J Physiol 592.16 (2014) pp 3403–3411 3403

SYMPOSIUM REVIEW Nociceptive primary afferents: they have a mind of their own

Susan M. Carlton

Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77554, USA

Neuroscience Abstract Nociceptive primary afferents have three surprising properties: they are highly complex in their expression of neurotransmitters and receptors and most probably participate in autocrine and paracrine interactions; they are capable of exerting tonic and activity-dependent inhibitory control over incoming nociceptive input; they can generate signals in the form of dorsal root reflexes that are transmitted antidromically out to the periphery and these signals can result in neurogenic inflammation in the innervated tissue. Thus, nociceptive primary afferents are highly complicated structures, capable of modifying input before it is ever transmitted to the central nervous system and capable of altering the tissue they innervate.

(Received 12 December 2013; accepted after revision 26 April 2014; first published online 30 May 2014) Corresponding author S. M. Carlton: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-1069, USA. Email: [email protected]

Abbreviations CAP, capsaicin; CGRP, calcitonin gene-related peptide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; c-SOM, cyclo-somatostatin; DRG, dorsal root ganglion; DRR, dorsal root reflex; GABA, γ-aminobutyric acid; LY, + + LY341495; mGluR, metabotropic glutamate receptor; NKCC, Na ,K ,Cl– cotransporter; NMDA, N-methyl-D-aspartate; SP, substance P; SST, somatostatin; TRPV1, transient receptor potential vanilloid 1; VT, volume transmission.

In the early years of studying sensory processing, the stimulus trajectory. Three hundred plus years of research peripheral afferent was thought to be no more than a later we know that this is not the case. On the contrary, the pipeline or a conduit that faithfully transported sensory study of nociceptive primary afferents has demonstrated information from the stimulated region to conscious that these fibres have many surprising properties, three levels. This belief is most clearly demonstrated in the of which will be discussed in this review. First, although drawing by Descartes published in the 1680s, where he nociceptive terminals appear simple and uncomplicated illustrated a boy experiencing burning pain as a result of (Fig. 1), immunohistochemical studies have demonstrated

The Journal of Physiology his toes coming in contact with fire (see Fig. 2 of Roper, that nociceptors are very complex in their expression of 2014, Introduction, this issue). An uninterrupted line is ligands, neurotransmitters and receptors. This allows for drawn from the toes to the brain, suggesting there is no nociceptors to have autocrine and paracrine interactions. modification of the fiery stimulus at any point along the Second, as a result of this complexity, they are able to

Susan M. Carlton is a native of upstate New York. She did her undergraduate studies at Mary Washington College (Fredericksburg, VA, USA) attaining a BS in Biology. She then moved to the Medical College of Virginia where she earned her PhD in anatomy, with a concentration in neuroanatomy. She did two postdoctoral fellowships, one at the University of Texus Medical Branch (UTMB) with Dr William D. Willis and one at Yale University (New Haven, CT, USA) with Dr Carole LaMotte. She became an assistant professor at UTMB, Department of Neuroscience and Cell Biology and rose through the ranks. She is now a tenured professor, teaching and researching in this department for over 30 years. Her area of expertise is the peripheral nociceptor, studying pain transmission in both acute and chronic pain states.

