**Interactions Between Glutamate Receptors and TRPV1 Involved in Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers**

You-Hong Jin1, Motohide Takemura2, Akira Furuyama3 and Norifumi Yonehara4 *1Department of Anatomy the Affiliated Stomatological, Hospital of Nanchang University, Nanchang, Jiangxi Province, 2Department of Oral Anatomy and Neurobiology, Osaka University Graduate School of Dentistry, 3Departments of Oral Physiology 4Oral Medical Science (Division of Dental Pharmacology), Ohu University School of Dentistry, Koriyama, Fukushima, 1China* 

#### *2,3,4Japan*

#### **1. Introduction**

42 Pharmacology

Wright RA, Arnold MB, Wheeler WJ, Ornstein PL and Schoepp DD (2000) Binding of [3H](2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III

Yamaguchi S and Ninomiya K (2000) Umami and food palatability. *J Nutr* 130(4S

Zhang F, Klebansky B, Fine RM, Xu H, Pronin A, Liu H, Tachdjian C and Li X (2008)

362(6):546-554.

Suppl):921S-926S.

105(52):20930-20934.

metabotropic glutamate receptor subtypes. *Naunyn Schmiedebergs Arch Pharmacol*

Molecular mechanism for the umami taste synergism. *Proc Natl Acad Sci U S A*

Glutamate (Glu) is a main excitatory neurotransmitter in the central nervous system. Concerning the existence of Glu in the small-diameter afferent fibers, their central (Westlund et al., 1989; Keast and Stephensen, 2000) and peripheral (Westlund et al., 1992; Keast and Stephensen, 2000) processes as well as dorsal root ganglion (DRG) cells (Battaglia and Rustioni, 1988; Keast and Stephensen, 2000) contain Glu. Recently, Glu has been shown to have a role in transduction of sensory input at the periphery (Carlton, 2001).

Electron microscope studies demonstrate that Glu receptors are transported from the DRG cell bodies into central and/or peripheral primary afferent terminals (Liu et al., 1994). The N-methyl-D-aspartic acid (NMDA), -amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and kainate receptors (NMDA/AMPA-kainate receptors) are localized on unmyelinated axons at the dermal-epidermal junction in the glabrous and hairy skin of the rat (Carlton et al., 1995; Coggeshall and Carlton, 1998), and in human hairly skin (Kinkelin et al., 2000). Approximately 20% of the fibers were immunostained in one of the receptor subtypes. As Sato et al. (1993) reported that virtually all DRG cells as well as their central (Laurie et al., 1995; Zou et al., 2002) and peripheral (Carlton et al., 1995) processes are positively labeled for the NMDA receptor, it is highly likely that two or more of the ionotropic Glu receptors are colocalized.

Interactions Between Glutamate Receptors and TRPV1 Involved in

**2.2 Release of Glu into the subcutaneous space** 

**2.1 Experimental procedures** 

food and water ad libitum.

**2.3 Amino acid analysis** 

quantitation.

value before stimulation.

**2.4 Drug administration** 

volume of 50 l into the s.c. of the neck.

**2.5 Behavioral assessments** 

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 45

Adult male Sprague-Dawley rats weighing between 200-300 g (CLEA Japan, INC. Tokyo, Japan) were used in all experiments. Rats were on a 12 hrs light/dark cycle and received

Animals were anesthetized with urethane (1 g/kg i.p.). A single loop catheter whose tip was covered with a 5000 molecular weight dialysis membrane (MS 0045, PSS® SELECT, Florida) was introduced into the s.c. space of the instep using a 2.2 mm outer diameter polyethylene tube as a guide. Ringer's solution was perfused at 15 l/min through this catheter with a micro syringe pump (EP-60, Eicom, Kyoto, Japan) and perfusate was collected into the tubes placed in an ice bath at intervals of 20 min. The samples were kept at -80°C until analysis.

