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The Journal of Neuroscience, July 1, 2000, 20(13):5076-5082
Functional Nicotinic Acetylcholine Receptors That Mediate
Ganglionic Transmission in Cardiac Parasympathetic Neurons
Steve
Bibevski1,
Yuefang
Zhou2,
J. Michael
McIntosh3,
Richard E.
Zigmond2, and
Mark E.
Dunlap1, 4
Departments of 1 Physiology and Biophysics and
2 Neurosciences, Case Western Reserve University,
Cleveland, Ohio 44106, 3 Departments of Biology and
Psychiatry, University of Utah, Salt Lake City, Utah 84112, and
4 Department of Medicine-Cardiology, Veterans Affairs
Medical Center, Cleveland, Ohio 44106
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ABSTRACT |
Nicotinic acetylcholine receptors (nAChRs) mediate ganglionic
transmission in the peripheral autonomic nervous system in mammals. Functional neuronal nAChRs have been shown to assemble from a combination of  and  subunits, including 3, 5, 7, 2,
and 4 in RNA-injected oocytes, but the subunit composition of
functional neuronal nAChRs in vivo in mammals remains
unknown. We examined the subunit composition of functional nAChRs in
the intracardiac parasympathetic ganglion in a physiologically intact
system in vivo. We report here that localized perfusion
of the canine intracardiac ganglion in situ with an
antagonist specific for nAChRs containing an 3/2 subunit
interface (-conotoxin MII 100-200 nM) resulted in
reversible attenuation of the sinus cycle length (SCL) response by
~70% to electrical stimulation of the preganglionic vagus nerve. Perfusion with antagonist specific for receptors containing an 3/4 subunit interface (-conotoxin AuIB 1 µM)
resulted in attenuation in SCL responses (~20%) compared with
baseline when applied by itself, but not in animals pretreated with
-conotoxin MII. Perfusion of the ganglion with -bungarotoxin (1 µM, which blocks 7 receptors) caused a reduction in
SCL response by ~30% compared with baseline when perfused on its own
and when added after blockade with MII and AuIB. Perfusion with
hexamethonium bromide resulted in complete blockade of ganglionic
transmission, confirming total perfusion of the ganglion and the
nicotinic nature of ganglionic transmission at this synapse.
Immunohistochemistry using monoclonal antibodies against specific
nicotinic subunits confirmed the presence of 3, 7, 2, and 4
subunits. We conclude that functional ganglionic transmission in the
canine intracardiac ganglion is mediated primarily by receptors
containing an 3/2 subunit interface, with a smaller contribution
by receptors containing 7 nAChRs. Despite the presence of 4
subunits in functional channels, a contribution of a distinct 3/4
receptor population that does not include an 3/2 subunit interface was less clear.
Key words:
nicotinic receptor; -conotoxin; neuronal; parasympathetic; cardiac; ganglion; acetylcholine
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INTRODUCTION |
Ganglionic transmission in
peripheral autonomic neurons is mediated by nicotinic acetylcholine
receptors (nAChRs) (Fee et al., 1987 ). nAChRs can be composed of
various combinations of heterologous subunits that impart different
ionic and ligand binding characteristics allowing for diverse
physiological functions (Patrick et al., 1993; McGehee and Role, 1995).
The composition of nicotinic receptors responsible for ganglionic
transmission in mammalian autonomic neurons currently is unknown.
Investigation into the subunit composition of functional receptors may
provide insight into pathogenic mechanisms of autonomic nervous
dysfunction and pharmacological targets in various disease states, as
well as increase understanding of the importance of nAChR diversity.
Neuronal nAChRs have been shown to assemble in vitro from a
combination of - and -subunits including 2-9 and
2-4 (Listerud et al., 1991; Vernallis et al., 1993). Rat
intracardiac neurons can express 2-9 and 2-4 subunits,
although not all neurons within a ganglion expressed all of these
subunits (Poth et al., 1997). In oocytes injected with mRNA for
different subunits, functional combinations of 3/2, 3/4,
3/2/4, 3/2/5, 3/4/5, and 7 homomers have
been detected, although the possibility of other combinations cannot be
excluded (Patrick et al., 1993; McGehee and Role, 1995; Wang et al.,
1996). The question as to whether neurons in vivo in
distinct anatomical sites and/or functional roles express and assemble
receptors from all or only a subset of these subunits has been
controversial and largely unanswered.
