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Physiol. Genomics 24: 252-263, 2006. First published November 22, 2005; doi:10.1152/physiolgenomics.00169.2005
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Received 12 July 2005; accepted in final form 13 November 2006.
Physiological Genomics 24:252-263 (2006)
American Physiological Society © 2006 American Physiological Society

Molecular profiling of murine sensory neurons in the nodose and dorsal root ganglia labeled from the peritoneal cavity

Pieter J. Peeters1, Jeroen Aerssens1, Ronald de Hoogt1, Andrzej Stanisz2, Hinrich W. Göhlmann3, Kirk Hillsley2, Ann Meulemans1, David Grundy2, Ronald H. Stead2 and Bernard Coulie1

1 Johnson and Johnson Pharmaceutical Research and Development, Department of Internal Medicine, Beerse, Belgium
2 Holburn Group of Companies, Bowmanville, Ontario, Canada
3 Johnson and Johnson Pharmaceutical Research and Development, Department of Functional Genomics, Beerse, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central nervous system. To elucidate molecular identities for these different properties, we compared gene expression profiles of neurons located in nodose ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of Alexa Fluor-488-conjugated cholera toxin B allowed enrichment for neurons projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG were isolated using laser-capture microdissection. Gene expression profiles of these afferent neurons, obtained by microarray hybridization, were analyzed using multivariate spectral map analysis, significance analysis of microarrays (SAM) algorithm, and fold-difference filtering. A total of 1,996 genes were differentially expressed in DRG vs. NG, including 41 G protein-coupled receptors and 60 ion channels. Expression profiles obtained on laser-captured neurons were contrasted to those obtained on whole ganglia, demonstrating striking differences and the need for microdissection when studying visceral sensory neurons because of dilution of the signal by somatic sensory neurons. Furthermore, we provide a detailed catalog of all adrenergic and cholinergic, GABA, glutamate, serotonin, and dopamine receptors; voltage-gated potassium, sodium, and calcium channels; and transient receptor potential cation channels present in afferents projecting to the peritoneal cavity. Our genome-wide expression profiling data provide novel insight into molecular signatures that underlie both functional differences and similarities between NG and DRG sensory neurons. Moreover, these findings will offer novel insight into mode of action of pharmacological agents modulating visceral sensation.

microarray; vagal and spinal afferents; nociception; spectral map analysis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE GASTROINTESTINAL (GI) tract receives dual extrinsic sensory innervation. Vagal afferents have their cell bodies in the nodose ganglia (NG) and project centrally to make synaptic connections in the brainstem, mainly at the level of the nucleus tractus solitarius, while spinal afferents arise from the dorsal root ganglia (DRG) and project into the dorsal horn of the spinal cord (22). These two types of neurons have different embryonic origins (epibranchial placode vs. neural crest), different dependencies on neurotrophic factors for development and survival (BDNF/NT3 vs. NGF/GDNF), and phenotypically distinct subpopulations that can be recognized by the presence or absence of certain peptides (particularly CGRP and substance P) (47).

Vagal and spinal afferents supplying the GI tract also differ in the pattern of their terminal innervation, which in part determines the stimulus-response properties of the peripheral endings (4). Vagal afferents terminate close to the mucosal epithelium, where they are exposed to chemicals (e.g., nutrients) absorbed from the lumen or mediators released from enteroendocrine cells or immune cells in the lamina propria. Vagal afferents also form intramuscular arrays and intraganglionic laminar endings that are thought to detect mechanical activity. Spinal afferents also innervate the mucosa, submucosa, and myenteric plexus. Additionally, projections of DRG neurons terminate in the serosa and mesenteric attachments, often in association with blood vessels. These endings are mechanosensitive, but the basis of this mechanosensitivity at the molecular level is unknown. Both vagal and spinal afferents respond to distension and contraction, but while vagal afferent endings respond to levels of distension that occur during the normal course of digestion, many spinal afferents have thresholds for activation that when applied in humans give rise to discomfort or pain (20).

These observations are the basis for the common view that vagal and spinal afferents have different functional roles: spinal afferents play a major role in nociception, while vagal afferents mediate physiological responses and behavioral regulation, particularly in relation to food intake, satiety, anorexia, and emesis. However, vagal and spinal afferents share a number of features: both have a large proportion of unmyelinated axons that can be ablated by capsaicin, and both express the capsaicin receptor (TRPV1), considered a hallmark of nociceptive neurons (44). Both NG and DRG neurons have been shown to become sensitized after inflammation, demonstrating plasticity in the mechanisms that regulate neuronal excitability, which has implications for pain processing (14).

