Serotonin transporter (5-HTT, SERT, SLC6A4) and sodium-dependent reuptake inhibitors (SRIs) as modulators of pain behaviors and analgesic responses

Abstract

The serotonin transporter (5-HTT, SERT, SLC6A4) modulates the activity of serotonin via sodium-dependent reuptake. Given the established importance of serotonin in the control of pain, 5-HTT has received much interest in studies of pain states and as a pharmacological target for serotonin reuptake inhibitors (SRIs). Animal models expressing varying levels of 5-HTT activity show marked differences in pain behaviors and analgesic responses, as well as many serotonin-related physiological effects. In humans, functional nucleotide variations in the SLC6A4 gene, which encodes the serotonin transporter 5-HTT, are associated with certain pathologic pain conditions and differences in responses to pharmacological therapy. These findings collectively reflect the importance of 5-HTT in the intricate physiology and management of pain, as well as the scientific and clinical challenges that need to be considered for optimization of 5-HTT related analgesic therapies.

Graphical Abstract

INTRODUCTION

Serotonin, or 5-hydroxytrptamine (5-HT), is a monoamine molecule that functions as a neurotransmitter within the central nervous system (CNS) and plays numerous roles in the periphery ⁷². The serotonergic system encompasses at least 15 serotonin receptors described to date, comprising 7 families of receptors, as well as the serotonin transporter (SERT, 5-HTT) ^5,7.

The 5-HTT is a principal component of the serotonergic system responsible for modulating activity of serotonin in the nerve synapse via sodium-dependent reuptake into the presynaptic nerve terminal ⁷². The reuptake activity of 5-HTT determines the kinetics of serotonin signaling and altered 5-HTT function can result in global adaptations in the serotonergic system. Changes in 5-HTT activity influence bioavailability of serotonin, as well as expression and function of the serotonin receptors ^72,76,107.

Therefore, 5-HTT function has significant implications in the extensive roles of the serotonin system in modulating behavior and perception. This review will cover pain-related phenotypes observed in several animal models of altered 5-HTT and experimental assays of sensitivity to different types of stimulation both at baseline and after injury, as well as associations with functional polymorphisms in the human SLC6A4 gene that encodes 5-HTT and various disease states involving chronic pain. Additionally reviewed will be the influence of 5-HTT genetic backgrounds in both animal models and human patients on differential responses to pharmacologic inhibitors targeting the transporter.

ROLE OF SEROTONIN IN MOOD AND NOCICEPTION

Nearly all aspects of cognition and affect involve the serotonergic system. Serotonin is well known to play a crucial role in mood regulation, where depression and anxiety disorders have become closely linked with serotonin dysfunction ^7,72. Additionally, serotonin has direct and often complicated roles in the perception of pain. Activity of the serotonergic system can either facilitate or modulate aspects of pain and analgesia depending on the subtypes of receptor involved, as well as location in the peripheral or central nervous system ^5,67. In the periphery, 5-HT is released after tissue injury and exerts primarily pro-inflammatory and neuroexcitatory effects. Much literature on functions of 5-HT receptor subtypes comes from agonist-antagonist paradigms and administration of exogenous 5-HT in animal models ^5,67,96. Peripheral sensory nerves express 5-HT1B/D, 5-HT2A-C, 5-HT3, 5-HT4, and 5-HT7 receptors ^5,67,96. Activity of most 5-HT receptor subtypes in the periphery produces a pro-nociceptive effect. The 5-HT1B and 5-HT1D receptors appear to be exceptions which attenuate inflammation and hyperalgesia after injury ^67,96. 5-HT2A, 5-HT3, and 5-HT4, and 5-HT7 receptors are upregulated in dorsal root ganglia following inflammation and facilitate nociceptive behavior after inflammatory injury ^5,96. 5-HT2A and 5-HT3 receptors are also important for facilitating mechanical hyperalgesia induced by chronic constrictive injury ⁵. Direct injection of exogenous 5-HT into tissue can temporarily produce pain and hyperalgesia in humans and animals ⁹⁶. Additionally, 5-HT appears to have more potency as an enhancer of pro-nociceptive effects of other inflammatory mediators and further interacts to regulate other peripheral signaling mechanisms ^67,96. Overall, 5-HT appears to promote nociception at the periphery, with important roles in sensitizing inflamed and injured tissue.

The effects of serotonin in the brain and spinal cord on nociception are more complex. 5-HT signaling in descending neural pathways originating from the rostroventral medullary area of the brainstem are activated by noxious stimuli and can modulate sensory processing at the level of the spinal dorsal horn ^5,96. However, whether descending 5-HT pathways inhibit or facilitate nociception can vary with mode of stimulus and with disease states. In healthy animals, 5-HT signaling in the spine overall has an inhibitory effect on nociception ^5,33,96. In rodents, the descending anti-nociceptive effects of 5-HT at the spinal dorsal horn are mediated by signaling through 5-HT1A, 5-HT1B, 5-HT2C, 5-HT3, and 5-HT4 receptors ³³. Some pro-nociceptive effects of central 5-HT signaling at baseline are observed, which are mediated through 5-HT1A, 5-HT2A, 5-HT2C, and 5-HT3 receptors ³³. The dual actions of 5-HT in central sensory processing depend on the receptor subtypes and their location in the neural network. Changes occur in these pathways in animal models of chronic neuropathic pain, shifting the overall effect of descending 5-HT signaling to become pro-nociceptive ³³. Spinal 5-HT levels are reduced after nerve injury ^33,96. Meanwhile, several adaptations in central 5-HT receptor subtypes occur after injury. 5-HT2A, 5-HT2B, and 5-HT3 receptors are upregulated ³³. 5-HT1F receptors, which have an anti-nociceptive effect after peripheral nerve injury, are downregulated in the spinal cord ¹⁰⁸. 5-HT3 receptor activity overall becomes pro-nociceptive, the anti-nociceptive effects of 5-HT4 receptors are reduced, while 5-HT7 receptors become differentially upregulated and contribute to inhibiting nociception after nerve injury ^33,103. A potential role for descending 5-HT input at the dorsal horn in facilitating increased mechanical sensitivity after chronic inflammation injury have also been shown ²⁹. Of note, some discrepancies are seen between studies, possibly related to differences in methodology. However, a generally anti-nociceptive activity of serotonin signaling is seen in the spinal cord of healthy animals, which appears to shift towards an overall pro-nociceptive effect after nerve injury, and this transition is mediated through differential changes in receptor subtype expression and activity.

