Molecular, Cellular and Circuit Basis of Cholinergic Modulation of Pain

Neuroscience xxx (2017) xxx–xxx

MOLECULAR, CELLULAR AND CIRCUIT BASIS OF CHOLINERGIC MODULATION OF PAIN PAUL V. NASER a* AND ROHINI KUNER a,b*

This article is part of a Special Issue entitled: SI: Pain Circuits. Ó 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

a

Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany b

Cell Networks Cluster of Excellence, Heidelberg University, Germany

Key words: acetylcholine, pain, muscarinic, cholinergic analgesia, nicotinic, cholinergic–opioidergic interaction.

Abstract—In addition to being a key component of the autonomic nervous system, acetylcholine acts as a prominent neurotransmitter and neuromodulator upon release from key groups of cholinergic projection neurons and interneurons distributed across the central nervous system. It has been more than forty years since it was discovered that cholinergic transmission profoundly modifies the perception of pain. Directly activating cholinergic receptors or extending the action of endogenous acetylcholine via pharmacological blockade of acetylcholine esterase reduces pain in rodents as well as humans; conversely, inhibition of muscarinic cholinergic receptors induces nociceptive hypersensitivity. Here, we aim to review the considerable progress in our understanding of peripheral, spinal and brain contributions to cholinergic modulation of pain. We discuss the distribution of cholinergic neurons, muscarinic and nicotinic receptors over the central nervous system and the synaptic and circuit-level modulation by cholinergic signaling. AchRs profoundly regulate nociceptive transmission at the level of the spinal cord via pre- as well as postsynaptic mechanisms. Moreover, we attempt to provide an overview of how some of the salient regions in the pain network spanning the brain, such as the primary somatosensory cortex, insular cortex, anterior cingulate cortex, the medial prefrontal cortex and descending modulatory systems are influenced by cholinergic modulation. Finally, we critically discuss the clinical relevance of cholinergic signaling to pain therapy. Cholinergic mechanisms contribute to several both conventional as well as unorthodox forms of pain treatments, and reciprocal interactions between cholinergic and opioidergic modulation impact on the function and efficacy of both opioids and cholinomimetic drugs.

INTRODUCTION Chronic pain remains one of the major challenges for today’s medicine (Woolf, 2011). Many pathological pain disorders are currently intractable because the available therapies are either inadequate or their usage is associated with debilitating side effects. Chronic pain patients show several deviations from normal nociception and pain sensitivity, ranging from hyperalgesia (exaggerated sensitivity to noxious stimuli), allodynia (pain in response to normally innocuous stimuli), dysesthesias (abnormal sensations, often abnormal unpleasant perceptions of touch), paraesthesias (tingling, tickling, pricking, numbness, burning, ‘pins and needles’, ‘falling-asleep’-sensations in cutaneous dermatomes) and spontaneously occurring pain. Despite considerable progress, the mechanisms underlying many chronic pain disorders are not wellunderstood and new therapies are still being intensively sought. Over the last years, there has been a major thrust in the molecular understanding of pain; in contrast, circuits and networks that are causally involved in the mediation and modulation of pain are still poorly understood (Kuner and Flor, 2017). Various activity-dependent and disease-related changes can occur over the peripheral and central components of the somatosensory nociceptive pathway during the course of pain chronicity. Nociceptive pathways are subject to modulation from a plethora of hormonal and neurotransmitter systems, including dopaminergic, serotonergic, adrenergic and cholinergic pathways. This review will focus on the considerable recent progress in our understanding of the cholinergic modulation of pain at the level of the central nervous system. Besides its modulatory quality, Acetylcholine (Ach) also acts as one of the most prominent neurotransmitters in both the central and peripheral nervous system. Peripherally, cholinergic neurons control the sympathetic and parasympathetic branches of the autonomous nervous system on the ganglionic

*Corresponding authors. E-mail addresses: [email protected] (P. V. Naser), [email protected] (R. Kuner). Abbreviations: 5-HT, Serotonin; Ach, acetylcholine; AchE, acetylcholinesterase; AChR, acetylcholine receptor; BLA, basolateral amygdala; CEA, central amygdala; ChAT, choline acetyltransferase; CNS, central nervous system; DRG, dorsal root ganglion; EPSC, excitatory postsynaptic current; GABA, c-aminobutyric acid; GABAA, caminobutyric acid receptor A; i.c.v., intracerebroventricular; LDTg, laterodorsal tegmental nucleus; mAchR, muscarinic AChR; mPFC, medial prefrontal cortex; nAchR, nicotinic AChR; SCS, spinal cord stimulation; VTA, ventral tegmental area.

https://doi.org/10.1016/j.neuroscience.2017.08.049 0306-4522/Ó 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1

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level. Additionally, parasympathetic terminals release Ach and thereby mediate the ‘rest and digest’ functionality in the autonomous nervous system (Tiwari et al., 2013). In the central nervous system (CNS), Ach acts as a neurotransmitter and neuromodulator upon release from key groups of cholinergic projection and interneurons in both brain as well as spinal cord, details of which are discussed below. Given space constraints and the fast pace and volume of new insights that are being gained on cholinergic modulation of pain, this review will focus exclusively on central mechanisms. Neuronally, two primary types of receptors respond to Ach. Neuronal nicotinic receptors (nAchRs) are - with the exception of a7 and a9 homopentamers heteropentameric ligand gated cation channels (Nemecz et al., 2016). In mammals, eleven of the known 16 different subunits (a2-7,9,10; b2-4) differentially combine to determine ion-selectivity as well as binding kinetics. Of the nAchRs widely expressed in the CNS, a7-containing subtypes show fast binding kinetics together with low affinity for nicotine and high a-bungaratoxin affinity. Conversely, b2 containing receptors bind nicotine with high affinity and a-bungaratoxin with very low affinity, possess significantly slower kinetics and experience more desensitization (Dani, 2015). The differential binding properties and their functional diversity provide scope for specific pharmacological manipulations (Albuquerque et al., 2009). While the depolarizing nAchR current is largely carried by sodium, calcium influx plays an important role in downstream intracellular metabolic signaling. This is especially important for the a7-homomer, its Ca2+ permeability being more than fivefold that of other, heteropentameric, variants (Dani, 2015). The five muscarinic AchRs (mAchRs; M1-5) belong to the a-branch of class A G-Protein coupled receptors (GPCRs). M1, M3 and M5 have been shown to couple to Gq/11, initiating the activation of PLCb and MAPK along with an increase in intracellular Ca2+ and a decrease in the M-current. M2 and M4 signal preferentially via Gi/o, inhibiting adenylycyclase activity, activating GIRK channels and inhibiting voltage gated Ca2+ channels (Wess et al., 2007) (Fig. 2). A tremendous amount of knowledge has been accumulated regarding the involvement of central cholinergic modulation in cognitive processes. Attention and memory, for example, are highly dependent on functional cholinergic input. This comes particularly evident in Alzheimer’s Disease, where impairment of cholinergic signaling evokes detrimental symptoms. Cholinergic modulation of pain, in contrast, has not been not a mainstream topic in the pain field and deserves further investigation given its promise in pain therapy. Scientifically, the cholinergic system is especially interesting due to its rich plasticity, exemplified by the changing expression of both nicotinic and muscarinic AchRs during developmental stages and old age, potentially underlying age-specific effects of pharmacological and recreational substances (Tayebati et al., 2004; Melroy-Greif et al., 2016). Modulation not only differs across age groups, but also across sexes. For example, the analgesic effect of inhibiting the

