Neurophysiology of Orofacial Pain

Transcription

1 Neurophysiology of Orofacial Pain Koichi Iwata, Mamoru Takeda, Seog Bae Oh, and Masamichi Shinoda Abstract It is well known that unmyelinated C-fibers and small-diameter Aδ-fibers innervate the orofacial skin, mucous membrane, orofacial muscles, teeth, tongue, and temporomandibular joint. Peripheral terminals consist of free nerve endings, and thermal and mechanical receptors such as transient receptor potential (TRP) channels and purinergic receptors exist in nerve endings. Ligands for each receptor are released from peripheral tissues following a variety of noxious stimuli applied to the orofacial region and bind to these receptors, following which action potentials are generated in these fibers and conveyed mainly to the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2). K. Iwata (*) M. Shinoda Department of Physiology, School of Dentistry, Nihon University, Tokyo, Japan iwata.kouichi@nihon-u.ac.jp; shinoda.masamichi@nihon-u.ac.jp M. Takeda Laboratory of Food and Physiological Sciences, Department of Food and Life Sciences, School of Life and Environmental Sciences, Azabu University, Sagamihara, Kanagawa, Japan m-takeda@azabu-u.ac.jp S.B. Oh Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea odolbae@snu.ac.kr # Springer International Publishing AG 2017 C.S. Farah et al. (eds.), Contemporary Oral Medicine, DOI / _8-3 Neurons receiving noxious inputs from the orofacial regions are somatotopically organized in the Vc and C1-C2. The third branch (mandibular nerve) of the trigeminal nerve innervates the dorsal portion of the Vc, and the first branch (ophthalmic nerve) of the trigeminal nerve innervates the ventral part of the Vc; the middle portion of them receives the second branch (maxillary nerve) of the trigeminal nerve. Various neurotransmitters such as glutamate and substance P (SP) are released from primary afferent terminals and bind to receptors such as AMPA and NMDA glutamate receptors and neurokinin 1 receptors in Vc and C1-C2 nociceptive neurons. Further, noxious information from the orofacial region reaching Vc and C1-C2 is sent to the somatosensory and limbic cortices via the ventral posterior medial thalamic nucleus (VPM) and medial thalamic nuclei (parafascicular nucleus, centromedial nucleus, and medial dorsal nucleus), respectively, and finally, orofacial pain sensation is perceived. It is also known that descending pathways in the brain act on Vc and C1-C2 nociceptive neurons to modulate pain signals. Under pathological conditions such as trigeminal nerve injury or orofacial inflammation, trigeminal ganglion (TG) neurons become hyperactive, and a barrage of action potentials is generated in TG neurons, and these are sensitized a long time 1

2 2 K. Iwata et al. after the hyperactivation of TG neurons. Furthermore, there is an increase in Vc and C1-C2 neuronal activities, and these neurons can be sensitized in association with TG-neuron sensitization, and then orofacial pain hypersensitivity can occur. Recent studies have also reported that glial cells are involved in pathological orofacial pain states related to trigeminal nerve injury and orofacial inflammation. Peripheral and central mechanisms of orofacial pain under physiologic and pathologic conditions are overviewed in this chapter, and future insights regarding the pathogenesis of persistent orofacial pain are discussed. Keywords Trigeminal nerve Orofacial pain Brainstem Medial system Lateral system Descending modulation Persistent pain Contents Introduction... 2 Primary Afferent Neurons... 3 Physiology of Orofacial Nociceptors... 3 Receptor Mechanisms of Trigeminal Primary Afferent Neurons... 5 Trigeminal Ganglion... 6 Pathological Changes in Trigeminal Ganglion Brainstem Nociceptive Neurons... 7 Trigeminal Spinal Nucleus and Upper Cervical Spinal Cord... 7 Physiology of Trigeminal Nociceptive Neurons Neurotransmitters in Brainstem Nociceptive Neurons Pathological Changes in Brainstem Nociceptive Neurons Higher Brain Function Regulating Orofacial Nociception Ascending Orofacial Pain Pathways Sensory-Discriminative and Motivational and Affective Aspects of Pain Human Brain Function for Orofacial Pain Sensation Descending Modulation of Orofacial Pain Descending Pathways Influencing Orofacial Pain Modulation of Trigeminal Nociceptive Neurons Pathological Changes in the Descending System Conclusion and Future Directions References Introduction The trigeminal nervous system is known to have unique structures and functions for processing orofacial nociception as well as non-noxious sensations in comparison to the spinal nervous system. Oral mucous membrane, tongue, tooth pulp, gingiva, and temporomandibular joints are innervated by small-diameter Aδ-fibers and unmyelinated C-fibers that process orofacial nociception. Peripheral terminals of these fibers are composed of free nerve endings, and various receptors are expressed on the membrane surface of nerve endings, and these receptors act as sensors responding to various noxious stimuli such as heat, cold, or chemical stimulus (Iwata et al. 2011a). For example, it has been well documented in recent decades that transient receptor potential vanilloid 1 (TRPV1) channel is cloned, and functions of this channel have been evaluated (Mickle et al. 2015). TRPV1 and TRPV2 channels are known to be involved in heat sensation and TRPV3 and TRPV4 in a warm sensation, whereas transient receptor potential ankyrin 1 (TRPA1) and M8 are known to participate in cool and cold sensations (Tominaga 2007). ATP and glutamate receptors have also been reported to be expressed in free nerve endings of C-fibers and are known to contribute to orofacial nociception (Sessle 2011). Though piezo receptors have been reported to take part in mechanical sensations, detailed mechanisms for mechanical sensation are still unknown (Woo et al. 2014). Neuronal activity is conveyed along the afferents to the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2), and nociceptive neurons in these areas are activated following various noxious stimuli applied to the orofacial regions (Sessle 2000). Vc and C1-C2 nociceptive neurons are classified as wide dynamic range (WDR) and nociceptive-specific (NS) neurons according to their response properties to mechanical stimulation of the receptive fields (Iwata et al. 1999, 2001). The nociceptive information is then conveyed to the somatosensory and limbic cortices via the ventral posteromedial thalamic nucleus (VPM) and medial

