New approaches to target glycinergic neurotransmission for the treatment of chronic pain

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New approaches to target glycinergic neurotransmission for the treatment of chronic pain Wendy L. Imlach Discipline of Pharmacology, School of Medical Sciences, Rm. W300, Blackburn D06, The University of Sydney, Sydney NSW 2006, Australia

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Article history: Received 24 August 2016 Received in revised form 13 December 2016 Accepted 13 December 2016 Available online xxx Keywords: Pain Inhibitory neurotransmission Glycine receptors Glycine transporters Dorsal horn

a b s t r a c t Inhibitory glycinergic neurotransmission in the spinal cord dorsal horn plays an important role in regulating nociceptive signalling by inhibiting neuronal excitation. Blocking glycinergic transmission in the dorsal horn causes normally innocuous stimuli to become painful (allodynia) and increases sensitivity to noxious stimuli (hyperalgesia). Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Management of neuropathic pain with current therapeutics is challenging and there is a great need for more effective treatments. Preclinical studies using drugs that increase glycinergic signalling by potentiating glycine receptor activity or inhibiting transporter activity suggest that targeting this system is a good therapeutic strategy. The spatially restricted expression of glycine receptors and transporters is an advantage for targeting specific pathologies such as pain. However, until recently there have been few pharmacological modulators identified and most of which do not specifically target glycinergic signalling. This mini-review provides an overview of recent advances in the development of therapeutics and novel approaches that aim to increase glycinergic neurotransmission for the treatment of persistent pain. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The tuning and regulation of sensory processing in the spinal cord is mediated through fast inhibitory neurotransmission [1]. Both glycine and ␥-aminobutyric acid (GABA) play important roles in limiting excitability in neural circuits in the dorsal horn and when the receptors of these neurotransmitters are inhibited in the spinal cord in vivo, this leads to the symptoms of pathological pain [2]. Studies using rat models of neuropathic pain show that loss of inhibitory GABAergic and glycinergic neurotransmission,

Abbreviations: ALX1393, (O [(2-benzyloxyphenyl-3AEA, anandamide; BN 50739, fluorophenyl)methyl]-l-serine); 4,7,8,10methyl-(chloro-2phenyl)6[dimethoxy3,4 tetrahydro[4 ,3 -4,5]thieno[3,2-f] triazolophenylthio]methylthiocarbonyl-9pyrido 1,2,4[4,3-a] diazepine-1,4; CBD, cannabidiol 2,6-DTBP 2,6-di-tert-butylphenol; eIPSC, evoked inhibitory postsynaptic current; GABA, ␥-amino butyric acid; GABAA R, GABAA receptor; GlyR, glycine receptor; GlyT, glycine transporter; IL-1␤, interleukin-1␤; ORG25543, 4-benzyloxy-3,5-dimethoxyPGE2 , prostaglandin N-[1-(dimethylaminocyclopently)-methyl]benzamide; E2; PAF, platelet-activating factor; siRNA, small interfering RNA; TCV-309, 3-bromo-5-[N-phenyl-N-[2-[[2-1,2,3,4-tetrahydro-2isoquinolylcarbonyloxy) ethyl]carbamoyl]ethyl]carbamoyl]-1-propylpyridinium nitrate); THC, tetrahydrocannabinol; WEB 2086, 3-[4-(2-chlorophenyl)-9-methyl-6H-thieno [3,2-f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2yl]-1-(4-morpholinyl)-1-propanone. E-mail address: [email protected]

