Department of Neurobiology, University of California, Los Angeles, California 90095-1763
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Shao, X. M. and
J. L. Feldman.
Acetylcholine Modulates Respiratory Pattern: Effects Mediated by
M3-Like Receptors in PreBötzinger Complex Inspiratory
Neurons.
J. Neurophysiol. 83: 1243-1252, 2000.
Perturbations of cholinergic neurotransmission in the
brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice
preparation from neonatal rat that spontaneously generates respiratory
rhythm, we patch-clamped inspiratory neurons in the preBötzinger
complex (preBötC), the hypothesized site for respiratory rhythm
generation, and simultaneously recorded respiratory-related motor
output from the hypoglossal nerve (XIIn). Most (88%) of the
inspiratory neurons tested responded to local application of
acetylcholine (ACh) or carbachol (CCh) or bath application of
muscarine. Bath application of 50 µM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the
cellular level, muscarine induced a tonic inward current, increased the
duration, and decreased the amplitude of the phasic inspiratory inward
currents in preBötC inspiratory neurons recorded under voltage
clamp at 60 mV. Muscarine also induced seizure-like activity evident
during expiratory periods in XIIn activity; these effects were blocked
by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preBötC inspiratory neurons induced an inward
current along with an increase in membrane conductance under voltage
clamp and induced a depolarization under current clamp. This response
was blocked by atropine in a concentration-dependent manner. Bath
application of 1 µM pirenzepine, 10 µM gallamine, or 10 µM
himbacine had little effect on the CCh-induced current, whereas 10 µM
4-diphenylacetoxy-N-methylpiperidine methiodide blocked
the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of
110 to
20 mV
and reversed at
11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was
unaffected when patch electrodes contained a high concentration of EGTA
(11 mM) or
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V
curve shifted to the left and the reversal potential shifted to
47 mV. Lowering extracellular Cl
concentration from 140 to
70 mM had no effect on the reversal potential. These results suggest
that in preBötC inspiratory neurons, ACh acts on M3-like ACh
receptors on the postsynaptic neurons to open a channel permeable to
Na+ and K+ that is not Ca2+
dependent. This inward cation current plays a major role in
depolarizing preBötC inspiratory neurons, including pacemakers,
that may account for the ACh-induced increase in the frequency of
respiratory motor output observed at the systems/behavioral level.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ACh is involved in central respiratory control (Burton et
al. 1994, 1995
; Gesell et al. 1943
;
Gillis et al. 1988
; Metz 1958
; Murakoshi et al. 1985
; Nattie and Li
1990
; Weinstock et al. 1981
), including central
chemosensitivity (Burton et al. 1997
; Dev and Loeschcke 1979
; Fukuda and Loeschcke 1979
;
Gonsalves and Borison 1980
; Haxhiu et al.
1984
; Monteau et al. 1990
; Nattie et al.
1989
). Defects in the ventral medullary muscarinic system may
play a role in disorders of respiratory control such as sudden infant death syndrome (SIDS) (Kinney et al. 1995a
). Central
cholinergic mechanisms contribute to respiratory failure caused by
organophosphate poisoning (Lotti 1991
). Muscarinic
receptor subtypes are distributed across brain stem respiratory regions
including the ventrolateral medulla (Kinney et al.
1995b
; Mallios et al. 1995
). Perturbations of
ACh synthesis, release, degradation, or activation of ACh receptors in
the brain stem result in perturbations of respiratory pattern both in
vivo (Foutz et al. 1987
; Gillis et al.
1988
; Nattie and Li 1990
) and in vitro in an en
bloc brain stem-spinal cord preparation in neonatal rats (Burton
et al. 1994
, 1995
; Monteau et al. 1990
; Murakoshi et al. 1985
). The activity of medullary
respiratory-related neurons are altered by ACh and cholinergic agonists
or antagonists (Böhmer et al. 1987
, 1989
;
Bradley and Lucy 1983
; Haji et al. 1996
;
Jordan and Spyer 1981
; Kirsten et al.
1978
). The cellular and synaptic mechanism of ACh actions and
how this cellular process relates to the behavioral changes in
ventilation is not understood.
