- Email: [email protected]
Electroencephalographic study of SR 95103, a GABAA antagonist: Interaction with inhibitory amino acids and muscimol
European Journal of Pharmacology, 114 (1985) 219-222
219
Elsevier
Short communication E L E C T R O E N C E P H A L O G R A P H I C S T U D Y OF SR 95103, A GABA A A N T A G O N I S T : I N T E R A C T I O N W I T H I N H I B I T O R Y A M I N O ACIDS AND M U S C I M O L V I N C E N T S A N T U C C I *, M I C H E L E F O U R N I E R , J E A N - P I E R R E C H A M B O N and K A T H L E E N BIZIERE
Department of Neurobiology, Sanofi Recherche, Rue du Pr. J. Blayac, 34082 Montpellier Cedex, France Received 10 June 1985, accepted 19 June 1985
V. SANTUCCI, M. FOURNIER, J.P. CHAMBON and K. BIZIERE, Electroencephalographic study of SR 95103, a GABA A antagonist: interaction with inhibitory amino acids and muscimol, European J. Pharmacol. 114 (1985) 219-222. SR 95103 has recently been described as a selective GABA A antagonist. In this study, the electroencephalographic (EEG) effects of SR 95103 were investigated as well as its central interaction with inhibitory amino acids and muscimol. Slow intravenous infusions of SR 95103 in rats induced epileptiform EEG activities which were antagonized by intracerebroventricularly injected muscimol, GABA and taurine whereas glycine did not modify and even facilitated the effects of SR 95103. These results suggest that the EEG effects of SR 95103 are due to the specific GABA A antagonistic properties of this compound. Electroencephalogram
GABA A antagonist
SR 95103
1. Introduction SR 95103 [2-(carboxy-3'-propyl)-3-amino-4methyl-6-phenyl pyridazinium chloride] has recently been shown to be a selective and competitive antagonist of G A B A at the GABA A receptor site (Chambon et al., 1985). The compound displaces [3H]GABA from rat brain membranes with a K~ of 2.2 /~M, antagonizes the GABA-elicited increase of [3H]diazepam binding and competitively antagonizes GABA-induced membrane depolarization in rat spinal ganglia. In vitro, the potency of SR 95103 is close to that of bicuculline. Moreover, SR 95103 does not interact with GABA B, benzodiazepine, strychnine and glutamate receptors. When injected intraperitoneally (i.p.) to mice, SR 95103 induces clonic-tonic seizures with a potency approximately 100 fold lower than that of bicuculline, suggesting either that the compound does not readily cross the blood-brain barrier or that it is rapidly metabolized. * To whom correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Muscimol
Taurine
Glycine
GABA antagonists such as bicuculline or picrotoxine typically elicit behavioral and electrocortical (EEG) paroxysms (Meldrum, 1975) which are antagonized by GABA agonists (Lloyd et al., 1979; Woodbury, 1980; Stone and Javid, 1981). The purpose of the present study was to evaluate the EEG effects of intravenously (i.v.) administered SR 95103 on the cortical seizure threshold of the rat. We also examined the effects of central inhibitory amino acids and muscimol on the EEG paroxysms induced by SR 95103.
2. Materials and methods 2.1. Procedure
Male, Sprague-Dawley C D rats (Charles River Breeding Laboratories, France), weighing 180-200 g, were anesthetized with ether and positioned in a David-Kopf stereotaxic apparatus, the ear bars of which were coated with lidocaine paste; 4 burr holes were drilled in the calvarium, 2 and 5 m m posterior to the coronal suture and bilaterally 2.5
220 m m from the sagittal suture; a cortical silver electrode was implanted through each burr hole. The animals were then immobilized with gallamine and artificially ventilated, according to the respiration parameters of the rat as described by Baker et al. (1979). Small amounts of lidocaine were injected around the wound margins. The E E G was recorded on paper with a Hewlett-Packard 7700 electroencephalograph. After the E E G was started, SR 95103 (15 m g / m l saline) was infused into the jugular vein with a Braun perfusor, at the speed of 0.2 m l / m i n . The dose of SR 95103 was chosen as that inducing the seizure with a maximum latency of 30 min. The infusion was stopped at the time the seizure occurred. Simultaneously, either GABA (1 mg), muscimol (MUS, 0.3 mg), taurine (TAU, 0.5 mg) or glycine (GLY, 1 and 0.1 mg) were injected into the left lateral ventricle over 2 min in a 10 ~1 saline solution under stereotaxic guidance according to De Groot's (1959) coordinates. 'Control' animals (SR 95103 alone) received 10 ~tl of saline intracerebroventricularly (i.c.v.). GABA, MUS, T A U and G L Y were assessed for their ability to modify the E E G variables of the epileptiform activity induced by SR 95103, namely: (i) the latency of occurrence of the first generalized spike (LFS); and (ii) the latency of seizure occurrence (LSO).
