JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 JPET ThisFast article Forward. has not been Published copyedited andon formatted.April 30, The 2008 final versionas DOI:10.1124/jpet.108.136267 may differ from this version.

JPET #136267

Spinal Anti-Allodynia Action of Transporter Inhibitors in

Neuropathic Pain Models in Mice Downloaded from

Katsuya Morita, Naoyo Motoyama, Tomoya Kitayama, Norimitsu Morioka, Koki jpet.aspetjournals.org

Kifune, and Toshihiro Dohi at ASPET Journals on September 26, 2021

Department of , Division of Integrated Medical Science, Hiroshima

University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku,

Hiroshima 734-8553, Japan (KM, NM, TK, NM, KK, TD)

1

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version.

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Running title: Anti-Allodynia and Glycine Transporter Inhibitors

Address correspondence to: Professor Toshihiro Dohi, Ph.D., Department of

Pharmacology, Division of Integrated Medical Science, Hiroshima University Graduate School of

Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan. E-mail Downloaded from address: [email protected]

Tel.: +81-82-257-5640; fax: +81-82-257-5644. jpet.aspetjournals.org

ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; ALX 1393, at ASPET Journals on September 26, 2021

(O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine); BDNF, brain-derived neurotrophic factor; CFA, complete Freund's adjuvant; DCK, 5,7-dichlorokynurenic acid (5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid); GlyR, glycine ; GlyT, glycine transporter; HVJ, hemagglutinating virus of the Japan; i.t., intrathecal; i.v., intravenous; L-701,324, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone;

NMDA, N-methyl-D-aspartate; ORG25543,

4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide;

ORG25935, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid; SDS, sodium dodecylsulfate; siRNA, small

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interfering RNA; STZ, streptozotocin. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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The number of text pages is 55 (figures not included).

The number of tables is 2.

The number of figures is 6.

The number of words

Abstract: 242 words Downloaded from

Introduction: 755 words

Discussion: 1708 words jpet.aspetjournals.org

The number of references: 50

Section: Behavioral Pharmacology at ASPET Journals on September 26, 2021

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ABSTRACT

Neuropathic pain is refractory against conventional analgesics and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the

regulation of pain signal transduction. Glycine transporter (GlyT) 1, present in glial Downloaded from cells, and GlyT2, located in neurons, play roles in modulating glycinergic

neurotransmission by clearing synaptically released glycine or supplying glycine to the jpet.aspetjournals.org neurons and thus could modify pain signal transmission in the spinal cord. Here we demonstrated that intravenous or intrathecal administration of GlyT1 inhibitors, ORG at ASPET Journals on September 26, 2021

25935 or , and GlyT2 inhibitors, ORG 25543 and ALX 1393, or knockdown of spinal GlyTs by siRNA of GlyTs mRNA produced a profound anti-allodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The anti-allodynia effect is mediated through spinal α3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. These manipulations to stimulate glycinergic neuronal activity, however, were without effect during the 4 days post-nerve injury, while manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of with their inhibitors should

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be considered, because glycinergic control of pain was reversed in the critical period of

3 to 4 days post-surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Downloaded from

jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Introduction

Neuropathic pain arising from peripheral or spinal nerve injury, diabetes, or infection with herpes virus is a result of the final product of an altered peripheral, spinal, and supraspinal process for which the usual analgesics are not effective and novel

analgesics are desired. Recent progress of research on the underlying mechanism of the Downloaded from pathology revealed more complexity depending on the cause and stage of ongoing

neuropathy. Among various mechanisms involved in the pathology, alterations of jpet.aspetjournals.org synaptic transmission within the spinal cord dorsal horn as well as peripheral nerves following peripheral nerve injury play key roles. In addition to the activation of at ASPET Journals on September 26, 2021 stimulatory spinal neurotransmission, disinhibition of inhibitory neurotransmission has been implicated in the generation of inflammatory and neuropathic pain (Woolf and

Mannion, 1999). Glycine as well as GABA serves as a major inhibitory neurotransmitter in the spinal cord of vertebrates. Actually, relief from glycinergic inhibition by an inhibitor of glycine release (Ito et al., 2001) or blockers of glycine receptors (Sivilotti and Woolf, 1994; Sherman et al., 1997; Loomis et al., 2001; Gomeza et al., 2003a) in the dorsal horn can elicit tactile allodynia, a major symptom of neuropathic pain. Recent studies suggested that reduction of the chloride gradient across the neuronal membrane, which in turn leads to reduction of anion reversal potential,

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occurred in neurons of lamina I of the superficial dorsal horn following peripheral nerve injury (Coull et al., 2003). The change in the driving force means that both glycine and

GABAA receptor-mediated inputs produce less hyperpolarization and could even paradoxically depolarize the neuron (Coull et al., 2003, 2005; Prescott et al., 2006). This

may be one of likely mechanism which contributes to the production of allodynia and Downloaded from hyperalgesia associated with neuropathic pain. In contrast, the enhancement of the

activity of glycinergic neurons exerts an anti-allodynia in a nerve injury model (Abdin jpet.aspetjournals.org et al., 2006). The evidence suggests the importance of glycinergic control of spinal pain processing. at ASPET Journals on September 26, 2021

Extracellular concentrations of glycine at glycinergic nerve terminals are regulated by Na+/Cl--dependent glycine transporters (GlyTs). GlyT1 is widely expressed in glial cells. The evidence suggests an essential role of GlyT1 in lowering extracellular glycine concentration at glycinergic synaptic cleft (Jursky and Nelson, 1996; Roux and

Supplisson, 2000). The inhibition of GlyT1 and enhancement of -sensitive glycine receptors would compromise the ability of GlyT1 inhibitors to be beneficial for pain control (Armer, 2000; Aragón and López-Corcuera, 2003). On the other hand,

GlyT1 prevents saturation of the glycine binding site on N-methyl-D-aspartate (NMDA) receptors and reduces the activity of excitatory synapses. The inhibition of this role of

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GlyT1 would counteract the ability of GlyT1 inhibitor to be beneficial for pain control.

GlyT2 is largely localized to the presynaptic terminals of glycinergic neurons in the spinal cord as well as in the brain stem and cerebellum (Zafra et al., 1995a, 1995b).

GlyT2 is present only in glycinergic neurons in association with postsynaptic glycine

receptors; therefore, there is a concept that GlyT2 mediates the clearance of Downloaded from postsynaptically released glycine at inhibitory synaptic cleft (Brasnjo and Otis, 2003). It

is expected that GlyT2 inhibitors would be useful in the treatment of pain by enhancing jpet.aspetjournals.org glycine receptor systems (Armer, 2000; Aragón and López-Corcuera, 2003). However, several studies have shown that inhibition of GlyT2 by drugs or GlyT2-deficient mice at ASPET Journals on September 26, 2021 did not significantly prolong or rather reduce the decay kinetics of glycinergic IPSCs in hypoglossal motoneurons (Oku et al., 1999; Singer et al., 1998; Gomeza et al., 2003b).

This may be explained by inefficient refilling of the synaptic vesicle with glycine by loss of GlyT2 function, resulting in decreased release of glycine. The evidence suggests that GlyT2 is essential for glycine uptake into the presynaptic cytosol for refilling synaptic vesicles with glycine rather than for clearing glycine at glycinergic synaptic cleft (Eulenburg et al., 2005); therefore, whether GlyT2 inhibitors might be beneficial or detrimental to pain control is not clear.

