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date: 26 May 2019

Dorsal Horn Pain Mechanisms

Abstract and Keywords

The spinal dorsal horn and its equivalent structure in the brainstem constitute the first sites of synaptic integration in the pain pathway. A huge body of literature exists on alterations in spinal nociceptive signal processing that contribute to the generation of exaggerated pain states and hence to what is generally known as “central sensitization.” Such mechanisms include changes in synaptic efficacy or neuronal excitability, which can be evoked by intense nociceptive stimulation or by inflammatory or neuropathic insults. Some of these changes cause alterations in the functional organization of dorsal horn sensory circuits, leading to abnormal pathological pain sensations. This chapter reviews the present state of this knowledge. It does not cover the contributions of astrocytes and microglia in detail as their functions are the subject of a separate chapter.

Keywords: pain, spinal cord, dorsal horn, plasticity, circuit, inflammation, neuropathy, GABA, glycine, disinhibition

Introduction

The spinal dorsal horn and its equivalent structure in the brainstem constitute the first sites of synaptic integration in the pain pathway. A huge body of literature exists on alterations in spinal nociceptive signal processing that contribute to the generation of exaggerated pain states and hence to what is generally known as “central sensitization.” This chapter reviews the present state of this knowledge.

Hallmarks of Pathological Pain

The ability to sense and respond to painful stimuli is essential for our survival. Once tissue damage has occurred, inflammation causes sensitization of our nociceptive system to reduce the risk of further damage and to promote healing. This sensitization includes increased sensitivity to painful stimuli limited to the site of the tissue damage or inflammation (primary hyperalgesia) and in the surrounding tissue (secondary hyperalgesia). Primary hyperalgesia can be caused by sensitization of peripheral nociceptors or by a sensitization of central neurons to nociceptive input arriving from the inflamed tissue. The latter process corresponds to homosynaptic plasticity. Secondary hyperalgesia may be induced by diffusion of soluble pronociceptive molecules from inflamed to neighboring noninflamed tissue, or it may result from increased responsiveness of central neurons to input arriving from noninflamed tissue (heterosynaptic plasticity). Sensitization beyond the site of the primary insult often also involves pain elicited by stimulation of nonnociceptive fibers. This condition is referred to as allodynia. It involves alterations in central sensory processing that lead to the excitation of pain signaling neurons by polysynaptic input from nonnociceptive fibers. Pain can also be spontaneous, occurring in the absence of any sensory stimulation, and likely reflects spontaneous activity in peripheral nerve fibers or central neurons. Such spontaneous pain is frequent in patients with neuropathy, where it may arise from nonphysiological discharges in peripheral nerves as a consequence of the primary nerve damage. In central neurons, it may be the consequence of plastic changes induced by pathological activity of peripheral nerve fibers or of immune reactions triggered by the peripheral insult. Finally, pain can be widespread, in extreme cases extending throughout the whole body, pointing to a general dysfunction of the nociceptive system and potentially to a defect in endogenous pain control mechanisms. While changes at all levels of the neuraxis likely contribute to pathological pain, the spinal cord deserves special attention as many of its elements have been shown to play crucial roles in the generation of pathological pain states.

Gross Organization and Innervation of the Spinal Dorsal Horn

Somatosensory and nociceptive input from the body periphery is conveyed to the central nervous system (CNS) via sensory nerve fibers that terminate in the spinal dorsal horn or the equivalent structure in the brainstem. According to Rexed (1952), the spinal dorsal horn can be divided into six laminae (I–VI). Most nociceptive nerve fibers (high-threshold mechanoreceptors and polymodal nociceptors) terminate in the superficial dorsal horn (laminae I and II), while nonnociceptive fibers preferentially innervate the deep dorsal horn (laminae III–VI) (Figure 1). At these sites, sensory nerve fibers form excitatory glutamatergic synapses with projection neurons and local excitatory and inhibitory interneurons.

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Figure 1. Gross organization of the spinal dorsal horn. (A) Schematic overview of the innervation of the spinal dorsal horn by sensory fibers, the localization of projection neurons in the superficial and deep dorsal horn. (B) Dorsoventral distribution of markers for the different dorsal laminae. CGRP, calcitonin gene–related peptide; IB4, isolectin IB4; MrgprD, mas-related GPCR D; NK1R, neurokinin 1 receptor; PKCγ, protein kinase Cγ; TH, tyrosine hydroxylase; VGLUT3, vesicular glutamate transporter 3. Adapted from Todd (2010).

Most nociceptors are either unmyelinated C fibers or thinly myelinated Aδ fibers. About half of the C fibers release the neuropeptides calcitonin gene–related peptide (CGRP) or substance P (SP) in addition to glutamate. These peptidergic C fibers terminate in lamina I and outer lamina II. Nonpeptidergic C fibers express the mas-related G protein–coupled receptor (Mrgpr) D, bind isolectin B4 (IB4), and terminate in inner lamina II. A third type of C fiber functions as low-threshold mechanoreceptors (C-LTMRs). They are characterized by expression of tyrosine hydroxylase and by vesicular glutamate transporter 3 (VGLUT3), whereas low-threshold Aβ mechanoreceptors (A-LTMRs) express VGLUT1. Nociceptive Aδ and C fibers express VGLUT2 in their central terminals. The C-LTMRs terminate at the border between laminae II and III overlapping with the layer of protein kinase Cγ–expressing excitatory interneurons.