2.2. Drugs
3. Results
3.1. EEG effects of SR 95103 SR 95103 showed potent irritating and seizure activity (fig. 1). About 1 min after the onset of infusion, low-voltage, desynchronised E E G patterns were observed, followed by the appearance of high-voltage, isolated spikes. These spikes be-
BEFORE INFUSION
600 uv I 5s
A
C
D J
SR 95103 was synthesized by C.G. Wermuth (Universit6 Louis Pasteur, Strasbourg, France). Muscimol was purchased from Fluka A G (Switzerland). GABA, taurine and glycine were purchased from Sigma (U.S.A.). Lidocaine (Xylocaine R) was purchased from Roger Bellon (France).
I
2.3. Statistics The values of the EEG variables were computed for mean and standard error (S.E.). The significance of the effects of GABA, MUS, T A U and G L Y on the E E G parameters was determined by analysis of variance. A level of P < 0.05 was accepted as significant.
Fig. 1. EEG effects of SR 95103. Beforeinfusion (up), low-voltage, desynchronized activity. From the onset of infusion: (A) hyperactivated, desynchronized EEG with voltage reduction and frequency increase; (B) isolated high-voltage brief spikes superimposed on a desynchronized background; (C) frequent, high-voltage spikes-and-waves with flattening of background EEG; and (D) seizure, consisting of 3-4 cycles/s high-voltage spikes with intermingled fast-frequency activities. Calibration for time and voltage is indicated on the right.
221 TABLE 1 Interaction of SR 95103 with i.c.v.-injected muscimol (MUS), GABA, taurine (TAU) and glycine (GLY). Treatment a
LFS (rain) b
LSO (min) b
SR 95103 SR 95103 + M U S 0.3 mg
5.6 + 0.4 5.9+0.4
13.5 +0.6 24.8_+1.7 ** ( + 83.5%)
SR 95103 SR 95103+ G A B A 1 mg
5.9 + 0.6 5.6-+0.6
15.3 _+0.9 18.6-+0.9 * ( + 21.5%)
SR 95103 SR 95103 + T A U 0.5 m g
8.1 _+0.9 9.5+_1.1
13.8 -+ 1.4 19.9+_2.0 * ( + 43.7%)
SR 95103 SR 95103 + G L Y 0.1 mg
5.5 _+0.3 5.2+_0.3
14.9 5=0.5 15.4+_0.8
SR 95103 SR 9 5 1 0 3 + G L Y 1 mg
5.9-+0.5 6.0+_0.3
13.9+_0.6 10.8-+0.3"* ( - 22.5%)
a Ten animals were used for each treatment, b MUS, GABA, T A U and G L Y were evaluated for their ability to modify the latency of occurrence of the first generalized spike (LFS) and the latency of seizure occurrence (LSO). The value of both parameters are indicated as the mean + S.E. Percent changes from SR 95103 alone are shown in parentheses. * P < 0.05; • * P < 0.001 (analysis of variance).
came synchronous on all the derivations approx. 5 min later and their frequency increased progressively. The final phase was the seizure itself (latency of about 14 min and 10 to 30 s duration) with 3-4 cycles/s high-voltage spikes, superimposed on a background of fast frequency activities.
3.2. Interaction stud), MUS, GABA and TAU antagonized the epileptogenic effects of SR 95103, as shown by a significant increase in LSO (table 1). GLY had no effect at the dose of 0.1 mg but significantly reduced LSO at 1 mg. None of the compounds tested affected LFS.
