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Keywords:

  • descending colon;
  • dorsal root ganglion;
  • immunohistochemistry;
  • primary spinal afferent;
  • retrograde labelling;
  • visceral pain

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Abstract  Visceral pain is the most common form of pain produced by disease and is thus of interest in the study of gastrointestinal (GI) complaints such as irritable bowel syndrome, in which sensory signals perceived as GI pain travel in extrinsic afferent neurones with cell bodies in the dorsal root ganglia (DRG). The DRG from which the primary spinal afferent innervation of the mouse descending colon arises are not well defined. This study has combined retrograde labelling and immunohistochemistry to identify and characterize these neurones. Small to medium-sized retrogradely labelled cell bodies were found in the DRG at levels T8-L1 and L6-S1. Calcitonin gene-related peptide (CGRP)- and P2X3-like immunoreactivity (LI) was seen in 81 and 32%, respectively, of retrogradely labelled cells, and 20% bound the Griffonia simplicifolia-derived isolectin IB4. CGRP-LI and IB4 were co-localized in 22% of retrogradely labelled cells, whilst P2X3-LI and IB4 were co-localized in 7% (vs 34% seen in the whole DRG population). Eighty-two per cent of retrogradely labelled cells exhibited vanilloid receptor 1-like immunoreactivity (VR1-LI). These data suggest that mouse colonic spinal primary afferent neurones are mostly peptidergic CGRP-containing, VR1-LI, C fibre afferents. In contrast to the general DRG population, a subset of neurones exist that are P2X3 receptor-LI but do not bind IB4.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Pain perception from the internal organs is a fundamental human physiological requirement, and visceral pain is the most common form of pain produced by disease. In irritable bowel syndrome (IBS), for example, it has been hypothesized that sensitization of mechanoreceptors, and perhaps chemoreceptors, in the gastrointestinal (GI) tract account for the symptoms of abdominal pain and discomfort.1 Understanding the anatomical and physiological basis of differences such as these, and indeed of the transmission of visceral pain in normal and diseased states, is an important and developing area of research.

The afferent nerves that innervate the GI viscera run alongside the efferent sympathetic and parasympathetic nerves, and in order to distinguish them from those efferent nerves, they are referred to by the name of the respective nerve (e.g. vagal or pelvic afferent). Visceral sensory fibres terminate either in the spinal cord (spinal afferents) with their cell soma in the dorsal root ganglia (DRG), or in the brainstem (vagal afferents) with their cell soma in the nodose ganglia. Although not dealt with here (as their cell bodies are not in the DRG), there are two additional afferent systems supplying the distal colon: intestinofugal fibres,2 with cell bodies in the enteric ganglia, and rectospinal afferents,3,4 which may contribute to high threshold mechanosensory visceral afferent input (see ref.5 for review).

Those sensory neurones that innervate the colon, and other viscera, are almost exclusively small diameter myelinated Aδ fibres or unmyelinated C fibres.5 Activity in most of these afferents does not result in perception of events in the GI tract, but provides regular central nervous system input to aid the regulation and control of the local environment. However, sensations of stool, urge to defecate, fullness, etc. are important regulators of conscious GI behaviours, and pain and nausea are protective signals of GI distress. It is perhaps when such sensations are perceived inappropriately that disease symptoms occur.

Whilst the mouse presents practical challenges to the experimenter due to its size (e.g. during surgery and dissection of ganglia), and despite the modest amount of available comparative data and neuroanatomical studies (only one previous study has used retrograde labelling in mouse lower bowel, albeit from the rectum,6) there are certain advantages in using this species. The most significant of these is the large, and growing, library of knockout animals. DRG taken from knockouts, when used in conjunction with retrograde labelling techniques described here, could provide an invaluable source of information regarding the cellular characteristics of specific visceral primary afferent populations. Furthermore, if it can be shown that data obtained in the mouse are comparable with that previously obtained in rats, both species may be used in parallel, thus combining the advantages of the mouse and the wider availability of good rat antibodies.

The aim of the current study was to identify those DRG neurones that innervate the mouse descending colon, to detail the spinal levels at which these are found, and to examine the properties of these neurones using immunohistochemistry. The colon was chosen to allow comparison with data available from studies in the rat and to allow correlation with functional studies using colorectal distension as a noxious stimulus. It is hoped that the properties of these colonic afferent neurones will later be examined using in vitro electrophysiological techniques, and the results consequently applied to studies of the mechanisms of visceral hypersensitivity.

