Background
The conceptualization of pain in humans recognizes several interrelated components. The components involves the encoding and perception of stimulus parameters (e.g., stimulus localization, intensity, and quality), and the component involves the encoding of the affective salience or unpleasantness of the noxious stimulus [
1]. Teasing apart the mechanisms that control the neural pathways mediating pain affect and sensation in nociceptive behavioral response is a challenge. Our series of publications have demonstrated that perigenual ACC (pACC) plays a critical role in the modulation of sensory aspect of visceral pain in viscerally hypersensitive rats [
2‐
4]. The ACC has been proposed to process information relating to pain- unpleasantness, and contribute to the avoidance learning that sometimes follows as a secondary reaction to pain [
5]. One of the first studies using a rodent visceral pain assay that combines the colorectal distension (CRD)-induced visceromotor response (VMR) with the conditioned place avoidance (CPA), we measured a learned behavior that directly reflects the affective component of visceral pain [
6]. When CRD was paired with a distinct environment context, the rats spent significantly less time in this compartment on the post-conditioning test days as compared with the pre-conditioning day. Effects were lasted for several days. Lesions of pACC or administration of glutamate receptors antagonists abolished CRD-induced CPA; these data indicated that pACC activation is critical for the memory processing involved in long-term negative affective state and prediction of aversive stimuli by contextual cue [
6].
Physiological and psychological events can either strengthen or weaken the formation of memories. Progress has been made and suggested that activation of vagal afferents plays a role in the behavioral control of nociception [
7] and memory storage processes [
8,
9]. Clark et al [
8] have shown that electrical vagus nerve stimulation (VNS) given immediately after training enhanced retention performance on an inhibitory-avoidance task in rats. In human patients VNS at an intensity similar to that effect in rodent enhanced retention of verbal learning (word recognition) performance [
10]. Subdiaphragmic vagotomy attenuates the memory modulation produced by post-training systemic injections of various substances [
11], including 4-OH amphetamine, leu-enkephalin [
12], and substance P [
13]. Cholecystokinin (CCK) is a gastrointestinal hormone released during feeding [
14‐
16]. Electrophysiological and functional studies have demonstrated that CCK receptors in the vagal afferent fibers mediate various physiological functions [
17‐
20]. Based on findings in rats and human, we hypothesize that CCK activation of vagal afferent facilitates the visceral pain memory consolidation involved in long-term negative affective state.
The current study used colorectal distension (CRD)-induced visceral pain with the conditioned place avoidance (CPA). We examined the learned behavior and aversive memory in conscious rats. In combination with CRD-CPA with administration of CCK this approach enables one to determine whether CCK modulates “aversiveness memory” of visceral pain. One question addressed in the current study concerns whether CCK-induced enhancement of affective component of pain memory is specifically with aversiveness of visceral nociceptor-activating stimuli, or is associated with aversive stimuli in general. We examined the effects of CCK on CPA induced by an aversive, but non-nociceptive-activating stimulus κ-opioid receptor agonist U69,593 [
21]. To identify the role of vagal afferent
C-fibers in the mediation of CCK’s action perivagal capsaicin studies were performed. To determine if endogenously released CCK also acts on capsaicin-sensitive vagal afferent to enhance pain memory, we showed that the similar effects were observed following intra-duodenal perfusion of peptone, which has been shown to postprandial release endogenous CCK [
16,
22]. The nociceptive response (visceromotor response [VMR]) to CRD was recorded to clarify that administration of CCK does not alter the visceral pain sensitivity. Our recent studies showed that ACC activation was critical for the memory processing involved in visceral long-term negative affective state [
6], however, electrophysiological recording of pACC neurons showed that administration of CCK did not change the ACC responses to CRD.
