As mentioned earlier, visceral pain perception and psychological processing is divergent to that of somatic pain (1). Importantly, there are clear distinctions which set visceral pain aside from all other pain modalities. These clinical features are crucial for the understanding of this complex physiological process. The characteristics of visceral pain were first outlined by Cervero and Laird (1) and have advanced our appreciation of this complex phenomenon. These characteristics are summarized in Table 1.

Visceral pain is the pain associated with a wide variety of disorders including gallstones, acute pancreatitis, acute appendicitis, diverticulitis, painful functional bowel syndromes such as irritable-bowel syndrome (IBS) and functional dyspepsia (FD), inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), interstitial cystitis/bladder pain syndrome (IC/BPS), male chronic pelvic pain syndrome, and gynecological pain [endometriosis, vulvodynia, menstrual pain, polycystic ovary syndrome (PCOS)] (16–20). Moreover, and less commonly known is that visceral pain also encompasses chronic chest pain and colic (21, 22).

Appreciating this wide array of disorders has allowed us to understand the complex nature of the pathophysiology of visceral pain but have also induced considerable hurdles when aiming to fully understand the distinct subset of molecular mechanisms which underpin this phenomenon. For the remainder of this review, we will specifically discuss gastrointestinal (GI) visceral pain.

Knowing that the clinical characteristics of visceral pain are distinct from that of somatic pain, it implies that the neurobiology must also be distinct between these modalities. Indeed this appears to be the case with numerous groups reporting both anatomical and physiological differences (23–26).

Pathways for visceral sensation are diffusely organized both peripherally and centrally. Communication of sensory information from the GI tract to the central nervous system (CNS) occurs via vagal, pelvic, and splanchnic nerve pathways (27–29). Vagal afferents innervate the GI tract from the esophagus to the transverse colon (8). Pelvic nerves innervate the remaining parts of the colon and rectum (8). A smaller group of afferents called the splanchnic afferents whose cell bodies arise from the thoracolumbar region of the spinal cord innervate the whole GI tract (27), Figure 1.

More specifically, primary afferent nerve fibers innervating the viscera, project into the CNS via three pathways: (1) the vagus nerve and its branches; (2) sympathetic pathways; and (3) the pelvic nerve (parasympathetic pathways) and its branches (26). Primary afferents signaling to the CNS reside primarily in the vagal nodose ganglion, which project to the nucleus tractus solitarius (NTS) located within the medulla of the brainstem (8), and in the T2–L2 and S1–5 dorsal root ganglia (30).

Visceral primary afferents have been demonstrated to enter the spinal cord and form synapses with dorsal horn neurons ipsilateral and contralateral to the site of entry. The result is extensive, diffuse CNS activation (31, 32). These axons form the postsynaptic dorsal column pathway. Interestingly, the dorsal column in itself has been shown to relay visceral nociceptive information and is now thought of as a visceral pain pathway in its own right. Numerous clinical studies have shown that lesioning the fibers of the dorsal columns significantly relieves pain and decreases analgesic requirements in patients suffering from cancer originating in the visceral organs (33, 34).

Visceral afferents terminate in laminae I, II_(outer), V–VII and X in the spinal cord. Laminae I and V form part of the spinothalamic tract, laminae VII and X constitute part of the dorsal column pathway (35, 36).

Second-order processing of visceral stimuli occurs at spinal segments and brainstem sites receiving primary afferent input. Figure 2 depicts the principal visceral afferent pathways projecting to the spinal cord, and then ascending to the thalamus and midbrain. These are the spinothalamic, spinoreticular, and spinomesencephalic tracts (37, 38). The spinothalamic tract transmits sensory information from the spinal cord to the reticular formation in the brainstem and terminates in the thalamus (medial and posterior) at which point the thalamocortical fibers project to the primary somatosensory cortex (38). This tract is responsible for sensory discrimination and localization of painful stimuli and thus the reflexive, affective, and motivational properties of noxious stimulation (38, 39).