Studies employing PCR, in situ hybridization, and immunohistochemistry have yielded some insight into the extent to which regulatory molecules form common pathways for the processing of GI sensory information, and those that are unique to vagal or spinal afferents. However, the advent of expression profiling and the availability of microarray chips that cover the mouse genome facilitate the exciting opportunity to examine the whole range of receptors, channels, transporters, and kinases that determine and shape sensory signal transduction. Here we have compared the expression profiles of NG and DRG neurons projecting to the mouse peritoneal cavity.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Retrograde Labeling of Sensory Neurons
Female balb-c mice (20 ± 2 g, n = 4) were anesthetized with ketamine, xylazine, and acepromazine [80:50:1 mg/kg, respectively, intraperitoneally (ip)]. Anesthesia was maintained by top-up doses of 20 mg/kg ketamine (ip) as required. After midline laparotomy, 5 cm of the jejunum were exposed to enable intramuscular injections of fluorescently labeled cholera toxin subunit B (CTB488; Molecular Probes, Eugene, OR). A Hamilton syringe was used to inject 2–4 µl of CTB488 (2.5 µg/µl) into the intestinal musculature at 10 sites bilaterally. After suturing of the incision and recovery, mice were injected (ip) with a contrasting fluorophore (CTB594, 250 µg; Molecular Probes). After 4 days, animals were euthanized. NG and DRG from T1–L4 were removed. Each ganglion was placed on a slide, and coverslips were used to squash the ganglia to enable counts of CTB488- and CTB594-labeled neurons, using a Leica fluorescence microscope equipped with TX2 (CTB594) and L5 (CTB488) filter blocks (Leica, Toronto, Canada). All protocols and surgical procedures were approved by an independent Institutional Animal Care and Use Committee at Holburn, in accordance with the guidelines of the American Association for Laboratory Animal Science.

Assessment of Numbers of Neurons in Sensory Ganglia
In a second of series of mice (n = 4), CTB488 was administered ip (250 µg in 100 µl), and the animals were euthanized 4 days later. One of each pair of DRG (from T10 to T13) or NG was frozen before sectioning on the cryostat at 10 µm. The paired ganglia from the contralateral side were squashed on slides beneath coverslips, as described above. Photomicrographs of at least 10 sections per ganglion and the squash preparations were prepared, and the numbers of fluorescent cells were counted from the resultant photomicrographs. Subsequently, the sections were stained with methylene blue, and the total numbers of neurons (ganglion cells containing a recognizable nucleus) were counted. From these measurements, the percentage of fluorescent neurons in the sections (no. of fluorescent neurons x 100/total no. of neurons) was determined.

Laser-Capture Microdissection
All ganglia for microarray studies were isolated 3–4 days after one ip injection of CTB488. NG and DRG (T10–T13) were harvested, placed in tissue-freezing medium (TFM; Triangle Biomedical Sciences, Durham, NC), frozen, and stored at –80°C.

Cryostat sections (12 µm) were attached to RNase-free PEN membrane-covered glass slides (PALM Microlaser Technologies, Bernried, Germany), fixed with 100% ethanol and air dried before laser-capture microdissection (LCM). Microdissection was performed on a PALM microbeam-equipped microscope (Axiovert 135; Zeiss, Gottingen, Germany). Fluorescent cells were excised after Nissl staining [0.5% cresyl violet acetate (Sigma-Aldrich, St. Louis, MO)/0.1 M sodium acetate (Fluka, Buchs, Switzerland)]. For each animal, a total of 200–300 cells were captured: 100–150 from DRG and 100–150 from NG. The microdissected cells from the DRG and the NG, respectively, were catapulted in two separate tubes, each one filled with 75 µl of RNeasy lysis buffer (RLT; Qiagen, Hilden, Germany) containing 0.14 M ß-mercaptoethanol and 200 ng polyinosinic acid (Sigma).

RNA Isolation
Laser-captured samples were incubated at 42°C for 20 min and then chilled on ice. An equal volume of 70% ethanol was added to each sample and then transferred to RNeasy MinElute Spin Columns (Qiagen). RNA was cleaned up according to the manufacturer's instructions, eluted in 14 µl of RNase-free water, and adjusted to 4 µl by vacuum drying.