or disinhibition, accompanies peripheral nerve-injury [2–5]. The importance of glycinergic neurons in the control of both nociceptive and itch circuits in the dorsal horn was recently demonstrated in studies where these neurons were either ablated or activated in mice, which caused pain and analgesia, respectively [6]. Since glycinergic transmission has such a profound influence on whether nociceptive signals are transmitted from the spinal cord to the brain where the signals are perceived as pain, this system is an attractive target for pain therapeutic development. Inhibitory glycine receptors (GlyRs) are anion-selective transmitter-gated ion channels of the Cys-loop family and are expressed in spinal cord, brainstem, retina and cerebellum. There have been four glycine receptor alpha subtypes identified to date (␣1–4) and one beta subunit, but most receptors in adult tissue are ␣1␤ heteromers in a 2:3 stoichiometry [7]. In the spinal cord dorsal horn, the ␣1␤ and ␣3␤ glycine receptors are highly expressed. Both receptor subtypes are known to play a key role in the regulation of pain signalling in this region, but the ␣3 containing GlyRs are particularly important in inflammatory pain where release of prostaglandin E2 (PGE2 ) activates EP receptors which leads to phosphorylation of the receptor and a decrease in signalling [8]. Therefore, therapeutics that potentiate the function of GlyR ␣1␤ and ␣3␤ receptors and reverse the loss of inhibition in pain states, have potential as analgesics. It has been recently

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shown that the GlyR␣2 subunit is expressed in the dorsal horn following the development of neuropathic pain [9]. This receptor is expressed embryonically and is important during development, but its expression declines rapidly postnatally [10]. This may be a response to injury where gene expression reverts to early or embryonic patterns in pathological states, and may provide a novel therapeutic target for pain treatment. Another strategy to increase glycinergic signalling in the dorsal horn is to increase the available synaptic glycine by decreasing re-uptake by the glycine transporter 2 (GlyT2), which is highly expressed on glycinergic neurons in the dorsal horn. Although we are becoming increasingly aware of the importance of glycinergic transmission in the symptoms and maintenance of chronic pain, there are few pharmacological modulators of glycinergic transmission that have been identified and most are not selective for glycine receptors. The spatially restricted expression of glycine receptors and transporters is an advantage for targeting specific pathologies, such as pain, that are caused by excessive neuronal excitability. This review focuses on where the field is currently at in the search for new drugs to target inhibitory glycinergic neurotransmission. This includes approaches to increase glycinergic transmission in spinal pain circuits with agonists and positive modulators of glycine receptors (GlyR␣1 and GlyR␣3), through inhibition of glycine transporters, gene therapy and through inhibition of inflammatory mediated pathways(Fig. 1). 2. High throughput screens for GlyR modulators that potentiate activity 2.1. Natural marine extracts The lack of specific glycine receptor modulators has led to investigators performing large scale screens using drug libraries and natural compounds in search of novel modulators. One of these screens was performed by a group in Queensland, Australia, who screened an extract library of more than 2500 southern Australian and Antarctic marine invertebrates and algae for glycine receptor modulators. This included a screen by Balansa and colleagues [11] on compounds from Irciniidae sponges, where a fluorescencebased anion influx assay using recombinantly expressed ␣1 and ␣3 GlyRs was performed, followed by patch-clamp electrophysiology of positive modulators. This led to the identification of subunit selective glycine receptor modulators including four GlyR ␣1 potentiators that increased currents with a maximal range of between 140% and 330%, two of which inhibited GlyR␣3 and one potentiated GlyR␣3 currents by 183%. The active compounds identified in this screen are a rare class of glycinyl lactam sesterterpenes [11,12]. Since these compounds potentiate the activity of GLyR␣1 and ␣3 they provide novel tools to probe GlyR function and may be useful for treating pain, or lead to the development of derivatives that are analgesic in the future. 2.2. Small molecule library screen for positive allosteric modulators In addition to the orthosteric glycine binding sites of GlyRs, there are allosteric sites that can modulate the activity of the receptor in a positive or negative way [13]. Recently, Stead et al. [14] screened a small molecule compound library consisting of compounds that have putative ion channel activity (56,558 compounds) and identified the first positive allosteric modulator (PAM) that has selectivity for the glycine receptor. Through this screen, they developed a suite of in vitro assays that screened compounds for activity on ␣3GlyR and ␣3␤-GlyRs which included high-throughput fluorescent membrane potential screens and medium-throughput automated