Recently, several specific inhibitors of GlyTs have been developed. ORG 25935 is a

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highly selective inhibitor of GlyT1 (IC50=100 nM) (Walker et al., 2001). Sarcosine competitively inhibits GlyT1 (Ki=30-60 µM), but not GlyT2 (Ki >1,000 µM)

(López-Corcuera et al., 1998; Palacín et al., 1998). ORG 25543 is a highly selective

inhibitor of GlyT2 (pIC50=7.8; IC50=16 nM) with negligible affinity for GlyT1 Downloaded from (pIC50<4; IC50>100 µM) and for the strychnine-sensitive glycine receptor (Caulfield et al., 2001). ALX 1393 is an inhibitor of GlyT2 (Ki < 1 µM) (Xu et al., 2005; Luccini and

Raiteri, 2007). The present study was undertaken to examine whether GlyT inhibitors jpet.aspetjournals.org are effective to regulate nociceptive responses in neuropathic pain models. at ASPET Journals on September 26, 2021

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Methods

Animal models. Experiments were performed on adult male ddY mice that were 5 weeks old and weighed 25-30 g at the beginning of the study. Mice were housed at 22 ±

2°C with free access to commercial food and tap water. All experimental procedures and

animal handling were performed according to both the Guiding Principles for the Care Downloaded from and Use of Laboratory Animals approved by the Japanese Pharmacological Society and

the guidelines of Hiroshima University, Hiroshima, Japan. Mice were anesthetised with jpet.aspetjournals.org (60 mg/kg, i.p.). Partial ligation of the sciatic nerve was performed by tying the distal 1/3 to 1/2 of the sciatic nerve, according to the procedure described in at ASPET Journals on September 26, 2021 rats by Seltzer et al. (1990) and adapted to mice by Malmberg and Basbaum (1998).

Briefly, the sciatic nerve was exposed at high thigh level through a small incision and was carefully cleared of surrounding connective tissue just distal to the bony prominence of the femur. An 8-0 silk suture was inserted into the nerve with a 3/8 curved, reversed-cutting mini-needle and tightly ligated so that the dorsal one-third to one-half of the nerve thickness was held within the ligature and the wound was closed. In sham-operated mice, the nerve was exposed using the same procedure, but without ligation. Diabetic mice were produced by an injection of streptozotocin (STZ) (200 mg/kg body weight) dissolved in 0.05 M sodium citrate buffer (pH 4.6) into the tail vein.

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The same volume of citrate buffer-injected age-matched mice was employed as a non-diabetic control group. One or 2 days after the injection of STZ, diabetes was confirmed by measuring urinary glucose with a Test-tape A (Shionogi Pharmaceutical,

Osaka, Japan), and by measuring glucose concentration in a blood sample obtained from

the tail vein with a GLUTEST E (Sanwa Chemical). Mice with blood glucose levels Downloaded from above 400 mg/dl were used as diabetic mice. The mechanical withdrawal threshold

began to decrease significantly 3 days after STZ injection. The threshold of control and jpet.aspetjournals.org diabetic mice at 10 days after injection was 0.733 ± 0.061 and 0.009 ± 0.001 gm (p<

0.0001), respectively. Allodynia presenting in diabetic mice persisted for 14 days after at ASPET Journals on September 26, 2021 injection. The experiments were carried out 10 days after the injection of STZ or vehicle.

For the preparation of complete Freund's adjuvant (CFA) model in mice, mice

(n = 6-10/group) were lightly anesthetized with ether and received 20 µl of CFA (1 mg/ml of heat-killed Mycobacterium tuberculosis in 85% paraffin oil and 15% mannide monooleate) subcutaneously in the intraplantar surface of the right hindpaw. The control groups received 20 µl of mineral oil in the ipsilateral paw. The dose of CFA produced a significant increase in paw volume and allodynia development (Ferreira et al., 2001;

Kassuya et al., 2003). CFA was injected 2 or 4 days before behavioral testing for allodynia as described above.

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Measurement of touch-evoked tactile allodynia. Tactile allodynia was assessed by lightly stroking the injured leg with a paintbrush. Tactile allodynia response was ranked as described by Minami et al. (1995): 0, no response; 1, mild squeaking with attempts to move away from the stroking probe; 2, vigorous squeaking, biting the stroking probe

and strong efforts to escape from the stroking probe. Values are the average of the total Downloaded from score evaluated 3 times at each time point (possible maximum score at each time point:

2/mouse). In some experiments, tactile allodynia was evaluated by measuring the paw jpet.aspetjournals.org withdrawal threshold in response to probing with a series of calibrated fine filaments.

Mice were further acclimated in individual clear Plexiglas boxes (9×7×11 cm) on an at ASPET Journals on September 26, 2021 elevated wire mesh platform to allow access to the planter surface of the injured hind paws. The von Frey filament was pressed perpendicularly against the mid-planter surface of the hind paw from below the mesh floor and held for 3–5 sec with it slightly buckled. The smallest filament that caused the animal to flinch or move the paw away from the stimulus five times out of five trials at intervals of 5 sec was determined to be the mechanical threshold. The filament number was converted to buckling force (gm).

Data analysis was conducted using analysis of variance and appropriate post-hoc comparisons (P< 0.05). For assessment of allodynia, injection and observation of behavior were performed by different persons with blind. Intrathecal injection was

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performed as described previously (Morita et al., 2004).

Behavioral tests. Spontaneous behaviors were assessed by posture, gait, activity of spontaneous movement and defection. Locomotor activity in a novel environment was measured using a computerized locomotion detection system equipped with

photosensors (SCANET MV-10MT; MATYS Co., Tokyo, Japan). Raising the glycine Downloaded from levels at spinal synapses in motor neurons should reduce spontaneous motor activity, as

demonstrated in GlyT1-deficient mice (Gomeza et al., 2003a). To test motor jpet.aspetjournals.org coordination and equilibrium, animals were placed on the rotating rod of the rotarod apparatus (Natume KN-75 RotaRod, Tokyo, Japan) on three successive days. For the at ASPET Journals on September 26, 2021 rotarod assay, mice were allowed a 30-min acclimation period in the testing room. Two days after the mice were habituated to the rotarod, they were evaluated by the rotarod, in which the rotational speed of the rod was kept constant at 8 rpm. Each session consisted of three 5 min trials a day, and each trial was separated by 30 min. The latency

(sec) to slip off the rod was automatically measured in each trial (maximal trial duration: 300 sec). The average performance in three trials was used in data analysis.

On the day of the experiment, the mice were given an intrathecal (i.t.) or intravenous

(i.v.) injection of GlyTs inhibitors (indicated doses) or artificial cerebrospinal fluid

(ACSF)/saline (control group). After 3 hrs, the mice were placed on the rotating rod.

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The latency to fall from the rod was recorded. The test was repeated three times at the

30-min intervals. Dysfunction of GlyT2 is crucial for insufficient transmitter loading of synaptic vesicles in glycinergic nerve terminals. Hyperekplexia is a human neurological disorder characterized by exaggerated startle reflexes and muscle stiffness in the

neonate and is typically caused by mutations in the gene encoding GlyRα1. Recently, Downloaded from mutations have been found in the gene encoding GlyT2 (SLC6A5) resulting in defective

GlyT2 function of hyperekplexia patients (Rees et al., 2006; Eulenburg et al., 2006). jpet.aspetjournals.org

Hindfeet clenching is associated with dominant hyperekplexia (Becker et al., 2002).

Qualitatively, motor deficiencies in animals were recognized by handling. Display of at ASPET Journals on September 26, 2021 hind feet clenching or limb clenching when picked up by the tail was determined.

Righting time was determined after bringing the animals into a supine position as described previously (Becker et al., 2000; Hartenstein et al., 1996).