Retrograde labelling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Five adult (6 weeks old) male BALB/c mice were used for surgical injection of the fluorescent retrograde neuronal tracer, Fast Blue (FB). All surgical procedures were performed under sterile conditions within a designated animal procedure room. Animals were anaesthetized with isofluorane and, following laparotomy, four to eight injections (approximately 5 μL, Hamilton syringe with a 25-gauge needle) of FB (2–5% in saline) were made at several sites around the wall of the descending colon. The viscera were carefully rinsed with sterile saline, to ensure that dye did not spread to areas other than the colon wall, before the muscle and skin were sutured. The animals were then allowed to recover, under constant observation, in a warm environment. Throughout all procedures, locally and nationally approved animal regulations were adhered to strictly.

Previous studies in the guinea pig have shown that injection of FB into the oesophageal or carotid artery, in contrast to labelling from the trachea, did not result in retrograde labelling of nodose ganglia neurones.7 This provides evidence for the specificity of FB as a retrograde label.

Dorsal root ganglia dissection and tissue preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

At 9–20 days postoperatively, DRG from bilateral spinal levels T4-S3, inclusive, were removed post-mortem from their respective foramina (S1-S3 were not analysed in one animal because the tissue was lost during dissection). The level of each was noted and recorded, using the costae fluctuantes as an anatomical guide. Dissections were carried out in a dissection chamber gravity-perfused with ice-cold gassed (95% O2 : 5% CO2) Dulbecco's phosphate-buffered saline (D-PBS) at a rate of approximately 4 mL min−1.

Any excess connective tissue and spinal roots were carefully dissected away before the ganglia were placed in 4% paraformaldehyde and stored at 4 °C for 2–4 h to fix the tissue. DRG were then rinsed in D-PBS before cryoprotection in sucrose (30%, 4 °C) for 2 days. Excess liquid was removed from each DRG, which were then snap frozen by brief immersion into 2-methylbutane (−30 °C) and subsequently stored individually at −70 °C.

Dorsal root ganglia were sectioned at −30 °C in a cryostat and four to eight DRG sections (10–14 μm) were cut and placed on individual slides. The sections from each ganglion were thaw-mounted non-serially onto six slides, thus ensuring each slide contained sections taken at least 50 μm lower in the ganglion than the previous section. This reduced the probability that any cell would appear on the same slide more than once. Slides were stored at −20 °C in the dark until required.

Cell counts and cell size analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

The number of FB-labelled cells in sections taken from each DRG level was analysed either by eye (at the microscope) or by viewing previously saved digital photomicrographs. In order to avoid errors caused by the variations in both ganglion size and the proportions of cell bodies to nerve fibres between different sections, the data were standardized within each section by calculating the numeric mean of the number of FB-labelled cells per unit area (mm2) of DRG neuronal cell bodies in a number of sections (four to 19) at each DRG level. The standardized counts were normalized to the DRG level containing the greatest number of FB-labelled cells within each animal and these values were averaged (numeric mean) to produce a final representation of the population.

Image analysis software (UTHSCSA, ImageTool v3.0, developed at the University of Texas Health Science Center, San Antonio, TX, USA) was used to measure the cross-sectional area of individual FB-labelled cell bodies and the total area of tissue containing neuronal cell bodies within each section.

