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. Author manuscript; available in PMC: 2021 Apr 26.
Published in final edited form as: Neuroscience. 2008 Mar 4;153(3):803–813. doi: 10.1016/j.neuroscience.2008.02.046

IDENTIFICATION AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF COLOSPINAL AFFERENT NEURONS IN THE RAT

S K SUCKOW a, R M CAUDLE a,b,*
PMCID: PMC8073176  NIHMSID: NIHMS1686590  PMID: 18424003

Abstract

The classification, morphology and function of enteric neurons have been extensively studied in the small and large intestine. However, little is known about enteric neurons that directly project to the CNS. Previous studies have identified these unique neurons in the rectum, rectospinal neurons, but little was done to characterize them. Therefore, the aim of this study was to identify and characterize enteric neurons in the rat colon that directly project to the CNS by using retrograde neuronal tracing and immunohistochemistry. By applying the retrograde tracers 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) and Fluorogold (FG) to the L6/S1 segments of the spinal cord, we identified these neurons in both the myenteric and submucosal plexuses of the colon. These neurons were immunoreactive for neurofilament (NF) a marker for Aδ-fibers and isolectin-B4 (IB4) a marker for C-fibers. These neurons expressed the enzyme neuronal nitric oxide synthase (nNOS) as well as peptides associated with sensory neurons such as substance P (SP) and vasoactive intestinal polypeptide (VIP) but did not express calcitonin gene-related peptide (CGRP). The N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2D and proteinase-activated receptor-2 (PAR2) were also found in these neurons. However they did not express the transient receptor potential receptor V1 (TRPV1) or neurokinin 1 receptor (NK1). The expression of the peptides and receptors suggests that there are at least two separate populations of neurons projecting from the colon to the CNS. The data suggest that these colospinal afferent neurons (CANs) might be involved in nociception. Whether sensory information from CANs is perceived by the animal or is part of the parasympathetic reflex is currently not known.

Keywords: enteric neurons, spinal cord, retrograde labeling, nociception, sensory neurons, fluorescence

Information about the state of tissues is transmitted to the CNS by afferent neurons residing in sensory ganglia such as the dorsal root ganglia (DRG) and vagal sensory ganglia (Furness, 2006a). In the gut, lumbosacral DRGs/pelvic afferents innervate the lower bowel and send projections up to the lumbosacral spinal cord whereas vagal ganglia, with projections to the brain stem, innervate the esophagus through the transverse colon (Al Chaer and Traub, 2002). The spinal sensory afferents from DRGs consist of both Aδ and C-fibers that respond to a variety of stimuli, including chemical, thermal, and mechanical (Duclaux et al., 1980; LaMotte and Thalhammer, 1982). Vagal afferents innervating the gut are very similar to pelvic afferents. However, they are also involved in the parasympathetic reflex that controls motor and secretomotor responses (Hubscher et al., 2004; Furness, 2006a).

Two types of afferent neurons are present in the gut, intrinsic primary afferent neurons (IPANs) and intestinofugal neurons (Furness et al., 2004). IPANs have their cell bodies in both the myenteric and submucosal plexuses and synapse on motor neurons and interneurons within the gut (Furness et al., 2004; Furness, 2006a). IPANs are involved in local reflexes that direct muscle movements, respond to mechanical stimuli of the mucosa and changes in luminal chemistry (Furness, 2006a). Intestinofugal neurons are also mechanosensory, but are involved in the entero-enteric reflex pathway. These neurons are unique in that their cell bodies lie in the myenteric plexus and extend their axons to the prevertebral ganglia that lie outside the gut wall. Activation of intestinofugal neurons results in modulation of smooth muscle contraction of the colon through the sympathetic nervous system (Szurszewski et al., 2002).

