**3. Results**

#### **3.1 Basal Glu release**

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

Interactions Between Glutamate Receptors and TRPV1 Involved in

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 49

Fig. 2. Effect of the ionotropic (A) and metabotropic (B) glutamate receptor antagonists on

subcutaneously injected together with capsaicin at the time indicated by the arrow. All data are presented as the mean ± S.E.M. obtained from 10 animals. #*P*<0.05 compare with the

capsaicin+vehicle group at each time measured. MK801, selective non-competitive NMDA receptor antagonist; NBQX, competitive kainate/AMPA receptor antagonist; CPCOOEt, group 1 mGlu receptor selective non-competitive mGlu1 receptor antagonist; MCCG, group

At the doses employed, CPCCOEt (5 mM) (Cap + CPCCOEt) showed remarkable inhibition in capsaicin-evoked Glu release. The average concentration of the released Glu was 1.46 ± 0.1 M/ 20 min after the co-injection of CPCCOEt with capsaicin. S.c. combined injection of MCCG (5 mM) (Cap + MCCG) or MSOP (5 mM) (Cap + MSOP) with capsaicin did not show significant decrease in Glu release compared to capsaicin injection alone. The average concentration of the released Glu was 3.68 ± 0.38 M / 20 min or 4.31 ± 0.60 M/ 20min in 2 fractions collected after the co-injection of MCCG or MSOP with capsaicin, respectively.

The mean withdrawal latencies to stimulation with radiant heat at pre-injection were 11.2 ± 0.3 s and 11.2 ± 0.3 s (n=40) on the left and right side, respectively (Fig.3-A). The withdrawal

the capsaicin-induced glutamate release. The glutamate receptor antagonists were

value prior to s.c. administration of capsaicin+vehicle. \**P*<0.05 compared with

II mGlu receptor antagonist; MSOP, selective group III mGlu receptor antagonist.

**3.4 Effects of mGluRs antagonists injection on capsaicin-induced Glu release** 

**3.5 Effects of capsazepine on capsaicin-induced thermal hypersensitivity** 

(Fig. 2-B)

#### **3.2 Effects of capsazepine on capsaicin-evoked Glu release**

The s.c. injection of capsaicin (3 mM) in the vicinity of the perfusion side evoked a significant increase in Glu release (Fig.1). The average concentration of the released Glu was 4.86 ± 0.48 M/20 min in 2 fractions collected after the injection of capsaicin. This augmentation of Glu release was last over 2 h. This effect was remarkably suppressed by preadministration of capsazepine (30 mg/kg, s.c.) 30 min before capsaicin injection (Fig.1). In the group of pretreatment with capsazepine, the average concentrations of the released Glu were 2.25 ± 0.4 M/20 min and 2.36 ± 0.31 M/20 min in 2 fractions collected after the injection of vehicle or capsaicin, respectively. S.c. injections of vehicle or capsazepine alone did not produce any significant changes in the levels of Glu in the perfusates.

Fig. 1. Effect of capsazepine on the capsaicin-induced glutamate release. Capsazepine (s.c.) was injected subcutaneously into the neck 30 min before capsaicin treatment. Capsazepine (30 mg/kg) or vehicle for capsazepine, and capsaicin (3 mM) or vehicle for capsaicin were subcutaneously injected at the time indicated by the arrows, ( ) and ( ), respectively. All data are presented as the mean ± S.E.M. obtained from 10 animals. #P<0.05 compare with the value prior to s.c. administration of capsaicin+vehicle. \*P<0.05 compared with capsaicin+vehicle (for capsazepine) group at each time measured.

#### **3.3 Effects of iGluRs antagonists injection on capsaicin-evoked Glu release**

The combined injection of capsaicin with MK801(1 mM) (Cap + MK-801) or NBQX (5 mM) (Cap + NBQX) into the perfusion region showed far less Glu release than injection of capsaicin alone (Fig 2-A). The average concentration of the released Glu was 1.20 ± 0.1 M/ 20 min or 1.70 ± 0.1 M/ 20min in 2 fractions collected after the co-injection of MK-801 or NBQX with capsaicin, respectively. These inhibitory effects of iGluRs antagonists sustained over 2.5 h.