In sum, serotonin activity appears to have important roles in facilitating peripheral nociception and sensitization after injury, as well as modulating nociception in the central nervous system, demonstrating adaptations in receptor expression and function in states of chronic neuropathy. Therefore, the serotonergic system—and key components such as the 5-HTT—has received significant interest in the investigation of pain processing, especially in chronic pain conditions which involve neuroplastic changes in the organism ^55,103. The influence of genetic variations that alter 5-HTT function have thus been investigated in relation to several pain disorders in humans, including fibromyalgia, trigeminal neuralgia, irritable bowel syndrome, migraine, and chronic tension-type headache. Animal models of chronic pain conditions have also been utilized to further investigate the roles of 5-HTT within the serotonergic system’s influence on nociception and post-injury sensitization.

COMORBIDITY OF PAIN AND DEPRESSION

Depression and pain pose significant epidemiological burdens. Depressive disorders were estimated by the Institute for Health Metrics and Evaluation to have a global prevalence of 3.4% in 2019, contributing to nearly 47 million disability-adjusted life years ⁴⁵. Meanwhile, prevalence of chronic pain can range up to 40% in some studies, with an estimated point prevalence of 20.4% within the United States in 2016 ^16,18. Furthermore, depression and pain have a reciprocal relationship. Patients with depression experience a significantly higher incidence of pain symptoms, with mean estimates up to 65% across studies, and have worse outcomes when it comes to treating pain ^4,21. This is supported by animal models which consistently demonstrate the ability of pain to induce depressive-like behavior in rats and mice ^21,60. Meanwhile, having a greater degree of pain symptoms predicts a higher risk of major depressive disorder and worse depressive symptoms and outcomes ⁴. A genetic predisposition to chronic pain also predisposes to depression, which may be especially linked with manifestations of pain at specific body sites⁹⁷.

The bi-directional link between depression and pain is likely due to similar underlying mechanisms. Disruption of common biologic pathways, including serotonin signaling and serotonin-mediated neuroplasticity, are implicated in the pathogenesis of both depression and pain ^4,21. Shared structural neural pathways including the thalamus, insula, and anterior cingulate cortex have been elucidated as well ⁶⁹. In addition to providing insight into the striking association between depression and pain, the evidence of shared pathways has helped guide exploration into how treatments may translate effectively between the two conditions, as well as approaches to addressing the challenging reciprocal negative influences on outcomes when depression and pain coexist.

THE 5-HTT AS A PHARMACOLOGIC TARGET FOR DEPRESSION AND PAIN

In conjunction with the popularization of the monoamine hypothesis of depression—which theorized that depression results from a deficiency of the monoamines, including serotonin—medications that produce a general increase in serotonin concentration have been widely used for treating depression for more than 60 years ³⁶. Many of these commonly used antidepressants act, at least partially, as serotonin reuptake inhibitors (SRIs). These agents inhibit the 5-HTT—thereby increasing serotonin availability at the synaptic cleft—and include classes with differing degrees of selectivity for the 5-HTT, such as tricyclic antidepressants (TCAs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and selective serotonin reuptake inhibitors (SSRIs) ^8,80. More recent studies suggest that drugs inhibiting serotonin reuptake may also help restore neuroplasticity mediated by serotonin activity, which is disrupted in patients with depression ⁵⁴. However, patient populations that do not respond to monoaminergic medications have been characterized, leading to exploration of additional pathways involved in depression such as the glutamatergic system, and the subsequent development of antidepressant medications targeting alternative pathways. Therefore, a modern interpretation of the monoamine hypothesis is that these systems may have more of a modulatory function—rather than a simple direct role—in depression, which is often multifactorial and often heterogeneous in pathogenesis ³⁶.

Given the high degree of comorbidity and similarity in underlying mechanisms between depression and pain, some subclasses of SRIs are used for the treatment of pain as well, either alone or concurrently with coexisting depression. SSRIs can elicit antinociceptive effects in experimental settings but have not been shown to have significant clinical efficacy in treating most chronic pain conditions ^8,80. Some of the less selective agents such as TCAs and SNRIs, however, appear to have more reliable analgesic properties in certain contexts, especially neuropathic pain ^8,80. While pain and depressive symptoms can simultaneously improve with SRIs and cognitive behavioral therapy, the analgesic and antidepressant activities of SRIs are distinct effects ^4,60. Notably, patients with chronic pain experience less relief of depressive symptoms with SRIs and patients with depression experience less reliable improvement in pain with SRIs ⁹⁰. Nonetheless, pharmacologic agents acting at the 5-HTT remain a mainstay of clinical depression treatment, as well as important tools for further studying the biological processes of mood and cognition. Here, variations in 5-HTT function appear relevant to the efficacy of SRI therapy. For example, endogenous 5-HTT activity levels influence SRI effects on depression-like behavior in animals ⁴⁷. Additionally, exploration of natural 5-HTT polymorphisms and experimental models of altered 5-HTT function provides insight into the role of the 5-HTT as an important regulator of the serotoninergic system and its extensive roles in cognition, mood, and pain perception.

PHYSIOLOGICAL FUNCTIONS OF 5-HTT/SLC6A4 AS DEFINED IN ANIMAL MODELS

Animal models such as the Slc6a4 knockout (null) mouse have been used to explore how altered 5-HTT function influences behavior and responses related to pain ^10,77. Mice with two copies of constitutively inactive Slc6a4 alleles (Slc6a4-/-; Slc6a4 null) exhibit absent reuptake of high-affinity serotonin markers, indicating effective knockout of transporter function ⁶. Clearance of extracellular serotonin is reduced in heterozygous Slc6a4+/- mice, and further reduced in homozygous Slc6a4-/- mice. This loss of function is supported by the effects of administering a selective serotonin reuptake inhibitor (SSRI), which targets the 5-HTT/SLC6A4 protein. SSRIs reduce 5-HT clearance in Slc6a4+/- mice more effectively than in wild type (Slc6a4+/+) mice, but has no effect in Slc6a4-/- mice ⁷³. These gene-dosage dependent effects provide genetic evidence for the specificity of SSRIs for 5-HTT/SLC6A4 protein.