There has been a question of whether enteric neurons can project directly to the CNS. Doerffler-Melly and Neuhuber (1988) demonstrated that enteric neurons residing in the rectum, rectospinal neurons, project directly to the spinal cord. Experiments injecting retrograde tracers into the dorsal horn of the lumbosacral spinal cord, dorsal root, and spinal nerve of segment S1 demonstrated that these neurons exist in the myenteric plexus of the rectum. Rectospinal neurons were not observed when horseradish peroxidase (HRP) was injected into the ventral root or when the dorsal root was cut (Doerffler-Melly and Neuhuber, 1988; Neuhuber et al., 1993). Immunohistochemical analysis showed that rectospinal neurons stained for vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). Furthermore, using antibodies raised against intermediate filaments it was shown that the rectospinal neurons were immunoreactive for both neurofilament (NF) and peripherin. The authors suggested that since rectospinal neurons send their axons to the dorsal horn of spinal cord and are immunoreactive for peptides associated with sensory neurons of the DRG that rectospinal neurons are indeed sensory neurons that take part in the visceral afferent pathway (Neuhuber et al., 1993). Similar to these previous studies, this project identifies a new group of neurons that send their axonal projections directly from the colon to the dorsal horn of the lumbosacral spinal cord. The data suggest that these neurons may be involved in nociception.

EXPERIMENTAL PROCEDURES

Experiments were performed on male Sprague–Dawley rats (n=5) weighing 200–250 g. They were housed in pairs with free access to food and water in the University of Florida’s animal care facility with a 12-h light/dark cycle. These facilities are AAALAC accredited. All experiments conformed to procedures to minimize potential pain, distress or discomfort as well as minimized the number of animals used. All experiments conformed to guidelines on the ethical use of animals as published by the International Association for the Study of Pain. All procedures were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee.

Laminectomy

Prior to the laminectomy, the animals were anesthetized with isoflurane (1–3% in O2). The dorsal portion of the L1 and/or L2 vertebrae was removed to expose the L6 and S1 segments of spinal cord. The dura covering the spinal cord was removed and a 5×2 mm piece of gel foam (Henry Schien, Melville, NY, USA) was placed onto the dorsal surface of the spinal cord. The gel foam was soaked in 40 μl of the retrograde tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR, USA; 2.5 mg/ml in DMSO) or 20 μl of the retrograde tracer Fluorogold (FG) (Courtesy of Dr. Wolfgang Streit, University of Florida, Gainesville, FL, USA). The wound was closed by suturing in layers. A triple antibiotic was applied to the wound site and buprenorphine (0.3 mg/kg) was given i.m. twice a day for approximately 3 days after surgery.

Perfusion fixation

After a survival time of 10 days, rats were given a lethal dose of pentobarbital i.p. and perfused through the heart with cold 0.9% saline followed immediately with cold 4% paraformaldehyde in phosphate-buffered saline (PBS). After fixation the spinal cord and colon of the animal were removed, post-fixed in 4% paraformaldehyde in PBS for 24 h at 4 °C, and then stored in 30% sucrose at 4 °C for at least 24 h.

Immunohistochemistry

Tissue was sectioned at 20 μm on a cryostat, serially mounted on a glass slide, and air-dried for 1 h. All preparations were washed three times (10 min each) in PBS, placed in blocking buffer containing 3% normal goat serum (NGS) with PBS for 1 h, and incubated in primary antibody in 3% NGS/0.3% tween-20/PBS (Table 1) for 24 h at 4 °C. The sections were then washed three times in PBS (10 min each) followed by 1 h incubation in secondary antibody Alexa Fluro 488 (1:1000; Molecular Probes, Boston, MA, USA) in 3% NGS/0.3% tween-20/PBS (Table 1). Tissue was then washed three times (10 min each) and coverslipped with ProLong® Antifade Kit mounting media (Molecular Probes) or Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA). For double labeling, preparations were washed three times (10 min each) in PBS, placed in blocking buffer containing 3% NGS with PBS for 1 h, and incubated in primary antibody in 3% NGS/0.3% tween-20/PBS (see Table 1) for 24 h at 4 °C. The sections were then washed three times in PBS (10 min each) in PBS followed by 1 h incubation in secondary antibody Alexa Fluro 488 (1:1000; Molecular Probes) and Alexa Fluro 350 (1:250; Molecular Probes) in 3% NGS/0.3% tween-20/PBS. Tissue was then washed three times (10 min each) and coverslipped with ProLong® Antifade Kit mounting media (Molecular Probes). The sections were visualized with filters for red, green, and DAPI excitation. Images were photographed on a Leica DM LB2 Fluorescence microscope (Leica, Wetzlar, Germany). Negative controls, where the secondary was applied to the sections in the absence of primary antibodies, were used to verify that non-specific binding of secondary antibodies to tissue did not occur. For double labeling experiments, the antibodies used were raised in different species to ensure that there was no cross-reactivity. All images were processed using the Adobe Photoshop program. For cell counts, every other section was counted. Approximately 100 cells per immunolabel were counted for analysis. Cell counts were done using the ImageJ program (NIH, Bethesda, MD, USA) where DiI positive cells (in red) were counted and marked; double-labeled cells were determined by superimposing the marked image with the label being investigated (in green).