RNA Amplification
As "spike-in" controls, the GeneChip Poly-A RNA control kit (Affymetrix, Santa Clara, CA) was used. Serial dilutions were made of the prokaryotic Poly-A control using the following dilution steps: 1:20, 1:50, 1:50, 1:20, and 1:10. First-strand cDNA was prepared as described by the Affymetrix two-cycle cDNA synthesis protocol, except for the use of Superscript III (Invitrogen, Carlsbad, CA) and incubation at 50°C for 30 min. Second-strand master mix consisted of 1 µl of 10x Bst polymerase buffer (Epicentre, Madison, WI), 1 µl of 10 mM dNTP (Invitrogen), 0.5 µl (1 U) thermostable RNaseH (Invitrogen), 1 µl (5 U) Bst DNA polymerase (Epicentre), and water to 10 µl. This master mix was added to the first-strand cDNA reaction and incubated at 65°C for 10 min before heat inactivation at 80°C for 3 min. Subsequently 2 µl of exonuclease mix were added containing ExoI and ExoVII and incubated at 37°C for 10 min, followed by heat inactivation at 80°C for 3 min. Double-stranded cDNA was transcribed at 42°C for 3 h using the AmpliScribe T7 High Yield Transcription Kit (Epicentre) in a total volume of 100 µl. The resulting amplified RNA was incubated with DNase I (4 U/µl) at 37°C for 15 min. Amplified RNA was purified after addition of 100 ng of polyinosinic acid using RNeasy MinElute Cleanup Kit (Qiagen). RNA was eluted in 14 µl of RNase-free water and adjusted to 4 µl by vacuum drying. The second round of amplification was performed as described above, except that 50 ng of random hexamer primers were used to prime the reverse transcription reaction and the second-strand cDNA reaction was primed with 0.25 ng of T7 oligonucleotide.

RNA Labeling and Microarray Hybridization
The third round of amplification, including biotin labeling, was performed on 500 ng of second round-amplified RNA. First-strand cDNA synthesis was performed as described above. Second-strand cDNA synthesis was performed using 1 µl of T7 oligonucleotide dT24 (Affymetrix, 100 pmol/µl) annealed for 5 min at 70°C, and the reaction was then incubated at 42°C for 10 min. A master mix was prepared, consisting of 10x second-strand buffer, dNTPs (200 mM final), Escherichia coli RNaseH (2 U), and 10 U of E. coli DNA polymerase (Invitrogen), and added to the first-strand reaction to obtain a 50-µl reaction volume. After incubation at 37°C for 10 min, denaturation was done at 80°C for 3 min. Cleanup was performed with Qiagen PCR purification kit. Synthesis of biotin-labeled RNA was performed using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY). Cleanup was done using the RNeasy Mini Kit (Qiagen). Labeled RNA was hybridized to either mouse genome MG-U74Av2 or MG-430_2.0 GeneChip arrays (Affymetrix). Hybridization of microarrays was performed using 12.5 µg of biotin-labeled RNA at 45°C for 16 h under continuous rotation. Arrays were stained in Affymetrix Fluidics stations using streptavidin-phycoerythrin (SAPE) followed by staining with anti-streptavidin antibody and a second SAPE staining. Subsequently, arrays were scanned with a Agilent Laserscanner (Affymetrix), and data were analyzed with the Microarray Suite (MAS) software version 5.0 (Affymetrix). No scaling or normalization was performed at this stage. All data are available at the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE2917.

Data Analysis and Selection of Genes
Normalization.
Genes that were called absent according to Affymetrix MAS 5.0 software (P value > 0.06) were removed from further analysis. Raw intensities were log2 transformed to get data normally distributed. Subsequently, all data were quantile normalized per type of ganglion (1). Following the groupwise quantile normalization, a second quantile normalization was carried out across both DRG and NG samples. This alignment sets the range of intensities of one array to the range measured across all arrays, compensating for array-to-array variations in hybridization, washing and staining, ultimately allowing a reasonable comparison between arrays.

Spectral map analysis.
This unsupervised multivariate projection method, applied to the normalized data set, helps to reduce the complexity of highly dimensional data (n genes vs. p samples) (46) and provides an unbiased means to visually identify the predominant clusters of genes or subjects in the data. The aim of the technique is to retrieve the most predominant differences in the data set, disregarding genes that do not contribute to the difference.

Significance analysis.
Individual genes with different expression levels between groups (NG vs. DRG) were identified using significance analysis of microarray data (SAM) (43), and the false discovery rate was determined using q-values (40). Whereas a P value is commonly used to evaluate a single significance test, the q-value allows for multiple tests performed simultaneously, as in microarray experiments. We applied a 1% threshold (q = 0.01) for our analysis.

Fold-difference filtering.
For comparison purposes, an arbitrary fold-difference (FD) filter was applied excluding all genes that exhibited a difference in expression below 50% (1.5 FD filter).

Effect of CTB488 Injection and Amplification on Gene Expression
Effect of CTB488.
The effect of CTB488 labeling on gene expression profiles was assessed by comparing ganglia isolated from three vehicle-treated animals with three combined intradermal- and ip-injected mice (labeling most neurons). Although a clear difference in expression profile was observed between NG and DRG (see below), no significant effect of the dye injection was noted (data not shown).

