Involvement of GABAB receptors in the antinociception induced by baclofen in the formalin test

Gen. Pharmac. Vol. 28, No. 4, pp. 611-615, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/97 $17.00+.00 PII S0306-3623(96)00241-8 All rights reserved ELSEVIER

Involvement of GABAB Receptors in the Antinociception Induced by Baclofen in the Formalin Test M. Shafizadeh, 1 S. Semnanian, 2. M. R. Zarrindast 3 and B. Hashemi 3 1DEPARTMENTOF PHYSIOLOGY,TARBIATMODARRESUNIVERSITY,TEHRAN, IRAN; 2DEPARTMENTOF PHYSIOLOGY,SHAHEEDBEHESHTIUNIVERSITYOF MEDICALSCIENCES, P. O. Box 19835-181, TEHUaN, leaN [Fax: (21 ) 6404680]; AND 3DEPARTMENTOF PHARMACOLOGY, TEHRAN UNIVERSITYOF MEDICALSCIENCES,TEHRAN, IRAN

ABSTRACT. 1. The effect of GABA receptor antagonists on baclofen-induced antinociception was examined in rats using the formalin test. Intraperitoneal (IP) administration of different doses of baclofen (2.5-10 mg kg l) to rats induced antinociception in both phases of the test. 2. The response was dose-dependent and the maximum response was observed with 10 mg kg 1 of the drug. Intracerebroventricular (ICV) injection of baclofen (0.5-20 ~tg/rat) also induced dose-dependent antinociception in the second phase of the formalin test. 3. The GABAs antagonist, phaclofen (1 mg kg -1, IP) but not the GABAA antagonists picrotoxin (1 mg kg -1, IP) and bicuculline (1.5 mg kg -1, IP), decreased the antinociception induced by both ICV and IP administration of baclofen. 4. It is concluded that baclofen antinociception in the formalin test is mediated through GABAB receptor activation. GEN PHARMAC28;4:611--615, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Baclofen, GABA antagonists, antinociception, rat INTRODUCTION The behavioral effects produced by the neurotransmitter G A B A stems from a cascade of events which follows the binding of the transmitter to its receptors. G A B A interacts with two types of sites in the central nervous system. These sites are referred to as GABAA and GABAB receptors (Bowery et al. 1980, 1981; Matsumoto, 1989). The two types of receptors differ in their pharmacologic profile, sensitivities to ions and nucleotides and anatomic distribution (Hill and Bowery, 1981). Although the mechanism of action of the two receptor types differs, GABA stimulation of both results in neuronal inhibition. G A B A mediates a variety of pharmacologic events, including analgesia (Enna, 1983; Sawynok, 1987; Sivam and Ho, 1983). It is shown that antinociception induced by G A B A agonists is not sensitive to naloxone, suggesting that it does not involve endogenous opioids (Kendall et al., 1982; Zarrindast and Djavdan, 1988). The results obtained by some investigators also indicate that GABAergic drugs share some of the classical properties of morphine, such as analgesia and inhibition of gastrointestinal motility, but they probably do so by different mechanisms (Sivam and Ho, 1983). Systemic injection of the GABAB agonist baclofen produces analgesia in tailflick, hot-plate, writhing and shock titration tests and in arthritis pain (Bartolini et al., 1981; Cutting and Jordan, 1975; Hill et al., 1981; Levy and Proudfit, 1977; Sawynok, 1983; Sawynok and Labella, 1982; Vaught et al., 1985). The GABAA receptor subtype has been implicated in antinocioception induced by baclofen in the tail-flick test (Zarrindast and Djavdan, 1988). Because of reports of the bicuculline-sensitiveaction of baclofen (Henry, 1982; Inoue et al., 1985) and the binding of baclofen to GABAA receptors in high concentration (Olsen et al., 1978) we intended to assess the mechanism of its effect on formalin-induced nociception. In the present *To whom correspondence should be addressed. Received 13 December 1995; accepted 9 May 1996.

work, the mechanism of baclofen-induced antinociception in the formalin test has been investigated. MATERIALS AND METHODS Animals Male NMRI rats (250-350 g) were used in the experiments. The animals were housed in groups of five in conditions of constant temperature (21+2°C) in a light-controlled room (light period, 07:00-19:00 h). Animals had free access to food and water, except during the experiments.