Previous work in chick ciliary ganglia has indicated that the
EPSC response to preganglionic stimulation can be attenuated by
antagonists to receptors composed of both homomeric 7 and 3/2
subunits (Ullian et al., 1997), suggesting that synaptic currents are
mediated by a combination of these two receptors. Despite the existence
of a postsynaptic current contributed by 7 receptors in the ciliary
ganglion (Zhang et al., 1996; Ullian et al., 1997), pharmacological
blockade of 7 receptors has failed to show any functional effect on
ganglionic transmission (Duggan et al., 1976; Loring et al., 1984;
Mandelzys et al., 1995; Sargent and Garrett, 1995). Furthermore, 7
receptors are primarily extrasynaptic in chick ciliary neurons and thus
are not likely to be anatomically positioned for a role in rapid
synaptic transmission (Jacob and Berg, 1983; Horch and Sargent, 1995).
This has raised the question over the past decade as to whether 7
receptors play a functional role in ganglionic transmission despite
their anatomical presence in neurons. More recently, the identification
of a novel, slowly desensitizing current mediated by 7 receptors in
neurons isolated from rat intracardiac ganglia has suggested that 7
may have different functional roles dependent on the physiology and
location of the neuronal population (Cuevas and Berg, 1998). In
addition, single-channel receptor currents that do not conform to 7
homomeric channels can be modified by antisense "knockout" of 7,
indicating that these subunits may contribute to cellular currents by
combining with other and subunits (Yu and Role, 1998a,b).
To date, these studies have shared the common limitation of being
conducted in isolated, "nonphysiological" conditions. Such conditions may mask and/or provide artifactual evidence for functional roles of subunits located at perijunctional or specialized synaptic sites as well as provide incomplete information based primarily on
contribution to EPSCs. Additionally, although the use of
immunohistochemistry and molecular biology has provided evidence for
the physical presence of subunits, very little information has been
gained about the relationship between subunit diversity and in
vivo receptor function and physiology. The purpose of this
investigation was to determine the functional contributions of various
nicotinic receptor subunits to ganglionic transmission in
vivo in parasympathetic neurons innervating the heart in a
physiologically intact system. We show here that a large component of
ganglionic transmission in canine intracardiac neurons is mediated by
distinct populations of receptors containing an 3/2 subunit
interface and 7. Although we also show a smaller functional role for
3/4-like receptors, the existence of this subunit combination as
a distinct population is less clear.
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MATERIALS AND METHODS |
Animals and surgery. All procedures were performed
according to guidelines for the care and use of laboratory animals at
Case Western Reserve University and the Cleveland Veterans Affairs Medical Center. Adult, male Beagle dogs (Covance, Kalamazoo, MI) were induced with sodium thiopental and anesthetized with
-chloralose until toe pinch reflex was absent. Supplemental
chloralose was given every 30 min as needed. After endotracheal
intubation, the dogs were placed on a microprocessor controlled
respirator (Engler Corporation, Hialeah, FL). Blood gas and pH
were tightly controlled, and a heating blanket maintained temperature
in the physiological range. The femoral artery and vein were cannulated
for continuous blood pressure monitoring and drug
administration. The vagus nerve trunks were dissected and isolated at
the cervical level for preganglionic stimulation through a single
midline incision. Each nerve trunk was ligated and sectioned to prevent
proximal conduction to the brain. A bipolar electrode was inserted into
the caudal remnant of the right vagus nerve for stimulation. The right
vagus was used for stimulation because it has preferential input to the sino-atrial (SA) node (Hamlin and Smith, 1968). A right thoracotomy was
made at the fourth intercostal space, and a bipolar electrode was
placed near the atrial appendage for recording of an electrogram (electrical bursts) that was used to measure sinus cycle length (SCL)
directly from the heart. SCL was used as an index of organized SA node
activity, which is predominantly under vagal and sympathetic control.
The sinus node artery, which arises from a branch of the right coronary
artery, perfuses the pulmonary vein complex fat pad, a site that
contains the ganglia of neurons specifically innervating the SA node
(Fee et al., 1987; Billman et al., 1989). This artery was cannulated to
provide the means for direct localized perfusion of the intracardiac
parasympathetic ganglion. We perfused the artery with oxygenated
standard physiological Tyrode's solution at a rate of ~2 ml/min,
which was sufficient to maintain forward flow in the perfused area
while generating a physiological pressure of 80-140 mmHg in most dogs.
Solution reservoirs were warmed to 37°C and gassed with 97%
O2-3% CO2. By turning a
stopcock, we could selectively switch from control solution to one
containing antagonists without changing any other parameters. Various
antagonists specific for different combinations of receptor subunits
were used.
Drugs and solutions. Antagonists used in this study included
-conotoxin MII 100-200 nM [antagonist specific for
nAChRs containing 3/2 interface (Cartier et al., 1996)],
-conotoxin AuIB 1 µM [antagonist for nAChRs
containing 3/4 interface (Luo et al., 1998)], 1 µM
-bungarotoxin (-BgTx, antagonist for 7 nAChRs), and
hexamethonium bromide (5 mg). The conotoxins were dissolved in 0.2 mg/ml BSA in the perfusion system to prevent the toxin from sticking to
the perfusion glassware and tubing. -BgTx was purchased from both
Sigma (St. Louis, MO) and Biotoxins (St. Cloud, FL). We used two
different sources because previous reports have shown that some
-BgTx may contain neuronal bungarotoxin (Loring and Zigmond, 1988).
