Background
Spinal central sensitization is suggested to be one of the key mechanisms underlying visceral hypersensitivity in response to visceral irritation and/or inflammation in patients and experimental animal models [
1‐
3]. Alterations in the expression and activity levels of neurochemical compounds and ion channels in the spinal dorsal horn underlie the molecular mechanisms of spinal central sensitization and is modulated by release of neurotransmitters from the primary sensory neurons located in the dorsal root ganglia (DRG) [
4,
5]. Following colonic inflammation such as colitis induced by chemicals including tri-nitrobenzene sulfonic acid (TNBS), zymosan, acetic acid, mustard oil, or dextran sulfate sodium, there are increased levels of neurotrophins and neuropeptides in the DRG and the spinal cord resulting in visceral hypersensitivity [
6‐
10]. As a result of zymosan-induced colitis, visceral hyperalgesia in rats is mediated, at least in part, by spinal
N-methyl-D-aspartate (NMDA) receptor and is attenuated by intrathecal administration of the non-competitive NMDA receptor channel pharmacological blocker [
7]. Peripheral tissue and nerve injuries recruit NMDA receptor at synapses within the pain-related spinal dorsal horn which is highly considered as an important process in spinal central sensitization under several physiologic and pathophysiologic conditions [
11‐
16].
The NMDA receptor has an ionotropic property which regulates Ca
2+ influx and Ca
2+-dependent physiological effects thereby regulating neuronal activity and synaptic efficacy. NMDA receptor forms a heterotetramer composed of two NMDA receptor 1 (NR1) and two NR2 subunits [
17]. NMDA receptor function may depend on phosphorylation of the subunits of the NMDA receptor [
18,
19]. The NR1 subunit can be phosphorylated at Ser
890 and Ser
896 by protein kinase C (PKC) and at Ser
897 by protein kinase A (PKA) [
20,
21]. This is also true in animal models. Phosphorylation of Ser
897 of the NR1 subunit in the spinal dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats is mediated by the PKA pathway and is sensitive to PKA inhibitors [
21]. Phosphorylation of Ser
896 of the NR1 subunit in the spinal dorsal horn following noxious heat stimulation of the rat hind paw is mediated by activation of PKC [
22]. In cystitis, both Ser
896 and Ser
897 sites of the NR1 subunit are phosphorylated in the spinal cord [
5]. The Ser
897 but not Ser
896 phosphorylation in the spinal cord is regulated by calcitonin gene-related peptide (CGRP) [
5]. We speculate that brain-derived neurotrophic factor (BDNF) may be involved in PKC-mediated NR1 Ser
896 phosphorylation in the spinal cord during visceral inflammation.
BDNF is a member of nerve growth factor family. The release of BDNF from the neuron somata in the DRG to the spinal dorsal horn, where BDNF binds high affinity receptor TrkB, influences associative synaptic plasticity and increases synaptic efficacy by refinement of neural connectivity [
9,
23,
24]. The involvement of BDNF in sensory hypersensitive is proved by a line of evidence and is well discussed in several animal models. In particular with colitis, systemic injection of BDNF neutralizing antibody reverses colonic hypersensitivity in response to colonic distention [
25]. Intrathecal infusion of BDNF neutralizing antibody via mini pump also attenuates colitis-associated bladder hyperactivity [
10]. In other animal models, the role of BDNF in mediating sensory sensitization is observed during peripheral inflammation-induced somatic pain [
26,
27], cancer-induced bone pain [
28], nerve injury, etc. [
29‐
32]. Traditionally, three signaling pathways are activated by BDNF in neurons: activation of the Ras-extracellular signal-regulated kinase (ERK) cascade leads to gene transcription promoting cellular growth, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has anti-apoptotic function, and activation of phospholipase C-gamma (PLCγ) initiates Ca
2+ release or influx via modulation of store activity or ion channels possibly through diacyl glycerol (DAG)-mediated PKC activation [
33]. These pathways are recently recognized as essential components in sensory hypersensitivity in several animal models [
34‐
38].