4. Discussion The present study showed that SR 95103 evoked in the EEG of the rat a typical epileptiform activ-
ity which was antagonized by MUS, GABA and TAU, but not by GLY. The EEG effects of SR 95103 are consistent with those reported for other GABA antagonists (Meldrum, 1975; Lloyd et al., 1979) and reflect a reduction of seizure threshold. The results of the interaction study were consistent with the specificity and selectivity of SR 95103 since compounds such as MUS, GABA and TAU, but not GLY, antagonize its epileptogenic effects. The anticonvulsant effects of TAU are in agreement with its reported affinity for the GABA receptor site (Olsen et al., 1978), although this affinity is approximately 200-fold lower than that of GABA itself. In addition, TAU stimulates GABA release in vitro (Leach, 1979) and this is likely to be the case in vivo also. The GLY-induced facilitation of SR 95103 effects was unexpected, since GLY is known to be inhibitory in both spinal cord and brain (Snyder et al., 1973, Davidson 1976). Since SR 95103 does not interact with the strychnine binding site (Chambon et al., 1985), it seems difficult to give a satisfactory explanation of the GLY effects observed in our study. One can only speculate that GLY may increase the bioavailability of SR 95103 or that it inhibits GABA release. In conclusion, the present study shows that SR 95103 exerts potent epileptogenic effects on the rat brain and that these effects were reversed by GABA agonists but not by glycine. SR 95103 could thus be a useful tool to investigate the brain dynamics of GABA-mediated behavioral phenomena.
Acknowledgements We wish to thank M. Heaulme and J.C. Michaud for helpful discussion of the results.
References Baker, H.J., J.R. Lindsey and S.H. Weisbroth, 1979, Selected Normative Data, in: The Laboratory Rat, eds. H.J. Baker, J.R. Lindsey and S.H. Weisbroth (Academic Press, New York) 1, p. 411. Chambon, J.P., P. Feltz, M. Heaulme, S. Restle, R. Schlichter, K. Bizi~re and C.G. Wermuth, 1985, An aryl-aminopyrida-
222 zine derivative of GABA is a new selective and competitive antagonist at the GABA A receptor site, Proc. Natl. Acad. Sci. U.S.A. 82, 1832. Davidson, N., 1976, Neurotransmitter amino acids (Academic Press, New York) p. 179. De Groot, J., 1959, The rat forebrain in stereotaxic coordinates (Elsevier, Amsterdam, New York). Leach, M.J., 1979, Effect of taurine on release of [3H]GABA by depolarizing stimuli from superfused slices of rat brain cerebral cortex in vitro, J. Pharm. Pharmacol. 31,533. Lloyd, K.G., P. Worms, H. Depoortere and G. Bartholini, 1979, Pharmacological profile of SL 76002, a new GABAmimetic drug, in: GABA Neurotransmitters, eds. P. Krogsgaard-Larsen, J. Scbeel-Kr~ger and H. Kofod, Alfred Benzon Symposium 12, (Munksgaard, Copenhagen) p. 308.
Meldrum, B.S., 1975, Epilepsy and T-aminobutyric acid-mediated inhibition, Int. Rev. Neurobiol. 17, 1. Olsen, R.W., M.K. Ticku, D. Greenlee and P. Van Ness, 1978, GABA receptor and ionophore binding sites: interaction with various drugs, in: GABA Neurotransmitters, eds. P. Krogsgaard-Larsen, J. Scheel-Kriager and H. Kofod, Alfred Benzon Symposium 12, (Munskgaard, Copenhagen) p. 165. Snyder, S.H., A.B. Young, J.P. Bennet and A.H. Mulder, 1973, Synaptic biochemistry of amino acids, Fed. Proc. 32, 2039. Stone, W.E. and M.J. Javid, 1981, Muscimol as an antagonist of chemical convulsants, Arch. Int. Pharmacodyn. 253, 294. Woodbury, D.M., 1980, Convulsant drugs: mechanisms of action, in: Antiepileptic Drugs: Mechanisms of Action, eds. G.H. Glaser, J.K. Penry and D.M. Woodbury (Raven Press, New York) p. 249.
