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Keywords:

  • action potential;
  • neuronal excitability;
  • retrograde labelling;
  • TNBS colitis;
  • viscerovisceral cross-sensitization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  Clinical studies reveal concomitant occurrence of several gastrointestinal and urologic disorders, including irritable bowel syndrome and interstitial cystitis. The purpose of this study was to determine the mechanisms underlying cross-organ sensitization at the level of dorsal root ganglion (DRG) after acute and subsided gastrointestinal inflammation. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) and Fast Blue were injected into the distal colon and urinary bladder of male rats, respectively. Convergent DRG neurons were found in L1-L3 and L6-S2 ganglia with an average distribution of 14% ± 2%. The resting membrane potential (RMP) of cells isolated from upper lumbar (UL) ganglia was −59.8 ± 2.7 mV, whereas lumbosacral (LS) neurons were more depolarized (RMP = −49.4 ± 2.1 mV, P ≤ 0.05) under control conditions. Acute trinitrobenzene sulfonic acid (TNBS) colitis (3 days) decreased voltage and current thresholds for action potential firing in LS but not UL convergent capsaicin-sensitive neurons. This effect persisted for 30 days in the absence of overt colonic inflammation. The current threshold for action potential (AP) firing in UL cells was also decreased from 165.0 ± 24.5 pA (control) to 85.0 ± 19.1 pA at 30 days (P ≤ 0.05), indicating increased excitability. The presence of a subpopulation of colon-bladder convergent DRG neurons and their persistent hyperexcitability after colonic inflammation provides a basis for pelvic organ cross-sensitization.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The occurrence and persistence of chronic pelvic pain is a common symptom of many functional and organic disorders including irritable bowel syndrome (IBS) and interstitial cystitis (IC). Clinical data show a high prevalence of increased urinary system problems such as painful urination, nocturia, urgency and difficulty of micturition in individuals with IBS and inflammatory bowel disease (IBD).1–5 Conversely, approximately one-third of patients diagnosed with IC complain of pain in the intestine and have symptoms of IBS.6,7 The clinical overlap of bowel and bladder dysfunctions suggests the involvement of the same or similar pathways of chronic pelvic pain occurrence and distribution within the abdominal cavity. Several recent studies, in both rats and mice, point towards viscerovisceral and viscerosomatic convergence of afferent inputs from the colon, bladder and underlying somatic receptive fields following acute inflammation.8–10 Pelvic organ cross-sensitization underlies the effects of acute cystitis on sensory thresholds of the distal colon to colorectal distention and changes in micturition parameters of the urinary bladder after acute colonic irritation.11

The precise mechanisms for this cross-talk are unclear at present but are thought to involve the peripheral and central nervous systems. Noxious stimuli are detected by specific nociceptors with their cell bodies located within a dorsal root ganglion (DRG). These neurons are primary candidates to serve as a substrate for direct neuronal connections between pelvic viscera. It has been shown that axons of DRG neurons can branch and, as a result, receive afferent inputs either from remote parts of the same organ12 or from two distinct visceral structures.13,14 Retrograde labelling technique allows for the identification of convergent cell bodies within the DRG receiving afferent stimuli from discrete pelvic organs. Convergent DRG neurons have been identified for both the colon and urinary bladder,8 uterus and urinary bladder,15 lumbar muscle and knee,16 lumbar disk and groin skin,17 thoracic and splancnic nerves.18

A variety of pathological factors including inflammation and injury alter excitability and firing rate of DRG nociceptors. The excitability of extrinsic primary afferents is orchestrated by activation of several voltage-gated ion channels. In the gastrointestinal tract acute inflammation of the colon,19 ileum20 and stomach21,22 increases excitability of respective DRG cell bodies mainly due to upregulation of NaV1.8 tetrodotoxin resistant (TTX-R) Na+ channel.

In the present study, we hypothesized that acute and/or subsided inflammation in the colon can evoke hypersensitivity of the urinary bladder via convergence of afferent information at the level of the DRG. To identify possible mechanisms underlying ‘cross-talk’ between the colon and urinary bladder, we used a retrograde labelling technique to detect convergent capsaicin-sensitive neurons within DRG and characterize their excitability after colonic acute and subsided inflammation.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Labelling of colon and urinary bladder DRG neurons and trinitrobenzene sulfonic acid-induced colitis