Discussion
Pain contains both sensory and affective dimensions. Studies of the perception of unpleasantness are only amenable to human experiments where self-report is possible, whereas the neural substrates of learning and motivated behavior are more easily studied in animals. Recently, we used behavioral paradigms visceromotor responses (VMR) evoked by colorectal distention (CRD) to assess visceral pain in the conscious rat [
2,
3]. Using such tasks and models, we have shown that CRD induced conditioned place avoidance (CPA) when paired with a distinct environment context [
6]. In conditioned avoidance procedures, animal avoids stimuli based on the formation of a negative association between the given stimuli and the environment. This tendency for animal to avoid environmental cues that have been deemed aversive is believed to be affective important [
27].
Current study used three days conditioning protocol; we were able to show that a negative affective state was maintained for 3 days following acute visceral pain insult in the absence of further conditioning. These results are consistent with the notion that acute pain insult leads to the persistence of negative affective state. It is particularly interesting in light of clinical data, which indicates how the emotional or motivational effects of visceral pain may live much longer than the pain itself [
28].
Physiological and psychological events can either strengthen or weaken the formation of memories. In the animal and human, available evidences indicated that vagal nerve stimulation enhanced memory and attention [
8,
10,
29]. Several studies have shown that subdiaphragmic vagotomy attenuates the memory modulation produced by post-training systemic injections of various substances, including 4-OH amphetamine, Leu-enkephalin [
12], and substance P [
13]. These findings suggest that peripherally acting substances may modulate memory storage processes via the activation of peripheral receptors that send neural messages to the brain via the vagus nerve.
An elegant study have demonstrated that when mice were fed immediately after aversive T-maze training, they remembered that task better than when access to food was delayed and intraperitoneally administration of CCK-8 enhanced memory retention in the mice after aversive training [
19]. Similarly, when humans are fed immediately after learning, they have better recall of a complex task 48 hours later. Children who have a 63% increase in the amount eaten at breakfast have enhanced short-term memory [
30]. Cholecystokinin-octapeptide (CCK-8), is a gastrointestinal hormone released during feeding [
16,
22]. Electrophysiological studies in rats have provided evidence that CCK stimulates vagal afferent [
18,
23] to induce satiety [
31] and decrease gastric emptying [
17].
The formation of memories was strengthened by physiological and psychological events. Some of these events may occur immediately before or during the perception of a stimulus, enabling an organism to learn about it more efficiently [
32]. In contrast, arousal may also occur shortly following a learning experience, that is, during memory consolidation [
33]. The current study demonstrated that intravenous infusion of CCK at physiological doses [
20] immediately following a visceral aversive learning experience (colorectal distension), improved conditional place avoidance performance in rats suggesting that activations of CCK during the memory consolidation period were capable to enhance memory storage, rather than the acquisition, of the information. The increases in CPA scores maintained for 5 days compared with 3 days in the rats subjected to sham treatment. The higher CPA scores throughout the post-conditioning days suggests the higher degree of aversiveness, which may mimic the serious negative affective state in human and decrease pain tolerance.
The vagus nerve, like all cranial nerves, contains three types of fibers (A–C), distinguished by their physical and electrical conductance properties. We have demonstrated previously, that subdiaphragmatic electric vagal stimulation reduced pain [
7], this effect only occurred through stimulation of Aδ-fibers and was absent when C-fibers were stimulated. Capsaicin is a specific activator of the transient receptor potential vanilloid type 1 (TRPV1) channel, a polymodal nociceptive transducer expressed predominantly in non-myelinated sensory afferents. Perivagal application of capsaicin has been shown to inhibit axonal transport of peptides including substance P and somatostatin [
24,
34]. Our published data have demonstrated that CCK-8 at physiological levels stimulated vagal afferent neurons, which in turn stimulated pancreatic secretion via a vago-vagal reflex [
20,
35]. Perivagal capsaicin treatment abolished CCK-8 stimulated vagal afferent neuronal responses and pancreatic secretion [
20,
23,
24,
35]. This treatment also suppressed the vagal neuronal responses induced by secretin and 5-HT [
18,
36]. To verify that perivagal application of capsaicin was effective in ablating capsaicin-sensitive vagal afferent fibers, using retrograde tracing, we showed that perivagal capsaicin treatment prevented uptake of True blue in the nodose ganglia [
24]. In the current study perivagal application of capsaicin abolished the effects of CCK and peptone on the enhancement of affective pain memory retention suggesting CCK acts on capsaicin-sensitive vagal afferent
C-fibers to enhance pain memory.