Considering the key role that the 5-HTT plays in serotonin signaling kinetics, adaptive changes throughout the serotonergic system are predicably seen as a result of effective 5-HTT knockout⁷⁶. Indeed, Slc6a4-/- mice exhibit increased serotonin concentrations in the extracellular fluid of brain regions, whereas concentrations in brain tissue, most peripheral organs, plasma, and platelets are decreased due to deficient serotonin reuptake ^{6,23,50,71,76}. Increased serotonin synthesis and turnover in the brain are also observed in Slc6a4-/- mice ^50,76, as adaptations in the expression and activity of serotonin receptor subtypes. For example, 5-HT1A receptor expression is decreased throughout several brain regions of Slc6a4-/- mice and similar changes are elicited by treating rat models with chronic SSRIs ^23,76. Meanwhile, 5-HT2A receptors are differentially increased or decreased in various brain regions and 5-HT2C receptors are downregulated in certain areas ⁷⁶. Additionally, the expression of other low-affinity monoamine transporters is increased in Slc6a4-/- mice as a possible mechanism to compensate for 5-HTT deficiency, although without significant efficacy ⁷⁶. While the many downstream changes in the serotonergic system elicited by 5-HTT loss-of-function highlight the potential complexity of determining precise mechanisms for phenotypic presentations, the murine Slc6a4-/- model nonetheless demonstrates effective knockout of 5-HTT function and provides a powerful tool for studying the role of Slc6a4 activity in numerous behavioral and physiological pathologies, including those related to pain ⁷⁷.

Characteristics that have been studied in Slc6a4 knockout mice include general sensitivity to pain, analgesic response to pharmacotherapy, and hyperalgesia; hyperalgesia reflects increased sensitivity to painful stimuli that occurs after tissue damage and involves sensitization of pain receptors ⁸⁸. Regarding baseline sensitivity to pain, male Slc6a4 null mice do not exhibit differences in mechanical, thermal, or formalin-induced nociception compared to wild type mice⁴⁸. However, differences in response to pharmacotherapy have been observed because female Slc6a4 knockout mice were reported to have decreased analgesia to heat upon administration of opioids—including morphine, meperidine, and tramadol—although the mechanisms for these differences were not explored ²⁴. Therefore, knockout of Slc6a4 in these mice appears important for analgesic response to opioids, without significantly influencing baseline nociception.

Several studies have found attenuation of increased sensitivity to noxious thermal stimulus, assessed by latency time to paw withdrawal, after injury in female Slc6a4 null mice compared to wild type using multiple methods of injury. Following constrictive injury of the sciatic nerve, increased ipsilateral sensitivity to noxious heat was observed in wild type mice, but absent in Slc6a4 null mice. This finding was correlated with lower levels of tissue serotonin in Slc6a4 null animals, although increased levels of serotonin from baseline were observed in the injured nerve in both genotypes ¹⁰⁴. Reduced thermal hypersensitivity was also observed in Slc6a null mice following hind paw inflammation induced by injection of complete Freund's adjuvant (CFA). This effect on heat sensitivity could be rescued by injection of exogenous serotonin. The latter experimental finding supports the association between decreased tissue serotonin levels and the protective effects of Slc6a4 inactivation on injury-induced reductions in paw withdrawal latency to noxious heat ⁸³.

A later study reprised the observed reduction in CFA-induced thermal hypersensitivity among Slc6a4 knockout mice and demonstrated that it is at least partially mediated by reduced levels of 5-HIAA, a serotonin metabolite, in the sciatic nerve of Slc6a4 null mice. Pretreating wild type mice with para-chlorophenylalanine (p-CPA), an inhibitor of serotonin synthesis, reduced 5-HIAA levels and attenuated the thermal hypersensitivity observed after CFA injection. Administration of exogenous 5-HIAA increased thermal hypersensitivity in Slc6a4 knockout mice and wild type mice pretreated with p-CPA, but not in wild type mice without p-CPA pretreatment. These phenotypic effects were observed regardless of concurrent broad pharmacologic blockade of serotonin receptors ¹². Similarly, vincristine-induced neuropathy produced thermal hypersensitivity in wild type, but not in Slc6a4-/- mice, without evidence of differences in neuronal damage or inflammation between genotypes ³⁰. Of note, distal sensory nerves in Slc6a4 null mice appeared to be more susceptible to injury following CFA ⁸³. However, these nerves were previously found to be only weakly correlated with thermal sensation ⁶². Overall, Slc6a4-/- mice exhibit decreased thermal hypersensitivity following either inflammatory or mechanical nerve injury. The observed injury-induced increases in sensitivity to noxious heat appears to be mediated at least in part by increased peripheral nerve serotonin levels following injury. Furthermore, the protective effects of Slc6a4 inactivation are in part associated with decreased levels of serotonin and its metabolite, 5-HIAA, in the nerves of Slc6a4 knockout mice. Meanwhile, the relative increased sensitization of distal nerves in Slc6a4-null mice after injury appears less relevant given weak associations with thermal sensation.

In addition, the biochemical effects of Slc6a4 knockout and resulting changes in the serotonergic system in the CNS may also play a role in mechanical allodynia. In these animal models, proxy measures for mechanical allodynia such as withdrawal threshold to application of mechanical force such as via filaments or pressure plates are used. While comparable rates of post-injury increases in sensitivity to mechanical stimulus were seen in Slc6a4-/- and wild type mice with each model of injury, the study using sciatic nerve crush injury did find more frequent occurrence of mechanical hypersensitivity on the contralateral, non-injured side in Slc6a4-/- mice. In addition, studies using vincristine-induced neuropathy found that Slc6a4 null mice recovered from mechanical hypersensitivity more quickly. The authors suggest that this observation may be related to a central effect of lower levels of 5-HT seen in the spinal cord of Slc6a4 knockout mice, and a resulting decrease in spinal inhibition ^12,30,83,104. While the effect of Slc6a4 inactivation may not influence the risk of developing mechanical hypersensitivity at the periphery, the central changes in serotonin concentration and receptor expression may play key roles in the development or recovery of contralateral mechanical hypersensitivity and may depend on the mode of nerve injury.