This review was presented at the symposium Sensory end organs: signal processing in the periphery, which took place at the annual meeting of the Society for Neuroscience, San Diego, CA, USA on 10 November 2013.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society DOI: 10.1113/jphysiol.2013.269654 3404 S. M. Carlton J Physiol 592.16 modify input before it reaches the central nervous system There are no classic synapses in relation to cutaneous (CNS), including inhibition of input so that signals are nociceptors (pre- and postsynaptic elements that dampened before ever leaving the peripheral terminal. communicate via a synaptic specialization within a Finally, these fibres can generate outgoing signals, termed synaptic cleft). Therefore, the best way to describe the dorsal root reflexes (DRRs), which alter the peripheral type of transmission that occurs in relation to nociceptors tissues they innervate. This antidromic activity contributes in the periphery is volume transmission (Zoli et al. 1998; to disease states. Fig. 2). This is when a ligand is released and through 3-dimensional diffusion in the extracellular space reaches its receptor. Travelling in this manner, a ligand is able Complexity of nociceptive primary afferents not only to reach a larger number of targets but also can In the late 1990s, Richard Coggeshall and I wrote two reach targets at an increased distance. Consistent with review articles that focused on neurotransmitter and volume transmission, several lines of evidence strongly receptor localizations in primary afferents. One article suggest that autocrine and paracine interactions can surveyed primary afferents in general (Coggeshall & occur at the level of the nociceptor (Fig. 2). This is Carlton, 1997) and the second focused on nociceptors exemplified by the fact that nociceptors can release in particular (Carlton & Coggeshall, 1998). As a glutamate (De Groot et al. 2000; Jin et al. 2009) and result of these endeavours, it became very clear that several receptors in the glutamate family (ionotropic integration of information at the level of the peripheral and metabotropic) are expressed by both nociceptive terminal was a real possibility and the oversimplified and non-nociceptive peripheral terminals (for review see view of the nociceptor as just a conduit for noxious Carlton, 2001; Neugebauer & Carlton, 2002). Thus, there is information was a gross misrepresentation. To date compelling evidence that this compartment of the nervous numerous ligands/neurotransmitters have been localized system, once thought to be simple and straightforward in nociceptive primary afferents (Fig. 2). Add to this list in its processing capabilities, is capable of integrating growth factors (nerve growth factor, brain-derived neuro- input before it ever reaches the first synapse in the trophic factor, glial-derived neurotrophic factor) and CNS. hormones (prolactin, somatostatin), and the capability of peripheral nociceptors to influence their micro- environment becomes quite impressive. Add to this an Nociceptors modify incoming input prior to array of receptors that are expressed on these terminals transmission to the CNS and one can begin to imagine that each terminal resembles There are now several lines of evidence that nociceptive a tiny brain! The complexity of nociceptors has led us to primary afferents can modify signals before they are think of them as having a ‘mind of their own’. transmitted centrally. This ability can change the way we perceive the outside world. There are comprehensive reviews written on nociceptor sensitization, processes through which nociceptors can amplify signals (Gold & Gebhart, 2010). In this review, I will discuss the concept of nociceptor-initiated inhibition: processes through which nociceptors can inhibit signals. Two receptor families known to have inhibitory control over nociceptor activity will be discussed, each using a different mechanism of action. These phenomena take the form of tonic inhibitory control and activity-dependent inhibitory control.

Somatostatin (SST). Subsets of dorsal root ganglion (DRG) cells express somatostatin peptide (Hokfelt¨ et al. 1975) and SST receptors (SST1, 2A, 2B, 3 and 4; Carlton et al. 2001a, 2004; Bar¨ et al. 2004). Nociceptors in particular express SST receptor 2A (Carlton et al. 2001a, 2004). Several lines of evidence demonstrate that SST agonists applied in the periphery will attenuate formalin or capsaicin (CAP)-induced nociceptive behaviours Figure 1. Immunostained nociceptive primary afferents and block bradykinin-induced nociceptor sensitization visualized as they penetrate the epidermis (Carlton et al. 2001a, 2004; Ji et al. 2006). The surprising Modified from Zylka et al. 2005, with permission. finding is that SST receptors maintain a tonic inhibitory

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society J Physiol 592.16 Nociceptive primary afferents 3405 control over nociceptors (Carlton et al. 2001b). In naive ient receptor potential vanilloid 1 (TRPV1) receptors rats, blockade of these receptors on nociceptors by intra- expressed on cutaneous nociceptors (Carlton et al. plantar injection of either the SST receptor antagonist 2004). Blockade of peripheral SST receptors with c-SOM cyclo-somatostatin (c-SOM) or a SST receptor anti- dramatically enhances TRPV1 agonist CAP-induced pain body, generates nociceptive behaviours such as flinching behaviours and nociceptor activity. SST receptors are the and lifting/licking. In rats in acute pain (formalin), first receptors shown to have a tonic inhibitory control injection of these same drugs produces enhancement over nociceptor processing in the periphery. of the formalin-induced nociceptive behaviours. These actions are blocked when c-SOM is co-applied with three different SST agonists (SRIF-14, octreotide, vapreotide). Metabotropic glutamate receptors (mGluRs). Our lab Tonic inhibitory control is confirmed at the single has a long history of studying glutamate receptors on fibre level using a glabrous skin–nerve preparation. nociceptors in the periphery (Carlton et al. 1995, 2009b; Application of c-SOM to the receptive field of identified Zhou et al. 1996; Davidson et al. 1997; Carlton, 2001; nociceptors innervating the skin results in the generation Du et al. 2001, 2003, 2006, 2008; Neugebauer & Carlton, of activity that is blocked by the addition of the 2002; Govea et al. 2012). We have been most inter- SST agonists. Additional studies demonstrate that SST ested in the group II and III mGluRs because when receptors maintain tonic inhibitory control over trans- activated, they produce neuronal depression/inhibition

SKIN DRG CORD

Ligands with Ligands: non-neural sources: SP Opioids ATP Adenosine ACh NPY Glutamate ATP CCK Somatostatin PGE Bomb Prolactin Opioids CGRP Adenosine Galanin Glutamate BK,HA,5HT