The s.c. injection of capsaicin (3 mM) in the vicinity of the perfusion side evoked a significant increase in Glu release (Fig.1). The average concentration of the released Glu was 4.86 ± 0.48 M/20 min in 2 fractions collected after the injection of capsaicin. This augmentation of Glu release was last over 2 h. This effect was remarkably suppressed by preadministration of capsazepine (30 mg/kg, s.c.) 30 min before capsaicin injection (Fig.1). In the group of pretreatment with capsazepine, the average concentrations of the released Glu were 2.25 ± 0.4 M/20 min and 2.36 ± 0.31 M/20 min in 2 fractions collected after the injection of vehicle or capsaicin, respectively. S.c. injections of vehicle or capsazepine alone did not produce any significant changes in the levels of Glu in the

Fig. 1. Effect of capsazepine on the capsaicin-induced glutamate release. Capsazepine (s.c.) was injected subcutaneously into the neck 30 min before capsaicin treatment. Capsazepine (30 mg/kg) or vehicle for capsazepine, and capsaicin (3 mM) or vehicle for capsaicin were subcutaneously injected at the time indicated by the arrows, ( ) and ( ), respectively. All data are presented as the mean ± S.E.M. obtained from 10 animals. #P<0.05 compare with

the value prior to s.c. administration of capsaicin+vehicle. \*P<0.05 compared with

**3.3 Effects of iGluRs antagonists injection on capsaicin-evoked Glu release** 

The combined injection of capsaicin with MK801(1 mM) (Cap + MK-801) or NBQX (5 mM) (Cap + NBQX) into the perfusion region showed far less Glu release than injection of capsaicin alone (Fig 2-A). The average concentration of the released Glu was 1.20 ± 0.1 M/ 20 min or 1.70 ± 0.1 M/ 20min in 2 fractions collected after the co-injection of MK-801 or NBQX with capsaicin, respectively. These inhibitory effects of iGluRs antagonists sustained

capsaicin+vehicle (for capsazepine) group at each time measured.

**3.2 Effects of capsazepine on capsaicin-evoked Glu release** 

perfusates.

over 2.5 h.

Fig. 2. Effect of the ionotropic (A) and metabotropic (B) glutamate receptor antagonists on the capsaicin-induced glutamate release. The glutamate receptor antagonists were subcutaneously injected together with capsaicin at the time indicated by the arrow. All data are presented as the mean ± S.E.M. obtained from 10 animals. #*P*<0.05 compare with the value prior to s.c. administration of capsaicin+vehicle. \**P*<0.05 compared with capsaicin+vehicle group at each time measured. MK801, selective non-competitive NMDA receptor antagonist; NBQX, competitive kainate/AMPA receptor antagonist; CPCOOEt, group 1 mGlu receptor selective non-competitive mGlu1 receptor antagonist; MCCG, group II mGlu receptor antagonist; MSOP, selective group III mGlu receptor antagonist.

#### **3.4 Effects of mGluRs antagonists injection on capsaicin-induced Glu release**

At the doses employed, CPCCOEt (5 mM) (Cap + CPCCOEt) showed remarkable inhibition in capsaicin-evoked Glu release. The average concentration of the released Glu was 1.46 ± 0.1 M/ 20 min after the co-injection of CPCCOEt with capsaicin. S.c. combined injection of MCCG (5 mM) (Cap + MCCG) or MSOP (5 mM) (Cap + MSOP) with capsaicin did not show significant decrease in Glu release compared to capsaicin injection alone. The average concentration of the released Glu was 3.68 ± 0.38 M / 20 min or 4.31 ± 0.60 M/ 20min in 2 fractions collected after the co-injection of MCCG or MSOP with capsaicin, respectively. (Fig. 2-B)

#### **3.5 Effects of capsazepine on capsaicin-induced thermal hypersensitivity**

The mean withdrawal latencies to stimulation with radiant heat at pre-injection were 11.2 ± 0.3 s and 11.2 ± 0.3 s (n=40) on the left and right side, respectively (Fig.3-A). The withdrawal

Interactions Between Glutamate Receptors and TRPV1 Involved in

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 51

Fig. 4. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of various concentration of the ionotropic glutamate receptor agonists; glutamate, NMDA and AMPA. The data for each group (at least 10 animals) are presented as the means

Fig. 5. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of various concentration of the metabotropic glutamate receptor agonists; (s)- DHPG, L-CCG-I and L-AP4. The data for each group (at least 10 animals) are presented as

the means ± S.E.M. \**P* <0.05 significantly different from vehicle-treated group.