Interesting observations on the influence of Slc6a4 activity on pain perception have also emerged from studies in rat models with a genetic knockout of Slc6a4, as well as Wistar-Zagreb 5-HT rat sublines exhibiting "high 5-HT" or "low 5-HT". The latter sublines were generated by selecting for extremes of platelet serotonin levels. High 5-HT animals exhibit increased levels of serotonin in platelets and whole blood, primarily due to increased expression and activity of Slc6a4 ^13,37,42. The extracellular concentration of 5-HT in blood might logically be expected to be lower in animals with higher activity of 5-HTT. Therefore, the finding of higher concentrations of extracellular 5-HTT in blood in these animals compared to animals with lower 5-HTT activity is of unclear mechanism, possibly due to inherent limitations of laboratory blood processing or yet unelucidated alterations in 5-HT homeostasis ¹⁴. Additionally, high 5-HT rats have increased serotonin levels in peripheral tissues including lung, spleen, heart, small bowel, and large bowel, as well as regional differences in CNS serotonin homeostasis compared to control and low 5-HT rats ¹⁴. No significant differences were seen in concentration of 5-HT, 5-HTT expression, 5-HT uptake, or density of 5-HT1A, 5-HT1B, and 5HT-2A receptors in brain regions of high 5-HT compared to low 5-HT rats. However, regional differences in metabolic ratios of 5-HIAA and 5-HT were observed, as well as increased binding of labeled SSRI in high 5-HT rats, suggesting possible differences in 5-HT hemostasis and cytoplasmic concentrations of 5-HTT ¹⁴. Compared to a complete knockout of 5-HTT function such as seen in Slc6a4 null mice, the rat lines selected for extremes of platelet 5-HTT function present with more modest differences in the serotonergic system. While the mechanisms for some of the disparate alterations in 5-HT metabolism compared to knockout models remain to be fully characterized, the Wistar-Zagreb 5-HT rat lines nonetheless offers a closer approximation of physiological genetic variations in 5-HTT function—and subsequent effects on behavioral and pain-related phenotypes—that might be seen with naturally occurring polymorphisms in animals and humans.

Some affective characteristics of the Wistar Zagreb 5-HT rat lines include higher degrees of anxiety-related traits and lower degrees of depression-related traits in high 5-HT rats compared to low 5-HT rats ¹⁴. Regarding pain, low 5-HT male rats exhibit increased baseline sensitivity to thermal stimuli compared to high 5-HT rats. Low 5-HT rats also display better analgesic responses upon acute administration of tramadol and prolonged administration of clomipramine, both of which inhibit the activity of the 5-HTT protein ⁴⁹. For comparison Slc6a4-/- mice exhibit no difference in baseline sensitivity compared to wild type mice, possibly due to compensatory changes in serotonin receptor sensitivity and expression. The increased thermal sensitivity in low 5-HT rats may be explained by physiological modulations in 5-HT levels due to variations in Slc6a4 activity compared to a Slc6a4 loss-of-function model ^2,25,49,89. Hence, while the findings with these distinct experimental rat models are clear, the interpretations are less definitive. Nonetheless, findings from the rat 5-HT sublines support the concept that Slc6a4 and adaptive changes in serotoninergic systems modulate baseline pain sensitivity and analgesic responses.

Animal models provide versatile in vivo tools to investigate specific pain phenomena related to diseases in human patients. For example, in an established rat model of ovariectomy-induced osteoporosis, altered activity of Slc6a4 protein and the central serotonin system have been associated with the analgesic effects of calcitonin. Following ovariectomy, decreased time until hind paw withdrawal to thermal stimuli was observed and associated with a decrease in thalamic Slc6a4 expression and activity, with increased 5-HT1A receptor expression. Concurrent administration of calcitonin led to partial reversal of thermal hypersensitivity, as well as a further decrease in thalamic Slc6a4 activity and a further increase in expression of the serotonin 1A receptor (5-HT1A receptor encoded by the HTR1A gene). However, whether this relationship between the serotonergic system and the anti-nociceptive effects of calcitonin in osteoporosis is a direct or indirect remains to be determined and physiological effects of calcitonin at peripheral nerves cannot be discounted ¹⁰⁶.

Slc6a4 null rats also permit modeling of other phenotypes relevant to human diseases. For instance, Slc6a4 knockout rats have been used to represent the colonic hypersensitivity that is observed in female patients with irritable bowel syndrome. Female, but not male, Slc6a4-/- rats exhibit increased visceral sensitivity to colorectal distension and increased excitability of colonic sensory neurons, which was correlated to increased extracellular 5-HT levels in the colon, but not in mucosal cells ²⁸. This result was replicated by a later study and found to be associated with increased 5-HT3 receptor activity in the spinal cord of female, but not male, Slc6a4-/- rats ²². These findings are in further support of the importance of Slc6a4 function, as well as adaptations in serotonergic concentration and receptor activity, in mediating sensitivity to pain. While such loss-of-function animal models are not capable of fully recapitulating the nuances and complexity of human disease, these models nonetheless demonstrate in exploring the underlying mechanisms behind pathologies clinically correlated with disease states in humans.

It is important to note that most of the studies reviewed here used only one sex of experimental animal. Given the relationships between sex and pain—such as differences between sexes in the prevalence and severity of most chronic pain conditions, with additional limited evidence for differences in pain sensitivity and analgesic response—that are seen in humans, sex-specific differences should be considered and further explored in the translation of preclinical findings ^70,81. However, the results of animal studies overall provide convincing experimental evidence for the striking role of Slc6a4 in modulating pathologic hypersensitivity following injury and differential responses to analgesic pharmacotherapy—often due to adaptations in the serotonergic system that result from altered Slc6a4 function—with possible additional roles in baseline sensitivity.

HUMAN GENETIC VARIATIONS THAT ALTER 5-HTT/SLC6A4 ACTIVITY IN PATIENTS

Natural nucleotide variation in human populations may alter the expression or activity of the SLC6A4 protein that could contribute to serotonergic dysfunction or psychiatric disorders. Numerous DNA polymorphisms have been described in the SLC6A4 gene and its promoter region, the 5-HTT-linked polymorphic region (5-HTTLPR) ^78,105. Of particular interest is a 43-bp insertion/deletion in the 5-HTTLPR, which creates a "long" 16-repeat allele (L) and a "short" 14-repeat allele (S), with the long allele producing higher transcriptional efficacy ^34,53,78. Phenotypic penetration of this effect appears to depend on an upstream single nucleotide polymorphism, rs25531. This polymorphism is characterized by an A to G substitution that is phenotypically linked to the L-allele and reduces transcriptional activity to levels similar to those seen with the S-allele ^43,105. Together, these two polymorphisms comprise the "tri-allelic" 5-HTTLPR genotype, that modulates 5-HTT gene expression. This tri-allele is divided into high (LA/LA), intermediate (LA/SA, LA/LG), and low-expressing groups (SA/SA, SA/LG, LG/LG), the latter of which have reduced levels of 5-HTT expression and function comparable to those of heterozygous (5-HTT+/- ) mice ⁷⁶. While the transcriptional and epigenetic regulatory mechanisms affected by natural nucleotide variation remain to be defined, genetic variation in the SLC6A4 gene may contribute to differences in serotonin levels and downstream responses within patient populations.