Knockdown of GlyT1, GlyT2 and GlyRα3 in the spinal cord. Knockdown of

GlyRα3 was performed as described previously (Morita et al., in press). The hemagglutinating virus of the Japan (HVJ) envelope vector system (HVJ Envelope

Vector Kit GenomONE; Ishihara Sangyo Kaisha, Ltd., Osaka, Japan) was used for in vivo siRNA transfer. This HVJ-Envelope Vector has been proven to be an effective oligodeoxynucleotide (ODNs) delivery system both in vitro and in vivo (Kaneda et al.,

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2002). siRNAs were incorporated into the HVJ-Envelope Vector according to the manufacturer’s instructions. Briefly, after mixing 40 µl (1 assay unit, AU) of

HVJ-Envelope Vector with 4 µl of the enclosing factor, the mixture was centrifuged

(10,000 × g, 10 min, 4°C), and the pellet suspended in 10 µl of buffer solution. Then,

10 µl of a mixture of 3 siRNAs solution (#1, #2 and #3, 1 µg/µl each) was added, and Downloaded from the mixture was kept on ice for 5 min. Sterile ACSF (10 µl) containing synthetic siRNA

duplexes (0.45 pmole/animal) was injected into the subarachnoid space between the L5 jpet.aspetjournals.org and L6 vertebrate of conscious mice. The sequences of siRNA (sense) were as follows:

GlyT1 (#1, 5’-GCUGUCAGACGACAUUGGAAACUUU-AG-3’; #2, at ASPET Journals on September 26, 2021

5’-GCAACCAGAUCGAGUUUGUACUGAC-AG-3; #3,

5’-GAGUGGAUUCUGCAGAAGAAGACCU-AG-3’; #4,

5’-GCUAUCAGACGACAGUGGACACUGU-AG-3); GlyT2 (#1,

5’-GGAACACACCAGAAUGCAAAGAUAA-AG-3’; #2,

5’-GCGAAAGGAAUUAAGUCAUCAGGAA-AG-3’; #3,

5’-CCCAAGAUACAGAUCAAGAACUCUA-AG-3’; #4,

5’-GGAGCACACUAGAAUGCACAGAUAG-AG-3’); GlyRα3 (#1,

5’-AGGUUUCGGCGAAAGAGAAAGAAUA-AG-3’; #2,

5’-GGUACUGCACUAAACACUACAAUAC-AG-3’; #3,

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5’-CCUUAGGCAUGAAGACAUUCAUCAU-AG-3’; #4,

5’-GAUACUGCACUACACACUACGAUAC-AG-3’). At the times indicated in the text, mice were anesthetized with pentobarbital, and the spinal cords were subjected to immunoblotting analysis. In the controls, we followed the same procedures; however,

we substituted an identical amount of mismatched siRNA#4 with HVJ-Envelope Downloaded from

Vector and only the HVJ-Envelope Vector without siRNA.

Western blot analysis. The lumbar regions of spinal cord (L2-L6) were obtained jpet.aspetjournals.org from untreated animals or animals injected with HVJ-Envelope Vector, GlyR siRNA or the mutant siRNA. The spinal cord tissues (4.8 - 5.0 mg) were homogenized in 20 mM at ASPET Journals on September 26, 2021

Tris-HCl (pH 7.5) containing 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 10 mM

β-glycerophosphate and 1 µg/ml of the various protease inhibitors (benzamdine, leupeptin and antipain). The homogenate was reacted with 0.6% NP-40 for 5 min at 4˚C, followed by centrifugation at 15,000 rpm for 5 min at 4˚C and the supernatant was obtained. The supernatant was mixed at a volume ration of 4:1 in 10 mM Tris-HCl (pH

6.8) containing 10% (v/v) glycerol, 2% sodium dodecylsulfate (SDS), 0.01% bromophenol blue and 0.5% β-mercaptoethanol, with subsequent boiling at 100˚C for

10 min.

To detect GlyT1, GlyT2 and GlyRα3, tissue lysates (30 µg of crude protein

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extracts/line) were electrophoresed on a polyacrylamide gels (stacking gel 3 % and separating gel 10 %), transferred onto polyvinylidene fluoride membranes, and Western blotted. The primary antibodies were followed: against GlyT1 (Alpha Diagnostic international, San Antonio, TX); GlyT2 (Alpha Diagnostic international, San Antonio,

TX); GlyRα3 (Sigma Aldrich Inc., St. Louis, MO). The secondary antibodies were as Downloaded from follows: for GlyT1 and GlyT2 antibodies, anti-rabbit IgG antibody conjugated with

HRP (Dakocytomation, Glostrup, Demark); for GlyRα3 antibody, biotinylated jpet.aspetjournals.org pan-specific secondary antibody (Vector Laboratories, Burlingame, CA).

To detect β-actin, lysates (15 µg of crude protein extracts/line) were separated on a at ASPET Journals on September 26, 2021

10 % SDS-polyacrylamide gel, transferred onto polyvinylidene fluoride membranes, and Western blotted. Blots were incubated with primary antibodies against β-actin obtained from IMGENEX Innovations in Functional Genomics (SanDiego, CA) and the secondary anti-rabbit IgG antibody conjugated with HRP was obtained from

Dakocytomation (Glostrup, Demark). All Western blots were developed using

Hyperfilm ECL and the ECL kit (Amersham BioScience) according to the manufacturer’s instructions, followed by exposure to X-ray films. Densitometry was carried out on these X-ray films with the aid of an ATTO Densitograph 4.0 (Atto

Corporation, Tokyo, Japan).

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Materials. ALX 1393 (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine),

5,7-dichlorokynurenic acid (5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid; DCK),

Gabapentin, L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone) and

Sarcosine (N-methyl glycine) was obtained from Sigma-Aldrich (St.Louis, MO). ORG

25543 (4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide) Downloaded from and ORG 25935 (cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl

methyl)amino methylcarboxylic acid) were donated by Organon Laboratories Ltd., jpet.aspetjournals.org

(Scotland, UK). ALX 1393 was dissolved in a solvent containing 25%

2-hydroxypropyl-β-cyclodextrin (RBI, Natick, MA) and distilled water, pH adjusted to ~6 at ASPET Journals on September 26, 2021 using 1 N NaOH. DCK and L-701,324 were dissolved in DMSO and diluted appropriately

(final concentration of DMSO was 0.1 %). Other reagents were dissolved in ACSF or

saline. ACSF composition (in mM) was NaCl 142, KCl 5, CaCl2•2H2O 2, MgCl2•6H2O 2,

NaH2PO4 1.25, D-glucose 10, HEPES 10, pH 7.4.

Results

Effects of GlyTs inhibitors on mechanical allodynia in neuropathic pain models in mice. Intravenous injection of GlyT1 inhibitors, ORG 25935 and sarcosine decreased the allodynia score and ameliorated the reduction of the withdrawal threshold in a

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partial sciatic nerve ligation-injury model (Fig. 1A, 1B). This effect appeared after a time lag of about 1.5 to 2 hrs post-injection of the drug. Intravenous injection of GlyT2 inhibitors, ORG 25543 and ALX 1393, ameliorated allodynia, and this anti-allodynia effect appeared without a time lag after injection of the drugs (Fig. 1C, 1D). Both GlyT1

and GlyT2 inhibitors ameliorated allodynia by i.t. injection and the effect was much Downloaded from

potent and longer than that of gabapentin as shown in ED50 values and the duration of jpet.aspetjournals.org action (ED50 estimated by the withdrawal threshold for ORG 25935, ORG 25543 and gabapentin were 5.3, 11.0 and 8,200 ng/mouse, respectively) (Table 1). The inhibitors of

GlyT1 and GlyT2 ameliorated mechanical allodynia in other neuropathic pain models at ASPET Journals on September 26, 2021 such as the STZ-induced diabetic model. Dose-response effects of ORG 25935 showed that the anti-allodynia effect peaked by i.t. injection of 3-300 ng in the partial sciatic nerve injury model and showed a bell-shaped effect with a peak effect at 0.1 mg/kg by i.v. injection at 10 µg-10 mg/kg in the STZ diabetic model, while ORG 25543 showed a dose-dependent anti-allodynia effect in this dose range (Table 1). Anti-allodynia effects of GlyTs inhibitors are relatively less in the CFA-induced chronic inflammation model

(Table 1). To further confirm the specific inhibition of GlyT1 and GlyT2 exerting an anti-allodynia effect, the effect of knockdown of GlyTs by siRNA of GlyTs mRNA on tactile allodynia was examined. Significant reduction of GlyT1 and GlyT2 expression in

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the spinal cord of mice treated with GlyT siRNA with HVJ-Envelope Vector compared with untreated, HVJ-Envelope Vector -injected or mutant-siRNA-injected control mice was confirmed by Western blotting analysis 3 days after treatment (Fig. 2A, 2B). On the