3 Neurophysiology of Orofacial Pain 3 thalamic nuclei, respectively. The VPM-somatosensory pathway is known to be involved in the sensory-discriminative aspect of pain, whereas medial thalamic nuclei-limbic cortices pathways are involved in the motivational and affective aspect of pain, and some noxious information is also conveyed to the limbic cortices via the parabrachial nucleus (Treede et al. 1999; Benarroch 2001). At each level of ascending pathways, various excitatory and inhibitory neurotransmitters are involved in synaptic transmission and modulatory processes of orofacial nociceptive information. Projection neurons are known to express excitatory transmitters, whereas most of the local circuit neurons are inhibitory interneurons expressing inhibitory transmitters. Both excitatory and inhibitory neurons are involved in the modulation of the neuronal excitability regarding orofacial nociception in higher central nervous system (CNS) regions. It is also well known that the descending system acts on nociceptive neurons at each level of the ascending pain pathways, and neuronal activity is modulated (Mason 2012). These ascending and descending pathways are thought to play a pivotal role involved in orofacial nociception. Under pathological conditions, functional and morphological changes are known to occur in the peripheral and central nervous system. Noticeable shifts in the peripheral nervous system are the enhancement of neuronal activity and up- or downregulation of various molecules within the trigeminal ganglion (TG) associated with trigeminal nerve injury or orofacial inflammation. It is well known that neuropeptides and potassium channels are downregulated in TG neurons following nerve transection, whereas sodium channels are upregulated and accumulated at the stump end of the nerve resection (Mulder et al. 1997; Pollema-Mays et al. 2013). High-frequency action potentials are generated in primary afferent neurons innervating orofacial territories after trigeminal nerve injury or orofacial inflammation. For a considerable time after trigeminal nerve damage or inflammation, TG neurons become hyperactive and sensitized. Furthermore, satellite cells are activated within the TG in association with hyperactivation of TG neurons (Tsuboi et al. 2004; Nakagawa et al. 2010). Activated satellite glial cells release various molecules, and TG neuronal activity can be further enhanced. High-frequency action potentials are generated in TG neurons and conveyed to the Vc and C1-C2 neurons following trigeminal nerve injury or orofacial inflammation (Iwata et al. 1999, 2001). Microglial and astroglial cells in Vc and C1-C2 are also activated in association with the hyperactivation of nociceptive neurons in these areas (Okada-Ogawa et al. 2009; Shibuta et al. 2012; Kiyomoto et al. 2013). Within the TG, the functional interaction between neurons and glial cells is also thought to be a key mechanism in the enhancement of neuronal activity under pathological conditions (Katagiri et al. 2012; Kaji et al. 2016). The enhanced nociceptive information within the TG is sent to the higher CNS regions via the trigeminal brain stem nuclei, resulting in severe pain in the orofacial region. It is crucial to know the detailed mechanisms underlying the pathogenesis of orofacial pain associated with trigeminal nerve injury or orofacial inflammation to formulate appropriate diagnosis and treatment of persistent orofacial pain states. In this chapter, peripheral mechanisms and the ascending and descending pain pathways regarding orofacial pain are overviewed, and new knowledge on persistent orofacial pain mechanisms under pathological conditions is considered, and the possible clinical relevance is discussed. Primary Afferent Neurons Physiology of Orofacial Nociceptors The orofacial area is mainly innervated by three main branches of the trigeminal nerve: ophthalmic, maxillary, and mandibular (Fig. 1a). The majority of trigeminal afferents are pseudounipolar with cell bodies lying in the TG, except proprioceptive afferents whose cell bodies lie in the mesencephalic trigeminal nucleus (Davies

4 4 K. Iwata et al. a Trigeminal ganglion Mandibular nerve Ophthalmic nerve Ethmoidal nerve Ciliary nerve Maxillary nerve Lingual nerve Inferior alveolar nerve b c Fig. 1 Trigeminal nerve and tooth pulp afferents. (a) Area innervated by trigeminal nerves (ophthalmic, maxillary, and mandibular nerves) (Revised in Purves et al., Neuroscience, 2004). (b) Molecular mechanisms of neural theory. Thermo-TRP channels are functionally expressed by dental primary afferents. Action potentials evoked by the influx of cations such as Ca 2+ and Na + through TRP channels transmit pain signals to the central nervous system. (c) Molecular mechanisms of hydrodynamic theory. The fluid movement initiated by diverse external stimuli eventually activates mechanoreceptors in dental primary afferents. Candidates of mechanosensitive molecules are listed. Little is known about how the activation of the low-threshold mechanoreceptor is eventually perceived as pain in the central nervous system (Reproduced and modified from Chung et al. (2013))

5 Neurophysiology of Orofacial Pain 5 Table 1 Conduction velocity of each type of nerve fiber Type Erlanger-Gasser classification Diameter (μm) Myelin Conduction velocity (m/s) Ia Aα Yes Ib Aα Yes II Aβ 6 12 Yes III Aδ 1 5 Thin 3 30 IV C No et al. 2010). Free nerve endings that detect noxious stimuli are dispersed over the orofacial area, innervating the oral mucous membrane, tongue, tooth pulp, gingiva, masticatory muscle, or temporomandibular joint in the trigeminal system (Sessle 2000). Nociceptors are specialized peripheral nerve terminals in a subset of sensory neurons and detect noxious stimuli that have potential to cause tissue damage, which is then transduced into electrical activity in nociceptors (Julius and Basbaum 2001). Nociceptors are associated with primary afferent axons with either light myelination (Aδ-fibers) or unmyelination (C-fibers) that have a relatively slow conduction velocity (3~30 m/s and <2 m/s, respectively), compared to Aβ-fibers (33~75 m/s) responsible for the conduction of activity evoked by innocuous mechanical stimuli such as proprioceptive and light touch (see Table 1). It is generally assumed that Aδ-fibers and unmyelinated C-fibers mediate first and second pain evoked by noxious stimuli due to the temporal difference in their respective conduction velocity: the rapid, acute, sharp pain and the delayed, more diffuse, dull pain. Nociceptors, mostly C-fibers, often respond to multiple kinds of stimuli including intense heat, intense mechanical stimuli, and various chemicals and are known as polymodal nociceptors. Nociceptors are usually divided into two groups: non-peptidergic neurons that bind with isolectin B (or IB4-positive nociceptors) and peptidergic neurons which express substance P (SP) and calcitonin generelated peptide (or IB4-negative nociceptors) (Basbaum et al. 2009). It is important to note that the nociceptive fibers do not exclusively conduct nociceptive information; some are also associated with the transmission of itch sensation. Cheek model of itch in the mouse provides a behavioral differentiation of chemicals that evoke predominantly itch in humans that elicit nociceptive sensations (Shimada and LaMotte 2008). The molecular mechanisms by which nociceptive fibers relay itch sensation to the CNS are now under extensive investigation. Receptor Mechanisms of Trigeminal Primary Afferent Neurons Nociceptors are unique in which they have the ability to detect multiple types of noxious stimuli, including those of a physical or chemical nature. Therefore, nociceptors are equipped with diverse repertoires of transduction molecules that give rise to painful sensations by thermal, mechanical, or chemical stimuli, although with different extents of sensitivity (Julius and Basbaum 2001). TRPV1, the first pain receptor cloned, previously known as VR1, is expressed in a subset of small- to medium-sized nociceptive trigeminal primary afferent neurons. TRPV1 is sensitive to heat temperature above 43 C and is also found to be activated not only by capsaicin and heat but also by low ph and inflammatory mediatorrelated molecules, such as products of lipoxygenases, anandamide, and other endocannabinoids. Other TRP channels, which are activated by a distinctive range of temperature, thermo-trp channels, are also found to be expressed by TG neurons. While TRPM8 and TRPA1 are activated by cool (<25 C) and cold (<17 C) temperature, respectively, TRPV3 and TRPV4 sense a warm range (Tominaga 2007). Given that TRPV1 and TRPA1 are activated by noxious temperature which produces pain in vivo,