All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center. Adult male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA, 250–300 g) were anaesthetized with sodium pentobarbital (60 mg kg−1) injected intraperitoneally. A midline laparotomy was performed to gain access to the pelvic organs. The distal colon (2.5–3.5 cm from rectum) was exposed with cotton swabs and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR, USA; 1.5% w/v in methanol) was injected into the colonic wall using a Hamilton syringe at 6–8 sites (Hamilton Company, Reno, NV, USA). The colon was placed back into the abdominal cavity and then the urinary bladder was exposed for injections. Fast Blue (Polysciences Inc., Warrington, PA, USA; 1.5% w/v in water) was injected into the urinary bladder wall as described for the colon. The total volume of dye injected into each organ was 20–25 μL. To avoid non-specific labelling of surrounding tissues and organs, several precautions were taken during injection procedure. These precautions included isolation of adjacent pelvic organs with gauze during dye injections; keeping the needle in place after the injection for 20–30 s and soaking up of dye reflux upon needle removal; washing off any traces of dye from the organ surface with sterile saline before placing the organ into the pelvic cavity. Incisions were sutured in layers under sterile conditions and rats were allowed to recover on a warm blanket and monitored for signs of pain or discomfort. In case of observed pain, buprenorphine (2.0 mg kg−1) was injected subcutaneously. After recovery from surgery (3–5 days), the rats were fasted for 12–18 h and briefly anaesthetized with isoflurane (5%). While sedated, the animals received 0.6–0.7 mL of either vehicle or trinitrobenzene sulfonic acid [TNBS; 12.5 mg mL−1 in 25% of ethanol (C2H5OH)] enema. To avoid significant loss of instilled liquid, the rats were kept elevated by the tail until they regained consciousness and then were returned to their cages.

Isolation of DRG neurons

Rats were killed by injection of an overdose of pentobarbital (100 mg kg−1, i.p.) followed by decapitation at 3 and 30 days after TNBS instillation. Dorsal root ganglia were dissected and removed bilaterally at the L1-L3 and L6-S2 levels. The specific ganglia were identified corresponding to the appropriate segment of the spinal column. The T13 segment was initially identified at the base of the rib cage and used as the reference point for the L1-L3 and L6-S2 segments. The ganglia were treated with collagenase (Worthington, type 2, Biochemical Corp., Lakewood, NJ, USA) in F-12 medium (Invitrogen, Carlsbad, CA, USA) for 90 min in an incubator with 95% O2 and 5% CO2 at 37 °C. Isolated ganglia were rinsed in phosphate-buffered saline (PBS) and incubated for 15 min in the presence of trypsin (Sigma, St Louis, MO, USA; 1 mg mL−1) at room temperature. The enzymatic reaction was terminated in DMEM media containing 10% fetal bovine serum. Single neurons were obtained by gentle trituration in DMEM with trypsin inhibitor (Sigma; 2 mg mL−1) and deoxyribonuclease (Sigma; DNase 1 mg mL−1) and plated on a poly-L-ornithine coated 35 mm Petri dishes. Isolated cells were maintained in an incubator at 37 °C with 95% O2/5% CO2 and were used for electrophysiological experiments within 24 h.

Myeloperoxidase assay, histology and immunohistochemistry

The distal colon and urinary bladder were removed from vehicle and TNBS treated animals, immediately frozen in liquid nitrogen and stored at −80 °C. The myeloperoxidase (MPO) assay was based on the method adopted from Pothoulakis et al.23 Briefly, colonic and urinary bladder tissues (20–22 mg of wet weight) were homogenized in 2 mL of phosphate buffer (PB, pH = 6.0, 50 millimol L−1) with hexadecyltrimethylammonium-bromide (HTAB, 0.5%; Sigma). Each homogenate (1 mL) was transferred to eppendorf tubes and underwent three cycles of freeze-thawing followed by sonication for 10 s. After 15 min of centrifugation at 12 000 g at 4 °C, supernatant was collected and used for sample dilutions 1 : 1 and 1 : 10 in HTAB. Reactions were run in substrate buffer containing 0.005% of H2O2in PB buffer with o-Dianisidine dihydrochloride (ODHC, 25 μL/50 mL of PB; Sigma). The optical density value of each sample was read at 450 nm on a Multiscan EX spectrophotometer (Thermo Electron Corporation, Marietta, OH, USA) and was converted into MPO values by using curves obtained from a standard sample of human MPO (Sigma).

For histological experiments the colon and urinary bladder tissues were fixed in 10%neutral buffered formalin (NBF), then embedded in paraffin and cut in 10 μm sections. These cross sections were stained with haematoxyline and eosin to define the severity of experimental colitis. Other histological sections of the colon and urinary bladder were checked for any signs of cross-leakage between the organs. After 7 days following dye injection, tissue samples from both organs were sectioned longitudinally. Plain sections were examined under fluorescent microscope for DiI and Fast Blue labelling within each organ.