The preBötzinger complex (preBötC) in the
rostroventrolateral medulla is hypothesized as the site for respiratory
rhythm generation in mammals (Rekling and Feldman 1998;
Smith et al. 1991
). There have been accumulating data
supporting this hypothesis (Connelly et al.
1992
; Funk et al. 1993
; Johnson
et al. 1994
; Koshiya and Guyenet 1996
;
Koshiya and Smith 1999
; Ramirez et al. 1996
,
1998
; Schwarzacher et al. 1995
; Solomon
et al. 1999
). In earlier studies of the role of cholinergic
neurotransmission in central chemosensitivity, cholinergic agents were
applied locally to the ventral medullary surface (Dev and
Loeschcke 1979
; Haxhiu et al. 1984
;
Nattie et al. 1989
), intravenously, or
intracerebroventricularly (Burton et al. 1997
;
Gonsalves and Borison 1980
) to anesthetized mammals or
by perfusion of the en bloc brain stem-spinal cord preparation in vitro
(Monteau et al. 1990
). Because the preBötC is
located close to the ventral medullary surface, it is difficult to
distinguish the direct effects of the agents on preBötC neurons or secondary effects through, for example, nearby (putative) central chemoreceptive areas. Results based on extracellular recording combined
with ionophoretic administration of cholinergic agents in vivo have
been controversial due to the variability (or lack of precise
information) concerning recording locations and the states of
anesthesia (Böhmer et al. 1987
, 1989
;
Bradley and Lucy 1983
; Foutz et al. 1987
;
Jordan and Spyer 1981
; Kirsten et al. 1978
; Salmoiraghi and Steiner 1963
). Using a
medullary slice from neonatal rat that generates respiratory-related
motor output, we can locate the preBötC precisely (Gray et
al. 1999
) and without anesthetic interference, then patch-clamp
physiologically classified respiratory neurons and record the system
motor output simultaneously in well-controlled conditions, and the
tissue is highly accessible for pharmacological manipulation. In this
paper, we address the following questions: which kinds of preBötC
neurons, if any, are affected by ACh? How does ACh modulate the
activity of these cells? What receptors mediate the ACh effects? What
is the relationship between the ACh effects on respiratory neurons and
its effects on the respiratory-related motor pattern? We illustrate the
ACh effects on respiratory-related motor pattern and identify the underlying synaptic and ionic mechanisms; we also illustrate the ACh
receptor pharmacology in preBötC inspiratory neurons including pacemaker neurons. Preliminary data have been published in abstract form (Shao and Feldman 1998
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Slice preparation
Experiments were performed on a medullary slice
preparation that retains functional respiratory networks and generates
respiratory rhythm (Smith et al. 1991). Briefly,
Sprague-Dawley neonatal rats (0- to 3-days old) were anesthetized by
hypothermia (by incubating on ice for ~3-4 min.) and decerebrated.
The neuraxis was isolated with care to preserve the XIIn roots, and the
cerebellum was removed. The brain stem was pinned down and mounted in
the specimen vise of a Vibratome (VT100, Technical Products
International) oriented vertically with rostral end upward. The brain
stem was sectioned serially in the transverse plane under a dissection
microscope until the landmarks at the rostral boundary of preBötC
were visible. One transverse slice (550- to 650-µm thick) was cut.
The slice was transferred to a recording chamber of 3 ml volume and
stabilized with a threaded frame. The dissection and slicing were
performed in an artificial cerebrospinal fluid (ACSF) plus 1.0 mM
ascorbic acid bubbled with 95% O2-5%
CO2 at room temperature. The ACSF contained (in
mM) 128 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 23.5 NaHCO3, 0.5 NaH2PO4, and 30 glucose.
During electrophysiological recording, the slice was continuously
superfused (~3 ml/min) with ACSF with increased KCl (9 mM) that was
recycled into a 200-ml reservoir equilibrated with 95%
O2-5%CO2 at
26-27°C. All slices had rhythmic activities from the
XIIn that were similar in frequency and in temporal pattern to the
respiratory activities recorded from en bloc brain stem-spinal cord
preparations (Smith et al. 1991
).