Effect of amplification.
Efficiency and sensitivity of amplification were assessed with spike-in controls. In agreement with previous reports, spike-in controls revealed a detection limit of 1 copy in 1,000,000 and a direct correlation between signal intensity and copy number (data not shown). Arrays that did not show this correlation were excluded from data analysis.

Quantitative RT-PCR
Microarray data were confirmed using real-time PCR analysis. First-strand cDNA synthesis was performed on 50 ng of second round-amplified RNA using random hexamer primers and SuperscriptII RT (Invitrogen). Quantitative PCR (qPCR) was performed on an ABI Prism 7900 cycler (Applied Biosystems, Foster City, CA) using a Taqman PCR kit (Applied Biosystems). Serial dilutions of cDNA were used to generate standard curves of threshold cycles vs. the logarithms of concentration for ATPSase and the genes of interest (see Table 1 for sequences of primers; Eurogentec, Seraing, Belgium).


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  		Table 1. Sequence of oligonucleotides used for qPCR

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Labeling of Visceral Sensory Neurons
Intramuscular (im) injection of abdominal tissues necessitates invasive surgery that is likely to alter the expression of a variety of genes. Initial experiments were thus performed to evaluate ip injection of label as an alternative. Injection of CTB488 im labeled DRG neurons from T2 to L1, with 61% of neurons labeled between T10 and T13 (Fig. 1). In comparison, ip injection of CTB594 labeled DRG neurons over a slightly larger range, from T1 to L4, but with 50% of neurons labeled still located between T10 and T13 (Fig. 1). Every neuron labeled after im injection of CTB488 was co-labeled by ip injection of CTB594 (Fig. 1). The total number of T10–T13 DRG neurons labeled after im injection was 37 ± 12 ganglion cells, which was only 6.4% of the neurons labeled after ip injection (580 ± 132 ganglion cells). There was also a similar percentage (8.2%) of NG neurons labeled with im injection compared with ip injection (32 ± 18 vs. 398 ± 62 neurons, respectively). All four levels of DRG gave similar results: 2.5–3% of neurons were labeled after ip injection. Because ip injection should avoid most of the alterations in neuronal expression that occur after surgery and im label injections, ip CTB was used for subsequent microarray studies.


Figure 1
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  		Fig. 1. Cholera toxin B (CTB) labeling of sensory neurons. A: the mean no. of neurons (n = 4–6 experiments) labeled by intramuscular (im) injection in dorsal root (DRG) and nodose ganglia (NG) neurons. B: the same data as in A, except after an intraperitoneal (ip) injection. C and D: all neurons that are labeled by ip injection are co-labeled by im injection (arrows). An example of a squash preparation of the same DRG illuminated through a fluorescein isothiocyanate (FITC) filter (C: im injection with CTB Green 488) and a cyanine-3 (Cy3) filter (D: ip injection with CTB Red 594).

 
Gene Expression Profiling of NG and DRG
Because only 3% of the neurons in DRG and NG are labeled from the abdominal cavity, we applied LCM to enrich for gut-specific neurons. Thus visceral afferent-specific gene expression profiles in DRG and NG were identified and compared with those obtained from whole ganglia.

Three separate microarray experiments were performed on RNA isolated from 1) whole ganglia on MG-U74Av2 GeneChips, 2) laser-captured neurons on MG-U74Av2 GeneChips, and 3) laser-captured neurons on MG-430v2.0 GeneChips.

1) Whole ganglia using MG-U74Av2 arrays.
RNA extracted from the entire DRG and NG was hybridized to MG-U74Av2 GeneChips. Graphical exploration of microarray data using spectral map analysis (SPM) revealed overt differences in gene expression between whole DRG and NG (Fig. 2). The first component of the principal component analysis explained 60% of the total variance present in the data set of 7,744 genes that were reliably detected, and this separated DRG from NG samples. To identify differentially expressed individual genes, SAM (q-value < 0.01) and FD filtering (>1.5 FD) were applied. Positioning of the genes at the extremities of the SPM biplot was also taken into account (data not shown). On the basis of these three criteria, 628 genes were identified to be differentially expressed in the entire DRG vs. NG, including 17 G protein-coupled receptors (GPCRs) and 18 ion channels (Table 2).