Chronic guide c a n n u l a i m p l a n t a t i o n Stainless-steel guide cannulas (21 gauge) were stereotaxically implanted under sodium pentobarbital (40 mg kg l, IP), 7 days before the experiments. The guide cannula was implanted in the left lateral cerebral ventricle at the following coordinates: A P = - 0 . 8 mm, L = - l . 6 mm (both with respect to bregma) and H=3.5 mm from the dura. It was then fixed to the skull with steel screws and dental cement. Each cannula was kept patent with a steel obturator. The drugs were injected in a volume of 5 ~xl over a period of I min, by means of an internal cannula (28 gauge) connected by polyethylene tubing to a 25-~1 Hamilton syringe, and the injection cannula was left in place for an additional 1 min before being slowly withdrawn.

Drugs The drugs used were baclofen (Ciba-Geigy), picrotoxin and bicuculline (Sigma) and phaclofen (Tocris Neuramin). The drugs were dissolved in distilled water. All drugs except baclofen were administered in a volume of 10 ml kg -1, IP. Baclofen was injected in a volume of 5 ~l/rat, ICV and 10 ml kg -1, IP. Baclofen was administered 15 rain and GABA antagonists 30 rain prior to formalin injection. Six animals were used for each dose of the drugs.

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Antinociception recording

45

Animals were allowed to acclimatize for at least 30 min before formalin injection in the experimental environment (temperature 23 - I°C). Fifty microliters of 2.5% formalin (1% formaldehyde) was injected subcutaneously into the plantar surface of the right hind paw of the rats using a microsyringe with a 26-gauge needle. Immediately after formalin injection, animals were placed individually in a wire mesh cage (21 ×21 cm) on a flat glass floor and a mirror was arranged in a 45 ° angle under the cage to allow for clear observation of the animals' paws. The severity of pain was recorded using the following scores: 0=rats walked or stood firmly on the injected paw; 1 =partially elevated or favored the paw; 2 = elevated the paw without contact with the floor; or 3 =licked, bit or shook the paw. Two distinct nociceptive time periods are shown to be induced following formalin injection, the first representing phasic pain and the second period representing tonic pain.

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Phaclofen, when used alone (2.4 txg/rat, ICV), decreased the nociceptive response in the first [F(2, 15) = 18.76, P<0.0001] and second IF(2, 15)= 64.45, P<0.0001] phase of the formalin test. Also, bicuculline administration (0.1, 0.5 txg/rat, ICV) induced antinociception in the first [F(2, 15)=7.63, P<0.01] and second [F(2, 15)=48.54, P<0.0001] phase of the test (data not shown). Figure 2 shows the effects of the GABAB antagonist, phaclofen, on IP administration of baclofen. When animals were administered phaclofen (1 mg kg-l, IP) 30 min before baclofen (IP) injection, a significant decrease in baclofen response in the first (A) [F(7, 40)=31.9, P<0.0001] and second (B) phase IF(7, 40)=666.9, P<0.0001] was found. Table 1 shows the responses of baclofen in the presence of picrotoxin and bicuculline. Different doses of baclofen (5 and 10 mg kg -1, IP) induced antinociception in the first [F(4, 25)=63.98, P<0.0001] and the second phase IF(4, 25)= 1260, P<0.0001] of the test. Pretreatment of animals with picrotoxin (1 mg kg -1, IP) and

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Effects of GABA antagonists on antinociceptive response induced by baclofen

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The formalin test score was calculated based on the method of Dubuisson and Dennis (1977). Total scores during periods of 0-5 and 15-45 min after formalin injections were measured as an indicator of nociception. Data were expressed as the mean+SEM. AN©VA followed by Newman-Keuls test were used for analysis of the data. Differences between means were considered statistically significant if P<0.05.

Figure 1 indicates the effect of IP injection of baclofen on formalininduced pain. Different doses of the drug (2.5, 5 and 10 mg kg-1) induced antinociception in the first (A) IF(3, 20)=402, P<0.0001] and the second (B) phase [F(3, 20)=2035, P<0.0001] of the test. The response was dose-dependent. Maximum response was achieved at 0-5 and 15-45 min after formalin administration. When the different doses of the drug (0.5, 1, 5 and 20 Ixg/rat) were administered ICV, antinociceptive response was induced only in the second phase of the test [F(4, 25)=43.4, P<0.0001] (data not shown).