In culture, BDNF is able to modulate NMDA receptor in activity-dependent manner [
39]. In the spinal cord, BDNF regulates the activity of the NR2B-containing NMDA receptor thereby participating in the development of neuropathic pain [
40,
41]. The interrelationship of BDNF with the NR1 subunit of the NMDA receptor has also been reported showing that BDNF can regulate NR1 protein synthesis in vivo and in culture [
28,
42]. Earlier studies demonstrate a role of BDNF in inducing NR1 phosphorylation on Ser
897, the traditional PKA site, via the ERK and PKC pathways in isolated spinal dorsal horn [
43,
44]. The present study is undertaken to examine the possibility of BDNF in regulating the NR1 subunit of the NMDA receptor by phosphorylating Ser
896, the traditional PKC site, in vivo and in spinal culture, and explore the signal transduction in an animal model of colitis-induced visceral hypersensitivity.
Experimental procedures
Animals and reagents
Adult male rats (150–200 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All experimental protocols involving animal use were approved by the Institutional Animal Care and Use Committee at the Virginia Commonwealth University. Animal care was in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress, or distress as well as to reduce the number of animals used. TNBS, β-actin antibody, and other chemicals used in this experiment were purchased from Sigma-Aldrich (St. Louis, MO). Antibody against p-NR1 Ser896 was from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p-Akt and total Akt were from Cell Signaling Technology (Danvers, MA). Secondary antibodies for western blot were from Pierce Biotechnology (Rockford, IL), and secondary antibodies for immunohistochemistry were from Molecular Probes (Eugene, OR).
Induction of colonic inflammation
Colonic inflammation was induced in fasted rats under anesthesia (2 % isoflurane). TNBS solution (60 mg/mL in 50 % EtOH) at a dose of 90 mg/kg (1.5 mL TNBS solution per kg body weight) was instilled into the lumen of the distal colon through a syringe-attached polyethylene catheter via the rectum 6 cm proximal to the anus. Control animals received similar volume of 50 % EtOH enema. Euthanasia of animals was performed on day 3 or day 7 after TNBS treatment. To ensure exposure of the distal colon to TNBS, rats were held head down by lifting up the tail for 1 min. This treatment regimen caused significant inflammatory responses identified by histology [
45] as well as the up-regulation of pro-inflammatory factor in the distal colon (Additional file
1: Supplemental data).
Treatment of animals with antagonists
Animals received antagonists via intravenous (i.v.) injection. Antagonists used in this study were BDNF neutralizing antibody (36 μg/kg body weight) and the PI3K inhibitor LY 294002 (50 μg/kg body weight). A single dose of antagonist was injected. When animals were examined on day 3 post colitis induction, the antagonist was injected on the same day and post-TNBS treatment. When animals were examined on day 7 post colitis induction, the antagonist was injected on day 3 after colitis induction. This treatment design was customized by us through preliminary studies.
Tissue collection
The L1 and S1 spinal cord segments were dissected out and used for western blot, immunostaining, or acute culture. For western blot, spinal segments were freshly minced and homogenized in T-PER buffer (Pierce Biotechnology, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma). For immunohistochemistry, spinal segments were fixed with 4 % paraformaldehyde in 0.1 M PBS (pH = 7.4) followed by 20 % sucrose for cryoprotection. For acute culture, spinal segments were freshly dissected out from naïve animals, transversely sectioned at a thickness of 250 μm, and randomly divided into groups for treatment.
Western blot
The protein extracts were subject to centrifugation at 20,200g for 10 min at 4 °C, and the supernatant was removed to a fresh tube. The protein concentration was determined using Bio-Rad DC protein assay kit. Proteins were then separated on a 10 % SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5 % milk in Tris-buffered saline for 1 h and then incubated with a specific primary antibody followed by HRP-conjugated secondary antibody. For internal loading control, the same membrane was stripped and re-probed with anti-β-actin antiserum or antibody to a non-phosphorylated form of the protein examined. The concentrations of antibodies used were p-NR1: 1:1000; p-Akt and Akt: 1:1000; and β-actin: 1:3000. The bands were visualized by enhanced chemiluminescence (ECL). Densitometric quantification of the immunoreactive bands was performed using the software FluorChem 8800 (Alpha Innotech, San Leandro, CA).
Immunohistochemistry
The spinal cord segments were sectioned transversely at a thickness of 30 μm and were immunostained by free-floating method. Generally, sections were incubated with blocking solution containing 5 % normal donkey serum (Jackson Immuno Research, West Grove, PA) in PBST (0.3 % Triton X-100 in 0.1 M PBS, pH 7.4) for 30 min followed by specific primary antibodies overnight at 4 °C. After rinsing (3 × 10 min with 0.1 M PBS), tissues were incubated with fluorescence-conjugated species-specific secondary antibody for 2 h at room temperature. Following washing, the sections were mounted to slides and coverslipped with Citifluor (Citifluor Ltd., London). The sections were then viewed and analyzed with a Zeiss AxioImage Z1 Apitome fluorescent microscope.