Amino acids in the dialysate were analyzed by a high-performance liquid chromatography (HPLC) system for automated analysis of amino acids using o-phthalaldehyde derivatization and fluorescence detection. Amino acids were quantified by reverse-phase chromatography using a C18 octadecylsilyl (ODS) silica-gel column (EICOMPAK SC-50DS 2.1 mm x 150 mm) with pre-column (EICOM PREPAKSET-AC 3 mm x 4 mm). An HPLC system (HTEC500, EICOM) attaching this column consists of a pump connected with a degasser, a sampling injector with a sample processor and a cool pump, a fluorescence HPLC monitor and a personal computer with the data processor (Power Chrom; EPC-500, EICOM). The mobile phase used for separation of amino acids was 100 mM, pH 6 phosphate buffer containing 30% methanol and 10 M EDTA. The flow rate was 0.23 ml/min. Peak areas of unknown substances were compared to those of control compounds for

To determine the effect of drugs on the level of Glu, the average amounts of Glu concentration in two 20-min fractions collected over periods of 40 min before and after local application of capsaicin cream were obtained and expressed as percentages of the control

While the animals were inside the small cage, drugs were administered into the s.c. left hindpaw in a volume of 50 l using a 100 l Hamilton syringe (Reno, NV, USA) with a 30 gauge needle without any anesthesia. The needle was inserted into the plantar skin proximal to the midpoint of the hindpaw. Capsazepine (30mg/kg) was injected in the

The Plantar Test (model 7370; Ugo Basile, Verese, Italy) was used in accordance with previously described methods (Yonehara et al., 1997) to determine whether the rats were hyperalgesic. In brief, prior to testing, the animals were placed in a small cage on a glass

Behavioral evidence supports a role for peripheral Glu receptors in normal nociceptive transmission. Intraplantar injection of L-Glu into the hindpaw evokes hyperalgesia in rats (Follenfant and Nakamura-Craig, 1992; Carlton et al., 1995). Futhermore, intraplantar injection of the specific Glu receptors agonists NMDA, AMPA or kainate results in mechanical hyperalgesia and allodynia that can be blocked by appropriate antagonists (Zhou et al., 1996). Hyperalgesia is induced by binding the released glutamate to NMDA receptor (Leem et al., 2001; Du et al., 2003), group I mGluR (Bhave et al., 2001; Zhou et al., 2001; Hu et al., 2002; Walker et al., 2001; Lee et al., 2007), but not group II mGluR (Yang and Gereau IV, 2003).

In addition to these behavioral and anatomical data, Omote et al. (1998) showed that subcutaneous administration of inflammatory substances such as formalin induced the release of peripheral EAAs (Glu and aspartate) on the ipsilateral side. We have already reported that local application of capsaicin cream evoked a marked increase in Glu level in the s.c. perfusate. In addition, electrical stimulation of the sciatic nerve or noxious heat stimulation (50C) also caused increase of Glu level in the s.c. space, and this capsaicinevoked Glu release was significantly decreased by daily high-dose pretreatment with capsaicin for three consecutive days (Jin et al., 2006).

The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), is located in a neurochemically heterogeneous population of small diameter primary afferent neurons (Tominaga et al., 1998). This receptor is sensitive to high temperature in the noxious range of 43°C to 50°C (Hardy, 1953; Beitel and Dubner, 1976; Caterina et al., 1997). Furthermore, repeated exposure to high-dose capsaicin selectively produces a prolonged influx of cations leading to desensitization of small-diameter sensory neurons to subsequent noxious stimulation (Yonehara et al., 1987; Lynn, 1990; Zhou et al., 1998; Caterina and Julius, 2001), while myelinated Afibers are insensitive to capsaicin (Jancso et al., 1977; Nagy et al., 1983; Michael and Priestly, 1999).