Table 1.

Primary antibodies for identification of DiI-labeled cells in rat colon using immunohistochemistry

Label Host Dilution Company
NR1 Mouse 1:500 BD Biosciences
NR2B Mouse 1:500 BD Biosciences
NR2D Rabbit 1:100 Santa Cruz
NK1r Mouse 1:500 Zymed
SP Rabbit 1:250 Chemicon
PAR-2 Chicken 1:500 Aves Lab, Inc.
TRPV1 Rabbit 1:500 Affinity Bioreagents
CGRP Rabbit 1:250 Courtesy of Dr. Michael Iadarola
nNOS Rabbit 1:250 Chemicon
VIP Rabbit 1:500 Chemicon
NF 200 Mouse 1:250 Sigma
IB4 Goat conjugated to IB4 1:1000 Invitrogen
FG Rabbit 1:500 Chemicon
Alexa Fluor 594 Chicken anti-rabbit 1:1000 Invitrogen
Alexa Fluor 488 Goat anti-rabbit 1:1000 Invitrogen
Alexa Fluor 488 Donkey anti-mouse 1:1000 Invitrogen
Alexa Fluor 488 Goat anti-chicken 1:1000 Invitrogen
Alexa Fluor 350 Goat anti-rabbit 1:250 Invitrogen
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RESULTS

Labeling in the spinal cord

Transverse sections through the L6 and S1 segments of the spinal cord showed DiI and FG labeling in the superficial laminae, lamina I and II (Fig. 1a′ and a″). The labeling was comparable on both sides of the spinal cord.

Fig. 1.

Fig. 1.

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Neuronal retrograde tracer labeling of spinal cord and neurons in the SM and M of colon. Application of the retrograde tracer DiI (a′) and FG (a″) to the dorsal horn of lumbosacral spinal cord resulted in labeling of laminae I and II of the spinal cord segment (a). Application of the retrograde tracer DiI (b′) and FG (b″) to the lumbosacral spinal cord resulted in DiI and FG labeling of enteric neurons in the SM and M of colon. Retrograde labeled DiI (c′) and FG (c″) enteric neurons co-labeled with DAPI. Scale bars=50 μm (b); 10 μm (c). LM: longitudinal muscle; M: myenteric plexus; CM: circular muscle; SM: submucosal plexus.

Neuronal labeling in the colon

The tracers DiI and FG labeled neuronal cell bodies in both the myenteric plexus and the submucosal plexus of colon (Fig. 1b′ and b″; Suppl. Fig. 1). DiI was located in the soma of the cell, where FG filled the entire cell and some dendrites. Cell diameters were consistent among all the cell bodies averaging 10–20 μm in diameter.

Immunohistochemistry

To verify that the labeling observed was in fact cell bodies, DiI and FG cells were labeled with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI). All DiI- and FG-labeled cells co-labeled with DAPI (Fig. 1c′ and c″). Furthermore, DiI cells were labeled with the sensory afferent markers NF for Aδ-fibers and isolectin-B4 (IB4) for C-fibers. Approximately 46% of the DiI labeled cells were NF immunoreactive (NF-IR) (Fig. 2a). Previous studies demonstrated that IPANs in the colon and in the DRG that are classified as nociceptors bind IB4 (Hind et al., 2005). Therefore we co-labeled DiI neurons in the colon for IB4. We found that 36% of the DiI-labeled cells were IB4-IR (Fig. 2b). We performed dual immunohistochemistry on control tissue to verify that NF and IB4 do not colocalize on the same neuron (Fig. 2c).