The brain regions innervated by these pathways, more commonly known as the pain matrix, which are activated in response to colorectal stimulation include the prefrontal cortex (dorsolateral), the insula, the thalamus, the amygdala, and the anterior cingulate cortex (ACC) (38). The ACC is a critical component in the pain matrix and is functionally divided into two discrete regions, the perigenual ACC and the midcingulate cortex (MCC), with the former involved in affect and the latter in behavioral response modification (38, 40). Moreover, the amygdala, in particular, the central nucleus of the amygdala (CeA), is a critical region in the limbic system and the pain matrix. The CeA integrates many signals for the processing of painful stimuli. It receives input from the brainstem, as well as more complex information from the thalamus and the cerebral cortex. Moreover, it has also been shown to have direct synaptic interactions with locus coeruleus (LC) neurons (41), highlighting a clear role of stress pathways in the development of visceral pain. Furthermore, the amygdala is also known to receive afferents from the spinal cord through the spino-(trigemino-)parabrachio-amygdaloid pathway (42). It is also involved in the descending pain pathway and is involved in the emotional, affective, and cognitive functions of pain processing. Numerous preclinical studies have shown the important role of the amygdala in pain processing (43–52).

This multicomponent integration of nociceptive information explains the variability in the experience and reporting of visceral pain (37, 53) and thus the difficulty in development of effective pharmacological treatments. Interestingly, diffuse noxious inhibitory controls (DNIC) are a phenomenon more commonly referred to as “pain inhibiting pain” whereby a painful stimulus is applied to a part of the body, distant from the actual site of pain, thus inhibiting neurones within the dorsal horn of the spinal cord that are actively responding to chronic (unexplained) pain as seen in visceral hypersensitivity (54). DNIC is frequently used to quantify the central pain sensitization in chronic pain patients such as in the case of IBS (55–58). IBS patients consistently show a deficit in DNIC which correlates with symptom severity. These findings elude to the hypothesis that chronic pain patients are not only hyper-sensitive to pain but they also demonstrate reduced DNIC, possibly because of dysfunction of endogenous pain inhibition systems (55).

Neurotransmitters, cytokines, and other mediators such as peptides and neuropeptides are thought to mediate visceral nociceptive signals from the periphery to the central pathways. Indeed, mediators released during peripheral inflammation and injury, are thought to influence spinal nociceptors resulting in increased nociceptive activity and central sensitization (27). For example, the inflammation and irritation associated with bladder infections is believed to cause the release of glutamate that sensitizes visceral primary afferents (59). Moreover, glutamatergic signaling in particular, metabotropic glutamate (mGlu) receptors and glutamate reuptake are fast becoming attractive areas of research in the context of visceral pain (60, 61). However, there are a whole host of other mediators and receptors that are involved in visceral pain processing including; neurotransmitter receptors [acetylcholine nicotinic receptors (62), cannabinoid receptors (63), opioid receptors (64, 65), GABA_A, GABA_B, and GABA_C receptors (66, 67), glutamate (ionotropic) receptors (68), glutamate (metabotropic) receptors (69, 70), glucocorticoid receptors (47, 71)], inflammatory receptors (bradykinin receptors, cholecystokinin receptors, cytokine receptors, leukotriene receptors, prostanoid receptors, tachykinin receptors, nitric oxide signaling, cyclooxygenase, lipoxygenase) (72, 73) and ion channel receptors [transient receptor potential vanilloid (TRPV) (74), purinergic (P2X) receptors (75), voltage-gated calcium channels (Ca_V), voltage-gated potassium (K_V) channels, voltage-gated sodium (Na_V) channels] (76). Due to the vast array of mediators and pathways, a clear pathophysiology of visceral pain remains to be elucidated thus hindering drug development.

Although most visceral pain disorders are not life-threatening (non-cancer pain), they have a considerable negative impact on the quality of lives of patients with increased psychological distress, increased work absenteeism and both sleep and sexual dysfunction (77, 78). There are currently no pharmacological treatments on the market specifically for visceral pain. This leads to persistent bouts of discomfort and possible debilitation for the patient but also results in recurring visits to clinicians, associated with a significant economic burden on both the patient and healthcare services (12). Many patients are treated with multiple drug combinations to no avail. This void of effective analgesics in the context of visceral pain is frustrating for all parties involved. The drive to develop new analgesics has started right back to the basic molecular mechanisms of which very little is known (60, 79). Basic science and animal models have proved crucial in this effort for future developments of novel visceral analgesics.

