Departments of Pharmacology, Georgetown University School of
Medicine (M.F., S.N.E., R.P.Y., C.M.B., M.I.D.-G., K.J.K., R.A.G.),
Washington, DC, and George Washington University School of Medicine
(D.C.P.), Washington, DC
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Introduction |
The
dorsal motor nucleus of the vagus (DMV) is comprised of mainly
preganglionic motoneurons whose axons innervate the stomach (Gillis et
al., 1989
; Miselis et al., 1989). Published reports have indicated that
nicotine will act on these motoneurons to affect gastric motor activity
(Nagata and Osumi, 1991; Ferreira et al., 2000). Hunt and
Schmidt (1978) demonstrated that, 125I-
-BGTx,
a radioligand that selectively binds to nicotinic receptors, exhibits
an intense degree of binding in the DMV. Subsequent studies have
demonstrated that the 125I--BGTx binding sites
in brain tissue are composed principally of the 7-nAChR subtype
(Dominguez del Toro et al., 1994; Breese et al., 1997).
Evidence of a functional 7-nAChR in the DMV has been published by
Zaninetti et al. (1999). These investigators used the techniques of in
vitro 125I--BGTx autoradiography and
whole-cell patch clamp recordings of DMV neurons in a slice
preparation. Specific labeling by 125I--BGTx
was described as "intense" in the DMV, but hardly detectable in the
surrounding structures. Acetylcholine-evoked currents from DMV neurons
were comprised of a methyllycaconitine (MLA)-sensitive component. MLA
is known to be a selective antagonist of the 7-homomeric nAChR when
used at the 10 nM concentration as by Zaninetti et al. (1999) (Alkondon
and Albuquerque, 1993). However, the MLA-sensitive current did not
display the typical rapid time course of activation observed in studies
of homomeric 7-nAChRs. The issue of whether native 7-nAChR
subtypes are homomeric, or are heteromeric assemblies of subunits is of
current controversy. Some data favor a homomeric structure (Chen and
Patrick, 1997), and other data favor an assembly of other subunits with
the 7-subunit(s) (Yu and Role, 1998a,b).
None of the above-mentioned studies have addressed the question of
whether the 7-nAChR subtype is located on DMV motoneurons whose
axons contribute to the vagus nerve. In addition, except for the
atypical time course of MLA-sensitive currents in DMV neurons
(Zaninetti et al., 1999), the issue of whether the 7-nAChR associated with DMV neurons is similar to the 7-homomeric nAChR is
unresolved. The purpose of the present study was to address these two questions.
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Materials and Methods |
Compounds.
-Chloralose, urethane, fast green dye, and
(
)-nicotine hydrogen tartrate salt were purchased from Sigma (St.
Louis, MO). MLA, -bungarotoxin (-BGTx), and strychnine were
purchased from Research Biochemicals International (Natick, MA).
Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ).
Xylazine was purchased from Haver Pharmaceuticals (Shawnee, KS). The
following compounds were purchased from other vendors: ketamine
(Phoenix Scientific, St. Joseph, MO), buprenorphine (Reckitt and Colman Pharmaceuticals, Richmond, VA), and Nembutal (Abbott Laboratories, North Chicago, IL). All compounds were dissolved in physiological saline (final pH 7.2-7.4). Because of solubility limitations, moderate
heating was required for the more concentrated dose of strychnine (1500 pmol).
Chronic Vagotomy Surgery.
Sprague-Dawley male rats weighing
260 to 300 g (Taconic Farms, Germantown, NY) were anesthetized
with a ketamine-xylazine solution (90-100 mg/kg ketamine in
combination with 5-10 mg/kg xylazine administered by the
intraperitoneal route). Body temperature was maintained at 37 ± 1°C with a heating lamp. Using aseptic technique, a small incision
was made on the ventral part of the neck. The fascia was teased away
and muscle layers were retracted, thus exposing the carotid sheath. The
cervical vagus nerve was cut. A 3-mm section of the nerve was removed
followed by cauterization of both cut ends (to prevent regeneration).
In the case of the sham control animals, the vagus nerve was separated
from the carotid sheath, but the nerve was never cut. The surgical site
was then rinsed with sterile saline and sutured with monofilament nylon 4.0. All rats were given buprenorphine (0.02-0.08 mg/kg i.p.), for
postoperative attenuation of pain. After recovery from surgery, rats
were followed for a minimum of 2 weeks (no more than 3 weeks) to allow
for adequate neuronal degeneration in the DMV. This time period has
been documented as an adequate time for the degeneration of vagal
motoneurons and loss of choline acetyltransferase, following cervical
vagotomy (Ruggiero et al., 1993). Brainstem tissue was then taken for
performing either in vitro 125I--BGTx
autoradiography or anti-7 immunohistochemical examination of the DMV
(see below).
125I--BGTx Autoradiography.