Multiple mood-related traits and diseases states have been associated with these SLC6A4 promoter polymorphisms, but it remains difficult to determine by what mechanisms these polymorphisms exert functional effects or whether associations are merely statistical in nature. Some notable associations involving low-expressing alleles include increased anxiety and depression-related characteristics, especially in patients with chronic pain, which have been supported by findings in mouse models ^{11,31,39,59,77,100,102}. Studies have explored associations between these variants and various chronic pain states. For example, while SLC6A4 has been linked to fibromyalgia via genomic analysis and some studies found an association between the S allele and susceptibility to fibromyalgia, possibly mediated by anxiety-related traits, a meta-analysis found no correlation between the polymorphisms in the 5-HTT promoter region and fibromyalgia ^3,15,58,84. Low-expressing 5-HTTLPR genotypes did, however, exhibit interactions with an allele of the mu-opioid receptor gene—proposed to increase the tonicity of endogenous opioid activity—to mediate greater exercise-induced modulation of pressure pain sensitivity in the low-expressing group among patients with fibromyalgia, as well as healthy controls ⁹⁸. The S/S bi-allelic genotype was more prevalent among patients with trigeminal neuralgia ¹⁷. Similarly, the S allele was more frequent in patients with chronic tension-type headache ⁸⁵. Meanwhile, 5-HTTLPR alleles were not overall associated with migraine ^65,94. The G-allele of rs25531 has been observed to significantly increase IBS risk and the 5-HTTLPR genotypes were closely associated with irritable bowel syndrome, particularly in female patients, and differentially correlated with either constipation or diarrhea as the predominant symptom. However, the actual direction of this correlation differed between studies, and these differences appeared to depend on ethnicity ⁴⁶. Overall, while the 5-HTTLPR polymorphisms do appear to be relevant to several chronic pain conditions, the occasionally contradictory findings between studies, as well as interactions with other biologic pathways and the ethnic background of subjects to produce differing influences, indicate a more subtle modulatory role for these polymorphisms in humans ^51,75.

Interestingly, serotonin ligand binding studies and neuroimaging analyses in humans do not consistently show an association between polymorphisms and differences in 5-HTT expression in the CNS ¹⁰¹. Nonetheless, functional brain changes related to 5-HTT polymorphisms are seen, such as enhanced activity of the amygdala and anterior cingulate cortex seen in s-allele carriers ^{26,32,75,82,86,91}. Overall, human 5-HTT alleles are associated with affective traits and pain-related states. More nuanced relations are observed in patients compared to animal models, as expected from increased neural and emotional complexity in human patients. Changes in regions that contribute to emotion and pain modulation resulting from functional 5-HTT polymorphisms form one possible mechanism by which genotypes may contribute to clinically relevant manifestations.

Regarding affective outcomes related to pain, one area of concern is how 5-HTT genotypes can influence the experience and natural modulation of pain for patients. One study using a picture-based paradigm for emotional modulation of muscular pain induced by saline injection found that concurrently viewing positive images inhibited pain and viewing negative images facilitated pain for individuals with high-expressing tri-allelic 5-HTTLPR genotypes (LA/LA), but not either the intermediate or low-expressing groups. Baseline pain sensitivity and emotional responses to pictures were consistent across groups ⁴¹. These findings do contrast with an earlier study using electrocutaneous stimuli to induce pain, which found that carriers of the short-allele exhibited both greater emotional facilitation and inhibition of pain in the picture-viewing paradigm than did l/l homozygotes, although the earlier study did not account for tri-allelic genotypes including the rs25531 polymorphism ⁸². Therefore, these findings appear to indicate that individuals with high-expressing tri-allelic 5-HTTLPR genotypes may experience a greater degree of emotional attenuation or facilitation of pain in response to visual stimuli, without exhibiting any differences in baseline sensitivity.

5-HTTLPR polymorphisms may also influence conditioned pain modulation, which is the inhibition of a primary pain response by applying a second noxious stimulus. This phenomenon is commonly referred to as "pain inhibits pain" and involves descending opioid and noradrenergic signaling pathways ^19,66. One study compared fibromyalgia patients with healthy controls regarding sensitivity to heat-induced pain, and the degree that it could be inhibited by a concurrent painful cold pressor. The authors found that fibromyalgia is associated with thermal hyperalgesia and decreased inhibition by the secondary noxious stimulus; there was no effect of the long v/s short 5-HTTLPR allele on thermal pain threshold, pain ratings during the cold pressor test, conditioned pain modulation efficacy, or pain catastrophizing ⁸⁷. However, a later study using the same investigative paradigm in healthy patients found that when a non-painful level is used in the cold pressor test, presence of the long allele additively increases the modulation of thermal pain; S/S homozygotes exhibited less modulation than both other phenotypes, but this effect was not seen with painful conditioned pain modulation ⁹⁹. Further, in a study including the rs25531 SNP, low-expressing individuals had significantly reduced conditioned pain modulation of both pressure- and heat-induced pain compared to high-expressing individuals, where the conditioned pain modulation was induced by application of concurrent submaximal tourniquet ⁶³. Although a direct serotonergic pathway has not been identified in conditioned pain modulation, these findings suggest a potential modulatory role for serotonergic signaling interacting with known descending signaling pathways.

An interesting negative finding is that 5-HTTLPR genotype does not appear to modulate the nociceptive flexion reflex, a spinally mediated withdrawal response, in either an emotional or conditioned pain model of pain modulation ^63,82. This finding suggests that the effects of 5-HTTLPR on pain modulation may primarily act at a supraspinal level such as at the amygdala and other brain regions. As noted earlier, these regions have been seen to exhibit functional changes related to 5-HTT genotypes, further supporting a possible supra-spinal role for these polymorphisms in exerting downstream effects.