1 Volume Transmission Ligand 2

Receptors:

ATP, NK1, NPY, ACh, PGE, CCK, 5HT, Bomb, BK, HA, Opioid, TRKs, TRPs, Piezo, Adenosine, mGluR, iGluR,

GABAA, GABAB, Mrgprd, Somatostatin, Adrenergic, Angio II

Figure 2. Schematic drawing of two nociceptors with their terminals expanded at the level of the skin The dorsal root ganglia (DRG) and central processes of these fibres (terminating in the spinal cord dorsal horn) are also sketched. Nociceptors express a large variety of receptors and channels and they contain numerous ligands, many of which they release. Receptor activation in the periphery occurs through volume transmission. This allows for autocrine (1) and paracrine (2) regulation of their activity. Ligands with non-neural sources are also listed. 5HT, serotonin; ACh, acetylcholine, ATP, adenosine triphosphate, Angio II, angiotensin II; Bomb, bombesin; BK, bradykinin; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; HA, histamine; iGluR, ionotropic glutamate receptor; mGluR, metabotropic glutamate receptors; mrgprd, MAS-related G protein-coupled receptor; NK1, neurokinin 1; NPY, neuropeptide Y; PGE, prostaglandin E; SP, substance P; TRPs, transient receptor potential receptors; TRKs, tyrosine kinase receptors. (Reproduced from Carlton & Coggeshall, 1998, with permission.)

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society 3406 S. M. Carlton J Physiol 592.16

(Schoepp & Conn, 1993; Conn & Pin, 1997), actions inhibitory control over nociceptors (Carlton et al. that would be important in controlling pain transmission 2011). Intraplantar injection of LY significantly enhances from the periphery to the CNS. When antibodies and CAP-induced nociceptive behaviours (Fig. 3). This is pharmacological tools became available to investigate confirmed at the single fibre level where CAP-induced the presence and action of mGluRs, another exciting activity is enhanced not only by LY but also possibility for modulation of painful input by peri- by (RS)-1-amino-5-phosphonoindan-1-carboxylic acid pheral nociceptors was elucidated. Anatomical studies (APICA), a selective group II antagonist and by demonstrate that a significant percentage of DRG cells 2R,4R-4-aminopyrrolidine-2,4-dicarboxyate (UBP), a express either group II, III or both receptors. Importantly, selective group III antagonist. In a dose-dependent 93% and 71% of DRG cells expressing TRPV1 express fashion, LY can induce calcium mobilization in DRG group II (Carlton et al. 2001b) or group III (Govea et al. cells when applied with CAP. The actions of LY on 2012), respectively. Thus, the anatomical substrate is pre- CAP-induced responses are attenuated by either group sent supporting an action of the two receptors on the same II or III selective agonists (Carlton et al. 2011). peripheral nociceptor. A series of experiments demonstrates that excess Do these mGluRs maintain a tonic inhibitory control, glutamate plays a role in inducing activity-dependent similartowhatisseenwithperipheralSSTreceptors? inhibition by group II/III mGluRs. Sensitivity to heat In contrast to what is observed following injection of does not develop following injection of glutamate alone an SST receptor antagonist, a group II/III antagonist (300 μM), but injection of this concentration plus LY LY341495 (LY, 100 μM) injected alone into the hind- (to prevent group II/III mGluR activation) produces a paw does not generate nociceptive behaviours (Du robust and prolonged sensitivity to heat evidenced by a et al. 2008). Thus, there is no tonic inhibitory control significant lowering of the paw withdrawal latency to a exhibited by group II/III mGluRs. This lack of effect heat stimulus (Carlton et al. 2011). A similar result is of LY alone and lack of tonic control is confirmed in observed at the single fibre level where 1 mM glutamate DRG cells using calcium imaging and in nociceptors alone does not change the discharge rate or the unit using single fibre recordings (Carlton et al. 2011). response to heat, but in the presence of LY there is a However, several lines of evidence demonstrate that 4-fold increase in the glutamate-induced discharge rate groups II and III maintain an activity-dependent and the threshold to activation is lower. The experiments

Figure 3. Behavioural data demonstrating group II/III involvement in activity-dependent inhibition of TRPV1 receptors Intraplantar capsaicin (CAP) alone results in flinching (A and B) and Lift/Lick (C and D) behaviour. This behaviour is enhanced when CAP is injected with LY341495 (LY), a group II/III antagonist. CAP in one hindpaw and LY in the other results in behaviour that is no different from CAP alone, confirming that LY does not become systemic but is having a local effect. (Reproduced from Carlton et al. 2011, with permission.)