6 6 K. Iwata et al. they might play important roles in heat- and coldinduced thermal pain. TRPV2 detects the highest temperature level among the thermo-trp channels with a threshold of 52 C. TRPA1 can be activated by pungent cysteine-reactive chemicals, such as isothiocyanates (mustard oil), cinnamaldehyde (cinnamon), and allicin (garlic), and also by many endogenous substances that are generated at the site of tissue injury and inflammation, for example, the highly reactive aldehyde 4-hydroxy-2-nonenal (4-HNE), 15-deoxy-12,14- prostaglandin J2, and reactive oxygen and nitrogen species. The activation of TRPA1 by these compounds directly excites nociceptors and thereby generates a warning signal to the organism to protect the body (Chung et al. 2011). A variety of thermo-trp channels are expressed in dental primary afferent neurons (Fig. 1b). Activation of these thermo-trp channels increases intracellular calcium and evokes cationic currents in subsets of neurons, as does the appropriate temperature changes (Chung et al. 2013). These results suggest that activation of thermo-trp channels expressed by dental afferent neurons contributes to dental pain evoked by temperature stimuli. Thermo-TRP channels have thereby been shown to be substantial sensory transducers that transfer signals elicited by external noxious thermal stimuli to the nociceptive neurons (Chung et al. 2013). Mechanoreceptors responding to mechanical stress such as direct pressure and tissue deformation are also involved in nociceptive processes in the trigeminal system. For example, hydrodynamic theory describes the cause of dental pain regarding mechanical forces generated by the movement of dentinal fluid (Fig. 1c). Low-threshold mechanoreceptors (so-called algoneurons ) in dental primary afferent nerve fibers could be involved in nociception, in contrast to conventional low-threshold mechanoreceptors thought to transduce light touch in other parts of the body (Fried et al. 2011). Piezo channels might be a candidate molecule (Woo et al. 2014). Further, trigeminal primary afferent neurons express P2X purinoreceptor 3 (P2X3) homomer and P2X2/3 heteromer, a subtype of the P2X receptor that detects adenosine triphosphate (ATP) released by damaged keratinocytes or in inflammatory sites. Chemical substances are also sources for nociceptors to transduce pain. Tissue acidosis associated with tissue inflammation or injury is the representative example. Acid-sensing ion channel 3 (ASIC3) and TRPV1 are two major acid sensors involved in proton-induced hyperalgesic priming (Sun and Chen 2016). Trigeminal Ganglion Given that cell bodies of trigeminal primary afferent neurons are located in the TG, TG neurons are involved in various sensory functions in the orofacial region such as innocuous or noxious mechanical, thermal, or chemical sensation (Goto et al. 2016). Following application of noxious stimuli to the orofacial region, a barrage of action potentials is generated in small-diameter primary afferent neurons, and those are conveyed to the small TG neurons. On the other hand, innocuous stimuli may cause action potentials in large-diameter nerve fibers. Various ion channel proteins and neuropeptides are up- and downregulated following non-noxious and/or noxious stimuli under physiological conditions (Goto et al. 2016). A recent RNA-Seq analysis of TG transcriptome reveals a comprehensive expression profile of all ion channels and G-protein-coupled receptors (GPCRs) in TG neurons and provides insight into both trigeminal sensings and the physiological and pathophysiological mechanisms of pain (Manteniotis et al. 2013). Pathological Changes in Trigeminal Ganglion The trigeminal sensory system represents a distinct and complex functional unit, with its wellcharacterized nociceptive and modulatory pathways (Sessle 2000). A range of severe facial pain syndromes, with varying etiologies, are associated with trigeminal neuropathy. Pain related to nociceptor activation within structures specific to the orofacial area, such as the tooth pulp and

7 Neurophysiology of Orofacial Pain 7 cornea, as well as the cranial dura, does not have clinical correlates in the spinal somatosensory system. As such, the treatment regime for trigeminal-related pains may differ from that of spinal neuropathic pain (Davies et al. 2010). Thus, changes in TG neurons following peripheral nerve injury or tissue inflammation are not always comparable with its counterpart in the spinal somatosensory system, dorsal root ganglion. Peripheral nerve injury or orofacial inflammation often causes changes in the excitability of TG neurons, thereby resulting in pain hypersensitivities such as allodynia and hyperalgesia. It is well known that excitability and molecular expression change in TG neurons under pathological conditions. Specifically, differential expression of voltage-gated Na +,K +, and Ca 2+ channels and hyperpolarization-activated cyclic nucleotidegated (HCN) channels following trigeminal nerve injury has been shown to contribute to the enhanced excitability of TG neurons. Further, changes in the expression of a variety of molecules such as TRPV1 and P2X3 channels in TG neurons are thought to be involved in orofacial sensory dysfunctions associated with trigeminal nerve injury or orofacial inflammation such as temporomandibular joint (TMJ) inflammation. Moreover, the activation of intra-ganglionic communication via nitric oxide (NO), nerve growth factor (NGF), or calcitonin gene-related peptide signaling plays a major role in the creation and maintenance of trigeminal pathological pain (Goto et al. 2016). Satellite glial cells have been shown to be involved in orofacial sensory abnormalities associated with trigeminal nerve injury or orofacial inflammation. It has recently been demonstrated that neuron-glia interactions such as those underlying a paracrine mechanism via SPneurokinin 1(NK1) receptor and IL-1β are critical processes involved in the modulation of the excitability of TG neurons (Dubner et al. 2014; Goto et al. 2016). Thus, interactions of TG neurons and satellite glial cells might play a key role in the manifestation of orofacial sensory dysfunctions such as ectopic pain hypersensitivity under pathological conditions. Tissue injury or inflammation in masticatory muscles leads to elevated extracellular excitatory amino acid concentrations and a high concentration of ATP in the TG. It has recently been demonstrated that both ATP- and N-methyl-Daspartate (NMDA)-induced mechanical hypersensitivities involve upregulation and sensitization of both TRPV1and TRPA1 (Asgar et al. 2015). It is possible that TRPV1 and TRPA1 function as a downstream integrator of various pronociceptive/inflammatory intracellular signals in masticatory muscles (Chung et al. 2011). Brainstem Nociceptive Neurons Trigeminal Spinal Nucleus and Upper Cervical Spinal Cord Noxious sensory information in the area innervated by the trigeminal nerve is relayed from trigeminal afferents to second-order neurons in the trigeminal sensory nuclear complex (TSNC) in the brainstem and the upper cervical (C1-C2) spinal cord (Jacquin et al. 1986b; Bereiter and Bereiter 2000; Sessle 2000) (Figs. 1a and 2a). The TSNC and C1-C2 are the primary sites of synaptic integration for sensory inputs from the specific craniofacial tissues, such as face and oral cavity (Waite and Tracy 1995). The TSNC consists of the principal sensory nucleus (PrV) and the trigeminal spinal nucleus. PrV is a relay station for non-noxious sensory information, but not noxious information, whereas the trigeminal spinal nucleus (SpV) is an important relay station in the transmission of orofacial noxious sensory information, and this nucleus is functionally subdivided into three nuclei (from rostral to caudal): oralis (Vo), interpolaris (Vi), and Vc (Olszewski 1950; Darian-Smith 1973). Since the laminar organization of neurons in the Vc is similar to that in the spinal dorsal horn, Vc is also termed the medullary dorsal horn (MDH) (Gobel 1978). There is evidence of intrasubnuclar connections between rostral and caudal portions of the TSNC (Ikeda et al. 1984; Jacquin 1986a; Hirata et al. 2003). For example, chemical blockade of Vc has been shown to reduce the excitability of Vo