219
Elsevier
Short communication E L E C T R O E N C E P H A L O G R A P H I C S T U D Y OF SR 95103, A GABA A A N T A G O N I S T : I N T E R A C T I O N W I T H I N H I B I T O R Y A M I N O ACIDS AND M U S C I M O L V I N C E N T S A N T U C C I *, M I C H E L E F O U R N I E R , J E A N - P I E R R E C H A M B O N and K A T H L E E N BIZIERE
Department of Neurobiology, Sanofi Recherche, Rue du Pr. J. Blayac, 34082 Montpellier Cedex, France Received 10 June 1985, accepted 19 June 1985
V. SANTUCCI, M. FOURNIER, J.P. CHAMBON and K. BIZIERE, Electroencephalographic study of SR 95103, a GABA A antagonist: interaction with inhibitory amino acids and muscimol, European J. Pharmacol. 114 (1985) 219-222. SR 95103 has recently been described as a selective GABA A antagonist. In this study, the electroencephalographic (EEG) effects of SR 95103 were investigated as well as its central interaction with inhibitory amino acids and muscimol. Slow intravenous infusions of SR 95103 in rats induced epileptiform EEG activities which were antagonized by intracerebroventricularly injected muscimol, GABA and taurine whereas glycine did not modify and even facilitated the effects of SR 95103. These results suggest that the EEG effects of SR 95103 are due to the specific GABA A antagonistic properties of this compound. Electroencephalogram
GABA A antagonist
SR 95103
1. Introduction SR 95103 [2-(carboxy-3'-propyl)-3-amino-4methyl-6-phenyl pyridazinium chloride] has recently been shown to be a selective and competitive antagonist of G A B A at the GABA A receptor site (Chambon et al., 1985). The compound displaces [3H]GABA from rat brain membranes with a K~ of 2.2 /~M, antagonizes the GABA-elicited increase of [3H]diazepam binding and competitively antagonizes GABA-induced membrane depolarization in rat spinal ganglia. In vitro, the potency of SR 95103 is close to that of bicuculline. Moreover, SR 95103 does not interact with GABA B, benzodiazepine, strychnine and glutamate receptors. When injected intraperitoneally (i.p.) to mice, SR 95103 induces clonic-tonic seizures with a potency approximately 100 fold lower than that of bicuculline, suggesting either that the compound does not readily cross the blood-brain barrier or that it is rapidly metabolized. * To whom correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Muscimol
Taurine
Glycine
GABA antagonists such as bicuculline or picrotoxine typically elicit behavioral and electrocortical (EEG) paroxysms (Meldrum, 1975) which are antagonized by GABA agonists (Lloyd et al., 1979; Woodbury, 1980; Stone and Javid, 1981). The purpose of the present study was to evaluate the EEG effects of intravenously (i.v.) administered SR 95103 on the cortical seizure threshold of the rat. We also examined the effects of central inhibitory amino acids and muscimol on the EEG paroxysms induced by SR 95103.
2. Materials and methods 2.1. Procedure
Male, Sprague-Dawley C D rats (Charles River Breeding Laboratories, France), weighing 180-200 g, were anesthetized with ether and positioned in a David-Kopf stereotaxic apparatus, the ear bars of which were coated with lidocaine paste; 4 burr holes were drilled in the calvarium, 2 and 5 m m posterior to the coronal suture and bilaterally 2.5
220 m m from the sagittal suture; a cortical silver electrode was implanted through each burr hole. The animals were then immobilized with gallamine and artificially ventilated, according to the respiration parameters of the rat as described by Baker et al. (1979). Small amounts of lidocaine were injected around the wound margins. The E E G was recorded on paper with a Hewlett-Packard 7700 electroencephalograph. After the E E G was started, SR 95103 (15 m g / m l saline) was infused into the jugular vein with a Braun perfusor, at the speed of 0.2 m l / m i n . The dose of SR 95103 was chosen as that inducing the seizure with a maximum latency of 30 min. The infusion was stopped at the time the seizure occurred. Simultaneously, either GABA (1 mg), muscimol (MUS, 0.3 mg), taurine (TAU, 0.5 mg) or glycine (GLY, 1 and 0.1 mg) were injected into the left lateral ventricle over 2 min in a 10 ~1 saline solution under stereotaxic guidance according to De Groot's (1959) coordinates. 'Control' animals (SR 95103 alone) received 10 ~tl of saline intracerebroventricularly (i.c.v.). GABA, MUS, T A U and G L Y were assessed for their ability to modify the E E G variables of the epileptiform activity induced by SR 95103, namely: (i) the latency of occurrence of the first generalized spike (LFS); and (ii) the latency of seizure occurrence (LSO).