Fig. 2.

Fig. 2.

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DiI-labeled neurons in rat colon immunoreactive for afferent specific neuronal markers. Photomicrographs of DiI-labeled neurons in the submucosal plexus of colon showing co-localization with neuronal afferent markers NF (a) for Aδ-fibers and IB4 (b) for C-fibers. Photomicrograph of control tissue showing that NF and IB4 do not co-localize (c). Arrows indicate double labeled neurons. Scale bars=20 μm.

Immunoreactivity for enzymes, neuron-associated peptides and receptors (Table 1) was tested in sections of colon. Immunohistochemistry was performed using the enzyme neuronal nitric oxide synthase (nNOS) and VIP antibodies. As previously reported (Neuhuber et al., 1993; Miampamba and Sharkey, 1998; Lomax and Furness, 2000), there was strong labeling of nNOS and VIP in both the myenteric plexus and submucosal plexus. In the DiI-labeled cells there was colocalization with nNOS in 67% of the neurons and VIP in 48% of the neurons (Fig. 3a, b).

Fig. 3.

Fig. 3.

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DiI-labeled neurons immunoreactive for neuronal markers associated with sensory afferents. Photomicrographs of DiI-labeled neurons of colon showing co-localization with nNOS (a) in the submucosal plexus, VIP (b) in the myenteric plexus, and substance P (SP) (c) in submucosal plexus of colon. Photomicrograph of DiI labeled neurons of colon showing no co-localization with CGRP (d). Arrows indicate double labeled neurons. Scale bars=20 μm.

In order to determine if the DiI-labeled cells could be sensory neurons immunohistochemistry was performed on DiI-labeled cells with antibodies to the peptides SP and CGRP. There was strong SP labeling in both the myenteric plexus and submucosa. Approximately 86% of DiI cells were SP-IR (Fig. 3c). In contrast to previous findings in rectospinal neurons (Neuhuber et al., 1993), there were no DiI-labeled cells that were CGRP-IR (Fig. 3d).

Immunohistochemistry was performed with antibodies to the receptors, transient receptor potential receptor V1 (TRPV1), proteinase-activated receptor-2 (PAR2), neurokinin 1 (NK1), and N-methyl-D-aspartate (NMDA) receptor subunits NR1, NR2B, and NR2D. Colocalization showed 92% of DiI cells were PAR2-IR (Fig. 4a). There was strong labeling with NR1 in both myenteric and submucosa neurons. Fifty-five percent of DiI neurons double labeled with NR1 (Fig. 4b). NR2D labeling was somewhat sparse in the myenteric plexus and submucosa; however, colocalization demonstrated that 40% of DiI cells were NR2D-IR (Fig. 4c). There was no DiI cell immunoreactivity with TRPV1; however, several DRG fibers innervating the colon were immunoreactive for the TRPV1 receptor (Fig. 5c). Furthermore, NK1 nor the NMDA subunit NR2B (Fig. 5) co-localized with DiI-labeled cells even though there was strong labeling in the submucosa and myenteric plexus as previously reported (Ward et al., 2003; Harrington et al., 2005; Zhou et al., 2006).

Fig. 4.

Fig. 4.

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DiI-labeled neurons immunoreactive for receptors involved in nociception. Photomicrographs of DiI-labeled neurons of colon showing co-localization with the PAR2 (a) in the submucosal plexus, the NMDA receptor subunit NR1 in the myenteric plexus (b) and the NMDA receptor subunit NR2D in submucosal plexus of colon (c). Arrows indicate double labeled neurons. Scale bars=20 μm.

Fig. 5.

Fig. 5.

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DiI-labeled neurons immunonegative for receptors involved in nociception. Photomicrographs of DiI-labeled neurons that do not co-localize with NMDA receptor subunit NR2B (a), NK1 (b), TRPV1 (c) in the submucosal plexus of colon. Arrows indicate non-double labeled neurons. Scale bars=20 μm.