For these studies
four animals underwent ipsilateral chronic vagotomy as described above,
and two animals underwent sham ipsilateral vagotomy also described
above. Two additional animals were used as control animals and their
brains studied (i.e., no vagotomy or sham surgery was performed). Two
to 3 weeks later, animals were sacrificed by rapid decapitation and
brains were rapidly removed and frozen on dry ice. Serial coronal
sections (20 µm) of the medulla oblongata encompassing the full
length of the DMV were cut with a cryostat, thaw mounted on
gelatin-coated slides, and stored at 80°C until used. For
autoradiography, tissue sections were allowed to equilibrate to room
temperature and then preincubated for 30 min (at room temperature) in
Tris-HCl buffer (50 mM, pH 7.3) containing 0.1% bovine serum albumin
(Sorenson and Chiappinelli, 1992). The sections were then incubated
with 125I--BGTx (116 Ci/mmol, 0.8 nM; NEN Life
Sciences, Boston, MA) for 60 min at room temperature, followed by three
10 min rinses in the same buffer (but ice-cooled), and then quickly
rinsed in ice-cold distilled water. Nonspecific binding of
125I--BGTx was determined by adding 100 µM
nicotine bitartrate to the preincubation and incubation buffers, and to
a control group of adjacent coronal sections. Nonspecific binding
(i.e., binding of 125I--BGTx that remained in
the presence of 100 µM nicotine bitartrate) was typically less than
10% of total binding. The slides were then dried under a stream of air
and desiccated overnight. To generate autoradiographs, slides
containing labeled brain sections were exposed to Hyperfilm 
max
(Amersham Pharmacia Biotech, Piscataway, NJ) for 3 to 7 days.
The exposed film was developed for 2 min in Kodak D-19 developer and
fixed for 4 min in Kodak rapid fixer. Film images were then digitized
using the Loats Inquiry System, and figures printed from these
digitized images. Brain regions were identified by light microscopic
examination of adjacent coronal sections stained with neutral red to
visualize the nuclear groups of the medulla. Confirmation of these
nuclear groups was made with reference to the rat brain atlas of
Paxinos and Watson (1998). In describing the results, only a rough
approximation of quantification of 125I--BGTx
has been made using descriptors such as very strong signal, moderate
signal, weak signal, and background signal.
7-Fluorescence Immunohistochemistry.
Immunofluorescent
staining procedures were performed essentially as described previously
(Ebert et al., 1994), with the following modifications. Adult male
Sprague-Dawley rats (vagotomized and sham) were anesthetized with
Nembutal (50 mg/kg) and their tissues fixed by intracardiac perfusion
with freshly prepared 2% paraformaldehyde in phosphate-buffered saline
(PBS), pH 7.3. The brainstem was then removed, transferred to a 30%
sucrose solution in PBS, pH 7.3, and allowed to equilibrate at 4°C
for at least 48 h. Brainstem sections 30-µm thick were prepared
and mounted onto Superfrost Plus (Fisher Scientific; Pittsburgh, PA)
microscope slides by FD Neurotechnologies, Inc. (Catonsville, MD).
For single-labeled immunofluorescent staining, an 7-nAChR-specific
mouse monoclonal antibody (mAb 306) was obtained from Research
Biochemicals International and used at a dilution of 1:200. Adjacent
sections were incubated with a choline acetyltransferase (ChAT)-specific mouse monoclonal antibody (mAb 305) (Chemicon, Inc.,
Temecula, CA) at a dilution of 1:2000. Primary antibody signals were
amplified using the cyanine 3 TSA-Direct kit from NEN Life Sciences
according to the manufacturer's instructions.
Dual-immunofluorescent labeling of brainstem neurons was performed
using a modified version of the above-described protocol. For the
primary antibody incubations, the 7-nAChR-specific antibody mAb 306 was used at dilutions of 1:20 to 1:50, whereas a rabbit anti-ChAT
antibody (Chemicon, Inc.) was used at dilutions of 1:200 to 1:500. No
amplification was used for the dual-labeling experiments. Instead,
secondary antibodies (FITC-conjugated donkey anti-mouse IgG and Texas
Red-conjugated donkey anti-rabbit IgG) were obtained from
Jackson/Immunoresearch (West Grove, PA) and used at a dilution of 1:200
(in PBS). All images were visualized and captured using an Olympus
Fluoview confocal laser-scanning microscope.
Positively stained neurons were quantitated for the purposes of
describing the effect of unilateral vagotomy and of comparing data
obtained from DMV nuclei with data obtained from hypoglossal nuclei.
For these purposes, only the fluorescently stained cells with a clearly
identifiable dark (unstained) nucleus were included. To ensure that the
same neurons were not counted more than once, neurons were counted from
every sixth section in a series through the DMV (at the rostral to
caudal extent of the area postrema) from three separate rat brains. In
addition, neuron counting was independently performed by two different
investigators, and the results were averaged.
Acute Anesthetized Rat Surgical Preparation.
Experiments
were performed on male Sprague-Dawley rats weighing 270 to 360 g
(Taconic Farms), and procedures used have been described in detail in
another article (Ferreira et al., 2000). Briefly, before each
experiment, food was withheld overnight but water was provided ad
libitum. Animals were anesthetized with an -chloralose (60 mg/ml)
and urethane (800 mg/ml) solution (dissolved in 0.9% saline) in a dose
of 3 ml/kg given intraperitoneally. Toe-pinch and corneal reflexes were
performed to assess depth of anesthesia. Body temperature was monitored
by a rectal thermometer and maintained at 37 ± 1°C with an
infrared heating lamp. The trachea and carotid artery were exposed, and
animals were intubated to facilitate breathing. The carotid artery was
cannulated to monitor arterial blood pressure. A laparotomy was
performed, and the stomach was gently pulled from the abdominal cavity
for insertion of an intragastric balloon (made from the finger of a
latex glove and tied to polyethylene 160 tubing). The balloon
was inflated with warm water to give a baseline pressure of
approximately 10 mm Hg. Dexamethasone (0.8 mg) was given subcutaneously
to prevent swelling of the brain. Both the arterial and intragastric
pressure lines were connected to pressure transducers. These
transducers were, in turn, connected to bridge amplifiers (Analog
Digital Instruments, Milford, MA), and each of these amplifiers was fed into a MacLab motherboard. Data were saved on an Apple Macintosh G3 for
analysis at a later time. Rats were then placed in a stereotaxic apparatus (David Kopf, Tujunga, CA), an incision was made on the dorsal
aspect of the cranium, the occipital plate was exposed, and the
atlanto-occipital membrane was cut and gently pulled away. The
occipital bone was removed, the dura was cut, and the cerebellum retracted to expose the area postrema and calamus scriptorius.