8 8 K. Iwata et al. a Cortex ACC/IC Thalamus Limbic system VPM SI/SII Noxious inputs b Midbrain Brainstem PAG RVM (-) PrV Vo V1 V2 V3 Trigeminal ganglia Orofacial area (-) Vi/Vc Vc Vc/C1-2 (+) (+) TSNC Vi Vc C1-2 Spinal cord C1-2 C1 C2 Fig. 2 Trigeminal sensory nuclear complex and pain pathways. (a) Central pain pathway for orofacial area innervated by the trigeminal nerve. ACC, anterior cingulate cortex; IC, insular cortex; VPM, posterior ventromedial thalamus; PAG, periaqueductal gray; RVM, rostral ventral medulla; TSNC, trigeminal sensory nuclear complex; PrV, principal sensory nucleus; Vo, spinal trigeminal nucleus oralis; Vi, spinal trigeminal nucleus interpolaris; Vc, Vo, spinal trigeminal nucleus caudalis; C1-C2, upper cervical dorsal horn. (b) Intrasubnuclar connections into the TSNC (Vo, Vi/Vc, Vc, and Vc/C1-C2) and C1-C2 regions neurons responsive to noxious stimulation (Greenwood and Sessle 1976; Chiang et al. 2002; Hirata et al. 2003). By contrast, there is a report that GABA A receptor agonist blockade of neuronal activity of Vi/Vc transition region facilitates nociceptive neurons of Vc (Hirata et al. 2003). Collectively, these findings suggest that both ascending and descending connections within the TSNC may contribute to the integration of noxious sensory input relevant to orofacial pain (Fig. 2b). Although it is known that nociceptive Vc neurons have similar properties to those at the spinal dorsal horn level, consistent with a prominent role in nociceptive processing (Dubner and Bennett 1983; Bereiter and Bereiter 2000; Sessle 2000), the contribution of the rostral portion of the TSNC to orofacial pain is less precise. Since the majority of sensory neurons in nociceptive defensive reflex (e.g., nociceptive jaw-opening reflex) are located in Vo and this projects to the trigeminal motor nucleus (Mizuno et al. 1975; Dubner 1978; Sugimoto and Takemura 1993), Vo has a significant role in the brainstem-central station underling trigeminal nociceptive reflexes. Among these three nuclei, Vc is the most important relay station for trigeminal nociceptive inputs from the orofacial area, as well as the C1-C2 dorsal horn (Dubner et al. 1978; Bereiter and Bereiter 2000). There is considerable evidence suggesting that Vc/C1-C2 junction region differs from the lower spinal cord. It has also been reported that the nociceptive inputs from receptors in deep craniofacial tissues are conveyed to the ventral Vi/Vc transition region (Vi/Vc) through the Vc/C1-C2 junction region (Bereiter et al. 2002a) (Fig. 2b). These findings suggest that the Vi/Vc transition zone plays a significant role in deep tissue pain processing, integrating nociceptive orofacial pain inputs, and the development of persisting orofacial pain (Ren and Dubner 2011). Since Vc/ C1-C2 neurons have widespread ascending

9 Neurophysiology of Orofacial Pain 9 connections to the hypothalamus, periaqueductal gray (PAG), and other brainstem regions (Keay et al. 1997; Burstein et al. 1998), they can be involved in the circuitry control of autonomic outflow and in endogenous pain modulation circuits (Bossut et al. 1992; Tanimoto et al. 2002). Over the last several decades, c-fos expression and extracellular signal-regulated kinase (ERK) phosphorylation have been known to occur in nociceptive neurons in Vc and C1-C2 following various noxious stimuli applied to the orofacial region (Nomura et al. 2002; Noma et al. 2008; Suzuki et al. 2013). After the excitation of nociceptive neurons, these molecules can be detected in Vc and C1-C2 neurons at 1 2 h for c-fos in cell nuclei and at less than 5 min for phosphorylated ERK (perk) in the cytoplasm after noxious stimuli (Noma et al. 2008). The number of c-fos or perk-ir cells is increased following an increase in the stimulus intensity, suggesting that these molecules are widely accepted as a reliable marker of activated neurons following various noxious stimuli. Since ERK phosphorylation occurs at a very early period of fewer than 5 min after noxious stimuli, perk-ir cells are thought to receive direct inputs from trigeminal nociceptive afferents. Further, ERK phosphorylation is blocked by MK801, indicating that NMDA receptor mechanism is involved in ERK signaling (Ji et al. 1999). Nociceptive neurons in the Vc and C1-C2 can be further classified as projection neurons or local circuit neurons. Most of the nociceptive neurons in Vc and C1-C2 regions have been shown to be local circuit neurons (Okada-Ogawa et al. 2015). Many of these local circuit neurons have inhibitory functions releasing inhibitory transmitters, GABA, and glycine (Fig. 3). These inhibitory interneurons are thought to be involved in the modulation of noxious ascending outputs conveying noxious information to the higher central nervous system. GABA and glycine are known to bind their receptors, and chloride influx is accelerated, and then membrane potentials become deeper than resting level resulting in a reduction of excitability. Projection neurons have a long axon ascending to the ventral posterior medial thalamic nucleus (VPM) thalamic nuclei and reticular formation, whereas local circuit interneurons have very short axons involved in local inhibitory and excitatory functions (Sessle 1999, 2000). Nociceptive neurons in Vc that project to the VPM originate mainly in laminae I and V (Ikeda et al. 2003). The majority of projection neurons are found in rostral Vc rather than caudal Vc/C1-C2 junction region and lamina I of Vc projects heavily to the VPM (Guy et al. 2005). It has been shown that nociceptive Vc and Vo neurons are markedly inhibited by direct stimulation of the PAG or ventromedial medulla (RVM) (Sessle et al. 1981; Chiang et al. 1989, 1994). However, afferent pathways from second-order Vc neurons to these endogenous pain control regions are not well defined. Compared to the significant inputs from upper cervical levels of the spinal cord to the PAG (Keay et al. 1997), projection from the TSNC to the PAG is sparse (Keay et al. 1997; Beitz 1982). These findings support the idea that laminae I II of Vc are critical regions processing nociceptive information relevant for multiple aspects of craniofacial pain. Local circuit interneurons have both inhibitory and excitatory functions. For example, in the local inhibitory circuits, there is evidence that local GABAergic mechanisms exert a tonic inhibition of mechanoreceptive transmission in the Vc neurons, and this effect may limit responsiveness and size of receptive fields of the Vc neurons (Takeda et al. 2000). The trigeminal afferents terminate somatotopically in Vc, with mandibular afferents ending dorsally and ophthalmic afferents ventrally in Vc; mandibular endings become more dorsomedial and ophthalmic endings more ventral (Waite and Tracy 1995) (Fig.4a). At the caudal level of TSNC, craniofacial tissues are represented in a series of semicircular bands that converge at the rostral midline of the face and by medial-lateral arrangement in which the head is inverted (Jacquin 1986; Shigenaga et al. 1986).