breakdown of Ach via the Acetylcholine Esterase (AchE) inhibitor neostigmine, was shown to be five times more effective in female versus male rats (Chiari et al., 1999). The availability of knockout models for all muscarinic and most nicotinic receptors has recently fueled research into central cholinergic modulation and predestines this topic for future exciting findings. Classical pharmacological studies in animal models and humans have laid a rich foundation for studying the role cholinergic modulation plays in pain states (Hama and Menzaghi, 2001; Cucchiaro et al., 2008). Donepezil, another AchE inhibitor, produces a dose-dependent analgesic effect while exerting relatively few side-effects when administered systemically in humans, potentially emerging as an effective therapeutic agent. Donepezil is also effective as a prophylactic treatment for migraine, superior to propranolol in reducing total hours spent in pain as well as the number of pain episodes (Nicolodi et al., 2002). Direct systemic nicotinic stimulation also exerts antinociceptive effects in animal models of acute as well as chronic pain states (Cucchiaro et al., 2008). This review will discuss current insights on cholinergic modulation of pain while focusing primarily on the modulation of the central nervous system by cholinergic mechanisms (Fig. 1).

SPINAL MODULATION AchRs profoundly regulate nociceptive transmission at the level of the spinal cord via pre- as well as postsynaptic mechanisms. Cumulative evidence implicates a strong clinical relevance for spinal cholinergic mechanisms. Elevation of Ach levels in the spinal cord induces analgesia whereas locally decreasing Ach levels or activity (via receptor blockade) potentiates nociceptive sensitivity, inducing hyperalgesia and allodynia (Zhuo and Gebhart, 1991; Rashid and Ueda, 2002; Matsumoto et al., 2007). The most salient known spinal mechanisms are discussed below. Expression of cholinergic markers in the spinal dorsal horn Early studies employed the expression of the Achproducing enzyme, Choline acetyltransferase (ChAT), to elucidate the organization of the local cholinergic circuitry in the spinal dorsal horn. ChAT is prominently expressed in cholinergic interneurons of the spinal dorsal horn and in motor neurons. In addition to the common (central) form (cChAT), a peripherally expressed alternatively spliced variant (pChAT) has also been described, and is believed to be the primary form expressed in a majority of neurons in the dorsal root ganglion (DRG) and the trigeminal nuclei (Bellier and Kimura, 2007; Matsumoto et al., 2007; Koga et al., 2013). More recently, reporter transgenic mice expressing enhanced green fluorescent protein specifically in cholinergic neurons (ChAT-EGFP) have yielded in-depth insights into the morphology, neurochemistry and firing properties of cholinergic interneurons in the spinal dorsal horn (Mesnage et al., 2011). Dorsal horn cholinergic

Please cite this article in press as: Naser PV, Kuner R. Molecular, cellular and circuit basis of cholinergic modulation of pain. Neuroscience (2017), https://doi.org/10.1016/j.neuroscience.2017.08.049

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tent with presynaptic modulation of primary afferent function by cholinergic signaling (discussed below). A few varicosities were also opposed to astrocytic processes, although the functional significance of this finding remains unclear. Thus, cumulative evidence suggests that the salient morphological features of dorsal horn cholinergic neurons are similar across mice, rats and monkeys (Pawlowski et al., 2013). Interestingly, the presence of ChAT-immunoreactive fibers and neuronal somata in the superficial laminae has also been reported in the spinal cord of some, but not all, humans (Gill et al., 2007). Cholinergic actions at the level of the spinal dorsal horn Early studies indicated a tonic cholinergic inhibition of spinal Fig. 1. Avenues in pain pathways that are subject to cholinergic modulation. Muscarinic (mAchRs) transmission in rats and nicotinic (nAchRs) acetylcholine receptors mediate cholinergic modulation on both spinal nociceptive (lower portion) and supraspinal (upper portion) levels. The resulting changes in function of diverse (Zhuo and Gebhart, 1991). It has also regions (depicted by black arrows) in somatosensory, cortical and limbic pathways are depicted. been suggested that a nerve injuryPeripheral changes are not shown. induced loss of this cholinergic tone underlies the analgesic effects of exogenously administered cholinergic interneurons are a sparse population of cells that send agonists in neuropathic pain (Rashid and Ueda, 2002; long axons and elongated dendrites in the rostro-caudal Matsumoto et al., 2007). axis, thereby building a plexus of cholinergic processes There is a large body of evidence pointing to the in the inner lamina II, a key lamina for nociceptive proimportance of muscarinic signaling in these functions cessing. Thus, although these cells are relatively rare (Fiorino and Garcia-Guzman, 2012). Directly activating and represent the main source of ACh in the spinal dorsal mAChRs reduces pain in rodents as well as humans horn, they contact a large number of neurons and are and conversely, inhibition of spinal mAChRs induces nociwell-positioned to collect segmental information. Imporceptive hypersensitivity. However, nAchRs have also tantly, immunohistochemical colabelling experiments been implicated in spinal modulation of pain (see Presyhave revealed that a majority of these cells are GABAernaptic cholinergic function and receptors on primary affergic and coexpress the nitric oxide synthase, both in rats ent terminals below). and in mice (Mesnage et al., 2011). Electrophysiological The antinociceptive effects of systemically recordings revealed that these cells show repetitive spikadministered Donepezil are reported to be reversed by ing patterns (Mesnage et al., 2011), and can thus moduthe spinal blockade of GABAergic inhibition as well as late nociceptive processing across several segments that by antagonism of muscarinic receptors (Kimura et al., they project over. 2013), suggesting that the GABAergic component acts The significance of these findings to higher mammals, downstream of muscarinic receptors, i.e. in cells targeted particularly in the context of pain therapy, was questioned by cholinergic neurons. Administration of Donepezil has by studies which suggested that this population of cells is also been reported to induce release of GABA (Kimura absent from the spinal cord of the monkey. However, a et al., 2013), suggesting that the inhibitory action attriburecent study has employed ChAT immunolabeling, both ted to cholinergic neurons may be mediated via GABAerwith light and electron microscopy (EM), to demonstrate gic signaling. So far, it remains unclear whether there is a the presence of a plexus of cholinergic fibers spanning link between these observations and the majority of spinal laminae II-III in the macaque monkey (Pawlowski cholinergic spinal neurons being GABAergic in nature, et al., 2013). All the varicosities of ChAT-expressing i.e. whether autocrine cholinergic signaling brings about spinal neurons were also immunoreactive for GABA, reninhibition via modulation of GABA release from cholinerdering it unlikely that they represent primary afferent gic neurons. fibers or terminals of preganglionic autonomic neurons. Intrathecally administered acetylcholine esterase Moreover, the varicosities formed symmetric axo(AchE) inhibitors, such as neostigmine, are also dendritic and occasionally axo-somatic synapses on reported to reduce inflammatory hypersensitivity, which spinal dorsal neurons, along the lines typically observed is sensitive to muscarinic antagonists (Yoon et al., for inhibitory interneurons, and some also engulfed glo2005). These effects are principally attributed to M2 recepmeruli of presynaptic primary afferent terminals, consistor activation, and are not blocked upon either M1 or M3 Please cite this article in press as: Naser PV, Kuner R. Molecular, cellular and circuit basis of cholinergic modulation of pain. Neuroscience (2017), https://doi.org/10.1016/j.neuroscience.2017.08.049