Following each experiment, animals were sacrificed with an overdose of
pentobarbital. Brains were removed and placed in a 4% paraformaldehyde
and 20% sucrose solution for at least 24 h. Brains were sectioned
into 50-µm sections. These sections were stained with neutral red to
visualize the nuclear groups of the medulla. The location of nuclear
groups was studied in relation to microinjection sites using the atlas
of Paxinos and Watson (1998). Camera lucida drawings of pipette tracts
were made and analyzed using a projection microscope. Animal care and
experimental procedures were performed in accordance with the National
Institutes of Health guidelines and with the approval of the Animal
Care and Utilization Committee of Georgetown University, Washington, DC.
Microinjection Technique.
Double-barreled pipettes were used
as described previously (Ferreira et al., 2000). All compounds were
dissolved in saline and the pH was brought to 7.2 to 7.3. In some
experiments, fast green (1 mg/ml) was dissolved in the drug or vehicle
solution to aid in viewing microinjection sites. Coordinates for the
DMV ranged from 0.3 to 0.5 mm rostral to calamus scriptorius,
medial-lateral 0.3-0.5 mm lateral from the midline, and dorsal-ventral
0.5-0.7 mm from the dorsal surface of the medulla. Coordinates for the medial subnucleus of the tractus solitarius (mNTS) ranged from 0.3 to
0.5 mm rostral to calamus scriptorius, medial-lateral 0.5-0.7 mm
lateral from the midline, and from 0.4 to 0.6 mm from the dorsal surface of the medulla. Microinjections were all made in 60-nl volumes
by observing the movement of the fluid meniscus of the pipette against
a reticule. In some experiments, vehicle (saline or saline with fast
green dye, 1 mg/ml) was microinjected into the DMV or mNTS. For the DMV
experiments, nicotine (100 pmol/60 nl) was microinjected to elicit a
response. When robust increases in intragastric pressure were elicited,
antagonists (-BGTx, MLA, and strychnine) were microinjected 15 min
later. Nicotine was then microinjected 5 min following microinjections
of the antagonists, to explore blockade of the response. For the mNTS,
nicotine (100 pmol/60 nl) was microinjected and when robust decreases
in blood pressure were elicited, antagonists were microinjected 5 min
later. Nicotine was then microinjected after 5 more min. When an
antagonist was able to block the response from the DMV, we tested the
same doses of the compounds against nicotine in the mNTS. This was done
to determine antagonist specificity for 7-containing nicotinic receptors.
Data Analysis.
Data were analyzed using the Chart Software
for data analysis made for MacLab (ADI Instruments, Milford,
MA). Before microinjections were performed, the lowest points of
the intragastric pressure trace were obtained over a 3-min baseline
period, and a single value was calculated as the mean of all of these
points. This value was used as an index of gastric tone. Phasic stomach
contractions (frequency of 2-5/min) were often noted in the
intragastric pressure trace but this index of gastric function
was not analyzed for drug effects in this study. Phasic contractions
will be measured more directly in future studies by extraluminal strain
gauge force transducers attached to specific areas of the stomach.
After microinjections into the DMV, the maximum value in the trace was
taken as the largest increase in gastric tone. The percentage of change
from baseline in intragastric pressure was then calculated. For studies using -BGTx, the percentage of the response that was blocked was
calculated by comparing the post-antagonist response to the pre-antagonist response. For the recovery experiments, the response to
nicotine before -BGTx microinjection was treated as a 100% normalized response. Each subsequent response to nicotine was compared
with this initial response to calculate the percentage of the response
that was blocked. For blood pressure calculations, the change in mean
blood pressure was taken (mm Hg). The mean blood pressure over a 3-min
period was taken before microinjections. These baseline values were
compared with the mean of the blood pressure trace 30 s following
microinjections (which corresponded to the peak response, in this case
a decrease in blood pressure) (Ferreira et al., 2000). Data appear as
means (% change from baseline for intragastric pressure and change in
mm Hg for blood pressure changes) ± standard error of the mean.
For the calculations of the IC50 value for
-BGTx, the inhibition curve was constructed using different
concentrations of the antagonist. The effects of varying doses of
-BGTx (0.01-300 pmol) to inhibit the increase in IGP elicited from
the DMV with 100 pmol of nicotine were included in the calculations,
and compared with the effect after microinjection of vehicle. These
values were used to define an approximate IC50
value, for the doses of BGTx tested in this study. The
IC50 value for -BGTx was determined by
nonlinear least-squares regression analysis (sigmoidal dose-response; GraphPad, San Diego, CA).
In all cases, statistical analysis was performed on both percentage of
change and on raw data. The data are presented here as percentage of
change from baseline (intragastric pressure) or raw changes (mm Hg,
blood pressure). Paired samples t test was performed when
animals served as their own controls. Independent-sample t
test was performed on data from separate control and experimental groups. Comparisons among more than two means from different groups of
rats were made by analysis of variance followed by Duncan's multiple
range test. Differences were considered significant at P < 0.05. All values are expressed as mean ± S.E.M.