To count the colon and bladder labelled neurons, L1-L3 and L6-S2 ganglia were isolated bilaterally on day 7 after dye injections and fixed with 4% paraformaldehyde in PBS, cryoprotected with 5% sucrose, embedded in optimal cutting temperature (OCT) compound (Sakura Tissue Teck, Torrance, CA, USA) and rapidly frozen in isopentane by liquid nitrogen. Frozen ganglia were sectioned on a cryotome at 10 μm increments and every fifth section was mounted onto slides. This ensured that counting of labelled neurons in adjacent sections was not repeated. All mounted sections were incubated with biotinylated lectin from Griffonia simplicifolia, isoform B4 (IB4) (Sigma) followed by streptavidin-Cy5 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Positive IB4 staining refers to the neurons receiving afferent input via non-peptidergic unmyelinated C-fibres.24,25 Sections were mounted in Vectashild (Vector Laboratories, Inc., Burlingame, CA, USA) and visualized using a Nikon Diaphot inverted microscope (Nikon, Lewisville, TX, USA) with a 20X multi-immersion objective lens and Roper Cool-Snap HQ camera (Roper Scientific, Tucson, AZ, USA), using the following filter cubes: DAPI for Fast Blue, Cy3 for DiI and Cy5 for the IB4 specific fluorescence. Images were acquired with MetaMorph®vs 6.2 software (Universal Imaging Corp., Downingtown, PA, USA).

Quantitative analysis of the photomicrographic images was carried out by using Adobe Photoshop software. Due to the large total number of neurons (labelled and unlabelled), only DiI labelled, Fast Blue (FB) labelled, dual labelled and IB4 positive cells were counted. Double labelled neurons were identified by the presence of both red and blue fluorescence. The percentage of dual labelled neurons was determined as a ratio of the sum of DiI and FB single labelled neurons. The percentage of IB4 positive cells was only defined for dual labelled neurons. Only cells with a clear nucleus and specific neuronal morphology were considered to be neurons.

Electrophysiological recordings

Labelled neurons were identified using specific filters for DiI (UV-1A, Nikon, Lewisville, TX, USA) and Fast Blue (UV-2A, Nikon) under an inverted fluorescent microscope (TE2000–5, Nikon). Only neurons exhibiting bright fluorescence under both filters (2–4 per dish) were studied using the standard whole-cell patch clamp technique. For current clamp experiments the external solution contained (in millimol L−1): NaCl 135, KCl 5.4, NaH2PO4 0.33, MgCl2 1, CaCl2 2, HEPES 5, D-glucose 5.5, adjusted with NaOH to pH 7.4. Pipette solution for these experiments consisted of (in millimol L−1): K+ aspartate 100, KCl 30, NaCl 5, MgCl2 2, Na-ATP 2, EGTA 1, HEPES 5 with pH 7.2 adjusted with KOH. Patch electrodes had resistances of 3–5 MΩ when filled with internal solution. All cells were examined for capsaicin-induced currents at the end of experiments to identify capsaicin-sensitive cell bodies. To determine the voltage threshold for the action potential (AP), current steps (30 ms duration) were applied in 20 pA increments from −20 pA (used to calculate the input resistance) to 300 pA. Current clamp steps of 500 ms duration were used to define the phasic or tonic character of AP firing. All experiments were performed at room temperature (23 °C) and recorded using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). pCLAMP software (Axon Instruments) was used for data acquisition and analysis.

Chemicals

All chemicals were obtained from Sigma with the exception of DiI (Molecular Probes) and Fast Blue (Polysciences, Warrington, PA, USA).

Statistical analysis

All data are expressed as mean ± SE. Statistical significance was assessed by Student's t-test. Data with P ≤ 0.05 were considered significantly different.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Distribution of convergent neurons in L1-L3 and L6-S2 dorsal root ganglia

Seven–ten days after injection of DiI into the colon and FB in the urinary bladder L1-L3 and L6-S2 dorsal root ganglia were removed bilaterally, sectioned and examined for DiI, FB and IB4 fluorescence. Single DiI or FB labelled neurons were found in all sectioned ganglia. Fig. 1 shows one of the cross sections of the L6 ganglion with colonic neurons (panel A), urinary bladder neurons (panel B) and IB4 labelled cells (panel C). Fig. 1D illustrates the merged image with bright fluorescence of dual labelled neurons depicted by arrows. Fig. 1E shows a merged image of the L2 segment with a larger population of single labelled cells. Double labelled (from here on also referred as convergent) cells were identified in all studied ganglia and their distribution varied from 7% in L1 to 20% in S1 ganglia (Fig. 1F). Convergent neurons represented about 14% ± 2% of all single labelled cells. Only 10–30% of double labelled neurons were IB4 positive (Fig. 1E) and represented the population of non-peptidergic unmyelinated cell bodies.