There is an evidence suggesting possibility that capsaicin-evoked pain responses might be regulated by peripheral GluRs. In this connection, Lam et al. (2005) demonstrated that peripheral NMDA receptor modulate jaw muscle electromyographic activity induced by capsaicin injection into the temporomandibular joint of rats.

This study, therefore, has been done to elucidate at large in what manner Glu receptors and Glu existing in the peripheral endings of small-diameter afferent fibers and their extracellular space, respectively, are involved in development and/or maintenance of nociception evoked by capsaicin. Additionally, in order to demonstrate a link between the increase of Glu levels in the extracellular space following noxious stimulation and pain behavior, the changes in thermal withdrawal latency and the expression of c-Fos protein in the dorsal horn were determined following subcutaneous (s.c.) injection of drugs associated with Glu receptors with/without capsaicin.

## **2. Materials and methods**

All surgical and experimental procedures for animals were reviewed and approved by the Ohu University Intramural Animal Care and Use Committee and conformed to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).

#### **2.1 Experimental procedures**

44 Pharmacology

Behavioral evidence supports a role for peripheral Glu receptors in normal nociceptive transmission. Intraplantar injection of L-Glu into the hindpaw evokes hyperalgesia in rats (Follenfant and Nakamura-Craig, 1992; Carlton et al., 1995). Futhermore, intraplantar injection of the specific Glu receptors agonists NMDA, AMPA or kainate results in mechanical hyperalgesia and allodynia that can be blocked by appropriate antagonists (Zhou et al., 1996). Hyperalgesia is induced by binding the released glutamate to NMDA receptor (Leem et al., 2001; Du et al., 2003), group I mGluR (Bhave et al., 2001; Zhou et al., 2001; Hu et al., 2002; Walker et al., 2001; Lee et al., 2007), but not group II mGluR (Yang and

In addition to these behavioral and anatomical data, Omote et al. (1998) showed that subcutaneous administration of inflammatory substances such as formalin induced the release of peripheral EAAs (Glu and aspartate) on the ipsilateral side. We have already reported that local application of capsaicin cream evoked a marked increase in Glu level in the s.c. perfusate. In addition, electrical stimulation of the sciatic nerve or noxious heat stimulation (50C) also caused increase of Glu level in the s.c. space, and this capsaicinevoked Glu release was significantly decreased by daily high-dose pretreatment with

The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), is located in a neurochemically heterogeneous population of small diameter primary afferent neurons (Tominaga et al., 1998). This receptor is sensitive to high temperature in the noxious range of 43°C to 50°C (Hardy, 1953; Beitel and Dubner, 1976; Caterina et al., 1997). Furthermore, repeated exposure to high-dose capsaicin selectively produces a prolonged influx of cations leading to desensitization of small-diameter sensory neurons to subsequent noxious stimulation (Yonehara et al., 1987; Lynn, 1990; Zhou et al., 1998; Caterina and Julius, 2001), while myelinated Afibers are insensitive to capsaicin (Jancso et al., 1977; Nagy et al., 1983;

There is an evidence suggesting possibility that capsaicin-evoked pain responses might be regulated by peripheral GluRs. In this connection, Lam et al. (2005) demonstrated that peripheral NMDA receptor modulate jaw muscle electromyographic activity induced by

This study, therefore, has been done to elucidate at large in what manner Glu receptors and Glu existing in the peripheral endings of small-diameter afferent fibers and their extracellular space, respectively, are involved in development and/or maintenance of nociception evoked by capsaicin. Additionally, in order to demonstrate a link between the increase of Glu levels in the extracellular space following noxious stimulation and pain behavior, the changes in thermal withdrawal latency and the expression of c-Fos protein in the dorsal horn were determined following subcutaneous (s.c.) injection of drugs associated

All surgical and experimental procedures for animals were reviewed and approved by the Ohu University Intramural Animal Care and Use Committee and conformed to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).

Gereau IV, 2003).