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Results |
Effect of Vagotomy on 125I--BGTx Binding in the
DMV.
We initially performed light microscopic autoradiographic
studies on the medulla oblongata of two rats using coronal sections taken at the level of the DMV. Similar findings were made in both rats,
i.e., specific labeling for 125I--BGTx was
very strong in the DMV. In contrast, the labeling signal was weak
and/or not detectable in nuclei in proximity to the DMV such as the
mNTS, the area postrema, and the hypoglossal nucleus. There was a
moderate signal in several ventrolateral structures, namely, the
inferior olive and the lateral reticular nucleus.
Binding of 125I--BGTx in coronal sections of
the medulla oblongata obtained from either sham-operated animals
(N = 2) or animals subjected to chronic vagotomy (one
cervical vagus nerve was sectioned and the other vagus nerve was
left intact) (N = 4) is compared in Fig.
1. The distribution of
125I--BGTx binding in the sham-operated animal
is similar to that found in the two control (non-operated) animals
(data not shown). A very strong signal was detected over the DMV
bilaterally (Fig. 1A). A moderate signal was noted for the lateral
reticular nucleus and the inferior olive. Very weak or rare signals
were noted in the mNTS, area postrema, and the hypoglossal nucleus.
Most importantly, in all rats subjected to chronic unilateral vagotomy
(N = 4), no signal for
125I--BGTx labeling was detectable in the
ipsilateral DMV (Fig. 1B). On the contralateral side, where the vagus
nerve was left intact, the DMV exhibited the usual very strong signal
125I--BGTx binding (Fig. 1B). Furthermore,
125I--BGTx binding in the inferior olive and
the lateral reticular nucleus, which exhibit a moderate signal, was
almost identical on both sides of the brain (Fig. 1B).
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Fig. 1.
Autoradiographic images of 125I--BGTx
binding in coronal sections of the rat medulla oblongata containing the
DMV. The coronal section labeled A was taken from a rat subjected to
sham surgery, and the coronal section labeled B was taken from a rat
subjected to right-side unilateral vagotomy. Note the very strong
signal for 125I--BGTx labeling of the DMV. A moderate
signal can be noted for the inferior olive (IO) and the lateral
reticular nucleus (LRt). See Results for details.
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Anti-7-Immunohistochemical Examination of the DMV of Sham and
Chronically Vagotomized Rats.
To obtain more definitive proof for
a postsynaptic (on the DMV cell bodies) location of the 7-subunit,
we performed immunofluorescence histochemical staining of brainstem
sections from rats that had been subjected to unilateral (right)
cervical vagotomy 2 to 3 weeks before fixation. We observed a
significant reduction (P < 0.01, N = 3) in the number of positively stained neurons in the right
(vagotomized side) DMV compared with the left (nonvagotomized control
side) DMV (Figs. 2A and 3A). Despite the
vagotomy, approximately one-third of the total number of 7-positive
neurons remained in the DMV (Fig. 3A). As
a control, we also counted 7-positive cells in the hypoglossal
regions from the same brainstem sections used to quantitate
7-expression in the DMV. The hypoglossal neurons serve as a useful
control in this regard because they are peripherally projecting via the
hypoglossal nerve and are not damaged by cervical vagotomy (Ruggiero et
al., 1993). As shown in Fig. 3A, no significant differences in the
number of 7-stained hypoglossal neurons were observed
(P > 0.5, N = 3).
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Fig. 2.
Effect of unilateral cervical vagotomy on
7-immunoreactivity (A) and ChAT immunoreactivity (B) in the DMV
(circled regions). CC, central canal; HG, hypoglossal. Control refers
to nonvagotomized side. See Results for details.
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Fig. 3.
Effect of right cervical vagotomy on
7-immunoreactivity (A) or ChAT-immunoreactivity (B) in the DMV and
hypoglossal nuclei in the rat. L- and R-, left and right, respectively;
hg, hypoglossal nucleus. **P < 0.01 and
***P < 0.001 using the Student's group
t test (N = 3/group).
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As an additional control, we stained alternate brainstem sections for
expression of the cholinergic neuronal marker ChAT. As shown in Figs.
2B and 3B, we observed nearly complete elimination of ChAT-labeled
neurons in the DMV ipsilateral to the vagotomy, whereas the
contralateral DMV contained clearly labeled neurons. No significant
differences were observed in the number of ChAT-expressing neurons in
either side of the hypoglossal region following unilateral cervical
vagotomy (Fig. 3A). In sham-operated animals (N = 3), no left-right differences were observed in the number of DMV or hypoglossal neurons labeled for either ChAT or 7-nAChRs (data not shown).
To determine whether 7-nAChRs are expressed on cholinergic neurons
in the DMV, coimmunofluorescent staining for 7-nAChR and ChAT was
undertaken. Before proceeding with the double-staining experiment,
however, we performed control experiments to confirm that there was no
cross-reactivity of secondary antibodies. For these experiments,
7-nAChR expression was visualized using an FITC-conjugated
anti-mouse secondary antibody and ChAT expression was identified via
staining with a Texas Red-conjugated anti-rabbit secondary antibody. As
shown in Fig. 4, 7-nAChR expression is only evident in the presence of the anti-7-nAChR primary antibody and the FITC-conjugated secondary antibody (Fig. 4A). No 7-nAChR staining was observed when the Texas Red-conjugated anti-rabbit antibody was used (Fig. 4B), despite the fact that the anti-7-nAChR primary antibody was present. Conversely, ChAT expression was only
observed when the Texas Red-conjugated anti-rabbit secondary antibody
was used following incubation with the rabbit anti-ChAT primary
antibody (Fig. 4, C and D). When neither primary antibody was present,
no staining was observed following incubation with both secondary
antibodies (Fig. 4, E and F). Thus, these results demonstrate that the
primary and secondary antibodies used for this study are specific and
do not show any appreciable cross-reactivity.