electrophysiology. Seven compounds of interest were further characterized using conventional manual patch clamp which gave maximum potentiation of ␣3-GlyR activity that ranged from 136% to 2710%. Of these, the most promising screening hit, 2,4-fluoroN-(2-(quinolin-8-yloxy)ethyl)benzenesulfonamide, did not show activity at the structurally related GABAA receptor and also lacked potency at the ␣1-GlyR. Although the selectivity profile of this compound requires more thorough investigation, there appears to be a lack of promiscuity as it had never shown activity in previous Pfizer screens and binding assays. This compound shows promise as a pharmacological tool and may be an effective way of increasing glycinergic activity without affecting GABAergic transmission. 3. Antinociceptive activity and mechanism of gelsemine Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants. Extracts have been used for many years to treat pain conditions including trigeminal neuralgia and migraine [15]. Gelsemine has been shown to reduce mechanical allodynia and thermal latency in a mouse neuropathic pain model, with no effect on motor performance [16]. Zhang et al. [17] showed that intrathecal administered gelsemine is effective at producing specific and potent antinociception in formalin-induced tonic pain, bone cancer-induced mechanical allodynia and nerve-injury induced painful neuropathy. In the bone-cancer model, multiple daily injections over a 7 day period did not produce tolerance to antinociceptive effects. Antinociception was blocked by strychnine in neuropathic and formalin-induced pain, which suggests activity is mediated through GlyR. Silencing GlyR␣3, but not GlyR␣1, with intrathecal small interfering RNA (siRNA) prevented gelsemineinduced antinociception in neuropathic pain, suggesting that the effect is mediated via the GlyR␣3 receptor – which is preferentially affected in inflammatory pain states [8]. A recent study by Lara et al. [18] shows that gelsemine has subunit specific effects on different GlyR subtypes and modulates spinal glycinergic synapses. In cultured cells expressing GlyRs, they found that this alkaloid changes the apparent affinity and open probability of the ion channel in a voltage-independent fashion. The reported effects on receptor subtypes were interesting as homomeric glycine receptors consisting of ␣2 or ␣3 subunits were inhibited by gelsemine, as were ␣1/␤ heteromeric receptors, but homomeric ␣1 receptors were potentiated. Since synaptic GlyRs are likely to be heteromeric [19], these results would suggest that gelsemine decreases glycinergic neurotransmission, which should increase spinal excitability. They show in spinal cord neurons that native GlyRs were more sensitive to gelsemine modulation than GABAA and AMPA receptors. Interestingly, they show gelsemine decreases spontaneous glycine release and glycine-evoked currents in spinal neurons, which may be expected to increase dorsal horn excitability leading to increased nociception. The authors speculate that the analgesic effects of this drug are due to the drug specifically targeting unidentified presynaptic glycine receptors, where they are likely to decrease the frequency of glutamatergic release. It has also been suggested that gelsemine may produce antinociception by activating the spinal ␣3 glycine/allopregnanolone pathway [20]. More investigation is required to explain the antinociceptive effects of gelsemine. 4. Targeting phosphorylated GlyR␣3 to increase signalling In chronic inflammatory pain, release of prostaglandin E2 (PGE2 ) activates EP receptors, which induces protein kinase A (PKA)-mediated phosphorylation of GlyR␣3 and in turn reduces glycinergic signalling mediated through this receptor [21]. Since PGE2 triggers central sensitization through inhibition of GlyR␣3 activation, targeting the EP receptor with an antagonist may be

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Fig. 1. Summary of new pharmacological approaches to increase glycinergic signalling for the treatment of pain. Nociceptive signalling can be reduced by increasing glycinergic inhibitory activity in the spinal cord dorsal horn through increased activation of glycine receptors and inhibition of the glycine transporter 2 (GlyT2). The section in this review that describes each approach is indicated by the number in brackets.

a good therapeutic strategy to prevent GlyR␣3 phosphorylation ˜ et al. [22] found that the and inhibition. In a recent study, Acuna propofol derivative 2,6-di-tert-butylphenol (2,6-DTBP) specifically acts on ␣␤GlyRs to reverse inflammation-mediated disinhibition. In spinal cord tissue, 2,6-DTBP increased synaptic glycine decay time, but only after the tissue had been primed with PGE2. Administration of this compound to a murine model of inflammatory pain reduced hyperalgesia in a dose-dependent manner, which suggests that potentiation of this receptor is a promising therapeutic approach for treating inflammatory pain conditions. Earlier studies using antagonists of the EP receptor have shown efficacy in models of inflammatory pain, for example GSK345, which reduces complete Freund’s adjuvant (CFA) induced joint pain in rat [23,24].