-BgTx from Biotoxins is physiologically assayed after purification
to ensure that there is no neuronal bungarotoxin present. We felt that
this was important because neuronal bungarotoxin blocks non-7
receptors, and this would give false evidence for a functional role of
7. Hexamethonium bromide blocks neuronal ganglionic nicotinic
receptors via competitive binding to the receptor site without
specificity for neuronal subunits and was used in this study as a
positive control for inducing ganglionic blockade.
Protocol. Vagal stimulations (preganglionic) were performed
in a dose-response manner at 3, 5, and 10 Hz using 8 V and a
pulse-width of 1 msec. Atrial electrogram and electrocardiogram
(ECG) signals were recorded for 30 sec of baseline, 30 sec of
stimulation, and 30 sec recovery for the assessment of postganglionic
responses to preganglionic stimulation. Sufficient time was given
between stimulations to allow heart rate to return to prestimulation
levels. After making recordings under control conditions, the perfusate reservoir was switched to one that contained one of the antagonists. Perfusion was maintained for at least 15 min to allow complete delivery
of the antagonist to the ganglion before repeating the stimulations.
Once recordings under antagonist-perfused conditions were obtained, we
then washed out the antagonist by switching back to plain Tyrode's
solution to ensure that the decreased response was not caused by
decreased viability of the preparation over time. The same antagonist
was then either reperfused to show that the effect was repeatable, or
the reservoir was switched to one that contained a different
antagonist. Hexamethonium was given at the completion of each protocol
via the same means to determine the completeness of perfusion of the ganglion.
Data capture and analysis. ECG and electrogram signals were
captured at 500 Hz with an analog-to-digital converter (Gould Instruments, Cleveland, OH), but only SCL was used for data
analysis. SCL, which is the time between successive spontaneous
electrical bursts from the sinus node, was graphically and numerically
plotted on-line in real time by built-in software macros. Quantitative analysis of sinus cycle length was made using SCL values averaged over
10 sec during baseline and 15 sec during stimulation. Statistical significance was determined by a paired or unpaired t test
where appropriate after testing for equality of variance using
SigmaStat software (SPSS, Chicago, IL). For samples with unequal
variance, statistical significance was determined by using a Wilcoxon
signed rank test.
Immunohistochemistry. Immediately after the heart was
excised, either the entire heart or a branch of the coronary artery supplying the ganglion was perfused with 4% paraformaldehyde. The
tissue then was placed in paraformaldehyde for 2-12 hr and transferred
to 15% sucrose (PBS) overnight. Before sectioning, the tissue was
immersed in 30% sucrose overnight for cryoprotection at 4°C. The
tissue was then frozen in OCT Compound (Miles, Elkhart, IN), and 10-20
µm sections were cut on a cryostat microtome at  24°C. The frozen
sections were placed onto gelatin-coated slides and frozen at 80°C.
Sections were incubated overnight with one antibody or a
combination of mAb210, mAb306, mAb313, mAb295, and polyclonal 4 at a
final concentration of 1:5000 or glial fibrillary acid protein (GFAP)
at 1:2500 (see Table 1). Fluorescence was imparted via Cy3- or
Cy2-conjugated donkey anti-rat, anti-mouse, or anti-rabbit secondary
antibody. A no-primary control was performed for each sample of tissue
for each secondary antibody, and background was determined by
comparison of the adjacent cardiac muscle with the ganglion.
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RESULTS |
Perfusion of the ganglion with -conotoxin MII
SCL response to preganglionic stimulation of the right cervical
vagus was examined before and after perfusion with -conotoxin MII,
an antagonist for nAChRs containing an 3/2 subunit interface (Fig. 1). Stimulations under both control
and conotoxin-perfused conditions resulted in an immediate increase of
sinus cycle length. There was no consistent difference in the rate of
onset of the response between antagonist-perfused and control
conditions. Figure 1B illustrates the mean group
change in SCL (from baseline) in response to vagal stimulation at three
frequencies of stimulation for both control and MII-perfused
conditions. The increase in SCL was reduced at all levels of
stimulation after drug treatment (control: 381 ± 90, 2349 ± 175, 6290 ± 267 msec vs MII perfused: 200 ± 70, 541 ± 279, 728 ± 252 msec, n = 9, p < 0.05 by paired t test at 3, 5, and 10 Hz stimulation), for
an average reduction of ~70%.
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Figure 1.