Another affective trait of interest related to pain is pain catastrophizing, which is the cognitive tendency to "magnify and ruminate about pain and having a helpless attitude toward actual or anticipated pain". This process has occasionally been associated with both pain sensitivity and recall ²⁷. One study found that carriers of the s-allele reported higher pain catastrophizing, as well as increased sensitivity thermal pain ⁵⁶. An earlier study, which also used bi-allelic evaluation of 5-HTTLPR, found no association between genotype and pain catastrophizing ⁸⁷. However, these studies applied contrasting methods. The study that observed a positive association assessed pain catastrophizing in the same session as applying a painful thermal stimulus. Therefore, while a possible influence of 5-HTTLPR polymorphisms on pain catastrophizing is suggested, further studies would be required to rule out confounding factors related to the context of assessment.

Of note, differences in baseline sensitivity to pain are not consistently observed across 5-HTTLPR variants. Although results and methodology vary between studies, these polymorphisms often are not associated with differences in baseline sensitivity to pain induced by heat, cold, saline injection, pressure, capsaicin, or the thermal grill illusion ^40,41,87,92. A role of 5-HTT in tonic sensitivity to pain is supported by results of functional studies, such as 5-HTT binding in the hypothalamus and insula associated with decreased tonic sensitivity to thermal pain. Yet, genetic association studies are inconclusive and emphasize the challenge of discerning the role of a single protein in the complex experience of human pain. The magnitude of this challenge is underscored by the observation that the association of genotypes with decreased pain sensitivity depends on sex or is restricted specifically to the context of chronic pain ^38,57,64,82.

Another particular area of interest is how 5-HTT genotypes may affect responses to a variety of pharmacological classes in acute and chronic pain states. For example, in a tri-allelic evaluation of 5-HTTLPR genotypes for thermal sensitivity, the low-expressing group exhibited better analgesia than the high-expressing group with the opioid remifentanil, although baseline sensitivity did not differ across groups ⁵². This finding was attributed to desensitization of 5-HT1A receptors in s/s homozygotes, which are important for morphine analgesia and have decreased activity in 5-HTT knockout mice and humans with the short-allele ^20,23,61. A study of bi-allelic 5-HTTLPR in adult males found that citalopram (a selective serotonin reuptake inhibitor that is clinically used as an anti-depressant) reduced pain-related neural MRI activity in response to electrical stimulus in l/l homozygotes only. Genotypes also differentially modulated the effect of individual neural response to treating subjective pain with citalopram. Stronger baseline neural responses in l/l homozygotes and weaker baseline responses in s/s homozygotes predicted, respectively, greater or lesser reduction of pain reports with citalopram. However, subjective pain ratings overall were not significantly reduced with citalopram and did not differ between genotypes⁶⁸. Additionally, among patients with idiopathic trigeminal neuralgia, s/s homozygotes were found to have higher pain and worse treatment response with carbamazepine compared to l/l homozygotes, but no effect of the rs25531 SNP was observed ¹⁷. Meanwhile, tri-allelic evaluation of 5-HTTLPR did not reveal any significant differences in the non-responsiveness of cluster headaches to triptans, which are clinically prescribed as anti-migraine medication ⁹³. Overall, the 5-HTTLPR genotypes appear to potentially influence individual response to analgesics of multiple categories, with the association depending on the condition. These genotypes may become particularly relevant for pharmacological strategies using different types of SRIs to target the serotonin transporter. Such therapies are actively considered and a number of studies show efficacy in chronic pain conditions such as migraine, tension-type headaches, diabetic neuropathy, fibromyalgia, post stroke central pain, and others ⁸⁰.

As with prescribing any pharmacologic treatment, however, the potential adverse effects relating to SRI therapy must be taken into consideration. Although side effect profiles vary between specific agents and classes of SRIs, some characteristic adverse effects that are common to TCAs, SNRIs, and SSRIs include drowsiness, insomnia, agitation, gastrointestinal toxicity, sexual dysfunction, and weight gain ⁸⁰. Considering the influence that functional 5-HTT polymorphisms have on pharmacologic response to SRIs, similar influences on the experience of adverse effects might also be expected. Indeed, one study did observe an association between the LA allele, compared to the LG and S alleles, of the HTTLPR and decreased overall burden of adverse effects in patients with major depressive disorder being treated with the SSRI citalopram as part of a clinical trial. Of note, the participants primarily consisted of White Americans, differential changes in specific adverse effects were not assessed, and differences in treatment response were not seen in association with the tri-allelic 5HTTLPR genotypes ⁴⁴. Nonetheless, the ability of functional 5-HTT polymorphisms to influence susceptibility to, and severity of, adverse effects warrants further investigation and consideration when utilizing drugs targeting the 5-HTT.

Other polymorphisms in the human SLC6A4 gene that have been studied in relation to pain disorders include a 17-bp variable number of 10 or 12 tandem repeats (VNTR) in the second intron. The 12/12 repeat genotype was associated with increased susceptibility to migraine, especially among those of European descent ^65,94. When comparing individuals with migraine with or without aura, these groups demonstrate significant variation in the presence of different repeat length alleles. This finding indicates that the VNTRs of the SLC6A4 gene may contribute to the manifestation of aura in patients with migraines ⁷⁹. Patients with temporomandibular joint disorder (TMJD) also have significantly different distributions of VNTR polymorphisms compared to control patients. The 10-repeat allele is more frequent in patients with TMJD, while the 12-repeat allele is more prevalent in the control group and is associated with higher somatization and anger scores. 5-HTTLPR genotypes are not significantly different between groups and are not associated with differences in psychiatric or somatization measurements ³⁵. Like the 5-HTTLPR alleles, this region of VNTR in the second intron appears to be a potentially relevant factor contributing to certain pathologic pain states such as migraine with aura and TMJD, warranting further investigation.

The rs1042173 (A v/s C alleles) and rs7224199 (G v/s T alleles) polymorphisms have also been evaluated in patients with chronic pain, and the rs1042173 polymorphism has been associated with painful TMJD among adolescents ⁹. Additionally, in patients with lung cancer, allele distributions for either polymorphism do not differ between patients with pain and those without pain. Yet, patients homozygous for the rs7224199 GG genotype are significantly more common among those with cancer pain, although no differences are evident in patient groups with different degrees of self-reported pain. However, the analgesic dose of morphine, corrected for weight and body surface area, is significantly lower for rs1042173 CC homozygotes, indicating analgesic sensitivity for this genotype ¹⁰⁹. Meanwhile, another polymorphism, rs3813034, with T and G alleles, was investigated in a cold and pressure threshold model of pain sensitivity; this paper was unable to ascertain significant difference in pain thresholds between genotypes ¹. Of these three additional alleles, rs1042173 and rs7224199 appear potentially relevant for TMJD and analgesic responses in chronic lung cancer pain. While rs1042173 is yet to be correlated with a clinically significant outcome, all three polymorphisms remain potential targets for investigation to obtain a comprehensive understanding of how genetic variations in 5-HTT can influence pain states and analgesia.

