 {
4 (Fig. 2B).
We then tested whether the chelator effects are primarily
due to buffering of transients or alteration of resting [Ca 2/ ]i
by buffering the internal solution to Ç150 nM with 10 mM
EGTA or 20 mM BAPTA and added Ca 2/ (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 mM muscarine in most cells tested (5
of 7 cells in EGTA plus Ca 2/ ; 5 of 5 cells in BAPTA plus
Ca 2/ ). The median modulation for these recordings was
20%. These data suggest that the primary effect of BAPTA
or EGTA is to lower [Ca 2/ ]i (Beech et al. 1991).
The magnitude of the muscarinic modulation was depen-
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hybrid then was removed by adding 1 ml RNase H (2 U/ ml) and
incubating for 20 min at 377C. 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 ml 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 mM primers,
2.5 U Taq DNA polymerase and buffer (Promega). The thermal
cycling program was 947C for 1 min, 587C for 1 min, and 727C
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.
MUSCARINE MODULATES CALCIUM CURRENTS
75
dent 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 mM muscarine blocked 6 { 1%, 12 { 2%, 18 { 3%,
26 { 4%, and 32 { 4 of the current, respectively. The maximum block was Ç32% (Fig. 4B).
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 mM muscarine blocked 7 { 1%, 19 { 5%, 30 {
6%, 39 { 7%, and 45 { 8% of the current, respectively.
Thus Ç45% of the current was blockable (data not shown).
The dose-response data were well fit with a single Langmuir isotherm of the form
I Å {A/1 / exp [2.3 ∗ log (dose)]/EC50 } n
The best fit (as determined by R 2 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 Å 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).
FIG . 2. Modulation could be separated into 2 kinetically different components. A: recorded with the 10
mM EGTA internal. Current was generated by a 10ms voltage step from 090 to 0 mV repeated every
second. Fast component had a tonset of 1.6 s in this cell
(median Å 1.3 s). Inset: box plot for tonset . B: recorded
with the 0.1 mM BAPTA internal. Currents were obtained by a 30-ms step from a holding potential of 080
to /10 mV, repeated every 5 s. Slow component had
a tonset of 10 s in this cell (median Å 17 s). Inset: box
plots for tonset .
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2/
FIG . 1. Muscarine reduces Ba
currents in a reversible manner. A: recorded with the 10 mM ethylene glycol-bis( b-aminoethyl ether)-N,N,N *,N *-tetraacetic acid
(EGTA) internal: 1 mM muscarine rapidly reduced the
current by Ç12% (current generated by a 30-ms voltage
step from 090 to /10 mV). Left: peak current vs. time
plot. Inset: box plot for the effect of 1 mM muscarine
(median reduction Ç12%). 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. r, 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 mM muscarine with minimal chelation
(median reduction Ç 20%). Right: representative traces
depicting the current reduction by muscarine. A and B
were taken at the times indicated in the plot at left.
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A. E. STEWART, Z. YAN, D. J. SURMEIER, AND R. C. FOEHRING
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 membranedelimited 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,
FIG . 4. Muscarinic modulation was dose dependent.
Current was generated by a voltage step from 090 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).
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FIG . 3. Slow component of the modulation was observed in the presence of chelators if resting [Ca 2/ ] i was
raised to Ç150 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 Ca 2/
(see METHODS ). Left: peak current vs. time plot illustrating an initial fast and subsequent slow phase to the modulation in response to 1 mM muscarine (maximum modulation was attained in Ç30 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 mM muscarine. B: similar data obtained with 10 mM
EGTA and 3 mM Ca 2/ 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 mM muscarine (maximum modulation was attained in
Ç30 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 mM muscarine.
MUSCARINE MODULATES CALCIUM CURRENTS
77
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 Ç24 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 mM muscarine reduced the calcium current (me-
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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.
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).
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, GTPgS (400 mM),
which should render a G-protein-mediated event irreversible.
With GTP in the internal solution, both the fast (n Å 15)
and slow (n Å 8) components of the muscarinic modulation
were readily reversible and could be repeatably induced (Fig.
6A). With GTPgS in the internal recording solution, both
the fast (EGTA internal; n Å 5) and slow components (low
BAPTA internal; n Å 4) of the modulation were seen in
response to 1 mM muscarine. In the first minute after attaining whole cell mode, these responses were reversible
(median reversibility Å 100%; data not shown). After
allowing GTPgS to dialyze into the cell for ¢8 min, the
fast component of the muscarinic effect became irreversible
(Fig. 6B; median % reversal Å 0%; significant difference:
P õ 0.006). The slow component also became irreversible
under these conditions (Fig. 6C; n Å 4; 0% median reversal).