A previous study demonstrated that sensory neurons expressing the PAR2 receptor also contain SP (Steinhoff et al., 2000). Immunohistochemistry was performed to determine if the DiI cells contained both PAR2 and SP within the same neuron. Co-localization showed 87% of DiI cells were immunoreactive for both PAR2 and SP (Fig. 6a). In addition, this co-localization occurred in other sensory neurons that did not contain DiI. Immunohistochemistry was also performed to determine if DiI cells contained both NR1 and VIP since both have been associated with Aδ-fibers (Basbaum and Glazer, 1983; Marvizon et al., 2002). It was observed that 33% of DiI cells were immunoreactive for both NR1 and VIP (Fig. 6b). This co-localization was observed in both the submucosal and myenteric plexus. NR1 and VIP were also co-localized on neurons that did not contain DiI.

Fig. 6.

Fig. 6.

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DiI-labeled neurons dual labeled for neuropeptides and receptors involved in nociception. Photomicrographs of DiI-labeled neurons (a) that underwent double immunofluorescence labeling show co-localization with PAR2 (a′) and SP (a″) on the same neuron in submucosal plexus of colon. Double immunofluorescence labeling (b) also showed co-localization with NMDA receptor subunit NR1 (b′) and VIP (b″) on the same neuron. Arrows indicate double labeling. Scale bars=10 μm.

Table 2 provides a summary of the immunohistochemical results previously discussed in this study. In addition, we identified the proportion of colospinal afferent neurons (CANs) within the enteric neuron population of the colon (Table 3). Immunohistochemical analysis suggested that CANs comprise approximately 6% of the colonic neuron population. This is comparable to the proportion of intrinsic sensory neurons in the submucosa (11%); however, intestinofugal neurons are a smaller proportion (<1%) (Furness, 2006b).

Table 2.

Percentage of DiI-labeled cells that co-localized with neuropeptides and receptors involved in sensory transmission

Label DiI double label/DiI cells Percentage
IB4 35/96 36
NF 44/95 46
SP 83/97 86
nNOS 60/90 67
VIP 42/88 48
PAR2 93/101 92
NR1 38/69 55
NR2D 48/121 40
NR2B 0/83 0
TRPV1 0/73 0
NK1 0/81 0
CGRP 0/70 0
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Immunohistochemical characterization of CANs resulted in the expression of peptides and receptors commonly thought to be involved in sensory processing. These data suggest there are at least two populations of CANs. The majority of CANs co-localized with SP, nNOS, and the PAR2 receptor. CANs also colocalized with VIP and the NMDA receptor.

Table 3.

Percentage of neuropeptides and receptors that co-localized with DiI-labeled cells

Label Labeled cells/DiI double labeled Percentage
IB4 129/16 8
NF 109/26 4
SP 119/23 5
nNOS 127/20 6
VIP 100/16 6
PAR2 104/25 4
NR1 110/23 5
NR2D 117/17 7
NR2B 81/0 0
TRPV1 90/0 0
NK1 92/0 0
CGRP 75/0 0
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Immunohistochemical characterization of colonic tissue determined the proportion of CANs within the colon. These data suggest that the population of CANs within the enteric neuron population totals approximately 6%.

DISCUSSION

CANs project directly to the CNS

The present study has identified a unique population of neurons, CANs, with a direct projection from the enteric nervous system to the CNS. Both DiI and FG are not known to cross synapses or enter the vascular circulation (Schmued and Fallon, 1986; Honig, 1993). Schmued and Fallon (1986) and Honig (1993) comment that these tracers were rarely seen in microglia and this only occurred after prolonged survival times following tracer application, typically being more than a month. Our study used survival times of 10 days which was sufficient time to label the pathway under investigation but shorter than the time it would take either tracer to potentially cross synapses. Furthermore, the proportion of CANs within the colonic neuron population is approximately 6% (Table 3); thus, if the retrograde tracers did cross synapses or enter the vascular circulation we would expect the proportion of labeled cells to be greater. Further studies investigating the pathway by performing a rhizotomy would further determine how projections from CANs innervate the spinal cord. However, due to the tracing properties of DiI and FG, we feel the presence of these markers in colonic neurons indicates that CANs project directly to the dorsal horn of the spinal cord. These neurons are present in both the myenteric and submucosal plexuses, extending their axons to laminae I and II of the dorsal horn of the spinal cord.