The vast majority of dorsal horn neurons are propriospinal neurons, most of which are local interneurons that remain confined to one spinal cord segment. Projection neurons make up less than 1% of all dorsal horn neurons. They are mainly found within lamina I and are scattered throughout the deeper laminae III–V. In nonperturbed conditions, projection neurons in lamina I are nociceptive specific; that is, they are only activated by input from primary nociceptors. Most other dorsal horn neurons, including the deep dorsal horn projection neurons, are wide dynamic range neurons that can be activated by both nociceptive and nonnociceptive input. While the role of the deep dorsal horn projection neurons for different pain states is not entirely clear, converging evidence supports a critical role of lamina I projection neurons in chronic pain states (Maiaru et al., 2018; Mantyh et al., 1997; Nichols et al., 1999).

In addition to sensory signals arriving to the spinal cord from the periphery, the dorsal horn receives signals descending from the brain. These descending projections modulate spinal nociception via both fast amino acid transmitters and slow-acting neuromodulators. In the following sections, we summarize the present knowledge of long-lasting and profound changes that occur via at least four principle mechanisms: (a) A change in the efficacy of transmission between primary nociceptors and second-order dorsal horn neurons; (b) an impaired balance of excitation and inhibition in intrinsic dorsal horn circuits; (c) altered functional connectivity in dorsal horn circuits; and (d) changes in descending pain control. Many of these changes also involve signaling by astrocytes and microglia. These are not discussed in detail here, as they are the subjects of a different chapter.

Plasticity at Primary Nociceptor Synapses

Intense nociceptive input to the dorsal horn induces short-term and long-term plasticity in the dorsal horn at the level of synapses, neurons, and circuits. Although excitability can decrease or increase, depending on the nature of the synapse studied and the stimulation paradigms, most work has focused on mechanisms that facilitate excitability. Windup is a phenomenon of short-term plasticity that has been extensively investigated in the past and has been revisited recently. Most of the recent work, however, has focused on long-term potentiation (LTP), which exists in different forms in dorsal horn neurons (Figure 2).

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Figure 2. Synaptic long-term plasticity at spinal nociceptor synapses. (A) Synaptic transmission between nociceptors and dorsal horn lamina I projection neurons undergoes (long-term potentiation) LTP on repetitive stimulation of C-fiber nociceptors, leading to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation and increased excitatory postsynaptic currents. (B) Depending on the projection area (periaqueductal gray [PAG] or lateral parabrachial nucleus [PbN]), induction of LTP requires low-frequency stimulation (LFS) or high-frequency stimulation (HFS) of nociceptor terminals, respectively (Ikeda et al., 2006). (C) Rises in intracellular Ca2+ via Ca2+ influx through N-methyl-d-aspartate (NMDA) receptors or through Ca2+ release from ryanodine receptors (RyR), activation of protein kinases (protein kinase C [PKC] and calmodulin-dependent protein kinase II [CaMKII]), and subsequent AMPA receptor phosphorylation are processes common to most forms of nociceptor LTP. Glial cells activated by ATP via purinergic P2X7 receptors, glia transmitters such as d-serine, and cytokines such as tumor necrosis factor α(TNFα) and interleukin beta (IL-β) contribute to heterosynaptic plasticity (Kronschlager et al., 2016). Brief intense activation of µ-opioid receptors (MORs) can depotentiate nociceptor synapses via dephosphorylation of AMPA receptors (Drdla-Schutting et al., 2012), while acute opioid removal induces “pharmacogenic” LTP (Drdla et al., 2009). PbN, parabrachial nucleus; EPSC, excitatory postsynaptic current; PP1, protein phosphatase 1.

Long-Term Potentiation

Long-term potentiation and long-term depression (LTD), the two main forms of long-lasting synaptic plasticity in the brain, also exist in the spinal dorsal horn. Early work (Randic, Jiang, & Cerne, 1993) found LTP and LTD occurring with similar frequencies in monosynaptic connections between C or Aδ fibers and unidentified dorsal horn neurons. Subsequent work has mainly focused on synaptic plasticity between C fibers and lamina I projection neurons retrogradely labeled from their projection targets in the brainstem or midbrain. This work has shown that intense excitatory input from nociceptors induces LTP at spinal nociceptor synapses (Ikeda, Heinke, Ruscheweyh, & Sandkühler, 2003). In those neurons projecting to the PAG, LTP could be elicited by stimulation of C-fiber nociceptors at frequencies that resemble their natural activity during prolonged activation (Ikeda et al., 2006).

It is important to note that, although many features of this LTP resemble homosynaptic (“Hebbian”) LTP, certain forms of LTP at synapses between C-fiber nociceptors and lamina I projection neurons lack synapse specificity and hence may also account for secondary hyperalgesia. This heterosynaptic LTP involves glial cells and cytokines such as tumor necrosis factor alpha (TNF-α) to communicate between conditioned and nonconditioned synapses (Kronschlager et al., 2016). In other cases, postsynaptic depolarization of lamina I neurons induces a rather generalized state of increased responsiveness to synaptic input (Naka, Gruber-Schoffnegger, & Sandkühler, 2013), involving even potentiation of γ-aminobutyric acid–ergic (GABAergic) input. This last form of plasticity requires activation of Class I metabotropic glutamate receptors and retrograde signaling via nitric oxide (NO) (Fenselau, Heinke, & Sandkühler, 2011). It may contribute to the maintenance of a physiological level of nociceptive sensitivity.