10th day post-surgery, introduction of either GlyT1 siRNA (Fig. 2C) or GlyT2 siRNA

(Fig. 2D) into the spinal cord ameliorated allodynia, as shown by reduction of the Downloaded from allodynia score and recovery from the decreased withdrawal threshold from 2 to 7 days

for GlyT1 siRNA or 2 to 9 days for GlyT2 siRNA after introduction. HVJ- Envelope jpet.aspetjournals.org

Vector or GlyT siRNA mutants had no effect (data not shown). No apparent behavioral change was observed during this period. at ASPET Journals on September 26, 2021

Phenotypic characteristics by blockade of GlyTs and GlyRs. All of GlyT inhibitors with doses displaying anti-allodynia effects or even higher doses, either by intravenous or intrathecal injection, had no effect on spontaneous behaviors, locomotor activity, the withdrawal threshold in normal mice, and motor behavior in the RotaRod test (Table 2). These treatments did not disturb the righting reflex and did not produce hind limb disorder, typical behaviors of hyperekplexia (Table 2). Strychnine produce hind limb disorder at 9 µg, i.t. which was much higher than doses for blocking the anti-allodynia effect of GlyT2 inhibitors (Table 2).

Effects of antagonists of the NMDA glycine site on the effects of GlyT1

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inhibitors. The present study demonstrated that GlyT1 inhibitors, ORG 25935 and sarcosine, but not GlyT2 inhibitors, ORG 25543 and ALX 1393, required a lag time before onset of the effect. To explore whether these effects of GlyT1 inhibitors are due to the action of glycine on NMDA receptor and subsequent activation of the receptor

function, the effects of inhibitors on the NMDA glycine binding site, L-701,324 and Downloaded from

DCK, were examined. ORG 25935, but not ORG 25543, by itself produced a slight and

transient reduction of the withdrawal threshold in sham-operated mice (Fig. 3A). jpet.aspetjournals.org

Pretreatment of L-701,324 or DCK protected ORG 25935 from reduction of the withdrawal threshold (Fig. 3A). L-701,324 abolished the lag time of ORG 25935 to at ASPET Journals on September 26, 2021 reduce the withdrawal threshold, and prolonged the anti-allodynia effect in a partial sciatic nerve ligation-injured model (Fig. 3B). Therefore, the activation of NMDA receptors by increased glycine through the inhibition of GlyT1 by GlyT1 inhibitors may delay the onset of and counteract the anti-allodynia effect of GlyT1 inhibitors.

Effect of blockade of glycine receptors by strychnine or knockdown of GlyRα3 by siRNA on anti-allodynia effect of GlyT inhibitors. To confirm that the anti-allodynia effect of ORG 25935 and ORG 25543 is due to the inhibition of GlyTs and the subsequent activation of postsynaptic glycine receptors, the effect of strychnine, an inhibitor of glycine receptors, and knockdown of glycine receptors containing the α3

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subunit (GlyRα3) was examined. The anti-allodynia effects of intravenously injected

ORG 25935 and ORG 25543 were reversed by intrathecal injection of strychnine at 5 hrs (Fig. 4A), 1, 2 and 3 days (Fig. 4C, D) after the injection of GlyT inhibitors.

Strychnine at the doses used, or even 10 ng, i.t. did not reduce significantly the

withdrawal threshold (Fig. 4B) and did not produce any sign of convulsions (Table 2). Downloaded from

The anti-allodynia effect of gabapentin, the mechanisms of which do not involve the

glycinergic system, was not reversed by strychnine (data not shown). These results jpet.aspetjournals.org suggest that GlyT inhibitors, injected systemically, act through the activation of glycinergic receptors in the spinal cord for anti-allodynia action. GlyRα3 is specifically at ASPET Journals on September 26, 2021 expressed in the superficial dorsal horn, while GlyRα1 is widely distributed in the dorsal horn. To explore the target molecule for GlyT inhibitors to induce anti-allodynia, the effect of knockdown of the GlyRα3 subunit by siRNA on the anti-allodynia effect of

GlyT inhibitors was examined. Significant reduction of GlyRα3 expression during 1 to

5 days with a peak reduction at 3 days after treatment with GlyRα3 siRNA in the spinal superficial layers of mice compared with untreated, HVJ-Envelope Vector-injected or mutant-siRNA-injected control mice was confirmed by immunohistochemical and

Western blotting analysis (Morita et al., in press). Both ORG 25935 (Fig. 4E) and ORG

25543 (Fig. 4F) by i.v. injection failed to ameliorate the allodynia response in

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nerve-injured mice transfected with siRNA of GlyRα3, while they produced an anti-allodynia effect in mice injected with HVJ-Envelope Vector or mutant siRNA.

Timing of the treatment of GlyT inhibitors. GlyT2 is suggested to play a role to supply glycine to synaptic vesicles in nerve terminals of glycinergic neurons rather than

to clear glycine in the synaptic cleft from the observation of tissues from Downloaded from

GlyT2-deficient mice or in in vitro experiments with continuous exposure of GlyT2

inhibitors to tissues from normal animals. If this occurred in glycinergic neurons jpet.aspetjournals.org regulating sensory neurons in the spinal cord in vivo, the repeated administration of

GlyT2 inhibitors would result in the reduction of glycine release and thus decrease of at ASPET Journals on September 26, 2021 the anti-allodynia effect. However, this may not have occurred in the present case, because 6 repeated injections of ORG 25543 every 4th day produced an anti-allodynia effect with similar potency (Fig. 5). The anti-allodynia effect of ORG 25543 1 day after the 6th injection was antagonized by i.t. injection of strychnine, suggesting a continuous anti-allodynia effect mediated by stimulation of glycine receptors even after repeated treatment with GlyT2 inhibitors.

It has been suggested that different mechanisms mediated by a variety of neuronal and neighbor cells, various neurotransmitters and mediators may be diversely concerned in the stage of formation of neuropathic pain, such as the initiation, development and

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maintenance of allodynia. Although GlyT inhibitors had an excellent anti-allodynia effect on established allodynia, it would be of more value if they could protect against the formation of neuropathic pain when treated in the early phase of nerve injury.

However, when ORG 25543 was treated before surgery, or 1 and 2 days post-surgery, it

never recovered the reduced withdrawal threshold during the 3 days post-surgery. Downloaded from

However, these treatments started to ameliorate the reduced withdrawal threshold after

the 4th day post-surgery, regardless of the different timing of injection (Fig. 6A). Effects jpet.aspetjournals.org of knockdown of GlyT2 on time-dependent appearance of allodynia after nerve injury was also examined. For this purpose, siRNA for GlyT2 mRNA was injected into the at ASPET Journals on September 26, 2021 spinal cord two times at 3 days and 3 hrs before surgery to maintain the reduced expression of GlyT2. These treatments did not ameliorate the reduced withdrawal threshold during 2 days post-surgery but the withdrawal threshold started to increase at

3 days and ameliorated at 4 days after surgery (Fig. 6B). These results suggest that even when the expression of GlyT2 protein was reduced, an allodynia response appeared during the 3 days post-surgery and became effective after 3 days, as shown by the increased threshold on the 3rd to 4th day. Re-reduction of the threshold on the 5th day may be due to recovery of the expression of GlyT2 protein. The treatment of siRNA on the 5th as well on the 15th day post-surgery produced a profound anti-allodynia effect

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(Fig. 6B). Mutant siRNA had no effect at any injection. Similar results were obtained with the injection of ORG 25935 and GlyT1 knockdown (Fig. 6C, D). The results clearly showed that the inhibition of GlyTs did not prevent the initiation of allodynia by sciatic nerve ligation nor antagonized the allodynic response during the early

pathological stage. The present evidence that GlyT inhibitors lack an inhibitory effect Downloaded from on allodynia in the early stage of allodynia development after nerve injury, while they

produce potent and long-acting anti-allodynia action against established allodynia may jpet.aspetjournals.org suggest that glycinergic inhibitory control is disordered in the early stage of developing allodynia after nerve ligation. Strychnine, a glycine ameliorated at ASPET Journals on September 26, 2021 allodynia at 20 hrs and 3 days post-surgery, while it had no effect after 4 days post-surgery in the partial sciatic nerve ligation-injury model (Fig. 6E). Again, when

GlyRα3 was knocked down by the injection of siRNA for GlyRα3 mRNA at 3 days and

3 hr before surgery on the sciatic nerve, allodynia was not initiated during the first 3 days post-surgery, and then reduction of the withdrawal threshold occurred on the 4th day (Fig. 6F). Injection of siRNA at 5th and 13th day post-surgery had no effect (Fig.