2.2. Drugs
3. Results
3.1. EEG effects of SR 95103 SR 95103 showed potent irritating and seizure activity (fig. 1). About 1 min after the onset of infusion, low-voltage, desynchronised E E G patterns were observed, followed by the appearance of high-voltage, isolated spikes. These spikes be-
BEFORE INFUSION
600 uv I 5s
A
C
D J
SR 95103 was synthesized by C.G. Wermuth (Universit6 Louis Pasteur, Strasbourg, France). Muscimol was purchased from Fluka A G (Switzerland). GABA, taurine and glycine were purchased from Sigma (U.S.A.). Lidocaine (Xylocaine R) was purchased from Roger Bellon (France).
I
2.3. Statistics The values of the EEG variables were computed for mean and standard error (S.E.). The significance of the effects of GABA, MUS, T A U and G L Y on the E E G parameters was determined by analysis of variance. A level of P < 0.05 was accepted as significant.
Fig. 1. EEG effects of SR 95103. Beforeinfusion (up), low-voltage, desynchronized activity. From the onset of infusion: (A) hyperactivated, desynchronized EEG with voltage reduction and frequency increase; (B) isolated high-voltage brief spikes superimposed on a desynchronized background; (C) frequent, high-voltage spikes-and-waves with flattening of background EEG; and (D) seizure, consisting of 3-4 cycles/s high-voltage spikes with intermingled fast-frequency activities. Calibration for time and voltage is indicated on the right.
221 TABLE 1 Interaction of SR 95103 with i.c.v.-injected muscimol (MUS), GABA, taurine (TAU) and glycine (GLY). Treatment a
LFS (rain) b
LSO (min) b
SR 95103 SR 95103 + M U S 0.3 mg
5.6 + 0.4 5.9+0.4
13.5 +0.6 24.8_+1.7 ** ( + 83.5%)
SR 95103 SR 95103+ G A B A 1 mg
5.9 + 0.6 5.6-+0.6
15.3 _+0.9 18.6-+0.9 * ( + 21.5%)
SR 95103 SR 95103 + T A U 0.5 m g
8.1 _+0.9 9.5+_1.1
13.8 -+ 1.4 19.9+_2.0 * ( + 43.7%)
SR 95103 SR 95103 + G L Y 0.1 mg
5.5 _+0.3 5.2+_0.3
14.9 5=0.5 15.4+_0.8
SR 95103 SR 9 5 1 0 3 + G L Y 1 mg
5.9-+0.5 6.0+_0.3
13.9+_0.6 10.8-+0.3"* ( - 22.5%)
a Ten animals were used for each treatment, b MUS, GABA, T A U and G L Y were evaluated for their ability to modify the latency of occurrence of the first generalized spike (LFS) and the latency of seizure occurrence (LSO). The value of both parameters are indicated as the mean + S.E. Percent changes from SR 95103 alone are shown in parentheses. * P < 0.05; • * P < 0.001 (analysis of variance).
came synchronous on all the derivations approx. 5 min later and their frequency increased progressively. The final phase was the seizure itself (latency of about 14 min and 10 to 30 s duration) with 3-4 cycles/s high-voltage spikes, superimposed on a background of fast frequency activities.
3.2. Interaction stud), MUS, GABA and TAU antagonized the epileptogenic effects of SR 95103, as shown by a significant increase in LSO (table 1). GLY had no effect at the dose of 0.1 mg but significantly reduced LSO at 1 mg. None of the compounds tested affected LFS.