10 10 K. Iwata et al. microglia Vc nociceptive neuron C-fos expression Na + Ca 2+ Glu NMDA-R ERK perk Ca 2+ Na + Ca 2+ Nav Na + Na + Na + Na + K + Na + Na + NK1R Cl - Cl- GABA B R K + Mg 2+ Na + Ca 2+ Na + Na + AMPA-R Ca 2+ GABA A R Cl - GABA astrocyte primary afferent neuron Glu SP C-fiber Ad fiber excitatory interneuron Inhibitory interneuron Fig. 3 Schematic diagram of the synaptic transmission of nociceptive neurons in Vc and C1-C2. Three types of nociceptive neurons exist in Vc and C1-C2, projection neuron, and inhibitory and excitatory interneurons. Each neuron is connected to each other with excitatory or inhibitory synapses. Glu, glutamate; SP, substance P; ERK, extracellular signal-regulated kinase; NK1-R, neurokinin 1; NMDA-R, N-methyl-D-aspartic acid receptor; AMPA- R, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; GABA, gamma-aminobutyric acid; Nav, voltage-gated sodium channel Physiology of Trigeminal Nociceptive Neurons Orofacial noxious information is conveyed to the higher CNS regions via Vc and C1-C2 neurons. As shown in Fig. 4b, there are two types of nociceptive neurons classified as nociceptive-specific (NS) neurons (superficial laminae I-II) and WDR neurons (both in superficial and deep laminae I II and IV V) in the SpVc and C1-C2 based on their sensitivity to mechanical stimulation applied to orofacial areas such as facial skin (Sessle 1999). NS neurons respond only to noxious stimulation (e.g., high-threshold mechanical stimulation) of the receptive field, suggesting that NS neurons encode stimulus localization (Sessle 1999). WDR neurons respond to both noxious (via Aδand C-fibers) and non-noxious (via Aβ-fiber) stimulations and have large receptive fields (Iwata et al. 2001; Takeda et al. 2011) (Fig. 2a). Graded noxious stimuli applied to the most sensitive area of the receptive field produce increased firing frequency of Vc WDR neurons in proportion to stimulus intensity, and many of them also respond to noxious heat stimulation (Fig. 4b, c), suggesting that WDR neurons encode stimulus intensity. The Vc and C1-C2 NS and WDR neurons also commonly receive convergent inputs from tooth pulp, facial skin, jaw, masseter muscle, and phrenic afferents (Razook et al. 1995; Sessle et al. 1986; Takeda et al. 2005). Thus they likely contribute to the phenomenon of referred pain,

11 Neurophysiology of Orofacial Pain 11 a b Vc V3 V2 V1 Lateral Dorsal Impulse NS neuron c Frequency of impulses Aδ-fiber C-fiber Aβ-fiber WDR neuron Non-noxious Ventral I II IV V NS neuron Noxious Mechanical stimulus intensity Frequency Impulse Frequency g Non-noxious Noxious WDR neurons g Non-noxious Noxious Mechanical stimulus intensity Fig. 4 General characteristics of trigeminal nociceptive-specific (NS) and wide dynamic range (WDR) neurons. (a) Connections between nociceptive and non-nociceptive trigeminal primary afferent fibers and layers (I V) of spinal trigeminal nucleus caudalis. V1, V2, and V3 indicate ophthalmic, maxillary, and mandibular nerves, respectively. (b) A typical example of discharge patterns in NS and WDR neurons responding to mechanical stimulation of receptive fields. (c) Stimulusresponse function for NS and WDR neurons whereby pain is associated with an injury affecting a visceral tissue. Further, the mechanical threshold in the center of the receptive field is low and that in the surrounding portion of that area is high (Iwata et al. 2002). Neurotransmitters in Brainstem Nociceptive Neurons Using immunohistochemistry and in situ hybridization histochemical studies has revealed that a variety of classical neurotransmitter substances and their receptors are associated with the subsets of trigeminal primary afferent neurons (Lazarov 2012). Among them are amino acids, glutamic acid (Glu), γ-aminobutyric acid (GABA), glycine) and monoamines (noradrenaline, dopamine, and serotonin (5-HT)), acetylcholine (Ach), and adenosine triphosphate (ATP). After the activation of nociceptive Aδ- and C-terminals associated with external noxious stimulation, nerve impulses are conducted to the central terminals and depolarize them which subsequently triggers the release of classical neurotransmitters. Neuroanatomical and molecular characterization of nociceptors has revealed further heterogeneity, particularly in C-fibers (Snider and McMahon 1998). For example, the peptidergic population of C nociceptors releases the neuropeptides substance P (SP) and calcitonin gene-related peptides (CGRP); they also express the tyrosine kinase A (trka) neurotrophin receptor which responds to NGF. The non-peptidergic population of C nociceptors expresses c-ret neurotrophic receptor that is targeted by a glial-

12 12 K. Iwata et al. a Inflammation tissue injury b (1)Increase in excitatory mechanism (2) Decrease in inhibitory mechanism Peripheral Senstization (trigeminal nerve) Central Senstization (SpVc/C1-C2) Aδ-/Cnociceptive afferent Nociceptive signal Glial activation (3) Neuroglial interaction Potentiation of NMDA-mediated excitatory signaling Glu SP CGRP BDNF Fractakine, Cytokine Vc/C1-2 neuron GABA/Glycine Disinhibition Augmented Nociceptive signal Pathological pain Spontaneous pain Allodynia Hyperalgesia c Pain No stimulus Normally non-painful stimulus Painful Stimulus Time Fig. 5 The mechanism underlying central sensitization and pathological pain. (a) Following inflammation and tissue injury of an area innervated by trigeminal nerves, large peripheral sensitization can trigger central sensitization, leading to chronic pathological pain states. (b) The mechanism of central sensitization depends on the following three postulated mechanisms: (1) increase in the excitatory mechanism, (2) loss of inhibitory mechanism, and (3) glialneuronal interaction. Following inflammation and tissue derived neurotrophic factor, as well as neurturin and artemin. Pathological Changes in Brainstem Nociceptive Neurons Peripheral tissue injury and inflammation can alter the properties of somatic sensory pathways, resulting in behavioral hypersensitivity and pain caused by both noxious stimuli (hyperalgesia) and normally innocuous stimuli (allodynia) (Scholz and Woolf 2002) (Fig. 5a). Thus, it can be injury, increased firing impulses conducted through Aδ-/Cfibers in the central terminals, then increase in excitatory mechanism and lose inhibition via glial-neuronal interaction evoked by augmented nociceptive signal information. These augmented nociceptive signals are associated with the primary symptom of pathological pain. (c) The main symptom of pathological pain: spontaneous pain caused by no stimulus. Abnormality caused by both noxious stimuli (hyperalgesia) and normally innocuous stimuli (allodynia) assumed that peripheral sensitization could trigger central sensitization, leading to chronic pathological pain states. Peripheral inflammation and/or injury activate intracellular signaling transduction pathways in nociceptor terminals and reduction of activation threshold and an increase in firing frequency of action potentials (Garry and Hargreaves 1992; Bereiter and Benetti 1996; Scholz and Woolf 2002). High-frequency impulses conduct to central terminals of primary afferents can induce neurotransmitter release and increase the excitability of second-order neurons via upregulation of postsynaptic receptors in Vc