Electrophysiological recording
Neurons within 100 µm from the upper surface of the slice are
visualized readily with infrared-differential interference contrast (IR-DIC) microscopy (×400, Axioskop, Zeiss), and the nucleus ambiguus is identified easily (Rekling and Feldman 1997). The
inspiratory neurons we recorded fired in phase with the inspiratory
bursts from XIIn were located ventral to the nucleus ambiguus about
halfway between the ventral surface of the slice and the nucleus
ambiguus. Patch electrodes were pulled from thick-wall (0.32 mm)
borosilicate glass on a horizontal puller (Model P-97, Sutter
Instruments). The tip size was ~1-1.5 µm (resistance: 4-6.5
M
). The electrode filling solution contained (in mM) 140 potassium
gluconate, 5.0 NaCl, 0.1 CaCl2, 1.1 EGTA, and 2.0 ATP (Mg2+ salt), pH adjusted to 7.3 with KOH. In
some experiments, the EGTA concentration of the patch electrode filling
solution was raised to 11 mM [or EGTA substituted by 10 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA)]and the concentration of CaCl2 was
raised to 1 mM (high EGTA solution). The electrode was mounted on a
hydraulic micromanipulator. Positive pressure of 100-150 mmHg was
applied to the back of the electrode as it was advanced. When the
electrode was positioned on the soma surface of the target neuron,
positive pressure was released, and negative pressure was applied to
form a gigaohm seal. The cell then was ruptured with short negative pressure pulses and/or with a voltage pulse produced by the patch-clamp amplifier. Intracellular signals were amplified with this amplifier (AXOPATCH 200A, Axon Instruments); whole cell capacitance was compensated, as was the series resistance (85-95%). A
10-mV
junction potential was determined experimentally; reported values of
potential are corrected values.
Respiratory-related nerve activity was recorded from the cut ends of
XIIn roots with a suction electrode, amplified 10,000-20,000 times and
band-pass filtered (3-3000 Hz) with an amplifier (Model P511K, GRASS
Instrument). Signals from intracellular recording and from XIIn roots
were recorded on videocassettes via pulse code modulation (Vetter
Instruments). Selected segments of intracellular signals were low-pass
filtered with an eight-pole Bessel filter (Frequency Device) and XIIn
nerve activity was rectified and low-pass filtered (Paynter filter,
= 15 ms), then digitized with DIGIDATA 1200 hardware and
CLAMPEX 7 software (Axon Instrument) on a Pentium computer on- or
off-line.
Data were analyzed with pCLAMP (Axon Instrument) or ORIGIN (V5,
Microcal Software) software. Averaged data are presented as means ± SD except the cases indicated with means ± SE,
n = number of cells (for whole cell recording) or
preparations (for XIIn motor output recording) are indicated. Paired
t-tests were used taking the electrophysiological
measurements during pharmacological manipulation versus those in
control conditions in the same cell or same preparation;
P 0 05 was the criterion for statistical significance.
In experiments in which Cl concentration was
changed, a 3 M KCl-agar bridge was used for grounding the bath solution
to avoid variation in junction potential between the bath solution and the AgCl grounding probe.
Drug application
Drugs were applied either by adding to the bath solution or by local pressure ejection. Single- or double-barrel pressure ejection micropipettes were mounted on a hydraulic micromanipulator, and the tip size of the ejection pipette was ~1-1.5 µm for each barrel with ~8-10 psi pressure applied to the back of the pipette. The ejection pipettes were positioned in the tissue ~30-40 µm away from the soma of the recorded neurons under an IR-DIC microscope. The space clamp problem inherent to voltage clamping in slice preparations was minimized by applying agonists in this way because the current elicited was spatially restricted to the soma or proximal dendrites, which were well clamped. Drugs were dissolved in a pipette solution containing (in mM) 150 NaCl, 9 KCl, 1.5 CaCl2, 1.0 MgSO4, 10 HEPES, and 30 glucose, pH adjusted to 7.4 with NaOH. In experiments in which the ionic components of the bath solution were changed (e.g., low Na+ solution), the composition of the pipette solution also changed correspondingly. Because the ejection pipette tip was small and in the tissue, the concentration of the locally applied drugs was not homogenous around the neuron. The drug concentration at the cell was estimated to be much lower than that in the pipette. We used higher concentrations (10 times higher than that one would use for isolated cells) in the pipette to ensure eliciting responses.