2086 have anti-allodynia effects in neuropathic pain. The potent and long-lasting reduction in allodynia in a mouse pain model when administered by intravenous or oral administration routes, was reduced by intrathecal administration of GlyR␣3 siRNA. This suggests that PAF antagonists induce analgesia by preventing PAF from decreasing signalling through glycine receptors containing ␣3 subunits. PAF antagonists are promising therapeutics for the treatment of bone cancer pain, which causes severe levels of pain which is resistant to current analgesics. A study by Morita and colleagues [27] shows a combination of opioids and PAF antagonists in a mouse model of bone cancer pain supressed pain behaviours and prolonged survival. 6. Modulation of GlyR activity by cannabinoids

5. Increasing GlyR␣3 activity with platelet-activating factor antagonists Platelet-activating factor (PAF) is known to play a role in the pathology of neuropathic pain and intrathecal administration of PAF induces allodynia. It has been suggested that PAF-induced tactile allodynia is mediated through the glutamate–NO–cGMP–PKG pathway in spinal cord neurons [25]. When the NO/cyclic GMP cascade is increased, GlyR␣3 function is reduced, which results in allodynia and hyperalgesia [25]. A recent study by Motoyama et al. [26], shows that the PAF antagonists, TCV-309, BN 50739 and WEB

Endocannabinoids were first reported to modulate GlyR in hippocampal neurons, where they were shown to retrogradely affect current amplitude and kinetics when released from postsynaptic neurons [28]. It was found that these cannabinoid-related compounds increased glycine activity by causing a leftward shift in the glycine concentration response curve, increasing sensitivity to the neurotransmitter. This activity is independent of cannabinoid receptor activation. Recently, a study by Xiong et al. [29] showed the GlyR␣3 knock out mice had reduced analgesia from (9)-tetrahydrocannabinol

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(THC), which was the first evidence that the effect of cannabinoids in vivo is mediated through glycine receptors. This study also identified a physical site for the THC-induced potentiating effect on glycine currents through functional mutagenesis of the glycine receptor. In another study, it was shown that sustained incubation with the endocannabinoid anandamide (AEA) increased glycine current amplitudes in cultured spinal neurons and HEK cells expressing ␣1 and ␣3 subunits [30]. Xiong et al. [31] showed that systemic and intrathecal administration of cannabidiol (CBD) is able to suppress symptoms of chronic inflammatory and neuropathic pain in rodents without causing tolerance. Dehydroxyl-CBD was also found to rescue PGE2 -induced inhibition of ␣3 GlyR function which also suggests that cannabinoid analgesia is mediated through the ␣3 GlyR-dependent pathway. One issue with cannabinoid-related molecules is their lack of specificity for the GlyR. Almost of these compounds interfere with the function of other ion channels in addition to their activity on the cannabinoid receptors. However, these molecules may be interesting lead compounds for the development of glycine receptor modulators. 7. Targeting interleukin-1 beta induced potentiation of GlyR on GABAergic neurons It was recently reported that long-term potentiation (LTP) occurs at glycinergic synapses on inhibitory GABAergic neurons in the dorsal horn following exposure to the inflammatory cytokine interleukin-1 beta (IL-1␤). This LTP is triggered by peripheral inflammation in vivo and is thought to reduce GABAergic signalling. This potentiation of GlyR activity results from an increase in the number of GlyR and/or a change in the biophysical properties of the receptor [32]. The proinflammatory cytokine IL-1␤ is a potent hyperalgesic agent, and following nerve-injury or inflammation, its levels are increased in the spinal cord [33]. Intrathecal delivery of an IL-1␤ antagonist was shown to block the development of allodynia in a rat model of inflammatory pain [34]. This suggests that inhibiting IL-1␤ in the spinal cord would be an effective therapeutic strategy for treating pain through a GlyR mediated mechanism. A recent study using hecogenin acetate, a steroidal sapogenin from the Agave plant that reduces IL-1␤, caused amelioration of mechanical hyperalgesia in mice [35]. Another recent study describes the reduction of mechanical allodynia in a rat nerve-injury model following administration of iridoid glycosides from the L. rotata plant, which corresponds to a decrease in IL-1␤ in treated animals [36]. Although these plant derived compounds are not directly acting on the GlyR, they do suggest that inhibiting the activity of IL-1␤ on glycine receptors may be an effective way of reducing pain signalling. A more specific strategy to target this pathway may be through direct inhibition the IL-1␤ site on the glycine receptor, rather than broadly blocking inflammation. 8. Potentiation of GlyR by ginkgolides and bilobalide derivatives Although ginkgolides (bioactive compounds from Ginkgo biloba) have been found to inhibit GlyR activity [7], Maleeva et al. [37] found that nanomolar concentrations of ginkgolic acid strongly potentiates the activity of ␣1 GlyRs expressed in cultured cells. This effect was specific to ␣1 GlyRs, with no effect observed on ␣3 GlyRs or GABAA Rs composed of ␣1/␤1/␥2 subunits and only a small inhibition of ␣2 GlyRs at much higher doses. Another bioactive Ginkgo biloba compound, bilobalide, has also been shown to inhibit GlyRs. However a derivative of this natural compound, NV-31, which was synthesized to increase stability, was found to potentiate GlyR activity. Similarly to ginkgolic acid, potentiation was most effective