The analysis of the immunoreactivity at the dorsal horn were done as previously reported by converting fluorescent images to a grayscale that ranged in intensity from 0 (black) to 255 (white) for the purpose of densitometry [
46]. The same number of standard sized rectangles was overlaid on the area of interest (i.e., superficial dorsal horn in this study) for each spinal section. Intensity measured within the rectangles was averaged as one point.
Spinal cord culture
Spinal cord segments were acutely cultured for 4–6 h in cell culture wells containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 200 units/mL penicillin, 200 mg/mL streptomycin, and 100 mg/mL gentamycin. BDNF (50 ng/mL) was added to the culture medium and incubated for designated time points. After incubation, tissues were collected for western blot analysis. All cultures were maintained in a 10 % CO2 environment at 37 °C.
Statistical analysis
Comparison between control and experimental groups was made by using Kruskal-Wallis non-parametric one-way ANOVA. For in vivo experiments, 4–6 animals were used for each experimental group. For culture, 3–4 independent experiments were performed. Results were presented as mean ± SE. Differences between means at a level of p ≤ 0.05 were considered to be significant.
Discussion
Colitis-induced spinal central sensitization is attributable to posttranscriptional and posttranslational regulation in the spinal dorsal horn and is modulated by mediators generated in the primary afferent neurons. The NMDA receptor is one of the key molecules in regulating neuronal excitability and synaptic plasticity. The present study investigated the signaling pathways by which colonic inflammation facilitated phosphorylation of the NR1 subunit of the NMDA receptor in the spinal dorsal horn and identified that endogenous and exogenous BDNF were able to elicit NR1 phosphorylation at Ser
896. In the spinal cord, colitis caused an increase in the level of NR1 Ser
896 phosphorylation which was attenuated by inhibition of endogenous BDNF action with a specific BDNF antibody. The level of NR1 phospho-Ser
896 was also reduced in BDNF
+/− rats treated with TNBS. The signaling pathways that transduced BDNF signal in the spinal cord leading to NR1 phosphorylation at Ser
896 included the PLCγ, PKC, and the PI3K/Akt pathways. Even though the ERK pathway was also activated in the spinal cord by BDNF during colitis [
9], the activation of this pathway did not lead to NR1 phosphorylation at Ser
896. The phosphorylation of NR1 at Ser
896 was traditionally recognized as the PKC site and was phosphorylated by PKC [
20]. The present study revealed an additional pathway, i.e., the PI3K/Akt pathway, in mediating NR1 Ser
896 phosphorylation in the spinal cord. The PI3K/Akt pathway was reported to mediate substance P (SP)-regulated NR2B subunit of the NMDA receptor [
4], but had no role in CGRP-induced NR1 phosphorylation at Ser
897 [
5]. Taken together, these results suggest that inflammation-induced spinal central sensitization characterized by site- and isoform-specific phosphorylation of the NMDA receptor is regulated by primary afferent release of neuropeptides and neurotransmitters such as BDNF that is examined in the present study. Specific signal transduction pathways are involved.
Visceral hypersensitivity is a highly complex entity that can occur due to hyperexcitability of the primary sensory afferents and dysregulation of spinal neurons (central sensitization) that modulate nociceptive transmission. Following noxious stimulation (e.g., colitis in the present study), excitatory neurotransmitters such as BDNF, substance P, and CGRP release centrally from the primary afferent neurons into the spinal dorsal horn [
46,
48‐
50] where they can bind to their respective receptors and facilitate signal transduction [
4,
9,
35,
46], ultimately resulting in pain hypersensitivity (central sensitization) [
51]. The mechanism and contribution of neuropeptides to central sensitization has been well discussed by Seybold [
52]. These neuropeptides are often stored in large dense core vesicles of unmyelinated (C) and small myelinated (Aδ) terminals, and their release are triggered by higher firing frequency-evoked intracellular Ca
2+ concentration and/or by persistent stimuli [
53‐
56]. Neuropeptides sometimes also co-store with small molecule transmitters such as glutamate which is released from both the large and small myelinated fibers that can synapse on NMDA receptor-containing neurons in the spinal cord in regulation of nociception [
57,
58].