Cholinergic agents pirenzepine dihydrochloride, gallamine triethiodide,
4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were obtained from RBI. (+)-Himbacine, ()-atropine sulfate,
ACh-chloride, carbachol, (+)-muscarine chloride, (
)-nicotine
(hydrogen tartrate salt), and tetrodotoxin were obtained from Sigma.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ninety-two preBötC inspiratory neurons in 75 slice preparations were examined. Inspiratory neurons exhibit periodic bursts of action potentials (under current clamp) or periodic inward currents (under voltage clamp) in phase with the inspiratory burst activities of XIIn. Eighty-one (88%) of these inspiratory neurons responded to local application of 2 mM ACh or CCh or bath application of 50 µM muscarine.
Bath application of 50 µM muscarine increased the respiratory
frequency by 50.3 ± 33.5%, increased inspiratory duration by 109.5 ± 90.1%, and increased the amplitude of integrated
inspiratory bursts from XIIn by 34.0 ± 31.6% (Table
1). In inspiratory neurons, 50 µM
muscarine induced a tonic inward current (21.5 ± 20.4 pA, n = 6), increased the duration of the phasic
inspiratory inward currents by 66.6 ± 59.9%,and decreased the
amplitude by 39.4 ± 14.0% in inspiratory neurons (Fig.
1A, a-c) under
voltage clamp at 60 mV (Table 1). Muscarine depolarized inspiratory
neurons under current clamp. Muscarine also induced seizure-like
activity apparent during the expiratory periods (Fig. 1B,
raw signal from XIIn). Notice that there was no corresponding activity
in inspiratory neurons during the seizure-like activity of XIIn (Fig.
1B). Similar responses also were observed in pacemaker
neurons (n = 3, see following text). Both the motor
output of the system, i.e., XIIn activity, and the activity of the
inspiratory neurons recovered after bath application of 10 µM
atropine (Fig. 1A). Muscarine increased the number of action
potentials from 14.7 ± 7.7 to 18.4 ± 8.7 per inspiratory
period under current clamp (Fig. 1C) but did not change the
number of action potentials per depolarizing current pulse applied
during the expiratory period (40 pA, 500 ms; Fig. 1D, Table
1). This suggests that the muscarine-induced increase in number of
action potentials per inspiratory drive resulted primarily from the
increased duration of inspiratory drives. Inspiratory neurons did not
exhibit adaptation during depolarizing current pulses in either control
conditions or during muscarine application (n = 6).
|
|
In the presence of TTX (0.5-1.0 µM), local application of 2 mM ACh or CCh with a short pressure pulse (~200-250 ms) induced a slowly activating and long-lasting (12.2 ± 3.2 s) inward current (43.2 ± 26.4 pA, n = 12) with an associated increase in whole cell current noise and membrane conductance (Fig. 2B). The profile of the response inward current did not change significantly during an 1-h recording period. The response slightly desensitized with a long application (10 s) of ACh (Fig. 2A). The desensitization was observed in both ACh- and CCh-induced responses. This inward current was blocked by atropine in a concentration-dependent manner (n = 7) and partially recovered after perfusion with standard recording solution (Fig. 2B). Local application of 0.5 or 1 mM nicotine in identical conditions (double-barrel ejection pipette, one with ACh and the other with nicotine) did not induce similar response (data not shown). These results, taken together, suggest that there are muscarinic receptors on preBötC inspiratory neurons and that the excitatory effect of ACh was mediated primarily by muscarinic receptors that open channels.
|
Pacemaker neurons are a subset of inspiratory neurons that have
voltage-dependent endogenous bursting properties, i.e.,
"pacemaker-like properties." They normally fire during inspiratory
bursts but also can be induced to fire rhythmic bursts during
expiratory periods when current is injected to keep the membrane
voltage at 45 to
55 mV (q.v., Smith et al. 1991
).
Figure 3A shows a pacemaker
and a nonpacemaker inspiratory neuron. In the presence of 0.5-1.0 µM
TTX, local application of 2 mM CCh induced virtually identical
responses in these neurons under voltage clamp at
60 mV (Fig.