± S.E.M. \**P* <0.05 significantly different from vehicle-treated group.

latency did not significantly change after injection of vehicle or low dose of capsaicin (0.6 mM). A quarter and one h after injection of capsaicin (3 mM and 6 mM), withdrawal latency to irradiation decreased to much shorter than that of vehicle injection, which was recorded at the same interval (P<0.05), and then recovered gradually to the level of vehicle injection by 4 h after injection of capsaicin. Pretreatment of capsazepine (30 mg/kg, s.c.) produced a marked inhibition against capsaicin-induced thermal hyperalgesia (Fig.3-B). We did not observed any signs of motor deficiency or other side effects for any of the doses of any drugs in all paradigms described here and below.

Fig. 3. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin, and co-injection of capsazepine with capsaicin. The data for each group (10 animals) are presented as the means ± S.E.M. The withdrawal latency per animal at respective time points was calculated as the average of the latencies obtained from 3 consecutive stimuli applied at intervals of 5 min. The value at time zero (pre) was obtained 1 h prior to s.c. injection of capsaicin. \* and # *P*<0.05 significantly different from vehicletreated group and capsaicin-treated group (3 mM), respectively.

#### **3.6 Thermal sensitivity after injection of iGluRs agonists**

S.c. injections of Glu, NMDA or AMPA produced dose-dependent decreases in withdrawal latency on the ipsilateral side 15 min after s.c. injection, and lasted for a few hours (Fig. 4). S.c. injection of vehicle did not produced any changes in thermalwithdrawal latency.

#### **3.7 Thermal sensitivity after injection of mGluRs agonists**

S.c. injection of (s)-DHPG caused a dose-dependent decrease in withdrawal latencies on the ipsilateral side from 15 min to 6 h, but L-CCG-I and L-AP4 did not show any significant changes (Fig. 5).

Interactions Between Glutamate Receptors and TRPV1 Involved in Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 51

50 Pharmacology

latency did not significantly change after injection of vehicle or low dose of capsaicin (0.6 mM). A quarter and one h after injection of capsaicin (3 mM and 6 mM), withdrawal latency to irradiation decreased to much shorter than that of vehicle injection, which was recorded at the same interval (P<0.05), and then recovered gradually to the level of vehicle injection by 4 h after injection of capsaicin. Pretreatment of capsazepine (30 mg/kg, s.c.) produced a marked inhibition against capsaicin-induced thermal hyperalgesia (Fig.3-B). We did not observed any signs of motor deficiency or other side effects for any of the doses of any

Fig. 3. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin, and co-injection of capsazepine with capsaicin. The data for each group (10 animals) are presented as the means ± S.E.M. The withdrawal latency per animal at respective time points was calculated as the average of the latencies obtained from 3 consecutive stimuli applied at intervals of 5 min. The value at time zero (pre) was obtained 1 h prior to s.c. injection of capsaicin. \* and # *P*<0.05 significantly different from vehicle-

S.c. injections of Glu, NMDA or AMPA produced dose-dependent decreases in withdrawal latency on the ipsilateral side 15 min after s.c. injection, and lasted for a few hours (Fig. 4). S.c. injection of vehicle did not produced any changes in thermal-

S.c. injection of (s)-DHPG caused a dose-dependent decrease in withdrawal latencies on the ipsilateral side from 15 min to 6 h, but L-CCG-I and L-AP4 did not show any significant

treated group and capsaicin-treated group (3 mM), respectively.

**3.6 Thermal sensitivity after injection of iGluRs agonists** 

**3.7 Thermal sensitivity after injection of mGluRs agonists** 

withdrawal latency.

changes (Fig. 5).

drugs in all paradigms described here and below.

Fig. 4. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of various concentration of the ionotropic glutamate receptor agonists; glutamate, NMDA and AMPA. The data for each group (at least 10 animals) are presented as the means ± S.E.M. \**P* <0.05 significantly different from vehicle-treated group.

