Background
The experience of pain is usually initiated by the activation of nociceptors, which are the peripheral terminations of nociceptive ganglion neurons. The central projections of these neurons enter the dorsal horn of the spinal cord to terminate on second order neurons [
1]. After strong nociceptive stimulation these neurons may show an enhanced responsiveness to afferent inputs, which may last for several hours [
2‐
4]. The mechanism underlying this enhanced responsiveness is similar to that of long-term potentiation (LTP) [
5], which is a form of activity dependent plasticity that has been investigated extensively in other parts of the CNS, especially in the hippocampus [
6]. Another form of activity dependent plasticity is long-term depression (LTD), a state of decreased sensitivity of neurons. Whether LTP or LTD is produced in the spinal nociceptive system depends on many variables, including the type of activity in nociceptive afferents [
2]. For long term changes to become persistent it is essential that activity regulated genes, including immediate early genes (IEG), orchestrate a cascade of transcriptions and subsequent protein synthesis [
7]. The first IEG that was found to be strongly increased in spinal neurons after a nociceptive stimulus is c-Fos [
8]. This IEG is now widely used for the identification of activated nociceptive neurons [
9]. Other IEGs that have been implicated in plastic changes are c-Jun, Jun-d, Krox 24 and Homer 1a [
10,
11]. Recently it has become clear that in cortex, hippocampus and other higher brain centers, the IEG named Arc/Arg3.1 (activity regulated cytoskeleton-associated protein/activity regulated gene 3.1) plays a crucial role in activity dependent synaptic plasticity [
12]. Moreover, Arc/Arg3.1 is critically involved in processes essential for synaptic structural rearrangement such as LTP, LTD and homeostatic scaling of AMPA receptors [
13,
14]. These mechanisms are also essential in spinal processing [
15], and dysfunctional forms of activity dependent plasticity such as LTP and LTD that lead to persistent changes in neuronal sensitivity, may underlie chronic pain disorders [
16]. Therefore, in this study we set out to investigate the role of Arc/Arg3.1 in nociceptive processing in the spinal cord.
Our findings show that Arc/Arg3.1 is not expressed at detectable levels in naïve spinal cord. However, after peripheral nociceptive stimulation we found de novo expression of Arc/Arg3.1 in a limited number of neurons in the superficial dorsal horn, depending on the type of stimulus. Further, Arc/Arg3.1 is predominantly expressed in spinal interneurons located in lamina II and many of these neurons also contain the opioid neurotransmitter enkephalin. Finally, we found that the pain behavior in Arc/Arg3.1 knockout (KO) mice after nociceptive stimuli was not significantly different from their wild type (WT) littermates.
Discussion
In this study we have used in situ hybridization (ISH) and immunohistochemistry (IHC) to show that nociceptive stimulation induced Arc/Arg3.1 mRNA and protein in the superficial dorsal horn of the spinal cord. Both techniques specifically identified Arc/Arg3.1 since standard controls, most notably nociceptively stimulated spinal cord of Arc/Arg3.1 knockout (KO) mice, did not show any specific labeling. In naïve or vehicle treated animals expression of Arc/Arg3.1 mRNA and protein was absent in the spinal cord, in agreement with a study using RT-PCR [
19]. This strongly indicates that in the spinal cord a nociceptive stimulus induces
de novo expression of Arc/Arg3.1, in contrast with other areas of the nervous system, like hippocampus [
17] and cortex [
20].
Arc/Arg3.1 mRNA and protein were induced in the superficial dorsal horn in the acute phases of all pain models that we tested, i.e. after nociceptive stimulation with capsaicin, CFA, formalin and mustard oil. Injection of CFA induces an inflammatory process [
21] that leads to the release of cytokines and other local messengers, all of which may activate different types of receptors on nociceptive fibers. Capsaicin, however, specifically activates nociceptive fibers expressing the transient receptor potential vanilloid-1 (TRPV1) [
22]. Further, mustard oil and formalin both specifically activate the TRPA1 receptor, although formalin may exert TRPA1-independent effects at higher concentrations [
23,
24]. The number of neurons producing Arc/Arg3.1 mRNA varied in the different pain models, and increasing the intensity of the pain stimulus resulted in an increased number of neurons expressing Arc/Arg3.1 as shown in the mustard oil experiments. Therefore, our data indicate that the number of neurons expressing Arc/Arg3.1 depends on the intensity of the stimulus, but is not limited to the activation of one specific receptor on peripheral nerves.