at ␣1 GlyRs, with less activity at the ␣1/␤ GlyRs and no effect on the ␣2 and ␣3 glycine receptors [38]. Although these compounds may have activity on other receptors and ion channels, their efficacy at the ␣1 GlyR suggests they are specific enhancers of these receptors and therefore may be a good lead for pain therapeutics. 9. RNA aptamers as positive modulators of glycine receptors RNA aptamers are single-stranded RNA or DNA oligonucleotides, usually consisting of between 50 and 100 nucleotides. These molecules are able to fold into three-dimensional structures and bind to identified targets with a complementary shape with a similar affinity to monoclonal antibodies. Shalaly et al. [39] generated five RNA aptamers to the human GlyR␣1 subunit that acted as positive modulators of glycine-activated chloride channels. These oligonucleotides were screened using fluorescence assays that detected changes in membrane potential and positive hits were tested using electrophysiological techniques to confirm the aptamer potentiated chloride currents. Although this approach to targeting glycine receptors is very new, this technology has been applied to other systems and in disease treatment where aptamers are used clinically, as reviewed in Yu et al. [40]. Since these modulators can be screened for specificity they are likely to be valuable tools for the development of assays for the identification of small molecular agonists and positive modulators, which may eventually lead to therapeutic development. 10. Potentiation of GlyR activity by tropeines Tropeines are 5-HT3 receptor antagonists that are also known to allosterically modulate glycine receptors. These drugs are used clinically to treat pain and as anti-emetics due to their ability to target 5-HT3 receptors. These drugs bind with high affinity to the GlyRs and have potent effects on activity, which make them promising candidates as therapeutics to treat pain. Tropisetron is the most characterized compound of the tropeines and it potentiates GlyR chloride currents at nanomolar concentrations [41]. This compound has been shown to potentiate GlyR␣1 currents at concentrations in the femtomolar concentration range, but higher (micromolar) concentrations cause inhibition [42,43]. 11. Gene therapy to increase GlyR1 expression Another approach for treating persistent pain through glycine receptor activation that may be complementary to pharmaceutical therapeutics is gene therapy. A number of spinal cord targets for gene therapy have been identified and tested in pre-clinical models of pain, including potassium channels, receptors and cytokines [44]. An advantage of these therapies are that they allow targeting of specific mechanisms in tissues of interest. To overcome the lack of temporal control (analgesia onset and duration) in these methods, Goss et al. [45] designed a therapeutic approach where human GlyR␣1 is expressed in sensory afferents, where they are not normally present, and activated by application of exogenous glycine. A herpes simplex viral (HSV) based vector was chosen because they are able to inject primary sensory neurons when inoculated at peripheral sites and from there are efficiently transported to the nucleus. In this study the exogenous glycine was administered through subcutaneous injection in the plantar surface of the foot, which is not ideal considering that the animal would be suffering from hyperalgesia in this region. However, if the method was developed to use less invasive administration, such as transdermal delivery through patches or creams, then this could be an interesting approach to controlling pain.