image

Figure 1.  Cross section of L6 ganglion after retrograde labelling of distal colon (DiI) and urinary bladder (FB). (A) DiI fluorescence of colonic neurons within dorsal root ganglion. (B) FB labelled neurons, corresponding to urinary bladder afferent cell bodies. Scale bar is 100 μm. (C) The same section shows IB4 fluorescence after immunohistochemical labelling. (D) Overlap image of all three colour channels reveals convergent DRG neurons and those arising from non-peptidergic unmyelinated C-fibres (arrows). (E) Merged image of a cross section from the L2 ganglion shows single labelled cells (pointed by arrows). (F) Ratio of double labelled cells vs single labelled. The percentage of convergent neurons was calculated from the total sum of single DiI and FB labelled cells (taken together as 100%). The number of IB4 positive cells is calculated as a percentage of convergent neurons.

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Longitudinal sections of tissue samples from injected colon and urinary bladder revealed no cross-organ dye leakage and no tissue cross-labelling 7 days after dye injections (Fig. 2). Fig. 2A–C shows the presence of DiI in the colon but not FB. Conversely, Fig. 2D–F illustrates the absence of DiI fluorescence in the bladder wall.

image

Figure 2.  Longitudinal sections of the colon and urinary bladder. Tissue samples of the colon and bladder were obtained 7 days after dye injections. The colon was injected with DiI and the bladder with Fast Blue. (A) Phase contrast image of the colon wall. Fluorescent image of the same section of the colon using (B) DiI filter and (C) Fast Blue filter. (D) Phase contrast image of the longitudinal section of the urinary bladder. Fluorescent images of the same section using (E) DiI filter and (F) Fast Blue filters. Scale bar is 100 μm.

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Electrophysiological characteristics of convergent DRG neurons

L1-L3 and L6-S2 isolated ganglia were digested to obtain single cells for patch clamp experiments as described in Methods. Fig. 3 shows isolated convergent cells in culture under the fluorescent microscope. Only neurons having bright colour when using both filters for DiI and FB were selected for patch clamp recordings. Convergent DRG neurons obtained from both L1-L3 and L6-S2 regions produced large inward and outward currents upon depolarization from −70 mV (data not shown). The current clamp protocol was then used to assess the excitability of convergent colon/bladder neurons. One micromol per litre of capsaicin was applied in the bath solution at the end of the recordings to identify the cells with nociceptor-like properties. Only neurons which responded to capsaicin were used for data analysis.

image

Figure 3.  Isolated convergent neurons in culture for patch clamp experiments. (A–C) panels illustrate an isolated convergent neuron from L1-L3 ganglia (cell capacitance 53 pF) under normal light (A), UV fluorescence for DiI (B) and Fast Blue (C). DiI was used to label bladder neurons and Fast Blue for colonic cells in this rat. Scale bar is 100 μm. Panels D–F demonstrate an isolated convergent neuron from L6-S2 ganglia (cell capacitance 26 pF) labelled with DiI (colon, E panel) and FB (urinary bladder, F).

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Electrophysiological parameters of convergent capsaicin-sensitive neurons isolated from L1-L3 and L6-S2 ganglia did not substantially differ between the two regions (Table 1). Average cell capacitance of these cells was approximately 60 pF in both groups consistent with these neurons belonging to medium rather then small size population of DRG cells. Voltage and current thresholds of action potential (AP) firing, AP upstroke velocity, AP amplitude and duration, afterhyperpolarization (AHP) amplitude and duration, and input resistance were not distinguishable between upper lumbar (UL; L1-L3) and lumbosacral (LS; L6-S2) convergent neurons. However, L6-S2 neurons were more depolarized when compared with L1-L3 cells [resting membrane potential (RMP)-value is −49.4 ± 2.1 mV, n = 7 vs−59.8 ± 2.7 mV, n = 12, respectively, P ≤ 0.05].

Table 1.   Electrophysiological parameters of convergent DRG neurons isolated from L1-L3 and L6-S2 ganglia
Electrophysiological parameters L1–L3 (n = 12) L6–S2 (n = 7)
  1. *P ≤ 0.05 compared with L1–L3 neurons.