In current study, we addressed a question concerning whether CCK modulate CRD-CPA by altering the visceral pain sensation. Behavioral visceral pain responses were examined. We showed that the rats subjected to immediately post-conditioning CCK infusion or sham treatment exhibited pressure-dependent increases in the CRD-induced VMR. These responses were not significantly changed after CCK administration suggesting CCK had no effects on the CRD-induced pain responses. Therefore, it is highly unlikely that the enhanced retention of CPA performance observed following CCK was due to alteration of the visceral pain sensitivity.
The findings of this study raise the question as to what physiological stimuli activate the vagal afferents that produce this modulation of visceral pain affection. To clarify if endogenously released CCK produce similar effects we performed study of intra-duodenal perfusion of peptone. In the rat, protein is the major dietary intestinal stimulus for CCK release. We have characterized that the release of CCK into the circulation is mediated by a “CCK-releasing peptide” secreted into the intestine of the rat [
22]. We showed in this study that intraduodenal infusion of peptone during conditioning training enhanced the pain memory retention. The effects were similar to the effects of exogenous CCK administration, and were abolished by CR-1409 suggesting postprandial released CCK played a role in modulating pain affective memory.
The anterior cingulated cortex (ACC) is a major cortical component of the limbic loop system, and its functional relationship to emotional and motivational responses have been well described [
5]. Performing single ACC neuronal activities in response to colorectal distension (CRD) our previous works have identified CRD-responsive neurons in the ACC and showed that persistence of a heightened visceral afferent nociceptive input to the ACC induced ACC sensitization [
26]. Moreover, recent studies demonstrated that neurons in the pACC are necessary for the “aversiveness” of visceral nociceptor stimulation [
6]. Since ACC activation is critical for the memory processing involved in long-term visceral negative affective state, it is important to clarify whether ACC is involved in the CCK-induced enhancement of pain aversive memory. In the present study, to determine if CCK enable to modulate ACC neuronal activities we performed electrophysiological recording of single ACC neuron in in vivo. We confirmed our previous reports that rostral ACC neurons were activated by CRD pressure-dependently. However, administration of CCK did not change the basal ACC neuronal firings. Simultaneously infusion of CCK during colorectal distension (CRD) had no effects on CRD-induced ACC neuronal responses. Thus, CCK enhances the visceral pain affective memory retention; this process appears not to be mediated by alteration of ACC neuronal responses to visceral noxious stimuli.
The most robust activation detected by fMRI following vagus nerve stimulation was seen in the thalami and insular cortices suggesting that these areas may play a role in modulating cerebral cortical activity [
37]. Currently, the mechanisms and neuronal circuitry responsible for mediating CCK-induced enhancement of visceral pain related aversiveness memory are unclear. The pain is likely to be reflected in a matrix of neuronal structures rather than in a fixed pain center. A ‘neuromatrix' incorporating, for example, the ACC and the prefrontal and insula cortices, amygdala and hippocampus may be involved in the processing of pain memory without any single region unto itself being necessary and sufficient for the pain experience [
38].
Our studies support the theory of the ongoing nature visceral pain-induced affective disorder observed in the clinic, such as the irritable bowel syndrome (IBS). Altered vagal function in patients with IBS has been reported. Findings in the current study, that feeding facilities visceral pain-related affective memory, underscores the importance of memory in visceral pain perception [
3,
28].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BC: electrophysiology study of ACC neuronal activity, technical support supervision; analysis and interpretation of data; XZ: animal surgery, VMR, interpretation of data and manuscript preparation; NY: conditioned place avoidance study, antagonist study; SC: perivagal application of capsaicin; YL: study concept, design, and supervision; analysis and interpretation of data, obtain funding and wrote the manuscript. All authors read and approved the final manuscript.