13 Neurophysiology of Orofacial Pain 13 and C1-C2. Consequently, sensitization of Vc and C1-C2 neurons may contribute to inflammationinduced spontaneous pain, hyperalgesia, and allodynia. The mechanism of central sensitization depends on the following three postulated mechanisms: (1) increase in the excitatory mechanisms, (2) loss of inhibitory mechanisms, and (3) glial-neuronal interaction (Fig. 5b). Under normal conditions, acute pain is signaled by the release of excitatory neurotransmitters such as glutamate from the central terminals of nociceptive afferents, generating excitatory postsynaptic potentials (EPSPs) in the secondorder neurons. EPSPs occur primarily through activation of postsynaptic AMPA ionotropic glutamate receptors. Summation of subthreshold EPSPs in the postsynaptic neurons eventually results in the firing of neuronal action potentials and transmission of the signals to higher-order neurons. Under these conditions, NMDA glutamate receptor is silent. Following persistent injury and inflammation, activated Aδ- and C-fibers release a variety of neurotransmitters [glutamate, SP, CGRP, brain-derived neurotrophic factor (BDNF), and ATP], and these transmitters act on receptors of second-order neurons. As a consequence, increased release of neurotransmitters from nociceptive afferents will sufficiently depolarize postsynaptic nociceptive neurons to activate normally silent NMDA glutamate receptors. The nociceptive neurons can signal, increase intracellular calcium, and activate Ca 2+ -dependent signaling pathways and second messengers including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and protein kinase A (PKA). This cascade of events can strengthen synaptic transmission (neoplastic changes) and increase the excitability of nociceptive neurons and facilitate the nociceptive transmission to higher CNS regions (Basbaum et al. 2009) (Fig. 5b, c). GABAergic and glycinergic inhibitory interneurons are densely distributed in the superficial spinal dorsal horn and Vc which postulates that loss of function of these inhibitory interneurons would result in increased pain (Merzack and Wall 1965). Under normal conditions, inhibitory interneurons continuously release GABA and/or glycine to decrease the excitability of nociceptive neurons or interneurons in Vc and modulate nociceptive transmission (inhibitory tone) (Basbaum et al. 2009; Malan et al. 2002; Sivilotti and Woolf 1994; Yaksh 1965; Takeda et al. 2000). In the setting of persistent injury and inflammation, this inhibition can be lost (disinhibition), resulting in hyperalgesia. Also, disinhibition can enable non-nociceptive myelinated Aβ-primary afferents to engage the nociceptive transmission circuitry such that normally non-noxious stimuli are now perceived as painful. Combined with NMDAmediated central sensitization, disinhibition enhances spinal cord outputs in response to painful and non-painful stimulation, contributing to mechanical allodynia (Keller et al. 2007; Torsney and MacDermott 2006) (Fig. 5b, c). A huge number of glial cells, microglia, astroglia and oligodendroglia are known to be distributed in the whole brain and have significant functions involved in nutrition, structure maintenance, and phagocytosis. Glial cells are thought to be involved in the modulation of neuronal activity by releasing various molecules such as glutamate, glutamine, neurotrophic factors, and cytokines following activation and also known to express various receptors involved in the modulation of excitabilities of nociceptive neurons receiving orofacial noxious inputs (Chiang et al. 2011; Iwata et al. 2011). Recent studies have reported that microglial cells are involved in the modulation of neuronal excitability as well as nourishing and supporting CNS structures (Cao and Zhang 2008; Milligan and Watkins 2009). Two types of glial cells (microglia and astroglia) are known to be hyperactive in the spinal dorsal horn following nerve injury and inflammatory conditions (Cao and Zhang 2008; Milligan and Watkins 2009). Under normal circumstances, the microglia function as resident macrophages of the CNS. Persistent injury and inflammation can promote the release of ATP and chemokine and fractalkine from the primary afferent terminals that stimulate microglial cells. In particular, activation of purinergic, fractalkine, and Toll-like receptors on microglia results in the release of BDNF which, through activation of trkb receptors expressed in the

14 14 K. Iwata et al. nociceptive neurons, promotes increased excitability and enhanced pain in response to non-noxious and noxious stimulation (hyperalgesia and allodynia). Activated microglia also release a host of cytokines, such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and other factors that contribute to central sensitization. On the other hand, astroglial cells may also become hyperactive and release glutamine which is taken up at presynaptic terminals via glutamine transporter, resulting in an increase in glutamate release from the presynaptic terminals and causing an enhancement of Vc and C1-C2 neuronal excitability (Chiang et al. 2007; Okada-Ogawa et al. 2009). Therefore, astroglial cell activation in the Vc and C1-C2 may also contribute to central sensitization of nociceptive Vc and C1-C2 neurons, resulting in orofacial hyperalgesia or allodynia following nerve injury and inflammation (Fig. 5b, c). Higher Brain Function Regulating Orofacial Nociception Ascending Orofacial Pain Pathways Projection neurons in the Vc and C1-C2 are known to send their axons to the thalamic nuclei (VPM and medial thalamic nuclei) and PBN (Iwata et al. 1992, 1998, 2011) (Fig. 6). Recent anatomical studies have demonstrated that projections from the Vc and C1-C2 to the PBN are much denser compared with those to the VPM and medial thalamic nuclei (Al-Khater and Todd 2009; Akiyama et al. 2016). Nociceptive neurons receiving noxious inputs from the orofacial region are somatotopically organized within the VPM but not in the medial thalamic nuclei and PBN. Intraoral noxious information is conveyed to the somatosensory cortex via the medial portion of the VPM and face and head via the lateral portion of the VPM (Willis et al. 2001). Nociceptive neurons in the VPM receiving inputs from the orofacial region send axons to the primary (SI) and secondary (SII) somatosensory cortical neurons whereas those of medial thalamic nuclei and PBN project to the limbic cortices such as anterior cingulate cortex (ACC) and insula cortex (Ins). Some previous studies have documented the electrophysiological characteristics of nociceptive neurons in the SI, SII, ACC, and Ins, and those in SI and SII are classified as WDR and NS neurons, and ACC neurons are NS and noxioustap neurons (Vogt 2005). Ins neurons are known to receive autonomic inputs (sympathetic and parasympathetic) and are involved in the modulation of autonomic responses, but there is no data regarding response properties of Ins nociceptive neurons (Ito 1998). Although it is evident that these cortical areas are involved in pain, the details of the involvement of these higher brain regions in pain are still largely unknown. Sensory-Discriminative and Motivational and Affective Aspects of Pain Pain is known to have very complicated aspects; sensory-discriminative and motivational and affective aspects (Dubner 1988). The sensorydiscriminative aspect of pain is analogous to non-noxious touch sensation and is thought to be involved in the discrimination of pain features such as its location, intensity, and quality. This sensory function is critical for survival. For example, when we receive a needle insertion into the finger, we can detect the location, intensity, and quality of the pain immediately after the injury. This sensory-discriminative aspect is necessary for humans to survive. SI and SII are thought to be involved in this aspect. On the other hand, the motivational and affective aspects of pain are believed to be related to emotional and autonomic responses due to long-lasting, intense noxious stimuli. This aspect is considered to be a feature of persistent pain associated, for example, with chronic inflammation or peripheral nerve injury. In this type of pain, it is often difficult to discriminate the localization, intensity, and quality of the pain. Another important issue is the prediction, cognition, and attention of pain. Nociceptive neurons in the ACC may be activated before a noxious stimulus is applied or when the subject predicts the noxious stimulus, indicating that nociceptive