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

After optimization experiments, selected slides were processed for immunohistochemistry. Unless stated, all stages were performed at room temperature, and all washes were in TNT wash buffer (120 mmol L−1 NaCl, 50 mmol L−1 pH 7.5 Trizma, 0.05% Tween-20; 1 mmol L−1 CaCl2 was also added for IB4 experiments) under gentle agitation (3 × 10 min). Once defrosted, slides were washed before an incubation of 30 min in 10% normal goat serum (NGS; Vector Laboratories Inc., Burlingame, CA, USA) to reduce non-specific binding, and then overnight at 4 °C in the primary antibody, or antibodies for double-labelling experiments (Sigma, St. Louis, MO, USA; CGRP: 1 : 10 000; In-House raised8 P2X3: 1 : 1000; Calbiochem, San Diego, CA, USA; Capsaicin Receptor: 1 : 50). Slides were then washed and incubated with the secondary antibody (1 : 200; Alexa Fluor 488 or Alexa Fluor 568; Molecular Probes, Inc., Eugene, OR, USA) or Isolectin IB4 from Griffonia simplicifolia Alexa Fluor 568 conjugate (2.5 μL mL−1; Molecular Probes Inc.) for 2 h. All antibodies were made up in 2% NGS, with 1 mmol L−1 CaCl2 added to the IB4 conjugate. One final wash was carried out before any excess fluid was shaken from the slides, which were then mounted and coverslipped. No specific labelling was observed in the absence of primary antibody. Slides were viewed on a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) with epifluorescence unit and A4, I3 and N2.1 filters for visualization of FB, Alexa Fluor 488 and Alexa Fluor 568 labelled cells, respectively. Digital images were captured using a Leica DC200 digital camera. Previous studies in mouse and rat tissues have shown the P2X3 primary antibody used in the present study to be a specific tool for the study of this receptor.8,9 BLAST searches of the mouse genome showed a high degree of epitope specificity for the calcitonin gene-related peptide (CGRP) primary antibody. Experiments in which the VR1 primary antibody was preincubated with its immunizing peptide showed no labelling of the DRG neurones, indicating the specificity of the antibody for this sequence.

Preliminary experiments, the data from which have been included in the present study, were carried out using CGRP primary antibody (1 : 10 000; Sigma), FluoroLinkTM CyTM 3-labelled streptavidin (1 : 300; Amersham Pharmacia Biotech, Little Chalfont, UK) and secondary antibody (biotinylated anti-rabbit IgG (H+L), affinity purified; Vector Laboratories Inc.) or a biotin-labelled IB4 antibody (Sigma; 10 μg mL−1) with Cy3. NGS was dissolved in Triton-X100 solution (0.3%; BDH, Poole, UK) to 2% final content, in which the primary antibodies, secondary antibody or Cy3 were dissolved to their required concentrations. Washes for these experiments were carried out in D-PBS and slides were mounted in VECTASHIELD® fluorescent mounting medium (Vector Laboratories Inc.). These sections were viewed with a Nikon Optiphot-2 epifluorescence microscope (Nikon Corporation, Tokyo, Japan) fitted with ultraviolet (UV)-2A and G-2A for observation of FB- and Cy3-labelled cells, respectively, and digital images were captured using a Nikon CoolPix 950 camera.

Fast Blue-labelled dorsal root ganglia: numbers and spinal levels

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

After injection of FB into the wall of the descending colon of five mice, the dissected DRG were sectioned entirely, revealing retrogradely labelled neurones with varying intensity blue fluorescence in the cytoplasm (Fig. 1). The number of FB-labelled cells varied between different levels, with some DRG containing many (e.g. T11; Fig. 1B) and some at other levels containing none (e.g. T5; Fig. 1B). The absence of labelling at certain levels was consistent between animals (data not shown), providing evidence that FB leakage did not occur and that the retrograde labelling in the DRG was specific to the colonic afferents under study (see Materials and methods). The highest number of FB-labelled cells seen in any one section was 37.

image

Figure 1. Retrogradely labelled neurones characterized by varying intensity blue fluorescence in the dorsal root ganglia (DRG) cell bodies. Fast Blue (FB) fluoresces under ultraviolet (UV) light enabling the visualization of retrogradely labelled DRG neurones. Panel A illustrates one DRG section viewed under UV light (i), and the same section viewed after haematoxylin and eosin (H&E) staining (ii), clearly detailing the section's histology. Panel B illustrates typical DRG sections taken at different spinal levels (white captions) of one mouse, after retrograde labelling from the colon with FB. There was a variation in the number of FB-labelled cell bodies between levels, and examples of DRG sections with many FB-labelled neurones (i) and no FB-labelled neurones (ii) are illustrated. Sections are 14 μm thick and scale bars are 100 μm.