Fig. 5. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of various concentration of the metabotropic glutamate receptor agonists; (s)- DHPG, L-CCG-I and L-AP4. The data for each group (at least 10 animals) are presented as the means ± S.E.M. \**P* <0.05 significantly different from vehicle-treated group.

Interactions Between Glutamate Receptors and TRPV1 Involved in

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 53

Fig. 7. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the metabotropic glutamate receptor antagonists; CPCCOEt and MPEP. The data for each group (at least 10 animals) are presented as the means ± S.E.M. \**P* <0.05 significantly different from capsaicin (3 mM)-treated group.

Fig. 8. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the metabotropic glutamate receptor antagonists; MCCG and MSOP. The data for each group (at least 10 animals) are presented as the means

± S.E.M

#### **3.8 Effect of iGluRs antagonists injection on capsaicin-induced thermal hypersensitivity**

When MK801 or CNQX were injected together with capsaicin (Cap+MK801 or Cap+CNQX), a dose-dependent increase in withdrawal latency was observed. These analgesic effects of MK801 or CNQX on capsaicin-induced thermal hyperalgesia lasted for more than 6 h (Fig. 6). The single injection of MK801 or CNQX into the hindpaw did not show changes in withdrawal latencies compared to vehicle injection.

Fig. 6. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the ionotropic glutamate receptor antagonists; MK801 and CNQX. The data for each group (at least 10 animals) are presented as the means

#### **3.9 Effect of mGluRs antagonists injection on capsaicin-induced thermal hypersensitivity**

± S.E.M. \**P* <0.05 significantly different from capsaicin (3 mM)-treated group.

Following s.c. injection of CPCCOEt (5 mM), MPEP (30 mM), MCCG (5 mM), and MSOP (5 mM) into hindpaw, there was no changes in withdrawal latencies compared to vehicle injection (Figs. 7 and 8). When CPCCOEt or MPEP were injected together with capsaicin (Cap+CPCCOEt or Cap+MPEP), withdrawal latencies showed a dose-dependent increase from 15 min to 2~3 h after the injection compared with when capsaicin was injected alone (P<0.05) (Fig. 7). The heat insensitivity evoked in ipsilateral side following Cap+CPCCOEt and Cap+MPEP injection continued for 5 h or more. S.c. injection of MCCG or MSOP combined with capsaicin did not show any significant changes in withdrawal latencies compared to capsaicin injection alone (Fig. 8).

When MK801 or CNQX were injected together with capsaicin (Cap+MK801 or Cap+CNQX), a dose-dependent increase in withdrawal latency was observed. These analgesic effects of MK801 or CNQX on capsaicin-induced thermal hyperalgesia lasted for more than 6 h (Fig. 6). The single injection of MK801 or CNQX into the hindpaw did not show changes in

Fig. 6. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the ionotropic glutamate receptor antagonists; MK801 and CNQX. The data for each group (at least 10 animals) are presented as the means

Following s.c. injection of CPCCOEt (5 mM), MPEP (30 mM), MCCG (5 mM), and MSOP (5 mM) into hindpaw, there was no changes in withdrawal latencies compared to vehicle injection (Figs. 7 and 8). When CPCCOEt or MPEP were injected together with capsaicin (Cap+CPCCOEt or Cap+MPEP), withdrawal latencies showed a dose-dependent increase from 15 min to 2~3 h after the injection compared with when capsaicin was injected alone (P<0.05) (Fig. 7). The heat insensitivity evoked in ipsilateral side following Cap+CPCCOEt and Cap+MPEP injection continued for 5 h or more. S.c. injection of MCCG or MSOP combined with capsaicin did not show any significant changes in withdrawal latencies

± S.E.M. \**P* <0.05 significantly different from capsaicin (3 mM)-treated group.

**3.9 Effect of mGluRs antagonists injection on capsaicin-induced thermal** 

**3.8 Effect of iGluRs antagonists injection on capsaicin-induced thermal** 

withdrawal latencies compared to vehicle injection.

**hypersensitivity** 

**hypersensitivity** 

compared to capsaicin injection alone (Fig. 8).