15 Neurophysiology of Orofacial Pain 15 Fig. 6 Two distinct ascending pathways, medial and lateral systems in orofacial pain perception. Orofacial noxious inputs are conveyed to Vc and C1-C2, and further relayed to somatosensory (SI-SII) and limbic cortices (ACC and Ins) via thalamic nuclei, VPM, and medial thalamic nuclei, respectively. VPM, ventral posteromedial thalamic nucleus; SI, primary somatosensory cortex; SII, secondary somatosensory cortex; ACC, anterior cingulate cortex; Ins, insular cortex Trigeminal spinal nucleus oralis interpolaris caudalis (Vc) Cervical spinal cord C1-C2 Medial system Limbic cortices ACC and Ins) Medial thalamic nuclei Trigeminal ganglion mid-line Lateral system Somatosensory cortices SI and SII VPM Trigeminothalamic pathway Noxious inputs from the orofacial region neurons in the ACC are involved in pain prediction. Some of the nociceptive neurons also decrease their activity when attention is moved to a different modality of stimuli such as light or sound, suggesting that these nociceptive neurons are involved in attention to the noxious stimuli (Koyama et al. 1998; Iwata et al. 2005). Pain cognition is also important, but there is no animal data regarding this aspect. It may be possible to get new insight regarding brain function involved in pain cognition by human functional MRI (fmri) studies (Davis and Stohler 2014). Human Brain Function for Orofacial Pain Sensation Various strategies to analyze human brain function have been dramatically developed in recent decades. fmri is considered a useful tool to discover brain areas involved in pain in humans. The thalamus, brainstem, SI and SII, ACC, and Ins in humans are activated following painful stimuli, and these regions are thought to be involved in pain perception (Davis and Stohler 2014; Vachon- Presseau et al. 2016). The SI region is strongly activated by hypnotic suggestions that pain intensity is increased without any noxious stimuli, whereas ACC area is activated by suggestions that emotion is enhanced (Rainville et al. 1999). In a human magnetoencephalography study, SI-evoked responses following painful stimuli have shorter latencies and smaller amplitudes compared with ACC, suggesting that ACC may have a larger contribution to pain perception compared with SI (Nakata et al. 2008). It is highly likely that SI is involved in the coding of pain intensity, and ACC and Ins are involved in motivational and affective aspects of pain. These functional relationships of SI, ACC, and Ins to pain perception are consistent with results from various animal studies. Some of the fmri studies have reported that the prefrontal cortex (PFC) is also activated by painful stimulation of the hand in humans (Nakata et al. 2008). It has not been evaluated if PFC neurons receive noxious inputs from the orofacial area in animal studies because most of the animal studies have been conducted under anesthesia. At present, although many papers have been published regarding orofacial pain structures in the human brain, the detailed mechanisms underlying orofacial pain in humans are not entirely understood (Nash et al. 2009; Moayedi et al. 2011; Davis and Stohler 2014).

16 16 K. Iwata et al. Descending Modulation of Orofacial Pain Descending Pathways Influencing Orofacial Pain The ascending pathway in the CNS related to orofacial pain consists of many complex neuronal structures. Firstly, as noted above, nociceptors which exist in the free nerve endings of primary afferent Aδ- and C-fibers respond to mechanical, thermal, or chemical noxious stimulation of the orofacial region, e.g., orofacial skin, oral mucosa, dental pulp, periodontal tissue, or TMJ. The noxious information is then conducted to the medulla where the central terminals of the Aδ- andc-fibers release a number of excitatory neurotransmitters, and nociceptive neurons are excited. These events initiate the central neural processing of nociceptive information to the cortical and limbic circuits for the sensory perception that is transmitted by way of several nuclei in the brainstem and the thalamic nuclei (Sessle 2011). The ascending noxious information can, however, be modulated by the descending pain modulation system through various mechanisms. Potent drugs clinically used for pain relief include the opioid family which is represented by morphine. Indeed, morphine microinjection into the PAG, RVM, amygdala, or anterior insular cortex produces an intensive analgesic effect (Yaksh and Rudy 1978). At present, three classical receptors (μ-, δ-, and κ-opioid receptors) and a fourth related receptor (opioid receptor-like (ORL1) receptor) have been identified as opioid receptors which are widely distributed in the brain such as PAG, RVM, and dorsolateral pontomesencephalic tegmentum (DLPT) and superficial laminae of the spinal dorsal horn (Mansour et al. 1995; Darland et al. 1998). Opioid receptors belong to members of the G-protein-coupled receptors (GPCRs). Moreover, the major classes of endogenous opioid peptides, β-endorphin, enkephalin, and the dynorphin, bind the above opioid receptors present in the CNS. These opioid peptides are derived from a large, usually inactive protein precursor. For instance, β-endorphin is cleaved from pro-opiomelanocortin which is synthesized from adrenocorticotrophic hormone or melanocyte-stimulating hormone. Though the various types of opioid receptors are distributed widely within the CNS and peripheral nervous system, the affinity for opioid receptors shows a characteristic pattern due to the types of opioid peptides. Enkephalin is the dominant ligand for δ-opioid receptors compared to μ-opioid receptors; β-endorphin has equal affinity for δ-opioid receptors and μ-opioid receptors and low affinity for κ-opioid receptors. Dynorphin has a high affinity for κ-opioid receptors. The opioid peptides signaling via various opioid receptors in the brain and spinal cord induce hyperpolarization and inhibition of spike activity in neurons by blocking neurotransmitter release through inhibition of calcium influx into the presynaptic terminal or opening potassium channels, resulting in the depression of neuronal activity (Nagi and Pineyro 2014; Ossipov et al. 2014). The midbrain PAG and RVM play a significant role in the major pain-modulating pathway (Lau and Vaughan 2014). Information from the frontal lobe or amygdala is conveyed via the hypothalamus to the PAG. In turn, neurons in the PAG communicate with serotonergic and nonserotonergic projection neurons in the RVM and also interact with noradrenergic projection neurons in the DLPT. These projection neurons modulate the excitability of Vc nociceptive neurons which carry nociceptive signals to higher levels of the CNS. Opioid signaling leads to changes in the excitability of these neurons, resulting in the enhancement of descending pain suppression. The μ-opioid or δ-opioid receptors are also abundant in primary nociceptive neurons (Minami et al. 1995; Brederson and Honda 2015). Intraplantar administration of morphine which is a μ-opioid receptor agonist exerts antihyperalgesic effects in a rat model of diabetic polyneuropathy (Schiene et al. 2015). These results indicate that the opioid signaling in primary nociceptive neurons via opioid receptors is involved in peripheral analgesia (Bakke et al. 1998; Stein and Zollner 2009).