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12. Inhibition of the glycine transporter 2 (GlyT2) to increase synaptic glycine

13. Future potential of therapeutics targeting spinal glycine transmission

Extracellular glycine concentrations at both inhibitory and excitatory synapses are regulated by the glycine transporters 1 and 2 (GlyT1 and GlyT2) which are part of the sodium-dependent solute carrier family 6 (SLCC6) transporters. GlyT1 is expressed by astrocytes at inhibitory and excitatory synapses, while GlyT2 expression is restricted to presynaptic terminals of inhibitory glycinergic neurons, where they regulate synaptic glycine levels and recycle glycine back to the presynaptic terminals to maintain high glycine concentrations [46]. Inhibitors of both transporters produce antinociceptive effects in neuropathic and inflammatory pain models [47] and both are expressed in the dorsal horn. GlyT2 is highly expressed in lamina III and is a more powerful transporter than GlyT1 as it is able to reduce synaptic glycine to the low nanomolar range, preventing low levels of GlyR activity [48]. The ability of GlyT2 to finely control low level synaptic glycine concentrations, suggests that inhibition of GlyT2 would enhance basal inhibitory neurotransmission by reducing the clearance of glycine, which may be beneficial for the treatment of neuropathic pain [49]. However, studies using GlyT2 knockout mice have shown that complete inhibition of GlyT2 has a detrimental effect on neuromuscular function and these mice develop severe motor deficiencies during the second postnatal week [50]. This is due a greatly diminished inhibitory glycine transmission due to the loss of intracellular glycine that is required for loading of synaptic vesicles, preventing synaptic glycine release. Further studies using partial knockdown of the GlyT2 gene with siRNAs showed that a reduction in GlyR2 activity corresponds with a reduction in allodynia in a mouse model of neuropathic pain [51]. Furthermore, these partial GlyT2 knockdown animals had no adverse motor or respiratory effects, which suggests that partial GlyT2 inhibition has therapeutic potential. This study also demonstrated that two GlyT2 inhibitors, ALX1393 and ORG25543, have antiallodynia effects when administered intravenously in the mouse neuropathic pain model [51]. A more recent study by Mingorance-Le Meur et al. [52] found that ALX1393 also inhibits GlyT1 at higher concentrations. This may limit the therapeutic potential of ALX1393, as inhibition of GlyT1 activity increases glycine at NMDA receptors, where it acts as an excitatory neurotransmitter, which may exacerbate pain symptoms by increasing spinal sensitization. The GlyT2 inhibitor ORG25543 has similar antinociceptive effects to ALX1393 in animal models of pain and it has shown to increase glycinergic tonic currents and eIPSC decay time in spinal cord slices [53]. However, after ten minutes of GlyT2 inhibition by ORG25543 in slices, glycinergic eIPSCs were greatly reduced, suggesting that the presynaptic glycine vesicle recycling is reduced to a level that does not support glycinergic synaptic signalling. This is likely to be due to the irreversible inhibition of GlyT2 by ORG25543 and is similar to what is observed in GlyT2 knockout mice, which has motor phenotypes that would not be acceptable as therapeutic side effects [50]. In a study by Wiles et al. [54], the endocannabinoid N-arachydonoyl-glycine (NAGly) was shown to selectively and reversibly inhibit GlyT2 over GlyT1 and GAT1. NAGly has reduces pain in inflammatory and neuropathic models of pain in animals through a mechanism that is independent of cannabinoid receptor activation [55,56]. Recently a number of lipid inhibitors such as Oleoyl-l-carnitine and Noleoyl-glycine (NOGly), which are based on the endogenous GlyT2 inhibitor NAGly, have been synthesized that may provide more specific and irreversible inhibition of GlyT2 [57]. This new generation of lipid inhibitors may lead to more effective therapeutics for targeting glycine transport to treat pathological pain.