3B). Selected traces of whole cell currents elicited by
hyperpolarizing and depolarizing voltage-clamp pulses (
110,
40, and
10 mV, 200 ms) in control and during local application of CCh are
shown in Fig. 3C, i and ii.
Subtraction of the elicited current during CCh from that of control
gives the net effects of CCh (Fig. 3Ciii). The
I-V relationships of the CCh-induced responses were very
similar between pacemaker and nonpacemaker inspiratory neurons (Fig.
3D).
|
The M3 muscarinic receptor antagonist 4-DAMP (10 µM) blocked the CCh
(2 mM, pressure ejection)-induced inward current in inspiratory neurons
under voltage clamp at 60 mV (bath solution contained 0.5 µM TTX).
There was partial recovery after a 10-min wash with standard recording
solution (Fig. 4A). Because
4-DAMP is not very specific for the M3 receptor subtype
(Dörje et al.1991
), we tested three other
antagonists: M1 antagonist pirenzepine (PZ, 1 µM), M2 antagonist
gallamine (10 µM), and M2/M4 antagonist himbacine (10 µM, Fig.
4A). They had little effect on the CCh-induced inward current compared with control. Figure 4C shows a summary of
antagonist effects on the amplitude of the CCh-induced inward current.
4-DAMP decreased this current by 83.4 ± 10.5% (from 32.9 ± 21.5 to 4.7 ± 2.9 pA, paired t-test, P = 0.021, n = 6). The effects of PZ (paired
t-test, P = 0.338, n = 6),
gallamine (P = 0.581, n = 3), or
himbacine (P = 0.144, n = 5) were not
statistically significant. Taken together, these results suggest that
CCh induced a 4-DAMP-sensitive current in preBötC inspiratory
neurons that is not mediated by M1, M2, or M4 receptors. It is most
likely mediated by M3-like acetylcholine receptors. The effects of
4-DAMP were the same in pacemaker (Fig. 4B) and nonpacemaker
inspiratory neurons. On the basis of this and the previous observations
(Fig. 3), we assume that ACh acts on the same muscarinic receptor
subtype and ionic channels in both pacemaker and nonpacemaker
inspiratory neurons, and therefore we pooled the data from these
neurons in the following experiments.
|
To investigate whether the CCh-induced response is Ca2+ dependent, we recorded the CCh-induced inward current with whole cell patch electrode containing a high concentration of EGTA (11 mM EGTA) or BAPTA (10 mM). The mean current amplitude was 39.9 ± 18.3 pA and mean duration was 12.9 ± 4.0 s (n = 11). There was no significant difference compared with controls with low EGTA patch electrodes (43.2 ± 26.4 pA and 12.2 ± 3.2 s, n = 12, P = 0.74 and 0.66, respectively).
The steady-state I-V relationship of preBötC
inspiratory neurons was obtained by applying a series of 200-ms voltage
pulses from a holding potential of 65 mV to a range from
110 to 0 mV. Local application of CCh increased the slope of the
I-V curve. The I-V curve during
application of CCh and that of the control intersected at about
10 mV
(Fig. 5A). The
I-V relationship of the response induced by local
application of CCh was determined by subtracting the steady-state
I-V curve in control conditions from that during
application of CCh (Figs. 5B and 3C). The
I-V curves were linear in the voltage range of
110 to
20 mV, and the reversal potential ECCh,
determined by extrapolation of the linear regression line, was
11.4
mV (Fig. 5B, mean I-V curve of 18 inspiratory neurons). In a bath solution containing a
low-Na+ solution (128 mM of NaCl was substituted by
equimolar Tris-Cl; the remaining Na+ concentration was 24 mM), the CCh-induced inward current under voltage clamp at
65 mV was
reduced greatly, and it partially recovered after switching back to
standard recording solution (Fig.
6A). This suggests that
CCh opens a channel permeable to Na+. Because the
CCh-induced current reversed at about
11 mV, between the equilibrium
potentials of Na+ (ENa) and
K+ (Ek), we assumed the channel
was permeable to both Na+ and K+. According to
the equation derived by Goldman (1943)
and
Hodgkin and Katz (1949)
, the permeability ratio of
K+ to Na+ can be calculated
![]() |
(1) |
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ACh affects the oscillatory and repetitive firing properties of
neurons (Gola et al. 1998; Klink and Alonso
1997a
; McCormick and Prince 1986b
). ACh and its
agonists increase respiratory frequency in vivo (Burton et al.