Cell capacitance (pF)61.9 ± 4.761.0 ± 8.5
RMP (mV)−59.8 ± 2.7−49.4 ± 2.1*
Voltage threshold (mV)−19.8 ± 2.1−16.5 ± 2.1
Rheobase (pA)165.0 ± 24.5224.3 ± 52.6
AP overshoot (mV)59.0 ± 3.163.1 ± 4.4
AP amplitude (mV)118.4 ± 4.8112.7 ± 5.3
AP duration at 0 mV (ms)2.9 ± 0.33.3 ± 0.2
AHP amplitude (mV)10.7 ± 1.412.8 ± 2.2
AHP duration (ms)28.6 ± 6.827.2 ± 6.9
Upstroke velocity (mV ms−1)32.5 ± 5.627.1 ± 2.2
Input Resistance (MΩ)181.3 ± 32.6200.7 ± 53.6

In current clamp, at least three subgroups of cells could be identified based on the sensitivity of AP to TTX (1 micromol L−1). In the first subgroup of cells (six of 19), TTX completely inhibited AP firing regardless of a five- to 10-fold increase in the amplitude of injected current. Fig. 4A shows representative raw traces of AP and their complete inhibition by TTX during short (30 ms) and long (500 ms) pulse protocols. Action potentials in the second subpopulation of convergent DRG neurons were unaffected by TTX (11 of 19 cells, Fig. 4B), whereas in third subgroup of cells (two of 19 cells) application of TTX had stimulating effect on AP firing (Fig. 4C). These three subpopulations of convergent neurons, based on the sensitivity of AP to TTX, were present in both L1-L3 and L6-S2 experimental groups. The graph in Fig. 4D reflects the percentage of cells with action potentials affected by TTX in UL and LS convergent neurons, showing that in almost 80% of cells the action potentials are TTX-resistant in LS ganglia, which was slightly greater than that in L1-L3 region. For this figure cells with TTX-R action potentials were pooled from subgroups 2 and 3.

image

Figure 4.  Action potentials (AP) in convergent DRG neurons. (A) Raw traces of AP elicited by short (30 ms, top panel) and long (500 ms, bottom panel) pulse protocols in the absence and presence of tetrodotoxin (1 micromol L−1). In this subgroup, TTX completely abolished AP firing. (B) Raw traces of AP elicited in the second subgroup of convergent cells. Application of TTX did not affect neuronal excitability. (C) TTX induced hyperexcitability in third subgroup of convergent neurons. (D) Percentage of cells with TTX resistant (TTX-R) and TTX sensitive (TTX-S) action potentials in L1-L3 and L6-S2 ganglia.

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Effects of colonic inflammation

The distal colon and the urinary bladder were removed on day 3 or 30 after vehicle (control group, Fig. 5A) or TNBS (inflamed groups) enema to assess the severity of colonic inflammation. Haematoxyline and eosin staining of the colon cross sections revealed visible changes in the structure of colonic wall including crypt segmentation, local haemorrhage and massive infiltration after 3 days post-TNBS-treatment (Fig. 5B). By day 30 after TNBS instillation, the cytoarchitecture of colon did not significantly differ from control tissue and no visible alterations in the colonic wall structure were detected (Fig. 5C). Histological sections from urinary bladder showed no signs of inflammation on either day 3 or day 30 following the TNBS enema (Fig. 5D–F).

image

Figure 5.  Haematoxyline and eosin staining of rat colon and urinary bladder from control and inflamed tissues. (A) Histological cross section of the normal colon. (B) TNBS-induced colitis partially disrupted the colonic crypts and evoked haemorrhagic infiltration in muscularis mucosa. (C) Cross section of the colon after 30 days of TNBS instillation. No visible signs of inflammation were detected. (D–F) panels represent cross sections of urinary bladder wall before and after 3 and 30 days of experimental colitis, respectively. No significant changes were observed in the cytoarchitecture of the bladder by TNBS application (Scale bar is 0.5 mm). (G) MPO activity levels in the colon and urinary bladder in control and inflamed groups.

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The MPO assay was performed to assess the possibility of bladder inflammation by TNBS through humoral transduction pathways. The activity of the MPO enzyme was measured in tissue samples removed from both the colon and the urinary bladder. The data show a twofold increase in MPO level in the colon on day 3 post-TNBS instillation, which was back to normal at day 30 post-TNBS (Fig. 5G). MPO levels obtained in bladder samples in all groups were unaffected by colonic inflammation. Taken together these data demonstrate substantial inflammation-induced changes in the distal colon at day 3 which subsided by day 30 post-TNBS treatment, without any effect on the urinary bladder.