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The numeric mean of the normalized number of FB-labelled cells per mm2 of neuronal DRG cell bodies (see Materials and methods) for each of the five mice was plotted as a histogram (Fig. 2), which revealed a distribution based around two peaks: one wide, covering spinal levels T8-L1, and one narrow, restricted to L6 and S1.

image

Figure 2. Retrograde FB labelling of mouse descending colon observed at each spinal dorsal root ganglia (DRG) level. The mean normalized number of Fast Blue (FB)-labelled cells per mm2 of neuronal DRG cell bodies is plotted against the spinal DRG level in which the count was observed. A distribution based around two peaks can be seen: one broad, covering T8-L1, and the other restricted to L6 and S1. The number of animals averaged in each calculation is indicated above its respective column.

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Cell size analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

The mean cross-sectional area of FB-labelled cells was 407 ± 6 μm2 (n = 1032). However, the frequency histogram (Fig. 3) provides a true representation of the range and distribution of FB-labelled cell size. This shows that the majority (92%) of FB-labelled cells had a cross-sectional area between 100 and 700 μm2 (grey bars in Fig. 3).

image

Figure 3. Cross-sectional area of Fast Blue (FB)-labelled cells in mouse dorsal root ganglia. This histogram shows the distribution of 1032 FB-labelled cells, selected from all spinal levels of five mice, and plotted as number of cells (frequency) observed in each of 50 μm2 bins of cross-sectional area. The grey bars indicate 92% of the population that are between 100 and 700 μm2.

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Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Two major subclasses of unmyelinated C fibre nociceptor have been suggested to exist on the basis of histological markers, namely one group that expresses pro-inflammatory peptides such as CGRP and another that expresses binding sites for IB4. It has further been suggested that the latter group co-express P2X3 receptors10,11 and so all three of these markers have been investigated in the present study in order to characterize FB-labelled primary afferent neurones in the DRG. The data generated in these studies are summarized in Tables 1 and 2.

Table 1.  CGRP, P2X3 and VR1 immunoreactivity and IB4 binding in mouse colonic spinal primary afferent neurones
MarkerRetrogradely labelled cellsWhole DRG population
  1. Percentage of cells, either from a population of retrogradely labelled cells, or from the whole DRG population (note that these are not necessarily from the same sample) that exhibited immunoreactivity or binding, where appropriate, for each of the four investigated markers. All percentages are rounded to the nearest whole number. CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia.

CGRP8127
P2X33242
IB4 binding2044
VR18242
Table 2.  Co-localization of P2X3- or CGRP-immunoreactivity with IB4 binding in retrogradely labelled mouse colonic primary spinal afferent neurones and the whole DRG
 Retrogradely labelled cellsWhole DRG population
  1. Percentages of cells that bound IB4 and expressed either CGRP-LI or P2X3-LI. All percentages are rounded to the nearest whole number. CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia.

CGRP & IB42210
P2X3 & IB4734

Calcitonin gene-related peptide  An antibody raised against CGRP was used to investigate whether this peptide was present in DRG neurones exhibiting FB labelling, and each FB-labelled cell with a visible nucleus was classified as either having CGRP-like immunoreactivity (CGRP-LI) or not. CGRP-LI was observed in 81% (477/589) of FB-labelled cells, the majority being small- to medium-sized. Fig. 4B illustrates CGRP-LI seen in a DRG section (red arrows) and panel 4A shows FB labelling in the same section. In contrast, in the whole DRG population only 27% (326/1206) of cells were CGRP-LI.

image

Figure 4. C fibre nociceptor marker immunoreactivity in retrogradely labelled mouse colonic primary spinal afferent neurones. The three sets of panels show representative dorsal root ganglia (DRG) sections that have been retrogradely labelled with Fast Blue (FB) (A, C, E) and also CGRP-LI (B), IB4 binding (D) and P2X3-LI (F). Red or green arrows indicate FB-labelled cells that were also labelled with each antibody (or bound IB4), whilst examples of FB-labelled cells that were not labelled by each antibody (or did not bind IB4) are indicated by white arrows. Note the characteristic ring pattern of IB4 binding. DRG sections illustrated are from DRG levels T13 (A, B), L1 (C, D) and T11 (E, F). Scale bars are 100 μm on all panels.

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Isolectin-B4 and P2X3 purinoceptor   In the population of FB-labelled cells examined, 20% (172/861) of FB-labelled cells exhibited IB4 binding (see green arrows in Fig. 4C,D) and 32% (114/357) of FB-labelled cells were P2X3-LI (see red arrow in Fig. 4E,F). In the whole DRG population, a larger proportion of cells was labelled with these markers; thus, 42% were P2X3-LI (428/1019) and 44% bound IB4 (979/2225). Both markers principally labelled small- and medium-diameter cells.