-A

2.5 5 10 Baelofen (mg/Kg)

FIGURE 1. Antinociceptive effects of baclofen on the formalin test. B a c l o f e n w a s injected intraperitoneally (IP) 15 min before formalin administration. Nociceptive behavior w a s recorded 0-5 (A) and 15-45 rain (B) after formalin injection. Each point is the m e a n + SEM of six experiments. * P < 0.01 statistical difference from saline-treated group.

bicuculline (1.5 mg kg -1, IP) 30 min prior to 1P injection of different doses of baclofen (5 and 10 mg kg 1) did not show a significant difference in the first [F(2, 15)=1.26, P>0.05] and second [F(2, 15)=0.24, P>0.05] phases of the test. Table 2 shows the effects of GABA antagonists on the response induced by ICV injection of baclofen. ICV administration of baclofen (20 ~g/rat) induced antinociception in the second phase of the formalin test [F(2, 15)=49.9, P<0.001]. Pretreatment with picrotoxin ( lmg kg -l) and bicuculline ( 1.5 mg kg -1) 30 min prior to ICV injection of baclofen (20 l~g/rat) did not show a significant difference in the first [F(2, 15)=0.0743, P>0.05] and second [F(2, 15)=0.135, P>0.05] phase of the formalin test; however phaclofen pretreatment (ling kg -~, IP, 30 rain) decreased the baclofen (ICV) response [F(3, 20)=32.1, P<0.0001).

DISCUSSION The formalin test (Dubuisson and Dennis, 1977) is an animal model of nociception associated with tissue injury and inflammation that is sensitive to a variety of opioid and nonopioid analgesics (Franklin and Abbott, 1989, 1993; Tjclsen et al., 1992). In the present work,

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Baclofen (mg/Kg) FIGURE 2. Effect of phaclofen on baclofen-induced antinociception. Animals were administered baclofen 15 min and phaclofen (1 mg kg -~) 30 min prior to formalin injection. Nociceptive behavior was recorded 0-5 (A) and 15-45 (B) min after formalin administration. Each point is the mean + SEM of six experiments. (O) Saline+baclofen; (O) phaclofen+baclofen. * P < 0.01 statistical difference from baclofen control groups.

the mechanism of antinociception induced by the GABAB agonist baclofen on the formalin test has been investigated. A number of pharmacologic studies suggest that the inhibitory regulation of the projection from the periaqueductal gray (PAG) to the nu-

cleus raphe magnus is in part GABAergic (Moreau and Fields, 1986; Reichling and Basbaum, 1990). High levels of GABAB receptors have been reported in the PAG and dorsal horn of the spinal cord (Gehlert et al., 1985; McCabe and Wamsley, 1986). These areas have been shown to be involved in both the transmission and inhibition of nociception (Levy and Proudfit, 1979; Matsumoto, 1989). The GABAB receptor agonist baclofen has been reported to produce analgesia in tail-flick, hot-plate and stress tests following systemic administration in mice and rats (Cutting and Jordan, 1975; Levy and Proudfit, 1977; Proudfit and Levy, 1978). The nature of the baclofen-induced antinociception has been proposed to be mediated through both spinal (Wilson and Yaksh, 1978) and supraspinal (Levy and Proudfit, 1979) mechanisms. Baclofen is a selective agonist at GABAB receptors (Bowery et al., 1983, 1984). However, some pharmacologic data support the involvment of GABAA receptors in the analgesic effects of baclofen in the tail-flick test (Zarrindast and Djavdan, 1988). In the present work, different doses of baclofen (ICV and IP) were examined by the formalin test. The drug induced antinociception dose-dependently in both ICV and IP administration. This may be in agreement with the findings of other investigators showing that supraspinal GABAergic mechanisms mediate the baclofen antinociceptive action. In the present study, the antinociceptive effect of baclofen was reduced by the GABAB antagonist phaclofen. Therefore, the GABAB receptor mechanism may be involved in the antinociceptive response of baclofen. Picrotoxin, the noncompetitive GABAA receptor antagonist (Ticku and Maksay, 1983) has been shown to induce analgesia in the hot-plate and tail-flick tests (Zarrindast and Djavdan, 1988). In another work, neither picrotoxin, nor bicuculline, the competitive GABAA receptor antagonist (Ticku and Maksay, 1983), induced any analgesic effect in the formalin test. Bicuculline or picrotoxin administration did not reduce the response induced by ICV or IP injection of baclofen. These results further support that the present antinociceptive response induced by baclofen in the formalin test is mediated through a GABAB mechanism. It has previously been shown that baclofen may induce analgesia through GABAA receptor sites indirectly (Zarrindast and Djavdan, 1988) in the tail-flick test. However, baclofen has been shown to increase the release of GABA (Kerwin and Pycock, 1978; Roberts et al., 1978) and displaces pH]GABA from conventional binding sites in brain membranes (Olsen et al., 1978; Waddington and Cross, 1979). Therefore, the GABA released by the drug may stimu-