Michael and Priestly, 1999).

capsaicin for three consecutive days (Jin et al., 2006).

capsaicin injection into the temporomandibular joint of rats.

with Glu receptors with/without capsaicin.

**2. Materials and methods** 

Adult male Sprague-Dawley rats weighing between 200-300 g (CLEA Japan, INC. Tokyo, Japan) were used in all experiments. Rats were on a 12 hrs light/dark cycle and received food and water ad libitum.

## **2.2 Release of Glu into the subcutaneous space**

Animals were anesthetized with urethane (1 g/kg i.p.). A single loop catheter whose tip was covered with a 5000 molecular weight dialysis membrane (MS 0045, PSS® SELECT, Florida) was introduced into the s.c. space of the instep using a 2.2 mm outer diameter polyethylene tube as a guide. Ringer's solution was perfused at 15 l/min through this catheter with a micro syringe pump (EP-60, Eicom, Kyoto, Japan) and perfusate was collected into the tubes placed in an ice bath at intervals of 20 min. The samples were kept at -80°C until analysis.

#### **2.3 Amino acid analysis**

Amino acids in the dialysate were analyzed by a high-performance liquid chromatography (HPLC) system for automated analysis of amino acids using o-phthalaldehyde derivatization and fluorescence detection. Amino acids were quantified by reverse-phase chromatography using a C18 octadecylsilyl (ODS) silica-gel column (EICOMPAK SC-50DS 2.1 mm x 150 mm) with pre-column (EICOM PREPAKSET-AC 3 mm x 4 mm). An HPLC system (HTEC500, EICOM) attaching this column consists of a pump connected with a degasser, a sampling injector with a sample processor and a cool pump, a fluorescence HPLC monitor and a personal computer with the data processor (Power Chrom; EPC-500, EICOM). The mobile phase used for separation of amino acids was 100 mM, pH 6 phosphate buffer containing 30% methanol and 10 M EDTA. The flow rate was 0.23 ml/min. Peak areas of unknown substances were compared to those of control compounds for quantitation.

To determine the effect of drugs on the level of Glu, the average amounts of Glu concentration in two 20-min fractions collected over periods of 40 min before and after local application of capsaicin cream were obtained and expressed as percentages of the control value before stimulation.

#### **2.4 Drug administration**

While the animals were inside the small cage, drugs were administered into the s.c. left hindpaw in a volume of 50 l using a 100 l Hamilton syringe (Reno, NV, USA) with a 30 gauge needle without any anesthesia. The needle was inserted into the plantar skin proximal to the midpoint of the hindpaw. Capsazepine (30mg/kg) was injected in the volume of 50 l into the s.c. of the neck.

#### **2.5 Behavioral assessments**

The Plantar Test (model 7370; Ugo Basile, Verese, Italy) was used in accordance with previously described methods (Yonehara et al., 1997) to determine whether the rats were hyperalgesic. In brief, prior to testing, the animals were placed in a small cage on a glass

Interactions Between Glutamate Receptors and TRPV1 Involved in

pH 9.5 carbonate buffer.

**2.8 Statistical analysis** 

**2.9 Abbreviations** 

N-methyl-D-aspartic acid.

**3.1 Basal Glu release** 

120 min (fraction 5~6 in control group in Fig.1).

**3. Results** 

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 47

(MCCG); selective group III mGlu receptor antagonist, (RS)-α-methylserine-O-phoephate (MSOP). These compounds of Glu receptors were obtained from Tocris (Ballwin, MO, USA). 8- Methyl-N-vanillyl-6-noneamide (capsaicin) was obtained from Sigma Chemical Co. (USA). All other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

In accordance with the product material safety data sheets, L-glutamate acid, L-CCG-I and L-AP4 were diluted in NaOH; and MK801, NMDA, (S)-3, 5,-DHPG, MCCG and MSOP were diluted in water. CNQX, CPCCOEt and MPEP were diluted in dimethyl sulphoxide. The other drugs except for these were dissolved in saline. Capsaicin was prepared as a 10 mg/ml solution in saline containing 10% ethanol and 10% Tween 80. The pH of all solutions was adjusted to 7.4. Capsazepine was dissolved in dimethyl formamide and then diluted with saline. O-phthalaldehyde was dissolved in methanol and adjusted to 4 mM with 0.1 M,