Further, we have identified several sensory neuron–associated peptides and receptors that are co-localized with CANs in both the submucosal and myenteric plexus of the colon. To ensure that the observed CANs were in fact sensory neurons, co-localization studies were performed with sensory afferent neuronal markers NF for Aδ fibers and IB4 for C-fibers. These markers have been found to bind primary afferent neurons of DRGs innervating the colon as well as IPANs (Lawson and Waddell, 1991; Streit et al., 1985; Ambalavanar and Morris, 1992; Hind et al., 2005). In the present study, CANs expressed both neuronal markers suggesting at least two populations of putative sensory afferents. We also found that CANs express SP but do not express CGRP, even though CGRP is present in both the submucosal and myenteric plexuses. However, it was demonstrated previously that rectospinal neurons express CGRP but not SP (Neuhuber et al., 1993). This suggests some differentiation between CANs and rectospinal neurons. In addition, half the population of CANs expresses the neuropeptide VIP and the enzyme nNOS. VIP and NOS are commonly found in inhibitory motor neurons in the enteric nervous system (Furness, 2006a); however, some studies have shown that NOS and VIP exist in sensory ganglia of DRGs as well as in the dorsal horn of the spinal cord. It is important to note that VIP has been shown to co-localize on both Aδ- and C-fibers (Basbaum and Glazer, 1983; Morgan et al., 1999). Papka et al. (1995) demonstrated that NOS positive DRG neurons (T13-S2) co-localize with either CGRP, SP or VIP. Similarly, Alm et al. (1995) demonstrated that DRG neurons that were immunopositive for NOS were mainly found in the thoracic and lumbar regions. Other studies have shown involvement of NOS and VIP in sensory neurons during an inflammatory response. In a model of TNBS-induced inflammation, Demedts et al. (2006) demonstrated that the number of cells in the myenteric plexus immunoreactive for nNOS decreased following inflammation. In addition, in a model of neuropathic pain it was demonstrated that VIP in DRG neurons increases following injury; thus, VIP is a possible contributor to central sensitization (Nahin et al., 1994). Since nNOS and VIP are found in CANs, it can be suggested that CANs are putative sensory neurons involved in sensory transmission during the inflammation process.

Approximately half the CANs were immunoreactive for NMDA receptor subunits NR1 and NR2D and were immunonegative for NMDA subunit NR2B. Our previous data demonstrated that only the NR2B and NR2D subunits are expressed in control colon (Valle-Pinero et al., 2007). On the other hand, PAR2 is expressed on almost all CANs and was found to be co-localized with CANs expressing SP. This is in agreement with other reports that found neurons expressing PAR2 also express SP (Steinhoff et al., 2000; Vergnolle et al., 2001). CANs were found to be immunonegative for NK1 and TRPV1 even though both these receptors were found to be expressed by other sensory neurons in the colon as well as primary sensory afferent fibers innervating the colon. Based on the data presented here, CANs might be involved in nociception as well as other modes of sensory transmission.

Two populations of CANs

Based on present findings, it seems that CANs can be separated into two populations of putative sensory neurons (Table 2). Immunoreactivity suggests that CANs are similar to both Aδ and C-fibers as shown by binding of NF and IB4 respectively. The majority of CANs express SP, PAR2, and nNOS which have all been found to be associated with the inflammatory response (Vergnolle et al., 1999; Steinhoff et al., 2000; Demedts et al., 2006). After colonic inflammation, Miampamba and Sharkey (1998) demonstrated a reduction in the distribution of SP and VIP in the colon where as Traub et al. (1999) found a reduction in SP distribution in DRG neurons suggesting that both peptides play a role in inflammatory nociception. In our study, dual immunoreactivity with CANs demonstrated that SP and PAR2 are present on the same neuron. This is consistent with the report that activation of the PAR2 receptor causes release of SP during a state of inflammation (Vergnolle et al., 2001; Steinhoff et al., 2000). Furthermore, approximately half of CANs express the NMDA receptor as well as VIP.