The LTP at dorsal horn nociceptor synapses is mainly considered a central mechanism of inflammatory hyperalgesia, but it may also contribute to other pain pathologies. For example, acute withdrawal of remifentanil, an ultrafast-acting opioid, from spinal cord slices induced LTP (Drdla, Gassner, Gingl, & Sandkühler, 2009) and may contribute to what is known as opioid-induced hyperalgesia and potentially also to opioid tolerance. Induction of this opioid withdrawal–induced LTP required postsynaptic G protein signaling, N-methyl-d-aspartate (NMDA) receptor activation, and an increase in intracellular Ca2+ concentration. It is interesting to note that, conversely, acute exposure of spinal cord slices to the same opioid at a very high dose was able to reverse LTP induced by low-frequency stimulation of primary nociceptor input (Drdla-Schutting, Benrath, Wunderbaldinger, & Sandkühler, 2012). There is some preliminary evidence that a brief high-dose infusion of remifentanil partially reverses some forms of chronic pain in humans (Prosenz et al., 2017).

Nociceptor synapses with dorsal horn projection neurons also show a different form of LTP called spike time–dependent plasticity, in which induction of LTP depends on the precise timing of the presynaptic action potential and the postsynaptic response. This spike time–dependent plasticity appears to depend on the transient production of prostaglandin E2 (PGE2) through cyclooxygenase 2 and activation of type 2 PGE2 (EP2) receptors (Li, Serafin, & Baccei, 2018). Although the consequences of this form of plasticity for pain have not yet been addressed, suppression of the underlying signaling pathway may contribute to the analgesic or antihyperalgesic properties of cyclooxygenase inhibitors.

In general, it is very difficult to establish a causal link between synaptic and cellular mechanisms discovered in animal experiments to human pain syndromes. However, it has been demonstrated that repetitive electrical stimulation of human peripheral nerves at C-fiber intensity evokes local long-lasting increases or decreases in pain sensitivity (Henrich, Magerl, Klein, Greffrath, & Treede, 2015; Klein, Magerl, Hopf, Sandkühler, & Treede, 2004), some but not all of these changes were prevented with the NMDA receptor antagonist ketamine, supporting a role of NMDA receptor–dependent plasticity (Klein et al., 2007). These reports are consistent with a contribution of long-term synaptic plasticity to increased pain sensitivity after electrical nerve stimulation, but increased nociceptive input may also trigger other changes in dorsal horn nociceptive processing, such as an activity-dependent reduction in inhibition.

Long-Term Depression

In addition to LTP, LTD has been found at synapses between nociceptors and dorsal horn neurons. Probably depending on the nature of the postsynaptic cell, different forms of LTD have been described that depend on NMDA receptor activation (Sandkühler, Chen, Cheng, & Randic, 1997), endocannabinoid signaling (Kato et al., 2012), or postsynaptic transient receptor potential channel vanilloid 1 (TRPV1) activation (Kim et al., 2012). In general, low-frequency stimulation of cutaneous nociceptors at a certain intensity range may lead to reduced pain perceptions (Ellrich & Schorr, 2004). The TRPV1-dependent LTD appears to occur specifically in GABAergic second-order neurons. This may contribute to spinal disinhibition and neuropathic pain (Kim et al., 2012).

Windup

Windup is another phenomenon that has been extensively investigated as a potential mechanism of activity-dependent hyperalgesia (Mendell & Wall, 1965). It refers to a process in which repetitive stimulation of nociceptive afferents leads to a gradual transient increase in the postsynaptic responses. This increase in responsiveness originates from progressive slow depolarization of the postsynaptic neuron. Neurokinin 1 (NK1) receptors activated by SP and NMDA receptors both contribute to this phenomenon (Dickenson & Sullivan, 1987; Xu, Dalsgaard, & Wiesenfeld-Hallin, 1992). Recent work suggests in addition that a “reverberatory” circuit of interconnected excitatory neurotensin-positive interneurons in the superficial dorsal horn underlies the gradual increase in postsynaptic excitation (Hachisuka et al., 2018). A gradual transient increase in pain sensitivity in response to repeated nociceptor stimulation is also observed in humans, but its contribution to pathological pain states in humans is unknown (Herrero, Laird, & Lopez-Garcia, 2000).

Primary Afferent Depolarization

GABA inhibits neurotransmitter release from spinal sensory neuron terminals via presynaptic inhibition and primary afferent depolarization. This process involves primarily GABAA receptors, but GABAB receptors are also present on nociceptor terminals; their activation also reduces transmitter release. Unlike mature CNS neurons, peripheral sensory neurons and their terminals are depolarized by GABA because the chloride importer Na-K-2Cl cotransporter (NKCC1) keeps intracellular chloride concentration above the electrochemical equilibrium potential (Alvarez-Leefmans, 2009). In the case of (nonnociceptive) low-threshold Aβ- and Aδ-fiber terminals, GABA is released directly onto these terminals from axons that make synaptic contacts with them (Todd & Koerber, 2006). Peptidergic terminals have few, if any, axoaxonic synapses (Ribeiro-da-Silva & Coimbra, 1982) but are nevertheless inhibited by GABA, likely via spillover from neighboring GABAergic synapses (Witschi et al., 2011). Under certain conditions, presynaptic depolarization can potentially become suprathreshold and evoke action potentials, which can then give rise to action potentials retrogradely invading peripheral nociceptors to support the spread of neurogenic inflammation (Willis, 1999).