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Fig. 4.
Control immunofluorescent staining in the DMV region
of the rat brainstem. A and B, anti-7-nAChR primary antibody. C and
D, anti-ChAT primary antibody. E and F, no primary antibody (null). All
sections were incubated with both secondary antibodies (FITC-conjugated
anti-mouse IgG and Texas Red-conjugated anti-rabbit IgG) and visualized
using laser-scanning confocal fluorescence microscopy using either the
488-nm emission filter (FITC, A, C, and E) or the 568-nm emission
filter (Texas Red, B, D, and F).
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Next, we performed simultaneous staining of rat brainstem sections with
anti-7 and anti-ChAT antibodies. Every DMV neuron that was clearly
stained for ChAT also showed 7-staining (Fig. 5). The patterns of expression for ChAT
and 7-nAChRs were different within the cells, but it is clear that
the same DMV neurons express both of these proteins (Fig. 5C).
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Fig. 5.
Dual-immunofluorescent staining of DMV neurons for
7-nAChRs and ChAT. A, 7-nAChR staining for seven to eight DMV
neurons. B, ChAT staining for these same DMV neurons. C, an overlay of
staining for both 7 (green) and ChAT (red). Note the yellow stain
represents neurons exhibiting stain for both 7 and ChAT.
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Pharmacological Studies Designed to Assess Whether the 7-nAChR
Subtype Resembles the Heterologously Expressed Homomeric
7-nAChR.
To explore whether the 7-nAChR is pharmacologically
similar to the homomeric 7-nAChR expressed in heterologous systems
(e.g., oocytes) we first characterized the full dose-response curve for the antagonist effect of -BGTx on DMV neurons. From our previous study we determined that 100 pmol/60 nl was very close to the ED50 value (89 pmol) for nicotine in evoking an
increase in intragastric pressure (Ferreira et al., 2000). We also
demonstrated that 100 pmol/60 nl BGTx could produce a significant
blockade of the nicotine-induced increase in IGP (Ferreira et al.,
2000). We also learned in this previous study that a 15-min interval
between microinjections of a 100-pmol dose of nicotine was sufficient
to allow reproducibility of the nicotine response. The new data
obtained in this study are the results from studying doses of 0.01 to
300 pmol/60 nl -BGTx (Fig. 6). As can
be noted from this figure, 0.01 pmol/60 nl -BGTx exerted no effect
on nicotine-induced increase in intragastric pressure, and 100 pmol/60
nl -BGTx evoked a maximal antagonistic effect. Furthermore, analysis
of data obtained with all six doses of -BGTx tested indicated an
IC50 value of 0.23 pmol/60 nl for -BGTx (Fig.
6).
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Fig. 6.
Dose-dependent inhibition of nicotine-induced
increases in IGP by -BGTx. Nicotine-induced (100 pmol) increases in
IGP were inhibited by increasing doses of -BGTx. The curve
represents a best fit to the data with half-maximal inhibition at 0.23 pmol of -BGTx and a maximal inhibition of 52%. The data point
coinciding with 4 on the log scale in B is data obtained with vehicle
(zero antagonist). Data are normalized to values obtained in the same
animals with microinjection of nicotine. The number of microinjection
sequences tested with each dose of -BGTx was four (0.01 pmol) to six
animals.
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The blocking effect observed with 100 pmol/60 nl -BGTx was
reversible (N = 5) (Fig.
7). In these studies 100 pmol/60 nl
nicotine was microinjected unilaterally into the DMV. This was followed 15 min later by a microinjection of 100 pmol/60 nl -BGTx. Nicotine, 100 pmol/60 nl, was again microinjected into the same site in the DMV 5 min after the -BGTx. As can be seen from the summarized data of Fig.
7A, a 60% block of nicotine's response occurred at this 5-min time
point. Over the next 1.5 h, the -BGTx-induced block gradually
wore off (Fig. 7). A representative experiment depicting the time of
peak antagonism with -BGTx and near restoration of the
nicotine-induced response 105 min later appears as Fig. 7B. As controls
for this type of experiment, nicotine, 100 pmol/60 nl, was
microinjected into the DMV of three additional animals, at
approximately four successive 15-min intervals. Intragastric pressure
responses were determined and -BGTx was replaced with saline (Fig.
7A). The initial intragastric pressure response was designated as 100%
response. The intragastric pressure responses evoked by the second,
third, fourth, and fifth administration of 100 pmol/60 nl nicotine into
the DMV were 114 ± 14, 101 ± 9, 112 ± 13, and
104 ± 10%, respectively. These results indicate a very
reproducible effect of successive doses of nicotine into the DMV, and
contrast the significant changes in intragastric pressure when
successive doses of nicotine are tested with -BGTx present (Fig. 7).
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Fig. 7.