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2003); however, the identity of these nAchR is not entirely clear. A neuronal subpopulation of the DRG expresses a7-nAChRs, transports them axonally to both the central terminal in the spinal cord and periphery. These neurons also functionally respond to a7-selective positive allosteric modulators with a calcium rise (Shelukhina et al., 2015) (Fig. 2). The functional significance of these findings remains unclear. By performing recordings on neonatal spinal cord slices, Genzen and McGehee report that activation of nAchRs via exogenously applied agonists potentiates excitatory transmission on a large proportion of spinal dorsal horn neurons via a7-nAChRs and NMDARs. Both short- and long-term potentiation was reported, suggesting a mechanism for exogenous nicotinic hyperalgesia. Importantly, experiments utilizing AchE inhibition supported the notion that endogenous nAchR signaling plays a similar role. In experiments measuring the release of glutamate or nociceptive peptides from DRG neurons, Shelukhina and colleagues did however not find evidence for modulation Presynaptic cholinergic function and receptors on of basal or evoked release by nAchR agonists primary afferent terminals (Shelukhina et al., 2015). Paradoxically, both hyper- as well as analgesic effects have been attributed to the actiA significant proportion of the effects described above vation of spinal nicotinic receptors (Genzen and may be attributed to presynaptic cholinergic receptors. McGehee, 2003). Spinal nicotine analgesia has been The literature on the nature of cholinergic modulation is reported to involve short- and long-term modulation of somewhat divided, with contrasting reports on spinal GABAergic transmission (Genzen and McGehee, potentiation of excitation versus inhibition of nociceptive 2005). Takeda et al. (2003) also reported the involvement transmission (Fig. 2). of nAchRs other than a7-nAChRs and a4b2-nAchRs in NAchRs are known to be expressed in mammalian presynaptic enhancement of inhibitory transmission in peripheral sensory neurons and transported to primary the spinal dorsal horn. Yet other studies report that afferent terminals (Steen and Reeh, 1993; Lang et al., a7-nAChR agonists demonstrate analgesic, anti-hyperalgesic and antiinflammatory effects, which have been attributed largely to the activation of peripheral receptors (Matsumoto et al., 2007). Thus, the precise role of nicotinic modulation may vary depending on the anatomical location as well as the context and warrants further elucidation. Muscarinic modulation of spinal nociceptive transmission has been well studied. Electrophysiological studies indicate that presynaptic muscarinic receptors expressed in the DRG and the trigeminal ganglia modulate primary afferent input on to spinal or medullary dorsal horn neurons, respectively. In rats, monosynaptic excitatory postsynaptic currents (EPSCs) recorded from medullary dorsal horn neurons upon electrical stimulation of the trigeminal tract are reversibly and Fig. 2. Mechanisms of cholinergic modulation of nociceptive transmission in the spinal cord. dose-dependently inhibited by Cholinergic interneurons (green) in the spinal cord release acetylcholine, which acts presynapmuscarine and Ach (Jeong et al., tically via muscarinic receptors (mAchRs) to regulate glutamate release from primary afferents 2013). Muscarinic agonists also (yellow). Expression of alpha7 nicotinic receptors (nAchRs) at primary afferent terminals has been increase the paired-pulse ratio and demonstrated, but functions are yet unknown. ACh reduces excitability of second order spinal reduce the frequency of miniature neurons (blue) via M2 and M4 mAchR activation and downstream activation of G-Protein-coupled inwardly rectifying potassium channels (GIRK). ACh is metabolized rapidly by acetylcholine EPSCs, thereby indicating a decrease receptor blockade (Yoon et al., 2005). Indeed, expression analyses indicate that among muscarinic receptors, M2 is the major mAChR subtype expressed in the spinal cord. Autoradiographic studies revealed significant binding to M2, M3 and M4 receptors in the spinal cord, with only background levels of M1 receptor binding (Ho¨glund and Baghdoyan, 1997). Recent experiments employing siRNAs to achieve specific receptor knockdown in spinal neurons in vivo in rats confirmed that M2 and M4 receptors predominantly mediate antinociception, whereas M1 and M3 receptors were not found to be appreciably involved (Cai et al., 2009). It should, however, be noted that the precise outcomes of pharmacological manipulations of muscarinic receptors are highly dependent on the species being tested. In particular, Pan and colleagues have reported extensively on species differences between mice and rats with respect to cholinergic pharmacology (Cai et al., 2009; Chen et al., 2010).

esterase (AchE) and AchE blockers thereby yield antinociceptive effects. Please cite this article in press as: Naser PV, Kuner R. Molecular, cellular and circuit basis of cholinergic modulation of pain. Neuroscience (2017), https://doi.org/10.1016/j.neuroscience.2017.08.049

