Department of Neurobiology, UCLA School of Medicine, Los Angeles, California 90095-1763
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ABSTRACT |
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Shao, Xuesi M. and
Jack L. Feldman.
Mechanisms Underlying Regulation of Respiratory Pattern by
Nicotine in PreBötzinger Complex.
J. Neurophysiol. 85: 2461-2467, 2001.
Cholinergic
neurotransmission plays a role in regulation of respiratory pattern.
Nicotine from cigarette smoke affects respiration and is a risk factor
for sudden infant death syndrome (SIDS) and sleep-disordered breathing.
The cellular and synaptic mechanisms underlying this regulation are not
understood. Using a medullary slice preparation from neonatal rat that
contains the preBötzinger Complex (preBötC), the
hypothesized site for respiratory rhythm generation, and generates
respiratory-related rhythm in vitro, we examined the effects of
nicotine on excitatory neurotransmission affecting inspiratory neurons
in preBötC and on the respiratory-related motor activity from
hypoglossal nerve (XIIn). Microinjection of nicotine into preBötC
increased respiratory frequency and decreased the amplitude of
inspiratory bursts, whereas when injected into XII nucleus induced a
tonic activity and an increase in amplitude but not in frequency of
inspiratory bursts from XIIn. Bath application of nicotine (0.2-0.5
µM, approximately the arterial blood nicotine concentration
immediately after smoking a cigarette) increased respiratory frequency
up to 280% of control in a concentration-dependent manner. Nicotine
decreased the amplitude to 82% and increased the duration to 124% of
XIIn inspiratory bursts. In voltage-clamped preBötC inspiratory
neurons (including neurons with pacemaker properties), nicotine induced
a tonic inward current of 19.4 ± 13.4 pA associated with an
increase in baseline noise. Spontaneous excitatory postsynaptic
currents (sEPSCs) present during the expiratory period increased in
frequency to 176% and in amplitude to 117% of control values; the
phasic inspiratory drive inward currents decreased in amplitude to 66%
and in duration to 89% of control values. The effects of nicotine were
blocked by mecamylamine (Meca). The inspiratory drive current and
sEPSCs were completely eliminated by
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence or absence
of nicotine. In the presence of tetrodotoxin (TTX), low concentrations
of nicotine did not induce any tonic current or any increase in
baseline noise, nor affect the input resistance in inspiratory neurons.
In this study, we demonstrated that nicotine increased respiratory
frequency and regulated respiratory pattern by modulating the
excitatory neurotransmission in preBötC. Activation of nicotinic
acetylcholine receptors (nAChRs) enhanced the tonic excitatory synaptic
input to inspiratory neurons including pacemaker neurons and at the
same time, inhibited the phasic excitatory coupling between these
neurons. These mechanisms may account for the cholinergic regulation of
respiratory frequency and pattern.
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INTRODUCTION |
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Acetylcholine (ACh)
plays a role in central respiratory control (Burton et al.
1995; Gesell et al. 1943
; Gillis et al.
1988
; Metz 1958
; Murakoshi et al.
1985
; Nattie and Li 1990
; Shao and Feldman 2000
; Weinstock et al. 1981
), which may
have important implications in some common disorders of breathing.
Impairment in cardiorespiratory control and the responsiveness to
hypoxia are hypothesized to be important mechanisms in sudden infant
death syndrome (SIDS). The incidence of SIDS is correlated to maternal smoking, smoking by the primary care giver, and nicotine exposure through breast milk (Haglund and Cnattingius 1990
;
Klonoff-Cohen et al. 1995
; Milerad and Sundell
1993
; Stepans and Wilkerson 1998
), all of which
produce elevated circulating levels of nicotine in infants. ACh in
brain stem regions associated with sleep is involved in state-dependent
respiratory depression (Lydic and Baghdoyan 1993
), and
smoking is a risk factor for sleep-disordered breathing characterized
by repeated episodes of breath cessation (apnea) and reduced
ventilation (hypopnea) during sleep (Wetter et al. 1994
). Cholinergic mechanisms also underlie central respiratory failure during organophosphate, e.g., nerve gas, poisoning
(Lotti 1991
). On the beneficial side, nicotine is being
investigated as a therapeutic agent for diseases such as Parkinson's
disease, Alzheimer's disease, and sleep apnea (Benowitz
1996
; Gothe et al. 1985
).