Recovery of -BGTx-induced blockade. A, data
showing reversibility of blockade with 100 pmol of -BGTx in the DMV
(N = 5). The 100% normalized response depicted on
the vertical is the increase in IGP with 100 pmol of nicotine, before
-BGTx. Times noted on the horizontal axis (below) refer to
nicotine-induced responses before (15) and after (5, 35, 105, and
135) -BGTx. Times noted on the horizontal axis (above) refer to
nicotine-induced responses before (15) and after (5, 20, 35, and 50)
saline (N = 3). B, representative traces from an
experiment showing -BGTx blockade and reversibility.
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-BGTx-induced antagonism of nicotine-induced increase in
intragastric pressure appeared to be specific for an 7-containing nAChR subtype. Evidence for this appears in Fig.
8. Nicotine microinjected into the mNTS
produced a dose-related decrease in mean arterial pressure (Ferreira et
al., 2000). Although the increase in IGP elicited by nicotine
microinjected into the DMV is blocked by -BGTx, the blood
pressure-lowering effect of nicotine is not (Fig. 8; Ferreira et al.,
2000). As can be noted, -BGTx in a dose (100 pmol/60 nl) that
produced maximal antagonism of nicotine-evoked increases in
intragastric pressure (Fig. 8A) had no effect on the nicotine-evoked
decrease in mean blood pressure (Fig. 8B, N = 3).
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Fig. 8.
Pharmacological characteristics of -BGTx (A) and
-BGTx-insensitive (B) responses to nicotine in the dorsal-medial
medulla. A, percentage of change of IGP due to microinjection of 100 pmol of nicotine into the DMV ( ) and the response due to a second
administration ( ), following various drug treatments. B, change of
mean blood pressure in response to microinjection of 100 pmol of
nicotine into the mNTS () and response due to a second
administration (), following various drug treatments. DMV data are
represented as the means ± S.E.M. for five microinjection
sequences. mNTS data are represented as the means ± S.E.M. for
three to five microinjection sequences. *P < 0.05 using the Student's group t test. +, vehicle-treated
group.
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We also tested MLA for antagonist activity against nicotine-induced
increases in intragastric pressure in doses that would most assuredly
block the homomeric 7-nAChR subtype, namely, 1 pmol/60 nl
(N = 5) and 10 pmol/60 nl (N = 5) (Fig.
8A) (Briggs and McKenna, 1996; Palma et al., 1996). As can be
noted from our summarized data, neither 1 nor 10 pmol of MLA
microinjected unilaterally into the DMV significantly antagonized the
effect of 100 pmol of nicotine microinjected into the same site. An
excessively high dose of MLA was also tested, e.g., 30 pmol/60 nl
(N = 5). This dose did exert a significant antagonistic
effect against nicotine's effect at the DMV (Fig. 8A). This dose was
clearly not selective for the 7-subtype because it also counteracted
nicotine's effect at the mNTS to lower mean blood pressure (Fig. 8B).
MLA antagonism was tested in the above-mentioned studies by retesting
nicotine 5 min following microinjection of the presumed antagonist
(under Materials and Methods). It is possible that MLA could
"wash out" quickly and not be present in adequate concentrations at
the 5-min time point. To avoid such a possibility five experiments were
performed wherein 10 pmol of MLA was coadministered with nicotine (100 pmol/60 nl) as a "cocktail" (Fig. 8A). The percentage of increase
in intragastric pressure with nicotine alone was 21.8 ± 2.8 and
the corresponding increase with the "cocktail" was 18.5 ± 3.0 (P > 0.05). Thus, 10 pmol/60 nl MLA was still
ineffective when coapplied with nicotine. In addition to these studies,
we conducted two experiments whereby following an initial nicotine dose
(100 pmol/60 nl, to verify DMV placement), MLA (10 pmol) was
microinjected 20 and 5 min before nicotine (100 pmol). In one
experiment, the percentage of increase in intragastric pressure elicited by nicotine was 19.2 versus 20.6 following MLA. In the second
experiment, nicotine elicited an 18.1% increase in intragastric pressure and a 17.9% increase following the two doses of MLA. Therefore, the 10-pmol/60 nl dose of MLA was still not able to block
nicotine's effects on the DMV, even after 20 min.
We attempted to block the nicotine-induced responses with two different
doses of strychnine, namely, 70 (N = 4) and 1500 pmol (N = 6). Data obtained are presented in Fig. 8A and
indicate that strychnine, 1500 pmol/60 nl, does counteract the effect
of nicotine on the 7-nAChR subtype in the DMV. This dose was clearly
acting specifically since it did not significantly counteract
nicotine's effect at the mNTS to lower mean blood pressure (Fig. 8B).
The 70-pmol/60 nl dose of strychnine had no effect. This finding is noteworthy because 67 pmol/60 nl has been reported to act in the NTS to
abolish cardiovascular responses induced by maximal doses of glycine
(Talman and Robertson, 1989). This dose of strychnine that has been
shown by others to block the glycine receptor had no effect on the
nicotine-evoked response; instead, a higher dose of strychnine (1500 pmol/60 nl) was required to counteract some of the nicotine-evoked
effect. The brainstem sites where these drug tests were performed are
depicted in Fig. 9.
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Fig. 9.
Microinjection sites for all studies. Two camera
lucida drawings of coronal sections of the medulla are depicted to
illustrate microinjection sites. Microinjection sites (tip of
micropipette) were located in these two rostral-caudal areas of the
medulla, near the obex (0.2 mm is 0.2 mm caudal to the obex). For
clarity, all mNTS injection sites are shown on the left and all DMV
sites are shown on the right. TS, tractus solitarius; CC, central
canal; HG, hypoglossal; AP, area postrema.