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society J Physiol 592.16 Nociceptive primary afferents 3407 described above use exogenous glutamate. To determine given its critical role in pain transmission (Tominaga if endogenous glutamate release has functional relevance et al. 1998; Caterina & Julius, 2001). Our studies show in relation to mGluR activation, a protocol is used that if mGluR activation is prevented, then prolonged that causes endogenous release of glutamate, namely enhancement of TRPV1 function occurs. Without the formalin injection (Omote et al. 1998). Formalin (2%) activity-dependent modulation of group II/III mGluRs, is injected alone or with LY. There is a 50% increase the TRPV1-induced peripheral sensitization is prolonged in formalin-induced nociceptive behaviours when it is and the nociceptive signal transmitted to the cord or accompanied by LY. This increase is prevented when a brainstem is amplified. The augmented signals would group II agonist is added. The data infer that release of end- impact dorsal horn neurons and most likely promote ogenous glutamate plays a pivotal role in engaging group central sensitization. This use-dependent modification II/III inhibition, which dampens formalin-induced pain by mGluRs is important, serving to prevent ‘runaway’ behaviours. Sources of endogenous glutamate include first excitation of nociceptors. If the system fails, this could and foremost the primary afferents themselves (Westlund enhance both peripheral and/or central sensitization that et al. 1989; Jeftinija et al. 1991; Omote et al. 1998; De is a hallmark of many chronic pain conditions. Thus, Groot et al. 2000; Keast & Stephensen, 2000), keratinocytes there is great potential for primary afferent nociceptors (Genever et al. 1999) and blood serum (Erdo, 1991). Based to protect us from exaggerated pain transmission that can on this series of experiments we conclude that group II/III produce, or transform into, chronic pain. mGluRs do not influence nociceptive afferents when at rest and they do not modulate responses following brief activation (i.e. 10 s heat pulse). The mGluR inhibitory Generation of outgoing signals: dorsal root reflexes influence becomes apparent after high frequency and/or prolonged stimulation (as occurs in response to algogenic Primary afferents can function as a two-way street. In substances like CAP or formalin; Carlton et al. 2011). addition to modifying input before it is transmitted to The data are compelling that group II/III mGluRs the CNS, they can also generate action potentials at their function as built-in negative modulators of peripheral central terminals which are then conducted to their peri- nociceptor activity. They have little or no role under basal, pheral terminals, causing release of neurotransmitters. quiescent conditions, or when nociceptors respond to brief Thus, they can transmit information into the CNS and stimuli. However, the mGluRs clearly regulate nociceptors carry input out of the CNS (Gotch & Horsley, 1891; undergoing vigorous excitation. Endogenous inhibitory Barron & Matthews, 1935). Because this activity was modulation of TRPV1 function is undoubtedly important originally recorded in dorsal roots that were disconnected

Figure 4. Producing primary afferent depolarization (PAD) in central terminals of sensory fibres The proposed circuitry underlying dorsal root reflex activity at the level of the primary afferent in the dorsal horn is shown. An axoaxonic contact is made by a GABAergic interneuron onto a primary afferent terminal. Release of GABA from the interneuron and activation of – GABAA receptors produces an efflux of Cl from the primary afferent terminal, resulting in its depolarization. The Cl– is concentrated in the primary afferent terminal by the Na+,K+,Cl– cotransporter symbolized by the dark oval in the membrane of the afferent. (Modified from Alvarez-Leefmans et al. 1998, with permission.)