ACh enhances respiratory motor activity and consequent minute
ventilation following intra-arterial injection or application to the
fourth ventricle of anesthetized dog in vivo; these effects are
potentiated by the cholinesterase inhibitor physostigmine (Gesell et al. 1943). In an en bloc brain stem-spinal
cord preparation from neonatal rat generating a rhythmic respiratory
motor output, bath application of ACh increases respiratory frequency;
this effect is diminished, but not completely abolished, by atropine. Further addition of the nicotinic antagonist dihydro-
-erythroidine (DH-
-E), can completely abolish the effect of ACh (Murakoshi et al. 1985
). These effects may be mediated by nAChR. nAChR
subunits
4,
2, and
7 are present in the ventrolateral medulla
(Dominguez del Toro et al. 1994
; Wada et al.
1989
). However, the mechanisms underlying the central effects
of nicotine on breathing are poorly understood. Basic physiological
questions include the following. Where does nicotine act to affect
breathing? Is part of this action via direct effects on the neurons
postulated to generate respiratory rhythm? How does nicotine affect
respiratory neurons and their interactions resulting in modulation of
respiratory pattern?
Insight into the cellular mechanisms for cholinergic actions on breathing would provide a physiological understanding of the central effects of smoking, would help to delineate the possible side effects of therapeutic application of nicotine, and could lead to better strategies for treatment of SIDS, sleep apnea, and central respiratory failure during organophosphate poisoning.
The preBötC is hypothesized to be the site for respiratory rhythm
generation (Gray et al. 1999; Rekling and Feldman
1998
; Smith et al. 1991
). A subpopulation of
inspiratory neurons in preBötC that have intrinsic pacemaker
properties coupled by excitatory synaptic connections are proposed to
be the kernel for respiratory rhythm generation (Butera et al.
1999
; Rekling and Feldman 1998
). The
voltage-dependent bursting properties of pacemaker neurons provide a
means for controlling frequency by tonic depolarizing or
hyperpolarizing input. There are M3-like acetylcholine receptors on
preBötC inspiratory neurons, including pacemaker neurons
(Shao and Feldman 2000
). Activation of these receptors
depolarizes inspiratory neurons, which may underlie the
cholinergic-induced increase of respiratory frequency. The purpose of
this study is to understand the mechanisms underlying the cholinergic
modulation of respiratory pattern mediated by nicotinic receptors.
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METHODS |
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Slice preparation
Experiments were performed on medullary slice preparations that
retain functional respiratory networks and generate respiratory rhythm
(Smith et al. 1991). Sprague-Dawley neonatal rats (0-3 days old) were anesthetized by hypothermia by placing them on crushed
ice for 3-4 min and then promptly decerebrated. The cerebellum was
removed, and the brain stem-spinal cord was isolated. The brain
stem-spinal cord was mounted in the specimen vise of a Vibratome (Technical Products International, VT 100) oriented vertically with
rostral end upward. The brain stem was sectioned serially in the
transverse plane under a dissection microscope until the landmarks at
rostral boundary of preBötC were visible. One transverse slice
(500-650 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) 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 (~2.5-3.5 ml/min) with ACSF with increased KCl (9 mM)
that was recycled into a reservoir equilibrated with 95%
O2-5% CO2. The ACSF in the
recording chamber was maintained at 27 ± 1°C during experiments. All slices studied had rhythmic activities from XIIn that
were similar in frequency and in temporal pattern to the respiratory
activities recorded from the en bloc brain stem-spinal cord
preparations (Smith et al. 1991
).
Electrophysiological recording
Neurons within 100 µm of the slice surface were visualized
with an infrared-differential interference contrast (IR-DIC)
microscope (×400, Axioskop, Zeiss). The respiratory neurons we
recorded in this study fired in phase with the inspiratory bursts from
XIIn and were located ventral to the nucleus ambiguus. Patch electrodes were pulled from thick wall (0.32 mm) borosilicate glass with tip size
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) with the pH adjusted to 7.3 with KOH.
Intracellular signals were amplified with a patch-clamp amplifier
(AXOPATCH 200A, Axon Instruments). A
10-mV junction potential was
determined experimentally; reported values of potential are corrected values.
The 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 (3-3,000 Hz). Both signals from intracellular recording and from XIIn roots were recorded on videocassettes via pulse code modulation (Vetter Instruments).
Data analysis
Selected segments of intracellular signal were low-pass filtered
at 1 kHz (except those for average analysis of phasic inspiratory drive current, indicated in figure legends) with an 8-pole Bessel filter (Frequency Devices), and XIIn nerve activity was rectified and
integrated (Paynter filter, = 15 ms); then both were digitized at sampling frequency 2.5 kHz with DIGIDATA 1200 and software CLAMPEX 8 (Axon Instruments).