Excitability of L1-L3 and L6-S2 convergent neurons after 3 days of TNBS colitis

Acute colonic inflammation elicited by TNBS, significantly affected the excitability of convergent neurons (Fig. 6A). L1-L3 cells had a cell capacitance value of 47.1 ± 3.6 pF (n = 8, P ≤ 0.05 to matching control) and 42.1 ± 3.4 pF for L6-S2 neurons (n = 9, P ≤ 0.05) on day 3 of the postinflammatory period. Colonic inflammation significantly reduced voltage threshold for AP firing in L6-S2 cells (−21.7 ± 1.3 mV vs−16.5 ± 2.1 mV in control group, P ≤ 0.05, Fig. 6B) and lowered rheobase from 224.3 ± 52.6 to 56.7 ± 7.8 pA (P ≤ 0.05, Fig. 6C). These changes were followed by enhanced AP upstroke velocity in LS cell bodies which was increased from 27.1 ± 2.2 to 37.6 ± 3.4 mV ms−1 (P ≤ 0.05) after 3 days of TNBS instillation. Lower voltage and current thresholds together with increased AP upstroke velocity suggest hyperexcitability of L6-S2 but not L1-L3 convergent neurons during acute inflammation. Enhanced excitability of L6-S2 DRG cells was evident in all groups of neurons firing either TTX-R or TTX-S action potentials. Colonic inflammation did not affect the RMP in both UL (−57.4 ± 2.3 mV) and LS (−50.8 ± 1.1 mV) DRG cells. Convergent neurons in the L6-S2 group remained more depolarized vs L1-L3 cells after colitis (Fig. 6D). Input resistance, AP and AHP amplitude and duration remained unchanged in all groups of DRG neurons after TNBS treatment.

image

Figure 6.  Excitability of convergent DRG neurons after 3 days of TNBS-induced colitis. (A) AP firing in control and inflamed groups induced by short (30 ms) and long (500 ms) current injection at rheobase in convergent cell bodies from L6-S2 ganglia. Note that rheobase was markedly decreased after inflammation. (B) Voltage threshold in convergent neurons. (C) Current threshold in L1-L3 and L6-S2 convergent cells after experimental colitis. (D) RMP was not affected at day 3 of TNBS-induced colitis in both L1-L3 and L6-S2 nociceptors.

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Electrophysiological characteristics of L6-S2 bladder labelled neurons before and after 3 days of TNBS-induced colitis

To address the question whether acute colonic inflammation differentially affects the excitability of convergent DRG neurons vs non-convergent cells, the action potential parameters from single labelled bladder neurons were measured. The electrophysiological characteristics of bladder labelled neurons are presented in Table 2. In general, bladder labelled neurons from the L6-S2 region had similar excitability to that of convergent cell bodies in this region under control conditions. The only statistically significant difference was the lower voltage threshold for the bladder capsaicin-sensitive cells (−28.1 ± 2.0 vs−16.5 ± 2.1 mV in convergent group, P ≤ 0,05). However, recordings from bladder labelled LS neurons made on day 3 post-TNBS instillation did not show statistically significant changes in their excitability unlike the convergent neurons which became hyperexcitable.

Table 2.   Electrophysiological characteristics of bladder DRG neurons isolated from L6-S2 ganglia
Electrophysiological parametersControl (n = 8)TNBS (n = 8)
  1. *P ≤ 0.05 compared with L6-S2 convergent neurons.

Cell capacitance (pF)52.5 ± 3.942.9 ± 5.7
RMP (mV)−56.8 ± 1.5−52.4 ± 1.8
Voltage threshold (mV)−28.1 ± 2.0 *−24.0 ± 1.8
Rheobase (pA)130.0 ± 30.681.3 ± 5.5
AP overshoot (mV)62.3 ± 3.955.4 ± 3.3
AP amplitude (mV)119.1 ± 4.4108.1 ± 3.9
AP duration at 0 mV (ms)2.3 ± 0.33.9 ± 1.0
Upstroke velocity (mV ms−1)78.5 ± 13.548.0 ± 8.7
Input Resistance (MΩ)175.7 ± 14.7306.9 ± 56.3

Effect of subsided inflammation on action potential characteristics and membrane properties of convergent DRG cells

Electrophysiological characteristics of convergent DRG neurons after 30 days of TNBS instillation are summarized in Table 2. Cell capacitance of UL and LS convergent somata was back to normal values; the amplitude and duration of both AP and AHP were also unchanged (Table 3). L6-S2 neurons still had lower voltage and current thresholds for AP firing and increased upstroke velocity 1 month after induction of inflammatory insult and were at the same level as observed after 3 days of postinflammatory period. These data show the persistence of hyperexcitability of LS convergent neurons 1 month after induced experimental colitis. In addition, 30 days post-TNBS instillation, L1-L3 neurons became more depolarized (−47.5 ± 1.9 vs−9.8 ± 2.7 mV, P ≤ 0.05 to control) and threshold for AP firing was significantly decreased from 165.0 ± 24.5 to 85.0 ± 19.1 pA (P ≤ 0.05 to control) which reflects an increase in excitability of L1-L3 convergent neurons 1 month after TNBS treatment.