Double labelling   Double labelling experiments were carried out with combinations of either anti-CGRP or anti-P2X3 antibodies together with the Alexa Fluor 568-IB4 conjugate. The results from these experiments are summarized in Table 2 and described below.

CGRP−IB4 double labelling  Eighty-one per cent of FB-labelled cells examined were CGRP-LI (see above); however, only 22% of the FB-labelled cells exhibited both CGRP-LI and IB4 binding. An example of such a cell is indicated by the white arrow in Fig. 5A. In the whole DRG population 27% of cells were CGRP-LI and 10% were both CGRP-LI and bound IB4. It is interesting to note that the intensity of IB4 staining seen in those FB-labelled neurones that were also CGRP-LI was weaker than the majority of the other IB4 binding cells in the DRG.

image

Figure 5. Double labelling of mouse colonic dorsal root ganglia (DRG) neurones. Panels A and B show DRG sections that have been immunostained for Alexa Fluor-conjugated IB4 and either CGRP-LI (A; DRG level T10) or P2X3-LI (B; DRG level L1). Panel A shows that the majority of Fast Blue (FB)-labelled cells exhibited CGRP-LI but not IB4 binding (yellow arrows), although some FB-labelled cells also exhibited CGRP-LI and IB4 binding (white arrows; note the weaker IB4 staining in these cells). The majority of FB-labelled cells shown in panel B did not exhibit IB4 binding or P2X3-LI whilst those remaining showed only P2X3-LI (yellow arrows). Note, however, the higher proportion of non-FB-labelled cells that showed IB4 binding with P2X3-LI (examples are indicated by purple arrows). Scale bars are 100 μm.

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P2X3−IB4 double labelling   Thirty-two per cent of FB-labelled cells examined were P2X3-LI (see above) but only 7% of FB-labelled cells exhibited both P2X3-LI and IB4 binding. This was in contrast to an analysis of 1019 cells in the whole DRG population, in which 34% of neurones contained both markers, as illustrated in Fig. 5B (purple arrows indicate some examples of these co-localized cells).

Immunohistochemistry: capsaicin receptor, VR1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

An antibody raised against the capsaicin receptor (vanilloid receptor 1, VR1) was used on some sections taken from thoracic and lumbar DRG levels that contained FB-labelled cells. Eighty-two per cent (185/225) of these labelled cells were shown to have VR1-like immunoreactivity (VR1-LI; Table 1) compared with only 42% (365/861) of the whole DRG population. Fig. 6 shows a typical section that was incubated with this antibody (red arrows indicate examples of VR1-LI FB-labelled cells).

image

Figure 6. The majority of retrogradely labelled mouse colonic primary spinal afferent neurones are immunoreactive for the capsaicin receptor, VR1. The representative dorsal root ganglia (DRG) section shown here (taken from DRG level T12) has been retrogradely labelled with Fast Blue (FB) and immunostained for VR1-LI (white captions). The majority of FB-labelled neurones were also VR1-LI (red arrows), an example of an FB-labelled cell that was not VR1-LI is also shown (yellow arrow). Scale bars are 100 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Using FB retrograde labelling, the present study shows that neurones innervating the mouse descending colon have cell bodies in DRG at specific spinal levels. These labelled DRG were distributed bimodally, with one broad population encompassing levels T8-L1 and a second population comprising only L6-S1. Interestingly, such bimodal distributions of sensory afferents are not unique to the colon, as previous studies have reported similar patterns of innervation, albeit at different DRG levels, of the urogenital organs in other mammals.12 Immunohistochemistry performed on DRG sections containing FB-labelled cells showed the majority to be CGRP-LI and the minority to be Isolectin B4 binding or P2X3-LI. Furthermore, double labelling studies showed that a proportion of mouse colonic afferents were both CGRP-LI and bound IB4. With respect to the pattern of innervation of the colon, and indeed the proportions of colonic afferents containing CGRP and IB4 binding sites, these data are similar to those reported previously in studies of both specific visceral afferents and the general DRG population (see later). However, two findings from this initial characterization of mouse colonic spinal afferents have provided some novel and interesting findings: whilst 34% of labelled cells examined in the whole DRG showed P2X3 and IB4 co-localization, only 7% of FB-labelled neurones showed the same co-localization, suggesting a difference in the afferent innervation of colonic (visceral) and other (e.g. somatic) structures (see below). Furthermore, the finding that 82% of FB-labelled neurones showed VR1-LI suggests a major role for this channel in the transmission of sensory signals from the gut to the spinal cord.