TABLE 1. Effects of baclofen in the presence or absence of GABA receptor antagonists on first (0-5 rain) and second phase (15-45 min) of the formalin test Pretreatment (mg kg-1; IP) -Bicuculline 1.5 Picrotoxin 1 Saline 10 ml kg -l Saline 10 ml kg-I Bicuculline 1.5 Bicuculline 1.5 Picrotoxin 1 Picrotoxin 1

Treatment (rag kg-1); IP Saline 10 ml kg -1 Saline 10 ml kg -1 Saline 10 ml kg -1 Baclofen 5 Baclofen 10 Baclofen 5 Baclofen 10 Baclofen 5 Baclofen 10

First phase (0-5 min)

Second phase (15-45 min)

41.5 38 37.2 19.7 6.7 17 7.5 16.7 8.5

240.5 + 4.2 237 _+ 1.9 246 + 1.9 137 _+3.6* 7.2 - 2.38* 138 _+ 2.1 12.7 4- 2.8 140 -+ 3.4 14.7 -+ 1.9

_+ 2.7 -+ 1.4 + 1.7 -4- 1.4" -+ 1.8" -+ 1.4 _+ 1.9 -+ 1.6 + 1.9

Animals were treated with saline (10 ml kg-1, IP) or baclofen (5 and 10 mg kg 1), picrotoxin (1 mg kg<) and bicuculline(1.5 mg kg<) 30 min prior to formalinadministration.Antinociceptive behaviorwas recorded during 0-5 and 15~.5 min after formalin injection. Each value is the mean -4- SEM of six experiments. * P < 0.01, statistical differencefrom respective groups.

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T A B L E 2. Effects of G A B A receptor antagonists on baclofen-induced antinociception at first

(0-5 rain) and second phase {15-45 min) of the formalin test. Pretreatment mg kg-l; IP) --Saline 10 ml kg 1 Picrotoxin 1 Bicuculline 1.5 Phaclofen 1

Treatment

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Second phase (15-45 min)

40.8 36.8 37 37.2 38.8 34.3

258 191.8 190.2 192 194 245

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_+ 4.3 _+ 6.5* _+ 5.4* _+ 5.3 -+ 4.8 _+ 2.7*

Animals were administered saline (5 p,l/rat, ICV) and baclofen (20 p.g/rat, ICV) alone, or baclofen (1CV) 30 rain after either saline (I0 ml kg 1, IP), picrotoxin (1 mg kg i IP), bicuculline (1 mg kg -1, 1P) or phaclofen ( 1 mg kg ~, IP) injection. Nociceptive behavior was recorded during 0-5 and 15-45 min after formalin injection. Each value is the mean -+ SEM of six experiments. *P < 0.001 statistical difference from respective groups.

late the G A B A ^ receptor. One possibility is that the drug shows different mechanisms in these different tests. In the present work, the antagonists, when used alone, also decreased the nociceptive response. This effect of antagonists may be due to presynaptic G A B A receptor blockade and in turn release of GABA, which may stimulate either GABAA or GABAB receptors and induce antinociception.

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615 Wilson P. R. and ¥aksh T. L. (1978) Baclofen is antinociceptive in the spinal intrathecal space of animals. Eur. J. Pharmac. 51,323-330. Zarrindast M. R. and Djavdan M. (1988) GABA antagonists and baclofen analgesia. Gen. Pharmac. 19, 703-706.