Table 3.   Membrane properties of rat convergent DRG neurons on day 30 post-TNBS instillation
Electrophysiological parametersL1–L3 (n = 6)L6–S2 (n = 10)
  1. *P ≤ 0.05 compared with matching control.

  2. P ≤ 0.05 compared with L1-L3 neurons.

Cell capacitance (pF)49.2 ± 3.760.7 ± 3.0
RMP (mV)−47.5 ± 1.9*−49.6 ± 1.6
Voltage threshold (mV)−17.7 ± 1.5−26.3 ± 1.7†
Rheobase (pA)85.0 ± 19.1*68.0 ± 15.5*
AP overshoot (mV)63.8 ± 2.669.8 ± 2.6
AP amplitude (mV)108.7 ± 4.8116.7 ± 4.3
AP duration at 0 mV (ms)3.4 ± 0.32.7 ± 0.2
AHP amplitude (mV)12.2 ± 0.510.3 ± 1.5
AHP duration (ms)19.8 ± 4.820.3 ± 3.8
Upstroke velocity (mV ms−1)29.3 ± 3.745.6 ± 3.9*
Input Resistance (MΩ)160.8 ± 22.1167.2 ± 17.1

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study we identified and characterized the properties of convergent sensory DRG neurons receiving afferent input from two different pelvic organs – the colon and the urinary bladder. The goal in this study was to address: (i) the distribution and percentage of DRG neurons receiving convergent afferent inputs from the distal colon and urinary bladder in the rat; (ii) whether there are differences in electrophysiological properties of convergent neurons from L1-L3 ganglia vs L6-S2 cells; and (iii) how colonic inflammation affects the excitability of convergent neurons at the early and late stages of the postinflammatory period. Data presented in this study demonstrate that: (i) there is a subpopulation of DRG neurons receiving afferent inputs from two pelvic organs, the distal colon and urinary bladder; the percent of convergent neurons in UL and LS ganglia varies from 7% to 20% depending on the ganglion; (ii) the major difference in passive and active membrane properties between L1-L3 and L6-S2 convergent DRG neurons under control conditions is RMP (L6-S2 neurons are more depolarized); (iii) acute experimental colitis enhances excitability of L6-S2 convergent neurons up to, at least, 30 days postinflammatory period whereas it does not affect the excitability of UL neurons after 3 days but significantly lowers the current threshold for AP firing by day 30 post-TNBS treatment. Taken together, these data suggest that convergent colon/bladder neurons receive afferent signals from both pelvic organs and the inflammatory insult to the colon results in long-term enhanced excitability of L6-S2 convergent neurons. These neurons may play a key role in colon/urinary bladder cross-sensitization and chronic pelvic pain pathways.

Using the retrograde labelling technique with two dyes (DiI and FB) we were able to identify, isolate and maintain in culture DRG neurons receiving sensory afferent information from the distal colon and urinary bladder. Fluorescent analysis of UL and LS DRG cryosections showed the presence of double labelled cell bodies within all investigated ganglia with a majority of convergent neurons in L6 and S1 ganglia. On average, the percentage of double labelled neurons as compared with the entire pool of single labelled cell bodies was 14% ± 2% which correlates with the number of colon/bladder convergent neurons determined in other studies. Furthermore, the absence of cross-labelling between two organs due to dye leakage indicates that the labelling of DRG neurons arises specifically from the target organs.

Immunohistochemical labelling of DRG sections with lectin IB4 (hallmark of non-peptidergic unmyelinated C-fibres) demonstrated that convergent DRG neurons and IB4 were co-localized in 10–30% of all double labelled cells, which is in agreement with published data for the descending colon19,26 and urinary bladder.27–30 The existence of convergent DRG neurons receiving afferent information from distinct visceral structures was previously found for the urinary bladder and uterus,15 colon and urinary bladder 8 and thoracic and visceral nerves.18 Our data suggest that sensory afferent information from the colon and urinary bladder, as probably between other visceral structures, may converge at the level of DRG and peripheral axons of neuronal cell bodies serve as direct neuronal connections between pelvic organs. However, it is not entirely clear how convergent neurons receive afferent inputs from two organs. One possibility is that each convergent cell body has either two axons or one dichotomizing (branching) axon projecting to different pelvic viscera.11,13,14 Additional studies are necessary to identify specific anatomical features supporting these suggestions.