Numbers and levels of retrogradely labelled dorsal root ganglia

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Fast Blue was retrogradely transported from the injection sites in the colon to the DRG, in which the cell bodies of the labelled neurones were visible under a UV light source. Some variation in the intensity of the labelling was observed between cells, which is most likely due to the concentration gradient of FB as it spreads from varying volume and depth injection sites. It should be emphasized that the retrograde labelling method used in the present study was not designed to provide a precise anatomical analysis of all spinal levels supplying afferent neurones to the colon, as has been the case in studies that labelled whole transected nerve bundles,13 as only discrete areas of the colon were injected with the retrograde label. The emphasis of the current study therefore concerns the characteristics of FB-labelled cells. Nevertheless, a gross analysis (performed as described above) provided a reasonable indicator of those spinal levels innervated, as our findings agreed well with those previously reported.

The number of cells labelled per section in the present study (mean range 0–24, n = 5) is comparable with previous studies that have reported five to 20 labelled cells in DRG sections after FB retrograde labelling of the rat bladder.14 However, the authors do not report how many injections were made in the bladder wall, only that 5–10 μL FB was used in each animal. This labelling was concentrated in DRG at spinal levels L6 and S1, which are levels that innervate both the colon and the bladder. FB retrograde labelling from both the distal colon and bladder has also been seen in the rat major pelvic ganglion.15 As would be expected, the number of FB-labelled cells varied between DRG taken from different spinal levels within each animal. The histogram plotted in Fig. 2 shows a clear bimodal distribution of FB labelling based around two peaks: one wide (T8-L1) and one more specific (L6-S1). Histograms plotted individually for each animal (data not shown here) showed similar patterns of labelling, providing persuasive evidence for the reproducibility of both the data and experimental methods.

With the exception of one retrogradely labelled mutant mouse study in which the rectum was labelled by injection of tracer through the anal orifice,6 and which focused solely on DRG levels L5-S3, there is currently no literature documenting such anatomical information in mice. However, if for the purpose of this discussion it is assumed that rat GI sensory neuroanatomy is comparable with that of the mouse, then the narrow peak of FB-labelled cells seen at spinal levels L6-S1 is likely to be due to innervation from the pelvic nerve.16 The lack of labelling either side of this area highlights this innervation particularly well. Similarly, the rat greater splanchnic nerve has shown to retrogradely label neurones with cell bodies in spinal DRG levels T3-T13, with the greatest density seen at T8-T12,17 which is likely to account for the FB labelling seen in the thoracolumbar DRG in the present study. Additionally, the lumbar colonic, lumbar splanchnic, intermesenteric and hypogastric nerves have neurones with cell bodies in the thoracolumbar DRG, which are likely to account for the larger numbers of labelled cells observed at these DRG levels.

Previous studies in the mouse have shown that the L6-S2 DRG provide sensory innervation of the lower bowel (rectum),6 which supports the L6-S1 pelvic nerve labelling peak in the present study. A similar picture is also seen in the rat, where previous studies have shown colonic retrograde labelling to be present in L6 and S1, and very little or no labelling in the adjacent L5 and S2 DRG.18–21 These studies also recorded labelling in levels L1 and L2 (probably innervation by the proximal hypogastric nerve,22) but not in any higher DRG. This is an interesting difference to the mouse data provided in the present study, which can most reasonably be explained by differences in injection site in the two species, as most injection sites may have been more distal in the rat studies. Additionally, whilst the present study was not designed to detect it, inter-species variation within the neuroanatomical innervation of the colon is another possible explanation.