In the clinical setting, treatment of visceral pain is extremely difficult. This is due to its complex nature, in that individuals can have many different triggering factors of their visceral pain and with no known cause, effective treatment strategies are difficult to identify. Treatment can differ from patient to patient and indeed treatment of the same patient over time may also change. As a result of this, a wide variety of pharmacological tools are used including a variety of analgesics [opioids (80), non-steroidal anti-inflammatory drugs (NSAIDS) (81), benzodiazepines (82)] and others (antibiotics, laxatives, serotonin modulators) (83, 84). Moreover, patients may also be treated with antispasmodics, particularly in the case of GI visceral pain and anti-depressants as well as others (84). Due to this heterogeneous pharmacological treatment profile, there are numerous and serious side effects including constipation, sedation, vomiting, tolerance, dependence, and addiction. However, none of the above are specific for the treatment of visceral pain per se, and mainly target some other features associated with chronic pain in general. However, in recent years, promising findings are emerging from both preclinical and clinical research which are nicely reviewed in Ref. (15, 83, 85, 86).

As with all chronic/severe pain disorders, opioids form the core of pain management for visceral pain conditions. However, as mentioned earlier, this class of analgesics are associated with the most serious side effects particularly over chronic use ranging from constipation (87) to analgesic tolerance (88–90) and nociceptive sensitization (91). More worryingly is the development of opioid-induced hyperalgesia after periods of prolonged opioid use (92–94).

Over the counter analgesics are routinely used by patients suffering with visceral pain. These include NSAIDS (aspirin, ibuprofen) and paracetamol. Again as with the case of most pharmacological agents used in the treatment of visceral pain, these agents do not specifically target visceral pain and only provide mild pain relief. Moreover, as these compounds are available over the counter they can be abused by consumers and carry a host of side effects not least GI bleeding, increased stomach acid, liver disease, liver failure, and even death in some cases (95).

Serotonergic compounds such as tegaserod (5-HT4 agonist) and alosetron (5-HT3 antagonist) (96–102) have been the main route of treatment for a range of visceral pain conditions, in particular IBS. However, tegaserod has since been removed from the market due to significant cardiovascular effects as outlined by the FDA. Alosetron was also withdrawn from the market in 2000 due to life-threatening adverse GI effects, however, in 2002, it was reintroduced but its availability and use were dramatically restricted.

Recent evidence from preclinical studies has pointed to a role of the transient receptor potential (TRP) channel family in the pathophysiology of visceral pain, which may lead to future development of novel therapeutics (103, 104). Moreover, both preclinical and clinical data have shown analgesic efficacy of pregabalin and gabapentin in acute and chronic visceral pain conditions, acting both at spinal and supra-spinal levels in particular at the level of the rostral ventromedial medulla (RVM) (60, 105–108). Furthermore, the efficacy of mGlu receptors and also glial glutamate transporters have revealed themselves to be very promising targets (59, 61, 70, 109–115).

As mentioned previously, there is an unmet need for effective pharmacological therapeutics in the context of visceral pain. However, there are still major challenges to be met, not least in the fact that we still lack a clear understanding of the etiopathogenesis of visceral pain disorders.

Psychiatric disturbances are the most frequent comorbidity of visceral pain (116–125). In particular, anxiety and depression are the most commonly reported comorbidities. This complex link between visceral sensation and psychological perceptions are mediated via the brain–gut axis.

Moreover, stress-related changes in bowel habit can attest to the fact that the brain can influence gut function and sensation (126). Several clinical studies have suggested that psychosocial comorbidity is a major contributor to the severity and impact on quality of life of visceral pain disorders such as IBS and somatic pain disorders such as fibromyalgia (127–131). These findings are reinforced by a considerable volume of experimental research that links stress, anxiety, and depression to altered GI sensory and motor function as well as altered pain processing (8, 132–138). Indeed, successful management of patients with visceral pain disorders requires careful attention to these psychosocial factors, often in consultation with mental health professionals.