Figure 2
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  		Fig. 2. Spectral map biplots. In each panel, the first 2 principal components (PC) of the weighted spectral map analysis (SPM) applied on normalized microarray data are plotted. Squares (DRG) and triangles (NG) depict different samples, whereas circles depict genes (size of the circle corresponds to the signal intensity of the gene on the array). Distances between squares and/or triangles are a measure for similarity between samples. A positive association of a gene with a given sample (i.e., an upregulation of that gene in that particular sample) results in the positioning of the gene and sample on a common line through the centroid (depicted by a cross). Genes that do not contribute to the differences between samples are indicated by gray dots around the centroid. The 10 most significantly contributing genes (measured by their distance from the centroid) to differences among samples are annotated with their gene symbol. A: SPM biplot of whole DRG vs. NG on MG-U74Av2 GeneChips. The first PC discriminates NG (n = 7) from DRG (n = 5) samples and explains 60% of the total variance in the data set. B: SPM biplot of laser-captured visceral DRG vs. NG neurons on MG-U74Av2 GeneChips. The first PC discriminates NG (n = 5) from DRG (n = 5) samples and explains 60% of the total variance in the data set. Examples of genes that contribute to the difference between DRG and NG neurons include neurotensin receptor (Ntsr) and kainate glutamate receptor 5 (Grik1). C: SPM biplot of laser-captured DRG vs. NG neurons on MG-430V2.0 GeneChips. The first PC discriminates NG (n = 11) from DRG (n = 9) samples and explains 43% of the total variance in the data set.

 

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  		Table 2. Summary of data analysis of the 3 microarray experiments

 
2) Laser-captured neurons using MG-U74Av2 arrays.
Laser-assisted microdissection was performed on 100–150 DRG or NG neurons labeled by ip CTB488. Similar to the whole ganglia experiments, SPM on amplified and labeled RNA from isolated cells clearly separated neurons derived from NG and DRG based on the overall expression pattern of 7,571 genes reliably detected on MG-U74Av2 arrays (Fig. 2). In applying the same three criteria as above for identifying significant differentially expressed genes, 616 were found. These included 13 GPCRs and 19 ion channels, of which 11 and 9, respectively, were also identified as differentially expressed in whole ganglia (Table 2).

Of the 4,956 genes that showed no differences in expression between whole NG and DRG (i.e., FD < 1.5 or q-value > 0.01), 467 were significantly different when measured in laser-captured cells. Striking examples of these differences include galanin, TrkC, and the {alpha}2c-adrenergic receptor (Fig. 3). Conversely, for some genes, such as the tetrodotoxin (TTX)-resistant, voltage-gated, Nav1.8 sodium channel, differences determined in whole ganglia were not found when comparing laser-captured NG and DRG neurons. In general, of 5,031 transcripts that showed no differences in expression in laser-captured cells, 579 were significantly different when measured in whole NG and DRG.


Figure 3
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  		Fig. 3. Gene expression in whole ganglia vs. laser-captured neurons. Gene expression profiles of galanin, TrkC, {alpha}2c-adrenergic receptor, and Nav1.8 in whole DRG and NG and in laser-captured visceral neurons derived of these ganglia. Expression levels are expressed as fluorescent signal intensity measured on the array after normalization. Expression levels are shown for individual animals (red circles = DRG, blue circles = NG). Black circles represent unreliable detections. Red and blue horizontal lines represent mean expression levels in DRG and NG, respectively.

 
3) Laser-captured neurons using MG-430V2.0 arrays.
Laser-captured material was hybridized to MG-430V2.0 whole genome arrays interrogating expression levels of 39,000 gene transcripts. In agreement with previous results, SPM on the 28,920 reliably detected genes showed a clear difference in expression between DRG and NG (Fig. 2). In applying the same criteria as above, 1,996 genes showed significant differences in expression between NG and DRG (Table 2). This genome-wide analysis allowed us to measure the relative abundance of mRNAs encoding members of several groups of nerve-related proteins such as adrenergic and cholinergic receptors; GABA, glutamate, serotonin, and dopamine receptors; voltage-gated potassium, sodium, and calcium channels; and transient receptor potential cation channels (Supplemental Fig. S1; available at the Physiological Genomics web site).1

As a proof of concept, the expression levels of several genes known to be differentially expressed in spinal vs. vagal visceral afferents were examined in the array data of the third experiment and confirmed by qPCR. Of the several 5-HT receptors present on the arrays, only mRNA for the 5-HT3A receptor was readily detected in both NG and DRG neurons (Fig. 4), with a ninefold higher abundance in NG. qPCR revealed a greater difference in expression (21-fold difference). Messenger RNA for the 5-HT3B receptor was clearly expressed in NG but not in DRG neurons, in contrast with mRNA levels for the 5-HT1D receptor, which were markedly higher in DRG neurons than in NG cells. Cholecystokinin (CCK)A receptor mRNA was also detected more abundantly in vagal compared with spinal afferents (Fig. 4). In total, 18 genes were evaluated for FD in expression level using qPCR and compared with array experiments (Table 3). In general, FD was higher with qPCR compared with array data. Very high differences (>8-fold) seem to level off in array experiments. However, a good correlation was present between FD measured by microarray and qPCR (Fig. 5).