1997
; Gesell et al. 1943
; Weinstock et al. 1981
) and in the en bloc brain stem-spinal cord preparation in vitro (Monteau et al. 1990
; Murakoshi et al.
1985
). In this study, we describe the actions of ACh (and its
agonists) on respiratory-related motor pattern in a medullary slice
preparation and on inspiratory neurons in the preBötC, its
pharmacology, and its ionic mechanisms. Our principal finding is that
in preBötC inspiratory neurons, ACh acts via M3-like receptors on
the postsynaptic neurons to open a cationic channel permeable to
Na+ and K+. This resultant
inward cation current depolarizes inspiratory neurons including
pacemaker neurons. It has been hypothesized that pacemaker(-like)
neurons in preBötC are the kernel for respiratory rhythm
generation (Feldman and Smith 1989
; Feldman et
al. 1990
; Smith et al. 1991
), and the
voltage-dependent bursting properties of the pacemaker neurons underlie
respiratory frequency regulation (Koshiya and Smith
1999
). We showed that when the membrane potentials of
inspiratory and pacemaker neurons were modulated by cholinergic agents,
the respiratory frequency was altered correspondingly. These results
are consistent with the pacemaker hypothesis. Our results do not
exclude the possibility of presynaptic effects of ACh on preBötC
respiratory neurons through presynaptic muscarinic and/or nicotinic receptors.
Possible role of premotoneurons and hypoglossal motoneurons
We observed that muscarine induced seizure-like activity in XIIn
with no corresponding activity in simultaneously recorded preBötC
inspiratory neurons (Fig. 1B). We also observed an increase in the amplitude of inspiratory bursts in XIIn, while the amplitude of
inspiratory drive recorded from the inspiratory neurons decreased and
their action potential frequency was unaffected. These seizures differ
from those induced under similar conditions by bicuculline where
seizure-like activity is seen in both XIIn and respiratory neurons in
preBötC (Shao and Feldman 1997). The
muscarine-induced seizure-like activity and the increase of inspiratory
amplitude may be due to excitatory effects of muscarine at the
motoneuron or premotoneuron level outside the preBötC. The
various changes in respiratory-related motor output recorded from XIIn
may result from the combined effects of muscarine on the central rhythm
generator and on the hypoglossal motoneurons. We identified, by
simultaneously recording from inspiratory neurons, that cholinergic
agents had effects on the rhythm generator in preBötC, and
differentiated the roles of the preBötC inspiratory neurons and
of the premotoneurons or hypoglossal motoneurons in cholinergic
regulation of the respiratory pattern.
Pharmacology of muscarinic receptor subtype
Five subtypes of muscarinic receptors, m1-m5, have been
identified by cDNA cloning (Bonner et al. 1987, 1988
;
Hulme et al. 1990
; Kubo et al. 1986
).
They correspond to the pharmacologically characterized subtype M1-M5
in animal tissues (Caulfield and Birdsall 1998
;
Lazareno et al. 1990
, Waelbroeck et al.
1990
). The affinity profiles of subtype specific antagonists
were characterized by expressing the five cloned receptors in Chinese
hamster ovary (CHO) cells (Dörje et al.1991
;
Lazareno and Birdsall 1993
). None of the tested
antagonists is specific for a single subtype, whereas each receptor
displayed a unique antagonist binding profile. Characterizing subtypes
of muscarinic receptors functionally in neurons in brain slices is
difficult compared with isolated neurons because the agonist
concentration applied by pressure ejection cannot be measured accurately. Although the concentration is known with bath application, the effect is very slow due to the time it takes for the agonists to
diffuse through the tissue to the recorded neurons; during this time,
agonist-induced desensitization may occur (Bünemann and
Hosey 1999
). Our data show that the response of preBötC
inspiratory neurons to local application of ACh or CCh, at least at
high concentration, is desensitized (Fig. 2). This prevents accurate
estimates for dose-response curves and antagonist affinity parameters.