17 Neurophysiology of Orofacial Pain 17 Modulation of Trigeminal Nociceptive Neurons 5-HT-containing neurons in RVM project to the Vc and C1-C2. Depending on the 5-HT receptor subtype, 5-HT in the Vc and C1-C2 acts as either inhibitor or facilitator (Suzuki et al. 2004; Dogrul et al. 2009), e.g., 5-HT2A, 5-HT3, and 5-HT4 are excitatory 5-HT receptors and 5-HT1A, 5-HT1B, and 5-HT1C are inhibitory 5-HT receptors expressed in the central terminals of nociceptive primary afferents. Therefore, 5-HT released from 5-HT-containing neurons can change the excitability of nociceptive neurons in the Vc and C1-C2. 5-HT1B is expressed in primary afferents, and the activation of presynaptic 5-HT1B reduces glutamate release from primary afferent terminals in the Vc and C1-C2, resulting in modulation of nociceptive neuronal activity (Choi et al. 2012). Furthermore, by acting on 5-HT1A which are inhibitory receptors expressed in second-order neurons, 5-HT exerts the suppressive effect on nociceptive neurons. 5-HT7, an excitatory 5-HT receptor, has also been identified in GABAergic interneurons in the Vc and C1-C2, as well as on central terminals of nociceptive primary afferents (Doly et al. 2005). The excitation of 5-HT7 enhances the release of GABA, resulting in depression of the excitability of secondary nociceptive neurons (Brenchat et al. 2009). The A5 (locus coeruleus), A6, and A7 (Kölliker-Fuse) noradrenergic nuclei are major sources of direct noradrenergic projections to medullary and spinal dorsal horns (Willis 1985; Holden and Proudfit 1998; Bajic and Proudfit 1999). Noradrenaline (NA) released from the medullary and spinal terminals and of these noradrenergic neurons plays a role in inhibition of the presynaptic and postsynaptic nociceptive transmission. Many previous studies have shown that NA signaling exerts a potent antinociceptive effect mediated by the activation of α2 receptors (Pertovaara 2006). Noradrenergic systems inhibit nociceptive transmission at the level of the medulla and spinal cord through presynaptic mechanisms. Released NA activates α2-adrenergic receptors expressed in the central terminal of nociceptive primary afferents, inhibiting release of excitatory neurotransmitters such as glutamate from the primary afferent terminals. Moreover, postsynaptic sites of second-order nociceptive neurons also express α2-adrenergic receptors; NA signaling induces a potent antinociceptive effect by acting on these α2-adrenergic receptors. Further, α1-adrenergic receptors in GABA interneurons produce depolarization, which results in the attenuation of the hyperexcitability of secondorder nociceptive neurons that occurs under pathological conditions. In this manner, the summation of 5-HT, NA, and GABA signaling in second-order nociceptive neurons in Vc and C1-C2 results in these descending systems acting as inhibitor or facilitator (Fig. 7). Although the precise medullary and spinal mechanisms involved in descending pathways modulating orofacial pain remain unclear, these descending systems work almost invariably as an inhibitor. Pathological Changes in the Descending System The descending pain inhibitory system can become dysfunctional in patients with a chronic pain condition (e.g., temporomandibular disorders), resulting in enhanced sensitivity to noxious stimuli (King et al. 2009). Recent studies have demonstrated that the descending inhibitory or facilitatory systems are modulated in experimental chronic pain models (Burgess et al. 2002; Dubner et al. 2014; Rahman et al. 2009; Wei et al. 2010). Orofacial neuropathic pain induced by infraorbital nerve injury or stress-induced TMJ pain is primarily dependent on the central mechanisms involving 5-HT derived from the RVM and activation of 5-HT3 receptors in the Vi/Vc (Okubo et al. 2013; Okamoto et al. 2015). Clinical studies have indicated that 5-HT3 receptor antagonist administration is useful for the treatment of neuropathic pain associated with fibromyalgia (Seidel and Muller 2011; Forster and Baron 2012). In human studies, μ-opioid receptor availability in the nucleus accumbens (which is an area

18 18 K. Iwata et al. Central nucleus of the amygdala PAG RVM Serotonergic neuron Vc LC Noradrenergic neuron Serotonergic neuron Noradrenergic neuron 5-HT 1A a 2 Primary neuron Secondary neuron Fig. 7 Descending pathways involved in pain modulation. Descending pain modulation is mediated through projections to the periaqueductal gray (PAG), which also receives inputs from other sites, including the hypothalamus. PAG communicates with the rostroventromedial medulla (RVM) that sends descending projections to the trigeminal spinal subnucleus caudalis (Vc). The noradrenergic locus coeruleus (LC) receives inputs from the PAG and sends descending noradrenergic inhibitory projections to Vc. The effect of 5-HTand noradrenaline in Vc can work as either inhibitor or facilitator, depending on its receptor subtype involved in pain modulation) is reduced in trigeminal neuropathic pain (DosSantos et al. 2012). While dysfunction of the descending pain inhibitory system in orofacial pain patients is not entirely understood, increased knowledge of the dysfunctional modality of descending pain inhibitory system offers approaches to develop improved orofacial pain therapy. Conclusion and Future Directions Peripheral and central mechanisms, ascending and descending pathways related to orofacial pain and its neuromodulation, and the pathogenesis of persistent orofacial pain have been reviewed and discussed in this chapter. Many of the trigeminal nerve fibers respond to cool or cold as well as heat and mechanical stimuli applied to the intraoral mucous membrane. A barrage of action potentials following noxious stimulation of the orofacial regions are conveyed to the Vc and C1-C2, and those are further relayed to the somatosensory or limbic cortices via medial and lateral thalamic pathways. Pain threshold is significantly lower in the orofacial regions compared with other body parts. Further, orofacial noxious inputs are represented by the widest areas in the VPM and somatosensory cortex, indicating that neurons receiving noxious inputs from the orofacial regions are distributed more densely and broadly than in other body parts. Under pathological conditions such as trigeminal nerve injury or orofacial inflammation, pain intensity becomes much stronger than that of other body parts. Persistent orofacial pain also causes a variety of deficits in the orofacial motor and sensory functions such as mastication, swallowing, and taste. It is crucial to know the underlying mechanism of pathological orofacial