Studies have shown that the NMDA receptor is present on both Aδ and C-fibers in the DRG neurons innervating the colon (Marvizon et al., 2002; Seagrove et al., 2004). Marvizon et al. (2002) demonstrated that the NR2A/B subunit was associated with Aδ-fibers shown by the binding of NF and the NR2C/D subunit was associated with C-fibers shown by the binding of IB4. We demonstrated that CANs co-localize with both the NR1 and NR2D subunits suggesting that these cells are associated with C-fibers. Furthermore, Morgan et al. (1999) demonstrated that unmyelinated neurons contain VIP in DRGs, dorsal roots and the sacral spinal cord. We demonstrated that the NMDA subunit NR1 and VIP were present in the same neurons further suggesting their association with unmyelinated neurons. Studies have demonstrated that the NMDA receptor and VIP are involved in mechanical nociception (Basbaum and Glazer, 1983; Parkman et al., 1993; Zhai and Traub, 1999; McRoberts et al., 2001). Specifically, Zhai and Traub (1999) as well as McRoberts et al. (2001) demonstrated that behavioral responses to colorectal distention (CRD) were attenuated after systemic application of NMDA receptor antagonists. Even though the antagonists were applied systemically, the data suggest that the NMDA receptor is involved in mechanical sensory input. Therefore these studies suggest that CANs immunoreactive for the NMDA receptor are putative mechanosensory neurons.

CANs share characteristics with spinal primary afferent neurons

Sensory information from the colon is thought to be processed exclusively by spinal primary afferent neurons of DRGs that innervate the colon and dorsal horn of the spinal cord (Chung et al., 1979). However, the presence of CANs suggests that sensory information can be transmitted from the gut to the spinal cord by neurons whose cell bodies reside outside the DRGs. This suggests a direct link between the enteric nervous system and CNS bypassing the DRGs. It was previously demonstrated that IB4 binding in DRGs occurs in small diameter neurons that have terminals in the dorsal horn of spinal cord (Silverman and Kruger, 1990; Kitchener et al., 1993). However, DRG neurons immunoreactive for NF are associated with thinly myelinated Aδ-fibers (Lawson and Waddell, 1991). In addition, Aδ and C-fibers project to laminae I and II of the lumbosacral spinal cord (Morris et al., 2004; Todd et al., 2005). In the present study, CANs express either IB4 or NF suggesting that they may be analogous to Aδ and C-fiber primary afferent fibers arising from the DRG.

It was previously demonstrated that C-fibers in DRGs that innervate the colon are immunoreactive to SP. These same neurons also detect thermal nociception and inflammation (Silverman and Kruger, 1990; Traub et al., 1999; Lawson, 2002). However, SP is not exclusively associated with C-fibers. McCarthy and Lawson (1989) have demonstrated that a small population of Aδ-fibers in DRGs is also immunoreactive to SP. In the present study, we demonstrated that the CAN population consists of both Aδ and C-fibers. Since there is a large population of CANs that are immunoreactive for SP, the data suggest that CANs containing SP may not be exclusively associated with C-fiber neurons. It has also been reported that DRG neurons release SP in response to mechanical stimulation (Lawson et al., 1997). Therefore, CANs might be involved in both inflammation as well as mechanical sensory transmission.

Immunohistochemical studies have shown that NMDA receptors, specifically the NR2B and NR2D subunits, are present on DRG neurons innervating the colon. These studies indicated that neurons containing NMDA receptors can detect mechanical stimuli (Zhai and Traub, 1999; Marvizon et al., 2002; Li et al., 2004). In this and a previous study, it was observed that neurons in the colon express both the NR2B and NR2D subunits of the NMDA receptor (Valle-Pinero et al., 2007); however, we demonstrated that CANs contain the NR2D subunit only. Therefore, the expression of the NMDA receptor on CANs suggests that these neurons might be mechanosensory. It is important to note that Zhou et al. (2006) found in a model of TNBS-induced colitis that inflammation induced changes in the expression of the NR1 splice variants. The authors hypothesized that these changes may contribute to visceral hypersensitivity. Therefore, CANs expressing the NMDA receptor may be involved in both inflammation and mechanical sensory transmission.