The average cell capacitance of UL and LS colon/bladder DRG neurons (around 60 pF) corresponded to medium sized cell bodies rather than small cells. The majority (approximately 80%) of convergent neurons responded to capsaicin, therefore, were considered as having the nociceptor-like properties. These results concur with other studies identifying nociceptors as small and medium sized DRG neurons giving rise to unmyelinated C- and slightly myelinated Aδ-fibres.26,31 Aβ fibres can also contribute to nociceptive perception but usually to a much lesser extent.32 Passive and active electrophysiological properties of colon/bladder cells from UL and LS ganglia did not significantly differ between two regions with one exception. Lumbosacral convergent neurons were 10 mV more depolarized when compared with L1-L3 subpopulation under control conditions. Differences in membrane properties between thoracolumbar and LS ganglia have been previously reported.33 Voltage threshold for AP firing in UL and LS convergent neurons was similar to that reported for the urinary bladder34 but more positive than in colonic afferent cell bodies.19,33 The higher voltage threshold may be due to the significant expression of voltage gated Ca2+ channels in convergent cells. For example, the T-type of Ca2+ channel is found to be expressed in DRG neurons and plays an important role in neuronal excitability and AP inflection.35

Acute colonic inflammation (3 days post-TNBS) enhanced the excitability of LS but not UL convergent neurons. The direct effect of either colonic19,36 or urinary bladder34 inflammation on the excitability of respective DRG cell bodies has been well established. Unchanged excitability of UL neurons after colonic inflammation can be partially explained by the fact that thoracolumbar neurons are less excitable in naïve animals. In vivo and in vitro studies, performed in the cat,37 murine,38 rat39,40 colon and rat uterus41 have shown that afferent fibres from hypogastric and lumbar splanchnic sympathetic nerves had higher thresholds to mechanical stimuli than those in pelvic nerve. These data fit with our observation that acute colonic inflammation increases the excitability of LS but not UL convergent neurons and we speculate that LS cell bodies may be primarily involved in mechanisms of inflammatory pain and hyperalgesia. Experiments on LS bladder labelled neurons did not reveal significant changes in excitability during the acute phase of colonic inflammation compared with convergent cells. In our previous study we showed that acute colonic inflammation increases the expression of TTX-R Na+ current in bladder neurons.42 Those experiments utilized the DSS model of experimental colitis (vs TNBS model presented here) and were performed on day 6–9 after starting the DSS treatment compared with day 3 post-TNBS in this study. The lower voltage threshold for AP firing in single labelled bladder neurons after inflammation, although statistically insignificant, is suggestive of hyperexcitability of urinary bladder neurons occurring at later time points or upon development of more severe inflammation. This would suggest that cross-talk between the colon and urinary bladder develops not only through the neural pathways but may also recruit other neurohumoral and/or neuroimmune mechanisms of noxious stimulus propagation within the pelvis.

Hyperexcitability of convergent LS nociceptors lasted for a month, a time frame at which colonic inflammation has significantly subsided and histological signs of inflammation were undetectable. Partial excitability of UL convergent neurons after 30, but not 3 days of post-TNBS treatment could account for delayed response of sympathetic afferents to acute colonic inflammation. In addition to significant and prolonged hyperexcitability of LS capsaicin-sensitive neurons, partly hyperexcitable UL neurons might also contribute to long-term persistent hypersensitivity of affected organs and underlie visceral hyperalgesia.

In summary, the present study identified and characterized a subpopulation of DRG neurons receiving afferent input from two pelvic organs: the colon and the urinary bladder. The ability of these neurons to converge afferent inputs from discrete target organs suggests their important role in making direct neuronal connections between the pelvic domains. The hyperexcitability of convergent DRG neurons following inflammatory insult may underlie the process of noxious stimulus transmission from a directly affected organ to an adjacent non-irritated structure. In our previous study we also showed that the excitability of non-convergent and convergent neurons in the LS segments of the dorsal horn in the spinal cord increases after colonic inflammation. The non-convergent spinal neurons received afferent input from the urinary bladder but not from the colon.10 As non-convergent bladder neurons were hyperexcitable in the spinal cord without direct input from convergent DRG, other factors may also contribute to the central sensitization of spinal neurons that participate in the pelvic organ cross-talk. Further studies need to be performed to understand the mechanisms of afferent convergence at the level of DRG and spinal cord in greater detail and their impact on pelvic organ cross-sensitization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Ashiq Zaman for help in analysing the DRG sections. This study was supported by NIH grants DK46367 (HIA), DK69628 (HIA) and NS35471 (RDF). Dr Akbarali's present address is Department of Pharmacology and Toxicology, Virginia Commonwealth University, MCV Campus, Richmond, VA, USA.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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