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Discussion |
Our new findings are 1) the 7-nAChR subtype in the DMV is
located postsynaptically on DMV neurons that project into the vagus nerve, and 2) the DMV 7-nAChR subtype's pharmacological
characterization and recovery profile from -BGTx blockade separates
it from the homomeric nAChR 7-subtype described in expression
systems (Anand et al., 1993).
Autoradiographic studies of 125I--BGTx
binding, combined with unilateral cervical vagotomy, clearly
demonstrated that binding sites for -BGTx disappear once DMV neurons
degenerate. The demonstration that -BGTx blocks most of the
nicotine-induced increases in intragastric pressure argues strongly for
a predominant influence of an 7-nAChR subtype receptor at the DMV
mediating a functional response.
Ashworth-Preece et al. (1998) also performed in vitro autoradiography
using 125I--BGTx on coronal sections taken
from the rat medulla oblongata of sham controls and of rats subjected
to chronic (2-week duration) unilateral vagotomy. Their technique for
"vagotomy" involved unilateral nodose ganglionectomy (in contrast,
we used unilateral cervical vagotomy, where the nerve was cut distal to
the nodose ganglion, with a 2-week recovery). These investigators
concluded that 125I--BGTx binding sites were
located presynaptically on vagal afferent terminals in the mNTS. Nodose
ganglionectomy removes cell bodies of afferent vagal nerves, resulting
in loss of vagal terminals in the NTS (Helke et al., 1983). Nodose
ganglionectomy also produces loss of cell bodies of efferent vagal
neurons in the medulla oblongata because efferent vagal fibers travel
through the nodose ganglia (Helke et al., 1983). Thus, using the
procedure of nodose ganglionectomy, it is impossible to tell whether
125I--BGTx binding is presynaptic on afferent
vagal nerve terminals in the mNTS, or postsynaptic on cell bodies of
efferent vagal neurons, i.e., postsynaptic on DMV neurons. We used the
unilateral cervical vagotomy to test the hypothesis that
125I--BGTx binding was postsynaptic on DMV
neurons. Indeed, unilateral cervical vagotomy in our study resulted in
nearly 100% loss of 125I--BGTx binding in the
DMV.
Additional evidence supporting the postsynaptic location of the
7-nAChR subtype was obtained with anti-7-immunohistochemical examination of the DMV of sham and chronically vagotomized rats. By
using antibodies against ChAT, we identified DMV neurons that project
into the vagus nerve and presumably innervate the abdominal viscera,
including the stomach. It is well accepted that the vagus nerve is
cholinergic in nature. Immunocytochemistry for ChAT demonstrates that
the DMV contains many cholinergic neurons (Ruggiero et al., 1993).
Consistent with this notion are our own data showing complete disappearance of ChAT-expressing neurons in the DMV following unilateral cervical vagotomy. Simultaneous staining of rat brainstem sections with anti-7 and anti-ChAT antibodies revealed that every DMV neuron stained for ChAT also showed 7-staining. Furthermore, following unilateral cervical vagotomy, no ChAT-positive neurons expressing 7-nAChRs remained in the DMV. Although roughly one-third of the 7-expressing cells were still present in the DMV following unilateral cervical vagotomy, none of these exhibited positive staining
for ChAT. These remaining DMV neurons probably do not project out of
the brainstem (McLean and Hopkins, 1982).
In the autoradiographic studies, the hypoglossal nucleus did not show
binding of 125I--BGTx, suggesting that the
7-nAChR subtype was not present. On the other hand,
immunofluorescent staining of hypoglossal neurons using an
anti-7-nAChR antibody indicated the presence of 7-subunits (our
data; Dominguez del Toro et al., 1994). Thus, we are left with the
dilemma that 125I--BGTx does not bind to
hypoglossal neurons but that 7-subunit antibody fluorescence is
present in the hypoglossal nucleus. Breese et al. (1997) reported
7-mRNA in the hypoglossal nucleus, showing that these neurons are
probably synthesizing the 7-protein, but they also reported near
background levels of -BGTx radiolabeling. One explanation may be
that 7-nAChRs produced in the soma of hypoglossal neurons do not
form functional -BGTx binding sites. Alternatively, 7-nAChRs
produced in hypoglossal neurons may not be accessible for
125I--BGTx binding until they are transported
out of the nucleus and reach nerve terminals.
The 7-nAChR subtype that is present in the DMV of the rat appears to
be functionally and pharmacologically distinct from heterologously
expressed homomeric 7-nAChRs and from 7-nAChRs characterized in
most neural tissue. Homomeric 7-nAChRs are nearly irreversibly
blocked by -BGTx (Couturier et al., 1990). In addition, these
7-nAChRs are blocked by MLA (Ward et al., 1990) and strychnine (Seguela et al., 1993). Current responses evoked by nAChR agonists at
7-nAChRs in hippocampal neurons (Alkondon and Albuquerque, 1993;
Frazier et al., 1998), in olfactory bulb neurons (Alkondon et al.,
1996), and in parasympathetic ganglion neurons (Vijayaraghavan et al., 1992) behave similarly.