Spontaneous excitatory postsynaptic current (sEPSC) data were analyzed
with a program written in AXOBASIC (Axon Instruments). This program
read the Axon Binary Files (ABF) containing two channels of digitized
data: the whole cell patch-clamp signal and the integrated XIIn
activity. The program detected sEPSCs during expiratory periods by
setting a threshold for the derivative of the membrane current signal
and then measured the time as well as the peak amplitude of sEPSCs. The
program also detected peaks of the integrated XIIn signal that mark
inspirations and muted these periods. The conventional statistical
methods for miniature EPSCs, e.g., using the Kolmogorov-Smirnov test to
analyze the inter-event intervals of two groups of EPSCs, are not
valid, because the expiratory periods are interrupted by variable
inspiratory periods. We are not able to obtain continuous inter-event
intervals for sEPSCs. To compare the rates between sEPSC series, we
assumed that each series was a Poisson process with mean rate . If
m1 and
m2 sEPSCs are observed in time
periods t1 and
t2, the estimate
1
2. The variance of
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Drug application
Nicotine was applied either by adding it to the perfusate or by
local pressure injection into preBötC or XII nucleus. For bath
application, the effects were measured immediately prior to adding
nicotine for control and 3.5-5 min after adding it for the tests. For
pressure injection, the effects were measured immediately prior to the
injection for control and ~1-2 s after the injection for tests.
Five-microliter calibrated glass pipettes (1 µl/division, Fisher
Scientific) were pulled and the tips were broken to a diameter of 6-9 µm. The injection pipette was mounted on a micromanipulator and advanced into XII nucleus or 50-150 µm ventral to nucleus ambiguus (XII nucleus and nucleus ambiguus can be identified
easily by their distinct anatomical location and the morphology of the neurons under IR-DIC microscopy), 100-200 µm below the
slice surface. The injection volume was monitored by the displacement
of the fluid meniscus with a microscope containing a calibrated
eyepiece reticule. For pressure injection, nicotine was dissolved in a pipette solution containing (in mM) 142 NaCl, 9.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 10 HEPES,
and 30 glucose (pH was adjusted to 7.4). The antagonists were applied
by adding them to the perfusate. The effects of the antagonists were
measured 4.5-6 min after adding them. ()-Nicotine (hydrogen
tartrate salt), mecamylamine hydrochloride, TTX, and
6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) were obtained from
SIGMA/RBI.
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RESULTS |
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Bath application of 0.2 or 0.5 µM nicotine (equivalent to the
arterial blood nicotine concentration shortly after a cigarette has
been smoked) (Henningfield et al. 1993) increased
respiratory frequency. To avoid possible confounding effects of
desensitization, only one concentration was used for each preparation.
Under control conditions, the frequency was 5.9 ± 1.7/min
(mean ± SD, n = 28). The frequency
increased to 9.7 ± 3.3/min (188 ± 28% of control) with 0.2 µM (n = 8) and to 16.6 ± 4.1/min (280 ± 61% of control) with 0.5 µM nicotine (n = 20; Figs.
1 and 3A). The amount of 0.5 µM nicotine also increased the duration of XIIn inspiratory bursts from 640 ± 185 ms to 800 ± 300 ms (124 ± 30% of
control, n = 20) and decreased the amplitude from
135 ± 82 µV to 110 ± 69 µV (82 ± 12% of control;
Table 1). These effects were maximal
within 3-5 min after nicotine was added to the perfusate.
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To determine the site of nicotinic action, we unilaterally pressure injected 10 nl nicotine (20 µM) into the preBötC and observed increases in frequency from 7.6 ± 2.0 to 29.4 ± 19.7/min (n = 6, paired t-test on the period, P = 0.0002), which were symmetric on both XII nerves (Fig. 2, A and B; data from both sides were pooled). We also observed a decrease in amplitude of inspiratory bursts (integrated) of XIIn from 143 ± 76 µV to 113 ± 67 µV (paired t-test, P = 0.006, Fig. 2, A and B). The effects were reduced or absent when the injections were in the vicinity of but outside the preBötC. When the same amount of nicotine was injected into the XII nucleus, there was an increase in tonic activity and an increase in amplitude of inspiratory bursts from 112 ± 80 µV to 127 ± 80 µV (P = 0.014, n = 5) of the ipsilateral but not the contralateral XIIn. There was no change in frequency of the rhythmic inspiratory bursts (Fig. 2, A and B). Bath application of the nicotinic antagonist Meca (1 µM) blocked the effects of nicotine injections into either preBötC or XII nucleus (Fig. 2, A and B).