Fig. 7. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the metabotropic glutamate receptor antagonists; CPCCOEt and MPEP. The data for each group (at least 10 animals) are presented as the means ± S.E.M. \**P* <0.05 significantly different from capsaicin (3 mM)-treated group.

Fig. 8. Time course of withdrawal latencies in response to noxious heat stimulation after s.c. injection of capsaicin in combination with the metabotropic glutamate receptor antagonists; MCCG and MSOP. The data for each group (at least 10 animals) are presented as the means ± S.E.M

Interactions Between Glutamate Receptors and TRPV1 Involved in

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 55

injection (I/II, 60 ± 5; III/IV, 22 ± 8). The numbers of capsaicin-induced c-Fosimmunopositive cells in laminae I/II (489 ± 34), but not in laminae III/IV (63 ± 18), were significantly decreased (P<0,005), when MK801 and CNQX were injected with capsaicin (Cap+MK801, I/II, 227 ± 32, III/IV, 14 ± 4; Cap+CNQX, I/II, 205 ± 40, III/IV, 11 ± 7) (Fig. 10 and Table 1). The numbers of capsaicin-induced c-Fos-immunopositive cells on the contralateral sides did not significantly change by any of drugs with/without capsaicin.

Fig. 10. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of capsaicin alone (A, B), combined with MK801 (C), and combined with MK801 CNQX (D). A, C and D: ipisilateral side. B: contralateral side. Solid line indicates

**3.12 Effects of metabotropic glu antagonists injeection on the capsaicin-induced c-**

compared to single injection of capsaicin, respectively (Fig. 11 and Table 1).

Few c-Fos-immunopositive cells in the ipsilateral laminae I/II and III/IV, and fewer cells in the contralateral sides, were observed with single injection of CPCCOEt (I/II, 59 ± 8, III/IV, 1 ± 1), MCCG (I/II, 63 ± 10, III/IV, 3 ± 2) and MSOP (I/II, 66 ± 16, III/IV, 5 ± 3). Co-injection of CPCCOEt with capsaicin (Cap+CPCCOEt) significantly decreased the number of capsaicin-induced c-Fos-immunopositive cells in the ipsilateral laminae I/II (236 ± 58), but not in laminae III/IV (6 ± 4) and contralateral laminae I/II and III/IV. There was no significant change in the number of c-Fos-immunopositive cells in the ipsilateral laminae I/II, and III/IV by administration of MCCG combined with capsaicin (Cap+MCCG) or by administration of MSOP combined with capsaicin (Cap+MSOP; I/II, 383 ± 21, III/IV, 22 ± 3)

100 μm.

**Fos expression** 

#### **3.10 Basal c-Fos expression in dorsal horn after injection of vehicle, capsaicin and Glu into hindpaw**

Immunoreactivity for c-Fos appeared gray-to-black and homogeneously labeled the oval or roundish nucleus of cells in spinal dorsal horn at L5 (Figs. 9-11). In all the experimental tests with injection of Glu, the maximum number of labeled cells occurred consistently in laminae I and II (I/II) of the spinal dorsal horn on the ipsilateral side (mean number ± S.E.M.=268 ± 21) (Figs. 9-11 and Table 1). Much smaller number of c-Fos immunopositive cells occurred in lamine III and IV (III/IV, 30 ± 7). The capsaicin-induced c-Fos expression in laminae I/II (489 ± 34) and laminae III/IV (63 ± 18) on the ipsilateral side was greater than that with Glu (Figs. 9, 10 and Table 1). The numbers of c-Fos-immunopositive cells on the contralateral side was modest either with glutamate (I/II, 16 ± 7; III/IV, 12 ± 6) or capsaicin (I/II, 44 ± 13; III/IV, 20 ± 9). In animals administered with vehicle, c-Fos-immunopositive cells were rarely distributed either in laminae I/II (60 ± 5) or in laminae III/IV (22 ± 8) on the ipsilateral side or on the contralateral side (I/II, 12 ± 4; III/IV, 7 ± 2) (Table 1).

Fig. 9. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of vehicle and glutamate. A and C: ipisilateral side. B and D: contralateral side. Solid line indicates 100 μm.