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society 3408 S. M. Carlton J Physiol 592.16 from the periphery, the phenomenon was termed dorsal (SP) from nerve endings (Maggi, 1995). CGRP induces root reflexes (DRRs; Barron & Matthews, 1935). While neurogenic inflammation as evidenced by vasodilatation recording from dorsal roots is the ‘cleanest’ way to isolate (flare) and SP induces plasma extravasation (oedema; for this antidromic activity and be confident of its origin, it review see Willis, 1999). Activation of TRPV1-expressing can also be recorded distal to DRGs in nerve branches C fibres produces changes in blood flow, a flare response (Toennies, 1938; Sluka et al. 1995a;Reeset al. 1996) with and neurogenic inflammation (Lin et al. 1999). Evidence the caveat that proper controls must be done to confirm the that these events are due in part to DRR activity comes origin and the fibre type carrying the output. Originally from the observation that acute dorsal rhizotomy nearly all sizes of myelinated axons were reported to carry DRRs, abolishes the blood flow changes and significantly reduces but not C fibres (Toennies, 1938; Lisney, 1979). However, the flare and neurogenic inflammation at the injection site. more recently, C fibres have been shown to carry DRRs as Furthermore, intrathecal bicuculline (GABAA antagonist) well (Lin et al. 2000; Weng & Dougherty, 2005). or CNQX (non-NMDA antagonist) dramatically reduces The DRR circuitry that has been proposed involves the all of these signs. Using the same CAP injection protocol, same circuitry underlying primary afferent depolarization DRRs can be recorded from the central stump of cut dorsal (PAD) (Willis, 1999) and involves the release of rootlets and this activity is significantly reduced with γ-aminobutyric acid (GABA) from dorsal horn inter- intrathecal bicuculline or CNQX (Lin et al. 1999). From neurons. This activates GABAA receptors on presynaptic these experiments it is concluded that DRRs play a major terminals of primary afferents, resulting in chloride role in the development of neurogenic inflammation. ions (Cl–) leaving the terminal. This depolarizes the In addition to CAP, electrical stimulation or noxious terminal, bringing the membrane potential closer to mechanical stimulation of cutaneous C fibres can also the threshold for generation of an action potential. evoke DRRs and the receptive field to drive the DRRs can If enough Cl– leaves the terminal and the membrane be as small as one digit or as large as the whole body reaches threshold, an action potential is generated and (Weng & Dougherty, 2005). Importantly, spread of DRR propagated antidromically out to the periphery. Why activity can occur over many cord segments, rostral and does activation of GABAA receptors lead to excitation caudal to the point of origin (Bagust et al. 1993). In animal here but to inhibition elsewhere in the CNS? The answer models of acute knee arthritis, DRRs are detected in the lies in the expression by primary afferents of a particular central stump of the cut dorsal root filaments or from the transporter: the Na+,K+,2Cl– or NKCC cotransporter proximal stump of the cut medial articular nerve which (Alvarez-Leefmans et al. 1988; Rocha-Gonzalez et al. innervates the knee joint (Rees et al. 1994; Sluka et al. 2008). This cotransporter allows afferent terminals 1995a). Sympathectomy does not change the DRR activity to sequester Cl– ions such that the concentration of but cutting the dorsal roots eliminates it confirming a CNS Cl– becomes greater inside the terminal compared origin. Delivery of non-NMDA antagonists via a micro- to outside (Alvarez-Leefmans et al. 1998; Sung et al. dialysis fibre threaded into the spinal cord dorsal horn also 2000). When GABAA receptors located on primary changes the outcome of knee joint inflammation: CNQX afferent terminals are activated, Cl– flows out of the reduces the joint swelling, prevents the major temperature terminal and the membrane equilibrium potential increase in the inflamed knee and attenuates the heat moves toward the threshold potential. This proposed circuitry is depicted in Fig. 4 (Alvarez-Leefmans et al. 1998). Non-NMDA receptors have also been shown to play a role in DRRs; it has been hypothesized that the first synapse in the spinal cord, where incoming input gains access to DRR circuitry, involves non-NMDA receptors since 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, but not DL-2-amino-7-phosphonoheptanoic acid (AP7), an NMDA antagonist, can block DRR activity (Sluka & Westlund, 1993; Sluka et al. 1995b). Additions have been made to this hypothesized circuitry which explain the phenomena of touch-evoked allodynia following peripheral injury (Cervero & Laird, 1996). Animal studies show us that DRRs, particularly those in Figure 5. Evidence of DRR activity in fibres in the median nerve following a T10 spinal cord injury C fibres, have an important efferent function that affects A, dorsal root reflex (DRR) activity is recorded from the proximal the functional state of peripheral tissues (Maggi et al. stump of the cut median nerve in vivo. B, trace of spontaneous DRRs 1987). This antidromic activity in C fibres results in release and evoked DRRs (produced by mechanical stimulation of the of calcitonin gene-related peptide (CGRP) and substance P forepaw) recorded from the median nerve proximal stump.