The biological responses to neuropeptide release in the spinal cord involve a series of changes in the intracellular components. Activation of kinases is a key process in receptor-mediated signal transduction. The present study characterized three major pathways that transduce the BDNF signaling in neurons: the MEK/ERK pathway, the PI3K/Akt pathway, and the PLCγ pathway. Among these three pathways, MEK/ERK pathway is activated by BDNF in the spinal cord during colitis [
9]; however, it is not involved in BDNF-induced NR1 phosphorylation at Ser
896 characterized in the present study. We have previously reported that the phosphorylation level of Akt is increased in the L1 and S1 spinal cord during colitis [
46]. In the present study, the PI3K/Akt pathway also mediates BDNF- and colitis-induced NR1 phosphorylation at Ser
896 in the spinal cord. This is an additional pathway to the widely accepted NR1 Ser
896 phosphorylation mechanism by which PKC is involved [
20]. Akt is traditionally considered as a survival factor targeting Bcl proteins, pro-caspase, and Forkhead [
59,
60]. Several lines of recent research have suggested a critical role of the PI3K/Akt pathway in mediating sensory sensitization in response to peripheral inflammation or injury [
4,
5,
61]. The activity of Akt is regulated by PI3K-facilitated formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) which results in Akt trafficking and activation [
62]. In the periphery, Akt is activated in the sensory neurons and regulates pain perception by activating TRPV1 receptor [
61,
63]. In the spinal cord, the Akt activity can be regulated by BDNF, CGRP, and SP in a PI3K-dependent manner [
4,
5,
46]. In terms of visceral inflammation, activation of the PI3K/Akt pathway is unable to regulate NR1 phosphorylation at Ser
897, but mediates NR1 phosphorylation at Ser
896 (the present study) in the spinal cord, suggesting that the PI3K/Akt pathway is important but not the sole pathway in visceral inflammation-induced NMDA receptor activation and spinal central sensitization.
The interrelationship of Akt activation and NMDA receptor activity has been demonstrated in several systems. A study in a formalin-induced hyperalgesia model shows that inhibition of the PI3K/Akt pathway blocks peripheral inflammation-induced phosphorylation of NR2 subunit of the NMDA receptor in the spinal cord [
4]. In cerebellar granule, cell culture activation of Akt is also able to mediate forskolin-induced phosphorylation of Ser
897 in the NR1 subunit [
64]. In inflammatory and neuropathic pain models, the NR1 phosphorylation is enhanced in the spinal cord [
65,
66], and in these studies, the antibodies used for NR1 phosphorylation are selective for either the Ser
897 site alone or both the Ser
897 and Ser
896 sites together but not Ser
896 alone. In our current study, we use an antibody that recognizes specifically Ser
896 of the NR1 and find that this site is phosphorylated in vivo during colitis. We have previously tested that CGRP is unable to increase NR1 phosphorylation levels at Ser
896 [
5]. The present study demonstrates that p-NR1 Ser
896 is regulated by BDNF-induced PLCγ and PKC cascade, consistent to the established findings [
20,
67]. The present study also reveals that BDNF- and colitis-induced NR1 phosphorylation at Ser
896 is regulated by the PI3K/Akt pathway. However, there is no biochemical evidence to show whether the PI3K and/or Akt can use Ser
896 of NR1 as a phosphorylation substrate. Akt serves as a convergent point in signaling network and is activated by growth factors and G-protein coupled receptors and also cross talks with other signaling pathways in cells and tissues. In the complex PI3K/Akt signaling network, the mammalian target of rapamycin complex can promote the stability and activity of PKC [
68]. In spinal slice culture, it takes 1 h for BDNF to stimulate NR1 phosphorylation, which implies that complicated signaling cross talk may exist within the spinal cord. Akt can be activated by a number of cytokines that are discovered in the spinal cord, such as the pro-inflammatory cytokine tumor necrosis factor (TNF) that is released from spinal glial cells [
34,
69]. The involvement of cytokines in persistent pain has been well discussed recently [
70]. These studies along with our current findings suggest a key modulatory role of the PI3K/Akt pathway in the generation and maintenance of visceral and somatic hypersensitivity and pain by being activated by a number of inflammatory and neuronal factors and regulating a number of ion channels.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ML, JCK, and LYQ designed and conducted most of the experiments. SS designed and conducted some of the experiments. ML and LYQ wrote most of the manuscript. All authors analyzed the data, revised the manuscript, and gave final approval for publication.