The etiology of visceral pain is most likely multi-factorial involving biological, psychological, and social factors leading many to describe visceral pain as a biopsychosocial disorder. Due to the array of comorbidities associated with visceral pain, it is clear that both the brain and viscera play significant roles through the brain–gut axis (139–142). Moreover, the emerging role of the gut microbiota on brain signaling has now led to the concept of the microbiota–brain–gut axis (143–145). Numerous other pathways and systems feed into this complex network of communication, including the stress axis and immune system. The role of the amygdala in IBS has been extensively reviewed in the context of brain–gut axis communication (146) and numerous clinical and preclinical trials have also highlighted an important contribution of the amygdala to visceral pain processing and IBS (52, 147, 148). Here, we discuss the pathophysiology of visceral pain in terms of signaling along the microbiota–brain–gut axis, the hypothalamic–pituitary–adrenal (HPA) axis, and the immune system. Many other factors such as gender, genetics, and epigenetics are implicated in these pathways which not only exacerbate visceral hyperalgesia but may also be predisposing factors for the development of visceral hypersensitivity in later life. Furthermore, we also discuss possible future targets for visceral analgesia including the modulation of mGlu receptors in addition to glial glutamate transporters and histone deacetylation.

The HPA axis is the main stress axis in mammals and its anatomical structures are located both in the CNS and in the periphery. The major components of the stress axis are localized in the paraventricular nucleus (PVN) of the hypothalamus, the pituitary gland (anterior lobe), and the adrenal gland (154). In response to stress, corticotrophin-releasing hormone (CRH) is released and travels to the anterior pituitary gland where binding of CRH to CRH receptors (CRHR1 and CRHR2) leads to the release of adrenocorticotropic hormone (ACTH) into systemic circulation. ACTH targets the adrenal cortex to produce and secrete glucocorticoids (154). Glucocorticoids are the main effector molecules of the stress response and regulate the physiological adaptations through binding to their intracellular receptors (162, 163). Dysregulation of the HPA axis via inadequate or excessive activation, is thought to contribute to the development of a wide array of pathologies (154, 162, 164). Indeed CRH and its receptors have been extensively investigated in the context of stress and visceral pain (165–177). Taken together, it is apparent that stringent regulation of the HPA axis is essential to a normal adaptive and efficient stress response.

The bidirectional signaling network that exists between the GI tract and the brain is vital for maintaining homeostasis and is regulated at the neural [both central and enteric nervous systems (ENS)], hormonal, and immunological levels. Perturbation of these systems results in alterations in the stress response and overall behavior (143, 207). The high rate of psychiatric comorbidities with GI disorders and vice versa (208–210) are further evidence of the importance of this network of communication. The brain–gut axis is by no means a new discovery however its role in many disorders outside of gastroenterology has become an area of intense research. Moreover, advances in biomedical research have allowed us to elucidate the role of the gut microbiota community in signaling along this axis, which is now more commonly referred to as the microbiota–brain–gut axis (Figure 3). The impact of the microbiota–brain–gut axis has become a rapidly advancing research topic encompassing broad domains of biomedical research including neuroscience, psychiatry, gastroenterology, and microbiology.

The classical construct of the brain–gut axis, describes the way in which complex bidirectional signals can transmit between the CNS and the GI tract. This axis is critical for preserving gut homeostasis, dysregulation of which has been implicated in an array of disorders and disease states including gut inflammation, chronic abdominal pain syndromes, and eating disorders (143–145, 210–217). The brain–gut axis is responsible for some of our most basic functions such as facilitating the central regulation of digestive function. Indeed, the role of this axis is a well-developed concept in the area of food intake and satiety (218, 219) and more recently obesity (220, 221).

Moreover, the role of the microbiota–brain–gut axis in the stress response via modulation of the HPA axis has been investigated by numerous independent research groups (222–228). Furthermore, many forms of psychological stress have been shown to alter the gut microbiota fingerprint with many prebiotics and probiotics showing beneficial effects against the negative impact of stress. Thus, it is now acknowledged that the gut microbiota themselves are critical mediators of information along this bidirectional communication network (145, 207, 211, 213, 229, 230).

Indeed, in IBS patient cohorts, numerous independent research groups have shown distinct gut microbiota populations when compared to healthy controls (231–236). This was recently reviewed by Mayer and colleagues (237). Moreover, probiotic interventions appear to be beneficial to IBS patients (238).