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society J Physiol 592.16 Nociceptive primary afferents 3409 hyperalgesia that normally develops in the ipsilateral hind- belief that primary afferents are waiting to conduct input paw (Sluka & Westlund, 1993). One explanation for this unmodified directly to the brain, we now know that they outcome is a CNQX-induced reduction in DRR activity take an active role in modifying the signal before it is ever and a consequent decrease in release of SP and CGRP in transmitted to the CNS. In terms of pain management, the the periphery (Sluka & Westlund, 1993). DRRs not only variety of receptors expressed by nociceptive afferents pre- have consequences in the periphery but also affect dorsal sents numerous targets for drug development. The ability horn neuron activity. Specifically, NKCC1 cotransporter of some receptors to decrease/dampen nociceptive input activity is functionally linked to sensitization of dorsal before it is transmitted to the CNS should move them to the horn nociceptive neurons (Pitcher & Cervero, 2010). top of the drug development list! Finally, the phenomena of Neurogenic inflammation occurs not only in skin and DRRs, their significant contributions to pain and swelling joints but also in the eye, middle ear, dura mater and following inflammation, and their ability to propagate to the respiratory, genito-urinary reproductive and digestive many segments rostral and caudal in the cord, suggests systems (see Geppetti & Holzer, 1996). DRRs could be contributing to many pain states but In an animal model of spinal cord injury (Carlton heretofore this influence has been undetected and/or et al. 2009a), the presence of DRR activity has been unrecognized. Clearly, the more we understand about how reported (Du & Carlton, 2007) and may contribute nociceptive primary afferents ‘work’, the better chance we to the pathophysiological pain observed following this have of developing evidence-based therapies for peripheral CNS injury. Thirty-five days after a thoracic 10 (T10) pain management. contusion, spontaneous and evoked DRRs are recorded in Aδ and C fibres in filaments teased from the median nerve in the forelimb (Fig. 5). A significantly higher percentage of units show DRR activity compared to References that found in shams (Fig. 6). There is no change in the DRR activity following dorsal or ventral rhizotomy Alvarez-Leefmans FJ, Gamino SM, Giraldez F & Nogueron I and no change following sympathectomy confirming that (1988). Intracellular chloride regulation in amphibian dorsal sensory fibres carry the output (S.M. Carlton, unpublished root ganglion neurones studied with ion-selective observations). The evoked DRR activity is attenuated microelectrodes. JPhysiol406, 225–246. Alvarez-Leefmans FJ, Nani A & Marquez S (1998). Chloride by intrathecal bicuculline and the rats develop forepaw transport, osmotic balance, and presynaptic inhibition. In oedema evidenced by an increase in paw volume at 35 days Presynaptic Inhibition and Neural Control,ed.RudominP, post-injury. Thus, DRR activity can develop not only after Romo R & Mendell L, pp. 50–79. Oxford University Press, a peripheral injury/inflammation but also after a CNS New York. injury. Bagust J, Chen Y & Kerkut GA (1993). Spread of the dorsal root reflex in an isolated preparation of hamster spinal cord. Exp Physiol 78, 799–809. Conclusions Bar¨ KJ, Schurigt U, Scholze A, Segond Von Banchet G, Stopfel N, Brauer¨ R, Halbhuber KJ & Schaible HG (2004). The Our understanding of primary afferent function has expression and localization of somatostatin receptors in grown exponentially since the 1600s. In contrast to the dorsal root ganglion neurons of normal and monoarthritic rats. Neuroscience 127, 197–206. Barron DH & Matthews BH (1935). Intermittent conduction in ABC fibres Aδ fibres the spinal cord. JPhysiol85, 73–103. 100 Carlton SM (2001). Peripheral excitatory amino acids. Curr *† 80 *† Opin Pharmacol 1, 52–56. 60 Carlton SM & Coggeshall RE (1998). Nociceptive integration: Does it have a peripheral component? Pain Forum 7, 71–78. 40 Carlton SM, Du J, Davidson E, Zhou S & Coggeshall RE 20 efferent activity (2001a). Somatostatin receptors on peripheral primary