Modulators of Neurotransmitter Release from Nociceptor Terminals

Numerous neuromodulators can decrease or increase neurotransmitter release from spinal nociceptor terminals. These include, among others, opioid peptides; noradrenaline (NA); serotonin (5-HT); purinergic agonists (adenosine triphosphate [ATP], adenosine diphosphate [ADP], adenosine); and cannabinoids. We discuss the effects of opioid peptides, cannabinoids, and GABA on transmitter release from nociceptor terminals in the material that follows. 5-HT and NA are discussed in the context of descending pain modulation, for which they are key players.

Opioid peptides, and their small molecule analogues, opiates and opioids, activate G protein–coupled receptors on the spinal sensory neuron terminals to inhibit presynaptic Ca2+ influx and to reduce glutamate and neuropeptide release. All three subtypes of classical opioid receptors (i.e., µ, δ, and κ receptors) are expressed on different subsets of peripheral sensory neurons, including not only peptidergic nociceptors but also low-threshold mechanoreceptors (A-LTMRs) (Bardoni et al., 2014; Scherrer et al., 2009; Snyder et al., 2018).

Opioid receptors are expressed in all parts of the central ascending and descending pain axis, including dorsal horn interneurons and neurons in the parabrachial nucleus, the rostral ventromedial medulla (RVM), and the PAG. Although direct application to the spinal cord evokes analgesia, descending pathways to the spinal cord were found to be crucial (Basbaum, Marley, O’Keefe, & Clanton, 1977). The corresponding GABA disinhibition hypothesis of opioid analgesia proposes that µ-opioid receptor activation reduces the tonic inhibitory tone of spinally projecting antinociceptive output neurons (Jensen & Yaksh, 1989; Lau & Vaughan, 2014).

In addition to the classical µ, δ, and κ opioid receptors, dorsal horn neurons express the nociceptin/orphanin FQ (N/OFQ) opioid peptide (NOP) receptor formally known as ORL-1 receptor. Activation of spinal NOP receptors inhibits glutamatergic neurotransmission in the dorsal horn (Liebel, Swandulla, & Zeilhofer, 1997). Accordingly, intrathecal injection of N/OFQ induces analgesia in rodents and monkeys (Erb et al., 1997; Ko & Naughton, 2009). Dorsal horn neurons express not only NOP receptors but also their endogenous ligand, the neuropeptide N/OFQ. Mice lacking NOP receptors or the N/OFQ neuropeptide precursor showed normal acute nociceptive thresholds but developed stronger hyperalgesia in inflammatory pain models (Depner, Reinscheid, Takeshima, Brune, & Zeilhofer, 2003).

Cannabinoid CB1 receptors and their endogenous ligands, the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide (AEA), are widely distributed in the CNS, where they regulate neuronal excitability. They are also present in the spinal cord, where they are concentrated in nociceptor synapses (Nyilas et al., 2009); in addition, they are found at GABAergic dorsal horn synapses (Pernía-Andrade et al., 2009). At both sites, CB1 receptors are located presynaptically to inhibit transmitter release. Intrathecal injection of CB1 receptor agonists produced antihyperalgesic effects in inflammatory and neuropathic pain models. Endocannabinoids (mainly 2-AG) contribute to short-term and long-term synaptic plasticity at nociceptor synapses (Kato et al., 2012). Postsynaptic depolarization and activation of Group I metabotropic glutamate receptors trigger 2-AG production and short-term depression of nociceptor input. They may contribute to stress-induced analgesia (Nyilas et al., 2009). CB1 receptors are also found on terminals of inhibitory dorsal horn interneurons, where they induce heterosynaptic depolarization-induced suppression of inhibition contributing to hyperalgesia under conditions of intense nociceptive input to the spinal cord (Carey et al., 2016; Pernía-Andrade et al., 2009).

Despite the presence of CB1 receptors and the endocannabinoid synthesis machinery in the dorsal horn nociceptor synapses, cannabinoid-mediated analgesia originates mainly from supraspinal sites, especially the RVM and the PAG. Microinjections of potent cannabinoid agonists into various brain regions were most effective when delivered to the dorsal PAG and the RVM (Martin, Patrick, Coffin, Tsou, & Walker, 1995), while ablation of CB1 receptors from the peripheral sensory neurons and the spinal cord had little effect on cannabinoid analgesia (Klinger-Gratz et al., 2018). Similar to opioid analgesia, analgesia by cannabinoids requires intact descending projections to the spinal cord (Seyrek, Kahraman, Deveci, Yesilyurt, & Dogrul, 2010).