CANs are a specialized group of putative sensory neurons in the colon

To date, sensory neurons of the colon include IPANs, intestinofugal neurons, and spinal primary afferent neurons (Furness, 2006a). However, the present study has identified CANs as another set of sensory neurons in the enteric nervous system. A possible role of CANs may be involvement in the transmission of nociception from the gut to the spinal cord as well as sensory input to the parasympathetic pathway. Evidence suggests that visceral pain of the colon is transmitted via afferents of the pelvic nerve that terminate in the dorsal horn of the lumbosacral spinal cord (de Groat et al., 1981; Traub et al., 1994). In addition, it was demonstrated that preganglionic neuronal cell bodies of the sacral parasympathetic nucleus (SPN) and pelvic nerve sensory fibers are located in the lumbosacral spinal cord (Nadelhaft and Booth, 1984). Since CANs extend their axons to the dorsal horn of lumbosacral spinal cord it is possible that CANs synapse on second order neurons in the dorsal horn in order to transmit sensory information from the colon to neurons in the CNS to provide information necessary for the parasympathetic reflex. However, further studies looking at projections of CANs in deeper laminae as well as their physiology are needed to further determine their involvement in the parasympathetic reflex and/or nociception.

CONCLUSION

In conclusion, the present study found a unique population of putative sensory neurons identified as CANs that project directly to the CNS from the myenteric and submucosal plexus of the colon. It was found that CANs also contain neuropeptides and receptors associated with nociception which suggests a possible role for CANs in pain sensory processing. Furthermore, immunohistochemical analysis suggests two populations of CANs: CANs involved in inflammation and CANs involved in mechanical sensory transmission.

Supplementary Material

Supplementary figure. The enteric plexus of a transverse section of colon. Photomicrograph of an H&E stained section of colon showing the muscle layers and nerve plexus. LM: longitudinal muscle; M: myenteric plexus; CM: circular muscle; SM: submucosal plexus.

Acknowledgments—

The authors thank Dr. Michael Iadarola, from the National Institute of Dental and Craniofacial Research (NIDCR) in Bethesda, MD and Dr. John Neubert from the Department of Orthodontics at the University of Florida for supplying the CGRP antibodies. The authors also thank Dr. Wolfgang Streit from the Department of Neuroscience at the University of Florida for supplying the retrograde tracer Flurogold. The work summarized here was supported by the National Institutes of Health NS045614.

Abbreviations:

CANs

colospinal afferent neurons

CGRP

calcitonin gene-related peptide

DAPI

4′6-diamidino-2-phenylindole

DiI

1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate

DRG

dorsal root ganglia

FG

Fluorogold

IB4

isolectin-B4

IPANs

intrinsic primary afferent neurons

NF

neurofilament

NGS

normal goat serum

NK1

neurokinin 1

NMDA

N-methyl-D-aspartate

nNOS

neuronal nitric oxide synthase

PAR2

proteinase-activated receptor-2

SP

Substance P

PBS

phosphate-buffered saline

TRPV1

transient receptor potential receptor V1

VIP

vasoactive intestinal polypeptide

Footnotes

APPENDIX

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuroscience.2008.02.046.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary figure. The enteric plexus of a transverse section of colon. Photomicrograph of an H&E stained section of colon showing the muscle layers and nerve plexus. LM: longitudinal muscle; M: myenteric plexus; CM: circular muscle; SM: submucosal plexus.
NIHMS1686590-supplement-Supplementary_figure__The_enteric_plexus_of_a_transverse_section_of_colon__Photomicrograph_of_an_H_E_stained_section_of_colon_showing_the_muscle_layers_and_nerve_plexus__LM__longitudinal_muscle__M__myenteric_plexus__CM.jpg (5.6MB, jpg)

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