All data are shown as mean ± S.E.M. In the study of Glu release, statistical analyses were performed using posthoc test of Fisher's protected least significant difference and P<0.05 was considered to be statistically significant. In the behavioral study, statistical analyses were performed with Dunnett's test for multiple comparison subsequent to analyses of variance. In the c-Fos immunohistochemichal study, a Student's test was used to test

AMPA-amino-3-hydroxy-4-isoxazole proprionic acid, Cap+MK801; Capsaicin combined with MK801, Cap+CNQX; Capsaicin combined with CNQX, Cap+NBQX; Capsaicin combined with NBQX, Cap+CPCCOEt; Capsaicin combined with CPCCOEt, Cap+MCCG; Capsaicin combined with MCCG, Cap+MSOP; Capsaicin combined with MSOP, CNQX; 6-Cyano-7 nitroquinoxaline-2,3-dione disodium, CPCCOEt; 7-(hydroxyimino) cyclopropa[b]chromen-1acarboxylate ethyl ester, (S)-3,5-DHPG; (S)-3,5- dihydroxyphenylglycine, DRG; dorsal root ganglion, Glu, glutamate; L-CCG-I; (2S,1'S,2'S)-2-(carboxycyclopropyl) glycine, L-AP4; L-(+)-2 amino-4-phosphonobutyric acid, MCCG; (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine, mGluRs; metabotropic glutamate receptors, (+)-MK-801; (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclo-hepten-5, 10-imine hydrogen maleate, MPEP; 2-Methyl-6- (phenylethynyl) pyridine hydrochloride, MSOP; (RS)-α-methylserine-O-phoephate, NBQX; 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, NMDA;

The concentration of Glu in the perfusate was initially high, but gradually decreased with time reaching a stable level after 2 hrs of perfusion, which was then maintained for at least 4.5 h. Glu was present at 1.95 ± 0.25 M (n=10, S.E.M.) in the resting state which is defined here as the mean of the two 20-min fraction collected from 80 min after starting perfusion to

significant differences of the c-Fos expression between the treatments.

plate. They were not restrained and could move about and explore freely. Radiant heat was beamed onto the plantar surface of the hindpaw. The intensity of the beam was controlled and adjusted prior to the experiments, and the cutoff latency was set at 24 sec. The beam was applied to the test and control foot in turns and the latency of the withdrawal reflexes was recorded. The mean of the four responses was determined (Figs 4-8), and the ratio of the test foot latency divided by control foot latency, multiplied by 100, was calculated and termed the "percentage withdrawal latency" (Fig.3), at hourly intervals, from 1 hr before injection of the drugs to 6 h after the injection, except for 15 min after the injection

#### **2.6 c-Fos immunohistochemistry**

Two hours after the drug injection, animals were deeply anesthetized with sodiumpentobarbital and perfused transcardially with 100ml of 0.9% saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) and the spinal cord was taken out, postfixed in the same fixative overnight at 4°C, and then immersed into 20% sucrose in 0.1M PB at 4°C until it sank. Serial transverse 60 µm thick sections at L4-6 were cut using a freezing microtome and collected in 0.02 M phosphate buffered saline (PBS). Sections were washed in PBS for 30 min and blocked with 1% normal goat serum for 30 min and then incubated in a rabbit antibody against c-Fos (1:7000 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA) for 60 min in room temperature and then for 12 hrs at 4°C. After washing in PBS for 30 min, sections were incubated in biotinylated goat anti-rabbit antiserum, and washed in PBS for 30 min and then immunohistochemically stained for 60 min using avidinbiotin-peroxidase complex (Vectastain, Vector Laboratories, Burlingame, CA, USA). To visualize peroxidase activity, sections were immersed in 0.05% diaminobenzidine tetrahydrochloride, 0.1% ammonium nickel sulfate and 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.2). Sections were washed in PBS for 30 min and then mounted on gelatin-coated slides, air-dried and coverslipped. The c-Fos-immunoreactive cells of 10 bestlabeled sections were counted in the L5 spinal dorsal horn. In all these tests a double blind procedure was used to prevent the observers from knowing the experimental groups.