Reduced GABAergic and Glycinergic Inhibition

In addition to increased excitation, diminished spinal inhibition is a hallmark of central pain sensitization (Figure 3). About 30% of all dorsal horn neurons are inhibitory interneurons. Most of them, especially those in the deep dorsal horn, use both GABA and glycine for fast synaptic inhibition, while the majority of those in lamina II release GABA but not glycine. A similar ventrodorsal gradient exists for the glycinergic-to-GABAergic contribution to inhibitory postsynaptic currents (Takazawa et al., 2017), but overall the glycinergic contribution to evoked synaptic inhibition also appears to be dominant in the superficial dorsal horn (Foster et al., 2015). Plenty of experiments using GABA or glycine receptor antagonists have shown that reduced GABA or glycine receptor function induces pathological pain states in rodents.

More recent experiments employing viral tools and bacterial toxins to interfere with the function of dorsal horn inhibitory neurons have shown that these neurons are indispensable for maintaining a physiological level of pain sensitivity. Compromising their function increases the responsiveness of lamina I projection neurons to nociceptive input, renders nociceptive projection neurons susceptible to activation by input from nonnociceptive fibers, and leads to spontaneous epileptiform activity of dorsal horn neurons (Keller, Beggs, Salter, & De Koninck, 2007; Miraucourt, Dallel, & Voisin, 2007; Ruscheweyh & Sandkühler, 2003).

On the behavioral level, ablation or silencing of inhibitory dorsal horn neurons induces signs of hyperalgesia (increased responses to nociceptor activation), allodynia (aversive responses to light mechanical stimulation), and spontaneous aversive behavior in the absence of sensory stimulation (Foster et al., 2015). Diminished inhibition thus recapitulates typical symptoms of chronic pain in humans. Direct support for a role of glycinergic inhibition in pain control in humans comes from hyperekplexia (or startle disease) patients, who have different genetic defects in glycinergic inhibition. The main symptoms in these patients are exaggerated startle responses, but a recent study has demonstrated that these patients also exhibit significantly decreased pain thresholds (Vuilleumier et al., 2018).

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Figure 3. Mechanisms of spinal disinhibition. (A) Schematic illustration of different types of dorsal horn inhibitory synapses that are involved in spinal disinhibitory pain mechanisms. (B) Signaling pathways that reduce inhibition in the different sites labeled in (A) with a–d. (Ba) Peripheral nerve injury leads to release of brain-derived neurotrophic factor (BDNF), which downregulates K-Cl cotransporter (KCC2) via activation of tropomyosin receptor kinase B (trkB). This downregulation leads to a depolarizing shift in the transmembrane chloride gradient in lamina I neurons, rendering GABAergic and glycinergic input less inhibitory (Coull et al., 2003). (Bb) Peripheral inflammation induces production of prostaglandin E2 (PGE2) in the spinal cord. PGE2 acts on type 2 PGE2(EP2) receptors, which stimulate cAMP production and a subsequent phosphorylation and inhibition of α3 glycine receptors (GlyRα3s) (Ahmadi et al., 2002; Harvey et al., 2004). (Bc) Peripheral nerve injury reduces the excitatory drive in inhibitory interneurons (Leitner et al., 2013). (Bd) Reduced excitatory drive to inhibitory interneurons may also reduce primary afferent depolarization and presynaptic inhibition. Glu, glutamate; PKA, protein kinase A.

Reduced Inhibition in Inflammatory Pain States

Peripheral inflammation induces the expression of cyclooxygenase 2 in the spinal cord (Beiche, Scheuerer, Brune, Geisslinger, & Goppelt-Struebe, 1996; Samad et al., 2001) and increases spinal prostaglandin concentrations (Reinold et al., 2005). Among the different prostaglandins, PGE2 is particularly relevant for spinal hyperalgesia. On binding to EP2 receptors, PGE2 causes protein kinase A–dependent phosphorylation of glycine receptors that renders them less responsive to glycine (Acuña et al., 2016; Ahmadi, Lippross, Neuhuber, & Zeilhofer, 2002; Reinold et al., 2005). This pathway is specific to glycine receptors containing the α3 subunit (α3GlyR) and does not affect GABAergic inhibition. Accordingly, inflammation leads to a selective loss of glycinergic inhibition (Takazawa et al., 2017), while GABAergic inhibition might even undergo a compensatory increase via inflammation-induced neurosteroid production (Poisbeau et al., 2005). Mutant mice lacking key signaling elements of PGE2-mediated glycine receptor inhibition exhibit strongly reduced inflammatory hyperalgesia (Harvey et al., 2004; Reinold et al., 2005). Because many classical nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase 2–selective coxibs effectively cross the blood–brain barrier (Dembo, Park, & Kharasch, 2005; Har-Even et al., 2014; Kokki et al., 2008; Kumpulainen et al., 2010; Valitalo et al., 2012), it is conceivable that the suppression of this pathway contributes to analgesia by cyclooxygenase inhibitors.

Reduced Inhibition in Neuropathic Pain States

Peripheral nerve damage activates microglia, triggering a cascade of events that eventually leads to downregulation of the KCC2 potassium chloride cotransporter, an increase in intracellular chloride concentration, and ultimately reduced inhibitory capacity of GABAergic and glycinergic input. Damaged nerve fibers release the chemokine (C-C motif) ligand 2 (CCL-2) to recruit and activate microglia, which release brain-derived neurotrophic factor (BDNF) on stimulation with ATP and activation of type 2X4 purinergic (P2X4) receptors. BDNF then binds to tropomyosin receptor kinase B (trkB) receptors to downregulate KCC2 (Coull et al., 2003, 2005). Whether the resulting increase in the intracellular chloride is strong enough to allow GABA-evoked action potentials is still a matter of debate. Additional disinhibitory mechanisms have been described that involve reduced activation of GABAergic neurons by peripheral sensory input (Kim et al., 2012; Leitner et al., 2013).