#### **3.11 Effects of ionotropic Glu receptors antagonists injection on the capsaicininduced c-Fos expression**

Few c-Fos-immunopositive cells were found in laminae I/II and laminae III/IV of the ipsilateral dorsal horn after each single injection of ionotropic Glu receptors antagonists MK-801 (I/II, 79 ± 3; III/IV, 11 ± 7) and CNQX (I/II, 70 ± 8; III/IV, 7 ± 3) similar to vehicle

Immunoreactivity for c-Fos appeared gray-to-black and homogeneously labeled the oval or roundish nucleus of cells in spinal dorsal horn at L5 (Figs. 9-11). In all the experimental tests with injection of Glu, the maximum number of labeled cells occurred consistently in laminae I and II (I/II) of the spinal dorsal horn on the ipsilateral side (mean number ± S.E.M.=268 ± 21) (Figs. 9-11 and Table 1). Much smaller number of c-Fos immunopositive cells occurred in lamine III and IV (III/IV, 30 ± 7). The capsaicin-induced c-Fos expression in laminae I/II (489 ± 34) and laminae III/IV (63 ± 18) on the ipsilateral side was greater than that with Glu (Figs. 9, 10 and Table 1). The numbers of c-Fos-immunopositive cells on the contralateral side was modest either with glutamate (I/II, 16 ± 7; III/IV, 12 ± 6) or capsaicin (I/II, 44 ± 13; III/IV, 20 ± 9). In animals administered with vehicle, c-Fos-immunopositive cells were rarely distributed either in laminae I/II (60 ± 5) or in laminae III/IV (22 ± 8) on the ipsilateral side

Fig. 9. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of vehicle and glutamate. A and C: ipisilateral side. B and D: contralateral

Few c-Fos-immunopositive cells were found in laminae I/II and laminae III/IV of the ipsilateral dorsal horn after each single injection of ionotropic Glu receptors antagonists MK-801 (I/II, 79 ± 3; III/IV, 11 ± 7) and CNQX (I/II, 70 ± 8; III/IV, 7 ± 3) similar to vehicle

**3.11 Effects of ionotropic Glu receptors antagonists injection on the capsaicin-**

**3.10 Basal c-Fos expression in dorsal horn after injection of vehicle, capsaicin and** 

or on the contralateral side (I/II, 12 ± 4; III/IV, 7 ± 2) (Table 1).

side. Solid line indicates 100 μm.

**induced c-Fos expression** 

**Glu into hindpaw** 

injection (I/II, 60 ± 5; III/IV, 22 ± 8). The numbers of capsaicin-induced c-Fosimmunopositive cells in laminae I/II (489 ± 34), but not in laminae III/IV (63 ± 18), were significantly decreased (P<0,005), when MK801 and CNQX were injected with capsaicin (Cap+MK801, I/II, 227 ± 32, III/IV, 14 ± 4; Cap+CNQX, I/II, 205 ± 40, III/IV, 11 ± 7) (Fig. 10 and Table 1). The numbers of capsaicin-induced c-Fos-immunopositive cells on the contralateral sides did not significantly change by any of drugs with/without capsaicin.

Fig. 10. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of capsaicin alone (A, B), combined with MK801 (C), and combined with MK801 CNQX (D). A, C and D: ipisilateral side. B: contralateral side. Solid line indicates 100 μm.

#### **3.12 Effects of metabotropic glu antagonists injeection on the capsaicin-induced c-Fos expression**

Few c-Fos-immunopositive cells in the ipsilateral laminae I/II and III/IV, and fewer cells in the contralateral sides, were observed with single injection of CPCCOEt (I/II, 59 ± 8, III/IV, 1 ± 1), MCCG (I/II, 63 ± 10, III/IV, 3 ± 2) and MSOP (I/II, 66 ± 16, III/IV, 5 ± 3). Co-injection of CPCCOEt with capsaicin (Cap+CPCCOEt) significantly decreased the number of capsaicin-induced c-Fos-immunopositive cells in the ipsilateral laminae I/II (236 ± 58), but not in laminae III/IV (6 ± 4) and contralateral laminae I/II and III/IV. There was no significant change in the number of c-Fos-immunopositive cells in the ipsilateral laminae I/II, and III/IV by administration of MCCG combined with capsaicin (Cap+MCCG) or by administration of MSOP combined with capsaicin (Cap+MSOP; I/II, 383 ± 21, III/IV, 22 ± 3) compared to single injection of capsaicin, respectively (Fig. 11 and Table 1).