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in the probability of glutamate release on to dorsal horn neurons (Jeong et al., 2013; Fig. 2). Similar results have been reported by Zhang et al. regarding muscarinic regulation of glutamate release on to spinal neurons from both primary afferents and spinal interneurons (Zhang et al., 2007). Both pharmacological agents as well as mAchR subtype specific single- and double-knockout mice have been employed to uncover the differential contributions of diverse muscarinic receptor subtypes. These studies have revealed that activation of M2 and M4 receptors inhibits the flow of excitatory synaptic inputs from primary afferents into the spinal cord (Chen et al., 2014). Mechanistically, this is believed to be accounted for by the inhibition of voltage-gated calcium channels in primary sensory neurons, thereby explaining the inhibitory effect of M2/M4 agonists on monosynaptic and polysynaptic EPSCs evoked in spinal neurons by dorsal root stimulation (Chen et al., 2014; Fig. 2). Both M2 and M4 receptors are expressed at primary afferent terminals; the M4 subtype appears to be particularly interesting in the context of chronic pain since its expression is increased in neuropathic pain states, such as in diabetic neuropathy (Cao et al., 2011). It should, however, be noted that another study did not find alterations in M4 expression in the spinal cord of animals with spinal nerve injury (Kang and Eisenach, 2003), indicating that these changes may be context-dependent and require further elucidation. A more complex role has been ascribed to the M5 subtype in regulating primary afferent input: positive effects directly mediated by M5 on primary afferent terminals stimulation, are accompanied by putative negative modulation through increased glutamate release from spinal interneurons and subsequent activation of group II/III metabotropic glutamate receptors (mGluRs) (Chen et al., 2014). The latter thus serves to restrain excessive primary afferent input into the spinal dorsal horn. Cholinergic signaling in brainstem nuclei and its contributions to descending modulation of spinal nociceptive processing Supraspinal centers such as the serotonergic raphe nuclei, adrenergic centers in the locus coeruleus and the rostroventral medulla (RVM) play a key role in the endogenous control of nociception via descending modulation of spinal function. There is evidence indicating nicotinic cholinergic signaling in the brainstem nuclei stimulates descending inhibitory pathways (Fig. 1) and mediates antinociceptive effects via a2 adrenergic, 5-HT1c/2 and 5-HT3 serotonergic, and M2 cholinergic receptor interactions in the lumbar spinal cord (Iwamoto and Marion, 1993). Recently, administration of nAChR agonists in the RVM has also been shown to elicit antinociception in naı¨ ve rats and reverse inflammatory hyperalgesia via local activation of a4b2-containing nAChRs (and to a lesser extent, a7-containing nAChRs) in the brainstem (Jareczek et al., 2017). Activation of muscarinic signaling in the raphe nucleus via microinjection of carbachol has

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also been suggested to activate descending inhibition (da Silva and Menescal-de-Oliveira, 2006). Overall, this topic has been sparsely studied and there is a need to better understand how supraspinal cholinergic circuits regulate pain perception and its plasticity in pathological states. Interestingly, by employing viral tracers in ChAT-Cre mice, a recent study has demonstrated direct projections of cholinergic neurons in the rostral ventrolateral medulla to lamina III of the spinal cord, suggesting direct descending control of spinal sensory transmission by brainstem cholinergic neurons (Stornetta et al., 2013).

SUPRASPINAL MODULATION Practically all structures of the mammalian brain are subject to cholinergic modulation either receiving or providing direct cholinergic input. Cholinergic agonists evoke stable hyporeflexia when applied intracerebroventricular (Vierck et al., 2016). AchE inhibition via physiostigmine i.c.v. was also shown to attenuate the early as well as late phases of the formalin-induced pain response in rodents (Mojtahedin et al., 2009). Thus, selective modulation of cholinergic signaling in the brain has an impact on nociception and pain. Muscarinic receptors are nearly ubiquitously expressed throughout the brain. M1 is the most abundant mAchR in the CNS, being postsynaptically expressed in neocortical as well as allo- and subcortical areas (Langmead et al., 2008). M2 are thought to autoinhibit the Ach release from cholinergic terminals by a negative feedback mechanism, however, in brainstem motor neurons, about 30% of the total M2-protein was shown to be located postsynaptically at glutamatergic synapses (Langmead et al., 2008; Csaba et al., 2013). In comparison, M3 and M5 receptor expression is much lower, with M3 being mainly located in neo- and allocortex and M5-expression tightly confined to the substantia nigra. M4- receptors are prominently found in striatal regions, controlling dopaminergic signaling and locomotion (Gomeza et al., 1999; Langmead et al., 2008). While muscarinic analgesic activity was previously shown to depend largely on M2-activation (Duttaroy et al., 2002), both pharmacological blocking via atropine as well as specific M1-knockdown are able to block this analgesic effect, suggesting involvement of both M1 and M2 type mAchRs (Mojtahedin et al., 2009; Bartolini et al., 2011). Emergence of selective muscarinic modulators additionally provides new perspectives for pain therapy (Fiorino and Garcia-Guzman, 2012). Although a bulk of the literature supports an antinociceptive role for cholinergic modulation in the brain, paradoxically, large scale cholinergic denervation of the cortex following basal forebrain lesioningreduces pain levels in rats (Vierck et al., 2016). This suggests that diverse local and long-range cholinergic pathways may differentially modulate pain processing, necessitating studies that target individual pathways. In general, two types of cholinergic neurons, namely local interneurons and projection neurons provide cholinergic input to various brain regions, the differential

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effects of which on pain-related areas will be discussed below. Contrary to most sensory modalities, such as vision or hearing, which are processed in their respective distinct brain areas, the percept of pain is not encoded solely in the somatosensory cortex. Rather, pain is the result of a network activity of multiple areas related to sensory, cognitive and emotional functions – below, we attempt to provide an overview of how some of the salient regions in the pain network are influenced by cholinergic modulation. Distribution of cholinergic neurons and receptors Striatal interneurons. Unlike other brain regions, cholinergic interneurons make up circa 2% of the striatal neuronal population. Firing in the 5-Hz range, these neurons provide spatially and, owing to the high expression of AchE, temporally restricted cholinergic tone (Zhou et al., 2002). Reports show these striatal cholinergic interneurons receive three times more GABAergic afferent input than glutamatergic, potentially suggesting a high level of tonic inhibition, which can be further increased in response to changing environments. (Gonzales and Smith, 2015). Earlier reports from immunohistochemistry against ChAT in the macaque brain suggested the highest density of cholinergic interneurons to be found in the ventral part of the striatum (Mesulam et al., 1984). In humans, it was shown that the associative dorsomedial striatal areas, however, harbor a substantially higher proportion of ChAT-positive cells than the sensory dorsolateral and limbic ventral areas suggesting significant differences in cholinergic signaling across functional territories of the striatum (Berna´cer et al., 2007). While the role of striatal cholinergic interneurons has not been rigorously assessed in the light of pain, the interconnectivity between cholinergic and dopaminergic signaling in the striatum is well-studied. In slices, dopamine release is reduced by 90% when lacking cholinergic input. This effect was shown to depend on the functional expression of the b2-nAchR-subunit (Zhou et al., 2001). Dopamine in turn, modulates the excitability of cholinergic interneurons by virtue of presynaptic inhibition via D2- receptors and postsynaptic excitation via D1 receptors (Pisani et al., 2000). Recently, the excitatory effect of corticostriatal afferents on dopaminergic neurons was shown to depend on b2-subunit-containing nAchRs, proposing a model of cholinergic gatekeeping. Glutamatergic afferents herein excite striatal cholinergic interneurons that, in turn, drive dopamine release from nAchR expressing dopaminergic cells (Kosillo et al., 2016). Studying the role of cholinergic interneurons with regard to pain is of great interest, especially since positron emission tomography has revealed drastic changes in striatal dopaminergic circuitry in chronic pain states (Hagelberg et al., 2004; Martikainen et al., 2015). Projection neurons. Cortical and subcortical brain regions receive cholinergic input from basal forebrain projection neurons. Since their first description as ‘unnamed medullary substance’ by Reil in the beginning