Apoptotic Cell Death

Death of local inhibitory interneurons is another potential source of disinhibition in the spinal dorsal horn. It has been reported that peripheral nerve injury in rats is accompanied by an apoptotic loss of GABA-expressing cells in the dorsal horn (Inquimbert et al., 2018; Moore et al., 2002; Scholz et al., 2005). However, other studies have shown that allodynia and hyperalgesia can occur in nerve-injured animals without any loss of inhibitory cells in the dorsal horn (Polgar, Gray, Riddell, & Todd, 2004; Polgar et al., 2003). Whether this loss of GABAergic neurons occurs after peripheral nerve injury and whether it contributes to neuropathic pain are therefore still unresolved.

Restoring GABAergic and Glycinergic Inhibition

Restoring compromised GABAergic or glycinergic inhibition alleviates inflammatory and neuropathic pain states in rodents. Because both transmitter systems control not only pain but also many important CNS functions, such as wakefulness, anxiety, respiration, reward, and motor control, it is important to restrict the increase in inhibition as much as possible to nociceptive CNS circuits. Certain GABAA and glycine receptor subtypes are concentrated in nociceptive circuits of the spinal dorsal horn.

Among the GABAA receptors, those that contain α2 and α3 subunits are strongly enriched in the superficial dorsal horn (Paul, Zeilhofer, & Fritschy, 2012), and among the inhibitory glycine receptors, α3GlyR is highly enriched in the superficial laminae (Harvey et al., 2004). Specific potentiation of α2/α3 GABAA receptors provides profound antihyperalgesia in inflammatory and neuropathic pain states in the absence of typical side effects of GABAA receptor modulation, such as sedation, tolerance development (i.e., loss of therapeutic activity during prolonged treatment), and impaired motor coordination (Knabl et al., 2008; Ralvenius, Benke, Acuña, Rudolph, & Zeilhofer, 2015; Ralvenius et al., 2018). Pronounced antihyperalgesic effects have also been reported for compounds that potentiate glycine receptors with varying degrees of selectivity (Acuña et al., 2016; Xiong et al., 2012); for a review, see the work of Zeilhofer, Acuña, Gingras, and Yevenes (2018).

Drug discovery efforts in industry have recently led to the discovery of a highly selective glycine receptor modulator with analgesic activity in a neuropathic pain model (Huang et al., 2017). Neurosteroids are endogenous positive allosteric modulators of GABAA receptors. They prolong synaptic GABAergic currents and enhance tonic GABAergic currents carried by extrasynaptic GABAA receptors (Mitchell, Gentet, Dempster, & Belelli, 2007). Synthetic neurosteroids possess significant analgesic activity after intrathecal injection (Charlet, Lasbennes, Darbon, & Poisbeau, 2008; Goodchild, Guo, & Nadeson, 2000). At present, it is unclear whether their spinal analgesic activity can be separated from their anesthetic actions. Restoring chloride extrusion capacity with compounds that act as positive allosteric modulators of KCC2 reduces neuropathic pain in animal models (Gagnon et al., 2013), but see also the work of Cardarelli et al. (2017).

Another opportunity to facilitate GABAergic and glycinergic inhibition may involve neuromodulators that enhance GABA and glycine release. Activation of nicotinic acetylcholine receptors on inhibitory interneuron terminals enhances glycine and GABA release (Takeda, Nakatsuka, Papke, & Gu, 2003) and may contribute to well-established antinociceptive effects of nicotine.

Besides pharmacological approaches, transplantation of GABAergic precursor neurons from the medial ganglionic eminence is able to restore proper inhibition and to reverse neuropathic hyperalgesia in mice (Braz et al., 2012).

Remodeling of Functional Dorsal Circuits in Pathological Pain Conditions

One of the hallmarks of pathological pain syndromes is pain experienced in response to light mechanical stimulation, in a clinical context also referred to as allodynia or touch-evoked pain. Strong evidence argues for changes in the functional dorsal horn circuitry as underlying causes. It is generally believed that input arriving from LTMRs in the dorsal horn does normally not excite nociceptive specific projection neurons of lamina I. Under physiological conditions, excitation of dorsal horn neurons evoked by Aβ-fiber input remained confined to the deep dorsal horn, whereas in slices taken from neuropathic mice, excitation spread to the superficial dorsal horn (Schoffnegger, Ruscheweyh, & Sandkühler, 2008). Blockade of synaptic inhibition with bicuculline and strychnine alone was sufficient for the spread of nonnociceptive signals to dorsal laminae (Miraucourt et al., 2007; Schoffnegger et al., 2008; Torsney & MacDermott, 2006). In support of this concept, ablation of a large population of dorsal horn inhibitory neurons has been shown to induce pathologically exaggerated pain states (Foster et al., 2015), while excitatory dorsal horn interneurons have been found essential for the generation of the full spectrum of nociceptive reactions (Wang et al., 2013). Current research efforts are focused on the identification of excitatory dorsal horn interneuron types that connect input from LTMRs to lamina I output neurons and on their inhibitory counterparts that normally prevent this pathological activation (Figure 4).