Retrogradely labelled cell size

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

The majority (92%) of the FB-labelled cells analysed had a cross-sectional area of 100–700 μm2, which, assuming that all cells are circular in profile, can be converted to diameters of 11–30 μm. This suggests that the FB-labelled cells seen in the present study can be classified as small- to medium-sized, and reinforces the suggestion that the majority of retrogradely labelled colonic neurones were Aδ or C fibre afferents. Previous studies in rat have reported the size range of retrogradely labelled colonic primary afferents as 12–30 μm diameter,20 12–64 μm diameter23 (for those neurones also expressing CGRP, substance P or somatostatin) and 300–900 μm2 area.18

Immunohistochemical cell markers

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

Calcitonin gene-related peptide expression is a well-documented histochemical marker for the peptidergic subpopulation of C fibre nociceptors24 that is also thought to express the neuropeptide substance P and the NGF receptor, TrkA. These neurones project to laminae I and IIo of the spinal cord25,26 and are not thought to express P2X3 or bind the plant lectin IB4. The second group (non-peptidergic) do not express inflammatory mediator peptides such as CGRP, but instead bind IB4 and express the P2X3 receptor, and project to lamina IIi of the spinal cord.10,26 However, there is some speculation regarding the suitability of IB4 as a marker for the non-peptidergic nociceptor population. Some previous studies have reported IB4 binding to be a selective marker of this subpopulation25–27 whilst others have reported IB4 binding in many TrkA mRNA-expressing DRG neurones.28 Furthermore, some studies have demonstrated low co-localization of IB4 binding and CGRP-LI27 whilst others have reported higher figures.29,30

Whilst the CGRP-positive neurones are thought to play a direct role in inflammation (and hence, perhaps, in peripheral inflammatory visceral pain), the non-peptidergic neurones are thought to be more involved in chronic neuropathic pain.24 As one proposed mechanism of peripheral hypersensitivity is via the release of endogenous inflammatory mediators such as CGRP, it would not be unreasonable to hypothesize that these cells may have a role in CGRP release upon stimulation to produce an inflammatory response.

In the present study, 81% of DRG neurones labelled from the colon expressed CGRP-LI, which is comparable with similar studies in both rat and mouse. Retrograde colonic labelling of rat L6-S1 and L1-L2 DRG has reported approximately 70 and 46%, respectively, of cells to be CGRP-LI.20 A similar FB retrograde labelling study of rat bladder found that 69% of labelled cells exhibited CGRP-LI.14 These previous studies show that, in agreement with the present findings in the mouse, the majority of visceral primary afferent neurones are CGRP-containing. This large percentage of CGRP-LI neurones is in contrast to the whole DRG population, in which only 27% of cells were found to be CGRP-LI, a figure that is not dissimilar to that reported previously in studies of rat lumbar DRG where 23–50% of neurones have been reported to be CGRP-LI.29,31

Binding of Alexa Fluor-conjugated IB4, the Griffonia simplicifolia-derived isolectin, was also examined in a similar way to that described above for CGRP. IB4 binding was seen in 20% of the FB-labelled cells examined as a fluorescent ‘ring’ pattern around the cell bodies, whereas cells that did not bind IB4 showed no fluorescence above the background level. To our knowledge, the present study is the first to have reported the localization of IB4 in mouse colonic afferents. However, a previous report has shown IB4 binding in 29% of an FB-labelled population of rat bladder afferents.14 A similar proportion of retrogradely labelled colonic cells showed co-localization of both CGRP-LI and IB4 binding to that reported from a previous study of unlabelled cells from L5 rat DRG, in which 25% of neurones were found to be labelled in this way.29 The apparent difference between the proportion of FB-labelled cells that showed co-localization of CGRP-LI and IB4 binding (22%), and the proportion of FB-labelled cells that bound IB4 alone (20%) is not significant, and likely to be due to variation in the sample sizes used for each study. Previous studies have reported both strong and weak intensity IB4 binding,28,30 and this was also observed in the present study. Weak labelling was consistently seen in FB-labelled cells that were also CGRP-LI (see white arrows in Fig. 5) and whilst the functional significance of this is not known, the potential that these cells could represent a specific subpopulation of colonic sensory afferent warrants further study.