A, Plot of raw data showing change
in SCL from baseline in response to preganglionic stimulation before
and after perfusion with -conotoxin MII in the same animal. Note
that there is no apparent difference in rate of onset or recovery from
stimulation between the two conditions. B, Group mean
postganglionic response to preganglionic stimulation at 3, 5, and 10 Hz
before and after -conotoxin MII (200 nM);
n = 9, p < 0.05 using paired
t test or Wilcoxon signed rank test at each stimulation
frequency. Vertical bars are SEM in this and other figures.
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Perfusion of the ganglion with -conotoxin AuIB
Figure 2 shows the group mean
response when conotoxin AuIB (3/4) was perfused at 1 µM (control: 468 ± 65, 762 + 109, 1574 + 266 vs
AuIB: 289 + 63, 479.8 + 90, 1227 + 297 msec at 3, 5, and 10 Hz,
respectively; n = 7). Perfusion with toxin AuIB
resulted in a reduction in ganglionic transmission (~ 20%,
p = <0.05 except at 10 Hz, p = 0.054 by paired t test). To examine the possibility that 3/4
subunits were part of receptors that also contained 2, we tested
AuIB after perfusion with MII (Fig. 3).
Perfusion with AuIB after MII did not have a significant effect on
ganglionic transmission (172.6 ± 12.4, 260 ± 41, 501.6 ± 122.8 msec before AuIB, vs 150 ± 49, 262 ± 137, 628 ± 373 msec after AuIB at 3, 5, and 10 Hz, p > 0.05 by
paired t test at each stimulation frequency), suggesting
that 3/4 subunits do not form a distinct population of receptors
that contribute significantly to ganglionic transmission in the
heart.
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Figure 2.
Group mean response to preganglionic stimulation
at 3, 5, and 10 Hz before and after -conotoxin AuIB (1 µM); n = 7, p < 0.05 using paired t test at 3 and 5 Hz and
p = 0.054 at 10 Hz.
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Figure 3.
Effects of multiple antagonists on ganglionic
transmission at 3 Hz stimulation. Cont,
Baseline response after 20 min perfusion with plain
Tyrode's solution; MII, response after perfusion with
200 nM -conotoxin MII for 20 min; MII + AuIB, response after perfusion with 1 µM
-conotoxin AuIB (coapplied with MII) for 20 min,
p > 0.05; MII + AuIB +
-BgTx, response after perfusion with 1 µM -bungarotoxin (coapplied with MII and AuIB) for 35 min; p < 0.05, n = 4.
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Perfusion of the ganglion with -bungarotoxin
To examine the potential role of 7 nAChRs in ganglionic
transmission, we recorded the SCL response to preganglionic nerve stimulation before and after perfusion with BgTx (Fig.
4). Perfusion of -BgTx showed an
average 30% attenuation of ganglionic transmission (363.2 ± 65, 580.7 ± 95, 1656.5 ± 352 msec at baseline, vs 229.2 ± 57, 397.25 ± 68, 1115 ± 250 msec after BgTx,
n = 5, p < 0.05 by one-tailed, paired
t test at each stimulation) and was not reversible at 1 hr
after switching back to control solution. To test whether the effect of
BgTx was independent of receptor populations blocked by MII, we
perfused BgTx after blockade with -conotoxin MII and AuIB together
(Fig. 3). The effect of BgTx persisted in the presence of MII (150 ± 49, 262 ± 137, 628 ± 373 msec before adding BgTx, vs
126 ± 21.5, 192.47, 332 ± 106 msec after BgTx, p < 0.05 by one-tailed t test at each
stimulation frequency), suggesting that 7 subunits comprise a
functionally distinct population of receptors.
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Figure 4.
Group mean response to preganglionic stimulation
at 3, 5, and 10 Hz before and after -BgTx (1 µM);
n = 5, p < 0.05 using paired
t test.
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Effects of hexamethonium bromide
To assess whether noncholinergic receptors contributed to
ganglionic transmission in our preparation, we perfused the ganglion at
the completion of each study with hexamethonium bromide. Perfusion with
hexamethonium totally abolished SCL response to preganglionic stimulation at 10 Hz (Fig. 5) in all
preparations (control 4960 ± 3500 vs 140 ± 90 msec at 10 Hz, n = 5, p < 0.01 using paired t test), confirming nAChR-mediated transmission.
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Figure 5.
Effect of hexamethonium bromide (5 mg) on
ganglionic transmission. Perfusion of the ganglion for 5 min with
hexamethonium completely abolished postganglionic response to
preganglionic stimulation confirming that the entire ganglion was
perfused and that synaptic transmission was completely mediated by
nAChRs. n = 5, p < 0.01 using
paired t test.