Figure 4
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  		Fig. 4. Expression profiles of serotonin and cholecystokinin (CCK)A receptors. Gene expression profiles of 5-HT3a, 5-HT3b, 5-HT1d, and CCKA receptors in laser-captured neurons derived from DRG and NG. Expression levels are expressed as fluorescent signal intensity measured on the array after normalization. Expression levels are shown for individual animals (red circles = DRG, blue circles = NG). Black circles represent unreliable detections. Red and blue horizontal lines represent mean expression levels in neurons derived from DRG and NG, respectively.

 

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  		Table 3. Log2 of fold difference in gene expression in DRG vs. NG neurons

 

Figure 5
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  		Fig. 5. Comparison of fold difference (FD) in quantitative PCR (qPCR) vs. microarray. Log2 FD of gene expression levels of 18 genes (Table 3) in DRG vs. NG visceral neurons observed with qPCR (x-axis) vs. microarray (y-axis). Array data for both MG-U74Av2 (triangles) and MG-430V2.0 (circles) GeneChips are represented. FDs are calculated as the ratio of the median relative expression (qPCR) or median signal intensity (array). A significant correlation was found with both arrays (P < 0.0001).

 
Sensory Transduction and Neurotransmission
We focused our further analysis on gene families that are involved in sensory transduction, excitability, and neurotransmission. With respect to signal transduction, expression profiles of members of the transient receptor potential (TRP) cation family of channels were analyzed more closely. From the 6 subfamilies, mRNA levels of 26 members are interrogated by different probe sets on the MG-430V2.0 microarrays. In visceral sensory neurons, only 14 of them are reliably detected (Fig. 6). Trpv1 was in both DRG and NG, with 3.8-fold higher levels in the former (q < 0.001, 9-fold by qPCR). Trpv2, the only other TrpV channel family member detected in visceral afferents, was more abundant in NG vs. DRG. Trpa1 showed high levels of expression in both NG and DRG, with sevenfold greater abundance in the latter (q < 0.001). Voltage-gated ion channels are important in determining membrane excitability. Over 100 different subunits of voltage-gated sodium, calcium, or potassium channels were detected in visceral neurons. When taking only sodium channels into consideration, nine {alpha}-subunits were reliably detected (Fig. 6). Messenger RNA for Nav1.8 (Scn10a) was equally present in vagal and spinal afferents, whereas Nav1.1 (Scn1a), Nav1.6 (Scn8a), Nav1.7 (Scn9a), and Nav1.9 (Scn11a) were enriched in DRG. The expression level of the sodium level-sensing channel Nax (encoded by Scn7a) was 6.5-fold higher in visceral DRG vs. NG neurons. In contrast, high levels of the cardiac channel Nav1.5 mRNA (Scn5a) were detected in vagal but not spinal afferents.


Figure 6
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  		Fig. 6. Overview of the relative expression levels of specific gene families in DRG vs. NG visceral neurons. For each gene, the corresponding Affymetrix identifier, gene symbol, possible aliases, and gene description are shown. Expression levels range from not detectable (–) to poorly expressed (+/–) to highly expressed (+ + +). This scaling is based on arbitrary intervals of the signal intensity as measured by array in log2 scale. FDs are calculated as the ratio of the median signal intensity. FDs >1.5 are color coded (red = enriched in DRG, blue = enriched in NG). A: transient receptor potential (TRP) cation channel genes. B: voltage-gated sodium channels. C: tachykinin receptor NK3 (Tacr3) and substance P (Tac1). D: glutamate receptors.

 
Analyses of expression profiles of genes encoding molecules involved in neurotransmission focused on tachykinergic and glutaminergic families. The Tac1 mRNA encodes both substance P and neurokinin A and was readily detected in both spinal and visceral afferents, being four times more abundant in DRG neurons (Fig. 6). Of all tachykinin receptors, only NK3 receptor mRNA was detected in NG neurons projecting to the viscera but not in DRG neurons (q = 0.01) (Fig. 6), while NK1 and NK2 mRNA levels were below detection limit. With respect to glutamate receptors, the most striking difference was observed for the presynaptic kainate receptor GluR5 (encoded by Grik1) (Fig. 6), the expression of which was 16-fold higher in DRG compared with NG neurons. The only other kainate receptor detected, KA1 (Grik4), was present in equal amounts in DRG and NG neurons. With regard to AMPA GluR1–4 (also called GluR-A-D) receptors, GluR1 (Gria1) was present in both DRG and NG, with levels six times higher in DRG, whereas GluR2 (Gria1), GluR3 (Gria1), and GluR4 (Gria4) were enriched in NG neurons (GluR3 is absent in DRG). Interestingly, N-methyl-D-aspartate (NMDA) receptor mRNA levels were around detection limit and did not show any differential expression between DRG and NG, with the exception of the NMDA-like receptor 1A (Grinl1a). Metabotropic glutamate receptors were mainly detected in DRG neurons, namely group III receptors mGluR4 (Grm4) and mGluR8 (Grm8) and the group I receptor mGluR5 (Grm5) (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present investigation is one of the first genome-wide studies of gene expression in sensory neurons supplying the abdominal viscera. Our aim was threefold: first, to validate expression profiling as a means of characterizing sensory neuronal populations and compare expression profiles of whole ganglia vs. the population of visceral sensory neurons; second, to assess the expression of key members of gene families responsible for sensory transduction, excitability, and transmission; and third, to compare and contrast expression between spinal and vagal sensory neurons. The results of this study set a baseline for expression profiling of sensory neurons in visceral disease models, particularly hypersensitivity.