#### **2.7 Drugs**

The list of drugs and chemicals were as follows: as Glu receptors agonist, L-glutamic acid; selective NMDA receptor agonist, NMDA; AMPA receptor agonist, -amino-3-hydroxy-4 isoxazoleproprionic acid (AMPA); selective group 1 mGlu receptor agonist, (S)-3,5 dihydroxyphenylglycine ((S)-3,5-DHPG); group II mGlu receptor agonist, (2S,1'S,2'S)-2- (carboxycyclopropyl) glycine (L-CCG-I); selective group III mGlu receptor agonist, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). The following drugs were used for Glu receptors antagonists, selective non-competitive NMDA receptor antagonist, (5S,10R)-(+)-5- Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclo-hepten-5, 10-imine hydrogen maleate ((+)-MK-801 hydrogen maleate); competitive kainite/AMPA receptor antagonist, 6-Cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) and 2,3-Dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX); group 1 mGlu receptor selective non-competitive mGlu1 receptor antagonist, 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt); group 1 mGlu receptor mGlu5 subtype-selective antagonist, 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP); group II mGlu receptor antagonist, ((2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG); selective group III mGlu receptor antagonist, (RS)-α-methylserine-O-phoephate (MSOP). These compounds of Glu receptors were obtained from Tocris (Ballwin, MO, USA). 8- Methyl-N-vanillyl-6-noneamide (capsaicin) was obtained from Sigma Chemical Co. (USA). All other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

In accordance with the product material safety data sheets, L-glutamate acid, L-CCG-I and L-AP4 were diluted in NaOH; and MK801, NMDA, (S)-3, 5,-DHPG, MCCG and MSOP were diluted in water. CNQX, CPCCOEt and MPEP were diluted in dimethyl sulphoxide. The other drugs except for these were dissolved in saline. Capsaicin was prepared as a 10 mg/ml solution in saline containing 10% ethanol and 10% Tween 80. The pH of all solutions was adjusted to 7.4. Capsazepine was dissolved in dimethyl formamide and then diluted with saline. O-phthalaldehyde was dissolved in methanol and adjusted to 4 mM with 0.1 M, pH 9.5 carbonate buffer.

#### **2.8 Statistical analysis**

46 Pharmacology

plate. They were not restrained and could move about and explore freely. Radiant heat was beamed onto the plantar surface of the hindpaw. The intensity of the beam was controlled and adjusted prior to the experiments, and the cutoff latency was set at 24 sec. The beam was applied to the test and control foot in turns and the latency of the withdrawal reflexes was recorded. The mean of the four responses was determined (Figs 4-8), and the ratio of the test foot latency divided by control foot latency, multiplied by 100, was calculated and termed the "percentage withdrawal latency" (Fig.3), at hourly intervals, from 1 hr before