6F). Thus, glycinergic neuronal activity reversed the control of pain signal transduction, depending on the development stage of neuropathy; exacerbation in the early stage and

reduction in the late stage. In agreement with this concept, , a GABAA

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receptor antagonist, also produced an anti-allodynia effect within 3 days and this effect

was reversed by isoguvacine, a GABAA receptor (Fig. 6G). Bicuculline lost its action after the 4th day post-surgery (Fig. 6G). Isoguvacine produced anti-allodynia which was sensitive to bicuculline antagonism only after the 4th day post-surgery (Fig.

6H). Of great interest, all of the data showed that the critical turning point of the reverse Downloaded from inhibitory regulation seemed to be between the 3rd and 4th day post-surgery. jpet.aspetjournals.org

Discussion

The present study demonstrated a profound and long-lasting anti-nociceptive effect at ASPET Journals on September 26, 2021 of the inhibitors of GlyT1 and GlyT2 in partial sciatic nerve ligation-induced neuropathic pain models, STZ-induced diabetic models and with less potency in the

CFA-induced inflammatory model in mice. Their different potency among the models may be due to the diverse mechanisms involved in the development of chronic pain in each model, especially between neuropathic pain and inflammation.

Considering the role of GlyT1 to lower extracellular glycine concentration at the glycinergic synaptic cleft (Cubelos et al., 2005; Roux and Supplisson, 2000; Jursky and

Nelson, 1996), the anti-allodynia effect of GlyT1 inhibitors, ORG 25935 and sarcosine, due to the blockade of glycine uptake, mostly by glial cells and partly by neurons,

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resulted in the accumulation of glycine at the site of GlyRs, which suppress the excitatory neuronal activity of target neurons for noxious signal transduction in the spinal cord. It is noteworthy that the anti-allodynia effect of GlyT inhibitors was lasted all day and was antagonized by i.t. injection of strychnine at any time, such as 1, 2 or 3

days after the intravenous injection of GlyT inhibitors. The evidence suggests that these Downloaded from inhibitors by single intravenous injection may produce an anti-allodynia effect by

stimulating glycine receptors at the spinal cord during the effective term. The possibility jpet.aspetjournals.org remains to be elucidated further that these compounds irreversibly block GlyTs during the period and/or thus cause down-regulation of the expression of GlyTs in the plasma at ASPET Journals on September 26, 2021 membrane. A limitation point of GlyT1 inhibitors as analgesics is the lag time of 1 to 2 hrs required after administration before developing an anti-allodynia effect, which was partly masked due to saturation of the glycine binding site on NMDA receptors by inhibiting glycine uptake with GlyT1 inhibitors, as shown by the specific antagonists of

NMDA receptor glycine site, L-701,324 or DCK, completely abolishing this lag time and potentiating the GlyT1 inhibitor-induced anti-allodynia effect. This explanation was supported by the ability of ORG 25935 but not ORG 25543 by i.t. injection itself to produce slight and transient reduction of the withdrawal threshold, which was inhibited by L-701,324 and DCK. The observations that the potency of the anti-allodynia effect of

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high dosages of ORG 25935 decreased and the period of the anti-allodynia effect by

GlyT1 knockdown was shorter than that of GlyT2 knockdown also may suggest a counteracting effect by the stimulation of NMDA receptors in the spinal cord and higher brain regions at higher doses of GlyT1 inhibitors, even though the time-dependent

changes of GlyT1 protein expression were similar to those of GlyT2. Actually, Downloaded from microdialysis perfusion of the lumbar dorsal spinal cord of rats reveals that ORG 24598,

a selective inhibitor of GlyT1, increases extracellular glycine accompanied with a jpet.aspetjournals.org progressive increase in citrulline, which resulted from activation of the NMDA receptor/NOS signaling cascade (Whitehead et al., 2004). GlyT1 is distributed widely in at ASPET Journals on September 26, 2021 the CNS and may have a role in controlling concentrations of glycine in the vicinity of

NMDA receptors. This affords an opportunity for GlyT1 inhibitors to enhance NMDA receptor function and may have relevance in addressing the hypoglutamatergic function associated with schizophrenia or depression (Tsai et al., 2004; Gomeza et al., 2006).

This effect may have the desired effect for the use of these compounds as analgesics in such psychotic patients, or undesired otherwise.

The restricted presence of GlyT2 in glycinergic neurons suggests its important role in the control of glycinergic neurotransmission (Jursky and Nelson, 1995); however, evidence supports that GlyT2 does not appear to play an important role in clearing

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glycinergic synaptic cleft. GlyT2 inhibitors do not affect the decay kinetics of glycinergic IPSPs in rat brain stem motoneurons (Titmus et al., 1996; Singer et al.,

1998). Hypoglossal motoneurons from GlyT2-deficient mice are shown to display glycinergic mIPSPs with a marked reduction in amplitude as compared to those

recorded from wild-type mice (Gomeza et al., 2003b), and those from GlyT1-deficient Downloaded from mice, in which the decay time constant of glycinergic mIPSPs was longer than that in

wild-type mice (Gomeza et al., 2003a). GlyT2 presents in glycinergic boutons adjacent jpet.aspetjournals.org to active zones (Spike et al., 1997); thus, it has been suggested that GlyT2 plays an important role in taking up glycine to refill synaptic vesicles with glycine and to at ASPET Journals on September 26, 2021 maintain exocytotic release of glycine in response to nerve stimulation (Oku et la.,

1999; Singer et al., 1998; Gomeza et al., 2003b). In this case, the inhibition of GlyT2 function would be expected to deteriorate nociceptive mechanisms. However, Bradaïa et al. (2004) demonstrated using whole cell voltage clamp recording from lamina X neurons in rat spinal cord slices that ORG 25543 as well as ORG 24598, a GlyT1 inhibitor, increase the decay time constants of miniature, electrically evoked and spontaneous IPSPs, although ORG 25543 decreased the frequency of mIPSP. The authors conclude that blocking glial and/or neuronal glycine transporters increased the level of glycine in the spinal cord, which in turn prolonged the duration of the

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glycinergic synaptic current. An increase in extracellular glycine without accompanying citrulline release was demonstrated by microdialysis perfusion with ORG 25543 of the lumbar dorsal spinal cord of rats (Whitehead et al., 2004). Evidence suggests that the pharmacological blockade of GlyT2 increases either glycinergic and/or GABAergic

neurotransmission in vivo in the spinal cord. The present evidence shows that the Downloaded from anti-allodynia effect of systemically administered (i.v.) GlyT2 inhibitors was

counteracted by i.t. injection of strychnine or knockdown of spinal GlyRα3, suggesting jpet.aspetjournals.org mediation by the activation of spinal GlyRα3 of the anti-allodynia effect of GlyT2 inhibitors. at ASPET Journals on September 26, 2021

The anti-allodynia effect of GlyT2 inhibitors, ORG 25543 and ALX 1393, appeared without a time lag after administration. ORG25543 showed a dose-dependent anti-allodynia effect with the same dose range as ORG25935, which was effective in a limited dose range. Together with the confined distribution and highest densities of

GlyT2 in boutons in lamina III of dorsal horn in comparison with the distribution of