of the 19th century, the exact identity of cholinergic BFB projection neurons has been much debated (Liu et al., 2015). Retrograde tracing studies revealed the identity of four rostrocaudally distinct, yet partially overlapping, populations of cholinergic projection neurons: Ch1-Ch4. The Ch1 and Ch2 groups project to the hippocampal formation, whereas Ch3 innervates the olfactory cortex. The Ch4 population is largely congruent with the more traditional term ‘Nucleus Basalis of Meynert’ and gives rise to the projections into higher cortical areas. Within the Ch4 itself, projections are topographically organized with more laterally located somata sending projections to lateral cortical areas (Wu et al., 2014; Liu et al., 2015). Retrograde tracing from auditory and somatosensory cortices revealed an even higher level of organization with partially overlapping, yet largely segregated, basal forebrain populations projecting to the two cortices (Chaves-Coira et al., 2016). Cholinergic projection neurons are among the most arborized nerve cells known. Single neuron tracing revealed axon trees spanning more than 50 cm in mice, translating to an estimated 100 m (!) in humans. Consequently, single neurons can modulate entire neuronal networks, essential for their function in fine tuning large networks (Wu et al., 2014). While the density of cholinergic terminals is relatively stable across different cortical areas, there are differences in the laminar distribution. For example, in the visual cortex, layer IV receives strongest cholinergic input, whereas in sensory-motor areas, the highest terminal density is observed in layers I and V and the lowest in layer IV (Eckenstein et al., 1988). Upon optogenetic stimulation of basal forebrain cholinergic neurons, a strong disruption of the cortical field potentials in the respective target areas can be observed, which is accompanied by a significant increase in the amplitude of evoked potentials (ChavesCoira et al., 2016). Cholinergic afferents also differentially target parvalbumin (PV), somatostatin (SOM) and vasointestinal peptide (VIP) subtypes of cortical GABAergic interneurons (Poorthuis et al., 2014). Rabies virus-mediated transsynaptic input tracing revealed that basal forebrain neurons themselves receive a wide variety of inputs from almost all brain areas. Thus, input from diverse pain-related areas as the insular cortex, the central amygdala and midbrain areas, like the RVM or periaquaeductal gray (PAG), could also mediate neuroplastic changes in basal forebrain projections (Hu et al., 2016). This is an exciting possibility that remains to be explored across different chronic pain states. Modulation of the primary sensory cortex by cholinergic transmission While cholinergic transmission has been shown to enhance the intensity of evoked potentials within the somatosensory cortex, the influence of cholinergic modulation on the alterations seen in chronic pain states has not been directly studied (Donoghue and Carroll, 1987). Dense cholinergic innervation from the basal forebrain largely originating in the region of the diagonal band

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of Broca qualitatively enhances neocortical coding, often portrayed as an improved signal-to-noise ratio, via a variety of mechanisms as described below (Chaves-Coira et al., 2016). When applying a tactile stimulus, ‘distraction’ by simultaneous application of a different stimulus to a different dermatome can reduce the cortical response evoked by the first stimulus – a phenomenon known as sensory interference. In the hindlimb area of the sensory cortex, this phenomenon was reduced after either ablating cholinergic afferents via 192 IgG saporin or by pharmacologically blocking muscarinic receptors, indicating that cholinergic projections from the basal forebrain to the somatosensory cortex importantly contribute to sensory interference via mAchR signaling (Alenda and Nun˜ez, 2004, 2007). On a circuit level, three main routes of modulation have been proposed to underlie the increase in the signal-to-noise ratio: First, basal forebrain cholinergic afferents drive layer I and II/III non-PV-interneurons via non-a7-nAchR. These, in turn, inhibit local inhibitory PV and SOM interneurons, reducing the net inhibitory input to pyramidal cells and resulting in increased excitability (Brombas et al., 2014; Hedrick and Waters, 2015). Second, Ach activates inhibitory mAchRs on pyramidal layer IV terminals, reducing the intra-cortical communication, thereby reducing the ‘background-noise’ (Eggermann and Feldmeyer, 2009). Third, initial glutamate release from thalamocortical afferents to layer IV is increased via presynaptically located b2-nAChR, boosting the incoming signal (Disney et al., 2007; Hedrick and Waters, 2015). Additionally, it was reported, that Ach can directly drive neocortical pyramidal neurons in a layer-specific fashion via nAchRs, with layer V showing the strongest response (Hedrick and Waters, 2015). In contrast, M4mediated direct inhibition of spiny stellate neurons receiving thalamocortical input was also reported from the rat sensory cortex (Eggermann and Feldmeyer, 2009; Picciotto et al., 2012). Furthermore whether M1 activation actually exerts a depolarizing or hyperpolarizing effect is reported to depend on the state of intracellular calcium storages, suggesting differential effects of Ach on excitability depending on the state of neuronal excitation (Picciotto et al., 2012). A contributing factor to these partially conflicting differential effects could be the experimental approach of electrical stimulation that is commonly used in most studies. Newer chemogenetic and optogenetic approaches targeted to genetically defined subpopulations will enable researchers to decipher the underlying circuitry mediating the differential effects of Ach with a higher precision. Structural and functional neuroplastic processes are the basis for the ability of the nervous system to adapt to changing environments. While extensive research has been conducted into maladaptive structural plasticity, the cellular and molecular mechanisms underlying this phenomenon in pain states, and especially during pain chronicity remain a black box within the field of pain research (for review see Kuner and Flor, 2017). The cholinergic system was suggested