Two hours after the drug injection, animals were deeply anesthetized with sodiumpentobarbital and perfused transcardially with 100ml of 0.9% saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) and the spinal cord was taken out, postfixed in the same fixative overnight at 4°C, and then immersed into 20% sucrose in 0.1M PB at 4°C until it sank. Serial transverse 60 µm thick sections at L4-6 were cut using a freezing microtome and collected in 0.02 M phosphate buffered saline (PBS). Sections were washed in PBS for 30 min and blocked with 1% normal goat serum for 30 min and then incubated in a rabbit antibody against c-Fos (1:7000 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA) for 60 min in room temperature and then for 12 hrs at 4°C. After washing in PBS for 30 min, sections were incubated in biotinylated goat anti-rabbit antiserum, and washed in PBS for 30 min and then immunohistochemically stained for 60 min using avidinbiotin-peroxidase complex (Vectastain, Vector Laboratories, Burlingame, CA, USA). To visualize peroxidase activity, sections were immersed in 0.05% diaminobenzidine tetrahydrochloride, 0.1% ammonium nickel sulfate and 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.2). Sections were washed in PBS for 30 min and then mounted on gelatin-coated slides, air-dried and coverslipped. The c-Fos-immunoreactive cells of 10 bestlabeled sections were counted in the L5 spinal dorsal horn. In all these tests a double blind procedure was used to prevent the observers from knowing the experimental groups.

The list of drugs and chemicals were as follows: as Glu receptors agonist, L-glutamic acid; selective NMDA receptor agonist, NMDA; AMPA receptor agonist, -amino-3-hydroxy-4 isoxazoleproprionic acid (AMPA); selective group 1 mGlu receptor agonist, (S)-3,5 dihydroxyphenylglycine ((S)-3,5-DHPG); group II mGlu receptor agonist, (2S,1'S,2'S)-2- (carboxycyclopropyl) glycine (L-CCG-I); selective group III mGlu receptor agonist, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). The following drugs were used for Glu receptors antagonists, selective non-competitive NMDA receptor antagonist, (5S,10R)-(+)-5- Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclo-hepten-5, 10-imine hydrogen maleate ((+)-MK-801 hydrogen maleate); competitive kainite/AMPA receptor antagonist, 6-Cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) and 2,3-Dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX); group 1 mGlu receptor selective non-competitive mGlu1 receptor antagonist, 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt); group 1 mGlu receptor mGlu5 subtype-selective antagonist, 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP); group II mGlu receptor antagonist, ((2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine

injection of the drugs to 6 h after the injection, except for 15 min after the injection

**2.6 c-Fos immunohistochemistry** 

**2.7 Drugs** 

All data are shown as mean ± S.E.M. In the study of Glu release, statistical analyses were performed using posthoc test of Fisher's protected least significant difference and P<0.05 was considered to be statistically significant. In the behavioral study, statistical analyses were performed with Dunnett's test for multiple comparison subsequent to analyses of variance. In the c-Fos immunohistochemichal study, a Student's test was used to test significant differences of the c-Fos expression between the treatments.

#### **2.9 Abbreviations**

AMPA-amino-3-hydroxy-4-isoxazole proprionic acid, Cap+MK801; Capsaicin combined with MK801, Cap+CNQX; Capsaicin combined with CNQX, Cap+NBQX; Capsaicin combined with NBQX, Cap+CPCCOEt; Capsaicin combined with CPCCOEt, Cap+MCCG; Capsaicin combined with MCCG, Cap+MSOP; Capsaicin combined with MSOP, CNQX; 6-Cyano-7 nitroquinoxaline-2,3-dione disodium, CPCCOEt; 7-(hydroxyimino) cyclopropa[b]chromen-1acarboxylate ethyl ester, (S)-3,5-DHPG; (S)-3,5- dihydroxyphenylglycine, DRG; dorsal root ganglion, Glu, glutamate; L-CCG-I; (2S,1'S,2'S)-2-(carboxycyclopropyl) glycine, L-AP4; L-(+)-2 amino-4-phosphonobutyric acid, MCCG; (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine, mGluRs; metabotropic glutamate receptors, (+)-MK-801; (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclo-hepten-5, 10-imine hydrogen maleate, MPEP; 2-Methyl-6- (phenylethynyl) pyridine hydrochloride, MSOP; (RS)-α-methylserine-O-phoephate, NBQX; 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, NMDA; N-methyl-D-aspartic acid.