Many calcium channel modulations have been shown to
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FIG . 6. Modulations became irreversible in the presence of the nonhydrolyzable GTP analogue, GTPgS. 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 Å 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 GTPgS (10 mM EGTA in electrode). Left:
plot of peak current vs. time. After Ç10 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 GTPgS (0.1 mM BAPTA in electrode). Left: plot
of peak current vs. time. After Ç8 min perfusion, the response to muscarine
(fast and slow modulations) became irreversible. Right: representative
traces from same cell as that providing data on left.
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A. E. STEWART, Z. YAN, D. J. SURMEIER, AND R. C. FOEHRING
dian Å 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 Å 0%; P õ 0.002; n Å 6;
Fig. 7A).
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 mM NEM. NEM alone
blocked current (10 { 2%; n Å 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 Å 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.
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1992), therefore a saturating dose of the selective N-type
channel antagonist v-conotoxin GVIA (CgTX; 1 mM) (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
mM muscarine reduced the current by 10 { 1% (median Å
10%; n Å 12 cells). CgTX reduced the current 34 { 4%
(median Å 35%) in these cells. The modulation after CgTX
was 5 { 1% (median Å 5%; P õ 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 Å 12; Fig. 8A). Therefore,
muscarine’s effect on the calcium channel current was partially due to modulation of N-type channels.
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; Mo-
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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 Å 14%; median modulation after PTX Å 0%). In this and subsequent scatter plots, each cell is represented by s.
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
mM) greatly reduced the fast modulation (median
modulation before NEM Å 11%; median modulation
after NEM Å 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 Å 8%; slow
before NEM Å 7%; fast modulation after NEM Å
0%; slow modulation after NEM Å 9%).
MUSCARINE MODULATES CALCIUM CURRENTS
79
gul 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 mM) reduced the
current by 12 { 2% (median Å 12%) before AgTX. AgTX
blocked 22 { 3% of the current. The modulation after AgTX
was 4 { 1% (median Å 4%; significant difference from
control: P õ 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 Å
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 mM muscarine reduced the current by 18 { 4% (median Å 17%) before the
addition of AgTX / CgTX. After the toxins were applied,
the modulation was reduced to 2 { 1% (median Å 0%;
significant: P õ 0.001; Fig. 8B). The combined toxins,
therefore, blocked virtually all of the modulation (median
reduction Å 100%; n Å 8).
The involvement of L-type channels was assessed using
its antagonist nifedipine (5 mM) (Lorenzon and Foehring
1995). Nifedipine had no effect on muscarine’s ability to
reduce calcium current with the 10 mM EGTA internal (n Å
4; data not shown).
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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. 1 B ) . We previously showed that with the EGTA
internal, AgTX / CgTX almost completely blocked the
fast component of the modulation ( n Å 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 mM CgTX and 25 nM AgTX
blocked the fast but not the slow portion of the modulation
in these cells ( 1 mM muscarine; n Å 3 ) . Before applying
the toxins, the fast modulation was 12 { 3% ( median Å
15% ) and the slow was 12 { 3% ( median Å 12%; for a
total modulation of 24 { 4%; median Å 24% ) . After the
toxins, the fast modulation was eliminated ( median Å
0% ) , and the slow remained unchanged ( median block Å
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 mM NEM, nifedipine blocked 100% of the
modulation in every cell tested, suggesting that the slow
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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 mM v-conotoxin-GVIA (CgTX) in this cell (median reduction in modulation Å 49%). Inset: box
plot of percent reduction in modulation by CgTx for
12 cells tested. B: combination of 25 nM v-agatoxinIVA (AgTX) and 1 mM 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 Å 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.
80
A. E. STEWART, Z. YAN, D. J. SURMEIER, AND R. C. FOEHRING
FIG . 9. Slow component targeted L-type channels.
A: plot of peak current vs. time for tail current measured
at Ç4 ms (and 070 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: t Å 19 ms in this cell) reduction of the
BayK-enhanced tail by 31% in this cell (median Å
21%). Box plot summarizes data for tonset 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 mM 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 030 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 Å 14%) to 2 { 1%
(median Å 2%; significant: P õ 0.001) by a prepulse to
/100 mV (5 mM muscarine; Fig. 10B).
