Department of Neurobiology, University of California, Los Angeles, California 90095-1763
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ABSTRACT |
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Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M2 and M3 mRNAs were observed in the first round of PCR amplification. All five subtypes, M1-M5, were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M2 and M3, with minor expression of M1, M4, and M5.
reverse transcription-polymerase chain reaction; neural control of respiration; medullary slice; neonatal rat; messenger ribonucleic acid
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INTRODUCTION |
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ACETYLCHOLINE plays a role in the central control of breathing (3, 16, 17, 19). Defects in the medullary muscarinic system may play a role in disorders of respiratory control such as sudden infant death syndrome (9) and after organophosphate (including "nerve gas") poisoning. ACh is also involved in state-dependent respiratory depression in the brain stem, which may contribute to the pathophysiology of sleep apnea (13). Five subtypes of muscarinic ACh receptors, M1-M5, have been identified by cDNA cloning (1, 4, 8, 10), and these subtypes are distributed throughout the brain (2, 22). Various subtypes are also distributed across brain stem respiratory regions including the ventrolateral medulla (14).
The rostral ventrolateral medulla is involved in a number of physiological functions including cardiovascular and respiratory regulation; it contains bulbospinal neurons that project to sympathetic preganglionic neurons in the thoracolumbar spinal cord (12, 15, 17). The pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation in mammals, is located in the rostral ventrolateral medulla ventral to the compact division of the nucleus ambiguus, midway between the facial nucleus and the obex (18, 20). The pre-BötC contains predominantly propriobulbar neurons (18, 20). Our laboratory (19) has shown, with the use of pharmacological tools, that pre-BötC inspiratory neurons express M3-like ACh receptors. These receptors mediate the postsynaptic excitatory effects of ACh on inspiratory neurons that may underlie the increase in respiratory frequency induced by ACh. However, the pharmacological tools currently available for identifying the subtypes of muscarinic receptors are limited. Because of their structural homology and pharmacological similarity, ligand-binding probes do not clearly distinguish among the subtypes. The purpose of this study was to identify muscarinic receptor subtype expression in the pre-BötC with molecular biological methods, primarily RT-PCR.
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METHODS |
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Tissue preparation.
Medullary slices (600- to 700-µm thick), which retain functional
respiratory networks and generate respiratory rhythm (20), were cut from Sprague-Dawley neonatal rats (~3 days old) with a
Vibratome (VT100, Technical Products International). The care and the use of laboratory animals were in accordance with National Institutes of Health guidelines and approved by the University of
California, Los Angeles Animal Research Committee. A region ventral to
the nucleus ambiguus containing the pre-BötC was micropunched with a 17-gauge needle (inner diameter 1.06 mm). The dissection and
slicing were performed in 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. The
micropunched tissue and the rest of the slice were placed in separate
1.5-ml Eppendorf tubes and frozen at 80°C for further experimentation.
mRNA extraction and RT-PCR.
Total mRNA was extracted from the pre-BötC tissue, the punched
medullary slice, and rat gastric smooth muscle with the Micro-FastTrack 2.0 mRNA extraction kit (Invitrogen, Carlsbad, CA) according to the
provided protocol. Briefly, the prepared tissue was incubated at 45°C
with lysis buffer (200 mM NaCl, 200 mM Tris, pH 7.5, 1.5 mM
MgCl2, 2% SDS, and protein/RNase degrader) for 5 min. The
lysate was then homogenized by passing it repeatedly through an
18-gauge needle fitted on a 1-ml syringe. The sample was centrifuged
briefly and the supernatant was collected. The supernatant was then
incubated with oligo(dT) cellulose for 20 min with slight agitation.
Next, the lysate-oligo(dT) mixture was centrifuged to remove the
supernatant, leaving the powder. A binding buffer [500 mM NaCl and 10 mM Tris · HCl, pH 7.5, in diethyl pyrocarbonate (DEPC)-treated
water] was added to the oligo(dT) bound with RNA. The mixture was then transferred to a spin column. The binding buffer was added to the
oligo(dT) powder, and the supernatant was removed by centrifugation. A
low-salt wash buffer (250 mM NaCl and 10 mM Tris · HCl, pH 7.5, in DEPC-treated water) was added to the oligo(dT) and removed by
centrifugation. Finally, RNA was eluted from the oligo(dT) powder by
the addition of an elution buffer (10 mM Tris · HCl, pH 7.5, in
DEPC-treated water). The eluted RNA was then precipitated with 2 M
sodium acetate and 100% ethanol and frozen at 80°C overnight. The
next day, the sample was thawed and centrifuged to produce a pellet.