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to play a major role in both the functional aspects as well as the structural changes. In conjunction with the aforementioned increase in signal-to-noise ratio, cholinergic modulation prolonged the neuronal ensemble activity evoked by thalamic inputs, while reducing the background firing in primary somatosensory neurons (Runfeldt et al., 2014). Structural plasticity also appears to highly depend on cholinergic modulation. In a simple model of sensory deprivation, the whiskers of rats are trimmed and the rapid expansion of the cortical columns that are still innervated into the deprived areas is measured. This reorganization is strongly reduced in animals whose basal forebrain cholinergic neurons were selectively lesioned (Sachdev et al., 1998; Zhu and Waite, 1998). Reciprocally, changes in sensory reorganization also shape cholinergic projections. For example, in trigeminally denervated rats, in addition to the acute reduction of Ach levels in the respective areas of the sensory cortex, a physical retraction of basal forebrain neurons was observed. While the drop in Ach could be avoided by continuous artificial stimulation of the nerve stump after transection, the retraction of cholinergic axons in the sensory cortex remained unchanged. As basal forebrain neurons are not reported to receive direct sensory input, this finding suggests a reciprocal cortico-basal pathway through which cortical regions to some extent autoregulate the level of cholinergic input they receive (Herrera-Rincon and Panetsos, 2014). The exact mechanisms of how this relationship dynamically changes over pain chronicity and other sensory disorders warrants thorough investigation. Cholinergic modulation of the insular cortex in pain states The insular cortex plays a multifunctional role at the intersection between the inner workings of the brain and the external environment. The neuroanatomical correlates thereof are extensive reciprocal connections between both sensory-motor cortices as well as the emotion-associated nuclei of the limbic system. Dysfunctional processing in the insula has been implicated in patients with the sensory deficits as well as hallucinations associated with schizophrenia (Wylie and Tregellas, 2010). With regard to pain, imaging studies indicate that the physical properties, such as the intensity and location of the noxious stimulus, are processed in the posterior insular cortex, followed by an emotional appraisal in the anterior portion. (Frot et al., 2014; Segerdahl et al., 2015). In rats, the expression of inhibitory M2-type mAchR was shown to be strongly upregulated in neuropathic pain states. Local muscarinic activation via oxotremorine as well as systemic donepezil induced strong analgesia during the late stage of neuropathic pain. Systemic donepezil-induced analgesia could likewise be partially reverted by local application of the M2-antagonist methoctramine. Neither donepezil nor oxotremorine exerted an effect on the baseline sensitivity. These results suggest, that the insula undergoes sensitization to the exposure of Ach in the pathological state,

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although the functional implications of this finding need to be further explored (Ferrier et al., 2015). Cholinergic modulation of the amygdala Located in the temporal lobe, the amygdala has been extensively studied for its involvement in emotional processing, especially in the context of fear and pain. Human imaging studies showed that pain, as a multidimensional experience, also reliably activates this area (Simons et al., 2014). Neuroplastic reorganization of the amygdala has been reported in several rodent models of chronic pain (Neugebauer, 2007; Simons et al., 2014). In the basolateral amygdala, the facilitation of synaptic transmission and increase in excitability associated with fear memory was shown to be modulated by cholinergic inputs, leading to a slowed extinction of learned fear. Additionally, endogenous Ach was shown to be crucially required for the initial fear learning (Jiang et al., 2016). This contrasts earlier reports of cholinergic inputs enhancing GABAergic inhibition via a7-nAchRs and thereby reducing the overall excitability (Unal et al., 2015). Via its reciprocal connections with the descending modulatory system, the amygdala can additionally induce acute contextual analgesia in states of fear or stress (Neugebauer et al., 2004). In guinea pigs, cholinergic stimulation by carbachol injection into the central nucleus of the amygdala (CEA) induced antinociception in a mAchR-dependent and opioid receptor-dependent manner, that was reversed by blockade of the pathway connecting the CEA to the ventrolateral PAG (Leite-Panissi et al., 2003, 2004). Further investigation of these divergent findings will be key in understanding not only fear, but also the aversive components of pain and the reciprocal interactions between fear and pain sensitivity that are observed in chronic pain states. Cholinergic modulation of the prefrontal cortex Cholinergic modulation of the prefrontal cortex has been widely studied in the context of attention and normal cognitive performance (for review, see Bloem et al., 2014). However, this structure also plays an important role in the processing of pain. In healthy adults, electrical oscillations between 20 and 80 Hz (c-band oscillations; GBO) are commonly seen as a proxy for cortical synchronicity underlying cognitive tasks such as memory formation. In the primary somatosensory cortex, GBO are a direct correlate of the encoding of the pain intensity (Zhang et al., 2012). Recently, GBOs were also found to encode tonic pain perception in the human medial prefrontal cortex (mPFC) (Schulz et al., 2015). Synchronization of pyramidal neurons by PV-type GABAergic interneurons is believed to represent the cellular basis of GBO generation (Cardin et al., 2009; Kim et al., 2015). Interestingly, in acute slice recordings from the primary somatosensory cortex, carbachol is reported to only elicit GBOs in conjunction with kainate (Buhl et al., 1998). In mPFC slices, however, the sole application of carbachol was sufficient to elicit and sustain

GBO, suggesting differential modes of action for cholinergic modulation of oscillatory activity patterns (Pafundo et al., 2013). As the precise circuitry underlying cholinergic modulation of GBO has not been elucidated, exploration of this topic will be of high interest in understanding pain given the putative importance of GBO in pain perception (Zhang et al., 2012; Schulz et al., 2015). In neuropathic pain states, the mPFC is one of the areas that are consistently deactivated. This phenomenon is believed to depend on glutamatergic input from the amygdala, driving tonic inhibition of mPFC activity by GABAergic interneurons (Ji and Neugebauer, 2011). Interestingly, nicotinic stimulation of local inhibitory interneurons within the mPFC can also reduce the mPFC net output in the short term (Bloem et al., 2014). Very recently, however, internalization of M1 muscarinic receptors in layer V mPFC neurons was also shown to be associated with a reduction in their excitability. Collectively, these results imply that apart from the GABAergic amygdala-mPFC interneuron pathway, cholinergic mechanisms, at least partially, underlie mPFC-inactivation in neuropathic pain states (Radzicki et al., 2017).

Cholinergic modulation of the anterior cingulate cortex The anterior cingulate cortex (ACC) is one of the brain regions which is most consistently and strongly activated during pain and has been reported to undergo significant plasticity in chronic pain patients (Bliss et al., 2016). Production of pain affect as well as sensory processing have been ascribed to the ACC. Pharmacological M1-mAchR stimulation in the rat ACC was recently shown to exert an analgesic effect in naı¨ ve animals by virtue of increasing the frequency and amplitude of GABAA-mediated IPSCs (Koga et al., 2017). Mechanistically, it remains unclear whether M1-mediated postsynaptic modulation, which has been reported in addition to presynaptic regulation of GABA release from inhibitory interneurons (Domı´ nguez et al., 2014; Koga et al., 2017), also plays a role in pain modulation.

CLINICAL RELEVANCE OF CHOLINERGIC SIGNALING TO PAIN THERAPY Cholinergic mechanisms contribute to several conventional as well as unorthodox forms of pain treatment. Below, we attempt to provide an overview and to classify these into spinally mediated or brainmediated actions, as far as possible. It is understood that some therapies and forms of antinociception employ both spinal and brain-related mechanisms. Table 1 furthermore provides an overview of various pharmacologically relevant agents acting on the cholinergic system. Cholinergic-opioidergic are discussed separately at the end of this section.