Overall, the multiple functional variations in the SLC6A4 gene and its promoter region show promise as potential genetic factors in several behavioral and pain-related pathologies, as well as in sensitivity to analgesics. While the manner in which these genotypes may influence physiologic outcomes is not as clear as those seen in animal models, some evidence for supra-spinal effects does present based on functional imaging studies and lack of effects on spinally mediated reflexes. This receptor gene and its polymorphisms remain interesting subjects of study and warrant continued investigation to gain a better understanding of pain physiology and the treatment of chronic pain states.

CONCLUSIONS

Animal and human studies provide both converging and diverging findings on the biological function of the 5-HHT/SLC6A4 protein in pain perception. Animal studies show a clear role of the Slc6a4 gene in pathologic pain states, where absent expression of Slc6a4 protects against hyperalgesia following several different modes of injury in mice but confers decreased analgesic response to opioids. Low expression of Slc6a4 in rats promotes increased baseline sensitivity to pain as well as better analgesic responses to tramadol and clomipramine. In contrast, 5-HTT/SCL6A4 plays a more nuanced role in human pain-related states, although functional imaging and the absence of effects on spinally mediated withdrawal responses may imply a higher-level role for 5-HTT in pain modulation in humans. The observed functional genotypes, particularly the "tri-allelic" 5-HTTLPR, produce different levels of expression and corresponding pain phenotypes. These phenotypes are associated with several chronic pain conditions and differences in the experience and processing of pain. In general, low-expressing groups have decreased modulation of pain, while differential sensitivity to analgesics is evident for certain polymorphic genotypes in chronic pain states. Apparent differences between studies do not necessarily indicate contradictions or false findings, but rather may reflect the considerable complexities in the physiology of pain perception, experimental set ups, and study assumptions that complicate logical interpretations. Together, these findings suggest that while 5-HTT phenotypes may play an important role in chronic pain states in humans, they likely represent only one part of an intricate biological phenomenon.

FUTURE PERSPECTIVES

The 5-HTT/SCL6A4 protein is an attractive pharmacological target that is amenable to inhibition via SRIs. Several studies have shown some benefit in various pain states, including migraine, diabetic neuropathy, fibromyalgia, and more ⁸⁰. Furthermore, the different 5-HTTLPR genotypes have been observed to exhibit different responses to certain analgesics including morphine, citalopram, remifentanil, and carbamazepine ^17,52,68,109.

Going forward, future experimental studies may enhance our fundamental understanding of the 5-HTT/SCL6A4 protein and the physiological role of serotonin levels in pain, while providing clinically important insights into the pathological experience and treatment of pain. 5-HTT/SCL6A4 genotypes provide a robust and promising tool for studying the involvement of serotonin signaling in animal models of physiologic and pathologic pain states. Knowledge of the functional importance of genetic polymorphisms and mutations may facilitate our understanding chronic pain states in humans and meet the considerable clinical challenge of providing safe treatments for pain relief.

Figure 1. Serotonin reuptake inhibitors block the reuptake of serotonin from the nerve synapse via the serotonin transporter.

Figure 2. The human SLC6A4 gene on chromosome 17, with the locations of functional polymorphisms and variations discussed in this review ^74,95.

*Both figures should be printed in color.

Table 1.

Summary of studies using animal models to investigate the influence of 5-HTT altered 5-HTT function on pain-related phenotypes.

Study Name/Year	Animal Model	Key Findings
(Kayser et al., 2007)	Slc6a4-/-mice	No baseline differences in sensitivity to noxious mechanical, thermal, or formalin-induced sensitivity in Slc6a4-/- mice compared to WT mice.
(Fox et al., 2009)	Slc6a4-/-mice	Decreased analgesia to heat after opioid administration (morphine, meperidine, tramadol) in female, compared to male, Slc6a4-/- mice.
(Vogel et al., 2003)	Slc6a4-/-mice	Increased sensitivity to noxious thermal stimulus after constrictive nerve injury in female Slc6a4-/- mice compared to WT. More frequent occurrence of mechanical hypersensitivity on the contralateral, non-injured side in Slc6a4-/- mice
(Palm et al., 2008)	Slc6a4-/-mice	Reduced thermal hypersensitivity after CFA-induced hind paw injury in Slc6a4-/- mice compared to WT. Effect rescued by injection of exogenous serotonin.
(Chen et al., 2011)	Slc6a4-/-mice	Reprised findings of Palm et al., 2008, demonstrated reduction in CFA-induced thermal hypersensitivity in Slc6a4-/- mice is partly mediated by reduced 5-HIAA in sciatic nerve.
(Hansen et al., 2011)	Slc6a4-/-mice	Thermal hypersensitivity in WT mice after vincristine-induced neuropathy, but not in Slc6a4-/- mice. No evidence of differences in neuronal damage or inflammation. Faster recovery from post-injury mechanical hypersensitivity in Slc6a4-/- mice.
(Kesic et al., 2015)	High 5-HT/Low 5-HT rats	Increased baseline sensitivity to thermal stimulus and better analgesic response to acute administration of tramadol or prolonged administration of clomipramine in low 5-HT ratscompared to 5-HT rats.
(Yeh et al., 2016)	WT rats	Ovariectomy-induced model of osteoporosis was associated with increased thermal sensitivity and decreased thalamic Slc6a4 expression and activity.
(Galligan et al., 2013)	Slc6a4-/-rats	Female, but not male, Slc6a4-/- rats demonstrate increased visceral sensitivity compared to WT rats, correlated with increased extracellular 5-HT levels in the colon.

Table 2.

Summary of studies investigating the influence of 5-HTTLPR polymorphisms in various pain conditions.