The two common properties that the 7-nAChR in the DMV has in common
with other 7-nAChRs and 7-nAChRs is that -BGTx and strychnine
are effective antagonists of the nicotine-induced response evoked from
the DMV. However, in contrast to the 7-nAChRs in the hippocampus
(Frazier at al., 1998), the 7-nAChR subtype in the DMV is not
irreversibly blocked by -BGTx. The -BGTx blockade of
nicotine-induced increases in intragastric pressure had faded within
1.5 h. It should be noted that the reversibility of -BGTx blockade in the Frazier et al. (1998) study was followed for only 35 min. Recent data reported by Cuevas and Berg (1998) and Cuevas et al.
(2000) indicate that there is a type of functional 7-nAChR that is
blocked by -BGTx but in a reversible manner. Our data raise the
important question of whether the 7-nAChR can be synaptically activated. Although not mentioned under Results, we did not
observe any change in intragastric pressure from microinjecting
-BGTx into the DMV in a dose that significantly antagonized
nicotine-induced increase in intragastric pressure. This would argue
against the existence of a tonic cholinergic input to neurons of the
DMV. In agreement with this finding are data of Schafer et al. (1998) using antibodies directed against the C terminus of rat vesicular acetylcholine transporter as a marker of intrinsic cholinergic innervation, which indicate little cholinergic innervation of the DMV
relative to nearby brainstem structures such as facial and hypoglossal
motor nuclei, and the nucleus tractus solitarius. Strychnine, an
antagonist of the 7-nAChR when used in doses known to be acting
selectively (Peng et al., 1994) is effective in blocking nicotine at
the DMV, whereas MLA (Alkondon and Albuquerque, 1993) is not. Taken
together, these findings raise the possibility that the native
7-nAChR at the DMV may exist as a heteromeric complex, comprised of
the 7 with an as yet unknown nicotinic receptor subunit. However, we
can not rule out post-translational changes in an 7-homomeric nAChR.
Evidence does exist that some neurons may express heteromeric
7-nAChRs (Anand et al., 1993; Pugh et al., 1995; Cuevas and Berg,
1998; Guo et al., 1998; Yu and Role, 1998a,b). Guo et al. (1998)
studied the presynaptic nicotinic receptors in the lateral geniculate
nucleus of the chick. Exposure to nicotinic receptor agonists resulted
in increases in the frequency of glutamatergic spontaneous postsynaptic
currents that were sensitive to -BGTx. This response, like
ours in the DMV, recovered from -BGTx blockade. MLA failed to alter
the response unless high non-7-selective doses were used. In
contrast to the present finding, Guo et al. (1998) found that
strychnine failed to block the -BGTx-sensitive response. However,
the strychnine concentration studied may have been too low to achieve
7-selective blockade. The data of Peng et al. (1994) using an
7-nAChR homomeric expression system created with chick gene product
indicate that a much higher concentration of strychnine than used by
Guo et al. (1998) is required for attenuating this receptor's
response. The strychnine concentration used by Guo et al. (1998) was 1 to 3 µM and was based on concentrations found to be effective for
attenuating -BGTx-sensitive currents in the hippocampus of rats
(Matsubayashi et al., 1998).
In our study, we microinjected micromolar concentrations of compounds
into the DMV. That is, nicotine was used as a dose of 100 pmol
contained in 60 nl (1.66 mM solution). Other investigators who perform
microinjection studies of nicotine into brain tissue of rats use
similar high concentrations (Nagata and Osumi, 1991; Tseng et al.,
1993). Nicotine will be diluted once it diffuses and equilibrates in
the brain tissue. According to Fu et al. (1999), a 1 mM concentration
of nicotine in the microdialysate would yield a tissue concentration of
17.6 µM. These data indicate a dilution factor of 57-fold, and
extrapolating back to our data using 1.66 mM nicotine, our dose of 100 pmol/60 nl would be anticipated to result in a tissue concentration of
approximately 27 µM. Compared with the data of Fu et al. (1999), 27 µM is in the range of concentrations that induces a significant
response in their experimental system.
We are not the first to suggest that the 7-nAChR subtype in the DMV
has different properties from 7-homomeric nAChRs. This MLA-sensitive
current evoked in the brain slice of 3- to 9-day-old rats had a
relatively long time-to-peak (Zaninetti et al., 1999). These
investigators were able to demonstrate that MLA was an antagonist for
currents evoked by acetylcholine. No clear explanation exists for why
we failed to observe an antagonist effect of MLA in our test system. It
is possible that the 7-nAChR subtype in 3- to 9-day-old rats may
differ from that found in adult rats. There are data indicating changes
in 7-subunit immunoreactivity between postnatal day 3 and postnatal
day 15 in rats (Dominguez del Toro et al., 1997). Thus, 7-nAChR
subunit expression is developmentally regulated. It is possible that
sensitivity of the 7-nAChR subtype to MLA varies with the stages of
development. Zaninetti et al. (1999) also reported that some DMV
neurons possess a non-7-containing nAChR. This finding fits the data
we obtained, indicating that -BGTx does not fully block
nicotine-induced increases in intragastric pressure (Fig. 7A).
We thank Drs. Yingxian Xiao and Niaz Sahibzada for expert
assistance with the manuscript.
This work was supported by a grant supplement (to M.F.) from
the National Institute of Diabetes and Digestive Diseases to research
Grant DK 29975 (to R.A.G.). This work was completed as part of a Ph.D.
thesis for Manuel Ferreira and previously presented (Ebert et al.,
1999).
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7-Neuronal Nicotinic Receptor Subtype
Located on Motoneurons of the Dorsal Motor Nucleus of the Vagus
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Abstract
Introduction