Manipulation of the gut microbiota through the use of probiotic and prebiotics treatments have shown that by augmenting so-called “good bacteria” such as Bifidobacteria and Lactobacillus, in the gut, visceral hypersensitivity can be reversed in preclinical models. A mixture of eight probiotic bacteria strains (VSL#3) was shown to have protective effects against development of visceral hypersensitivity in the neonatal maternal separation model. Moreover, TPH1, tryptophan hydroxylase 1, the gene for the enzyme responsible for synthesizing serotonin, a key neurotransmitter involved in IBS treatment, was markedly up-regulated by neonatal maternal separation and this effect was reversed by VSL#3 intervention (239). Moreover, the same cocktail of probiotics was shown to prevent visceral hypersensitivity induced by inflammation via intra-colonic instillation of 4% acetic acid when given prophylactically (240). Bifidobacterium species particularly, Bifidobacterium infantis 35624 has been shown to be particularly effective at ameliorating visceral hyperalgesia in both stress-induced visceral hypersensitivity and colitis (241–243). Moreover, Lactobacillus species have also displayed efficacy in visceral pain models (244–247).

Furthermore, antibiotic-induced visceral hypersensitivity again underpins a role of the gut microbiota in the pathophysiology of visceral pain (244, 248). Interestingly, rifaximin, a semisynthetic, non-absorbable antibiotic that demonstrates no clinically relevant bacterial resistance has also shown positive effects in the treatment of IBS (249–257). These findings may seem contradictory, however, rifaximin is particularly efficacious in cases of small bowel bacterial overgrowth found in IBS patients. These findings add to the growing literature that microbiota dysfunction may be a key player in the pathophysiology of IBS and may lead to future novel therapeutic interventions.

Microglia represent the first line of defense for the CNS, acting as a sensor for pathological events (268). The process of central sensitization and the subsequent changes in synaptic plasticity has long been thought to play a major role in nociceptive processes both in the context of chronic pain as well as acute, in both somatic and visceral modalities (269, 270). In the last decade, the role of microglia, both spinal and supra-spinal, has become an area of interest in the context of nociception (271–273). In animal models of both inflammatory and neuropathic pain, activation of microglia is a key step in the onset and maintenance of hypersensitivity and allodynia (274–279).

The role of spinal microglia in visceral pain has only recently been addressed and reviewed nicely by Lu (269). Saab and colleagues were first to report increased activated microglia in a rat model of chronic visceral hyperalgesia, namely the neonatal colon irritation model (280). Moreover, minocycline, a second-generation tetracycline compound known to interrupt microglia activation and its associated pro-inflammatory response, reversed the visceral hypersensitivity in adulthood (269, 280).

Furthermore, enhanced microglia activation was reported in the hippocampus, in a model of trinitrobenzene sulfonic acid (TNBS)-induced colitis, concomitant with increased tumor necrosis factor-α (281). However, this study did not assess nociceptive behavior, but one could predict visceral hyperalgesia as observed by others in the TNBS-model (282). More recently, it was shown that the activation of spinal microglia plays a critical role in the initiation and maintenance of visceral hypersensitivity in the TNBS-induced chronic pancreatitis (CP) rat model (283). Intrathecal injection of minocycline attenuated visceral hypersensitivity and phospho-p38 levels in CP rats.

In other non-inflammatory models of visceral hyperalgesia, it has also been shown that chronic psychological stress leads to microglia activation in the lumbar spinal cord (284). Moreover, this stress-induced increase in activated microglia could be blocked by SB203580, a p38 inhibitor or minocycline. Other compounds known to alter the microglia phenotype such as FKN, a microglia activator, was shown to induce visceral hypersensitivity in naïve rats when administered spinally, an effect which was blocked by minocycline (269), thus adding further evidence to support the role of spinal microglia in mediating stress-induced visceral hypersensitivity (269). Taken together, these studies suggest that microglia may play an important role in the pathogenesis of visceral pain.

However, it is also pertinent to note that exact role of inflammation and indeed micro-inflammation still remains controversial in the context of IBS. Indeed, no effect of anti-inflammatory drugs has been shown in IBS such as prednisolone (285).