Spinal sensory neurons supplying somatic and visceral receptive fields have a number of features in common but differ in terms of morphology, conduction parameters, and expression of ion channels and receptors (29, 36). Vagal neurons are also heterogeneous in their terminal innervation to cardiac, pulmonary, and GI structures and express a wide range of ion channels and receptors, some of which are specific for different populations (47). Our fiber-tracing experiments provide quantitative information on the extent to which abdominal viscera-projecting neurons contribute to the total pool of sensory neurons in DRG and NG. In T10–T13 DRG, ~3% of neurons were labeled after ip CTB injection. Previous studies in the rat estimate the proportion of visceral afferents to be <7% of the total (10). As demonstrated before, ip administration of tracer allows enrichment of sensory afferents projecting within the ip cavity (2, 32). Clearly, the extent to which endings in the peritoneal lining are labeled cannot be determined (42). All im CTB-labeled neurons were also labeled by ip injection, and extrapolation of the number of neurons that supply a 5-cm segment of jejunum to the whole bowel gives a close approximation of the total population labeled by ip injection. The same holds true for NG. The importance of this information is twofold. First, it gives confidence that a specific subset of sensory neurons that supply the abdominal viscera can be labeled, harvested, and enriched for without any prior surgical intervention. Such surgical procedures are used to elicit paralytic ileus, which recent studies have shown to be dependent on an inflammatory response within the bowel wall (15, 16). Because inflammation leads to long-term changes in sensory neuron excitability, we felt it was necessary to develop a method of labeling that was relatively noninvasive to establish a baseline of gene expression in nodose and DRG neurons. Second, it gives an estimate of the extent to which expression from these signals is likely to be diluted when whole ganglia expression is assessed. In this respect, the comparison of data obtained from whole DRG and NG with data obtained from laser-captured neurons using the same microarrays is important. Even in whole DRG vs. NG, a large number of genes are differentially expressed (8% of the reliably detected genes). This may be not surprising, given that DRG and NG neurons have different embryonic origins (neural crest and epibranchial placodes, respectively; Refs. 13, 30), innervate different target organs (largely somatic structures vs. visceral organs), and generate different sensory signals (conscious perception vs. reflex and behavior control; Ref. 9). Notable genes that appear in the analysis of whole ganglia reflect these differences. These include the 5-HT3A receptor, the Nav1.8 sodium channel, and the CCKA receptor. Some of these differences in gene expression observed in whole ganglia are preserved when laser-captured neurons are compared (e.g., 5-HT3A and CCKA). Other genes drop out (e.g., Nav1.8), illustrating the extent to which somatic neurons dominate expression in whole ganglia. A third category of genes is not differentially expressed when whole ganglia are compared, but is clearly different in laser-captured material. This reflects the extent to which dilution of the signal can obscure important differences in the specific population of neurons that innervate the abdominal viscera.

Important validation of our study comes from expression profiles of genes such as those encoding 5-HT3A, Nav1.8 and CCKA, for which the presence in visceral afferents has previously been documented (5, 7, 37). In addition, we have shown a good correlation between expression measured by qPCR and microarray technology for 18 genes.

In the further discussion below, we have highlighted genes that play a role in sensory signaling. One example is the capsaicin receptor Trpv1, considered important in nociception (8). Its expression in both DRG and NG confirms earlier immunohistochemical data (25, 35, 44) and may indicate a role for both vagal and spinal afferents in nociception. Trpa1 was also readily detectable in both DRG and NG neurons. This channel is gated by mechanical stimuli as well as by cold temperatures, mustard oil, and cannabinoids (12, 27). As such, Trpa1 could confer mechanosensitivity on the peripheral endings of both spinal and vagal afferents.