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To further investigate the cellular mechanisms underlying nicotinic
effects on respiratory frequency and pattern, we whole cell
patch-clamped preBötC inspiratory neurons while simultaneously recording the respiratory-related motor output from XIIn. Bath application of 0.5 µM nicotine induced an inward current of
19.4 ± 13.4 pA (n = 16) in voltage-clamped
(
60 mV) preBötC inspiratory neurons (Fig.
3A) and induced depolarization
under current clamp (data not shown). There was also an increase in
baseline noise (Fig. 3, A and B). The frequency
of sEPSCs during the expiratory periods was 2.7 ± 1.2/s, and the
amplitude was
19.6 ± 5.0 pA under control conditions with
neurons voltage-clamped at
60 mV (Table 1). Nicotine increased the
frequency of these sEPSCs to 4.3 ± 1.8/s and the amplitude to
22.2 ± 5.7 pA (n = 15, Fig. 3, A, B,
D, and E, Table 1). Statistical analyses for each
neuron were done assuming that each series of sEPSCs was a Poisson
process (see METHODS). The increase in frequency was
statistically significant in 10 of 15 neurons, and the increase in
amplitude of sEPSCs was significant (Kolmogorov-Smirnov test) in 9 of
15 neurons. Most neurons (8 of 10) that showed a sEPSC frequency
increase with nicotine also exhibited an increase in amplitude. All
five neurons that did not show a sEPSC frequency increase with nicotine
exhibited the nicotine-induced inward current and increase in baseline
noise. Three pacemaker neurons (neurons that fired rhythmic bursts of action potentials during the normally silent expiratory period if
depolarized to
45 to
55 mV) (Smith et al. 1991
) were
among these 15 inspiratory neurons. Nicotine increased the frequency and amplitude of sEPSCs in all three of these pacemaker neurons. In
inspiratory neurons including the pacemaker neurons, nicotine decreased
the amplitude from
57.7 ± 38.3 pA to
38.2 ± 26.8 pA (66 ± 12% of control) and the duration from 935 ± 191 ms
to 824 ± 197 ms (89 ± 16% of control) of the inspiratory
drive current in phase with the inspiratory bursts of XIIn
(n = 17, Fig. 3C; Table 1). These effects on
respiratory motor output and respiratory neurons were blocked by Meca
(1-10 µM, n = 3, Fig. 3, A-E). The increase in frequency of sEPSCs induced by nicotine was decreased significantly by Meca (n = 3), and the average
amplitude was decreased significantly in two of these three neurons.
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To identify the neurotransmitter systems for the endogenous excitatory input to the preBötC inspiratory neurons, we added CNQX to the perfusate (20 µM). Under control conditions, the sEPSC frequency was 2.6 ± 1.9/s (n = 6). CNQX completely eliminated sEPSCs (frequency = 0, Fig. 4, A and B); subsequent bath application of 0.5 µM nicotine did not induce sEPSCs. CNQX also eliminated the phasic inspiratory drive current in these neurons (n = 7, Fig. 4, A and B) and rhythmic motor activity from XIIn. The sEPSCs, phasic inspiratory drive current, and respiratory-related rhythmic motor activity from XIIn recovered after CNQX was washed out with fresh ACSF. One of these seven neurons had pacemaker-like properties: it generated rhythmic bursts during expiratory periods when it was moderately depolarized in the control condition under current clamp, and it also generated voltage-dependent rhythmic bursts after blockade of excitatory synaptic input by CNQX.
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With the bath solution containing TTX (0.5 µM), bath application of 0.5 µM nicotine did not induce any current or any increase in baseline noise, nor did it affect the whole cell input resistance in voltage-clamped inspiratory neurons (n = 7, Fig. 5). Two of these seven neurons were pacemaker neurons.
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DISCUSSION |
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By modulating synaptic transmission, nAChRs plays an important
role in a variety of brain functions (McGehee et al.