Neurons expressing Arc/Arg3.1 in the spinal cord are most likely driven by direct input from afferent nociceptive fibers that use glutamate as their main neurotransmitter [
25]. Apart from glutamate and various neuropeptides, these fibers may also contain growth factors like BDNF [
26] or GDNF [
27]. We found that intrathecal injection of NMDA or BDNF induced Arc/Arg3.1 mRNA in spinal dorsal horn neurons. This is in line with Arc/Arg3.1 expression in cultured neurons following BDNF application [
18]. The same study showed a significantly enhanced expression of Arc/Arg3.1 mRNA when NBQX, a potent AMPA receptor blocker, was applied together with BDNF. However, in the present study a significant increase in the number of Arc/Arg3.1 mRNA expressing neurons could not be confirmed after intrathecal injection of BDNF and NBQX together. Taken together, our findings are in line with the idea that release of glutamate and/or BDNF from activated nociceptive fibers are at least partly responsible for Arc/Arg3.1 induction in the spinal dorsal horn.
Following nociceptive stimulation, Arc/Arg3.1 was often expressed in activated neurons as identified by c-Fos. Especially after nociceptive stimulation with capsaicin, and after chronic inflammatory pain, the number of neurons expressing Arc/Arg3.1 is low as compared to those showing c-Fos expression. This finding may be interpreted to indicate that Arc/Arg3.1 is only expressed in activated neurons that received the strongest input from nociceptive fibers. This assumption is in line with our finding that Arc/Arg3.1 expression is intensity dependent. On the other hand, there may be specific subpopulations of spinal nociceptive neurons that are capable of producing Arc/Arg3.1, while others are not. In search of such a neuronal subpopulation that specifically expressed Arc/Arg3.1, we focused on neurons that were characterized by the expression of the neurokinin-1 (NK-1) receptor, Protein Kinase C gamma (PKC-γ), calbindin, GAD67 or preproenkephalin. We found a high percentage of Arc/Arg3.1 expressing neurons (68%) to contain preproenkephalin, while percentages of colocalization with other markers were less prominent (19% for NK-1; 8.5% for PKC-γ; 3.6% for GAD67; 10% for calbindin). NK-1 expressing neurons project to supraspinal sites [
28] and are essential for the initiation and maintenance of chronic neuropathic and inflammatory pain [
29], and neurons expressing PKC-γ are considered critically important for the development of neuropathic pain after peripheral nerve injury [
30]. The finding that only a small number of Arc/Arg3.1 positive neurons also expressed NK-1 or PKC-γ indicates that Arc/Arg3.1 is not strongly involved in pain processing by the NK-1 or PKC-γ subpopulations of dorsal horn neurons. This is remarkable since especially the NK-1 expressing neurons projecting to the parabrachial area or periaqueductal grey show LTP formation after high or low frequency stimulation, respectively [
31]. Our finding indicates that Arc/Arg3.1 dependent long term changes may occur preferentially in local interneurons rather than in projection neurons. Further, we found low colocalization with GAD67, the marker for GABAergic neurons, indicating that the expression of Arc/Arg3.1 is low in the total subpopulation of dorsal horn inhibitory neurons since glycinergic neurons are virtually absent in the superficial dorsal horn [
32‐
34], and, if present, also contain GABA [
35]. In the hippocampal and neocortical neurons expression of Arc/Arg3.1 in GABAergic positive neurons is also low but this is not the case in the dorsal striatum [
20]. Together, NK-1, PKC-γ and/or preproenkephalin constitute more than 90% of the Arc/Arg3.1 expressing neurons. Since to date there is no evidence for the colocalization of these substances with each other, we conclude that Arc/Arg3.1 is preferentially expressed in the subpopulation of enkephalinergic neurons. Preproenkephalin mRNA is the precursor of both Met- and Leu-enkephalin, which are both expressed by neurons in the spinal cord and mainly exert their effect on the δ-opioid receptor (DOR) [
36]. Also, preproenkephalin mRNA in the spinal cord is increased after peripheral inflammation and is also present in neurons that express c-Fos after nociceptive stimuli [
37]. Further, using VgluT2 immunohistochemistry for identifying glutamatergic terminals, it was shown [
38] that 85% of the enkephalin containing terminals in the dorsal horn use glutamate as transmitter. However, a study [