Other gene families of particular importance for sensory neurons are those that determine membrane excitability. Sodium and potassium channels are particularly important, especially given recent experimental data showing hypersensitivity resulting from upregulation of sodium and downregulation of potassium channels after inflammation or injury (5, 26, 39). For the purpose of this discussion, we have focused only on voltage-gated sodium channels. The {alpha}-subunits determine much of the biophysical and pharmacological properties, and nine {alpha}-subunits have been identified for expressed mammalian sodium ion channels (34). In unselected DRG, Nav1.7 and, to a lesser extent, Nav1.1, Nav1.2, and Nav1.6 carry much of the TTX-sensitive current (6). In GI-projecting DRG and NG neurons, the TTX-sensitive current has biophysical properties resembling Nav1.7 (encoded by Scn9a). In this respect, Scn9a is expressed in both DRG and NG. A recent study using the Cre-loxP system to generate nociceptor-specific knockouts for Nav1.7 found increased thresholds to mechanical and thermal pain in the Nav1.7-deficient mice (33). TTX-resistant channels Nav1.8 and Nav1.9 have received a great deal of attention recently because of their preferential localization to small, unmyelinated, nociceptor-like neurons (18). Scn10a (Nav1.8) mRNA expression and immunocytochemical staining for Nav1.8 have been demonstrated in DRG neurons innervating the colon, ileum and stomach, and Nav1.8-like currents are present in most DRGs innervating these organs (41). Only one study has systematically looked for persistent Nav1.9-like currents in GI afferents, showing that only a small proportion (~13%) of colonic DRGs had a persistent, low threshold current (5). Thus it seems likely that Nav1.8 carries the major TTX-resistant current in GI afferents; however, evidence demonstrating either mRNA or immunohistochemically detectable Nav1.8 or Nav1.9 in gut-specific neurons is lacking. In our study we found that Nav1.9 (Scn11a) mRNA is abundant in DRG and, to a lesser extent, also expressed in NG, while Nav1.8 mRNA expression level is comparable in both neuronal populations. The functional significance of Nav1.5 expression in NG neurons needs to be explored, as it has been described as the cardiac sodium channel (17). Of note, mRNA for one of the auxiliary ß-subunits (Scn3b) that alters the kinetics of Nav1.5 is also enriched in NG neurons (19). Detection of Nav1.5 mRNA expression in NG but not in DRG visceral neurons may indicate that this channel contributes to the sodium ion current in visceral vagal afferents (38).

Glutamate is a major transmitter at first-order synapses in the brainstem and spinal cord. There are at least six NMDA receptors, nine non-NMDA receptors, and eight metabotropic glutamate receptors (mGluRs) (11, 45). Both pre- and postsynaptic mGluRs have been implicated in shaping autonomic signal transmission in the nucleus tractus solitarius (21, 24). Similarly, NMDA and non-NMDA receptors influence the release of both glutamate and substance P from spinal nociceptive neurons (3, 31). Therefore, an examination of the expression profile of glutamate receptors in DRG and NG neurons is important for understanding sensory transmission. The abundant expression of the kainate receptor GluR5 (Grik1) in DRG compared with NG neurons strongly supports the view that this is the predominant presynaptic regulator of visceral afferent input to the spinal cord (28). Gria1, encoding the AMPA receptor GluR1, is also more abundant in DRG compared with NG. Differential incorporation of specific AMPA subunits has been shown to modulate pain-related behavior (23). Our expression data suggest that, in visceral afferents, heteromers of GluR1/GluR2 or GluR1/GluR4 might predominate at the spinal level, whereas at the vagal level, GluR3-containing heteromers may be more common. Specific mGluR subtypes present at the synapses also modulate glutaminergic transmission. In general, group I mGluRs (subtypes-1 and -5) increase neuronal excitability, while group II (subtypes-2 and -3) and group III (subtypes-4, -6, -7, and -8) receptors inhibit glutamate release to reduce synaptic transmission (11). Our current findings suggest that the presynaptic mechanisms controlling transmitter release may be different for DRG and NG neurons, with mGluR5 and mGluR8 being dominant in the spinal transmission and none of the mGluRs being enriched in vagal afferents.

In conclusion, the present study provides a genome-wide insight into the molecular signatures that underlie both functional differences and similarities between NG and DRG sensory neurons projecting to the peritoneal cavity. These divergences may underlie the contrasting roles of NG and DRG and contribute to differences in the control of excitability between these visceral sensory neurons. Moreover, these findings will offer novel insight into the mode of action of pharmacological agents modulating visceral sensation and pave the way for additional, more directed studies investigating the role of novel proteins abundantly expressed in visceral afferents.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: P. J. Peeters, Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica NV, Turnhoutseweg 30, B-2340 Beerse, Belgium (e-mail: ppeeter3{at}prdbe.jnj.com).

1 The Supplemental Material for this article (Supplemental Fig. S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00169.2005/DC1. Back


   REFERENCES
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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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