In this study, the ACh-induced inward current in the preBötC
inspiratory neurons (including pacemaker neurons) was blocked by 4-DAMP
(Fig. 4, A and B). Because 4-DAMP exhibits
similar affinity to m1 and m3 receptors (Doods et al.
1987
; Dörje et al. 1991
), we also tested
other antagonists: gallamine (selective for the m2 receptor), himbacine
(similar affinity to both the m2 and m4 receptors), and pirenzepine
(very potent at m1 receptors) (Doods et al. 1987
; Dörje et al. 1991
; Lazareno and Birdsall
1993
). They each had little effect on CCh-induced inward
current, suggesting that this 4-DAMP-sensitive ACh-induced response in
preBötC inspiratory neurons is not mediated by M1, M2, or M4
receptors. It is most likely primarily mediated by M3-like receptors
(Fig. 4, A and C). Of course, we cannot rule out
the possibility of coexpression of m1, m2, m4, or m5 receptors in these
neurons. The genetic subtype of the receptors should be confirmed by
molecular biological methods. Our result is consistent with that of
Nattie and Li (1990)
from anesthetized cats, who
concluded that respiratory regulation by ACh involved predominantly M3
receptors, whereas cardiovascular regulation involved the M2 subtype.
Ionic mechanism of muscarinic receptor-mediated response
The excitatory effect of muscarinic receptor activation in
neurons has been attributed to switching off one or more
K+ currents such as: M current (Brown and
Adams 1980; Coggan et al. 1994
; McCormick
and Prince 1986a
), Ca2+-activated
K+ current (Madison et al. 1987
;
McCormick and Prince 1986a
), inward rectifier
K+ current (Uchimura and North
1990
; Wang and McKinnon 1996
), and a "leak
current" (Benson et al. 1988
). Excitatory effects of
muscarinic receptors mediated by opening channels have been reported
recently including: voltage-sensitive Na+ current
(Delmas et al. 1996
; Gola et al. 1998
),
nonselective cation current (Guérineau et al.
1995
; Haj-Dahmame and Andrade 1996
; Shen
and North 1992
), and Ca2+-dependent
cation current (Fraser and MacVicar 1996
; Klink
and Alonso 1997b
). We found that activation of muscarinic
receptors in preBötC inspiratory neurons at resting potential
induced an inward current with an increase in membrane conductance. The
current was linear in the voltage range between
110 and
20 mV and
reversed at
11.4 mV. A high concentration of EGTA or BAPTA in the
patch electrode had no effect on this current. A bath solution
containing a low concentration of Na+ shifted the
I-V curve for this current to the left and shifted the
reversal potential to
47 mV, which matched the value calculated with
the Goldman equation (Goldman 1943
; Hodgkin and
Katz 1949
) if we assume the channel is permeable to both
Na+ and K+ ions. A bath
solution containing a low concentration of Cl
did not shift the reversal potential of this current. Thus activation of muscarinic receptors in preBötC inspiratory neurons appears to
open a nonselective cation channel permeable to both
Na+ and K+, which is
neither Ca2+ nor voltage dependent in the range
of
110 to
20 mV. The characteristics of this current resemble the
ACh-activated nonselective cation current in CA3 pyramidal neurons in
rat hippocampus (Guérineau et al. 1995
). This kind
of muscarine-activated cation current also is observed in rat locus
coeruleus neurons (Shen and North 1992
) and in canine
pyloric circular muscle cells (Vogalis and Sanders
1990
). CCh also induced a slowly activated current at the
voltages more positive than
20 mV in preBötC inspiratory neurons (Fig. 3C iii,
10-mV voltage trace). Multiple
channels presumably are involved here (Shao and Feldman
1998
).
![]() |
ACKNOWLEDGMENTS |
---|
We thank Drs. Lawrence Kruger and Nicholas Mellen for assistance with this manuscript.
![]() |
FOOTNOTES |
---|
Address for reprint requests: X. M. Shao, Dept. of Neurobiology, Box 951763, UCLA School of Medicine, Los Angeles, CA 90095-1763.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 July 1999; accepted in final form 26 October 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|