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Immunofluorescence identification of nicotinic
receptor subunits
To confirm the presence of the various subunits tested in the
pharmacological studies, we conducted immunohistochemical studies using
monoclonal antibodies specific for nAChR subunits (Table 1). mAb313, mAb295, and mAb306 were used
to confirm the anatomical presence of 3, 2, and 7 subunits,
respectively. Figure 6 shows results
obtained with these three monoclonal antibodies and a polyclonal
antibody for 4 subunits. The pattern of staining was different for
each of the antibodies used, but signal was strong for each subunit
compared with background muscle staining for each antibody. No-primary
controls for each secondary antibody showed no evidence for nonspecific
binding. To ensure that the labeling was exclusively neuronal, we
conducted double staining with GFAP. As seen in Figure
7, GFAP and mAb210 (specific for 1,
3, and 5) were localized in distinct regions without any overlap.
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Figure 6.
Immunohistochemistry showing anatomical presence
of multiple nAChR subunits with cyanine-labeled monoclonal antibodies.
A, mAb313 (3 subunit); B, mAb295 (2
subunit); C, mAb306 (7 subunit); D,
polyclonal 4 (4 subunit). Background staining of cardiac muscle
can be seen in the top right corner of B.
No-primary controls did not show nonspecific binding for any of the
secondary antibodies. Scale bar, 25 µm.
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Figure 7.
Image showing double staining immunofluorescence
for GFAP and mAb210. A, mAb210 only; B,
GFAP only; C, both GFAP and mAb210. Note that GFAP
totally surrounds and envelopes the neuron with a mesh-like covering.
There is no overlap of GFAP and mAb210, confirming that the
fluorescence seen with mAb210 was localized to neurons only. Scale bar,
25 µm.
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DISCUSSION |
This study aimed to determine the functional nAChR subtypes
responsible for ganglionic transmission in peripheral parasympathetic neurons in dogs. Our results show that nAChRs responsible for a large
portion of ganglionic transmission in canine intracardiac parasympathetic neurons contain an 3/2 subunit interface. In addition, a functional contribution from receptors that contain 7
was evident, but a functional role for receptors that contain 3/4
without 2 could not be demonstrated.
The large contribution to ganglionic transmission by 3/2-like
receptors is consistent with previous findings in chick ciliary ganglion (Ullian et al., 1997). This study indicated that blockade of
3/2 receptors using MII abolished a major component of the EPSC
and blocked ganglionic transmission. Our results show that ganglionic
transmission was attenuated by ~70% at concentrations within the
range of specificity for receptors containing 3/2 (Cartier et
al., 1996; Harvey et al., 1997). It is possible that the component not
blocked by -conotoxin MII was mediated by a different population of
receptors or by neurons that were outside the perfused region. To
exclude this latter possibility, we perfused the region with
hexamethonium bromide, a nicotinic receptor antagonist that blocks all
neuronal nicotinic receptors. This resulted in complete blockade,
confirming that all transmission at the synapse was mediated by nAChRs
and that the entire ganglion was being perfused without alternate
inputs to the SA node. Another possibility for the incomplete blockade
is that acetylcholine released from the presynaptic nerve terminal may
have been able to displace MII from its binding site in a competitive
fashion. It was not feasible to increase the concentration of MII
beyond 250 nM to test this possibility because of the loss
of specificity of MII at a higher concentration of MII. Although
evidence in chick ciliary ganglion shows that EPSCs mediated by
3/2 nAChRs can be abolished with 50 nM MII (Ullian et
al., 1997), these studies were conducted in isolated neurons in
vitro, which is not comparable to our in vivo system.
In view of our finding that higher stimulation intensities elicited
greater responses in the presence of MII, it seems likely that
competitive displacement of MII could have contributed to the
incomplete blockade of ganglionic transmission in these experiments.
The notion that pharmacological blockade of receptors containing
3/4 subunits would result in attenuation of ganglionic transmission is supported by previous findings. Vernallis et al. (1993) have shown previously that AChRs in ganglionic
extracts that contained immunoreactivity to 3 and 2 subunits also
contained 4 subunits. The presence of 4 subunits also has been
described previously in mammalian intracardiac neurons (Poth et al.,
1997), and the 4 subunit has been shown to assemble with the 3
subunit (Vernallis et al., 1993), suggesting that 3/4
receptor should comprise a functional receptor population. However, we
found no significant contribution of receptors containing an 3/4
subunit interface to functional ganglionic transmission after blockade of 3/2 receptors. Biophysical data in Xenopus oocytes
suggests that 3/4 receptors generate a larger current than
3/2 (McGehee and Role, 1995). Therefore, if this receptor subtype
assembled to the same degree as 3/2 as a distinct population, and
assuming similar regulation, we should have seen a greater attenuation in ganglionic transmission with toxin AuIB. Our data therefore imply
that receptors containing an 3/4 subunit interface may not be
formed to any large degree as a distinct population and/or may only
assemble into functional channels along with 3, 2, or other
subunits. Another possibility is that 3/4 is assembled in the
cytoplasm, but the receptor is not transported to the membrane to a
significant degree. Our current data, and a recent report showing that
mice deficient in the 4 subunit do not show gross autonomic
dysfunction until cross-bred with 2-deficient mice, suggest that
2 and 4 subunits may coassemble with 3 to form functional
nAChRs (Xu et al., 1999).