The supernatant was removed, and the pellet was resuspended in elution buffer.
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Cloning and sequencing of muscarinic receptor PCR products. Bands on the agarose gel corresponding to the muscarinic receptor PCR products were excised, and the DNA was extracted from the agarose with a gel extraction kit (QIAGEN, Valencia, CA). The extracted DNA fragments were then ligated into the pCR2.1-TOPO vector with the TOPO TA (Taq-amplified) cloning kit (Invitrogen). One Shot Competent cells (Invitrogen) were transformed with the TOPO TA vector by heat shocking at 42°C and plated on tryptone-yeast extract agar plates containing ampicillin for selection. Colonies were picked, and Miniprep cultures were grown. Miniprep DNA was then obtained and digested with EcoRI to determine positive clones. Positive clones were ascertained, and the bacterial cultures were inoculated in 50 ml of tryptone-yeast medium and grown overnight at 37°C. A large amount of DNA was purified from the bacteria with the Midi kit (QIAGEN) according to the provided protocol. The DNA samples were then sequenced. The DNA sequences obtained were compared with the sequences database in GenBank.
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RESULTS |
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In the first round of amplification, with the first-round primers
specific for the five muscarinic receptor genes
(M1-M5; Table 1), bands corresponding to
M1, M2, and M3 were observed in
product from the medullary slice. For the rat smooth muscle, M2, M3, and M5 were observed, with
M5 showing a very faint band. For the punched
pre-BötC tissue, M2 and M3 were observed.
The negative control without tissue yielded no bands (Fig.
1). No PCR product was detectable in the
samples without RT.
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In the second round of amplification, with the second-round primers
specific for the five muscarinic receptor genes
(M1-M5; Table 1), bands corresponding to
all five muscarinic subtypes, M1-M5, were
observed for all three tissues: medullary slice, rat gastric smooth
muscle, and pre-BötC. The negative control without tissue once
again yielded no bands. The other control experiment, in which the
entire RT-PCR procedure was carried out for the brain tissue but RT
reagents were omitted, yielded no PCR product either. The negative
results of these control experiments confirm the absence of amplified
products from genomic DNA or other contamination accidentally
introduced into the samples (Fig. 2,
Table 2).
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DNA sequencing of the excised PCR products for M1-M5 receptors from pre-BötC tissue confirmed that the sequences matched those in the sequence database in GenBank.
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DISCUSSION |
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Neurotransmitter receptor mRNA such as that for the glucocorticoid and ATP receptors was detected by RT-PCR from micropunched tissue in the rostral ventrolateral medulla (5). In the pre-BötC, we observed M2 and M3 receptor mRNA. M1, M4, and M5 receptor mRNAs were not observed in the first-round PCR but were observed after the reamplification round. This suggests that M2 and M3 are the predominant muscarinic subtypes in the pre-BötC. All five subtypes of muscarinic receptor mRNA have been found in every gross anatomic region in the central nervous sytem of the rat, including the cerebral cortex, hippocampus, striatum, thalamus-hypothalamus, pons medulla, cerebellum, and spinal cord, although they are expressed at relatively different levels in these regions (22). The current study focused on a specific area, the pre-BötC, and provides information relating the muscarinic receptor subtypes to the function of respiratory modulation. Our results are consistent with a previous study by our laboratory (19) that identified M3-like ACh receptors mediating the postsynaptic excitatory effects of ACh on pre-BötC inspiratory neurons.