% units with evoked units% with 0 afferent terminals: inhibition of sensitized nociceptors. Pain Naive Sham SCI Naive Sham SCI 90, 233–244. n= 7/35 17/44 50/73 4/20 8/26 25/32 CarltonSM,DuJ,TanHY,NesicO,HargettGL,BoppAC, Yamani A, Lin Q, Willis WD & Hulsebosch CE (2009a). Figure 6. Evidence that DRR activity increases in spinal Peripheral and central sensitization in remote spinal cord cord-injured (SCI) animals compared to naive and sham regions contribute to central neuropathic pain after spinal Histograms show the increased percentage of C (A)andAδ (B) fibres cord injury. Pain 147, 265–276. exhibiting DRR activity in spinal cord-injured rats compared to naive Carlton SM, Du J & Zhou S (2009b). Group II metabotropic and sham. ∗P < 0.05 compared to naive; †P < 0.05 compared to glutamate receptor activation on peripheral nociceptors sham, one-way ANOVA. modulates TRPV1 function. Brain Res 1248, 86–95.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society 3410 S. M. Carlton J Physiol 592.16

Carlton SM, Du J, Zhou S & Coggeshall RE (2001b). Tonic Gold MS & Gebhart GF (2010). Nociceptor sensitization in control of peripheral cutaneous nociceptors by somatostatin pain pathogenesis. Nat Med 16, 1248–1257. receptors. JNeurosci21, 4042–4049. Gotch F & Horsley V (1891). On the mammalian nervous Carlton SM, Hargett GL & Coggeshall RE (1995). Localization system, its functions and their localizations, determined by and activation of glutamate receptors in unmyelinated axons an electrical method. Philos Trans R Soc Lond B Biol Sci 182, of rat glabrous skin. Neurosci Lett 197, 25–28. 267–526. Carlton SM, Zhou S, Du J, Hargett GL, Ji G & Coggeshall RE Govea RM, Zhou S & Carlton SM (2012). Group III (2004). Somatostatin modulates the transient receptor metabotropic glutamate receptors and transient receptor potential vanilloid 1 (TRPV1) ion channel. Pain 110, potential vanilloid 1 co-localize and interact on nociceptors. 616–627. Neuroscience 217, 130–139. Carlton SM, Zhou S, Govea R & Du J (2011). Group II/III Hokfelt¨ T, Elde R, Hohansson O, Luft R & Arimura A (1975). metabotropic glutamate receptors exert endogenous Immunohistochemical evidence for the presence of activity-dependent modulation of TRPV1 receptors on somatostatin, a powerful inhibitory peptide, in some peripheral nociceptors. JNeurosci31, 12727–12737. primary sensory neurons. Neurosci Lett 1, 231–235. Caterina MJ & Julius D (2001). The vanilloid receptor: a Jeftinija S, Jeftinija K, Liu F, Skilling SR, Smullin DH & Larson molecular gateway to the pain pathway. Ann Rev Neurosci 24, AA (1991). Excitatory amino acids are released from rat 487–517. primary afferent neurons in vitro. Neurosci Lett 125, Cervero F & Laird JM (1996). Mechanisms of touch-evoked 191–194. pain (allodynia): a new model. Pain 68, 13–23. Ji GC, Zhou ST, Shapiro G, Reubi JC, Jurczyk S & Carlton SM Coggeshall RE & Carlton SM (1997). Receptor localization in (2006). Analgesic activity of a non-peptide the mammalian dorsal horn and primary afferent neurons. imidazolidinedione somatostatin agonist: In vitro and in Brain Res Brain Res Rev 24, 28–66. vivo studies in rat. Pain 124, 34–49. Conn PJ & Pin JP (1997). Pharmacology and functions of Jin YH, Yamaki F, Takemura M, Koike Y, Furuyama A & metabotropic glutamate receptors. Annu Rev Pharmacol Yonehara N (2009). Capsaicin-induced glutamate release is Toxicol 37, 205–237. implicated in nociceptive processing through activation of Davidson EM, Coggeshall RE & Carlton SM (1997). Peripheral ionotropic glutamate receptors and group I metabotropic NMDA and non-NMDA glutamate receptors contribute to glutamate receptor in primary afferent fibers. JPharmacol nociceptive behaviors in the rat formalin test. Neuroreport 8, Sci 109, 233–241. 941–946. Keast JR & Stephensen TM (2000). Glutamate and aspartate De Groot JF, Zhou S & Carlton SM (2000). Peripheral immunoreactivity in dorsal root ganglion cells supplying glutamate release in the hindpaw following low and high visceral and somatic targets and evidence for peripheral intensity stimulation. Neuroreport 11, 497–502. axonal transport. JCompNeurol424, 577–587. Du J & Carlton SM (2007). Spinal cord injury pain: Further Lin Q, Wu J & Willis WD (1999). Dorsal root reflexes and evidence that dorsal root reflexes play a critical role in cutaneous neurogenic inflammation after intradermal sensory abnormalities arising after a T10 contusion. injection of capsaicin in rats. J Neurophysiol 82, Program No. 77.19. 2007 Neuroscience Meeting Planner. 2602–2611. Society for Neuroscience, San Diego, CA, USA. Online. Lin Q, Zou X & Willis WD (2000). Aδ and C primary afferents Du J, Koltzenburg M & Carlton SM (2001). Glutamate-induced convey dorsal root reflexes after intradermal injection of excitation and sensitization of nociceptors in rat glabrous capsaicin in rats. J Neurophysiol 84, 2695–2698. skin. Pain 89, 187–198. Lisney SJ (1979). Evidence for primary afferent depolarization Du J, Zhou S & Carlton SM (2006). Kainate-induced excitation of single tooth-pulp afferents in the cat. JPhysiol288, and sensitization of nociceptors in normal and inflamed rat 437–447. glabrous skin. Neuroscience 137, 999–1013. Maggi CA (1995). Tachykinins and calcitonin gene-related Du J, Zhou S & Carlton SM (2008). Group II metabotropic peptide (CGRP) as co-transmitters released from peripheral glutamate receptor activation attenuates peripheral endings of sensory nerves. Prog Neurobiol 45, sensitization in inflammatory states. Neuroscience 154, 1–98. 754–766. Maggi CA, Borsini F, Santicioli P, Geppetti P, Abelli L, Du J, Zhou S, Coggeshall RE & Carlton SM (2003). Evangelista S, Manzini S, Theodorsson-Norheim E, Somma N-methyl-D-aspartate-induced excitation and sensitization V, Amenta F, Bacciarelli C & Meli A (1987). Cutaneous of normal and inflamed nociceptors. Neuroscience 118, lesions in capsaicin-pretreated rats. A trophic role of 547–562. capsaicin-sensitive afferents? Naunyn-Schmiedebergs Arch Erdo SL (1991). Excitatory amino acid receptors in the Pharmacol 336, 538–545. mammalian periphery. Trends Pharmacol Sci 12, 426–429. Neugebauer V & Carlton SM (2002). Peripheral metabotropic Genever PG, Maxfield SJ, Kennovin GD, Maltman J, Bowgen glutamate receptors as drug targets for pain relief. Expert CJ, Raxworthy MJ & Skerry TM (1999). JInvestDerm112, Opin Ther Targets 6, 349–361. 337–342. Omote K, Kawamata T, Kawamata M & Namiki A (1998). Geppetti P & Holzer P (eds) (1996). Neurogenic Inflammation. Formalin-induced release of excitatory amino acids in the CRC Press, New York. skin of the rat hindpaw. Brain Res 787, 161–164.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society J Physiol 592.16 Nociceptive primary afferents 3411