GlyT1 in astrocytes in lamina I and II in the spinal cord (Zafra et al., 1995a), these results may suggest fewer side effects of these compounds. However, the deletion of mouse GlyT2 results in a hyperekplexia phenotype (Gomeza et al., 2003b). Thus, dysfunction of GlyT2 function is crucial for insufficient transmitter loading of synaptic

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vesicles in glycinergic nerve terminals. However, there was no sign of hyperekplexia by the administration of GlyT2 inhibitors. Again, strychnine required about 3,000 times higher doses to produce hind limb disorder than those for blocking the anti-allodynia effect of GlyT2 inhibitors. Thus, it seems likely that glycinergic control of signal

transduction in sensory nerves is more sensitive to manipulation by drugs than that in Downloaded from motor neurons. Therefore, it does not seem probable that GlyT2 inhibitors by treatment

for pain control may produce hyperekplexia-like disorders. In addition, reproduction of jpet.aspetjournals.org the anti-allodynia effect by repeated treatment of ORG 25543 (i.v.) with similar potency each time suggests that glycine refilling was not impaired in glycinergic nerve terminals at ASPET Journals on September 26, 2021 by pharmacological manipulation in vivo. Tricyclic antidepressants are one of the major clinical medicaments for neuropathic pain, and some can inhibit GlyTs. Sarcosine blocks GlyT1 (Morow et al., 1998) and inhibits GlyT2 more selectively

(Núñez et al., 2000). Doxepin, amitriptyrine and nortriptyline block both GlyT1 and

GlyT2 (Núñez et al., 2000). We have achieved the knockdown of spinal GlyT1 and

GlyT2 in vivo by introducing the corresponding siRNAs into the mouse spinal cord.

Evidence that either knockdown of GlyT1 or GlyT2 effectively ameliorated allodynia further supports the anti-allodynia effect by inhibition of GlyTs.

It is interesting that with pre-treatment or treatment immediately after surgery in a

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nerve injury model, both GlyT1 and GlyT2 inhibitors failed to produce an anti-allodynia effect until 4 days after surgery; therefore, it is assumed that glycinergic inhibitory controls are impaired in this early stage of the development of neuropathic pain. Recent attention has focused on relief from the inhibitory systems of spinal neurons as a

mechanism for neuropathic pain. Coull et al. (2003) demonstrated that the reduced Downloaded from expression of K+Cl-(KCC) cotransporter, KCC2, reduced anion reversal potential in

spinal lamina I neurons in the dorsal spinal cord after peripheral nerve injury. The jpet.aspetjournals.org

change in the driving force means that both glycine and GABAA receptor-mediated inputs produce less hyperpolarization and could even paradoxically depolarize neurons at ASPET Journals on September 26, 2021

(Coull et al., 2005; Prescott et al., 2006). This contributes to cellular hyperexcitability producing allodynia after peripheral nerve injury.

The present evidence that glycine receptor blockade by strychnine or knockdown of

the GlyRα3 or GABAA receptor antagonist bicuculline inhibited the development of tactile allodynia in the early stage post-surgery suggests that the activation of

glycinergic receptors, especially GlyRα3 and GABAA receptors, may lead to the induction of allodynia. There was the critical turning point at 3 to 4 days post-surgery; stimulation of the glycinergic system by GlyT inhibitors and knockdown of GlyTs, or

stimulation of the GABAergic system by GABAA receptor agonist isoguvacin started to

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ameliorate allodynia after 4 days post-surgery. It is also suggested that blockade of the excitatory influence of glycinergic and GABAergic systems in the early stage can not cure the pathological cause, because allodynia developed after the 4th day-post surgery under continuous treatment. Coull et al. (2005) have proposed that microglia and neuron

interaction via brain-derived neurotrophic factor (BDNF) released from microglia Downloaded from through TrkB leads to a reduction of the expression of KCC2 and a depolarization shift

in the anion reversal potential in the spinal cord. Reduction of the anion reversal jpet.aspetjournals.org potential in spinal lamina I neurons in the dorsal spinal cord in the present model and

glycine and GABAA receptor-mediated depolarization of the neurons as a function of at ASPET Journals on September 26, 2021 time after peripheral nerve injury remain to be confirmed.

In summary, the present study demonstrated that the inhibition of GlyT1 and GlyT2 produced an anti-allodynia effect in neuropathic pain models in mice. This effect was potent and long-lasting. The mechanism of their anti-allodynia action was enhancement of the glycinergic inhibitory pathway through GlyRα3 in the spinal cord. Therefore,

GlyTs seem to be a hopeful target for the development of medicaments for neuropathic pain. The present study also implied that the timing of medication is very important.

Reversal of the inhibitory regulation of glycinergic and GABAergic systems to excitatory regulation at a critical point 3 to 4 days post-surgery also provided important

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information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Acknowledgments

The authors wish to thank Dr. Glenn Walker (Organon Laboratories Ltd, Scotland Downloaded from

UK) for providing the ORG 25935 and ORG 25543. jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Footnotes

This work was supported in part by Grants-in aid for Scientific Research (B) of

Japan and by a grant from the Japanese Smoking Research Association, and was carried out with equipment from the Research Facilities for Laboratory Animal Science. Some

of these results have been presented in abstract form (Dohi et al., 2007; Morita et al., Downloaded from

2007).

Dohi T, Morita K, Motoyama N, Kitayama T and Morioka N (2007) Spinal glycine jpet.aspetjournals.org

receptor α3 subunit (GlyRα3) involvement of inhibitor of glycine

transporter-induced anti-allodynia effects in neuropathic pain models in mice. at ASPET Journals on September 26, 2021

37th Annual Meeting of Society for Neuroscience, Presentation number: 287.10.

Morita K, Motoyama N, Kitayama T, Morioka N and Dohi T (2007) Anti-allodynia

effects of glycine transporter inhibitors in neuropathic pain models in mice. 37th

Annual Meeting of Society for Neuroscience, Presentation number: 287.9.

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Legends for Figures

Fig. 1. ORG 25935 and sarcosine, specific inhibitors of GlyT1, and ORG 25543 and

ALX 1393, specific inhibitors of GlyT2, ameliorated the mechanical allodynia assessed after partial sciatic nerve ligation injury in the mice. Mechanical allodynia was assessed

by lightly stroking the flank of each mouse with a paintbrush on the side of the Downloaded from nerve-injured paw ( ). Each point represents the average score evaluated at each

time point. Mechanical allodynia was assessed by measuring the paw withdrawal jpet.aspetjournals.org threshold in response to probing with von Frey hairs ( ). ORG 25935 (A), sarcosine

(B), ORG 25543 (C) and ALX 1393 (D) were administered intravenously at time 0. at ASPET Journals on September 26, 2021

Data are expressed as the mean ± S.E.M. n=7-15 mice per group. Control mice were injected with vehicle; artificial cerebrospinal fluid (ACSF) or 25%

2-hydroxypropyl-β-cyclodextrin. Mechanical allodynia developed after partial sciatic nerve ligation injury in mice was not affected by vehicle treatments. *P<0.01 compared with the corresponding control values just prior to the injection of ORG 25935 (A), sarcosine (B), ORG 25543 (C) or ALX 1393 (D), as determined by analysis of variance followed by Tukey’s test.

Fig. 2. Knockdown of GlyT1 and GlyT2 by siRNA ameliorated mechanical

46 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version.

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allodynia assessed after partial sciatic nerve ligation injury in the mice. A, B: Western blotting analysis of spinal GlyT1 (A) and GlyT2 (B) after transfection of siRNAs for

GlyT1 and GlyT2 into the spinal cord of mice. As controls, HVJ-Envelope Vector

(HVJ-E) or mutant siRNA was injected into the spinal cord. The levels of GlyT1 and

GlyT2 were normalized to β-actin and represented as % induction compared with the Downloaded from values of untreated mice. Data are expressed as the mean of % of naïve control ± S.E.M

(n=4-8 mice per group). *P<0.05 compared with the corresponding control, as jpet.aspetjournals.org determined by analysis of variance followed by Fisher’s PLSD. C, D: Effects of treatment with GlyT1 siRNA and mutant siRNA (C) or GlyT2 siRNA and mutant at ASPET Journals on September 26, 2021 siRNA (D) on mechanical allodynia assessed after partial sciatic nerve ligation injury in mice. The experiments were carried out 10 days post-nerve ligation. *P<0.01 compared with the corresponding control, as determined by analysis of variance followed by

Tukey’s test.