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P. V. Naser, R. Kuner / Neuroscience xxx (2017) xxx–xxx Table 1. List of analgesic drugs/treatments with proposed involvement of cholinergic receptors/signaling/circuits Drug/treatment Brain action Morphine Morphine withdrawal Caffeine 5HT-Agonists Gabapentin Stress-induced analgesia Spinal action Donepezil Gabapentin Gabapentin + Donepezil Clonidine Nicotine Opioids Spinal cord stimulation (SCS) SCS + Clonidine Sildenafil Melatonin Aspirin and other non-steroidal antiinflammatory drugs Natural plant products and animal toxins (e.g. Cobrotoxin and Cobratoxin) that exert analgesic actions

Reference(s) Romano and Shih (1983) Pinsky et al. (1973) Ghelardini et al. (1997) Bianchi et al. (1990), Ghelardini et al. (1996) Takasu et al. (2006), Hayashida et al. (2007) Romano and Shih (1983) Hayashida et al. (2007) Takasu et al. (2006), Hayashida et al. (2007) Hayashida et al. (2007) e.g. Chen and Pan (2001) Jareczek et al. (2017) Yang et al. (1998) Schechtmann et al. (2008), Prager (2010) Schechtmann et al. (2008) Park et al. (2011) Shin et al. (2011) Hamza and Dionne (2009) Chen et al. (2006)

Spinal cholinergic contributions to analgesia produced by diverse agents/treatments Modulation of pain by Ach has been validated in humans and has led to an increasingly large usage of epidural injections of acetylcholinesterase inhibitors, such as neostigmine, in analgesic therapy for postoperative and labor-induced pain patients. Moreover, the endogenous cholinergic system of the spinal cord has been demonstrated to be involved in the effects of clinically used analgesics, such as morphine (discussed below) and clonidine, which activates a2-adrenergic receptors (Chen and Pan, 2001). In animal models of neuropathic pain, analgesia induced by a2-adrenoceptor stimulation has been hypothesized to involve spinal stimulation of acetylcholine release, as opposed to inhibition of acetylcholine release by adrenergic signaling under normal circumstances (Hayashida and Eisenach, 2010). Furthermore, cholinergic contributions are now being described in an increasing number of analgesic therapies or manipulations (see Table 1). Thus, analgesia induced by gabapentin, a key drug employed in diverse forms of neuropathic pain, has been suggested to involve spinal Ach release (Takasu et al., 2006; Hayashida et al., 2007)). Analgesia induced by gabapentin, both via oral as well as i.c.v. routes of administration, is reversed by spinal blockade of muscarinic receptors (Hayashida et al., 2007). Coadministration of donepezil and gabapentin was also shown to exhibit a synergistic effect in neuro-

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pathic pain patients (Basnet et al., 2014). Moreover, drugs with entirely diverse targets, such as sildenafil (phosphodiesterase inhibitor), melatonin (involved in the regulation of sleep-wake cycles), nicotine (via smoking), among others, are associated with Ach release in the spinal cord. It has been suggested that chronic pain patients smoke tobacco in order to obtain pain relief (Jareczek et al., 2017). Interestingly, electrical stimulation of the spinal cord (SCS), which is employed for chronic intractable pain and exerts beneficial effects on sensory, motor and vascular components of chronic neuropathic pain disorders, has been recently suggested to involve activation of inhibitory GABA-ergic and cholinergic spinal interneurons (Prager, 2010). In neuropathic rats, intrathecal administration of the a2-adrenoceptor agonist, clonidine, which induces Ach release in the spinal dorsal horn, potentiates analgesia induced by SCS. Accordingly, SCS selectively elevates spinal Ach levels in rats responding beneficially to SCS and SCS-induced antinociception is reversed by inhibition of spinal muscarinic, but not nicotinic, receptors (Schechtmann et al., 2008). Brain cholinergic contributions to analgesia produced by diverse non-cholinergic agents In addition to the aforementioned analgesic effects produced by direct modulation of the spinal cholinergic system, or cholinergic–opioidergic interactions discussed below, some unorthodox other treatments seem to, in part, depend on cholinergic mechanisms. For example, the antinociceptive effect exerted by caffeine was shown to depend on cholinergic, nonopioidergic transmission, being susceptible to blockade by cholinergic antagonists, but not naloxone, suggesting amplification of cholinergic transmission underlying this phenomenon (Ghelardini et al., 1997). Furthermore, serotonergic signaling has been also suggested to directly modulate the cholinergic output of projection neurons in the cortex, as shown by the steep rise in Ach levels induced by 5-HT1A and decrease of Ach levels by 5-HT3 agonists (Bianchi et al., 1990). 5-HT4 agonists furthermore produce analgesic effects in a Ach-dependent manner, suggesting a close interconnection between the tryptaminergic and cholinergic systems in the brain (Ghelardini et al., 1996). Stress-induced analgesia is largely thought to be mediated by opioidergic circuits. However, cold waterinduced analgesia was shown in rats to modulate cortical Ach levels differentially from direct morphine application, suggesting a parallel mode of action (Romano and Shih, 1983). Cholinergic–opioidergic interactions Interactions with opioidergic pathways is one of the scientifically highly interesting and clinically most relevant aspects of cholinergic signaling in pain. Interactions between opioidergic and cholinergic mechanisms occur at various avenues in the central nervous system. Opioidergic–cholinergic interactions are