1995; Role and Berg 1996
; Wonnacott
1997
). Low concentrations of nicotine enhance glutamatergic
excitatory synaptic transmission in hippocampus by a presynaptic action
(Gray et al. 1996
). In this study, we showed that
nicotine acted on the preBötC, resulting in increases in
respiratory frequency and changes in respiratory-related motor output
in vitro. Nicotine induced a tonic inward current, an increase in
baseline noise, and an increase in sEPSC frequency and amplitude in
inspiratory neurons including those with pacemaker-like properties. Nicotine did not induce these effects nor induce changes in input resistance when action potential-dependent synaptic transmission was
blocked by TTX. These results suggest that nicotine affects inspiratory
neurons primarily by modulating excitatory neurotransmission. By
systematically analyzing sEPSCs during the expiratory period as well as
the phasic inspiratory drive current, we demonstrated that activation
of nAChRs enhanced tonic excitatory synaptic input to and inhibited
excitatory coupling between inspiratory (including pacemaker) neurons.
These results suggest that the nicotinic modulation of excitatory
neurotransmission underlies cholinergic regulation of respiratory
frequency and pattern.
Medullary sites of cholinergic modulation of respiratory pattern
Determination of the sites for cholinergic modulation of
respiratory pattern has been difficult in in vivo experiments
(Gesell et al. 1943; Nattie and Li 1990
)
as well as in the in vitro en bloc brain stem-spinal cord preparation
(Murakoshi et al. 1985
). Taking advantage of the slice
preparation that retains functional respiratory networks and generates
respiratory-related rhythmic motor output (Smith et al.
1991
), we can locate the preBötC precisely and make
targeted injections of cholinergic drugs. Unilateral injection of
nicotine into the preBötC increased respiratory frequency. In
contrast, injection of nicotine into the XII nucleus increased the
tonic activity and the amplitude of inspiratory bursts of XIIn but did
not affect the frequency of rhythmic activity (Fig. 2). These results
are consistent with the hypothesis that the preBötC is the site
for respiratory rhythm generation. The results from nicotine injection
into the XII nucleus, in addition to serving as a control for the
injection into the preBötC, consistent with the observation
(Haxhiu et al. 1984
) that nicotine injected intravenously, into the lateral ventricle or applied onto the ventral
surface of the medulla increases the activity of XIIn and genioglossus
muscle (a dilator upper airway muscle) in cat in vivo, provide a
physiological rationale for the investigation of the clinical use of
nicotinic agonists in the treatment of obstructive sleep apnea
(Gothe et al. 1985
), which involves sleep-related loss
of tone in the genioglossus muscle.
Differential modulation of excitatory neurotransmission
Excitatory neurotransmission in the preBötC is essential for
respiratory rhythm generation in vitro (Funk et al.
1993; Greer et al. 1991
). Coupled glutamatergic
inspiratory neurons with pacemaker properties are hypothesized as the
kernel of respiratory rhythm generation (Rekling and Feldman
1998
). The mutual phasic excitatory interactions between
inspiratory neurons synchronize the bursting activity of these neurons.
These neurons also receive tonic excitatory input that maintains the
membrane potential in the range for oscillation and controls the
oscillation frequency (Koshiya and Smith 1999
). In this
study, we showed that the phasic excitatory drive and the tonic
excitatory input to preBötC inspiratory (including pacemaker)
neurons can be differentially modulated. Low concentrations of nicotine
decreased the amplitude and duration of the phasic inspiratory inward
current, suggesting a suppression of the phasic excitatory interaction
between inspiratory neurons. Nicotine also increased the frequency and
amplitude of sEPSCs, indicating an enhancement of the tonic excitatory
input. These effects on sEPSCs may be due to activation of nAChRs on
neurons that provide tonic excitation to preBötC inspiratory
neurons. Some of these sEPSCs may also come from other inspiratory
neurons that are depolarized by nicotine and generate spikes randomly
during expiration. The nicotine-induced inward current associated with
an increase in baseline noise in inspiratory neurons also indicates a
facilitation in tonic synaptic input since these effects disappeared in
the presence of TTX (Fig. 5). The synaptic input is likely from distal dendrites; thus they cannot be identified as separate sEPSCs.
Butera et al. (1999) proposed computational models of
rhythm generation based on the preBötC pacemaker hypothesis
(Smith et al. 1991
). Their model contains a
heterogeneous population of voltage-dependent bursting neurons coupled
by excitatory synapses and receiving tonic excitatory inputs.