Fig. 3. (A) Effect of GlyT inhibitors and specific antagonists of the glycine site of

NMDA receptors L-701,324 and 5,7-dichlorokynurenic acid (DCK) on withdrawal threshold in sham-operated mice. (B) Effects of L-701,324 and DCK on the anti-allodynia effect of ORG 25935 in partial sciatic nerve ligation injury. L-701,324

47 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version.

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and DCK were injected i.t. 30 min before the injection of ORG 25935. ORG 25935 was administered intrathecally at 12 days post-surgery. Data are expressed as the mean ±

S.E.M. n=10 mice per group. *P<0.01 compared with the corresponding control values just prior to the injection of ORG 2543, as determined by analysis of variance followed

by Tukey’s test. Downloaded from

Fig. 4. Effect of glycine receptor blockade by strychnine and GlyRα3 knockdown jpet.aspetjournals.org on GlyT inhibitor-induced anti-allodynic effect in partial sciatic nerve ligation injury.

Glycine receptor antagonist, strychnine, was injected i.t. 0.125 (3 hr), 0.208 (5 hr), 1, at ASPET Journals on September 26, 2021

1.5, 2, 2.5, 3 days after the injection of ORG 25935 i.v. or ORG 25543 i.v. in nerve-injured mice (A, C, D) or sham-operated mice (B). ORG 25935 (E) and ORG

25543 (F) were administered 3 days after the injection of siRNA or mutant siRNA of

GlyRα3 or the vector. Experiments were carried out at 12 days post-nerve ligation. Data are expressed as the mean ± S.E.M. n=6-12 mice per group. *P<0.01 compared with the corresponding control values just prior to the injection of strychnine (A, B, C, D), ORG

25935 (E) or ORG 25543 (F), as determined by analysis of variance followed by

Tukey’s test.

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Fig. 5. Reproduction of anti-allodynia effect of ORG 25543 by repeated injection of

ORG 25543 in partial sciatic nerve ligation-injury mice. Mechanical allodynia was assessed in mice using von Frey hairs on the ipsilateral paws 11, 15, 19, 23, 27, 31 days after surgery when animals received an i.v. injection of ORG 25543. Strychnine was

administered intravenously 24 hr after the last injection of ORG 25543. Pre-operative Downloaded from basal values were obtained on day 0. Data are expressed as the mean ± S.E.M. n=8 mice

per group. jpet.aspetjournals.org

Fig. 6. Post-surgery period time-dependent anti-allodyninc effect of GlyT inhibitors, at ASPET Journals on September 26, 2021

GlyT knockdown, strychnine, bicuculline and isoguvacin in partial sciatic nerve ligation-injury mice. Mechanical allodynia was assessed in mice using von Frey hairs on ipsilateral paws. (A) ORG 25543 and (C) ORG 25935 were administered i.v. one day and 30 min before surgery, and 2 and 4 days post-surgery. (B) siRNA or mutant siRNA of GlyT2 mRNA and (D) siRNA or mutant siRNA of GlyT2 mRNA were injected into the spinal cord 3 days and 3 hrs before, 5 days and 15 days after nerve ligation. (E)

Strychnine was injected i.t. 1, 3, 4.5 or 13 days post-nerve ligation. (F) siRNA or mutant siRNA of GlyRα3 mRNA was injected into the spinal cord 3 days and 3 hrs before or 5 and 12 days post-nerve ligation. (G) Bicuculline was injected i.t. 1.5, 3, 4.5 or 12 days

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post-nerve ligation. Isoguvacin (Isg) was injected 40 min after the injection of bicuculline. (H) Isoguvacin was injected i.t. 1.5, 3, 4.5 or 12 days post-nerve ligation.

Bicuculline (Bicu) was injected 40 min after isoguvacin injection. Data are expressed as the mean ± S.E.M. n=5-10 mice per group. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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TABLE 1 Effects of GlyTs inhibitors on mechanical allodynia in various neuropathic pain models in mice. Anti-allodynia effects von Frey (withdrawal threshold)

Treatments Dose AUC Peak response Duration ED50 (%) (hr) PSNI model ACSF 5 µl i.t. 0 0 0 Sarcosine 20 ng i.t. 22.4 ± 9.2* 70.6 ± 22.3* 45.5 ORG 25935 3 ng i.t. 5.29 ± 1.2* 25.0 ± 5.8* 44.0 5.3 ng 30 ng i.t. 33.8 ± 6.0* 101.1 ± 11.8* 68.0 Downloaded from 300 ng i.t. 32.3 ± 3.5* 82.7 ± 9.3* 92.0 ALX 1393 10 ng i.t. 37.0 ± 10.7* 105.9 ± 23.2* 72.0 ORG 25543 3 ng i.t 2.8 ± 1.1* 9.9 ± 2.5* 47.0 11 ng 30 ng i.t. 26.4 ± 9.5* 67.4 ± 14.0* 71.0 300 ng i.t. 44.7 ± 3.8* 100.0 ± 8.1* 95.0 jpet.aspetjournals.org Gabapentin 0.1 µg i.t. 0.04 ± 0.01 5.4 ± 0.6* 1.0 8.2 µg 1 µg i.t. 0.31 ± 0.10* 15.8 ± 2.3* 2.5 10 µg i.t. 0.85 ± 0.15* 54.8 ± 8.1* 3.0 Saline 5 ml/kg i.v. 0 0 0 Sarcosine 0.3 mg/kg i.v. 51.5 ± 8.5* 112.3 ± 14.8* 92.0

ORG 25935 0.3 mg/kg i.v. 37.5 ± 3.9* 102.3 ± 8.3* 92.0 at ASPET Journals on September 26, 2021 ALX 1393 10 µg/kg i.v. 50.9 ± 8.0* 101.1 ± 10.7* 95.0 ORG 25543 0.3 mg/kg i.v. 44.4 ± 3.2* 103.7 ± 7.6* 95.0 Gabapentin 75 mg/kg i.v. 0.94 ± 0.20* 59.3 ± 14.1* 2.5

STZ diabetic model ACSF 5 µl i.t. 0 0 0 Sarcosine 20 ng i.t. 12.6 ± 2.8* 64.3 ± 11.1* 45.0 ORG 25935 3 ng i.t. 1.0 ± 0.3* 13.9 ± 4.5* 20.0 70 ng 30 ng i.t. 2.7 ± 0.6* 35.7 ± 7.5* 44.0 300 ng i.t. 12.1 ± 1.9* 74.4 ± 6.6* 68.0 ALX 1393 1 ng i.t. 8.2 ± 1.5* 32.7 ± 3.5* 47.0 1.9 ng 3 ng i.t. 21.6 ± 5.6* 63.6 ± 4.1* 71.0 10 ng i.t. 32.9 ± 4.6* 92.0 ± 6.5* 71.0 ORG 25543 3 ng i.t 3.1 ± 0.8* 13.9 ± 4.5* 47.0 42 ng 30 ng i.t. 8.0 ± 1.6* 35.7 ± 7.5* 47.0 300 ng i.t. 21.1 ± 2.2* 84.4 ± 6.6* 71.0 Gabapentin 0.1 µg i.t. 0.08 ± 0.06 6.7 ± 3.5 0 5.2 µg 1 µg i.t. 0.26 ± 0.06* 19.4 ± 2.8* 1.0 10 µg i.t. 0.94 ± 0.20* 68.5 ± 10.7* 2.0 Saline 5 ml/kg i.v. 0 0 0 Sarcosine 3 µg/kg i.v. 3.3 ± 0.6* 17.3 ± 3.2* 20.0 38 µg/kg 30 µg/kg i.v. 4.9 ± 0.7* 45.2 ± 8.7* 20.0 0.3 mg/kg i.v. 11.2 ± 3.0* 83.3± 17.5* 44.0