Interactions Between Glutamate Receptors and TRPV1 Involved in

neither acute noxious stimulation nor inflammation.

**4. Discussion** 

days (Jin et al., 2006).

Folkow, 1953).

terminal (Lam et al., 2003; Lam et al., 2004).

Nociceptive Processing at Peripheral Endings of Primary Afferent Fibers 57

We confirmed a large release of Glu immediately after the introduction of the catheter, followed by a rapid decrease, like in our previous study (Yonehara et al., 1987; Yonehara et al., 1992; Yonehara et al., 1995). Insertion of the polyethylene tube into the s.c. space of the rat instep did not evoke any inflammatory responses such as extravasation (Yonehara et al., 1995). All these data suggest that the basal levels of Glu in the s.c. perfusate were caused by

Topical application of capsaicin cream to the instep evoked a marked increase in Glu level in the s.c. perfusate, similar to the results in our previous study (Jin et al., 2006). In addition, electrical stimulation of the sciatic nerve or noxious heat stimulation (50C) also caused an increase of Glu level in the s.c. space, and this capsaicin-evoked Glu release was significantly decreased by daily high-dose pretreatment with capsaicin for three consecutive

The TRPV1 is located in a neurochemically heterogeneous population of small diameter primary afferent neurons (Tominaga et al., 1998). Furthermore, repeated exposure to highdose 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). These findings and the present results suggest that the activation of capsaicin-sensitive afferent fibers by capsaicin causes release of Glu from the peripheral endings via activation of peripheral TRPV1, particularly from those of small-diameter fibers possibly through a mechanism such as the axon-reflex pathway, or autocrine and/or paracrine. It is reasonable to speculate that axon-reflex mechanism is involved in capsaicin-induced Glu release observed in Figs. 1 and 2, as only nociceptive afferent fibers have the axon-reflex mechanism which is localized on superficial tissues exposed to noxious influences (Celander and

Amount of capsaicin-induced Glu release was remarkably decreased by concomitant administration of ionotropic Glu receptors antagonists; MK801 and NBQX, and mGluR I antagonist; CPCCOEt in the hindpaw, but not by administration of group II and III mGluR antagonist; MCCG and MSOP. These results suggest that peripheral ionotropic Glu receptors and group I mGluR appear to play a role in mediating capsaicin-evoked increases in Glu release. The Glu release through the activation of TRPV1 could then further activate ionotoropic Glu receptors and group I mGluR on the same neuronal terminal or adjacent neighboring peripheral terminals. In this connection, there were evidences supporting the co-localization of peripheral NMDA and TRPV1 receptors on the same primary afferent

Activation of peripheral Glu receptors could lead to enhance the Glu release in the peripheral tissues and might alter TRPV1 receptor responsiveness to reinforce nociceptive responses. As it is necessary to investigate the interaction between TRPV1 and glutamate receptors by using specific receptor antagonists of TRPV1 in detail, the mechanism to account for the antagonism of peripheral Glu receptors contributes to inhibit capsaicininduced Glu release remains unanswered. However, it may be possible that glutamate receptors play a pivotal role for the activation of TRPV1 in the peripheral terminals. This

Fig. 11. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of capsaicin alone (A), combined with CPCCOEt (B), with MCCG (C), with MSOP (D). A, B, C and D: ipisilateral side. Solid line indicates 100 μm .


Table 1. Mean value of c-Fos-positive neurons in the dorsal horn of L5 2 h after s.c. injection of Glu receptors agonists and antagonists. The value in each group was represented mean ± S.E.M. obtained from at least 10 animals, and the difference of the means was analyzed with the Student's t-test. \* Significant difference at P< 0.05 between vehicle and capsaicin, or glutamate-treated group. # Significant difference at P< 0.05 between capsaicin and capsaicin+MK801, or capsaicin+CNQX, or capsaicin+CPCCOEt-treated group