The finding that blockade of 7 receptors did have an effect on
ganglionic transmission is also surprising in light of findings previously reported in the literature. It has been shown that blockade
of 7 receptors with -BgTx does not block compound action potentials in chick ciliary ganglion (Loring et al., 1989), presumably because synaptic currents in the presence of 7 blockade are
sufficient to reach threshold (Zhang et al., 1996). Other reports have
shown clearly that there is a rapid desensitizing current that is
blocked with -BgTx (Zhang et al., 1996; Ullian et al., 1997) and
that currents are modified by blockade of the 7 subunit (Yu and
Role, 1998a). More recently it was shown that 7 receptors mediate a slowly desensitizing current in response to acetylcholine in neurons isolated from the intracardiac ganglion of the rat (Cuevas and Berg,
1998) and that blockade of 7 receptors in chick ciliary ganglion can
lead to failure of transmission when stimulated at higher frequencies
(Chang and Berg, 1999). These findings have lead investigators to
postulate that 7 may play a physiological role in ganglionic
transmission depending on the function and environment of the neuron. A
concern in the former study was that with artificial delivery of
acetylcholine to the dissociated neuron there might be a
nonphysiological activation of 7 receptors, which has been shown by
a number of groups to be perisynaptic (Jacob and Berg, 1983; Loring et
al., 1985). On the other hand, it is possible that the role of 7
subunits is species, developmentally, and/or anatomically dependent and
the 7 subunit contributes to a greater component of an EPSC in
cardiac neurons compared with adult chick ciliary ganglion. The
contribution of 7 to ganglionic transmission in this study supports
the latter.
Another possibility raised by the anatomical localization of 7
subunits outside the synaptic region is that 7 may play a role at
higher intensities of stimulation. In such a model, 7 receptors are
not activated at lower stimulation intensity (firing rate) because any
released acetylcholine is quickly bound to receptors in the main
synaptic region and/or rapidly degraded by acetylcholinesterase. At
higher stimulation intensity, more acetylcholine is available for
longer periods of time and therefore able to diffuse to the anatomically distant 7 receptors. Although it did not reach
significance in our study, blockade of these receptors tended to result
in greater attenuation of ganglionic transmission at higher stimulation intensities (Fig. 4), suggesting that 7 may contribute a greater component to postsynaptic currents at higher levels of the
physiological range.
Immunohistochemistry confirmed the presence of 3, 7, 2, and
4 subunits. 3 and 4 subunits appeared diffusely distributed throughout the cytoplasm, whereas 2 was predominantly periplasmic. An interesting observation was the localization of 7 subunits, which
appeared punctate, suggesting storage or transport of the subunit in
vesicles. The implications of this finding are unclear, but the
different intracellular localization may represent a physiologically distinct function of 7 receptors. A particularly intriguing
possibility is the potential specialized localization of 7 receptors
at somatic spines where increased calcium handling may occur through
these receptors. Under these circumstances, 7 receptors could be
securely compartmentalized to specific regions, as suggested by Shoop
and colleagues (1999).
The mechanism(s) by which perfusion with the various antagonists
produces blockade warrants some discussion. Possible mechanisms that
may explain the results reported here can be described by two
hypotheses. Hypothesis A is that decreased ganglionic transmission after perfusion with antagonist is caused by an elimination of a
population of receptors that contributes to the EPSC, thereby resulting
in threshold being reached less frequently. Current evidence in support
of this hypothesis lies in the finding that multiple subtypes of
receptor can contribute to the EPSC on single neurons (Ullian et al.,
1997). Hypothesis B is that individual neurons predominantly express
one type of receptor, and perfusion with antagonist results in total
blockade of those neurons, leaving other neurons that express different
receptor populations active to contribute to the EPSC. Evidence to
support this hypothesis comes from the finding that not all neurons
express the same population of nicotinic receptor, and that some
receptor subunits are expressed in only a small percentage of total
neurons (Poth et al., 1997). On the other hand, a combination of these
two mechanisms may be the most likely explanation for our findings,
although our data support hypothesis A because all neurons in the
ganglia stained positive for each subunit tested.
A limitation of our study is that although we are able to block various
combinations of nicotinic subunits, this does not exclude the
possibility that other subunits are present within the same pentamer.