Toll-like receptors (TLRs) are a family of pattern-recognition receptors of the innate immune system (Figure 4). There are 10 human TLRs and 12 mouse TLRs (286). TLR signaling consists of at least two distinct pathways: (1) the MyD88-dependent pathway, which leads to a pro-inflammatory phenotype, and (2) the MyD88-independent pathway, which leads to the production of interferon-β and the maturation of dendritic cells. The MyD88-dependent pathway is common to all TLRs, except TLR3 (287). TLRs are important players in the maintenance of mucosal and commensal homeostasis within the gut via innate host defense mechanisms. Intestinal dysbiosis and inflammation underlie several disease states affecting the GI tract such as IBS and IBD (288, 289). Indeed, reports have shown the involvement of peripheral TLR4 in patients suffering from IBS (290, 291) and in animal models of visceral pain (289, 292, 293). Moreover, the presence of TLR4 in the ENS and in the dorsal root ganglia indicate a role for TLR4 in the transmission of sensory information from the GI tract (294, 295). Furthermore, TLR4 is also expressed within the CNS, predominately in microglia (296), which have been discussed above and their role in visceral pain. Moreover, it is now emerging as a possible therapeutic target for neuropathic pain (297). Taken together, these findings suggest TLR4 as a promising novel target for the treatment of visceral pain.

N-Methyl-D-aspartate (NMDA) receptors are not only known for their important role in excitatory synaptic transmission but also for their role in pain (346). Glutamate found in vagal and spinal afferents contributes to nociceptive signaling via NMDA and non-NMDA receptors (347). Though evidence for direct links between glutamate and visceral nociception is lacking, NMDA receptor antagonists have been shown to reduce the response of vagal and pelvic afferents in the colon and other viscera to mechanical stimuli (348). A study by Coutinho and colleagues (349) investigated the role of glutamate receptors in the RVM in visceral hyperalgesia using a colonic irritation model. They found that activation of NMDA receptors by colonic inflammation facilitated visceral hyperalgesia while non-NMDA receptors mediate inhibitory effects (349). Moreover, NMDA receptors may also be involved in the transmission of visceral nociception in the non-inflamed gut (350). It was observed that activated peripheral NMDA receptors in the colon caused the release of pro-inflammatory peptides, calcitonin gene-related peptide (CGRP) and substance-P, which have significant roles in the mediation of chronic and severe pain (350).

Metabotropic glutamate receptors with the exception of mGlu6 receptor are expressed in all areas of the pain matrix from the spinal cord to supra-spinal sites. The action of mGlu receptors can be pro-nociceptive or anti-nociceptive depending on the subtype and site of action (351).

In the last decade, the role of mGlu receptors has gained attention in the realm of visceral pain. To our knowledge, the first study explicitly investigating the role of these receptors in visceral nociceptive processes was performed by Chen et al. (352). They found that antagonism of group 1 mGlu receptors with LY393053 reduced nociceptive behaviors in the mouse acetic acid writhing test. Furthermore, administration of a group 1 mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the CeA by microdialysis increased the responses to innocuous visceral stimulation; an effect that was reversed by a reactive oxygen species (ROS) scavenger phenyl-N-t-butyl nitrone (PBN) and a superoxide dismutase mimetic (TEMPOL). In the same study, mGlu receptor 1 antagonist LY367385 was also found to decrease the responses to visceral stimulation (353).

Moreover, work by Lindstrom and colleagues investigated specifically the role of mGlu5 receptor in a rat model of visceral hypersensitivity (111). Here they found that mGlu5 receptor antagonism with MPEP [2-Methyl-6-(phenylethynyl)-pyridine] and MTEP [3-(2-Methyl-1,3-thiazol-4-yl)ethynyl]pyridine was sufficient to reduce the visceromotor response (VMR) in conscious Sprague-Dawley (SD) rats without altering colonic compliance. Moreover, mGlu5 receptor antagonism reduced colorectal distension (CRD)-evoked increases in heart rate and blood pressure (111). In this study, the effects seen could not be conclusively due to a peripheral or central site of action. Indeed, it has also been found that noxious colonic stimulation increases the number of Fos-positive neurons in the dorsal horn of the thoracic and lumbar spinal cord. Moreover, pre-treatment with MPEP significantly attenuated this (354).

Visceral pain originating from the bladder has also been shown to be mediated via mGlu receptors; specifically mGlu receptor 5 activation in the CeA induces bladder pain sensitization by increasing CeA output, an effect that was reversed by intra-amygdala MPEP treatment and lentivirus-mediated conditional disruption of mGlu5 receptor in the CeA (70).