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reciprocal in nature, impacting the function and efficacy of both opioids and cholinomimetic drugs, and were discovered as early as the 1970s. It was found that morphine increases the levels of acetylcholine in mouse striatum at doses that induce analgesia and with a time frame consistent with analgesic actions (e.g. Green et al., 1976). In contrast, the effects on epinephrine and dopamine levels were not significant at these doses, suggesting that cholinergic mechanisms contribute largely to opioid analgesia. Subsequent pharmacological studies demonstrated that coadministration of AchE inhibitors can potentiate opioid analgesia. The relative contributions of nicotinic and muscarinic receptors to cholinergic modulation of opioid analgesia are debated. Some studies suggest that nicotinic receptors in the nucleus accumbens play an important role in opioid analgesia in nicotine-naı¨ ve rats, and that this role is attenuated in the nicotine-dependent state (Schmidt et al., 2001). Others observed a lack of involvement of cholinergic nicotinic neurotransmission in antinociceptive effects of acute morphine or the development of antinociceptive tolerance to morphine (e.g. Bajic et al., 2015). Recently, antagonists at muscarinic cholinergic receptors, but not nicotinic cholinergic receptors, were found to reverse the enhancing effects of AChE inhibitors on opioid analgesia (Gawel et al., 2017). Using knockout mice, a study reported that M5 receptor activity modulates morphine reward and withdrawal, but not the analgesic efficacy of morphine, suggesting a potential for M5 modulators in the treatment of opiate addiction (Basile et al., 2002). Collectively, the current literature suggests differential contributions of nicotinic and muscarinic signaling to morphine reward and dependence versus morphine analgesia and analgesic tolerance. Issues with drug specificity and lack of selectivity of targeting individual pathways and circuits limit our current understanding of these critical questions. This is further confounded by studies suggesting morphine and several opioidergic drugs directly bind AchE and negatively modulate its activity (Motel et al., 2013). Moreover, different opioids are reported to share many common structural elements with drugs that act as allosteric potentiating ligands of nAchRs (Motel et al., 2013). Conversely, opioidergic mechanisms also alter responses to nicotinic drugs. Thus, interactions not only impact on analgesia and analgesic tolerance, but also likely mediate cross-dependence for opioids and nicotine. Clinically, the use of nicotine products and opioid analgesics is frequent among chronic pain patients (Jareczek et al., 2017) and smokers show a higher frequency of prescription opioid use (Yoon et al., 2015). Studies indicate that tobacco smoking is also a strong predictor of risk for nonmedical use of prescription opioids (Yoon et al., 2015). Thus, opioidergic–cholinergic interactions not only contribute to therapeutic usage and efficacy of opioids and cholinergic agents, but also present a complicating factor in drug abuse, addiction and tolerance. So far, most studies embraced pharmacological manipulations that fall short of providing cellular specificity, while pathways and circuits underlying the

therapeutically beneficial as well as the deleterious aspects of opioidergic–cholinergic interactions have not been widely studied. A few studies have addressed brain regions involved in the effects of opioids and nicotine on reward and addiction. It has recently been described that chronic morphine administration increases the turnover of the cholinergic transmission in the laterodorsal tegmental nucleus (LDTg)/pedunculopontine tegmentum – neurons in this area are known to provide the only cholinergic (excitatory) inputs to dopaminergic neurons in the ventral tegmental area (VTA), which in turn project to the nucleus accumbens (Bajic et al., 2015). This has been suggested to be a pathway mediating the impact of cholinergic transmission on motivated behavior, reward pathways, and addiction (Fig. 1). However, several other areas and pathways may also play a role. For example, morphine changes the activity of local cholinergic interneurons in the nucleus accumbens and the striatum; accordingly, enhanced local cholinergic neurotransmission in the nucleus accumbens has been suggested to inhibit morphine addiction. Thus, the cholinergic system may have complex and opposing effects on morphine addiction that are likely related to different brain areas that are recruited (Bajic et al., 2015). Importantly, the circuits mediating the complex effects of cholinergic transmission on opioid analgesia, behavioral sensitization and antinociceptive tolerance remain to be elucidated in detail. Cholinergic neurons in the LDTg/pedunculopontine tegmentum are reported to modulate behavioral (locomotor) sensitization to morphine, but do not affect acute antinociception or antinociceptive tolerance, which likely depend upon recruitment of descending modulatory systems (Bajic et al., 2015). Modulation of spinal opioidergic neurons by Ach has also been described. Furthermore, elevated Ach levels spinally increases the concentration of Lenkephalin, b-endorphin and dynorphin A1-13, while spinal inhibition of muscarinic receptors decreases the concentration of these opioid peptides in rats (Yang et al., 2012). Interestingly, one area that has not received due attention is the nucleus basalis of Meynert, which is the key source of cholinergic inputs to the cortex. Although early studies indicated that lesioning the nucleus basalis of Meynert changes the opioid receptor distribution across key cortical and hippocampal regions, very little is known about how this pathway may contribute to opioid analgesia (Ofri et al., 1992). Overall, pathways and specific circuits mediating opioidergic–cholinergic interaction warrant detailed, causal analysis.

CONCLUSIONS AND OUTLOOK As described above, there is substantial evidence supporting a role for endogenous cholinergic modulation of pain as well as a basis for the therapeutic usage of cholinomimetics in pain relief. Particularly, the use of acetylcholine esterase inhibitors and muscarinic agonists may constitute a highly promising line of therapy to study and pursue in greater detail – accordingly, there is a need for intensifying clinical trials

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in diverse types of chronic pain patients and to particularly test the efficacy of cholinomimetics in augmenting pain relief by conventional analgesics, such as opioids. Opioidergic–cholinergic interactions present both a challenge and opportunity in this regard. They are likely to be of high relevance in improving pain management; however, cross-tolerance and particularly, crossdependence may constitute a problem with respect to nicotinic modulation. Selective pharmacological modulation of muscarinic signaling may represent a better option in this regard. Several avenues in the periphery, spinal cord and brain contribute to these phenomena, but a precise understanding of circuits and mechanisms involved still eludes us. Importantly, delineating the role of endogenous cholinergic modulation versus the impact of exogenous (artificial) activation of cholinergic signaling with pharmacological agents has remained challenging owing to the limited availability of studies that have addressed this question in a systematic and scientifically sound manner. Therefore, although pharmacological studies have contributed very prominently to our current understanding of the topic, there is an urgent need to clarify precise endogenous contributions and elucidate regions and cholinergic pathways that modulate diverse components of pain. The advent of new technologies for reversibly activating and silencing specific pathways, such as optogenetics and chemogenetics, will be particularly gainful in this regard. Several key sources of cholinergic modulation, such as the nucleus basalis of Meynert, remain unexplored. Moreover, it will be important to study how affective, cognitive and sensory aspects of pain are regulated by cholinergic transmission in chronic pain states. Once pathways and their targets have been validated, it will be imperative to address cellular mechanisms of cholinergic modulation in greater detail. In turn, cholinergic pathways may themselves be the target of modulation in chronic pain states; therefore, it will be important to address how cholinergic circuits and function are dynamically changed over the course of pain chronicity. Taken together, these approaches will enable intensifying the clinical relevance and efficacy of cholinergic modulation in chronic pain and help overcome potential side effects of current therapies.

Acknowledgments—The authors are grateful to Rose LeFaucheur for secretarial assistance and to the members of the laboratory and the scientists in the Collaborative Research Center 1158 (SFB1158, Heidelberg Pain Consortium) in Heidelberg/ Mannheim for valuable discussions. The authors acknowledge funding in form of SFB1158 grants from the Deutsche Forschungsgemeinschaft (DFG) to R.K. and European Research Council (ERC) Advanced Investigator grant to R.K. (Pain Plasticity 294293). P.V.N. received support from SFB1158 and a fellowship from the German National Merit Foundation. The authors apologize to those colleagues in the field whose work could not be discussed and cited owing to space restrictions.

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(Received 22 April 2017, Accepted 29 August 2017) (Available online xxxx)

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