Dorsal Horn Pain MechanismsClick to view larger

Figure 4. Rearrangement of dorsal horn circuits in chronic pain. Shown is dorsal horn signal relay in nonsensitized conditions and in inflammation or neuropathy. Enhanced excitation and reduced inhibitory control change the functional organization of dorsal horn sensory circuits, permitting suprathreshold excitation of lamina I projection neurons by input from low-threshold mechanoreceptors. Light red and light blue, respectively, indicate nociceptive and tactile signal relay under nonsensitized conditions. Light yellow indicates the polysynaptic connections of tactile, low-threshold input to normally nociceptive-specific lamina I neurons, which become unmasked during inflammation and neuropathy. The dashed box illustrates a “reverberating” microcircuit potentially underlying the windup phenomenon. Green circles, excitatory neurons; red circles, inhibitory neurons; solid blue line, low-threshold sensory fibers; solid red line, nociceptive sensory fiber. CR, calretinin; NK1r, neurokinin 1 receptor; NTS, neurotensin; PKCγ, protein kinase Cγ; PrP; prion promoter green fluorescent protein–labeled cells; PV, parvalbumin; TrC, transient central cell; ver, vertical cell; transient vesicular glutamate transporter 3-expressing cells, VGLUT3; WDR neuron, wide dynamic range neuron. Scheme adapted from Peirs et al. (2015).

Current research efforts are focused on the identification of excitatory dorsal horn interneuron types that connect input from LTMRs to lamina I output neurons and on their inhibitory counterparts that normally prevent this pathological activation. Neuronal ablation and silencing of genetically defined subpopulations of dorsal horn neurons have identified critical contributions of three excitatory interneuron types: neurons that transiently express VGLUT3 during development, protein kinase Cγ (PKCγ) positive, and calretinin-positive excitatory interneurons. Neurons transiently expressing VGLUT3 receive excitatory input from LTMRs and relay these signals further to PKCγ-positive neurons, which are located at strategic sites in laminae II and III, and calretinin-positive excitatory interneurons (Peirs et al., 2015). Work based on simultaneous (paired) recordings of two neurons in spinal cord slices has led to the proposition of similar models involving three types of excitatory dorsal horn interneurons (PKCγ, transiently firing neurons with a central-like morphology, and vertical cells) that are connected in series to relay signals arriving from LTMRs to lamina I projection neurons (Lu et al., 2013). This work has also established feedforward inhibition of PKCγ neurons by glycinergic interneurons in lamina III.

In addition, several smaller inhibitory interneuron populations have been identified that control this circuit, including parvalbumin-positive interneurons in lamina III, islet cells in lamina II, and dynorphin-positive interneurons in lamina II (Peirs et al., 2015). Inhibition of parvalbumin-positive and dynorphin-positive interneurons can induce allodynic states, indicating their importance in preventing activation of nociceptive projections neurons in uninjured conditions (Duan et al., 2014; Petitjean et al., 2015).

Several signaling pathways activated in inflammation or neuropathy compromise inhibition in dorsal horn circuits and contribute to unmasking the connection from LTMRs to nociceptive pathways. There is, however, in addition good evidence that facilitated excitation within this polysynaptic pathway also contributes to this unmasking. Not only does ablation of PKCγ neurons reduce neuropathic pain, but also deletion of PKCγ gene has been found to strongly reduce neuropathic pain (Malmberg, Chen, Tonegawa, & Basbaum, 1997).

Changes in Descending Pain Control

Spinal nociceptive processing is under strong influence by descending supraspinal projections, which are responsible for the so-called top-down processing of pain (Figure 5). Many of these descending projections contain neuromodulators such as 5-HT and NA, which are released into the spinal cord from axons descending from the RVM and pontine nuclei, including the locus coeruleus (LC), respectively. The RVM also contains GABA/glycine and enkephalin-containing projections to the spinal cord, which are important for controlling mechanical sensitivity (Francois et al., 2017; Zhang et al., 2015).

Dorsal Horn Pain MechanismsClick to view larger

Figure 5. Changes in descending pain control. (A) Origin of descending input from the brain to the spinal dorsal horn. Important areas include the primary somatosensory cortices S1 and S2, the anterior cingulate cortex (ACC), the locus coeruleus (LC), and the rostral ventromedial medulla (RVM). (B) Dorsoventral organization of the termination zones of descending supraspinal projections with their known neurotransmitters indicated. (C) Distribution of the different receptor types activated by descending input. GPCR, G protein–coupled receptor; 5HT, serotonin; DOR, δ-opioid receptor; MOR, µ-opioid receptor; NA, noradrenaline.