Due to the negative side effects (psychomimetic activity) seen with modulation of ionotropic receptors, compounds which target these receptors are not suitable for long-term treatment of pain. To redress this and in the drive to develop better analgesics, other mechanisms such as glutamate reuptake may provide more effective treatments in controlling glutamate neurotransmission and thereby exert anti-nociceptive effects (109).

One of the first clear demonstrations that glutamate transport [via excitatory amino-acid transporters (EAATs)] may be implicated in pain processing was performed by Liaw and colleagues where they showed that selective inhibition of glutamate transporters with dl-threo-β-benzyloxyaspartate (TBOA) and dihydrokainate (DHK) produced a dose-dependent spontaneous nociceptive behavior response. These behaviors included licking, shaking, and caudally directed biting (355). Moreover, TBOA administered intrathecally was also found to induce visceral nociceptive behaviors in naïve rats (114). To further unravel the role of glutamate transport in visceral nociception, in particular stress-induced visceral hypersensitivity, the neuroprotective drug riluzole known to activate glutamate reuptake, was investigated to see whether it could attenuate visceral hypersensitivity induced by maternal separation of rats. It was found that riluzole reduces visceral hypersensitivity in stressed animals only, having no effect in non-separated animals. Moreover, EAAT-1 expression was also found to be reduced in the lumbar region of the spinal cord in hyper-sensitive animals. As mentioned previously, riluzole does not affect visceral sensitivity in naïve animals, further emphasizing the role of glutamate transport in pathological pain (114).

EAAT-2 is the main glial transporter for glutamate reuptake and its experimental over-expression in animal models has recently been found to be effective in reducing visceral pain (59, 112, 113). A study by Lin and colleagues (112) found the over-expression of EAAT-2 with the use of cephalosporin antibiotic ceftriaxone, in wildtype mice, attenuated the visceral nociceptive response to CRD. Moreover, similar effects were seen in the EAAT-2 overexpressing transgenic mouse (112). To further support these findings, systemic and intrathecal administration of DHK, a selective EAAT-2 inhibitor blocks the ceftriaxone-induced attenuation of visceral pain (112, 113). In subsequent studies performed by the same group assessing the efficacy of glutamate reuptake in animal models of colitis, it was shown that adeno-associated virus-mediated EAAT-2 over-expression was effective to mitigate VMR to 60 mmHg CRD (113). Interestingly, overexpression of EAAT-2 has been shown to reduce bladder nociception (59). However, colon irritation may affect afferents innervating the bladder thus giving a plausible mechanism for cross-organ sensitization (59). In the same study, it was also found that enhanced expression of EAAT-2 by ceftriaxone also reduced the VMR to bladder distension caused by colonic irritation (59).

As stated earlier, stress is one of the main predisposing factors for the development of visceral pain. Interestingly, glutamate transport has also been shown to be altered due to stress, with both early-life stress and stress in adulthood both showing significant alteration in EAAT expression (114, 115). Taken together, these findings provide evidence for an important role of glutamate transport in visceral pain states (271, 356). Moreover, glutamatergic signaling is critical to the process of central sensitization. This term describes the way in which excessive glutamatergic function within neurons and circuits, in particular pain circuits, leads to increases in membrane excitability and synaptic efficacy as well as reduced inhibition. These changes in synaptic function manifest as altered plasticity within the somatosensory nervous system in response to activity, inflammation, and neural injury (357).

Colorectal distension is the most widely used method to assess visceral sensation both preclinically and clinically. This technique involves insertion of a balloon into the colorectal cavity of the human subject or animal, and with the aid of a barostat, distending in a repeated or ascending phasic manner. Although the main premise of the technique is essentially identical, the way in which it is performed varies between laboratories and researchers. This can be due to numerous factors including the model employed; whether it be human, rat, or mouse, the parameter of interest; baseline visceral sensation, pain threshold, and tolerance to painful stimuli. CRD has been characterized in humans, rats, and mice with the bulk of the work being performed in rats due to ease of use and robust reproducibility (408).

In 1988, Ness and Gebhart were the first to describe a technique used to assess visceral sensitivity in the preclinical setting. The technique was based on the assessment of pseudoaffective and behavioral responses to controlled isobaric distensions of the GI tract. This has now become the mainstay for assessing visceral pain both clinically and preclinically (409). CRD in rats induces an array of autonomic and behavioral outputs termed pseudoaffective reflexes. These reflexes include; alterations in blood pressure and heart rate, passive avoidance behaviors, and contraction of the abdominal musculature (8), the latter of which is more commonly referred to as the VMR. VMR is the most widely used parameter of the visceral pain response (8, 408).