Simulation studies of this model suggest that 1)
facilitation of the tonic excitatory input to pacemaker neurons
increases respiratory frequency, and 2) decreases in the
strength of excitatory synaptic coupling between pacemaker neurons
(paradoxically) increases respiratory frequency. We observed that
nicotine induced both 1) and 2) in inspiratory,
including pacemaker, neurons concurrent with a dramatic increase in
respiratory frequency. These observations are supportive of this model
and suggest that nicotinic modulation of excitatory neurotransmissions at the cellular level can account for the cholinergic modulation of
respiratory frequency observed at the systems level.
Bath application of nicotine decreased the amplitude and duration of the phasic inspiratory drive to preBötC inspiratory neurons, suggesting that nicotine inhibited excitatory coupling between inspiratory neurons. At the same time, we observed an increase in duration and a decrease in amplitude of the inspiratory bursts in XIIn; the decrease in amplitude was also observed when nicotine was locally injected into the preBötC. Inhibition of excitatory coupling between inspiratory neurons resulting in their desynchronizion could account for the observed decrease in amplitude and the increase in duration of inspiratory bursts in the respiratory motor output.
Both the excitatory coupling between inspiratory neurons and the tonic
excitatory input to inspiratory neurons in the preBötC are
proposed to be glutamatergic (Funk et al. 1993;
Greer et al. 1991
; Koshiya and Smith
1999
). By systematically analyzing sEPSCs in preBötC
inspiratory neurons, we found that sEPSCs during the expiratory period
were blocked by CNQX (Fig. 4). There are two possibilities:
1) the sEPSCs are from various sources that use different
excitatory neurotransmitters. However, the sEPSCs disappeared (frequency = 0) after application of CNQX, making this unlikely. 2) The tonic excitatory input arises from a source that
connects to preBötC inspiratory neurons through an oligosynaptic
pathway. If such a pathway exists, our results suggest that at least
part of this pathway is glutamatergic and mediated by ionotropic
non-N-methyl-D-aspartate (non-NMDA) receptors.
Cholinergic synapses are unlikely to be one of the primary transmitters
mediating the tonic excitatory input because the sEPSCs persisted in
the presence of Meca.
In the presence of TTX, low concentrations of nicotine did not induce a
postsynaptic current, increase the baseline noise, or affect the input
resistance. This does not exclude the possibility that postsynaptic
nicotine-gated channels are present in these neurons. The concentration
of nicotine (0.5 µM) may have been too low to evoke a detectable
current; alternatively, an evoked postsynaptic current may have quickly
desensitized because bath application of nicotine was slow. However, we
observed a long-lasting increase in respiratory frequency and other
responses at this low concentration of nicotine without fast
desensitization, suggesting that rapidly desensitizing postsynaptic
nicotine-gated channels that mediate fast ACh synaptic transmission, if
any, are not involved in this nicotinic regulation of respiratory
frequency and respiratory pattern. Our results do not exclude the
possibilities that electrical coupling through gap junctions or
inhibitory neurotransmission may also be involved in the nicotinic
modulation of respiratory pattern. Rekling et al. (2000)
show that electrical coupling is present between preBötC
rhythmogenic neurons. The inhibitory neurotransmitters GABA and glycine
regulate respiratory pattern (Shao and Feldman 1997
),
and nicotine can modulate GABA release in the hippocampus
(Alkondon et al. 1997
) and in the lateral geniculate nucleus (Guo et al. 1998
).
In summary, our major findings are as follows. 1) Low concentrations of nicotine act on the preBötC and regulate respiratory frequency and pattern. 2) Activation of nAChRs differentially modulates excitatory neurotransmission. The tonic excitatory input to inspiratory neurons including pacemaker neurons is enhanced, and the excitatory coupling between these neurons is inhibited by nicotine. 3) The tonic excitatory input to preBötC inspiratory neurons modulated by nicotine is likely glutamatergic and mediated by non-NMDA glutamate receptors. 4) Modulation of excitatory neurotransmission via nAChRs may be a mechanism that underlies the cholinergic regulation of respiratory frequency and pattern. Whether the modulatory effects of nicotine on excitatory neurotransmission in preBötC inspiratory neurons are pre- and/or postsynaptic or preterminal remains to be determined.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Shane Saywell for valuable comments on the manuscript.
This research was supported by National Heart, Lung, and Blood Institute Grant HL-40959.
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FOOTNOTES |
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Address for reprint requests: X. M. Shao, Dept. of Neurobiology, Box 951763, UCLA School of Medicine, Los Angeles, CA 90095-1763 (E-mail: mshao{at}ucla.edu).
Received 21 December 2000; accepted in final form 14 March 2001.
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REFERENCES |
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