Descending Projections to the Spinal Dorsal Horn Originating from the Brainstem

The RVM, which includes the nucleus raphe magnus (RMg), is important for the control of spinal nociception. Early studies established that electrical stimulation of this region inhibits nociceptive responses in the hind limbs, and that this stimulation-produced analgesia is prevented by naloxone. The descending fiber tracts from this region project through the dorsolateral funiculus, which is required for the analgesic effect of morphine as well as many endogenous forms of analgesia (Millan, 2002). The monoamines 5-HT and NA play a complex role in the descending control of pain due to the variety of monoamine receptor types expressed in the spinal cord, with both inhibition and facilitation of pain being reported. These two monoamine pathways are also involved in diffuse noxious inhibitory control (DNIC), a phenomenon by which pain in one area can be inhibited by pain generated in a distinct region. This endogenous pain control system can also be studied in humans and is referred to as conditioned pain modulation (CPM), in which a conditioning stimulus is given before a noxious test stimulus. This paradigm can be used to determine whether patients with chronic pain have a deficiency in their pain inhibition system and can also be used to predict a patient’s responsiveness to particular drugs, such as those that prevent reuptake of monoamines.

Serotonergic neurons have long been associated with pain facilitation because 5-HT–containing neurons in the hindbrain are activated during chronic pain, and activation of these neurons using optogenetics can induce pain sensitization in naïve mice (Cai, Wang, Hou, & Pan, 2014; Suzuki, Morcuende, Webber, Hunt, & Dickenson, 2002). This facilitation is due to the activation of spinal 5-HT3 receptors, and depletion of 5-HT from this projection can occlude the hypersensitivity produced by induction of neuropathic pain (Wei et al., 2010). In human studies of CPM, low 5-HT transporter expression, and hence presumably higher synaptic 5-HT, was associated with reduced endogenous pain inhibition (Treister et al., 2011). However, 5-HT can also inhibit pain through activation of 5-HT7 receptors, with intrathecal 5-HT7 agonists providing pain relief in many pain models (Brenchat et al., 2010). Furthermore, blockade of 5-HT7 receptors at the spinal level can prevent morphine- and cannabinoid-induced analgesia and endogenous mechanisms of analgesia (such as stress-induced analgesia) in a dose-dependent manner (Dogrul, Ossipov, & Porreca, 2009; Yesilyurt et al., 2015). Earlier studies in rats found that blockade of 5-HT receptors at the spinal level reduced DNIC efficacy, whereas increasing 5-HT levels potentiated DNIC (Chitour, Dickenson, & Le Bars, 1982). Together, these results demonstrate that 5-HT can both facilitate and inhibit pain, and its ultimate effect depends on many variables, including the type of 5-HT receptor engaged.

The release of NA into the spinal cord produces analgesia via α2-adrenergic receptor activation (Reddy & Yaksh, 1980). Decreasing the excitability of the NA-containing projections produced thermal hyperalgesia in rats, suggesting that there is normally ongoing activity of this pathway (Howorth et al., 2009). Furthermore, studies of endogenous pain control and studies of analgesics have demonstrated that spinal α2-adrenergic receptor activation is essential for mediating their effectiveness (Bannister, Patel, Goncalves, Townson, & Dickenson, 2015).

Both monoamine pathways undergo changes during chronic pain conditions, reducing the efficacy of DNIC, and suggest that the hypersensitivity that accompanies neuropathic pain is caused, in part, by descending facilitation (Bannister et al., 2015). In a clinical setting, chronic pain conditions can reduce the endogenous pain control of patients, demonstrated by CPM studies in healthy subjects and chronic pain patients (Lewis, Rice, & McNair, 2012). In addition, some conditions, such as fibromyalgia, display pain facilitation in response to CPM testing, indicating a dysfunction of pain control mechanisms in these patients (Potvin & Marchand, 2016).

Corticospinal Input to the Spinal Dorsal Horn

Recent studies have functionally characterized the direct projections to the dorsal horn from cortical brain regions, such as the anterior cingulate cortex (ACC) and the somatosensory cortex (S1) (Chen et al., 2018; Liu et al., 2018). These areas have been associated with the affective and the sensory discriminant component of pain, respectively. Corticospinal projections from S1 to the spinal dorsal horn play an important role in controlling tactile sensation in both normal and neuropathic pain states (Liu et al., 2018). These projections are critical for setting mechanical sensory thresholds, as evidenced by the reduction of static and dynamic mechanical sensitivity when this pathway is ablated or silenced. Furthermore, interference with this projection could be used to reduce the mechanical allodynia induced by the spared nerve injury model of neuropathic pain.

The ACC is another cortical area that contains direct projections to the spinal dorsal horn (Chen et al., 2018). These projections terminate in laminae I–III of the spinal cord, are activated by nerve injury, and can undergo LTP in response to nerve ligation of the common peroneal nerve. Furthermore, activation of these projections can facilitate mechanical pain, whereas their inhibition can alleviate mechanical allodynia produced by common peroneal nerve ligation. Together, these results have identified exciting new potential targets for the treatment of chronic pain and have expanded our view of descending pain control to include cortical structures when we consider spinal mechanisms of pain.

Conclusion and Future Directions

The spinal dorsal horn harbors a plethora of neuronal and synaptic plasticity mechanisms potentially related to pathological pain states. Work in rodents, mainly mice, has shown that such plasticity mechanisms have direct implications for the functional connectivity in dorsal horn neuronal circuits and for pain-related behaviors. Although we are confident that the basic principles are conserved in humans it is unknown to what extent they contribute to different clinical pain syndromes. We view the translation of this knowledge to patients as one of the biggest current challenges in pain research.

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