In the last decade, the use of electromyographic (EMG) signals and its numerous applications have allowed many to assess visceral sensitivity in conscious animals. This procedure allows recording from electrodes which are implanted in the musculature and externalized through the skin, primarily on the abdomen or neck (410–412). They can also be connected to radiotelemetric implants in the abdominal cavity (413, 414). Moreover, the development of manometric recordings measuring changes in the pressure of the balloon inserted into the GI tract, have also allowed for VMR to be assessed in freely moving animals (134, 175, 311, 408, 415, 416). Other groups have implemented other indirect approaches such as manual scoring of the abdominal withdrawal reflex (408, 417), operant behavioral assays (409), and functional brain imaging tools such as functional MRI (44, 418).

Mood disorders are commonly comorbid with IBS (432–434). Genetic predisposition to such disorders has been implicated in the development of IBS in later life. These genetic factors may not lead directly to the development of IBS per se but may indirectly, through heightened stress responsivity, cause altered GI function and IBS symptomatology. The availability of a catalog of rodent strains has led to the advent of genetic studies assessing the exact contribution of genetic factors to disease presentation and progression. Moreover, the use of transgenic rodent strains allows us to specifically investigate single genes implicated in disease pathology.

Using different rat strains of known levels of baseline anxiety: low-anxiety SD and Fisher-344 (F344), and high-anxiety WKY rats, Gunter and colleagues were able to demonstrate a link between anxiety and visceral hypersensitivity. Specifically, high-anxiety WKY animals had increased response to CRD compared to low-anxiety strains (132). Moreover, WKY rats exhibited an exaggerated response to acetic acid instillation into the colon, a peripheral sensitization model, compared with low-anxiety strains, SD and F344 (132).

The role of the stress response and the development of IBS was again shown to be intrinsically linked when CRHR1 (CRHR1^−/−) knockout animals were shown to have an altered VMR to CRD. VMR to CRD was only observed at the highest distension pressure (60 mmHg). Moreover, pharmacological CRHR1 antagonism decreased the VMR to CRD in CRHR1^+/+ mice (167).

Small interfering RNA (siRNA) technologies have now become one of the most widely used approaches to selectively suppress gene expression and is a powerful tool to assess gene function. This technology has been used extensively to suppress stress-related genes in specific brain areas and models are continuously being developed (435–440). Whilst genetically modified mice have been the mainstay of behavioral genetics to date there is a growing utility for genetically modified rats (441). Moreover, other genetic tools such as optogenetic and Designer Receptor Exclusively Activated by Designer Drugs (DREADD)-based manipulations allows for both species to be used in the future (442).

The role of the microbiota–brain–gut axis on health and disease is an area of biomedicine that is receiving much media attention of late. The complex communication between the gut microbial population and the CNS has far reaching implications not least in the area of visceral pain and psychiatric comorbidities. Modulation of the gut microbiota via prebiotic/probiotic treatment has been shown to have positive effects on visceral pain behaviors as discussed earlier (222, 240–242). Antibiotic treatment has long been known to alter GI function, effects which are reversed upon probiotic treatment (524). Thus, it is logical that antibiotic-induced visceral pain may be a future model used to investigate the underlying mechanism of such a phenomenon. Indeed work from our own lab has recently demonstrated that early-life antibiotic-induced disruption of the colonizing microbiota is associated with visceral hypersensitivity and altered spinal signaling in adulthood. These results indicate that a temporary alteration in the composition of the GI microbiota during a crucial time-window in the neonatal rat has a long-lasting impact on nociceptive pathways and that the developing pain systems are subject to modulation by the microbiota (248).

Moreover, intriguing studies to assess the exact contribution of the gut microbiota to the development of visceral hypersensitivity and IBS can be achieved through fecal transplantation studies whereby fecal matter from donor IBS patients can be used to inoculate recipient animals and subsequent behaviors can be assessed. This paradigm has already shown its merit in transferring other behaviors specifically anxiety-related behaviors in a mouse model (525).