The rostral ventrolateral medulla contains heterogeneous populations of neurons and plays a role in a number of regulatory functions (12, 15). The pre-BötC is in this area and contains neurons hypothesized to generate respiratory rhythm (7, 18). Although we tried our best to isolate the pre-BötC accurately during slicing (which determined the rostral-caudal boundary of the punch) and micropunching (which approximated the boundary of the pre-BötC in the transection plane), we are not able to exclude the possibility that the micropunched tissue contained tissue from neighboring structures, such as the BötC, rostral ventral respiratory group, or nucleus ambiguus, and neurons involved in functions other than regulation of respiration, e.g., cardiovascular control. Our observation that M2 and M3 receptor mRNAs are expressed at relatively high levels in the pre-BötC tissue is consistent with the results of Nattie and Li (17), who demonstrated that in the cat, rostral ventrolateral medulla M2 receptors are involved in cardiovascular regulation and M3 receptors are involved in respiratory regulation. In this study, low levels of mRNA for the other three muscarinic subtypes (M1, M4, and M5) were found in the pre-BötC. Their physiological role and functional relationship with other muscarinic subtypes in the pre-BötC remains to be determined.
We observed M2 and M3 in the first-round PCR for rat gastric smooth muscle along with a very faint signal corresponding to M5, consistent with previous studies done by Lin et al. (11), who also observed M2 and M3 in rat gastric smooth muscle cells. (They did not perform PCR for M5.) After reamplification, all five muscarinic subtypes were found to exist in rat smooth muscle. Previous studies, such as that of Lin et al. (11), only performed one round of PCR amplification and therefore may not have detected subtypes expressed in the tissue at low levels. We were able to detect previously undetected muscarinic subtypes in various tissues because we performed a second round of PCR amplification.
In our procedure, after the first round of PCR was finished, one-fiftieth of the PCR product was used as the template for a second round of PCR. For the second round of PCR, a second set of nested primers, whose sequence immediately followed the sequence of the first-round PCR primers, was used. The use of nested primers greatly increased the specificity of the PCR; only DNA specific to the muscarinic receptors amplified in the first-round PCR was further amplified in the second round. As a control for the second round of PCR, the PCR product from the first round for two kinds of negative control samples (see METHODS) was used as a template. The negative results from these controls indicate that no contamination or artifact was introduced in the second round of PCR. Although our procedure was not "quantitative" RT-PCR (6), the dilution control experiment (control experiment 3 in METHODS) showed that the intensity of the bands for PCR products correlated to the amount of cDNA in our experimental conditions, and the second round of PCR did amplify small amounts of cDNA that were not detectable in the first-round PCR. The limitation of this procedure is that the variation in RT or PCR efficiencies of the different subtypes of muscarinic receptor mRNA may affect the amount of cDNA and the PCR product. After the second round of PCR, we analyzed the PCR products on an agarose gel. We then cut out the appropriate bands (see Fig. 2) and extracted the DNA. The isolated PCR fragments were then cloned into a vector that allowed for sequencing. The sequences matched perfectly with the muscarinic receptor sequences found in GenBank.
To minimize the possibility that genomic DNA was introduced into the system when mRNA extraction was performed and, as a result, was amplified in subsequent PCRs, two procedures were performed. First, the tissues were homogenized by passage through an 18-gauge syringe needle many times. Genomic DNA is sheared by this process. Second, after lysis of the tissue, the lysate was incubated with an oligo(dT) powder. Only mRNAs bind to the oligo(dT) powder because of the strong hybridization of the poly(A) tail of mRNA to the dT matrix. Genomic DNA is not able to bind unless it has long stretches of As. Thus we are reasonably sure that our samples were genomic DNA free. The control experiment in which no RT reagent was added during the RT process showed no band after either the first or second PCR. This provides further evidence that the positive bands did not arise from contamination by genomic DNA.
In this study, we determined the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from neonatal rat medullary slices. M2 and M3 subtypes of ACh receptor mRNA were observed in the first round of PCR amplification. All five subtypes, M1-M5, were observed in the second round of amplification. These results suggest that the majority of muscarinic receptor subtypes in the pre-BötC, and probably in some neighboring structures of rostral ventrolateral medulla, are M2 and M3, with minor expression of M1, M4, and M5.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-40959.
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FOOTNOTES |
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* J. Lai and X. Shao contributed equally to this work.
Address for reprint requests and other correspondence: J. L. Feldman, Dept. of Neurobiology, UCLA, Box 951763, Los Angeles, CA 90095-1763 (E-mail: feldman{at}ucla.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 May 2001; accepted in final form 3 August 2001.
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