This possibility has been explored previously in vitro in
some detail, and it appears that functional receptors can be composed
from multiple subunit species in various combinations (Listerud et al.,
1991; Yu and Role, 1998a). Data from our study suggest that 3, 2,
and 4 can coassemble to form a functional receptor but that distinct
populations of 3/4 are not formed. In addition, the possibility
exists that the results reported here are a combination of effects on
presynaptic as well as postsynaptic nAChR populations; however, we did
not test this issue.
Why do neurons express multiple subtypes of receptor and use different
populations to differing degrees in mediating ganglionic transmission?
One possibility is that the different characteristics of various
subunit populations may allow for an increased spectrum of information
to be passed on to the postsynaptic neuron. The total number of vagal
neurons innervating the SA node in dogs is approximately 200 (Yuan et
al., 1994), which is remarkable in view of the critical and powerful
influence that the parasympathetic nervous system has over heart rate
control. In addition, it has become evident more recently that the
intrinsic cardiac innervation may involve a complex network analogous
to the intrinsic nervous system of the gut (Armour et al., 1998). This
type of system may use different populations of nicotinic receptors for
relay of information from divergent sources. In addition, the nature of nerve traffic varies in different anatomical and functional roles. It
may be possible that the subunit composition of nAChRs at different anatomic sites is dictated by the intensity and nature of nerve impulses needed for functional efficiency. Although such a function for
nicotinic receptors is speculative at this stage, it clearly identifies
the need for investigating the in vivo role of nicotinic receptor function in the future.
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FOOTNOTES |
Received Jan. 13, 2000; revised April 5, 2000; accepted April 11, 2000.
This work was supported by the Medical Research Service of the
Department of Veterans Affairs and National Institutes of Health Grants
HL50669 (M.E.D.), NS12651 (R.E.Z.), MH53631 (J.M.M.), and GM48677
(J.M.M.).
Correspondence should be addressed to Dr. Mark Dunlap, Veterans Affairs
Medical Center Medical Research Service 151W, 10701 East Boulevard,
Cleveland OH 44106. E-mail: med3{at}po.cwru.edu.
| |
REFERENCES |
-
Armour JA,
Collier K,
Kember G,
Ardell JL
(1998)
Differential selectivity of cardiac neurons in separate intrathoracic autonomic ganglia.
Am J Physiol
274:R939-R949[Abstract/Free Full Text].
-
Billman GE,
Hoskins RS,
Randall DC,
Randall WC,
Hamlin RL,
Lin YC
(1989)
Selective vagal postganglionic innervation of the sinoatrial and atrioventricular nodes in the non-human primate.
J Auton Nerv Syst
26:27-36[Web of Science][Medline].
-
Cartier GE,
Yoshikami D,
Gray WR,
Luo S,
Olivera BM,
McIntosh JM
(1996)
A new alpha-conotoxin which targets alpha3/beta2 nicotinic acetylcholine receptors.
J Biol Chem
271:7522-7528[Abstract/Free Full Text].
-
Chang KT,
Berg DK
(1999)
Nicotinic acetylcholine receptors containing alpha7 subunits are required for reliable synaptic transmission in situ.
J Neurosci
19:3701-3710[Abstract/Free Full Text].
-
Cuevas J,
Berg DK
(1998)
Mammalian nicotinic receptors with alpha7 subunits that slowly desensitize and rapidly recover from alpha-bungarotoxin blockade.
J Neurosci
18:10335-10344[Abstract/Free Full Text].
-
Duggan AW,
Hall JG,
Lee CY
(1976)
Alpha-bungarotoxin, cobra neurotoxin and excitation of Renshaw cells by acetylcholine.
Brain Res
107:166-170[Web of Science][Medline].
-
Fee JD,
Randall WC,
Wurster RD,
Ardell JL
(1987)
Selective ganglionic blockade of vagal inputs to sinoatrial and/or atrioventricular regions.
J Pharmacol Exp Ther
242:1006-1012[Abstract/Free Full Text].
-
Hamlin RL,
Smith CR
(1968)
Effects of vagal stimulation on S-A and A-V nodes.
Am J Physiol
215:560-568[Free Full Text].
-
Harvey S,
McIntosh J,
Cartier G,
Maddox F,
Luetje C
(1997)
Determinants of specificity for alpha-conotoxin MII on alpha3/beta2 neuronal nicotinic receptors.
Mol Pharmacol
51:336-342[Abstract/Free Full Text].
-
Horch HL,
Sargent PB
(1995)
Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion.
J Neurosci
15:7778-7795[Abstract].
-
Jacob MH,
Berg DK
(1983)
The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons.
J Neurosci
3:260-271[Abstract].