As both IB4 binding and P2X3-LI are suggested as markers for the same type of C fibre neurone, it was interesting to observe that, whilst 34% of neurones in the general DRG population expressed both these non-peptidergic neuronal markers, only 7% of FB-labelled neurones showed the same pattern. This may suggest a more prominent role of those P2X3 receptor-containing, non-IB4 binding afferent neurones in FB-labelled cells than in the general population, and is a potentially important finding with respect to visceral vs somatic sensory processing. Furthermore, this finding suggests that the current hypothesis of purine-mediated mechanosensory transduction in the gut, in which intense distension of the gut wall is thought to release ATP that, in turn, acts upon extrinsic sensory afferent nerve fibres that bind IB4 and express P2X3 and/or P2X2/3 receptors,36 may need to be extended to include this neuronal population. Indeed, although it is not clear to what extent IBS patients experience cutaneous as well as visceral hyperalgesia,32–34 it is clear that visceral and somatic pain exhibit different characteristics. Unlike the skin, for example, the viscera are insensitive to many forms of stimulation: the liver and kidneys are insensitive to any form of stimulation, and the hollow organs, including the colon, whilst being highly sensitive to distension or inflammation, are insensitive to burning or cutting stimuli (see ref.35 for review).

The findings concerning the general DRG population described above are also consistent with previous studies in rat that report 35–40% of lumbar DRG neurones to be P2X3-LI10,11,37 and 49–65% to bind IB4.28,29 Those cells labelled in the present study were similarly sized to those reported in previous studies.10,11,37

The vanilloid receptor 1 capsaicin receptor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References

The functional capsaicin receptor, VR1, is activated by noxious heat and low pH38,39 as well as by capsaicin itself, and is a non-selective cation channel. VR1 is also reported to be expressed by both the peptidergic and non-peptidergic subpopulations of C fibre nociceptor 39–41 and is thought to have a role in the transduction of noxious chemical and thermal stimuli. It is thus an interesting receptor to investigate in the context of visceral pain mechanisms.

Previous studies in the rat have suggested that capsaicin-sensitive neurones may be involved in hyper-reflexia and referred hyperalgesia following bladder inflammation,42 and systemic capsaicin has also been shown to prevent sensitization after acetic acid and colorectal distension-induced visceral pain.43 Similarly, studies have also shown that neonatal pretreatment with capsaicin reduces abdominal contractions, and thus perhaps the degree of visceral pain, induced by intraperitoneal injections of either acetic acid or CGRP.44 Xylene-induced (chemogenic) visceral pain also involves peptidergic capsaicin-sensitive sensory nerves.45 More directly, cannula-delivered capsaicin into the mouse colon evokes dose-dependent visceral pain behaviour and referred hyperalgesia46 and capsaicin can induce significant increases of CGRP and substance P release from mouse colon.47 The finding that 82% of colonic afferent neurones have VR1 receptors strongly suggests that the channel might have an important role in transmission of noxious signals from the colon.

In conclusion, the present study has shown that retrograde tracing using FB is very reproducible, uses only the minimum of animal tissue, and is a valuable technique with which to study the primary afferent innervation of the mouse colon. It has shown that spinal primary afferent neurones innervating the mouse colon are found in DRG at levels T8-L1, L6 and S1 and are small- to medium-sized. These neurones are mostly CGRP-containing, probably C fibre, afferents that express VR1, with smaller populations that express P2X3 or bind IB4. Additionally, those neurones that are P2X3-containing appear to differ from the general DRG population in that a much smaller proportion also bind IB4. The results presented here in mouse appear to correlate well with previous retrograde colonic labelling studies in the rat, suggesting that the combined study of both mouse and rat retrogradely labelled colonic spinal primary afferents, in conjunction with immunohistochemical analysis, could be a very powerful tool for future research into sensory systems of the viscera.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Retrograde labelling
  6. Dorsal root ganglia dissection and tissue preparation
  7. Cell counts and cell size analysis
  8. Immunohistochemistry
  9. Data analysis and statistics
  10. Results
  11. Fast Blue-labelled dorsal root ganglia: numbers and spinal levels
  12. Cell size analysis
  13. Immunohistochemistry: neuronal subtype markers in Fast Blue-labelled cells
  14. Immunohistochemistry: capsaicin receptor, VR1
  15. Discussion
  16. Numbers and levels of retrogradely labelled dorsal root ganglia
  17. Retrogradely labelled cell size
  18. Immunohistochemical cell markers
  19. The vanilloid receptor 1 capsaicin receptor
  20. Acknowledgments
  21. References
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