In recent years the role of spinal glycinergic inhibitory neurotransmission in nociception has been a focus of attention in the field of pain research. Although we are aware of the importance of glycinergic signalling in normal and pathological states, there are few pharmacological modulators available that target this system for use as research tools, or for future therapeutic development. As described in this review, there are an increasing number of ways to target glycinergic signalling in the spinal cord, including targeting orthosteric and allosteric sites on receptors, transporters, and inflammatory mediators. The therapeutic approaches that are being discovered are also diverse, from designer oligonucleotides, natural products, bioactive lipids to gene therapy. Targeting glycinergic transmission in the spinal cord is a potentially lucrative strategy for analgesic drug discovery and the search for novel pharmacological modulators that target glycinergic signalling is still in its infancy, but this mini-review provides an overview of recent research of strategies and drugs that may increase glycinergic signalling in pain circuits – which is the first step in the development of pain therapeutics in the future. Conflict of interest None. Funding This work was supported by a Rolf Edgar Lake Research Fellowship. References [1] H.U. Zeilhofer, H. Wildner, G.E. Yévenes, Fast synaptic inhibition in spinal sensory processing and pain control, Physiol. Rev. 92 (January (1)) (2012) 193–235, http://dx.doi.org/10.1152/physrev.00043.2010, Review. PubMed PMID: 22298656; PubMed Central PMCID: PMC3590010. [2] T.L. Yaksh, Behavioral and autonomic correlates of the tactile evoked allodynia produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists, Pain 37 (1989) 111–112. [3] S.E. Sherman, C.W. Loomis, Strychnine-dependent allodynia in the urethane-anesthetized rat is segmentally distributed and prevented by intrathecal glycine and betaine, Can. J. Physiol. Pharmacol. 73 (1995) 1698–1705. [4] S.E. Sherman, C.W. Loomis, Strychnine-sensitive modulation is selective for non-noxious somatosensory input in the spinal cord of the rat, Pain 66 (1996) 321–330. [5] L. Sivilotti, C.J. Woolf, The contribution of GABAA and glycine receptors to central sensitization: disinhibition and touch-evoked allodynia in the spinal cord, J. Neurophysiol. 72 (1994) 169–179. [6] E. Foster, H. Wildner, L. Tudeau, S. Haueter, W.T. Ralvenius, M. Jegen, H. Johannssen, L. Hösli, K. Haenraets, A. Ghanem, K.K. Conzelmann, M. Bösl, H.U. Zeilhofer, Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch, Neuron 85 (March (6)) (2015) 1289–1304, http://dx.doi.org/10.1016/j.neuron. 2015.02.028, PubMed PMID: 25789756; PubMed Central PMCID: PMC4372258. [7] T.I. Webb, J.W. Lynch, Molecular pharmacology of the glycine receptor chloride channel, Curr. Pharm. Des. 13 (23) (2007) 2350–2367, Review. PubMed PMID: 17692006. [8] V.L. Harvey, A. Caley, U.C. Müller, R.J. Harvey, A.H. Dickenson, A selective role for alpha3 subunit glycine receptors in inflammatory pain, Front. Mol. Neurosci. 2 (November (14)) (2009), http://dx.doi.org/10.3389/neuro.02.014. 2009, eCollection 2009. PubMed PMID: 19915732; PubMed Central PMCID: PMC2776487. [9] W.L. Imlach, R.F. Bhola, S.A. Mohammadi, M.J. Christie, Glycinergic dysfunction in a subpopulation of dorsal horn interneurons in a rat model of neuropathic pain, Sci. Rep. 6 (2016) 37104, http://dx.doi.org/10.1038/ srep37104, PMID:27841371. [10] M.L. Malosio, B. Marquèze-Pouey, J. Kuhse, H. Betz, Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain, EMBO J. 10 (September (9)) (1991) 2401–2409, PubMed PMID: 1651228; PubMed Central PMCID: PMC452935.

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