-
Listerud M,
Brussaard AB,
Devay P,
Colman DR,
Role LW
(1991)
Functional contribution of neuronal AChR subunits revealed by antisense oligonucleotides.
Science
254:1518-1521[Abstract/Free Full Text][Erratum (1992) 255:12].
-
Loring R,
Zigmond R
(1988)
Characterization of neuronal nicotinic receptors by snake venom neurotoxins.
Trends Neurosci
11:73-78[Medline].
-
Loring R,
Chiappinelli V,
Zigmond R,
Cohen J
(1984)
Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion.
Neuroscience
11:989-999[Web of Science][Medline].
-
Loring R,
Dahm L,
Zigmond R
(1985)
Localization of alpha-bungarotoxin binding sites in the ciliary ganglion of the embryonic chick: an autoradiographic study at the light and electron microscopic level.
Neuroscience
14:645-660[Web of Science][Medline].
-
Loring RH,
Aizenman E,
Lipton SA,
Zigmond RE
(1989)
Characterization of nicotinic receptors in chick retina using a snake venom neurotoxin that blocks neuronal nicotinic receptor function.
J Neurosci
9:2423-2431[Abstract].
-
Luo S,
Kulak JM,
Cartier GE,
Jacobsen RB,
Yoshikami D,
Olivera BM,
McIntosh JM
(1998)
-Conotoxin AuIB selectively blocks 3/4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release.
J Neurosci
18:8571-8579[Abstract/Free Full Text].
-
Mandelzys A,
De Koninck P,
Cooper E
(1995)
Agonist and toxin sensitivities of ACh-evoked currents on neurons expressing multiple nicotinic ACh receptor subunits.
J Neurophysiol
74:1212-1221[Abstract/Free Full Text].
-
McGehee DS,
Role LW
(1995)
Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons.
Annu Rev Physiol
57:521-546[Web of Science][Medline].
-
Patrick J,
Sequela P,
Vernino S,
Amador M,
Leutje C,
Dani JA
(1993)
Functional diversity of neuronal nicotinic acetylcholine receptors.
Prog Brain Res
98:113-120[Web of Science][Medline].
-
Poth K,
Nutter TJ,
Cuevas J,
Parker MJ,
Adams DJ,
Luetje CW
(1997)
Heterogeneity of nicotinic receptor class and subunit mRNA expression among individual parasympathetic neurons from rat intracardiac ganglia.
J Neurosci
17:586-596[Abstract/Free Full Text].
-
Sargent PB,
Garrett EN
(1995)
The characterization of alpha-bungarotoxin receptors on the surface of parasympathetic neurons in the frog heart.
Brain Res
680:99-107[Web of Science][Medline].
-
Shoop RD,
Martone ME,
Yamada N,
Ellisman MH,
Berg DK
(1999)
Neuronal acetylcholine receptors with 7 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia.
J Neurosci
19:692-704[Abstract/Free Full Text].
-
Ullian EM,
McIntosh JM,
Sargent PB
(1997)
Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors.
J Neurosci
17:7210-7219[Abstract/Free Full Text].
-
Vernallis AB,
Conroy WG,
Berg DK
(1993)
Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes.
Neuron
10:451-464[Web of Science][Medline].
-
Wang F,
Gerzanich V,
Wells GB,
Anand R,
Peng X,
Keyser K,
Lindstrom J
(1996)
Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits.
J Biol Chem
271:17656-17665[Abstract/Free Full Text].
-
Xu W,
Orr-Urtreger A,
Nigro F,
Gelber S,
Sutcliffe CB,
Armstrong D,
Patrick JW,
Role LW,
Beaudet AL,
De Biasi M
(1999)
Multiorgan autonomic dysfunction in mice lacking the 2 and the 4 subunits of neuronal nicotinic acetylcholine receptors.
J Neurosci
19:9298-9305[Abstract/Free Full Text].
-
Yu CR,
Role LW
(1998b)
Functional contribution of the alpha5 subunit to neuronal nicotinic channels expressed by chick sympathetic ganglion neurones.
J Physiol (Lond)
509.3:667-681[Abstract/Free Full Text].
-
Yu CR,
Role LW
(1998a)
Functional contribution of the alpha7 subunit to multiple subtypes of nicotinic receptors in embryonic chick sympathetic neurones.
J Physiol (Lond)
509.3:651-665[Abstract/Free Full Text].
-
Yuan BX,
Ardell JL,
Hopkins DA,
Losier AM,
Armour JA
(1994)
Gross and microscopic anatomy of the canine intrinsic cardiac nervous system.
Anat Rec
239:75-87[Medline].
-
Zhang ZW,
Coggan JS,
Berg DK
(1996)
Synaptic currents generated by neuronal acetylcholine receptors sensitive to alpha-bungarotoxin.
Neuron
17:1231-1240[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20135076-07$05.00/0
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