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TABLE 1 continued Anti allodynia effects von Frey (withdrawal threshold)

Treatments Dose AUC Peak response Duration ED50 (%) (hr) ORG 25935 10 µg/kg i.v. 1.6 ± 0.4* 10.4 ± 2.4* 20.0 42 µg/kg 0.1 mg/kg i.v. 13.0 ± 1.4* 68.0 ± 5.5* 44.0 1 mg/kg i.v. 10.9 ± 3.3* 36.9 ± 13.1* 68.0 10 mg/kg i.v. 0.2 ± 0.1* 2.5 ± 0.4* 0 ALX 1393 10 µg/kg i.v. 8.3 ± 2.7* 28.5 ± 9.7* 71.0 22 µg/kg 0.1 mg/kg i.v. 32.7 ± 15.5* 88.1 ± 11.8* 71.0 Downloaded from ORG 25543 10 µg/kg i.v. 1.6 ± 0.2* 11.7 ± 2.1* 23.0 90 µg/kg 0.1 mg/kg i.v. 15.4 ± 7.9* 57.8 ± 11.9* 47.0 1 mg/kg i.v. 21.1 ± 5.1* 76.4 ± 7.3* 71.0 10 mg/kg i.v. 46.2 ± 10.4* 95.2 ± 14.5* 95.0

jpet.aspetjournals.org CFA model ACSF 5 µl i.t. 0 0 0 Sarcosine 20 ng i.t. 7.5 ± 1.0* 38.1 ± 8.7* 44.0 ORG 25935 300 ng i.t. 8.9 ± 1.8* 52.4 ± 7.1* 44.0 ALX 1393 10 ng i.t. 7.4 ± 1.2* 58.3 ± 10.3* 47.0

ORG 25543 300 ng i.t. 8.0 ± 1.7* 45.3 ± 8.7* 47.0 at ASPET Journals on September 26, 2021 Gabapentin 10 µg i.t. 1.0 ± 0.2* 58.3 ± 10.4* 2.5 i.t.: intrathecal injection, i.v.: intravenous injection

AUC: The area under the withdrawal threshold against time curve (AUC) was calculated by the trapezoidal method. Large value of AUC represents the effectiveness of the compound. Peak response: % of peak values of withdrawal threshold (gm) after the administration of compounds to the values before administration. Duration: Values are time during significant reduction of withdrawal threshold. ED50 values were estimated from the peak response by using least-squares linear regression. Values are the mean ± S.E.M. n=5-34 of mice per group. *P<0.01 compared with the corresponding control values, as determined by analysis of variance followed by

Tukey’s test.

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TABLE 2 Phenotypic characteristics by the administration of GlyTs and GlyRs inhibitors and GlyTs siRNS in normal mice. a) b) c) RotaRod test Locomotor activity Allodynia Convulsion Righting reflex Hind limb This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Treatments Dose Length of time Counts/60 min % incidence Timed) (sec) disordere)

(sec) JPET FastForward.PublishedonApril30,2008asDOI:10.1124/jpet.108.136267 Exp. 1 ACSF 5 µl i.t. 3 hr after 209.4 ± 19.2 – 0 < 1 – Sarcosine 20 ng i.t. 3 hr after 207.5 ± 27.5 – 0 < 1 – ORG 25935 300 ng i.t. 3 hr after 214.3 ± 40.8 – 0 < 1 – ALX 1393 10 ng i.t. 3 hr after 176.0 ± 37.9 – 0 < 1 – ORG 25543 300 ng i.t. 3 hr after 177.2 ± 20.0 – 0 < 1 – 1 µg i.t. 3 hr after – 0 < 1 – 3 µg i.t. 3 hr after 188.3 ± 26.2 – 0 < 1 – ACSF 5 ml/kg i.v. 3 hr after 155.1 ± 15.3 19005 ± 1377 – 0 < 1 – Sarcosine 0.3 mg.kg i.v. 3 hr after 180.8 ± 21.7 24599 ± 1733 – 0 < 1 – ORG 25935 0.3 mg/kg i.v. 3 hr after 187.9 ± 44.6 18999 ± 1779 – 0 < 1 – 10 mg/kg i.v. 3 hr after 205.4 ± 26.1 20345 ± 1324 – 0 < 1 – ALX 1393 0.1 mg/kg i.v. 3 hr after 179.7 ± 22.5 22426 ± 3087 – 0 < 1 – ORG 25543 0.3 mg/kg i.v. 3 hr after 144.9 ± 25.4 23069 ± 2343 – 0 < 1 – 1 mg/kg i.v. 3 hr after – 0 < 1 – 3 mg/kg i.v. 3 hr after – 0 < 1 – 10 mg/kg i.v. 3 hr after 176.5 ± 25.0 24316 ± 1919 – 0 < 1 – ACSF 5 ml/kg i.v. 3 hr after 222.5 ± 25.3 20455 ± 1998 – 0 < 1 – Sarcosine 30 mg/kg i.v. 3 hr after 220.5 ± 28.2 18110 ± 1805 – 0 < 1 – ALX 1393 3 mg/kg i.v. 3 hr after 196.1 ± 26.6 21478 ± 2296 – 0 < 1 – Exp. 2 ACSF 5 µl i.t. 10 min after – 0 < 1 – Strychnine 3 ng i.t. 10 min after – 0 < 1 – 10 ng i.t. 10 min after – 0 < 1 – 30 ng i.t. 10 min after ++ 0 < 1 –

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TABLE 2 continued a) b) c) RotaRod test Locomotor activity Allodynia Convulsion Righting reflex Hind limb This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Treatments Dose Length of time Counts/60 min % incidence Timed) (sec) disordere)

(sec) JPET FastForward.PublishedonApril30,2008asDOI:10.1124/jpet.108.136267 Strychnine 300 ng i.t. 10 min after +++ 0 < 1 – 3 µg i.t. 10 min after +++ 0 < 1 – 9 µg i.t. 10 min after 12 < 1 + 18 µg i.t. 10 min after 100 >30 + Exp. 3 HVJ-E 1 day after 143.2 ± 28.6 – 0 < 1 – 2 day after 128.6 ± 34.4 – 0 < 1 – 3 day after 110.3 ± 30.9 19752 ± 1373 – 0 < 1 – GlyT1 siRNA, 0.45 pmol i.t. 1 day after 142.0 ± 30.8 – 0 < 1 – 2 day after 130.8 ± 36.6 – 0 < 1 – 3 day after 105.3 ± 12.0 21029 ± 2661 – 0 < 1 – GlyT2 siRNA, 0.45 pmol i.t. 1 day after 185.7 ± 43.2 – 0 < 1 – 2 day after 146.3 ± 32.0 – 0 < 1 – 3 day after 130.9 ± 38.1 20855 ± 1867 – 0 < 1 – 5 days after – 0 < 1 – 7 days after – 0 < 1 –

The compound was injected intrathecally (i.t.) or intravenously (i.v.) at the indicated times before behavioral testing. Unless otherwise noted, all experimental and control groups contained at least seven animals per group. a) Total amount of locomotor activity in 60 min after inhibitors of GlyTs and specific siRNA of GlyTs. There were no significant differences between animals pretreated with vehicle and with mutant siRNA. All values represent the mean locomotor activity in 60 min

± S.E.M.

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b) The intensity of mechanical allodynia was mentioned as: –, (score 0-0.5); +, (score 0.5-1); ++, (score 1-1.5); +++, (score 1.5-2). This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. c) Seizures induced by drugs were characterized by ataxia, brief loss of the righting reflex, and clonic and tonic convulsions. The percentage of JPET FastForward.PublishedonApril30,2008asDOI:10.1124/jpet.108.136267 animals that exhibited convulsions in each treatment group was determined. d) The time required to right after being turned on to its back. < 1: less than 1 second. e) Hindfeet clenching or limb clenching phenotype when picked up by the tail. – negative, + positive.

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Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021