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 090 mV between a prepulse to /120 mV and
the test pulse to 030 mV. Reinhibition of the current oc-
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curred with a t of 69 { 14 ms (median Å 68 ms; n Å 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 mM NEM.
Before the prepulse, the median reduction by 5 mM muscarine was 15% (n Å 15). After the prepulse, the modulation
was unchanged (median Å 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 Å 92%; n Å
10) of the modulation was voltage dependent by this criteria,
as compared with only 35 { 17% (median Å 20%; n Å 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 Å 0%; n Å 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 Ca 2/ current elicited by a 30-ms voltage step from
090 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 mM), and no significant differences were obtained for
two dosage ranges ( ¢5 and °2 mM). The average tactivation
for control for the low dose group (n Å 28 cells) was
1.3 { 0.1 s (median Å 1.2 s) as compared with that of
muscarine which had an average tactivation of 1.2 { 0.1 s
(median Å 1 s; data not shown). In the high-dose group
(n Å 63 cells), the activation t was 1.1 { 0.1 s (median Å
0.9 s) in control versus 1.3 { 0.2 (median Å 1.1 s) in
muscarine (data not shown). At a test step of 020 mV,
similar data were obtained: the tactivation was 1.9 ms in control
solutions and 1.8 ms in 10 mM muscarine (n Å 5 cells).
DISCUSSION
The excitability and firing behavior of cortical pyramidal
cells can be altered by the activation of muscarinic receptors
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phase of the modulation is due to the modulation of L-type
channels (n Å 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 mM). 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 BayKenhanced slow tails would indicate that the modulator is
acting on L-type currents. Muscarine (5 mM) reduced the
BayK-enhanced tail by 24 { 9% in the four neurons tested
(median Å 21%; n Å 4; Fig. 9). The modulation of the
BayK-enhanced tail was slow in onset ( tonset was 13 { 3 s,
median Å 14 s, n Å 4) requiring Ç25 ms (median) to reach
steady state (Fig. 9A). When the EGTA internal was used,
muscarine had no effect on the BayK-enhanced tail (n Å 3;
data not shown).
MUSCARINE MODULATES CALCIUM CURRENTS
81
(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 Ca 2/ 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 [ Ca 2/ ]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 [ Ca 2/ ]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
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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 [ Ca 2/ ]i was buffered to Ç150
nM, suggesting that the effect of chelators is mediated
primarily through effects on resting [ Ca 2/ ]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 õ1, 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 M2class 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 modu-
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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 Å 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 Å 10)
or the 0.1 mM BAPTA (n Å 8) internals. In the
presence of NEM, the slow component is isolated
and is not voltage dependent. B: kinetics of reinhibition (at 090 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 030
mV after various intervals at 090 mV (see protocol in inset at right). Left: exponential fit to data
measured from traces shown at right (Cd 2/ -subtracted). treinhibition in this cell was 81 ms. Inset:
box plot illustrating treinhibition for 8 cells measured
(median Å 68 ms). Right: representative traces
illustrating the time dependence of reinhibition.
Inset: voltage protocol.
82
A. E. STEWART, Z. YAN, D. J. SURMEIER, AND R. C. FOEHRING
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high concentrations of the transmitter (i.e., 100 mM) 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 Ca 2/ , although we cannot completely
rule out effects on intracellular Mg 2/ .
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 serotonin1A receptors
(Foehring 1996) and a2-adrenergic receptors (Foehring and
Lorenzon 1993) on these cells. Muscarinic receptor activation also initiates a slower, cytoplasmic modulation of Ltype 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
Ca 2/ -dependent K / currents that underlie the medium and
slow afterhyperpolarization (Pineda et al. 1998). L-type
channels do not activate Ca 2/ -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 Ca 2/ channels also may affect the integration
of dendritic synaptic inputs. For example, HVA Ca 2/ 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.
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).
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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lation 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 GTPgS. 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 tonset of the fast phase of the modulation in neocortical
pyramidal cells was rapid and within the range of membranedelimited 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 membranedelimited modulations (Bean 1989; Elmslie and Jones 1994;
Foehring 1996; Howe and Surmeier 1995; Yan and Surmeier
1996) as was the rate of reinhibition at 090 mV (Ehrlich
and Elmslie 1995; Ikeda 1991). Slowed activation kinetics
in the presence of muscarine were not obvious in our data
at /10 or 020 mV. However, the membrane-delimited modulation of calcium currents in this same cell type by serotonin
was only associated with slowed activation kinetics when
MUSCARINE MODULATES CALCIUM CURRENTS
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