Pitcher MH & Cervero F (2010). Role of the NKCC1 Toennies JF (1938). Reflex discharge from the spinal cord over co-transporter in sensitization of spinal nociceptive neurons. the dorsal roots. J Neurophysiol 1, 378–390. Pain 151, 756–762. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Rees H, Sluka KA, Lu Y, Westlund KN & Willis WD (1996). Skinner K, Raumann BE, Basbaum AI & Julius D (1998). Dorsal root reflexes in articular afferents occur bilaterally in The cloned capsaicin receptor integrates multiple a chronic model of arthritis in rats. J Neurophysiol 76, pain-producing stimuli. Neuron 21, 531–543. 4190–4193. Weng HR & Dougherty PM (2005). Response properties of Rees H, Sluka KA, Westlund KN & Willis WD (1994). Do dorsal root reflexes in cutaneous C fibers before and after dorsal root reflexes augment peripheral inflammation? intradermal capsaicin injection in rats. Neuroscience 132, Neuroreport 5, 821–824. 823–831. Rocha-Gonzalez HI, Mao S & Alvarez-Leefmans FJ (2008). Westlund KN, McNeill DL & Coggeshall RE (1989). Glutamate Na+,K+,2Cl– cotransport and intracellular chloride immunoreactivity in rat dorsal roots. Neurosci Lett 96, regulation in rat primary sensory neurons: thermodynamic 13–17. and kinetic aspects. J Neurophysiol 100, Willis WD Jr (1999). Dorsal root potentials and dorsal root 169–184. reflexes: a double-edged sword. Exp Brain Res 124, 395–421. Roper SD (2014). Sensory End Organs: Signal Processing in the Zhou S, Bonasera L & Carlton SM (1996). Peripheral Periphery: a symposium presented at the 2013 annual administration of NMDA, AMPA or KA results in pain meeting of the Society for Neuroscience, San Diego, CA, behaviors in rats. Neuroreport 7, 895–900. USA. JPhysiol592, 3383–3385. ZoliM,TorriC,FerrariR,JanssonA,ZiniI,FuxeK&Agnati Schoepp DD & Conn PJ (1993). Metabotropic glutamate LF (1998). The emergence of the volume transmission receptors in brain function and pathology. Trends Pharmacol concept. Brain Res Brain Res Rev 26, 136–147. Sci 14, 13–20. Zylka MJ, Rice FL & Anderson DJ (2005). Topographically Sluka KA, Rees H, Westlund KN & Willis WD (1995a). Fiber distinct epidermal nociceptive circuits revealed by axonal types contributing to dorsal root reflexes induced by joint tracers targeted to Mrgprd. Neuron 45, 17–25. inflammation in cats and monkeys. J Neurophysiol 74, 981–989. Sluka KA & Westlund KN (1993). Centrally administered non-NMDA but not NMDA receptor antagonists block Additional information peripheral knee joint inflammation. Pain 55, 217–225. Competing interests Sluka KA, Willis WD & Westlund KN (1995b). The role of None declared. dorsal root reflexes in neurogenic inflammation. Pain Forum 4, 141–149. Sung KW, Kirby M, McDonald MP, Lovinger DM & Delpire E Funding (2000). Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl Funding for studies done in the Carlton lab: NIH grants R01 cotransporter null mice. JNeurosci20, 7531–7538. NS027910 and R01 NS054765.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society