39] using cultured dorsal horn neurons showed 42% colocalization of immunohistochemically identified GAD and enkephalin. A more recent study [
40] using preproenkephalin green fluorescent protein transgenic mice, showed that 43% of the fluorescent enkephalin neurons also expressed immunohistochemically identified GABA. Colocalization of enkephalin with VgluT2 was not explored in these studies. Since we found a low level of colocalization of Arc/Arg3.1 with GABAergic neurons, it is not unlikely that several of the enkephalinergic neurons in the spinal cord that express Arc/Arg3.1 also use glutamate as a transmitter. The functional role of glutamate in these fibers is unclear, since it is not known whether they activate inhibitory or excitatory (i.e. anti- or pro-nociceptive) circuits in the spinal cord, nor is it known under which circumstances enkephalin and/or glutamate is released from these fibers. Since the activation of the delta opioid receptor (DOR), through which enkephalin exerts its effect, decreases pain behavior during chronic peripheral inflammation [
41], we tend to conclude that the overall effect of Arc/Arg3.1 expressing enkephalinergic neurons is anti-nociceptive.
In order to understand the functional role of Arc/Arg3.1 in enkephalinergic neurons at the behavioral level, we employed Arc/Arg3.1 KO mice and their WT littermates. The only significant difference between these mice was that in the hotplate test the thermal threshold of naïve Arc/Arg3.1 KO mice was significantly higher as compared to naïve WT mice. This finding is difficult to interpret since naïve WT mice, like their KO littermates, do not show Arc/Arg3.1 expression in the spinal cord. One explanation may be that there is a very low basal expression of Arc/Arg3.1 that we and others [
19] were not able to detect, and that the permanent lack of Arc/Arg3.1 in the KO mice may have altered spinal processing of nociceptive thermal stimuli over time. Alternatively there may be supraspinal changes in nociceptive processing. After nociceptive stimuli, we did not find any difference in the pain behavior between the KO and WT mice in the formalin test and chronic inflammatory pain model. We therefore conclude that Arc/Arg3.1 KO mice do not show a clear phenotypic change that can be attributed to pain transmission in the spinal cord.
Several studies have shown that in hippocampus knockdown of Arc/Arg3.1 leads to enhanced LTP in the early phase but impaired consolidation of LTP and long term depression (LTD) in the late phase [
13]. In the spinal cord, LTP is one of the major components of central sensitization [
16], especially in lamina I projecting neurons [
31]. LTP leads to enhanced responsiveness of spinal nociceptive neurons, which is important for maintenance of hyperalgesia and allodynia during acute and chronic pain. Our finding that Arc/Arg3.1 KO mice develop hypersensitivity in acute and chronic pain models in the same way as their WT littermates, suggests that the LTP formation that contributes to central sensitization and subsequent developing hyperalgesia is unaffected by the lack of Arc/Arg3.1. It seems therefore that Arc/Arg3.1 is not critically involved in LTP as occurring in the dorsal horn projection neurons, which in line with our result that few NK-1 positive neurons express Arc/Arg3.1.
The low number of spinal projection neurons that express Arc/Arg3.1 may be explained by the fact that, in contrast to other areas of the brain, structural long-term changes in the excitability of these spinal neurons are counterproductive if they persist after the healing process has been completed. Our finding that Arc/Arg3.1 is expressed predominantly in enkephalinergic neurons may suggest that in these neurons long term changes are actually consolidated. However, Arc/Arg3.1 KO mice that lack consolidation of long term changes show normal pain behavior. This would not exclude that enkephalinergic neurons, which have an inhibitory effect on pain transmission, may serve as an anti-nociceptive mechanism against strong nociceptive inputs that may occur in the future.
Competing interests
The authors declare that they have no competing interests
Authors' contributions
MH performed or contributed to all experiments, analyzed data and drafted the paper. JLMJ contributed to experiments and analysis. KB contributed to experiments. DK provided KO mice and gave advice. JCH conceived and supervised the project and edited the manuscript. All authors read and approved the final manuscript.