4. Discussion The present study showed that SR 95103 evoked in the EEG of the rat a typical epileptiform activ-
ity which was antagonized by MUS, GABA and TAU, but not by GLY. The EEG effects of SR 95103 are consistent with those reported for other GABA antagonists (Meldrum, 1975; Lloyd et al., 1979) and reflect a reduction of seizure threshold. The results of the interaction study were consistent with the specificity and selectivity of SR 95103 since compounds such as MUS, GABA and TAU, but not GLY, antagonize its epileptogenic effects. The anticonvulsant effects of TAU are in agreement with its reported affinity for the GABA receptor site (Olsen et al., 1978), although this affinity is approximately 200-fold lower than that of GABA itself. In addition, TAU stimulates GABA release in vitro (Leach, 1979) and this is likely to be the case in vivo also. The GLY-induced facilitation of SR 95103 effects was unexpected, since GLY is known to be inhibitory in both spinal cord and brain (Snyder et al., 1973, Davidson 1976). Since SR 95103 does not interact with the strychnine binding site (Chambon et al., 1985), it seems difficult to give a satisfactory explanation of the GLY effects observed in our study. One can only speculate that GLY may increase the bioavailability of SR 95103 or that it inhibits GABA release. In conclusion, the present study shows that SR 95103 exerts potent epileptogenic effects on the rat brain and that these effects were reversed by GABA agonists but not by glycine. SR 95103 could thus be a useful tool to investigate the brain dynamics of GABA-mediated behavioral phenomena.
Acknowledgements We wish to thank M. Heaulme and J.C. Michaud for helpful discussion of the results.
References Baker, H.J., J.R. Lindsey and S.H. Weisbroth, 1979, Selected Normative Data, in: The Laboratory Rat, eds. H.J. Baker, J.R. Lindsey and S.H. Weisbroth (Academic Press, New York) 1, p. 411. Chambon, J.P., P. Feltz, M. Heaulme, S. Restle, R. Schlichter, K. Bizi~re and C.G. Wermuth, 1985, An aryl-aminopyrida-
222 zine derivative of GABA is a new selective and competitive antagonist at the GABA A receptor site, Proc. Natl. Acad. Sci. U.S.A. 82, 1832. Davidson, N., 1976, Neurotransmitter amino acids (Academic Press, New York) p. 179. De Groot, J., 1959, The rat forebrain in stereotaxic coordinates (Elsevier, Amsterdam, New York). Leach, M.J., 1979, Effect of taurine on release of [3H]GABA by depolarizing stimuli from superfused slices of rat brain cerebral cortex in vitro, J. Pharm. Pharmacol. 31,533. Lloyd, K.G., P. Worms, H. Depoortere and G. Bartholini, 1979, Pharmacological profile of SL 76002, a new GABAmimetic drug, in: GABA Neurotransmitters, eds. P. Krogsgaard-Larsen, J. Scbeel-Kr~ger and H. Kofod, Alfred Benzon Symposium 12, (Munksgaard, Copenhagen) p. 308.
Meldrum, B.S., 1975, Epilepsy and T-aminobutyric acid-mediated inhibition, Int. Rev. Neurobiol. 17, 1. Olsen, R.W., M.K. Ticku, D. Greenlee and P. Van Ness, 1978, GABA receptor and ionophore binding sites: interaction with various drugs, in: GABA Neurotransmitters, eds. P. Krogsgaard-Larsen, J. Scheel-Kriager and H. Kofod, Alfred Benzon Symposium 12, (Munskgaard, Copenhagen) p. 165. Snyder, S.H., A.B. Young, J.P. Bennet and A.H. Mulder, 1973, Synaptic biochemistry of amino acids, Fed. Proc. 32, 2039. Stone, W.E. and M.J. Javid, 1981, Muscimol as an antagonist of chemical convulsants, Arch. Int. Pharmacodyn. 253, 294. Woodbury, D.M., 1980, Convulsant drugs: mechanisms of action, in: Antiepileptic Drugs: Mechanisms of Action, eds. G.H. Glaser, J.K. Penry and D.M. Woodbury (Raven Press, New York) p. 249.