Authors/Year	Key Findings
(Lee et al., 2012)	Meta-analysis found no correlation between polymorphisms in the 5-HTT promoter region and fibromyalgia.
(Arnold et al., 2013)	Genomic association study linked fibromyalgia to chromosomal regions including SLC6A4 (17p11.2-q11.2) in multi-case families with fibromyalgia.
(Tour et al., 2017)	Case-control study found low-expressing 5-HTTLPR genotypes interacted with mu-opioid receptor gene to mediate greater exercise-induced modulation of pain in patients with fibromyalgia as well as healthy controls.
(Cui et al., 2014)	Case-control study found S/S genotype of 5-HTTLPR was more prevalent in patients with trigeminal neuralgia, associated with higher pain severity and worse analgesic response to carbamazepine compared to L/L genotype.
(Park et al., 2004)	Case-control study found S allele of 5-HTTLPR was more frequent in patients with chronic tension-type headache.
(Schürks et al., 2010)	Meta-analysis found 5-HTTLPR alleles not overall associated with migraine. However, increased risk of migraine for S allele carriers among European women.
(Liu et al., 2011)	Meta-analysis found no increased risk of migraine with 12/12 genotype of the 5-HTT VNTR, but no overall association between 5-HTTLPR or rs2020942 polymorphisms and migraine.
(Jin et al., 2016)	Narrative review indicated that 5-HTTLPR genotypes are associated with irritable bowel syndrome. However, the direction of correlation varied between studies and appeared to depend on ethnicity.
(Palit et al., 2011)	Carriers of S allele of 5-HTTLPR exhibited greater emotional facilitation and inhibition of pain with picture-viewing paradigm compared to L/L homozygotes. Did not account for tri-allelic genotypes including the rs25531 polymorphism.
(Horjales-Araujo et al., 2013)	High-expressing 5-HTTLPR genotypes, but not low or intermediate-expressing, experienced inhibition of saline injection-induced muscular pain with emotional modulation by viewing positive images. Baseline sensitivity and emotional responses to pictures were similar across groups.
(Potvin et al., 2010)	No effect of long v/s short 5-HTTLPR allele on thermal pain threshold, pain ratings during cold pressor test, conditioned pain modulation efficacy with concurrent cold pressor, or pain catastrophizing in patients with fibromyalgia and healthy controls.
(Treister et al., 2011)	Using painful thermal stimulus and concurrent cold pressor, similar to Potvin et al., 2010, found that presence of L alleles of 5-HTTLPR additively increased modulation of thermal pain when using a non-painful level of concurrent cold pressor. This effect was not seen when using a painful stimulus level for conditioned pain response.
(Lindstedt et al., 2011)	Low-expressing tri-allelic 5-HTTLPR phenotypes exhibited reduced conditioned pain modulation of pressure- and heat-induced pain with concurrent submaximal tourniquet compared to high-expressing phenotypes.
(Kunz et al., 2016)	Carriers of S allele of 5-HTTLPR reported higher pain catastrophizing and increased sensitivity to thermal pain, assessed in same session, as compared to Potvin et al., 2010, which found no significant effect on pain catastrophizing.
(Schaldemose et al., 2014)	Tri-allelic differentiation of 5-HTTLPR high-expressing and low-expressing phenotypes among healthy volunteers found no significant differences in pain thresholds to pressure or cold and warm stimuli, presence of paradoxical heat sensation, perception of thermal grill illusion, or capsaicin-induced pain and hyperalgesia.
(Kosek et al., 2009)	Healthy low-expressing tri-allelic 5-HTTLPR individuals exhibited better analgesia of heat-induced pain with intravenous remifentanil compared to high-expressing individuals. No baseline differences in sensitivity.
(Ma et al., 2016)	Citalopram reduced MRI neural activity induced by painful electrical stimulus only in L/L homozygotes in bi-allelelic evaluation of 5-HTTLPR. Stronger baseline neural responses in l/l homozygotes and weaker baseline responses in s/s homozygotes predicted, respectively, greater or lesser reduction of pain reports with citalopram. Pain ratings overall were not reduced by citalopram and did not differ between genotypes.
(Schürks et al., 2014)	Tri-allelic evaluation of 5-HTTLPR in patients with cluster headache did not find significant differences in non-responsiveness to triptans.
(Hu et al., 2007)	LA allele of 5-HTTLPR associated with decreased overall burden of adverse effects, compared to LG and S alleles, in patients with major depressive disorder being treated with citalopram.
(Herken et al., 2001)	The 10-repeat allele of the VNTR was found to be more frequent than the 12-repeat allele in patients with temporomandibular joint disorder compared to control patients.
(Brancher et al., 2019)	Cross-sectional sample of adolescents ages 10–14 found association between the rs1042173 polymorphism and painful temporomandibular disorder.
(Zhu et al., 2020)	G/G homozygotes for rs7224199 polymorphism were found more frequently among those with cancer pain. While C/C homozygotes exhibited lower morphine dose requirements.
(Aoki et al., 2010)	No significant direct effect of T and G alleles of polymorphism rs 3813034 on pain thresholds for cold or pressure stimuli among healthy volunteers.

HIGHLIGHTS.

• Serotonin levels are controlled by a specific transporter (5-HTT, SERT, SLC6A4).

• This transporter mediates the re-uptake of serotonin in the pre-synapse.

• Pain perception is controlled by serotonin.

• Serotonin re-uptake inhibitors (SRIs) permit management of pain.

• Scientific and clinical challenges limit optimization of 5-HTT related analgesic therapies.

PERSPECTIVES.

The serotonin transporter 5-HTT/SCL6A4 is sensitive to pharmacological serotonin re-uptake inhibitors. Experimental studies on the physiological functions of serotonin, as well as genetic mouse models and clinical phenotype/genotype correlations of nucleotide variation in the human 5-HTT/SCL6A4 gene provide new insights for the use of SRIs in chronic pain management.

Abbreviations

5-HT

5-hydroxytryptamine, serotonin

5-HTT

5-hydroxytryptamine transporter, serotonin transporter

5-HIAA

5-hydroxyindoleacetic acid

HTR1A

5-hydroxytryptamine receptor 1A, 5-HT1A receptor, serotonin 1A receptor

HTR3A

5-hydroxytryptamine receptor 3A, 5-HT3A receptor, serotonin 3A receptor

5-HTTLPR

serotonin-transporter-linked promoter region

SERT

serotonin transporter

SLC6A4

Solute Carrier Family 6 Member

4SRI

serotonin reuptake inhibitor

TCA

tricyclic antidepressant

SNRI

serotonin and norepinephrine reuptake inhibitor

SSRI

selective serotonin reuptake inhibitor

CFA

complete Freund's adjuvant

p-CPA

para-chlorophenylalanine

VNTR

variable number of tandem repeats

TMJD

temporomandibular joint disorder